Anti-TNFR2 antibodies and uses thereof

Abstract

Anti-TNFR2 (tumor necrosis factor receptor 2) antibodies, antigen-binding fragments, and uses thereof are provided.

Description

[Technical field]

[0001] Claiming priority This application claims the benefit of International Application No. PCT / CN2021 / 140487, filed December 22, 2021. The entire contents of the foregoing are incorporated herein by reference.

[0002] (Technical field) The present disclosure relates to anti-TNFR2 (Tumor Necrosis Factor Receptor 2) antibodies, and uses thereof. [Background technology]

[0003] Cancer is currently one of the diseases with the highest mortality rates among humans. According to statistics from the World Health Organization, in 2012, the incidence and mortality rates of cancer worldwide reached 14 million and 8.2 million, respectively. In China, the number of newly diagnosed cancer cases was 3.07 million, and the number of deaths was 2.2 million.

[0004] The recent clinical and commercial success of anti-cancer antibodies has generated considerable interest in antibody-based therapies, and there is a need for the development of antibodies for use in a variety of antibody-based therapeutic approaches to treat cancer or autoimmune diseases. Summary of the Invention

[0005] The present disclosure relates to anti-TNFR2 antibodies, antigen-binding fragments thereof, and uses thereof.

[0006] In one aspect, the disclosure provides a polypeptide comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence, The CDR1, 2, and 3 amino acid sequences and the selected VL CDR1, 2, and 3 amino acid sequences are as follows: (1) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 6, 7, and 8, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 9, 10, and 11, respectively; (2) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 12, 13, and 14, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 15, 16, and 17, respectively; (3) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 18, 19, and 20, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 21, 22, and 23, respectively; (4) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 24, 25, and 26, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 27, 28, and 29, respectively; (5) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 30, 31, and 32, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 33, 34, and 35, respectively; (6) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 42, 43, and 44, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 45, 46, and 47, respectively; (7) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 48, 49, and 50, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 51, 52, and 53, respectively; (8) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 54, 55, and 56, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 57, 58, and 59, respectively; (9) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 60, 61, and 62, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 63, 64, and 65, respectively; or (10) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 66, 67, and 68, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 69, 70, and 71, respectively; One of the The present invention relates to an antibody or an antigen-binding fragment thereof that binds to TNFR2.

[0007] In some embodiments, according to Kabat numbering, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 9, 10, and 11, respectively.

[0008] In some embodiments, according to Kabat numbering, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 15, 16, and 17, respectively.

[0009] In some embodiments, according to Kabat numbering, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 21, 22, and 23, respectively.

[0010] In some embodiments, according to Kabat numbering, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 24, 25, and 26, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 27, 28, and 29, respectively.

[0011] In some embodiments, according to Kabat numbering, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 30, 31, and 32, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively.

[0012] In some embodiments, the antibody or antigen-binding fragment specifically binds to human TNFR2.

[0013] In some embodiments, the antibody or antigen-binding fragment is a human antibody or antigen-binding fragment thereof (eg, a human IgG1 antibody).

[0014] In some embodiments, the antibody or antigen-binding fragment is a single chain variable fragment (scFv).

[0015] In one aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: (1) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively, 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; (2) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NO: 9, 10, and 11, respectively, wherein the VL binds to TNFR2 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 36; (3) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, 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; (4) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 15, 16, and 17, respectively, wherein the VL binds to TNFR2 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 38; (5) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, respectively, 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; (6) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 21, 22, and 23, respectively, wherein the VL binds to TNFR2 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 40; (7) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 24, 25, and 26, respectively, 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; (8) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NO: 27, 28, and 29, respectively, wherein the VL binds to TNFR2 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 72; (9) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NO: 30, 31, and 32, respectively, 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 (10) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NO: 33, 34, and 35, respectively, wherein the VL binds to TNFR2 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 74; The present invention relates to a nucleic acid comprising a polynucleotide encoding a polypeptide comprising: In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively.

[0016] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof, comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 10, and 11, respectively.

[0017] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively.

[0018] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof, comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 15, 16, and 17, respectively.

[0019] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, respectively.

[0020] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof, comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 21, 22, and 23, respectively.

[0021] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 24, 25, and 26, respectively.

[0022] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof, comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 27, 28, and 29, respectively.

[0023] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, comprising a VH comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 30, 31, and 32, respectively.

[0024] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof, comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively.

[0025] In some embodiments, the VH, when paired with the VL, specifically binds human TNFR2, or the VL, when paired with the VH, specifically binds human TNFR2.

[0026] 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.

[0027] In some embodiments, the nucleic acid encodes a single chain variable fragment (scFv).

[0028] In some embodiments, the nucleic acid is cDNA.

[0029] In one aspect, the disclosure relates to a vector comprising one or more of the nucleic acids described herein.

[0030] In one aspect, the disclosure relates to a vector comprising two of the nucleic acids described herein, the vector encoding a VH region and a VL region that both bind to TNFR2.

[0031] In one aspect, the disclosure relates to a pair of vectors, each vector comprising one of the nucleic acids described herein, which together encode a VH region and a VL region that both bind to TNFR2.

[0032] In one aspect, the disclosure relates to a cell comprising a vector described herein or a pair of vectors described herein. In some embodiments, the cell is a CHO cell.

[0033] In one aspect, the disclosure relates to a cell comprising one or more of the nucleic acids described herein.

[0034] 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 both bind to TNFR2.

[0035] In one aspect, the disclosure relates to a method of producing an antibody or antigen-binding fragment thereof, comprising: (a) culturing a cell described herein under conditions sufficient for the cell to produce the antibody or antigen-binding fragment; and (b) harvesting the antibody or antigen-binding fragment produced by the cell.

[0036] In one aspect, the disclosure includes 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: (1) the selected VH sequence is SEQ ID NO: 36 and the selected VL sequence is SEQ ID NO: 37; (2) the selected VH sequence is SEQ ID NO: 38 and the selected VL sequence is SEQ ID NO: 39; (3) the selected VH sequence is SEQ ID NO: 40 and the selected VL sequence is SEQ ID NO: 41; (4) the selected VH sequence is SEQ ID NO: 72 and the selected VL sequence is SEQ ID NO: 73; or (5) the selected VH sequence is SEQ ID NO: 74 and the selected VL sequence is SEQ ID NO: 75; The present invention relates to an antibody or an antigen-binding fragment thereof that binds to TNFR2, which is one of the above.

[0037] In some embodiments, the VH comprises the sequence of SEQ ID NO:36 and the VL comprises the sequence of SEQ ID NO:37.

[0038] In some embodiments, the VH comprises the sequence of SEQ ID NO:72 and the VL comprises the sequence of SEQ ID NO:73.

[0039] In some embodiments, the VH comprises the sequence of SEQ ID NO:38 and the VL comprises the sequence of SEQ ID NO:39.

[0040] In some embodiments, the VH comprises the sequence of SEQ ID NO:40 and the VL comprises the sequence of SEQ ID NO:41.

[0041] In some embodiments, the VH comprises the sequence of SEQ ID NO:74 and the VL comprises the sequence of SEQ ID NO:75.

[0042] In some embodiments, the antibody or antigen-binding fragment specifically binds to human TNFR2.

[0043] In some embodiments, the antibody or antigen-binding fragment is a human antibody or antigen-binding fragment thereof.

[0044] In some embodiments, the antibody or antigen-binding fragment is a single chain variable fragment (scFv).

[0045] 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.

[0046] 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) that comprises a VH CDR1, a VH CDR2, and a VH CDR3 identical to the VH CDR1, a VH CDR2, and a VH CDR3 of a selected VH sequence, and a light chain variable region (VL) that comprises a VL CDR1, a VL CDR2, and a VL CDR3 identical to the VL CDR1, a VL CDR2, and a VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are selected from the following: (1) the selected VH sequence is SEQ ID NO: 36 and the selected VL sequence is SEQ ID NO: 37; (2) the selected VH sequence is SEQ ID NO: 38 and the selected VL sequence is SEQ ID NO: 39; (3) the selected VH sequence is SEQ ID NO: 40 and the selected VL sequence is SEQ ID NO: 41; (4) the selected VH sequence is SEQ ID NO: 72 and the selected VL sequence is SEQ ID NO: 73; or (5) the selected VH sequence is SEQ ID NO: 74 and the selected VL sequence is SEQ ID NO: 75; It is one of them.

[0047] In one aspect, the disclosure relates to an antibody drug conjugate comprising an antibody or antigen-binding fragment thereof described herein covalently linked to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent.

[0048] 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.

[0049] In some embodiments, the subject has colorectal cancer, ovarian cancer, acute myeloid leukemia, Lewis lung carcinoma, 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.

[0050] In one aspect, the disclosure relates to a method of reducing tumor growth rate, the method comprising contacting tumor cells with an effective amount of a composition comprising an antibody or antigen-binding fragment thereof described herein, or an antibody drug conjugate described herein.

[0051] In one aspect, the disclosure relates to a method of killing a tumor cell, the method 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.

[0052] In one aspect, the disclosure relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein and a pharma- ceutically acceptable carrier.

[0053] In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody drug conjugate described herein and a pharma- ceutically acceptable carrier.

[0054] As used herein, the term "cancer" refers to cells with autonomous growth potential. Examples of such cells include cells with an abnormal state or condition characterized by rapid proliferation of cell growth. The term is meant to include cancerous growths, e.g., tumors, oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of the type of tissue change or stage of invasiveness. Also included are malignant tumors of various organ systems, e.g., head and neck, respiratory, cardiovascular, renal, reproductive, hematological, nervous system, hepatic, gastrointestinal, and endocrine systems, as well as most colon cancers, renal cell carcinoma, prostate and / or testicular tumors, non-small cell carcinoma of the lung, small intestine cancer, glioma, and small intestine cancer. "Naturally occurring" cancers include any cancer that is not experimentally induced by implanting cancer cells in a subject, such as naturally occurring cancers, cancers caused by exposing a patient to a carcinogen, cancers caused by the insertion of a transgenic oncogene or knockout of a tumor suppressor gene, and cancers caused by infectious diseases, e.g., viral infections. The term "carcinoma" is art-recognized and refers to a malignant tumor of epithelial or endocrine tissue. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissue. "Adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is art-recognized and refers to a malignant tumor of mesenchymal origin. The term "hematopoietic neoplastic disease" includes diseases involving hyperplastic / neoplastic cells of hematopoietic origin. Hematopoietic neoplastic diseases may arise from myeloid, lymphoid, or erythroid lineages, or from their precursor cells. Hematologic cancers are cancers that begin in blood-forming tissues, such as the bone marrow, or in cells of the immune system. Examples of hematologic cancers include, for example, leukemia, lymphoma, and multiple myeloma.

[0055] As used herein, the term "antibody" refers to any antigen-binding molecule that contains at least one (e.g., one, two, three, four, five, or six) complementarity determining regions (CDRs) (e.g., any of the three CDRs from an immunoglobulin light chain or any of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding to an epitope. Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody can contain an Fc region of a human antibody. The term antibody also includes derivatives, such as bispecific antibodies, single-chain antibodies, diabodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0056] As used herein, the term "antigen-binding fragment" refers to a portion of a full-length antibody, which portion of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a heavy chain variable domain or a light chain variable domain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab)2, and Fv fragments.

[0057] As used herein, the term "human antibody" refers to an antibody encoded by endogenous nucleic acid derived from a human (e.g., a rearranged human immunoglobulin heavy or light chain locus). In some embodiments, a human antibody is recovered from a human or produced in human cell culture (e.g., in human hybridoma cells). In some embodiments, a human antibody is produced in a non-human cell (e.g., a mouse or hamster cell line). In some embodiments, a human antibody is produced in a bacterial cell or a yeast cell. In some embodiments, a human antibody is produced in a transgenic non-human animal (e.g., a cow) that contains unrearranged or rearranged human immunoglobulin loci (e.g., a heavy or light chain human immunoglobulin locus).

[0058] As used herein, the term "chimeric antibody" refers to an antibody that contains sequences present in at least two different species (e.g., an antibody from two different mammalian species, such as a human and a murine antibody). A non-limiting example of a chimeric antibody is an antibody that contains variable domain sequences (e.g., all or a portion of the light and / or heavy chain variable domain sequences) of a non-human (e.g., murine) antibody and the constant domain of a human antibody. Further examples of chimeric antibodies are described herein and are well known in the art.

[0059] As used herein, the term "humanized antibody" refers to a non-human antibody that contains minimal sequence derived from non-human (e.g., murine) immunoglobulin and contains sequence derived from human immunoglobulin. In a non-limiting example, a humanized antibody is a human antibody (recipient antibody) in which residues from hypervariable (e.g., CDR) regions of the recipient antibody are replaced by residues from hypervariable (e.g., CDR) regions of a non-human antibody (e.g., donor antibody), such as a mouse, rat, or rabbit antibody, having the desired specificity, affinity, and capacity. In some embodiments, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human (e.g., murine) immunoglobulin residues. In some embodiments, humanized antibodies can contain residues that are not found in the recipient antibody or in the donor antibody. These modifications can be made to further refine antibody performance. In some embodiments, a humanized antibody contains nearly all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops (CDRs) corresponding to those of a non-human (e.g., murine) immunoglobulin and all or substantially all of the framework regions being those of a human immunoglobulin sequence. The humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Humanized antibodies can be produced using molecular biology techniques well known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.

[0060] As used herein, the term "single chain antibody" means a single polypeptide containing at least two immunoglobulin variable domains (e.g., the variable domains of a mammalian immunoglobulin heavy or light chain) capable of specifically binding to an antigen. Non-limiting examples of single chain antibodies are described herein.

[0061] As used herein, the term "multimeric antibody" refers to an antibody that contains four or more (eg, six, eight, or ten) immunoglobulin variable domains.

[0062] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification and refer to an animal, human, or non-human to which treatment according to the methods of the invention is provided. Veterinary and non-veterinary applications are contemplated in this disclosure. A human patient can be an adult human or a juvenile human (e.g., a human under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. For example, patients include non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, porcines (e.g., pigs, minipigs), equines, canines, felines, bovines, and other domestic, livestock, and zoo animals.

[0063] As used herein, the phrases "specifically binding" and "specifically binds" when referring to an antibody means that the antibody interacts with its target molecule (e.g., TNFR2) preferably in preference to other molecules because the interaction is dependent on the presence of a particular structure (i.e., an antigenic determinant or epitope) on the target molecule, in other words, the reagent generally recognizes and binds to a molecule that contains a particular structure rather than to the entire molecule. An antibody that specifically binds to a target molecule may also be referred to as a target-specific antibody. For example, an antibody that specifically binds to a TNFR2 molecule may also be referred to as a TNFR2-specific antibody or an anti-TNFR2 antibody.

[0064] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to polymers of amino acids of any length, of at least two amino acids.

[0065] As used herein, the terms "polynucleotide," "nucleic acid molecule," and "nucleic acid sequence" are used interchangeably herein and refer to polymers of nucleotides of any length, of at least two nucleotides, including, but not limited to, DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art can also be used.Materials, methods, and examples are illustrative only and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will control.

