Anti-TNFR2 antibodies and uses thereof

Abstract

An antibody or an antigen-binding fragment thereof that can specifically bind to TNFR2. The antibody or the antigen-binding fragment thereof can regulate the function of immune cells and can be used as a drug to treat diseases related to immune abnormalities, such as tumors.

Description

[0001] This application claims the priority of the Chinese patent application filed with the China Patent Office on August 2, 2019, with application number 201910713742.1, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to an anti-TNFR2 antibody or an antigen-binding fragment thereof, a pharmaceutical composition comprising the TNFR2 antibody or the antigen-binding fragment thereof, and uses thereof. Background Art

[0003] Immunity is a protective response of the body, which is affected by many genes, proteins and cells. Immune abnormalities can cause many diseases, including tumors, immunodeficiency (such as AIDS), allergies and rheumatoid arthritis. In the past few years, tumor immunotherapy, as a new treatment method, has become a hot topic in the field of tumor treatment research. Antagonistic antibodies targeting immune checkpoint proteins, such as anti-PD-1 and anti-CTLA-4 monoclonal antibodies, have been used to treat many types of cancer and have achieved revolutionary results, greatly prolonging the survival of patients with malignant tumors. However, there are still many cancer patients who do not respond to the treatment of antagonistic antibodies to immune checkpoint proteins or develop resistance or drug resistance after a short period of treatment. Therefore, it is necessary to develop new drugs for the treatment of cancer, which can be used alone or in combination with other tumor treatment methods, including antagonistic antibodies to immune checkpoint proteins, to further improve efficacy and safety. Summary of the invention

[0004] The inventors found that human TNFR2 is overexpressed on the surface of human Treg and various human tumor cells, suggesting that human TNFR2 may promote tumorigenesis in patients and mediate immunosuppression and immune escape in the tumor microenvironment.

[0005] Regulating the function of Treg cells through TNFR2, thereby inhibiting the occurrence of tumors, may be a very promising anti-tumor strategy. The inventors prepared an antagonistic antibody against TNFR2, which can 1) specifically bind to TNFR2, block the binding of TNFR2 and its ligand TNFα, thereby inhibiting the proliferation of Treg cells and their mediated inhibitory function, promote the expansion of T cells and the anti-tumor function mediated by T cells and other immune cells. In addition, 2) since TNFR2 is highly expressed in human tumor cell lines, this antibody can also directly mediate the killing effect on tumor cells with high TNFR2 expression.

[0006] The inventors also found that related experiments in mice further verified the potential function of the TNFR2 target. The results of the mouse colorectal cancer CT26 model showed that TNFR2 antagonist antibodies, compared with PD-1 antibodies or PD-L1 monoclonal antibodies, can significantly inhibit the growth of PD-1 antibody-resistant tumors, reduce tumor size, and effectively increase CD8 + T / Treg ratio and partial reversal of CD8 + The immune exhaustion state of T cells. Therefore, TNFR2 may become a new target for tumor immunotherapy, and TNFR2 antagonist antibodies are expected to help change the tumor microenvironment, and can be used alone and / or in combination with existing immune checkpoint antagonist antibodies, with broad application prospects. For this purpose, the inventors prepared a variety of TNFR2 antibodies, and completed the present invention on this basis.

[0007] In the first aspect, the present invention discloses an antibody or an antigen-binding fragment thereof that specifically binds to TNFR2, which can regulate the function of immune cells, including regulating the function of Treg cells and / or MDSC, and can be used as a drug to treat diseases related to immune abnormalities, such as tumors.

[0008] In one embodiment, the regulation includes inhibiting the proliferation and / or activation of Treg cells and / or myeloid derived suppressor cells (MDSC). In another embodiment, the regulation is achieved by blocking the binding of TNFα to TNFR2.

[0009] In a specific embodiment, the antibody or antigen-binding fragment thereof comprises:

[0010] (1) a heavy chain CDR combination of CDR1-VH, CDR2-VH and CDR3-VH,

[0011] The CDR1-VH, CDR2-VH and CDR3-VH have any sequence combination selected from the following or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the sequence combination:

[0012]

[0013]

[0014] and / or,

[0015] (2) light chain CDR combinations of CDR1-VL, CDR2-VL and CDR3-VL,

[0016] The CDR1-VL, CDR2-VL and CDR3-VL have a sequence combination selected from any of the following sequence combinations or a sequence combination having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the sequence combination:

[0017]

[0018]

[0019] In particular, for example, the antibodies or antigen-binding fragments thereof of the present invention comprise a combination of heavy chain CDRs and light chain CDRs selected from the group consisting of: VH1+VL1, VH2+VL2, VH3+VL3, VH4+VL4, VH5+VL5, VH6+VL6, VH7+VL7, VH8+VL8, VH9+VL9, VH10+VL10, VH11+VL11, VH12+VL12, VH13+VL14, VH14+VL15, VH15+VL16, VH16+VL17, VH17+VL18, VH18+VL19, VH19+VL20, VH20+VL21, VH21+VL22, VH22+VL23, VH23+VL24, VH24+VL25 +VL13, VH14+VL14, VH15+VL15, VH16+VL16, VH17+VL17, VH18+VL18, VH19+VL19, VH20+VL20, VH21+VL21 and VH22+VL22, as well as CDR combinations having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions compared to the sequences of the heavy and light chain CDR combinations.

[0020] In another specific embodiment, the present invention provides an antibody or antigen-binding fragment thereof, wherein

[0021] 1) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO:1 and SEQ ID NO:2, respectively, or sequences having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequences shown;

[0022] 2) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO:3 and SEQ ID NO:4, respectively, or sequences having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequences shown;

[0023] 3) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO:5 and SEQ ID NO:6, respectively, or sequences having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequences shown;

[0024] 4) the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO:7 and SEQ ID NO:8, respectively, or sequences having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto;

[0025] 5) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO:9 and SEQ ID NO:10, respectively, or sequences having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequences shown;

[0026] 6) the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO:11 and SEQ ID NO:12, respectively, or sequences having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequences shown;

[0027] 7) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively, or sequences having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequences shown;

[0028] 8) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively, or sequences having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequences shown;

[0029] 9) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18, respectively, or sequences having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequences shown;

[0030] 10) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 19 and SEQ ID NO: 20, respectively, or sequences having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity thereto; or

[0031] 11) The heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO:21 and SEQ ID NO:22, respectively, or sequences with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity with the sequences shown.

[0032] In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention binds to human TNFR2 with a dissociation constant (KD) of no greater than 5 nM and to cynomolgus monkey TNFR2 with a dissociation constant (KD) of no greater than 5 nM.

[0033] In a preferred embodiment, the antibodies or antigen-binding fragments thereof of the present invention are chimeric or humanized or fully human.

[0034] In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a constant region selected from human IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD, and the antibody or antigen-binding fragment thereof:

[0035] 1) Specifically bind to cells expressing TNFR2 on their surface;

[0036] 2) Specific binding to Treg cells;

[0037] 3) Inhibit the binding of TNFα to TNFR2 protein;

[0038] 4) Inhibit the binding of TNFα to TNFR2 expressed on the cell surface;

[0039] 5) Inhibit TNFα-mediated Treg proliferation and / or Treg function;

[0040] 6) mediating ADCC function against TNFR2 expressing cells; or / and

[0041] 7) Inhibit tumor growth.

[0042] In another preferred embodiment, the antigen-binding fragment of the present invention is selected from F(ab)2, Fab', Fab, Fv, scFv, bispecific antibody, nanobody and antibody minimal recognition unit.

[0043] In another embodiment, the antibody or antigen-binding fragment thereof of the invention can compete with an antibody selected from Nos. 001, 088, 125, 133, 219, 224, 226, 309, 352, 365 and 395 for binding to TNFR2.

[0044] In a second aspect, the present invention provides a polynucleotide encoding the antibody or antigen-binding fragment thereof or any combination thereof as described in the first aspect above.

[0045] In a third aspect, the present invention provides an expression vector comprising the polynucleotide as described in the second aspect above.

[0046] In a fourth aspect, the present invention provides a cell comprising the expression vector described in the third aspect above. The cell is, for example, a prokaryotic cell or a eukaryotic cell, including Chinese hamster ovary cells, yeast cells, insect cells, Escherichia coli and Bacillus subtilis.

[0047] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in the first aspect, the polynucleotide as described in the second aspect, the expression vector as described in the third aspect, or the cell as described in the fourth aspect, and a pharmaceutically acceptable carrier.

[0048] In one embodiment, the pharmaceutical composition of the present invention further comprises an additional anti-tumor agent. For example, the additional anti-tumor agent is selected from chemotherapeutic agents, targeted therapeutic agents and immunotherapeutic agents, such as anti-PD-1 / PD-L1 therapeutic agents (including anti-PD-1 antibodies and anti-PD-L1 antibodies) and anti-CTLA-4 therapeutic agents (including anti-CTLA-4 antibodies).

[0049] In a sixth aspect, the present invention provides a kit comprising the antibody or antigen-binding fragment thereof as described in the first aspect, the polynucleotide as described in the second aspect, the expression vector as described in the fourth aspect, or the cell as described in the fourth aspect, and instructions for use.

[0050] In the seventh aspect, the present invention provides a method for treating and / or preventing an immune abnormality-related disease, comprising administering to a subject in need thereof the antibody or antigen-binding fragment thereof as described in the first aspect, the polynucleotide as described in the second aspect, the expression vector as described in the third aspect, the cell as described in the fourth aspect, or the pharmaceutical composition as described in the fifth aspect. The immune abnormality-related disease is, for example, an immune abnormality disease characterized by abnormal sTNFR2, including diseases related to Treg cells and / or MDSC functions, specifically, such as tumors.

[0051] In one embodiment, the method of the present invention further comprises administering to the subject an additional anti-tumor therapy, including chemotherapy, radiotherapy, targeted therapy and immunotherapy, such as anti-PD-1 / PD-L1 therapy such as anti-PD-1 / PD-L1 antibodies, anti-CTLA-4 therapy such as anti-CTLA-4 antibodies.

[0052] In one embodiment, the related disease is a tumor, preferably selected from:

[0053] 1) Ovarian cancer, advanced epidermal T-cell lymphoma, stage III / IV metastatic colorectal cancer, triple-negative breast cancer and / or pancreatic cancer; or,

[0054] 2) Metastatic melanoma or other possible advanced solid tumors resistant to CTLA-4 and PD-1 therapy.

[0055] In an eighth aspect, the present invention provides a pharmaceutical product corresponding to the method described in the seventh aspect, which is used in the prevention and / or treatment method described in the seventh aspect. In addition, there is provided a use of preparing a drug or a kit corresponding to the method described in the seventh aspect, wherein the product of the first to fifth aspects is used for manufacturing, and the drug or kit is used in the prevention and / or treatment method of the seventh aspect.

[0056] In a ninth aspect, the present invention provides a method for detecting sTNFR2, comprising contacting a sample suspected of containing sTNFR2 with the antibody or antigen-binding fragment thereof described in the first aspect. The detection method can be used for the diagnosis of immune abnormality diseases characterized by abnormal sTNFR2, for example, diagnosing whether the sample source subject suffers from immune abnormality-related diseases (such as Treg cell and / or MDSC function-related diseases) or has a risk of developing the disease. Accordingly, the present invention also provides a use of a reagent for detecting sTNFR2 to prepare a diagnostic kit, which is used to diagnose whether the sample source subject suffers from immune abnormality-related diseases (such as immune abnormality diseases associated with abnormal TNFR2 expression) or has a risk of developing the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The above aspects and other aspects of the present invention are clearly described below by detailed description of the present invention and accompanying drawings. The accompanying drawings herein are for illustrating some preferred embodiments of the present invention, however, it is to be understood that the present invention is not limited to the specific embodiments disclosed.

[0058] Figure 1 The expression of TNFR2 in 13 human tumor cell lines is shown. The positive ratio is determined by the detection value after flow cytometry fluorescence sorting (FACS) staining of negative antibodies with the same subtype fluorescence control. The percentage shown in the figure is the expression percentage of TNFR2.

[0059] Figure 2 The expression of TNFR2 in Treg, CD8 + T cells and CD4 + CD25 - Expression on Tcon cells. Figure 2 A is Treg cells, CD8 +Flow cytometric staining and gating strategy of T cells and Tcon cells and flow cytometric scatter plot of the expression ratio of TNFR2 on cells; Figure 2 B is activated Treg (CD4 + CD25 hi FoxP3 hi Treg), non-activated Treg (CD4 + CD25 hi FoxP3 + Treg), Tcon (CD4 + CD25 - T cells) and CD8 + The expression ratio and mean fluorescence intensity (MFI) value of TNFR2 in T cells.

[0060] Figure 3 The binding ability of the tested antibodies to CHO cells that highly express human TNFR2 (CHO-TNFR2) is shown. Figure 3 A is the expression verification of human TNFR2 on the surface of CHO-TNFR2 cells; Figure 3 B is the verification of the cell surface target binding ability of 11 test articles, among which anti human IgG1 Fc-PE only and anti-hen egg white lysozyme (anti-Hel) isotype control antibody (hen egg white lysozyme is an intracellular antigen and does not exist in the human body, so it is a good irrelevant control antibody, outsourced by Biotron), are both negative controls of this experiment, and Unstained is a blank cell control without any antibody added.

