Tnfr2 antibodies and uses thereof

Abstract

This invention relates to the field of antibody drug technology, and more particularly to TNFR2 antibodies and their applications. The TNFR2 antibody of this invention binds specifically to human TNFR2 and monkey TNFR2 with high affinity, and specifically blocks the binding of the ligand TNFα to TNFR2. It can also inhibit the proliferation of Treg cells in vitro and promote the expansion of Teff cells. In vivo experiments in mice show that this molecule significantly inhibits tumor growth. Its antitumor effect is mainly achieved by selectively inhibiting the activity of TNFR2 in immunosuppressive cells (Tregs), thereby weakening the inhibition of T effector cells by Tregs, promoting the activation and expansion of T effector cells, alleviating the immunosuppressive microenvironment, and thus enhancing the antitumor immune response.

Description

Technical Field

[0001] This invention relates to the field of antibody drug technology, and more particularly to TNFR2 antibody and its applications. Background Technology

[0002] The use of naturally occurring and genetically engineered T lymphocytes is a prominent example of improving various human pathologies. In recent years, adoptive immunotherapy has seen a resurgence in cancer treatment. Tumor-infiltrating lymphocyte (TIL) therapy, in particular, utilizes T cells that target different antigens and possess strong cytotoxicity to directly kill tumor cells or stimulate the body's immune response to kill tumor cells, offering cancer patients a range of options. Despite the promising future of T lymphocyte-based immunotherapy, the development of this treatment platform has been hampered by the natural tendency of the immune system to suppress its own cellular immune attacks. Regulatory T cells (Treg cells) can suppress the activity of T cells that respond to "self" MHC antigens, playing a crucial role in maintaining peripheral tolerance. However, these cells' ability to regulate the activity of self-reactive T cells also disrupts adoptive immunotherapy and the innate immune response by inhibiting the activity of tumor-reactive T lymphocytes. Therefore, how to release the inhibition of the immune system and enhance the immune response against tumors has become another challenge for researchers. Obtaining a drug that can inhibit Treg-mediated T cell inhibition can greatly improve the scope and efficacy of adoptive cancer immunotherapy and enhance the immune system's ability to eradicate pathogens that cause infectious diseases.

[0003] TNFR2, a member of the TNF receptor superfamily, is selectively expressed as a surface oncogene in various tumor types, promoting tumor cell proliferation. TNFR2 is also expressed in suppressive immune cells within the tumor microenvironment, including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), and is a potential driver of immune escape, tumor growth, and checkpoint blockade resistance. Treg cells highly expressing TNFR2 in the tumor microenvironment exhibit the greatest inhibitory activity, making the development of TNFR2 antagonistic antibodies one solution to these challenges. TNFR2 is activated by membrane-bound TNFα, triggering effective signal transduction, thereby promoting cell survival and immunosuppression. Currently, the development of TNFR2 antagonistic antibodies appears to have the following advantages: 1) Targeting Tregs in the tumor microenvironment, releasing the inhibitory effect of Tregs on T effector cells, mainly CD8+ T cells, and enhancing the killing effect of CD8+ T cells on tumor cells; 2) Simultaneously inhibiting the secretion of soluble TNFR2, alleviating the immunosuppressive microenvironment, thereby enhancing the anti-tumor immune response; 3) Moreover, inhibiting TNFR2 can inhibit cell proliferation and directly kill TNFR2+ tumor cells or tumor-supporting TNFR2+ cells.

[0004] There are currently no TNFR2 antagonistic antibodies on the market globally, and TNFR2 development is in its early stages. Most products under development are in clinical or preclinical phases, including BI-1808 (NCT04752826) developed by BioInvent (Sweden) and SIMC235 (NCT05569057) developed by Simcere Pharmaceutical (China). Exploring and developing TNFR2 can expand the population that can benefit from immunotherapy. This invention aims to develop a TNFR2 antagonistic antibody, primarily used in combination with PD-1 antibodies, to selectively inhibit TNFR2 activity in immunosuppressive cells (Tregs), thereby reducing the inhibition of T effector cells by Tregs, promoting T effector cell activation and expansion, alleviating the immunosuppressive microenvironment, and enhancing the anti-tumor immune response. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a TNFR2 antibody and its application.

[0006] The TNFR2 antibody or antigen-binding molecule provided by this invention,

[0007] The three CDR regions of its heavy chain each have an amino acid sequence as shown in SEQ ID NO:11, 12 or 13, or have a sequence based on the amino acid sequence shown in SEQ ID NO:11, 12 or 13 by substitution, deletion, addition and / or replacement of one or more amino acids; or have a sequence with more than 80% (85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%) homology with the amino acid sequence shown in SEQ ID NO:11, 12 or 13.

[0008] The three CDR regions of its light chain each have an amino acid sequence as shown in SEQ ID NO:14, 15 or 16, or a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in SEQ ID NO:14, 15 or 16; or a sequence that has more than 80% (85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%) homology with the amino acid sequence shown in SEQ ID NO:14, 15 or 16.

[0009] In some embodiments, the amino acid sequence of the heavy chain CDR1 of the TNFR2 antibody or antigen-binding molecule provided by the present invention is: GYTFTSNTMH (SEQ ID NO:11);

[0010] In some embodiments, the amino acid sequence of the heavy chain CDR2 of the TNFR2 antibody or antigen-binding molecule provided by the present invention is: AIYPGNX1DTSYNQKFKG, X1 = G or E. (SEQ ID NO:12);

[0011] In some embodiments, the amino acid sequence of the heavy chain CDR3 of the TNFR2 antibody or antigen-binding molecule provided by the present invention is: QTLYDPFAY (SEQ ID NO:13);

[0012] In some embodiments, the amino acid sequence of the light chain CDR1 of the TNFR2 antibody or antigen-binding molecule provided by the present invention is: RASESVDTYGSSFMH (SEQ ID NO:14);

[0013] In some embodiments, the amino acid sequence of the light chain CDR2 of the TNFR2 antibody or antigen-binding molecule provided by the present invention is: RASNLES (SEQ ID NO:15);

[0014] In some embodiments, the amino acid sequence of the light chain CDR3 of the TNFR2 antibody or antigen-binding molecule provided by the present invention is: QQSNEDPWT (SEQ ID NO:16);

[0015] In some specific embodiments:

[0016] The amino acid sequences of the three CDR regions of the heavy chain of the TNFR2 antibody or antigen-binding molecule are GYTFTSNTMH (SEQ ID NO:11), AIYPGNGDTSYNQKFK (SEQ ID NO:12, X1 is G) and QTLYDPFAY (SEQ ID NO:13), respectively, and the amino acid sequences of the three CDR regions of the light chain are RASESVDTYGSSFMH (SEQ ID NO:14), RASNLES (SEQ ID NO:15), and QQSNEDPWT (SEQ ID NO:16), respectively.

[0017] Alternatively, the amino acid sequences of the three CDR regions of the heavy chain of the TNFR2 antibody or antigen-binding molecule are, in order, GYTFTSNTMH (SEQ ID NO:11), AIYPGNEDTSYNQKFKG (SEQ ID NO:12, X1 is E) and QTLYDPFAY (SEQ ID NO:13), and the amino acid sequences of the three CDR regions of its light chain are, in order, RASESVDTYGSSFMH (SEQ ID NO:14), RASNLES (SEQ ID NO:15) and QQSNEDPWT (SEQ ID NO:16).

[0018] The TNFR2 antibody or antigen-binding molecule provided by this invention,

[0019] The amino acid sequences of the four FR regions of the heavy chain each have an amino acid sequence as shown in SEQ ID NO:17, 18, 19 or 20; or, have a sequence based on the amino acid sequence shown in SEQ ID NO:17, 18, 19 or 20 by substitution, deletion, addition and / or replacement of one or more amino acids; or have a sequence with more than 80% homology (85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%) to the amino acid sequence shown in SEQ ID NO:17, 18, 19 or 20.

[0020] The amino acid sequences of the four FR regions of its light chain have the amino acid sequences shown in SEQ ID NO:21, 22, 23 and 24, respectively; or have sequences based on the amino acid sequences shown in SEQ ID NO:21, 22, 23 and 24 by substitution, deletion, addition and / or replacement of one or more amino acids; or have sequences with more than 80% homology (85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%) to the amino acid sequences shown in SEQ ID NO:21, 22, 23 and 24.

[0021] Alternatively, in the TNFR2 antibody or antigen-binding molecule provided by this invention

[0022] The amino acid sequences of the four FR regions of its heavy chain have the amino acid sequences shown in SEQ ID NO:25, 26, 27 or 28, respectively; or have sequences based on the amino acid sequences shown in SEQ ID NO:25, 26, 27 or 28 by substitution, deletion, addition and / or replacement of one or more amino acids; or have sequences with more than 80% homology to the amino acid sequences shown in SEQ ID NO:25, 26, 27 or 28.

[0023] The amino acid sequences of the four FR regions of its light chain have the amino acid sequences shown in SEQ ID NO:29, 22, 30 and 24, respectively; or have sequences based on the amino acid sequences shown in SEQ ID NO:29, 22, 30 and 24 by substitution, deletion, addition and / or replacement of one or more amino acids; or have sequences with more than 80% homology to the amino acid sequences shown in SEQ ID NO:29, 22, 30 and 24.

[0024] In some embodiments, the amino acid sequence of the heavy chain FR1 of the TNFR2 antibody or antigen-binding molecule provided by the present invention is: QVQLQQPGAELVKPGASVKMSCKAS (SEQ ID NO:25); or EVQLVQSGAEVKKP GSSVKVSCKAS (SEQ ID NO:17).

[0025] In some embodiments, the amino acid sequence of the heavy chain FR2 of the TNFR2 antibody or antigen-binding molecule provided by the present invention is WVKQTPGQGLEWIG (SEQ ID NO:26) or WVRQAPGQGLEWMG (SEQ ID NO:18).

[0026] In some embodiments, the amino acid sequence of the heavy chain FR3 of the TNFR2 antibody or antigen-binding molecule provided by the present invention is: KATLTADKSSSTAYMHLSSLTSEDSAVYYCAR (SEQ ID NO:27), or RVTITADKSTSTAYMELSSLRSEDTAVYYCAR (SEQ ID NO:19).

[0027] In some embodiments, the amino acid sequence of the heavy chain FR4 of the TNFR2 antibody or antigen-binding molecule provided by the present invention is: WGQGTLVTVSA (SEQ ID NO:28) or WGQGTLVTVSS (SEQ ID NO:20).

[0028] In some embodiments, the amino acid sequence of the light chain FR1 of the TNFR2 antibody or antigen-binding molecule provided by the present invention is: DIVLTQSPASLAVSLGQRATISC (SEQ ID NO:29), or DIVMTQSPX2SLAVSLGE RATINC,X2=A or D (SEQ ID NO:21).