[0067] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. [Brief description of the drawings]

[0068] [Figure 1] FIG. 13 is a graph showing 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). [Diagram 2] FIG. 13 is a graph showing 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-1F10-IgG1 (G3), BC-1F4-IgG1 (G4), anti-mPD-1 (G5), anti-mCTLA4 (G6). [Diagram 3] FIG. 13 is a graph showing tumor size over time in mice injected with MC38 cancer cells and treated with PBS as a control (G1), antibody BC-1C3-lgG1 (G2), anti-mPD-1 (G3), anti-mCTLA4 (G4). [Figure 4] FIG. 13 is a graph showing 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-lgG1 (G3), BC-1F10-lgG1 (G4), BC-1F4-lgG1 (G5), BC-1B6-lgG1 (G6). [Diagram 5] FIG. 1 is a graph showing tumor size over time in mice injected with MC38 cancer cells and treated with PBS as a control (G1), BC-1A8-IgG1 (G2), BC-1C3-IgG1 (G3), BC-1F10-IgG1 (G4), BC-1F4-IgG1 (G5), BC-1B6-IgG1 (G6). [Figure 6]FIG. 13 is a graph showing tumor size over time in hTNFα / hTNFR2 mice injected with MC38 cancer cells and treated with PBS as a control (G1), BC-1C3-lgG1 (G2), anti-mPD-1 (G3). [Figure 7A-7B] The results of tests of blood biochemistry indices (AST, ALT) in the peripheral blood of mice 5 days (D5) after group allocation are shown. [Figure 8] 1 shows epitope correlations between anti-hTNFR2 antibodies. [Figure 9] Cytotoxicity data for BC-1C3-IgG1, BC-1C3-IgG1-SI, BC-1C3-IgG1-LALA, and human IgG1 are shown. [Figure 10] 1 is a drug concentration-time curve showing the change in antibody drug concentration in serum of humanized TNFR2 mice after injection of different antibody drugs. [Figure 11A] Fluorescence signals indicating activation of reporter cells (Jurkat-GFP-TNFR2 cells) are shown. [Figure 11B] Fluorescence signals indicating activation of reporter cells (Jurkat-GFP-TNFR2 cells) are shown, TNFα is not shown. [Figure 12] Fluorescence signals indicating activation of reporter cells (Jurkat-GFP-TNFR2 cells) are shown. [Figure 13A-13B] The test results of blood biochemistry indicators (AST, ALT) in mouse peripheral blood are shown. [Figure 14] Listed below are the CDR sequences for anti-TNFR2 antibodies BC-1A8 ("1A8"), BC-1B6 ("1B6"), BC-1C3 ("1C3"), BC-1F4 ("1F4"), and BC-1F10 ("1F10"), as defined by the Kabat numbering scheme. [Figure 15] The CDR sequences of anti-TNFR2 antibodies BC-1A8 ("1A8"), BC-1B6 ("1B6"), BC-1C3 ("1C3"), BC-1F4 ("1F4"), and BC-1F10 ("1F10") are listed as defined by the Chothia numbering scheme. [Figure 16]The amino acid sequences of the heavy chain variable region and light chain variable region of anti-TNFR2 antibodies (1A8, 1B6, 1C3, 1F4, and 1F10) are listed. [Figure 17] Certain related amino acid sequences are listed. [Figure 18] FIG. 13 is a graph showing tumor size over time in mice injected with MC38 cancer cells and treated with PBS (G1), BC-1C3-lgG1 (G2), anti-mPD-1 (G3), a combination of BC-1C3-lgG1 and anti-mPD1 (G4), an atezolizumab analog (G5), or a combination of BC-1C3-lgG1 and an atezolizumab analog (G6). [Figure 19] FIG. 13 is a graph showing 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). [Figure 20] FIG. 1 is a graph showing 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). [Figure 21] FIG. 1 is a graph showing tumor size over time in mice injected with MC38 cancer cells and treated with PBS (G1), BC-1C3-lgG1 (G2), or h600-25-71 analog (G3). [Figure 22] FIG. 1 is a graph showing the proliferation of hCD8a+ T cells as measured by flow cytometry. [Figure 23] FIG. 1 is a graph showing the proliferation of hCD8a+ T cells as measured by flow cytometry. [Figure 24A] 1 is a graph showing the release of human IL12. [Figure 24B] 1 is a graph showing the release of human IFNγ. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] Tumor necrosis factor (TNF) is generally considered to be the main 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)). During inflammatory processes (including the cancer microenvironment), TNF is one of the first inflammatory mediators secreted and produced. It supports the production of cytokine cascades and promotes the production of other inflammatory mediators (e.g., transcription factors, interleukin (IL)-1, IL-6). There are two types of TNF receptors (TNFR1 and TNFR2) that are localized on the cell surface. Experiments with inflammation-associated cancers revealed that TNFR2 was upregulated in preference to TNFR1, and treatment with anti-TNF monoclonal antibodies reduced the number and size of tumors. Thus, the TNF-TNFR2 axis is implicated in suppressing immune responses and affecting tumor progression and metastasis.

[0070] The present disclosure provides examples of antibodies, antigen-binding fragments thereof, that bind to TNFR2 (tumor necrosis factor receptor 2). TNFR2 and Cancer T cells have become an important focus of cancer immunotherapy due to their ability to directly kill tumor cells when they are CD8+ cytotoxic T lymphocytes (CTLs), also known as CD8+ effector T cells (Teff). However, Treg cells can suppress Teff cells, thereby halting the appropriate host immune response to clear tumors. Thus, inhibition of suppressive Treg cells and simultaneous activation of cytotoxic CD8+ Teff could be a potential strategy for treating cancer (Vanamee, Eva S., et al. "TNFR2: a novel target for cancer immunotherapy." Trends in molecular medicine 23.11(2017):1037-1046).

[0071] TNFR2 is a member of the TNFR superfamily (TNFRSF) and is activated by TNF. It is a cell surface receptor that controls cell survival and proliferation, and targeting this receptor has emerged as a potential next-generation cancer therapeutic approach. Certain human tumor cells can aberrantly express TNFR2, and tumor infiltration is dominated by highly suppressive TNFR2+ Treg cells.

[0072] TNFR2 is expressed primarily in cells of the immune system, particularly regulatory T (Treg) cells, and by endothelial cells, and preferentially binds transmembrane TNF (tmTNF). TNFR1 and TNFR2 are single-pass transmembrane glycoproteins with 28% homology, mainly in the extracellular domain, composed of four cysteine-rich motifs. However, the intracellular domains of the TNF receptors are largely unrelated and lack homologous sequences, suggesting that different signaling functions originate 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), which is primarily involved in cell death signaling. TNFR2 does not contain a cytoplasmic DD, but interacts with TNF-associated factor 2 (TRAF2), mainly resulting in cell survival. The signaling circuitry of TNFR2 is distinct from that of other TNFRs. TNFR1 contains an intracellular death domain and can activate either apoptotic or inflammatory pathways, whereas TNFR2 binds to TNF receptor-associated factors (TRAFs) and can activate both the canonical and non-canonical NF-κB pathways to control cell survival and proliferation in both humans and mice.

[0073] TNFR2 has 461 amino acids, of which amino acids 1-22 are a signal peptide, amino acids 23-257 are an extracellular domain, amino acids 258-287 are a transmembrane domain, and amino acids 288-461 are a cytoplasmic domain with a TRAF2 binding site. TRAF2 can bind to TRAF1 and TRAF3, which are inhibitor of apoptosis protein 1 (cIAP1) and inhibitor of apoptosis protein 2 (cIAP2).

[0074] Antagonistic anti-TNFR2 antibodies can block ligand binding and lock membrane receptors in a resting (non-signaling) antiparallel dimer configuration, whereas agonistic cross-linking antibodies can stabilize the parallel TNF-TNFR2 complex, i.e., resulting in structural stabilization of the active signaling network. Furthermore, it is now well recognized that TNFR2 contributes to the stabilization of the CD4+Foxp3+Treg phenotype in an inflammatory environment.

[0075] Detailed descriptions of TNFR2 and its functions can be found, for example, in 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; Orti-Casan, et al., "Targeting TNFR2 as a novel therapeutic strategy for Alzheimer's disease," Frontiers in neuroscience 13 (2019): 49; and 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.

[0076] 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. Anti-TNFR2 antibodies and antigen-binding fragments 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 and thus increase the immune response. In some embodiments, these antibodies can initiate complement-dependent cytotoxicity (CMC) or antibody-dependent cellular cytotoxicity (ADCC).

[0077] The 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, humanized, and human antibodies.

[0078] CDR sequences for 1A8 and antibodies derived from 1A8 (e.g., humanized antibodies) include the heavy chain variable domain CDRs SEQ ID NOs: 6, 7, 8, and the light chain variable domain CDRs SEQ ID NOs: 9, 10, 11, as defined by Kabat numbering. CDRs can also be defined by the Chothia system. Under Chothia numbering, the heavy chain variable domain CDR sequences are set forth in SEQ ID NOs: 42, 43, 44, and the light chain variable domain CDR sequences are set forth in SEQ ID NOs: 45, 46, 47.

[0079] CDR sequences for 1B6 and 1B6-derived antibodies include the heavy chain variable domain CDRs SEQ ID NOs: 12, 13, 14 and the light chain variable domain CDRs SEQ ID NOs: 15, 16, 17 as defined by Kabat numbering. Under Chothia numbering, the heavy chain variable domain CDR sequences are set forth in SEQ ID NOs: 48, 49, 50 and the light chain variable domain CDRs are set forth in SEQ ID NOs: 51, 52, 53.

[0080] CDR sequences for 1C3 and antibodies derived from 1C3 include the heavy chain variable domain CDRs SEQ ID NOs: 18, 19, 20 and the light chain variable domain CDRs SEQ ID NOs: 21, 22, 23 as defined by Kabat numbering. Under Chothia numbering, the heavy chain variable domain CDR sequences are set forth in SEQ ID NOs: 54, 55, 56 and the light chain variable domain CDRs are set forth in SEQ ID NOs: 57, 58, 59.

[0081] CDR sequences for 1F4 and antibodies derived from 1F4 include the heavy chain variable domain CDRs SEQ ID NOs: 24, 25, 26 and the light chain variable domain CDRs SEQ ID NOs: 27, 28, 29 as defined by Kabat numbering. Under Chothia numbering, the heavy chain variable domain CDR sequences are set out in SEQ ID NOs: 60, 61, 62 and the light chain variable domain CDRs are set out in SEQ ID NOs: 63, 64, 65.

[0082] CDR sequences for 1F10 and antibodies derived from 1F10 include the heavy chain variable domain CDRs SEQ ID NOs: 30, 31, 32 and the light chain variable domain CDRs SEQ ID NOs: 33, 34, 35 as defined by Kabat numbering. Under Chothia numbering, the heavy chain variable domain CDR sequences are set out in SEQ ID NOs: 66, 67, 68 and the light chain variable domain CDRs are set out in SEQ ID NOs: 69, 70, 71.

[0083] The amino acid sequence for the heavy chain variable region of the 1A8 antibody is set forth in SEQ ID NO: 36. The amino acid sequence for the light chain variable region of the 1A8 antibody is set forth in SEQ ID NO: 37.

[0084] The amino acid sequence for the heavy chain variable region of the 1B6 antibody is set forth in SEQ ID NO: 38. The amino acid sequence for the light chain variable region of the 1B6 antibody is set forth in SEQ ID NO: 39.

[0085] The amino acid sequence for the heavy chain variable region of the 1C3 antibody is set forth in SEQ ID NO: 40. The amino acid sequence for the light chain variable region of the 1C3 antibody is set forth in SEQ ID NO: 41.

[0086] The amino acid sequence for the heavy chain variable region of the 1F4 antibody is set forth in SEQ ID NO: 72. The amino acid sequence for the light chain variable region of the 1F4 antibody is set forth in SEQ ID NO: 73.

[0087] The amino acid sequence for the heavy chain variable region of the 1F10 antibody is set forth in SEQ ID NO: 74. The amino acid sequence for the light chain variable region of the 1F10 antibody is set forth in SEQ ID NO: 75.

[0088] Also provided are amino acid sequences for the heavy and light chain variable regions of the modified antibodies. In some embodiments, the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 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, both of which bind to TNFR2.

[0089] Percent humanization refers to the percent identity of a heavy or light chain variable region sequence compared to human antibody sequences in the International Immunogenetics Database (IMGT). A top hit means that a heavy or light chain variable region sequence is closer to a particular species than to other species. For example, a top hit to human means that the sequence is closer to human than to other species. A top hit to human and cynomolgus monkey means that the sequence has the same percent identity to human sequence and cynomolgus monkey sequence, and these percent identity are the highest compared to sequences of other species. In some embodiments, the percent humanization is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%. Detailed descriptions of how to determine the humanization percentage and how to determine the top hits are well known in the art and are described, for example, in Jones, et al. "The INNs and outs of antibody nonproprietary names." MAbs. Vol. 8. No. 1. Taylor & Francis, 2016, which is incorporated by reference in its entirety. A high humanization percentage often has various advantages, such as being safer and more effective in humans, more likely to be tolerated by human subjects, and / or less likely to have side effects. In some embodiments, the variable regions are fully human, e.g., derived from human heavy chain immunoglobulin locus sequences (e.g., a combination of human IGHV, IGHD, and IGHJ genes) and / or human kappa chain immunoglobulin locus sequences (e.g., a combination of human IGKV and IGKJ genes).

[0090] Furthermore, in some embodiments, the antibodies or antigen-binding fragments thereof described herein may also contain one, two, or three heavy chain variable region CDRs selected from the group consisting of SEQ ID NOs: 6-8, 12-14, 18-20, 24-26, and 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, 15-17, 21-23, 27-29, and 33-35 (Kabat numbering).

[0091] In some embodiments, an antibody may have a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2 and 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VH CDR3 amino acid sequence. In some embodiments, the antibody may have a light chain variable region (VL) comprising CDR1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90% or 95% identical to a selected VL CDR3 amino acid sequence. Selected VH CDR1, 2, 3 amino acid sequences and selected VL CDR1, 2, 3 amino acid sequences are shown in Figure 14 (Kabat CDRs) and Figure 15 (Chothia CDRs).

[0092] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO:6 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO:7 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO:8 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0093] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 12 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 13 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 14 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0094] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 18 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 19 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 20 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0095] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO:24 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO:25 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO:26 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0096] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a heavy chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 30 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 31 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 32 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0097] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two or three of the CDRs of SEQ ID NO:9 with 0, 1 or 2 amino acid insertions, deletions or substitutions, SEQ ID NO:10 with 0, 1 or 2 amino acid insertions, deletions or substitutions, or SEQ ID NO:11 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0098] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two, or three of the CDRs of SEQ ID NO: 15 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; SEQ ID NO: 16 with 0, 1, or 2 amino acid insertions, deletions, or substitutions; or SEQ ID NO: 17 with 0, 1, or 2 amino acid insertions, deletions, or substitutions.

[0099] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two or three of the CDRs of SEQ ID NO:21 with 0, 1 or 2 amino acid insertions, deletions or substitutions, SEQ ID NO:22 with 0, 1 or 2 amino acid insertions, deletions or substitutions, or SEQ ID NO:23 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0100] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two or three of the CDRs of SEQ ID NO: 27 with 0, 1 or 2 amino acid insertions, deletions or substitutions, SEQ ID NO: 28 with 0, 1 or 2 amino acid insertions, deletions or substitutions, or SEQ ID NO: 29 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0101] In some embodiments, the antibodies or antigen-binding fragments described herein may contain a light chain variable domain containing one, two or three of the CDRs of SEQ ID NO: 33 with 0, 1 or 2 amino acid insertions, deletions or substitutions; SEQ ID NO: 34 with 0, 1 or 2 amino acid insertions, deletions or substitutions; or SEQ ID NO: 35 with 0, 1 or 2 amino acid insertions, deletions or substitutions.

