Cd40 antibodies or antigen-binding fragments and uses thereof

Abstract

The application discloses a CD40 antibody or antigen binding fragment and application thereof. The CD40 antibody or antigen binding fragment has strong binding capacity with hCD40, can activate a CD40 / CD40L signal transduction pathway and downstream regulation signals thereof, can induce human B cell activation and significantly up-regulate CD95 protein expression, can promote B cell proliferation, can induce human PBMC-derived iDC cell activation and secrete a cytokine IL-12. The CD40 antibody or antigen binding fragment does not have ADCC effect, ADCP effect and CDC effect, can significantly reduce tumor volume / weight without affecting mouse body weight, prolong the survival time of mice, has good drug metabolism kinetics, and can be used for detecting hCD40 expression level and treating tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a CD40 antibody or antigen-binding fragment and its application. Background Technology

[0002] CD40 protein, also known as B cell surface antigen 40, is a member of the TNF receptor superfamily 5 (TNFRSF5). It is mainly expressed on the surface of B cells, antigen-presenting cells (APCs), dendritic cells (DCs), macrophages, and monocytes. In addition, it is also widely expressed on the surface of endothelial cells, platelets, and tumor cells. CD40 protein mainly mediates cellular immune responses and inflammatory responses, including T cell-dependent immunoglobulin type switching, and the maturation and development of memory B cells. Early reports indicated two ligands for CD40: heat shock protein 70 (HSP70) (Wang Y, Kelly CG, Karttunen JT, Whittall T, Lehner PJ, Duncan L, et al. CD40 is a cellular receptor mediating mycobacterial heatshock protein 70 stimulation of CC-chemokines. Immunity. 2001; 15(6):971-83.) and C4b-binding protein (C4BP) (Brodeur SR, Angelini F, Bacharier LB, Blom AM, Mizoguchi E, Fujiwara H, et al. C4b-binding protein (C4BP) activates B cells through the CD40 receptor. Immunity. 2003; 18(6):837-48.). Another important ligand for CD40 is CD40L, a 32-39 kDa type II transmembrane protein belonging to the TNF superfamily, primarily expressed on activated CD40. +T cells, activated B cells, and platelets (Elgueta R, Benson MJ, de Vries VC, Wasik A, Guo Y, Noelle RJ. Molecular mechanism and function of CD40 / CD40L engagement in the immune system. Immunological reviews. 2009; 229(1):152-72.) monocytes, NK cells, mast cells, and basophils can be induced to express CD40 under inflammatory stimulation. The secretory soluble form of CD40L mainly activates the CD40 pathway. Because CD40 lacks an intracellular kinase domain, it requires the participation of members of the TNF receptor-associated factor family for intracellular signal transduction. Its various transcriptosomes have significant differences and distinctions, which may be closely related to its function.

[0003] From the perspective of the mechanism of action of CD40-CD40L, blocking the direct binding between the two will block the direct signal transduction between APC cells and T cells, leading to the loss of the second signal for antigen presentation and the autonomous activation and allergic reaction of T cells, manifested as the occurrence of autoimmune diseases. This blocking effect is not only manifested in the failure of antigen presentation but also in the impairment of T cell recognition. Many studies have reported that CD40 is widely expressed on the surface of tumor cells, especially in all B-cell tumors and 70% of solid tumors. The CD40 pathway can exert a dual role in promoting and inhibiting tumor growth, mainly depending on the tumor microenvironment. Animal experiments have shown that when tumor cells highly express CD40L protein, it induces cytotoxic killing and immune rejection in the T cell phase of experimental animals, and the tumor shrinks significantly. In the KPC model study of mouse pancreatic cancer, T cell activation and infiltration were found to be very low, and there was no tumor antigen presentation response. This KPC model has become a good model for evaluating T cell activation. After CD40 antibody treatment, the tumor shrank significantly. In particular, the combination of CD40 antibodies and PD-1 / PD-L1 in the treatment of tumors has achieved better results than the latter alone (Ngiow SF, Young A, Blake SJ, Hill GR, Yagita H, Teng MW, et al. Agonistic CD40 mAb-Driven IL12 Reverses Resistance to Anti-PD1 in aT-cell-Rich Tumor. Cancer Research. 2016; 76(21):6266-77.; Zippelius A, Schreiner J, Herzig P, Muller P. Induced PD-L1 expression mediates acquired resistance to agonistic anti-CD40 treatment. Cancer Immunology Research. 2015; 3(3):236-44.). Furthermore, animal experiments using CTLA4 and CD40 antibodies in combination have also shown very good therapeutic effects. Therefore, CD40 activating antibodies can enhance the anti-tumor effect of immune checkpoint antibodies. On the one hand, CD40 antibodies activate T cell proliferation and differentiation; on the other hand, immune checkpoint antibodies such as PD-1 / PD-L1 and CTLA4 may be immunomodulatory adjuvants for CD40. In studies of combining chemotherapy drugs with CD40 antibodies for tumor treatment, if T cells are defective or absent, or BATF3 is knocked out (an essential protein for DC cell antibody presentation), or the CD40 gene is knocked out, there is no therapeutic effect from the combination. Therefore, B cells and macrophages are essential for the combined use of CD40 antibodies and chemotherapy drugs in tumor treatment.Although the specific mechanisms are still not fully understood, the combination of CD40 antibodies and chemotherapy drugs for the treatment of solid tumors is already in the clinical research stage (O'Hara MH, O'Reilly EM, Mick R, et al. 2019. A phase 1b study of CD40 agonistic monoclonal antibody APX005M together with gemcitabine and nab-paclitaxel with or without nivolumab in untreated metastatic pancreatic ductal adenocarcinoma (PDAC) patients. Paper presented at the American Association for Cancer Research Annual Meeting, Atlanta, GA, Mar. 29–Apr. 3, Abstr. CT004.).

[0004] Currently, clinical trials of CD40 monoclonal antibodies include Roche's Selicrelumab (RO7009789), a fully humanized IgG2 monoclonal antibody; Celldex's CDX-1140, also a fully humanized IgG2 monoclonal antibody; and Apexigen's APX005M (Sotigalimab), a rabbit-derived humanized antibody. Due to Fc region mutations, the cross-linking ability and affinity of each CD40 antibody differ. APX005M and ADC-1013 require FcR cross-linking, while the other antibodies do not. This method of enhancing signal through cross-linking with FcR has been proven feasible in in vitro and in vivo experiments (Dahan R, Barnhart BC, Li F, Yamniuk AP, Korman AJ, Ravetch JV. Therapeutic Activity of Agonistic, Human Anti-CD40 Monoclonal Antibodies Requires Selective FcgammaR Engagement. Cancer Cell. 2016; 29(6):820-31.). This is based on research concerns that CD40 antibodies may have difficulty reaching the target at safe doses, therefore the mechanism of action of CD40 mAb-FcR cross-linking is used to increase the therapeutic effect (Knorr DA, Dahan R, Ravetch JV. Toxicity of an Fc-engineered anti-CD40 antibody is abrogated by intratumoral injection and results in durable antitumor immunity. Proceedings of the National Academy of Sciences. 2018; 115(43):11048-53.). Sotigalimab is a novel humanized monoclonal antibody that stimulates anti-tumor immune responses and targets CD40, a key co-stimulatory receptor that activates both the innate and adaptive immune systems. When Sotigalimab binds to CD40 on antigen-presenting cells, it induces a multifaceted immune response, combining several components of the immune system to attack cancer cells. It is also an Fc-domain mutated humanized IgG1 subtype antibody, requiring cross-linking with FcRs to exhibit enhanced activity and competing for the CD40L binding site.However, based on the results of most clinical trials, no correlation has been found between the presence or absence of FcR crosslinking and the quality of treatment.

