Antibody specifically binding to TNFR2 and application thereof

Abstract

Described are novel regulatory antibody molecules that specifically bind to TNFR2 on a target cell and thereby block the binding of TNF-alpha to TNFR2 and block TNFR2 signalling, having the effects of down-regulating the proportion of Tregs, simultaneously activating and promoting effector and killer T cell proliferation, generally up-regulating the proportion of Tegs / Tregs in the tumor microenvironment, and, in the tumor microenvironment, at the same time activating and promoting the proliferation of effector T cells and killer T cells. The anti-tumor immune level of the organism can be enhanced. The use of such antibody molecules in the treatment of cancer is also described.

Description

Technical Field

[0001] The present invention relates to the fields of biomedicine and bioengineering, and in particular, to a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to TNFR2, a composition comprising the same and use thereof in preparing a drug, and a method for using the same to increase the Teffs / Tregs ratio for cancer immunotherapy. Background Art

[0002] Tumor necrosis factor receptor superfamily (TNFR) proteins (TNFR1 & TNFR2) are key to the coordination of innate / adaptive immune cells. These proteins play an important role in the body's immunity, including activation of immunity, suppression of immunity, and proliferation and apoptosis of immune cells. TNFR2 (Tumor Necrosis Factor Receptor 2) is a type II transmembrane protein and a member of the TNF receptor superfamily. It is mainly expressed on the surface of tumor cells and inhibitory immune cells in the tumor microenvironment, but not expressed or rarely expressed on normal tissue cells and peripheral immune cells in the human body. After activation, it inhibits the body's immunity. Studies have shown that TNFR2 has become a potential cause of tumor immune escape, tumor growth, and checkpoint blockade drug resistance: 1) TNFR2 is selectively expressed as a surface oncogene in many types of tumors and has the function of promoting tumor cell proliferation. In tumors such as acute myeloid leukemia, lung cancer, and ovarian cancer, abnormally elevated TNFR2-positive cell subpopulations can participate in the formation of an immunosuppressive tumor microenvironment, weaken the body's anti-tumor immune response, assist tumor immune escape, and are associated with poor prognosis. 2) TNFR2 is specifically expressed in suppressive immune cells in the tumor microenvironment, including regulatory T cells (Tregs) and myeloid-derived suppressor cells, which weakens the body's anti-tumor immune response and assists tumor immune escape. It is a potential driving force for immune escape, tumor growth, and checkpoint blockade resistance.

[0003] Based on the unique expression pattern and function of TNFR2 in biological organisms, antibodies that block the binding of TNFR2 to TNF-α and have enhanced FC function can specifically inhibit the proliferation of Treg cells that highly express TNFR2 in the tumor microenvironment, while specifically killing tumor cells. It is currently one of the best targets in tumor treatment based on Treg.

[0004] Since the first immune checkpoint inhibitor was approved in 2011, immunotherapy has become one of the important treatments for malignant tumors and has been upgraded to the first-line treatment for a variety of advanced malignant tumors. The most commonly used immune checkpoint blockade therapies are anti-CTLA-4 immunotherapy and anti-PD-1 / PD-L1 immunotherapy. However, at present, only about 20% to 40% of patients can actually benefit from PD-1 / PD-L1 inhibitor treatment, and there is no effective treatment for patients who progress after receiving anti-PD-(L)1 monoclonal antibody treatment. Therefore, there is still a huge unmet clinical need for cancer immunotherapy, and new immunotherapy methods are urgently needed. As one of the best targets in tumor treatment based on Treg, the development of regulatory anti-TNFR2 antibodies that can be used to regulate cell signaling of the TNF-TNFR2 axis, activate effector T cell function and enhance T cell proliferation and / or downregulate and inhibit Tregs cell function has broad prospects for clinical therapeutic efficacy, or can be combined with PD-1 inhibitors to further meet the current clinical needs of immunotherapy. Summary of the invention

[0005] The present invention solves the above needs by providing antibodies and antigen-binding fragments thereof that specifically bind to anti-tumor necrosis factor receptor 2 (TNFR2). These antibodies and antigen-binding fragments thereof are characterized by a unique CDR amino acid sequence combination, specific binding to human TNFR2, and cross-reactivity with cynomolgus monkey TNFR2. More specifically, the present invention relates to antibodies that bind to human TNFR2, and their use in the preparation of immunotherapeutic drugs for treating cancer based on their activities in regulating the TNF-TNFR2 axis, inhibiting Tregs activity, downregulating the number of Tregs, activating and promoting the proliferation of effector T cells and killer T cells, increasing the tumor killing activity of killer T cells, and increasing the body's tumor immune killing level.

[0006] The purpose of the present invention is to provide a regulatory anti-TNFR2 antibody or its antigen-binding fragment that can activate and promote the proliferation of effector T cells and activate and promote the proliferation of cytotoxic T cells and enhance their cell tumor killing activity by downregulating the proportion of Tregs, thereby enhancing the body's tumor immunity level, and a composition comprising the anti-TNFR2 antibody or its antigen-binding fragment, and verify its activity and use.

[0007] In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to TNFR2, the antibody or the antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises VHCDR1, VHCDR2 and VHCDR3 comprising the amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, or comprising amino acid sequences that are not less than 95% identical to the amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; and the VL comprises VLCDR1, VLCDR2 and VLCDR3 comprising the amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively, or comprising amino acid sequences that are not less than 95% identical to the amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0008] In some embodiments, the VH comprises an amino acid sequence selected from any one of SEQ ID NOs: 7 and 11, or an amino acid sequence having at least 85% sequence identity compared to the amino acid sequence shown in any one of SEQ ID NOs: 7 and 11, or an amino acid sequence having 1, 2, 3, 4, 5 or more amino acid insertions, deletions and / or substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 7 and 11.

[0009] In some embodiments, the VL comprises an amino acid sequence selected from any one of SEQ ID NOs: 8 and 12, or an amino acid sequence having at least 85% sequence identity compared to the amino acid sequence shown in any one of SEQ ID NOs: 8 and 12, or an amino acid sequence having 1, 2, 3, 4, 5 or more amino acid insertions, deletions and / or substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 8 and 12.

[0010] In some embodiments, the amino acid insertion, deletion and / or substitution occurs in the framework regions (VHFRs and VLFRs) of VH or / and VL; in some embodiments, the substitution is a conservative amino acid substitution.

[0011] In certain embodiments, in the antibodies or antigen-binding fragments thereof described in the present invention, the VH is a functional conservative variant of a VH sequence comprising a CDR that is completely identical to any one of SEQ ID NOs: 7 and 11, and a FR that has at least 85% sequence identity; the VL comprises a functional conservative variant of a VL sequence comprising a CDR that is completely identical to any one of SEQ ID NOs: 8 and 12, and a FR that has at least 85% sequence identity.

[0012] In certain embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise VH and VL selected from the following groups:

[0013] (1) a VH having the amino acid sequence of SEQ ID NO: 7 and a VL having the amino acid sequence of SEQ ID NO: 8;

[0014] (2) a VH having the amino acid sequence of SEQ ID NO: 11 and a VL having the amino acid sequence of SEQ ID NO: 12;

[0015] In certain embodiments, the antibody or antigen-binding fragment thereof described in the present invention is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, and a full-length antibody.

[0016] In certain embodiments, the antibodies described herein are monoclonal antibodies.

[0017] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof is one of a human-mouse chimeric antibody, a humanized antibody, and a human antibody.

[0018] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain constant region (CH) and / or a light chain constant region (CL).

[0019] In certain preferred embodiments, the CH is a hIgG1 type CH.

[0020] In certain preferred embodiments, the CL is a kappa-type CL.

[0021] In a second aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to TNFR2, comprising a heavy chain (HC) and a light chain (LC), wherein the heavy chain (HC) comprises an amino acid sequence selected from any one of SEQ ID NOs: 9 and 13, or an amino acid sequence having at least 85% sequence identity compared to the amino acid sequence shown in any one of SEQ ID NOs: 9 and 13, or an amino acid sequence having 1, 2, 3, 4, 5 or more amino acid insertions, deletions and / or substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 9 and 13.

[0022] In some embodiments, the light chain (LC) comprises an amino acid sequence selected from any one of SEQ ID NOs: 10 and 14, or an amino acid sequence having at least 85% sequence identity compared to the amino acid sequence shown in any one of SEQ ID NOs: 10 and 14, or an amino acid sequence having 1, 2, 3, 4, 5 or more amino acid insertions, deletions and / or substitutions compared to the amino acid sequence shown in any one of SEQ ID NOs: 10 and 14.

[0023] Optionally, the substitution is a conservative amino acid substitution.

[0024] In certain embodiments, the antigen-binding fragment thereof is Fab, Fab', F(ab')2, Fd, single-chain Fv or scFv, DVD-Ig, Fcab, mAb2, (scFv)2 or scFv-Fc, disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrafunctional antibody, trifunctional antibody, bifunctional antibody, or single domain antibody.

[0025] In a third aspect, the present invention provides a defucosylated variant of the antibody or antigen-binding fragment thereof that specifically binds to TNFR2 according to the first aspect.

[0026] In some embodiments, the antibodies or antibody fragments thereof of the first and second aspects of the present invention, and the defucosylated variants of the third aspect, alone or in combination, exhibit one or more of the following structural and functional features:

[0027] The antibody or antigen-binding fragment thereof that specifically binds to TNFR2 according to the first and second aspects of the present invention and the defucosylated variant according to the third aspect have one or more of the following specific binding and inhibitory activities:

[0028] (1) having high affinity binding to human and / or cynomolgus monkey TNFR2 proteins in vitro;

[0029] (2) Binding to human TNFR2 expressed on the cell surface;

[0030] (3) inhibiting the binding of TNF-α ligand to human TNFR2 in vitro;

[0031] (4) inhibiting the interaction / binding between TNF-α and human TNFR2 expressed on cells;

[0032] (5) substantially no cross-reactivity with mouse TNFR2;

[0033] (6) There is virtually no cross-binding reaction with the same family proteins CD40 and 4-1BB protein.

[0034] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof binds to human TNFR2 with an ELISA binding EC50 of less than about 6 ng / mL and binds to cynomolgus monkey TNFR2 with an ELISA binding EC50 of less than about 15 ng / mL.

[0035] In some embodiments, the humanized antibody or antigen-binding fragment thereof or defucosylated variant thereof that specifically binds to TNFR2 of the present invention has one or more of the following characteristics:

[0036] (1) Targeting selectivity and activity in the tumor microenvironment: It selectively binds to CD4+CD25+ cells with medium to high levels of TNFR2 protein expression, and its binding in TIL is significantly higher than that in peripheral blood and spleen, and it basically does not bind to CD4+CD25-Tcon cells; in the tumor microenvironment, both wild-type and defucosylated antibodies can downregulate the tumor infiltration of Tregs cells, and the defucosylated antibody downregulates the tumor infiltration of Tregs cells to a higher degree, and can also increase the tumor infiltration of CD4+T and CD8+T cells. At the same time, it has no lymphocyte toxicity in peripheral blood and can upregulate the immune level;

[0037] (2) Agonistic activity: The wild-type humanized antibodies and the defucosyl modified humanized antibodies in the coated fixed state and FAB antibody cross-linked state have a certain agonistic effect on the TNFR2 downstream signaling activity NF-KB signaling pathway stimulated by the ligand TNF-α; the single Fc cross-linked antibody Fab has no agonistic effect on the TNFR2 signaling system;

[0038] (3) Can mediate downregulation of the proportion of Tregs cells in PBMCs:

[0039] (4) It can activate CD4+T cells and CD8+T cells in vitro; promote the proliferation of CD4+T cells and CD8+T cells, upregulate the proportion of CD25+T cells and the level of CD25 protein and PD-1 membrane protein; promote the secretion of INF-γ and improve the tumor killing level of CD4+T cells and CD8+T cells;

[0040] (5) It can mediate the killing of tumor cells by PBMC in vitro, and the in vitro efficacy of the defucosyl modified antibody in tumor cells is better than that of the wild-type HU6 antibody; high concentration levels do not mediate the killing of CD4+ cells and CD8+ cells in PBMC;

[0041] (6) It has an in vivo anti-tumor effect in the MC38-transplanted hTNFR2 humanized mouse tumor model, in which the anti-tumor effect of the defucosylating humanized antibody HU6-AF is significantly stronger than that of the wild-type humanized HU6 antibody;

[0042] In a third aspect, the present invention provides a bispecific antibody comprising an antigen binding region of the antibody or antigen binding fragment thereof according to the first aspect or the defucosylated variant according to the second aspect and a second different antigen binding region.

[0043] In a fourth aspect, the present invention provides a nucleic acid encoding the heavy chain variable region and / or light chain variable region of the antibody or antigen-binding fragment described in the first aspect.

[0044] In a fifth aspect, the present invention provides a pharmaceutical composition, wherein the composition comprises:

[0045] (1) the antibody or antigen-binding fragment thereof according to the first aspect, the variant according to the second aspect, the bispecific antigen-binding molecule according to the third aspect, or the nucleic acid according to the fourth aspect of the present invention;

[0046] (2) Pharmaceutically acceptable carriers, diluents or adjuvants.

[0047] Preferably, the composition further comprises an additional anti-tumor agent, an immunotherapeutic agent or an immunosuppressant; preferably, the additional anti-tumor agent is selected from PD-1 antibody, PD-L1 antibody or CTLA-4 antibody.

[0048] In the sixth aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof described in the first aspect, or the defucosylated variant antibody described in the second aspect, or the bispecific antibody described in the third aspect, wherein the method comprises culturing a host cell containing the nucleic acid described in the fourth aspect, and isolating the antibody or antigen-binding fragment thereof or multi-specific antigen-binding molecule expressed by the host cell.

[0049] In the seventh aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof described in the first aspect, the variant antibody described in the second aspect, the bispecific antibody described in the third aspect, the nucleic acid described in the fourth aspect, and the pharmaceutical composition described in the fifth aspect in the preparation of drugs for treating and / or preventing cancer or tumors and immune abnormality-related diseases.

[0050] In an eighth aspect, the present invention provides the antibodies or antigen-binding fragments thereof described in the first and second aspects of the invention, the variant antibodies described in the second aspect, the bispecific antibodies described in the third aspect, the nucleic acids described in the fourth aspect, and the pharmaceutical compositions described in the fifth aspect, which are used to treat and / or prevent diseases associated with abnormal immune abnormalities such as abnormally high expression of TNFR2 and an excessively low ratio of effector T cells / regulatory T cells (Teffs / Tregs).

[0051] In the ninth aspect, the present invention provides a method for down-regulating the number of regulatory T cells (Tregs) of a subject while promoting the proliferation of effector T cells and killer T cells of the subject, improving tumor killing activity, and ultimately increasing the Teffs / Tregs ratio and improving the patient's anti-tumor level, which comprises administering to the subject an effective amount of the antibody, bispecific antibody or immunoconjugate described in the preceding aspects to achieve an increase in T cell proliferation or a decrease or consumption of the number of regulatory T cells or co-stimulation of effector T cells.

[0052] In the tenth aspect, the present invention provides a method for treating and / or preventing tumors and immune abnormality-related diseases, the method comprising administering to a subject an effective amount of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the variant antibody described in the second aspect, the bispecific antigen-binding molecule described in the third aspect, the nucleic acid described in the fourth aspect, or the composition described in the fifth aspect.

[0053] Preferably, the method further provides for administering to the subject an additional anti-tumor therapeutic agent, such as a chemotherapeutic agent, a targeted therapeutic agent, and an immunotherapeutic agent.

[0054] In the eleventh aspect, the present invention provides a method for inhibiting tumor immune response mediated by Tregs cells in a human body, the method comprising administering to the human the antibody or antigen-binding fragment thereof described in the first aspect, the variant antibody described in the second aspect, the bispecific antigen-binding molecule described in the third aspect, the nucleic acid described in the fourth aspect, or the composition described in the fifth aspect. .

[0055] In a twelfth aspect, the present invention provides a method for treating a cell proliferation disorder in a human, the method comprising administering to the human the antibody or antigen-binding fragment thereof of the first aspect, the variant antibody of the second aspect, the bispecific antigen-binding molecule of the third aspect, the nucleic acid of the fourth aspect, or the composition of the fifth aspect.

[0056] Preferably, the disease is an immune abnormality-related disease.

[0057] In certain embodiments, the method further comprises administering an immune checkpoint antagonist.

[0058] Preferably, the immune abnormality-related disease is cancer or an autoimmune disease;

[0059] Preferably, the immune abnormality-related disease is cancer;

[0060] Preferably, the cancer or tumor is a cancer associated with abnormal function of Tregs cells and / or MDSCs.

[0061] In some embodiments, the cancer is a cancer selected from the group consisting of leukemia, lymphoma, liver cancer, bone cancer, lung cancer, brain cancer, bladder cancer, gastrointestinal cancer, breast cancer, cardia cancer, cervical cancer, uterine cancer, head and neck cancer, gallbladder cancer, laryngeal cancer, lip and oral cancer, eye cancer, melanoma, pancreatic cancer, prostate cancer, colorectal cancer, testicular cancer, and laryngeal cancer.

[0062] Preferably, the cancer is selected from ovarian cancer, advanced epidermal T-cell lymphoma, stage III / IV metastatic colorectal cancer, triple-negative breast cancer, pancreatic cancer, non-small cell lung cancer and / or advanced solid tumors resistant to CTLA-4 and PD-1 therapy, such as metastatic melanoma.

[0063] Preferably, the cancer or tumor is a cancer associated with abnormal function of Tregs cells and / or MDSCs.

[0064] Preferably, the cancer to be treated is non-small cell lung cancer, breast cancer, ovarian cancer or colorectal cancer.

[0065] In certain embodiments, the immune checkpoint is a PD-1 / PD-L1 immune checkpoint.

[0066] In certain embodiments, the immune checkpoint antagonist is an antibody or antigen-binding fragment thereof specific for PD-1 or PD-L1.

[0067] In certain embodiments, the method further comprises administering to the patient a chemotherapeutic agent, an anti-angiogenic agent, a growth inhibitory agent, a tumor immunotherapy agent, and / or an anti-neoplastic composition.

[0068] Beneficial Effects

[0069] The antibody or antigen-binding fragment thereof specifically binding to TNFR2 of the present invention and its defucosylated variant can selectively bind to TNFR2+Tregs and reduce the proportion of Tregs in the tumor microenvironment, and can activate and promote the proliferation of CD4+T cells and CD8+T cells, increase the INF-γ level and tumor killing activity, and can mediate the in vitro killing of tumor cells in PBMC without mediating the killing of CD4+ and CD8+ cells, and can inhibit tumor growth in vivo in a mouse model of tumor, especially colon cancer transplantation, and has the prospect of being developed into a tumor-targeted immunotherapeutic agent with better tumor treatment effect; its monoclonal antibody, bispecific antibody, conjugate thereof with therapeutic drugs, and composition containing the same are expected to provide a new tumor immunotherapy strategy for tumor patients with unsatisfactory PD-1 targeted tumor treatment effect and Tregs regulation-related tumor patients, which can not only target and block antigen and ligand signaling pathways, but also activate the patient's immune system to promote its spontaneous elimination of tumors without directly acting on normal tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] When read in conjunction with the accompanying drawings, the foregoing summary of the invention and the specific embodiments of the present disclosure below will be better understood. For the purpose of illustrating the present disclosure, shown in the drawings are currently preferred embodiments. However, it should be understood that the present disclosure is not limited to the precise arrangements, embodiments and means shown.

[0071] Figure 1 . Shows the binding activity of the chimeric antibody 21E11-CHI (A shows the results of ELISA-binding activity analysis of binding to TNFR2-His; B shows the results of FACS-binding activity analysis of binding to CHOK1 cells M5 overexpressing TNFR2).

[0072] Figure 2 .Shows the activity of chimeric antibody 21E11-CHI in antagonizing the binding of TNF-α to protein TNFR2 (A. ELISA antagonism IC50 curve of antagonizing the binding to TNFR2-His determined by ELISA, B. FACS antagonism IC50 curve of antagonizing the binding of TNF-α to CHO-K1 cells M5 overexpressing TNFR2 determined by FACS).

[0073] Figure 3 . Shows the binding activity of humanized antibody HU6 (A shows the results of ELISA-binding EC50 analysis of binding to TNFR2-His; B shows the results of FACS-binding EC50 analysis of binding to TNFR2-overexpressing CHO-K1 cells M5).

[0074] Figure 4. Shows the affinity test results of the chimeric antibody 21E11-CHI and the humanized antibody HU6 binding to the antigen TNFR2-His (A is the affinity determination curve of the chimeric antibody 21E11, and B is the affinity determination curve of the humanized antibody HU6).

[0075] Figure 5 . ELISA test results showing the cross-reactivity of the chimeric antibody 21E11-CHI and the humanized antibody HU6 of the present invention with mouse and cynomolgus monkey TNFR2 proteins (A is the ELISA binding EC50 curve for binding to mouse TNFR2-His, and B is the ELISA binding EC50 curve for binding to cynomolgus monkey TNFR2-His protein).

[0076] Figure 6 The results of binding activity test of the chimeric antibody 21E11-CHI and the humanized antibody HU6 of the invention with proteins of the same family are shown (A is the binding activity curve with CD40, and B is the binding activity curve with 4-1BB).

[0077] Figure 7 The activity of humanized antibody HU6 in antagonizing the binding of ligand TNF-α to protein TNFR2 is shown (A. IC50 curve of ELISA for antagonizing the binding of ligand TNF-α to TNFR2-His in vitro, B. IC50 curve of FACS for antagonizing the binding of TNF-α to CHO-K1 cells M5 overexpressing TNFR2).

[0078] Figure 8 The results of constructing the reporter system for the TNFR2 receptor signaling pathway are shown (A shows that the JURKAT-NF-KB-M45-D1 clone selected by flow cytometry overexpressed the TNFR2 receptor; B shows that the addition of soluble and coated TNF-α-biotin to the constructed reporter cell line JURKAT-NF-KB-M45-D1 can stimulate the NF-KB signal downstream of the TNFR2 receptor).

[0079] Fig. 9 It is shown that the soluble humanized HU6 antibody of the present invention does not have the activity of stimulating the TNFα pathway, but can inhibit the downstream signaling activity of TNFR2 stimulated by the ligand TNF-α.

[0080] Fig.10The humanized antibody HU6 of the present invention shows the agonistic effect on the NF-KB signaling pathway (A shows that the coated and fixed humanized HU6 antibody has an agonistic effect on the NF-KB signaling pathway, and the measurement results of different antibody concentrations of 0-150μg / mL, B shows that a single FC cross-linked antibody Fab has no agonistic effect on the TNFR2 signaling system, while the humanized HU6 antibody cross-linked with the FAB antibody has obvious agonistic activity, and is positively correlated with the antibody concentration, and C shows the agonistic activity of the THP-1 cell cross-linked defucosylated humanized HU6 antibody, which is also significantly lower than 10ng / mL).

[0081] Fig.11 The results showed that TNFR2 protein was expressed specifically in the tumor microenvironment.

[0082] Fig.12 Shown are the results of FACS evaluation of the binding selectivity of the humanized antibody HU6 to CD4+CD25+ T cells.

[0083] (A shows that the level of CD25 is positively correlated with the expression of TNFR2, B shows that the binding activity of HU6 to CD4+CD25+ cells is significantly stronger than that of #001, and C shows that the humanized antibodies HU6 and #001 basically do not bind to CD4+CD25- cells).

[0084] Fig.13 It shows that the humanized antibody HU6 mediates the downregulation of the proportion of Tregs cells in PBMCs in vitro (A, B, C and D are the mediation assay results in B.P121120602C, P122121106C, CAPB01230501C and P122110501C, respectively).

[0085] Fig.14 The results show that the defucosylating humanized antibody HU6-AF mediates downregulation of the proportion of Tregs cells in PBMCs in vitro (A, B, C and D are the results of the mediation assays in P122121106C, CAPB01230501C and P122110501C, respectively).

[0086] Fig.15 Flow cytometric analysis results showing in vitro activation of CD4 T cells by wild-type humanized antibody HU6 (A and B show that after CD4 T cells were treated with antibody HU6, the proportion of CD25+ cells was significantly upregulated compared with the IgG1 control antibody, and 5C shows that the CD25 protein fluorescence level of CD25+ positive cells also increased).

[0087] Fig.16.The results of flow cytometric analysis showed that wild-type humanized antibody HU6 significantly promoted CD4+T cell proliferation after treating CD4+T cells (A is the PB fluorescence graph of flow cytometry analysis (five-finger peak graph), B shows that the proliferation ratio of CD4T cells in the HU6 antibody group is 43.25% (A right), which is significantly higher than the proliferation ratio of 23.67% (A left) in the IgG1 control group; C shows that compared with the IgG1 control group, the 10μg / mL HU6 antibody group significantly promoted the secretion level of INF-γ).

[0088] Fig.17 . Flow cytometric analysis results of the wild-type humanized antibody HU6-mediated upregulation of PD-1 expression in CD4+ T cells in vitro (A and B show the IgG1 control antibody, the proportion of PD-1+ cells in the antibody HU6-treated group was significantly upregulated; (C shows that the fluorescence level of PD-1 membrane protein in CD25+ positive cells also increased).

[0089] Fig.18 .Flow cytometric analysis results of the defucosylating humanized antibody HU6-AF promoting CD4+T cell proliferation in vitro (A shows that the addition of CD3 / CD28 can stimulate the activation and proliferation of CD4+T cells, B shows that the proliferation ratio of CD4+T cells in the IgG1 control group is 9.33%, C shows that the proliferation ratio of CD4+T cells in the HU6-AF antibody group is 35.14%, and D is a bar graph showing that the division ratio of CD4+T cells is positively correlated with the antibody concentration).

[0090] Fig.19 . Results showing that wild-type humanized HU6-AF up-regulated the proportion and expression level of CD25+ positive cells in a concentration-dependent manner (A shows that the proportion of CD25+ positive cells is up-regulated in a concentration-positive manner, B shows that the expression level of CD25+ positive cells is up-regulated in a concentration-positive manner, C and D show that HU6-AF up-regulated the proportion of PD-1+ positive cells in a concentration-positive manner ( Fig.19 C) and expression levels).