[0102] Insertions, deletions, and substitutions can occur within the CDR sequences or at either or both ends of the CDR sequences. In some embodiments, the CDRs are determined according to the Kabat numbering scheme. In some embodiments, the CDRs are determined according to the Chothia numbering scheme. In some embodiments, the CDRs are determined according to a combination of the Kabat and Chothia numbering schemes.

[0103] The present disclosure also provides an antibody or antigen-binding fragment thereof that binds to TNFR2. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising or consisting of an amino acid sequence 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 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.

[0104] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment for comparison, and non-homologous sequences may be ignored). The length of the reference sequence aligned for comparison is at least 80% of the length of the reference sequence, and in some embodiments, at least 90%, 95%, or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap. For example, sequence comparisons and determination of percent identity between two sequences can be performed using a Blossum62 scoring matrix, with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0105] The disclosure also provides a nucleic acid comprising a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or an immunoglobulin light chain, the immunoglobulin heavy chain or immunoglobulin light chain comprising the CDRs shown in Figure 14 or Figure 15, or having the sequence shown in Figure 16. When the polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds to TNFR2.

[0106] Anti-TNFR2 antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of antibodies or antibody fragments, as well as multispecific (e.g., bispecific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal, monoclonal, multimeric, multispecific (e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, intracellularly produced antibodies (i.e., intrabodies), and antigen-binding fragments thereof. The antibodies or antigen-binding fragments thereof can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibodies or antigen-binding fragments thereof are IgG antibodies or antigen-binding fragments thereof.

[0107] Antibody fragments are suitable for use in the methods provided, so long as they retain the desired affinity and specificity of the full-length antibody. Thus, antibody fragments that bind to TNFR2 retain the ability to bind to TNFR2. Fv fragments are antibody fragments that contain a complete antigen recognition and binding site. This region is composed of a tightly associated dimer of one heavy chain variable domain and one light chain variable domain, which can be essentially covalent, for example in scFv. In this configuration, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Overall, the six CDRs or a subset thereof confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) can have the ability to recognize and bind to an antigen, but usually with a lower affinity than the entire binding site. Single-chain Fv or (scFv) antibody fragments comprise the VH and VL domains (or regions) of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0108] The present disclosure also provides an antibody or antigen-binding fragment thereof that cross-competes with any of the antibodies or antigen-binding fragments thereof described herein. Cross-competition assays are well known in the art and are described, for example, in Moore et al., "Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein." Journal of Virology 70.3 (1996): 1863-1872, which is incorporated herein by reference in its entirety. In one aspect, the present disclosure also provides an antibody or antigen-binding fragment thereof that binds to the same epitope or region as any of the antibodies or antigen-binding fragments described herein. Epitope binding assays are well known in the art and are described, for example, in Estep et al., "High throughput solution-based measurement of antibody-antigen affinity and epitope binning." MAbs. Vol. 5. No. 2. Taylor & Francis, 2013, which is incorporated herein by reference in its entirety. Antibodies and antigen-binding fragments The present disclosure provides various antibodies and antigen-binding fragments thereof derived from the anti-TNFR2 antibodies described herein. Generally, antibodies (also called immunoglobulins) are composed of two classes of polypeptide chains, light chains and heavy chains. A non-limiting example of an antibody of the present disclosure can be an intact four immunoglobulin chain antibody, including two heavy chains and two light chains. The heavy chain of the antibody can be of any isotype, including IgM, IgG, IgE, IgA, or IgD, or a 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 include two identical copies of the light chain and two identical copies of the heavy chain, each of which contains one variable domain (or variable region, V HThe heavy chains each contain one variable domain (or variable region, V) and multiple constant domains (or constant regions) that are linked together via disulfide bonds within their constant domains to form the "stem" of the antibody. L Each light chain, which contains a constant domain (or region) and one light chain, is bound to one heavy chain via disulfide bonds. The variable region of each light chain aligns with the variable region of the heavy chain to which it is bound. The variable regions of both the light and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FR).

[0109] The hypervariable regions, known as complementarity determining regions (CDRs), form the loops that comprise the antigen-binding surface of the antibody. The four framework regions mostly conform to a β-sheet structure, and the CDRs form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs of each chain are held in close proximity by the framework regions and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding region.

[0110] Methods for identifying CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known, and several definitions of CDR are commonly used: the Kabat definition is based on sequence variability, and the Chothia definition is based on the location of the structural loop regions. These methods and definitions are described, for example, in Martin, "Protein sequence and structure analysis of antibody variable domains," Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan, et al., "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains," Molecular immunology 45.14 (2008): 3832-3839; Wu, TT and Kabat, EA (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203: 121-53 (1991); Morea et al., Biophys Chem. 68 (1-3): 9-16 (Oct. 1997); Morea et al., J Mol Biol. 275 (2): 269-94 (Jan. .1998), Chothia et al., Nature 342(6252):877-83 (Dec.1989), and Ponomarenko and Bourne, BMC Structural Biology 7:64(2007), each of which is incorporated by reference in its entirety.

[0111] CDRs are important for recognizing the epitope of an antigen. As used herein, an "epitope" is the smallest part of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope can be about 3, 4, 5, 6, or 7 amino acids, but these amino acids do not need to be in a contiguous linear sequence in the primary structure of the antigen, since the epitope can depend on the three-dimensional structure of the antigen based on the secondary and tertiary structure of the antigen.

[0112] In some embodiments, antibodies are intact immunoglobulin molecules (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved and differ in their constant regions, particularly the hinge and upper CH2 domains. The sequences and differences of IgG subclasses are well known in the art and are described, for example, in Vidarsson, et al., "IgG subclasses and allotypes: from structure to effector functions." Frontiers in immunology 5 (2014); Irani, et al., "Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases." Molecular immunology 67.2 (2015): 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016, each of which is incorporated by reference in its entirety.

[0113] An antibody can also be an immunoglobulin molecule from any species (e.g., human, rodent, mouse, camelid). Antibodies disclosed herein include, but are not limited to, polyclonal, monoclonal, monospecific, multispecific antibodies, and chimeric antibodies comprising an immunoglobulin binding domain fused to another polypeptide. The term "antigen-binding domain" or "antigen-binding fragment" refers to any portion of an antibody that retains the specific binding activity of the intact antibody, i.e., that is capable of specifically binding to an epitope on the target molecule of the intact antibody. This includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, an antibody or antigen-binding fragment thereof can be, for example, an scFv, Fv, Fd, dAb, bispecific antibody, bispecific scFv, diabody, linear antibody, single chain antibody molecule, multispecific antibody formed from antibody fragments, and any polypeptide that includes a binding domain that is an antibody binding domain, or is homologous thereto. Non-limiting examples of antigen-binding domains include, for example, the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or individual CDRs derived from either the heavy or light chain of an intact antibody.

[0114] Also provided are fragments of antibodies 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 domain (CH1) of the heavy chain. F(ab')2 antibody fragments comprise a pair of Fab fragments, generally covalently linked near their carboxy termini by hinge cysteines between them. Other chemical linkages of antibody fragments are well known in the art.

[0115] Diabodies are small antibody fragments that contain two antigen-binding sites, comprising a VH connected to a VL (VH and VL) in the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains can be forced to pair with the complementary domains of another chain to generate two antigen-binding sites.

[0116] Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions. Linear antibodies may be bispecific or monospecific.

[0117] The antibodies and antibody fragments of the disclosure can be modified within the Fc region to confer desired effector functions or serum half-life.

[0118] Antibody multimerization can be achieved by natural aggregation of antibodies or by chemical or recombinant conjugation techniques well known in the art. For example, a percentage of purified antibody preparations (e.g., purified IgG1 molecules) naturally form protein aggregates containing antibody homodimers and other higher order antibody multimers.

[0119] Alternatively, antibody homodimers can be formed by chemical coupling techniques well known in the art. For example, heterobifunctional crosslinkers including, but not limited to, SMCC (succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate) and SATA (N-succinimidyl S-acetylthio-acetate) can be used to form antibody multimers. An exemplary procedure for forming antibody homodimers is described in Ghetie et al. (Proc. Natl. Acad. Sci. USA 94:7509-7514, 1997). Antibody homodimers can be converted to Fab'2 homodimers by pepsin digestion. Another method for forming antibody homodimers is to use the autophilic T15 peptide described in Zhao et al. (J. Immunol. 25:396-404, 2002).

[0120] In some embodiments, the multispecific antibody is a bispecific antibody. Bispecific antibodies can be produced by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell culture. For example, the interface can contain at least a portion of the CH3 domain of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is created in the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other unwanted end products such as homodimers. This method is described, for example, in WO 96 / 27011, which is incorporated by reference in its entirety.

[0121] Bispecific antibodies include cross-linked, or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be bound to avidin, and the other to biotin. Heteroconjugate antibodies can also be made using any convenient cross-linking method. Suitable cross-linking agents and techniques are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.

[0122] Any of the antibodies or antigen-binding fragments described herein can be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or antigen-binding fragment thereof in a subject or in solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). Conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of the antibody or antigen-binding fragment in vitro (e.g., when stored in tissue culture medium or as a pharmaceutical composition) or in vivo (e.g., in humans).

[0123] In some embodiments, the antibodies or antigen-binding fragments described herein can be conjugated to a therapeutic agent. The antibody drug conjugates comprising the antibodies or antigen-binding fragments thereof can be covalently or non-covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, maytansinoids (such as DM-1 and DM-4), dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs).

[0124] In some embodiments, the antigen-binding fragment can form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of a single chain variable fragment (scFv) described herein fused to the CD3ζ transmembrane and endodomain. In some embodiments, the chimeric antigen receptor also comprises intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor comprises multiple signaling domains, e.g., CD3z-CD28-41BB, or CD3z-CD28-OX40, for increased potency. Thus, in one aspect, the present disclosure further provides a cell (e.g., a T cell) expressing a chimeric antigen receptor described herein.

[0125] In some embodiments, an scFv has one heavy chain variable domain and one light chain variable domain, hi some embodiments, an scFv has two heavy chain variable domains and two light chain variable domains. Antibody characteristics 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 having cells expressing TNFR2.

[0126] In some embodiments, the antibody (or antigen-binding fragment thereof) binds to TNFR2 (human TNFR2, mouse TNFR2, monkey TNFR2, canine TNFR2, chimeric TNFR2) at a concentration of 0.1 s -1 Less than 0.01s -1 Less than 0.001s -1 Less than 0.0001s -1 Less than or equal to 0.00001s -1In some embodiments, the koff is less than 0.01 s -1 Super, 0.001s -1 Super, 0.0001s -1 Super, 0.00001s -1 Greater than or equal to 0.000001s -1 It's super.

[0127] In some embodiments, the kinetic association rate (k) is 1×10 2 / Ms super, 1×10 3 / Ms super, 1×10 4 / Ms super, 1×10 5 / Ms or 1×10 6 In some embodiments, the kinetic association rate (k) is greater than 1×10 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 / Ms.

[0128] Affinity can be estimated from the quotient of kinetic rate constants (KD=koff / kon). In some embodiments, KD is 1×10 -6 Less than M, 1×10 -7 Less than M, 1×10 -8 Less than M, 1×10 -9 Less than M or 1 x 10 -10 In some embodiments, the KD is less than 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, the KD is less than 1 x 10 -7 Super M, 1×10 -8 Super M, 1×10 -9 Super M, 1×10 -10 Super M, 1×10 -11 More than M or 1×10 -12 It's super M.

[0129] Common techniques for measuring the affinity of an antibody to an antigen include, for example, ELISA, RIA, and surface plasmon resonance (SPR). In some embodiments, the antibody binds to human 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.

[0130] TNFR2 has four cysteine-rich domains (CRDs). CRD1 is 39aa to 76aa of SEQ ID NO: 1, CRD2 is 77aa to 118aa of SEQ ID NO: 1, CRD3 is 119aa to 162aa of SEQ ID NO: 1, and CRD4 is 168aa to 196aa of SEQ ID NO: 1. In some embodiments, the antibody (or antibody-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.

[0131] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are capable of blocking the inhibition of CD8+ T cell proliferation by Treg cells.

[0132] 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%.

[0133] 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 IL-2 release by CD8+ T cells. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can enhance IL-2 release 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 IFN-γ release by CD8+ T cells. In some embodiments, the antibodies or antigen-binding fragments thereof described herein can enhance IFN-γ release 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%.

[0134] In some embodiments, the antibodies or antigen-binding fragments thereof described herein induce a tumor growth inhibition (TGI) percentage of greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. TV In some embodiments, the antibody has a tumor growth inhibition percentage of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. TV Percentage of tumor growth inhibition (TGI) can be measured, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the start of treatment, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the start of treatment. TV %) is calculated using the following formula: T.G.I. TV(%) = [1-(Ti-T0) / (Vi-V0)] x 100% Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume in the treatment group on day 0. Vi is the mean tumor volume in the control group on day i. V0 is the mean tumor volume in the control group on day 0.

[0135] In some embodiments, the antibodies or antigen-binding fragments thereof described herein have cytotoxicity, for example, against cells expressing TNFR2. Methods for measuring cytotoxicity are well known in the art. In some embodiments, cytotoxicity is calculated by the following formula:

[0136]

number

[0137] Wherein, experimental means the absorbance value of the experimental group (effector cells + target cells + test product), and effector means the absorbance value of the effector cells (effector cells only). Target means the absorbance value of the target cells (target cells only). Spontaneous release means the absorbance value of the autofluorescence of the cell culture medium (cell culture medium only, no effector cells, no target cells). Target maximum means the maximum absorbance value of the target cells (target cells + cell lysate). Spontaneous release* means the absorbance value of the cell culture medium volume control well (medium only + cell lysate). 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.

[0138] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are TNFR2 antagonists. In some embodiments, the antibodies or antigen-binding fragments reduce TNFR2 signal transduction in target cells that express TNFR2 (e.g., T cells, such as Tregs).

[0139] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are non-toxic, in some embodiments, no significant difference in body weight can be observed between treatment and control groups, for example, at 0.3 mg / kg, 1 mg / kg, 10 mg / kg, or 25 mg / kg.

[0140] In some embodiments, the antibody or antigen-binding fragment can bind to tumor cells expressing TNFR2, hi some embodiments, the antibody or antigen-binding fragment can induce complement dependent cytotoxicity (CMC) and / or antibody dependent cellular cytotoxicity (ADCC) and kill the tumor cells.

[0141] In some embodiments, the antibody or antigen-binding fragment has a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector function of the functional Fc region is ADCC and phagocytosis.

[0142] In some embodiments, the antibody or antigen-binding fragment is capable of inducing complement-mediated cytotoxicity (CMC).

[0143] In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4, hi some embodiments, the antibody is a human IgG1 antibody.