[0005] Previous clinical trial results have shown that the toxic side effects of CD40 antibody treatment for tumors are generally mild to moderate. The main toxic side effect is the occurrence of cytokine storm (CRS), which occurs several minutes to several hours after administration, and clinical manifestations include weakness, fatigue, and chills. The occurrence of CD40 antibody-related CRS may be mainly related to the release of IL-6 (Vonderheide RH, Flaherty KT, Khalil M, Stumacher MS, Bajor DL, Hutnick NA, et al. Clinical activity and immune modulation in cancer patients treated with CP-870,893, a novel CD40 agonist monoclonal antibody. Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2007; 25(7):876-83.). Multiple clinical trials have not used hormones to alleviate the response to CRS, except in emergency situations, because studies suggest that CD40-related CRS has a fundamentally different mechanism from CRS caused by CAR-T therapy, resulting in a much milder response. Furthermore, hormone therapy can inhibit the efficacy of CD40 antibody therapy. CD40 antibody treatment can also cause moderate, transient liver dysfunction, grade 1-3. Some studies suggest this may be related to CD40 protein expression in Kupffer cells of the liver, but the exact mechanism remains unclear. In mouse animal experiments, injection of CD40 monoclonal antibodies caused liver cell damage, suggesting that the therapeutic dose window for CD40 antibodies may be too narrow; high doses can easily damage liver function, while low doses are insufficient to achieve therapeutic effects. This is a key point that requires additional attention in the development and clinical application of CD40 monoclonal antibodies.Treatment with CD40 antibodies has also resulted in a transient decrease in platelet count (Vonderheide RH, Flaherty KT, Khalil M, Stumacher MS, Bajor DL, Hutnick NA, et al. Clinical activity and immune modulation in cancer patients treated with CP-870,893, a novel CD40 agonist monoclonal antibody. Journal of Clinical Oncology: official journal of the American Society of Clinical Oncology. 2007; 25(7):876-83.). This decrease in platelet count at CD40 antibody treatment doses is common and unrelated to baseline platelet levels. CD40 monoclonal antibodies do not significantly differ from PD-1 / PD-L1, CTLA4, and other monoclonal antibodies in terms of anti-antibody production; some patients develop anti-antibodies, while others do not. These results are currently difficult to evaluate because clinical trials are still in their early stages, and accurate results are not yet readily available.

[0006] Therefore, there is an urgent need for an antibody or antigen-binding fragment that can effectively bind to CD40. Summary of the Invention

[0007] The first aspect of the present invention is to provide a CD40 antibody or antigen-binding fragment.

[0008] A second aspect of the present invention is to provide a nucleic acid molecule encoding a CD40 antibody or antigen-binding fragment of the first aspect of the present invention.

[0009] A third aspect of the present invention aims to provide gene expression cassettes, recombinant vectors, or transgenic cell lines comprising nucleic acid molecules of the second aspect of the present invention.

[0010] A fourth aspect of the present invention is to provide an immunoconjugate.

[0011] The fifth aspect of this invention aims to provide the application of the CD40 antibody or antigen-binding fragment of the first aspect of this invention, the nucleic acid molecule of the second aspect of this invention, the gene expression cassette of the third aspect of this invention, the recombinant vector or transgenic cell line and / or the immunoconjugate of the fourth aspect of this invention.

[0012] The sixth aspect of this invention aims to provide a pharmaceutical composition.

[0013] The object of the eighth aspect of the present invention is to provide a method for treating tumors.

[0014] To achieve the above objectives, the present invention provides the following technical solution.

[0015] In a first aspect, the present invention provides a CD40 antibody or antigen-binding fragment, said CD40 antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein:

[0016] (1) The amino acid sequence of CDR1 in the heavy chain variable region is as shown in SEQ ID NO: 2, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 2; the amino acid sequence of CDR2 in the heavy chain variable region is as shown in SEQ ID NO: 3, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3; the amino acid sequence of CDR3 in the heavy chain variable region is as shown in SEQ ID NO: 4, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4; and the amino acid sequence of CDR1 in the light chain variable region is as shown in SEQ ID NO: 7, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 7; the amino acid sequence of CDR2 in the light chain variable region is as shown in SEQ ID NO: 8, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; the amino acid sequence of CDR3 in the light chain variable region is as shown in SEQ ID NO: 9, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 9.

[0017] or

[0018] (2) The amino acid sequence of CDR1 in the heavy chain variable region is as shown in SEQ ID NO: 12, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 12; the amino acid sequence of CDR2 in the heavy chain variable region is as shown in SEQ ID NO: 13, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 13; the amino acid sequence of CDR3 in the heavy chain variable region is as shown in SEQ ID NO: 14, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 14; and the amino acid sequence of CDR1 in the light chain variable region is as shown in SEQ ID NO: 17, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 17; the amino acid sequence of CDR2 in the light chain variable region is as shown in SEQ ID NO: 18, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 18; the amino acid sequence of CDR3 in the light chain variable region is as shown in SEQ ID NO: 19, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 19. The amino acid sequence shown in NO:19 has at least 90% sequence identity.

[0019] Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[0020] Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 11, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 11.

[0021] Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 21, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 21.

[0022] Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 23, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 23.

[0023] Preferably, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 6, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 6.

[0024] Preferably, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 16, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 16.

[0025] Preferably, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 22, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 22.

[0026] Preferably, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 24, or has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 24.

[0027] In a second aspect, the present invention provides a nucleic acid molecule encoding a CD40 antibody or antigen-binding fragment of the first aspect of the present invention.

[0028] A third aspect of the present invention provides a gene expression cassette comprising the nucleic acid molecule of the second aspect of the present invention.

[0029] In a fourth aspect, the present invention provides a recombinant vector comprising a nucleic acid molecule of the second aspect of the present invention or a gene expression cassette of the third aspect of the present invention.

[0030] In a fifth aspect, the present invention provides a transgenic cell line comprising a nucleic acid molecule of the second aspect of the present invention, a gene expression cassette of the third aspect of the present invention, or a recombinant vector of the fourth aspect of the present invention.

[0031] Preferably, the transgenic cell line does not include plant or animal varieties.

[0032] A sixth aspect of the present invention provides an immunoconjugate comprising: a CD40 antibody or antigen-binding fragment of the first aspect of the present invention; and a conjugation portion.

[0033] Preferably, the conjugated portion is at least one of a detectable marker, drug, toxin, cytokine, antibody, antibody Fc fragment, antibody scFv fragment, radionuclide, enzyme, gold nanoparticles / nanoran, magnetic nanoparticles, and viral capsid proteins. More preferably, the conjugated portion is a drug, toxin, and / or therapeutic isotope.

[0034] Preferably, the detectable marker is a fluorescent marker or a luminescent marker.

[0035] Preferably, the radionuclide is a diagnostic isotope or a therapeutic isotope.

[0036] Preferably, the diagnostic isotope is at least one selected from Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, and Re-188.

[0037] Preferably, the therapeutic isotope is at least one selected from Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, and Yb-177.

[0038] Preferably, the drug is a cytotoxic drug. More preferably, the cytotoxic drug is at least one of the following: anti-microtubule drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, and vinca alkaloids; further, it is at least one of auristatins, camptothecins, duocarmycins, etoposides, maytansines, and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines, or drugs containing benzodiazepines. (e.g., at least one of pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, oxazolidinobenzodiazepines, and vincaalkaloids).

[0039] A seventh aspect of the present invention provides a detection product comprising a CD40 antibody or antigen-binding fragment of the first aspect of the present invention, or an immunoconjugate of the sixth aspect of the present invention. The detection product may be at least one of a reagent, a detection plate, and a kit.

[0040] Preferably, the testing product is used to test CD40.

[0041] An eighth aspect of the present invention provides a pharmaceutical composition comprising a CD40 antibody or antigen-binding fragment of the first aspect of the present invention, a nucleic acid molecule of the second aspect of the present invention, a gene expression cassette of the third aspect of the present invention, a recombinant vector of the fourth aspect of the present invention, a transgenic cell line of the fifth aspect of the present invention, or an immunoconjugate of the sixth aspect of the present invention.