[0029] In some embodiments, the amino acid sequence of the light chain FR2 of the TNFR2 antibody or antigen-binding molecule provided by the present invention is: WYQQKPGQPPKLLIY (SEQ ID NO:22).

[0030] In some embodiments, the amino acid sequence of the light chain FR3 of the TNFR2 antibody or antigen-binding molecule provided by the present invention is: GIPARFSGSGSRTDFTLTINPVEADDVAAYYC (SEQ ID NO:30), or GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO:23).

[0031] In some embodiments, the amino acid sequence of the light chain FR4 of the TNFR2 antibody or antigen-binding molecule provided by the present invention is: FGGGTKVEIK (SEQ ID NO:24) or FGGGTKLEIK (SEQ ID NO:39).

[0032] In some specific embodiments:

[0033] The amino acid sequences of the four FR regions of the heavy chain of the TNFR2 antibody or antigen-binding molecule are, in order: EVQLVQSGAEVKKPGSSVKVSCKAS, WVRQAPGQGLEWMG, RVTITADKSTSTAYMELSSLRSEDT AVYYCAR, and WGQGTLVTVSS.

[0034] Alternatively, the amino acid sequences of the four FR regions of the heavy chain of the TNFR2 antibody or antigen-binding molecule are QVQLQQPGAELVKPGASVKMSCKAS, WVKQTPGQGLEWIG, KATLTADKSSSTAYMHLSSLT SEDSAVYYCAR, and WGQGTLVTVSA, respectively.

[0035] In some specific embodiments:

[0036] The amino acid sequences of the four FR regions of the light chain of the TNFR2 antibody or antigen-binding molecule are, in order: DIVMTQSPDSLAVSLGERATINC, WYQQKPGQPPKLLIY, GVPDRFSGSGSGTDFTLTISSLQAEDVA VYYC, and FGGGTKVEIK.

[0037] Alternatively, the amino acid sequences of the four FR regions of the light chain of the TNFR2 antibody or antigen-binding molecule are, in order, DIVMTQSPASLAVSLGERATINC, WYQQKPGQPPKLLIY, GVPDRFSGSGSGTDFTLTISSLQAED VAVYYC, and FGGGTKVEIK.

[0038] Alternatively, the amino acid sequences of the four FR regions of the light chain of the TNFR2 antibody or antigen-binding molecule are, in order, DIVLTQSPASLAVSLGQRATISC, WYQQKPGQPPKLLIY, GIPARFSGSGSRTDFTLTINPVEADDV AAYYC, and FGGGTKLEIK.

[0039] In some specific embodiments, the heavy chain variable region of the TNFR2 antibody or antigen-binding molecule provided by the present invention has an amino acid sequence as shown in any one of SEQ ID NO:31-32; and the light chain variable region has an amino acid sequence as shown in any one of SEQ ID NO:33-34.

[0040] More specifically, the amino acid sequence of the heavy chain variable region of the TNFR2 antibody or antigen-binding molecule is shown in SEQ ID NO:31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:33;

[0041] The amino acid sequence of the heavy chain variable region of the TNFR2 antibody or antigen-binding molecule is shown in SEQ ID NO:31, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:34.

[0042] Or the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:32, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:33.

[0043] Or the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:32, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:34.

[0044] Or the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:35, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:36.

[0045] The TNFR2 antibody or antigen-binding molecule provided by this invention has a constant region of heavy chain consisting of any one of human IgG1, IgG2a, IgG2b or IgG3; and a constant region of light chain consisting of kappa type.

[0046] In some embodiments, the heavy chain constant region of the antibody or antigen-binding molecule of said TNFR2 has an amino acid sequence as shown in SEQ ID NO:37, or a sequence based on the amino acid sequence shown in SEQ ID NO:37 by substitution, deletion, addition and / or replacement of one or more amino acids; or has a sequence with more than 80% homology to the amino acid sequence shown in SEQ ID NO:37.

[0047] The light chain constant region of the antibody or antigen-binding molecule of said TNFR2 has an amino acid sequence as shown in SEQ ID NO:38, or a sequence based on the amino acid sequence shown in SEQ ID NO:38 with substitution, deletion, addition and / or replacement of one or more amino acids; or has a sequence with more than 80% homology to the amino acid sequence shown in SEQ ID NO:38.

[0048] Furthermore, the present invention also provides a nucleic acid encoding the TNFR2 antibody or antigen-binding molecule described above.

[0049] The nucleic acid described in this invention is a nucleic acid encoding the CDR region of the antibody as described above, or a nucleic acid encoding the variable region, or a nucleic acid encoding the full length of the antibody. This invention does not limit the specific nucleic acid to this type.

[0050] Furthermore, the present invention also provides a plasmid vector comprising a backbone vector and nucleic acid as described above.

[0051] In this invention, the backbone vector of the plasmid vector is a mammalian cell expression vector, for example, pMD19-T, pCDNA5, pTT5_hIgG1.G1m3 or pTT5_hKappa.Km3.

[0052] Furthermore, the present invention also provides a host cell that is transformed or transfected with the plasmid vector as described above, or whose genome integrates the nucleic acid described above.

[0053] In this invention, the host is a mammalian cell, such as human embryonic kidney HEK293 cells or Chinese hamster ovary cells CHOK1.

[0054] Furthermore, the present invention also provides a method for preparing TNFR2 antibodies or antigen-binding molecules, comprising: culturing host cells as described above to obtain a product containing TNFR2 antibodies or antigen-binding molecules.

[0055] The preparation method further includes steps of enriching, extracting and / or purifying the culture.

[0056] Furthermore, the present invention also provides the use of the TNFR2 antibody or antigen-binding molecule, the nucleic acid, the plasmid vector, and / or the host cell in the preparation of a treatment for TNFR2-related diseases.

[0057] In some embodiments, the TNFR2-related disease is a tumor, particularly a tumor that overexpresses TNFR2.

[0058] Furthermore, the present invention also provides a drug comprising the TNFR2 antibody or antigen-binding molecule, the nucleic acid, the plasmid vector, and / or the host cell.

[0059] In this invention, the drug also includes pharmaceutically acceptable excipients.

[0060] In this invention, the dosage form of the drug includes, but is not limited to, oral preparations, injections, aerosols, or suppositories. Oral preparations include, but are not limited to, tablets, capsules, pills, oral liquids, or dispersants. Injections include, but are not limited to, injectable solutions or powders for injection.

[0061] Furthermore, the present invention also provides a pharmaceutical composition comprising the aforementioned drug and other therapeutic agents.

[0062] The other therapeutic agents are antitumor drugs. As a feasible example, the antitumor drugs include, but are not limited to, at least one of the following: cisplatin, carboplatin, oxaliplatin, paclitaxel, vinorelbine, gefitinib, fluorouracil, doxorubicin, erlotinib, rituximab, trastuzumab, pertuzumab, nivolumab, pembrolizumab, atezolizumab, olaparib, niraparib, etoposide, tamoxifen, toremifene, fulvestrant, or letrozole.

[0063] In the pharmaceutical composition, multiple drugs may exist in a mixture or independently; this invention does not limit this. Multiple drugs may be administered simultaneously or sequentially; this invention also does not limit this.

[0064] The present invention also provides a method for treating TNFR2-related diseases, comprising administering the drug or pharmaceutical composition described herein.

[0065] The administration methods of the drug or drug composition include, but are not limited to: oral administration, inhalation spray, buccal administration, nasal administration, vaginal administration, rectal administration, topical administration, and parenteral administration such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion, or administration via an external implantation device. Oral, intraperitoneal, or intravenous administration is preferred.

[0066] Furthermore, the present invention also provides labeled antibodies obtained by binding the TNFR2 antibody or antigen-binding molecule to a label, wherein the label is: a fluorescent indicator, a chemiluminescent indicator, an isotope, a colloidal indicator, biotin, avidin, or an enzyme label.

[0067] In some specific embodiments, the fluorescent indicator is selected from one or more of AF350, AF488, AF532, AF546, AF555, AF568, AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, OregonGreen 488, Pacific Blue dye, Pacific Orange dye, Texas Red, or PerCP dye.

[0068] In some specific embodiments, the chemiluminescent indicator is selected from one or more of acridine ester, acridine sulfonamide and its derivatives, luminol, isoluminol, isoluminol isothiocyanate and its derivatives, N-(4-aminobutyl)-N-ethyl isoluminol, 4,5-diaminophthalic acid hydrazide or aminobutylethyl benzoyl hydrazide.

[0069] In some specific embodiments, the isotopes are selected from... 125 I, 1 31 I, 124 I, 3 H, 14 C 111 In、 89 Zr or 32 One or more of P.

[0070] In some specific embodiments, the colloidal indicator is selected from one or more of colloidal gold, colloidal carbon, or colloidal selenium.

[0071] In some specific embodiments, the enzyme label is selected from one or more of horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase-antiperoxidase bridge, alkaline phosphatase-antialkaline phosphatase bridge, and β-galactosidase-antiβ-galactosidase bridge.

[0072] Furthermore, the present invention also provides a medium coated with the aforementioned TNFR2 antibody or antigen-binding molecule; the medium is an enzyme-labeled plate, magnetic beads, or latex microspheres. In the present invention, the antibody and the medium can be linked by an amide bond or by a streptavidin-biotin system, and the present invention is not limited thereto.

[0073] Furthermore, the present invention also provides the application of the TNFR2 antibody or antigen-binding molecule, the labeled antibody, or the medium in the preparation of TNFR2 detection reagents and / or TNFR2-related disease diagnostic reagents.

[0074] The present invention also provides a reagent comprising the TNFR2 antibody or antigen-binding molecule, the labeled antibody, or the medium.

[0075] This invention also provides a method for detecting TNFR2, which includes detecting a sample using the reagents described in this invention. The detection method includes, but is not limited to, ELISA, flow cytometry, and / or immunohistochemistry. The samples include, but are not limited to, cells, blood, sections, or tissue homogenates.

[0076] This invention also provides a diagnostic method for TNFR2-related diseases, the diagnosis including detection after processing a sample, or detection or imaging after processing a subject with the reagents described above. The samples include, but are not limited to, cells, blood, sections, or tissue homogenates. Administration to the subject includes, but is not limited to, administration via the gastrointestinal tract, intravenous administration, or intraperitoneal injection, and imaging includes, but is not limited to, detection using fluorescent or radioactive substances.