[0144] In some embodiments, the antibody or antigen-binding fragment does not have a functional Fc region. For example, the antibody or antigen-binding fragment is a Fab, Fab', F(ab')2, or Fv fragment. In some embodiments, the Fc region has a LALA mutation (L234A and L235A mutations in EU numbering) or a LALA-PG mutation (L234A, L235A, P329G mutations in EU numbering).

[0145] In some embodiments, the Fc has SI mutations (S239D and I332E mutations in the EU numbering). Method for producing anti-TNFR2 antibody An isolated fragment (e.g., the extracellular region) of human TNFR2 can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be raised in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of the antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species to be immunized. Animals can be injected with the antigenic peptide or protein two or more times (e.g., two, three, or four times).

[0146] 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 generated against the peptide form specific immune complexes with the protein. As previously mentioned, the full-length sequence of human TNFR2 is well known in the art (SEQ ID NO: 1). In some embodiments, an Fc-tagged human TNFR2 protein (the Fc fusion protein contains the human TNFR2 extracellular domain at positions 23-257 of SEQ ID NO: 1) is used as an immunogen.

[0147] An immunogen is typically used to prepare antibodies by immunizing a suitable subject (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). An appropriate immunogenic preparation can contain, for example, a recombinantly expressed or chemically synthesized polypeptide (e.g., a fragment of human TNFR2). The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulant.

[0148] Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a TNFR2 polypeptide or an antigenic peptide thereof (e.g., a portion of TNFR2, such as the extracellular domain) 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 the blood) and further purified by well-known techniques, such as protein A of protein G chromatography, to obtain an IgG fraction. At an appropriate time after immunization, e.g., when the specific antibody titer is at its highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al. (Nature 256:495-497, 1975), the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96, 1985), or the trioma technique. Techniques for producing hybridomas are well known (see generally Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, NY)). For example, hybridoma cells producing monoclonal antibodies are detected by screening hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest using a standard ELISA assay.

[0149] Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding the human, humanized, or chimeric antibodies, or antibodies or antigen-binding fragments thereof described herein, or by peptide synthesis. Such variants include, for example, deletion, insertion, or substitution of residues within the amino acids of the sequence that make up the antigen-binding site or domain of the antibody. In such a population of variants, some antibodies or antigen-binding fragments have increased affinity for the target protein, e.g., TNFR2. Any combination of deletions, insertions, and / or combinations can be achieved in antibodies or antigen-binding fragments thereof with increased binding affinity for the target. Antibodies or antigen-binding fragments can be altered or new post-translational modifications can be introduced into the antibodies or antigen-binding fragments by changing the number (e.g., increasing or decreasing) of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence so that a different sugar is attached by an enzyme present in the cell), or introducing new glycosylation sites.

[0150] The antibodies disclosed herein can be derived from any species of animal, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates, such as monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including transgenic rodents that have been genetically engineered to produce human antibodies.

[0151] Human and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequences derived from) human germline immunoglobulin sequences. Human antibodies can include, for example, amino acid residues within the CDRs that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0152] A humanized antibody typically has a human framework (FR) into which non-human CDRs have been grafted. Thus, a humanized antibody has one or more amino acid sequences introduced into a human from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can essentially be performed, for example, by substituting rodent CDRs or CDR sequences with the corresponding sequences of a human antibody. These methods are described, for example, in Jones et al. "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 by reference herein in its entirety. Thus, a "humanized" antibody is a chimeric antibody in which substantially less than an intact human V-domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically murine antibodies in which some CDR residues and some FR residues have been substituted by residues from analogous sites in human antibodies.

[0153] The selection of human VH and VL domains used in the production of humanized antibodies is very important to reduce immunogenicity. According to the so-called "best-fit" method, the sequence of the V domain of a mouse antibody is screened against the entire library of known human domain sequences. The human sequence that is closest to the mouse sequence is then accepted as the human FR for the humanized antibody (Sims et al. "A humanized CD18 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).

[0154] Furthermore, it is important that antibodies be humanized while retaining high specificity and affinity for the antigen, as well as other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that can illustrate and display possible three-dimensional conformations of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the possible role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences to achieve the desired antibody characteristic, e.g., increased affinity for the target antigen.

[0155] Typically, an amino acid sequence variant of a human, humanized, or chimeric anti-TNFR2 antibody contains an amino acid sequence that has a percent identity of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% with a sequence present in the light or heavy chain of the original antibody.

[0156] In some embodiments, a mouse having a humanized heavy chain immunoglobulin locus and a humanized kappa chain immunoglobulin locus (e.g., RenMab mouse) is used to generate antibodies. The heavy chain immunoglobulin locus is a region on a chromosome that contains genes for the heavy chain of an antibody. The locus can include, for example, a human IGHV (variable) gene, a human IGHD (diversity) gene, a human IGHJ (joining) gene, and a mouse heavy chain constant domain gene. The kappa chain immunoglobulin locus is a region on a chromosome that contains genes encoding the light chain (kappa chain) of an antibody. The kappa chain immunoglobulin locus can include, for example, a human IGKV (variable) gene, a human IGKJ (joining) gene, and a mouse light chain constant domain gene. A detailed description of the RenMab mouse can be found in PCT / CN2020 / 075698, which is incorporated by reference in its entirety. The antibody generated by the mouse has a fully human VH, a fully human VL, and a mouse constant region. In some embodiments, the human VH and human VL bind to a human IgG constant region (e.g., IgG1, IgG2, IgG3, and IgG4). In some embodiments, the constant region has a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 76, 77, 78, or 90.

[0157] Identity or homology to an original sequence is usually the percentage of amino acid residues present in a candidate sequence that are identical to a sequence present in a human, humanized, or chimeric anti-TNFR2 antibody or fragment, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without taking into account conservative substitutions as part of the sequence identity.

[0158] Further modifications can be made to the anti-TNFR2 antibody or antigen-binding fragment. For example, cysteine ​​residues can be introduced into the Fc region to allow interchain disulfide bond formation in this region. The homodimeric antibodies thus generated can have some 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, for example, as described by Wolff et al. Wolff et al. ("Monoclonal antibody homodimers: enhanced antitumor activity in nude mice." Cancer research 53.11 (1993): 2560-2565). Alternatively, an antibody with dual Fc regions can be engineered.

[0159] In some embodiments, covalent modifications can be made to the anti-TNFR2 antibodies or antigen-binding fragments thereof. These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of antibodies or antibody fragments are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with organic derivatizing agents capable of reacting with selected side chains or with the N- or C-terminal residues.

[0160] In some embodiments, antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibody compositions may be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid, and high mannose structures) attached to Asn297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at position 297 (position 314 in the Eu numbering of Fc region residues, or Kabat numbering) in the Fc region, however, Asn297 may also be located approximately ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. In some embodiments, the Fc region of the antibody is further engineered to replace the asparagine at position 297 with alanine (N297A) to reduce glycan heterogeneity.

[0161] In some embodiments, the Fc region of the antibody is further engineered to replace the serine at position 228 (EU numbering) of IgG4 with a proline (S228P) to promote production efficiency by avoiding Fab-arm exchange. A detailed description of the S228 mutation is provided, for example, in Silva et al., "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation." Journal of Biological Chemistry 290.9(2015):5462-5469, which is incorporated by reference in its entirety. Recombinant Vectors The present disclosure also provides recombinant vectors (e.g., expression vectors) comprising the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors have been introduced (i.e., such that the host cell contains the polynucleotides and / or vectors containing the polynucleotides), and the production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.

[0162] As used herein, a "vector" is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An "expression vector" is capable of delivering and expressing one or more polynucleotides of interest as encoded polypeptides in a host cell into which the expression vector is introduced. Thus, in an expression vector, a polynucleotide of interest is positioned for expression in the vector by being operably linked to control elements, such as promoters, enhancers, and / or polyA tails, either in the vector or in the genome of the host cell at, near, or adjacent to the integration site of the polynucleotide of interest, such that the polynucleotide of interest is translated in the host cell into which it is introduced with the expression vector.

[0163] Vectors can be introduced into host cells by methods well known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., by recombinant viruses). Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

[0164] In some embodiments, a polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein) is introduced using a viral expression system (e.g., variola or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication-competent virus, or may use a replication-incompetent virus. In the latter case, viral propagation generally occurs only in complementary viral packaging cells. For example, Fisher-Hoch et al.,1989,Proc.Natl.Acad.Sci.USA 86:317-321, Flexner et al.,1989,Ann.NYAcad Sci.569:86-103, Flexner et al. al., 1990, Vaccine, 8:17-21, U.S. Patent No. 4,603,112, U.S. Pat. 91 / 02805, Berkner-Biotechniques, 6:616-627, 1988; Rosenfeld et al. Suitable systems are disclosed in: Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. The DNA can also be "naked," as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749, and Cohen, 1993, Science, 259:1691-1692. Uptake of naked DNA can be increased by coating the DNA onto biodegradable beads that are efficiently transported into cells.

[0165] For expression, a DNA insert comprising an antibody-encoding or polypeptide-encoding polynucleotide disclosed herein can be operably linked to a suitable promoter (e.g., a heterologous promoter), such as the phage lambda PL promoter, E. coli lac, trp and tac promoters, SV40 early and late promoters, and promoters of retroviral-type LTRs, to name a few. Other suitable promoters will be known to those skilled in the art. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. The expression construct can further contain sites for transcription initiation and termination, and, within the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct can include a translation initiation at the beginning and a termination codon (UAA, UGA or UAG) positioned approximately at the end of the polypeptide to be translated.

[0166] As indicated, the expression vector can include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for E. coli and other bacterial culture. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium cells, fungal cells such as yeast cells, insect cells such as Drosophila melanogaster S2 and Spodoptera litura Sf9 cells, animal cells such as CHO, COS, Bowes melanoma, and HK 293 cells, and plant cells. Appropriate culture media and conditions for the host cells described herein are well known in the art.

[0167] Non-limiting vectors for use in bacteria include pQE70, pQE60 and pQE-9 available from Qiagen, pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG available from Stratagene, and pSVK3, pBPV, pMSG, and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to those of skill in the art.

[0168] Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters, and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, promoters of retroviral-type LTRs such as those of Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0169] In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, can be used.

[0170] Introduction of the construct into the host cell can be accomplished by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, or other methods, which are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which are incorporated herein by reference in their entirety.

[0171] Transcription of DNA encoding the antibody of the present disclosure in more eukaryotic organisms can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10-300 bp, that serve to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer, located at base pairs 100-270 on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0172] To allow secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals can be incorporated into the expressed polypeptide. The signals can be endogenous to the polypeptide or the signals can be heterologous signals.

[0173] Polypeptides (e.g., antibodies) can be expressed in modified forms, such as fusion proteins (e.g., GST fusions) or with histidine tags, and can contain not only secretion signals, but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of a polypeptide to improve stability and durability in the host cell during purification or during subsequent handling and storage. Peptide moieties can also be added to the polypeptide to facilitate purification. Such regions can be removed before final preparation of the polypeptide. It is well known and routine in the art to add peptide moieties to polypeptides to, among other things, effect secretion or excretion, improve stability, and facilitate purification. Treatment method The antibodies or antigen-binding fragments thereof of the present disclosure can be used for a variety of therapeutic purposes.

[0174] In one aspect, the present disclosure provides a method of treating cancer in a subject, reducing the rate of increase in tumor volume in a subject over time, reducing the risk of developing metastases, or reducing the risk of developing further metastases in a subject. In some embodiments, the treatment can halt, slow, prevent, or inhibit the progression of cancer. In some embodiments, the treatment can result in a reduction in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0175] In one aspect, the disclosure relates to a method comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, cancer, e.g., breast cancer (e.g., triple-negative breast cancer), carcinoid cancer, cervical cancer, uterine cancer, nervous system cancer, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or a malignant hematological disorder). In some embodiments, the cancer is unresectable or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the subject is afflicted with a solid tumor. In some embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN), renal cell carcinoma (RCC), triple-negative breast cancer (TNBC), or colon cancer. In some embodiments, the subject has Hodgkin's lymphoma. In some embodiments, the subject has triple-negative breast cancer (TNBC), gastric cancer, urothelial carcinoma, Merkel cell carcinoma, or head and neck cancer. In some embodiments, the cancer is melanoma, pancreatic cancer, mesothelioma, glioma, hematological malignancies, particularly non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia, or advanced solid tumors.

[0176] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk for cancer. Patients suffering from cancer can be identified in a variety of ways well known in the art.

[0177] In some aspects, the present disclosure relates to a method of treating an autoimmune disease or inflammation, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof described herein, or a composition comprising an antibody drug conjugate described herein.

[0178] In one aspect, the disclosure provides a method for treating, preventing, or reducing the risk of developing aberrant or unwanted immune responses, e.g., disorders associated with autoimmune diseases, e.g., by affecting Treg function, such as 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, nodular polyarthritis, cardiomyopathy, polychondritis, celiac sprue dermatitis, polyglandular syndrome, chronic fatigue syndrome (CFIDS), polymyalgia rheumatica, chronic inflammatory demyelination, polymyositis and dermatomyositis, chronic inflammatory polyneuropathy, primary agammaglobulinemia, Churg-Strauss syndrome, primary biliary tract disease, and primary inflammatory polyneuropathy. These conditions include, but are not limited to, idiopathic cirrhosis, cicatricial pemphigoid, psoriasis, CREST syndrome, Raynaud's phenomenon, cold agglutinin disease, Reiter's syndrome, Crohn's disease, rheumatic fever, discoid lupus, rheumatoid arthritis, cryoglobulinemic sarcoidosis, fibromyalgia, scleroderma, Graves' disease, Sjogren's syndrome, Guillain-Barre, stiff-man syndrome, Hashimoto's thyroiditis, Takayasu's arteritis, idiopathic alveolar fibrosis, temporal arteritis / giant cell arteritis, idiopathic thrombocytopenic purpura (ITP), ulcerative colitis, IgA nephropathy, uveitis, diabetes mellitus (e.g., type I), vasculitis, lichen planus, and vitiligo. Anti-TNFR2 antibodies or antigen-binding fragments thereof can also be administered to a subject to treat, prevent, or reduce the risk of developing disorders associated with an aberrant or unwanted immune response associated with cell, tissue, or organ transplantation, such as kidney, liver, and heart transplants, such as graft-versus-host disease (GVHD), or to prevent allograft rejection. In some embodiments, the subject suffers from Crohn's disease, ulcerative colitis, or type 1 diabetes. In some embodiments, the antibodies or antigen-binding fragments can be used to treat inflammation.

[0179] In some embodiments, the antibody or antigen-binding fragment thereof is a TNFR2 agonist.

[0180] In some aspects, the present disclosure relates to a method of inhibiting an immune response in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof described herein, or a composition comprising an antibody drug conjugate described herein, hi some embodiments, the subject suffers from an autoimmune disease.

[0181] As used herein, an "effective amount" refers to an amount or dosage sufficient to bring about a beneficial or desired result, including halting, slowing, preventing, or inhibiting the progression of a disease, e.g., cancer. The effective amount will vary depending on, for example, the age and weight of the subject to whom the antibody, antigen-binding fragment, polynucleotide encoding the antibody, vector comprising the polynucleotide, and / or composition is administered, the severity of the symptoms, and the route of administration, and thus dosing can be determined on an individual basis.

