Anti-tnfr2 antibodies and uses thereof

Abstract

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

Description

[0001] This application claims priority to international patent application PCT / CN2021 / 140487, filed on 2021 / 12 / 22. The full text of the aforementioned Chinese patent application is incorporated herein by reference. Technical Field

[0002] This disclosure relates to anti-TNFR2 (tumor necrosis factor receptor 2) antibodies and their uses. Background Technology

[0003] Cancer is one of the leading causes of death worldwide. According to statistics from the World Health Organization, in 2012, there were 14 million new cancer cases and 8.2 million cancer deaths globally. In China, there were 3.07 million newly diagnosed cancer cases and 2.2 million cancer deaths.

[0004] The recent clinical and commercial successes of anticancer antibodies have sparked great interest in antibody-based therapies. There is a need to develop antibodies for use in various antibody-based therapies to treat cancer or autoimmune diseases. Summary of the Invention

[0005] This disclosure relates to anti-TNFR2 antibodies, their antigen-binding fragments, and their uses.

[0006] In one aspect, this disclosure relates to an antibody or antigen-binding fragment thereof that binds to TNFR2, the antibody or antigen-binding fragment comprising: a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2 amino acid sequence, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3 amino acid sequence; and a light chain variable region (VL) comprising CDRs 1, 2, and 3, wherein the VL CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR1 amino acid sequence, the VL CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR2 amino acid sequence, and the VL CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VL CDR3 amino acid sequence, wherein these selected VH CDRs 1, 2, and 3 amino acid sequences and these selected VL CDRs... The amino acid sequences 1, 2, and 3 are one of the following:

[0007] (1) The selected VH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO:6, 7, 8, and the selected VL CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO:9, 10, 11, respectively;

[0008] (2) The selected VH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO:12, 13, 14, and the selected VL CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO:15, 16, 17, respectively;

[0009] (3) The selected VH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO:18, 19, 20, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO:21, 22, 23, respectively;

[0010] (4) The selected VH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO:24, 25, 26, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO:27, 28, 29, respectively;

[0011] (5) The selected VH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO:30, 31, 32, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO:33, 34, 35, respectively;

[0012] (6) The selected VH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO:42, 43, 44, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO:45, 46, 47, respectively;

[0013] (7) The selected VH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO:48, 49, 50, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO:51, 52, 53, respectively;

[0014] (8) The selected VH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO:54, 55, 56, respectively, and the selected VL CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO:57, 58, 59, respectively;

[0015] (9) The selected VH CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 60, 61, and 62, respectively, and the selected VL CDR 1, 2, and 3 amino acid sequences are shown in SEQ ID NO: 63, 64, and 65, respectively; or

[0016] (10) The selected VH CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO:66, 67, 68, and the selected VL CDR 1, 2, 3 amino acid sequences are shown in SEQ ID NO:69, 70, 71, respectively.

[0017] In some embodiments, according to the Kabat number, VH comprises CDR 1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 6, 7, and 8, respectively, and VL comprises CDR 1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 9, 10, and 11, respectively.

[0018] In some embodiments, according to the Kabat number, VH comprises CDR 1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 12, 13, and 14, respectively, and VL comprises CDR 1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 15, 16, and 17, respectively.

[0019] In some embodiments, according to the Kabat number, VH comprises CDR 1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 18, 19, and 20, respectively, and VL comprises CDR 1, 2, 3 having the amino acid sequences shown in SEQ ID NO: 21, 22, and 23, respectively.

[0020] In some embodiments, according to the Kabat number, VH comprises CDR 1, 2, 3 having the amino acid sequences shown in SEQ ID NO:24, 25, and 26, respectively, and VL comprises CDR 1, 2, 3 having the amino acid sequences shown in SEQ ID NO:27, 28, and 29, respectively.

[0021] In some embodiments, according to the Kabat number, VH comprises CDR 1, 2, 3 having the amino acid sequences shown in SEQ ID NO:30, 31, and 32, respectively, and VL comprises CDR 1, 2, 3 having the amino acid sequences shown in SEQ ID NO:33, 34, and 35, respectively.

[0022] In some embodiments, the antibody or antigen-binding fragment specifically binds to human TNFR2.

[0023] In some embodiments, the antibody or antigen-binding fragment is a human antibody or its antigen-binding fragment (e.g., a human IgG1 antibody).

[0024] In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFV).

[0025] In one aspect, this disclosure relates to nucleic acids comprising polynucleotides encoding polypeptides, the polypeptides comprising:

[0026] (1) An immunoglobulin heavy chain or a fragment thereof containing a heavy chain variable region (VH), the heavy chain variable region comprising complementarity-determining regions (CDRs) 1, 2 and 3 respectively containing the amino acid sequences shown in SEQ ID NO: 6, 7 and 8, and wherein the VH binds TNFR2 when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO: 37.

[0027] (2) An immunoglobulin light chain or a fragment thereof containing VL, wherein the VL comprises CDR 1, 2 and 3 respectively containing the amino acid sequences shown in SEQ ID NO:9, 10 and 11, and wherein the VL binds TNFR2 when paired with VH containing the amino acid sequence shown in SEQ ID NO:36;

[0028] (3) An immunoglobulin heavy chain or a fragment thereof containing a heavy chain variable region (VH), wherein the heavy chain variable region comprises CDR 1, 2 and 3 respectively containing the amino acid sequences shown in SEQ ID NO:12, 13 and 14, and wherein the VH binds TNFR2 when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO:39.

[0029] (4) An immunoglobulin light chain or fragment thereof containing VL, wherein the VL comprises CDR 1, 2 and 3 respectively containing the amino acid sequences shown in SEQ ID NO:15, 16 and 17, and wherein the VL binds TNFR2 when paired with VH containing the amino acid sequence shown in SEQ ID NO:38;

[0030] (5) An immunoglobulin heavy chain or a fragment thereof containing a heavy chain variable region (VH), wherein the heavy chain variable region comprises CDR 1, 2 and 3 respectively containing the amino acid sequences shown in SEQ ID NO:18, 19 and 20, and wherein the VH binds TNFR2 when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO:41.

[0031] (6) An immunoglobulin light chain or fragment thereof containing VL, wherein the VL comprises CDR 1, 2 and 3 respectively containing the amino acid sequences shown in SEQ ID NO:21, 22 and 23, and wherein the VL binds TNFR2 when paired with VH containing the amino acid sequence shown in SEQ ID NO:40;

[0032] (7) An immunoglobulin heavy chain or a fragment thereof containing a heavy chain variable region (VH), the heavy chain variable region comprising CDR 1, 2 and 3 respectively containing the amino acid sequences shown in SEQ ID NO:24, 25 and 26, and wherein the VH binds TNFR2 when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO:73.

[0033] (8) An immunoglobulin light chain or a fragment thereof containing a VL, wherein the VL comprises CDRs 1, 2 and 3, respectively, containing the amino acid sequences shown in SEQ ID NO:27, 28 and 29, and wherein the VL binds to TNFR2 when paired with a VH containing the amino acid sequence shown in SEQ ID NO:72;

[0034] (9) An immunoglobulin heavy chain or a fragment thereof containing a heavy chain variable region (VH), wherein the heavy chain variable region comprises CDRs 1, 2, and 3, respectively, containing the amino acid sequences shown in SEQ ID NO:30, 31, and 32, and wherein the VH binds TNFR2 when paired with a light chain variable region (VL) containing the amino acid sequence shown in SEQ ID NO:75; or

[0035] (10) An immunoglobulin light chain or a fragment thereof containing a VL, wherein the VL comprises CDR 1, 2 and 3, respectively, containing the amino acid sequences shown in SEQ ID NO:33, 34 and 35, and wherein the VL binds TNFR2 when paired with a VH containing the amino acid sequence shown in SEQ ID NO:74.

[0036] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof containing a VH, wherein the VH comprises CDR1, 2 and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 6, 7 and 8.

[0037] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof containing a VL, the VL comprising CDRs 1, 2 and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 9, 10 and 11.

[0038] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof containing a VH, wherein the VH comprises CDRs 1, 2 and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 12, 13 and 14.

[0039] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof containing a VL, the VL comprising CDRs 1, 2 and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 15, 16 and 17.

[0040] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof containing a VH, wherein the VH comprises CDRs 1, 2 and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 18, 19 and 20.

[0041] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof containing a VL, the VL comprising CDRs 1, 2 and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 21, 22 and 23.

[0042] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof containing a VH, wherein the VH comprises CDRs 1, 2 and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 24, 25 and 26.

[0043] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or fragment thereof containing a VL, the VL comprising CDRs 1, 2 and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 27, 28 and 29.

[0044] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof containing a VH, wherein the VH comprises CDRs 1, 2 and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 30, 31 and 32.

[0045] In some embodiments, the nucleic acid comprises a polynucleotide encoding a polypeptide comprising an immunoglobulin light chain or a fragment thereof containing a VL, the VL comprising CDRs 1, 2 and 3, respectively, containing the amino acid sequences shown in SEQ ID NO: 33, 34 and 35.

[0046] In some embodiments, VH specifically binds to human TNFR2 when paired with VL, or VL specifically binds to human TNFR2 when paired with VH.

[0047] In some embodiments, the immunoglobulin heavy chain or a fragment thereof is a human immunoglobulin heavy chain or a fragment thereof, and the immunoglobulin light chain or a fragment thereof is a human immunoglobulin light chain or a fragment thereof.

[0048] In some embodiments, nucleic acids encode single-stranded variable fragments (scFv).

[0049] In some embodiments, the nucleic acid is cDNA.

[0050] In one respect, this disclosure relates to vectors that contain one or more of the nucleic acids described herein.

[0051] In one aspect, this disclosure relates to a vector comprising two of the nucleic acids described herein, wherein the vector encodes a VH region and a VL region, which together bind TNFR2.

[0052] In one aspect, this disclosure relates to vector pairs, each of which contains one of the nucleic acids described herein, wherein the vector pair co-encodes a VH region and a VL region that together bind TNFR2.

[0053] In one aspect, this disclosure relates to cells that comprise the vectors described herein, or pairs of vectors described herein. In some embodiments, the cells are CHO cells.

[0054] In one respect, this disclosure relates to cells that contain one or more of the nucleic acids described herein.

[0055] In one aspect, this disclosure relates to cells containing two of the nucleic acids described herein. In some embodiments, the two nucleic acids co-encode a VH region and a VL region, which together bind TNFR2.

[0056] In one aspect, this disclosure relates to a method for generating an antibody or an antigen-binding fragment thereof, the method comprising (a) culturing the cell under conditions sufficient to cause the cell described herein to generate an antibody or an antigen-binding fragment; and (b) collecting the antibody or antigen-binding fragment generated by the cell.

[0057] In one aspect, this disclosure relates to an antibody or antigen-binding fragment thereof that binds to TNFR2, the antibody or antigen-binding fragment comprising: a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region (VH) comprising an amino acid sequence that is at least 80% identical to a selected VH sequence, and the light chain variable region (VL) comprising an amino acid sequence that is at least 80% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0058] (1) The selected VH sequence is SEQ ID NO:36, and the selected VL sequence is SEQ ID NO:37;

[0059] (2) The selected VH sequence is SEQ ID NO:38, and the selected VL sequence is SEQ ID NO:39;

[0060] (3) The selected VH sequence is SEQ ID NO:40, and the selected VL sequence is SEQ ID NO:41;

[0061] (4) The selected VH sequence is SEQ ID NO:72, and the selected VL sequence is SEQ ID NO:73; or

[0062] (5) The selected VH sequence is SEQ ID NO:74, and the selected VL sequence is SEQ ID NO:75.

[0063] In some embodiments, VH contains the sequence of SEQ ID NO:36, and VL contains the sequence of SEQ ID NO:37.

[0064] In some embodiments, VH comprises the sequence of SEQ ID NO:72, and VL comprises the sequence of SEQ ID NO:73.

[0065] In some embodiments, VH contains the sequence of SEQ ID NO:38, and VL contains the sequence of SEQ ID NO:39.

[0066] In some embodiments, VH contains the sequence of SEQ ID NO:40, and VL contains the sequence of SEQ ID NO:41.

[0067] In some embodiments, VH contains the sequence of SEQ ID NO:74, and VL contains the sequence of SEQ ID NO:75.

[0068] In some embodiments, the antibody or antigen-binding fragment specifically binds to human TNFR2.

[0069] In some embodiments, the antibody or antigen-binding fragment is a human antibody or its antigen-binding fragment.

[0070] In some embodiments, the antibody or antigen-binding fragment is a single-chain variable fragment (scFV).

[0071] In one respect, this disclosure relates to antibodies or antigen-binding fragments thereof that cross-compete with the antibodies or antigen-binding fragments thereof described herein.

[0072] In one aspect, this disclosure relates to an antibody or antigen-binding fragment thereof that binds to TNFR2, the antibody or antigen-binding fragment comprising: a heavy chain variable region (VH) containing VH CDR1, VH CDR2, and VH CDR3 identical to selected VH sequences; and a light chain variable region (VL) containing VL CDR1, VL CDR2, and VL CDR3 identical to selected VL sequences, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0073] (1) The selected VH sequence is SEQ ID NO:36, and the selected VL sequence is SEQ ID NO:37;

[0074] (2) The selected VH sequence is SEQ ID NO:38, and the selected VL sequence is SEQ ID NO:39;

[0075] (3) The selected VH sequence is SEQ ID NO:40, and the selected VL sequence is SEQ ID NO:41;

[0076] (4) The selected VH sequence is SEQ ID NO:72, and the selected VL sequence is SEQ ID NO:73; or

[0077] (5) The selected VH sequence is SEQ ID NO:74, and the selected VL sequence is SEQ ID NO:75.

[0078] In one aspect, this disclosure relates to antibody-drug conjugates comprising an antibody or antigen-binding fragment thereof, as described herein, covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or a cell growth inhibitor.

[0079] In one aspect, this disclosure relates to a method of treating a subject with cancer, the method comprising administering a therapeutically effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein, or an antibody-drug conjugate as described herein.

[0080] In some embodiments, the subject has colorectal cancer, ovarian cancer, acute myeloid leukemia, Lewis lung cancer, breast cancer, hepatocellular carcinoma, and colon cancer. In some embodiments, the subject has renal cell carcinoma, multiple myeloma, colon cancer, ovarian cancer, or cutaneous T-cell lymphoma. In some embodiments, the cancer is colon cancer or ovarian cancer.

[0081] In one aspect, this disclosure relates to a method for reducing tumor growth rate, the method comprising: contacting tumor cells with an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein, or an antibody-drug conjugate as described herein.

[0082] In one aspect, this disclosure relates to a method for killing tumor cells, the method comprising: contacting the tumor cells with an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein, or an antibody-drug conjugate as described herein.

[0083] In one aspect, this disclosure relates to pharmaceutical compositions comprising an antibody or an antigen-binding fragment thereof as described herein, and a pharmaceutically acceptable carrier.

[0084] In one aspect, this disclosure relates to pharmaceutical compositions comprising the antibody-pharmaceutical conjugates described herein, and pharmaceutically acceptable carriers.

[0085] As used herein, the term “cancer” refers to cells capable of autonomous growth. Examples of such cells include cells exhibiting an abnormal state or condition characterized by rapidly proliferating cell growth. The term is intended to encompass cancerous growth, such as tumors; carcinogenic processes, metastatic tissue, and malignantly transformed cells, tissues, or organs, regardless of histopathological type or degree of invasion. It also includes malignancies of various organ systems, such as those of the head and neck, respiratory, cardiovascular, renal, reproductive, hematologic, nervous, hepatic, gastrointestinal, and endocrine systems; and adenocarcinomas, which include malignancies such as most colon cancers, renal cell carcinomas, prostate and / or testicular tumors, non-small cell lung cancer, and small bowel cancer. “Naturally occurring” cancer includes any cancer other than cancer experimentally induced by implanting cancer cells into a subject, and includes, for example, spontaneously occurring cancer, cancer caused by patient exposure to one or more carcinogens, cancer caused by the insertion of a transgenic oncogene or the knockout of a tumor suppressor gene, and cancer caused by infection (e.g., viral infection). The term “cancer” is recognized in the art and refers to a malignant disease of epithelial or endocrine tissue. The term also includes carcinosarcomas, which are malignant tumors composed of cancerous and sarcomatous tissue. "Adenocarcinoma" refers to cancer originating from glandular tissue or cancer in which tumor cells form identifiable glandular structures. The term "sarcoma" is generally accepted in the art and refers to a mesenchymal-derived malignant tumor. The term "hematopoietic neoplastic disorder" includes diseases involving proliferative cells / tumor cells of hematopoietic origin. Hematopoietic neoplastic disorders can be caused by bone marrow, lymphoid, or erythroid cells or their precursor cells. Hematologic cancers are cancers that begin in blood-forming tissues (such as bone marrow) or in cells of the immune system. Examples of hematologic cancers include, for example, leukemia, lymphoma, and multiple myeloma.

[0086] As used herein, the term "antibody" refers to any antigen-binding molecule containing at least one (e.g., one, two, three, four, five, or six) complementarity-determining regions (CDRs) (e.g., any one of the three CDRs from the immunoglobulin light chain or any one of the three CDRs from the immunoglobulin heavy chain) and capable of specifically binding to an epitope. Non-limiting examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody may contain the Fc region of a human antibody. The term antibody also includes derivatives such as bispecific antibodies, single-chain antibodies, biantibodies, linear antibodies, and multispecific antibodies formed from antibody fragments.

[0087] As used herein, the term "antigen-binding fragment" refers to a portion of a full-length antibody that is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of the heavy chain or a variable domain of the light chain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0088] As used herein, the term "human antibody" refers to an antibody encoded by an endogenous nucleic acid derived from a human being (e.g., a rearranged heavy or light chain human immunoglobulin locus). In some embodiments, human antibodies are collected from humans or generated in human cell cultures (e.g., human hybridoma cells). In some embodiments, human antibodies are generated in non-human cells (e.g., mouse or hamster cell lines). In some embodiments, human antibodies are generated in bacterial or yeast cells. In some embodiments, human antibodies are generated in transgenic non-human animals (e.g., bovine animals) containing unrearranged or rearranged human immunoglobulin loci (e.g., heavy or light chain human immunoglobulin loci).

[0089] As used herein, a "chimeric antibody" refers to an antibody containing sequences present in at least two different species (e.g., antibodies from two different mammalian species, such as a human antibody and a mouse antibody). A non-limiting example of a chimeric antibody is an antibody containing all or part of a variable domain sequence (e.g., all or part of a light chain and / or heavy chain variable domain sequence) of a non-human (e.g., mouse) antibody and a constant domain of a human antibody. Further examples of chimeric antibodies are described herein and are known in the art.

[0090] As used herein, the term "humanized antibody" refers to a non-human antibody containing a minimal sequence derived from a non-human (e.g., mouse) immunoglobulin and a sequence derived from a human immunoglobulin. In a non-limiting example, a humanized antibody is a human antibody (receptor antibody) in which hypervariable region (e.g., CDR) residues of the receptor antibody are replaced with hypervariable region (e.g., CDR) residues of a non-human antibody (e.g., donor antibody) having the desired specificity, affinity, and capability, such as a mouse, rat, or rabbit antibody. In some embodiments, Fv framework residues of a human immunoglobulin are replaced with corresponding non-human (e.g., mouse) immunoglobulin residues. In some embodiments, a humanized antibody may contain residues not found in the receptor antibody or donor antibody. These modifications can further improve antibody performance. In some embodiments, a humanized antibody contains at least one, and typically substantially all, of two variable domains, wherein all or substantially all of the hypervariable loops (CDRs) correspond to the hypervariable loops of a non-human (e.g., mouse) immunoglobulin, and all or substantially all of the framework regions are framework regions of a human immunoglobulin. Humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), typically the human immunoglobulin constant region. Humanized antibodies can be generated using molecular biology methods known in the art. Non-limiting examples of methods for generating humanized antibodies are described herein.