[0061] Figure 4 The binding ability of the tested antibodies and Treg cells is shown. Figure 4 A shows Foxp3 used in the experiment + The purity of Treg cells was 91.4%, and the expression ratio of TNFR2 was 99.5% (the hollow peak graph was the anti-Hel isotype control antibody of TNFR2 antibody, and the solid peak graph was the anti-TNFR2 PE antibody); Figure 4 B shows the binding ability of each of the 11 antibodies to Treg cells (solid peaks), human IgG1 Fc-PE only and Unstained (blank cell control without any antibody added) are negative controls, and the antibody concentration is 200 ng / ml.

[0062] Figure 5The test antibody inhibits the interaction between human TNFα and human TNFR2 on CHO-TNFR2 cells. 002#Ab is a tool antibody, which is a positive control antibody in this experiment; anti-Hel isotype control is a negative isotype control antibody; the cell control is CHO-K1 (the mother cell used to construct CHO-TNFR2, which does not express human TNFR2).

[0063] Figure 6 The test antibody shows the inhibitory effect of the test antibody on the inhibitory activity of Treg on Tcon cell proliferation. The vertical axis is different antibodies, and the horizontal axis is (the proliferation ratio of effector cells under each antibody-the proliferation ratio of effector cells under the control antibody) / the proliferation ratio of effector cells under the control antibody × 100%. The control antibody is anti-Hel, and the experimental concentration of each antibody is 12.5ug / ml. The effector cells used are CD4 + CD25 - Tcon cells.

[0064] Figure 7 The effect of the tested antibodies on the expression of sTNFR2 in the cell supernatant of the Treg function inhibition activity experiment is shown. The vertical axis is different antibodies, and the horizontal axis is (the expression of sTNFR2 in the cell supernatant corresponding to each antibody - the expression of sTNFR2 in the cell supernatant by the control antibody) / the expression of sTNFR2 in the cell supernatant by the control antibody × 100%, the control antibody is anti-Hel, and the experimental concentration of each antibody is 12.5ug / ml.

[0065] Figure 8 The results show that the tested antibodies inhibit the proliferation of Treg cells induced by TNFα. Figure 8 A shows the proliferation of Treg cells after 3 days of culture without the addition of cytokines, with the addition of IL-2 alone, or with the addition of IL-2 and TNFα. Figure 8 B is the inhibitory effect of different antibodies on TNFα-induced Treg proliferation. The vertical axis is different antibodies, and the horizontal axis is (the inhibitory ability of each antibody on TNFα-induced Treg proliferation-the inhibitory ability of the control antibody on TNFα-induced Treg proliferation) / the inhibitory ability of the control antibody on TNFα-induced Treg proliferation×100%, the control antibody is anti-Hel, and the experimental concentration of each antibody is 12.5ug / ml.

[0066] Fig. 9 ELISA test of the test antibody against IL-2 and TNF-α induced CD4 +Blockade of TNFR2 secretion by T cells. Statistical analysis method: Graphpad prism6, One-way ANOVA, **P<0.01, ***P<0.001.

[0067] Fig.10 The ADCC (antibody-dependent cellular cytotoxicity) killing effect of the test antibody on Treg. Fig.10 A is the expression of TNFR2 on target cells, namely Treg; Fig.10 B is the verification of the purity of effector NK cells separated and enriched from human peripheral blood cells PBMC; Fig.10 C is the strength of the ADCC effect of the tested antibody, where sample #52 is the ADCC positive control antibody screened for the TNFR2 target, and anti-Hel isotype control is the isotype antibody negative control.

[0068] Fig.11 The ADCC killing effect of the tested antibodies on CHO-TNFR2 cells that highly express human TNFR2 is shown. Among them, #219, #224, and #001 are the tested antibodies, and anti-Hel isotype control is the negative control antibody.

[0069] Fig.12 The tumor volume of each drug-treated group in the in vivo efficacy experiment of CT26 tumor cells resistant to PD-1 antibody (mm) 3 The change curve of (12A) and tumor growth inhibition rate (TGI%) (12B) and the change curve of mouse body weight (12C).

[0070] Fig.13 Figure 13A and 13B show the analysis of various immune cell subsets in the tumor tissue-infiltrated lymphocytes (TIL) in the mouse in vivo CT 26 tumor cell in vivo drug efficacy experiment. Figure 13A and 13B show the gating strategies of panel 1 and panel 2 in FACS immunophenotyping. Figure 13C shows the analysis of CD4 + T, CD8 + Study on the ratio of T and Treg cells; Fig.13 D shows the FACS immunophenotyping of total CD8 + Study on the ratio of T cell number to Treg cell number; Fig.13 E shows the FACS immunophenotyping of memory CD8 +Study on the ratio of T cell number to Treg cell number; Fig.13 F shows FACS immunophenotyping of CD8 + Study on the levels of PD-1, LAG-3 and TNFR2 expressed by T cells.

[0071] Fig.14 In vivo efficacy experiment of CT26 tumor cells in mice: comparison of tumor volume (mm) in four experimental groups using anti-TNFR2 antibody or anti-PD-1 antibody alone or in combination with the two antibodies 3 The change curve of (14A) and the change of tumor growth inhibition rate (TGI%) (14B), as well as the survival curve of mice (14C). DETAILED DESCRIPTION

[0072] Unless otherwise specified, the terms used herein have the meanings commonly understood by those of ordinary skill in the art. For terms that are explicitly defined herein, the meaning of the term is subject to the definitions.

[0073] As used herein, the term "antibody" (Ab) refers to an immunoglobulin molecule that specifically binds to or is immunoreactive with a target antigen, including polyclonal, monoclonal, genetically engineered and other modified forms of antibodies (including but not limited to chimeric antibodies, humanized antibodies, fully human antibodies, heterologous coupling antibodies (e.g., bispecific, trispecific and tetraspecific antibodies, diabodies, triantibodies and tetraantibodies), antibody conjugates) and antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG and scFv fragments). In addition, unless otherwise indicated, the term "monoclonal antibody" (mAb) is meant to include complete antibody molecules that are capable of specifically binding to a target protein as well as incomplete antibody fragments (e.g., Fab and F(ab')2 fragments, which lack the Fc fragment of a complete antibody (cleared more quickly from animal circulation), and therefore lack Fc-mediated effector functions (see Wahl et al., J. Nucl. Med. 24:316, 1983; the contents of which are incorporated herein by reference).

[0074] As used herein, the term "antigen-binding fragment" refers to one or more antibody fragments that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. An antibody fragment can be a Fab, F(ab')2, scFv, SMIP, diabody, triabody, affibody, nanobody, aptamer or domain antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH domains; (ii) a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fab fragment consisting of the VH and CH domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb comprising the VH and VL domains; (vi) a dAb fragment consisting of the VH domain (Ward et al., Nature 341:544-546, 1989); (vii) a dAb consisting of a VH or VL domain; (viii) isolated complementarity determining regions (CDRs); and (ix) combinations of two or more isolated CDRs, which may optionally be linked by a synthetic linker. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by independent genes, the two domains can be joined by a linker using recombinant methods, which enables them to be made into a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called a single-chain Fv (scFv); see, for example, Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as intact antibodies. Antigen binding fragments can be produced by recombinant DNA technology, enzymatic or chemical cleavage of intact immunoglobulins, or in some embodiments by chemical peptide synthesis procedures known in the art.

[0075] As used herein, the term "TNFR2" refers to tumor necrosis factor receptor 2, also known as tumor necrosis factor receptor superfamily member 1B (TNFRSF1B) or CD120b, which is a membrane receptor that binds tumor necrosis factor-α (TNFα). The TNFR2 is preferably human TNFR2.

[0076] As used herein, the terms "anti-TNFR2 antibody", "TNFR2 antibody", "anti-TNFR2 antibody", "TNFR2 antibody", "anti-TNFR2 antibody portion" and / or "anti-TNFR2 antibody fragment" refer to any protein or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule that can specifically bind to TNFR2 (e.g., but not limited to, at least one complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework region or any portion thereof). TNFR2 antibodies also include an antibody-like protein scaffold (e.g., the tenth fibronectin type III domain (10Fn3)) that contains BC, DE and FG structural loops that are similar in structure and solvent accessibility to antibody CDRs. The tertiary structure of the 10Fn3 domain is similar to that of the IgG heavy chain variable region, and by replacing the residues of the BC, DE, and FG loops of 10Fn3 with residues from the CDR-H1, CDR-H2, or CDR-H3 regions of the TNFR2 monoclonal antibody, one skilled in the art can graft, for example, the CDRs of the TNFR2 monoclonal antibody onto the fibronectin scaffold.

[0077] As used herein, the term "bispecific antibody" refers to an antibody, typically a human or humanized antibody, that has monoclonal binding specificities for at least two different antigens. In the present invention, one of the binding specificities can be detected for an antigenic epitope of TNFR2, and the other can be detected for another antigenic epitope of TNFR2 or any other antigen, such as a cell surface protein, a receptor, a receptor subunit, a tissue-specific antigen, a virus-derived protein, a virus-encoded envelope protein, a bacterial-derived protein, or a bacterial surface protein.

[0078] As used herein, the term "chimeric" antibody refers to an antibody having variable sequences of an immunoglobulin derived from one source organism (e.g., rat or mouse) and constant regions of an immunoglobulin derived from a different organism (e.g., human). Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719): 1202-7; Oi et al., 1986, Bio Techniques 4: 214-221; Gillies et al., 1985 J Immunol Methods 125: 191-202; the above are incorporated herein by reference.

[0079] As used herein, the term "complementarity determining region" (CDR) refers to a hypervariable region found in both the light chain and heavy chain variable domains. The more highly conserved portion of the variable domain is called a framework region (FR). As understood in the art, the amino acid position representing the hypervariable region of an antibody can vary according to the context and various definitions known in the art. Some positions within the variable domain can be considered as hybrid hypervariable positions, because these positions can be considered to be within the variable region under a set of standards (such as IMGT or KABAT), and are considered to be outside the variable region under different sets of standards (such as KABAT or IMGT). One or more of these positions can also be found in an extended variable region. The present invention includes antibodies comprising modifications in these hybrid variable positions. The variable domains of native heavy and light chains each include four framework regions that are primarily in a sheet configuration, which are connected by three CDRs (CDR1, CDR2, and CDR3), which form a loop connecting the sheet structure, and in some cases form a part of the sheet structure. The CDRs in each chain are closely held together by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and contribute to the formation of the antigen binding site of the antibody with the CDRs from other antibody chains (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, Md. 1987; which is incorporated herein by reference). For example, in this article, CDR1-VH, CDR2-VH and CDR3-VH refer to the first CDR, the second CDR and the third CDR of the heavy chain variable region (VH), respectively, and these three CDRs constitute the CDR combination (VHCDR combination) of the heavy chain (or its variable region); CDR1-VL, CDR2-VL and CDR3-VL refer to the first CDR, the second CDR and the third CDR of the light chain variable region (VL), respectively, and these three CDRs constitute the CDR combination (VLCDR combination) of the light chain (or its variable region).

[0080] As used herein, the term "antibody conjugate" refers to a coupling / conjugate formed by chemically bonding an antibody molecule directly or through a linker to another molecule, such as an antibody-drug conjugate (ADC), in which the drug molecule is the other molecule.

[0081] As used herein, the term "monoclonal antibody" refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), without limitation to the method by which the antibody is produced.

[0082] As used herein, the term "VH" refers to the variable region of an immunoglobulin heavy chain (including the heavy chain of Fv, scFv or Fab) of an antibody. The term "VL" refers to the variable region of an immunoglobulin light chain (including the light chain of Fv, scFv, dsFv or Fab).

[0083] As used herein, the term "regulatory T cells" or "Treg", also formerly known as suppressor T cells, is a group of lymphocytes that negatively regulate the body's immune response to maintain tolerance to self-antigens, control excessive immune responses, avoid immune damage to normal cells, and prevent the occurrence of autoimmune diseases. Treg expresses the following biomarkers: CD4, FOXP3, and CD25, and is believed to be derived from the same lineage as naive CD47 cells. Treg plays an extremely important role in the occurrence of tumors. Many studies have shown that the number of Treg cells in the tumor microenvironment increases significantly, including melanoma, ovarian cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, etc. At the same time, the number of Treg cells is closely related to the survival rate of tumor patients. In addition, tumor cells induce the proliferation of tumor-infiltrating Treg cells, and the proliferated Treg cells secrete a large amount of immunosuppressive factors such as TGF-β, inhibiting CD8 + The function of immune cells such as T cells hinders the killing effect of immune cells on tumors, which is an important resistance mechanism for the failure of immunotherapy for various solid tumors and hematological tumors. Recent studies have shown that the immune tolerance of patients treated with immunotherapy such as PD-1 / PD-L1 is also closely related to Treg.