[0182] An effective amount can be administered in one or more administrations. For example, an effective amount of an antibody or antigen-binding fragment is an amount sufficient to ameliorate, stop, stabilize, reverse, inhibit, slow, and / or delay the progression of an autoimmune disease or cancer in a patient, or an amount sufficient to ameliorate, stop, stabilize, reverse, slow, and / or delay the proliferation of a cell (e.g., a biopsy cell, any of the cancer cells described herein, or a cell line (e.g., a cancer cell line)) in vitro. As is understood in the art, an effective amount of an antibody or antigen-binding fragment may vary depending on other factors, such as, inter alia, the medical history of the patient, as well as the type (and / or dose) of antibody used.

[0183] Effective amounts and schedules for administering the antibodies, antibody-encoding polynucleotides, and / or compositions disclosed herein can be determined empirically, and making such determinations is within the skill of one in the art. One of skill in the art will understand that the dosage required to be administered will vary depending on, for example, the mammal receiving the antibodies, antibody-encoding polynucleotides, and / or compositions disclosed herein, the route of administration, the particular type of antibody, antibody-encoding polynucleotide, antigen-binding fragment, and / or composition disclosed herein that is used, and other agents administered to the mammal.

[0184] A typical daily dose of an effective amount of antibody is 0.01 mg / kg to 100 mg / kg (mg per kg of patient body weight). In some embodiments, the dose 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 can 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.

[0185] In any of the methods described herein, at least one antibody, antigen-binding fragment thereof, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding fragments, or pharmaceutical compositions described herein) and, optionally, at least one additional therapeutic agent can be administered to a subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day). In some embodiments, at least two different antibodies and / or antigen-binding fragments are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, at least one additional therapeutic agent is administered as a pill, tablet, or capsule. In some embodiments, the at least one additional therapeutic agent is administered in a sustained release oral formulation.

[0186] In some embodiments, one or more additional therapeutic agents can be administered to the subject before or after administration of at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein). In some embodiments, the one or more additional therapeutic agents and the at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, or pharmaceutical compositions described herein) are administered to the subject such that there is an overlap in the subject between the period of biological activity of the one or more additional therapeutic agents and the period of biological activity of the at least one antibody or antigen-binding fragment (e.g., any of the antibodies or antigen-binding fragments described herein).

[0187] In some embodiments, the subject can be administered 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) for an extended period of time (e.g., for a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional can determine the length of time for treatment using any of the methods described herein to diagnose or track the effectiveness of the treatment (e.g., to observe at least one symptom of cancer). As described herein, a skilled medical professional can also vary (e.g., increase or decrease) the identity and number of antibodies or antigen-binding antibody fragments (and / or one or more additional therapeutic agents) administered to a subject, and can adjust (e.g., increase or decrease) the dosage or frequency of administration of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) to a subject based on an evaluation of the effectiveness of the treatment (e.g., using any of the methods described herein and well known in the art).

[0188] In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agents can include one or more inhibitors selected from the group consisting of inhibitors of B-Raf, EGFR inhibitors, inhibitors of MEK, inhibitors of ERK, inhibitors of K-Ras, inhibitors of c-Met, inhibitors of anaplastic lymphoma kinase (ALK), inhibitors of phosphatidylinositol 3-kinase (PI3K), inhibitors of Akt, inhibitors of mTOR, dual PI3K / mTOR inhibitors, inhibitors of Bruton's tyrosine kinase (BTK), and inhibitors of isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2). In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2,3-dioxygenase-1 (IDO1) (e.g., epacadostat).

[0189] In some embodiments, the additional therapeutic agent can include one or more inhibitors selected from the group consisting of inhibitors of HER3, inhibitors of LSD1, inhibitors of MDM2, inhibitors of BCL2, inhibitors of CHK1, inhibitors of the activated hedgehog signaling pathway, and agents that selectively degrade the estrogen receptor.

[0190] In some embodiments, the additional therapeutic agent is trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, leolysin, Alimta, Dicaida, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, pazopanib, IMA-901, AGS-00 3, cabozantinib, vinflunine, Hsp90 inhibitors, Ad-GM-CSF, temozolomide, IL-2, IFNa, vinblastine, thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, lenalidomide, bortezomib, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0191] In some embodiments, the additional therapeutic agent can comprise one or more therapeutic agents selected from the group consisting of an adjuvant, a TLR agonist, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-6 antagonist (e.g., the 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 selectin agonist.

[0192] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI is administered to the subject.

[0193] 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. Pharmaceutical Compositions and Routes of Administration Any one or more (e.g., 1, 2, 3, or 4) of any of the antibodies or antigen-binding fragments described herein can be present in a pharmaceutical composition in any combination. Any two or more (e.g., 2, 3, or 4) of any of the antibodies or antigen-binding fragments described herein can be present in a pharmaceutical composition in any combination. The pharmaceutical compositions can be formulated in any manner known in the art.

[0194] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The compositions may contain a sterile diluent (e.g., sterile water or saline), fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antibacterial or antifungal agents (e.g., benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffers (e.g., acetate, citrate, or phosphate), and isotonic agents (e.g., sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride)), or any combination thereof. Liposomal suspensions may also be used as pharma- ceutically acceptable carriers. The preparation of the composition can be formulated and enclosed in ampoules, disposable syringes, or multiple dose vials. Where necessary (e.g., in injectable formulations), proper fluidity can be maintained, for example, by the use of a coating such as lecithin, or a surfactant. Absorption of the antibody or its antigen-binding fragment can be prolonged by including an agent that delays absorption (e.g., aluminum monostearate and gelatin). Alternatively, sustained release can be achieved by implants and microencapsulated delivery systems, which can include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid).

[0195] Compositions containing one or more of any of the antibodies or antigen-binding fragments described herein can be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in unit dosage form (i.e., physically discrete units containing a predetermined amount of active compound for ease of administration and uniformity of dosage).

[0196] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions may be provided in unit dosage form (i.e., a dose for a single administration). Pharmaceutical compositions may be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the chosen route of administration. For injection, the antibody may be formulated in an aqueous solution, preferably in a physiologically compatible buffer, to reduce discomfort at the injection site. The solution may contain formulating agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the antibody may be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0197] The toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). For example, the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) can be determined, and the therapeutic index is the ratio of LD50:ED50. Drugs that exhibit a high therapeutic index are preferred. If a drug exhibits undesirable side effects, care should be taken to minimize the potential for harm (i.e., to reduce the undesirable side effects). The toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

[0198] Data obtained from cell culture assays and animal studies can be used in formulating an appropriate dose of any given agent for use in a subject (e.g., a human). A therapeutically effective amount of one or more (e.g., one, two, three, or four) antibodies or antigen-binding fragments thereof (e.g., any of the antibodies or antibody fragments described herein) is an amount that treats the disease in a subject (e.g., a human subject identified as having cancer) or at risk of developing the disease (e.g., a subject who previously developed cancer but has now been cured), (e.g., an amount that reduces the severity, frequency, and / or duration of one or more symptoms of the disease in the subject (e.g., a human). The efficacy and administration of any of the antibodies or antigen-binding fragments described herein can be determined by a health care professional or veterinary professional using methods well known in the art, as well as by observing one or more symptoms of the disease in the subject (e.g., a human). Certain factors can affect the dosage and timing required to effectively treat a subject (e.g., the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and the presence of other diseases).

[0199] Exemplary doses include amounts (milligrams or micrograms) of any of the antibodies or antigen-binding fragments described herein per kilogram of subject body weight (e.g., about 1 μg / kg to about 500 mg / kg, about 100 μg / kg to about 500 mg / kg, about 100 μg / kg to about 50 mg / kg, about 10 μg / kg to about 5 mg / kg, about 10 μg / kg to about 0.5 mg / kg, 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). While these doses cover a wide range, one of skill in the art will understand that the efficacy and effective amounts of therapeutic agents comprising antibodies and antigen-binding fragments thereof can be determined by methods well known in the art. Typically, a relatively low dose is administered initially, and the attending health care professional or veterinary professional (for therapeutic applications) or researcher (if still working in the development stage) can subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend on a variety of factors, including the activity of the specific compound used, the age, weight, general health, sex, and diet of the subject, the time of administration, the route of administration, the rate of excretion, and the half-life of the antibody or antibody fragment in vivo.

[0200] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.The present disclosure also provides methods of making the antibodies, or antigen-binding fragments thereof, for the various uses described herein. Working Example The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. Example 1. Generation of anti-hTNFR2 antibodies To generate antibodies against human TNFR2 (TNFR2, SEQ ID NO: 1), RenMab mice were immunized with human TNFR2. Anti-TNFR2 antibodies were produced by the method described below.

[0201] The RenMab mouse has both a humanized heavy chain immunoglobulin locus and a humanized kappa chain immunoglobulin locus. The heavy chain immunoglobulin locus is a region on a chromosome that contains genes for the heavy chain of an antibody. The locus includes the IGHV (variable), IGHD (diversity), IGHJ (joining), and heavy chain constant domain genes. The kappa chain immunoglobulin locus is a region on a chromosome that contains genes encoding the light chain (kappa chain) of an antibody. The kappa chain immunoglobulin locus includes the IGKV (variable), IGKJ (joining), and light chain constant domain genes. A detailed description of the RenMab mouse can be found in PCT / CN2020 / 075698, which is incorporated by reference in its entirety. Immunization of mice RenMab mice were immunized with Fc-tagged human TNFR2 protein (the Fc fusion protein contains the human TNFR2 extracellular domain at positions 23aa-257aa of SEQ ID NO:1). The Fc-tagged human TNFR2 protein was emulsified with adjuvant and injected into four sites 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 an interval of at least 14 days between every two injections. Seven days after the third injection or booster immunization, blood (serum) was collected and antibody titers were analyzed using fluorescence-activated cell sorting (FACS).

[0202] In another experiment, some mice were immunized by injecting them with an expression plasmid encoding human TNFR2. The antigen-encoding plasmid was injected into the tibialis anterior muscle (intramuscular, im injection). At least four injections were performed, with at least 14 days between every two injections. Blood (serum) was collected 7 days after the last immunization, and the serum was tested for antibody titers by ELISA.

[0203] At least 14 days after immunization, a procedure to enhance immunization (either by injecting plasmids or proteins) was performed. CHO cells expressing the TNFR2 antigen on their surface were injected intravenously into mice through the tail vein. Four days after injection, immune system organs (e.g., bone marrow, lymph nodes, spleen, etc.) were harvested.

[0204] Spleen cells were collected and fused with mouse myeloma cells to form hybridoma cell lines while maintaining viability. Hybridoma cells were screened and selected to identify cell lines that produced TNFR2-specific antibodies. Using this technology and the aforementioned immunogens, several anti-TNFR2 chimeric antibodies (i.e., antibodies with human variable domains and mouse constant domains) were obtained. Specifically, after stimulatory immunization, immune organs of mice were collected and plasma cells were separated by magnetic beads. Using hybridoma fusion technology, monoclonal hybridoma cells secreting antigen-specific monoclonal antibodies were selected and excluded. The antibody light and heavy chain V region sequences were obtained by reverse transcription and PCR sequencing on the selected monoclonal hybridoma cells. The antibody light and heavy chain V region sequences were constructed in an antibody expression vector and verified by Expi CHO-STM CELL expression system. The cells were transfected into a 24-well system, and the antibodies were collected on the third day in serum. FACS was used to verify the specificity of binding between the antibody and TNFR2. Using this technique, several anti-TNFR2 chimeric antibodies (i.e., antibodies with human variable domains and mouse constant domains) were obtained. The constant domains of 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"). As an example, when the antibody VH\VL connects to a different subtype, such as the IgG1 subtype, using 14-4A9-hHvKv, the antibody is named 14-4A9-hHvKv-IgG1.

[0205] Antigen-positive B cells were also isolated directly from the immunized mice without fusion with myeloma cells. Anti-TNFR2 antibodies were further isolated from the antigen-positive B cells. Antibody light and heavy chain V-region sequences were isolated directly from the antigen-positive B cells. For example, single cell technology (e.g., Beacon® Optofluidic System, Berkeley Lights Inc.) was used to screen and find plasma cells secreting antigen-specific monoclonal antibodies. Antibody V-region sequences were obtained using reverse transcription and PCR sequencing. Antibodies were expressed. FACS was used to verify the binding between the antibodies 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"). Take BC-1F4 as an example, if the antibody VH\VL connects to a different subtype, such as IgG1 subtype, the antibody is named BC-1F4-IgG1. Examples of other subtypes are: BC-1F4-IgG1-SI, BC-1F4-IgG1-LALA.

[0206] The heavy and light chain CDR1, CDR2, and CDR3 amino acid sequences for 1A8 are shown in SEQ ID NOs: 6 to 11 (Kabat numbering) or SEQ ID NOs: 42 to 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.

[0207] The heavy and light chain CDR1, CDR2, and CDR3 amino acid sequences for 1B6 are shown in SEQ ID NOs: 12 to 17 (Kabat numbering) or SEQ ID NOs: 48 to 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.

[0208] The heavy and light chain CDR1, CDR2, and CDR3 amino acid sequences for 1C3 are shown in SEQ ID NOs: 18 to 23 (Kabat numbering) or SEQ ID NOs: 54 to 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.

[0209] The heavy and light chain CDR1, CDR2, and CDR3 amino acid sequences for 1F4 are shown in SEQ ID NOs: 24 to 29 (Kabat numbering) or SEQ ID NOs: 60 to 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.

[0210] The heavy and light chain CDR1, CDR2, and CDR3 amino acid sequences for 1F10 are shown in SEQ ID NOs: 30 to 35 (Kabat numbering) or SEQ ID NOs: 66 to 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. Preparation of antibodies The positive antibody sequences from the sequence verification stage were subjected to plasmid extraction and transfected into 25mL systems. After 10-12 days of cell culture, the expression supernatant was collected and subjected to affinity chromatography. The obtained antibody samples were used in in vitro testing and screening. Example 2. In vitro testing of anti-TNFR2 antibodies Blocking the binding of human TNFR2 to TNFα A blocking assay was performed to determine whether anti-TNFR2 antibodies could block the binding of TNFR2 to its ligand, hTNFα.

[0211] Specifically, 30 μL of CHO cells (1 × 10 5 A total of 1000 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. Titrated antibodies were added to each well at 30 μL per well and incubated for 30 minutes at 4°C.

[0212] 30 μL of biotin-hTNFα (Acro Biosystems, Catalog Number: TNA-H82E1) was added to each well (at a final concentration of 0.5 μg / mL per well). Cells with biotin-hTNFα and antibodies were incubated at 4° C. for 30 minutes.

[0213] After washing twice with phosphate buffered saline (PBS), 50 μL of a 1:100 dilution of PE-labeled and anti-human IgG Fc antibody (PE anti-human IgG, Rc, Jackson ImmunoResearch, Catalog No.: 109-115-098) and a 1:500 dilution of Alexa Fluor® 647-labeled streptavidin (AF647 streptavidin, Jackson ImmunoResearch Catalog No.: 016-600-084) were added to each well and incubated for 15 min at 4° C., followed by washing with PBS. Signals for AF647 and PE were determined by flow cytometry (Thermo Attune NX).