[0077] The antibody TNFR2 of this invention binds specifically to human TNFR2 and monkey TNFR2 with high affinity, and specifically blocks the binding of the ligand TNFα to TNFR2. It also inhibits the proliferation of Treg cells in vitro and promotes the expansion of Teff cells. In vivo experiments in mice show that this molecule significantly inhibits tumor growth. Its antitumor effect is mainly achieved by selectively inhibiting the activity of TNFR2 in immunosuppressive cells (Tregs), thereby weakening the inhibition of T effector cells by Tregs, promoting the activation and expansion of T effector cells, alleviating the immunosuppressive microenvironment, and thus enhancing the antitumor immune response. Attached Figure Description

[0078] Figure 1 This demonstrates the expression of TNFR2 in the HEK293 TNFR2 FL cell line;

[0079] Figure 2 The images show the binding of chimeric antibodies to human TNFR2 protein and monkey TNFR2 protein. In the images, A / B represents the binding of chimeric antibodies to human TNFR2 protein, and C / D represents the binding of chimeric antibodies to monkey TNFR2 protein.

[0080] Figure 3 The activity assessment of the chimeric antibody is shown in Figure A, where A represents the binding of the chimeric antibody to HEK293 TNFR2 FL cells overexpressing the antibody; B represents the blocking activity of the chimeric antibody against ligand receptor binding at the protein level; C represents the blocking activity of the chimeric antibody against ligand receptor binding at the cellular level; and D represents the ADCC killing activity of the chimeric antibody.

[0081] Figure 4 Assess the protein-level binding activity of humanized antibodies;

[0082] Figure 5 Assessment of the protein-level blocking activity of humanized antibodies;

[0083] Figure 6 Detection of binding between humanized antibodies and overexpressing cells;

[0084] Figure 7 Evaluation of the blocking activity of humanized antibodies at the cellular level;

[0085] Figure 8Assessment of ADCC activity of humanized antibodies;

[0086] Figure 9 The binding of humanized antibodies to TNFR1 was detected;

[0087] Figure 10 The binding of humanized antibodies to human monkey TNFR2 protein was detected;

[0088] Figure 11 Evaluation of the blocking effect of humanized antibodies on human TNFR2 protein;

[0089] Figure 12 The binding of humanized antibodies to HEK293 TNFR2-overexpressing FL cells was detected.

[0090] Figure 13 Evaluation of the blocking activity of humanized antibody against ligand and HEK293 TNFR2 FL cell binding;

[0091] Figure 14 Assessment of ADCC activity of humanized antibodies;

[0092] Figure 15 The effect of anti-TNFR2 antibody on Treg proliferation and Teff amplification was shown in the following: A, treatment with different anti-TNFR2 antibodies at 20 μg / ml in the presence of 200 IU / ml IL-2 and / or 20 ng / ml TNFα, replicates, and flow cytometry analysis of the proportion of Tregs (CD4+CD25hiFoxP3+) after incubation; data are expressed as mean ± SEM. B, changes in Treg proliferation and percentage of inhibition after different antibody treatments compared to wells treated with IL-2 and TNFα simultaneously. C, changes in Treg proliferation and percentage of inhibition after incubation with 200 IU / ml IL-2 and / or 20 ng / ml TNFα. In the presence of TNFα, different anti-TNFR2 antibodies at 20 μg / ml were used for treatment. After incubation, changes in Teffs (CD4+CD25hiFoxP3-) were analyzed by flow cytometry. Data are expressed as Mean ± SEM. D represents the change in the percentage of Teff amplification after different antibody treatments compared to wells treated with both IL-2 and TNFα. E and F represent the effects of different antibody concentrations on Treg proliferation (E) and Teff amplification (F) in the presence of 200 IU / ml IL-2 and 20 ng / ml TNFα. Data are expressed as Mean ± SEM. Independent samples t-tests were used for comparisons between groups. *: P < 0.05; **: P < 0.01; ***: P < 0.001.

[0093] Figure 16The changes in tumor volume in mice of different groups are shown in Figure A, where: A represents the changes in tumor volume after treatment with different doses of anti-TNFR2 antibody; B represents the changes in tumor volume after combined treatment with 2 mpk anti-TNFR2 antibody and 1 mpkm anti-PD-1 antibody; C and J represent the changes in tumor volume in individual mice from G1 to G8, respectively. Data are expressed as mean ± SEM. Independent samples t-tests were used to compare the groups. *: P < 0.05; **: P < 0.01; ***: P < 0.001.

[0094] Figure 17 The changes in tumor weight and body weight of mice in different groups are shown. In this figure, A represents the tumor weight of mice under different treatments, and B represents the body weight changes of mice in different groups. Note: Data are expressed as mean ± SEM. Detailed Implementation

[0095] This invention provides a TNFR2 antibody and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0096] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0097] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural.

[0098] In this application, the terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0099] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0100] In this application, "specific binding" refers to the fact that antigen-binding molecules (e.g., antibodies) typically bind antigens specifically to substantially the same antigens with high affinity, but do not bind unrelated antigens with high affinity. Affinity is usually reflected by the equilibrium dissociation constant (KD), where a lower KD indicates higher affinity. For example, for antibodies, high affinity typically refers to a KD of approximately 10. -6 M or lower, approximately 10 -7 M or lower, approximately 10 -8 M or lower, approximately 1×10 -9 M or lower, approximately 1×10 -10 M or lower, 1×10 -11 M or lower or 1×10 -12 M or lower KD. KD is calculated as follows: KD = Kd / Ka, where Kd represents the dissociation rate and Ka represents the binding rate. The equilibrium dissociation constant KD can be measured using methods known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis.

[0101] In this application, "antigen-binding molecule" refers to a molecule that specifically binds to an antigen. For example, the antigen-binding molecule includes, but is not limited to, antibodies or antibody mimics. "Antibody mimic" refers to a biomolecule that mimics the structure and function of a natural antibody through non-natural synthetic methods. For example, antibody mimics include, but are not limited to, affibody, affitin, affilin, designed ankylosing spondylamine repeat (DARPin), nucleic acid aptamers, or Kunitz-type domain peptides.

[0102] In this application, "antibody" includes a typical "quadruple-chain antibody," which belongs to the immunoglobulin class composed of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain composed of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the N-terminal to C-terminal direction. Optionally, when the full-length antibody is an IgE isotype, it also includes a heavy chain constant region CH4 domain. The light chain is a polypeptide chain composed of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. Heavy chains are linked to each other and to each other with disulfide bonds, forming a "Y"-shaped structure. Because the amino acid composition and sequence of the immunoglobulin heavy chain constant region differ, their antigenicity also differs. Based on this, the "immunoglobulins" in this article can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain can further lead to different subclasses. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA into IgA1 and IgA2. Light chains are classified as κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can possess either a κ or λ chain.

[0103] In this application, "antibody" encompasses various forms and structures, including complementary-determining regions (CDRs) and antibody framework regions (FRs). CDRs are hypervariable regions of the antibody, located at the amino termini of the heavy chain (VH) and light chain (VL). These regions contain highly variable amino acid sequences, allowing for precise binding of the antibody to the antigen. The three main CDR regions are CDR-H1, CDR-H2, and CDR-H3 (in the heavy chain) and CDR-L1, CDR-L2, and CDR-L3 (in the light chain). FRs are the regions in the antibody heavy and light chains other than the CDRs. Although FRs are not directly involved in antigen binding, they significantly influence the conformation of the CDRs and the specificity of antigen binding.

[0104] In this application, "antibody" also includes antibodies that do not contain a light chain or antibodies that do not contain a constant region.

[0105] In this application, the "antibody" can be derived from any animal, including but not limited to humans and non-human animals. The non-human animals can be selected from primates, mammals, rodents and vertebrates, such as camels, llamas, ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g., sharks).

[0106] In this application, "chimeric antibody" refers to an antibody that has a variable sequence of immunoglobulin derived from one source organism (such as rat, mouse, rabbit, or alpaca) and a constant region of immunoglobulin derived from a different organism (such as human). Chimeric antibodies are typically prepared using genetic engineering, which involves splicing the V region gene of a monoclonal antibody from one source organism with the C region gene of an antibody from another organism to form a chimeric gene, which is then inserted into a vector and transfected into antibody molecules expressed in myeloma tissue.

[0107] In this application, "humanized antibody" refers to a non-human antibody that has been genetically engineered to improve its amino acid sequence homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody originates from a non-human antibody (donor antibody), while all or part of the non-CDR region (e.g., the variable region FR and / or constant region) originates from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain or partially retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to enhance immune cell activity, and ability to strengthen immune responses.

[0108] In this application, the "heavy chain constant region" refers to the carboxyl-terminal portion of the antibody heavy chain, which does not directly participate in the binding of the antibody to the antigen but exhibits effector functions, such as interaction with the Fc receptor. It has a more conserved amino acid sequence compared to the variable domains of the antibody. The "heavy chain constant region" can be selected from: the CH1 domain, the hinge region, the CH2 domain, the CH3 domain, or variants or fragments thereof. The "heavy chain constant region" includes a "full-length heavy chain constant region" and a "heavy chain constant region fragment," the former having a structure substantially similar to the natural antibody constant region, while the latter includes only a portion of the full-length heavy chain constant region. Exemplarily, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include the CH1 domain. Exemplarily, a typical "heavy chain constant region fragment" can be selected from the Fc or CH3 domain.

[0109] In this application, the "light chain constant region" refers to the carboxyl terminus of the antibody light chain, which does not directly participate in the binding of the antibody to the antigen. The light chain constant region can be selected from the constant κ domain or the constant λ domain.

[0110] In this application, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations of amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0111] In this application, "identity" can be calculated as follows: To determine the percentage of "identity" between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position. Taking into account the number of vacancies that need to be introduced for optimal alignment of the two sequences and the length of each vacancy, the percentage of identity between the two sequences varies with the common positions of the sequences.

[0112] In this application, "nucleic acid" includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented as 5′ to 3′.

[0113] In this application, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising mixtures of two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, and single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derived sugar or phosphate backbones bonded or chemically modified residues. Nucleic acid molecule also encompasses DNA and RNA molecules suitable as carriers for the direct expression of the antibodies of the present invention in vitro and / or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) carriers can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, thereby allowing the mRNA to be injected into the subject to generate antibodies in vivo (see, for example, Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038 / nm.4356 or EP 2101823 B1).

[0114] In this application, "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid linked to it. This term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which the vector has been introduced. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors".

[0115] In this application, "host" or "host cell" refers to a cell in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their progeny, regardless of the number of passages. Progeny may not be completely identical to parental cells in their nucleic acid contents and may contain mutations. This document includes mutant progeny with the same function or biological activity as those screened or selected in the initially transformed cells.

[0116] In this application, "medicine" allows the biologically active ingredients contained therein to exist in an effective form and does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the pharmaceutical composition.