[0084] As used herein, the term "myeloid-derived suppressor cells" or "MDSCs" is a heterogeneous cell population of the immune system composed of immature neutrophils, monocytes, and dendritic cells that has the effect of suppressing immune responses and tumor immune responses. MDSCs regulate the activity of a variety of effector cells and antigen-presenting cells (such as T cells, NK cells, dendritic cells, and macrophages, etc.). Myeloid-derived suppressor cells are distinguished by their gene expression profile and express all or part of the proteins and small molecules selected from the following group: B7-1 (CD80), B7-H1 (PD-L1), CCR2, CD1d, CD1d1, CD2, CD31 (PECAM-I), CD43, CD44, complement component C5aR1, F4 / 80 (EMR1), FcγRIII (CD16), FcγRII (CD32), FcγRIIA (CD32a), FcγRIIB (CD32b), FcγRIIB / C (CD32b / c), FcγRIIC (CD32c), FcγRIIIA (CD16A), FcγRIIIB (CD16b), galectin-3, GP130, Gr-1 (Ly-6G), ICAM-1 (CD54), IL-1RI, IL-4Ra, IL-6Ra, integrin α4 (CD49d), integrin αL (CDlla), integrin αM (CDllb), M-CSFR, MGL1 (CD301a), MGL1 / 2 (CD301a / b), MGL2 (CD301b), nitric oxide, PSGL-1 (CD162), L-selectin (CD62L), sialolectin-3 (CD33), transferrin receptor (TfR), VEGFR1 (Flt-I) and VEGFR2 (KDR or Flk-1). In particular, MDSCs do not express proteins selected from the group consisting of B7-2 (CD86), B7-H4, CD11c, CD14, CD21, CD23 (FcεRII), CD34, CD35, CD40 (TNFRSF5), CD117 (c-kit), HLA-DR, and Sca-I (Ly6).

[0085] As used herein, the term "percent (%) sequence identity" refers to the percentage of amino acid (or nucleotide) residues of a candidate sequence that are identical to the amino acid (or nucleotide) residues of a reference sequence after aligning sequences and introducing gaps (if necessary) to achieve maximum percent sequence identity (e.g., for optimal alignment, gaps can be introduced in one or both of the candidate and reference sequences, and for the purpose of comparison, non-homologous sequences can be ignored). For the purpose of determining percent sequence identity, alignment can be achieved in a variety of ways well known to those skilled in the art, such as using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAIi) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm that requires maximum alignment over the full length of the compared sequence. For example, a reference sequence for comparison with a candidate sequence can show that the candidate sequence exhibits from 50% to 100% sequence identity over the full length of the candidate sequence or a selected portion of the continuous amino acid (or nucleotide) residues of the candidate sequence. The length of the candidate sequence compared for comparison purposes can be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

[0086] As used herein, the term "specific binding" refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules that are specifically recognized, for example, by an antibody or its antigen-binding fragment. An antibody or its antigen-binding fragment that specifically binds to an antigen will bind to the antigen with a KD of less than 100 nM. For example, an antibody or its antigen-binding fragment that specifically binds to an antigen will bind to the antigen with a KD of up to 100 nM (e.g., between 1 pM and 100 nM). An antibody or its antigen-binding fragment that does not show specific binding to a specific antigen or its epitope will show a KD of greater than 100 nM (e.g., greater than 500 nM, 1 μM, 100 μM, 500 μM, or 1 mM) to the specific antigen or its epitope. A variety of immunoassays can be used to select antibodies that specifically immunoreact with a specific protein or carbohydrate. For example, solid phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with proteins or carbohydrates. See, Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), which describe immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0087] As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors (such as plasmids), RNA vectors, viruses or other suitable replicons (such as viral vectors). A variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. The expression vector of the present invention contains polynucleotide sequences and additional sequence elements, such as for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express the antibodies and antibody fragments of the present invention include plasmids containing regulatory sequences (such as promoters and enhancer regions) that direct gene transcription. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES) and polyadenylation signal sites to direct the efficient transcription of genes carried on the expression vector. The expression vector of the present invention may also contain the following polynucleotides, which encode markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseothricin.

[0088] As used herein, the terms "subject," "object," and "patient" refer to an organism that is being treated for a particular disease or condition as described herein, such as cancer or an infectious disease. Examples of subjects and patients include mammals such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, guinea pigs, members of the Bovidae family (such as cattle, bison, buffalo, elk, and yaks, etc.), cattle, sheep, horses, and bison, etc., that are being treated for a disease or condition (e.g., a cell proliferative disorder such as cancer or an infectious disease).

[0089] As used herein, the term "treatment" refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or lesions in the treatment subject, such as the progression of cell proliferative disorders (such as cancer or infectious diseases). Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of disease severity, stabilization of the disease state (i.e., no deterioration), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Subjects in need of treatment include subjects who already have a disease or disease, as well as subjects who are susceptible to a disease or disease or subjects who intend to prevent a disease or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, and alleviating, their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0090] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0091] Example 1. Preparation of TNFR2 monoclonal antibody

[0092] Female SJL mice (purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) or Balb / c mice (purchased from Shanghai Slake Laboratory Animal Co., Ltd.) aged 6-8 weeks were injected with complete Freund's adjuvant (CFA, purchased from SIGMA, catalog number: F5881) mixed with human TNFR2 recombinant protein (purchased from SinoBiological, catalog number: 10417-H08H and Novoprotein, catalog number: C830) for the first immunization, and incomplete Freund's adjuvant (IFA, purchased from SIGMA, catalog number: F5506) and CpGODN1826 (synthesized from Shanghai Shenggong Biology) were used as adjuvants for the next three immunizations. In particular, the first and second immunizations were injected into the hind foot pad and back, and the third and fourth immunizations were injected into the subcutaneous part of the tail and back, so as to obtain high titer, high affinity, and high specificity antiserum and specific immune cells. On the 7th day after the last immunization (the fourth immunization), the mice were euthanized and the spleen was removed aseptically. The mouse spleen lymphocytes were isolated and extracted aseptically, and the cells were frozen in liquid nitrogen in aliquots. The cells were revived when needed later. The TNFR2-specific single B cells in the spleen or lymph nodes of the immunized mice were sorted into a 96-well plate using a BD Aria III flow sorter, and the mRNA of the single cells was reverse transcribed into cDNA. Then, nested PCR was performed using cDNA as a template to amplify the antibody heavy chain and light chain, respectively. The antibody heavy chain variable region and light chain variable region were amplified and cloned into the heavy chain expression vector and the light chain expression vector, respectively, by homologous recombination. The constant regions of the heavy chain expression vector and the light chain expression vector are both from human IgG1. The complete heavy chain expression sequence is signal peptide-VH-CH1-hinge region-CH2-CH3, and the complete light chain expression sequence is signal peptide-Vκ-Cκ. The above-mentioned single B cell antibody cloning and expression were all carried out in a 96-well plate in a high-throughput manner to achieve rapid identification and discovery of antibodies. After a series of physical and chemical and functional screening of 523 pairs of cloned expressed antibody heavy chains and light chains, a total of 11 positive candidate antibody molecules were obtained, and the CDRs of their sequences were analyzed by IMGT and KABAT software, respectively. The corresponding sequence information is shown in Tables 1 to 3 below, where Table 1 shows the VH and VL sequences of the candidate antibody molecules, Table 2 shows the IMGT analysis results of the candidate antibody molecules, and Table 3 shows the KABAT analysis results of the candidate antibody molecules).

[0093] Table 1. VH and VL sequences of candidate antibody molecules

[0094]

[0095]

[0096]

[0097] Table 2. IMGT analysis results of candidate antibodies

[0098]

[0099] Table 3. KABAT analysis results of candidate antibodies

[0100]

[0101]

[0102] Example 2. FACS detection of TNFR2 expression in tumor cell lines

[0103] Well-growing cells were collected and counted, and 2×10 cells of each tumor were obtained. 6 cells were used for isotype control group and TNFR2 staining group (1×10 6 cells / test). Wash the cells twice with PBS, centrifuge at 300g×5min, discard the supernatant, add 1×Live / Dead working solution (Zombie violet L / D, Biolegend, catalog number: 423114) 100μl / test, and stain for 20 minutes at room temperature. After 20 minutes, wash the cells twice with FACS buffer (DPBS+2%FBS), centrifuge at 300g×5min, discard the supernatant, add 1× staining antibody working solution 100μl / test as follows, and incubate at 4℃ in the dark for 30 minutes.

[0104] For human tumor cells: 1) isotype control group: add 1μl PE rat IgG2b, Kisotype control (BD Pharmingen, catalog number: 553989) / 100μl FACS buffer / test; 2) TNFR2 staining group: add 8μl PE Rat anti-human CD120b (BD Pharmingen, catalog number: 552418) / 100μl FACS buffer / test to human tumor cells. After incubation, wash the cells twice with FACS buffer, centrifuge at 300g×5min, discard the supernatant, resuspend the cells with 300μl FACS buffer to the cell suspension, wait for the flow cytometer to detect (Invitrogen, Attune NxT), export the data and analyze it with Flowjo software to draw the following figure Figure 1 As shown, different tumor cells have different expression levels of TNFR2, and the results are summarized in Table 4.

[0105] Table 4. TNFR2 expression results of 13 human tumor cell lines

[0106]

[0107] Example 3. FACS detection of TNFR2 expression levels on different human T cells

[0108] Commercially frozen human PBMC (Hemacare) was resuscitated or activated Treg cells were expanded using anti-CD3 / CD28 Dynabeads (Gibco, Cat. No. 11129D) and cultured overnight. The next day, human PBMC were centrifuged at 300×g for 10 min and the supernatant was discarded. After counting, 2×10 6 PBMC cells were used for isotype control group and TNFR2 staining group (1×10 6 cells / test). Wash the cells twice with PBS, centrifuge at 300g×5min, discard the supernatant, add 1×Live / Dead working solution (Zombie violet L / D, Biolegend, catalog number: 423114) 100μl / test, and stain for 20 minutes at room temperature. After 20 minutes, wash the cells twice with FACS buffer (DPBS+2%FBS), centrifuge at 300g×5min, discard the supernatant, add 1× staining antibody working solution 100μl / test as follows, and incubate at 4℃ in the dark for 30 minutes.

[0109] For PBMC cells: 1) Fluorescence Minus One control (FMO control) group: add 1 μl of the antibody shown in the table below / 100 μl FACS buffer / test;

[0110] Product Number Fluorescent dyes / labels clone BD Pharmingen-557943 Alexa fluor 700CD3 UCHT1 BD Bioscience-560650 PerCP / Cy5.5 CD4 RPA-T4 BD Bioscience-335789 PE / Cy7 CD25 2A3 BD Bioscience-557714 Alexa fluor 647mouse IgG1,κ MOPC-21 Biolegend-423114 Zombie violet Live / Dead Biolegend-300926 APC / Cy7 CD8 HIT8a BD / R&D PE rat IgG2b,κ A95-1 ;

[0111] 2) TNFR2 staining group: add 1 μl of the antibody shown in the table below / 100 μl FACS buffer / test;

[0112] Product Number Fluorescent dyes / labels clone BD Pharmingen-557943 Alexa fluor 700CD3 UCHT1 BD Bioscience-560650 PerCP / Cy5.5 CD4 RPA-T4 BD Bioscience-335789 PE / Cy7 CD25 2A3 BD Bioscience-560045 Alexa fluor 647FoxP3 259D / C7 Biolegend-423114 Zombie violet Live / Dead Biolegend-300926 APC / Cy7 CD8 HIT8a BD / R&D PE TNFR2 hTNFR-M1 .

[0113] For Treg cells: 1) FMO control group: add 1 μl of the antibody shown in the table below / 100 μl FACS buffer / test;

[0114]

[0115]

[0116] 2) TNFR2 staining group: add 1 μl of the antibody shown in the table below / 100 μl FACS buffer / test;

[0117] Product Number Fluorescent dyes / labels clone BD Pharmingen-557943 Alexa fluor 700CD3 UCHT1 BD Bioscience-560650 PerCP / Cy5.5 CD4 RPA-T4 BD Bioscience-335789 PE / Cy7 CD25 2A3 BD Bioscience-560045 Alexa fluor 647FoxP3 259D / C7 Biolegend-423114 Zombie violet Live / Dead BD / R&D PE TNFR2 hTNFR-M1 .

[0118] After the incubation, the cells were washed twice with FACS buffer, centrifuged at 300g×5min, the supernatant was discarded, and the cells were resuspended in 300μl FACS buffer to make a cell suspension, and then detected by flow cytometry (Invitrogen, Attune NxT). After exporting the data, the Flowjo software was used for analysis and plotting. Figure 2 As shown, TNFR2 is expressed in CD4 + CD25 - Tcon cells and CD8 + The expression level on T cells is much lower than that on Treg cells, but it is highly expressed on both unactivated and activated Treg cells, and the expression level on activated Treg cells is the highest.

[0119] Example 4. BIAcore detection of specific binding of TNFR2 antibody to human and cynomolgus monkey TNFR2 protein

[0120] Biacore was used to detect the specific binding between the 11 TNFR2 antibodies obtained in Example 1 and human and monkey TNFR2 proteins. The experiment used a Protein A chip, and the time required for the chip to capture the diluted antibody was determined by manual operation (manual run) so that the saturated binding antigen Rmax was 50RU. Human (Human TNFR2, Sino10417-H08H) and cynomolgus (Cynomolgus TNFR2, Sino 90102-C08H) TNFR2 were gradiently diluted to 32, 16, 8, 4, and 2nM. The affinity of the antibody and the antigen was measured using multi-cycle kinetics. In each cycle, after the antibody was injected, a gradient concentration of TNFR2 was injected to allow the antigen to bind and dissociate. After each cycle, the Protein A chip was regenerated with Glycine pH1.5 (to remove the protein on the chip). The affinity KD of the antibody and antigen was fitted using BIAcore T200 analysis software.

[0121] Table 5. Biacore results of specific binding of TNFR2 antibodies to human or monkey TNFR2 protein

[0122]

[0123] From the results in Table 5, it can be seen that the 11 anti-TNFR2 antibodies obtained in Example 1 specifically bind to human and monkey TNFR2 proteins with high affinity levels.