[0042] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0043] A ninth aspect of the present invention provides the use of the CD40 antibody or antigen-binding fragment of the first aspect of the present invention, or the nucleic acid molecule of the second aspect of the present invention, or the gene expression cassette of the third aspect of the present invention, or the recombinant vector of the fourth aspect of the present invention, or the transgenic cell line of the fifth aspect of the present invention, or the immunoconjugate of the sixth aspect of the present invention in the preparation of a medicament for the treatment and / or prevention of tumors.

[0044] Preferably, the tumor is a solid tumor or a non-solid tumor.

[0045] Preferably, the solid tumor is any one of the following: non-small cell lung cancer, small cell lung cancer, colorectal cancer, melanoma, breast cancer, esophageal cancer, gastric tumor, bladder cancer, endometrial cancer, head and neck cancer, and kidney cancer. More preferably, the tumor is colorectal cancer or breast cancer.

[0046] Preferably, the non-solid tumor is a hematologic malignancy.

[0047] The tenth aspect of the present invention provides the use of the CD40 antibody or antigen-binding fragment of the first aspect of the present invention, or the nucleic acid molecule of the second aspect of the present invention, or the gene expression cassette of the third aspect of the present invention, or the recombinant vector of the fourth aspect of the present invention, or the transgenic cell line of the fifth aspect of the present invention, or the immunoconjugate of the sixth aspect of the present invention in the preparation of a reagent for detecting CD40.

[0048] Preferably, the CD40 is a human CD40 or a monkey CD40. More preferably, the CD40 is a human CD40.

[0049] The eleventh aspect of the present invention provides the use of the CD40 antibody or antigen-binding fragment of the first aspect of the present invention, or the nucleic acid molecule of the second aspect of the present invention, or the gene expression cassette of the third aspect of the present invention, or the recombinant vector of the fourth aspect of the present invention, or the transgenic cell line of the fifth aspect of the present invention, or the immunoconjugate of the sixth aspect of the present invention in the preparation of a drug for blocking the CD40L-CD40 pathway.

[0050] The twelfth aspect of the present invention provides a CD40 antibody or antigen-binding fragment of the first aspect of the present invention, or a nucleic acid molecule of the second aspect of the present invention, or a gene expression cassette of the third aspect of the present invention, or a recombinant vector of the fourth aspect of the present invention, or a transgenic cell line of the fifth aspect of the present invention, or an immunoconjugate of the sixth aspect of the present invention, for the preparation of a diagnostic tool for CD40-related diseases.

[0051] According to a thirteenth aspect of the present invention, a method for producing a protein is provided, comprising the following steps:

[0052] (1) Cultivate the transgenic cell line according to claim 13, so that the transgenic cell line expresses the CD40 antibody or antigen-binding fragment according to any one of claims 1 to 9;

[0053] (2) Recover the expressed protein.

[0054] In a fourteenth aspect of the present invention, a method for treating tumors is provided, comprising administering to a patient an effective amount of the CD40 antibody or antigen-binding fragment of the first aspect of the present invention, or a nucleic acid molecule of the second aspect of the present invention, or a gene expression cassette of the third aspect of the present invention, or a recombinant vector of the fourth aspect of the present invention, or a transgenic cell line of the fifth aspect of the present invention, or an immunoconjugate of the sixth aspect of the present invention.

[0055] The beneficial effects of this invention are as follows:

[0056] This invention provides a CD40 antibody or antigen-binding fragment that strongly binds to hCD40, activating the CD40 / CD40L signaling pathway and its downstream regulatory signals. This induces human B cell activation and significantly upregulates CD95 protein expression, promoting B cell proliferation and inducing activation of human PBMC-derived iDC cells, leading to the secretion of the cytokine IL-12. This antibody does not exhibit ADCC, ADCP, or CDC effects. It significantly reduces tumor volume / weight in colon cancer MC38 cell line-bearing mice without affecting mouse body weight (i.e., without side effects), prolonging mouse survival time. Furthermore, it possesses good pharmacokinetic characteristics and can be used to detect hCD40 expression levels and treat tumors. Attached Figure Description

[0057] Figure 1 This is a graph showing the binding activity of hCD40 antibodies with the hCD40-his antigen in Example 3. In Figure A, the binding activity of murine hCD40 antibodies m36F11 and m72F10 with the hCD40-his antigen is shown; in Figure B, the binding activity of humanized hCD40 antibodies 36F11-H4L2 and 72F10-H3L3 with the hCD40-his antigen is shown.

[0058] Figure 2 This is a graph showing the binding activity of hCD40 antibodies with hCD40 on the surface of 293F-hCD40 cells in Example 3. Specifically, A shows the binding activity of murine hCD40 antibodies m36F11 and m72F10 with hCD40 on the surface of 293F-hCD40 cells; B shows the binding activity of humanized hCD40 antibodies 36F11-H4L2 and 72F10-H3L3 with hCD40 on the surface of 293F-hCD40 cells.

[0059] Figure 3 This is a graph showing the competitive activity of hCD40 antibodies and CD40L protein in binding to hCD40 on the surface of 293F-hCD40 cells in Example 4. In Figure A, the competitive activity of hCD40 murine antibodies m36F11 and m72F10 in binding to hCD40 on the surface of 293F-hCD40 cells with CD40L protein is shown; Figure B shows the competitive activity of hCD40 humanized antibodies 36F11-H4L2 and 72F10-H3L3 in binding to hCD40 on the surface of 293F-hCD40 cells with CD40L protein is shown.

[0060] Figure 4 This is a graph showing the binding activity of hCD40 antibodies with monkey and mouse CD40 proteins in Example 5. Specifically, A shows the binding activity of hCD40 mouse antibodies m36F11 and m72F10 with mouse CD40 protein; B shows the binding activity of hCD40 humanized antibodies 36F11-H4L2 and 72F10-H3L3 with mouse CD40 protein; C shows the binding activity of hCD40 mouse antibodies m36F11 and m72F10 with monkey CD40 protein; and D shows the binding activity of hCD40 humanized antibodies 36F11-H4L2 and 72F10-H3L3 with monkey CD40 protein.

[0061] Figure 5 This is a graph showing the detection results of the activation ability of hCD40 antibodies against the CD40 / CD40L signal transduction pathway in Example 6. In this graph, A shows the detection results of the activation ability of hCD40 murine antibodies m36F11 and m72F10 against the CD40 / CD40L signal transduction pathway; B shows the detection results of the activation ability of hCD40 humanized antibodies 36F11-H4L2 and 72F10-H3L3 against the CD40 / CD40L signal transduction pathway.

[0062] Figure 6This is a graph showing the results of hCD40 antibody-induced B cell activation and CD95 protein expression in Example 7. In this graph, A is a bar chart showing the results of hCD40 murine antibodies m36F11 and m72F10 inducing B cell activation and CD95 protein expression; B is a flow cytometry bar chart showing the results of hCD40 humanized antibodies 36F11-H4L2 and 72F10-H3L3 inducing B cell activation and CD95 protein expression.

[0063] Figure 7 This is a graph showing the results of the hCD40 humanized antibody on B cell proliferation in Example 8. The results are the flow cytometry results of hCD40 humanized antibodies 36F11-H4L2 and 72F10-H3L3 on B cell proliferation converted into a bar graph.

[0064] Figure 8 This is a diagram showing the results of hCD40 antibody activating and inducing IL-12 secretion in PBMC-derived iDCs in Example 9. Specifically, A shows the results of hCD40 murine antibodies m36F11 and m72F10 activating and inducing IL-12 secretion in PBMC-derived iDCs; B shows the results of hCD40 humanized antibodies 36F11-H4L2 and 72F10-H3L3 activating and inducing IL-12 secretion in PBMC-derived iDCs.