[0117] In this application, "prevention and treatment" includes prevention and / or treatment. "Treatment" refers to surgical or therapeutic treatment aimed at preventing, mitigating (reducing) undesirable physiological changes or lesions, such as cancer and tumors, in the treated individual. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, disease severity reduction, disease stability (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of disease status, and remission (whether partial or complete), whether detectable or undetectable. Individuals requiring treatment include those already suffering from the condition or disease, those susceptible to the condition or disease, or those intending to prevent the condition or disease. When terms such as mitigation, reduction, weakening, mitigation, and relief are used, they also include elimination, disappearance, and non-occurrence.

[0118] In this application, "subject" refers to an organism receiving treatment for a specific disease or symptom as described in this invention. Exemplarily, "subject" includes mammals receiving treatment for a disease or symptom, including bovine, equine, sheep, suidae, canine, feline, rodent, and primate animals, with preferred mammals being humans, cats, dogs, or pigs.

[0119] In this application, "cancer" refers to or describes a physiological condition in mammals characterized by unregulated cell growth. This definition includes both benign and malignant cancers. The terms "tumor" or "tumor" herein refer to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used herein.

[0120] In this application, "imaging" refers to the process of converting certain substances or energy into observable or measurable signals to demonstrate their morphology, properties, structure, or distribution. For example, in this invention, after treating samples with antibodies bearing chemical or biological labels, commonly used medical imaging techniques such as X-rays, MRI, CT scans, and ultrasound are used to distinguish cells expressing TNFR2 from other cells, or tissues expressing TNFR2 from other tissues.

[0121] In this application, "ADCC" refers to antibody-dependent cell-mediated cytotoxicity, which means that the Fab fragment of an antibody binds to the antigenic epitope of virus-infected cells or tumor cells, and its Fc fragment binds to the FcR on the surface of killer cells (NK cells, macrophages, etc.), mediating the direct killing of target cells by killer cells.

[0122] In this application, antibody blocking activity refers to the ability of an antibody to bind to a specific antigen, thereby preventing that antigen from binding to its corresponding receptor or ligand. Antibody blocking activity can be detected by various experimental methods, such as competitive binding assays and cell function assays.

[0123] The test materials used in this invention are all commercially available products. The sequence information involved in this invention is as follows:

[0124] Table 1. Antigen and Control Antibody Sequences

[0125]

[0126]

[0127] Table 2. CDR and FR regions of 5G15H-7G2K chimeric antibodies and humanized antibodies

[0128]

[0129]

[0130] Table 35G15H-7G2K Variable Region Sequence After Humanization Modification

[0131]

[0132] Table 4. Variable region sequences of chimeric antibodies (5G15H-7G2K)

[0133]

[0134] Note that the bold black text in the table above indicates the CDR area, and the underlined areas indicate amino acid mutations.

[0135] Heavy chain constant regions of chimeric and humanized antibodies:

[0136] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:37)

[0137] Light chain constant regions of chimeric and humanized antibodies:

[0138] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:38)

[0139] The present invention will be further illustrated below with reference to the embodiments:

[0140] Example 1: Preparation of TNFR2 recombinant overexpressing cell line

[0141] The reading frame of the TNFR2 gene was cloned from a vector containing human TNFR2 cDNA using PCR. The reading frame of the TNFR2 gene (the correct sequence has been verified by sequencing; the nucleotide and amino acid sequences are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively) was cloned into a stable expression vector containing a glutamyl synthase (GS) selection gene by enzyme digestion. The cells were electrotransfected (Nucleofector IIb, Lonza) into HEK293 cells. The transfected cells were placed in DMEM medium (Gibco, Cat:-11960-044) with a pressure of 50 μM MSX (Sigma, Cat: M5379) and seeded in 96-well cell culture plates. The cells were incubated statically at 37°C and 5% CO2 for 2-3 weeks. Single-cell growth wells obtained from the pre-screening under a microscope were selected by MSX pressure screening and scaled up to 24-well cell culture plates. Finally, TNFR2 high-expression clones were selected by flow cytometry (FACS) for further culture and cryopreservation.

[0142] The cell line overexpressing TNFR2 was named HEK293 TNFR2 FL cells. TNFR2 expression in the cell line was identified using flow cytometry with positive antibodies. The TNFR2 positive control antibodies used were all constructed and expressed based on the variable region sequences of antibody molecules in existing TNFR2 patents, and are as follows:

[0143] BioInvent 1H10-IgG (US20220073634A1, VH amino acid sequence SEQ ID NO:3, VH amino acid sequence SEQ ID NO:4).

[0144] Nanjing Weilizhibo's TNFR2 antibody hu32C-IgG (VH / VL amino acid sequences are SEQ ID NO:5 and SEQ ID NO:6, respectively);

[0145] hu3E-IgG (VH / VL amino acid sequences are SEQ ID NO:7 and SEQ ID NO:8 respectively) (WO2021249542A1).

[0146] The results of TNFR2 expression in HEK293 TNFR2 FL cells are as follows: Figure 1 This cell line can specifically overexpress TNFR2 in an antibody concentration-dependent manner.

[0147] Example 2: Animal Immunization and Hybridoma Screening

[0148] Six- to eight-week-old female Balb / c mice were immunized with recombinant human TNFR2 protein in groups of six. Specifically, each mouse received a primary immunization via subcutaneous injection of 50 μg of a mixture of recombinant TNFR2 his protein (Novoprotein, Cat#CI39) and Freund's complete adjuvant (CFA). A total of four immunizations were administered. The subsequent three immunizations were performed using 50 μg, 30 μg, and 25 μg of recombinant TNFR2 his protein mixed with adjuvant (CFA / IFA / no adjuvant), respectively, with each immunization spaced 2-3 weeks apart. Starting with the third immunization, orbital blood was collected one week after immunization. The obtained serum was used for antigen-specific binding and binding blockade tests. Mice with high binding activity and blocking activity were selected for hybridoma fusion.

[0149] Spleens were harvested from two selected mice after booster immunization. The resulting cells were ground and electrofused with mouse myeloma cells SP20. After culturing in HAT and HT media for 10-14 days, the hybridoma supernatant was screened. First, protein binding and blocking screening were performed using ELISA. For human TNFR2 protein binding screening, 1 μg / ml human TNFR2his (Acrobiosystems, Cat#TN2-H5227) protein was coated onto 384-well plates at 40 μL / well. After overnight incubation, washing and blocking, 30 μL of hybridoma supernatant was added and incubated before binding detection. A total of 929 clones (OD650nm>0.5) were screened for further blocking assays. To assess the blocking effect of TNFα-TNFR2 binding, 1 μg / ml human TNFR2 his protein was coated onto 384-well plates at 40 μL / well and incubated overnight. After washing and blocking, 30 μL of hybridoma supernatant was added and incubated for 1 hour. After washing, 100 μL / well of 0.5 μg / ml Biotin TNF-α (Acrobiosystems, Cat#TNA-H8211) was added and incubated at room temperature for 1 hour. Binding assay: After incubation and washing of the hybridoma supernatant, 1:30000 dilution of goat anti-mouse IgG F(ab')2 fragment-specific HRP antibody (Jackson ImmunoResearch, Cat#115-036-146) was added. Blocking assay: After incubation and washing of the hybridoma supernatant, 1:10000 dilution of HRP-Conjugated Streptavidin (Thermo Fisher Scientific, Cat#N100) was added. After washing with PBST, 100 μL of TMB substrate was added to each well to detect antibody binding, and the reaction was terminated with an equal volume of 1N HCl. The OD650 nm reading was measured using a Spectra M5e instrument. Fifty-eight clones with blocking activity (OD650 < 1) were obtained through screening. These 58 positive clones were then subjected to subcloning, and after 7 days of culture, cellular-level binding and protein-level blocking assays were performed, ultimately yielding 20 clones that specifically bound and exhibited blocking activity.

[0150] Example 3: Production and Identification of Human-Mouse Chimeric Antibodies

[0151] (I) Extraction of candidate cloned genes

[0152] 1) Extraction of total RNA and synthesis of cDNA from hybridoma cells

[0153] After culturing monoclonal hybridoma cells to the logarithmic growth phase, cells were collected (approximately 1E6 cells / clone) and utilized... Total RNA was extracted from hybridoma cells using RNAPlus (MN, Cat#740984.250). Using 1 μg of extracted RNA, [the following steps were performed]: III RT SuperMix for qPCR (+gDNAwiper) (Vazyme, Cat#R323-01) was used for cDNA synthesis.

[0154] 2) Gene amplification of antibody VH and VL sequences and T vector cloning

[0155] Using the synthesized cDNA as a template, specific amplification of the cDNA was performed using primers Primer A+S mix (containing universal heavy and light chain primers Primer A, and primers for the constant regions of human hIgG1, 2a, 2b, and 3, as well as specific primers for human kappa) and Ex Taq enzyme (TaKaRa, Cat#RR902A). The PCR reaction system and cycles are as follows. The PCR products were purified using a gel extraction kit after 1% agarose gel electrophoresis. Gel and PCR Clean-up (MN, Cat#740609.250) were used to recover the target fragment.

[0156]

[0157] The recovered fragment was then cloned into the pMD19-T (TaKaRa, Cat#3271) vector using Solution I (TaKaRa, Cat#6022Q). The cloned plasmid was then transformed into competent DH5α cells (Yestern, Cat#FYE607-80VL) using a heat stimulation method and evenly spread on 2YT solid plates containing ampicillin. The plates were then sent to Genewiz sequencing company for sequencing using the universal primer PMAL-C2X-R.

[0158] (II) Construction, expression and purification of candidate cloned human-mouse chimeric expression vectors

[0159] 1) Construction of expression carrier

[0160] Analyze the antibody VH and VL sequencing sequences, select TA clone plasmids with correct antibody sequencing, and amplify the target fragment using the antibody VH / VL universal primer mix (containing the expression vector signal peptide) under the action of the high-fidelity enzyme PrimeSTAR (TaKaRa, Cat#R045) and primers (PCR procedure as follows). After electrophoresis, the target fragment is recovered using a gel recovery kit (MN, Cat#740609.250).

[0161]

[0162]

[0163] The recovered fragments were inserted into the corresponding linearized vectors (pTT5_hIgG1.G1m3 / pTT5_hKappa.Km3) under the action of recombinase (Vazyme, Cat#C112-02); the recombinant vectors were transformed into competent DH5α cells (Yestern, Cat#FYE607-80VL) and evenly spread on 2YT solid plates containing ampicillin. The plates were sent to Genewiz sequencing company for sequencing, and the sequencing primers were pTT5-F and pTT5-R.