[0091] Fig. 20 Flow cytometry results showing the effect of defucosylating humanized antibody HU6-AF on activating CD8+T cell proliferation (A left, middle and right respectively show that blank group CD3 / CD28 can stimulate CD8+T cell activation and proliferation, IgG1 control group stimulates CD8+T cell proliferation ratio of 20.69%, HU6-AF antibody group stimulates CD8+T cell proliferation ratio of 51.00%, B shows that the activation effect of antibody on CD4 T cells is positively correlated with antibody concentration, C and D respectively show that after HU6-AF treatment of CD8 T cells, compared with IgG1 control antibody, the proportion of CD25+ cells and the fluorescence level of CD25 protein in CD25+ positive cells increased).

[0092] Fig.21 The results show that the defucosylating humanized antibody HU6-AF antibody promotes the killing of CD8+T cells (A and B respectively show that compared with the control group IgG1, the HU6-AF antibody group significantly upregulated the expression levels of INF-γ and granulamycin B in a concentration-positively correlated manner).

[0093] Fig. 22 The results of the pharmacodynamic evaluation showed that the antibodies HU6 and HU6-AF of the present invention significantly mediated the killing of tumor cells by PBMC in vitro.

[0094] Fig.23 The ADCC results of the antibodies HU6 and HU6-AF of the present invention on activated CD4+T cells are shown (A shows that the ADCC of the defucosyl antibody HU6-AF on CD4+T activated cells is stronger than that of the wild-type antibody HU6, and B is a bar graph showing the ADCC killing rates mediated by the blank control hIgG1 group, the humanized antibody HU6, the defucosyl antibody HU6-AF and the HU6-IgG4 subtype (ADCC function defective)).

[0095] Fig.24 The results show that the antibodies HU6 and HU6-AF of the present invention do not mediate the killing of CD4+ and CD8+ cells in PBMC (A and B respectively show the proportions of CD4+ and CD8+ cells after HU6-AF and HU6-treated activated PBMC).

[0096] Fig.25 These are the results of in vivo anti-tumor efficacy tests of HU6 and HU6-AF in the MC38-transplanted hTNFR2 humanized mouse model.

[0097] Fig.26 These are the results of measuring the effects of HU6 and HU6-AF on the infiltration levels of lymphocytes in the periphery and tumor microenvironment in the MC38 tumor model. DETAILED DESCRIPTION

[0098] The detailed aspects of the present invention are further and separately described in each section below. However, it should be understood that any embodiment of the present invention (including the embodiment described only in the examples or drawings and the embodiment described only in one section below) can be combined with any other embodiment of the present invention.

[0099] Provided herein are antibodies or antigen-binding fragments thereof, bispecific antibodies, pharmaceutical compositions, and combination therapeutic drugs that specifically bind to TNFR2 (e.g., human TNFR2). Also provided are methods for preparing antibodies, and uses of the antibodies or antigen-binding fragments thereof, bispecific antibodies, and pharmaceutical compositions in the preparation of drugs for inhibiting tumor growth and treating cancer.

[0100] I. Antigen, ligand and regulatory antibody strategies

[0101] The ligand tumor necrosis factor-α (TNF-α) exists in two biologically active forms, transmembrane TNF-α (tmTNF-α) and soluble TNF-α (sTNF-α). Transmembrane TNF (tmTNF-α) is the major ligand of TNFR2 and the only form that effectively activates TNFR2. It contains the conserved carboxyl-terminal homology domain of the TNF homology domain (THD).

[0102] The terms "tumor necrosis factor receptor 2" and "TNFR2", TNFRII, "CD120b", "TNFR1B" and "TNFR80". and are used interchangeably. In the tumor microenvironment, TNFR2 is abnormally highly expressed mainly in tumor cells and suppressive immune cells such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). TNFR2 mediates the stimulatory activity of TNF on CD4+FoxP3+Tregs, and binding to tmTNF-α activates downstream signaling pathways, thereby promoting the proliferation and activation of Tregs and MDSCs, and inhibiting the function of tumor-functional cytotoxic T cells. TNFR2 can participate in the formation of an immunosuppressive tumor microenvironment (TME) in many types of tumors, and is also a potential driving force for checkpoint blockade resistance. For both PD-1-resistant and PD-1-sensitive models, TNFR2 monoclonal antibodies have significant tumor inhibitory effects.

[0103] TNFR2 is also a co-stimulatory member of the tumor necrosis factor receptor superfamily (TNFRSF). TNFR2 is expressed at low levels in naive CD4+T and CD8+T cells, but TNFR2 is an effective co-stimulatory molecule expressed on the surface of activated CD8+T and CD4+T cells in the tumor microenvironment. In the presence of TCR stimulation, binding to cross-linked TNFR2 antibodies promotes the activation, proliferation and production of tumor-killing active cytokines such as INF-γ of CD4+T cells and CD8+T cells.

[0104] The antibody of the present invention can downregulate the infiltration ratio of Tregs cells in the tumor microenvironment, induce CD8+ and CD4+ T cell proliferation, and upregulate the ratio of CD8+ and CD4+ T cells. The antibody of the present invention preferentially binds to TCR-activated CD8+ T cells and CD4+ T cells, and enhances CD3 / CD28-induced T cell activation and proliferation; and increases the secretion level of INF-γ and the tumor killing activity based on INF-γ.

[0105] In the present invention, TNFR2 is a human TNFR2 protein comprising an amino acid sequence as shown in SEQ ID NO: 17, and an encoding nucleic acid comprising a polynucleotide sequence as shown in SEQ ID NO: 18:

[0106] The amino acid sequence of human TNFR2 membrane protein is:

[0107] MAPVAVWAALAVGLELWAAAHALPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNR ICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSDVVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPE PSTAPSTSFLLPMGPSPPAEGSTGDFALPVGLIVGVTALGLLIIGVVNCVIMTQVKKKPLCLQREAKVPHLPADKARGTQGPEQQHLLITAPSSSSSSLESSASALDRRAPTRNQP QAPGVEASGAGEARASTGSSDSSPGGHGTQVNVTCIVNVCSSSDHSSQCSSQASSTMGDTDSSPSESPKDEQVPFSKEECAFRSQLETPETLLGSTEEKPLPLGVPDAGMKPS(SEQ ID NO:17).

[0108] The encoding nucleic acid sequence of human TNFR2 membrane protein is:

[0109]

[0110] Among many immunotherapies, targeting and eliminating abnormal numbers of TNFR2+Tregs to revitalize the patient's immune system or inhibiting the binding of ligands to TNFR2 on the surface of tumor cells to inhibit the immune escape of tumor cells is a classic tumor treatment option with great potential. TNFR2 is a promising therapeutic target for tumor immunotherapy in recent years. TNFR2 expression on regulatory and effector T cells in the tumor microenvironment is associated with T cell exhaustion and resistance to immune checkpoint blockade. In a variety of tumor microenvironments such as acute myeloid leukemia, lung cancer, and ovarian cancer, TNFR2+Treg subpopulations and TNFR2+MDSCs with abnormally elevated TNFR2 expression can participate in the formation of an immunosuppressive tumor microenvironment, weaken the body's anti-tumor immune response, and assist in the immune escape of tumors.

[0111] The term "myeloid-derived suppressor cells" or "MDSC" refers to a heterogeneous population of immature myeloid cells that regulate the activity of various effector cells and antigen-presenting cells of the immune system, such as T cells, NK cells, dendritic cells, and macrophages. MDSC express FcγRs and have a significant ability to suppress innate and adaptive immune responses in vitro and in vivo.

[0112] The term "regulatory T cells" or "Tregs", also known as suppressor T cells, is a group of lymphocytes that have a regulatory effect and negatively regulate the body's immune response by suppressing / inhibiting the proliferation, activation and cytotoxicity of other immune cells (such as CD8-positive (CD8+) effector T cells). They express biomarkers CD4, FOXP3 and CD25, are immune cells with high expression of TNFR2, and can infiltrate into tumors to inhibit the activity of the immune system. Therefore, they play an extremely important role in the occurrence of tumors. Tumor cells induce the proliferation of tumor-infiltrating Tregs cells. The infiltrating Tregs proliferating cells will secrete a large amount of immunosuppressive factors such as TGF-β, inhibit the function of effector T cells (Teffs) such as CD8+T cells, and hinder the killing effect of immune cells on tumors. It is an important resistance mechanism for the failure of immunotherapy for various solid tumors and hematological tumors. Therefore, regulating the function of Tregs in the tumor microenvironment is a proven effective cancer treatment strategy.

[0113] The regulatory antibodies that specifically bind to TNFR2 of the present invention can restore immune balance and kill tumor cells by clearing Tregs and activating Teffs at the same time. The TNFR2-specific regulatory antibodies of the present invention may have specific recognition of the tumor microenvironment, and may even be more targeted in treating tumors than other immunotherapies. The key to Tregs-related anti-tumor treatment strategies is whether it is possible to effectively and accurately downregulate the number of Tregs and stimulate and promote the proliferation and killing activity of effector T cells at the tumor site, and the regulatory antibodies targeting TNFR2 of the present invention can selectively downregulate the number of Tregs and increase the proportion and killing activity of effector T cells in the tumor microenvironment, thereby improving the anti-tumor treatment effect of patients, and are more specific than other immune checkpoint inhibitors. In general, the TNFR2 regulatory antibodies of the present invention show great medicinal potential at the tumor microenvironment level.

[0114] II. Exemplary regulatory antibodies that specifically bind to TNFR2

[0115] The present invention provides antibodies or antigen-binding fragments thereof that specifically bind to human TNFR2, which can be used as anticancer agents. Although not wishing to be bound by any particular theory, it is believed that the anti-TNFR2 antibody of the present invention downregulates the number of Tregs in tumor tissues and inhibits tumor immune escape while enhancing the tumor killing activity of effector T cells and cytotoxic T cells, and is a regulatory antibody.

[0116] The anti-TNFR2 antibodies (eg, isolated anti-human TNFR2 antibodies) disclosed herein are characterized by particular functional and structural features (eg, CDRs, variable regions, heavy and light chains).

[0117] The following section provides a description of exemplary features of the antibodies and antigen-binding fragments thereof that specifically bind to TNFR2 of the present invention, as well as their use in the preparation of medicaments and methods of treating diseases.

[0118] On the one hand, the invention provides an antibody or an antigen-binding fragment thereof that specifically binds to TNFR2, which comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises VHCDR1, VHCR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NOs: 1, 2 and 3, respectively, and the VL comprises an antigen-binding fragment thereof, which comprises a heavy chain variable region (VH) and a light chain variable region (VL).

[0119] Table 1. Antibodies or antigen-binding fragments specifically binding to TNFR2 of the present invention

[0120]

[0121] In the present invention, the term "CDR" or "complementarity determining region" refers to the non-continuous antigen binding sites found in the variable regions of both heavy and light chain polypeptides, which are short polypeptide sequences in the variable regions of heavy and light chain polypeptides that are primarily responsible for mediating specific antigen recognition, including amino acids in CDRs or complementary determining regions identified using sequence-based or structure-based methods. In certain embodiments, the term "CDR" is a CDR defined by Kabat based on sequence comparison. There are three CDRs (called CDR1, CDR2, and CDR3) in each VL and each VH. Unless otherwise specified herein, CDRs and framework regions are annotated according to the Kabat numbering scheme.

[0122] In some embodiments, the antibody that specifically binds to TNFR2 includes a monoclonal antibody or a polyclonal antibody.

[0123] Exemplary anti-TNFR2 antibodies include the murine antibody 21E11, the murine chimeric antibody 21E11-CHI, and the humanized antibody HU6.

[0124] In some embodiments, the antibody that specifically binds to TNFR2 is a monoclonal antibody.

[0125] In some embodiments, exemplary monoclonal antibodies of the invention that specifically bind to TNFR2 are murine antibodies.

[0126] The amino acid sequences of the exemplary murine antibodies VHCDR1, VHCR2 and VHCDR3 are shown in Table 1, respectively. The amino acid sequences of VLCDR1, VLCR2 and VLCDR3 are shown in Table 1, respectively. The exemplary murine monoclonal antibody is named 21E11.

[0127] The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0128] In some embodiments, an exemplary murine monoclonal antibody that specifically binds to TNFR2 of the present invention comprises a heavy chain variable region (VH1) having an amino acid sequence as shown in SEQ ID NO:7 in Table 2.

[0129] In some embodiments, an exemplary murine monoclonal antibody that specifically binds to TNFR2 of the present invention comprises a light chain variable region (VL1) having an amino acid sequence as shown in SEQ ID NO: 8 in Table 2.

[0130] In some exemplary embodiments, the exemplary monoclonal antibody that specifically binds to TNFR2 of the present invention is a murine chimeric antibody, comprising a heavy chain variable region (VH1) having an amino acid sequence as shown in SEQ ID NO:7 shown in Table 2 and a light chain variable region (VL1) having an amino acid sequence as shown in SEQ ID NO:8, and the exemplary murine chimeric antibody is named 21E11-CHI.

[0131] In some embodiments, exemplary monoclonal antibodies of the invention that specifically bind to TNFR2 are humanized antibodies.

[0132] In some embodiments, an exemplary human monoclonal antibody that specifically binds to TNFR2 of the present invention comprises a heavy chain variable region (VH2) having an amino acid sequence as shown in SEQ ID NO: 11 in Table 2.

[0133] In some embodiments, an exemplary human monoclonal antibody that specifically binds to TNFR2 of the present invention comprises a light chain variable region (VL2) having an amino acid sequence as shown in SEQ ID NO: 12 in Table 2.

[0134] In some exemplary embodiments, the exemplary humanized monoclonal antibody that specifically binds to TNFR2 of the present invention comprises a heavy chain variable region (VH2) having an amino acid sequence as shown in SEQ ID NO: 11 and a light chain variable region (VL2) having an amino acid sequence as shown in SEQ ID NO: 12 as shown in Table 2, and the exemplary humanized antibody is named HU6.

[0135] Table 2 Variable regions of exemplary anti-TNFR antibodies

[0136]

[0137] Constant region

[0138] In some embodiments, the antibody or antigen-binding fragment thereof comprises or does not comprise a heavy chain constant region (CH) and / or a light chain constant region (CL);

[0139] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) and / or a light chain constant region (CL);

[0140] Preferably, the heavy chain constant region (CH) comprises a full-length heavy chain constant region or a fragment thereof, wherein the fragment is selected from a CH1 domain, an Fc domain or a CH3 domain;

[0141] In some embodiments, the monoclonal antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL).

[0142] In some embodiments, the C-terminus of the heavy chain variable region (VH) and the N-terminus of the heavy chain constant region (CH) in the monoclonal antibody are connected to form a heavy chain (HC).

[0143] Preferably, the heavy chain constant region can be selected from an IgG heavy chain constant region, such as one of an IgG1 heavy chain constant region, an IgG2 heavy chain constant region, an IgG3 heavy chain constant region or an IgG4 heavy chain constant region.

[0144] Preferably, the heavy chain constant region can be selected from one of the human IgG1 heavy chain constant region, the human IgG2 heavy chain constant region, the human IgG3 heavy chain constant region or the human IgG4 heavy chain constant region.

[0145] CH2 and CH3 combine to form an Fc structure that can interact with certain Fc receptors to exert effector functions. "Effector functions" include but are not limited to C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions (such as ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) and downregulation of cell surface receptors. Such effector functions generally require the Fc region to be combined with an antigen-binding domain (e.g., an antibody variable domain).

[0146] In some embodiments, the amino acid changes described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0147] In some embodiments, the light chain constant region (CL) is selected from one of human kappa and lambda type light chain constant regions.

[0148] In some embodiments, the light chain constant region (CL) is a human kappa light chain constant region.

[0149] In some embodiments, the variable region (VH or VL) of the anti-TNFR2 antibodies of the invention can be covalently linked to at least one other antibody domain or fragment thereof at the C-terminal amino acid. Thus, for example, the VH domain present in the variable region domain can be linked to an immunoglobulin CH1 domain or fragment thereof. Similarly, the VL domain can be linked to a CL domain at the C-terminus.

[0150] Light and heavy chains

[0151] In some embodiments, the C-terminus of the heavy chain variable region (VH) and the N-terminus of the heavy chain constant region (CH) in the monoclonal antibody are connected to form a heavy chain (HC).

[0152] In some embodiments, the heavy chain constant region (CH) comprises the amino acid sequence shown in SEQ ID NO:15.

[0153] In some embodiments, the C-terminus of the light chain variable region (VL) and the N-terminus of the light chain constant region (CL) in the monoclonal antibody are connected to form a light chain (LC).

[0154] In some embodiments, the light chain constant region (CL) comprises the amino acid sequence shown in SEQ ID NO:16.

[0155] In some embodiments, the monoclonal antibody includes a murine chimeric antibody or a humanized antibody.

[0156] In some embodiments, the monoclonal antibody is a murine chimeric antibody.

[0157] In some embodiments, an exemplary murine chimeric antibody that specifically binds to TNFR2 of the present invention comprises a heavy chain 1 (HC1) having an amino acid sequence as shown in SEQ ID NO:9 in Table 3.

[0158] In some embodiments, an exemplary murine single chimeric antibody that specifically binds to TNFR2 of the present invention comprises the light chain 1 (LC1) of the amino acid sequence shown in SEQ ID NO: 10 in Table 3.

[0159] In some exemplary embodiments, the exemplary murine chimeric monoclonal antibody that specifically binds to TNFR2 of the present invention comprises a heavy chain 1 (HC1) having an amino acid sequence as shown in SEQ ID NO:9 and a light chain 2 (LC2) having an amino acid sequence as shown in SEQ ID NO:10, and is named In some embodiments, the exemplary murine chimeric monoclonal antibody that specifically binds to TNFR2 of the present invention is named murine clone 21E11-CHI.

[0160] In some embodiments, the monoclonal antibody is a humanized antibody.

[0161] In some embodiments, an exemplary human monoclonal antibody that specifically binds to TNFR2 of the present invention comprises a heavy chain 2 (HC2) having an amino acid sequence as shown in SEQ ID NO: 13 in Table 3.

[0162] In some embodiments, an exemplary human monoclonal antibody that specifically binds to TNFR2 of the present invention comprises light chain 2 (LC2) having an amino acid sequence as shown in SEQ ID NO: 14 in Table 3.

[0163] In some exemplary embodiments, an exemplary humanized monoclonal antibody that specifically binds to TNFR2 of the present invention comprises a heavy chain 2 (HC2) having an amino acid sequence as shown in SEQ ID NO:13 and a light chain 2 (LC2) having an amino acid sequence as shown in SEQ ID NO:14, and is named In some embodiments, an exemplary murine monoclonal antibody that specifically binds to TNFR2 of the present invention is named HU6.

[0164] Table 3. Heavy and light chains of exemplary anti-TNFR2 antibodies

[0165]

[0166] IV. Antigen Binding Fragment

[0167] Exemplary antigen-binding fragments of the invention that specifically bind to TNFR2 include: (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments connected by a disulfide bridge at the hinge region; (iii) Fd fragments consisting of VH and CH domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody, (v) dAb fragments consisting of the VH domain; (vi) isolated complementarity determining regions (CDRs) and (vii) combinations of two or more isolated CDRs, which may be optionally connected by synthetic linkers. The original binding fragments contain the aforementioned VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3, respectively, as shown in SEQ ID NOs: 1-6.

[0168] III. Antibody variants that specifically bind to TNFR2

[0169] In yet another aspect, the present invention provides a functional conservative variant of the antibody or antigen-binding fragment that specifically binds to TNFR2 according to the first aspect, wherein the functional conservative variant comprises a heavy chain variable region variant (VH') and a light chain variable region variant (VL'), wherein the VH' comprises any one, two or three of VH'CDR1, VH'CDR2 and VH'CDR3, and the VL' comprises any one, two or three of VL'CDR2, VL'CDR2 and VL'CDR3;

[0170] (1) the VH'CDR1, VH'CDR2 and VH'CDR3 respectively comprise:

[0171] a. an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity compared to the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, or,

[0172] b. An amino acid sequence selected from the group consisting of 1 or 2 amino acid insertions, deletions and / or substitutions compared to the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3;

[0173] (2) the VL'CDR2, VL'CDR3 and VL'CDR3 respectively comprise:

[0174] c. a VL sequence having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity compared to the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, or,

[0175] d. An amino acid sequence having 1 or 2 amino acid insertions, deletions and / or substitutions compared to the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

[0176] The VH CDR3 motif of the antibody or antigen-binding fragment that specifically binds to TNFR2 is conserved, while other positions within this CDR can be varied without losing affinity for TNFR2 and antagonistic function.

[0177] In some embodiments, the VH' comprises:

[0178] a. an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity compared to the amino acid sequence of SEQ ID NO: 7 or 11, or,

[0179] b. A conservatively modified amino acid sequence having 1, 2, 3, 4, 5 or more amino acid insertions, deletions and / or substitutions compared to the amino acid sequence shown in SEQ ID NO: 7 or 11.

[0180] In some embodiments, the VL' comprises:

[0181] c. an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity compared to the amino acid sequence of SEQ ID NO: 8 or 12, or,

[0182] d. A conservatively modified amino acid sequence having 1, 2, 3, 4, 5 or more amino acid insertions, deletions and / or substitutions compared to the amino acid sequence shown in SEQ ID NO: 8 or 12.

[0183] Optionally, the amino acid insertion, deletion and / or substitution occurs in the FR region of the heavy chain variable region or / and the light chain variable region;

[0184] Optionally, the substitution is a conservative amino acid substitution.

[0185] In some embodiments, the functional conservative variants of the antibody or antigen-binding fragment that specifically binds to TNFR2 comprise a heavy chain variant (HC') and a light chain variant (LC').

[0186] In some embodiments, the HC' comprises:

[0187] a. an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity compared to the amino acid sequence of SEQ ID NO: 9 or 13, or,

[0188] b. A conservatively modified amino acid sequence having 1, 2, 3, 4, 5 or more amino acid insertions, deletions and / or substitutions compared to the amino acid sequence shown in SEQ ID NO: 9 or 13.

[0189] In some embodiments, the LC' comprises:

[0190] c. an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity compared to the amino acid sequence of SEQ ID NO: 10 or 14, or,

[0191] d. A conservatively modified amino acid sequence having 1, 2, 3, 4, 5 or more amino acid insertions, deletions and / or substitutions compared to the amino acid sequence shown in SEQ ID NO: 10 or 14.

[0192] The term "sequence identity" or "sequence identity percentage (%)" refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those of a reference sequence after the sequences are aligned and (if necessary) gaps are introduced to obtain the maximum sequence identity percentage. Alignment for the purpose of determining the sequence identity percentage can be achieved in various ways within the technical scope of the art, such as using publicly available computer software, such as BLAST, ALIGN software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve 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 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.

[0193] Optionally, the amino acid insertion, deletion and / or substitution occurs in the heavy chain variable region and / or the light chain variable region.

[0194] Optionally, the substitution is a conservative amino acid substitution.

[0195] The skilled artisan will further appreciate that the light chain variable region and the heavy chain variable region can be independently selected or mixed and matched to prepare a functional conservative variant of an anti-TNFR2 antibody comprising a combination of a variable heavy chain and a variable light chain other than the above identified pairs. The variable region domains of the functional conservative variants of the present invention will generally comprise one or two or more of the corresponding CDR variants of VH'CDR1, VH'CDR2 and VH'CDR3, VL'CDR1, VL'CDR2 and VL'CDR3 described herein, and which are adjacent to or within the framework of one or more framework sequences.

[0196] Thus, in some embodiments, function conservative variants of the modulatory TNFR2 antibodies of the invention comprise a variant VH' and a variant VL'. In some embodiments, the variant VH' and variant VL' comprise at least one variant CDR as described herein. As described herein, fragments of antibody variants may comprise at least two, three, four, five or six variant CDRs.

[0197] The antibody variant may further comprise at least one of the VH' and VL' described herein.

[0198] "Conservatively modified amino acids" refer to amino acid modifications that do not significantly affect or change the binding properties of the antibody containing the amino acid sequence. Conservative modifications include amino acid substitutions, additions, and deletions. Conservative substitutions refer to replacement of an amino acid with an amino acid residue having a similar side chain. "Conservatively modified amino acids" are a family of amino acid residues with similar side chains.

[0199] "Conservative amino acids" generally refer to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). Exemplarily, the amino acids in each of the following groups belong to conservative amino acid residues of each other, and the replacement of amino acid residues in the group belongs to the replacement of conservative amino acids:

[0200] Conservative amino acid families include, for example, (i) G, A, V, L, I, P, and M; (ii) D and E; (iii) C, S, and T; (iv) H; K and R; (v) N and Q; and (vi) F, Y; and A conservative mutation or substitution is thus a mutation or substitution in which an amino acid replaces members of the same amino acid family (e.g., Ser replaces Thr or Lys replaces Arg).

[0201] Functionally conservative variants of the antibodies that specifically bind to TNFR2 comprising a variable heavy chain variant (VH') and a variable light chain variant (VL') sequence retain the functional activity of specifically binding to TNFR2.

[0202] IV. Functional activity of antibodies

[0203] In some embodiments, the level of binding of the antibodies of the invention to TNFR2 can be assessed using standard binding assays, such as surface plasmon resonance (SPR), biofilm interferometry (BLI), ELISA, Western blot, immunofluorescence, flow cytometry analysis, chemotaxis assays, and cell migration assays. In some aspects, the ability of the generated antibodies to block / inhibit TNFα / TNFR binding interactions in solution or on the surface of cells can also be assessed.

[0204] The wild-type and defucosylated humanized anti-TNFR2 antibodies of the present invention have been functionally verified to have enhanced specific binding to human TNFR2, blocking of TNF-TNFR binding, binding selectivity to tumor microenvironment, and immune effector cell activation and tumor killing functional activity. The selected antibodies also show cross-reactivity to monkey orthologs of human TNFR2 and almost no binding to the same family CD40 protein and 4-1BB protein, which will be a feature that is beneficial to toxicity studies of human therapeutic agents in animals.

[0205] The antibody of the present invention downregulates the infiltration ratio of Tregs cells in tumor microenvironment, induces CD8+ and CD4+ T cell proliferation in the tumor microenvironment, and upregulates the ratio of CD8+ and CD4+ T cells. The antibody of the present invention preferentially binds to TCR-activated CD8+ T cells and CD4+ T cells, and enhances CD3 / CD28-induced T cell activation and proliferation; as well as the secretion level of INF-γ and the tumor killing activity based on INF-γ.

