TW202229342A - Anti-tnfr2 antibodies and uses thereof - Google Patents

Abstract

Provided is an antibody capable of specifically binding to TNFR2 or antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof can regulate the function of immune cells and can be used as drugs to treat diseases related to immune abnormalities, such as tumors.

Description

Anti-TNFR2 antibody and use thereof

The present invention relates to anti-TNFR2 antibodies or antigen-binding fragments thereof, pharmaceutical compositions comprising the TNFR2 antibodies or antigen-binding fragments thereof, and uses thereof.

Immunity is a protective response of the body and is affected by many genes, proteins and cells. Immunity abnormalities can cause many diseases, including tumors, immune deficiencies (such as AIDS, etc.), allergies, and rheumatoid arthritis. In the past few years, tumor immunotherapy, as a new treatment method, has become a hot spot in the field of tumor treatment research. Antagonistic antibodies targeting immune checkpoint proteins, such as anti-PD-1 and anti-CTLA-4 mAbs, have been used to treat many types of cancer with revolutionary results, greatly prolonging the survival of patients with malignant tumors. lifetime. However, many cancer patients still do not respond to treatment with antagonistic antibodies against immune checkpoint proteins or develop resistance or resistance after brief 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 against immune checkpoint proteins, to further improve the efficacy and safety.

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 immune suppression and immune escape in the tumor microenvironment.

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

The inventors also found that the relevant experiments in mice further verified the potential function of the TNFR2 target. The results of the CT26 model of colorectal cancer in mice showed that TNFR2 antagonistic antibodies, compared with PD-1 antibody or PD-L1 monoclonal antibody, could significantly inhibit the growth of PD-1 antibody-resistant tumors, reduce tumor size, and effectively improve CD8 ⁺ T/Treg ratio and partially reverse the immunodepleted state of CD8 ⁺ T cells. Therefore, TNFR2 may become a new target for tumor immunotherapy, and TNFR2 antagonistic antibodies are expected to help change the tumor microenvironment. They can be used alone and/or in combination with existing immune checkpoint antagonistic antibodies, and have broad application prospects. To this end, the inventors have prepared a variety of TNFR2 antibodies, and completed the present invention on this basis.

In a first aspect, the present disclosure provides an antibody or 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 MDSCs, and can be used as a drug therapy and immune system. Abnormal-related diseases, such as tumors.

In one embodiment, the modulating comprises by inhibiting the proliferation and/or activation of Treg cells and/or myeloid derived suppressor cells (MDSCs). In another embodiment, the modulation is achieved by blocking the binding of TNFα to TNFR2.

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

(1) heavy chain CDR combinations of CDR1-VH, CDR2-VH and CDR3-VH,

The CDR1-VH, CDR2-VH and CDR3-VH have any sequence combination selected from the following or have 1, 2, 3 or more amino acid insertions, deletions compared to the sequence combination and/or alternate sequence combinations:

and / or,

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

Said 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 said sequence combination:

In particular, for example, an antibody or antigen-binding fragment thereof of the invention comprises 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+VL13, VH14+VL14, VH15+VL15, VH16+VL16, VH17+VL17, VH18+VL18, VH19 +VL19, VH20+VL20, VH21+VL21 and VH22+VL22, and CDRs with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the sequence of the heavy and light chain CDRs combined combination.

In another specific embodiment, the present invention provides such antibodies or antigen-binding fragments thereof, wherein

1) The variable region of the heavy chain and the variable region of the light chain have the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or more identical sequences;

2) The variable region of the heavy chain and the variable region of the light chain have the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or more identical sequences;

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 have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or more identical sequences;

4) The variable region of the heavy chain and the variable region of the light chain have the sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or more identical sequences;

5) The variable region of the heavy chain and the variable region of the light chain have the sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or more identical sequences;

6) The variable region of the heavy chain and the variable region of the light chain have the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or more identical sequences;

7) The variable region of the heavy chain and the variable region of the light chain have the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or more identical sequences;

8) The variable region of the heavy chain and the variable region of the light chain have the sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or more identical sequences;

9) The variable region of the heavy chain and the variable region of the light chain have the sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18, respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or more identical sequences;

10) The variable region of the heavy chain and the variable region of the light chain have the sequences shown in SEQ ID NO: 19 and SEQ ID NO: 20, respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or more identical sequences; or

11) The variable region of the heavy chain and the variable region of the light chain have the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or more identical sequences.

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

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention is chimeric or humanized or fully human.

In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention comprises A constant region selected from the group consisting of human IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD, and the antibody or antigen-binding fragment:

1) Specific binding to cells expressing TNFR2 on the cell surface;

2) Specific binding to Treg cells;

3) Inhibit the binding of TNFα and 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) mediate ADCC function against TNFR2 expressing cells; or/and

7) Inhibit tumor growth.

In another preferred embodiment, the antigen-binding fragment of the present invention is selected from the group consisting of F(ab) ₂ , Fab', Fab, Fv, scFv, diabodies, nanobodies and antibody minimal recognition units.

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

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

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

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

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 above, the polynucleotide as described in the second aspect, the polynucleotide as described in the third aspect An expression vector, or a cell as described in the fourth aspect, and a pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutical compositions of the present invention further comprise additional antineoplastic agents. For example, the additional anti-tumor agent is selected from chemotherapeutic agents, targeted therapeutic agents and immunotherapeutic agents, eg Anti-PD-1/PD-L1 therapeutics (including anti-PD-1 antibodies and anti-PD-L1 antibodies) and anti-CTLA-4 therapeutics (including anti-CTLA-4 antibodies).

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

In a 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 described in the first aspect, the second aspect The polynucleotide, the expression vector described in the third aspect, the cell described in the fourth aspect, or the pharmaceutical composition described in the fifth aspect. The immune abnormality-related diseases are, for example, immune abnormal diseases characterized by abnormal sTNFR2, including diseases related to the function of Treg cells and/or MDSCs, particularly tumors.

In one embodiment, the methods of the invention further comprise administering to the subject additional anti-tumor treatments, including chemotherapy, radiotherapy, targeted therapy, and immunotherapy, eg, anti-PD-1/PD-L1 treatments such as anti-PD-1 /PD-L1 antibody, anti-CTLA-4 therapy such as anti-CTLA-4 antibody.

In one embodiment, the associated disease is a tumor, preferably 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) Metastatic melanoma or other potentially advanced solid tumors resistant to CTLA-4 and PD-1 therapy.

In an eighth aspect, the present invention provides a pharmaceutical product corresponding to the method of the seventh aspect above, for use in the prophylactic and/or therapeutic method of the seventh aspect. In addition, there is also provided the use of the preparation of a medicine or a kit corresponding to the method of the seventh aspect, wherein the products of the first to fifth aspects above are used for manufacture, and the medicine or kit is used for the prevention and/or the above seventh aspect. or treatment methods.

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 of the first aspect. The detection method can be used for the diagnosis of abnormal immune diseases characterized by abnormal sTNFR2, such as Diagnose whether the subject from which the sample is derived has an immune abnormality-related disease (such as Treg cell and/or MDSC function-related disease) or is at risk of developing the disease. Correspondingly, the present invention also provides a use of a reagent for detecting sTNFR2 to prepare a diagnostic kit, the kit is used for diagnosing whether a sample source object suffers from an immune abnormality-related disease (such as an immune abnormality disease related to abnormal TNFR2 expression) or disease risk.

The foregoing and other aspects of the present invention are clearly illustrated by the following detailed description and drawings of the present invention. The drawings herein are intended to illustrate some preferred embodiments of the invention, it being understood, however, that the invention is not limited to the particular embodiments disclosed.