[0065] Figure 9 This refers to the ADCC effect of the hCD40 humanized antibodies 36F11-H4L2 and 72F10-H3L3 in Example 10.

[0066] Figure 10 This refers to the ADCP effect of the hCD40 humanized antibodies 36F11-H4L2 and 72F10-H3L3 in Example 11.

[0067] Figure 11 This is the CDC effect of the humanized hCD40 antibodies 36F11-H4L2 and 72F10-H3L3 in Example 12. In this diagram, A shows the CDC effect of the humanized hCD40 antibodies 36F11-H4L2 and 72F10-H3L3 against target cells 293T-hCD40; B shows the CDC effect of the humanized hCD40 antibodies 36F11-H4L2 and 72F10-H3L3 against target cells ramos.

[0068] Figure 12The results show the affinity of the hCD40 humanized antibodies for CD32a and CD32b in Example 13. Figure A shows the affinity of hCD40 humanized antibodies 36F11-H4L2 and 72F10-H3L3 for CD32a; Figure B shows the affinity of hCD40 humanized antibodies 36F11-H4L2 and 72F10-H3L3 for CD32b.

[0069] Figure 13 This describes the activation effect of the humanized hCD40 antibody on human platelets in Example 14. The results are flow cytometry results of the activation effect of the humanized hCD40 antibodies 36F11-H4L2 and 72F10-H3L3 on human platelets, converted into bar chart results.

[0070] Figure 14 This is a graph showing the effects of hCD40 antibodies 36F11-H4L2 and 72F10-H3L3 on MC-38 tumor-bearing C57BL / 6-hCD40 transgenic mice in Example 15: A and B are the effects of hCD40 humanized antibodies 36F11-H4L2 and 72F10-H3L3 on tumor volume in MC-38 tumor-bearing C57BL / 6-hCD40 mice, respectively; C and D are survival curves of MC-38 tumor-bearing mice treated with hCD40 humanized antibodies; E and F are the effects of hCD40 humanized antibodies 36F11-H4L2 and 72F10-H3L3 on body weight in mice; G and H are the effects of hCD40 humanized antibodies on tumor volume in tumor-bearing mice.

[0071] Figure 15 This is a pharmacokinetic diagram of hCD40 antibodies 36F11-H4L2 and 72F10-H3L3 in Example 16. Detailed Implementation

[0072] The present invention will be further described in detail below through specific embodiments.

[0073] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0074] Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, the materials and reagents used in each example are commercially available. The nomenclature and techniques described herein for cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization are well-known and commonly used in the art. Unless otherwise specified, the methods and techniques of the present invention are generally performed according to conventional methods well-known in the art. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions or as is commonly known in the art. Standard techniques are used for chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and patient treatment.

[0075] Unless otherwise defined herein, scientific and technical terms used in connection with this invention shall have meanings commonly understood by one of ordinary skill in the art. The meanings and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definitions.

[0076] As used herein, "antibody" refers to a type of glycosyl globulin synthesized and secreted by B cells after being stimulated by an antigen to differentiate and proliferate into plasma cells. Antibodies mainly include five types: IgG, IgM, IgA, IgD, and IgE. This invention preferably uses IgG antibodies.

[0077] The basic structure of an immunoglobulin (Ig) molecule consists of four peptide chains: two identical, smaller peptide chains (light chains) and two identical, larger peptide chains (heavy chains). The light and heavy chains are linked by disulfide bonds, as are the two heavy chains. Each heavy chain and each light chain includes an N-terminal variable region (V region) and a C-terminal constant region (C region). The variable region is divided into a hypervariable region (HVR) and a backbone region (FR). The hypervariable region is the binding site between the antibody and the antigen, and is called the "complementarity determining region" (CDR). The HVR1, HVR2, and HVR3 of VL and VH are respectively called CDR1, CDR2, and CDR3.

[0078] As used in this article, "antibody" refers to a polypeptide fragment that can recognize and bind to a specific antigen. "CD40 antibody" refers to an antibody that can recognize and target CD40.

[0079] As used herein, an "antigen-binding fragment" refers to a portion of a full-length antibody, wherein said portion of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of the heavy chain or a variable domain of the light chain). Non-limiting examples of antibody fragments include, for example, Fab, Fab', F(ab')2, and Fv fragments.

[0080] As used herein, a "monoclonal antibody (mAb)" is a highly homogeneous antibody produced by cloning a single B cell (whose genes encode only one type of antibody) that targets only a specific antigenic epitope. Antibodies can be, for example, murine, chimeric, or humanized monoclonal antibodies.

[0081] As used in this article, “humanized antibody” refers to an antibody that specifically binds to a relevant antigen (such as human CD40) and contains a framework region with an amino acid sequence of human antibody and a complementarity-determining region with an amino acid sequence of non-human antibody.

[0082] As used herein, “recycling” refers to the process of separating a chemical substance, such as a peptide, using protein purification techniques well known in the art, so that the substance is essentially free of its naturally bound components.

[0083] Example 1 - Preparation of human CD40 mouse antibody

[0084] The specific steps for producing human CD40 mouse monoclonal antibodies using hybridoma technology are as follows:

[0085] Balb / c mice (purchased from Guangdong Yaokang Biotechnology Co., Ltd.) were immunized with the extracellular fragment sequence of hCD40 (NP_001241.1) as an antigen. The immunized mice were immunized four times with the purified antigen and either complete or incomplete Freund's adjuvant. The intensity of the immune response was assessed after blood was collected via the tail vein. Serum titers were screened by ELISA and flow cytometry to obtain mice with anti-human CD40 immunoglobulin. Spleen cells from mice with the highest anti-CD40 immunoglobulin levels were then fused with mouse myeloma cells SP2 / 0 (ATCC code CRL-1581).

[0086] The fused hybridoma cells were screened for antibodies to obtain human CD40 mouse antibodies: m36F11 and m72F10.

[0087] Sequencing was performed on human CD40 murine antibodies: m36F11 and m72F10.

[0088] The amino acid sequence of the heavy chain variable region of m36F11 is shown in SEQ ID NO: 1:

[0089] QVQAKESGPGLVAPSQSLSITCTVS GFSLIDNG VTWIRQPPGKGLEWLGV IWGGGST YYKAALESRLSISKDNSKSQVFLRMNSLQTEDTAMYYC VKHDNWAY WGQGTLVTVSA

[0090] The underlined parts are CDR1 (SEQ ID NO: 2), CDR2 (SEQ ID NO: 3), and CDR3 (SEQ ID NO: 4) in sequence.

[0091] The nucleotide sequence encoding the heavy chain variable region of m36F11, as shown in SEQ ID NO: 1, is shown in SEQ ID NO: 5.

[0092] CAGGTGCAGGCGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACAGTCTCAGGGTTCTCATTAATCGACAATGGTGTAACCTGGATTCGCCAGCCTCCAGGGAAGGGTCTGGAGTGGCTGGGAGTAATCTGGGGTGGTGGAAGCACA TACTATAAAAGCAGCTCTCGAATCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTGAGAATGAACAGTCTGCAAACTGAAGACACAGCCATGTACTACTGTGTCAAACATGACAACTGGGCCTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0093] The amino acid sequence of the light chain variable region of m36F11 is shown in SEQ ID NO: 6:

[0094] EIVLTQSPTTMAASPGEKITITCSAS SSISSNY LHWYQQKPGFSPKLLIY RTS NLASGVPARFSGSGSGTSHSLTIGTMEAEDVATYYC QQGSSMPYT FGGGTKLEMKRADA

[0095] The underlined parts are CDR1 (SEQ ID NO: 7), CDR2 (SEQ ID NO: 8), and CDR3 (SEQ ID NO: 9) in sequence.