[0206] Through functional verification, the antibody or antigen-binding fragment specifically binding to TNFR2 of the present invention, or its function-conservative variant and defucosylated variant have the following functional activities:

[0207] 1. Ability to bind to human TNFR2 antigen in vitro and at the cellular level

[0208] In some embodiments, the antibodies or antigen-binding fragments of TNFR2 of the present invention specifically bind to human TNFR2 with high affinity in vitro.

[0209] In some embodiments, the antibodies of the invention are chimeric antibodies.

[0210] In some embodiments, the chimeric anti-TNFR2 antibodies of the invention are capable of specifically binding to TNFR2-His in vitro.

[0211] In some preferred embodiments, the chimeric antibodies of the present invention can bind to human TNFR2 in vitro in an ELISA test with an ELISA binding EC50 of less than about 6.187 ng / mL, or preferably less than about 6.164 ng / mL, or preferably less than about 6.146 ng / mL, or more preferably less than about 6.115 ng / mL, or more preferably less than about 6.089 ng / mL, or more preferably less than about 6.068 ng / mL, or more preferably less than about 6.042 ng / mL, or more preferably less than about 6.025 ng / mL, or most preferably less than about 6.015 ng / mL.

[0212] In some embodiments, the chimeric antibodies of the invention are capable of binding to cells that overexpress human TNFR2.

[0213] In some preferred embodiments, in FACS assay, the chimeric antibodies of the present invention can bind to cells overexpressing human TNFR2 with a FACS binding EC50 of less than about 46.38 ng / mL, or preferably less than about 46.03 ng / mL, or preferably less than about 45.81 ng / mL, or preferably less than about 45.69 ng / mL, or more preferably less than about 45.52 ng / mL, or more preferably less than about 45.38 ng / mL, or more preferably less than about 45.15 ng / mL, or more preferably less than about 45.01.ng / mL, or most preferably less than about 44.90 ng / mL.

[0214] In some preferred embodiments, the cell overexpressing human TNFR2 is CHO-K1-TNFR2-M5.

[0215] In some embodiments, the anti-TNFR2 antibodies of the invention are humanized antibodies.

[0216] In some embodiments, in the affinity assay based on biolayer interferoc-Metry (BLI) for detecting the antibody of the present invention and the antigen, the humanized anti-TNFR2 antibody of the present invention has a KD (M) of preferably less than about 5.52E-09, or more preferably less than about 5.37E-09, or more preferably less than about 5.26E-09, or more preferably less than about 5.12E-09, or more preferably less than about 4.93E-09, or more preferably less than about 4.78E-09, or more preferably less than about 4.63E-09 in vitro. Preferably, the TNFR2-His binding protein is less than about 4.51E-09, or more preferably less than about 4.39E-09, or more preferably less than about 4.93E-09, or more preferably less than about 4.78E-09, or more preferably less than about 4.63E-09, or more preferably less than about 4.51E-09, or more preferably less than about 4.39E-09, or more preferably less than about 4.27E-09, or more preferably less than about 4.16E-09, or more preferably less than about 4.12E-09, or most preferably less than about 4.08E-09.

[0217] In some embodiments, the humanized anti-TNFR2 antibodies of the invention specifically bind to TNFR2-his in vitro with an ELISA binding EC50 comparable to that of the parent chimeric antibody.

[0218] In some embodiments, the humanized anti-TNFR2 antibodies of the invention specifically bind to human TNFR2-his in vitro in an ELISA assay with an ELISA binding EC50 of less than about 3.056 ng / mL, or preferably less than about 3.031 ng / mL, or preferably less than about 3.017 ng / mL, or more preferably less than about 2.986 ng / mL, or more preferably less than about 2.971 ng / mL, or more preferably less than about 2.951 ng / mL, or more preferably less than about 2.936 ng / mL, or more preferably less than about 2.917 ng / mL, or most preferably less than about 2.908 ng / mL.

[0219] In some embodiments, the humanized anti-TNFR2 antibodies of the invention specifically bind to cell surface TNFR2 with a FACS binding EC50 comparable to that of the parent chimeric antibody.

[0220] In some preferred embodiments, in a FACS assay, the humanized anti-TNFR2 antibodies of the present invention can bind to cells overexpressing human TNFR2 with a FACS binding EC50 of less than about 18.96 ng / mL, or preferably less than about 18.83 ng / mL, or preferably less than about 18.611 ng / mL, or preferably less than about 18.45 ng / mL, or more preferably less than about 18.31 ng / mL, or more preferably less than about 18.12 ng / mL, or more preferably less than about 17.94 ng / mL, or more preferably less than about 17.82 ng / mL, or most preferably less than about 17.76 ng / mL.

[0221] In some preferred embodiments, the cell overexpressing human TNFR2 is CHO-K1-TNFR2-M5.

[0222] 2. Cross-binding activity

[0223] In some preferred embodiments, the chimeric and humanized anti-TNFR2 antibodies of the invention have cross-reactivity with cynomolgus monkey TNFR2 protein, but not with murine TNFR2 protein.

[0224] In some preferred embodiments, the chimeric and humanized anti-TNFR2 antibodies of the present invention have no cross-binding activity with proteins of the same family.

[0225] In some preferred embodiments, the same family proteins include CD40 and 4-1BB proteins

[0226] 3. Antagonize the binding of TNF-α ligand to TNFR2 in vitro and at the cellular level

[0227] In some embodiments, the chimeric anti-TNFR2 antibodies of the invention are capable of antagonizing the binding of the ligand TNF-α to TNFR2-His in vitro.

[0228] In some embodiments, the chimeric anti-TNFR2 antibodies of the invention have an ELISA antagonist IC50 of less than about 893.4 ng / mL, or preferably less than about 862.5 ng / mL, or preferably less than about 849.6 ng / mL, or more preferably less than about 831.3 ng / mL, or more preferably less than about 816.6 ng / mL, or more preferably less than about 802.5 ng / mL, or more preferably less than about 799.8 ng / mL, or more preferably less than about 796.5 ng / mL, or most preferably less than about 794.1 ng / mL for antagonizing the binding of the ligand TNF-α to TNFR2-His in vitro.

[0229] In some embodiments, the chimeric anti-TNFR2 antibodies of the invention are capable of antagonizing the binding of the ligand TNF-α to the membrane protein TNFR2.

[0230] In some embodiments, the chimeric anti-TNFR2 antibodies of the invention have a FACS antagonism IC50 of antagonizing the binding of the ligand TNF-α to the membrane protein TNFR2 of less than about 596.8 ng / mL, or preferably less than about 571.4 ng / mL, or preferably less than about 557.8 ng / mL, or more preferably less than about 538.9 ng / mL, or more preferably less than about 513.2 ng / mL, or more preferably less than about 496.7 ng / mL, or more preferably less than about 481.2 ng / mL, or more preferably less than about 465.8 ng / mL, or most preferably less than about 463.9 ng / mL.

[0231] In some embodiments, the humanized anti-TNFR2 antibodies of the invention are capable of antagonizing the binding of the ligand TNF-α to TNFR2-His in vitro.

[0232] In some embodiments, the humanized anti-TNFR2 antibodies of the invention have an ELISA antagonism IC50 of less than about 381.6 ng / mL, or preferably less than about 373.5 ng / mL, or preferably less than about 368.2 ng / mL, or more preferably less than about 361.4 ng / mL, or more preferably less than about 353.7 ng / mL, or more preferably less than about 346.8 ng / mL, or more preferably less than about 342.4 ng / mL, or more preferably less than about 341.9 ng / mL, or most preferably less than about 341.6 ng / mL for antagonizing the binding of the ligand TNF-α to TNFR2-His in vitro, which is comparable to the ELISA antagonism IC50 of the parent chimeric antibody.

[0233] In some embodiments, the humanized anti-TNFR2 antibodies of the present invention are capable of antagonizing the binding of the ligand TNF-α to the membrane protein TNFR2.

[0234] In some embodiments, the humanized anti-TNFR2 of the present invention is capable of antagonizing the binding of the ligand TNF-α to the membrane protein TNFR2 in vitro with a FACS antagonism IC50 of less than about 155.3 ng / mL, or preferably less than about 153.1 ng / mL, or preferably less than about 150.2 ng / mL, or more preferably less than about 148.4 ng / mL, or more preferably less than about 145.3 ng / mL, or more preferably less than about 142.5 ng / mL, or more preferably less than about 139.4 ng / mL, or more preferably less than about 136.7 ng / mL, or most preferably less than about 133.8 ng / mL.

[0235] As used herein, the term "about" refers to a value that is no more than 10% above or below the described value. For example, the term "about 5 nM" indicates a range of 4.5 nM to 5.5 nM.

[0236] In some embodiments, the anti-TNFR2 antibody is a regulatory antibody that can selectively inhibit the proliferation of Tregs cells in the tumor microenvironment; after binding to TNFR2 in a cross-linked state, it can activate the NF-KB signaling pathway downstream of TNFR2 in the cell, promoting the proliferation of T effector cells and killer T cells (Teff). As described herein, the ability to deplete activated Tregs cell populations can reduce the attenuation of cytotoxic CD8+T cells, thereby enabling effector cells (Teff) to launch an immune response against cancer cells.

[0237] 4. Agonist activity on TNFR2 and downstream signaling pathways

[0238] In some embodiments, the soluble wild-type humanized antibody of the present invention does not have agonistic activity, but can inhibit the downstream signaling activity of TNFR2 stimulated by the ligand TNF-α, and the degree of inhibition increases with the increase of the antibody concentration.

[0239] In some embodiments, the coated and fixed wild-type humanized anti-TNFR2 antibody of the present invention has a certain agonistic effect on the NF-KB signaling pathway downstream of TNFR2, thereby increasing the activity of NF-kB.

[0240] For example, as assessed by a NF-kB reporter cell line (e.g., a NF-kB reporter cell line engineered to express human TNFR2), the coated and immobilized wild-type humanized anti-TNFR2 antibody of the invention increases NF-kB activity, e.g., by at least 2-fold, at least 3-fold, relative to a control (e.g., an isotype control antibody or a NF-kB reporter cell line that does not express human TNFR2).

[0241] In some embodiments, a single FC cross-linked antibody Fab has no agonistic effect on the TNFR2 signaling system, while the wild-type humanized anti-TNFR2 antibody of the present invention cross-linked with the FAB antibody has significant agonistic activity on the NF-KB signaling pathway, and the agonistic activity is positively correlated with the antibody concentration. In some embodiments, the wild-type humanized anti-TNFR2 antibody of the present invention cross-linked with the FAB antibody increases the NF-kB activity, for example, by at least 4 times, at least 5 times, or at least 10 times, relative to a control (e.g., an isotype control antibody or a NF-kB reporter gene cell line that does not express human TNFR2). In some embodiments, the agonistic activity of the wild-type humanized anti-TNFR2 antibody of the present invention cross-linked with the FAB antibody on the NF-KB signaling pathway is significantly lower than the agonistic activity of 10 ng / mL TNF-α.

[0242] In some embodiments, the defucosylated humanized anti-TNFR2 antibody of the present invention cross-linked with CD32a on human monocytic leukemia cells (THP-1 cells) has the activity of agonizing NF-KB signaling downstream of TNFR2.

[0243] In some embodiments, the activity of the defucosylated humanized anti-TNFR2 antibody in agonizing the TNFR2 signaling pathway at a low concentration has a positive correlation with the concentration.

[0244] In other embodiments, the humanized anti-TNFR2 antibody of the invention cross-linked with a defucosylated humanized anti-TNFR2 antibody of the invention by THP-1 cells increases NF-kB activity, e.g., by at least 2-fold, relative to a control (e.g., an isotype control antibody or a NF-kB reporter cell line that does not express human TNFR2).

[0245] In some embodiments, the THP-1 cell cross-linked defucosylated humanized anti-TNFR2 antibody of the invention has active agonist activity that agonizes the TNFR2 signaling pathway at a concentration of less than 0.08 μg / mL.

[0246] In some embodiments, the agonistic activity of THP-1 cell cross-linked defucosylated humanized anti-TNFR2, such as HU6 antibody, is significantly lower than the agonistic activity of TNF-α at a concentration of 10 ng / mL.

[0247] 5. Combined regioselectivity

[0248] The expression and distribution of TNFR2 protein are tissue, organ and cell specific. It is expressed at high concentrations in the tumor microenvironment. The TNFR2-positive cell rates and membrane protein levels in different lymphocyte populations in peripheral blood, spleen and tumor infiltration are different.

[0249] In some embodiments, FACS binding analysis shows that the humanized anti-TNFR2 antibodies of the present invention have selective and specific binding to CD4+CD25+ T cells, and do not substantially bind to CD4+CD25- cells.

[0250] In some embodiments, FACS binding analysis shows that the FACS binding EC50 of the wild-type humanized anti-TNFR2 antibody of the present invention for CD4+CD25+ T cells is less than about 10.61 ng / mL, or less than about 10.46 g / mL, or less than about 10.38 ng / mL, or less than about 10.27 ng / mL, or less than about 10.15 ng / mL, or less than about 10.03 ng / mL, or less than about 9.895 ng / mL, or less than about 9.75 ng / mL, or less than about 9.67 ng / mL, which is significantly stronger than the positive control; and at the same time, it does not substantially bind to CD4+CD25- cells.

[0251] 6. Can mediate downregulation of the proportion of Tregs cells in PBMC in vitro

[0252] In some embodiments, the wild-type humanized anti-TNFR2 antibody and the defucosyl humanized TNFR2 antibody of the present invention can mediate downregulation of the proportion of Tregs cells in PBMCs in vitro.

[0253] In some embodiments, the wild-type humanized anti-TNFR2 antibody mediates a greater downregulation of the proportion of Treg cells in PBMCs relative to a control (eg, a no antibody control or an isotype antibody control).

[0254] In some embodiments, the degree of downregulation of the proportion of Tregs cells in PBMCs mediated by the defucosyl humanized TNFR2 antibody of the present invention in vitro is higher than that mediated by the wild-type humanized TNFR2 antibody.

[0255] 7. Stimulate effector T cell proliferation and promote tumor killing ability

[0256] In some embodiments, the humanized anti-TNFR2 antibody and the defucosyl humanized anti-TNFR2 antibody of the present invention can activate immune effector cells such as CD4+T cells in vitro; upregulate the proportion of CD25+T cells; stimulate the activation and proliferation of CD4+T cells after the addition of CD3 / CD28; promote the secretion of INF-γ, and enhance the killing function of CD4+T.

[0257] In some embodiments, the wild-type humanized anti-TNFR2 antibody of the present invention activates CD4 T cells in vitro, promotes CD4 T cell proliferation, promotes INF-γ secretion, and can upregulate the expression level of CD25 protein and PD-1 membrane protein level of CD25+T cells. In some embodiments, the humanized anti-TNFR2 antibody of the present invention can activate CD4+T cells and increase the content of CD25+T cells at a concentration of 10μg / ml, or 1μg / ml, or 0.1μg / ml, and the higher the antibody concentration, the higher the percentage of CD25+T cells.

[0258] In some embodiments, after the action of 10 μg / ml of the humanized anti-TNFR2 antibody of the present invention, the content of CD25+ T cells reaches about 80%, which is 15% higher than the content of CD25+ T cells after the action of the no antibody control or the isotype antibody control, and the proportion of CD25+ cells is significantly increased.

[0259] In some embodiments, compared with the IgG1 control antibody, 10 μg / mL of wild-type humanized anti-TNFR2 antibody such as HU6 significantly promoted CD4+T cell proliferation after treating CD4+T cells, and the proliferation ratio of CD4+T cells was about 43.25% ( Fig.16 A right), which was significantly higher than the proliferation ratio of 23.67% in the IgG1 control group.

[0260] In some embodiments, the humanized anti-TNFR2 antibody of the present invention can promote the secretion level of INF-γ at a concentration of 10 μg / ml, or 1 μg / ml, or 0.1 μg / ml, and the secretion level of INF-γ is positively correlated with the concentration of the wild-type humanized TNFR2 antibody.

[0261] In some embodiments, the humanized anti-TNFR2 antibody of the present invention can promote the secretion of INF-γ at a concentration of 10 μg / ml, and the concentration of INF-γ reaches 380 ng / ml, which is significantly higher than the concentration of the isotype antibody control that promotes the secretion of INF-γ ( Fig.16 C).

[0262] In some embodiments, after the CD4+T cells added with CD3 / CD28 were treated with 50 μg / mL of the defucosylated TNFR2 antibody of the present invention, the proliferation ratio of CD4+T cells was 35.14%, which was significantly higher than the proliferation ratio of 9.33% of the IgG1 control group, and the activation effect on CD4 T cells was positively correlated with the antibody concentration, indicating that the defucosylated antibody of the present invention significantly promoted the proliferation of CD4+T cells.

[0263] In some embodiments, after CD4+T cells are treated with the humanized TNFR2 antibody of the present invention, the fluorescence level of CD25 protein in CD25+ cells increases compared to that of the IgG1 control antibody.

[0264] In some embodiments, the humanized TNFR2 antibody of the present invention can increase the fluorescence level of CD25 protein of CD25+ positive cells by 3.8×10 5 , which was significantly higher than the fluorescence level of CD25 protein in CD25+ positive cells in the isotype antibody control group (2.4×10 5 , which is about 2.1×10 higher than the fluorescence level of CD25 protein in CD25+ positive cells treated with 0.1μg / ml and 10.1μg / ml concentrations. 5 .

[0265] In some embodiments, after the humanized TNFR2 antibody of the present invention treats CD4+T cells, the proportion of PD-1+ cells is about 70%, compared with the treatment result of the IgG1 control antibody, the proportion of PD-1+ cells is about 51%, and the proportion of PD-1+ cells is significantly increased.

[0266] In some embodiments, after the humanized TNFR2 antibody of the present invention treats CD4+T cells, the PD-1 membrane protein fluorescence level of CD25+ positive cells reaches 3.0×10 4 , which is higher than that of 0.1μg / ml and 10.1μg / ml. The PD-1 membrane protein fluorescence level of CD25+ positive cells increased to about 1.6-1.8×10 4 .

[0267] In some embodiments, the defucosylating humanized anti-TNFR2 antibody of the present invention also activates CD4+ T cells, promotes CD4 T cell proliferation and upregulates PD-1 expression in vitro.

[0268] In some embodiments, after CD4+T cells are treated with 50 μg / mL of the defucosylating humanized anti-TNFR2 antibody of the present invention, the proliferation rate of CD4+T cells reaches 35.14%, which is significantly higher than the proliferation rate of 9.33% of the IgG1 control group, and the activation effect on CD4+T cells is positively correlated with the antibody concentration; in addition, the proportion and expression level of CD25+positive cells are upregulated in a manner positively correlated with the concentration, and the proportion and expression level of PD-1+positive cells are also upregulated in a manner positively correlated with the concentration.

[0269] In some embodiments, the defucosylated humanized anti-TNFR2 antibody of the present invention upregulates the proportion of CD25+ positive cells and the proportion of PD-1+ positive cells in a concentration-positively correlated manner, and the defucosylated antibody at a concentration of 50 μg / mL upregulates the proportion of CD25+ positive cells by 60% ( Fig.19 A) and the CD25 membrane protein fluorescence level reached 2.75×105μg / ml. The same concentration of defucosylated antibody upregulated the proportion of PD-1+ positive cells to 65% and the PD-1 membrane protein fluorescence level was higher than 1.6-1.8×10 4 .

[0270] 8. It can activate cytotoxic CD8+T cells in vitro, promote CD8+T cell proliferation, and enhance the killing ability of CD8+T cells

[0271] In some embodiments, the defucosylating humanized TNFR2 antibody of the present invention can activate cytotoxic CD8+T cells in vitro and promote CD8+T cell proliferation, and the activation effect on CD8+T cells is positively correlated with the antibody concentration; it upregulates the expression level of INF-γ and the expression level of granulamycin B, and promotes the killing ability of CD8+T cells.

[0272] In some embodiments, on the basis that CD3 / CD28 can stimulate the activation and proliferation of CD8 T cells, 50 μg / mL of the defucosylated antibody of the present invention can significantly promote the proliferation of CD8+T cells after treating CD8+T cells, and the proliferation ratio of CD8+T cells reaches 51.00%, which is significantly higher than the proliferation ratio of 20.69% of the IgG1 control group, and the activation effect on CD8+T cells is positively correlated with the antibody concentration.

[0273] In some embodiments, the defucosylated antibodies of the present invention can increase the proportion of CD25+CD8+ T cells to nearly 80% at a concentration of 50 μg / ml.

[0274] In some embodiments, increasing the concentration of the fucosylated antibody of the present invention does not significantly increase the fluorescence level of the CD25 protein of CD25+CD8+ T cells.

[0275] In some embodiments, compared with the control group IgG1, the defucosylated antibody group of the present invention significantly upregulates the expression level of INF-γ in a concentration-positively correlated manner, and can upregulate the expression level of INF-γ to a concentration of nearly 40% and the expression level of granulamycin B to a concentration of up to 75% at a concentration of 50 μg / ml.

[0276] 9. It can mediate PBMC killing of tumor cells in vitro.

[0277] In some embodiments, the in vitro efficacy of the defucosyl humanized anti-TNFR2 antibody of the present invention on TNFR2+ tumor cells is significantly better than that of the wild-type humanized antibody, and the in vitro killing efficacy of the defucosyl anti-TNFR2 antibody and the wild-type humanized antibody on TNFR2+ tumor cells is better than that of the anti-PD-1 antibody.

[0278] 10. Antibody-dependent cellular cytotoxicity (ADCC) killing effect on activated CD4+ lymphocytes

[0279] In some embodiments, the wild-type and defucosylated humanized TNFR2 antibodies of the present invention have a weaker ADCC-mediated level on CD4+ T activated cells, and a lower antibody concentration is required to reach the upper plateau.

[0280] In some embodiments, the defucosyl humanized TNFR2 antibody of the present invention mediates ADCC of NK cells against activated CD4+ T cells more strongly than the wild type.

[0281] In some embodiments, the killing rate of activated CD4+T cells by the wild-type humanized antibody of the present invention is only about 30%-40% at most. High concentrations of the humanized and defucosyl modified antibodies of the present invention cannot mediate the killing of CD4+ and CD8+ cells in PBMCs, which may be because the humanized antibodies and defucosyl modified antibodies of the present invention have the effect of activating and promoting the proliferation of effector T cells and killer T cells, and the promoting effect is enhanced with the increase of antibody concentration.

[0282] In some embodiments, the wild-type and defucosyl-modified antibodies of the present invention cannot activate ADCC killing of CD4+ and CD8+ cells in PBMCs.

[0283] 11. It has the efficacy of inhibiting tumor growth in vivo in the hTNFR2 humanized mouse xenograft model.

[0284] In some embodiments, the hTNFR2 humanized mouse xenograft model is a mouse colon cancer cell xenograft model.

[0285] In some embodiments, in a mouse colon cancer cell xenograft model, the tumor inhibition rate (TGI) of the wild-type humanized anti-TNFR2 antibody of the present invention is 16.5%.

[0286] In some embodiments, in a mouse colon cancer cell xenograft model, the tumor inhibition rate (TGI) of the de-fucosyl modified humanized anti-TNFR2 antibody variant of the present invention is 81.%.

[0287] 12. Defucosylated humanized anti-TNFR2 antibody can significantly increase the tumor infiltration of CD4+T and CD8+T cells

[0288] In some embodiments, the defucosyl modified humanized antibody of the present invention increases the proportion of tumor infiltration of CD4+ / CD45+T cells and CD8+ / CD45+T cells after stimulation, which is significantly higher than the stimulation result of the wild-type antibody.

[0289] The antibody or antigen-binding fragment thereof that specifically binds to TNFR2 of the present invention can inhibit TNFR2 activation by blocking TNFR2 from binding to TNFα; it can inhibit the activation of TNFR2 on tumor cells that overexpress TNFR2 by binding to TNFR2 receptors highly expressed on the outer surface of Tregs cells in the tumor microenvironment, cancer cells that abnormally highly express TNFR2, or myeloid-derived suppressor cells (MDSC), thereby preventing the protein from recruiting its cognate ligand TNFα to bind to TNFR2 and curb receptor activation, inhibiting the growth and proliferation of Tregs, downregulating the content of Tregs, and reducing the level of tumor immune escape; on the other hand, it can preferentially bind to TCR-activated primary CD8+T cells and CD4+T cells in a cross-linked state, an immobilized state, and a cell-bound state, and enhance CD3 / CD28-induced CD8+T cell activation and proliferation and increase the level of secreted INF-γ and the tumor killing activity based on INF-γ, and ultimately can overall increase the number and activity of immune cells in patients, increase Teffs / Tregs, and increase the level of anti-tumor immunity in patients.

[0290] The term "tumor microenvironment" refers to the cancer cells that form a tumor as well as the population of non-cancerous cells, molecules, and / or blood vessels within or at the borders of the tumor or surrounding the cancer cells.

[0291] VI. Multispecific and bispecific antibodies

[0292] In some embodiments, an antibody of the invention that specifically binds to TNFR2 is a bispecific antibody, a trispecific antibody, a multispecific antibody, a minibody, or a polypeptide that contains at least a portion of an immunoglobulin sufficient to specifically bind TNFR2 to the polypeptide.

[0293] In some embodiments, the multispecific antibodies (e.g., bispecific antibodies) provided herein include at least a binding affinity for TNFR2 (e.g., human TNFR2) described herein, and at least one other binding specificity. The multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 antibodies).

[0294] In a specific embodiment, a multispecific antibody comprises a first antibody (or a binding portion thereof) that binds to a target epitope on TNFR2 that is derived or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or a ligand of a receptor) to produce a multispecific molecule that binds to an epitope on TNFR2 and another target molecule. Antibodies can be derived or linked to more than one other functional molecule to produce multispecific molecules that bind to more than two different binding sites and / or target molecules. To create a multispecific molecule, the antibodies disclosed herein can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent binding or other means) to one or more other binding molecules (e.g., another antibody, antibody fragment, peptide or binding mimetic) to produce a multispecific molecule.

[0295] In some embodiments, the multispecific molecule comprises at least one antibody or antibody fragment thereof as a binding specificity, including, for example, Fab, Fab', F(ab')2, Fv or single-chain Fv. The antibody may also be a light chain or heavy chain dimer, or any minimal fragment thereof, such as an Fv or single-chain construct.

[0296] Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the multispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab')2 or ligand x Fab fusion protein. The multispecific molecule can be a single chain molecule comprising a single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. The multispecific molecule can comprise at least two single chain molecules. Methods for preparing multispecific molecules.