[0091] As used herein, a "single-chain antibody" refers to a single polypeptide containing at least two immunoglobulin variable domains (e.g., variable domains of the heavy or light chains of mammalian immunoglobulins) capable of specifically binding to an antigen. Non-limiting examples of single-chain antibodies are described herein.

[0092] As used in this article, "multimeric antibody" refers to an antibody containing four or more (e.g., six, eight, or ten) immunoglobulin variable domains.

[0093] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe human or non-human animals to whom treatment is administered according to the method of the invention. This disclosure covers both veterinary and non-veterinary applications. Human patients can be adults or minors (e.g., persons under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. This includes, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., piglets, miniature pigs), horses, dogs, felines, bovines, and other domesticated, farm, and zoo animals.

[0094] As used herein, when referring to antibodies, the phrases "specifically binds to" and "specifically binds to..." mean that the antibody interacts with its target molecule (e.g., TNFR2), preferably with other molecules, because this interaction depends on the presence of a specific structure on the target molecule (i.e., an antigenic determinant or epitope); in other words, the reagent typically recognizes and binds to molecules that include a specific structure, not all molecules. Antibodies that specifically bind to a target molecule can be called target-specific antibodies. For example, antibodies that specifically bind to TNFR2 molecules can be called TNFR2-specific antibodies or anti-TNFR2 antibodies.

[0095] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably and refer to an amino acid polymer of any length having at least two amino acids.

[0096] As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid sequence” are used interchangeably and refer to a nucleotide polymer of any length having at least two nucleotides, including but not limited to DNA, RNA, DNA / RNA hybrids, and their modifications.

[0097] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. This document describes the methods and materials used in this invention; other suitable methods and materials known in the art may also be used. These materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail.

[0098] Other features and advantages of the present invention will become apparent from the following detailed description, the accompanying drawings, and the claims. Attached Figure Description

[0099] Figure 1 This graph shows the changes in tumor size over time in mice injected with MC38 cancer cells and treated with the following: PBS (phosphate-buffered saline) as control (G1), anti-hTNFR2 antibody 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).

[0100] Figure 2This is a graph showing the change in tumor size over time in mice injected with MC38 cancer cells and treated with the following: PBS as control (G1), antibody BC-1A8-IgG1 (G2), BC-1F10-IgG1 (G3), BC-1F4-IgG1 (G4), anti-mPD-1 (G5), and anti-mCTLA4 (G6).

[0101] Figure 3 This is a graph showing the change in tumor size over time in mice injected with MC38 cancer cells and treated with the following: PBS as control (G1), antibody BC-1C3-lgG1 (G2), anti-mPD-1 (G3), and anti-mCTLA4 (G4).

[0102] Figure 4 This graph shows the changes in tumor size over time in mice injected with MC38 cancer cells and treated with the following: PBS as control (G1), antibody BC-1A8-IgG1 (G2), BC-1C3-lgG1 (G3), BC-1F10-lgG1 (G4), BC-1F4-lgG1 (G5), and BC-1B6-lgG1 (G6).

[0103] Figure 5 This graph shows the changes in tumor size over time in mice injected with MC38 cancer cells and treated with the following: PBS as control (G1), BC-1A8-IgG1 (G2), BC-1C3-IgG1 (G3), BC-1F10-IgG1 (G4), BC-1F4-IgG1 (G5), and BC-1B6-IgG1 (G6).

[0104] Figure 6 This is a graph showing the change in tumor size over time in hTNFα / hTNFR2 mice injected with MC38 cancer cells and treated with the following: PBS as control (G1), BC-1C3-lgG1 (G2), and anti-mPD-1 (G3).

[0105] Figures 7A-7B The results of blood biochemical parameters (AST, ALT) in peripheral blood of mice on day 5 (D5) after grouping are shown.

[0106] Figure 8 This demonstrates epitope correlations between anti-hTNFR2 antibodies.

[0107] Figure 9 This presentation shows cytotoxicity data for BC-1C3-IgG1, BC-1C3-IgG1-SI, BC-1C3-IgG1-LALA, and human IgG1.

[0108] Figure 10It is a drug concentration-time curve showing the changes in antibody drug concentration in the serum of humanized TNFR2 mice after injection of different antibody drugs.

[0109] Figure 11 The display shows fluorescent signals indicating activation of reporter cells (Jurkat-GFP-TNFR2 cells).

[0110] Figure 12 The display shows fluorescent signals indicating activation of reporter cells (Jurkat-GFP-TNFR2 cells).

[0111] Figures 13A-13B This displays the test results of blood biochemical indicators (AST, ALT) in the peripheral blood of mice.

[0112] Figure 14 The CDR sequences of the 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 are listed.

[0113] Figure 15 The CDR sequences of the anti-TNFR2 antibodies BC-1A8 (“1A8”), BC-1B6 (“1B6”), BC-1C3 (“1C3”), BC-1F4 (“1F4”), and BC-1F10 (“1F10”) as defined by the Chothia numbering scheme are listed.

[0114] Figure 16 The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-TNFR2 antibodies (1A8, 1B6, 1C3, 1F4 and 1F10) are listed.

[0115] Figure 17 The amino acid sequences of human TNFR2, mouse TNFR2, monkey TNFR2, dog TNFR2, human-mouse chimeric TNFR2, IgG1 heavy chain constant region, IgG1 heavy chain constant region with SI mutation, IgG1 heavy chain constant region with LALA mutation, and human TNFα protein are listed. Detailed Implementation

[0116] Tumor necrosis factor (TNF) is generally considered a major pro-inflammatory 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). In inflammatory processes, including the cancer microenvironment, TNF is the first inflammatory mediator to be produced and secreted. It promotes the production of cytokine cascades and other inflammatory mediators, such as transcription factors, interleukin (IL)-1, and IL-6. Two types of TNF receptors (TNFR1 and TNFR2) are located on the cell surface. Experiments in inflammation-related cancers have shown that TNFR2 is preferentially upregulated over TNFR1, and anti-TNF monoclonal antibody therapy reduces tumor number and size. Therefore, the TNF-TNFR2 axis is involved in the suppression of the immune response and influences tumor progression and metastasis.

[0117] This disclosure provides examples of antibodies that bind to TNFR2 (tumor necrosis factor receptor 2) and their antigen-binding fragments.

[0118] TNFR2 and Cancer

[0119] If the T cells are CD8+ cytotoxic T lymphocytes (CTLs) (also known as CD8+ effector T cells (Teff)), they are a key focus of cancer immunotherapy due to their ability to directly kill tumor cells. However, Treg cells can suppress Teff cells, thereby preventing the appropriate host immune response to eliminate the tumor. Therefore, inhibiting suppressor Treg cells while simultaneously activating cytotoxic CD8+ Teff cells could be a potential strategy for treating cancer (Vanamee, S. et al., “TNFR2: a novel target for cancer immunotherapy.” Trends in Molecular Medicine 23.11(2017):1037-1046.

[0120] TNFR2 is a member of the TNFR superfamily (TNFRSF) and is activated by TNF. It is a cell surface receptor that regulates cell survival and proliferation, and targeting this receptor has become a potential next-generation cancer treatment. Some human tumor cells can abnormally express TNFR2, and tumor infiltration is dominated by highly repressed TNFR2+ Treg cells.

[0121] TNFR2 is primarily expressed in cells of the immune system, particularly regulatory T (Treg) cells and endothelial cells, and preferentially binds to transmembrane TNF (tmTNF). TNFR1 and TNFR2 are single-pass transmembrane glycoproteins, sharing 28% homology primarily in their extracellular domain, which consists of four cysteine-rich motifs. However, the intracellular domains of the TNF receptor are largely unrelated, lacking homologous sequences, suggesting that different signaling functions originate from two distinct receptors. TNFR1 contains an intracellular death domain (DD), which binds to the TNFR1-associated death domain protein (TRADD) of the Fas-associated death domain (FADD) and is primarily involved in cell death signaling. While TNFR2 lacks cytoplasmic DD, it interacts with TNF-associated factor 2 (TRAF2) and primarily contributes to cell survival. The signaling circuitry of TNFR2 differs from that of other TNFRs. TNFR1 contains an intracellular death domain and can activate apoptosis or inflammatory pathways, while TNFR2 binds to TNF receptor-associated factor (TRAF) and can activate both typical and atypical NF-κB pathways to control cell survival and proliferation in humans and mice.

[0122] TNFR2 has 461 amino acids. Amino acids 1-22 are the signal peptide, amino acids 23-257 are the extracellular domain, amino acids 258-287 are the transmembrane domain, and amino acids 288-461 are the cytoplasmic domain with a TRAF2 binding site. TRAF2 can bind to inhibitors of TRAF1, TRAF3, cIAP1 (cIAP1), and cIAP2 (cIAP2).

[0123] Antagonistic anti-TNFR2 antibodies can block ligand binding and lock membrane receptors into a stationary (non-signal transduction) antiparallel dimer arrangement, while agonistic cross-linked antibodies can stabilize the parallel TNF-TNFR2 complex, thus providing structural stability to the active signal transduction network. Furthermore, it is now widely accepted that TNFR2 contributes to the stability of the CD4+Foxp3+Treg phenotype in an inflammatory environment.

[0124] Detailed descriptions of TNFR2 and its functions can be found in, for example, Al-Hatamleh et al., “A perspective review on the role of nanomedicine in the modulation of TNF-TNFR2 axis in breast cancer immunotherapy.” Journal of Oncology 2019; Vanamee et al., “TNFR2: a novel target for cancer immunotherapy.” Trends in Molecular Medicine 23.11(2017):1037-1046; Ortí- Chen et al., “Targeting TNFR2 as a novel therapeutic strategy for Alzheimer's disease.” Frontiers in Neuroscience 13(2019):49; Chen et al., “Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4+CD25+ T regulatory cells.” The Journal of Immunology 179.1(2007):154-161; These are cited in their full text and included here.

[0125] This disclosure provides anti-TNFR2 antibodies, their antigen-binding fragments, and methods for using these anti-TNFR2 antibodies and antigen-binding fragments to inhibit tumor growth and treat various diseases, including, for example, cancer.

[0126] Anti-TNFR2 antibody and antigen-binding fragment

[0127] This disclosure provides antibodies that specifically bind to TNFR2 (e.g., human TNFR2) and their antigen-binding fragments. The antibodies and antigen-binding fragments described herein are capable of binding to TNFR2. In some embodiments, these antibodies can block the TNFR2 signaling pathway, thereby increasing the immune response. In some embodiments, these antibodies can induce complement-dependent cytotoxicity (CMC) or antibody-dependent cytotoxicity (ADCC).

[0128] This 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 antibodies modified thereof (including, for example, chimeric antibodies, humanized antibodies, and human antibodies).

[0129] As defined by Kabat numbers, the CDR sequences of 1A8 and 1A8-derived antibodies (e.g., humanized antibodies) include CDRs of the heavy chain variable domain (SEQ ID NO: 6, 7, 8) and CDRs of the light chain variable domain (SEQ ID NO: 9, 10, 11). CDRs can also be defined by the Chothia system. According to Chothia numbers, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 42, 43, 44, and the CDR sequences of the light chain variable domain are shown in SEQ ID NO: 45, 46, 47.

[0130] As defined by the Kabat designation, the CDR sequences of 1B6 and 1B6-derived antibodies include CDRs of the heavy chain variable domain (SEQ ID NO: 12, 13, 14) and CDRs of the light chain variable domain (SEQ ID NO: 15, 16, 17). According to the Chothia designation, the CDR sequences of the heavy chain variable domain are shown by SEQ ID NO: 48, 49, 50, and the CDR sequences of the light chain variable domain are shown by SEQ ID NO: 51, 52, 53.

[0131] As defined by the Kabat designation, the CDR sequences of 1C3 and 1C3-derived antibodies include CDRs of the heavy chain variable domain (SEQ ID NO: 18, 19, 20) and CDRs of the light chain variable domain (SEQ ID NO: 21, 22, 23). According to the Chothia designation, the CDR sequences of the heavy chain variable domain are shown by SEQ ID NO: 54, 55, 56, and the CDR sequences of the light chain variable domain are shown by SEQ ID NO: 57, 58, 59.

[0132] As defined by the Kabat designation, the CDR sequences of 1F4 and 1F4-derived antibodies include CDRs of the heavy chain variable domain (SEQ ID NO: 24, 25, 26) and CDRs of the light chain variable domain (SEQ ID NO: 27, 28, 29). According to the Chothia designation, the CDR sequences of the heavy chain variable domain are shown by SEQ ID NO: 60, 61, 62, and the CDR sequences of the light chain variable domain are shown by SEQ ID NO: 63, 64, 65.

[0133] As defined by the Kabat designation, the CDR sequences of 1F10 and 1F10-derived antibodies include CDRs of the heavy chain variable domain (SEQ ID NO: 30, 31, 32) and CDRs of the light chain variable domain (SEQ ID NO: 33, 34, 35). According to the Chothia designation, the CDR sequences of the heavy chain variable domain are shown in SEQ ID NO: 66, 67, 68, and the CDR sequences of the light chain variable domain are shown in SEQ ID NO: 69, 70, 71.

[0134] The amino acid sequence of the heavy chain variable region of the 1A8 antibody is shown in SEQ ID NO:36. The amino acid sequence of the light chain variable region of the 1A8 antibody is shown in SEQ ID NO:37.

[0135] The amino acid sequence of the heavy chain variable region of the 1B6 antibody is shown in SEQ ID NO:38. The amino acid sequence of the light chain variable region of the 1B6 antibody is shown in SEQ ID NO:39.

[0136] The amino acid sequence of the heavy chain variable region of the 1C3 antibody is shown in SEQ ID NO:40. The amino acid sequence of the light chain variable region of the 1C3 antibody is shown in SEQ ID NO:41.

[0137] The amino acid sequence of the heavy chain variable region of the 1F4 antibody is shown in SEQ ID NO:72. The amino acid sequence of the light chain variable region of the 1F4 antibody is shown in SEQ ID NO:73.

[0138] The amino acid sequence of the heavy chain variable region of the 1F10 antibody is shown in SEQ ID NO:74. The amino acid sequence of the light chain variable region of the 1F10 antibody is shown in SEQ ID NO:75.

[0139] The amino acid sequences of the heavy chain variable region and the light chain variable region of the modified antibody are also provided. 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 may pair with the corresponding light chain variable region sequence, and together they bind to TNFR2.

[0140] Humanization percentage refers to the percentage identity of the heavy or light chain variable region sequence to human antibody sequences in the International Immunogenetic Information System (IMGT) database. Top hit refers to the sequence's heavy or light chain variable region being more closely related to a specific species relative to other species. For example, the top hit sequence for humans is more closely related to humans compared to other species. The top hit sequences for humans and Macaca fasciculars have the same percentage identity as human and Macaca fascicular sequences, and these percentage identities are the highest compared to sequences from other species. In some embodiments, the humanization percentage is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%. Detailed descriptions of how to determine the percentage of humanization and how to determine the highest hit are known in the art and are described, for example, in Jones et al., “The INNs and outs of antibody nonproprietary names.” MAbs. [Monoclonal Antibodies] Vol. 8. No. 1. Taylor & Francis [Taylor & Francis Group] 2016, which is incorporated herein by reference in its entirety. A high percentage of humanization generally has several advantages, such as greater safety and efficacy in humans, better tolerability in human subjects, and / or less likelihood of side effects. In some embodiments, the variable region is fully human, for example, derived from human heavy chain immunoglobulin locus sequences (e.g., recombinants of human IGHV, IGHD, and IGHJ genes), and / or human κ chain immunoglobulin locus sequences (e.g., recombinants of human IGKV and IGKJ genes).

[0141] In addition, in some embodiments, the antibodies or antigen-binding fragments thereof described herein may also contain one, two or three heavy chain variable regions (CDRs) selected from the group consisting of: SEQ ID NO:6-8, SEQ ID NO:12-14, SEQ ID NO:18-20, SEQ ID NO:24-26 and SEQ ID NO:30-32 (Kabat numbers); and / or one, two or three light chain variable regions (CDRs) selected from the group consisting of: SEQ ID NO:9-11, SEQ ID NO:15-17, SEQ ID NO:21-23, SEQ ID NO:27-29 and SEQ ID NO:33-35 (Kabat numbers).

[0142] In some embodiments, the antibody may have a heavy chain variable region (VH) comprising complementarity-determining regions (CDRs) 1, 2, and 3, wherein the CDR1 region contains or is composed of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to or constitutes the amino acid sequence of a selected VH CDR1, the CDR2 region contains or is composed of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to or constitutes the amino acid sequence of a selected VH CDR2, and the CDR3 region contains or is composed of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to or constitutes the amino acid sequence of a selected VH CDR3. In some embodiments, the antibody may have light chain variable regions (VLs) comprising 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 or constitutes the amino acid sequence of a selected VL CDR1, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to or constitutes the amino acid sequence of a selected VL CDR2, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to or constitutes the amino acid sequence of a selected VL CDR3. Figure 14 (Kabat CDR) and Figure 15 The selected amino acid sequences of VH CDR 1, 2, 3 and the selected amino acid sequences of VL CDR 1, 2, 3 are shown in (Chothia CDR).

[0143] In some embodiments, the antibody or antigen-binding fragment described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO:6 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO:7 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO:8 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0144] In some embodiments, the antibody or antigen-binding fragment described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO:12 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO:13 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO:14 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0145] In some embodiments, the antibody or antigen-binding fragment described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO:18 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO:19 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO:20 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0146] In some embodiments, the antibody or antigen-binding fragment described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO:24 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO:25 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO:26 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0147] In some embodiments, the antibody or antigen-binding fragment described herein may contain a heavy chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO:30 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO:31 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO:32 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0148] In some embodiments, the antibody or antigen-binding fragment described herein may contain a light chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO:9 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO:10 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO:11 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0149] In some embodiments, the antibody or antigen-binding fragment described herein may contain a light chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO:15 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO:16 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO:17 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0150] In some embodiments, the antibody or antigen-binding fragment described herein may contain a light chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO:21 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO:22 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO:23 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0151] In some embodiments, the antibody or antigen-binding fragment described herein may contain a light chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO:27 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO:28 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO:29 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0152] In some embodiments, the antibody or antigen-binding fragment described herein may contain a light chain variable domain containing one, two, or three of the following CDRs: SEQ ID NO:33 having zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO:34 having zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO:35 having zero, one, or two amino acid insertions, deletions, or substitutions.

[0153] Insertions, deletions, and substitutions can occur within the CDR sequence, or at one or both ends of the CDR sequence. In some embodiments, the CDR is determined based on the Kabat numbering scheme. In some embodiments, the CDR is determined based on the Chothia numbering scheme. In some embodiments, the CDR is determined based on a combination of the Kabat and Chothia numbering schemes.

[0154] This disclosure also provides antibodies or antigen-binding fragments thereof that bind to TNFR2. The antibody or antigen-binding fragment contains a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising or consisting of at least 80%, 85%, 90%, or 95% of an amino acid sequence identical to or composed of a selected VH sequence, and the light chain variable region comprising or consisting of at least 80%, 85%, 90%, or 95% of an amino acid sequence identical to or composed of 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.