[0096] The nucleotide sequence encoding the light chain variable region of m36F11, as shown in SEQ ID NO: 6, is shown in SEQ ID NO: 10 below:

[0097] GAAATTGTGCTCACTCAGTCTCCAACCACCATGGCTGCATCTCCCGGGGAGAAGATCACTATCACCTGCAGTGCCAGCTCAAGTATAAGTTCCAATTACTTGCATTGGTATCAGCAGAAGCCAGGATTCTCCCCTAAACTCTTGATTTATAGGACATCCAATCTGGCT TCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTCACTCTCTCACAATTGGCACCATGGAGGCTGAAGATGTTGCCACTTACTACTGCCAGCAGGGTAGCAGTATGCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATGAAACGGGCTGATGCT

[0098] The amino acid sequence of the heavy chain variable region of m72F10 is shown in SEQ ID NO: 11 below:

[0099] QVQAQQSGAELVRPGVSVKISCKGS GYTFSDYA MHWVKQSHAKSLEWIGV ISSYYGDD SYNQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYC ARSFPSTKGYGMDY WGQGTSVTVSS

[0100] The underlined parts are CDR1 (SEQ ID NO: 12), CDR2 (SEQ ID NO: 13), and CDR3 (SEQ ID NO: 14) in sequence.

[0101] The nucleotide sequence encoding the heavy chain variable region of m72F10, as shown in SEQ ID NO: 11, is shown in SEQ ID NO: 15 below:

[0102] CAGGTCCAAGCGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGTCTCAGTGAAGATTTCCTGCAAGGGTTCTGGCTACACATTCTCTGATTATGCTATGCACTGGGTGAAGCAGAGTCATGCAAAGAGTCTAGAGTGGATTGGAGTTATTAGTTCTTACTATGGTGATGATAGCTACA ACCAGAAGTTCAAGGGCAAGGCCACAATGACTGTAGACAAATCCTCCAGTACAGCCTATATGGAACTTGCCAGACTGACATCTGAGGATTCTGCCATCTATTACTGTGCAAGATCCTTCCCCTCGACGAAGGGGTATGGTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

[0103] The amino acid sequence of the light chain variable region of m72F10 is shown in SEQ ID NO: 16 below:

[0104] DIQGAQTTSSLSASLGDRVTISCRAS QDISNY LNWFQQRPDGTVKLLIY FTS RLHSGAPSRFSGSGSGTEYSLTISNLDQEDIATYFC QQGKTLPFT FGGGTKLEIKRADA

[0105] The underlined parts are CDR1 (SEQ ID NO: 17), CDR2 (SEQ ID NO: 18), and CDR3 (SEQ ID NO: 19) in sequence.

[0106] The nucleotide sequence encoding the light chain variable region of m72F10, as shown in SEQ ID NO: 16, is as follows: SEQ ID NO: 20:

[0107] GATATCCAAGGTGCACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAAACTGGTTTCAGCAGAGACCAGATGGAACTGTTAAGCTCCTGATCTACTTCACATCAAGATTACACT CAGGAGCCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGAATATTCTCTCACCATTAGCAACCTGGACCAAGAAGATATTGCCACTTACTTTTGCCACAGGGTAAAACGCTTCCGTTCACGTTCGGAGGGGGGACCAAACTGGAAATAAAACGGGCTGATGCT

[0108] Example 2 - Preparation of humanized CD40 antibody

[0109] Referring to the light chain variable region and heavy chain variable region sequences of the m36F11 and m72F10 antibodies, respectively, human antibody frameworks whose sequences are closest to their murine counterparts were selected. The CDR regions were then transplanted, and nucleotide sequences were synthesized for humanized antibody expression. The murine antibody CDR regions were transplanted onto the selected humanized templates, replacing the CDR regions of the human templates. Then, based on the three-dimensional structure of the murine antibodies, reverse mutations were performed on embedded residues, residues that directly interact with the CDR regions, and residues that significantly affect the conformation of VL and VH, yielding the humanized antibodies 36F11-H4L2 and 72F10-H3L3.

[0110] The sequence of the heavy chain variable region of humanized antibody 36F11-H4L2 is as follows (SEQ ID NO: 21):

[0111] QVQLQESGPGLVKPSQTLSLTCTVSGFSLIDNGVTWIRQPPGKGLEWIGVIWGGGSTYYNPSLKSRVTISVDTSKNQVSLKLSSVTAADTAVYYCVKHDNWAYWGQGTTVTVSS

[0112] The sequence of the light chain variable region of humanized antibody 36F11-H4L2 is as follows (SEQ ID NO: 22):

[0113] DIQMTQSPSSSLSASVGDRVTITCRASSSISSNYLHWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGSSMPYTFGGGTKLEIKRTVA

[0114] The sequence of the heavy chain variable region of the humanized antibody 72F10-H3L3 is as follows (SEQ ID NO: 23):

[0115] QVQLVQSGAEVKKPGASVKVSCKGSGYTFSDYAMHWVRQAPGQRLEWMGVISSYYGDDSYSQKFQGRVTITVDTSASTAYMELSSLRSEDTAVYYCARSFPSTKGYGMDYWGQGTTVTVSS

[0116] The sequence of the light chain variable region of the humanized antibody 72F10-H3L3 is as follows (SEQ ID NO: 24):

[0117] DIQMTQSPSSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYFTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGKTLPFTFGGGTKLEIKRTVA.

[0118] The heavy chain constant region sequence of the humanized antibody 36F10-H4L2 is as follows (SEQ ID NO: 25).

[0119] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0120] The light chain constant region sequence of humanized antibody 36F10-H4L2 is as follows (SEQ ID NO: 26).

[0121] APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0122] The heavy chain constant region sequence of the humanized antibody 72F10-H3L3 is as follows (SEQ ID NO: 27).

[0123] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0124] The light chain constant region sequence of the humanized antibody 72F10-H3L3 is as follows (SEQ ID NO: 28).

[0125] APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0126] Using gene synthesis and molecular cloning techniques, the nucleic acid sequences encoding the heavy or light chain of the aforementioned antibodies were inserted into the expression vector pTT5 (purchased from Addgene), respectively, to obtain recombinant plasmids that could be used to express the complete antibody. The corresponding heavy and light chain plasmids were co-transfected into CHO-k1 cells using the PEI Max transfection vector (purchased from Sigma-Aldrich) for transient expression. The transfected cells were then cultured in shake flasks for 7 days. After culture, the cell culture supernatant was harvested, and the purified antibody was obtained using antibody purification methods.

[0127] Example 3 - Binding activity of hCD40 antibody to hCD40

[0128] The binding activity of hCD40 mouse antibodies m36F11 and m72F10, humanized antibodies 36F11-H4L2 and 72F10-H3L3, and positive control PcAb to hCD40-His protein was detected by ELISA. The positive control PcAb was Sotigalimab (after sequence synthesis, it was expressed and purified according to existing literature guidelines (antibody expression and purification methods are described in the following references: Longo PA, Kavran JM, Kim MS, Leahy DJ. Transient mammalian cell transfection with polyethylenimine (PEI). Methods in enzymology. 2013; 529:227-40.; Andrew SM, Titus JA. Purification of immunoglobulin G. Current protocols in immunology. 2001; Chapter 2: Unit 2 7.)).

[0129] The hCD40-his protein was diluted to 0.25 μg / mL with CBS buffer and coated into microplates at 50 μL / well, incubated overnight at 4°C. The plates were washed three times with PBST buffer (200 μL / well) and blotted dry. Then, 1% BSA (dissolved in PBS) was added at 200 μL / well, and the plates were incubated at 37°C for 1 hour to block. After incubation, the plates were washed three times with PBST (200 μL / well) and blotted dry. The positive control PcAb (Sotigalimab) and hCD40 antibodies m36F11, m72F10, 36F11-H4L2, and 72F10-H3L3 were diluted to 3 μg / mL with PBST. These were serially diluted in columns 1 to 11 of the dilution plate using a 3-fold dilution method, resulting in 11 groups. Group 12 was treated with 1×PBST as a blank control. Transfer 100 μL of the solution to a microplate using a multipipeline and incubate at 37°C for 1 hour. After incubation, wash the plate three times with PBST (200 μL / well) and blot dry. Dilute the HRP-labeled anti-mouse IgG antibody or anti-human IgG antibody (1:5000 dilution) with 1% BSA (dissolved in PBST), 100 μL / well. Incubate at 37°C for 1 hour; after incubation, wash the plate three times with PBST (200 μL / well) and blot dry. Add 50 μL of TMB chromogenic solution to each well for colorimetric reaction, and incubate in the dark for 5 minutes. After 5 minutes of incubation, add 50 μL of hydrochloric acid stop solution to stop the reaction. Measure the absorbance (OD450) at 450 nm using the absorbance mode of a microplate reader.