Figure 1 shows the expression of TNFR2 in 13 human tumor cell lines. The positive ratio is determined according to the detection value of the negative antibody of the same isotype fluorescence control after flow cytometry sorting (FACS) staining. The percentage shown in the figure is the percentage of TNFR2 expression.

Figure 2 shows the expression of TNFR2 on Treg, CD8 ⁺ T cells and CD4 ⁺ CD25 ^- Tcon cells of human peripheral blood mononuclear cells (PBMC). Figure 2A shows the flow cytometric staining of Treg cells, CD8 ⁺ T cells and Tcon cells and the flow scatter plot of the gating strategy and the expression ratio of TNFR2 on the cells; Figure 2B is the activated Treg (CD4 ⁺ CD25 ^hi FoxP3 ^hi Treg ), the expression ratio and mean fluorescence intensity (MFI) of TNFR2 in non-activated Treg (CD4 ⁺ CD25 ^hi FoxP3 ⁺ Treg), Tcon (CD4 ⁺ CD25 ^- T cells) and CD8 ⁺ T cells.

Fig. 3 shows the binding ability of the test antibody to CHO cells (CHO-TNFR2) highly expressing human TNFR2. Figure 3A is the verification of the expression of human TNFR2 on the surface of CHO-TNFR2 cells; Figure 3B is the verification of the cell surface target binding ability of 11 samples, among which anti human IgG1 Fc-PE only, and anti-chicken protein lysozyme (anti-Hel ) isotype control antibody (chicken protein lysozyme is an intracellular antigen and does not exist in the human body, so it is a good irrelevant control antibody, which is outsourced by Biotron), which are all negative controls for this experiment. Unstained is a Blank cell control without any antibody added.

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

Figure 5 shows that test antibodies inhibit the interaction of human TNF[alpha] with 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; cells do not express human TNFR2).

Fig. 6 shows the inhibitory effect of the test antibody on the inhibitory activity of Treg on the proliferation of Tcon cells. The ordinate is for different antibodies, the abscissa is (proliferation ratio of effector cells under each antibody-proliferation ratio of effector cells under control antibody)/proliferation ratio of effector cells under control antibody*100%, control antibody is anti-Hel, each The experimental concentration of each antibody was 12.μg/ml, and the effector cells used were CD4 ⁺ CD25 ^- Tcon cells.

Figure 7 shows the effect of the test antibody on the expression of sTNFR2 in the cell supernatant of the Treg function inhibitory activity assay. The ordinate is the different antibodies, and the abscissa is (the expression of sTNFR2 in the supernatant of cells corresponding to each antibody - the expression of sTNFR2 in the supernatant of the cell by the control antibody) / the expression of sTNFR2 in the supernatant of the cell by the control antibody * 100% , the control antibody is anti-Hel, and the experimental concentration of each antibody is 12.5 μg/ml.

Figure 8 shows inhibition of TNFα-induced Treg cell proliferation by test antibodies. Figure 8A shows the proliferation of Treg cells without cytokines, or with IL-2 alone, or with IL-2 and TNFα added for 3 days. Figure 8B shows the inhibitory effect of different antibodies on TNFα-induced Treg proliferation. The ordinate is the different antibodies, and the abscissa is (the inhibitory ability of each antibody to TNFα-induced Treg proliferation - the control antibody's ability to inhibit TNFα-induced Treg proliferation)/the control antibody's ability to inhibit TNFα-induced Treg proliferation*100%, the control antibody For anti-Hel, the experimental concentration of each antibody was 12.5 μg/ml.

Figure 9 shows the blocking effect of the test antibodies on IL-2 and TNF-α anti-induced CD4 ⁺ T cells to secrete sTNFR2 by ELISA. Statistical analysis method: Graphpad prism6, One-way ANOVA, **P<0.01, ***P<0.001.

Figure 10 shows the ADCC (antibody-dependent cellular cytotoxicity) killing effect of test antibodies on Treg. Figure 10A is the expression of TNFR2 on target cells, namely Treg; Figure 10B is the verification of the purity of effector NK cells isolated and enriched from human peripheral blood cells PBMC; Figure 10C is the strength of the test antibody ADCC, wherein #52 sample is directed ADCC positive control antibody screened by TNFR2 target, anti-Hel isotype controlt is the negative control of isotype antibody.

Fig. 11 shows the ADCC killing effect of test antibodies on CHO-TNFR2 cells highly expressing human TNFR2. Among them, #219, #224, and #001 are the test antibodies, and anti-Hel isotype control is the negative control antibody.

Figure 12 shows the change curve of tumor volume mm ³ and the change of tumor growth inhibition% (TGI%) in each administration group in the in vivo efficacy test of PD-1 antibody-resistant CT26 tumor cells (12B) And the change curve of mouse body weight (12C).

Figure 13 shows the analysis of each immune cell subset in the tumor infiltrated lymphocytes (TIL) in the mouse in vivo in the in vivo efficacy test of CT26 tumor cells in mice. 13A and 13B are the gating strategies for panel 1 and panel 2 in FACS immunophenotyping. 13C shows the study of the proportion of CD4 ⁺ T, CD8 ⁺ T and Treg cells by FACS immunophenotyping; FIG. 13D shows the study of the ratio of the total number of CD8 ⁺ T cells to the number of Treg cells by FACS immunophenotyping; 13E shows the study of the ratio of the number of memory CD8 ⁺ T cells to the number of Treg cells by FACS immunophenotyping; FIG. 13F shows the level of PD-1, LAG-3 and TNFR2 expressed by CD8 ⁺ T in FACS immunophenotyping Research.

Figure 14 shows the in vivo efficacy test of CT26 tumor cells in mice with anti-TNFR2 antibody or anti-PD-1 antibody as a single drug or a combination of two antibodies to compare the change curve of tumor volume mm ³ in four experimental groups (14A) and tumor growth inhibition rate ( TGI%) (14B), and mouse survival curves (14C).

Unless otherwise defined, terms used herein have the meanings commonly understood by those of ordinary skill in the art. For terms expressly defined herein, the meanings of such terms shall be governed by the stated definitions.

As used herein, the term "antibody" (Ab) refers to an immunoglobulin molecule that specifically binds 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, heteroconjugated antibodies (e.g. bispecific, trispecific and tetraspecific antibodies, diabodies, triabodies and tetrabodies), antibody conjugates and Antigen-binding fragments of antibodies (including, for example, Fab', F(ab') ₂ , Fab, Fv, rIgG, and scFv fragments). Furthermore, unless otherwise specified, the term "monoclonal antibody" (mAb) is meant to include those capable of specific Intact antibody molecules that bind the target protein sexually as well as incomplete antibody fragments (e.g. Fab and F(ab') ₂ fragments, which lack the Fc fragment of an intact antibody (faster clearance from animal loops) and thus lack Fc-mediated The effector function of (see Wahl et al., J. Nucl. Med. 24:316, 1983; the contents of which are incorporated herein by reference).

As used herein, the term "antigen-binding fragment" refers to one or more antibody fragments that retain the ability to specifically bind a target antigen. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Antibody fragments can be Fab, F(ab')2, scFv, SMIP, diabodies, tribodies, affibodies, nanobodies, aptamers or domain antibodies. Examples of binding fragments encompassing the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) Fab fragments, a monovalent fragment consisting of VL, VH, CL and CH domains; (ii) F(ab) ₂ fragments , a bivalent fragment comprising two Fab fragments connected in the hinge region by disulfide bonds; (iii) a Fab fragment composed of VH and CH domains; (iv) a Fab fragment composed of the VL and VH domains of the antibody one-arm Fv fragments; (V) dAbs comprising VH and VL domains; (vi) dAb fragments comprising VH domains (Ward et al., Nature 341:544-546, 1989); (vii) VH or VL domains (viii) discrete complementarity determining regions (CDRs); and (ix) a combination of two or more discrete CDRs, which may optionally be linked by synthetic linkers. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, the two domains can be joined using recombinant methods by a linker that enables it to be made in which the VL and VH regions are paired to form A single protein chain of a monovalent molecule (called a single-chain Fv (scFv); see eg, 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 techniques, enzymatic or chemical cleavage of intact immunoglobulins, or in some embodiments by chemical peptide synthesis procedures known in the art.