[0164] 2) Expression and purification of chimeric antibodies

[0165] Analyze the sequencing sequences, amplify the plasmids with correct sequencing results, and transfect the heavy and light chain plasmids at a 2:3 ratio using PEI reagent (Polysciences, Cat#24885) (1 μg plasmid: 4 μg PEI) into HEK293 cells at a density of 2E6 / ml. Incubate the transfected cells at 37°C in a 5% CO2 incubator for 5–7 days. Centrifuge the culture supernatant and filter it through a 0.22 μm filter. Purify the supernatant using a Mabselect SuRe agarose gel column (GE Healthcare Bio-sciences, 17543802). First, equilibrate the column with 1×PBS (pH 7.4). Load the filtered culture supernatant onto the column, wash the column with 1×PBS (pH 7.4), elute the sample with elution solution (50 mM sodium citrate, pH 2.5), and neutralize the eluted sample with 1 M Tris-HCl, pH 9.0 solution. The neutralized sample was replaced with 1×PBS (pH 7.4), filtered through a 0.22µm filter for sterilization, and the concentration of the purified antibody was determined using Nanoone (Thermo Fisher Scientific Inc).

[0166] (III) Activity identification of human-mouse chimeric antibodies

[0167] 1) The specific binding of anti-human TNFR2 chimeric antibody to human TNFR2 and monkey TNFR2.

[0168] ELISA binding assays were performed using recombinant human TNFR2 mFc protein and monkey TNFR2 his protein. Specifically, 1 μg / ml of human TNFR2 mFc protein (Kaikai Biotechnology, Cat#TNF-HM3R2) and 1 μg / ml of monkey TNFR2 his protein (Kaikai Biotechnology, Cat#TNF-CM1R2) were coated into 96-well plates, 100 μL / well, and incubated overnight at 4°C. The next day, the coated 96-well plates were washed three times with 1×PBST, then blocked with 1% BSA (prepared with 1×PBST) and incubated at 37°C for 1 hour. After incubation, the plates were washed three times with 1×PBST, and serially diluted purified antibody was added and incubated at 37°C for 1 hour. 1H10-IgG and 1F02 were used as positive control antibodies (1F02, BioInvent activating antibody, US20220073634A1, VH amino acid sequence SEQ ID NO:9, VL amino acid sequence SEQ ID NO:10). After incubation and washing, 1:30000 diluted goat anti-human IgG Fcγ fragment specific HRP antibody (Jackson ImmunoResearch, Cat#109-035-098) was added and incubated at 37°C for 1 hour. After washing with PBST, 100 μL of TMB substrate was added to each well to detect antibody binding, and the reaction was terminated with an equal volume of 1N HCl. The results were detected using a Spectra M5e instrument at OD 450 nm.

[0169] The results show ( Figure 2 Except for 26N14 and 14K16, the other eight chimeric antibodies can specifically bind to human and monkey TNFR2 protein.

[0170] 2) Affinity analysis of anti-human TNFR2 chimeric antibodies

[0171] Antibody affinity was assessed by detecting antibody kinetic binding using the Octet Red 96 assay (Forte Bio, Serial No. FB-50482). Specifically, chimeric antibodies were captured using an AHC sensor (Forte Bio, REF Num: 18-5060), followed by loading recombinant human TNFR2 His protein (Acrobiosystems, Cat#TN2-H5227, 2-fold dilution, 200 nM–12.5 nM) or recombinant monkey Cyno TNFR2 His protein (Kaikai Bio, Cat#TNF-CM1R2, 2-fold dilution, 200 nM–12.5 nM). The binding constant (Kon) and dissociation constant (Koff) were analyzed using the Octet Red 96 assay in 1:1 binding mode to determine the equilibrium dissociation constant KD (the ratio of Koff / Kon). The results are shown in Table 5. The chimeric antibodies exhibited different levels of affinity for human-monkey TNFR2. Among them, 5G15, 8P15, and 10D14 showed human TNFR2 affinity levels comparable to the control antibody 1H10-IgG, while antibodies 36B10 and 16H23 showed superior affinity compared to the control antibody 1H10-IgG. The monkey affinity of all tested antibodies was superior to that of the control antibody 1H10-IgG. Furthermore, based on the sequence similarity between 5G15 and 7G2 VH / VL, an antibody combining 5G15 VH and 7G2 VL, 5G15H-7G2K, was constructed. This antibody showed superior human TNFR2 affinity compared to the maternal antibody, and its monkey affinity was also significantly superior to that of the control antibody 1H10-IgG, comparable to that of the control antibodies hu3E-IgG and hu32C-IgG.

[0172] Table 5. Affinity results of chimeric antibodies against human and monkey TNFR2.

[0173]

[0174] Based on the protein binding and affinity analyses of the antibodies, as well as the similarity of the antibody variable region sequences, six antibodies (5G15H-7G2K, 10D14, 8P15, 36B10, 16H23, and 25G12H0L0) were selected for further evaluation.

[0175] 3) Specific binding of anti-human TNFR2 chimeric antibody to human TNFR2 FL cells

[0176] Cellular-level binding assays were performed using HEK293 TNFR2 FL cells with an anti-human TNFR2 chimeric antibody. Specifically, the antibody was diluted 11 times in 3-fold increments, starting at 20 μg / ml. HEK293 TNFR2 FL cells at the growth index stage were collected, and the cell density was adjusted to 2E6 cells / ml. Cells were seeded into 96-well plates at 50 μl / well. Cells were resuspended in serially diluted chimeric antibody at 50 μl / well, and the plates were incubated at 4°C for 1 hour. The positive control antibody, 1H10-IgG, was diluted and incubated at the same level. After incubation, the plates were centrifuged as before, washed three times with PBS, and 100 μl / well of a 1:1000 dilution of goat anti-human Fcγ fragment-specific APC antibody (Jackson ImmunoResearch, 109-135-098) was added. The plates were incubated at 4°C in the dark for 30 minutes. The cells were washed three times with PBS and then resuspended in 80 μL of PBS. Cell flow cytometry readings were performed using iQue Screener PLUS, and the results were analyzed using GraphPad Prism. Figure 3 The results in Table A and Table 6 show that all six antibodies can specifically bind to HEK293TNFR2 FL cells, with activity comparable to the control antibody.

[0177] 4) Blocking activity of anti-human TNFR2 chimeric antibody at the protein level

[0178] The ELISA method was used to evaluate the blocking effect of the chimeric antibody on the binding of human TNFα and human TNFR2 proteins. Specifically, 1 μg / ml human TNFR2 his protein (Acrobiosystems, Cat#TN2-H5227) was coated into 96-well plates at 100 μl / well and incubated overnight at 4°C. The next day, the coated 96-well plates were washed three times with 1×PBST, then blocked with 1% BSA (prepared with PBST at 1°C) and incubated at 37°C for 1 hour; after incubation, the plates were washed three times with 1×PBST, serially diluted purified antibody was added, and the plates were incubated at 37°C for 1 hour; after incubation and washing, 0.5 μg / ml Biotin TNFα (Acrobiosystems, Cat#TNA-H8211) at 100 μl / well was added and incubated at room temperature for 1 hour. After washing, HRP-Conjugated Streptavidin antibody (Thermo Fisher Scientific, Cat#N100) was diluted 1:10000. After washing with PBST, 100 μL of TMB substrate was added to each well to detect antibody binding. The reaction was terminated with an equal volume of 1N HCl. The OD reading at 450 nm was measured using a Spectra M5e instrument. The results were analyzed using non-lin fit analysis with a GraphPad Prism. Figure 3The results in Table B and Table 6 show that all six antibodies can specifically block the binding of human TNFα and human TNFR2 proteins, and their activity is comparable to that of the control antibody.

[0179] 5) Blocking activity of anti-human TNFR2 chimeric antibody at the cellular level

[0180] Cellular-level blocking assays were performed using HEK293 TNFR2 FL cells with an anti-human TNFR2 chimeric antibody. Specifically, the antibody was diluted 11 times in 3-fold increments, starting at 20 μg / ml. Recombinant HEK293 TNFR2 FL cells at the growth index stage were collected, and the cell density was adjusted to 2E6 cells / ml. Cells were seeded into 96-well plates at 50 μl / well. Cells were resuspended in serially diluted chimeric antibody at 50 μl / well, and the plates were incubated at 4°C for 1 hour. The positive control antibody, 1H10-IgG, was diluted and incubated similarly. After incubation, the plates were centrifuged as before, washed three times with PBS, and 0.5 μg / ml Botin TNF-α (50 μl / well) was added and incubated for 1 hour. After washing three times with PBS, APC-Streptavidin antibody (Invitrogen, Cat#SA1005) diluted 1:500 (100 μl / well) was added and incubated at 4°C in the dark for 30 minutes. The cells were washed three times with PBS and then resuspended in 40 μL of PBS. Cell flow cytometry readings were performed using iQue Screener PLUS, and the results were analyzed using GraphPad Prism. Figure 3 The results in Table C and Table 6 show that all six antibodies can specifically inhibit the binding of TNF-α to HEK293TNFR2 FL cells, and the activities of 5G15H-7G2K and 25G12 H0L0 are comparable to those of the control antibody 1H10-IgG.

[0181] 6) Assessment of ADCC activity of anti-human TNFR2 chimeric antibody

[0182] The antibody-dependent cell-mediated cytotoxicity (ADCC) method was used to evaluate the cytotoxic activity of TNFR2 antibody against TNFR2-overexpressing cells. The effector cells were human ADCC effector cells (Jurkat FcγIIIa / NFAT cells, purchased from Jimon Biotechnology). The mechanism of action is that the Fc fragment of the antibody binds to FcγRIIIa on the surface of engineered effector cells, activating the expression of the luciferase reporter gene in the effector cells. After the addition of the substrate, chemiluminescence corresponding to the intensity of ADCC can be detected on a microplate reader. Specifically, target cells HEK293 TNFR2 FL and effector cells human ADCC were collected and their cell densities were adjusted to 1E6 / ml and 6E6 / ml, respectively, using culture medium (RPMI 1640 containing 10% FBS). 25 μl of each cell type (E:T ratio of 6:1) was added to each well of a 96-well plate. Antibody was serially diluted 4-fold at 10 spots, starting at 50 μg / ml, with 25 μl / well. The plates were incubated at 37°C for 17 hours. After incubation, 75 μl of Bio-Glo Luciferase Assay System reagent (Promega, Cat#G7940) was added to each well, and the plates were shaken for 5 minutes. Relative fluorescence unit (RLU) values ​​were then detected using a Spectra M5e instrument. Figure 3 The results in Table D and Table 6 show that the five antibodies exhibited specific ADCC killing activity. The activities of 5G15H-7G2K and 10D14 were comparable to those of the control antibody 1H10-IgG, while the top values ​​of 8P15, 16H23 and 36B10 were slightly better than those of the control antibody.