[0130] ELISA test results as follows Figure 1 As shown. By Figure 1 As shown in Figure A on the left, the murine hCD40 antibodies m36F11 (EC50 = 0.005098 μg / mL) and m72F10 (EC50 = 0.01262 μg / mL) exhibit strong binding affinity to the hCD40-His protein. Both the obtained murine antibodies m36F11 (EC50 = 0.005098 μg / mL) and m72F10 (EC50 = 0.01262 μg / mL) demonstrate strong binding activity against hCD40. Figure 1 As shown in Figure B on the right, the humanized hCD40 antibodies 36F11-H4L2 and 72F10-H3L3 also have a strong binding ability to the hCD40-His protein. The binding activities of 36F11-H4L2 (EC50 = 0.002523 μg / mL) and 72F10-H3L3 (EC50 = 0.001233 μg / mL) are similar to those of the positive control antibody (EC50 = 0.001594 μg / mL).

[0131] The binding activity of antibodies to cell surface hCD40 was detected by flow cytometry.

[0132] The binding affinity of antibodies to hCD40 on the cell surface was detected using 293F-hCD40 cells (product of Beijing Kangyuan Bochuang Co., Ltd., #KC-0222).

[0133] The 293F-hCD40 cell line was digested, terminated, centrifuged, resuspended, and counted to adjust the cell density to 4 × 10⁻⁶ cells / year. 6 Count / mL, at 50μL / tube, take 20×10 4 Cells were added to 1.5 mL EP tubes. Prepared hCD40 mouse antibodies m36F11, m72F10, 36F11-H4L2, 72F10-H3L3, or positive control PcAb were added. hCD40 antibodies and PcAb were diluted to an initial concentration of 60 μg / mL, and serially diluted 1:3 to seven concentrations. 50 μL of each diluted antibody was added to the corresponding tube. For the blank control, 50 μL of PBS was added. The cells were gently mixed by pipetting and incubated on ice for 1 h. After incubation, each cell was washed once with 500 μL of PBS by centrifugation at 1000 rpm for 5 min, and the supernatant was discarded. Add anti-mouse fluorescent secondary antibody or anti-human fluorescent secondary antibody (divide Alexa 488 fluorescently labeled anti-mouse IgG antibody or anti-human IgG antibody with PBS at a volume ratio of 1:500, add 200 μL / tube to the corresponding tube), gently pipette to mix, and incubate on ice in the dark for 30 min. After 30 min of incubation, detect the signal using flow cytometry. Analyze the binding activity of hCD40 antibody or positive control sample PcAb to obtain EC50 values. Detect the surface hCD40 binding activity of hCD40 antibody and positive control PcAb on 293F-hCD40 cells using flow cytometry (FCM).

[0134] FCM test results are as follows Figure 2 As shown. By Figure 2 As shown in Figure A on the left, the murine hCD40 antibodies m36F11 (EC50 = 2.782 μg / mL) and m72F10 (EC50 = 3.023 μg / mL), along with the positive control PcAb (EC50 = 3.092 μg / mL), exhibit strong binding affinity to hCD40 on the cell surface. The binding activities of the murine antibodies m36F11 and m72F10 are comparable to those of the positive control. Figure 2 As shown in Figure B on the right, the humanized hCD40 antibodies 36F11-H4L2 (EC50 = 1.027 μg / mL) and 72F10-H3L3 (EC50 = 1.077 μg / mL) and the positive control PcAb (EC50 = 1.053 μg / mL) have a strong binding ability to hCD40 on the cell surface. The binding activities of the humanized antibodies 36F11-H4L2 and 72F10-H3L3 are comparable to those of the positive control.

[0135] Example 4 - Competitive binding activity of hCD40 antibody and ligand

[0136] The 293F-hCD40 cell line was digested, terminated, centrifuged, resuspended, and counted. 20 × 10⁶ cells were collected. 4 Cells were added to 1.5 mL EP tubes, 50 μL / tube. hCD40 mouse antibodies m36F11 and m72F10, or humanized antibodies 36F11-H4L2 and 72F10-H3L3, or PcAb were diluted to an initial concentration of 75 μg / mL, and seven concentrations were obtained using a 1:3 serial dilution method, with 50 μL added to each corresponding tube. For the control group and rhCD40L-His group, 50 μL of PBS was added, and the mixture was gently pipetted to mix. The tubes were then incubated on ice for 15 min. After 15 min, prepared hCD40L-hFc protein (20 μg / mL) was added to each experimental group: 10 μL / tube for the control group and 10 μL of PBS for the blank group. The mixture was gently pipetted to mix, and the tubes were incubated on ice for another 1 h. After incubation, each tube was washed once with 500 μL of PBS by centrifugation at 1000 rpm for 5 min, and the supernatant was discarded. Dilute the His-tag AF647-labeled antibody with PBS (1:2400). Add 200 μL / tube to the corresponding tube, gently pipette to mix, and incubate on ice in the dark for 30 min. After 30 min of incubation, detect the signal using flow cytometry.

[0137] Figure 3 The results show the competitive activity of hCD40 antibody against CD40L protein in binding to hCD40 on the surface of 293F-hCD40 cells. The antagonistic activity results of the mouse antibodies m36F11 and m72F10 show that both antibodies have similar antagonistic activity to the positive control antibody. Figure 3 As shown in Figure A, the competitive activity of each antibody exhibits a concentration-dependent relationship. The IC50 values ​​for m36F11, m72F10, and PcAb are 3.632 μg / mL, 3.163 μg / mL, and 3.522 μg / mL, respectively. Figure 3 As shown in Figure B, the competitive activity of each humanized antibody exhibits a concentration-dependent relationship. The IC50 values ​​for 36F11-H4L2, 72F10-H3L3, and PcAb are 3.854 μg / mL, 9.288 μg / mL, and 8.508 μg / mL, respectively.

[0138] Example 5 - Cross-binding activity of hCD40 antibody with monkey and mouse CD40 proteins

[0139] The binding activities of hCD40 murine antibodies m36F11, m72F10, humanized antibodies 36F11-H4L2, 72F10-H3L3, and positive control PcAb with rhesus monkey (accession number: NP_001252791.1) or mouse CD40 protein (accession number: NP_035741.2) were analyzed by indirect ELISA, using the same method as in Example 3.

[0140] Based on the homology of CD40 protein sequences in humans, monkeys, and mice, human CD40 proteins show high homology with monkey CD40 proteins, while their homology with mouse CD40 proteins is low.

[0141] The results are as follows Figure 4 As shown. By Figure 4 As shown in A, the hCD40 murine antibodies m36F11 and m72F10, and the positive control PcAb, all exhibit very weak binding affinity to murine CD40 protein, and can be considered as having no affinity. Figure 4 As shown in Figure B, the humanized hCD40 antibodies 36F11-H4L2 and 72F10-H3L3 exhibit very weak binding activity to mouse CD40 protein, weaker than the binding activity of the reference antibody PcAb to mouse CD40 protein. Figure 4 As shown in Figure C, the hCD40 murine antibodies m36F11, m72F10, and the positive control PcAb all exhibited binding activity to monkey CD40 protein. m36F11 and PcAb showed higher binding activity to monkey CD40 protein, while m72F10 showed weaker affinity. The EC50 values ​​of m36F11, m72F10, and PcAb for monkey CD40 were 0.005024 μg / mL, 0.01737 μg / mL, and 0.001111 μg / mL, respectively. Figure 4 As shown in D, the binding activity of the humanized antibody 36F11-H4L2 to monkey CD40 protein was slightly weaker than that of the reference control PcAb to monkey CD40 protein, with EC50 values ​​of 0.01054 μg / mL and 0.003951 μg / mL, respectively; the binding activity of the 72F10-H3L3 antibody to monkey CD40 protein was also weak, with an EC50 of 0.8881 μg / mL.