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, a membrane receptor that binds tumor necrosis factor-alpha (TNFα). body. The TNFR2 is preferably human TNFR2.

As used herein, the terms "anti-TNFR2 antibody", "TNFR2 antibody", "anti-TNFR2 antibody", "TNFR2 antibody", "anti-TNFR2 antibody portion" and/or "anti-TNFR2 antibody" "TNFR2 antibody fragment" and the like refers to any immunoglobulin molecule comprising at least a portion capable of specifically binding TNFR2 (such as, 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 chain chain or light chain variable regions, heavy or light chain constant regions, framework regions or any portion thereof) protein- or peptide-containing molecules. TNFR2 antibodies also include antibody-like protein scaffolds (eg, the tenth fibronectin type III domain (10Fn3)) that contain BC, DE, and FG structural loops similar in structure and solvent accessibility to the antibody CDRs. The tertiary structure of the 10Fn3 domain is similar to the tertiary structure of the IgG heavy chain variable region, and by combining the residues of the BC, DE and FG loops of 10Fn3 with CDR-H1, CDR-H2 or CDRs from the TNFR2 monoclonal antibody - Residue substitutions in the H3 region, one skilled in the art can graft, for example, the CDRs of a TNFR2 monoclonal antibody onto a fibronectin scaffold.

As used herein, the term "bispecific antibody" refers to an antibody, typically a human or humanized antibody, having monoclonal binding specificities for at least two different antigens. In the present invention, one of the binding specificities can be detected against an epitope of TNFR2, the other can be against another epitope of TNFR2 or any other antigen, such as for cell surface proteins, receptors, Receptor subunits, tissue-specific antigens, virus-derived proteins, virus-encoded envelope proteins, bacterial-derived proteins or bacterial surface proteins are detected.

As used herein, the term "chimeric" antibody refers to an antibody having variable sequences of immunoglobulins derived from one source organism (eg, rat or mouse) and immunoglobulins derived from a different organism (eg, human) globulin constant region. Methods for producing chimeric antibodies are known in the art. See, eg, 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; into this article.

As used herein, the term "complementarity determining regions" (CDRs) refers to hypervariable regions found in both light and heavy chain variable domains. The more conserved parts of the variable domains are called framework regions (FRs). As understood in the art, the amino acid positions representing the hypervariable regions of an antibody can vary depending on the context and various definitions known in the art. Some positions within a variable domain can be considered as heterozygous hypervariable positions, as these positions can be considered within the variable region under one set of criteria (such as IMGT or KABAT), while those within a different set of outside the variable regions under standards such as KABAT or IMGT. One or more of these positions can also be found in extended variable regions. The present invention includes antibodies comprising modifications in these hybrid variable positions. The variable domains of native heavy and light chains each comprise four framework regions predominantly adopting a sheet configuration, connected by three CDRs (CDR1, CDR2 and CDR3) that form loops connecting the sheet structure , and in some cases form part of the lamellar structure. The CDRs in each chain are held tightly together by the FR regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and with CDRs from other antibody chains contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Protein sof Immunological Interest, National Institute of Health, Bethesda, Md. 1987; incorporated herein by reference). For example, in this context, 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, which constitute the heavy chain variable region (VH). The CDR combination (VHCDR combination) of the chain (or its variable region); CDR1-VL, CDR2-VL and CDR3-VL refer to the first CDR, the second CDR, Two CDRs and a third CDR, these three CDRs constitute the CDR combination of the light chain (or its variable region) (VLCDR combination).

As used herein, the term "antibody conjugate" refers to a conjugate/conjugate in which an antibody molecule is chemically bonded to another molecule, either directly or through a linker. For example, antibody-drug conjugates (ADCs), where the drug molecule is the other molecule in question.

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

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

As used herein, the term "regulatory T cells" or "Treg", also known as suppressor T cells, are a group of lymphocytes that negatively regulate the body's immune response to maintain tolerance to self-antigens Sex, control the excessive immune response, avoid immune damage to normal cells, and prevent the occurrence of autoimmune diseases. Tregs express the following biomarkers: CD4, FOXP3 and CD25 and are thought 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 is significantly increased, including melanoma, ovarian cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, etc. At the same time, Treg cells The number of tumor patients is also closely related to the survival rate of tumor patients. In addition, tumor cells will induce the proliferation of tumor-infiltrating Treg cells, and the proliferating Treg cells will massively secrete immunosuppressive factors such as TGF-β, inhibit the function of immune cells such as CD8 ⁺ T cells, and hinder the killing effect of immune cells on tumors. Important mechanisms of resistance to immunotherapy failure in a variety of solid and hematological tumors. Recent studies have shown that the immune tolerance of PD-1/PD-L1 and other immunotherapy patients is also closely related to Treg.

As used herein, the term "myeloid-derived suppressor cells" or "MDSC" is a heterogeneous population of cells of the immune system composed of immature neutrophils, monocytes, and dendritic cells that suppress immune responses and tumors The role of immune response. MDSCs regulate the activity of various effector cells and antigen-presenting cells such as T cells, NK cells, dendritic cells, and macrophages, among others. marrow Derived suppressor cells are distinguished by their gene expression profiles and express all or a portion of proteins and small molecules selected from the group consisting of B7-1 (CD80), B7-H1 (PD-L1), CCR2, CD1d, CD1dl, CD2 , CD31(PECAM-I), CD43, CD44, complement components 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 a4 (CD49d), integrin aL (CD11a), integrin aM (CD11b), M-CSFR, MGL1 (CD301a), MGL1/2 (CD301a/b), MGL2 (CD301b), monoxide Nitrogen, 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 a protein 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).

As used herein, the term "percent (%) sequence identity" refers to aligning sequences and introducing gaps (if needed) for maximum percent sequence identity (eg, for optimal alignment, the candidate and reference After the introduction of gaps in one or both of the sequences, and for comparison purposes, non-homologous sequences may be ignored), the amino acid (or nucleotide) residues of the candidate sequence differ from the amino acid (or nucleotide) residues of the reference sequence. ) residues that are identical. For purposes of determining percent sequence identity, alignment can be accomplished in a variety of ways well known to those skilled in the art, for example 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 algorithms required to achieve maximal alignment over the full length of the sequences being compared. For example, a reference sequence aligned for comparison to a candidate sequence may show that the candidate sequence exhibits from 50% to 100% over the full length of the candidate sequence or a selected portion of contiguous amino acid (or nucleotide) residues of the candidate sequence % sequence identity. The length of candidate sequences aligned 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 a 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.

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 such as antibodies or antigen-binding fragments thereof specific identification. An antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of less than 100 nM. For example, an antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of up to 100 nM (eg, between 1 pM and 100 nM). An antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or epitope thereof will exhibit a KD of greater than 100 nM (eg, greater than 500 nM, 1 μM, 100 μM, 500 μM, or 1 mM) for that particular antigen or epitope. Various immunoassay modalities can be used to select antibodies that specifically immunoreact with a particular protein or carbohydrate. For example, solid phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive 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 methods that can be used to determine specificity Immunoassay formats and conditions for immunoreactivity.