[0297] In one aspect, the present invention provides a bispecific antibody comprising a first antigen binding domain that specifically binds to TNFR2 and a second antigen binding domain that specifically binds to another antigen.

[0298] In some embodiments, the second antigen can be any antigen of interest, for example, selected from tumor antigens (such as tumor-associated antigens and tumor-specific antigens), immunomodulatory receptors and immune checkpoint molecules. Herein, "tumor-associated antigens" refer to antigens that are highly expressed in tumor cells and also exist in healthy cells but have low expression levels. Herein, "tumor-specific antigens" refer to antigens that are specifically expressed in tumor cells and hardly expressed in healthy cells. Non-limiting examples of tumor-associated antigens may include CD19, CD20, EGFR, GPC3, HER-2, and FOLR1. Non-limiting examples of immune checkpoint molecules may include CTLA-4, LAG-3, and TIM-3. Immunomodulatory receptors may include, for example, immune activating receptors (such as CD27, CD137, CD40, GITR, and OX40) and immunosuppressive receptors (such as BTLA, CTLA4, and LAG-3).

[0299] In one embodiment, the second antigen binding domain is an antibody or antigen binding fragment thereof specific for PD-1 or PD-L1.

[0300] In one embodiment, the antibody is an anti-PD-1 antibody, such as cemiplimab, nivolumab or pembrolizumab. In one embodiment, the antibody is an anti-PD-L1 antibody, such as avelumab, durvalumab, atezolizumab, KN035 or CK-301.

[0301] VI. Encoding nucleic acid, vector and host cell containing the same, and method for preparing antibody

[0302] In one aspect, the present invention provides biological materials for preparing the above-mentioned antibody or antigen-binding fragment that specifically binds to TNFR2.

[0303] In some embodiments, the biological material comprises nucleic acids encoding the heavy chain variable region and / or light chain variable region, or the heavy chain and / or light chain of any of the above antibodies or fragments thereof that specifically bind to TNFR2.

[0304] In some embodiments, the biological material comprises one or more vectors carrying the nucleic acid, and the vector is an expression vector, such as a eukaryotic expression vector.

[0305] In some embodiments, the eukaryotic vector includes, but is not limited to, a virus, a plasmid, a cosmid, a lambda phage, or a yeast artificial chromosome (YAC).

[0306] In some embodiments, the eukaryotic expression vector is a eukaryotic expression plasmid.

[0307] In addition, one or more markers of the host cell that allows to select transfected can be introduced to select the cell that has stably incorporated DNA into its chromosome. The marker can for example provide prototrophy, biocidal resistance (for example, antibiotic) or heavy metal (such as copper) resistance etc. to the auxotrophic host. The selectable marker gene can be directly connected to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be needed so that the best synthetic mRNA can be obtained. These elements can include splicing signals, as well as transcription promoters, enhancers and termination signals.

[0308] Once an expression vector or DNA sequence for expression has been prepared, the expression vector can be transfected or introduced into an appropriate host cell.

[0309] In some embodiments, a host cell comprising the nucleic acid or the vector is provided. In some embodiments, the host cell is eukaryotic. In other embodiments, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or HEK293 cells) or other cells suitable for preparing antibodies or their antigen-binding fragments.

[0310] In a preferred embodiment, the mammalian cell line grown in suspension is Chinese hamster ovary cell (CHO cell), CHO-S cell or NSO cell. In a more preferred embodiment, the mammalian cell line grown in suspension is CHO-S cell.

[0311] In a more preferred embodiment, the host cell is an expiCHOS cell.

[0312] In a more preferred embodiment, the host cell is an expiCHOS cell lacking the function of fucosyltransferase 8 (FUT8).

[0313] VII. Methods for Producing Antibodies

[0314] In yet another aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment that specifically binds to TNNFR2, or the deglycosylated modified antibody according to the first aspect.

[0315] The method of preparing an anti-TNFR2 antibody or antibody fragment thereof can be performed by any method known in the art.

[0316] In some embodiments, the method comprises a hybridoma method and a genetic engineering method.

[0317] In an exemplary embodiment, the anti-TNFR2 antibody is a murine antibody.

[0318] In some embodiments, the murine anti-TNFR2 antibodies of the invention are prepared using a hybridoma method.

[0319] The present invention immunizes Balb / c mice with human TNFR2 recombinant protein emulsified with a rapid immune adjuvant to obtain high-titer, high-affinity, high-specificity antiserum and specific immune cells; then extracts mouse spleen lymphocytes, generates hybridomas with mouse myeloma cells by an electrofusion method, and screens hybridoma mother clones that can bind to human TNFR2; and further separates mouse monoclones with TNFR2 binding activity by a limiting dilution method, and obtains the heavy chain variable region sequence and light chain variable region sequence of the mouse clone by sequencing.

[0320] For example, the immunogen that can be used to immunize the recipient may include: DNA encoding human TNFR2 or a fragment thereof, a fusion protein comprising the full-length extracellular domain of TNFR2, or a recombinant cell engineered to overexpress human TNFR2. Any suitable immunization method may be used. Such methods may include adjuvants, other immunostimulants, repeated booster immunizations, and the use of one or more immunization routes.

[0321] In some embodiments, the anti-TNFR2 antibodies of the present invention are chimeric antibodies or humanized antibodies, which are prepared by genetic engineering recombinant expression methods. In order to recombinantly express antibodies, host cells can be transfected with one or more recombinant expression vectors carrying DNA fragments encoding immunoglobulin light and heavy chains of the antibodies, so that the light and heavy chains are expressed in the host cells and optionally secreted into the culture medium in which the host cells are cultured, and the antibodies can be recovered from the culture medium. Standard recombinant DNA methods are used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors and introduce the vectors into host cells.

[0322] In an exemplary embodiment, the antibody is a murine chimeric antibody.

[0323] In order to reduce the immunogenicity of the antibody, the chimeric antibody of the present invention is modified by replacing its constant region with a constant region from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing an antigen while having reduced immunogenicity in humans as compared to a mouse antibody.

[0324] One strategy that can be used to design the chimeric antibodies of the present invention is to construct a heavy chain expression plasmid that introduces a mouse VH encoding nucleic acid and a light chain expression plasmid that introduces a mouse VL encoding nucleic acid, transiently transfect host cells, screen for recombinant cells expressing heavy and light chains, harvest the cells after culture, purify and affinity chromatograph, and obtain a chimeric antibody in which the heavy chain constant region and the light chain constant region are replaced with humanized constant regions relative to the mouse antibody.

[0325] The inventors cloned the heavy chain variable region sequence and the light chain variable region encoding nucleic acid of the mouse monoclonal antibody into an expression vector containing a human IgG1 heavy chain constant region encoding nucleic acid and an expression vector containing a human κ light chain constant region encoding nucleic acid, respectively, and then transiently transfected the generated recombinant heavy chain expression vector and recombinant light chain expression vector into expiCHOS cells, cultured the obtained recombinant host cells, collected the cell supernatant, filtered it, and then purified it by affinity chromatography to obtain a chimeric antibody that specifically binds to TNFR2.

[0326] In an exemplary embodiment, the amino acid sequences of the heavy chain variable region (VH1) and the light chain variable region (VL1) of the murine chimeric antibody 21E11-CHI can be obtained by sequencing the murine monoclonal antibody named 21E11 obtained by the hybridoma method. The conversion of the encoding nucleic acid sequence according to the amino acid sequence of a known protein is a skill that ordinary technicians in the antibody field are proficient in, such as the PCR method.

[0327] In some embodiments, the humanized antibodies of the present invention comprise VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2 and VLCDR3 of the exemplary antibodies in the aforementioned aspects, whose amino acid sequences are shown in SEQ ID NOs: 1-6, respectively.

[0328] In an exemplary embodiment, the antibody is a humanized antibody.

[0329] In order to further improve the safety and efficacy of antibodies in the human body, it is necessary to optimize the structure and function of non-human antibodies.

[0330] "Humanized antibody" refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In some embodiments, all or substantially all of the CDRs in the humanized antibody correspond to the CDRs of non-human antibodies such as mouse antibodies, and all or substantially all of the FRs correspond to the FRs of human antibodies. The humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.

[0331] VH and VL can be produced by a variety of recombinant and synthetic techniques, such as by recombinant gene expression or solid phase peptide synthesis procedures described herein or known in the art. When produced by chemical synthesis methods, native chemical ligation can be optionally used as a strategy for synthesizing long peptides (e.g., greater than 50 amino acids).

[0332] In an exemplary embodiment, a humanized TNFR2-specific binding antibody of the present invention is produced using genetic engineering methods.

[0333] In an exemplary embodiment, the method can be based on the generation of a sequence as shown in SEQ ID NO: 11.

[0334] Design and synthesis of the heavy chain variable region (VH2) encoding nucleic acid of a humanized antibody according to the structure of VHFR1-VHCDR1-VHFR2-VHCDR2-VHFR3-VHCDR3-VHFR4, according to the generation of the nucleic acid comprising the sequence shown in SEQ ID NO: 12

[0335] Design of the structure of VLFR1-VLCDR1-VLFR2-VLCDR2-VLFR3-VLCDR3-VLFR4 and synthesis of nucleic acid encoding the light chain variable region (VL2) of a humanized antibody.

[0336] Those skilled in the art of antibodies can express and produce humanized heavy chain variable regions and light chain variable regions containing these CDRs by designing polynucleotides encoding the structures and amino acid sequences shown in, for example, the above-mentioned VH2 and VL2, and then using genetic engineering methods. Optionally, the CDRs can be separated by spacers, such as by framework regions (FRs) (e.g., framework regions of the framework sequences described herein or the germline consensus sequences of human antibodies) or flexible linkers (polyglycine or glycine / serine linkers as described herein or known in the art).

[0337] In some embodiments of the antibody field, certain amino acid residues of FR can participate in non-covalent interactions with antigens and thus contribute to the affinity of the antibody for the target antigen. In some embodiments, the amino acid residues of a single FR or a combination thereof can modulate the conformation of the CDR and thus indirectly affect the interaction of the antibody with the antigen. Alternatively, certain combinations of amino acid residues of murine FR can form an interface between the VH and VL domains and thus can contribute to the overall antibody structure required for antigen binding. In the case described above, retaining certain key FR amino acid residues of the murine anti-TNFR2 antibody may be beneficial for the antagonist antibodies and antigen-binding fragments thereof (such as humanized antibodies) of the present invention, because the amino acid residues of various framework regions (FRs) can promote high epitope affinity and improved biochemical activity of the antibody or antigen-binding fragment thereof.

[0338] In some embodiments, in addition to the CDR sequence from a TNFR2 antibody, such as the murine chimeric antibody 21E11 (ch), a human antibody framework region (FR) residue of a specific sequence selected by comparison and having high homology with the chimeric antibody FR region is introduced into the heavy chain and / or light chain variable domain of the humanized antibody. Exemplarily, the inventors humanized the heavy chain variable region (VH) encoding nucleic acid and the light chain variable region (VL) encoding nucleic acid of the chimeric antibody specifically binding to TNFR2 obtained above by CDR transplantation and back mutation at the nucleic acid level, respectively, to obtain a CDR containing a murine antibody, but containing a human antibody common framework region (human common FR) in the VH and VL domains, and further made multiple amino acid substitutions in the human antibody common FR with reference to the framework region (FR) of the murine antibody, and designed and produced the VH encoding nucleic acid sequence and VL encoding nucleic acid sequence of the humanized anti-TNFR2 antibody of the present invention. Certain amino acid site mutations can be achieved, for example, by designing the encoding nucleic acid sequences of VH2 and VL2 using CDR grafting and back mutation methods known in the art or described herein, constructing a recombinant expression plasmid, and recombinantly expressing the recombinant expression plasmid containing the encoding polynucleotides encoding the heavy and light chains of the humanized antibody in a host cell.

[0339] "Common human antibody framework region" or "human consensus framework" can be used interchangeably and refers to the framework of the most common amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences.

[0340] Once DNA fragments encoding VH and VL containing humanized anti-TNFR2 antibodies are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. For example, the isolated DNA encoding the VH region of the anti-TNFR2 antibody of the present invention is converted into a full-length heavy chain gene (and a Fab heavy chain gene) by operably linking the DNA encoding VH to another DNA molecule encoding a heavy chain constant region domain (CH1, CH2, CH3, and optionally CH4). The sequences of human heavy chain constant region genes are known in the art. And DNA fragments covering these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1 constant region in certain embodiments. For example, the isolated DNA encoding the VL region of an anti-TNFR2 antibody can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operably linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region CL. The sequences of human light chain constant region genes are known in the art.

[0341] In some embodiments, in order to express the regulatory antibody or antigen-binding fragment thereof that specifically binds to TNFR2 of the present invention, the polynucleotides encoding VH and VL can be inserted into a eukaryotic expression vector containing a nucleic acid encoding a human IgG1 heavy chain constant region and a eukaryotic expression vector containing a nucleic acid encoding a human κ-type light chain constant region, respectively, by genetic engineering operations, so as to obtain a recombinant humanized heavy chain (e.g., HC2) gene and a humanized light chain (e.g., LC2) encoding nucleic acid, respectively, which are operably linked to a humanized heavy chain recombinant expression vector and a humanized light chain recombinant expression vector, wherein the selected eukaryotic expression vector and expression control sequence are compatible with the expression host cell used.

[0342] After transfecting a recombinant expression vector containing a gene encoding an antibody that specifically binds to TNFR2 of the present invention into a mammalian host cell, the host cell is cultured for a period of time sufficient to allow the antibody to be expressed in the host cell or secreted into the culture medium in which the host cell is grown to produce the antibody.

[0343] A variety of techniques can be used to achieve this goal, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection or other conventional techniques.

[0344] In some preferred embodiments, the eukaryotic expression plasmid vector containing the anti-humanized anti-TNFR2 antibody encoding nucleic acid described in the above aspects is transfected into a mammalian host cell by electroporation. The methods and conditions for culturing the produced transfected cells and for recovering the produced antibody molecules are known to those skilled in the art and can be varied or optimized based on the present specification and methods known in the prior art according to the specific expression vector and mammalian host cell used.

[0345] In some embodiments, the host cell is an expiCHOS cell.

[0346] Furthermore, the inventors used CHO-K1-FUT8(-) host cells to express recombinant heavy chain expression vectors and recombinant light chain expression vectors to obtain a humanized TNFR2 antibody that has the ability to downregulate the number of Tregs and the level of tumor immune escape, activate effector T cells and killer T cells, increase their number and killing ability, increase the ratio of effector T cells / Tregs, mediate the enhancement of T cell tumor killing ability, and defucosylate CD4+T cells and CD8+T cells.

[0347] The polypeptide (eg, single chain polypeptide, antibody or antigen-binding fragment thereof) can be recovered from the culture medium using standard protein purification methods. The host cells can also be used to produce portions of intact antibodies.

[0348] Once an anti-TNFR2 antibody or antigen-binding fragment thereof of the invention has been produced by recombinant expression, it can be purified by any method known in the art (e.g., methods applicable to the purification of immunoglobulin molecules), such as by chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly by affinity for TNFR2 after selection by Protein A or Protein G, and size fractionation column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. In addition, the anti-TNFR2 polypeptides of the invention or fragments thereof can be fused to heterologous polypeptide sequences or drugs described herein or otherwise known in the art to facilitate purification or to produce therapeutic conjugates.

[0349] Once isolated, the anti-TNFR2 single chain polypeptide, antibody or antigen-binding fragment thereof can be further purified, if desired, for example, by high performance liquid chromatography or by gel filtration chromatography on, for example, a Protein A column.

[0350] In some exemplary embodiments, the genetic engineering method for preparing the chimeric antibody or humanized antibody comprises the steps of:

[0351] (1) obtaining nucleic acid encoding a heavy chain variable region (VH) and a light chain variable region (VL);

[0352] (2) Enzymatically cleaving the nucleic acid encoding the heavy chain variable region (VH) and the expression vector 1 containing the nucleic acid encoding the human IgG1 heavy chain constant region, inserting the nucleic acid encoding the heavy chain variable region (VH) into the 5' end of the nucleic acid encoding the human IgG1 heavy chain constant region of the expression vector 1, thereby generating a recombinant expression vector 1 encoding the full length of the human IgG1 heavy chain; enzymatically cleaving the nucleic acid encoding the heavy chain variable region (VH) and the expression vector 2 containing the nucleic acid encoding the human κ light chain constant region, inserting the nucleic acid encoding the light chain variable region (VL) into the 5' end of the nucleic acid encoding the human κ light chain constant region of the expression vector 2, thereby generating a recombinant expression vector 2 encoding the full length of the human κ light chain;

[0353] (3) transiently transfecting recombinant expression vectors 1 and 2 into mammalian host cells to produce recombinant host cells expressing anti-TNFR2 antibodies;

[0354] (4) Cultivating the recombinant host cells and purifying the regulatory antibodies of the present invention that are secreted.

[0355] In some exemplary embodiments, the anti-TNFR2 antibody is a chimeric antibody, wherein the heavy chain variable region VH1 comprises the amino acid sequence set forth in SEQ ID NO:7, and the light chain variable region VL1 comprises the amino acid sequence set forth in SEQ ID NO:8.

[0356] In some exemplary embodiments, the anti-TNFR2 antibody is a humanized antibody, wherein the heavy chain variable region VH2 comprises the amino acid sequence shown in SEQ ID NO:11, and the light chain variable region VL2 comprises the amino acid sequence shown in SEQ ID NO:12.

[0357] In some embodiments, those skilled in the art of antibodies can also prepare recombinant antibodies using gene editing methods such as transposon method, (CRISPR) / Cas system method and zinc finger nuclease and transcription activator-like effector nuclease (TALEN) and the like.

[0358] Methods for preparing multispecific antibodies are well known in the art. For example, the production of full-length multispecific antibodies can be based on the co-expression of two paired immunoglobulin heavy chain-light chains, wherein the two chains have different specificities. For example, multispecific antibodies can be produced using leucine zippers. Another strategy for preparing multispecific antibody fragments by using single-chain Fv (sFv) dimers has also been reported.

[0359] VIII. Pharmaceutical Compositions

[0360] In one aspect, the present invention provides a pharmaceutical composition comprising any antibody or antigen-binding fragment that specifically binds to TNFR2 as described in the aforementioned aspects, or a defucosylated modified variant thereof, or a nucleic acid encoding the same.

[0361] In some embodiments, the pharmaceutical composition is used to treat a tumor.

[0362] In some embodiments, pharmaceutical compositions are prepared by mixing the active ingredient (eg, an anti-TNFR2 antibody described herein) having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers using standard methods known in the art.

[0363] In some embodiments, the pharmaceutical composition may be suitable for parenteral administration, comprising aqueous and / or non-aqueous sterile injection solutions, which may contain antioxidants and / or buffers and / or bacteriostats and / or solutes that render the formulation isotonic with the blood of the intended recipient; and / or aqueous and / or non-aqueous sterile suspensions, which may contain suspending agents and / or thickening agents.

[0364] Pharmaceutical compositions containing antibodies or antigen-binding fragments thereof that specifically bind to TNFR2 of the present invention can be prepared using methods known in the art.

[0365] Water is a preferred carrier when the pharmaceutical composition is administered intravenously.The pharmaceutical formulations of the TNFR2 binding molecules described herein are preferably prepared in the form of lyophilized formulations or aqueous solutions.

[0366] Preferred pharmaceutical compositions are sterile compositions, compositions suitable for injection and sterile compositions suitable for injection by the desired route of administration, such as by intravenous injection.

[0367] The antibody molecules, nucleotide sequences, plasmids, cells and / or pharmaceutical compositions of the present invention can be presented in unit-dose or multi-dose containers (e.g., sealed ampoules and vials) and can be stored in a freeze-dried (i.e., lyophilized) condition, requiring only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use.

[0368] Sterile injectable solutions can be prepared by incorporating the desired amount of monoclonal antibody and one or a combination of the desired ingredients listed above into an appropriate solvent followed by sterilization microfiltration. Typically, dispersions are prepared by incorporating the antibody into a sterile vehicle comprising an alkaline dispersion medium and the desired other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze drying (lyophilization), which produce a powder of the active ingredient plus any other desired ingredients from its previously sterile filtered solution.

[0369] It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suited as unitary dosages for subjects to be treated; each unit containing a predetermined quantity of antibody calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms provided herein are dictated by and directly dependent on: (a) the unique characteristics of the antibody and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such antibodies with respect to therapeutic sensitivity in individuals.

[0370] In some embodiments, the pharmaceutical composition contains an active agent (e.g., an antagonist anti-TNFR2 antibody or fragment thereof) at a concentration effective for treatment. For example, a pharmaceutical composition of the invention can contain at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%) of the active agent by weight (w / w).

[0371] In some embodiments, the active agent (e.g., an antibody of the invention that specifically binds to TNFR2) that can be incorporated into a pharmaceutical formulation can itself have a desired level of purity. For example, an antibody of the invention or antigen-binding fragment thereof can be characterized by a degree of purity following isolation of the antibody from cell culture medium or following chemical synthesis of, for example, a single-chain antibody fragment (e.g., scFv) by established solid phase peptide synthesis methods or native chemical ligation as described herein. Prior to incorporating the antibody into a pharmaceutical composition, a modulatory TNFR2 antibody of the invention can be at least 10% pure (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or 100% pure).

[0372] Parenteral administration is the most common route of administration for therapeutic compositions comprising antibodies. The formulations can conveniently be present in unit dosage form and can be prepared by any method known in the pharmaceutical art. The amount of the antibody that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the specific mode of administration. The amount of antibody is generally an amount sufficient to produce a therapeutic effect. Typically, out of 100%, this amount accounts for about 0.001% to about 90% of the antibody mass, preferably about 0.005% to about 70%, and most preferably about 0.01% to about 30%.

[0373] The therapeutic composition must generally be sterile and stable under the conditions of manufacture and storage. The composition can be formulated into a solution, microemulsion, liposome or other ordered structure suitable for high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and a suitable mixture thereof.

[0374] In some embodiments, the pharmaceutical composition of the antibody that specifically binds to TNFR2 of the present invention can be prepared for storage as a lyophilized preparation or aqueous solution by mixing the antibody having the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (e.g., buffers, stabilizers, preservatives, isotonic agents, non-ionic detergents, antioxidants and other miscellaneous additives) commonly used in the art. Such additives must be non-toxic to the recipient at the dosage and concentration used.

[0375] Buffers help maintain pH in a range close to physiological conditions. They can be present in a concentration ranging from about 2 mM to about 50 mM. Buffers suitable for use with the modulatory TNFR2 polypeptides (e.g., single-chain polypeptides, antibodies, and antigen-binding fragments thereof) of the invention include organic and inorganic acids and salts thereof, such as phosphate buffers, histidine buffers, and trimethylamine salts such as Tris.

[0376] Isotonifiers, sometimes referred to as "stabilizers", may be added to ensure the isotonicity of the liquid composition of the present invention and include polyol sugar alcohols, such as trivalent or higher sugar alcohols, such as glycerol, sorbitol and mannitol. Stabilizers may be present in the range of 0.1 to 10,000 weight per weight of active protein.

[0377] Antibody molecules of the present invention, encoding nucleic acid, plasmid, cell can be lyophilized for storage before use and reorganized in suitable carrier. Any suitable lyophilization method (for example, spray drying, cake drying (cakedrying)) and / or recombinant technology can be adopted. It will be appreciated by those skilled in the art that lyophilization and reorganization may cause antibody activity loss in varying degrees (for example, utilizing conventional immunoglobulin, the activity loss of IgM antibody is often larger than IgG antibody), and may have to adjust the use level upwards to compensate. In some embodiments, when rehydrating, the activity (before lyophilization) of freeze-dried (freeze-dried) polypeptide binding part loss is no more than about 20%, or no more than about 25%, or no more than about 30%, or no more than about 35%, or no more than about 40%, or no more than about 45%, or no more than about 50%.

[0378] Regardless of the route of administration selected, the compositions provided herein can be used in a suitable hydrated form, and they can be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0379] In some embodiments, a pharmaceutical composition (or medicament) comprising an antibody molecule described herein will contain the anti-TNFR2 antibody molecule at a concentration between about 2 mg / ml and 150 mg / ml or between about 2 mg / ml and 200 mg / ml.

[0380] In some embodiments, the antibody molecules, nucleotide sequences, plasmids, viruses, cells and / or pharmaceutical compositions described herein are suitable for delivery by a route selected from the group consisting of: intravenous (IV or iv); intramuscular (IM or im); subcutaneous (SC or sc) or intratumoral.

[0381] X. Use in the preparation of drugs for treating diseases

[0382] In one aspect, the present invention provides the use of the above-mentioned antibodies or antigen-binding fragments that specifically bind to TNFR2, their function-conservative variants, defucosylated variants, bispecific antibodies, nucleic acids, and pharmaceutical compositions in the preparation of drugs for treating and / or preventing diseases related to immune abnormalities.

[0383] In one embodiment, the immune abnormality-related disease is a disease associated with a high content of TNFR2+Tregs cells.

[0384] In one embodiment, the drug induces apoptosis of Tregs.

[0385] In one embodiment, the drug is a drug having a Tregs proliferation inhibitory function.

[0386] In one embodiment, the drug is a drug that blocks the TNF-TNFR2 tumor signaling pathway.

[0387] In one embodiment, the drug is a drug that enhances NFκB signal transduction and induces up-regulation of NFκB downstream target gene expression, thereby activating the NFκB downstream signaling pathway.

[0388] In one embodiment, the drug is a drug that activates and promotes Teff cell proliferation and differentiation and / or enhances tumor-killing biological activity.

[0389] In a preferred embodiment, the cancer is a cancer associated with the regulatory function of Tregs cells and / or MDSCs.

[0390] In a preferred embodiment, the patient is a patient with a tumor that does not express TNFR2, but anti-TNFR2 will stimulate an immune response and cause an increase in TNFR2 in tumor-infiltrating immune cells.

[0391] In one embodiment, the drug is a drug that targets tumor cells for killing.

[0392] The tumor can be a solid tumor or a liquid tumor. In one embodiment, the cancer is a solid tumor.

[0393] In some embodiments, the immune abnormality-related disease is a cancer that forms a tumor microenvironment with abnormally high overexpression of TNFR2, for example, bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, esophageal cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, sex and reproductive organ cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumors, neuroectodermal cancer, spinal axis tumors, gliomas, meningiomas and pituitary adenomas.