[0214] Table 1 below shows the percentage of test cells with streptavidin signal in flow cytometry analysis. If the percentage of test cells (AF647) with streptavidin signal increases while the antibody concentration decreases, the antibody has blocking affinity (indicates strong binding affinity). Based on the data, BC-1A8-IgG1, BC-1F4-IgG1, BC-3B7-IgG1, and BC-1F10-IgG1 had a strong blocking effect. However, BC-1B6-IgG1, BC-1C3-IgG1, 14-1B3-hHvKv-IgG1, and 14-4A9-hHvKv-IgG1 cannot effectively block the binding between TNFR2 and TNFα. [Table 1]

[0215] Binding affinity of anti-TNFR2 antibodies to human TNFR2 and TNFR1 The binding affinity of anti-TNFR2 antibodies to human TNFR2 and TNFR1 (tumor necrosis factor receptor 1) was measured using surface plasmon resonance (SPR) using a Biacore (Biacore, INC, Piscataway NJ) 8K biosensor equipped with a pre-immobilized Protein A sensor chip.

[0216] Purified anti-TNFR2 antibodies were diluted to 1 μg / mL and then injected into a Biacore 8K biosensor at 10 μL / min for about 50 seconds to achieve the desired protein density (e.g., about 50 response units (RU)). Subsequently, His-tagged human TNFR1 (Human TNFR1 / CD120a / TNFRSF1A protein, His tag, Beijing Acrobiosystems CO.LTD., Catalog No.: TN1-5222) or TNFR2 (Human TNFR2 / CD120b / TNFRSF1B protein, His tag, Beijing Acrobiosystems CO.LTD., Catalog No.: TN2-5227) at concentrations of 200, 100, 50, 25, 6.25, or 1.56 nM was injected at 30 μL / min for 120 seconds. Dissociation was monitored for 600 seconds. After the last injection of each titration, the chip was regenerated with glycine (pH 2.0, 30 μL / min for 30 s).

[0217] Kinetic association rates (k) and dissociation rates (k) were obtained simultaneously by fitting the entire data to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology 6.99-110) using the Biacore 8K Evaluation software 3.0. The affinity was estimated from the quotient of the kinetic rate constants (K = k / k).

[0218] The same method was performed for each test antibody, with appropriate adjustments to parameters (e.g., antibody concentration), as would be understood by one of skill in the art. The results for the test antibodies are summarized in Table 2 below. [Table 2]

[0219] The results show that these human antibodies have 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) can bind to TNFR1. Binding affinity of anti-TNFR2 antibodies to monkey TNFR2 Similar to the binding affinity experiment described above, the binding affinity of anti-TNFR2 antibodies BC-1B6-IgG1, BC-1C3-IgG1, and BC-1F4-IgG1 to His-tagged monkey (cynomolgus) TNFR2 (fTNFR2-His, Sino Biological, Catalog No.: 90102-C08H) was measured. The results are summarized in Table 3 below, which shows that all three anti-TNFR2 antibodies can bind to monkey TNFR2 with good binding affinity. [Table 3]

[0220] Cross-reactivity of anti-TNFR2 antibodies to mouse, canine, and monkey TNFR2 In each experiment, CHO cells were transfected with EGFP and human TNFR2 (TNFR2, SEQ ID NO: 1), mouse TNFR2 (mTNFR2, SEQ ID NO: 2), fTNFR2, or dog (canine) TNFR2 (dTNFR2, SEQ ID NO: 4).

[0221] 30 μL of CHO cells (1 × 10 5 30 μL of purified anti-TNFR2 antibodies (10 μg / mL) (as listed in Table 4) were added to each well and incubated at 4° C. for 30 minutes.

[0222] After washing twice with PBS (1600 rpm, 6 min), 50 μL of Alexa Fluor-labeled anti-human IgG Fc antibodies (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) were added to each well at a dilution of 1:500 and incubated at 4° C. for 15 min, followed by washing with PBS (1200 rpm, 5 min). Signals against AF647 were detected by flow cytometry.

[0223] The cross-reactivity of the tested antibodies with human (TNFR2), mouse (mTNFR2), monkey (fTNFR2), and dog (dTNFR2) TNFR2 is summarized in the table below. [Table 4]

[0224] Epitope correlation analysis of purified anti-hTNFR2 antibodies The relative location of the target protein epitopes between pairs of purified anti-TNFR2 monoclonal antibodies was analyzed by surface plasmon resonance (SPR) competition experiments. A total of five monoclonal antibodies were used to study the binding inhibition (blocking) effect of each antibody with the other antibodies: 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 experiments. Anti-His antibody was immobilized on the surface of a series S sensor chip CM5 by amino group coupling to generate an anti-His chip (i.e., CM5-anti-His-channel 1,8-chip). Then, 1 M ethanolamine (pH 8.5) was injected to block the remaining active carboxyl groups on the chip surface, followed by equilibration with HBS-EP+ buffer for 2 hours. Recombinant human TNFR2 protein with His tag (1 μg / mL) 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 (each 200 nM) was subsequently injected into the chip at 30 μL / min. The first antibody injected (analyte 1) had a binding time of 250 seconds, followed by the second antibody (analyte 2) with a binding time of 250 seconds. After antibody injection in each analysis cycle, the chip was regenerated twice with glycine buffer (pH 1.7, 30 s, 30 μL / min). When each monoclonal antibody was paired with another antibody, each pair of monoclonal antibodies went through the same experimental steps to obtain binding inhibition data.

[0225] 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 and each pair of antibodies was compared. The binding ratio was defined by the binding value of the second antibody (sample 2) divided by the binding value of the first antibody (sample 1). Statistical software was also used for cluster analysis. The epitope correlation was analyzed and the five anti-hTNFR2 antibodies were classified into four epitope clusters (Figure 8). In summary, 1A8 and 1F4 shared the same or overlapping epitopes. 3B7, 1C3, and F10 showed no epitope correlation with other antibodies.

[0226] 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 structural domains. A secondary antibody (Alexa Fluor® 647 AffiniPure F(ab')2 fragment goat anti-human IgG, Fcγ fragment specific, Jackson ImmunoResearch Laboratories, Inc., Catalog No. 151524) was used. The fluorescently labeled secondary antibody can bind to the Fc region of anti-hTNFR2 antibodies, allowing the anti-hTNFR2 antibodies bound to ΔCRD cells to be detected by FACS. Specifically, ΔCRD1-TNFR2 protein (CRD1 deleted), ΔCRD2-TNFR2 protein (CRD2 deleted), ΔCRD3-TNFR2 protein (CRD3 deleted), or ΔCRD4-TNFR2 protein (CRD4 deleted) were expressed in CHO-S cells, respectively, and then binding between the cells and different anti-hTNFR2 antibodies was detected. The TNFR2 protein is shown in SEQ ID NO: 1, where CRD1 is 39aa to 76aa of the sequence, CRD2 is 77aa to 118aa of the sequence, CRD3 is 119aa to 162aa of the sequence, and CRD4 is 168aa to 196aa of the sequence. The results of the flow cytometry experiments are shown in Table 5 below.

[0227] The results show that 1A8 and 1F4 do not bind to TNFR2 lacking CRD3, and the positive binding rate to TNFR2 lacking CRD4 is also significantly reduced, indicating that 1A8 and 1F4 have similar binding epitopes on human TNFR2, which are likely to be located at CRD3 (class I epitope). 1B6 and 1C3 do not bind to TNFR2 lacking CRD3 or CRD4, indicating that 1B6 and 1C3 have similar binding epitopes on human TNFR2, which are likely to be located at the junction of CRD3 and CRD4 (class III epitope). The positive binding rate of 1F10 to TNFR2 lacking CRD3 or CRD4 is significantly reduced, indicating that the binding epitope for 1F10 is different from class I and class III epitopes (belongs to class IV epitope). The results were consistent with the characterization of the SPR epitope. [Table 5]

[0228] In vitro ADCC detection experiment An experiment was conducted to evaluate the ADCC effect of anti-TNFR2 antibodies, using BC-1A8-IgG1, BC-1C3-IgG1, BC-1F4-IgG1, BC-1F10-IgG1, BC-1B6-IgG1, and an isotype control, human IgG1 (Crown Bioscience Inc., C0001-3).

[0229] Lactate dehydrogenase (LDH) is a cytoplasmic enzyme found in many different cell types and is released into cell culture medium after plasma membrane injury. The CyQUANT LDH Cytotoxicity Detection Kit (Invitrogen, Cat. No.: C20301) was used to accurately and quantitatively measure extracellular LDH to assess the ADCC effect of antibodies.

[0230] Anti-TNFR2 antibodies were serially diluted (10-fold) with a maximum concentration of 100 μg / mL. Target cells (MC38 cells overexpressing human TNFR2) were plated in 96-well plates (cell density 2 × 10 4 The effector cells (peripheral blood mononuclear cells (PBMCs)) were resuscitated (cell density 2 × 10 4 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 values ​​at 490 nm and 680 nm were detected by a microplate reader and used to calculate the target cell killing (% cytotoxicity) by each group of antibodies. EC50 values ​​were calculated using a nonlinear fit using antibody concentration as the horizontal axis and cytotoxicity as the vertical axis.

[0231]

number

[0232] In the above formula, experimental means the absorbance value of the experimental group, effector means the absorbance value of the effector cells, target means the absorbance value of the target cells, spontaneous release means the absorbance value of the autofluorescence of the cell culture medium, target maximum means the maximum absorbance value of the target cells, and spontaneous release* means the absorbance value of the cell culture medium volume control well.

[0233] The EC50 results are shown in Table 6. Compared to the isotype control hIgG1, target cell killing (% 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. [Table 6]

[0234] In another similar experiment, the ADCC effects of anti-TNFR2 antibodies (BC-1C3-IgG1, BC-1C3-IgG1-SI, and BC-1C3-IgG1-LALA) on target cells (MC38 cells overexpressing human TNFR2) and effector cells (FcR-TANK (CD16a-158V) cell line, ImmuneOnco Biopharmaceuticals (Shanghai) Co., Ltd.) were evaluated.

[0235] Cytotoxicity data is shown in Figure 9. EC50 results are shown in Table 7. Compared to the isotype control hIgG1, target cell killing (% cytotoxicity) 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 all have more potent ADCC activity. [Table 7]

[0236] Example 3. In vivo testing of anti-hTNFR2 antibodies To test antibodies in vivo and predict the effects of these antibodies 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 human TNFR2 extracellular domain. 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).

[0237] Humanized TNFR2 mouse models (e.g., B-TNFR2 mice) provide a novel tool for testing novel therapeutic treatments in clinical settings by significantly reducing the difference in clinical outcomes between human and normal mice expressing mouse TNFR2. A detailed description of the humanized TNFR2 mouse model can be found in PCT / CN2020 / 113618, which is incorporated by reference in its entirety.

[0238] Anti-hTNFR2 antibodies were tested in TNFR2-humanized mice (B-TNFR2) to demonstrate their effect on tumor growth in vivo. In vivo efficacy of B-TNFR2 with MC38, 10 mg / kg MC-38 tumor cells (colon adenocarcinoma cells) were injected subcutaneously into B-TNFR2 mice. The tumor volume in the mice was 100 mm 3 ~150mm 3 Once tumor volume was reached, the mice were randomly divided into different groups (6 mice per group) based on tumor volume.

[0239] 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, INC., Catalog No. BE0146). Anti-mPD-1, which has been shown to be effective in mice, was used as a positive control. Antibodies were given by intraperitoneal injection at 10 mg / kg for three weeks, on days 1 and 4 of each week (total of six injections).

[0240] The body weight of the mice was monitored throughout the treatment period. The body weights of the mice in the different groups all increased, and no statistically significant differences were found (P>0.05). On the day of group assignment (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 group assignment, D28), the average body weight of each group ranged from 22.7 g to 24.7 g, and the body weight change ranged from 107.4% to 116.5%. The results showed that the anti-hTNFR2 antibody was well tolerated and non-toxic to mice.

[0241] Tumor size (Figure 1) showed significant differences in the groups treated with anti-hTNFR2 antibody. TGI on day 28 (28 days after group assignment) for each treatment group TV The results are shown in Table 8 below. The positive control (anti-mPD-1, G9) showed good therapeutic efficacy (TGI 0.01, ... TV %=78.5%). Several antibodies, including BC-1F4-IgG1 (G2), BC-1F10-IgG1 (G4), BC-1A8-IgG1 (G6), and BC-1C3-IgG1 (G7), showed good therapeutic effects (TGI TV %>60%). Except for G3 and G5, the TGI of TNFR2 antibodies TV The TGI was over 80%. TV % is the positive control group (G19 / anti-mPD-1 TGI TV %=78.5%) was greater. [Table 8]

[0242] B-TNFR2 mice with MC38, in vivo efficacy at 1 mg / kg Similar to the in vivo drug efficacy experiment described above, after establishing the 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, INC., Catalog No.: BE0164). Antibodies were given at 1 mg / kg by intraperitoneal injection on the first and fourth days of each week for three weeks (total of six injections).

[0243] The weight of the mice was monitored throughout the experiment. The weights of the mice in the different groups all increased, and no significant differences were found between the groups (P>0.05). At the time of group assignment (D0), the mean weight of each group ranged from 20.4g to 20.8g. At the end of the experiment (24 days after group assignment, D24), the mean weight of each group ranged from 21.6g to 23.7g, and the weight change ranged from 102.5% to 114.7%. Similar to the previous experiment, the results showed that the anti-hTNFR2 antibody was well tolerated and non-toxic to mice.

[0244] Tumor size showed significant differences in the groups treated with anti-TNFR2 antibody (Figure 2). In particular, tumor sizes in G3, G4, and G5 were significantly smaller than those in G1 (G3: P = 0.005, G7: P = 0.001, G8: P = 0.011). TGI on day 24 (24 days after group assignment) for each treatment group TV The tumor inhibition (TGI) was also calculated as shown in Table 9 below. G2, G3, and G4 showed better tumor inhibition (TGI) than the positive control (G5, G6). TV %) was shown. [Table 9]

[0245] B-TNFR2 mice with MC38, in vivo efficacy at 0.3mg / kg In another similar experiment, the dose of anti-hTNFR2 antibody tested was reduced to 0.3 mg / kg. 3 Upon reaching age 18, mice (bearing MC38 tumors) were randomly assigned to different groups (6 mice per group).

[0246] Mice were then injected with PBS as a control (G1), anti-hTNFR2 antibody BC-1C3-lgG1 (G2), anti-mPD-1 (G3), anti-mCTLA4 (G4). Antibodies were given at 0.3 mg / kg by intraperitoneal injection on days 1 and 4 of each week for 3 weeks (total of 6 injections).

[0247] The weight of the mice was monitored throughout the experiment. The weight of the mice in the different groups all increased, with no significant differences between the groups (P>0.05). The mean weight of each group at the time of group assignment (D0) ranged from 19.5 g to 19.8 g. At the end of the experiment (25 days after group assignment, D25), the mean weight ranged from 21.6 g to 23.3 g, with weight changes ranging from 108.9% to 118.2%. Similar to previous results, the results showed that the anti-TNFR2 antibody was well tolerated and non-toxic to mice.

[0248] Tumor size (Figure 3) showed significant differences in the groups treated with anti-hTNFR2 antibody. TGI on day 25 (25 days after group assignment) for each treatment group TV The percentages were calculated as shown in Table 10 below. BC-1C3-IgG1 (G2) showed the best efficacy at this dose, and efficacy was better than the positive control (G3, G4). [Table 10]

[0249] B-TNFR2 mice with B16F10, in vivo efficacy at 10 mg / kg B16F10 tumor cells (melanoma cells) were injected subcutaneously into B-TNFR2 mice. The tumor volume in the mice was 100±50 mm 3Once tumor volume was reached, mice were randomly assigned to different groups (6 mice per group) based on tumor volume.