As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors (eg, plasmids), RNA vectors, viruses, or other suitable replicons (eg, viral vectors). Various vectors have been developed for the delivery of polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells. The expression vectors of the present invention contain polynucleotide sequences and additional sequence elements, eg, 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 invention include plasmids containing regulatory sequences (eg, activator 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 efficient transcription of genes carried on expression vectors. Expression vectors of the present invention may also contain polynucleotides encoding markers for selection of cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseothricin.

As used herein, the terms "subject", "subject" and "patient" refer to an organism receiving treatment for a particular disease or disorder (eg, cancer or infectious disease) as described herein. Examples of subjects and patients include mammals such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, Guinea pigs, bovid family members (such as domestic cattle, bison, buffalo, elk and yak, etc.), sheep, and horses, etc.

As used herein, the term "treatment" refers to surgical or therapeutic treatment for the purpose of preventing, slowing (reducing) an undesired physiological change or pathology, such as a cell proliferative disorder (eg, cancer), in a subject being treated or infectious disease). Beneficial or desirable clinical outcomes include, but are not limited to, reduction of symptoms, reduction in disease severity, stable disease state (ie, no worsening), delayed or slowed disease progression, improvement or alleviation of disease state, and remission (whether partial remission or complete remission), whether detectable or undetectable. Those in need of treatment include those already suffering from the disorder or disease as well as those prone to develop the disorder or disease or for whom the disorder or disease is to be prevented. When referring to terms such as alleviation, alleviation, weakening, alleviation, alleviation, etc., the meanings also include elimination, disappearance, non-occurrence, etc.

The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

Example 1. Preparation of TNFR2 monoclonal antibody

6-8 weeks old female SJL mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) or Balb/c mice (purchased from Shanghai Slack Laboratory Animal Co., Ltd.) were used for the first immunization injection. Freund's complete adjuvant (completefreund'sadjuvant, CFA, purchased from SIGMA, product number: F5881) mixed with human TNFR2 recombinant protein (purchased from SinoBiological, product number: 10417-H08H and Novoprotein, product number: C830), adjuvant for the last three immunization injections then use Incomplete Freund's adjuvant (incompletefreund'sadjuvant, IFA, purchased from SIGMA, product number: F5506) and CpGODN1826 (synthesized from Shanghai Sangon Biotechnology). In particular, the first and second immunizations were injected into the footpad and back, and the third and fourth immunizations were injected subcutaneously and on the back of the tail to obtain high-titer, high-affinity, high-specificity antiserum and specific immunity. cell. On the 7th day after the last immunization (the fourth immunization), the mice were euthanized and the spleen was aseptically removed. The spleen lymphocytes of the mice were aseptically isolated and extracted, and the cells were frozen and stored in liquid nitrogen. The TNFR2-specific single B cells in the spleen or lymph nodes of the immunized mice were sorted into 96-well plates with a BDAriaIII flow sorter, and the mRNA of the single cells was reverse transcribed into cDNA. Then, nested PCR was performed using the cDNA as a template, and the antibody heavy chain and light chain were amplified respectively. The variable region of the heavy chain and the variable region of the light chain of the antibody are obtained by amplification, and are respectively strained into the heavy chain expression vector and the light chain expression vector by the method of homologous recombination. The constant regions of both the heavy chain expression vector and the light chain expression vector are derived 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-CK. The single B cell antibody strains and expressions mentioned above are all in a 96-well plate to achieve rapid identification and discovery of antibodies in a high-throughput manner. After a series of physicochemical and functional screening of the antibody heavy and light chains expressed by 523 pairs of strains, a total of 11 positive candidate antibody molecules were obtained. 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. 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.

Example 2. FACS detection of the expression of TNFR2 in tumor cell lines

The well-grown cells were collected and counted, and 2×10 ⁶ cells were obtained from each tumor cell, which were used in the isotype control group and the TNFR2 staining group (1×10 ⁶ cells/test). The cells were washed twice with PBS, centrifuged at 300g × 5min, the supernatant was discarded, 1×Live/Dead working solution (Zombie violet L/D, Biolegend, Cat. No.: 423114) 100μl/test was added, and the cells were stained at room temperature for 20 minutes. After 20 minutes, wash the cells twice with FACS buffer (DPBS+2% FBS), centrifuge at 300g × 5min, discard the supernatant, add 100μl/test of 1× staining antibody working solution as follows, and incubate at 4°C for 30 minutes in the dark .

For human tumor cells: 1) isotype control group: add 1 μl PE rat IgG2b, κ isotype control (BD Pharmingen, product number: 553989)/100 μl FACS buffer/test; 2) TNFR2 staining group: add 8 μl PE Rat anti to human tumor cells -human CD120b (BD Pharmingen, Cat. No. 552418)/100 μl FACS buffer/test. After the incubation, 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 a cell suspension, which was detected by a flow cytometer (Invitrogen, Attune NxT), and the data was exported. As shown in Figure 1, different tumor cells have different expression levels of TNFR2, and the results are summarized in Table 4.

Example 3. Detection of TNFR2 expression levels on different human T cells by FACS

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

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

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

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

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

After the incubation, 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 a cell suspension, which was detected by a flow cytometer (Invitrogen, Attune NxT), and the data was exported. As shown in Figure 2A-2B, the expression level of TNFR2 on CD4 ⁺ CD25 ^- Tcon cells and CD8 ⁺ T cells is much lower than that of Treg, and it is highly expressed on both unactivated and activated Treg cells. , and the activated Treg cells expressed the highest level.

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

The specific binding between the 11 strains of TNFR2 antibodies obtained in Example 1 and human and monkey TNFR2 proteins was detected by Biacore. In this experiment, Protein A wafer was used, and the time required for the wafer to capture the diluted antibody was determined by manual operation, so that the Rmax of the saturated binding antigen was 50RU. Both human (Human TNFR2, Sino 10417-H08H) and cynomolgus (Cynomolgus TNFR2, Sino 90102-C08H) TNFR2 were serially diluted to 32, 16, 8, 4, 2 nM. The affinity of the antibody to the antigen was measured using multi-cycle kinetics. In each cycle, the antibody was injected and then a gradient concentration of TNFR2 was injected to make the antigen and antibody combine and dissociate. Regeneration of Protein A wafers (removal of protein from wafers) was performed with Glycine pH 1.5 after each cycle. Apply BIAcore T200 analysis software to fit the affinity KD of antibody antigen.

It can be seen from the results in Table 5 that the 11 anti-TNFR2 antibodies obtained in Example 1 have specific binding with human and monkey TNFR2 proteins with a high level of affinity.

Example 5. Detection of specific binding of TNFR2 antibody to human and monkey TNFR2 protein by ELISA

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

Remarks: 1. 002# is a tool antibody (from SBT-002 in WO2017/083525 A1)

2. Only a single concentration point of the antibody was measured, which was expressed by the OD value, among which the concentration of #088 and #309 antibodies was 12ng/mL, and the concentration of other antibodies was 28ng/mL.