[0394] In some embodiments, the cancer in which the tumor microenvironment abnormally overexpresses TNFR2 is selected from: ovarian cancer, melanoma, prostate cancer, intestinal cancer, gastric cancer, esophageal cancer, breast cancer, lung cancer, kidney cancer, pancreatic cancer, uterine cancer, liver cancer, bladder cancer, cervical cancer, oral cancer, brain cancer, testicular cancer, skin cancer, colorectal cancer, malignant glioma, thyroid cancer or related tumors.

[0395] In a preferred embodiment, the solid tumor is selected from ovarian cancer, advanced epidermal T-cell lymphoma, stage III / IV metastatic colorectal cancer, triple-negative breast cancer, pancreatic cancer, non-small cell lung cancer and / or advanced solid tumors resistant to CTLA-4 and PD-1 therapy, such as metastatic melanoma.

[0396] XIII. In vitro effects and therapeutic methods

[0397] In one aspect, the invention provides a method of inhibiting tumor growth, comprising administering to a subject a therapeutically effective amount of an anti-TNFR2 antibody described herein in an effective amount (eg, a therapeutically effective amount) to inhibit tumor growth.

[0398] In one aspect, the invention provides a method of inhibiting tumor cell growth, comprising administering to a subject a therapeutically effective amount (eg, a therapeutically effective amount) of an anti-TNFR2 antibody described herein to inhibit tumor cell growth.

[0399] In one aspect, the anti-TNFR2 antibodies described herein induce long-term anti-cancer effects.

[0400] In one aspect, the present invention provides a method for treating and / or preventing tumor diseases associated with immune abnormalities, the method comprising administering to a subject an effective amount of the aforementioned antibody or antigen-binding fragment thereof that specifically binds to TNFR2, or its defucosylated variant, bispecific antibody, nucleic acid fragment, pharmaceutical composition or combined therapeutic combination product of the present invention.

[0401] The term "effective amount" or "therapeutically effective amount" refers to an amount of a drug that is effective in treating a disease or condition in a subject. In some embodiments, an effective amount refers to an amount that is effective to achieve the desired therapeutic or preventive result at the desired dosage and time period. The therapeutically effective amount of an antibody of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antagonist to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which the therapeutically beneficial effects outweigh any toxic or deleterious effects of the antibody of the invention.

[0402] In a specific embodiment, the pharmaceutical composition is used as a therapeutic agent for treating patients with primary or metastatic cancers originating from epithelial cells. In a specific embodiment, a therapeutically effective amount of the composition described herein is administered to a cancer patient to kill tumor cells. In some embodiments, the composition described herein can be used to treat patients with tumors characterized by the presence of cancer cells that express or overexpress TNFR2. In some embodiments, the composition can be used to treat patients with tumors that do not express TNFR2, but anti-TNFR2 will stimulate an immune response and cause an increase in TNFR2 in tumor-infiltrating immune cells.

[0403] A "disorder" or "disease" is any condition that would benefit from treatment with one or more modulator TNFR2 antibodies of the invention, including those pathological conditions that predispose the mammal to the disorder in question. Non-limiting examples of disorders to be treated herein include cancer or tumors.

[0404] In one embodiment, the immune abnormality-related disease is a disease related to TNFR2+Tregs cell activity.

[0405] In one embodiment, the immune abnormality-related disease is a disease related to Tregs cells and / or MDSC function.

[0406] In some embodiments, the immune abnormality-related disease is a cancer that overexpresses TNFR2.

[0407] "Diseases associated with excessive levels of suppressive regulatory T lymphocytes (Tregs)" refers to any disease (non-autoimmune) in which the suppressive activity of regulatory T lymphocytes plays a role, specifically by promoting the development or persistence of the disease, and more specifically refers to cancers in which the suppressive activity of Tregs plays a role.

[0408] In the present invention, the term "cancer" is used to refer to a population of tumor cells that exhibit abnormally high levels of proliferation and growth. Cancer can be benign (also known as a benign tumor), precancerous or malignant. Cancer cells can be solid cancer cells (i.e., forming solid tumors) or leukemic cancer cells. The term "cancer growth" is used herein to refer to the proliferation or growth of one or more cells comprising a cancer that results in a corresponding increase in the size or extent of the cancer.

[0409] In the present invention, in the context of a cell population (such as a TNFR2+ cell population (e.g., Tregs, MDSCs, or TNFR2+ cancer cells)), the term "proliferation" refers to the mitosis and cell division of a cell to produce multiple cells. Cell proliferation can be, for example, demonstrated by finding an increase in the number of cells (e.g., TNFR+ cells) in a cell sample over a given period of time (e.g., over the course of one or more days). In the present invention, cell proliferation is considered to be "inhibited" when the rate of proliferation of a cell population (e.g., a population of TNFR2+ cells contacted with an antagonist anti-TNFR2 antibody described herein) relative to a control cell population (e.g., a population of TNFR2+ cells not contacted with an antagonist anti-TNFR2 antibody) is reduced.

[0410] In some embodiments, the cancer described herein includes, but is not limited to, solid tumors, blood cancers, soft tissue tumors, and metastatic lesions.

[0411] In certain embodiments, the tumor is an immunogenic tumor. In certain embodiments, the tumor is non-immunogenic.

[0412] In certain embodiments, the tumor is, for example, bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, esophageal cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, cancer of the sex and reproductive organs, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) tumors, neuroectodermal cancer, spinal axis tumors, gliomas, meningiomas, and pituitary adenomas.

[0413] In certain embodiments, non-limiting examples of cancers for treatment include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, glioma, gastric cancer, renal cancer, ovarian cancer, liver cancer, colorectal cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, breast cancer, head and neck cancer, melanoma, bone cancer, uterine cancer, and other hematological malignancies derived from either of the two major blood cell lineages (e.g., myeloid cell lineage or lymphocyte cell lineage).

[0414] In some embodiments, the solid tumor expressing or overexpressing TNFR2 is exemplified by breast cancer, head and neck cancer, lung cancer, melanoma (including uveal melanoma), colon cancer, kidney cancer, ovarian cancer, liver cancer, and prostate cancer.

[0415] In certain embodiments, the cancer is a hematological cancer (such as AML and DLBCL).

[0416] The subject can be examined during treatment to monitor the efficacy of the anti-TNFR2 antibody in reducing cancer progression (eg, as reflected by a reduction in the volume of one or more tumors).

[0417] In the present invention, the terms "subject", "object" and "patient", "subject in need of treatment" include subjects who already have a condition or disease, as well as subjects who are susceptible to a condition or disease or are intended to prevent a condition or disease. When referring to terms such as mitigation, alleviation, attenuation, alleviation, relief, etc., their meanings also include elimination, disappearance, non-occurrence, etc. of an organism receiving treatment for a specific disease or condition as described herein (such as cancer or infectious diseases). Examples of subjects and patients include mammals, such as humans, who are receiving treatment for a disease or condition (e.g., a cell proliferative disorder, such as cancer or an infectious disease).

[0418] The terms subject and patient are used interchangeably.

[0419] In some embodiments, the subject can be a mammal, e.g., a human (e.g., a patient suffering from a disease described herein or at risk of suffering from a disease described herein). In one embodiment, the subject suffers from a disease described herein (e.g., a tumor as described herein) or at risk of suffering from a disease described herein. In certain embodiments, the subject receives or has received other treatments, such as chemotherapy and / or radiation therapy.

[0420] In some embodiments, the patient (or subject) is a human who has been diagnosed with cancer and / or exhibits symptoms of cancer that expresses abnormally high TNFR2 or a low ratio of TRNFR2+Tregs / Teffs.

[0421] In some embodiments, the patient (or subject) is a patient with high TNFR2 expression in diseased tissue. In this context, high expression means a higher level of TNFR2 expression compared to corresponding healthy tissue.

[0422] 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 subject of treatment, to slow down (reduce) the targeted pathological disease or condition, and, for example, where the goal is to inhibit the recurrence of the disease or condition, such as the progression of a cell proliferative disorder (such as cancer or an infectious disease). Beneficial or desired clinical results include, but are not limited to, relief of symptoms, attenuation of disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or complete relief), whether detectable or undetectable; also includes reducing the severity of any phenotypic feature and / or reducing the incidence, degree or likelihood of the feature. Those in need of treatment include those who already have the disease and those at risk of recurrence of the disease or patients to be prevented or slowed of the recurrence of the disease.

[0423] In some embodiments, an anti-TNFR2 antibody described herein inhibits tumor volume by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, relative to the tumor volume prior to initiation of anti-TNFR2 antibody treatment.

[0424] In some respects, the treatment of cancer is an area where combination strategies are particularly needed, as the combined action of two, three, four or even more cancer drugs / therapies often produces synergistic effects that are much stronger than the impact of single treatment approaches.

[0425] In some embodiments, the treatment methods described herein further comprise administering to the subject or individual a modulatory TNFR2 antibody or pharmaceutical composition or combination product of the present invention in combination with one or more other therapies, such as other treatment modalities and / or other therapeutic agents.

[0426] In some embodiments, the other treatment modalities and / or other therapeutic agents include one or more of an immunotherapeutic agent, a chemotherapeutic agent, a radiotherapy, and a modulatory TNFR2 antibody or antigen-binding fragment thereof of the present invention. In the combined treatment methods of the present invention, the modulatory TNFR2 antibody or antigen-binding fragment thereof described herein can be co-administered (e.g., mixed) or administered separately with other therapeutic agents or therapies.

[0427] In some embodiments, in the case of separate administration, administration of the TNFR2 binding molecules of the invention, etc. can be carried out before, simultaneously with, and / or after administration of the other therapy.

[0428] In some embodiments, the regulatory antibody or antigen binding fragment thereof that specifically binds to TNFR2 is administered to the patient before the immunotherapeutic agent is administered to the patient. Alternatively, the regulatory antibody or antigen binding fragment thereof may be administered to the patient after the immunotherapeutic agent. For example, the regulatory antibody or antigen binding fragment thereof may be administered to the patient after the immunotherapeutic agent has failed. A skilled physician in the art can monitor the efficacy of immunotherapy treatment using methods described herein and known in the art to determine whether the therapy has successfully improved the pathology being treated (such as cancer or infectious disease, such as the cancer or infectious disease described herein).

[0429] In some embodiments, the therapeutic agent comprises one or more of the following: anti-tumor agents, chemotherapeutic agents, growth inhibitors, cytotoxic agents, immune checkpoint inhibitors, co-stimulatory molecules, kinase inhibitors, angiogenesis inhibitors, small molecule targeted therapeutic drugs and multi-epitope strategies. Therefore, in another embodiment of the present disclosure, cancer treatment can be effectively combined with various other drugs.

[0430] In some embodiments, the modulatory TNFR2 antibodies, antigen-binding fragments thereof described herein can be co-administered (e.g., mixed) or separately administered with a chemotherapeutic agent for treating cancer. For example, the modulatory TNFR2 antibodies, antigen-binding fragments thereof of the invention can be administered to a patient, such as a human patient with cancer, at the same time or at different times.

[0431] As used herein, "chemotherapeutic agent" refers to any chemical agent that is therapeutically useful in treating cancer, such as the cancers described herein. Chemotherapeutic agents encompass both chemical agents and biological agents. These agents may act to inhibit cell activity, which cancer cells depend on for continued survival. Classes of chemotherapeutic agents include alkylating / alkaloid agents, antimetabolites, hormones, hormone analogs, and anti-tumor drugs.

[0432] Cytotoxic agents useful in combination with the anti-TNFR2 antibodies of the invention for treating cancer include alkylating agents, antimetabolites, and other art-recognized antiproliferative agents.

[0433] Exemplary alkylating agents include nitrogen mustards, ethyleneimine derivatives, alkyl sulfonates, nitrosoureas and triazenes, such as uracil mustard, chlormethine, cyclophosphamide (CYTOXANTM), fosfamide, melphalan, chlorambucil, pipobroman, triethylene melamine, triethylene thiophosphoramide, busulfan, carmustine, lomustine, streptozotocin, dacarbazine and temozolomide. Exemplary antimetabolites include folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors, such as methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin and gemcitabine.Other suitable antiproliferative agents for use in combination with the anti-TNFR2 antibodies described herein include, for example, taxanes, paclitaxel (paclitaxel is commercially available as TAXOL™), docetaxel, discodermolide (DDM), dictyostatin (DCT), peloruside A, epothilones, epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, furanoepothilone D, deoxyepothilone epothilone B1,

[17] -dehydrodeoxyepothilone B,

[18] dehydrodeoxyepothilone B, C12,13-cyclopropyl-epothilone A, C6-C8 bridged epothilone A, trans-9,10-dehydroepothilone D, cis-9,10-dehydroepothilone D, 16-demethylepothilone B, epothilone B10, discoderomolide, patupirone (EPO-906), KOS-862, KOS-1584, ZK-EPO, ABJ-789, XAA296A (spongiolide), TZT-1027 (soli soblidotin), ILX-651 (tasidotin hydrochloride), halichondrin B, eribulin mesylate (E-7389), hemiasterlin (HTI-286), E-7974, cyrptophycins, LY-355703, maytansinoid immunoconjugate (DM-1), MKC-1, ABT-751, Tl-38067, T-900607, SB-7159 92 (Ispins), SB-743921, MK-0731, STA-5312, eleutherobin, 17β-acetoxy-2-ethoxy-6-oxo-B-homo-estra-l,3,5(10)-trien-3-ol, cyclostreptin, isolaulimalide, laulimalide, 4-epi-7-dehydroxy-14,16-didemethyl-(+)-spinolactone and cryptothilone 1.

[0434] "Chemotherapeutic agents" are chemical compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and (cyclophosphamide); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; polyacetylene glycols (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan).

[0435] In some embodiments, a modulatory TNFR2 antibody, antigen-binding fragment thereof, or construct described herein) is administered to a patient prior to administration of a chemotherapeutic agent to the patient; alternatively, a modulatory TNFR2 antibody, antigen-binding fragment thereof may be administered to a patient after a chemotherapeutic agent; for example, a modulatory TNFR2 antibody, antigen-binding fragment thereof may be administered to a patient after failure of chemotherapy treatment.

[0436] In the present invention, "immunotherapeutic agent" refers to a compound as described herein, such as an antibody or an antigen-binding fragment thereof, that can specifically bind to an immune checkpoint receptor or ligand and exert an antagonistic or agonistic effect on the receptor or ligand, thereby regulating the signal transduction of the receptor or ligand and regulating the level of tumor immune response.

[0437] In some embodiments, the aforementioned anti-TNFR2 antibodies can be combined with immunogenic agents (tumor vaccines), such as cancer cells, purified tumor antigens (including recombinant proteins, peptides and carbohydrate molecules). By lowering the T cell activation threshold through TNFR2 activation, tumor responses in the host can be activated, thereby treating non-immunogenic tumors with limited immunogenicity.

[0438] In some embodiments, exemplary immunotherapeutic agents that can be used in combination with the compositions and methods of the present invention include, but are not limited to, anti-CTLA-4 agents, anti-PD-1 agents, anti-PD-L1 agents, anti-PD-L2 agents, anti-TNF-α cross-linkers, anti-TRAIL cross-linkers, anti-CD27 agents, anti-CD30 agents, anti-CD40 agents, anti-4-1BB agents, anti-GITR agents, anti-OX40 agents, anti-TRAILR1 agents, and anti-TRAILR2 agents.

[0439] In some preferred embodiments, the other active ingredients are antibodies, such as anti-PD-1 antibodies, anti-PD-L1 antibodies, etc. The active ingredients are present in combination in amounts effective for the intended use.

[0440] In some embodiments, the other conventional treatment methods or therapeutic agents are combined or synergistic with the antibodies of the present invention to treat subjects with tumors or cancer. For example, the anti-TNFR2 antibodies described herein can be used in combination with immune checkpoint blockers. Suitable immune checkpoint blockers used in combination with the anti-TNFR2 antibodies described herein include, for example, anti-PD1 antibodies, anti-PD-L1 antibodies, anti-LAG-3 antibodies, anti-CTLA-4 antibodies, anti-TIGIT antibodies, or anti-TIM3 antibodies.

[0441] In some embodiments, exemplary other antibodies include but are not limited to inhibitors of immune checkpoint molecules (e.g., exemplary immunotherapeutic agents include but are not limited to anti-CTLA-4 agents, anti-PD-1 agents, anti-PD-L1 agents, anti-PD-L2 agents, anti-TNF-α cross-linkers, anti-TRAIL cross-linkers, anti-CD27 agents, anti-CD30 agents, anti-CD40 agents, anti-4-1BB agents, anti-GITR agents, anti-OX40 agents, anti-TRAILR1 agents, anti-TRAILR2 agents.

[0442] In some embodiments, anti-TNFR2 antibodies can be used in combination with checkpoint inhibitors (such as PD1 / PDL1 blockers) and other therapies that can overcome tumor immune escape (such as PDL1 / TGFb traps. In animal models, targeting TNFR2 has a synergistic effect with anti-PD-1), indicating that TNFR2 co-stimulation and PD1 blockade can have an enhanced anti-tumor immune response compared to PD1 monotherapy.

[0443] Preferably, the other antibodies are selected from anti-PD-1 antibodies and / or anti-PD-L1 antibodies. More preferably, the anti-PD-1 antibodies are Nivolumab from Bristol-Myers Squibb (BMS) and Pembrolizumab from Merck; the anti-PD-L1 antibodies are atezolizumab developed by Roche, avelumab jointly developed by Merck KGaA and Pfizer, and durvalumab developed by AstraZeneca.

[0444] In cases where it is desired to quiesce abnormally proliferating cells in conjunction with or prior to treatment with an anti-TNFR2 antibody described herein, hormones and steroids (including synthetic analogs), such as 17a-ethinylestradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, drostanolone propionate, testolactone, megestrol, methylprednisolone, methyltestosterone, prednisolone, triamcinolone, chlorfenestrol, hydroxyprogesterone, aminoglutethimide, estramustine, medroxyprogesterone acetate, leuprolide, flutamide, toremifene, ZOLADEX™, may also be administered to the patient. When the methods or compositions described herein are employed, other agents used to modulate tumor growth or metastasis in a clinical setting, such as antimimetic drugs, may also be administered as needed.

[0445] A physician skilled in the art can monitor the efficacy of chemotherapy treatment using methods described herein and known in the art to determine whether the therapy has successfully improved the pathology being treated (such as cancer as described herein). For example, a physician skilled in the art can monitor the number of cancer cells in a sample (e.g., a blood sample or a biopsy sample) isolated from a patient (such as a human patient), for example, using flow cytometry or FACS analysis.

[0446] The finding that the number of cancer cells, the size of the tumor, and / or the number or concentration of one or more tumor antigens present in the patient or in a sample isolated from the patient does not decrease, e.g., by a statistically significant amount, after administration of a chemotherapeutic agent within a specific time period (e.g., 1 day to 6 months, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, or 6 months) can indicate that chemotherapy treatment has failed to improve the cancer. Based on this indication, a physician skilled in the art can administer an antagonist TNFR2 polypeptide of the invention, such as a single chain polypeptide, antibody, or antigen-binding fragment thereof as described herein.

[0447] Additionally or alternatively, the modulatory TNFR2 antibodies, antigen-binding fragments thereof described herein can be administered simultaneously with radiation therapy or separately from radiation therapy. In some embodiments, an antagonist TNFR2 polypeptide (e.g., a single-chain polypeptide, antibody, antigen-binding fragment thereof, or construct) is administered to a patient after radiation therapy has failed. A physician skilled in the art can monitor the efficacy of radiation therapy using, for example, the methods described herein to determine whether the therapy has successfully improved the pathology being treated (e.g., a cancer described herein).

[0448] A physician skilled in the art can monitor the progression of a cancer (such as a cancer described herein) by evaluating the amount and / or concentration of tumor biomarkers in a patient, such as the amount and / or concentration of cell surface-bound tumor-associated antigens or secreted tumor antigens present in the patient's blood as an indicator of the presence of a tumor. The finding that the number of cancer cells, the size of the tumor, and / or the amount or concentration of one or more tumor antigens present in a patient or in a sample isolated from a patient do not decrease, for example, by a statistically significant amount, after administration of radiation therapy within a specific time period (e.g., 1 day to 6 months, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, or 6 months) may indicate that radiation therapy has failed to improve the cancer.

[0449] Based on this indication, a physician skilled in the art can administer an antagonist TNFR2 polypeptide of the invention, such as an antibody or antigen-binding fragment thereof that specifically binds to TNFR2 as described herein.

[0450] In some embodiments, to ensure that the therapeutic composition of the present invention crosses the blood-brain barrier (BBB) ​​(if necessary), the therapeutic composition can be formulated into, for example, liposomes, which can contain one or more moieties that are selectively transported to specific cells or organs to enhance targeted drug delivery, exemplary targeting moieties include, for example, folic acid or biotin.

[0451] Route of administration and dosage

[0452] In some embodiments, the modulatory TNFR2 antibodies and antigen-binding fragments thereof of the present invention, pharmaceutical compositions comprising the same, or combination therapeutic products can be administered to mammalian subjects (eg, humans) by a variety of routes.

[0453] The TNFR2 binding molecules of the present invention (and pharmaceutical compositions comprising the same) can be administered by any suitable method, including parenteral administration, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration, intrapulmonary administration and intranasal administration. Depending on whether the medication is short-term or long-term, the medication can be administered by any suitable route, such as by injection, such as intravenous or subcutaneous injection. Various medication schedules are contemplated herein, including but not limited to single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

[0454] For the prevention or treatment of disease, the appropriate dosage of an antibody of the invention that specifically binds to TNFR2 or a defucosylated modified variant thereof (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of TNFR2 binding molecule, the severity and course of the disease, whether the TNFR2 binding molecule is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the TNFR2 binding molecule, and the discretion of the attending physician. The TNFR2 binding molecule is suitably administered to the patient as a single treatment or over a series of treatments. The dosage and treatment regimen of the TNFR2 binding molecule can be determined by a skilled artisan.

[0455] It is understood that any of the above-mentioned prevention or treatment can be carried out using the pharmaceutical composition or combination product of the present invention instead of an antibody that specifically binds to TNFR2.

[0456] An effective dose of an anti-TNFR2 polypeptide of the invention may be in the range of about 0.0001 to about 100 mg / kg body weight per single, multiple, or continuous administration, or to achieve a serum concentration of 0.0001-5000 μg / mL per single, multiple, or continuous administration, or any effective range or value therein, depending on the condition being treated, the route of administration, and the age, weight, and condition of the subject. In certain embodiments, for example, for the treatment of cancer, each dose may be in the range of about 0.0001 mg to about 500 mg / kg body weight. For example, the pharmaceutical compositions of the invention may be administered at a daily dose in the range of 0.001-100 mg / kg (body weight). The dose may be administered to a mammalian subject (e.g., a human) in need thereof once or more (e.g., 2-10 times) daily, weekly, monthly, or yearly.

[0457] For parenteral administration to human patients, the daily dosage level of the anti-TNFR2 antibody molecule in the pharmaceutical composition will generally be from 1 mg / kg to 20 mg / kg of patient body weight, or in some cases even up to 100 mg / kg in a single dose or divided doses. In special cases, for example, in conjunction with chronic administration, lower doses may be used. In any case, the physician will determine the actual dose most suitable for any individual patient, and the actual dose will vary with the age, weight and response of the particular patient. The above dosages are examples of general situations. Of course, there may be individual cases where higher or lower dosage ranges are warranted, and such dosages are within the scope of the present invention.

[0458] The most suitable route of administration in any given case will depend on the specific polypharmacy being administered, the patient, the method of drug formulation, the method of administration (e.g., time of administration and route of administration), the patient's age, weight, sex, severity of the disease being treated, the patient's diet, and the patient's excretion rate. The actual dosage level of the antibody in the pharmaceutical composition provided herein can be varied to obtain an amount of active ingredient that effectively achieves the desired therapeutic response for a specific patient, composition, and mode of administration without causing toxicity to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the specific composition used or its ester, salt, or amide, the route of administration, the time of administration, the excretion rate of the specific compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition used, the age, sex, weight, condition, general health, and past medical history of the patient being treated, and similar factors well known in the medical field. A physician or veterinarian with ordinary skills in the art can easily determine and prescribe the effective amount of the desired composition. For example, a doctor or veterinarian can start the dosage of the antibody at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. Typically, the suitable daily dose of the compositions provided herein will be the amount of antibody at the lowest dose that effectively produces a therapeutic effect. This effective dose generally depends on the above factors. Preferably, it is administered intravenously, intramuscularly, intraperitoneally or subcutaneously, preferably near the target site. If necessary, the effective daily dose of the therapeutic composition can be administered in two, three, four, five, six or more subdoses at appropriate intervals throughout the day, optionally in unit dosage form. Although the antibody can be administered alone, it is preferably administered in the form of a preparation (composition).

[0459] It will be appreciated that any of the above-described prevention or treatments can be performed using the compositions or combination products of the present invention in place of the TNFR2 binding molecule.

[0460] The therapeutic composition can be administered using medical devices known in the art. For example, in one embodiment, the therapeutic composition of the present invention can be administered using a needle-free subcutaneous injection device. Therapeutic devices for administering drugs through the skin, drug infusion pumps for delivering drugs at precise infusion rates, variable flow implantable infusion devices for continuous drug delivery, osmotic drug delivery systems with multiple chamber compartments; and patents have reported osmotic drug delivery systems, and many other such implants, delivery systems, and modules are known to those skilled in the art.

[0461] In some embodiments, a method of treating a tumor is provided, the method comprising: testing a subject (e.g., a sample) (e.g., a subject sample comprising cancer cells) for the presence of TNFR2, thereby determining a TNFR2 value, comparing the TNFR2 value to a control value (e.g., the value of TNFR2 in a sample from a healthy individual), and if the TNFR2 value is greater than the control value, administering to the subject a therapeutically effective amount of a TNFR2 antibody (e.g., a TNFR2 antibody described herein), optionally in combination with one or more other therapies, thereby treating the tumor.

[0462] In some embodiments, provided herein is a method of blocking the binding of TNFα to TNFR2 in a cell, comprising contacting the cell with an effective amount of an anti-TNFR2 antibody described herein.

[0463] In some embodiments, provided herein is a method of activating TNFR2-mediated signal transduction in a cell, comprising contacting the cell with an effective amount of an anti-TNFR2 antibody described herein.