[0142] Example 6: The ability of the hCD40 antibody to activate the CD40-NFκB-Luciferase reporter gene pathway.

[0143] 293T-hCD40 cells were recombined with the CD40-NFκB-Luciferase reporter gene vector. The activation ability of candidate hCD40 antibodies on the hCD40 protein-related signaling pathway in 293T-hCD40 cells was detected by Luciferase assay. The stronger the fluorescence intensity detected in the experiment, the stronger the activation ability of the hCD40 mouse or human antibody on the hCD40 protein-related signaling pathway.

[0144] The 293T-hCD40 cell line was digested, terminated, centrifuged, resuspended, and counted. 2.0 × 10^4 cells / 50 μL / well were added to 96-well plates. Prepared CD40 antibody was added. CD40 candidate murine antibody or candidate derivatized antibody and reference control pcAb were all serially diluted 1:3 from an initial concentration of 30 μg / mL. The dilution medium was DMEM complete medium (containing 10% FBS). DMEM complete medium was added to the blank control group. 50 μL / well was added to the corresponding well. Each sample was tested in duplicate, with each well containing P / S double antibody (1:100). The 96-well plates were incubated at 37°C in a 5% CO2 cell culture incubator for 6 h. After incubation, the 96-well plates were removed and incubated at room temperature for 30 min. Then, 50 μL / well of luciferase assay reagent, equilibrated to room temperature, was added and incubated at room temperature for approximately 3 min. The chemiluminescence signal was detected using the luminescence mode of a multi-mode microplate reader. Use GraphPadPrism 5 data processing software to process the data and create a bar chart.

[0145] Experimental results are as follows Figure 5 As shown. By Figure 5 As shown in A, the activation abilities of hCD40 murine antibodies m36F11 and m72F10 on the hCD40 protein-related signaling pathway are comparable to those of the reference control PcAb. Figure 5 As shown in B, the humanized antibody 72F10-H3L3 has the strongest activation ability on the hCD40 protein-related signaling pathway, while 36F11-H4L2 and PcAb have comparable activation abilities on the hCD40 protein-related signaling pathway.

[0146] Example 7 - hCD40 antibody induces CD95 expression in Ramos cells

[0147] Upregulation of CD95 protein in B cells is one of the important markers of CD40 signaling pathway activation.

[0148] In this embodiment, the ability of hCD40 antibody to induce the expression of CD95 in Ramos cells (B cell-derived tumor cells) was detected by flow cytometry (FCM), thereby determining the activation ability of candidate antibodies on the CD40 signaling pathway.

[0149] The experimental method is briefly described below:

[0150] Ramos cells in good growth condition were digested, terminated, centrifuged, resuspended, and counted. Cells were added to 24-well plates at a concentration of 1.0 × 10⁵ cells / 400 μL / well. Prepared hCD40 antibody was added. hCD40 candidate murine antibody or candidate-derived antibody and reference control PcAb were all diluted starting at an initial concentration of 4.5 μg / mL, followed by three 1:3 serial dilutions. The dilution medium was 1640 complete medium (containing 10% FBS). For the control group, 100 μL of 1640 complete medium was added to each well. Each well contained P / S double antibody (1:100). The 24-well plates were incubated at 37°C in a 5% CO₂ cell culture incubator for 16 h. After incubation, cells were collected into corresponding 1.5 mL EP tubes, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. PE-Cy5-labeled CD95 antibody (1:50 dilution) was added to each tube at a concentration of 50 μL / tube. Incubate at 4°C in the dark for approximately 40 minutes. After incubation, add 200 μL of PBS to each tube. Detect signals using a flow cytometer. Process the data using FlowJo and GraphPad Prism 5 software and generate bar charts.

[0151] Experimental results are as follows Figure 6 As shown. By Figure 6 As shown in Figure A, the hCD40 murine antibodies m36F11 and m72F10 exhibit strong induction activity against CD95 expression, and this induction is concentration-dependent. Figure 6 As shown in B, the humanized hCD40 antibodies 36F11-H4L2 and 72F10-H3L3 also exhibit strong induction activity for CD95 expression, and this induction is concentration-dependent.

[0152] Example 8 - hCD40 antibody induces B cell activation and proliferation

[0153] CD19 is one of the most reliable surface biomarkers of B cells. It is expressed from precursor B cells until differentiation into plasma cells, at which point expression is lost. The density of CD19 on the B cell membrane is highly regulated during B cell development and maturation, with its expression level in mature B cells being three times higher than that in immature B cells. Activation of antigen-presenting cells such as B cells can further promote the activation of anti-tumor T cells, thereby enhancing the body's ability to clear tumor cells.

[0154] In this embodiment, the expression level of CD19 on B cells was detected in vitro by flow cytometry (FCM) to reflect the ability of candidate hCD40 humanized antibody to induce B cell activation, thereby determining the anti-tumor ability of the candidate antibody.

[0155] The experimental method is briefly described below:

[0156] First, B cell purification was performed. PBMCs were collected and purified using the Dynabeads Untouched Human B Cells Kit according to the manufacturer's instructions. B cells were then resuspended in 1640 complete medium (containing 10% FBS) and counted. Cells were seeded into 24-well plates at a concentration of 3.0 × 10^5 cells / 450 μL / well. The corresponding prepared antibodies were added: hCD40 antibody, negative control IgG2, and reference controls PcAb and PcAb2, all diluted starting at a concentration of 150 μg / mL, with four concentration gradients at a 1:3 ratio. Only one concentration of 150 μg / mL was prepared for the control IgG2, and the first three concentration gradients for the reference control PcAb2 were prepared. 1640 complete medium was used as the diluent. 50 μL of each cell culture medium was added to the wells. For the CD19- group, 1640 complete medium was added. Each well contained a 1:100 dilution of the penicillin antibody P / S. The 24-well plates were returned to a 37°C, 5% CO2 cell culture incubator and cultured for 48 h. After culture, cells were collected into corresponding 1.5 mL EP tubes, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. For each tube (except the CD19- group), 50 μL of CD19-APC (1:50) solution was added to the cell pellet, and the cells were resuspended and mixed. For the CD19- group, 50 μL of IgG1-APC (1:100) solution was added, and the cells were resuspended and mixed. The cells were incubated at 4°C in the dark for 30 min. After incubation, 1 mL of PBS was added, mixed, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. Cells were resuspended in 150 μL of PBS and analyzed using flow cytometry. Data were processed and plotted using FlowJo software.

[0157] Statistical results are as follows Figure 7 As shown. By Figure 7 This is the result of converting flow cytometry scatter plots into a bar chart. The results show that, compared with IgG2 control, 36F11-H4L2, 72F10-H3L3, and PcAb all effectively induced and activated B cells, with the order of their activation ability being: 72F10-H3L3 > 36F11-H4L2 > PcAb. PcAb2, being a CD40 antagonist, inhibited the induction and activation of B cells.

[0158] Example 9 - hCD40 antibody induces PBMC-derived iDCs to secrete IL-12 cytokine.

[0159] CD40 activates dendritic cells (DCs) to promote the secretion of key stimulating factors and adoptive immune responses. IL-12 secreted by activated DCs is an important factor in the activation of CD8+ T cells, and IL-12 is considered one of the indicators of the response to CD40 agonist antibody therapy.