Example 6. FACS detects the binding of antibody to human TNFR2 on the surface of CHO-TNFR2 cells

The CHO stable cells transfected with the TNFR2 high expression plasmid were taken, and the transfected plasmid was purchased from Sino Biological (Cat. No.: HG10417-UT). The experiments were carried out when the cell density did not exceed 80%. The cell culture medium was discarded, rinsed with PBS, and digested with 1 ml of trypsin for 2 minutes. The digestion was terminated with HamFBS medium containing 10% FBS, and the cell suspension was made. After counting, take an appropriate amount of cell suspension, centrifuge at 350×g, discard the supernatant, add a corresponding volume of blocking solution (10% FBS+PBS) at a density of 1×10 ⁷ cells/ml to resuspend the cells at 4°C Incubate for 30 minutes. After the incubation, centrifuge at 350×g to remove the supernatant, resuspend the cells with staining buffer (2% FBS+PBS) to a density of 2×10 ⁶ cells/ml, and plate them into a 96-well plate, and add 50 μl of Cell suspension, ready to use. Dilute the antibody to 80 ng/ml with PBS, add the diluted antibody to the well containing 50 μl of cell suspension, place it on a microplate shaker and shake at 500 rpm for 1 minute to fully mix the antibody with the cells and incubate at 4°C 1h. After incubation, cells were washed twice with staining buffer: 100 μl per well, centrifuged at 350 × g for 5 min, and the supernatant was discarded. Dilute PE goat anti-Human IgG Fc antibody (ebioscience, product number: 12-4998-82) 250 times with staining buffer, add 100 μl per well to the washed cell wells, mix well, and stain at 4°C for 30 minute. After staining, the cells were also 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. As shown in Figure 3A, PE Rat-IgG2b PE Rat-IgG2b (Biolegend, Cat. No. 400636) and PE Rat anti-hTNFR2 (BD Biosciences, Cat. No. 552418) were used to detect the expression of TNFR2 on the surface of CHO-TNFR2 cells. It was 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 detected, and the results were all positive, as shown in FIG. 3B .

Example 7. Binding experiment of FACS detection antibody and human TNFR2 on the surface of Treg cells

Human Treg cells were isolated from human PBMCs using a sorting kit (Stemcell, Cat. No. 18063), stimulated and expanded in vitro by Dynabeads Human Treg Expander (Gibco, Cat. No. 11129D) for 17 days, and then were obtained and frozen in aliquots. The Treg cells isolated and expanded in vitro were recovered 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 min, the supernatant was removed, the cell concentration was adjusted to 1 × 10 ⁷ cells/ml with blocking solution, and incubated at 4°C for 30 min. The blocked cells were centrifuged at 300×g for 5 min, the supernatant was discarded, the cell density was adjusted to 2×10 ⁶ /ml with staining buffer, and plated in a 96-well plate with 50 μl per well. Take the 11 test antibodies #001, #088, #125, #133, #219, #224, #226, #309, #352, #365, #395 of Example 1 and the control antibody anti-Hel isotype ( Each was diluted to 200 ng/ml with PBS, the total amount was 100 μl). Each sample was added to each well, 50 μl per well. Place the well plate with the cell suspension and the antibody on a microplate shaker, shake at 500 rpm for 1 min, and mix the cells and the antibody well. After mixing, the plate was placed in a refrigerator at 4°C and incubated for 60 min. After incubation, add 100 μl of staining buffer to each well, centrifuge at 350 × g for 5 min, and discard the supernatant; add 200 μl of staining buffer to each well to resuspend the cells, centrifuge at 350 × g for 5 min, and discard the supernatant. Add the staining buffer to PE goat anti-Human IgG Fc at a ratio of 250:1 (staining buffer: dye) to prepare a staining solution, mix well and add 100 μl per well; place the well plate on a microplate Shake on a shaker at a speed of 500 rpm for 1 min to thoroughly mix the cells with the staining solution. After mixing, it was placed in a 4°C refrigerator and incubated for 30 min. The cells were washed twice, and finally 200 μl of PBS was added to each well to resuspend the cells to detect the signal. The CD25 ⁺ FoxP3 ⁺ cells shown in FIG. 4A 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 4B shows that the control antibody anti-Hel isotype does not bind to Treg cells, while all 11 antibodies of Example 1 can effectively bind to Treg cells.

Example 8. ELISA detection of anti-TNFR2 antibody blocking the binding of TNFα/TNFR2

The ELISA plate was pre-coated with 1 μg/ml 100 μl/well human TNFR2 (Novoprotein, Cat. No. C830), each TNFR2 antibody of Example 1 was diluted to 40nM and 4nM, and the diluted antibodies were mixed with 15ng/ml human TNFa (Acro Biosystems). , Cat. No. TNA-H82E3), mix in equal volume, add 100 μl/well to the ELISA plate, incubate with shaking at room temperature for 2.0 h; add Streptavidin-HRP working solution (1:10000 dilution) after washing the plate, 100 μl/well, shake at room temperature Incubate for 40min; wash the plate again, add The substrate TMB of HRP was added for color development, and the stop solution was added to stop the reaction, and the absorbance value was read with a microplate 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 values of tool antibody 002#. The higher the percentage value, the stronger the inhibitory ability. The data in Tables 7a-7b show that the 11 antibodies of Example 1 have the activity of inhibiting the binding of TNFa to TNFR2 at both 20nM and 2nM concentrations. Compared with 002#Ab, the inhibitory activity was high or low (Tables 7a-7b).

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.

Example 9. Anti-TNFR2 antibody blocks the binding of TNFα to TNFR2 on CHO cells expressing high human TNFR2

Experiments were performed using CHO stable cells expressing high human TNFR2. The cells were recovered and passaged to a good state, and the human TNFR2 expression level of the cells was detected; compared with the isotype control, the mean fluorescence intensity (MFI) shift multiples of more than 100 times can be detected.

After CHO-TNFR2 cells were digested, washed twice with DPBS, stained with Live/Dead (L/D) (20min at room temperature), plated at 1×10 ⁵ cells/50 μl/well; anti-TNFR2 antibody of Example 1 Dilute with staining buffer to 40nM as the initial concentration, 3-fold gradient dilution, a total of 7 concentration points; 50μl/well of the diluted antibody is added to the plated cells, and the final concentration of the antibody is mixed by gentle pipetting. For 20, 6.67, 2.22, 0.74, 0.247, 0.08, 0.027 and 0 nM, incubate for 30 min at 4°C. Then, 100 μl/well of human TNFα-biotin diluted to 100 ng/ml was added and mixed by gentle pipetting, and incubated at 4°C for 30 min after mixing. After washing twice with staining buffer, 100 μl/well of PE-streptavidin was added and incubated at 4°C for 30 min. After washing twice with staining buffer, resuspend in 150 μl and test on the machine. Taking the logarithmic value of the antibody concentration as the abscissa, and the corresponding MFI value as the ordinate, the inhibition curve of the antibody was drawn, and the four-parameter fitting was performed to calculate the IC50 value. The smaller the IC50 value, the stronger the ability of the antibody to inhibit the binding of human TNFα to human TNFR2. Since the shape of the blocking curve of some antibodies is different, the blocking effect of all antibodies is normalized to 002# AUC. The higher the percentage value, the better the inhibitory effect of the antibody. The blocking curves of the 11 antibodies are shown in Figure 5, and the inhibitory activities are shown in Table 8.

It can be seen from Figure 5 and Table 8 that all 11 antibodies of Example 1 can significantly inhibit the binding of TNFα to TNFR2 on CHO-TNFR2 cells. Compared with the tool antibody 002#, the inhibitory activities of 11 antibodies were high or low.