[0183] Table 6 Summary of chimeric antibody activity results

[0184]

[0185]

[0186] Based on the chimeric antibody’s binding at the cellular level, its blocking activity at the protein and cellular levels, and its ADCC activity, and taking into account the similarity of the antibody variable region sequence, four antibodies (5G15H-7G2K, 10D14, 8P15, 25G12 H0L0) were selected for further evaluation.

[0187] Example 4: Preliminary humanization of anti-TNFR2 antibody and evaluation of antibody activity

[0188] 1) Humanized design and antibody production of anti-human TNFR2 antibodies

[0189] Antibody humanization was performed using a CDR (Cellular Root Deposition) region transplantation method. Specifically, an antibody structural model was established based on the VH / VL sequence (MOE software). The human antibody with the highest homology was selected to provide the antibody framework. The CDR regions from the mouse anti-VH / VL antibody, based on CCG nomenclature, were transplanted into the human antibody framework, forming the humanized antibody variable region sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Simultaneously, based on the established antibody variable region structural model, potential reversion mutations were selected to ensure the activity of the humanized antibody. Reversion mutations were performed primarily based on the following three principles: first, amino acids located at the VH-VL interface in the framework region, close to or directly interacting with CDRs; second, amino acids embedded within the protein were selected for reversion to reduce immunogenicity; and third, mutations that lower molecular energy were selected to ensure stability and expression levels.

[0190] Humanized antibodies are shown in Table 7. The letters H and L represent different backbone regions, with H representing heavy chains and L representing light chains. For example, H0, H1...H8 represent 9 different heavy chains, and L0, L1...L12 represent 13 different light chains.

[0191] By testing the binding of humanized antibodies containing different mutations to cells expressing TNFR2, assessing their affinity for human and monkey TNFR2, and evaluating their ligand receptor binding blocking activity, humanized antibodies with affinity, antibody characterization, and activity comparable to or better than the original antibodies were selected. Based on the sequence similarity of 10D14 VL and 7G2 VL, 10D14 VH and 7G2VL were combined to form the chimeric antibody 10D14H-7G2K, aiming to obtain a molecule with better activity. This antibody was also humanized. The production and purification of the chimeric antibody were carried out according to the method described in Example 3(II)2), for further activity evaluation.

[0192] 2) Preliminary binding and blocking activity of humanized anti-human TNFR2 antibody at the protein level

[0193] ELISA binding and blocking assays were performed using recombinant TNFR2 his protein (Acrobiosystems, Cat#TN2-H5227). Specifically, 1 μg / ml of human TNFR2 his protein was coated into 96-well plates (100 μL / well) and incubated overnight at 4°C. The next day, the coated 96-well plates were washed three times with 1×PBST, then blocked with 1% BSA (prepared with 1×PBST) and incubated at 37°C for 1 hour. After incubation, the plates were washed three times with 1×PBST, and serially diluted purified antibody was added and incubated at 37°C for 1 hour. Binding assay: After antibody incubation and washing, a 1:30000 dilution of goat anti-human IgG Fcγ fragment-specific HRP antibody (Jackson ImmunoResearch, Cat#109-035-098) was added and incubated at 37°C for 1 hour. Blocking assay: After incubation and washing, add 100 μL / well of 0.5 μg / ml Biotin TNF-α (Acrobiosystems, Cat#TNA-H8211) and incubate at room temperature for 1 h; after washing, add 1:10000 diluted HRP-Conjugated Streptavidin (Thermo; Cat#N100) and incubate at 37°C for 1 h. After secondary antibody incubation, wash with PBST and add 100 μL of TMB substrate to each well to detect binding. Terminate the reaction with an equal volume of 1N HCl. OD450 nm readings were measured using a Spectra M5e instrument. Non-lin fit analysis was performed using a GraphPad Prism. Figure 4 , Figure 5 The results in Table 7 show that most humanized antibodies exhibited binding and blocking activities comparable to those of chimeric or control antibodies, with a decrease in blocking activity after humanization of 16H23. Based on the protein-level binding and blocking activity results, humanized antibodies of 10D14, 10D14H-7G2K, 5G15H-7G2K, 8P15, and 25G12 were selected for further cellular-level evaluation.

[0194] 3) Preliminary binding and blocking activity of humanized anti-TNFR2 antibodies at the cellular level

[0195] Cellular-level binding and blocking assays were performed using HEK293 TNFR2 FL cells with an anti-human TNFR2 chimeric antibody. Specifically, the antibody was diluted 11 times in 3-fold increments, starting at 20 μg / ml. Human TNFR2 FL cells at the growth index stage were collected, and the cell density was adjusted to 2E6 cells / ml. Cells were seeded into 96-well plates at 50 μl / well. Cells were resuspended with serially diluted antibody at 50 μl / well, and the plates were incubated at 4°C for 1 hour. Positive control antibodies were 1H10-IgG and hu3E-IgG, which were also diluted and incubated. Binding assessment: After incubation, the cell plates were centrifuged as above, washed three times with PBS, and 100 μl / well of a 1:1000 dilution of goat anti-human Fcγ fragment-specific APC antibody (Jackson ImmunoResearch, 109-135-098) was added. The plates were incubated at 4°C in the dark for 30 minutes. Blockade assessment: After incubation, the cell plates were centrifuged as before, washed three times with PBS, and 0.5 μg / ml BotinTNF-α (50 μL / well) was added and incubated for 1 hour. After washing three times with PBS, APC-Streptavidin antibody (Invitrogen, Cat#SA1005) diluted 1:500 (100 μL / well) was added and incubated at 4°C in the dark for 30 min. After secondary antibody incubation, the cell plates were washed three times with PBS and then resuspended with 40 μL of PBS. Cell flow cytometry readings were performed using iQue Screener PLUS, and the results were analyzed using GraphPad Prism. Figure 6 , Figure 7 The results in Table 7 show that all humanized antibodies specifically bound to HEK293 TNFR2 FL cells overexpressing TNF-α. Among the 8P15 humanized antibodies, only H1L0 maintained inhibitory activity comparable to the control antibody 1H10-IgG, indicating that changes in individual amino acids affected this activity. Other humanized antibodies effectively inhibited the binding of TNF-α to HEK293 TNFR2 FL cells, showing blocking activity essentially equivalent to the control antibody 1H10-IgG. The control antibody hu3E-IgG showed partial blocking activity comparable to the patent results.

[0196] 4) Preliminary ADCC activity of humanized anti-human TNFR2 antibody

[0197] Similar to Example 3(III)6), the ADCC method was used to evaluate the cytotoxic activity of the humanized TNFR2 antibody against TNFR2-overexpressing cells. Figure 8 The results in Table 7 show that the humanized antibodies all exhibited specific ADCC killing activity, which was comparable to that of the control antibody 1H10-IgG.

[0198] Table 7 Summary of preliminary humanized antibody binding, blocking, and ADCC activity assessment results

[0199]

[0200]

[0201]

[0202] NT: Not detected; NA: Not applicable

[0203] 5) Preliminary detection of non-specific binding of humanized anti-human TNFR2 antibody

[0204] TNFR1 (p55, TNFRSF1A) and TNFR2 (p75, TNFRSF1B) are two closely homologous proteins that activate two independent intracellular signaling pathways for gene transcription. TNFR1 is expressed in almost all cells of the body, including the entire lymphatic system. Functionally, TNF primarily relies on TNFR1 for apoptosis and on TNFR2 for functions related to T cell survival. TNFR2 signaling promotes the transcription of pro-survival genes via the nuclear transcription factor NF-κB. Therefore, to avoid the non-specific effects of antibodies, we performed antibody-TNFR1 binding assays at the protein level. Specifically, 1 μg / ml of human TNFR1 his protein (Sino Biological, Cat#10872-H08H) was coated into 96-well plates, 100 μL / well, and incubated overnight at 4°C. The next day, the coated 96-well plates were washed three times with 1×PBST, then blocked with 1% BSA (prepared with 1×PBST) and incubated at 37°C for 1 hour. After incubation, the plates were washed three times with 1×PBST, and antibody was added in 30ug / ml serially diluted 3-fold, and incubated at 37°C for 1 hour. TNFR1 mIgG antibody (sino biological; Cat#10872-MM04) was used as a positive antibody specifically binding to TNFR1, diluted and incubated as above. After antibody incubation and washing, 1:30000 dilution of goat anti-human IgG Fcγ fragment-specific HRP antibody (Jackson ImmunoResearch, Cat#109-035-098) or 1:30000 dilution of goat anti-mouse IgG F(ab')2 fragment-specific HRP antibody (Jackson ImmunoResearch, Cat#115-036-146) was added, and the plates were incubated at 37°C for 1 hour. After incubation with secondary antibody and washing with PBST, 100 μL of TMB substrate was added to each well to detect binding. The reaction was terminated with an equal volume of 1N HCl, and the OD450 nm reading was measured using a Spectra M5e instrument. Non-lin fit analysis was performed using a GraphPad Prism. Figure 9The results showed that none of the humanized antibodies bound to the TNFR1 protein, indicating that these antibodies are TNFR2-specific binding antibodies.

[0205] Example 5: Humanization of anti-TNFR2 antibody, PTM site and activity assessment

[0206] 1) Humanization of anti-human TNFR2 antibody: PTM site removal design and production

[0207] Based on preliminary humanization CDR grafting and reverse mutation, post-translational modification sites (NS, DS, NG, etc.) in the sequence were mutated to reduce the impact of humanization on the physicochemical properties of the antibody and maintain antibody stability. By testing the binding of humanized antibodies with different mutations to cells expressing TNFR2, evaluating ligand receptor binding blocking activity and ADCC activity, and assessing affinity with human and monkey TNFR2, humanized antibodies with affinity, antibody characterization, and activity functions comparable to or better than the unmodified antibodies were selected.