[0124] Example 5. ELISA to detect the specific binding of TNFR2 antibodies to human and monkey TNFR2 proteins

[0125] The ELISA plate was pre-coated with 100 μl / well of 0.5 μg / ml human TNFR2 or cynomolgus monkey TNFR2 (the same antigen protein as in Example 4); the purified anti-TNFR2 antibody obtained in Example 1 was diluted to 28 ng / ml (corresponding to the EC80 of the binding curve of antibody #1), 100 μl / well was added, and the plate was incubated at room temperature with shaking for 1.5 hours; after washing the plate, mouse anti-human IgGFc-HRP working solution (1:10000 dilution) was added, 100 μl / well was added, and the plate was incubated at room temperature with shaking for 1.0 hours; the plate was washed again, HRP substrate TMB was added for color development, and the stop solution was added to stop the reaction and the absorbance was read with an ELISA reader. The data in Table 6 show that all 11 antibodies can specifically bind to the monkey TNFR2 protein, but not to the mouse TNFR2 or the control human CREG-His protein.

[0126] Table 6. ELISA results of specific binding of TNFR2 antibodies to human and monkey TNFR2 proteins

[0127]

[0128]

[0129] Note: 1. 002# is a tool antibody (SBT-002 from WO2017 / 083525 A1)

[0130] 2. Only a single concentration point was measured for the antibody, which was expressed as OD value. The concentration of #088 and #309 antibodies was 12 ng / mL, and the concentration of the remaining antibodies was 28 ng / mL.

[0131] Example 6. FACS detection of antibody binding to human TNFR2 on the surface of CHO-TNFR2 cells

[0132] Take CHO stable cells transfected with TNFR2 high expression plasmid, the transfection plasmid was purchased from Sino Biological (Cat. No.: HG10417-UT), and conduct the experiment when the cell density does not exceed 80%. Discard the cell culture medium, rinse with PBS and add 1ml of trypsin for 2 minutes, and terminate the digestion with HamFBS medium containing 10% FBS to prepare a cell suspension. After counting, take an appropriate amount of cell suspension, centrifuge at 350×g, discard the supernatant, and precipitate at 1×107 Cells were resuspended in a corresponding volume of blocking solution (10% FBS + PBS) at a density of 10 cells / ml and incubated at 4°C for 30 minutes. After incubation, the cells were centrifuged at 350 × g to remove the supernatant and resuspended in staining buffer (2% FBS + PBS) at a density of 2 × 10 6 The cells were plated into a 96-well plate at a density of 1 cell / ml, and 50 μl of cell suspension was added to each well for later use. The antibody was diluted to 80 ng / ml with PBS, and the diluted antibody was added to the wells containing 50 μl of cell suspension. The cells were placed on a microplate oscillator and shaken at 500 rpm for 1 minute to fully mix the antibody and cells, and incubated at 4°C for 1 hour. After the incubation, the cells were washed twice with staining buffer: 100 μl per well, centrifuged at 350 × g for 5 minutes, and the supernatant was discarded. The PEgoat anti-Human IgG Fc antibody (ebioscience, catalog number: 12-4998-82) was diluted 250 times with staining buffer, and added to the washed cell wells at a volume of 100 μl per well, mixed evenly, and stained at 4°C for 30 minutes. After the staining is completed, the cells were washed twice with staining buffer, and finally the cells were resuspended with 200 μl of staining buffer. The signal intensity was detected by flow cytometry. The stronger the signal, the stronger the binding ability of the antibody to TNFR2. Figure 3 As shown in A, PE Rat-IgG2b PE Rat-IgG2b (Biolegend, Catalog No.: 400636) and PE Rat anti-hTNFR2 (BD Biosciences, Catalog No.: 552418) were used to detect that the expression intensity of TNFR2 on the surface of CHO-TNFR2 cells was about 134 times that of the isotype control; on this basis, the ability of the 11 TNFR2 antibodies of Example 1 to bind to cell surface TNFR2 was tested, and the results were all positive, as shown in FIG. Figure 3 As shown in B.

[0133] Example 7. FACS detection of the binding of antibodies to human TNFR2 on the surface of Treg cells

[0134] Human Treg cells were isolated from human PBMC using a sorting kit (Stemcell, Catalog No.: 18063), and obtained after 17 days of in vitro stimulation and expansion using Dynabeads Human Treg Expander (Gibco, Catalog No.: 11129D) and cryopreserved. The Treg cells isolated and expanded in vitro were revived overnight, centrifuged at 300×g for 5 minutes the next day, resuspended in DPBS to obtain a cell suspension, and counted. The number of cells required for the experiment was added to a centrifuge tube, centrifuged at 300×g for 5 minutes, the supernatant was removed, and the cell concentration was adjusted to 1×10 7The blocked cells were centrifuged at 300×g for 5 min, the supernatant was discarded, and the cell density was adjusted to 2×10 cells / ml with staining buffer. 6 / ml, spread on a 96-well plate, 50μl per well. Take the 11 test antibodies #001, #088, #125, #133, #219, #224, #226, #309, #352, #365, #395 and the control antibody anti-Helisotype in Example 1 (diluted to 200ng / ml with PBS, total amount is 100μl). Add each sample to each well, 50μl per well. Place the well plate with cell suspension and antibody on a microplate oscillator, oscillate at 500rpm for 1min, and mix the cells and antibodies thoroughly. After mixing, place the well plate in a 4℃ refrigerator and incubate for 60min. After the incubation, add 100μl of staining buffer to each well, centrifuge at 350×g for 5min, and discard the supernatant; add 200μl of staining buffer to each well to resuspend the cells, centrifuge at 350×g for 5min, and discard the supernatant. Add PE goat anti-Human IgG Fc to the staining buffer at a ratio of 250:1 (staining buffer: dye) to make a staining solution. Mix well and add to the cell wells, 100μl per well; place the well plate on a microplate oscillator and oscillate at 500rpm for 1min to fully mix the cells and the staining solution. After mixing, place in a 4℃ refrigerator and incubate for 30min. Wash the cells twice, and finally add 200μl of PBS to each well to resuspend the cells and detect the signal. Figure 4 A shows CD25 + FoxP3 + The cells are human Treg cells, and the expression ratio of TNFR2 on the circled Treg cells reaches 99.5%, indicating that human Treg cells highly express TNFR2. Figure 4 B shows that the control antibody anti-Hel isotype does not bind to Treg, while the 11 antibodies in Example 1 can effectively bind to Treg cells.

[0135] Example 8. ELISA to detect the blocking effect of anti-TNFR2 antibodies on TNFα / TNFR2 binding

[0136] The ELISA plate was pre-coated with 1 μg / ml 100 μl / well human TNFR2 (Novoprotein, item number C830), and each TNFR2 antibody of Example 1 was diluted to 40 nM and 4 nM. The diluted antibodies were mixed with 15 ng / ml human TNFa (Acro Biosystem, item number TNA-H82E3) in equal volumes, added to the ELISA plate at 100 μl / well, and incubated at room temperature for 2.0 h; after washing the plate, Streptavidin-HRP working solution (1:10000 dilution) was added, 100 μl / well, and incubated at room temperature for 40 min; the plate was washed again, and the substrate TMB of HRP was added for color development, and the stop solution was added to terminate the reaction and the absorbance was read with an ELISA reader. The lower the OD value, the stronger the ability of the antibody to inhibit the binding of TNFa to TNFR2. Finally, the OD values ​​of all antibodies were normalized to the OD value of tool antibody 002#, and the higher the percentage value, the stronger the inhibition ability. The data in Table 7 show that the 11 antibodies of Example 1 have the activity of inhibiting the binding of TNFa to TNFR2 at the concentrations of 20 nM and 2 nM. Compared with Ab #002, the inhibitory activity is higher and lower (Tables 7a-7b).

[0137] Table 7a. ELISA results-1, some of the tested antibodies blocked the binding of TNFα and TNFR2

[0138]

[0139] Table 7b. ELISA results-2, some of the tested antibodies blocked the binding of TNFα and TNFR2

[0140]

[0141] Note: 1. 002# is the tool antibody; 2. % to 002# OD = ELISA OD for 002# (20nM or 2nM) / ELISA OD for antibody (20nM or 2nM) × 100.

[0142] Example 9. Anti-TNFR2 antibodies block the binding of TNFα to TNFR2 on CHO cells that highly express human TNFR2

[0143] The experiment was conducted using CHO stable cells that highly expressed human TNFR2. The cells were revived and passaged until they were in good condition, and the expression level of human TNFR2 in the cells was detected; the detection could be performed when the mean fluorescence intensity (MFI) shifted by more than 100 times compared with the isotype control.

[0144] After digestion, CHO-TNFR2 cells were washed twice with DPBS and stained with Live / Dead (L / D) (room temperature for 20 min) and then stained with 1×105 Cells / 50μl / well were plated; the anti-TNFR2 antibody of Example 1 was diluted to 40nM with staining buffer as the starting concentration, and diluted 3 times in a gradient, with a total of 7 concentration points; the diluted antibody was added to the plated cells at 50μl / well, and gently pipetted to mix. At this time, the final concentrations of the antibodies were 20, 6.67, 2.22, 0.74, 0.247, 0.08, 0.027 and 0nM, respectively, and incubated at 4°C for 30min. Then, 100μl / well of human TNFα-biotin diluted to 100ng / ml was added and gently pipetted to mix, and then incubated at 4°C for 30min. After washing twice with staining buffer, 100μl / well of PE-streptavidin was added and incubated at 4°C for 30min. After washing twice with staining buffer, 150μl was resuspended and tested on the machine. The inhibition curve of the antibody was drawn with the logarithm of the antibody concentration as the horizontal axis and the corresponding MFI value as the vertical axis. The IC50 value was calculated by four-parameter fitting. The smaller the IC50 value, the stronger the ability of the antibody to inhibit the binding of human TNFα to human TNFR2. Since the blocking curve shapes of some antibodies are different, the blocking effects of all antibodies are normalized to 002#AUC. The higher the percentage value, the better the antibody inhibition effect. The blocking curves of the 11 antibodies are shown in Figure 5 The inhibitory activity is shown in Table 8.

[0145] from Figure 5 As shown in Table 8, all 11 antibodies in Example 1 can significantly inhibit the binding of TNFα to TNFR2 on CHO-TNFR2 cells. Compared with tool antibody 002#, the inhibitory activities of the 11 antibodies are higher and lower.

[0146] Table 8. TNFR2 antibodies block the binding of TNFα to cell surface TNFR2

[0147]

[0148]

[0149] Note: AUC% = AUC for 002# ÷ AUC for antibody × 100.

[0150] Example 10. Inhibition of Treg function by TNFR2 antibody

[0151] By measuring the inhibitory function of Treg cells on effector (responder) T cells under the condition of TNFR2 antibody, the interference effect of antibody on the inhibitory function of Treg cells was determined. + CD25 + FoxP3 +T cells) and Tcon effector cells (CD4 + CD25 - T cells) were revived overnight, centrifuged at 400×g for 5 minutes the next day, resuspended in RPMI1640 medium (Gibco, catalog number 72400047) to obtain a cell suspension, and counted. Tcon cells were stained with CellTrace CFSE cell proliferation kit (Biolegend catalog number 423801), and 4×10 6 Use 1 μl of the storage solution to prepare 1 ml of staining solution. Stain for 5 min, neutralize the reaction with an equal volume of serum, let stand for 5 min, and centrifuge at 400×g for 5 min. Resuspend the cells in culture medium, centrifuge again and discard the supernatant. 5 The cell density was 1×10 5 The cells were resuspended at a density of 50 μl culture medium and transferred to a 50 mL centrifuge tube. Treg cells were prepared at the same time. Treg cells and Tcon cells were co-cultured at a ratio of 1:1, 1:2, 1:4, and 1:8. Each well of each 96-well plate corresponds to a different Treg cell density, and each well is resuspended in 50 μl culture medium. According to the total number of Tcon cells, the corresponding anti-CD3 / CD28 Dynabeads (Gibco, catalog number: 11129D) were prepared at a ratio of 1 / 8. The culture medium of Treg and Tcon was added at a volume of 50 μl per well. The antibody to be tested was added to each well, and the final concentration of the antibody was 12.5 μg / ml. Two parallel wells were set for each condition, and the above well plates were mixed and placed in a 37°C incubator for 4 days for FACS detection. The results showed that especially the #001 antibody and the #224 antibody were able to significantly inhibit the inhibitory effect of Treg on Tcon cells and promote the proliferation of Tcon under different Treg / Tcon ratios ( Figure 6 ).

[0152] Example 1 The calculation formula for the effect of the test antibody and the control antibody anti-Hel isotype on the inhibitory function of Treg cells is: (the proliferation capacity of effector T cells in the presence of the test antibody - the proliferation capacity of effector T cells in the presence of the anti-Hel antibody) / the proliferation capacity of effector T cells in the presence of the anti-Hel antibody × 100%.

[0153] Example 11. Effect of TNFR2 Antibody on sTNFR2 Expression in Cell Supernatant in Treg Function Inhibition Experiment

[0154] The inhibitory effect of TNFR2 antibody on Treg function was determined by measuring the expression of sTNFR2 in the cell supernatant. After treating Treg and effector T cells with the antibody of Example 1 for 4 days, the culture supernatant was collected as a sample, and 100 μl of supernatant was taken from each well. After the supernatant was diluted 5 times, sTNFR2 was measured. The method for measuring sTNFR2 refers to the humanTNFRII / TNFRSF1B kit of R&D Company (Cat. No. DRT200). The results showed that #001, #088, #125, #219, #224, #226 and #309 were able to significantly downregulate the expression level of sTNFR2 in the cell supernatant at different Treg:Tcon ratios ( Figure 7 ).