[0155] To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps are introduced in one or both of the first and second amino acid sequences or the first and second nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). The length of the reference sequence for alignment for comparison purposes is at least 80% of the reference sequence length, and in some embodiments at least 90%, 95%, or 100% of the reference sequence length. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecule is identical at that position. Taking into account the number of gaps and the length of each gap, the percentage identity between two sequences is a function of the number of common positions shared by these sequences, which need to be introduced for optimal alignment of the two sequences. For example, the comparison of two sequences and the determination of the percentage identity between the two sequences can be achieved using a Blosum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0156] This disclosure also provides a nucleic acid comprising a polynucleotide encoding a polypeptide, wherein the polypeptide comprises an immunoglobulin heavy chain or an immunoglobulin light chain. The immunoglobulin heavy chain or immunoglobulin light chain comprises, as in... Figure 14 or Figure 15 The CDR shown, or having as Figure 16The sequence is shown. When a polypeptide pairs with a corresponding polypeptide (e.g., the corresponding heavy chain variable region or the corresponding light chain variable region), these paired polypeptides bind to TNFR2.

[0157] 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. Other antibodies described herein include polyclonal antibodies, monoclonal antibodies, multimeric antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, intracellularly generated antibodies (i.e., intracellular antibodies), and their antigen-binding fragments. Antibodies or their antigen-binding fragments can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody or its antigen-binding fragment is an IgG antibody or its antigen-binding fragment.

[0158] An antibody fragment is suitable for use with the provided methods, provided it retains the desired affinity and specificity of the full-length antibody. Therefore, an antibody fragment binding TNFR2 will retain the ability to bind TNFR2. An Fv fragment is an antibody fragment containing both a complete antigen recognition site and a binding site. This region consists of a tightly bound dimer of a heavy chain variable domain and a light chain variable domain, which can be covalent in nature, such as scFv. In this structure, the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. In summary, 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 to the antigen) has the ability to recognize and bind to the antigen, although typically with lower affinity than the entire binding site. Single-chain Fv or (scFv) antibody fragments contain the VH and VL domains (or regions) of the antibody, where these domains are present within a single polypeptide chain. Typically, the scFv peptide further includes a peptide linker between the VH and VL domains, which enables the scFv to form the desired antigen-binding structure.

[0159] This disclosure also provides antibodies or antigen-binding fragments thereof that cross-compete with any antibody or antigen-binding fragment as described herein. Cross-compete assays are known in the art, for example, as described in Moore et al., "Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exteriorenvelope glycoprotein." Journal of Virology 70.3(1996):1863-1872, which is incorporated herein by reference in its entirety. In one aspect, this disclosure also provides antibodies or antigen-binding fragments thereof that bind to the same epitope or region as any antibody or antigen-binding fragment as described herein. Epitope binning assays are known in the art, for example, as described in Estep et al., "High throughput solution-based measurement of antibody-antigen affinity and epitope binning." MAbs. [Monoclonal Antibodies] Vol. 5. No. 2. Taylor and Francis, 2013, which is incorporated herein by reference in its entirety.

[0160] Antibody and antigen binding fragment

[0161] This disclosure provides various antibodies and their antigen-binding fragments derived from the anti-TNFR2 antibody described herein. Typically, antibodies (also called immunoglobulins) consist of two types of polypeptide chains: light chains and heavy chains. Non-limiting examples of antibodies disclosed herein may include antibodies containing two heavy chains and two light chains of all four immunoglobulin chains. The heavy chain of the antibody may be any isotype (including IgM, IgG, IgE, IgA, or IgD) or subisotype (including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc.). The light chain may be a κ light chain or a λ light chain. The antibody may contain two identical copies of the light chain and two identical copies of the heavy chain. Each contains a variable domain (or variable region, V...). H Heavy chains containing multiple constant domains (or constant regions) are linked together via disulfide bonds within their constant domains to form the "stem" of the antibody. Each contains a variable domain (or variable region, V...). LA light chain with a constant structural domain (or constant region) is bonded to a heavy chain via disulfide bonds. The variable region of each light chain pairs with the variable region of the heavy chain it is bonded to. The variable regions of both the light and heavy chains contain three highly variable regions sandwiched between more conservative framework regions (FRs).

[0162] These hypervariable regions (called complementarity-determining regions (CDRs)) form loops that contain the antigen-binding surface of the antibody. The four framework regions primarily adopt a β-sheet conformation, and the CDRs form loops that connect the β-sheet structures, and in some cases, these loops form part of the β-sheet structure. The CDRs in each chain are closely spaced through the framework regions and, together with CDRs from other chains, form the antigen-binding region.

[0163] Identifying the CDR region of an antibody by analyzing its amino acid sequence is a well-known method, and many definitions of CDR are commonly used. The Kabat definition is based on sequence variability, while the Chothia definition is based on the location of the structural loop region. These methods and definitions are described in, for example, Martin, "Protein sequence and structure analysis of antibody variable domains", Antibody Engineering, Springer Berlin Heidelberg, 2001, 422-439; Abhinandan et al., "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains", Molecular Immunology 45.14(2008):3832-3839; Wu, TT and Kabat, EA (1970) J. Exp. Med. 132:211-250; Martin et al., Methods Enzymol. [Enzymological Methods] 203:121-53 (1991); Morea et al., BiophysChem. 68(1-3):9-16 (October 1997); Morea et al., J Mol Biol. 275(2):269-94 (January 1998); Chothia et al., Nature 342(6252):877-83 (December 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007); each of these is incorporated herein by reference in its entirety.

[0164] CDRs are important for recognizing antigenic epitopes. As used in this article, an "epitaph" is the smallest part of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The smallest size of an epitope may be about three, four, five, six, or seven amino acids, but these amino acids do not necessarily have to be located in a continuous linear sequence of the primary structure of the antigen, as the epitope may depend on the three-dimensional conformation of the antigen based on its secondary and tertiary structures.

[0165] In some embodiments, antibodies are complete immunoglobulin molecules (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differing only in their constant regions, particularly their hinge and CH2 upper domain. The sequences and differences of IgG subclasses are known in the art and 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 these is incorporated herein by reference in its entirety.

[0166] Antibodies can also be immunoglobulin molecules derived from any species (e.g., humans, rodents, mice, camels). The antibodies disclosed herein also include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, and chimeric antibodies including an immunoglobulin-binding domain fused to another polypeptide. The term "antigen-binding domain" or "antigen-binding fragment" refers to the portion of an antibody that retains the specific binding activity of the intact antibody; that is, any portion of the antibody 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 its antigen-binding fragment can be, for example, scFv, Fv, Fd, dAb, bispecific antibodies, bispecific scFv, biantibodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed from antibody fragments, and any polypeptide including a binding domain that is an antibody-binding domain or homologous to it. Non-limiting examples of antigen-binding domains include, for example, heavy chain and / or light chain CDRs of an intact antibody, variable regions of heavy chain and / or light chain of an intact antibody, full-length heavy chain or light chain of an intact antibody, or individual CDRs derived from the heavy chain or light chain of an intact antibody.

[0167] Antibody fragments suitable for use in the methods described herein are also provided. The Fab fragments contain a variable and a constant domain of the light chain and a variable and a first constant domain (CH1) of the heavy chain. The F(ab')2 antibody fragment comprises a pair of Fab fragments, which are typically covalently linked near their carboxyl ends via a hinge cysteine ​​residue between them. Other chemical conjugations of antibody fragments are also known in the art.

[0168] Biantibodies are small antibody fragments with two antigen-binding sites, containing a VL linked to a VH (VH and VL) on the same polypeptide chain. By using a linker that is too short to pair between the two domains on the same chain, these domains are forced to pair with the complementary domain of the other chain, creating two antigen-binding sites.

[0169] Linear antibodies contain a pair of tandem Fd regions (VH-CH1-VH-CH1), which together with a complementary light chain polypeptide form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0170] The antibodies and antibody fragments disclosed herein can be modified in the Fc region to provide desired effector function or serum half-life.

[0171] Antibody polymerization can be achieved through the natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, a certain percentage of purified antibody products (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody polymers.

[0172] Alternatively, antibody homodimers can be formed using chemical linking techniques known in the art. For example, heterobifunctional crosslinkers (including, but not limited to, SMCC (succinimide-4-(maleimide-methyl)cyclohexane-1-carboxylate) and SATA (N-succinimide-S-ethylthio-acetate)) can be used to form antibody polymers. Exemplary methods for forming antibody homodimers are described in: Ghetie et al. (Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 94:7509-7514, 1997). Antibody homodimers can be converted to Fab'2 homodimers by digestion with pepsin. Another way to form antibody homodimers is by using an autophilic T15 peptide described in: Zhao et al. (J. Immunol. [Journal of Immunology] 25:396-404, 2002).

[0173] In some embodiments, the multispecific antibody is a bispecific antibody. Bispecific antibodies can be prepared by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell cultures. For example, the interface may contain at least a portion of the CH3 domain of the antibody's constant structural domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensating "cavity" of the same or similar size as the one or more large side chains is created at the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers relative to other unwanted end products as dimers. This method is described, for example, in WO96 / 27011, which is incorporated herein by reference in its entirety.

[0174] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one antibody in a heteroconjugate may be conjugated to avidin, while the other may be conjugated to biotin. Heteroconjugate antibodies can also be prepared using any conventional cross-linking method. Suitable cross-linking agents and techniques are well known in the art and are disclosed in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.

[0175] Any antibody or antigen-binding fragment described herein may be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or its antigen-binding fragment in a subject or solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). Conjugation to a stabilizing molecule may increase the half-life of the antibody or antigen-binding fragment or enhance its biological activity in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in the human body).

[0176] In some embodiments, the antibodies or antigen-binding fragments described herein may be conjugated to a therapeutic agent. Antibody-drug conjugates comprising antibodies or their antigen-binding fragments may covalently or non-covalently bind a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or a cell growth inhibitor (e.g., cytochalasin B, bacitracin D, ethidium bromide, ipecacine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, anthraxin dihydroxy, maytansinoids (e.g., DM-1 and DM-4), diketones, mitoxantrone, styraxin D, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogues).

[0177] In some embodiments, the antigen-binding fragment may form part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor is a fusion of a single-stranded variable fragment (scFv) as described herein with a CD3-ζ transmembrane domain and an intracellular domain. In some embodiments, the chimeric antigen receptor further comprises intracellular signaling domains from multiple co-stimulatory protein receptors (e.g., CD28, 41BB, ICOS). In some embodiments, the chimeric antigen receptor comprises multiple signaling domains, such as CD3z-CD28-41BB or CD3z-CD28-OX40, to enhance potency. Therefore, in one aspect, this disclosure further provides cells (e.g., T cells) expressing chimeric antigen receptors as described herein.

[0178] In some embodiments, the scFV has one heavy-chain variable structural domain and one light-chain variable structural domain. In some embodiments, the scFV has two heavy-chain variable structural domains and two light-chain variable structural domains.

[0179] Antibody characteristics

[0180] The antibodies or antigen-binding fragments thereof described herein can block the binding between TNFR2 and TNFR2 ligands. In some embodiments, antibodies can inhibit the TNFR2 signaling pathway by binding to TNFR2. In some embodiments, antibodies can upregulate the immune response. In some embodiments, antibodies can reduce tumor volume in animals with tumors expressing TNFR2.

[0181] In some embodiments, the antibody (or its antigen-binding fragment) specifically binds to TNFR2 (human TNFR2, mouse TNFR2, monkey TNFR2, dog TNFR2, chimeric TNFR2), wherein the dissociation rate (koff) is less than 0.1 s. -1 Less than 0.01s -1 Less than 0.001s -1 Less than 0.0001s -1 Or less than 0.00001s -1 In some embodiments, the dissociation rate (koff) is greater than 0.01 s. -1 Greater than 0.001s -1 Greater than 0.0001s -1 Greater than 0.00001s -1 or greater than 0.000001s -1 .

[0182] In some embodiments, the dynamic association rate (kon) is greater than 1 × 10⁻⁶. 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×10 5 / Ms or greater than 1×10 6 / Ms. In some embodiments, the kinetic association rate (kon) is less than 1 × 10⁻⁶. 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 / Ms.

[0183] The affinity (KD = koff / kon) can be derived from the quotient of the kinetic rate constant. In some embodiments, KD is less than 1 × 10⁻⁶. -6 M, less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M or less than 1×10 -10 M. In some embodiments, 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, KD is greater than 1 × 10⁻⁶.-7 M, greater than 1×10 -8 M, greater than 1×10 -9 M, greater than 1×10 -10 M, greater than 1×10 -11 M, or greater than 1×10 -12 M.

[0184] Common techniques for measuring antibody affinity for antigens 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.

[0185] TNFR2 has four cysteine-rich domains (CRDs). CRD1 is 39aa-76aa of SEQ ID NO:1, CRD2 is 77aa-118aa of SEQ ID NO:1, CRD3 is 119aa-162aa of SEQ ID NO:1, and CRD4 is 168aa-196aa of SEQ ID NO:1. In some embodiments, the antibody (or its antigen-binding fragment) specifically binds to CRD1, CRD2, CRD3, and / or CRD4. In some embodiments, the epitope is located at the junction of CRD3 and CRD4.

[0186] In some embodiments, the tumor growth inhibition percentage (TGI) of the antibody TV The percentage of tumor growth inhibition by the antibody is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the percentage of tumor growth inhibition by the antibody is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. For example, TGI can be determined at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the start of treatment, or at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the start of treatment. TV%. As used in this article, the tumor growth inhibition percentage (TGI) is calculated using the following formula. TV %):

[0187] TGI TV (%)=[1-(Ti-T0) / (Vi-V0)]×100

[0188] 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 zero. 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 zero.

[0189] In some embodiments, antibodies or antigen-binding fragments thereof, as described herein, are cytotoxic, for example, to cells expressing TNFR2. Methods for measuring cytotoxicity are known in the art. In some embodiments, cytotoxicity is calculated using the following formula:

[0190]

[0191] The experimental data refers to the absorbance value of the experimental wells (effect cells + target cells + test product); the effector data refers to the absorbance value of the effector cells (effect cells only); the target data refers to the absorbance value of the target cells (target cells only); the spontaneous data refers to the autofluorescence absorbance value of the cell culture medium (cell culture medium only, without effector cells or target cells); the maximum target data refers to the highest absorbance value of the target cells (target cells + lysate); and the spontaneous * data refers to the absorbance value of the cell culture medium volume control wells (culture medium only + lysate). EC50 can also be calculated. In some embodiments, EC50 is less than 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 μg / mL.

[0192] In some embodiments, the antibody or antigen-binding fragment thereof, as described herein, is a TNFR2 antagonist. In some embodiments, the antibody or antigen-binding fragment reduces TNFR2 signaling in target cells expressing TNFR2 (e.g., T cells such as Tregs).

[0193] In some embodiments, the antibody or its antigen-binding fragment as described herein is non-toxic. In some embodiments, such as at 0.3 mg / kg, 1 mg / kg, 10 mg / kg, or 25 mg / kg, no significant difference in body weight was observed between the treatment and control groups.

[0194] In some embodiments, the antibody or antigen-binding fragment can bind to tumor cells expressing TNFR2. In some embodiments, the antibody or antigen-binding fragment can induce complement-dependent cytotoxicity (CMC) and / or antibody-dependent cytotoxicity (ADCC) and kill tumor cells.

[0195] In some embodiments, the antibody or antigen-binding fragment has a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector functions of the functional Fc region are both ADCC and phagocytosis.

[0196] In some embodiments, antibodies or antigen-binding fragments can induce complement-mediated cytotoxicity (CMC).

[0197] In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the antibody is a human IgG1 antibody.

[0198] 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 LALA mutations (EU-numbered L234A and L235A mutations) or LALA-PG mutations (EU-numbered L234A, L235A, and P329G mutations).

[0199] In some embodiments, Fc has SI mutations (S239D and I332E mutations numbered in EU).

[0200] Methods for preparing anti-TNFR2 antibodies

[0201] Standard techniques for preparing polyclonal and monoclonal antibodies can be used to generate antibodies by using isolated fragments of human TNFR2 (e.g., extracellular regions) as immunogens. Polyclonal antibodies can be generated in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of antigenic peptides or proteins. In some embodiments, the antigenic peptide or protein is injected together with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an immunogenic agent in the species to be immunized. The antigenic peptide or protein can be injected into the animal more than once (e.g., twice, three times, or four times).

[0202] Full-length polypeptides or proteins can be used; alternatively, their antigenic peptide fragments can be used as immunogens. The antigenic peptide of the protein contains at least eight (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence of TNFR2 and covers the epitope of the protein, such that the generated antibody against the peptide forms a specific immune complex with the protein. As described above, the full-length sequence of human TNFR2 is known in the art (SEQ ID NO:1). In some embodiments, an Fc-tagged human TNFR2 protein (an Fc fusion protein containing the human TNFR2 extracellular domain, positions 23-257 of SEQ ID NO:1) is used as an immunogen.

[0203] Immunogens are commonly used to prepare antibodies by immunizing suitable subjects (e.g., humans or transgenic animals expressing at least one human immunoglobulin locus). Suitable immunogenic formulations may contain, for example, recombinantly expressed peptides or chemically synthesized peptides (e.g., fragments of human TNFR2). The formulation may further contain adjuvants, such as Freund's complete or incomplete adjuvants, or similar immunostimulants.

[0204] As described above, polyclonal antibodies can be prepared by immunizing suitable subjects with a TNFR2 peptide or its antigenic peptide (e.g., a portion of TNFR2 such as the extracellular region) as an immunogen. Changes in antibody titers over time in immunized subjects can be monitored using standard techniques, such as enzyme-linked immunosorbent assay (ELISA) using immobilized TNFR2 peptides or peptides. If desired, antibody molecules can be isolated from mammals (e.g., from blood) and further purified using well-known techniques (e.g., protein A or protein G chromatography) to obtain the IgG fraction. At an appropriate time after immunization, such as when the specific antibody titer is at its highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies using standard techniques such as hybridoma technology originally described by Kohler et al. (Nature 256:495-497, 1975), human B-cell hybridoma technology (Kozbor et al., Immunol. Today 4:72, 1983), EBV hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985), or trioma technology. Techniques for generating hybridomas are well-known (see generally Current Protocols in Immunology, 1994, Coligan et al. (eds.), John Wiley & Sons, Inc., NY, New York). For example, standard ELISA assays can be used to screen for antibodies that bind to target peptides or epitopes in the supernatant of hybridoma cultures, and to detect hybridoma cells that produce monoclonal antibodies.

[0205] Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding the human antibodies, humanized antibodies, or chimeric antibodies, or their antigen-binding fragments, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence constituting the antigen-binding site or antigen-binding domain of the antibody. Within this population of variants, some antibodies or antigen-binding fragments will have increased affinity for target proteins (e.g., TNFR2). Any combination of deletions, insertions, and / or combinations can be made to obtain antibodies or their antigen-binding fragments with increased binding affinity to the target. Amino acid changes introduced into the antibody or antigen-binding fragment can also alter or introduce new post-translational modifications into the antibody or antigen-binding fragment, such as altering (e.g., increasing or decreasing) the number of glycosylation sites, altering the type of glycosylation sites (e.g., altering the amino acid sequence so that enzymes present in the cell link different sugars), or introducing new glycosylation sites.