[0464] In some embodiments, provided herein is a method of activating NF-κB signaling in a cell or a subject, comprising contacting the cell or administering to the subject an effective amount of an anti-TNFR2 antibody described herein to activate NF-κB signaling.

[0465] In some embodiments, provided herein is a method of promoting (e.g., increasing) T cell proliferation (e.g., CD4+ T cells, CD8+ T cells, or both CD4+ T cells and CD8+ T cells) in vitro (e.g., in culture) or in vivo (i.e., in a subject), comprising contacting cells (e.g., T cells) with or administering to a subject an effective amount of an anti-TNFR2 antibody described herein to promote T cell proliferation.

[0466] In some embodiments, provided herein is a method of co-stimulating T cells in vitro (e.g., in culture) or in vivo (i.e., in a subject), comprising contacting a cell (e.g., a T cell) with or administering to a subject an effective amount of an anti-TNFR2 antibody described herein to co-stimulate the T cell.

[0467] In some embodiments, provided herein is a method of reducing the abundance of regulatory T cells (e.g., in a T cell compartment), comprising contacting a cell (e.g., a T cell) or administering to a subject an effective amount of an anti-TNFR2 antibody described herein to reduce the abundance of regulatory T cells. In some embodiments, the reduction in the abundance of regulatory T cells involves ADCC. In other embodiments, the reduction in the abundance of regulatory T cells involves inhibiting or reducing proliferation or inducing cell death.

[0468] It is understood that the various embodiments described in various parts of the present invention, such as diseases, therapeutic agents, treatment methods and administration, are also applicable to the embodiments of other parts of the present invention, or can be combined with the embodiments of other parts. The embodiments described in various parts of the present invention that are applicable to the properties, uses and methods of TNFR2 binding molecules are also applicable.

[0469] Table 14. Sequence Listing

[0470]

[0471]

[0472]

[0473] Example

[0474] The following examples are put forth in order to provide one of ordinary skill in the art with a description of how the compositions and methods claimed herein may be performed, prepared, and evaluated and are intended to be purely illustrative of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0475] Example 1. Preparation of TNFR2 monoclonal antibody

[0476] By injecting 6-8 week old female Balb / c mice (purchased from Sibeifu Biotechnology Co., Ltd.) with human TNFR2 recombinant protein (purchased from SinoBiological, catalog number: 10417-H08H and Novoprotein, catalog number: C830) (amino acid sequence shown in SEQ ID NO: 17) emulsified with rapid immune adjuvant (Biolong, KX0210041), the first immunization was performed in three ways: intramuscular, subcutaneous, and intraperitoneal. The second, third, and fourth times, the antigen-adjuvant mixture was immunized intramuscularly and intraperitoneally to obtain high-titer, high-affinity, and high-specific antiserum and specific immune cells. On the third day after the last immunization (shock immunization), the mice were euthanized and the spleen was aseptically removed. The mouse spleen lymphocytes were aseptically separated and extracted, and hybridomas were produced by electrofusion with SP20 myeloma cells (Nanjing Kebai, CBP60881). After the hybridomas were grown in a 96-well cell culture plate (corning, catalog number #3599) for 10 days, the mother clone 21E11 that could bind to human TNFR2 was screened by ELISA and FACS methods, and the monoclonal clone with TNFR2 binding activity was isolated by limiting dilution and sequenced. The heavy chain variable region sequence and light chain variable region sequence of the mouse clone 21E11 were obtained by sequencing. The CDR and variable region amino acid sequences of the mouse clone 21E11 are shown in Table 1. Then, the heavy chain variable region sequence and light chain variable region of 21E11 were cloned into the heavy chain expression vector and the light chain expression vector, respectively. The constant regions of the heavy chain expression vector and the light chain expression vector were derived from human IgG1. The variable region and the amino acid sequences of the full-length heavy chain and light chain of the chimeric antibody 21E11 (CHI) are shown in Table 4.

[0477] Table 1. CDR amino acid sequences of antibodies or antigen-binding fragments of the present invention that specifically bind to TNFR2

[0478]

[0479] Table 4. Amino acid sequences of mouse chimeric antibodies

[0480]

[0481] Plasmid DNA encoding anti-TNFR2 antibody 21E11 was transiently transfected into expiCHOS cells (ThermoFisher) using the ExpiFectamineCHO transfection kit (Gibco) as described by the manufacturer, and cultured in ExpiCHO-S expression medium for 10 days. The cell supernatant was collected and filtered (using a 0.22 μm filter) and then purified by affinity chromatography. Affinity chromatography capture: MabSelectSureLX filler (cytiva) was selected, with a retention time of 6 minutes. The fermentation supernatant was loaded onto the chromatography column with a loading of 40 mg protein per ml filler. The target protein was in the eluent at pH 3.8. The specific experimental steps are shown in Table 5.

[0482] Table 5 Purification scheme of mouse chimeric antibody 21E11 (ch)

[0483] step Buffer Column Volume (CV) balance 20 mM Pb, 150 mM NaCl, pH 7.4 5 Loading Cell culture medium / balance 20 mM Pb, 150 mM NaCl, pH 7.4 2 Rinse 1 20 mM NaAC, 0.5 M NaCl, pH 5.0 5 Rinse 2 20 mM NaAC, pH 5.0 4 Elution 20 mM NaAC, pH 3.8 6 CIP 0.2MNaOH 2 balance 20 Mm PB, 150 Mm NaCl, pH 7.4 2 save 20% ethanol 2

[0484] The obtained antibody solution was subjected to buffer exchange (buffer exchanged to PBS) and concentration through a 30KD ultrafiltration tube. The concentration of the obtained protein was tested by Nanodrop, and the purity was tested by HPLC-SEC, and the results showed that the purity was between 98% and 100%.

[0485] Example 2 Binding activity of anti-TNFR2 chimeric antibody and antigen protein

[0486] TNFR2-his (Bipsys, TN2-H82E3) was diluted to 1 μg / mL with coating solution (8 ml of 0.2 mol / L Na2CO3, 17 ml of 0.2 mol / L NaHCO3, 75 ml of distilled water, and pH adjusted to 9.6), and 50 μL / well of coating was added to the wells of the ELISA plate (corning, catalog number #3590), and incubated at 37°C for 2 hours. The liquid in the well was discarded, and the plate was washed 5 times with 200 μL / time of washing solution (PBST, 0.05% Tween-20 was added to the PBS diluent). 200 μL of blocking solution (PBST + 5% skim milk powder) was added to each well and incubated overnight at 4°C. The liquid in the well was discarded, and the plate was washed 5 times with 200 μL / time of washing solution (PBST + 5% skim milk powder) using a plate washer (tecan, model Hydrospeed). Add 50 μL of antibodies 21E11 and #001 (positive reference, sequence from WO2021023098A1 of Xiansheng Pharmaceutical) to each well at a concentration of 20 μg / mL, 5-fold dilution, 7 gradient antibodies, and set up a blank control at the same time; incubate at 37°C for 60 minutes; discard the liquid in the well, wash 5 times with 200 μL / time of washing solution using a plate washer. Add 50 μL of the second antibody Goat-anti-human-IgG-Fc-Secondary-Antibody (Jackson, 109-035-170, 1:10000 dilution) to each well, incubate at 37°C for 60 minutes; discard the liquid in the well, wash 5 times with 200 μL / time of washing solution using a plate washer. Add 50 μL / well of colorimetric solution and incubate at 37°C for 15 minutes. Add 50 μL / well of 2 mol / L H2SO4 to terminate the reaction, and read the OD450 value on an ELISA reader. The results of the ELISA-binding activity analysis of TNFR2-His are shown in Table 6. Figure 1 As shown in A, the ELISA binding activity of the chimeric antibody 21E11 is slightly better than that of Innovent's #001. The EC50 values ​​of #001 and 21E11 binding are not much different, which are 6.033 ng / ml and 6.005 ng / ml respectively; however, the maximum signal value (OD value) of 21E11 binding is higher than that of #001.

[0487] Table 6 ELISA-binding activity and FACS binding activity analysis results for TNFR2-His

[0488] EC50(ng / mL) #001 21E11 ELISA-Binding 6.033 6.005 FACS-binding 83.53 44.84

[0489] Example 3 Anti-TNFR2 chimeric antibody binds to CHO-K1 cells overexpressing TNFR2 membrane protein

[0490] In order to detect the specificity of the antibody of the present invention binding to the TNFR2 membrane protein on the cell surface, we stably overexpressed the human TNFR2 membrane protein (amino acid sequence as shown in SEQ ID NO: 17, encoding nucleic acid sequence as shown in SEQ ID NO: 18) in CHO-K1 cells (hamster ovary cell substrain, ATCC#CCL-61) by plasmid electroporation and drug addition screening. The CHO-K1-TNFR2-M5 cells overexpressing human TNFR2 were digested and counted, resuspended in a 96-U well plate (3E5 cells were added to each well), and washed with PBS (twice); a blank control group and an experimental group were set up on the 96-well plate, and antibodies 21E11 and #001 (at a concentration of 20 μg / mL) were added to the wells of the experimental group and diluted to 7 gradients with a 5-fold concentration by gradient dilution method; the 96-well plate was placed at 4°C for incubation for 30 minutes, centrifuged (1000 rpm, 2 minutes), and the liquid in the wells was removed and washed with PBS. S wash (200 μL / time, 2 times); PE-anti-human-IgG-Fc-Secondary-Antibody (Jackson, Catalog No. 109-116-098, 1:10000 dilution, 50 μL) was added to each well, incubated at 4°C for 30 mins, centrifuged (1000 rpm, 2 mins), and washed with PBS after removing the liquid (200 μL / time, 2 times); PBS was added to each well (200 μL / well), and the cells were resuspended and read using a flow cytometer. The results of FACS-binding activity analysis are shown in Table 6 and Figure 1 As shown in B, the FACS-cell binding activity of the chimeric antibody 21E11 is better than that of Innovent's #001. The EC50 values ​​of the binding of #001 and 21E11 are 44.84 ng / ml and 83.53 ng / ml, respectively.

[0491] The amino acid sequence of human TNFR2 membrane protein is shown in SEQ ID NO: 17:

[0492] MAPVAVWAALAVGLELWAAAHALPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCE

[0493] DSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAPLRKCRPGFGVARPGTETSD

[0494] VVCKPCAPGTFSNTTSSTDICRPHQICNVVAIPGNASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAP

[0495] STSFLLPMGPSPPAEGSTGDFALPVGLIVGVTALGLLIIGVVNCVIMTQVKKKPLCLQREAKVPHLPADKARGTQGPEQ

[0496] QHLLITAPSSSSSSLESSASALDRRAPTRNQPQAPGVEASGAGEARASTGSSDSSPGGHGTQVNVTCIVNVCSSSDHSSQCSSQASSTMGDTDSSPSESPKDEQVPFSKEECAFRSQLETPETLLGSTEEKPLPLGVPDAGMKPS(SEQ ID NO:17).

[0497] The coding nucleic acid sequence of human TNFR2 membrane protein is shown as SEQ ID NO:18:

[0498] ATGGCGCCCGTCGCCGTCTGGGCCGCGCTGGCCGTCGGACTGGAGCTCTGGGCTGCGGCGCACGCCTTGCCCGCCCAGG

[0499] TGGCATTTACACCCTACGCCCCGGAGCCCGGGAGCACATGCCGGCTCAGAGAATACTATGACCAGACAGCTCAGATGTG

[0500] CTGCAGCAAGTGCTCGCCGGGCCAACATGCAAAAGTCTTCTGTACCAAGACCTCGGACACCGTGTGTGACTCCTGTGAG

[0501] GACAGCACATACACCCAGCTCTGGAACTGGGTTCCCGAGTGCTTGAGCTGTGGCTCCCGCTGTAGCTCTGACCAGGTGG

[0502] AAACTCAAGCCTGCACTCGGGAACAGAACCGCATCTGCACCTGCAGGCCCGGCTGGTACTGCGCGCTGAGCAAGCAGGA

[0503] GGGGTGCCGGCTGTGCGCGCCGCTGCGCAAGTGCCGCCCGGGCTTCGGCGTGGCCAGACCAGGAACTGAAACATCAGAC

[0504] GTGGTGTGCAAGCCCTGTGCCCCGGGGACGTTCTCCAACACGACTTCATCCACGGATATTTGCAGGCCCCACCAGATCT

[0505] GTAACGTGGTGGCCATCCCTGGGAATGCAAGCATGGATGCAGTCTGCACGTCCACGTCCCCCACCCGGAGTATGGCCCC

[0506] AGGGGCAGTACACTTACCCCAGCCAGTGTCCACACGATCCCAACACACGCAGCCAACTCCAGAACCCAGCACTGCTCCA

[0507] AGCACCTCCTTCCTGCTCCCAATGGGCCCCAGCCCCCCAGCTGAAGGGAGCACTGGCGACTTCGCTCTTCCAGTTGGAC

[0508] TGATTGTGGGTGTGACAGCCTTGGGTCTACTAATAATAGGAGTGGTGAACTGTGTCATCATGACCCAGGTGAAAAAGAA

[0509] GCCCTTGTGCCTGCAGAGAGAAGCCAAGGTGCCTCACTTGCCTGCCGATAAGGCCCGGGGTACACAGGGCCCCGAGCAG

[0510] CAGCACCTGCTGATCACAGCGCCGAGCTCCAGCAGCAGCTCCCTGGAGAGCTCGGCCAGTGCGTTGGACAGAAGGGCGC

[0511] CCACTCGGAACCAGCCACAGGCACCAGGCGTGGAGGCCAGTGGGGCCGGGGAGGCCCGGGCCAGCACCGGGAGCTCAGA

[0512] TTCTTCCCCTGGTGGCCATGGGACCCAGGTCAATGTCACCTGCATCGTGAACGTCTGTAGCAGCTCTGACCACAGCTCA

[0513] CAGTGCTCCTCCCAAGCCAGCTCCACAATGGGAGACACAGATTCCAGCCCCTCGGAGTCCCCGAAGGACGAGCAGGTCC

[0514] CCTTCTCCAAGGAGGAATGTGCCTTTCGGTCACAGCTGGAGACGCCAGAGACCCTGCTGGGGAGCACCGAAGAGAAGCCCCTGCCCCTTGGAGTGCCTGATGCTGGGATGAAGCCCAGTTAA (SEQ ID NO: 18).

[0515] Example 4 Anti-TNFR2 chimeric antibodies antagonize the binding of ligand TNF-α to target protein TNFR2

[0516] 4.1 Antagonizing the binding of TNF-α to TNFR2 protein

[0517] TNFR2-his (Bipsys, TN2-H82E3) was diluted to 1 μg / mL with coating solution (8 ml of 0.2 mol / L Na2CO3, 17 ml of 0.2 mol / L NaHCO3, 75 ml of distilled water, and pH adjusted to 9.6), and coated into the wells of the ELISA plate at a volume of 50 μL / well, incubated at 37°C for 2 hrs, and washed with washing solution (PBST, 0.05% Tween-20 was added to the PBS diluent) after the liquid was removed (200 μL / time, 5 times); 200 μL of blocking solution was added to each well, and incubated at 4°C overnight, and the liquid was removed and washed with washing solution (200 μL / time, 5 times); a blank control group and an experimental group were set up on the ELISA plate, and 25 μL of 20 ng / mL Tween was added to the wells of the experimental group. NF-α (Biopsy, catalog number TNA-H82E3), and antibodies 21E11 and #001 at a concentration of 20 μg / mL were diluted into 7 gradients with a 5-fold concentration using the gradient dilution method, incubated at 37°C for 120 mins, and washed with washing solution (200 μL / time, 5 times) after removing the liquid; 50 μL of streptavidin-HRP (ACRO, catalog number #STN-NH913, dilution factor 1:10000) secondary antibody was added to each well, and washed with washing solution (200 μL / time, 5 times) after removing the liquid; color development solution (50 μL / well) was added to each well, and after incubation at 37°C for 15 mins, H2SO4 aqueous solution (50 μL / well, 2 mol / L) was added to terminate the reaction, and the OD450 value was read on the microplate reader. The results of ELISA-blocking activity analysis are shown in Table 7 and Figure 2 As shown in A, 21E11-CHI has good ELISA antagonistic activity, and its IC50 value is 793.7 ng / ml, which is slightly lower than the 916.7 ng / ml of the reference antibody #001.

[0518] 4.2 Antagonizing the binding of TNF-α to membrane protein TNFR2

[0519] CHOK1-TNFR2 overexpressing cells M5 were digested and counted, resuspended in a 96-well plate (3E5 cells were added to each well), and washed with PBS (twice); a blank control group and an experimental group were set up on the 96-well plate, and TNF-α (concentration of 200 ng / mL, 25 μL) and dual antibodies 21E11 and #001 (concentration of 40 μg / mL) were added to the wells of the experimental group and diluted into 7 gradients according to 5-fold concentration; the 96-well plate was placed at 4°C for 30 mins, centrifuged (1000 rpm, 2 mins), and the After removing the liquid from the wells, wash with PBS (200 μL / time, 2 times); add 50 μL of diluted R-phycoerythrinstreptavidin (Jackson, 016-110084, 1:200 dilution) to each well, incubate at 4°C for 30 mins, centrifuge (1000 rpm, 2 mins), remove the liquid and wash with PBS (200 μL / time, 2 times); add PBS (200 μL / well) to each well, resuspend the cells, and read with a flow cytometer. The FACS-analysis results are shown in Table 7 and Figure 2 As shown in B, at the cellular level, 21E11-CHI also has good antagonistic TNF-α ligand binding activity, with an IC50 value of 463.4 ng / ml, which is significantly lower than 2274 ng / ml of the reference antibody #001, indicating that the antagonistic ligand binding activity of the chimeric antibody 21E11 is stronger than that of Innovent's #001.

[0520] Table 7 ELISA and FACS analysis results of antagonistic activity of 21E11-CHI against ligand TNFα binding

[0521] IC50(ng / mL) #001 21E11 ELISA-Blocking 916.7 793.7 FACS-blocking 2274 463.4

[0522] Example 5 Binding activity of humanized antibody HU6 to TNFR2 protein

[0523] To reduce the immunogenicity of the antibody, we humanized and combined the light and heavy chains of the 21E11 antibody. The sequence of the variable region of the 21E11 antibody was used to identify the human germline sequence with the highest homology to it, using CDR transplantation and back mutation. The humanized antibody has the CDRs of the mouse antibody, but has multiple substitutions in the V domain framework region.

[0524] The amino acid sequence of the humanized antibody HU6 is shown in Table 8.

[0525] Table 8 Amino acid sequence of humanized antibody HU6

[0526]

[0527]

[0528] 5.1 The ELISA binding activity of humanized antibody HU6 is not inferior to that of chimeric antibody 21E11

[0529] Dilute TNFR2-his to 1 μg / mL with coating solution (take 8 ml of 0.2 mol / L Na2CO3, mix 17 ml of 0.2 mol / L NaHCO3, add 75 ml of distilled water, adjust pH to 9.6), add 50 μL / well of coating to the wells of the ELISA plate, and incubate at 37°C for 2 hours. Discard the liquid in the wells, wash 5 times with a plate washer, and use 200 μL / time of washing solution (PBST, 0.05% Tween-20 added to the PBS diluent). Add 200 μL of blocking solution (PBST + 5% skim milk powder) to each well and incubate overnight at 4°C. Discard the liquid in the wells, and use a plate washer, and use 200 μL / time of washing solution to wash 5 times. Add 50 μL of antibodies 21E11 and HU6 at a concentration of 20 μg / mL to each well, 5-fold dilution, 7 gradient antibodies, and set up a blank control; incubate at 37°C for 60 minutes; discard the liquid in the wells, wash 5 times with a plate washer, 200 μL / time of washing solution. Add 50 μL of the secondary antibody Goat-anti-human-IgG-Fc-Secondary-Antibody (Jackson, 1:10000 dilution) to each well, incubate at 37°C for 60 minutes; discard the liquid in the wells, wash 5 times with a plate washer, 200 μL / time of washing solution. Add 50 μL / well of colorimetric solution and incubate at 37°C for 15 minutes. Add 50 μL / well of 2 mol / L H2SO4 to terminate the reaction, and read the OD450 value on the microplate reader. The results of ELISA-binding activity analysis are shown in the following table. Figure 3 As shown in A, the ELISA binding activity of the humanized antibody HU6 is not weaker than that of the chimeric antibody 21E11, and the binding EC50 values ​​of antibodies HU6 and 21E11 are 2.902 and 4.550 ng / ml, respectively.

[0530] 5.2 FACS binding activity of humanized antibody HU6 is comparable to that of chimeric antibody 21E11

[0531] The method is similar to that of Example 3. CHO-K1-TNFR2-M5 cells overexpressing human TNFR2 were digested and counted, resuspended in a 96-well plate (3E5 cells were added to each well), and washed with PBS (twice); a blank control group and an experimental group were set up on the 96-well plate, antibodies 21E11 and HU6 (at a concentration of 20 μg / mL) were added to the wells of the experimental group and diluted into 7 gradients with a 5-fold concentration by gradient dilution method; the 96-well plate was placed at 4°C for 30 minutes, centrifuged (1000 rpm, 2 minutes), and the After removing the liquid from the wells, wash with PBS (200 μL / time, 2 times); add PE-anti-human-IgG-Fc-Secondary-Antibody (Jackson, 1:200 dilution, 50 μL) to each well, incubate at 4°C for 30 mins, centrifuge (1000 rpm, 2 mins), remove the liquid and wash with PBS (200 μL / time, 2 times); add PBS (200 μL / well) to each well, resuspend the cells, and read with a flow cytometer. The results of FACS-binding activity analysis are shown in Table 9 and Figure 3 As shown in B, the FACS binding activity of the humanized antibody HU6 was comparable to that of the chimeric antibody 21E11, and the binding EC50 values ​​of antibodies HU6 and 21E11 were 17.05 and 17.71 ng / ml, respectively.

[0532] Table 9 ELISA and FACS analysis results of the binding activity of humanized antibody HU6 to target antigen human TNFR2

[0533] EC50(ng / mL) HU6 21E11-CHI ELISA-Binding 2.902 4.550 FACS-binding 17.05 17.71

[0534] Example 6 Affinity detection of the antibody of the present invention and antigen

[0535] Based on the biolayer interferoc-Metry (BLI) technique, the affinity between the antibody of the present invention and the antigen was detected. TNFR2-His was gradiently diluted from 100 nM with PBST, and the gradient dilution method was used to dilute it 2 times to 7 concentration points, and a zero concentration reference well was set at the same time. Antibodies 21E11 and HU6 were diluted to 5 μg / mL respectively. The molecular interaction instrument (Fortebio, manufacturer: Sartorius, model: OctetR8) was set up with the operating conditions of temperature 30°C and Shakespeed 1000 rpm. The coated AHC2 probe (Sartorius, item number 18-5142) was used to capture the antibody, and the capture time was 180 s; the gradient diluted TNFR2-HIS sample was combined with a binding time of 120 s; the dissociation time was 300 s; and the regeneration buffer (10 mM glycine, PH1.7) was regenerated 3 times, each time for 30 s. The on-machine detection was performed using ForteBio's Octet System. After obtaining the sensorgram data, the binding constant (ka) and dissociation constant (kd) were analyzed using OctetBLIAnalysis software to fit the ideal binding and dissociation curves, such as Figure 4 The equilibrium dissociation constant KD (kd / ka) between the antibody and antigen was calculated as shown in Table 10. The results show that the binding dissociation constant values ​​(KD values) of the chimeric antibody 21E11 and the humanized antibody HU6 are comparable, 4.20 nM and 4.06 nM, respectively.

[0536] Table 10 Binding dissociation constant values ​​(KD values) of chimeric antibody 21E11 and humanized antibody HU6 determined by BLI method

[0537] Antibody Name KD value 21E11 4.20E-09 hu6 4.06E-09

[0538] Example 7 Detection of cross-reactivity between the antibody of the present invention and mouse and cynomolgus monkey TNFR2 proteins

[0539] The cross-species binding reaction was detected by ELISA. The recombinant proteins mouse TNFR2-his (Bipusaisi, TN2-M52H8) and cynomolgus monkey TNFR2-his (Bipusaisi, TN2-C52H7) were diluted to 2 μg / mL with coating solution, and 50 μL / well was added to the wells of the ELISA plate and incubated at 37°C for 2 hours. The liquid in the well was discarded, and the plate was washed 5 times with 200 μL / time of washing solution. 200 μL of blocking solution was added to each well at 4°C overnight. The liquid in the well was discarded, and the plate was washed 5 times with 200 μL / time of washing solution. 50 μL of antibodies BT461, 21E11 and HU6 were added to each well at a concentration of 20 μg / mL, 5-fold dilution, 7 gradient antibodies, and a blank control was set at the same time; incubated at 37°C for 60 minutes; the liquid in the well was discarded, and the plate was washed 5 times with 200 μL / time of washing solution. Add 50 μL of Goat-anti-human-IgG-Fc-Secondary-Antibody (1:10000 dilution) to each well and incubate at 37°C for 60 min. Discard the liquid in the well and wash 5 times with a plate washer, 200 μL / time of washing solution. Add 50 μL / well of color development solution and incubate at 37°C for 15 min. Add 50 μL / well of 2 mol / L H2SO4 to terminate the reaction and read the OD450 value on an ELISA reader. The results are as follows: Figure 5 As shown, the antibodies 21E11 and HU6 of the present invention have cross-reactivity with cynomolgus monkey TNFR2 protein, but not with mouse TNFR2 protein.