[0155] Example 12. TNFR2 Antibody Inhibits TNFα-Induced Treg Cell Proliferation

[0156] The inhibitory effect of the antibody on TNFα-induced Treg cell proliferation was determined by measuring the proliferation of TNFα-induced Treg cells in the presence of TNFR2 antibody (Zaragoza B et al., Nat Med. 2016 Jan; 22(1): 16-7). The Treg cells that were cryopreserved after in vitro amplification and separation were revived overnight, centrifuged at 400×g for 5 minutes the next day, resuspended in culture medium to obtain a cell suspension, and counted. Treg cells were stained with CFSE, and 4×10 6 Use 1 μl of the storage solution to prepare 1 ml of staining solution. Stain for 5 minutes, neutralize the reaction with an equal volume of serum, let stand for 5 minutes, and centrifuge at 400×g for 5 minutes. Resuspend the cells in culture medium and wash them once, centrifuge again to obtain the supernatant. 1×10 cells per well of a 96-well plate 5 The cell density was 1×10 5 The cells were resuspended in 50 μl of culture medium and transferred to a 96-well plate. The antibody to be tested in Example 1 was added to each well, the final concentration of the antibody was 12.5 μg / ml, and the plate was incubated in an incubator for 30 minutes. Subsequently, 50 μl of culture medium containing IL-2 (final concentration of 300 IU) and 50 μl of culture medium with a final concentration of TNFα of 50 ng / ml were added to each well to make up the final volume of each well to 200 μl. Three parallel wells were set for each condition, and the well plates were mixed and placed in a 37°C incubator for three days for FACS detection. The inhibitory effect of the antibody on TNFα-induced Treg proliferation was determined by the proportion of Treg proliferation. The results are shown in the figure. Figure 8The results showed that compared with the control anti-Hel antibody, the following antibodies effectively inhibited TNFα-induced Treg cell proliferation: #001 (inhibition 17.1%), #088 (inhibition 6.9%), #125 (inhibition 5.2%), #133 (inhibition 9.7%), #219 (inhibition 13.6%), #226 (inhibition 10.3%), #365 (inhibition 11.0%). The formula for calculating the ratio of Treg cell proliferation induced by the test antibody and the control antibody is as follows: (ratio of Treg proliferation induced by TNFα in the presence of the test antibody - ratio of Treg proliferation induced by TNFα in the presence of the anti-Helisotype antibody) / ratio of Treg proliferation induced by TNFα in the presence of the anti-Helisotype antibody × 100%. Figure 8 A shows that Treg proliferation increased significantly after adding IL-2 (20% vs. 4%), and the proliferation ratio was further increased after adding TNFα (40% vs. 20%). Figure 8 B shows the inhibitory effect of 11 antibodies on the inducible proliferation of Treg after the addition of IL-2 and TNFα. Except for #224, #352 and #395, the other 8 candidate antibodies can inhibit the proliferation of Treg induced by IL-2 and TNFα.

[0157] Example 13. ELISA detection of TNFR2 antibody to CD4 + Effect of sTNFR2 expression level in T cell culture supernatant

[0158] Commercially frozen human PBMC cells were resuscitated and cultured overnight. The next day, human PBMCs were centrifuged at 300×g for 10 min, the supernatant was discarded, and the cells were washed twice with Easybuffer, the separation buffer of the kit, and then the cells were counted. + Instructions for use of the T cell isolation kit (Stemcell, EasySepTM Human CD4 + T cell Isolation Kit, Catalog No. 17952) for human CD4 + T cell separation. + T cells were re-counted and resuspended to an appropriate concentration, and the cells were divided into 4 × 10 5Cells / well were seeded in a 96-well round-bottom plate, 50 μl / well. Antibody working solution (50 μg / ml, 4×) was prepared, 50 μl / well was added to the plate, and incubated at 37°C, 5% CO2 for 30 min. Next, IL-2 working solution (800 IU / ml, 4×) and TNFα working solution (200 ng / ml, 4×) were prepared and added at 50 μl / well respectively. The experimental groups were: no stimulation group, IL-2 stimulation group alone, TNFα stimulation group alone, IL-2+TNFα co-stimulation group, and antibody+IL-2+TNFα co-stimulation group (i.e., the sample group to be tested). The plate after plating was placed in an incubator and cultured at 37°C, 5% CO2 for 3 days. After 3 days, the culture supernatant was collected for sTNFR2 detection.

[0159] Prepare human sTNFR2 standard, take out the cell supernatant from the above experiment, and operate according to the instructions of the ELISA kit (R&D System, Soluble TNF Receptor II Human ELISA Kit, Catalog No. DRT200). The steps are as follows: 1) Add 100 μl / well of cell supernatant and standard to the high affinity plate, and incubate at room temperature (18-25°C) with slight shaking for 2.5 hours; 2) Discard the supernatant and wash with washing solution (Wash 1) After absorbing the water, add 100 μl / well of 1× Biotin-labeled soluble human TNFR2 antibody, and incubate at room temperature (18-25°C) with slight shaking for 1 hour; 2) After discarding the supernatant, wash the plate 4 times with washing buffer, 300 μl / well; 3) After absorbing the water, add 100 μl / well of 1× HRP-Streptavidin solution, and incubate at room temperature (18-25°C) with slight shaking for 45 minutes; 4) After absorbing the water, add 100 μl / well of 1× HRP-Streptavidin solution, and incubate at room temperature (18-25°C) with slight shaking for 45 minutes; 5) After absorbing the water, add 100 μl / well of TMB substrate solution, and incubate at room temperature (18-25°C) and in the dark with slight shaking for 30 minutes; 6) After absorbing the water, add 100 μl / well of TMB substrate solution, and incubate at room temperature (18-25°C) and in the dark with slight shaking for 30 minutes; 7) After absorbing the water, add 50 μl / well of reaction stop solution, and immediately detect the OD450 reading in an ELISA reader.

[0160] The experimental results are as follows Fig. 9 As shown, compared with the negative control Anti-Hel isotype group, all 11 antibodies in Example 1 can inhibit the secretion of sTNFR2 in the cell supernatant. + T cells are activated under the co-stimulation of IL-2 and TNFα, which leads to increased secretion of sTNFR2 in the cell supernatant. All 11 antibodies can block the effects of IL-2 and TNFα on CD4 +The activation effect of T cells, among which the inhibitory effect of antibodies numbered #88, #219, #001 (P<0.001), #125, #224 (P<0.01) was particularly significant, with statistically significant differences. The lower the OD450 reading, the lower the expression level of sTNFR2, that is, the stronger the antagonistic activity of the antibody.

[0161] Example 14. FACS detection of ADCC activity of TNFR2 antibody mediated on Treg cells

[0162] One day in advance, PBMC and frozen Treg cells were cultured in complete medium (RPMI1640-Glutamax+10% FBS+1×P / S+1×ITS+50μl MPM-mercaptoethanol), and PBMC needed to be supplemented with 100IU / ml IL-2. On the day of the experiment, NK cells were separated from PBMC according to the requirements of the separation kit (Stemcell, Cat. No. 17955), and resuspended in complete medium (without IL-2) at a density of 0.45×10 6 / ml. Treg cells were labeled with Cell Trace violet reagent, and the density was adjusted to 0.3×10 6 / ml. Use complete culture medium to gradient dilute 11 candidate antibodies to 4 times the final concentration and set aside. Seed the target cells into the cell plate according to the experimental plate distribution diagram, 50μl / ml, then spread the diluted drugs into the corresponding wells, and incubate with the target cells for 30 minutes at 37°C. After the incubation, add 100μl of effector cell suspension to the wells where effector cells need to be added, and add 100μl of culture medium to the unneeded wells, and incubate for 4h at 37°C. Add 1μl of PI dye to each well and detect on the machine. The stronger the PI signal in the target cell, the more significant the ADCC effect. ADCC experimental results ( Fig.10 Table 9) shows that the negative control anti-Hel isotype has no ADCC killing effect due to the lack of target, while the positive control sample #52 shows a very obvious ADCC action curve. All 11 tested antibodies showed weak ADCC activity. Fig.10 A shows that human Treg highly expresses TNFR2, with an 80-fold shift compared to anti-Hel isotype control antibody staining. Fig.10 B shows that the proportion of NK cells in PBMCs before separation using the kit was 14.5%, and the purity of NK cells after separation using the kit reached 88.3%. Fig.10C shows the ADCC effects of 11 antibodies, #52 antibody is the positive control antibody, anti-Hel isotype antibody is the negative control antibody, and Target alone is the negative control with only target cells Treg but no NK cells.

[0163] Table 9. Comparison of the AUC (Area under the ROC Curve) of the ADCC killing effect curve of the tested antibodies on Treg

[0164]

[0165] Note: The AUC value of the ADCC action curve of the test antibody was compared with that of the positive control #52 antibody sample to obtain the relative ADCC activity.

[0166] Example 15. Detection of ADCC activity of TNFR2 antibody-mediated CHO-TNFR2 cells by reporter gene method

[0167] One day in advance, the target cells (CHO-TNFR2) were plated in a transparent bottom white 96-well plate (Corning, Catalog No.: 3610), and 10,000 cells were plated in each well using detection buffer (RPMI1640 (Gibco, Catalog No. 22400105) + 0.5% FBS (Gibco, Catalog No.: 10099141) + 1×P / S (Gibco, Catalog No.: 15140-122)). The cells were allowed to adhere overnight. The next day, the drug was gradiently diluted to 2 times the final concentration with detection buffer for standby use. The detection buffer in the plate was discarded first, and then 25 μl of new detection buffer was added to each well. Then the diluted drug was plated into the corresponding wells, 25 μl per well, and incubated with the target cells for 1 hour at 37°C. After the incubation, add 25 μl of 2× drug to the corresponding wells, and then add 25 μl of ADCC cell suspension (BPS, catalog number 60541) to the designated wells, with 75,000 cells per well. At this time, there is 100 μl of mixed solution in each well, which is placed in the incubator for incubation for 6 hours. After the incubation, place the well plate at room temperature for 10 minutes, add Bright-Glo (Promega, catalog number: 2620) reagent that has been pre-thawed to room temperature, 100 μl per well, place the well plate on an oscillator and oscillate at 500 rpm for 10 minutes, and then detect the luminescence value on the machine. The stronger the luminescence signal, the more significant the ADCC effect. The results of the ADCC experiment showed that the negative control anti-Helisotype isotype control antibody had no target, and no ADCC killing effect was detected; while the three anti-TNFR2 antibodies #001, #219 and #224 tested all had ADCC effects ( Fig.11 ).

[0168] Example 16. Epitope binning of anti-TNFR2 antibodies binding to human TNFR2 protein

[0169] This method uses Biacore to detect whether there is competition between TNFR2 antibodies when binding to human TNFR2 protein to group antibodies and preliminarily determine which epitope bins they belong to (Abdiche YN et al., Plos One. Mar 20, 2014). Human TNFR2 is fixed by amino coupling, and the selected fixed antibody is first injected, and then other antibodies are further injected separately, and then the injection order of the fixed antibody and other antibodies is alternated. By comparing and analyzing whether there is a difference in the binding signal of the fixed antibody and other antibodies with human TNFR2 in the first and second cases, the relationship between the binding epitopes of the antibodies on the human TNFR2 protein can be determined. Specifically, in each cycle, all antibodies are measured with a concentration of 300nM and injected for 180s, and the antibodies to be tested are eluted with 650mM HCl solution.

[0170] The results in Table 10 show that the TNFR2 antigen epitope bound by antibody 224 is different from that of the other ten antibodies.

[0171] Table 10. Results of Biacore Epitope binning of TNFR2 antibodies on human TNFR2 protein

[0172]

[0173]

[0174] Note: Y: epitope competition; N: no epitope competition

[0175] Example 17. In vivo efficacy testing of anti-mouse TNFR2 surrogate antibodies

[0176] Since the 11 anti-human TNFR2 antibodies in Example 1 specifically bind to TNFR2 of humans and cynomolgus monkeys, but not to TNFR2 of mice, anti-mTNFR2 antibodies were used as surrogate antibodies to establish a PD-1 / PD-L1 therapy-resistant CT26.WT colon cancer animal model in Balb / c mice to conduct TNFR2 target-related efficacy experiments. The surrogate antibody anti-mTNFR2 was purchased from BioXcell (Cat. No. BE0247). 1×10 5 CT26 colon cancer cells (set as day 0 of the experiment). When the tumor volume reaches ~110mm 3At the time of the experiment, mice with moderate tumor volume were selected for enrollment. The animals were randomly divided into four experimental groups according to the tumor volume using the random grouping method based on EXCEL random numbers (G1 was the control group, G2 was the anti-mTNFR2 alternative antibody group, G3 was the anti-mPD-L1 (BioXcell, Catalog No. BE0101) antibody group, and G4 was the anti-mPD-1 (BioXcell, Catalog No. BE0146) antibody group, with 8 mice in each group. The drug was administered on the day of grouping (Day 13). The dosages were anti-mTNFR2 5 mg / kg (mpk), anti-mPD-L1 10 mg / kg (mpk), and anti-mPD-1 10 mg / kg (mpk), respectively, and the drugs were administered by intraperitoneal injection twice a week. The tumor volume and body weight were measured three times at fixed times each week.