[0206] The antibodies disclosed herein can be derived from any animal species, including mammals. Non-limiting examples of natural antibodies include those derived from: humans, primates (e.g., monkeys and apes), cattle, pigs, horses, sheep, camels (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including genetically engineered rodents to produce human antibodies.

[0207] Human and humanized antibodies include antibodies having variable and constant regions derived from human immunoglobulin sequences (or having the same amino acid sequence as that derived from human immunoglobulin sequences). Human antibodies may include amino acid residues not encoded by human immunoglobulin sequences (e.g., mutations introduced by in vitro random mutagenesis or site-specific mutagenesis or by in vivo somatic mutations), such as in CDR.

[0208] Humanized antibodies typically possess a human frame (FR) with a non-human core vector (CDR). Therefore, humanized antibodies have one or more amino acid sequences introduced from a non-human source. These non-human amino acid residues are often referred to as "input" residues, and they are typically derived from an "input" variable domain. Humanization is essentially performed by replacing, for example, a rodent CDR or CDR sequence with the corresponding sequence of the human antibody. These methods are described, for example, by Jones et al., "Replacing the complementarity-determining regions in a human antibody with those from a mouse." Nature 321.6069(1986):522; Riechmann et al., "Reshaping human antibodies for therapy." Nature 332.6162(1988):323; Dall'Acqua et al., "Antibody humanization by framework shuffling." Methods 36.1(2005):43-60; each of which is incorporated herein by reference in its entirety. Thus, a "humanized" antibody is a chimeric antibody in which essentially less than the complete human V domain is replaced by a corresponding sequence from a non-human species. In practice, humanized antibodies are typically mouse antibodies, in which some CDR residues and some FR residues are replaced by residues from similar sites in human antibodies.

[0209] Selecting the human VH and VL domains for preparing humanized antibodies is crucial for reducing immunogenicity. Using the so-called "best-fit" method, sequences of the V domains of mouse antibodies are screened against an entire library of known human domain sequences. The human sequence 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).

[0210] Further important is the humanization of antibodies while retaining high specificity and affinity for antigens, as well as other advantageous biological properties. To achieve this, humanized antibodies can be prepared by analyzing the parental sequence and various conceptual humanization products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs illustrating and demonstrating the possible three-dimensional conformations of selected candidate immunoglobulin sequences are available. Examination of these displays allows analysis of the possible roles of residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues affecting the candidate immunoglobulin's ability to bind its antigens. In this way, FR residues can be selected and combined from the receptor and input sequences to achieve desired antibody characteristics, such as increased affinity for one or more target antigens.

[0211] Typically, amino acid sequence variants of human anti-TNFR2 antibodies, humanized anti-TNFR2 antibodies, or chimeric anti-TNFR2 antibodies will contain an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequence present in the light or heavy chain of the original antibody.

[0212] In some embodiments, antibodies are generated using mice (e.g., RenMab mice) that have a humanized heavy chain immunoglobulin locus and a humanized κ chain immunoglobulin locus. The heavy chain immunoglobulin locus is a region of a chromosome containing an antibody heavy chain gene. This locus may include, for example, a human IGHV (variable) gene, a human IGHD (diversity) gene, a human IGHJ (linking) gene, and a mouse heavy chain constant domain gene. The κ chain immunoglobulin locus is a region of a chromosome containing a gene encoding an antibody light chain (κ chain). The κ chain immunoglobulin locus may include, for example, a human IGKV (variable) gene, a human IGKJ (linking) 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 herein by reference in its entirety. The antibodies generated by the mice have an intact human VH, an intact human VL, and a mouse constant region. In some embodiments, the human VH and human VL are linked to human IgG constant regions (e.g., IgG1, IgG2, IgG3, and IgG4). In some embodiments, the constant region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same sequence as SEQ ID NO:76, 77, or 78.

[0213] The identity or homology of the original sequence is typically defined as the percentage of amino acid residues in the candidate sequence that are identical to those in a human anti-TNFR2 antibody, humanized anti-TNFR2 antibody, or chimeric anti-TNFR2 antibody or fragment, after the sequence has been aligned and vacancy has been introduced (if necessary) to achieve the maximum percentage of sequence identity without incorporating any conserved substitutions as part of the sequence identity.

[0214] Anti-TNFR2 antibodies or antigen-binding fragments can be further modified. For example, one or more cysteine ​​residues can be introduced into the Fc region, allowing interchain disulfide bonds to form in that region. The resulting homodimer antibody can have any increased in vitro and / or in vivo half-life. Homodimer antibodies with increased in vitro and / or in vivo half-life can also be prepared using heterobifunctional crosslinkers, such as those described by Wolff et al. ("Monoclonal antibody homodimers: enhanced antitumor activity in nude mice." Cancer Research 53.11(1993):2560-2565). Alternatively, antibodies having two Fc regions can be engineered.

[0215] In some embodiments, anti-TNFR2 antibodies or their antigen-binding fragments may be covalently modified. These covalent modifications may be performed by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications to antibodies or antibody fragments may be introduced into the molecule by reacting the target amino acid residues of the antibody or fragment with an organic derivatizer capable of reacting with selected side chains or N-terminal or C-terminal residues.

[0216] In some embodiments, antibody variants are provided having a carbohydrate structure lacking (directly or indirectly) fucose attached to the Fc region. For example, the amount of fucose in such antibody compositions can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. For example, as described in WO 2008 / 077546, the amount of fucose is determined by calculating the average amount of fucose within the Asn297 glycan relative to the sum of all glycan structures (e.g., complex, heterozygous, and high-mannose structures) attached to Asn 297 as measured by MALDI-TOF mass spectrometry. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (Eu number of the Fc region residues, or position 314 in Kabat numbering); however, due to minor sequence variations in the antibody, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. These fucosylated variants can possess improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody can be further engineered to replace the asparagine at position 297 with alanine (N297A).

[0217] In some embodiments, to improve production efficiency by avoiding Fab-arm exchange, the Fc region of the antibody is further engineered to replace serine at position 228 (EU number) of IgG4 with proline (S228P). A detailed description of the S228 mutation is given below: e.g., Silva et al., "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation." Journal of Biological Chemistry 290.9(2015):5462-5469, which is incorporated herein by reference in its entirety.

[0218] Recombinant vector

[0219] This disclosure also provides recombinant vectors (e.g., expression vectors) including the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells incorporating the recombinant vectors (i.e., host cells containing polynucleotides and / or vectors containing polynucleotides), and recombinant antibody polypeptides or fragments thereof generated by recombinant technology.

[0220] As used herein, a “vector” is any construct capable of delivering one or more target polynucleotides to a host cell when introduced into that host cell. An “expression vector” is capable of delivering and expressing one or more target polynucleotides as encoded polypeptides in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the target polynucleotide is expressed within the vector by operatively linking to regulatory elements such as promoters, enhancers, and / or polyadenylate tails located within the vector or at, near, or flanking the integration site of the target polynucleotide in the genome of the host cell, such that the target polynucleotide will be translated in the host cell into which the expression vector has been introduced.

[0221] Vectors can be introduced into host cells by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., with recombinant viruses). Therefore, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, granules, phage vectors, and DNA or RNA expression vectors associated with cationic condensers.

[0222] In some embodiments, the polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein) are introduced using a viral expression system (e.g., vaccinia or other poxviruses, retroviruses, or adenoviruses). This may involve the use of a non-pathogenic (deficient), reproducible virus, or a replication-deficient virus. In the latter case, viral replication typically occurs only in complementary viral packaging cells. Suitable systems are disclosed in, for example, Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 86:317-321; Flexner et al., 1989, Ann. NYAcad. Sci. [Annual Report of the New York Academy of Sciences] 569:86-103; Flexner et al., 1990, Vaccine, 8:17-21; U.S. Patent Nos. 4,603,112, 4,769,330 and 5,017,487; WO 89 / 01973; U.S. Patent No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91 / 02805; Berkner-Biotechniques, 6:616-627, 1988; Rosenfeld et al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for integrating DNA into such expression systems are well known to those skilled in the art. DNA can also be “naked,” as described, for example, in Ulmer et al., 1993, Science, 259:1745-1749, and Cohen, 1993, Science, 259:1691-1692. The uptake of naked DNA can be increased by coating it onto biodegradable beads, which can be efficiently transported into cells.

[0223] For expression, the DNA insert sequence containing a polynucleotide encoding an antibody or a polypeptide disclosed herein can be operatively ligated to a suitable promoter (e.g., a heterologous promoter), such as the bacteriophage λPL promoter, the *E. coli* lac promoter, the *E. coli* trp promoter, and the *E. coli* tac promoter, the SV40 early and late promoters, and promoters of retroviral LTRs, to name just a few. Other suitable promoters are known to those skilled in the art. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. The expression construct may further contain sites for transcription initiation and termination, and (in the transcribed region) ribosome-binding sites for translation. The coding portion of the mature transcription expressed by the construct may include a translation initiation codon at the beginning and a stop codon (UAA, UGA, or UAG) appropriately located at the end of the polypeptide to be translated.

[0224] As indicated, the expression vector may include at least one optional marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culture in *E. coli* and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as *E. coli* cells, *Streptomyces* cells, and *Salmonella typhimurium* cells; fungal cells such as yeast cells; insect cells such as *Drosophila* S2 cells and *Spodoptera* Sf9 cells; animal cells such as CHO cells, COS cells, Bowes melanoma cells, and HK 293 cells; and plant cells. Appropriate culture media and conditions for the host cells described herein are known in the art.

[0225] Non-restricted vectors for use in bacteria include pQE70, pQE60, and pQE-9 from Qiagen; pBS, Phagescript, Bluescript, pNH8A, pNH16a, pNH18A, and pNH46A from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 from Pharmacia. Non-restricted eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG from Stratagene; and pSVK3, pBPV, pMSG, and pSVL from Pharmacia. Other suitable vectors will be readily apparent to those skilled in the art.

[0226] Suitable non-restrictive bacterial promoters include the *E. coli* lacI and lacZ promoters, T3 and T7 promoters, gpt promoters, λPR and PL promoters, and trp promoters. Suitable eukaryotic promoters include CMV (immediate early promoter), HSV thymidine kinase promoters, SV40 early and late promoters, promoters of retroviral LTRs such as Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0227] In the yeast Saccharomyces cerevisiae, a variety of vectors containing constitutive or inducible promoters such as α-factors, alcohol oxidases, and PGH can be used.

[0228] Constructs can be introduced into host cells via calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0229] The transcription of DNA encoding the disclosed antibodies in higher eukaryotes can be increased by inserting enhancer sequences into vectors. Enhancers are cis-acting elements of DNA, typically about 10 to 300 bp, that increase promoter transcriptional activity in a given host cell type. Examples of enhancers include the SV40 enhancer (located 100 to 270 base pairs after the origin of replication), the cytomegalovirus early promoter enhancer, the polyomavirus enhancer located posterior to the origin of replication, and the adenovirus enhancer.

[0230] To secrete translated proteins into the endoplasmic reticulum lumen, periplasmic space, or extracellular environment, appropriate secretion signals can be integrated into the expressed peptide. These signals can be endogenous or heterologous to the peptide.

[0231] Peptides (e.g., antibodies) can be in modified forms, such as fusion proteins (e.g., GST fusions), or express histidine tags, and can include not only secretion signals but also additional heterologous functional regions. For example, additional amino acid regions, particularly charged amino acid regions, can be added to the N-terminus of the peptide to improve stability and persistence in host cells during purification or subsequent processing and storage. Similarly, peptide moieties can be added to the peptide to facilitate purification. Such regions can be removed prior to the final preparation of the peptide. Adding peptide moieties to the peptide to induce secretion or excretion, thereby improving stability and facilitating purification, is a technique particularly well-known and conventional in the art.

[0232] Treatment

[0233] The antibodies or antigen-binding fragments disclosed herein can be used for a variety of therapeutic purposes.

[0234] In one aspect, this disclosure provides methods for treating cancer in a subject, methods for reducing the rate of increase in tumor volume over time in a subject, methods for reducing the risk of metastasis, or methods for reducing the risk of further metastasis in a subject. In some embodiments, the treatment may stop, slow, delay, or inhibit the progression of cancer. In some embodiments, the treatment may result in a reduction in the number, severity, and / or duration of one or more symptoms of cancer in the subject.

[0235] In one aspect, this disclosure is characterized by methods comprising administering a therapeutically effective amount of the antibody disclosed herein or an antigen-binding fragment thereof to a subject in need (e.g., a subject who has, is identified as having, or is diagnosed with, cancer), the cancer being, for example, breast cancer (e.g., triple-negative breast cancer), carcinoid tumor, cervical cancer, endometrial cancer, neurocancer, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, stomach cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or a hematologic malignancy. In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-resistant prostate cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is head and neck squamous cell carcinoma (SCCHN), renal cell carcinoma (RCC), triple-negative breast cancer (TNBC), or colorectal cancer. In some embodiments, the subject has Hodgkin lymphoma. In some embodiments, the subject has triple-negative breast cancer (TNBC), gastric cancer, urothelial carcinoma, Merkel cell carcinoma, or head and neck cancer. In some embodiments, the cancer is melanoma, pancreatic cancer, mesothelioma, hematologic malignancy, especially non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia, or advanced solid tumor.

[0236] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk of cancer. Patients with cancer can be identified using a variety of methods known in the art.

[0237] In some respects, this disclosure relates to methods for treating autoimmune diseases or inflammation, the method comprising administering to a subject an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein, or an antibody-drug conjugate as described herein.

[0238] In one aspect, this disclosure provides methods for treating, preventing, or reducing the risk of developing disorders associated with abnormal or unwanted immune responses (such as autoimmune disorders) by influencing Treg function, for example. These autoimmune disorders include, but are not limited to, alopecia areata, lupus, ankylosing spondylitis, Meniere's disease, antiphospholipid syndrome, mixed connective tissue disease, autoimmune Addison's disease, multiple sclerosis, autoimmune hemolytic anemia, myasthenia gravis, autoimmune hepatitis, pemphigus vulgaris, Behcet's disease, pernicious anemia, bullous pemphigoid, polyarthritis nodosa, cardiomyopathy, polychondritis, celiac disease-dermatitis, polygonococcal syndrome, chronic fatigue syndrome (CFIDS), polymyalgia rheumatica, chronic inflammatory demyelinating disease, polymyositis and dermatomyositis, chronic inflammatory polyneuropathy, primary agammaglobulinemia, and Churg-Strauss syndrome. Syndrome), primary biliary cirrhosis, cicatricial pemphigoid, psoriasis, CREST syndrome, Raynaud's phenomenon, cold agglutinin disease, Lightell's syndrome, Crohn's disease, rheumatic fever, discoid lupus, rheumatoid arthritis, cryoglobulinemia sarcoidosis, fibromyalgia, scleroderma, Graves' disease, Sjögren's syndrome, Guillain-Barré disease, stiff-person syndrome, Hashimoto's thyroiditis, Takayasu arteritis, idiopathic pulmonary fibrosis, temporal arteritis / giant cell arteritis, idiopathic thrombocytopenic purpura (ITP), ulcerative colitis, IgA nephropathy, uveitis, diabetes (e.g., type I), vasculitis, lichen planus, and vitiligo. Anti-CD40 antibodies or their antigen-binding fragments can also be administered to subjects to treat, prevent, or reduce the risk of developing abnormal or unwanted immune responses associated with cell, tissue, or organ transplantation (e.g., kidney, liver, and heart transplantation), or to prevent allogeneic graft rejection. In some embodiments, the subject has Crohn's disease, ulcerative colitis, or type 1 diabetes. In some embodiments, the antibody or antigen-binding fragment can be used to treat inflammation.

[0239] In some embodiments, the antibody or its antigen-binding fragment is a TNFR2 agonist.

[0240] In some aspects, this disclosure relates to a method for suppressing the immune response of a subject, the method comprising administering to the subject an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein, or an antibody-drug conjugate as described herein. In some embodiments, the subject has an autoimmune disease.

[0241] As used herein, “effective amount” means an amount or dose sufficient to achieve a beneficial or desired outcome, including stopping, slowing, delaying, or inhibiting the progression of a disease (e.g., cancer). The effective amount will depend on, for example, the age and weight of the subject to be administered the antibody, antigen-binding fragment, polynucleotide encoding the antibody, carrier containing the polynucleotide, and / or a combination thereof, the severity of symptoms, and the route of administration, and therefore can be determined on an individual basis.

[0242] An effective amount may be administered once or multiple times. For example, an effective amount of an antibody or antigen-binding fragment is an amount sufficient to improve, terminate, stabilize, reverse, inhibit, slow, and / or delay the progression of an autoimmune disease or cancer in a patient, or an amount sufficient to improve, stop, stabilize, reverse, slow, and / or delay the proliferation of cells in vitro (e.g., biopsy cells, any cancer cells described herein, or cell lines (e.g., cancer cell lines)). As understood in the art, the effective amount of an antibody or antigen-binding fragment may vary and may depend on patient history and other factors, such as the type (and / or dosage) of the antibody used.

[0243] Effective amounts and regimens for administering the antibodies, antibody-encoding polynucleotides, and / or compositions disclosed herein can be determined empirically, and such determinations are within the scope of the art. Those skilled in the art will understand that the necessary dosage will depend, for example, on the mammal to which the antibodies, antibody-encoding polynucleotides, and / or compositions disclosed herein will be received, the route of administration, the specific type of antibody, antibody-encoding polynucleotide, antigen-binding fragment, and / or composition disclosed herein used, and any other drugs administered to the mammal.

[0244] The typical daily dose of an effective amount of antibody is 0.01 mg / kg to 100 mg / kg (mg / kg patient body weight). In some embodiments, the dose may be less than 100 mg / kg, 50 mg / kg, 40 mg / kg, 30 mg / kg, 25 mg / kg, 20 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.3 mg / kg, or 0.1 mg / kg. In some embodiments, the dose may be greater than 50 mg / kg, 40 mg / kg, 30 mg / kg, 20 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.3 mg / kg, 0.1 mg / kg, 0.05 mg / kg, or 0.01 mg / kg. In some embodiments, the dosage is approximately 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.

[0245] In any of the methods described herein, at least one antibody, its antigen-binding fragment, or pharmaceutical composition (e.g., any antibody, antigen-binding fragment, or pharmaceutical composition described herein), and optionally at least one additional therapeutic agent may be administered to a subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day). In some embodiments, at least two different antibodies and / or antigen-binding fragments are administered 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 in the form of pills, tablets, or capsules. In some embodiments, at least one additional therapeutic agent is administered in a sustained-release oral formulation.

[0246] In some embodiments, one or more additional therapeutic agents may be administered to a subject before or after administration of at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any antibody, antigen-binding antibody fragment, or pharmaceutical composition described herein). In some embodiments, administration of one or more additional therapeutic agents and at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any antibody, antigen-binding antibody fragment, or pharmaceutical composition described herein) to a subject such that the biological activity periods of the one or more additional therapeutic agents and the at least one antibody or antigen-binding fragment (e.g., any antibody or antigen-binding fragment described herein) overlap in the subject.

[0247] In some embodiments, at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any antibody, antigen-binding antibody fragment, or pharmaceutical composition described herein) may be administered to a subject over an extended period of time (e.g., at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years). A skilled medical professional may determine the duration of treatment using any of the methods described herein for diagnosing or monitoring treatment effectiveness (e.g., observing at least one cancer symptom). As described herein, a skilled medical professional may also vary the type and amount (e.g., increase or decrease) of the antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to the subject, and may also adjust (e.g., increase or decrease) the dose or frequency of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to the subject based on an assessment of treatment effectiveness (e.g., using any methods described herein and known in the art).