[0250] Mice were then injected with PBS as a control (G1), or with the anti-hTNFR2 antibodies BC-1A8-IgG1 (G2), BC-1C3-lgG1 (G3), BC-1F10-lgG1 (G4), BC-1F4-lgG1 (G5), and BC-1B6-lgG1 (G6). Antibodies were given at 10 mg / kg by intraperitoneal injection on days 1 and 4 of each week for 2 weeks (3 injections in total).

[0251] The body weight of the mice was monitored throughout the experiment. The body weight of the mice in different groups all increased, and no difference was found between the groups (P>0.05). At the time of group assignment (D0), the average body weight of each group ranged from 19.2g to 19.7g. Ten days after group assignment (D10), the average body weight of each group ranged from 22.1g to 24.5g, and the body weight change ranged from 114.3% to 126.8%. The results showed that the anti-hTNFR2 antibody was well tolerated and non-toxic to mice.

[0252] Tumor size showed significant differences in the groups treated with anti-TNFR2 antibodies (Figure 4). BC-1A8-IgG1 (G2), BC-1C3-IgG1 (G3), BC-1F10-lgG1 (G4), and BC-1B6-lgG1 (G6) all showed antitumor efficacy in the B16F10 melanoma model. TGI at day 10 (10 days after grouping) for each treatment group. TV The percentages are shown in Table 11 below. [Table 11]

[0253] B-TNFR2 mice bearing MC38, in vivo efficacy and toxicity at 25mg / kg In another similar experiment, the dose of the anti-hTNFR2 antibody to be tested was adjusted to 25 mg / kg to test the in vivo efficacy and toxicity. MC38 cancer tumor cells (colon adenocarcinoma cells) were subcutaneously injected into B-TNFR2 mice. The tumor volume in the mice was 100±50 mm 3 Once tumor volume was reached, mice were randomly assigned to different groups (4 mice per group) based on tumor volume.

[0254] Mice were then injected with PBS as a control (G1) or with the anti-hTNFR2 antibodies BC-1A8-IgG1 (G2), BC-1C3-IgG1 (G3), BC-1F10-IgG1 (G4), BC-1F4-IgG1 (G5), and BC-1B6-IgG1 (G6) at 25 mg / kg intraperitoneal injection, administered on the day of group assignment (D0) and 3 days after group assignment (D3) (total of 2 injections).

[0255] The weight of the mice was monitored throughout the experiment. The weights of the mice in the different groups all increased, and no significant differences were found between the groups. The average weight of each group ranged from 18.5 g to 19.2 g at the time of group assignment. At the end of the experiment (21 days after group assignment, D21), the average weight of each group ranged from 21.1 g to 23.3 g, and the weight change was 110.0% to 122.7%. Five days after group assignment (D5), the peripheral blood of the mice was collected and the blood biochemical indexes (AST, ALT) were tested. The results of the biochemical index test at D5 (Figures 7A-7B) showed that ALT and AST did not change significantly compared to the control. Similar to the previous results, the results showed that 25 mg / kg of anti-hTNFR2 antibody was well tolerated and non-toxic to mice.

[0256] Tumor size (Figure 5) showed significant differences in the groups treated with anti-TNFR2 antibodies. Compared to the control group G1, tumor volume was significantly reduced in 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). TGITV The TGI on day 21 (21 days after group assignment) for each treatment group was 81.6% to 98.4%, indicating a clear tumor suppression effect. TV The percentages were also calculated as shown in Table 12 below. [Table 12]

[0257] hTNFα / hTNFR2 mice with MC38, in vivo efficacy and toxicity at 3 mg / kg The humanized TNFα mouse model was engineered to express human TNFα protein (SEQ ID NO: 79), in which the coding sequence of mouse TNFR2 protein was replaced with the corresponding human coding sequence. A double humanized TNFα / TNFR2 mouse model (B-hTNFα / hTNFR2 mouse) was also generated by crossing TNFα humanized mouse with TNFR2 humanized mouse. A detailed description of the humanized TNFα mouse model can be found in PCT / CN2020 / 072714, which is incorporated by reference in its entirety herein.

[0258] Similar to previous in vivo drug efficacy experiments, these anti-hTNFR2 antibodies were tested for their effect on in vivo tumor growth in a double humanized TNFα / TNFR2 mouse model. In each group, double humanized TNFα / TNFR2 mice were injected intraperitoneally (ip) with phosphate buffered saline (PBS, G1), BC-1C3-IgG1 (G2), and anti-mPD-1 (G3). Antibodies were administered at 3 mg / kg by ip injection twice a week for three weeks (total of six injections).

[0259] Body weight and tumor size were monitored throughout the experiment. The body weight of mice in different groups all increased, with no significant differences between groups (P>0.05). The mean body weight of each group at the time of group assignment (D0) 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 body weight changes ranging from 115.7% to 127.2%. Similar to previous results, the results showed that the anti-hTNFR2 antibody was well tolerated and non-toxic to mice.

[0260] Tumor size (Figure 6) showed significant differences in the groups treated with anti-hTNFR2 antibody. TV The % was calculated as shown below in Table 13. BC-1C3-IgG1 (G2) showed the best efficacy at this dose, and efficacy was better than the positive control (G3). [Table 13]

[0261] B-TNFR2 mice with MC38, in vivo efficacy at 3mg / kg Similar to the in vivo drug efficacy experiments described above, MC38 cancer tumor cells were injected subcutaneously into B-TNFR2 mice. 3 Once tumor volume reached 100 mg / mL, mice were randomly assigned to different groups (6 mice per group) based on tumor volume. Mice were injected with PBS as control (G1), 3 mg / kg BC-1C3-IgG1 (G2), 3 mg / kg anti-mPD-1 (G3), a combination of 3 mg / kg BC-1C3-lgG1 and 3 mg / kg anti-mPD-1 (G4), 3 mg / kg atezolizumab analog (G5), or a combination of 3 mg / kg BC-1C3-IgG1 and 3 mg / kg atezolizumab analog (G6). The dosing frequency was twice weekly (6 doses in total).

[0262] Atezolizumab is a humanized anti-PD-L1 monoclonal antibody (VH SEQ ID NO: 80, VL SEQ ID NO: 81) developed by Genentech.

[0263] Body weight and tumor size were monitored throughout the experiment. The body weight of mice in different groups all increased, with no significant differences between groups (P>0.05). The mean body weight of each group at the time of group assignment (D0) ranged from 18.9g to 19.3g. At the end of the experiment (D24), the mean body weight ranged from 22.4g to 24.2g, with body weight changes ranging from 118.6% to 125.3%. Similar to previous results, the results showed that the anti-hTNFR2 antibody was well tolerated and non-toxic to mice.

[0264] Tumor sizes in antibody-treated groups are shown in Figure 18. TGI at D24 for each treatment group TV The percentages were calculated as shown in Table 14 below. Compared with the control group, the tumor growth in the treatment groups was inhibited to different degrees. Among the treatment groups, the combination of anti-PD-1 antibody and anti-hTNFR2 antibody (G4) inhibited tumor growth with superior efficacy. Similarly, the combination of anti-PD-L1 antibody atezolizumab analog and anti-hTNFR2 antibody (G6) also showed superior tumor growth inhibition than anti-hTNFR2 antibody (G2) and anti-PD-L1 antibody atezolizumab analog (G5). [Table 14]

[0265] B-TNFR2 mice with GL261, in vivo efficacy at 1-10 mg / kg GL261 tumor cells (glioblastoma cells) were injected subcutaneously into B-TNFR2 mice. The tumor volume in the mice was approximately 80 mm. 3 Once tumor volume was reached, the mice were randomly divided into different groups (6 mice per group) based on tumor volume.

[0266] 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 given by intraperitoneal injection on days 1 and 4 of each week for 3 weeks (total of 6 injections).

[0267] The body weight of the mice was monitored throughout the treatment period. The body weights of the mice in the different groups all increased, and no statistically significant differences were found (P>0.05). On the day of group assignment (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 group assignment, D24), the average body weight of each group ranged from 22.5 g to 24.2 g, and the body weight change ranged from 109.4% to 118.3%. The results showed that the anti-hTNFR2 antibodies were well tolerated and non-toxic to mice.

[0268] Tumor size data for antibody-treated groups is shown in Figure 19. TGI at D24 for each treatment group TV The percentages were calculated as shown in Table 15 below. At a dose of 1 mg / kg, BC-1C3-IgG showed better anti-tumor effects than anti-mPD-1 antibody. In addition, BC-1C3-IgG1 showed dose-dependent anti-tumor effects (the higher the dose level, the better the anti-tumor effect). [Table 15]

[0269] B-TNFR2 mice with MC38, in vivo efficacy at 3mg / kg BI-1808 (VH SEQ ID NO:82, VL SEQ ID NO:83) is a monoclonal antibody developed by BioInvent targeting tumor necrosis factor receptor superfamily member 1B (TNFR2). The product is in early clinical development as a single agent and in combination with pembrolizumab to treat solid tumors and cutaneous T-cell lymphoma (CTCL).

[0270] h600-25-108 (VH SEQ ID NO: 84, VL SEQ ID NO: 85) is a humanized anti-TNFR2 monoclonal antibody developed by Apexigen.

[0271] HFB3-1hz6-hG1 (VH SEQ ID NO: 86, VL SEQ ID NO: 87) is a monoclonal anti-TNFR2 agonist antibody in Phase I clinical trials from HiFiBiO Therapeutics for the treatment of advanced solid tumors.

[0272] Similar to the in vivo drug efficacy experiments described above, MC38 tumor cells were injected subcutaneously into B-TNFR2 mice. 3 Once tumor volume reached 100 mg / kg, mice were randomly divided into different groups (6 mice per group) based on tumor volume. Mice were injected with PBS (G1), 3 mg / kg BC-1C3-lgG1 (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 weekly (6 administrations in total).

[0273] Body weight and tumor size were monitored throughout the experiment. The body weight of mice in different groups all increased, with no significant differences between groups (P>0.05). The mean body weight of each group at the time of group assignment (D0) ranged from 20.1 g to 20.4 g. At the end of the experiment (D28), the mean body weight ranged from 22.9 g to 24.9 g, with body weight changes ranging from 113.2% to 122.4%. Similar to previous results, the results showed that the anti-hTNFR2 antibody was well tolerated and non-toxic to mice.

[0274] Tumor sizes are shown in Figure 20. TGI at D28 for each treatment group TVThe percentages were calculated as shown below in Table 16. The results showed that the BC-1C3-IgG1 antibody (G2) showed significantly better in vivo efficacy than the BI-1808 analog (G3), the h600-25-108 analog (G4), or the HFB3-1hz6-hG1 analog (G5) at the same dose level. [Table 16]

[0275] In vivo efficacy of B-hTNFa / hTNFR2 with MC38, 3mg / kg h600-25-71 (VH SEQ ID NO: 88, VL SEQ ID NO: 89) is a humanized anti-TNFR2 monoclonal antibody developed by Apexigen.

[0276] Similar to the in vivo drug efficacy experiments described above, MC38 tumor cells were injected subcutaneously into B-hTNFa / hTNFR2 mice. 3 Once tumor volume reached 100 mg / kg, mice were randomly divided into different groups (6 mice per group) based on tumor volume and injected with PBS (G1), 3 mg / kg BC-1C3-lgG1 (G2), or 3 mg / kg h600-25-71 analog (G3). The dosing frequency was twice weekly (6 doses in total).

[0277] Body weight and tumor size were monitored throughout the experiment. The body weight of mice in different groups all increased, with no significant differences between groups (P>0.05). The mean body weight of each group at the time of group assignment (D0) ranged from 21.0g to 21.1g. At the end of the experiment (D24), the mean body weight ranged from 23.8g to 24.8g, with body weight changes ranging from 113.3% to 117.5%. Similar to previous results, the results showed that the anti-hTNFR2 antibody was well tolerated and non-toxic to mice.

[0278] Tumor sizes are shown in Figure 21. TGI at D24 versus values ​​for each treatment group TVThe percentages are shown below in Table 17. The results showed that BC-1C3-IgG1 demonstrated significantly better in vivo efficacy than the h600-25-71 analogue. [Table 17]

[0279] Example 4. PK and TILS Analysis B-TNFR2 mice with MC38 model, 10mg / kg, PK analysis The pharmacokinetic clearance rate of anti-TNFR2 antibodies was measured in humanized TNFR2 mice. MC38 cells (5 × 10 5 ) was subcutaneously injected into humanized TNFR2 mice, and tumors were grown to 300 mm 3 Once the mice had grown to 10 mg / kg, they were divided into 8 groups (n=4). 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 h, 24 h, 3 days, 5 days, and 7 days after administration.

[0280] Serum levels of human antibodies were measured by sandwich ELISA enzyme-linked immunoassay. Briefly, goat polyclonal anti-human IgG (Fc specific) capture antibody (Jackson ImmunoResearch, Catalog No.: 109-036-098) was diluted in 0.1 M carbonate buffer (pH 9.6) to a final concentration of 2.0 μg / mL, added at 100 μL / well to a 96-well plate (ELISA plate) 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, Cat. No.: 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 the 96-well plate at 100 μL / well as a substrate to react with HRP. After incubation at room temperature in the dark, 100 μL of stop solution (Beyotime, Cat. No.: P0215) was added to each well. The absorbance value of each well was read at wavelengths of 450 nm and 630 nm using a microplate reader. The data was analyzed using the data analysis software Gen5. A four-parameter standard curve was prepared using the absorbance values ​​of the calibration samples adjusted by each test product and the corresponding concentrations. The antibody concentration of each serum sample was then calculated using the standard curve. Drug concentration-time curves were generated using the calculated sample concentrations at each time point. Pharmacokinetic parameters were calculated using Phoenix winnolin 8.3. The results are shown in Table 18 below. [Table 18]

[0281] The above results show that after injection of different antibodies, the antibody concentration in the serum of TNFR2 humanized mice decreased over time (see FIG. 10), which is consistent with the pharmacokinetic characteristics. The longest half-life of the BC-1C3-IgG1(G2) antibody in mice was 2.99 days. The shortest half-life of the BC-1B6-IgG1-SI(G7) in mice was 0.86 days. The half-lives of the other antibodies in mice were relatively close, ranging from 1.69 days to 2.38 days. By the end of the 7th day of sampling, the area under the curve (AUC) of the drug concentration-time curve of the BC-1C3-IgG1(G2) antibody was 250.93 h*μg / mL, which was longer than the AUC of the other antibodies (AUC0-7day 77.90-168.89 h*μg / mL). The clearance rate (CL) of the BC-1C3-IgG1(G2) antibody was 28.85 mL / h / kg, while the CL of the other antibodies ranged from 47.07 to 61.19 mL / h / kg. The results showed that compared with the other antibodies, BC-1C3-IgG1(G2) had a lower clearance efficiency, and the metabolism of the BC-1C3-IgG1(G2) antibody in mice was slower. Example 5. Reporter cell activity and binding assays Activation effect in reporter cells Experiments were performed to test whether anti-TNFR2 antibodies could activate the TNFR2 pathway.