[0177] On the 21st day after tumor inoculation, the tumor volume growth of the anti-mTNFR2 group and the anti-mPD-L1 group was significantly inhibited compared with the control group, and there was a statistical difference (P<0.05); and the anti-mTNFR2 group had a larger TGI value than the anti-mPD-L1 group. The results show that the anti-mTNFR2 antibody has a better efficacy in this tumor model than the anti-PD-1 or anti-PD-L1 antibody ( Fig.12 AB).

[0178] The animals in each group were active and ate well during the administration period, and their body weight increased to a certain extent. The results show that the anti-mTNFR2 antibody has good safety ( Fig.12 C).

[0179] 24 h after the last administration, most mice in the G1 control group and G2 anti-mTNFR2 antibody group were selected for TIL isolation and FACS immunophenotyping. The gates of all cell populations were based on the FMO control ( Fig.13 AB), the results showed that the CD8 + The number of T cells in the control group was significantly higher than that in the control group (p<0.01). Fig.13 C); Total CD8 + T and memory CD8 + The ratio of T cells to Treg cells (CD8 + T / Treg) was also significantly higher than that in the control group, with statistical significance (p<0.001) ( Fig.13 DE); anti-mTNFR2 treatment group CD8 +The average fluorescence intensity of PD-1 expressed by T cells was lower than that of the isotype control group, with a significant difference (p<0.001) ( Fig.13 F). The above data suggest that the anti-tumor effect of anti-mTNFR2 antibodies may be achieved by increasing CD8 + T / Treg ratio and partial reversal of CD8 + The immune exhaustion state of T cells is achieved.

[0180] The calculation formula of tumor volume (TV) is: V = 1 / 2 × a × b 2 . Where a and b represent length and width, respectively. The relative tumor volume (RTV) was calculated based on the measurement results, and the calculation formula was: RTV = Vt / V0. Where V0 is the tumor volume measured at the time of cage administration (i.e. Day 0), and Vt is the tumor volume at each measurement. TGI% = [1-RTV (experimental group) / RTV (control group)] × 100%.

[0181] Example 18. In vivo efficacy test of anti-mouse TNFR2 surrogate antibody combined with anti-mouse PD-1 surrogate antibody

[0182] Mice were inoculated subcutaneously on the right side with 1×10 5 CT26.WT colon cancer cells (set as day 0 of the experiment). When the tumor volume reaches ~100mm 3 At the time of the study, mice with moderate tumor volume were selected for enrollment. The animals were randomly divided into 4 experimental groups (G1 as control group, G2 as anti-mTNFR2 antibody group, G3 as anti-mPD-1 antibody group, G4 as anti-mTNFR2+anti-mPD-1 antibody group) according to the tumor volume using the random grouping method based on EXCEL random numbers. There were 9 mice in each of the 4 groups for in vivo pharmacodynamic studies. The drug was administered on the day of grouping (day 14). The dosages were anti-mTNFR2 5mpk and anti-mPD-1 10mpk, respectively, and the drugs were administered by intraperitoneal injection twice a week. The tumor volume and body weight were measured three times a week at fixed times.

[0183] On the 28th day after tumor inoculation, compared with the control group, the tumor volume growth of the G2 anti-mTNFR2 group and the G3 anti-mPD-1 group was significantly inhibited, and there was a statistical difference (P<0.05); and the anti-mTNFR2 group had a larger TGI value than the anti-mPD-1 group. The results showed that the anti-mTNFR2 antibody had a better efficacy in this animal model; the G4 combined treatment group had the best efficacy, and compared with each single drug group, there was a statistical difference (p<0.01, p<0.001), indicating that the combined administration of anti-mTNFR2 antibody and anti-mPD-1 antibody has a synergistic effect ( Fig.14 AB).