[0540] Example 8 Detection of the binding activity of the antibody of the present invention with proteins of the same family

[0541] CD40 (Bipusai, CD0-H5228) or 4-1BB (CD137, Bipusai, 41B-H53H3) was diluted to 1μg / mL and 2μg / mL respectively with coating solution (8ml of 0.2mol / LNa2CO3, 17ml of 0.2mol / LNaHCO3, 75ml of distilled water, and pH adjusted to 9.6), and 100μL / well of coating was added to the wells of the ELISA plate, and incubated at 37°C for 2 hours. The liquid in the well was discarded, and the plate was washed 5 times with 200μL / time of washing solution (PBST, 0.05% Tween-20 was added to the PBS diluent). 200μL of blocking solution (PBST+5% skim milk powder) was added to each well at 4°C overnight. The liquid in the well was discarded, and the plate was washed 5 times with 200μL / time of washing solution (Tecan, model Hydrospeed). Add 50 μL of antibodies 21E11 and HU6 at a concentration of 20 μg / mL to each well, 5-fold dilution, 7 gradient antibodies, and set up a blank control; incubate at 37°C for 60 minutes; discard the liquid in the wells, wash 5 times with 200 μL / time of washing solution using a plate washer. Add 50 μL of the secondary antibody Goat-anti-human-IgG-Fc-Secondary-Antibody (Jackson, 1:10000 dilution) to each well, incubate at 37°C for 60 minutes; discard the liquid in the wells, wash 5 times with 200 μL / time of washing solution using a plate washer. Add 50 μL / well of colorimetric solution and incubate at 37°C for 15 minutes. Add 50 μL / well of 2 mol / L H2SO4 to terminate the reaction, and read the OD450 value on an enzyme reader. The results of ELISA-binding activity analysis are as follows. Figure 6 As shown, the antibodies of the present invention do not bind to CD40 and 4-1BB.

[0542] Example 9 Humanized Antibodies Antagonize Binding of Ligand TNF-α to Target Protein TNFR2

[0543] 9.1 Antagonizing the binding of TNF-α to TNFR2 protein

[0544] TNFR2-his (Bipsys, TN2-H82E3) was diluted to 1 μg / mL with coating solution (8 ml of 0.2 mol / L Na2CO3, 17 ml of 0.2 mol / L NaHCO3, 75 ml of distilled water, pH adjusted to 9.6), and coated into the wells of the ELISA plate at 50 μL / well, incubated at 37°C for 2 hrs, and washed with washing solution (PBST, 0.05% Tween-20 added to the PBS diluent) (200 μL / time, 5 times) after removing the liquid; 200 μL of blocking solution was added to each well, and incubated at 4°C overnight, and washed with washing solution (200 μL / time, 5 times) after removing the liquid. L / time, 5 times); a blank control group and an experimental group were set up on the ELISA plate, 25 μL of TNF-α (Bipsis, catalog number TNA-H82E3) with a concentration of 20 ng / mL and antibodies 21E11 and HU6 with a concentration of 20 μg / mL were added to the wells of the experimental group, and the gradient dilution method was used to dilute the concentration by 5 times to 7 gradients, incubated at 37°C for 120 mins, and the liquid was removed and washed with washing solution (200 μL / time, 5 times); 50 μL of streptavidin-HRP (ACRO, catalog number #STN-NH913, dilution factor 1:10000) secondary antibody was added to each well. After removing the liquid, wash with washing solution (200 μL / time, 5 times); add color development solution (50 μL / well) to each well, incubate at 37°C for 15 minutes, add H2SO4 aqueous solution (50 μL / well, 2 mol / L) to terminate the reaction, and read the OD450 value on the microplate reader. The results of ELISA-blocking activity analysis are shown in Table 11 and Figure 7 As shown in A, the ELISA antagonistic activity of the humanized antibody HU6 was comparable to that of the chimeric antibody 21E11, and the antagonistic IC50 values ​​of antibodies HU6 and 21E11 were 341.3 and 374.7 ng / ml, respectively.

[0545] 9.2 Antagonizing the binding of TNF-α to membrane protein TNFR2

[0546] CHOK1-TNFR2 overexpressing cells M5 were digested and counted, resuspended in a 96-well plate (3E5 cells were added to each well), and washed with PBS (twice); a blank control group and an experimental group were set up on the 96-well plate, and TNF-α (concentration of 200 ng / mL, 25 μL) and dual antibodies 21E11 and HU6 (concentration of 40 μg / mL) were added to the wells of the experimental group and diluted into 7 gradients according to 5-fold concentration; the 96-well plate was placed at 4°C for 30 minutes and centrifuged (1000 rpm, 2 min s), remove the liquid in the plate wells and wash with PBS (200 μL / time, 2 times); add R-phycoerythrinstreptavidin (Jackson, 016-110084, 1:200 dilution) to each well, incubate at 4°C for 30 mins, centrifuge (1000 rpm, 2 mins), remove the liquid and wash with PBS (200 μL / time, 2 times); add PBS (200 μL / well) to each well, resuspend the cells, and read with a flow cytometer. FACS-analysis results are shown in Table 11 and Figure 7 As shown in B, at the cellular level, the FACS antagonistic activity of the humanized antibody HU6 was comparable to that of the chimeric antibody 21E11, and the antagonistic IC50 values ​​of antibodies HU6 and 21E11 were 132.1 and 188.5 ng / ml, respectively.

[0547] Table 11 ELISA and FACS analysis results of humanized antibody HU6 in vitro and cellular levels to antagonize the activity of ligand TNFα binding to TNFR2

[0548] IC50(ng / mL) HU6-2 21E11-CHI ELISA-Blocking 341.3 374.7 FACS-blocking 132.1 188.5

[0549] Example 10 Construction of a reporter system for TNFR2 receptor signaling pathway

[0550] 10.1 Construction of Jurkat-NF-κB luciferase reporter gene stably transfected cell line

[0551] By electroporation, a Jurkat cell line (Kebai Bio, CBP60520) with stable expression of NF-κB luciferase reporter gene was constructed. Jurkat cells were cultured at 37°C and 5% CO2. When the cell density reached 1-2x106 / mL, the cells were collected, washed once with RPMI1640, and resuspended in FBS-free medium to adjust the cell density to 7.5x106 / mL. Take 20μg of plasmid (NF-κB luciferase reporter plasmid, yeasen, catalog number 11501ES03) in a 1.5mL fine tube, a total of 2 tubes, add 400μL of cell suspension to each tube, and mix well. Slowly add the cell suspension to the electroporation cup to avoid adding bubbles. On the machine, adjust the parameters, transfer 1 tube each at 240V and 250V, and transfer 1 tube without plasmid. Take a 6-well plate, add 2.5ml complete medium to each well, and gently transfer the electroporated cells into the well plate. After 24 hours, add 600 μg / ml G418 to the well plate cells, and take a small amount of cells to detect the transfection status. Continue to culture for about 10 days. When all negative control cells die and the polyclonal viability recovers to more than 80%, take the cell suspension for fluorescence detection and freeze the positive polyclonal.

[0552] The monoclonal clones were selected by limiting dilution and expanded. The polyclonal wells with relatively high expression were spread on 2 plates at a density of 1 cell / well and 5 cells / well in a 96-well plate. After 4-7 days, a portion of the cell suspension was taken for fluorescence detection after the clones grew up. After the detection, 6 cell lines (M12 / M13 / M41 / M45 / M50 / M72) with stimulating activity were selected for expansion and culture, frozen, and JURKAT-NF-KB-M45 was expanded to build a library.

[0553] 10.2 TNFR2 receptor reporter system detection

[0554] JURKAT-NF-KB-M45 cells were revived and passaged, and the culture conditions were 1640 medium + 10% FBS + 1% double antibody + 300μg / mL G418, 37°C, 5% carbon dioxide. Take the lentivirus Lenti-TNFR2-Puro (virus titer 1.3×108PFU / mL) and place it on ice to melt; Jurkat-NF-KBM45 cells were plated into six-well plates (2E6 / well), and lentivirus venom was added to each well, lentivirus: cell = 6:1, and cultured in the incubator for 48 hours. After 48 hours, the complete medium with puromycin was replaced for screening and expansion of the culture. Single clones were selected by the limiting dilution method, and the cells were diluted at a density of 10 / mL and plated into 96-well flat-bottom plates, and cultured in the incubator; Figure 8 As shown in A, JURKAT-NF-KB-M45-D1 clone was selected by flow cytometry to overexpress TNFR2 receptor.

[0555] 10.3Verification of Jurkat-NF-KB-M45TNFR2 signaling pathway activation

[0556] Four experimental groups were set up: TNF-α-biotin 0, 10 ng / mL (free), 1000 ng / mL (free) and coated wells (TNF-α was coated on a 96-well white bottom plate one day in advance). The 24h group was added at 16:00, and PBS was added to the coated wells during the 24h overnight period. The next day, Jurkat NFKBM45-D1 cells were added, 5E4 / well, and the cells were resuspended in 1640 culture medium with 1% inactivated serum. The cells were treated for 6h, 4h and 2h, and the plates were equilibrated to room temperature. 100 μL of Bright-Lite Luciferase Assay System detection reagent was added to each well, and the shaker was shaken at 300 rpm / min for 3min; CTG mode detection was selected. The results are shown in Figure 8 As shown in B, in our constructed reporter cell line JURKAT-NF-KB-M45-D1, the addition of soluble and coated TNF-α-biotin can stimulate the NF-KB signal downstream of the TNFR2 receptor, and is positively correlated with the treatment time within 2 to 6 hours.

[0557] Example 11 Soluble humanized HU6 antibody has no agonistic activity but can block TNF-α activity

[0558] It can be seen from Example 9 that the antibody of the present invention can antagonize the binding of the ligand TNF-α to TNFR2. In order to further explore whether the humanized antibody HU6 can antagonize the effect of TNF-α on the receptor signaling pathway, we set up 12 blank wells and TNF-α10ng / mL coated wells, and coated TNF-α to a 96-well white-bottom plate one day in advance at a concentration of 10ng / mL. The liquid in the wells was discarded on the second day and rinsed once with PBS. Dilute the antibody HU6, the antibody was set to 40μg / mL, 4μg / mL, 0.4μg / mL and 0μg / mL, the dilution buffer was 1640 culture medium with 1% inactivated serum, and 50μL was added to each well. JURKAT-NF-KB-M45-D1 cells were centrifuged and resuspended in 1640 culture medium with 1% inactivated serum, the concentration was diluted to 1E6 / mL, 50μL was added to each well, 5E4 cells / well. Detection was performed after culturing for 6 hours at 37°C and 5% CO2. The results are as follows Fig. 9 As shown, the soluble humanized HU6 antibody has no agonistic activity but can inhibit the downstream signaling activity of TNFR2 stimulated by the ligand TNF-α.

[0559] Example 12 The coated and fixed humanized HU6 antibody has an agonistic effect on the NF-KB signaling pathway

[0560] As can be seen from Example 11, the free humanized antibody HU6 has no agonistic activity and can antagonize TNF-α activity. We further explored whether the fixed HU6 antibody protein has agonistic activity.

[0561] Antibody HU6 and control antibody IgG1 were diluted with PBS gradient, and coated on a 96-well white bottom plate one day in advance. The highest concentration was 100 μg / mL, and 6 points were diluted with 3-fold gradient concentration. There were 8 points in total including the blank well, and 2 replica wells were set up. The liquid in the wells was discarded on the second day, and PBS was rinsed once. JURKAT-NF-KB-M45-D1 cells were resuspended by centrifugation in 1640 culture medium with 1% inactivated serum, and the concentration was diluted to 5E5 / mL. 100 μL was added to each well, 5E4 cells / well. The cells were cultured at 37°C and 5% CO2 for 6 hours before detection. The results are as follows Fig.10 As shown in A, the coated and fixed humanized HU6 antibody has a certain agonistic effect on the NF-KB signaling pathway.

[0562] Example 13 FAB antibody cross-linked humanized HU6 antibody has agonistic activity

[0563] Furthermore, we used the anti-FC FAB antibody to cross-link the humanized antibody HU6 to explore the mechanism of its agonistic effect. TNF-α coated wells were set up, and TNF-α was coated on a 96-well white bottom plate one day in advance at a concentration of 10 ng / mL. The liquid in the well was discarded on the second day and rinsed once with PBS. In addition, the test group (HU6+cross-linked antibody Fab) was configured as follows: dilute antibodies HU6 and IgG1, and the antibodies were set to 320μg / mL, 80μg / mL, 20μg / mL and 0μg / mL, and the dilution buffer was 1640 culture medium with 1% inactivated serum. Dilute the anti-FC FAB antibody, and the antibody was set to 320μg / mL, 80μg / mL, 20μg / mL and 0μg / mL. According to the same concentration, the detection antibody and the cross-linked antibody Fab were mixed in a volume of 1:1. In addition, a FAB 160 μg / mL control sample was set up. The test and control samples were added to a 96-well white bottom plate, and 1% inactivated serum 1640 medium was added to the TNF-α coated wells, and 50 μL was added to each well. JURKAT-NF-KB-M45-D1 cells were centrifuged and resuspended in 1% inactivated serum 1640 medium, diluted to a concentration of 1E6 / mL, and 50 μL was added to each well, 5E4 cells / well. The cells were cultured at 37°C and 5% CO2 for 6 hours before testing. The results are shown in the figure. Fig.10 As shown in B, a single FC-crosslinked antibody Fab has no agonistic effect on the TNFR2 signaling system, while the humanized HU6 antibody crosslinked with the FAB antibody has obvious agonistic activity and is positively correlated with the antibody concentration. However, its agonistic activity is significantly lower than that of 10 ng / mL TNF-α.

[0564] Example 14 THP-1 cell cross-linking defucosylated humanized HU6 antibody has agonistic activity

[0565] In addition, we also used CD32a on THP-1 cells to cross-link the defucosylated humanized HU6 antibody. Using CHO-K1 (FUT8 knockout) cell line, the defucosylated humanized antibody HU6-AF was expressed according to the steps of Example 1.

[0566] Similarly, a TNF-α coated well was set up. TNF-α was coated on a 96-well white bottom plate one day in advance at a concentration of 10 ng / mL. The liquid in the well was discarded on the second day and rinsed once with PBS. Antibodies HU6 and IgG1 were diluted to a maximum concentration of 100 μg / mL. The dilution buffer was 1640 medium with 1% inactivated serum. There were 6 points of 5-fold gradient dilution, plus a total of 8 points with the blank well. The test group samples were added to the 96-well white bottom plate, 50 μL was added to each well, and 1640 medium with 1% inactivated serum was added to the TNF-α well. JURKAT-NF-KB-M45-D1 cells and THP1 (monocyte cell line of human peripheral blood) cells were centrifuged and resuspended in 1640 medium with 1% inactivated serum and mixed. The concentration of the two cells (1:1 mixture) was diluted to 1E6 / mL, 50 μL was added to each well, and 5E4 cells / well were cultured at 37°C and 5% CO2 for 6 hours before detection. The results are as follows Fig.10 As shown in Figure C, compared with hIgG1, the defucosylated humanized antibody HU6 has the effect of stimulating NF-KB signaling, especially low concentrations of HU6-AF (less than 0.08μg / mL) have a concentration-positively correlated stimulating TNFR2 signaling activity, and the subsequent observation of NF-KB fluorescence signal downregulation with increasing antibody concentration may be due to the ADCP killing effect of THP-1 cells on reporter cells. In addition, the stimulating activity of THP-1 cell cross-linked defucosylated humanized HU6 antibody is also significantly lower than the stimulating activity of 10ng / mL TNF-α.

[0567] Example 15 Evaluation of the expression specificity of TNFR2 protein in the tumor microenvironment

[0568] Select NSG mice that have passed the quarantine, adjust the PBMC concentration to 2.5E7 / ml, and inject intraperitoneally, 0.2ml per mouse, for PBMC reconstruction. One week after PBMC reconstruction, resuspend A375 cells in the logarithmic growth phase, adjust the concentration to 1E8 / ml, and mix with equal volume of matrix gel 1:1 before inoculation. Each mouse is subcutaneously inoculated with 0.1ml of the cell mixture in the right anterior axilla. After 30 days, the PE / CY7-anti-TNFR2 (Jackson, Cat. No. 109-006-098) antibody is used to analyze the TNFR2 positive cell rate and membrane protein level of different lymphocyte populations in peripheral blood, spleen, and tumor infiltration.

[0569] Peripheral blood lymphocyte analysis experimental method:

[0570] Collect 100 μl of orbital blood from each mouse through the medial canthal venous plexus in a 1.5 ml anticoagulant tube, centrifuge at 7500 rpm at 4°C for 5 min, and discard the supernatant. Add 1 ml of red blood cell lysis buffer, mix well, lyse at room temperature for 10 min, centrifuge at 3000 rpm at 4°C for 5 min, and discard the supernatant. Add 1 ml of PBS, mix well, centrifuge at 3000 rpm at 4°C for 5 min, and discard the supernatant. Add 100 μl of antibody mixture (containing 1 μl PerCP / Cyanine5.5anti-humanCD45Antibody+1 μl APCanti-humanCD8Antibody+1 μl FITCanti-humanCD4Antibody+96 μl PBS+1 μl PE / cy7-anti-TNFR2), and incubate at 4°C for 30 min. After incubation, add 100 μl of PBS solution and mix well, centrifuge at 3000 rpm at 4°C for 5 min, and discard the supernatant. Add 100 μl of PBS solution and mix well, and detect on a flow cytometer.

[0571] Splenic lymphocyte analysis

[0572] After the animals were euthanized, the spleen was removed and single cell suspension was prepared. The ratio of CD4+CD25+\TNFR2+ and CD4+CD25-\TNFR2+ and the ratio of CD8+\TNFR2+ in spleen cells were determined by flow cytometry. The detection method is as follows:

[0573] (1) Place the spleen in a 40 μm cell strainer and grind it using the stopper end of a 1 ml syringe to obtain a single cell suspension. Transfer the filtered single cell suspension into a 50 mL centrifuge tube and centrifuge at 7500 rpm for 5 min at 4°C, then discard the supernatant.

[0574] (2) The remaining steps are the same as the detection steps of the above peripheral blood lymphocyte analysis experimental method.

[0575] Analysis of tumor infiltrating lymphocytes

[0576] Tumor tissue processing

[0577] Take an appropriate amount of tumor tissue and remove necrotic tissue. Cut the tumor into pieces as small as possible in a suitable container, with the size of the tissue pieces being about 1 mm. Add 200 μl of 0.05% trypsin and place it at 37°C for digestion for 3 minutes. Then add 400 μl of 1640 + 10% FBS and mix thoroughly to terminate digestion. Filter through a 40um filter. You can use the rubber stopper end of the syringe to grind the incompletely digested tissue fragments in the filter to obtain more single cells. Transfer the filtered single-cell suspension into a 50mL centrifuge tube, centrifuge at 1500rpm for 7 minutes, and discard the supernatant. Resuspend the cells in 5-6ml PBS and count. Take 1E6 cells for subsequent staining operations.

[0578] Flow cytometry antibody staining for lymphocyte detection

[0579] Centrifuge the cells at 1500rpm for 7min and discard the supernatant. Dilute the flow cytometry antibodies: CD45+CD3+CD4+CD8+CD25+TNFR2+1:50 with detection buffer (PBS+1% BSA), dilute LiveDye (1:1000), add 100μl antibody mixture to each tube and incubate at 4 degrees for 30min. Add 400μl detection buffer to each tube, centrifuge the cells at 1500rpm for 7min and discard the supernatant. Resuspend the cells with 100μl detection buffer and perform flow cytometry.

[0580] The results are as follows Fig.11 As shown, TNFR2 protein is mainly expressed at medium to high levels in some CD4+CD25+ cells; and the expression level in TIL is significantly higher than that in peripheral blood and spleen.

[0581] Example 16 FACS evaluation of the binding selectivity of humanized antibody HU6 to CD4+CD25+ T cells

[0582] I further used enrichment Cells were used to evaluate the selectivity of the antibodies of the present invention for CD4+CD25+T cells. CD4T cell activation: Rinse the coated wells of a 6-well plate (flat-bottom plate TCP011006, Jie Te) with PBS in advance. Dilute the antibody with PBS containing 5μg / mL CD3 antibody, 100μl per well, and coat overnight at 4°C. Then take the coated plate out from 4°C and wash it three times with PBS, 100μl each time; after washing, open the lid and air dry, air dry for about 10 minutes and put it back to four degrees for use. The CD4T cells that had been revived one day in advance were centrifuged at 1600rpm for 8 minutes, and the cells were resuspended in 1640 culture medium containing 10% inactivated serum and 1% PS, and CD28 was added to a concentration of 1μg / mL, and placed in a cell culture incubator for 3 days.

[0583] CD4 T cells were collected 3 days after activation, resuspended in 1640 medium containing 1% FBS, transferred to a 96-well U-shaped plate, 2E5 / well, centrifuged at 1600rpm for 8min, then added 100μl of gradient diluted HU6 antibody and #001 antibody, incubated at 4°C for 30mins, centrifuged (1500rpm, 8mins), removed the liquid and washed with PBS (200μL / time, 2 times); PBS (100μL / well) was added to each well, the cells were resuspended, and read using a flow cytometer. FACS-analysis results are shown in Figure 2. Fig.12 As shown, the level of CD25 is positively correlated with the expression of TNFR2 ( Fig.12 A), by drawing the CD4+CD25+ cell binding curves of antibodies HU6 and #001, it was found that the binding activity of humanized antibody HU6 to CD4+CD25+ cells was significantly stronger than that of #001 ( Fig.12 B), the EC50 of antibodies HU6 and #001 were 9.061 and 220.1 ng / mL, respectively (Table 12). And the experimental results showed that humanized antibodies HU6 and #001 did not bind to CD4+CD25- cells ( Fig.12 C).

[0584] Table 12 FACS binding EC50 results of humanized antibody HU6 binding selectivity to CD4+CD25+T cells

[0585] EC50(ng / mL) HU6 #001 CD4+CD25+ 9.061 220.1

[0586] Example 17 Humanized antibody HU6 mediates downregulation of the proportion of Tregs cells in PBMC in vitro

[0587] PBMC (Miaoshun) was resuscitated and resuspended in 10 mL of 1640 medium containing 10% inactivated serum, and then cultured overnight at 37°C and 5% CO2. Plated the next day. Preparation of antibody diluent: (1) First, CD3 / 28 beads (manufacturer Miltenyi, item number 130-111-160) were added to 1640 medium containing 10% inactivated serum at a ratio of 1:50 (150 μl was reserved as the NC group), and then IL-2 was added to it to a final concentration of 20 ng / mL; (2) 600 μL of the above medium was taken, and 0.6 μL of 0.2 mg / mL TNF-α was added to a final concentration of 200 ng / mL. Antibody dilution: The antibodies were diluted with the above two groups of culture media to a final concentration of 100 μg / mL; 50 μL of antibody solution was added to each well of the 96-well U-bottom plate. Preparation of cell suspension: PBMCs were centrifuged at 1600rpm for 8 minutes, resuspended in 1640 medium containing 10% inactivated serum, and the cell concentration was adjusted to 2E6 / mL. 50μL of cell suspension was added to each well, so that the number of PBMCs in each well of the 96-well U-bottom plate was 1E5, and cultured in the incubator for three days. After three days, the expression of CD4, CD25 and FOXP3 was detected. The culture plate was centrifuged at 1600rpm for 5 minutes, the supernatant was discarded, and the plate was washed once with PBS. The dyes of CD4 and CD25 were prepared at a ratio of 1:100, and the dye dilution buffer was PBS containing 1% BSA (PBSA1%). The cells were resuspended with 100μL of dye in each well and incubated at 4°C for 30 minutes. The supernatant was discarded after centrifugation, and the plate was washed once with PBS. BD Cytofix / Cytoperm TM Fixation and Permeabilization Solution was used for cell fixation. 100 μL of resuspended cells were added to each well and incubated at 4°C for 20 min. Centrifuged at 1600 rpm for 5 min, the supernatant was discarded, and the cells were washed twice with PBS. TM Perm / WashBuffer was used for cell permeabilization. 100 μL of per well was added to resuspend the cells and incubated at 4°C for 20 min. After centrifugation, the supernatant was discarded and FOXP3 dye was diluted with PBSA1% at a ratio of 1:100. 100 μL of dye was added to each well to resuspend the cells and incubated at 4°C for 30 min. The proportion of Tregs was detected by the machine.

[0588] The results are as follows Fig.13 As shown, in 4 PBMC donors (Miao Shun, P121120602C, P122121106C, CAPB01230501C, P122110501C), humanized antibody HU6 was able to significantly mediate the downregulation of Tregs proportion regardless of whether TNF-α was added.

[0589] Example 18: Defucosylating humanized antibody HU6-AF also mediates downregulation of the proportion of Tregs cells in PBMC in vitro

[0590] The detection method of this example is consistent with that of Example 17. The effect of the defucosylating humanized antibody HU6-AF on the proportion of Tregs cells in PBMC in vitro was detected in three PBMC donors (Miao Shun, P122121106C, P122120710C, and P121120307C).

[0591] The results are as follows Fig.14 As shown, regardless of whether TNF-α was added or not, the defucosyl humanized antibody HU6-AF also mediated the downregulation of the proportion of Tregs cells in PBMCs in vitro, and the downregulation level was more significant than that of wild-type HU6.

[0592] Example 19 Humanized antibody HU6 activates CD4 T cells in vitro and promotes CD4 T cell proliferation

[0593] CD4T cells were revived and resuspended in 10 mL of 1640 medium containing 10% inactivated serum, and then cultured overnight at 37°C and 5% CO2. Plated the next day. Coating CD3 and humanized antibodies: CD3 (CDE-M120a-500 μg, Biopsy) antibody was diluted to 5 μg / mL with PBS (300 μL was set aside as a blank control), and then the hIgG1 and Hu6 antibodies were diluted with the above solution containing 5 μg / mL CD3 antibody to make the final concentrations of Hu6 0.1, 1 and 10 μg / mL, respectively. The concentration of hIgG1 was only set to 10 μg / mL as a control. 100 μL of antibody diluent was added to each well of a 96-well plate (flat-bottom plate 3599, corning), and coated overnight at 4°C. The antibody solution was aspirated the next day, washed three times with PBS, and air-dried in a clean bench for 15 minutes. Cell PB staining: The CD4T cells that had been revived one day in advance were centrifuged at 1600rpm for 8min, washed once with PBS, stained with PB (C34571, Invitrogen, dissolved in 200μL PBS) (0.4uL / mL, cell concentration 1E6 / mL, staining buffer was PBS), stained at 37℃ for 10min, and the staining was terminated with 2 times the volume of complete medium of the dye, centrifuged at 1600rpm for 8min, washed once with PBS, resuspended the cells with 1640 medium containing 10% inactivated serum, adjusted the cell concentration to 1E6 / mL, and added 100μL of cell suspension to each well of the 96-well plate coated with the antibody, so that the number of cells in each well was 1E5. Dilute CD28 antibody: Dilute CD28 antibody with 1640 medium containing 10% inactivated serum to a concentration of 2ug / mL, and add 100μCD28 solution to each well where the cells were added to make the final concentration 1ug / mL. Incubate in an incubator for three days.