[0282] Human TNFα protein (Sino Biological Inc., Catalog No.: 10602-HNAE) was serially diluted (3-fold) with 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) with a maximum concentration of 60 μg / mL. Jurkat-GFP-TNFR2 cells were seeded in 96-well plates (cell density 1 × 10 5After incubation, 100 μL of human TNFα protein or 100 μL of anti-TNFR2 antibody was added to each well and incubated at 37°C overnight. 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.

[0283] As shown in Figure 11A, no fluorescent signal was detected in the presence of anti-TNFR2 antibodies BC-1F4-IgG1, BC-1C3-IgG1, BC-1B6-IgG1, and BC-1F10-IgG1, whereas BC-1C3-IgG1 showed weak reporter cell activation (Figure 11B). Blocking effect in reporter cells Experiments were performed to test whether anti-TNFR2 antibodies could block the binding of TNFR2 to its ligand, TNFα.

[0284] Reporter Jurkat-GFP-TNFR2 cells were seeded in 96-well plates (cell density 1 × 10 5 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) with 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 and the cells were resuspended. The signal for GFP was measured by flow cytometry.

[0285] As shown in FIG. 12, when the concentrations of the anti-TNFR2 antibodies BC-1F10-IgG1 and BC-1F4-IgG1 were increased, the GFP signal (indicating cells that bind TNFα) decreased (y-axis), suggesting that the binding between human TNFα and TNFR2 was blocked by the anti-TNFR2 antibodies BC-1F10-IgG1 and BC-1F4-IgG1. Example 6. In vivo toxicity experiments (tumor-free model) TNFR2 humanized mice (6-8 weeks old) were randomly divided into control and treatment groups (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 doses of anti-hTNFR2 antibody and CTLA4 antibody were 30 mg / kg or 100 mg / kg. The administration frequency was once a week for a total of four administrations. The specific doses, modes, and frequencies of administration are shown in Table 19 below. In the experiment, changes in body weight and any abnormalities were monitored. Blood biochemical indicators were monitored 1 day, 8 days, 15 days, 22 days, and 28 days after group assignment. Blood biochemical indices 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 28 days after group assignment. Blood routine examinations included tests on white blood cell count (WBC), red blood cell (RBC), hemoglobin (HGB), hematocrit (HTC), mean red blood cell volume (MCV), mean red blood cell hemoglobin content (MCH), mean red blood cell hemoglobin concentration (MCHC), PLT (platelet count), lymphocytes (LYMPH#), lymphocyte percentage (LYMPH%), monocytes (MONO#), monocyte percentage (MONO%), and neutrophil percentage (NEUT%). At the end of the experiment, the heart, liver, spleen, lungs, kidneys, and intestines of the mice were immersed in formalin for HE staining, and the weights of the liver, spleen, and kidneys were measured. [Table 19]

[0286] The results showed that the body weight of all mice in the control and treatment groups showed an upward trend throughout the experimental period, and there was no significant difference in body weight change between groups. Blood biochemistry index test results (see Figures 13A-13B for exemplary blood biochemistry indexes at day 28) and blood routine test results showed no significant difference compared to 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. Example 7. TNFR2 antibodies block Treg cell inhibition of CD8+ T cell proliferation Human IgG1 (control) and anti-TNFR2 antibodies BC-1C3-IgG1 and HFB3-1hz6-hG1 analog were diluted to a final concentration of 20ng / mL. Anti-CD3 antibody (CD3, Acro Biosystems, Cat. No.: CDE-M120a) was used to coat 96-well plates (10μg / mL, 100μL per well) overnight at 4℃. CFSE (CellTrace TM 100 μL of PBMC cells (AllCells, Catalog No.: PB003F-C) labeled with CFSE Cell Proliferation Kit, Thermo Fisher, Catalog No.: CDE-M120a) and 50 μL of Treg cells (OriCell, Catalog No.: FPB009-4F-2) were added to each well. 40 μL of BC-1C3-IgG1, 40 μL of human IgG1, or 40 μL of HFB3-1hz6-hG1 analogs were added to each well and incubated for 120 hours at 37°C, 5% CO2. After 48 hours of incubation, 10 μL of IL-2 (Acro Biosystems, Catalog No.: IL-2-H4113) was added to each well. After 120 hours of incubation, cells were harvested and hCD8+ cells were measured by flow cytometry.

[0287] As shown in FIG. 22, BC-1C3-IgG1 blocked the inhibition of CD8+ T cell proliferation by Treg cells. Example 8. CD8+ T cell activation assay 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 and anti-TNFR2 antibodies were used to coat 96-well plates overnight at 4° C. CFSE (CellTrace TM CD8+ T cells (OriCell) labeled with CFSE Cell Proliferation Kit (Thermo Fisher, Catalog Number: CDE-M120a) were added to each well (1 × 10 5 hCD28 (BioXcell, Cat#: BE0248) was added to each well (1 μg / mL, 100 μL per 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.

[0288] As shown in Figure 23, CD8+ T cells increased with increasing concentration of anti-TNFR2 antibody BC-1C3-IgG1. Figures 24A-24B show that the secretion of human IL2 and IFNγ increased after the addition of anti-TNFR2 antibody. The above results indicate that BC-1C3-IgG1 can promote the proliferation and activation of CD8+ T cells. Other embodiments Although the present invention has been described in conjunction with its detailed description, it should be understood that the above description is for illustrative purposes only and is not intended to limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to TNFR2, a heavy chain variable region (VH) comprising complementarity determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; a light chain variable region (VL) comprising CDR1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence; The selected VH CDR1, 2, and 3 amino acid sequences and the selected VL CDR1, 2, and 3 amino acid sequences are as follows: (1) (1-a) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 18, 19, and 20, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 21, 22, and 23, respectively; or (1-b) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 54, 55, and 56, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 57, 58, and 59, respectively; (2) (2-a) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 6, 7, and 8, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 9, 10, and 11, respectively; or (2-b) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 42, 43, and 44, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 45, 46, and 47, respectively; (3) (3-a) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 12, 13, and 14, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 15, 16, and 17, respectively; or (3-b) the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 48, 49, and 50, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 51, 52, and 53, respectively; (4) (4-a) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 24, 25, and 26, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 27, 28, and 29, respectively; or the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 60, 61, and 62, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 63, 64, and 65, respectively; or (5) (5-a) The selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 30, 31, and 32, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 33, 34, and 35, respectively; or the selected VH CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 66, 67, and 68, respectively, and the selected VL CDR1, 2, and 3 amino acid sequences are set forth in SEQ ID NOs: 69, 70, and 71, respectively. An antibody or antigen-binding fragment thereof.

2. (1) (1-a) According to Kabat numbering, the VH comprises CDRs 1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, respectively, and the VL comprises CDRs 1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 21, 22, and 23, respectively; or (1-b) According to Chothia numbering, the VH comprises CDRs 1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 54, 55, and 56, respectively, and the VL comprises CDRs 1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 57, 58, and 59, respectively; (2) (2-a) According to Kabat numbering, the VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 9, 10, and 11, respectively; or (2-b) According to Chothia numbering, the VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 42, 43, and 44, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 47, respectively; (3) (3-a) According to Kabat numbering, the VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 15, 16, and 17, respectively; or (3-b) According to Chothia numbering, the VH comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 48, 49, and 50, respectively, and the VL comprises CDR1, 2, and 3 having the amino acid sequences set forth in SEQ ID NOs: 51, 52, and 53, respectively; (4) (4-a) According to Kabat numbering, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 24, 25, and 26, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 27, 28, and 29, respectively; or (4-b) According to Chothia numbering, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 60, 61, and 62, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 63, 64, and 65, respectively; or (5) (5-a) According to Kabat numbering, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 30, 31, and 32, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, respectively; or (5-b) According to Chothia numbering, the VH comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 66, 67, and 68, respectively, and the VL comprises CDR1, 2, 3 having the amino acid sequences set forth in SEQ ID NOs: 69, 70, and 71, respectively; The antibody or antigen-binding fragment thereof described in claim 1. (i) the antibody or antigen-binding fragment specifically binds to human TNFR2; (ii) the antibody or antigen-binding fragment is a human antibody or antigen-binding fragment thereof (e.g., a human IgG1 antibody); (iii) the antibody or antigen-binding fragment is a single-chain variable fragment (scFv). The antibody or antigen-binding fragment thereof of claim 1, having one or more characteristics selected from the following:

4. A nucleic acid comprising a polynucleotide encoding a polypeptide, (1) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, or SEQ ID NOs: 54, 55, and 56, respectively, 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; (2) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 21, 22, and 23, or SEQ ID NOs: 57, 58, and 59, respectively, wherein the VL binds to TNFR2 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 40; (3) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8, or SEQ ID NOs: 42, 43, and 44, respectively, 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; (4) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 10, and 11, or SEQ ID NOs: 45, 46, and 47, respectively, wherein the VL binds to TNFR2 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 36; (5) An immunoglobulin heavy chain or a fragment thereof, comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 12, 13, and 14, or SEQ ID NOs: 48, 49, and 50, respectively, 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; (6) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 15, 16, and 17, or SEQ ID NOs: 51, 52, and 53, respectively, wherein the VL binds to TNFR2 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 38; (7) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 24, 25, and 26, or SEQ ID NOs: 60, 61, and 62, respectively, 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; (8) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 27, 28, and 29, or SEQ ID NOs: 63, 64, and 65, respectively, wherein the VL binds to TNFR2 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 72; (9) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region (VH) comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 30, 31, and 32, or SEQ ID NOs: 66, 67, and 68, respectively, 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 (10) An immunoglobulin light chain or a fragment thereof comprising a VL comprising CDR1, 2, and 3 comprising the amino acid sequences set forth in SEQ ID NOs: 33, 34, and 35, or SEQ ID NOs: 69, 70, and 71, respectively, wherein the VL binds to TNFR2 when paired with a VH comprising the amino acid sequence set forth in SEQ ID NO: 74; A nucleic acid comprising a polynucleotide encoding a polypeptide comprising:

5. said immunoglobulin heavy chain or fragment thereof is a human immunoglobulin heavy chain or fragment thereof and said immunoglobulin light chain or fragment thereof is a human immunoglobulin light chain or fragment thereof; and / or the nucleic acid encodes a single chain variable fragment (scFv), and / or the nucleic acid is cDNA; The nucleic acid of claim 4.

6. 6. A nucleic acid sequence comprising one or more of the nucleic acids of claim 4 or 5, Optionally, the vector comprises two of said nucleic acids, said vector encoding said VH region and said VL region that together bind to TNFR2.

7. A pair of vectors, each vector comprising one of the nucleic acids of claim 4 or 5, and together encoding the VH and VL regions that bind to TNFR2. A pair of vectors.

8. A method for the preparation of a nucleic acid sequence comprising: (i) one or more of the nucleic acids of claim 4 or 5; (ii) a vector comprising one or more of the nucleic acids; or (iii) a pair of vectors, each vector comprising one of the nucleic acids and together with the pair of vectors encoding the VH and VL regions that bind to TNFR2. A cell, Optionally, the cell is a CHO cell. cell.

9. 1. A method for producing an antibody or antigen-binding fragment thereof, comprising: (a) culturing the cell of claim 8 under conditions sufficient to produce the antibody or antigen-binding fragment thereof; (b) harvesting the antibody or antigen-binding fragment produced by the cell; and method.

10. An antibody or antigen-binding fragment thereof that binds to TNFR2, 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 as follows: (1) the selected VH sequence is SEQ ID NO: 40 and the selected VL sequence is SEQ ID NO: 41; (2) the selected VH sequence is SEQ ID NO: 36 and the selected VL sequence is SEQ ID NO: 37; (3) the selected VH sequence is SEQ ID NO: 38 and the selected VL sequence is SEQ ID NO: 39; (4) the selected VH sequence is SEQ ID NO: 72 and the selected VL sequence is SEQ ID NO: 73; or (5) the selected VH sequence is SEQ ID NO: 74 and the selected VL sequence is SEQ ID NO: 75; One of the The antibody or antigen-binding fragment thereof. (1) the VH comprises the sequence of SEQ ID NO: 40, and the VL comprises the sequence of SEQ ID NO: 41; (2) the VH comprises the sequence of SEQ ID NO: 36, and the VL comprises the sequence of SEQ ID NO: 37; (3) the VH comprises the sequence of SEQ ID NO: 38, and the VL comprises the sequence of SEQ ID NO: 39; (4) the VH comprises the sequence of SEQ ID NO: 72 and the VL comprises the sequence of SEQ ID NO: 73; or (5) The VH comprises the sequence of SEQ ID NO: 74, and the VL comprises the sequence of SEQ ID NO:

75. The antibody or antigen-binding fragment thereof described in claim 10. (i) the antibody or antigen-binding fragment specifically binds to human TNFR2; (ii) the antibody or antigen-binding fragment is a human antibody or antigen-binding fragment thereof; (iii) the antibody or antigen-binding fragment is a single-chain variable fragment (scFv). The antibody or antigen-binding fragment thereof of claim 10, having one or more characteristics selected from the following:

13. An antibody or antigen-binding fragment thereof that binds to TNFR2, a heavy chain variable region (VH) comprising VH CDR1, VH CDR2, and VH CDR3 identical to the VH CDR1, VH CDR2, and VH CDR3 of the selected VH sequence; and a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3 identical to the VL CDR1, a VL CDR2, and a VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are: (1) the selected VH sequence is SEQ ID NO: 40 and the selected VL sequence is SEQ ID NO: 41; (2) the selected VH sequence is SEQ ID NO: 36 and the selected VL sequence is SEQ ID NO: 37; (3) the selected VH sequence is SEQ ID NO: 38 and the selected VL sequence is SEQ ID NO: 39; (4) the selected VH sequence is SEQ ID NO: 72 and the selected VL sequence is SEQ ID NO: 73; or (5) the selected VH sequence is SEQ ID NO: 74 and the selected VL sequence is SEQ ID NO: 75; One of the The antibody or antigen-binding fragment thereof.

14. comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 3, 10 to 12 and 13 covalently linked to a therapeutic agent; Optionally, the therapeutic agent is a cytotoxic or cytostatic agent. Antibody drug conjugates.

15. A pharmaceutical composition for treating a subject with cancer, for reducing tumor growth rate and / or for killing tumor cells, comprising the antibody or antigen-binding fragment thereof, or antibody-drug conjugate according to any one of claims 1 to 3, 10 to 12 and 13; the antibody drug conjugate comprises the antibody or antigen-binding fragment thereof covalently linked to a therapeutic agent; Pharmaceutical compositions.

16. the subject has colorectal cancer, ovarian cancer, acute myeloid leukemia, Lewis lung carcinoma, breast cancer, hepatocellular carcinoma, nervous system cancer, glioma, and colon cancer; or the subject has renal cell carcinoma, multiple myeloma, colon cancer, ovarian cancer, glioma, or cutaneous T-cell lymphoma; or the subject has colon cancer, glioma, or ovarian cancer; 16. The pharmaceutical composition of claim 15.

17. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, 10 to 12, and 13, and a pharmaceutically acceptable carrier. Pharmaceutical compositions.

18. 15. An antibody-drug conjugate comprising the antibody-drug conjugate of claim 14 and a pharmaceutically acceptable carrier. Pharmaceutical compositions.

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