[0184] After stopping the drug administration on the 28th day, the first four groups of mice entered the observation phase after drug withdrawal, and the tumors of the mice were recorded at least once a week. After drug withdrawal, the tumor volumes of the G2 anti-mTNFR2 group and the G3 anti-mPD-1 group began to grow slowly, and the mice in the two groups died completely at the latest on the 55th day, while there were still mice surviving in the G4 combination treatment group on the 68th day, and the survival rate of this group was always higher than 50% (55.6%); the tumor volumes of 3 mice in the G4 combination treatment group gradually subsided and did not grow, which was significantly different from the single drug group and the control group (p<0.001, p<0.0001), indicating that the combination group had better efficacy than each single drug group ( Fig.14 C). Sequence Listing <110> Shandong Innovent Biological Pharmaceutical Co., Ltd. <120> Anti-TNFR2 antibodies and uses thereof <130> MP21005308P <150> 201910713742.1 <151> 2019-08-02 <160> 154 <170> SIPOSequenceListing 1.0 <210> 1 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(121) <223> Heavy chain variable region of positive clone Clone ID 1 <400> 1 Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Asn Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Thr Tyr 20 25 30 Asp Leu Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Tyr Ile Asn Asn Gly Gly Ile Ser Thr Tyr Tyr Ser Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Val Arg Gly Pro Phe Tyr Gly Ser Ala Asn Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 2 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(107) <223> Light chain variable region of Clone ID 1 of positive clone <400> 2 Glu Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ser Leu Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Thr Ser Glu Ser Ile Tyr Ser Asn 20 25 30 Leu Pro Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asp Ala Thr Lys Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Glu Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His Phe Trp Val Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 3 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(121) <223> Heavy chain variable region of clone ID 88 <400> 3 Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Asn Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Thr Tyr 20 25 30 Asp Leu Ser Trp Val Arg Gln Thr Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Tyr Ile Asn Asn Gly Gly Ile Ser Thr Tyr Tyr Ser Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Thr Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Val Arg Gly Pro Phe Tyr Gly Ser Ala Asn Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 4 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(107) <223> Light chain variable region of positive clone Clone ID 88 <400> 4 Asp Ile Leu Met Thr Gln Ser Pro Ala Ser Leu Ser Leu Phe Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asp Ala Thr Lys Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Glu Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His Phe Trp Val Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 5 <211> 121 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(121) <223> Heavy chain variable region of clone ID 125 <400> 5 Glu Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Asn Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ile Tyr 20 25 30 Asp Leu Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Tyr Ile Asn Asn Gly Gly Ile Ser Thr Tyr Tyr Ser Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Val Arg Gly Pro Phe Tyr Gly Ser Ala Asn Tyr Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 6 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(107) <223> Light chain variable region of positive clone Clone ID 125 <400> 6 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Leu Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asp Ala Thr Lys Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Glu Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Gly Thr Tyr Tyr Cys Gln His Phe Trp Val Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 7 <211> 119 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(119) <223> Heavy chain variable region of clone ID 133 <400> 7 Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Val Arg Ser Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Ile Gln Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asp Pro Glu Ser Gly Asn Thr Lys Tyr Ala Pro Lys Phe 50 55 60 Gln Asp Lys Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Asn Ala Tyr Tyr Asp Tyr Asp Gly Ser Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 8 <211> 113 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(113) <223> Light chain variable region of positive clone Clone ID 133 <400> 8 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe 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 Thr Asp Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Thr Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 9 <211> 123 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(123) <223> Heavy chain variable region of positive clone Clone ID 219 <400> 9 Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Ser Asp Thr Glu Thr Arg Leu Asn Gln Asn Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ser Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Glu Gly Leu Gly Ala Ala Arg Ser Val Ser Met Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 10 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(107) <223> Light chain variable region of positive clone Clone ID 219 <400> 10 Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Val Leu Ile 35 40 45 Tyr Tyr Thr Ala Ile Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 11 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(120) <223> Heavy chain variable region of clone ID 224 <400> 11 Glu Val Gln Leu Gln Glu Ser Gly Pro Glu Ile Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Asn Lys Asp Ile 20 25 30 Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Thr Gly Asn Thr Lys His Asp Pro Lys Phe 50 55 60 Gln Asp Lys Ala Thr Leu Ser Ser Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Phe Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala His Ser Pro Tyr Gly Asp Phe Gly Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 12 <211> 108 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(108) <223> Light chain variable region of positive clone Clone ID 224 <400> 12 Asp Ile Gln Met Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Asn 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Pro Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Tyr His Arg Ser Pro 85 90 95 Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 13 <211> 123 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(123) <223> Heavy chain variable region of clone ID 226 <400> 13 Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Glu Gly Leu Gly Ala Ala Arg Ser Val Ser Met Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 14 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(107) <223> Light chain variable region of positive clone Clone ID 226 <400> 14 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Val Leu Ile 35 40 45 Tyr Tyr Thr Ala Leu Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 15 <211> 120 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(120) <223> Heavy chain variable region of clone ID 309 <400> 15 Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile His Trp Val Lys Leu Arg Pro Gly Gln Gly Phe Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Asn Asn Gly Gly Thr Asp Tyr Asn Glu Lys Phe 50 55 60 Lys Arg Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Thr Ile Asp Ser Met Ile Thr Thr Thr Trp Phe Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 16 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(107) <223> Light chain variable region of positive clone Clone ID 309 <400> 16 Glu Ile Val Met Thr Gln Ser Gln Lys Leu Met Ser Thr Val Val Gly 1 5 10 15 Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn 20 25 30 Val Ala Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile 35 40 45 Asn Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 17 <211> 119 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(119) <223> Heavy chain variable region of positive clone Clone ID 352 <400> 17 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Val Val Ile Trp Ser Asp Gly Gly Thr Thr Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Arg His Asp Asp Asp Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 18 <211> 106 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(106) <223> Light chain variable region of positive clone Clone ID 352 <400> 18 Asp Ile Val Leu Thr Gln Ser Pro Ser Leu Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Gly Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Val Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asp Pro Phe Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 19 <211> 119 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(119) <223> Heavy chain variable region of positive clone Clone ID 365 <400> 19 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Leu Ile Trp Ser Asp Gly Ser Pro Asp Tyr Ser Ala Ala Phe Ile 50 55 60 Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg Asn Asp Asp Gly Gly Asp Tyr Val Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 20 <211> 106 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(106) <223> Light chain variable region of positive clone Clone ID 365 <400> 20 Glu Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys 100 105 <210> twenty one <211> 119 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(119) <223> Heavy chain variable region of positive clone Clone ID 395 <400> twenty one Glu Val Gln Leu Gln Glu Ser Gly Pro Val Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Ser Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser Asp Gly Gly Thr Asp Tyr Ser Ala Ala Phe Ile 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Arg Asn Asp Asp Gly Gly Asp Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> twenty two <211> 106 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(106) <223> Light chain variable region of positive clone Clone ID 395 <400> twenty two Glu Ile Val Leu Thr Gln Ser Pro Ala Leu Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys 100 105 <210> twenty three <211> 5 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(5) <223> CDR1-VH of positive clone Clone ID 1 analyzed by KABAT <400> twenty three Thr Tyr Asp Leu Ser 1 5 <210> twenty four <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(17) <223> CDR2-VH of positive clone Clone ID 1 analyzed by KABAT <400> twenty four Tyr Ile Asn Asn Gly Gly Ile Ser Thr Tyr Tyr Ser Asp Thr Val Lys 1 5 10 15 Gly <210> 25 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(12) <223> CDR3-VH of positive clone Clone ID 1 analyzed by KABAT <400> 25 Gly Pro Phe Tyr Gly Ser Ala Asn Tyr Phe Asp Tyr 1 5 10 <210> 26 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(8) <223> CDR1-VH of positive clone Clone ID 1 analyzed by IMGT <400> 26 Gly Phe Ala Phe Ser Thr Tyr Asp 1 5 <210> 27 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(8) <223> CDR2-VH of positive clone Clone ID 1 analyzed by IMGT <400> 27 Ile Asn Asn Gly Gly Ile Ser Thr 1 5 <210> 28 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(14) <223> CDR3-VH of positive clone Clone ID 1 analyzed by IMGT <400> 28 Val Arg Gly Pro Phe Tyr Gly Ser Ala Asn Tyr Phe Asp Tyr 1 5 10 <210> 29 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(11) <223> CDR1-VL of positive clone Clone ID 1 analyzed by KABAT <400> 29 Arg Thr Ser Glu Ser Ile Tyr Ser Asn Leu Pro 1 5 10 <210> 30 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(7) <223> CDR2-VL of positive clone Clone ID 1 analyzed by KABAT <400> 30 Asp Ala Thr Lys Leu Ala Glu 1 5 <210> 31 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(9) <223> CDR3-VL of positive clone Clone ID 1 analyzed by KABAT <400> 31 Gln His Phe Trp Val Thr Pro Trp Thr 1 5 <210> 32 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(6) <223> CDR1-VL of positive clone Clone ID 1 analyzed by IMGT <400> 32 Glu Ser Ile Tyr Ser Asn 1 5 <210> 33 <211> 3 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(3) <223> CDR2-VL of positive clone Clone ID 1 analyzed by IMGT <400> 33 Asp Ala Thr 1 <210> 34 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(9) <223> CDR3-VL of positive clone Clone ID 1 analyzed by IMGT <400> 34 Gln His Phe Trp Val Thr Pro Trp Thr 1 5 <210> 35 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(5) <223> CDR1-VH of positive clone Clone ID 88 analyzed by KABAT <400> 35 Thr Tyr Asp Leu Ser 1 5 <210> 36 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(17) <223> CDR2-VH of positive clone Clone ID 88 analyzed by KABAT <400> 36 Tyr Ile Asn Asn Gly Gly Ile Ser Thr Tyr Tyr Ser Asp Thr Val Lys 1 5 10 15 Gly <210> 37 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(12) <223> CDR3-VH of positive clone Clone ID 88 analyzed by KABAT <400> 37 Gly Pro Phe Tyr Gly Ser Ala Asn Tyr Phe Asp Tyr 1 5 10 <210> 38 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(8) <223> CDR1-VH of positive clone Clone ID 88 analyzed by IMGT <400> 38 Gly Phe Ala Phe Ser Thr Tyr Asp 1 5 <210> 39 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(8) <223> CDR2-VH of positive clone Clone ID 88 analyzed by IMGT <400> 39 Ile Asn Asn Gly Gly Ile Ser Thr 1 5 <210> 40 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(14) <223> CDR3-VH of positive clone Clone ID 88 analyzed by IMGT <400> 40 Val Arg Gly Pro Phe Tyr Gly Ser Ala Asn Tyr Phe Asp Tyr 1 5 10 <210> 41 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(11) <223> CDR1-VL of positive clone Clone ID 88 analyzed by KABAT <400> 41 Arg Ala Ser Glu Asn Ile Tyr Ser Asn Leu Ala 1 5 10 <210> 42 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(7) <223> CDR2-VL of positive clone Clone ID 88 analyzed by KABAT <400> 42 Asp Ala Thr Lys Leu Ala Glu 1 5 <210> 43 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(9) <223> CDR3-VL of positive clone Clone ID 88 analyzed by KABAT <400> 43 Gln His Phe Trp Val Thr Pro Trp Thr 1 5 <210> 44 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(6) <223> CDR1-VL of positive clone Clone ID 88 analyzed by IMGT <400> 44 Glu Asn Ile Tyr Ser Asn 1 5 <210> 45 <211> 3 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(3) <223> CDR2-VL of positive clone Clone ID 88 analyzed by IMGT <400> 45 Asp Ala Thr 1 <210> 46 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(9) <223> CDR3-VL of positive clone Clone ID 88 analyzed by IMGT <400> 46 Gln His Phe Trp Val Thr Pro Trp Thr 1 5 <210> 47 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(5) <223> CDR1-VH of positive clone Clone ID 125 analyzed by KABAT <400> 47 Ile Tyr Asp Leu Ser 1 5 <210> 48 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(17) <223> CDR2-VH of positive clone Clone ID 125 analyzed by KABAT <400> 48 Tyr Ile Asn Asn Gly Gly Ile Ser Thr Tyr Tyr Ser Asp Thr Val Lys 1 5 10 15 Gly <210> 49 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(12) <223> CDR3-VH of positive clone Clone ID 125 analyzed by KABAT <400> 49 Gly Pro Phe Tyr Gly Ser Ala Asn Tyr Phe Asp Tyr 1 5 10 <210> 50 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(8) <223> CDR1-VH of positive clone Clone ID 125 analyzed by IMGT <400> 50 Gly Phe Ala Phe Ser Ile Tyr Asp 1 5 <210> 51 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(8) <223> CDR2-VH of positive clone Clone ID 125 analyzed by IMGT <400> 51 Ile Asn Asn Gly Gly Ile Ser Thr 1 5 <210> 52 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(14) <223> CDR3-VH of positive clone Clone ID 125 analyzed by IMGT <400> 52 Val Arg Gly Pro Phe Tyr Gly Ser Ala Asn Tyr Phe Asp Tyr 1 5 10 <210> 53 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(11) <223> CDR1-VL of positive clone Clone ID 125 analyzed by KABAT <400> 53 Arg Ala Ser Glu Asn Ile Tyr Ser Asn Leu Ala 1 5 10 <210> 54 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(7) <223> CDR2-VL of positive clone Clone ID 125 analyzed by KABAT <400> 54 Asp Ala Thr Lys Leu Ala Glu 1 5 <210> 55 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(9) <223> CDR3-VL of positive clone Clone ID 125 analyzed by KABAT <400> 55 Gln His Phe Trp Val Thr Pro Trp Thr 1 5 <210> 56 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(6) <223> CDR1-VL of positive clone Clone ID 125 analyzed by IMGT <400> 56 Glu Asn Ile Tyr Ser Asn 1 5 <210> 57 <211> 3 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(3) <223> CDR2-VL of positive clone Clone ID 125 analyzed by IMGT <400> 57 Asp Ala Thr 1 <210> 58 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(9) <223> CDR3-VL of positive clone Clone ID 125 analyzed by IMGT <400> 58 Gln His Phe Trp Val Thr Pro Trp Thr 1 5 <210> 59 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(5) <223> CDR1-VH of positive clone Clone ID 133 analyzed by KABAT <400> 59 Asp Tyr Tyr Ile Gln 1 5 <210> 60 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(17) <223> CDR2-VH of positive clone Clone ID 133 analyzed by KABAT <400> 60 Trp Ile Asp Pro Glu Ser Gly Asn Thr Lys Tyr Ala Pro Lys Phe Gln 1 5 10 15 Asp <210> 61 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(10) <223> CDR3-VH of positive clone Clone ID 133 analyzed by KABAT <400> 61 Tyr Tyr Asp Tyr Asp Gly Ser Met Asp Tyr 1 5 10 <210> 62 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(8) <223> CDR1-VH of positive clone Clone ID 133 analyzed by IMGT <400> 62 Gly Phe Asn Ile Lys Asp Tyr Tyr 1 5 <210> 63 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(8) <223> CDR2-VH of positive clone Clone ID 133 analyzed by IMGT <400> 63 Ile Asp Pro Glu Ser Gly Asn Thr 1 5 <210> 64 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(12) <223> CDR3-VH of positive clone Clone ID 133 analyzed by IMGT <400> 64 Asn Ala Tyr Tyr Asp Tyr Asp Gly Ser Met Asp Tyr 1 5 10 <210> 65 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(16) <223> CDR1-VL of positive clone Clone ID 133 analyzed by KABAT <400> 65 Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 1 5 10 15 <210> 66 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(7) <223> CDR2-VL of positive clone Clone ID 133 analyzed by KABAT <400> 66 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 67 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(10) <223> CDR3-VL of positive clone Clone ID 133 analyzed by KABAT <400> 67 Ser Gln Ser Thr His Val Pro Thr Trp Thr 1 5 10 <210> 68 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(11) <223> CDR1-VL of positive clone Clone ID 133 analyzed by IMGT <400> 68 Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr 1 5 10 <210> 69 <211> 3 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(3) <223> CDR2-VL of positive clone Clone ID 133 analyzed by IMGT <400> 69 Lys Val Ser 1 <210> 70 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(10) <223> CDR3-VL of positive clone Clone ID 133 analyzed by IMGT <400> 70 Ser Gln Ser Thr His Val Pro Thr Trp Thr 1 5 10 <210> 71 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(5) <223> CDR1-VH of positive clone Clone ID 219 analyzed by KABAT <400> 71 Asn Tyr Trp Met Asn 1 5 <210> 72 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(17) <223> CDR2-VH of positive clone Clone ID 219 analyzed by KABAT <400> 72 Met Ile His Pro Ser Asp Thr Glu Thr Arg Leu Asn Gln Asn Phe Lys 1 5 10 15 Asp <210> 73 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(14) <223> CDR3-VH of positive clone Clone ID 219 analyzed by KABAT <400> 73 Gly Glu Gly Leu Gly Ala Ala Arg Ser Val Ser Met Asp Tyr 1 5 10 <210> 74 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(8) <223> CDR1-VH of positive clone Clone ID 219 analyzed by IMGT <400> 74 Gly Tyr Ser Phe Thr Asn Tyr Trp 1 5 <210> 75 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(8) <223> CDR2-VH of positive clone Clone ID 219 analyzed by IMGT <400> 75 Ile His Pro Ser Asp Thr Glu Thr 1 5 <210> 76 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(16) <223> CDR3-VH of positive clone Clone ID 219 analyzed by IMGT <400> 76 Ala Arg Gly Glu Gly Leu Gly Ala Ala Arg Ser Val Ser Met Asp Tyr 1 5 10 15 <210> 77 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(11) <223> CDR1-VL of positive clone Clone ID 219 analyzed by KABAT <400> 77 Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn 1 5 10 <210> 78 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(7) <223> CDR2-VL of positive clone Clone ID 219 analyzed by KABAT <400> 78 Tyr Thr Ala Ile Leu His Ser 1 5 <210> 79 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(9) <223> CDR3-VL of positive clone Clone ID 219 analyzed by KABAT <400> 79 Gln Gln Gly Asn Thr Leu Pro Trp Thr 1 5 <210> 80 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(6) <223> CDR1-VL of positive clone Clone ID 219 analyzed by IMGT <400> 80 Gln Asp Ile Ser Asn Tyr 1 5 <210> 81 <211> 3 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(3) <223> CDR2-VL of positive clone Clone ID 219 analyzed by IMGT <400> 81 Tyr Thr Ala 1 <210> 82 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(9) <223> CDR3-VL of positive clone Clone ID 219 analyzed by IMGT <400> 82 Gln Gln Gly Asn Thr Leu Pro Trp Thr 1 5 <210> 83 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(5) <223> CDR1-VH of positive clone Clone ID 224 analyzed by KABAT <400> 83 Asp Ile Tyr Met His 1 5 <210> 84 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(17) <223> CDR2-VH of positive clone Clone ID 224 analyzed by KABAT <400> 84 Arg Ile Asp Pro Ala Thr Gly Asn Thr Lys His Asp Pro Lys Phe Gln 1 5 10 15 Asp <210> 85 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(11) <223> CDR3-VH of positive clone Clone ID 224 analyzed by KABAT <400> 85 Ser Pro Tyr Gly Asp Phe Gly Ala Met Asp Tyr 1 5 10 <210> 86 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(8) <223> CDR1-VH of positive clone Clone ID 224 analyzed by IMGT <400> 86 Gly Phe Asn Asn Lys Asp Ile Tyr 1 5 <210> 87 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(8) <223> CDR2-VH of positive clone Clone ID 224 analyzed by IMGT <400> 87 Ile Asp Pro Ala Thr Gly Asn Thr 1 5 <210> 88 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(13) <223> CDR3-VH of positive clone Clone ID 224 analyzed by IMGT <400> 88 Ala His Ser Pro Tyr Gly Asp Phe Gly Ala Met Asp Tyr 1 5 10 <210> 89 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(12) <223> CDR1-VL of positive clone Clone ID 224 analyzed by KABAT <400> 89 Thr Ala Ser Ser Ser Val Ser Ser Asn Tyr Leu His 1 5 10 <210> 90 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(7) <223> CDR2-VL of positive clone Clone ID 224 analyzed by KABAT <400> 90 Ser Thr Ser Asn Leu Pro Ser 1 5 <210> 91 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(9) <223> CDR3-VL of positive clone Clone ID 224 analyzed by KABAT <400> 91 His Gln Tyr His Arg Ser Pro Trp Thr 1 5 <210> 92 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(7) <223> CDR1-VL of positive clone Clone ID 224 analyzed by IMGT <400> 92 Ser Ser Val Ser Ser Asn Tyr 1 5 <210> 93 <211> 3 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(3) <223> CDR2-VL of positive clone Clone ID 224 analyzed by IMGT <400> 93 Ser Thr Ser 1 <210> 94 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(9) <223> CDR3-VL of positive clone Clone ID 224 analyzed by IMGT <400> 94 His Gln Tyr His Arg Ser Pro Trp Thr 1 5 <210> 95 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(5) <223> CDR1-VH of positive clone Clone ID 226 analyzed by KABAT <400> 95 Asn Tyr Trp Met Asn 1 5 <210> 96 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(17) <223> CDR2-VH of positive clone Clone ID 226 analyzed by KABAT <400> 96 Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 97 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(14) <223> CDR3-VH of positive clone Clone ID 226 analyzed by KABAT <400> 97 Gly Glu Gly Leu Gly Ala Ala Arg Ser Val Ser Met Asp Tyr 1 5 10 <210> 98 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(8) <223> CDR1-VH of positive clone Clone ID 226 analyzed by IMGT <400> 98 Gly Tyr Ser Phe Thr Asn Tyr Trp 1 5 <210> 99 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(8) <223> CDR2-VH of positive clone Clone ID 226 analyzed by IMGT <400> 99 Ile His Pro Ser Asp Ser Glu Thr 1 5 <210> 100 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(16) <223> CDR3-VH of positive clone Clone ID 226 analyzed by IMGT <400> 100 Ala Arg Gly Glu Gly Leu Gly Ala Ala Arg Ser Val Ser Met Asp Tyr 1 5 10 15 <210> 101 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(11) <223> CDR1-VL of positive clone Clone ID 226 analyzed by KABAT <400> 101 Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn 1 5 10 <210> 102 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(7) <223> CDR2-VL of positive clone Clone ID 226 analyzed by KABAT <400> 102 Tyr Thr Ala Leu Leu His Ser 1 5 <210> 103 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(9) <223> CDR3-VL of positive clone Clone ID 226 analyzed by KABAT <400> 103 Gln Gln Gly Asn Thr Leu Pro Trp Thr 1 5 <210> 104 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(6) <223> CDR1-VL of positive clone Clone ID 226 analyzed by IMGT <400> 104 Gln Asp Ile Ser Asn Tyr 1 5 <210> 105 <211> 3 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(3) <223> CDR2-VL of positive clone Clone ID 226 analyzed by IMGT <400> 105 Tyr Thr Ala 1 <210> 106 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(9) <223> CDR3-VL of positive clone Clone ID 226 analyzed by IMGT <400> 106 Gln Gln Gly Asn Thr Leu Pro Trp Thr 1 5 <210> 107 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(5) <223> CDR1-VH of positive clone Clone ID 309 analyzed by KABAT <400> 107 Ser Tyr Trp Ile His 1 5 <210> 108 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(17) <223> CDR2-VH of positive clone Clone ID 309 analyzed by KABAT <400> 108 Glu Ile Asn Pro Asn Asn Gly Gly Thr Asp Tyr Asn Glu Lys Phe Lys 1 5 10 15 Arg <210> 109 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(11) <223> CDR3-VH of positive clone Clone ID 309 analyzed by KABAT <400> 109 Asp Ser Met Ile Thr Thr Thr Trp Phe Ala Tyr 1 5 10 <210> 110 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(8) <223> CDR1-VH of positive clone Clone ID 309 analyzed by IMGT <400> 110 Gly Tyr Thr Phe Thr Ser Tyr Trp 1 5 <210> 111 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(8) <223> CDR2-VH of positive clone Clone ID 309 analyzed by IMGT <400> 111 Ile Asn Pro Asn Asn Gly Gly Thr 1 5 <210> 112 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(13) <223> CDR3-VH of positive clone Clone ID 309 analyzed by IMGT <400> 112 Thr Ile Asp Ser Met Ile Thr Thr Thr Trp Phe Ala Tyr 1 5 10 <210> 113 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(11) <223> CDR1-VL of positive clone Clone ID 309 analyzed by KABAT <400> 113 Lys Ala Ser Gln Asn Val Gly Thr Asn Val Ala 1 5 10 <210> 114 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(7) <223> CDR2-VL of positive clone Clone ID 309 analyzed by KABAT <400> 114 Ser Ala Ser Tyr Arg Tyr Ser 1 5 <210> 115 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(9) <223> CDR3-VL of positive clone Clone ID 309 analyzed by KABAT <400> 115 Gln Gln Tyr Asn Ser Tyr Pro Phe Thr 1 5 <210> 116 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(6) <223> CDR1-VL of positive clone Clone ID 309 analyzed by IMGT <400> 116 Gln Asn Val Gly Thr Asn 1 5 <210> 117 <211> 3 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(3) <223> CDR2-VL of positive clone Clone ID 309 analyzed by IMGT <400> 117 Ser Ala Ser 1 <210> 118 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(9) <223> CDR3-VL of positive clone Clone ID 309 analyzed by IMGT <400> 118 Gln Gln Tyr Asn Ser Tyr Pro Phe Thr 1 5 <210> 119 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(5) <223> CDR1-VH of positive clone Clone ID 352 analyzed by KABAT <400> 119 Ser Tyr Gly Val His 1 5 <210> 120 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(16) <223> CDR2-VH of positive clone Clone ID 352 analyzed by KABAT <400> 120 Val Ile Trp Ser Asp Gly Gly Thr Thr Tyr Asn Ser Ala Leu Lys Ser 1 5 10 15 <210> 121 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(11) <223> CDR3-VH of positive clone Clone ID 352 analyzed by KABAT <400> 121 His Asp Asp Asp Gly Tyr Tyr Ala Met Asp Tyr 1 5 10 <210> 122 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(8) <223> CDR1-VH of positive clone Clone ID 352 analyzed by IMGT <400> 122 Gly Phe Ser Leu Thr Ser Tyr Gly 1 5 <210> 123 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(7) <223> CDR2-VH of positive clone Clone ID 352 analyzed by IMGT <400> 123 Ile Trp Ser Asp Gly Gly Thr 1 5 <210> 124 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(13) <223> CDR3-VH of positive clone Clone ID 352 analyzed by IMGT <400> 124 Ala Arg His Asp Asp Asp Gly Tyr Tyr Ala Met Asp Tyr 1 5 10 <210> 125 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(10) <223> CDR1-VL of positive clone Clone ID 352 analyzed by KABAT <400> 125 Ser Ala Ser Ser Ser Val Gly Tyr Met Tyr 1 5 10 <210> 126 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(7) <223> CDR2-VL of positive clone Clone ID 352 analyzed by KABAT <400> 126 Leu Thr Ser Asn Leu Ala Ser 1 5 <210> 127 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(9) <223> CDR3-VL of positive clone Clone ID 352 analyzed by KABAT <400> 127 Gln Gln Trp Ser Ser Asp Pro Phe Thr 1 5 <210> 128 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(5) <223> CDR1-VL of positive clone Clone ID 352 analyzed by IMGT <400> 128 Ser Ser Val Gly Tyr 1 5 <210> 129 <211> 3 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(3) <223> CDR2-VL of positive clone Clone ID 352 analyzed by IMGT <400> 129 Leu Thr Ser 1 <210> 130 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(9) <223> CDR3-VL of positive clone Clone ID 352 analyzed by IMGT <400> 130 Gln Gln Trp Ser Ser Asp Pro Phe Thr 1 5 <210> 131 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(5) <223> CDR1-VH of positive clone Clone ID 365 analyzed by KABAT <400> 131 Ser Tyr Gly Val His 1 5 <210> 132 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(16) <223> CDR2-VH of positive clone Clone ID 365 analyzed by KABAT <400> 132 Leu Ile Trp Ser Asp Gly Ser Pro Asp Tyr Ser Ala Ala Phe Ile Ser 1 5 10 15 <210> 133 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(11) <223> CDR3-VH of positive clone Clone ID 365 analyzed by KABAT <400> 133 Asn Asp Asp Gly Gly Asp Tyr Val Met Asp Tyr 1 5 10 <210> 134 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(8) <223> CDR1-VH of positive clone Clone ID 365 analyzed by IMGT <400> 134 Gly Phe Ser Leu Thr Ser Tyr Gly 1 5 <210> 135 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(7) <223> CDR2-VH of positive clone Clone ID 365 analyzed by IMGT <400> 135 Ile Trp Ser Asp Gly Ser Pro 1 5 <210> 136 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(13) <223> CDR3-VH of positive clone Clone ID 365 analyzed by IMGT <400> 136 Ala Arg Asn Asp Asp Gly Gly Asp Tyr Val Met Asp Tyr 1 5 10 <210> 137 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(10) <223> CDR1-VL of positive clone Clone ID 365 analyzed by KABAT <400> 137 Ser Ala Ser Ser Ser Val Ser Tyr Met Tyr 1 5 10 <210> 138 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(7) <223> CDR2-VL of positive clone Clone ID 365 analyzed by KABAT <400> 138 Leu Thr Ser Asn Leu Ala Ser 1 5 <210> 139 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(9) <223> CDR3-VL of positive clone Clone ID 365 analyzed by KABAT <400> 139 Gln Gln Trp Thr Ser Asn Pro Leu Thr 1 5 <210> 140 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(5) <223> CDR1-VL of positive clone Clone ID 365 analyzed by IMGT <400> 140 Ser Ser Val Ser Tyr 1 5 <210> 141 <211> 3 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(3) <223> CDR2-VL of positive clone Clone ID 365 analyzed by IMGT <400> 141 Leu Thr Ser 1 <210> 142 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(9) <223> CDR3-VL of positive clone Clone ID 365 analyzed by IMGT <400> 142 Gln Gln Trp Thr Ser Asn Pro Leu Thr 1 5 <210> 143 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(5) <223> CDR1-VH of positive clone Clone ID 395 analyzed by KABAT <400> 143 Ser Tyr Ser Val His 1 5 <210> 144 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(16) <223> CDR2-VH of positive clone Clone ID 395 analyzed by KABAT <400> 144 Val Ile Trp Ser Asp Gly Gly Thr Asp Tyr Ser Ala Ala Phe Ile Ser 1 5 10 15 <210> 145 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(11) <223> CDR3-VH of positive clone Clone ID 395 analyzed by KABAT <400> 145 Asn Asp Asp Gly Gly Asp Tyr Ala Met Asp Tyr 1 5 10 <210> 146 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(8) <223> CDR1-VH of positive clone Clone ID 395 analyzed by IMGT <400> 146 Gly Phe Ser Leu Thr Ser Tyr Ser 1 5 <210> 147 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(7) <223> CDR2-VH of positive clone Clone ID 395 analyzed by IMGT <400> 147 Ile Trp Ser Asp Gly Gly Thr 1 5 <210> 148 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(13) <223> CDR3-VH of positive clone Clone ID 395 analyzed by IMGT <400> 148 Ala Arg Asn Asp Asp Gly Gly Asp Tyr Ala Met Asp Tyr 1 5 10 <210> 149 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(10) <223> CDR1-VL of positive clone Clone ID 395 analyzed by KABAT <400> 149 Ser Ala Ser Ser Ser Val Ser Tyr Met Tyr 1 5 10 <210> 150 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(7) <223> CDR2-VL of positive clone Clone ID 395 analyzed by KABAT <400> 150 Leu Thr Ser Asn Leu Ala Ser 1 5 <210> 151 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(9) <223> CDR3-VL of positive clone Clone ID 395 analyzed by KABAT <400> 151 Gln Gln Trp Ser Ser Asn Pro Leu Thr 1 5 <210> 152 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(5) <223> CDR1-VL of positive clone Clone ID 395 analyzed by IMGT <400> 152 Ser Ser Val Ser Tyr 1 5 <210> 153 <211> 3 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(3) <223> CDR2-VL of positive clone Clone ID 395 analyzed by IMGT <400> 153 Leu Thr Ser 1 <210> 154 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <222> (1)..(9) <223> CDR3-VL of positive clone Clone ID 395 analyzed by IMGT <400> 154 Gln Gln Trp Ser Ser Asn Pro Leu Thr 1 5