[0248] In some embodiments, one or more additional therapeutic agents may be administered to the subject. These additional therapeutic agents may comprise one or more inhibitors selected from the group consisting of: B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, PI3K / mTOR dual inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) inhibitors and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In some embodiments, the additional therapeutic agent is an indoleamine 2,3-dioxygenase-1 (IDO1) inhibitor (e.g., epacadostat).

[0249] In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of: HER3 inhibitors, LSD1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, hedgehog signaling pathway inhibitors, and agents that selectively degrade estrogen receptors.

[0250] In some embodiments, additional therapeutic agents may comprise one or more of the following: trabectedin, nab-paclitaxel, trebananib, pazopanib, sildenafil, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, enalapril, ceritinib, sunitinib, sirolimus, axitinib, everolimus, sorafenib, Votrient, Pazopanib, IMA-901, AGS-003, Cabozantinib, Vinpocetine, Hsp90 inhibitors, Ad-GM-CSF, Temozolomide, IL-2, IFNa, Vincristine, Thalidomide, Dacarbazine, Cyclophosphamide, Lenalidomide, Azacytidine, Lenalidomide, Bortezomid, Amrubicin, Carfilzomib, Pralatrexate, and Enzastaurin.

[0251] In some embodiments, the additional therapeutic agent may comprise one or more therapeutic agents selected from the group consisting of: adjuvants, TLR agonists, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-6 antagonists (e.g., IL-6 receptors), IL-13 antagonists, IL-17 antagonists, HVEM antagonists, ICOS agonists, therapies targeting CX3CL1, therapies targeting CXCL9, therapies targeting CXCL10, therapies targeting CCL5, LFA-1 agonists, ICAM1 agonists, and selective agonists.

[0252] In some embodiments, the subject is administered carboplatin, albumin-bound paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI.

[0253] In some embodiments, additional therapeutic agents are anti-OX40 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-LAG-3 antibodies, anti-TIGIT antibodies, anti-BTLA antibodies, anti-CTLA-4 antibodies, or anti-GITR antibodies.

[0254] Pharmaceutical Compositions and Routes of Administration

[0255] This document also provides pharmaceutical compositions containing at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described herein. Two or more (e.g., two, three, or four) of any of the antibodies or antigen-binding fragments described herein may be present in the pharmaceutical composition in any combination. The pharmaceutical compositions may be formulated in any manner known in the art.

[0256] The pharmaceutical composition is formulated to be compatible with its intended route of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The composition may include sterile diluents (e.g., sterile water or sterile saline), fixative oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents, antibacterial or antifungal agents (e.g., benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffers (e.g., acetates, citrates, or phosphates), and isotonic agents (e.g., sugars such as glucose), polyols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride) or any combination thereof. Liposome suspensions may also be used as pharmaceutically acceptable carriers. Formulations of the composition may be prepared and packaged in ampoules, disposable syringes, or multi-dose vials. If desired (e.g., in injectable formulations), adequate flowability can be maintained by, for example, using a coating (such as lecithin or a surfactant). The absorption of antibodies or their antigen-binding fragments can be prolonged by incorporating agents that delay absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved through implants and microencapsulated delivery systems, which may include biodegradable biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid).

[0257] Compositions containing one or more of any of the antibodies or antigen-binding fragments described herein can be formulated for parenteral (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dose units (i.e., physically discrete units containing a predetermined amount of active compound for ease of administration and uniform dosing).

[0258] Pharmaceutical compositions intended for parenteral administration are preferably sterile and substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. The pharmaceutical composition may be provided in unit dosage forms (i.e., a single-dose dose). The pharmaceutical composition 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, antibodies may be formulated in aqueous solutions, preferably in physiologically compatible buffers to minimize injection site discomfort. The solution may contain formulations such as suspending agents, stabilizers, and / or dispersants. Alternatively, antibodies may be in lyophilized form prior to use for formulation with a suitable medium (e.g., sterile, pyrogen-free water).

[0259] The toxicity and therapeutic efficacy of the composition can be determined in cell cultures or laboratory animals (e.g., monkeys) using standard pharmaceutical procedures. For example, the LD50 (lethal dose for 50% of the population) and ED50 (therapeutic effective dose for 50% of the population) can be determined: the therapeutic index is the ratio of LD50 to ED50. Agents exhibiting a high therapeutic index are preferred. When an agent exhibits adverse side effects, care should be taken to minimize potential harm (i.e., reduce undesirable side effects). Toxicity and therapeutic efficacy can be determined using other standard pharmaceutical procedures.

[0260] Data obtained from cell culture assays and animal studies can be used to formulate appropriate doses 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 their antigen-binding fragments (e.g., any antibody or antibody fragment described herein) will be the amount by which the subject (e.g., a human subject identified as having cancer or a subject identified as being at risk of developing a disease, such as a subject who previously had cancer but is now cured) treats the subject's disease (e.g., kills cancer cells) and reduces the severity, frequency, and / or duration of one or more symptoms in the subject (e.g., a human). The effectiveness and dosing of any antibody or antigen-binding fragment described herein can be determined by a healthcare professional or veterinary professional using methods known in the art, and by observing one or more symptoms in a subject (e.g., a human). Certain factors (e.g., the severity of the disease or disorder, prior treatment, the subject's general health condition and / or age, and other pre-existing conditions) can influence the dosage and duration required for effective treatment of the subject.

[0261] Exemplary dosages include milligrams or micrograms per kilogram of subject weight for any antibody or antigen-binding fragment described herein (e.g., about 1 μg / kg to about 500 mg / kg; about 100 μg / kg to about 500 mg / kg; about 100 μg / kg to about 50 mg / kg; about 10 μg / kg to about 5 mg / kg; about 10 μg / kg to about 0.5 mg / kg; about 1 μg / kg to about 50 μg / kg; about 0.3 mg / kg to about 25 mg / kg; about 1 mg / kg to about 10 mg / kg; or about 1 mg / kg to about 5 mg / kg). While these dosage ranges are broad, those skilled in the art will understand that therapeutic agents, including antibodies and their antigen-binding fragments, vary in efficacy and that effective amounts can be determined by methods known in the art. Typically, a relatively low dose is administered first, and the attending healthcare professional or veterinary professional (in the case of therapeutic applications) or researcher (while still in the developmental stage) may subsequently and gradually increase the dose until an appropriate response is obtained. Furthermore, it should be 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 subject's age, weight, general health condition, sex, and diet, the time of administration, the route of administration, the rate of excretion, and the in vivo half-life of the antibody or antibody fragment.

[0262] Pharmaceutical compositions may be included in containers, packages, or dispensers together with instructions for use. This disclosure also provides methods for manufacturing antibodies or antigen-binding fragments thereof for the various uses described herein.

[0263] Example

[0264] The invention is further described in the following examples, which do not limit the scope of the invention as described in the claims.

[0265] Example 1. Production of anti-hTNFR2 antibody

[0266] To generate antibodies against human TNFR2 (TNFR2; SEQ ID NO:1), RenMab mice were immunized with human TNFR2. Anti-TNFR2 antibodies were prepared by the method described below.

[0267] RenMab mice possess both humanized heavy chain immunoglobulin loci and humanized κ chain immunoglobulin loci. The heavy chain immunoglobulin loci are regions on chromosomes containing genes encoding antibody heavy chains. These loci include IGHV (variable), IGHD (diversity), IGHJ (linking), and heavy chain constant domain genes. The κ chain immunoglobulin loci are regions on chromosomes containing genes encoding antibody light chains (κ chains). These loci include IGKV (variable), IGKJ (linking), and light chain constant domain genes. A detailed description of RenMab mice can be found in PCT / CN2020 / 075698, which is incorporated herein by reference in its entirety.

[0268] mouse immunization

[0269] RenMab mice were immunized with Fc-tagged human TNFR2 protein (the Fc fusion protein contains the extracellular domain of human TNFR2, positions 23aa-257aa of SEQ ID NO:1). The Fc-tagged human TNFR2 protein was emulsified with adjuvant and injected into four sites on the back of the mice. For the first subcutaneous injection (sc), the diluted antigen was emulsified with an equal volume of complete Freund's adjuvant (CFA). In subsequent subcutaneous injections, the protein was emulsified with an equal volume of incomplete Freund's adjuvant (IFA). At least four injections were performed, with at least 14 days between each injection. Seven days after the third injection or booster immunization, blood (serum) was collected and antibody titers were analyzed using fluorescence-activated cell sorting (FACS).

[0270] In another experiment, several mice were immunized by injecting an expression plasmid encoding human TNFR2 into them. The plasmid encoding the antigen was injected into the tibialis anterior muscle (intramuscular injection; IM). At least four injections were performed, with at least 14 days between each injection. Blood (serum) was collected seven days after the last immunization, and the antibody titer of the serum was tested by ELISA.

[0271] In addition, a booster immunization procedure was performed at least fourteen days after immunization (by injecting plasmids or proteins). CHO cells expressing the TNFR2 antigen on their surface were injected intravenously into mice via the tail vein. Immune system organs (e.g., bone marrow, lymph nodes, spleen, etc.) were then collected four days after injection.

[0272] Spleen cells were collected and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. Hybridoma cells were screened and selected to identify cell lines producing TNFR2-specific antibodies. Using this technique and the aforementioned immunogen, several anti-TNFR2 chimeric antibodies (i.e., antibodies with human variable domains and mouse constant domains) were obtained. Specifically, mouse immune organs were harvested after pulse immunization, and plasma cells were separated using magnetic beads. Monoclonal hybridoma cells secreting antigen-specific monoclonal antibodies were screened using hybridoma fusion technology. The antibody light chain and heavy chain V region sequences were obtained from the selected monoclonal hybridoma cells by reverse transcription and PCR sequencing. The antibody light chain and heavy chain V region sequences were constructed into antibody expression vectors and validated using the Expi CHO-STM cell expression system. Cells were transfected in a 24-well system, and antibodies were collected from the supernatant on day 3. FACS was used to verify the specificity of antibody binding to 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 in these antibodies can be readily replaced to obtain fully human anti-TNFR2 antibodies (anti-hTNFR2 antibodies). Exemplary fully human antibodies obtained by this method are named as follows: 14-1B3-hHvKv (“14-1B3” or “1B3”) and 14-4A9-hHvKv (“14-4A9” or “4A9”), etc. Taking 14-4A9-hHvKv as an example, when antibody VH / VL is linked to different subtypes such as IgG1, the antibody is named 14-4A9-hHvKv-IgG1.

[0273] Antigen-positive B cells were also directly isolated from immunized mice without fusing with myeloma cells. Anti-TNFR2 antibodies were further isolated from the antigen-positive B cells. The light chain and heavy chain V region sequences of the antibody were obtained directly from the antigen-positive B cells. For example, single-cell techniques (e.g., The Optofluidic system (BerkeleyLights Inc.) is used to screen and discover plasma cells that secrete antigen-specific monoclonal antibodies. Antibody V region sequences are obtained using reverse transcription and PCR sequencing. Antibodies are expressed. FACS is used to verify the binding between the antibody and TNFR2. Exemplary antibodies obtained by this method include: BC-1A8 (“1A8”), BC-1B6 (“1B6”), BC-1C3 (“1C3”), BC-1F4 (“1F4”), BC-1F10 (“1F10”), and BC-3B7 (“3B7”). Taking BC-1F4 as an example, when antibody VH / VL is conjugated to different subtypes such as IgG1, the antibody is named BC-1F4-IgG1. Examples of other subtypes are: BC-1F4-IgG1-SI and BC-1F4-IgG1-LALA.

[0274] The amino acid sequences of the heavy and light chains CDR1, CDR2, and CDR3 of 1A8 are shown in SEQ ID NO:6-11 (Kabat number) or SEQ ID NO:42-47 (Chothia number). The human heavy chain variable region and the human light chain variable region of the antibody are shown in SEQ ID NO:36 or SEQ ID NO:37.

[0275] The amino acid sequences of the heavy and light chains CDR1, CDR2, and CDR3 of 1B6 are shown in SEQ ID NO:12-17 (Kabat number) or SEQ ID NO:48-53 (Chothia number). The human heavy chain variable region and the human light chain variable region of the antibody are shown in SEQ ID NO:38 or SEQ ID NO:39.

[0276] The amino acid sequences of the heavy and light chains CDR1, CDR2, and CDR3 of 1C3 are shown in SEQ ID NO:18-23 (Kabat number) or SEQ ID NO:54-59 (Chothia number). The human heavy chain variable region and the human light chain variable region of the antibody are shown in SEQ ID NO:40 or SEQ ID NO:41.

[0277] The amino acid sequences of the heavy and light chains CDR1, CDR2, and CDR3 of 1F4 are shown in SEQ ID NO:24-29 (Kabat number) or SEQ ID NO:60-65 (Chothia number). The human heavy chain variable region and the human light chain variable region of the antibody are shown in SEQ ID NO:72 or SEQ ID NO:73.

[0278] The amino acid sequences of the heavy and light chains CDR1, CDR2, and CDR3 of 1F10 are shown in SEQ ID NO:30-35 (Kabat number) or SEQ ID NO:66-71 (Chothia number). The human heavy chain variable region and the human light chain variable region of the antibody are shown in SEQ ID NO:74 or SEQ ID NO:75.

[0279] Antibody preparation

[0280] The positive antibody sequences from the sequence validation phase were extracted into plasmids and transfected into a 25 mL system. After 10–12 days of cell culture, the expression supernatant was collected and subjected to affinity chromatography. The obtained antibody samples were used for the following in vitro tests and screening.

[0281] Example 2. In vitro testing of anti-TNFR2 antibodies

[0282] Blocking the binding of human TNFR2 to TNFα

[0283] A blocking assay was performed to determine whether the anti-TNFR2 antibody could block the binding between TNFR2 and its ligand hTNFα.

[0284] Specifically, 30 μl of CHO cells (1 × 10⁻⁶) transiently transfected with human TNF receptor 2 (TNFR2) were used. 5 (Number of cells) were added to each well in the plate. The purified antibody was titrated to final concentrations of 10, 2.5, 0.625, 0.1565, and 0.039 μg / ml. The titrated antibody was added to each well at 30 μl / well at 4°C and incubated for 30 minutes.

[0285] Add 30 μl of biotin-hTNFα (Acro Biosystems, catalog number: TNA-H82E1) to each well (final concentration in each well: 0.5 μg / ml). Incubate cells containing biotin-hTNFα and antibody at 4°C for 30 minutes.

[0286] After washing twice with phosphate-buffered saline (PBS), 50 μl of PE-labeled anti-human IgG Fc antibody diluted 1:100 (PE anti-human IgG Fc, Jackson Immuno Research, catalog number: 109-115-098) and Alexa antibody diluted 1:500 were added. AF647-labeled streptavidin (AF647 streptavidin, Jackson Immunological Research, catalog number: 016-600-084) was added to each well and incubated at 4°C for 15 min, followed by washing with PBS. AF647 and PE signaling were determined by flow cytometry (Thermo Attune NX).

[0287] Table 1 below shows the percentage of cells tested with streptavidin signaling in flow cytometry analysis. If the percentage of tested cells with streptavidin signaling (AF647) increases while the antibody concentration decreases, the antibody has blocking affinity (indicating strong binding affinity). Based on the data, BC-1A8-IgG1, BC-1F4-IgG1, BC-3B7-IgG1, and BC-1F10-IgG1 showed strong blocking activity. However, BC-1B6-IgG1, BC-1C3-IgG1, 14-1B3-hHvKv-IgG1, and 14-4A9-hHvKv-IgG1 were not effective in blocking the binding between TNFR2 and TNFα.

[0288] Table 1

[0289] BC-1A8-IgG1 12.0％ 29.9％ 58.6％ 67.4％ 62.8％ have BC-1B6-IgG1 75.1％ 87.2％ 80.4％ 82.7％ 78.9％ none BC-1C3-IgG1 71.1％ 81.5％ 77.3％ 74.3％ 77.0％ none BC-1F4-IgG1 18.5％ 59.5％ 61.9％ 76.2％ 84.6％ have BC-1F10-IgG1 51.9％ 54.5％ 60.2％ 55.2％ 59.0％ have BC-3B7-IgG1 19.4％ 46.6％ 69.7％ 70.1％ 64.5％ have 14-1B3-hHvKv-IgG1 66.6％ 65.1％ 62.5％ 63.2％ 63.8％ none 14-4A9-hHvKv-IgG1 66.1％ 65.7％ 60.7％ 63.2％ 63.0％ none

[0290] The binding affinity of anti-TNFR2 antibodies to human TNFR2 and TNFR1

[0291] Using a Biacore (Biacore, INC., Piscataway, NJ) 8K biosensor (equipped with a pre-immobilized protein A sensor chip), the binding affinity of anti-TNFR2 antibodies to human TNFR2 and TNFR1 (tumor necrosis factor receptor 1) was measured by surface plasmon resonance (SPR).

[0292] The purified anti-TNFR2 antibody was diluted to 1 μg / ml and then injected into the Biacore 8K biosensor at a rate of 10 μL / min for approximately 50 seconds to achieve the desired protein density (e.g., approximately 50 response units (RU)). Then, His-tagged human TNFR1 (human TNFR1 / CD120a / TNFRSF1A protein, His-tagged, Beijing Acrobiosystems CO.LTD., catalog number: TN1-5222) or TNFR2 (human TNFR2 / CD120b / TNFRSF1B protein, His-tagged, Beijing Acrobiosystems CO.LTD., catalog number: TN2-5227) was injected at concentrations of 200, 100, 50, 25, 6.25, or 1.56 nM at a rate of 30 μL / min for 120 seconds. Dissociation was monitored for 600 seconds. The chip is regenerated after the last injection of each glycine titration (pH 2.0, 30 μL / min, 30 seconds).

[0293] By using Biacore 8K evaluation software 3.0, the data were fitted to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B., 1994. Methods Enzymology, 6.99-110), thereby simultaneously obtaining the kinetic association rate (kon) and dissociation rate (koff). The affinity (KD = koff / kon) was derived from the quotient of the kinetic rate constant.

[0294] As those skilled in the art will understand, the same method was used for each antibody test, with parameters (e.g., antibody concentration) adjusted appropriately. Table 2 below summarizes the results of the tested antibodies.

[0295] Table 2

[0296]

[0297] The results showed that these human antibodies had a very high binding affinity to human TNFR2. 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) failed to bind to TNFR1.

[0298] Cross-reactivity of anti-TNFR2 antibodies against TNFR2 in mice, dogs, and monkeys

[0299] In each experiment, CHO cells were transfected with EGFP and human TNFR2 (TNFR2, SEQ ID NO:1), mouse TNFR2 (mTNFR2, SEQ ID NO:2), monkey (crab-eating monkey) TNFR2 (fTNFR2, SEQ ID NO:3), or dog (canine) TNFR2 (dTNFR2, SEQ ID NO:4).

[0300] 30 μl of CHO cells (1×10⁻⁶) 5 Add 30 μl of purified anti-TNFR2 antibody (10 μg / ml) (as listed in Table 3) to each well and incubate at 4 °C for 30 min.

[0301] 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) diluted 1:500 was added to each well. The cells were incubated at 4 °C for 15 min, followed by washing with PBS (1200 rpm, 5 min). AF647 signal was detected by flow cytometry.

[0302] The table below summarizes the cross-reactivity of the tested antibodies with human (TNFR2), mouse (mTNFR2), monkey (fTNFR2), and dog (dTNFR2) TNFR2.