[0208] 2) Binding and blocking activity of humanized anti-human TNFR2 antibodies at the protein level

[0209] The same detection method was used as for the preliminary humanized antibody, namely, ELISA binding and blocking assays were performed using recombinant TNFR2 his protein (Acrobiosystems, Cat#TN2-H5227) and monkey TNFR2 his protein (Kaikai Biotechnology, Cat#TNF-CM1R2). Specifically, 1 μg / ml of human TNFR2 his protein or 1 μg / ml of monkey TNFR2 his protein was coated into a 96-well plate, 100 μL / well, and incubated overnight at 4°C. The next day, the coated 96-well plates were washed three times with 1×PBST, then blocked with 1% BSA (prepared with 1×PBST) and incubated at 37°C for 1 hour. After incubation, the plates were washed three times with 1×PBST, serially diluted purified antibody was added, and the plates were incubated at 37°C for 1 hour. Binding assay: After antibody incubation and washing, 1:30000 diluted goat anti-human IgG Fcγ fragment-specific HRP antibody (Jackson ImmunoResearch, Cat#109-035-098) was added, and the plates were incubated at 37°C for 1 hour. Blocking assay: After incubation and washing, 0.5ug / ml Biotin TNFα (Acrobiosystems, Cat#TNA-H8211) 100ul / well was added, and the plates were incubated at room temperature for 1 hour. After washing, 1:10000 diluted HRP-Conjugated Streptavidin (Thermo Fisher Scientific; Cat#N100) was added, and the plates were incubated at 37°C for 1 hour. After incubation with the secondary antibody, the cells were washed with PBST, and 100 μL of TMB substrate was added to each well to detect binding. The reaction was terminated with an equal volume of 1N HCl, and the OD reading at 450 nm was measured using a Spectra M5e instrument. Non-lin fit analysis was performed using a GraphPad Prism. Figure 10 , Figure 11 The results in Table 8 show that, at the protein level, the humanized antibody exhibited binding and blocking activity comparable to the chimeric antibody or the control antibody 1H10-IgG, while the control antibody hu3E-IgG was a partial blocking antibody. Further evaluation of the humanized antibody at the cellular level is recommended.

[0210] 3) Binding and blocking activity of humanized anti-TNFR2 antibodies at the cellular level

[0211] The binding and blocking of anti-human TNFR2 humanized antibodies against HEK293 TNFR2 FL cells were detected at the cellular level. Specifically, the antibody was diluted 11 times in 3-fold increments, starting at 20 μg / ml. Human TNFR2 FL cells in the growth index stage were collected, and the cell density was adjusted to 2E6 cells / ml. The cells were seeded into 96-well plates at 50 μl / well. The cells were resuspended with serially diluted antibodies at 50 μl / well, and the plates were incubated at 4°C for 1 hour. Positive control antibodies, 1H10-IgG, hu3E-IgG, and hu32C-IgG, were incubated at the same dilution. Binding assessment: After incubation, the cell plates were centrifuged as above, washed three times with PBS, and 100 μl / well of a 1:1000 dilution of goat anti-human Fcγ fragment-specific APC antibody (Jackson ImmunoResearch, Cat#109-135-098) was added. The plates were incubated at 4°C in the dark for 30 minutes. Blockade assessment: After incubation, the cell plates were centrifuged as before, washed three times with PBS, and 0.5 μg / ml Botin TNF-α (50 μL / well) was added and incubated for 1 hour. After washing three times with PBS, 100 μL / well of APC-Streptavidin antibody (Invitrogen, Cat#SA1005) diluted 1:500 was added and incubated at 4°C in the dark for 30 min. After secondary antibody incubation, the cell plates were washed three times with PBS and then resuspended with 40 μL of PBS. Cell flow cytometry readings were performed using iQue Screener PLUS, and the results were analyzed using GraphPad Prism. Figure 12 , Figure 13 The results in Table 8 show that the humanized antibodies can specifically bind to HEK293 TNFR2 FL cells overexpressing the antibody and can effectively inhibit the binding of TNF-α to HEK293 TNFR2 FL cells, showing blocking activity comparable to the control antibodies 1H10-IgG and hu32C-IgG.

[0212] Table 8 summarizes the results of PTM-derived humanized antibody binding, blocking, and ADCC activity assessment.

[0213]

[0214]

[0215] Table 8 (continued)

[0216]

[0217]

[0218] 4) ADCC activity of humanized anti-human TNFR2 antibody

[0219] Similar to Example 3(III)6), the ADCC method was used to evaluate the cytotoxic activity of the humanized TNFR2 antibody against TNFR2-overexpressing cells. Figure 14 The results in Table 4 show that all humanized antibodies exhibited specific ADCC killing activity, which was comparable to that of the control antibody 1H10-IgG.

[0220] 5) Human-monkey affinity assay for anti-human TNFR2 humanized antibody

[0221] Similar to Example 3(III)2) chimeric antibody affinity detection, antibody affinity was assessed by detecting antibody kinetic binding using Octet Red 96 (Forte Bio, Serial No. FB-50482). Specifically, a 10 μg / ml humanized antibody was captured using an AHC sensor (Forte Bio, REF Num: 18-5060), and then recombinant human TNFR2His protein (Acrobiosystems, Cat#TN2-H5227, 2-fold dilution, 200 nM-12.5 nM) or recombinant monkey CynoTNFR2His protein (Kaikai Bio, Cat#TNF-CM1R2, 2-fold dilution, 200 nM-12.5 nM) was loaded for binding-dissociation curve detection, or 100 nM of human TNFR2 protein and monkey TNFR2 protein were directly used for single-point binding-dissociation detection of antibody-protein binding. The Octet Red 96 analysis software was used to analyze the binding constant (Kon) and dissociation constant (Koff) in a 1:1 binding mode, and the equilibrium dissociation constant KD (the ratio of Koff / Kon) was obtained. The results are shown in Table 9. The humanized antibodies showed different levels of affinity for human and monkey TNFR2. Among them, the partially humanized antibodies 10D14H-7G2K and 5G15H-7G2K showed better affinity for human TNFR2, with KD reaching sub nM; the KD for binding to monkey TNFR2 reached the nM level. After subsequent candidate molecules were mass-produced and evaluated, the binding of 5G15H-7G2K H4L3 to human and monkey proteins reached the 10-10 nM level.

[0222] Table 9 Affinity results of humanized antibodies

[0223]

[0224]

[0225] Based on the sequence modification, binding, blocking, ADCC, and affinity results of all the above-mentioned humanized antibodies, 5G15H-7G2KH4L3 and 25G12 H0L12 were selected for subsequent activity evaluation and / or animal experiments.

[0226] Example 6: Effect of anti-TNFR2 antibody 5G15H-7G2K H4L3 on the proliferation of Treg and Teff cells.

[0227] Regulatory T cells (Tregs) are a group of immunosuppressive cells expressing TNFR2. To further evaluate the functional activity of TNFR2 and anti-TNFR2 antibodies on Tregs, the effects of antibodies on the proliferation of Tregs (CD4+CD25+Foxp3+) and T effector cells (Teff, CD4+CD25+Foxp3-) were examined. We assessed the effects of different anti-TNFR2 antibodies on Treg proliferation and Teff amplification in the presence of IL-2 and / or TNFα. CD4 T cells were extracted from peripheral blood mononuclear cells (PBMCs) using a CD4 T cell isolation kit (Stemcell, Cat#17952). Freshly isolated CD4+ T cells were seeded at a density of 2E5 / ml into 96-well round-bottom plates (corning, Cat#3799), 50 μl / well. IL-2 (Tetracycline Biotech) and TNFα (Biolegend, Cat#570104) were added to a final concentration of 200 IU / ml, along with 20 μg / ml anti-TNFR2 antibody, 5G15H-7G2KH4L3, and 25G12 H0L12. Control antibodies included 1H10-IgG (BioInvent, blocking antibody) and 1F02-IgG (BioInvent activating antibody) or different concentrations of anti-TNFR2 antibody. hIgG1 served as a negative control. Results Figure 13 As shown in Figures A and B, antibodies 5G15H-7G2KH4L3 and 25G12H0L12 can both inhibit the proliferation of IL-2-activated Treg cells, and in the presence of TNFα, they can also significantly inhibit the proliferation of Treg cells; correspondingly Figure 15 The expansion of Teff cells in both C and D cells was also correspondingly increased. The control antibody 1H10-IgG inhibited Treg proliferation and promoted Teff cell expansion in both the presence of IL-2 alone and in the presence of both IL-2 and TNFα. The activating control antibody 1F02-IgG promoted Treg proliferation in the presence of IL-2, and this promotion was more pronounced in the presence of both IL-2 and TNFα; correspondingly, Teff cell expansion was inhibited. The activity of the negative control hIgG1 was comparable to that in the presence of both IL-2 and TNFα. Figure 15Treatment with different concentrations of antibodies in antibodies E and F showed similar amplification activity for Tregs and Teffs as described above, and the candidate molecules 5G15H-7G2K H4L3 and 25G12 H0L12 exhibited superior inhibitory activity against Tregs compared to the control antibody 1H10-IgG. These results indicate that the candidate molecules 5G15H-7G2K H4L3 and 25G12 H0L12 can specifically inhibit Treg proliferation and promote Teff amplification in vitro, thereby enhancing the immune response.

[0228] Example 7: PK evaluation of anti-TNFR2 antibody 5G15H-7G2K in mice.

[0229] To preliminarily assess the drug metabolism of antibody drugs in mice, a pharmacokinetic (PK) assay was designed. Six- to eight-week-old female Balb / c mice were used. A single intravenous administration of either 5G15H-7G2KH4L3 antibody or 25G12 H0L12 antibody (200 μg / mouse, 4 mice in total) was administered. Serum samples were collected at 0h, 1h, 4h, 8h, 24h, 48h, 96h, 120h, 144h, and 192h after administration. The drug metabolism in mice was assessed using an ELISA method. Two assays were used: one coated with an antibody-binding antigen to observe the metabolism of the active antibody in mice, and the other coated with an anti-human antibody to observe the metabolism of the human antibody. Theoretically, the blood drug concentrations at the same time point are comparable for both methods, and the calculated T1 / 2 (drug half-life) is consistent. t1 / 2 was calculated using K (elimination rate constant), i.e., t1 / 2 = 0.693 / K. The K value of the drug was obtained from the ELISA results, and the T1 / 2 of the drug was calculated. The results are shown in Table 10. The metabolic trends and T1 / 2 of the 5G15H-7G2K H4L3 antibody evaluated by the two methods were similar. Under the same method, the T1 / 2 of the 25G12 H0L12 antibody was much lower than that of the 5G15H-7G2K H4L3 antibody, meaning that under the same conditions, the 25G12 H0L12 antibody was metabolized faster than the 5G15H-7G2KH4L3 antibody.

[0230] Table 10. PK results of anti-TNFR2 antibodies 5G15H-7G2K H4L3 and 25G12H0L12 in mice.

[0231]

[0232]

[0233] Example 8: Activity assessment of anti-TNFR2 antibody 5G15H-7G2K H4L3 in mice.

[0234] The inhibitory effects of anti-TNFR2 antibody monotherapy and its combination with mPD-1 on tumor growth were evaluated using a BALB / c-hTNFR2 mouse CT26.WT colon cancer cell model. Both BALB / c-hTNFR2 mice and CT26.WT cells were provided by Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. Logarithmic growth phase CT26.WT cells were collected, the culture medium was removed, and the cells were washed twice with DPBS before inoculation at a dose of 1×10⁻⁶. 6 / 100μL / animal. When the average tumor volume reaches 80-100mm. 3 Around [time period missing], mice bearing tumors were randomly divided into 8 groups of 6 mice each, according to the experimental design. The coefficient of variation (CV) for tumor volume was defined as not exceeding 1 / 3. Day 0 (i.e., day 11 post-inoculation) was defined as the day of grouping, and drug administration began on day 0. Dosage volume: The intraperitoneal (ip) volume was calculated as 10 μL / g × mouse body weight (g). The specific dosing regimen is shown in the table below.