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to TNFR2, can regulate the function of immune cells, and can be used as a drug to treat diseases related to immune abnormalities; An antibody or antigen-binding fragment thereof comprising: (1) a heavy chain CDR combination of CDR1-VH, CDR2-VH and CDR3-VH, The CDR1-VH, CDR2-VH and CDR3-VH are selected from any combination of the following sequences: and, (2) light chain CDR combinations of CDR1-VL, CDR2-VL and CDR3-VL, The CDR1-VL, CDR2-VL and CDR3-VL are selected from any combination of the following sequences: in: VH5 and VL5 were coded according to the common analysis method of IMGT; VH16 and VL16 were coded according to the popular analysis method of KABAT; The heavy chain CDR and light chain CDR combination is: VH5+VL5 or VH16+VL16.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the regulation comprises inhibiting the proliferation and / or activation of regulatory T cells (Treg cells) and / or myeloid derived suppressor cells (MDSCs).

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the modulation is achieved by blocking the binding of TNFα to TNFR2.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region and the light chain variable region have the sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

5. The antibody or antigen-binding fragment thereof of claim 1, which has a dissociation constant (KD) of no greater than 5 nM for binding to human TNFR2 and a dissociation constant (KD) of no greater than 5 nM for binding to cynomolgus monkey TNFR2. The antibody or antigen-binding fragment thereof according to claim 1 , which is a chimeric antibody or a humanized antibody.

7. The antibody or antigen-binding fragment thereof of claim 1, comprising a constant region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD, and wherein: 1) Specifically bind to cells expressing TNFR2 on their surface; 2) Specific binding to Treg cells; 3) Inhibit the binding of TNFα to TNFR2 protein; 4) Inhibit the binding of TNFα to TNFR2 expressed on the cell surface; 5) Inhibit TNFα-mediated Treg proliferation and / or Treg function; 6) mediating ADCC function against TNFR2 expressing cells; or / and 7) Inhibit tumor growth.

8. The antibody or antigen-binding fragment thereof of claim 1, wherein the antigen-binding fragment is selected from F(ab)2, Fab', Fab, Fv, scFv or a bispecific antibody.

9. The antibody or antigen-binding fragment thereof according to claim 1, which can compete with the antibody selected from No. 219 for binding to TNFR2.

10. A polynucleotide encoding the antibody or antigen-binding fragment thereof or any combination thereof according to any one of claims 1 to 9. An expression vector comprising the polynucleotide according to claim 10 . A cell comprising the expression vector according to claim 11 .

13. The cell of claim 12, comprising a prokaryotic cell or a eukaryotic cell.

14. The cell of claim 12, wherein the cell comprises one or more of a Chinese hamster ovary cell, a yeast cell, an insect cell, Escherichia coli, and Bacillus subtilis.

15. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the polynucleotide according to claim 10, the expression vector according to claim 11, or the cell according to any one of claims 12 to 14, and a pharmaceutically acceptable carrier.

16. The pharmaceutical composition of claim 15, further comprising an additional anti-tumor agent.

17. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 9, the polynucleotide of claim 10, the expression vector of claim 11, or the cell of any one of claims 12 to 14, and instructions for use.

18. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the polynucleotide according to claim 10, the expression vector according to claim 11, the cell according to any one of claims 12 to 14, or the pharmaceutical composition according to claim 15 or 16 in the preparation of a medicament for treating a disease related to immune abnormality; The immune abnormality-related disease is a tumor; The tumor is selected from: 1) Ovarian cancer, advanced epidermal T-cell lymphoma, stage III / IV metastatic colorectal cancer, triple-negative breast cancer, and / or pancreatic cancer; or 2) Advanced solid tumors resistant to CTLA-4 and PD-1 therapy, such as metastatic melanoma.

19. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 in the preparation of a kit for detecting soluble TNFR2 (sTNFR2).

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