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

Example 10. Inhibition of Treg function by TNFR2 antibody

By measuring the inhibitory function of Treg cells on responder T cells under the condition of containing TNFR2 antibody, the interference effect of the antibody on the inhibitory function of Treg cells was determined. The Treg (CD4 ⁺ CD25 ⁺ FoxP3 ⁺ T cells) and Tcon effector cells (CD4 ⁺ CD25 ^- T cells) required for the experiment were recovered overnight, centrifuged at 400 × g for 5 minutes the next day, and washed with RPMI1640 medium (Gibco, Cat. No. 72400047) The cell suspension was resuspended and counted. Tcon cells were stained with CellTrace CFSE cell proliferation kit (Biolegend product number 423801), and 1 μl of stock solution was used for every 4*10 ⁶ cells to make 1 ml of staining solution. After staining for 5 min, neutralize the reaction with the same volume of serum, let stand for 5 min, and centrifuge at 400 × g for 5 min. Cells were washed once by resuspending in medium, and the supernatant was collected by centrifugation again. Plate the cells at a density of 1 x 10 ^{5 cells per well of a 96-well plate, resuspend cells at a density of 50 μl of medium per 1 x 10 5 cells} , and transfer to a 50 mL centrifuge tube. Treg cells were prepared at the same time. Treg cells and Tcon cells were co-cultured at the ratio of 1:1, 1:2, 1:4, 1:8. Each well of each 96-well plate corresponds to a different density of Treg cells, and each well is resuspended in 50 μl of medium. The corresponding anti-CD3/CD28 Dynabeads (Gibco, Cat. No. 11129D) were prepared at a ratio of 1/8 according to the total number of Tcon cells. A volume of 50 μl per well was added to the medium of Treg and Tcon. 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-mentioned well plates were mixed and placed in a 37°C incubator for 4 days for FACS detection. The results showed that, in particular, #001 antibody and #224 antibody could significantly inhibit the inhibitory effect of Treg on Tcon cells and promote the proliferation of Tcon under different Treg/Tcon ratio conditions (Figure 6).

Example 1 The calculation formula of the effect of the test antibody on the inhibitory function of Treg cells compared with the anti-Hel isotype of the control antibody: (Proliferation ability of effector T cells in the presence of the test antibody-proliferation of effector T cells in the presence of anti-Hel antibody ability)/proliferation ability of effector T cells in the presence of anti-Hel antibodies*100%.

Example 11. Effect of TNFR2 antibody on expression of sTNFR2 in cell supernatant in Treg function inhibition experiment

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 the supernatant was taken from each well. After the supernatant was diluted 5-fold, the measurement of sTNFR2 was performed. The determination method of sTNFR2 refers to the humanTNFRII/TNFRSF1B kit (Cat. No. DRT200) of R&D Company. The results showed that #001, #088, #125, #219, #224, #226 and #309 could significantly down-regulate the expression level of sTNFR2 in cell supernatants at different Treg:Tcon ratios (Figure 7).

Example 12. TNFR2 antibody inhibits TNFα-induced Treg cell proliferation

By measuring the proliferation of Treg cells induced by TNFα in the presence of TNFR2 antibody, the inhibitory effect of the antibody on the proliferation of Treg cells induced by TNFα was determined (Zaragoza B et al., Nat Med. 2016 Jan; 22(1): 16-7). The cryopreserved Treg cells after in vitro expansion and separation were recovered overnight, centrifuged at 400 × g for 5 minutes the next day, and resuspended in medium to obtain a cell suspension and counted. Treg was stained with CFSE, and 1 μl of stock solution was used to make 1 ml of staining solution per 4×10 ⁶ cells. After staining for 5 min, neutralize the reaction with the same volume of serum, stand still for 5 min, and centrifuge at 400 × g for 5 min. Cells were washed once by resuspending in medium, and the supernatant was collected by centrifugation again. Plate the cells at a density of 1 x 10 ^{5 cells per well in a 96-well plate, resuspend cells at a density of 50 μl of medium per 1 x 10 5 cells} , and transfer 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 incubator was incubated for 30 minutes. Then, 50 μl of medium containing IL-2 (final concentration of 300 IU) and 50 μl of medium containing 50 ng/ml of TNFα were added to each well, and the final volume of each well was 200 μl. Three parallel wells were set for each condition, and the above-mentioned 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 judged by the proportion of Treg proliferation. The results are shown in Figure 8. Compared with the control anti-Hel antibody, the following antibodies effectively inhibited TNFα-induced Treg cell proliferation: #001 (17.1% inhibition), #088 (6.9% inhibition), #125 (5.2% inhibition), # 133 (9.7% inhibition), #219 (13.6% inhibition), #226 (10.3% inhibition), #365 (11.0% inhibition). The formula for calculating the proportion of Treg cell proliferation inhibited by TNFα induced by the test antibody compared with the control antibody: (the proportion of Treg proliferation induced by TNFα in the presence of the test antibody - the proportion of Treg proliferation induced by TNFα in the presence of anti-Hel isotype antibody)/anti -Proportion of TNFα-induced Treg proliferation in the presence of Hel isotype antibody*100%. Figure 8A shows that the proliferation of Treg was significantly increased after the addition of IL-2 (20% vs. 4%), and the proliferation ratio was further increased after the addition of TNFα (40% vs. 20%). Figure 8B shows the inhibitory effect of 11 antibodies on the inducibility of Treg proliferation after adding IL-2 and TNFα, except #224, #352 and #395, the other 8 candidate antibodies can inhibit IL-2 and TNFα-induced Treg proliferation.

Example 13. ELISA detection of TNFR2 antibody against CD4 ⁺ Effect of sTNFR2 expression level in T cell culture supernatant

Commercially cryopreserved human PBMC cells were thawed 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 the kit's separation solution, Easybuffer, and then the cells were counted. Human CD4 ⁺ T cell isolation was performed according to the instruction manual of the human CD4 ⁺ T cell isolation kit (Stemcell, EasySepTM Human CD4 ⁺ T cell Isolation Kit, Cat. No. 17952). The isolated CD4 ⁺ T cells were recounted, resuspended to an appropriate concentration, and seeded at 4×10 ⁵ cells/well in a 96-well round bottom plate, 50 μl/well. Prepare antibody working solution (50 μg/ml, 4×), add 50 μl/well to the plate, and incubate at 37°C under 5% CO ₂ for 30 min. Next, IL-2 working solution (800 IU/ml, 4×) and TNFα working solution (200 ng/ml, 4×) were prepared and added in 50 μl/well respectively. The experimental groups were: non-stimulation group, IL-2 alone stimulation group, TNFα group alone stimulation group, IL-2+TNFα costimulation group, and antibody+IL-2+TNFα costimulation group (ie, the test sample group). Put the well-plated plate into an incubator and culture at 37°C under 5% CO ₂ for 3 days. After 3 days, the culture supernatant was collected for sTNFR2 detection.

The human sTNFR2 standard was prepared, and the cell supernatant in the above experiment was taken out and operated according to the instructions of the ELISA kit (R&D System, Soluble TNF Receptor II Human ELISA Kit, product number DRT200). The steps are as follows: 1) Add 100 μl/well of cell supernatant and standard substance to the high-affinity plate, and incubate with slight shaking at room temperature (18-25°C) for 2.5 hours; 2) Discard the supernatant and wash with Wash buffer Plate 4 times, 300 μl/well; 3) After blotting dry, add 100 μl/well of 1× Biotin-labeled soluble human TNFR2 antibody, and incubate for 1 h at room temperature (18-25°C) with slight shaking; 4) Discard the supernatant, Wash the plate 4 times with washing solution, 300 μl/well; 5) After blotting up 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; 6) Discard the supernatant , wash the plate 4 times with washing solution, 300 μl/well; 7) After blotting up the water, add 100 μl/well of TMB substrate solution, and incubate for 30 minutes at room temperature (18-25°C) and in the dark with slight shaking; 8) Add 50 μl/well of the reaction stop solution, and immediately detect the OD450 reading in the microplate reader.

The experimental results are shown in Figure 9. Compared with the negative control Anti-Hel isotype group, all 11 antibodies of Example 1 can inhibit the secretion of sTNFR2 in the cell supernatant. The increased secretion of sTNFR2 in the cell supernatant was caused by the activation of CD4 ⁺ T cells upon co-stimulation with IL-2 and TNFα. All 11 antibodies can block the activating effect of IL-2 and TNFα on CD4 ⁺ T cells to varying degrees, and the numbers are #88, #219, #001 (P<0.001), #125, #224 (P<0.01 ), the inhibitory effect was particularly significant, with a statistically significant difference. The lower the OD450 reading, the lower the expression level of sTNFR2, that is, the stronger the antagonistic activity of the antibody.