[0303] Table 3

[0304] BC-1A8-IgG1 have none have none BC-1B6-IgG1 have none have none BC-1C3-IgG1 have none have none BC-1F4-IgG1 have none have none BC-1F10-IgG1 have none have none BC-3B7-IgG1 have none have none 14-1B3-hHvKv-IgG1 have none have none 14-4A9-hHvKv-IgG1 have none have none

[0305] Epitope correlation analysis of purified anti-hTNFR2 antibody

[0306] The relative positions of target protein epitopes between a pair of purified anti-TNFR2 monoclonal antibodies were analyzed using surface plasmon resonance (SPR) competition assays. Five monoclonal antibodies were used to investigate the binding inhibition (blocking) effect of each antibody on the other: BC-1A8-IgG1, BC-1F4-IgG1, BC-3B7-IgG1, BC-1C3-IgG1, and BC-1F10-IgG1. HBS-EP+ buffer (10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 150 mM NaCl, 3 mM EDTA, and 0.05% P20, pH 7.4) was used as the run buffer throughout the experiments. Anti-His antibodies were immobilized on the surface of the S-series sensor chip CM5 via amino-coupling to generate an anti-His chip (i.e., CM5-anti-His-channel 1,8-chip). Then, 1M ethanolamine (pH 8.5) was injected to block the remaining active carboxyl groups on the chip surface, followed by equilibration with HBS-EP+ buffer for 2 hours. Recombinant human TNFR2 protein with a 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 (200 nM each) were sequentially injected onto the chip at 30 μL / min. The binding time for the first injected antibody (analyte 1) was 250 seconds, followed by the binding time for the second antibody (analyte 2) for 250 seconds. After antibody injection in each analytical cycle, the chip was regenerated twice with glycine buffer (pH 1.7; 30 μL / min for 30 seconds). The same experimental steps were performed for each pair of monoclonal antibodies to obtain binding inhibition data when each monoclonal antibody was paired with another antibody.

[0307] Binding values ​​for each antibody were obtained using Biacore Insight evaluation software. To quantify the interference of one antibody binding to another, binding rates were calculated to compare each antibody pair. The binding rate was defined as the binding value of the second antibody (analyte 2) divided by the binding value of the first antibody (analyte 1). Statistical software was also used for cluster analysis. Epitope correlations were analyzed, and the five anti-hTNFR2 antibodies were classified into four epitope clusters (…). Figure 8 In summary, 1A8 and 1F4 share the same or overlapping epitopes. 3B7, 1C3, and F10 do not show epitope correlations with other antibodies.

[0308] TNFR2 possesses four cysteine-rich domains (CRDs). In another experiment, FACS was used to detect the binding of anti-hTNFR2 antibodies to TNFR2 proteins with different domains. Secondary antibodies (Alexa) were used. 647 AffiniPure F(ab')2 fragment goat anti-human IgG, Fcγ fragment specific, Jackson ImmunoResearch Laboratories, Inc. (Catalogue No.: 151524). Fluorescently labeled secondary antibodies can attach to the Fc region of anti-hTNFR2 antibodies, allowing for FACS detection of anti-hTNFR2 antibodies binding to ΔCRD cells. Specifically, ΔCRD1-TNFR2 protein (CRD1 deletion), ΔCRD2-TNFR2 protein (CRD2 deletion), ΔCRD3-TNFR2 protein (CRD3 deletion), or ΔCRD4-TNFR2 protein (CRD4 deletion) are expressed in CHO-S cells, and the binding between cells and different anti-hTNFR2 antibodies is then detected. SEQ ID NO:1 shows the TNFR2 protein, with CRD1 being sequence 39aa-76aa, CRD2 being sequence 77aa-118aa, CRD3 being sequence 119aa-162aa, and CRD4 being sequence 168aa-196aa. The results of the flow cytometry experiments are shown in Table 4 below.

[0309] The results showed that 1A8 and 1F4 did not bind to TNFR2 lacking CRD3, and the positive rate of binding to TNFR2 lacking CRD4 was also significantly reduced, indicating that 1A8 and 1F4 have similar binding epitopes for human TNFR2, and these epitopes are likely located at CRD3 (class I epitopes). 1B6 and 1C3 did not bind to TNFR2 lacking either CRD3 or CRD4, indicating that 1B6 and 1C3 have similar binding epitopes for human TNFR2, and these epitopes are likely located at the junction of CRD3 and CRD4 (class III epitopes). The positive rate of binding between 1F10 and TNFR2 lacking either CRD3 or CRD4 was significantly reduced, indicating that the binding epitope of 1F10 is different from class I and class III epitopes (belonging to class IV epitopes). The results are consistent with SPR epitope characterization.

[0310] Table 4

[0311]

[0312] In vitro ADCC detection experiment

[0313] Experiments were conducted to evaluate the ADCC effect of the anti-TNFR2 antibody. In the experiments, BC-1A8-IgG1, BC-1C3-IgG1, BC-1F4-IgG1, BC-1F10-IgG1, BC-1B6-IgG1, and isotype control human IgG1 (Crown Bioscience Inc., C0001-3) were used.

[0314] Lactate dehydrogenase (LDH) is a cytoplasmic enzyme found in many different cell types and is released into the cell culture medium after plasma membrane damage. Extracellular LDH was accurately quantified using the CyQUANT LDH Cytotoxicity Assay Kit (Invitrogen, catalog number: C20301) to assess the ADCC effect of antibodies.

[0315] The anti-TNFR2 antibody was serially diluted (10-fold) to a maximum concentration of 100 μg / mL. Target cells (MC38 cells overexpressing human TNFR2) were seeded in 96-well plates (cell density 2 × 10⁶ cells / well). 4 100 μL of cells / well was incubated at 37°C for 3–4 hours. Effector cells (peripheral blood mononuclear cells (PBMCs)) were then resuscitated (cell density 2 × 10⁶ cells / well, 100 μL) and incubated at 37°C for 3–4 hours. Effector cells (peripheral blood mononuclear cells (PBMCs)) were then revived (cell density 2 × 4 (Cells / well). An equal 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. Absorbance values ​​at 490 nm and 680 nm were measured using a microplate reader and used to calculate the cell killing effect (cytotoxicity %) of each antibody group on the target cells. The EC50 value was calculated using a non-linear fitting method with antibody concentration as the x-axis and cytotoxicity as the y-axis.

[0316]

[0317] In the above formula, "experiment" refers to the absorbance value of the experimental well; "effect cell" refers to the absorbance value of the effector cell; "target" refers to the absorbance value of the target cell; "spontaneous" refers to the autofluorescence absorbance value of the cell culture medium; "target maximum" refers to the highest absorbance value of the target cell; and "spontaneous*" refers to the absorbance value of the control well (cell culture medium volume).

[0318] EC50 results are shown in Table 5. Compared with the isotype control hIgG1, the killing effect (cytotoxicity%) of anti-TNFR2 antibodies (BC-1A8-IgG1, BC-1C3-IgG1, BC-1F4-IgG1, BC-1F10-IgG1, or BC-1B6-IgG1) on target cells increased with increasing antibody dose, indicating that BC-1A8-IgG1, BC-1C3-IgG1, BC-1F4-IgG1, BC-1F10-IgG1, and BC-1B6-IgG1 all possess ADCC activity.

[0319] Table 5

[0320]

[0321] In another similar experiment, the ADCC effect of anti-TNFR2 antibodies (BC-1C3-IgG1, BC-1C3-IgG1-SI, and BC-1C3-IgG1-LALA) was evaluated using target cells (MC38 cells overexpressing human TNFR2) and effector cells (FcR-TANK (CD16a-158V) cell line, ImmuneOnco Biopharmaceuticals (Shanghai) Co., Ltd.).

[0322] Cytotoxicity data in Figure 9 The results are shown in Table 6. Compared with the isotype control hIgG1, the killing effect (cytotoxicity%) of anti-hTNFR2 antibodies (BC-1C3-IgG1 and BC-1C3-IgG1-SI) on target cells increased with increasing antibody dose, indicating that BC-1C3-IgG1-SI has strong ADCC activity.

[0323] Table 6

[0324]

[0325] Example 3. In vivo testing of anti-hTNFR2 antibody

[0326] To test antibodies in vivo and predict their effects in humans, a humanized TNFR2 mouse model was developed. This humanized TNFR2 mouse model was engineered to express a chimeric TNFR2 protein (SEQ ID NO:5), wherein the extracellular region of the mouse TNFR2 protein was replaced with the corresponding extracellular region of human TNFR2. Amino acid residues 33-260 of the mouse TNFR2 (SEQ ID NO:2) were replaced with amino acid residues 33-259 of human TNFR2 (SEQ ID NO:1).

[0327] Humanized TNFR2 mouse models (e.g., B-TNFR2 mice) provide a new tool for testing novel therapeutics in clinical settings by significantly reducing the differences in clinical outcomes between humans and normal mice expressing mouse TNFR2. A detailed description of humanized TNFR2 mouse models can be found in PCT / CN2020 / 113618, which is incorporated herein by reference in its entirety.

[0328] Anti-hTNFR2 antibodies were tested in TNFR2 humanized mice (B-TNFR2) to demonstrate their effect on tumor growth in vivo.

[0329] In vivo efficacy in β-TNFR2 mice with MC38 and 10 mg / kg

[0330] MC-38 tumor cells (colon adenocarcinoma cells) were subcutaneously injected into B-TNFR2 mice. When the tumor volume in the mice reached 100 mm², the tumors were induced to multiply. 3 -150 mm 3 At that time, the mice were randomly divided into different groups (six mice in each group) according to the size of the tumor.

[0331] Mice were then injected with PBS as a control (G1), anti-hTNFR2 antibody BC-1F4-IgG1 (G2), BC-3B7-IgG1 (G3), BC-1F10-IgG (G4), 14-1B3-hHvKv-IgG1 (G5), BC-1A8-IgG1 (G6), BC-1C3-IgG1 (G7), 14-4A9-hHvKv-IgG1 (G8), and anti-mPD-1 (G9) (BIO X CELL, catalog number: BE0146). Anti-mPD-1, proven effective in mice, was used as a positive control. Antibodies were administered intraperitoneally at a dose of 10 mg / kg on the first and fourth days of each week for 3 weeks (a total of 6 injections).

[0332] Mice body weight was monitored throughout the treatment period. Body weight increased in all groups without statistically significant differences (P>0.05). On the day of grouping (Day 0; "D0"), the mean body weight of each group ranged from 20.3g to 21.4g. At the end of the experiment (28 days after grouping, D28), the mean body weight of each group ranged from 22.7g to 24.7g, with a body weight change ranging from 107.4% to 116.5%. The results showed that the anti-hTNFR2 antibody was well tolerated and non-toxic to mice.

[0333] In the group treated with anti-hTNFR2 antibodies, tumor size ( Figure 1 Significant differences were observed in TGI levels on day 28 (28 days after grouping) in each treatment group. TV The percentages are shown in Table 7 below. The positive control (anti-mPD-1, G9) showed good therapeutic efficacy at the above doses and frequencies (TGI). TV % = 78.5%. Multiple 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 % greater than 80%. This TGI TV % better than the positive control group (G19 / anti-mPD-1TGI) TV (% = 78.5%).

[0334] Table 7

[0335]

[0336]

[0337] In vivo efficacy in β-TNFR2 mice with MC38 and 1 mg / kg

[0338] Similar to the aforementioned in vivo efficacy experiments, after establishing the tumor model, mice were injected with PBS as a control (G1), anti-hTNFR2 antibody BC-1A8-IgG1 (G2), BC-1F10-IgG1 (G3), BC-1F4-IgG1 (G4), anti-mPD-1 (G5), and anti-mCTLA4 (G6) (BIO X CELL, catalog number: BE0164). The antibodies were administered intraperitoneally at a dose of 1 mg / kg on the first and fourth days of each week for 3 weeks (a total of 6 injections).

[0339] Mice body weight was monitored throughout the experiment. Body weight increased in all groups, with no significant difference between groups (P>0.05). At grouping (D0), the mean body weight of each group ranged from 20.4g to 20.8g. At the end of the experiment (24 days after grouping, D24), the mean body weight of each group ranged from 21.6g to 23.7g, with a body weight change ranging from 102.5% to 114.7%. Similar to previous experiments, the results showed that the anti-hTNFR2 antibody was well tolerated and non-toxic to mice.

[0340] Significant differences in tumor size were observed in the group treated with anti-TNFR2 antibodies. Figure 2 Specifically, the tumor size of G3, G4, and G5 was significantly smaller than that of G1 (G3: P = 0.005, G7: P = 0.001, G8: P = 0.011). As shown in Table 8 below, the TGI was also calculated for each treatment group on day 24 (24 days after grouping). TV %. Compared with the positive controls (G5, G6), G2, G3, and G4 showed better tumor suppression (TGI). TV %)

[0341] Table 8

[0342]

[0343] In vivo efficacy in MC38, 0.3 mg / kg B-TNFR2 mice

[0344] In another similar experiment, the dose of the anti-hTNFR2 antibody was reduced to 0.3 mg / kg. When the tumor volume in mice reached 100 ± 50 mm... 3At that time (six mice per group), the mice (with MC38 tumors) were randomly divided into different groups.

[0345] Mice were then injected with PBS as a control (G1), anti-hTNFR2 antibody BC-1C3-lgG1 (G2), anti-mPD-1 (G3), and anti-mCTLA4 (G4). Antibodies were administered intraperitoneally at a dose of 0.3 mg / kg on the first and fourth days of each week for 3 weeks (a total of 6 injections).

[0346] Mouse body weight was monitored during the experiment. Body weight increased in all groups, with no significant difference between groups (P>0.05). At grouping (D0), the mean body weight of each group ranged from 19.5g to 19.8g. At the end of the experiment (25 days after grouping, D25), the mean body weight ranged from 21.6g to 23.3g, with a body weight change of 108.9%–118.2%. Similar to previous results, the results showed that the anti-TNFR2 antibody was well tolerated and non-toxic to mice.

[0347] In the group treated with anti-hTNFR2 antibodies, tumor size ( Figure 3 Significant differences were observed. Table 9 below shows the TGI for each treatment group on day 25 (25 days after grouping). TV % BC-1C3-lgG1 (G2) showed the best efficacy at this dose and was superior to the positive controls (G3, G4).

[0348] Table 9

[0349]

[0350] In vivo efficacy of B16 in 10 mg / kg B-TNFR2 mice

[0351] B16F10 tumor cells (melanoma cells) were subcutaneously injected into B-TNFR2 mice. The tumor volume in the mice reached 100±50 mm. 3 At that time, the mice were randomly divided into different groups (six mice in each group) according to the size of the tumor.

[0352] Mice were then injected with PBS as a control (G1), and 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 administered intraperitoneally at a dose of 10 mg / kg on the first and fourth days of each week for two weeks (a total of three injections).

[0353] Mouse body weight was monitored during the experiment. Body weight increased in all groups, with no significant difference between groups (P>0.05). At grouping (D0), the mean body weight of each group ranged from 19.2g to 19.7g. On day 10 (D10), the mean body weight of each group ranged from 22.1g to 24.5g, with a body weight change ranging from 114.3% to 126.8%. The results showed that the anti-hTNFR2 antibody was well tolerated and non-toxic to mice.

[0354] Significant differences in tumor size were observed in the group treated with anti-TNFR2 antibodies. Figure 4 BC-1A8-IgG1 (G2), BC-1C3-IgG1 (G3), BC-1F10-IgG1 (G4), and BC-1B6-IgG1 (G6) all demonstrated antitumor efficacy in the B16F10 melanoma model. The TGI in each treatment group on day 10 (10 days after grouping) was... TV % is shown in Table 10 below.

[0355] Table 10

[0356]

[0357] In vivo efficacy and toxicity in β-TNFR2 mice with MC38 and 25 mg / kg

[0358] In another similar experiment, the dose of the anti-hTNFR2 antibody was adjusted to 25 mg / kg to test its efficacy and toxicity in vivo. MC38 cancer cells (colon adenocarcinoma cells) were subcutaneously injected into B-TNFR2 mice. The tumor volume in the mice reached 100 ± 50 mm. 3 At that time, the mice were randomly divided into different groups (four mice in each group) according to the size of the tumor.

[0359] Mice were then injected with PBS as a control (G1), and anti-hTNFR2 antibodies BC-1A8-IgG1 (G2), BC-1C3-IgG1 (G3), BC-1F10-IgG1 (G4), BC-1F4-IgG1 (G5), and BC-1B6-IgG1 (G6). Antibodies were administered intraperitoneally at a dose of 25 mg / kg on the day of grouping (D0) and on day 3 post-grouping (D3) (a total of two injections).

[0360] Mice body weight was monitored during the experiment. Body weight increased in all groups, with no significant difference between groups. At the time of grouping, the average body weight of each group ranged from 18.5g to 19.2g. At the end of the experiment (21 days after grouping), the average body weight of each group ranged from 21.1g to 23.3g, with a body weight change between 110.0% and 122.7%. Peripheral blood was collected from mice on day 5 (D5) to test blood biochemical parameters (AST, ALT). The biochemical parameter test results on D5 are as follows (…). Figures 7A-7B The results showed no significant changes in ALT and AST compared to the control. Similar to previous results, the 25 mg / kg anti-TNFR2 antibody was well-tolerated and non-toxic in mice.

[0361] In the group treated with anti-TNFR2 antibodies, tumor size ( Figure 5 Significant differences were observed. Compared with the control group G1, the 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). TGI TV The percentages ranged from 81.6% to 98.4%, demonstrating a significant tumor-suppressive effect. As shown in Table 11 below, the TGI (tumor cytokinesiology) was also calculated for each treatment group on day 21 (21 days after grouping). TV %.

[0362] Table 11

[0363]

[0364] In vivo efficacy and toxicity of hTNFα / hTNFR2 in mice with MC38 and 3 mg / kg

[0365] A humanized TNFα mouse model was engineered to express human TNFα protein (SEQ ID NO:79), wherein the coding sequence of mouse TNFR2 protein was replaced with the corresponding human coding sequence. A dual-humanized TNFα / TNFR2 mouse model was also generated by crossing TNFα-humanized mice with TNFR2-humanized mice.

[0366] A detailed description of the humanized TNFα mouse model can be found in PCT / CN2020 / 072714, which is incorporated herein by reference in its entirety.

[0367] Similar to previous in vivo efficacy studies, the effects of these anti-hTNFR2 antibodies on tumor growth in a dual-humanized TNFα / TNFR2 mouse model were tested. In each group, dual-humanized TNFα / TNFR2 mice were administered phosphate-buffered saline (PBS, G1), BC-1C3-lgG1 (G2), and anti-mPD-1 (G3) via intraperitoneal (ip) injection. The antibodies were administered twice weekly via intraperitoneal injection at a dose of 3 mg / kg for 3 weeks (6 injections in total).

[0368] Weight and tumor size were monitored throughout the experiment. Mice in all groups showed weight gain, with no significant difference between groups (P>0.05). At group assignment (D0), the mean weight of each group ranged from 20.0g to 20.6g. At the end of the experiment (D28), the mean weight ranged from 23.5g to 25.1g, with a weight change of 115.7%–127.2%. Similar to previous results, the anti-hTNFR2 antibody was well tolerated and non-toxic to mice.

[0369] In the group treated with anti-hTNFR2 antibodies, tumor size ( Figure 6 The results showed significant differences. Table 12 below shows the TGI on day 28 for each treatment group. TV % BC-1C3-lgG1 (G2) showed the best efficacy at this dose and was superior to the positive control (G3).