[0594] 19.1 Antibody HU6 activates CD4 T cells in vitro

[0595] CD4 T cells were collected and subjected to flow cytometry analysis using the same method as in Example 15 to analyze the ratio of CD4+ and CD25+ cells. Fig.15 As shown, after the HU6 antibody treated CD4 T cells, the proportion of CD25+ cells was significantly increased compared with the IgG1 control antibody ( Fig.15 A and Fig.15 B), and the CD25 protein fluorescence level of CD25+ positive cells also increased ( Fig.15 C).

[0596] 19.2 Promoting CD4 T cell proliferation

[0597] CD4 T cells were collected for flow cytometry analysis using the same method as in Example 15, and the PB fluorescence graph (five-finger peak graph) was analyzed. The results are as follows: Fig.16 A and Fig.16 As shown in B, compared with the IgG1 control antibody, 10 μg / mL of antibody HU6 treated CD4 T cells significantly promoted CD4 T cell proliferation. The proliferation ratio of CD4 T cells in the HU6 antibody group was 43.25% ( Fig.16 A right), significantly higher than 23.67% of the IgG1 control group ( Fig.16 A (left) Proliferation ratio.

[0598] 19.3 Promoting the secretion of INF-γ

[0599] The 96-well plate was centrifuged, and the supernatant was taken and diluted 4 times. The INF-γ detection kit: Human IFN-γ Precoated ELISA Kit (Dayou, Cat. No. 1110002) was used to detect the INF-γ level in the supernatant of CD4 T cells treated with HU6 antibody for 3 days. The results are shown in Fig.16 As shown in C, compared with the IgG1 control group, the 10 μg / mL HU6 antibody group significantly promoted the secretion level of INF-γ.

[0600] Example 20 Humanized antibody HU6 mediates upregulation of PD-1 expression in CD4 T cells in vitro

[0601] The experimental method of this example is the same as above (Example 19), and CD4 T cells were treated with HU6 antibody and IgG1 antibody for 3 days. Flow cytometry analysis showed that compared with the IgG1 control antibody, the proportion of PD-1+ cells in the HU6 antibody treatment group was significantly increased ( Fig.17 A and Fig.17 B), and the PD-1 membrane protein fluorescence level of CD25+ positive cells also increased ( Fig.17 C).

[0602] Example 21: The defucosylating humanized antibody HU6-AF also activates CD4 T cells in vitro, promotes CD4 T cell proliferation, and upregulates PD-1 expression

[0603] Referring to Examples 19 and 20, HU6 wild-type antibody can activate CD4 T cells in vitro, promote CD4 T cell proliferation and upregulate the expression level of PD-1. We further evaluated the effect of defucosyl humanized HU6-AF antibody on CD4 T cells, and the experimental method was referred to Examples 19 and 20. The results are as follows Fig.18 and Fig.19 As shown, the defucosyl humanized antibody HU6-AF also activated CD4 T cells in vitro ( Fig.19 A and 19B), promote CD4 T cell proliferation ( Fig.18 ), upregulating PD-1 expression ( Fig.19 C and 19D).

[0604] As Fig.18 The experimental results show that compared with the blank group ( Fig.18 A), adding CD3 / CD28 can stimulate the activation and proliferation of CD4T cells, and on this basis, 50μg / mL of antibody HU6-AF treated CD4T cells significantly promoted CD4T cell proliferation. The proliferation ratio of CD4T cells in the HU6-AF antibody group was 35.14% ( Fig.18 C), significantly higher than 9.33% in the IgG1 control group ( Fig.18 B) proliferation ratio. And the study found that the activation effect of the antibody of the present invention on CD4T cells is positively correlated with the antibody concentration ( Fig.18 D).

[0605] same, Fig.19 The experimental results showed that HU6-AF upregulated the proportion of CD25+ positive cells in a concentration-positive manner ( Fig.19 A) and expression levels ( Fig.19 B); Similarly, HU6-AF upregulated the proportion of PD-1+ positive cells in a concentration-positive manner ( Fig.19 C) and expression levels ( Fig.19 D).

[0606] Example 22: Fucose-free humanized antibody HU6-AF activates CD8 T cells in vitro and promotes the function of cytotoxic T cells

[0607] CD8T cells were revived and resuspended in 10 mL of 1640 medium containing 10% inactivated serum, and then cultured overnight at 37°C and 5% CO2, and plated the next day. Coating CD3 and humanized antibodies: CD3 (CDE-M120a-500 μg, Biopsy) antibody was diluted to 2.5 μg / mL with PBS (300 μL was reserved as a blank control), and then hIgG1 and Hu6AF antibodies were diluted with the above solution containing 2.5 μg / mL CD3 antibody to make the final concentrations of 2, 10 and 50 μg / mL, respectively. 100 μL of antibody diluent was added to each well of a 96-well plate (flat-bottom plate 3599, corning), and coated overnight at 4°C. The antibody solution was aspirated the next day, washed three times with PBS, and air-dried in a clean bench for 15 minutes. Cell PB staining: The CD8T cells that had been revived one day in advance were centrifuged at 1600rpm for 8min, washed once with PBS, stained with PB (C34571, Invitrogen, dissolved in 200μL PBS) (0.4uL / mL, cell concentration 1E6 / mL, staining buffer was PBS), stained at 37°C for 10min, and the staining was terminated with 2 times the volume of complete medium of the dye, centrifuged at 1600rpm for 8min, washed once with PBS, resuspended the cells with 1640 medium containing 10% inactivated serum, adjusted the cell concentration to 1E6 / mL, and added 100μL of cell suspension to each well of the 96-well plate coated with the antibody, so that the number of cells in each well was 1E5. Dilute CD28 antibody: Dilute CD28 antibody with 1640 medium containing 10% inactivated serum to a concentration of 2ug / mL, and add 100μCD28 solution to each well where cells were added to make a final concentration of 1ug / mL. Incubate in an incubator for three days.

[0608] 22.1 Defucosylated HU6-AF antibody activates CD8 T cells and promotes CD8 T cell proliferation

[0609] CD8 T cells were collected for flow cytometry analysis using the same method as in Example 15 to analyze the ratio of CD8+ and CD25+ cells. Fig. 20 As shown, after the HU6-AF antibody was used to treat CD8 T cells, the proportion of CD25+ cells was significantly increased compared with the IgG1 control antibody ( Fig. 20 C), and the CD25 protein fluorescence level of CD25+ positive cells also increased ( Fig. 20 D).

[0610] The method for evaluating the effect of HU6-AF antibody on CD8 T cell proliferation was similar to that in Example 19.2, and the results were as follows Fig. 20 As shown, compared with the blank group ( Fig. 20A left), adding CD3 / CD28 can stimulate the activation and proliferation of CD8T cells, and on this basis, 50μg / mL of antibody HU6-AF treated CD48T cells significantly promoted the proliferation of CD8T cells. The proliferation ratio of CD8T cells in the HU6-AF antibody group was 51.00% ( Fig. 20 A right), significantly higher than 20.69% in the IgG1 control group ( Fig. 20 A) proliferation ratio. And the study found that the activation effect of the antibody of the present invention on CD4T cells is positively correlated with the antibody concentration ( Fig. 20 B).

[0611] 22.2 Defucosylated HU6-AF antibody promotes the killing ability of CD8 T cells

[0612] Collect cells 3 days after antibody inoculation, add PMA at a final concentration of 100 ng / ml and monesin A at 1:1000 (diluted with complete medium) to each well, and culture for 4-6 hours. Transfer cells to a 96-well U-bottom plate, centrifuge at 1600 rpm for 5 minutes, discard the supernatant, and wash once with PBS. TM Fixation and Permeabilization Solution was used for cell fixation. 100 μL of resuspended cells were added to each well and incubated at 4°C for 20 min. Centrifuged at 1600 rpm for 5 min, the supernatant was discarded, and the cells were washed twice with PBS. TM Perm / Wash Buffer was used for cell permeabilization. 100 μL of per well was added to resuspend the cells and incubated at 4°C for 20 min. After centrifugation, the supernatant was discarded and Granzyme B (372208, BioLegend) and IFN-γ (502538, BioLegend) dyes were diluted with PBSA1% at a ratio of 1:100. 100 μL of dye was added to resuspend the cells in each well and incubated at 4°C for 30 min. The cells were tested on the instrument.

[0613] The results are as follows Fig.21 As shown, compared with the control group IgG1, the HU6-AF antibody group significantly upregulated the expression level of INF-γ in a concentration-positive manner ( Fig.21 A) and the expression level of granulamycin B ( Fig.21 B).

[0614] Example 23 Evaluation of in vitro killing of tumor cells by PBMC mediated by the defucosylated antibody HU6-AF

[0615] ASPC1 and Miapaca-2 cells were revived using 1640 complete medium, and after stable culture for 3 generations, they were used for in vitro killing evaluation experiments. PBMCs were revived using 1640 medium containing 10% inactivated serum, and 10 mL of medium was resuspended and cultured overnight at 37°C, 5% CO2. Antibody dilution: CD3&28 beads and 20 ng / mL of IL-2 were added to 1640 medium containing 10% inactivated serum at a ratio of 1:25. The antibody was diluted with this medium to a concentration of 40 μg / mL, and 100 μL of the diluted antibody was added to each well of the 96-well U-bottom plate. Preparation of tumor cell suspension: After digestion of cells, wash once with PBS, adjust the concentration of tumor cells to 1E6 / mL, add 0.5μL / mL of PB dye, stain at 37℃ for 10min, add two times the volume of complete medium to terminate staining, wash tumor cells twice with PBS, resuspend cells in 1640 medium containing 10% inactivated serum, adjust the cell concentration to 2E5 / mL, and add 50μL of tumor cell suspension to each well of 96-well U-bottom plate. Preparation of PBMC suspension: Centrifuge at 1600rpm for 10min, resuspend PBMC in 1640 medium containing 10% inactivated serum, adjust the cell concentration to 1E6 / mL, and add 50μLPBMC cell suspension to each well of 96-well U-bottom plate. Set up control wells for single tumor cells and co-culture of tumor cells and PBMCs, and add the same concentration of CD3&28 beads and IL-2 to the control group. The final total volume of each well was 200 μL, and the conditions of each component were 1E4 tumor cells / well, 5E4 PBMC / well, CD3 / CD28 beads ratio was 1:50, and IL-2 concentration was 10 ng / mL. Culture was carried out at 37°C and 5% CO2 for 5 days.

[0616] After 5 days, the killing of tumor cells was detected. Apoptosis detection: After 5 days, the culture plate was removed from the incubator, and all cells were transferred to a new 96-well U-bottom plate. After the cells were washed twice with PBS, apoptosis staining was performed using the AnnexinV / 7-AAD apoptosis detection kit (APK10448-F, Sino Biological). The cells were resuspended in 25μL 1× binding buffer for staining; 1.2mL dye (912μL 1× binding buffer + 144μL AnnexinV + 144μL 7-AAD) was prepared, 25μL dye was added to each well and mixed evenly, and the staining was carried out at room temperature in the dark for 15 minutes, and then 150μL 1× binding buffer was added to terminate the staining, and the detection was carried out on the machine.

[0617] The results are as follows Fig. 22 As shown, the antibody of the present invention significantly mediated the in vitro killing of tumor cells by PBMC, and the in vitro efficacy of the defucosylating antibody HU6-AF in ASPC1 and Miapaca-2 cells was better than that of the wild-type HU6 antibody.

[0618] Example 24 In vitro evaluation of the ADCC killing effect of the antibodies of the present invention on activated CD4+ lymphocytes

[0619] As can be seen from Examples 15 and 16, the expression level of TNFR2 on CD4+CD25+ cells is the highest, especially on tumor-infiltrating CD4+CD25+ cells, so the antibodies of the present invention are most enriched in this type of cells. We then evaluated the ADCC killing effect mediated by the antibodies of the present invention on this type of cells.

[0620] 24.1 Evaluation of ADCC on Activated CD4 T Cells Using an ADCC Reporter System

[0621] CD4 T cell activation: CD4 T cells (Miaoshun, No. P422071105C-2) were revived by centrifugation at 1600 rpm for 8 min, resuspended in 1640 culture medium containing 10% inactivated serum and 1% PS, and activated by adding CD3 / CD28 beads, and cultured in a cell culture incubator for 2 days.

[0622] Antibody incubation: Resuspend the activated CD4 T cells in preheated ADCC detection buffer (1640 cell culture medium (without phenol red) + 1% FBS (inactivated at 56°C for 30 min)) and adjust the volume to 4E6 / ml. Add to white bottom plate ( 96-well all-white flat-bottom polystyrene microplate, 3917) 25 μL, add 25 μL of the gradient diluted antibody to the white bottom plate and incubate with CD4 T cells, incubate in a 37°C, 5% CO2 incubator for 30 minutes.

[0623] Inoculate ADCCBioassay effector cells (Rhinogen, Catalog No. RA-CK01): (1) Determine the density and viability of ADCCBioassay effector cells, and collect sufficient number of effector cells by centrifugation at room temperature at 1000 rpm for 5 min. (2) Discard the supernatant, wash the cells once with 10 mL preheated PBS, and centrifuge again. (3) Discard the supernatant, resuspend the cells in preheated ADCC assay buffer, adjust to 5E5 cells / mL, and inoculate 50 μL / well into a 96-well white plate, 25,000 cells / well.

[0624] Incubation: Cover the plate and incubate in a 37°C, 5% CO2 incubator for 4 h.

[0625] The results are as follows Fig.23 As shown in A, the ADCC reporting system evaluates ADCC of CD4 T activated cells. The ADCC of the defucosylated antibody HU6-AF is stronger than that of the wild-type antibody HU6. The overall ADCC level is weak, and the antibody concentration required to reach the upper platform is low. The signal basically does not increase subsequently.

[0626] 24.2 Antibody-dependent cellular cytotoxicity (ADCC) of NK cells to activated CD4 T cells mediated by the antibodies of the present invention

[0627] Resuscitation of hPBCD4+T lymphocytes: Resuscitate hPBCD4+T lymphocytes (Miaoshun, No. P422071105C-2), resuspend the cells in complete medium and culture overnight.

[0628] Coating CD3: Dilute CD3 to 5 μg / ml using 2 ml PBS, add to a T25 culture flask and place in a refrigerator at 4 degrees overnight.

[0629] CD4+T lymphocyte activation for 48 hours: CD4T cells revived one day in advance were centrifuged at 1600rpm for 8 minutes, resuspended in 1640 culture medium containing 10% inactivated serum and 1% PS, and CD28 was added to a concentration of 1μg / mL, and cultured in a cell culture incubator for 72 hours.

[0630] Recovery of NK cells: 48 h after CD4+ T lymphocyte activation, NK cells were recovered and resuspended in complete culture medium and cultured overnight (10 ng / ml hIL2 was added to the culture medium).

[0631] CD4+ T lymphocytes were collected, and the cell density was adjusted to 2E5 cells / ml using complete culture medium. The cell suspension was taken and 100 μl of cells were added to each well of a flat-bottomed transparent 96-well plate.

[0632] Antibody incubation: dilute antibodies hIgG1, HU6, HU6-AF, and HU6-IgG4 to corresponding concentrations, add 50 μl to each well of the corresponding well plate and mix well. Fill the blank control wells with 50 μl 1640 culture medium, mix well, and place in a 37°C, 5% CO2 incubator for 30 mins.

[0633] Effector cell plating: Collect NK cells, adjust the cell density to 5E5 / ml using complete medium, and add 100 μl of cells to each well according to the table above. Mix gently and place in a 37°C, 5% CO2 incubator for 5 hours.

[0634] Secondary antibody addition: After the incubation, centrifuge and wash once with 4°C pre-cooled flow cytometry solution (weigh 0.3g BSA, add to 30ml PBS, mix well), add anti-human CD4 and PI working solution to each well to resuspend the cells. Incubate at 4°C in the dark for 30 minutes.

[0635] After washing once with 4°C precooled flow cytometry solution, resuspend the cells in 150 μl flow cytometry solution, aspirate 50 μl per well for flow cytometry, analyze the proportion and number of live T cells in the system, and calculate the ADCC killing rate of each experimental group relative to the blank control well.

[0636] The results are as follows Fig.23 As shown in B, the ADCC mediated by the defucose antibody HU6-AF is stronger than that of wild-type HU6 and even stronger than that of HU6-IGG4 subtype (ADCC functional defect), but the maximum killing rate of the antibody of the present invention on activated CD4+T cells can only reach about 30%-40%.

[0637] Example 25 The antibodies of the present invention do not mediate the killing of CD4+ and CD8+ cells in PBMC

[0638] Resuscitate PBMC (Miaoshun) and activate: Use 1640 medium containing 10% inactivated serum, resuspend in 10 mL of medium, add CD3 / 28 beads (manufacturer Miltenyi, catalog number 130-111-160) at a ratio of 1:50 (leave 150 μl as the NC group), and then add IL-2 to a final concentration of 10 ng / mL; culture at 37°C, 5% CO2 for 48 hours.

[0639] Resuspend the activated PBMC cells, adjust the cell density to 1E6 cells / ml using complete culture medium, take the cell suspension, and add 100 μl of cells to each well of a flat-bottomed transparent 96-well plate.

[0640] Antibody incubation: dilute antibodies hIgG1, HU6, HU6-AF, and HU6-IgG4 to corresponding concentrations, add 50 μl to each well of the corresponding well plate and mix well. Fill the blank control wells with 50 μl 1640 culture medium, mix well, and place in a 37°C, 5% CO2 incubator for 24 hours.

[0641] Secondary antibody addition: After the incubation, transfer to a 96-well U-bottom plate, centrifuge, wash once with 4°C precooled flow cytometry solution (weigh 0.3g BSA, add to 30ml PBS, mix well), add anti-human CD4, CD8, PI working solution to each well to resuspend the cells. Incubate at 4°C in the dark for 30 minutes.

[0642] After washing once with 4°C precooled flow cytometry solution, resuspend the cells with 150 μl flow cytometry solution, pipette 50 μl into each well for flow cytometry, and analyze the proportion and number of live T cells in the system.

[0643] The results are as follows Fig.24As shown, compared with the IgG1 control group, no difference in the proportion of CD4+ and CD8+ cells was found in PBMCs activated by HU6-AF or HU6.

[0644] Example 26 In vivo anti-tumor efficacy of the antibody of the present invention in the MC38-transplanted hTNFR2 humanized mouse model

[0645] The in vivo anti-tumor activity of the anti-bispecific antibody was evaluated in a mouse xenograft model (hTNFR2 humanized mice, Biocytogen) of MC38 (mouse colon cancer cells, Sean Bio, Cat. No. SNL-505).

[0646] DMEM + 10% FBS + 1% NEAA + 1% P / S was used as complete culture medium. MC38 cells were propagated as monolayer culture in vitro. Cells in the logarithmic growth phase were digested and resuspended in serum-free culture medium / Matrigel (1:1) to obtain a suspension. Cells were cultured at 5×10 5 The mice were inoculated subcutaneously (sc) on the right side of the back to form tumors. Ten days after implantation, the mice were divided into three groups according to the tumor volume. The average tumor volume in each group was 80-150mm 3 Around 2:00 pm, the drug was started. IgG1 or antibody molecule HU6-AF or HU6 antibody was intraperitoneally (IP) administered twice a week for three weeks. Body weight and tumor diameter were measured twice a week. Tumors were dissected and weighed after euthanasia.

[0647] The results are shown in Table 13 and Fig.25 As shown in Figure 2, in the MC38 tumor model, the defucosylating humanized antibody HU6-AF has a significant anti-tumor effect ( Fig.25 A and Fig.25 C), and was significantly stronger than the wild-type humanized HU6 antibody. The tumor inhibition rate (TGI) of the HU6-AF antibody reached 81.6%, while the tumor inhibition rate (TGI) of the HU6 antibody was only 16.5%.

[0648] Table 13 In vivo anti-tumor efficacy of wild-type humanized antibody HU6 and defucosyl modified antibody HU6-AF in MC38-transplanted hTNFR2 humanized mouse model

[0649] TGI (tumor inhibition rate%) after 21 days of administration HU6 16.5 HU6-AF 81.6

[0650] Example 27 Detection of the effect of the antibody of the present invention on lymphocyte typing in the peripheral and tumor microenvironment in the MC38 tumor model

[0651] The hTNFR2 humanized mice administered with the drug for 21 days in Example 26 were used to collect peripheral blood and dissect the tumors after euthanasia and digest them into single cells (the method was the same as in Example 15). The proportions of peripheral and tumor-infiltrating mCD45+, mCD4+, mCD8+, and Tregs (mCD25+, mCD127(Foxp3)+) cells were analyzed by flow cytometry.

[0652] The results are as follows Fig.26 As shown in A, no lymphocytotoxicity of HU6 antibody or HU6-AF antibody was observed in peripheral blood. It was also found that the immune level of the HU6 antibody and HU6-AF antibody administration groups was upregulated, especially HU6-AF. Fig.26 B. In the tumor microenvironment, the tumor infiltration of Tregs cells in the HU6-AF antibody-treated group was significantly downregulated, and the tumor infiltration of CD4+T and CD8+T cells was significantly increased; in the HU6 antibody-treated group, only the tumor infiltration of Tregs cells was downregulated, and the extent of the downregulation was not as significant as that in the HU6-AF antibody-treated group.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to TNFR2, comprising a heavy chain variable region (VH) and a light chain variable region (VL), characterized in that: The VH comprises VH CDR1, VH CDR2 and VH CDR3 of amino acid sequences as shown in SEQ ID NOs: 1, 2 and 3, respectively, or amino acid sequences with no less than 95% sequence identity to the amino acid sequences as shown in SEQ ID NOs: 1, 2 and 3, respectively; the VL comprises VL CDR1, VL CDR2 and VL CDR3 of amino acid sequences as shown in SEQ ID NOs: 4, 5 and 6, respectively, or amino acid sequences with no less than 95% sequence identity to the amino acid sequences as shown in SEQ ID NOs: 4, 5 and 6, respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The VH comprises an amino acid sequence as shown in any one of SEQ ID NOs: 7 and 11, or an amino acid sequence having at least 85% sequence identity with the amino acid sequence as shown in any one of SEQ ID NOs: 7 and 11.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The VL comprises an amino acid sequence as shown in any one of SEQ ID NO: 8 or 12, or an amino acid sequence having at least 85% sequence identity compared to the amino acid sequence shown in any one of SEQ ID NO: 8 and 12.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein The VH and the VL respectively comprise the amino acid sequence combination of (1) or (2): (1) the amino acid sequence combination as shown in SEQ ID NO: 7 and 8, (2) The amino acid sequence combination as shown in SEQ ID NO: 11 and 12.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, characterized in that: The antibody is a monoclonal antibody, and the monoclonal antibody is selected from a mouse antibody, a human-mouse chimeric antibody, and a humanized antibody.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that: The human-mouse chimeric antibody and humanized antibody comprise a human heavy chain constant region (CH) and / or a human light chain constant region (CL); the human heavy chain constant region is selected from one of the human IgG1 heavy chain constant region, the human IgG2 heavy chain constant region, the human IgG3 heavy chain constant region or the human IgG4 heavy chain constant region.

7. An antibody or antigen-binding fragment thereof that heterosexually binds to TNFR2, comprising a heavy chain (HA) and a light chain (LA), characterized in that: The antibody is a human-mouse chimeric antibody or a humanized antibody, wherein the HA comprises an amino acid sequence as shown in any one of SEQ ID NOs: 9 and 13, or an amino acid sequence having no less than 85% sequence identity with the amino acid sequence as shown in any one of SEQ ID NOs: 9 and 13, and the LA comprises an amino acid sequence as shown in any one of SEQ ID NOs: 10 and 14, or an amino acid sequence having no less than 85% sequence identity with the amino acid sequence as shown in any one of SEQ ID NOs: 10 and 14.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, characterized in that: The antibody or antigen-binding fragment thereof has one or more of the following characteristics: (1) It can block the binding between TNFα and TNFR2; (2) the soluble antibody of the antibody or antigen-binding fragment thereof can block the activation activity of TNF-α on the downstream NF-KB signaling pathway; (3) the antibody or antigen-binding fragment thereof fixed or cross-linked with anti-FC antibody (Fab) or cross-linked with FC using THP-1 cells has an activation effect on the NF-KB signaling pathway downstream of TNFR2 (e.g., human or cynomolgus monkey TNFR2), but the activation ability is lower than that of TNF-α; (4) In the PBMC system, the antibody or antigen-binding fragment thereof can downregulate the TREG ratio but has no effect on CD4+T or CD8+T cells; (5) It can promote the activation, proliferation and secretion of INF-γ (pro-inflammatory factor) of effector T cells (such as CD4Tconv T cells), and upregulation of PD-1 expression level was observed; (6) It can directly activate CD8+ naive T cells and promote their proliferation, as well as the secretion of INF-γ (a pro-inflammatory factor) and granulomycin B (GzmB); (7) The antibody or its antigen-binding fragment comprises a defucosyl-modified antibody, which mediates increased tumor infiltration of CD4+T and CD8+T cells and down-regulation of tumor infiltration of TREG cells in TNFR2 humanized mice, and can inhibit tumor growth in vivo.

9. A bispecific antibody comprising the antigen-binding region of the antibody of any one of claims 1 to 8 and a second different antigen-binding region.

10. A pharmaceutical composition, characterized in that The composition comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the bispecific antibody according to claim 9, and a pharmaceutically acceptable carrier, diluent or adjuvant; or optionally, the drug combination further comprises a second active drug.

11. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, the bispecific antibody according to claim 9, or the pharmaceutical composition according to claim 10 in the preparation of a medicament for treating and / or preventing cancer associated with immune abnormality.

12. A method for treating cancer, comprising administering a therapeutically effective amount of the antibody of any one of claims 1 to 8, the bispecific antibody of claim 9, and / or the pharmaceutical composition of claim 10 to a subject in need thereof.

13. The use according to claim 11, the method according to claim 12, wherein the cancer is a cancer associated with abnormally high expression of TNFR2 and an excessively high ratio of TNFR2+Treg / Teff, such as ovarian cancer, advanced epidermal T-cell lymphoma, stage III / IV metastatic colorectal cancer, triple-negative breast cancer, pancreatic cancer, non-small cell lung cancer, and / or advanced solid tumors resistant to CTLA-4 and PD-1 therapy, such as metastatic melanoma, non-small cell lung cancer, breast cancer, and ovarian cancer.

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