Example 14. FACS detection of TNFR2 antibody-mediated ADCC activity on Treg cells

One day in advance, PBMC and cryopreserved Treg cells were cultured in complete medium (RPMI1640-Glutamax+10% FBS+1×P/S+1×ITS+50μl MPM mercaptoethanol), in which PBMC needed to add 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 after labeling. The 11 candidate antibodies were serially diluted to 4 times the final concentration in complete medium, and set aside. The target cells were seeded into the cell plate according to the distribution diagram of the experimental plate, 50 μl/ml, and then the diluted drug was spread into the corresponding wells, and incubated with the target cells for 30 minutes at 37°C. After the incubation, 100 μl of effector cell suspension was added to the wells to be added to effector cells, and 100 μl of culture medium was added to the unneeded wells, and incubated for 4 h at 37°C. 1 μl of PI dye was added to each well and detected on the machine. The stronger the PI signal in the target cells, the more significant the ADCC effect. The results of the ADCC experiment (Figure 10 and Table 9) showed that the negative control anti-Hel isotype did not have ADCC killing effect because it had no target; while the positive control sample #52 showed a clear ADCC effect curve. All 11 tested antibodies showed weak ADCC activity. Figure 10A shows that human Treg highly expresses TNFR2, with an 80-fold shift compared to anti-Hel isotype control antibody staining. Figure 10B shows that the ratio of NK cells in PBMCs before isolation with the kit was 14.5%, and the purity of NK cells after isolation with the kit reached 88.3%. Figure 10C shows the ADCC effect of 11 antibodies, #52 antibody is a positive control antibody, anti-Hel isotype antibody is a negative control antibody, and Target alone is a negative control of cells without NK cells and only target cells Treg.

Remarks: Compare the AUC value of the ADCC action curve of the tested antibody to the positive control #52 antibody sample to obtain the relative ADCC activity.

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

Target cells (CHO-TNFR2) were plated into clear bottom white 96-well plates (Corning, Cat. No. 3610) one day in advance, using assay buffer (RPMI1640 (Gibco, Cat. No. 22400105) + 0.5% FBS (Gibco, Cat. No. 10099141) +1×P/S (Gibco, Cat. No. 15140-122)) was plated at 10,000 cells per well, and the cells were allowed to adhere overnight. The next day, the drug was serially diluted to 2 times the final concentration with assay buffer, and set aside. First discard the detection buffer in the plate, then add 25 μl of new detection buffer to each well, then spread the diluted drug into the corresponding well, 25 μl per well, and incubate with the target cells for 1 hour at 37°C. After the incubation, 25 μl of 2* drug was added to the corresponding well, and then 25 μl of ADCC cell suspension (BPS, Cat. No. 60541) was added to the designated well, with 75,000 cells per well. At this time, there is 100 μl of mixed solution in each well, which is placed in an incubator for 6 hours. After the incubation, place the plate at room temperature for 10 minutes, add Bright-Glo (Promega, Catalog No.: 2620) pre-thawed to room temperature, 100 μl per well, and place the plate on a shaker at 500 rpm for 10 minutes. minutes, and then check 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-Hel isotype control antibody did not detect the killing effect of ADCC because it had no target; while the three anti-TNFR2 antibodies #001, #219 and #224 had ADCC effects (Fig. 11).

Example 16. Anti-TNFR2 Antibody Binding Epitope Binning on Human TNFR2 Protein (Epitope binning)

This method uses Biacore to detect whether the TNFR2 antibody competes when it binds to human TNFR2 protein to group the antibody, and it is preliminarily determined to belong to several epitope bins (Abdiche YN et al., Plos One. Mar 20, 2014 ). Human TNFR2 was immobilized by amino-conjugation, and the selected immobilized antibody was injected first, and then the other antibody was further injected separately, and then the injection sequence of the immobilized antibody and the other antibody was alternated. By comparing and analyzing whether there is a difference in the binding signal between the immobilized antibody and other antibodies and human TNFR2, the relationship between the binding epitopes of the antibodies on the human TNFR2 protein is determined. Specifically, in each cycle, all antibodies were measured at a concentration of 300 nM for 180 s, and 650 mM HCl solution was used to elute the antibodies to be tested.

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

Note: Y: with epitope competition; N: without epitope competition

Example 17. In vivo efficacy detection of anti-mouse TNFR2 surrogate antibody

Since the 11 anti-human TNFR2 antibodies in Example 1 specifically bind to human and cynomolgus monkey TNFR2, but none of them bind to mouse TNFR2, anti-mTNFR2 antibodies were used as surrogate antibodies to establish PD-1 in Balb/c mice /PD-L1 therapy-resistant CT26.WT colon cancer animal model for TNFR2 target-related efficacy experiments. The surrogate antibody anti-mTNFR2 was purchased from BioXcell (Cat. No. BE0247). The right side of the mice was subcutaneously inoculated with 1×10 ⁵ CT26 colon cancer cells (set as experimental day 0). When the tumor volume reached ~ ^110mm3 , the mice with moderate tumor volume were selected into the group, and the animals were randomly assigned to 4 experimental groups (G1 for the control group and G2 for the anti-tumor group based on the random number of EXCEL) according to the tumor volume. -mTNFR2 surrogate antibody group, G3 is anti-mPD-L1 (BioXcell, cat. No. BE0101) antibody group, G4 is anti-mPD-1 (BioXcell, cat. No. BE0146) antibody group, 8 mice in each group, grouped on the day of the 13th day Begin administration. The dosages are anti-mTNFR2 5mg/kg (mpk), anti-mPD-L1 10mg/kg (mpk), anti-mPD-1 10mg/kg (mpk), intraperitoneal injection twice a week The tumor volume and body weight were measured three times a week at a fixed time.

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

During the administration period, the experimental animals in each group were in a good state of activity and eating, and their body weights increased to a certain extent. The results indicated that the anti-mTNFR2 antibody was safe (Fig. 12C).

Twenty-four hours after the last administration, most of the mice in the G1 control group and the G2 anti-mTNFR2 antibody group were selected for TIL isolation and FACS immunophenotyping. All cell populations were gated based on the FMO control (Fig. 13A-13B), the results showed that the CD8 ⁺ T cells in the anti-mTNFR2 replacement antibody treatment group were significantly more than those in the control group and there was a significant difference (p<0.01) (Figure 13C); at the same time, the total CD8 ⁺ T cells in the anti-mTNFR2 treatment group The ratio of the number of memory CD8 ⁺ T cells to the number of Treg cells (CD8 ⁺ T/Treg) was also significantly higher than that of the control group, and there was a statistical difference (p<0.001) (Figure 13D-13E); anti-mTNFR2 treatment group CD8 The mean fluorescence intensity of PD-1 expressed by ⁺ T was lower than that of the isotype control group, with a significant difference (p<0.001) ( FIG. 13F ). The above data suggest that the anti-tumor effect of anti-mTNFR2 antibody may be achieved by increasing the ratio of CD8 ⁺ T/Treg and partially reversing the immune exhaustion state of CD8 ⁺ T cells.

The calculation formula of tumor volume (TV) is: V=1/2×a×b ² . where a and b represent the length and width, respectively. The relative tumor volume (RTV) was calculated according to the measurement results, and the calculation formula was: RTV=Vt/V0. Wherein V0 is the tumor volume measured at the time of administration in separate cages (ie, Day 0), and Vt is the tumor volume at each measurement. TGI%=[1-RTV(experimental group)/RTV(control group)]*100%.