[0370] Table 12

[0371]

[0372] Example 4. PK and TILS Analysis

[0373] PK analysis of MC38 model, 10 mg / kg B-TNFR2 mice

[0374] The pharmacokinetic clearance rate of anti-TNFR2 antibodies was determined in humanized TNFR2 mice. MC38 cells (5 × 10⁻⁶) were used. 5 Subcutaneous injection into humanized TNFR2 mice, until the tumor grows to 300 mm 3 Mice were divided into 8 groups (n=4). 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 intravenously at 10 mg / kg. Blood samples were collected at 15 min, 6 h, 24 h, day 3, day 5, and day 7 after administration.

[0375] Serum levels of human antibodies were determined using a sandwich ELISA assay. In brief, goat polyclonal anti-human IgG (Fc-specific) capture antibody (Jackson Immunological Research, Catalog No. 109-036-098) was diluted to a final concentration of 2.0 μg / mL with 0.1 M carbonate buffer (pH 9.6) and 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, horseradish peroxidase (HRP)-conjugated goat polyclonal anti-human IgG (Fc-specific) antibody (Jackson Immunological Research, Catalog No. 109-005-088) was added at 100 μL / well to each well of the ELISA plate and incubated at 37°C for 1 hour. After washing, tetramethylbenzidine (TMB) solution was added to each well of a 96-well plate at 100 μL / well as a substrate for HRP reaction. After incubation at room temperature in the dark, 100 μL of stop solution (Beyotime, catalog number: P0215) was added to each well. The absorbance values ​​of each well were read at 450 nm and 630 nm using a microplate reader. The data were analyzed using Gen5 software. Standard curves for the four parameters were created using the absorbance values ​​and corresponding concentrations of calibration samples prepared from each test product. The antibody concentrations of each serum sample were calculated using the standard curves. Drug concentration-time curves were created using the sample concentrations calculated at each time point. Pharmacokinetic parameters were calculated using Phoenix Winnolin 8.3. The results are shown in Table 13 below.

[0376] Table 13

[0377]

[0378]

[0379] T 1 / 2 Terminal half-life;

[0380] C max Maximum concentration;

[0381] AUC 0-7天 Area under the blood concentration-time curve from 0 to 7 days

[0382] CL: Clearance

[0383] The above results indicate that the antibody concentration in the serum of TNFR2 humanized mice decreased over time after injection of different antibodies (see [link to relevant documentation]). Figure 10This is consistent with the pharmacokinetic characteristics. The longest half-life (T1 / 2) of the BC-1C3-IgG1(G2) antibody in mice was 2.99 days. The shortest half-life of the BC-1B6-IgG1-SI(G7) antibody in mice was 0.86 days. The half-lives of other antibodies in mice ranged from 1.69 days to 2.38 days, which were relatively close. By the end of the seventh day of sampling, the area under the curve (AUC) of the drug concentration-time curve for the BC-1C3-IgG1(G2) antibody was 250.93 hr*ug / ml, which was greater than the AUC of other antibodies (AUC 0-7 days 77.90-168.89 hr*ug / ml). The clearance rate (CL) of the BC-1C3-IgG1(G2) antibody was 28.85 ml / hr / kg, while the CL of other antibodies ranged from 47.07 to 61.19 ml / hr / kg. The results showed that BC-1C3-IgG1(G2) had lower clearance efficiency compared to other antibodies, and that the BC-1C3-IgG1(G2) antibody was metabolized more slowly in mice.

[0384] Example 5. Reporter Cell Activation and Binding Assay

[0385] Activation of reporter cells

[0386] This experiment was conducted to test whether anti-TNFR2 antibodies could activate the TNFR2 pathway.

[0387] Human TNFα protein (Sino Biological Inc., catalog number: 10602-HNAE) was serially diluted (3-fold) to a maximum concentration of 10 ng / ml as a positive control. Anti-TNFR2 antibodies BC-1F4-IgG1, BC-1C3-IgG1, BC-1B6-IgG1, and BC-1F10-IgG1 were serially diluted (3-fold) to a maximum concentration of 60 μg / ml. Jurkat-GFP-TNFR2 cells were seeded in 96-well plates (cell density: 1 × 10⁻⁶). 5 Cells were added to each well (100 μL per well), and then 100 μL of human TNFα protein or 100 μL of anti-TNFR2 antibody was added to each well. The plates were incubated overnight at 37°C. After incubation, the plates were removed and transferred to 96-well plates. 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 plates were then placed in a luminescent detector to detect the fluorescence signal. If the antibody could activate TNFR2, the reporter cells would report a GFP signal.

[0388] like Figure 11As shown, 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. None of the anti-TNFR2 antibodies BC-1F4-IgG1, BC-1C3-IgG1, BC-1B6-IgG1, and BC-1F10-IgG1 showed reporter cell activation. None of the anti-TNFR2 antibodies activated the TNFR2 pathway in Jurkat-GFP-TNFR2 cells.

[0389] Blocking effect on reporter cells

[0390] Experiments were conducted to test whether anti-TNFR2 antibodies could block the binding between TNFR2 and its ligand TNFα.

[0391] Jurkat-GFP-TNFR2 cells were seeded in 96-well plates (cell density 1×10⁶). 5 Cells / well. Dilute TNFα protein to 1 ng / mL. Serially dilute (3-fold) anti-TNFR2 antibodies BC-1F4-IgG1, BC-1C3-IgG1, BC-1B6-IgG1, and BC-1F10-IgG1 to a maximum concentration of 10 μg / mL. Add 50 μl of human TNFα protein and 50 μl of antibody to each well and incubate at 37°C for 24 h. After incubation, remove the plate and transfer it to a 96-well plate. Wash each well with 150 μl of PBS. Discard the supernatant. Add 100 μL of PBS to each well to resuspend the cells. Determine GFP signaling by flow cytometry.

[0392] like Figure 12 As shown, when the concentrations of anti-TNFR2 antibodies BC-1F10-IgG1 and BC-1F4-IgG1 increased, the GFP signal (indicating cell binding to TNFα) decreased (y-axis), indicating that the binding between human TNFα and TNFR2 was blocked by anti-TNFR2 antibodies BC-1F10-IgG1 and BC-1F4-IgG1.

[0393] Example 6. In vivo toxicity experiment (non-tumor-bearing model)

[0394] Humanized TNFR2 mice (6-8 weeks old) were randomly assigned to a control group and a treatment group (n=4 mice per group) based on their body weight. The control group received an equal volume of PBS, while the treatment group received either anti-hTNFR2 antibody (BC-1C3-IgG1, BC-1F4-IgG1, or BC-1B6-IgG1) or CTLA4 antibody (anti-mCTLA4). The doses of anti-hTNFR2 and CTLA4 antibodies were 30 mg / kg or 100 mg / kg, respectively. Administration was once weekly for a total of four weeks. Specific dosages, methods, and frequencies are shown in Table 14 below. Body weight changes and any abnormalities were monitored throughout the experiment. Blood biochemical parameters were monitored on days 1, 8, 15, 22, and 28 post-grouping. Blood biochemical indicators included: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatine kinase (CK), albumin (ALB), total protein (TP), amylase (AMY), urea (UREA), creatinine (CREA), glucose (GLU), triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), calcium (Ca), and inorganic phosphorus (P). Complete blood count (CBC) tests were performed on day 28 after grouping. The complete blood count (CBC) included the following tests: white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin content (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), lymphocytes (LYMPH#), lymphocyte percentage (LYMPH%), monocytes (MONO#), monocyte percentage (MONO%), and neutrophil percentage (NEUT%). At the end of the experiment, the mouse's heart, liver, spleen, lungs, kidneys, and intestines were stained with hematoxylin and eosin (HE) in formalin, and the weights of the liver, spleen, and kidneys were measured.

[0395] Table 14

[0396] G1 PBS Intraperitoneal injection, once a week for a total of 4 times. G2 BC-1C3-IgG1 30 mg / kg, intraperitoneal injection, once a week for a total of 4 times. G3 BC-1C3-IgG1 100 mg / kg, intraperitoneal injection, once a week for a total of 4 times. G4 BC-1F4-IgG1 30 mg / kg, intraperitoneal injection, once a week for a total of 4 times. G5 BC-1F4-IgG1 100 mg / kg, intraperitoneal injection, once a week for a total of 4 times. G6 BC-1B6-IgG1 30 mg / kg, intraperitoneal injection, once a week for a total of 4 times. G7 BC-1B6-IgG1 100 mg / kg, intraperitoneal injection, once a week for a total of 4 times. G8 Anti-mCTLA4 30 mg / kg, intraperitoneal injection, once a week for a total of 4 times. G9 Anti-mCTLA4 100 mg / kg, intraperitoneal injection, once a week for a total of 4 times.

[0397] The results showed that the body weight of all mice in both the control and treatment groups increased throughout the experiment, and there were no significant differences in body weight changes among the groups. Blood biochemical parameters test results (see...) Figures 13A-13B Exemplary blood biochemical parameters and routine blood tests on day 28 showed no significant differences compared to the control. Similar to previous results, in vivo toxicity results showed that the anti-hTNFR2 antibody was well tolerated and non-toxic in mice.