[0235] Table 11 Dosing regimens for in vivo animal studies

[0236]

[0237] After drug administration began, tumor volume was measured 2-3 times per week in each group of mice, and mouse weight was measured 2-3 times per week. The formula for calculating tumor volume is: Tumor volume (mm²) 3 = 0.5 × tumor long diameter × tumor short diameter 2 Animals were euthanized at the end of the experiment (21 days after grouping, D21) or when the tumor size exceeded 3000 mm3, and the tumor was removed and weighed. Tumor volume change (TGI) was analyzed at the end of the experiment. TV Tumor weight (TGI) TW (2) Changes in body weight. Results are expressed as mean ± standard error (Mean ± SEM). Independent samples t-tests (T-tests) were used to compare two groups. Data were analyzed using SPSS, and p < 0.05 was considered statistically significant.

[0238] 1) Changes in tumor volume in mice

[0239] Tumor volume changes and data analysis in different groups of mice, as follows Figure 16 As shown in Tables 12 and 13. Based on the statistical analysis of D21 tumor volume data, compared with the control group G1 (hIgG1, 5mpk), the TGI values ​​of the G3 group (5G15H-7G2K H4L3, 2mpk), G4 group (5G15H-7G2K H4L3, 5mpk), G6 group (1H10-IgG, 5mpk), and G7 group (anti-mPD1+5G15H-7G2K H4L3, 1mpk+2mpk) were significantly higher. TVThe values ​​were 53.66%, 73.54%, 77.56%, and 74.85%, respectively. Tumor volume was significantly reduced in all four treatment groups (G3 vs G1, P<0.05; G4 vs G1, P<0.001; G6 vs G1, P<0.001; G7 vs G1, P<0.001). The TGI values ​​for the G2 group (anti-mPD1, 1mpk), G5 group (1H10-IgG, 2mpk), and G8 group (anti-mPD1+1H10-IgG, 1mpk+2mpk) were also significantly reduced. TV The values ​​were -19.43%, 27.63%, and 52.41%, respectively; there was no statistically significant difference in tumor volume among the three treatment groups (P>0.05). Compared with the low-dose 5G15H-7G2K H4L3 group (G3, 2mpk), there was no statistically significant difference in tumor volume after treatment in the high-dose group (G4, 5mpk) (P>0.05); compared with the low-dose 1H10-IgG group (G5, 2mpk), the tumor volume after treatment in the high-dose group (G6, 5mpk) was significantly reduced (P<0.01). These results indicate that anti-TNFR2 antibodies can inhibit tumor growth in a dose-dependent manner. The antibody 5G15H-7G2K H4L3 showed significant antitumor activity at a low dose of 2mpk, which was superior to the same dose of the control antibody 1H10-IgG; the high-dose 5mpk treatment showed extremely significant antitumor activity, comparable to the same dose of the control antibody 1H10-IgG. Moreover, there was no significant difference between the low-dose and high-dose groups of 5G15H-7G2K H4L3 treatment. Figure 16 (Table A and Tables 12 and 13).

[0240] Statistical analysis of D21 tumor volume data showed that, compared with the anti-mPD1 monotherapy group, the tumor volume of the combination therapy groups G7 (anti-mPD1 + 5G15H-7G2K H4L3, 1mpk + 2mpk) or G8 (anti-mPD1 + 1H10-IgG, 1mpk + 2mpk) was significantly reduced. Compared with the monotherapy groups at the same dose, the combination therapy groups showed increased inhibitory activity, but there was no statistically significant difference (G3 vs G7, p>0.05; G5 vs G8, p>0.05). This may be due to the lack of antitumor activity of PD-1 antibody monotherapy in this trial, which is speculated to be related to individual mice and antibody batches. Consistent with the 2mpk monotherapy activity, the 2mpk dose of antibody 5G15H-7G2K H4L3 combined with mPD-1 showed superior antitumor activity compared to the combination of 1H10-IgG + mPD-1. Figure 16 (See Tables B and 12 and 13).

[0241] Table 12 Changes in tumor volume inhibition rate (TGI) in different groups Tv )

[0242]

[0243] Note: 1. TGI Tv All calculations were compared with G1. 2. For comparisons between groups, an independent samples t-test was used: *: P < 0.05; **: P < 0.01; ***: P < 0.001.

[0244] Table 13 Statistical analysis of tumor volume P-value in different groups

[0245]

[0246]

[0247] Note: Independent samples t-tests were used for comparisons between groups. *: P<0.05; **: P<0.01; ***: P<0.001.

[0248] 2) Changes in tumor weight in mice

[0249] Changes in tumor weight in different groups of mice at the end of the experiment are as follows: Figure 17 As shown. Based on the statistical analysis of D21 tumor weight data, compared with the control group G1 (hIgG1, 5mpk), the TGI of the G3 group (5G15H-7G2KH4L3, 2mpk), G4 group (5G15H-7G2KH4L3, 5mpk), G6 group (1H10-IgG, 5mpk), and G7 group (anti-mPD1+5G15H-7G2KH4L3, 1mpk+2mpk) were significantly higher. TW The tumor reduction rates were 50.54%, 76.58%, 77.35%, and 73.10%, respectively; all tumors showed significant reduction (G3 vs G1, P<0.05; G3 vs G1, P<0.01; G3 vs G1, P<0.01; G3 vs G1, P<0.01). The TGI values ​​of the G2 group (anti-mPD1, 1mpk), G5 group (1H10-IgG, 2mpk), and G8 group (anti-mPD1+1H10-IgG, 1mpk+2mpk) were also significantly reduced. TW The percentages were -23.59%, 0.26%, and 45.34%, respectively (P>0.05); there was no statistically significant difference in tumor weight among the three groups.

[0250] Statistical analysis of tumor weight data from D21 showed that, compared with the anti-mPD1 monotherapy group, the tumor weight of the combination therapy groups G7 (anti-mPD1 + 5G15H-7G2K H4L3, 1mpk + 2mpk) or G8 (anti-mPD1 + 1H10-IgG, 1mpk + 2mpk) was significantly reduced (G7 vs G2: P < 0.001, G7 vs G2: P < 0.05). Compared with the low-dose 5G15H-7G2K H4L3 group (G3, 2mpk), there was no statistically significant difference in tumor weight after treatment in the high-dose group (G4, 5mpk) (P > 0.05); compared with the low-dose 1H10-IgG group (G5, 2mpk), the tumor weight after treatment in the high-dose group (G6, 5mpk) was significantly reduced (P < 0.001).

[0251] Changes in tumor weight in mice were consistent with changes in tumor volume. Similarly, 5G15H-7G2K H4L3 showed statistically significant antitumor activity at low doses of 2mpk, and significant antitumor activity at high doses of 5mpk. Furthermore, the combination of 2mpk and mPD-1 showed extremely significant antitumor activity.

[0252] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. TNFR2 antibody or antigen-binding molecule, The three CDR regions of its heavy chain each have an amino acid sequence as shown in SEQ ID NO:11, 12 or 13, or have a sequence based on the amino acid sequence shown in SEQ ID NO:11, 12 or 13 by substitution, deletion, addition and / or replacement of one or more amino acids; or have a sequence with more than 80% homology to the amino acid sequence shown in SEQ ID NO:11, 12 or 13. The three CDR regions of its light chain have amino acid sequences as shown in SEQ ID NO:14, 15 or 16, or have sequences based on the amino acid sequences shown in SEQ ID NO:14, 15 or 16 by substitution, deletion, addition and / or replacement of one or more amino acids; or have sequences with more than 80% homology to the amino acid sequences shown in SEQ ID NO:14, 15 or 16.

2. The TNFR2 antibody or antigen-binding molecule according to claim 1, characterized in that, Its heavy chain variable region has an amino acid sequence as shown in any one of SEQ ID NO:31-32; Its light chain variable region has an amino acid sequence as shown in any one of SEQ ID NO:33-34.

3. The TNFR2 antibody or antigen-binding molecule according to claim 2, characterized in that, The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:31, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:33; Or the amino acid sequence of the heavy chain variable region of the TNFR2 antibody or antigen-binding molecule is as shown in SEQ ID NO:31, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:34; Or the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:32, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:33; Or the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:32, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:

34. Or the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:35, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:

36.

4. The TNFR2 antibody or antigen-binding molecule according to any one of claims 1 to 3, characterized in that, Its heavy chain constant region is any one of human IgG1, IgG2a, IgG2b or IgG3; the light chain constant region is kappa type.

5. A nucleic acid encoding the TNFR2 antibody or antigen-binding molecule as described in any one of claims 1 to 4.

6. A plasmid vector, including a backbone vector and the nucleic acid as described in claim 5.

7. A host cell that is transformed or transfected with the plasmid vector of claim 6, or whose genome is integrated with the nucleic acid of claim 7.

8. A method for preparing the TNFR2 antibody or antigen-binding molecule according to any one of claims 1 to 4, comprising: Culture the host cells as described in claim 7 to obtain a product containing TNFR2 antibody or antigen-binding molecule.

9. The use of the TNFR2 antibody or antigen-binding molecule according to any one of claims 1 to 4, the nucleic acid according to claim 5, the plasmid vector according to claim 6, and / or the host cell according to claim 7 in the preparation of a treatment for TNFR2-related diseases.

10. The application according to claim 9, characterized in that, The TNFR2-related diseases mentioned are tumors that overexpress TNFR2.

11. A drug comprising the TNFR2 antibody or antigen-binding molecule according to any one of claims 1 to 4, the nucleic acid according to claim 5, the plasmid vector according to claim 6, and / or the host cell according to claim 7.

12. The TNFR2 antibody or antigen-binding molecule according to any one of claims 1 to 4, wherein the marker is: a fluorescent indicator, a chemiluminescent indicator, an isotope, a colloidal indicator, biotin, avidin, or an enzyme-labeled substance.

13. A medium coated with the TNFR2 antibody or antigen-binding molecule according to any one of claims 1 to 4; wherein the medium is an enzyme-labeled plate, magnetic beads, or latex microspheres.

14. The use of the TNFR2 antibody or antigen-binding molecule according to any one of claims 1 to 4, the labeled antibody according to claim 12, or the medium according to claim 13 in the preparation of TNFR2 detection reagents and / or TNFR2-related disease diagnostic reagents.

15. A reagent comprising the TNFR2 antibody or antigen-binding molecule according to any one of claims 1 to 4, the labeled antibody according to claim 12, and / or the medium according to claim 13.

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