Example 18. In vivo efficacy detection of anti-mouse TNFR2 surrogate antibody combined with anti-mouse PD-1 surrogate antibody

The right side of the mouse was subcutaneously inoculated with 1×10 ⁵ CT26.WT colon cancer cells (set as experimental day 0). When the tumor volume reached ~100 ^mm3 , the mice with moderate tumor volume were selected into the group, and the animals were randomly assigned to 4 experimental groups (G1 for the control group, G2 for the anti- -mTNFR2 antibody group, G3 is anti-mPD-1 antibody group, G4 is anti-mTNFR2+anti-mPD-1 antibody group), 9 mice in each group were used for in vivo pharmacodynamic studies. Dosing began on the day of the group (day 14). The doses were anti-mTNFR2 5mpk and anti-mPD-1 10mpk, administered by intraperitoneal injection twice a week. Tumor volume and body weight were measured three times a week at fixed times.

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 anti-mTNFR2 group had greater TGI values than the anti-mPD-1 group. The results showed that the anti-mTNFR2 antibody had better efficacy in the animal model; the G4 combination therapy group had the best efficacy, and compared with the single drug groups, there were statistical differences (p<0.01, p<0.01). 0.001), indicating that the combined administration of anti-mTNFR2 antibody and anti-mPD-1 antibody has a synergistic effect (Figure 14A-14B).

After the drug was stopped on the 28th day, the mice in the 4 groups entered the observation period after drug withdrawal, and the tumor of the mice was recorded at least once a week. After the drug was stopped, the tumor volume of the G2 anti-mTNFR2 group and the G3 anti-mPD-1 group Started to grow slowly, and at the latest on the 55th day, the two groups of mice died completely, while the G4 combined treatment group still had mice alive on the 68th day, and the survival rate of this group was always higher than 50% (55.6%); G4 The tumor volume of 3 mice in the combination treatment group gradually receded and did not grow. Compared with the single drug group and the control group, there were significant differences (p<0.001, p<0.0001). The single-drug group had better efficacy (Figure 14C).

Claims (22)

An antibody that specifically binds to TNFR2 or its antigen-binding fragment can regulate the function of immune cells, and can be used as a drug to treat diseases related to immune abnormalities, such as tumors. The antibody or antigen-binding fragment thereof of claim 1, wherein the regulation comprises inhibiting the proliferation of regulatory T cells (regulatory T cells, Treg cells) and/or myeloid derived suppressor cells (MDSCs) and / or activation. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the modulation is achieved by blocking the binding of TNF to TNFR2. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, comprising: (1) heavy chain CDR combinations of CDR1-VH, CDR2-VH and CDR3-VH, 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 :

and, (2) light chain CDR combinations of CDR1-VL, CDR2-VL and CDR3-VL, Said 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 said sequence combination:

The antibody or antigen-binding fragment thereof of claim 4, comprising 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+VL13, VH14+VL14, VH15+VL15, VH16+VL16, VH17+VL17, VH18+VL18, VH19+ VL19, VH20+VL20, VH21+VL21 and VH22+VL22, and CDRs with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to the sequence of the heavy and light chain CDR combinations combination. The antibody or antigen-binding fragment thereof of any one of claims 4 to 5, wherein 1) The variable region of the heavy chain and the variable region of the light chain have the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or more identical sequences; 2) The variable region of the heavy chain and the variable region of the light chain have the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively, or 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or more identical sequences; 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 have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or more identical sequences; 4) The variable region of the heavy chain and the variable region of the light chain have the sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or more identical sequences; 5) The variable region of the heavy chain and the variable region of the light chain have the sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or more identical sequences; 6) The variable region of the heavy chain and the variable region of the light chain have the sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or more identical sequences; 7) The variable region of the heavy chain and the variable region of the light chain have the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or more identical sequences; 8) The variable region of the heavy chain and the variable region of the light chain have the sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or more identical sequences; 9) The variable region of the heavy chain and the variable region of the light chain have the sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18, respectively column, or a sequence that is 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence shown; 10) The variable region of the heavy chain and the variable region of the light chain have the sequences shown in SEQ ID NO: 19 and SEQ ID NO: 20, respectively, or have 70%, 75%, 80%, 85%, 90% of the sequences shown , 95%, 96%, 97%, 98%, 99% or more identical sequences; or 11) The variable region of the heavy chain and the variable region of the light chain have the sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively, or have 70%, 75%, 80%, 85%, 90% of the sequence shown , 95%, 96%, 97%, 98%, 99% or more identical sequences. The antibody or antigen-binding fragment thereof of any one of claims 1 to 6, which binds to human TNFR2 with a dissociation constant (KD) of not more than 5 nM and binds to cynomolgus monkey TNFR2 with a dissociation constant (KD) of not more than 5 nM. The antibody or antigen-binding fragment thereof of any one of claims 1 to 7, which is a chimeric antibody, a humanized antibody, or a fully human antibody. The antibody or antigen-binding fragment thereof of any one of claims 1 to 8, comprising a constant region selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD, and the antibody or antigen-binding fragment: 1) Specific binding to cells expressing TNFR2 on the cell surface; 2) Specific binding to Treg cells; 3) Inhibit the binding of TNFα and 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) mediate ADCC function against TNFR2 expressing cells; or/and 7) Inhibit tumor growth. The antibody or antigen-binding fragment thereof of any one of claims 1 to 9, said antigen-binding fragment selected from the group consisting of F(ab) ₂ , Fab', Fab, Fv, scFv, diabody, nanobody, and antibody minimum recognition unit . The antibody or antigen-binding fragment thereof of any one of claims 1 to 10, which can compete for binding to TNFR2 with an antibody selected from the group consisting of numbers 001, 088, 125, 133, 219, 224, 226, 309, 352, 365 and 395. A polynucleotide encoding the antibody or antigen-binding fragment thereof or any combination thereof according to any one of claims 1 to 11. An expression vector comprising the polynucleotide of claim 12. A cell comprising the expression vector of claim 13, such as prokaryotic cells or eukaryotic cells, including Chinese hamster ovary cells, yeast cells, insect cells, Escherichia coli and Bacillus subtilis. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 11, the polynucleotide of claim 12, the expression vector of claim 13, or the cell of claim 14, and pharmaceutically acceptable Carrier. The pharmaceutical composition of claim 15, further comprising an additional antineoplastic agent. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 11, the polynucleotide of claim 12, the expression vector of claim 13, or the cell of claim 14, and instructions for use. A method of treating and/or preventing an immune abnormality-related disease, comprising administering to a subject in need thereof The antibody or antigen-binding fragment thereof of any one of claims 1 to 11, the polynucleotide of claim 12, the expression vector of claim 13, the cell of claim 14, or the pharmaceutical composition of claim 15, such as the immunization Abnormal-related diseases Diseases are diseases related to Treg cell and/or MDSC function, such as tumors in particular. The method of claim 18, further comprising administering to the subject additional anti-tumor treatments, such as chemotherapeutics, targeted therapeutics, and immunotherapeutics, including anti-PD-1/PD-L1 treatments such as anti-PD-1/PD -L1 antibody, anti-CTLA-4 therapy such as anti-CTLA-4 antibody. The antibody or antigen-binding fragment thereof of any one of claims 1 to 11, the polynucleotide of claim 12, the expression vector of claim 13, the cell of claim 14, or the pharmaceutical composition of claim 15 are prepared for the treatment of immune disorders Use in medicines for related diseases, for example, the immune abnormality related diseases are Treg cell and/or MDSC function related diseases, particularly tumors. The method of claim 18 or 19, the use of claim 20, wherein 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. A method for detecting soluble TNFR2 (sTNFR2), comprising contacting a sample suspected of containing sTNFR2 with the antibody or antigen-binding fragment thereof of any one of claims 1 to 11.

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