[0398] Other embodiments

[0399] It should be understood that although the invention has been described in conjunction with specific embodiments, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. SEQUENCE LISTING <110> Baochuan Biomedical Technology (Shanghai) Co., Ltd. <120> Anti-TNFR2 antibodies and their uses <130> P22011607CF <150> PCT / CN2021 / 140487 <151> 2021-12-22 <160> 79 <170> PatentIn version 3.5 <210> 1 <211> 461 <212> PRT <213> Artificial Sequence <220> <223> Human TNFR2 protein <400> 1 Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu 1 5 10 15 Trp Ala Ala Ala His Ala Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr 20 25 30 Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln 35 40 45 Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys 50 55 60 Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp 65 70 75 80 Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys 85 90 95 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg 100 105 110 Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu 115 120 125 Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg 130 135 140 Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val 145 150 155 160 Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr 165 170 175 Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly 180 185 190 Asn Ala Ser Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser 195 200 205 Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser 210 215 220 Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser 225 230 235 240 Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly 245 250 255 Asp Phe Ala Leu Pro Val Gly Leu Ile Val Gly Val Thr Ala Leu Gly 260 265 270 Leu Leu Ile Ile Gly Val Val Asn Cys Val Ile Met Thr Gln Val Lys 275 280 285 Lys Lys Pro Leu Cys Leu Gln Arg Glu Ala Lys Val Pro His Leu Pro 290 295 300 Ala Asp Lys Ala Arg Gly Thr Gln Gly Pro Glu Gln Gln His Leu Leu 305 310 315 320 Ile Thr Ala Pro Ser Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala Ser 325 330 335 Ala Leu Asp Arg Arg Ala Pro Thr Arg Asn Gln Pro Gln Ala Pro Gly 340 345 350 Val Glu Ala Ser Gly Ala Gly Glu Ala Arg Ala Ser Thr Gly Ser Ser 355 360 365 Asp Ser Ser Pro Gly Gly His Gly Thr Gln Val Asn Val Thr Cys Ile 370 375 380 Val Asn Val Cys Ser Ser Ser Asp His Ser Ser Gln Cys Ser Ser Gln 385 390 395 400 Ala Ser Ser Thr Met Gly Asp Thr Asp Ser Ser Pro Ser Glu Ser Pro 405 410 415 Lys Asp Glu Gln Val Pro Phe Ser Lys Glu Glu Cys Ala Phe Arg Ser 420 425 430 Gln Leu Glu Thr Pro Glu Thr Leu Leu Gly Ser Thr Glu Glu Lys Pro 435 440 445 Leu Pro Leu Gly Val Pro Asp Ala Gly Met Lys Pro Ser 450 455 460 <210> 2 <211> 474 <212> PRT <213> Artificial Sequence <220> <223> Mouse TNFR2 Protein <400> 2 Met Ala Pro Ala Ala Leu Trp Val Ala Leu Val Phe Glu Leu Gln Leu 1 5 10 15 Trp Ala Thr Gly His Thr Val Pro Ala Gln Val Val Leu Thr Pro Tyr 20 25 30 Lys Pro Glu Pro Gly Tyr Glu Cys Gln Ile Ser Gln Glu Tyr Tyr Asp 35 40 45 Arg Lys Ala Gln Met Cys Cys Ala Lys Cys Pro Pro Gly Gln Tyr Val 50 55 60 Lys His Phe Cys Asn Lys Thr Ser Asp Thr Val Cys Ala Asp Cys Glu 65 70 75 80 Ala Ser Met Tyr Thr Gln Val Trp Asn Gln Phe Arg Thr Cys Leu Ser 85 90 95 Cys Ser Ser Ser Cys Thr Thr Asp Gln Val Glu Ile Arg Ala Cys Thr 100 105 110 Lys Gln Gln Asn Arg Val Cys Ala Cys Glu Ala Gly Arg Tyr Cys Ala 115 120 125 Leu Lys Thr His Ser Gly Ser Cys Arg Gln Cys Met Arg Leu Ser Lys 130 135 140 Cys Gly Pro Gly Phe Gly Val Ala Ser Ser Arg Ala Pro Asn Gly Asn 145 150 155 160 Val Leu Cys Lys Ala Cys Ala Pro Gly Thr Phe Ser Asp Thr Thr Ser 165 170 175 Ser Thr Asp Val Cys Arg Pro His Arg Ile Cys Ser Ile Leu Ala Ile 180 185 190 Pro Gly Asn Ala Ser Thr Asp Ala Val Cys Ala Pro Glu Ser Pro Thr 195 200 205 Leu Ser Ala Ile Pro Arg Thr Leu Tyr Val Ser Gln Pro Glu Pro Thr 210 215 220 Arg Ser Gln Pro Leu Asp Gln Glu Pro Gly Pro Ser Gln Thr Pro Ser 225 230 235 240 Ile Leu Thr Ser Leu Gly Ser Thr Pro Ile Ile Glu Gln Ser Thr Lys 245 250 255 Gly Gly Ile Ser Leu Pro Ile Gly Leu Ile Val Gly Val Thr Ser Leu 260 265 270 Gly Leu Leu Met Leu Gly Leu Val Asn Cys Ile Ile Leu Val Gln Arg 275 280 285 Lys Lys Lys Pro Ser Cys Leu Gln Arg Asp Ala Lys Val Pro His Val 290 295 300 Pro Asp Glu Lys Ser Gln Asp Ala Val Gly Leu Glu Gln Gln His Leu 305 310 315 320 Leu Thr Thr Ala Pro Ser Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala 325 330 335 Ser Ala Gly Asp Arg Arg Ala Pro Pro Gly Gly His Pro Gln Ala Arg 340 345 350 Val Met Ala Glu Ala Gln Gly Phe Gln Glu Ala Arg Ala Ser Ser Arg 355 360 365 Ile Ser Asp Ser Ser His Gly Ser His Gly Thr His Val Asn Val Thr 370 375 380 Cys Ile Val Asn Val Cys Ser Ser Ser Asp His Ser Ser Gln Cys Ser 385 390 395 400 Ser Gln Ala Ser Ala Thr Val Gly Asp Pro Asp Ala Lys Pro Ser Ala 405 410 415 Ser Pro Lys Asp Glu Gln Val Pro Phe Ser Gln Glu Glu Cys Pro Ser 420 425 430 Gln Ser Pro Cys Glu Thr Thr Glu Thr Leu Gln Ser His Glu Lys Pro 435 440 445 Leu Pro Leu Gly Val Pro Asp Met Gly Met Lys Pro Ser Gln Ala Gly 450 455 460 Trp Phe Asp Gln Ile Ala Val Lys Val Ala 465 470 <210> 3 <211> 463 <212> PRT <213> Artificial Sequence <220> <223> Monkey TNFR2 protein <400> 3 Gly His His Ser Ala Ser Trp Gly Ala Met Gly Val Gln Leu Val Glu 1 5 10 15 Thr Trp Ala Ser Gly Met Ala Glu Pro Arg Ala Val Ala Phe Thr Pro 20 25 30 Tyr Ala Pro Glu Pro Gly Gly Thr Cys Arg Leu Arg Glu Tyr Tyr Asp 35 40 45 Gln Thr Ala Gln Met Cys Cys Ser Lys Cys Pro Pro Gly Gln His Ala 50 55 60 Lys Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu 65 70 75 80 Asp Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser 85 90 95 Cys Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr 100 105 110 Arg Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala 115 120 125 Leu Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Gln Leu Arg Lys Cys 130 135 140 Arg Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val 145 150 155 160 Val Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser 165 170 175 Thr Asp Ile Cys Arg Pro His Gln Ile Cys His Val Val Ala Ile Pro 180 185 190 Gly Asn Ala Ser Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg 195 200 205 Ser Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg 210 215 220 Ser Gln His Thr Gln Pro Thr Pro Ala Pro Ser Thr Ala Pro Gly Thr 225 230 235 240 Ser Phe Leu Leu Pro Val Gly Pro Ser Pro Pro Ala Glu Gly Ser Thr 245 250 255 Gly Asp Ile Val Leu Pro Val Gly Leu Ile Val Gly Val Thr Ala Leu 260 265 270 Gly Leu Leu Ile Ile Gly Val Val Asn Cys Val Ile Met Thr Gln Val 275 280 285 Lys Lys Lys Pro Leu Cys Leu Gln Arg Glu Thr Lys Val Pro His Leu 290 295 300 Pro Ala Asp Lys Ala Arg Gly Ala Gln Gly Pro Glu Gln Gln His Leu 305 310 315 320 Leu Thr Thr Val Pro Ser Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala 325 330 335 Ser Ala Leu Asp Arg Arg Ala Pro Thr Arg Asn Gln Pro Gln Ala Pro 340 345 350 Gly Ala Glu Lys Ala Ser Gly Ala Gly Glu Ala Arg Ala Ser Thr Gly 355 360 365 Ser Ser Asp Ser Ser Pro Gly Gly His Gly Thr Gln Val Asn Val Thr 370 375 380 Cys Ile Val Asn Val Cys Ser Ser Ser Asp His Ser Ser Gln Cys Ser 385 390 395 400 Ser Gln Ala Ser Ser Thr Met Gly Asp Thr Asp Ala Ser Pro Ser Gly 405 410 415 Ser Pro Lys Asp Glu Gln Val Pro Phe Ser Lys Glu Glu Ser Ala Phe 420 425 430 Arg Ser Gln Leu Glu Thr Pro Glu Thr Leu Leu Gly Ser Thr Glu Glu 435 440 445 Lys Pro Leu Pro Leu Gly Val Pro Asp Ala Gly Met Lys Pro Ser 450 455 460 <210> 4 <211> 482 <212> PRT <213> Artificial Sequence <220> <223> Dog TNFR2 protein <400> 4 Met Thr Tyr Val Gly Ala Gly Val Asn Glu Gly Thr His Glu Val Pro 1 5 10 15 Ser Ala Val Cys Ile Gln Gln Val Phe Ile Leu Gly Pro Arg Pro Cys 20 25 30 Pro His Leu Ser Asp Ser Val Asn Arg Ala Thr Gln Leu Pro Tyr Val 35 40 45 Pro Asp Pro Glu Leu Gly Ser Ser Cys Gln Gln Ser Glu Tyr Phe Asp 50 55 60 Gln Arg Thr Gln Met Cys Cys Ser Met Cys Pro Pro Gly Ser His Ala 65 70 75 80 Arg Leu Phe Cys Thr Lys Thr Ser Asn Thr Val Cys Ala Arg Cys Glu 85 90 95 Asn Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser 100 105 110 Cys Gly Ser Arg Cys Gly Ala Asp Gln Val Glu Thr Gln Ala Cys Thr 115 120 125 Arg Glu Gln Asn Arg Ile Cys Ser Cys Lys Ser Gly Trp Tyr Cys Thr 130 135 140 Leu Arg Arg Gln Gly Gly Cys Arg Leu Cys Ala Pro Leu Arg Arg Cys 145 150 155 160 Arg Pro Gly Phe Gly Val Ala Lys Pro Gly Thr Ala Thr Ser Asp Val 165 170 175 Val Cys Ala Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser 180 185 190 Thr Asp Thr Cys Arg Pro His Arg Ile Cys Ser Ser Val Ala Val Pro 195 200 205 Gly Asn Ala Ser Val Asp Ala Val Cys Ser Pro Ala Pro Pro Thr Val 210 215 220 Arg Thr Ala Pro Arg Pro Ala Ser Thr Arg Gln Pro Gly Ser Thr Gln 225 230 235 240 Pro Arg Pro Ala Glu Pro Thr Pro Gly Pro Ser Thr Pro Pro Arg Thr 245 250 255 Ser Val Leu Phe Pro Ala Val Pro Ser Pro Pro Ala Glu Gly Leu Ser 260 265 270 Thr Gly Asp Ile Ser Leu Pro Ile Gly Leu Ile Val Gly Val Thr Thr 275 280 285 Leu Gly Leu Leu Leu Ile Gly Leu Val Asn Cys Val Ile Val Thr Gln 290 295 300 Lys Lys Lys Lys Pro Phe Cys Leu Gln Gly Glu Ala Lys Val Pro His 305 310 315 320 Leu Pro Ala Asp Lys Ala His Gly Gly Pro Gly Pro Glu Gln Gln His 325 330 335 Leu Leu Thr Thr Ala Pro Ser Ser Ser Ser Ser Ser Leu Glu Ser Ala 340 345 350 Ala Ser Ser Ala Asp Gly Arg Ala Pro Pro Arg Ala Gln Pro Pro Ala 355 360 365 Pro Gly Thr Gly Lys Ala His Gly Ser Gly Glu Ala Gln Ala Ser Ser 370 375 380 Ser Ser Ser Glu Pro Ser Cys Gly Gly His Gly Thr Gln Val Asn Val 385 390 395 400 Thr Cys Ile Val Asn Val Cys Ser Ser Ser Gly Ser Asp His Gly Pro 405 410 415 Gln Cys Ser Ser Gln Ala Ser His Thr Thr Gly Asp Val Asp Ala Gly 420 425 430 Pro Ser Ser Ser Pro Asp Asp Gln Gln Val Pro Phe Ser Gln Glu Glu 435 440 445 Cys Pro Phe Gln Phe Gln Pro Gly Ala Leu Glu Thr Leu Leu Glu Asn 450 455 460 Pro Glu Asp Lys Pro Leu Pro Leu Gly Val Pro Asp Ala Gly Met Lys 465 470 475 480 Ser Ser <210> 5 <211> 473 <212> PRT <213> Artificial Sequence <220> <223> Chimeric TNFR2 protein (33 - 259aa is human sequence) <400> 5 Met Ala Pro Ala Ala Leu Trp Val Ala Leu Val Phe Glu Leu Gln Leu 1 5 10 15 Trp Ala Thr Gly His Thr Val Pro Ala Gln Val Val Leu Thr Pro Tyr 20 25 30 Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln 35 40 45 Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys 50 55 60 Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp 65 70 75 80 Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys 85 90 95 Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg 100 105 110 Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu 115 120 125 Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg 130 135 140 Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val 145 150 155 160 Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr 165 170 175 Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly 180 185 190 Asn Ala Ser Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser 195 200 205 Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser 210 215 220 Gln His Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser 225 230 235 240 Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly 245 250 255 Asp Phe Ala Leu Pro Ile Gly Leu Ile Val Gly Val Thr Ser Leu Gly 260 265 270 Leu Leu Met Leu Gly Leu Val Asn Cys Ile Ile Leu Val Gln Arg Lys 275 280 285 Lys Lys Pro Ser Cys Leu Gln Arg Asp Ala Lys Val Pro His Val Pro 290 295 300 Asp Glu Lys Ser Gln Asp Ala Val Gly Leu Glu Gln Gln His Leu Leu 305 310 315 320 Thr Thr Ala Pro Ser Ser Ser Ser Ser Ser Leu Glu Ser Ser Ala Ser 325 330 335 Ala Gly Asp Arg Arg Ala Pro Pro Gly Gly His Pro Gln Ala Arg Val 340 345 350 Met Ala Glu Ala Gln Gly Phe Gln Glu Ala Arg Ala Ser Ser Arg Ile 355 360 365 Ser Asp Ser Ser His Gly Ser His Gly Thr His Val Asn Val Thr Cys 370 375 380 Ile Val Asn Val Cys Ser Ser Ser Asp His Ser Ser Gln Cys Ser Ser 385 390 395 400 Gln Ala Ser Ala Thr Val Gly Asp Pro Asp Ala Lys Pro Ser Ala Ser 405 410 415 Pro Lys Asp Glu Gln Val Pro Phe Ser Gln Glu Glu Cys Pro Ser Gln 420 425 430 Ser Pro Cys Glu Thr Thr Glu Thr Leu Gln Ser His Glu Lys Pro Leu 435 440 445 Pro Leu Gly Val Pro Asp Met Gly Met Lys Pro Ser Gln Ala Gly Trp 450 455 460 Phe Asp Gln Ile Ala Val Lys Val Ala 465 470 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 of BC-1A8 (Kabat) <400> 6 Ser Asn Ser Ala Ala Trp Asn 1 5 <210> 7 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 of BC-1A8 (Kabat) <400> 7 Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Leu Ser Val 1 5 10 15 Lys Ser <210> 8 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 of BC-1A8 (Kabat) <400> 8 Glu Asp Gly Val Gly Gly Thr Lys Asp Tyr Phe Asp Tyr 1 5 10 <210> 9 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 of BC-1A8 (Kabat) <400> 9 Gln Ala Ser Gln Asp Ile Asp Asn Tyr Leu Asn 1 5 10 <210> 10 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL CDR2 of BC-1A8 (Kabat) <400> 10 Asp Val Ser Asn Leu Glu Ile 1 5 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VL CDR3 of BC-1A8 (Kabat) <400> 11 Gln Gln Tyr Asp Asn Leu Pro Leu Thr 1 5 <210> 12 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 of BC-1B6 (Kabat) <400> 12 Asn Phe Gly Met His 1 5 <210> 13 <211> 17 <212> PRT <213> Artificial Sequence<​​<223> VH CDR2 of BC-1B6 (Kabat) <400> 13 Val Ile Trp Tyr Glu Gly Ser Asp Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 14 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 of BC-1B6 (Kabat) <400> 14 Asp Ser Ser Trp Gly Phe Leu Leu Tyr Gly Met Asp Val 1 5 10 <210> 15 <211> 161] <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 of BC-1B6 (Kabat) <400> 15 Arg Ser Ser Arg Ser Leu Glu Tyr Ser Asp Gly Asn Thr Tyr Leu Asn 1 5 10 15 <210> 16 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL CDR2 of BC-1B6 (Kabat) <400> 16 Lys Val Ser Asn Arg Asp Ser 1 5 <210> 17 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VL CDR3 of BC-1B6 (Kabat) <400> 17 Met Gln Gly Thr His Trp Pro Pro Thr 1 5 <210> 18 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 of BC-1C3 (Kabat) <400> 18 Ile Tyr Gly Met His 1 5 <210> 19 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 of BC-1C3 (Kabat) <400> 19 Leu Ile Ser Tyr Asp Gly Asn Asp Lys Tyr Phe Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 20 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 of BC-1C3 (Kabat) <400> 20 Ala Val Asp Thr Ala Val Val Thr Phe Phe Asp Tyr 1 5 10 <210> 21 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 of BC-1C3 (Kabat) <400> 21 Arg Ala Ser Gln Ser Val Ser Ser Asn Leu Ala 1 5 10 <210> 22 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL CDR2 of BC-1C3 (Kabat) <400> 22 Ser Ala Ser Thr Arg Ala Thr 1 5 <210> 23 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VL CDR3 of BC-1C3 (Kabat) <400> 23 Gln Gln Tyr Ser Asn Trp Pro Phe Thr 1 5 <210> 24 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 of BC-1F4 (Kabat) <400> 24 Ser Asp Ser Ser Ala Trp Asn 1 5 <210> 25 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 of BC-1F4 (Kabat) <400> 25 Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Thr Val Ser Val 1 5 10 15 Lys Ser <210> 26 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 of BC-1F4 (Kabat) <400> 26 Glu Asp Gly Val Gly Gly Thr Lys Asp Tyr Phe Asp Tyr 1 5 10 <210> 27 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 of BC-1F4 (Kabat) <400> 27 Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn 1 5 10 <210> 28 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL CDR2 of BC-1F4 (Kabat) <400> 28 Asp Ala Ser Asn Leu Glu Thr 1 5 <210> 29 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VL CDR3 of BC-1F4 (Kabat) <400> 29 Gln Gln Tyr Asp Asn Leu Pro Ile Thr 1 5 <210> 30 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 of BC-1F10 (Kabat) <400> 30 Gly Asp Tyr Trp Ser 1 5 <210> 31 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 of BC-1F10 (Kabat) <400> 31 Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 32 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 of BC-1F10 (Kabat) <400> 32 Gly His Trp Asn Tyr Asp Tyr Tyr Tyr Gly Met Asp Val 1 5 10 <210> 33 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> BC-1F10's VL CDR1 (Kabat) <400> 33 Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp 1 5 10 15 <210> 34 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> BC-1F10's VL CDR2 (Kabat) <400> 34 Leu Asn Ser Asn Arg Ala Ser 1 5 <210> 35 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> BC-1F10's VL CDR3 (Kabat) <400> 35 Met Gln Ala Arg Gln Thr Pro Leu Thr 1 5 <210> 36 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> 1A8 people heavy chain variable region <400> 36 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30 Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala 50 55 60 Leu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Glu Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Arg Glu Asp Gly Val Gly Gly Thr Lys Asp Tyr Phe 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 37 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> 1A8 Human Light Chain Variable Region <400> 37 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Asp Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Val Ser Asn Leu Glu Ile Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 38 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> 1B6 heavy chain variable region <400> 38 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Glu Gly Ser Asp Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Ser Trp Gly Phe Leu Leu Tyr Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 39 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> 1B6 human light chain variable region <400> 39 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15[[ID=४४]] Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Arg Ser Leu Glu Tyr Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Lys Ile 65 70 75 80 Ser Gly Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95 Thr His Trp Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 40 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 1C3 human heavy chain variable region <400> 40 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val His Pro Gly Lys 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Ile Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Ser Tyr Asp Gly Asn Asp Lys Tyr Phe Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ala Val Asp Thr Ala Val Val Thr Phe Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 41 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> 1C3 human light chain variable region <400> 41 Glu Ile Val Met Thr Gln Ser Pro Val Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Asn Trp Pro Phe 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105 <210> 42 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 of BC-1A8 (Chothia) <400> 42 Gly Asp Ser Val Ser Ser Asn Ser Ala Ala Trp Asn 1 5 10 <210> 43 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 of BC-1A8 (Chothia) <400> 43 Tyr Tyr Arg Ser Lys Trp Tyr 1 5 <210> 44 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 of BC-1A8 (Chothia) <400> 44 Glu Asp Gly Val Gly Gly Thr Lys Asp Tyr Phe Asp Tyr 1 5 10 <210> 45 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 of BC-1A8 (Chothia) <400> 45 Gln Ala Ser Gln Asp Ile Asp Asn Tyr Leu Asn 1 5 10 <210> 46 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL CDR2 of BC-1A8 (Chothia) <400> 46 Asp Val Ser Asn Leu Glu Ile 1 5 <210> 47 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VL CDR3 of BC-1A8 (Chothia) <400> 47 Gln Gln Tyr Asp Asn Leu Pro Leu Thr 1 5 <210> 48 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 of BC-1B6 (Chothia) <400> 48 Gly Phe Thr Phe Ser Asn Phe Gly Met His 1 5 10 <210> 49 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 of BC-1B6 (Chothia) <400> 49 Trp Tyr Glu Gly Ser Asp 1 5 <210> 50 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 of BC-1B6 (Chothia) <400> 50 Asp Ser Ser Trp Gly Phe Leu Leu Tyr Gly Met Asp Val 1 5 10 <210> 51 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 of BC-1B6 (Chothia) <400> 51 Arg Ser Ser Arg Ser Leu Glu Tyr Ser Asp Gly Asn Thr Tyr Leu Asn 1 5 10 15 <210> 52 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL CDR2 of BC-1B6 (Chothia) <400> 52 Lys Val Ser Asn Arg Asp Ser 1 5 <210> 53 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VL CDR3 of BC-1B6 (Chothia) <400> 53 Met Gln Gly Thr His Trp Pro Pro Thr 1 5 <210> 54 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 of BC-1C3 (Chothia) <400> 54 Gly Phe Thr Phe Asn Ile Tyr Gly Met His 1 5 10 <210> 55 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 of BC-1C3 (Chothia) <400> 55 Ser Tyr Asp Gly Asn Asp 1 5 <210> 56 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 of BC-1C3 (Chothia) <400> 56 Ala Val Asp Thr Ala Val Val Thr Phe Phe Asp Tyr 1 5 10 <210> 57 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 of BC-1C3 (Chothia) <400> 57 Arg Ala Ser Gln Ser Val Ser Ser Asn Leu Ala 1 5 10 <210> 58 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL CDR2 of BC-1C3 (Chothia) <400> 58 Ser Ala Ser Thr Arg Ala Thr 1 5 <210> 59 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VL CDR3 of BC-1C3 (Chothia) <400> 59 Gln Gln Tyr Ser Asn Trp Pro Phe Thr 1 5 <210> 60 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 of BC-1F4 (Chothia) <400> 60 Gly Asp Ser Val Ser Ser Asp Ser Ser Ala Trp Asn 1 5 10 <210> 61 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 of BC-1F4 (Chothia) <400> 61 Tyr Tyr Arg Ser Lys Trp Tyr 1 5 <210> 62 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 of BC-1F4 (Chothia) <400> 62 Glu Asp Gly Val Gly Gly Thr Lys Asp Tyr Phe Asp Tyr 1 5 10 <210> 63 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 of BC-1F4 (Chothia) <400> 63 Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn 1 5 10 <210> 64 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL CDR2 of BC-1F4 (Chothia) <400> 64 Asp Ala Ser Asn Leu Glu Thr 1 5 <210> 65 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VL CDR3 of BC-1F4 (Chothia) <400> 65 Gln Gln Tyr Asp Asn Leu Pro Ile Thr 1 5 <210> 66 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> VH CDR1 of BC-1F10 (Chothia) <400> 66 Asp Gly Ser Phe Ser Gly Asp Tyr Trp Ser 1 5 10 <210> 67 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> VH CDR2 of BC-1F10 (Chothia) <400> 67 Asn His Ser Gly Ser 1 5 <210> 68 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> VH CDR3 of BC-1F10 (Chothia) <400> 68 Gly His Trp Asn Tyr Asp Tyr Tyr Tyr Gly Met Asp Val 1 5 10 <210> 69 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> VL CDR1 of BC-IB10 (Chothia) <400> 69 Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp 1 5 10 15 <210> 70 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> VL CDR2 of BC-1F10 (Chothia) <400> 70 Leu Asn Ser Asn Arg Ala Ser 1 5 <210> 71 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> VL CDR3 of BC-1F10 (Chothia) <400> 71 Met Gln Ala Arg Gln Thr Pro Leu Thr 1 5 <210> 72 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> 1F4 Human Heavy Chain Variable Region <400> 72 Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asp 20 25 30 Ser Ser Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu 35 40 45 Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Thr 50 55 60 Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80 Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Cys Ala Arg Glu Asp Gly Val Gly Gly Thr Lys Asp Tyr Phe 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 73 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> 1F4-person light chain variable zone <400> 73 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Asp Asn Leu Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 74 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 1F10-person heavy chain variable zone <400> 74 Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Ile Cys Ala Val Tyr Asp Gly Ser Phe Ser Gly Asp 20 25 30 Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly His Trp Asn Tyr Asp Tyr Tyr Tyr Gly Met Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 75 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> 1F10 human light chain variable region <400> 75 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Asn Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Leu Tyr Tyr Cys Met Gln Ala 85 90 95 Arg Gln Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 76 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> IgG1 heavy chain constant region <400> 76 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 77 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> IgG1 heavy chain constant region with SI mutation <400> 77 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 78 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> IgG1 heavy chain constant region with LALA mutation <400> 78 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 79 <211> 233 <212> PRT <213> Artificial Sequence <220> <223> Human TNFα protein <400> 79 Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala 1 5 10 15 Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys Leu Phe 20 25 30 Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr Thr Leu Phe 35 40 45 Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg Glu Glu Phe Pro 50 55 60 Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln Ala Val Arg Ser Ser 65 70 75 80 Ser Arg Thr Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro 85 90 95 Gln Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu 100 105 110 Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser 115 120 125 Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly 130 135 140 Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala 145 150 155 160 Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro 165 170 175 Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro Trp Tyr Glu 180 185 190 Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu 195 200 205 Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly 210 215 220 Gln Val Tyr Phe Gly Ile Ile Ala Leu 225 230

Claims

1. An antibody or antigen-binding fragment thereof that binds to TNFR2, the antibody or antigen-binding fragment comprising: The heavy chain variable region (VH) contains complementarity-determining regions (CDRs) 1, 2, and 3, and the light chain variable region (VL) contains CDRs 1, 2, and 3; according to the Kabat number, the VH contains the amino acid sequences CDRs 1, 2, and 3 as shown in SEQ ID NO: 12, 13, and 14, respectively, and the VL contains the amino acid sequences CDRs 1, 2, and 3 as shown in SEQ ID NO: 15, 16, and 17, respectively.

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

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

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

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

6. A nucleic acid comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof that binds TNFR2, said antibody or antigen-binding fragment thereof comprising: (1) An immunoglobulin heavy chain containing a heavy chain variable region (VH), the heavy chain variable region comprising CDRs 1, 2, and 3 of the amino acid sequences shown in SEQ ID NO: 12, 13, and 14, respectively, and wherein the VH binds TNFR2 upon pairing with a light chain variable region (VL) of the amino acid sequence shown in SEQ ID NO: 39; and, (2) An immunoglobulin light chain containing a VL comprising CDRs 1, 2 and 3 of amino acid sequences as shown in SEQ ID NO: 15, 16 and 17, respectively, wherein the VL binds TNFR2 when paired with a VH of an amino acid sequence as shown in SEQ ID NO:

38.

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

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

9. The nucleic acid according to claim 6, wherein the nucleic acid encodes a single-stranded variable fragment (scFv).

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

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

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

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

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

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

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

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

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

19. A method for generating an antibody or an antigen-binding fragment thereof, the method comprising: (a) The cells are cultured under conditions sufficient to induce the cells according to any one of claims 14-18 to produce the antibody or antigen-binding fragment; and (b) Collect the antibody or antigen-binding fragment produced by the cell.

20. An antibody or antigen-binding fragment thereof that binds to TNFR2, the antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH is the sequence of SEQ ID NO: 38 and the VL is the sequence of SEQ ID NO:

39.

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

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

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

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

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

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

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