[0160] The experimental method is briefly described below:

[0161] Collect adherent PBMCs and count them. Seed the cells in 24-well plates at approximately 5.0 × 10⁵ cells / 450 μL / well. Add 50 μL / well of rhGM-CSF (final concentration 100 ng / mL) and rhIL-4 (final concentration 50 ng / mL) prepared in 1640 complete medium (containing 10% FBS). Incubate the 24-well plates at 37°C in a 5% CO₂ incubator for 3–4 days. After incubation, tilt the plate and aspirate 50% of the supernatant from each well. Add an equal volume of fresh 1640 medium (containing 200 ng / mL rhGM-CSF and 100 ng / mL rhIL-4). Return the 24-well plates to the 37°C, 5% CO₂ incubator and incubate for another 2 days. After incubation, tilt the plate and aspirate 50% of the supernatant from each well. Add the prepared corresponding antibodies: hCD40 antibody or reference antibody PcAb, diluted starting at a concentration of 150 μg / mL, and serially diluted 1:3. The dilution buffer is 1640 complete medium. For the blank control group, add 1640 complete medium. Add 50 μL / well to each well. Finally, top up each well with 1640 complete medium to a final volume of 500 μL / well, ensuring each well contains the penicillin antibody P / S (1:100). Incubate the 24-well plate at 37°C in a 5% CO2 cell culture incubator for 48 h. After 48 h, remove the 24-well plate and transfer the supernatant to a 1.5 mL EP tube. Centrifuge at 1000 rpm for 5 min and collect the supernatant. Detect human cytokine IL-12p40 according to the Human IL-12 / IL-23p40 precoated ELISA kit instructions. Measure the absorbance at 450 nm (OD450) using a microplate reader. The expression levels of human IL-12p40 in iDCs stimulated with control standard PcAB and CD40 antibody were plotted and analyzed using GraphPad Prism5 data processing software.

[0162] Experimental results are as follows Figure 8 As shown. By Figure 8 As shown in Figure A, the murine antibodies m36F11 and m72F10 were less effective than the reference antibody PcAb in inducing iDCs to secrete IL-12p40. Figure 8As shown in B, the humanized antibody 36F11-H4L2 induced iDCs to secrete IL-12p40 significantly better than the reference antibody PcAb and 72F10-H3L3, while 72F10-H3L3 was weaker than the reference antibody PcAb.

[0163] Example 10: ADCC effect of hCD40 antibody

[0164] The chemiluminescent reporter gene approach was used to mimic antibody-dependent cell-mediated killing (ADCC) effects of T cells or NK cells. This was achieved by constructing Jurkat cells that exogenously express the FcγRIIIA (CD16A) receptor and its downstream NFAT transcription factor, along with the binding domain of this transcription factor and a luciferase reporter gene capable of transcription. When the constructed Jurkat-NFAT-Luc2-CD16a cells were co-incubated with 293F-hCD40 expressing hCD40, the antibody activated the CD16a signaling pathway in the Jurkat-NFAT-Luc2-CD16a cells, activating the downstream transcription factor NFAT for gene transcription. Luciferase, as a recombinant NFAT regulatory gene, was regulated by the NFAT transcription factor.

[0165] The experimental design uses the positive control antibody Rituximab to bind to the CD20 protein on the surface of Ramos cells, and the reference antibody PcAb and the detection antibody to bind to the CD40 protein, thereby detecting the ADCC effect of the antibody.

[0166] The experimental method is briefly described below:

[0167] First, prepare Ramos cells and Jurkat-NFAT-Luc2-CD16a-V158 cells. Collect and count the cells, adjusting the cell density to 5.0 × 10^5 cells / mL. Mix the two cell lines at a 1:1 ratio and seed each well in a 96-well plate (opaque, unfiltered) at 50 μL / well, resulting in 2.5 × 10^4 cells / well for each cell line. Add the corresponding prepared antibodies: hCD40 antibody, control antibody PcAb, and rituximab, all starting at a concentration of 30 μg / mL and serially diluted 1:3. The dilution medium is 1640 complete medium (containing 10% FBS). For the control group, add 1640 complete medium. Add 50 μL / well to each 96-well plate. Two replicates are used for each sample. Each well contains a 1:100 dilution of the penicillin antibody P / S. Incubate the cells at 37°C in a 5% CO2 cell culture incubator for 6 hours. After 6 hours of incubation, the 96-well white plate was removed and equilibrated at room temperature for 15 minutes. Then, 50 μL of luciferase assay reagent, equilibrated to room temperature, was added to each well, and the plate was incubated at room temperature for approximately 3 minutes. The chemiluminescence signal was detected using a microplate reader in luminescence mode. Four-parameter curves of the fluorescence signals of the reference control Sotigalimab and the CD40 candidate antibody were obtained using data processing software, thus yielding the EC50 value of antigen-antibody-dependent cell killing.

[0168] The results are as follows Figure 9 As shown, because the Fc domain of the candidate antibodies was engineered to lack ADCC function, the candidate-derived antibodies 36F11-H4L2 and 72F10-H3L3 did not mediate the ADCC effect in cells; similarly, the reference control PcAb was also an antibody with an Fc domain modified to lack ADCC effect. The CD20 antibody rituximab showed a significant ADCC effect, consistent with the latter's characteristics.

[0169] Example 11 - ADCP effect of hCD40 humanized antibody

[0170] Jurkat cells recombinantly express human FcγRIIA (CD32a) protein and integrate luciferase, a promoter of the NFAT transcription factor binding sequence, as a reporter gene. This reporter gene cell system can mimic the antibody-dependent phagocytic signal transduction pathway, thereby reflecting antibody-dependent phagocytosis. Recombinant Jurkat-NFAT-Luc2-CD32a-R167 cells (from Kangyuan Bocheng Biotechnology (Beijing) Co., Ltd., catalog number: KC-1524) were co-incubated with Ramos cells expressing hCD40. Under the mediation of the antibody, the antibody activated the signal transduction pathway of Jurkat-NFAT-Luc2-CD32a-R167 cells, demonstrating the antibody-dependent phagocytic effect of monocytes or macrophages on target cells.

[0171] The experimental method is briefly described below:

[0172] First, prepare Ramos cells and Jurkat-NFAT-Luc2-CD32a-R167 cells. Collect and count the cells, adjusting the cell density to 5.0 × 10^5 cells / mL. Mix the two cell lines at a 1:1 ratio and seed each well in a 96-well plate (opaque, unfiltered) at 50 μL / well, resulting in 2.5 × 10^4 cells / well for each cell line. Add the corresponding prepared antibodies: hCD40 antibody, control antibody PcAb, and rituximab, all starting at a concentration of 30 μg / mL and serially diluted 11 times at a 1:3 ratio. The dilution medium is 1640 complete medium (containing 10% FBS). For the control group, add 1640 complete medium. Add 50 μL of each antibody to a 96-well plate. Perform two replicates per sample. Each well contains a 1:100 dilution of the penicillin antibody P / S. Incubate the cells at 37°C in a 5% CO2 cell culture incubator for 6 hours. After 6 hours of incubation, the 96-well white plate was removed and equilibrated at room temperature for 15 minutes. Then, 50 μL of luciferase assay reagent, equilibrated to room temperature, was added to each well, and the plate was incubated at room temperature for approximately 3 minutes. The chemiluminescence signal was detected using a microplate reader in luminescence mode. Four-parameter curves of the fluorescence signals of the reference control Sotigalimab and the CD40 candidate antibody were obtained using data processing software, thus yielding the antibody-dependent phagocytic EC50 value.

[0173] Experimental results are as follows Figure 10 As shown, the candidate hCD40 humanized antibodies 36F11-H4L2 and 72F10-H3L3 and the control antibody PcAb did not show significant ADCP effects; the CD20 antibody rituximab showed significant ADCP effects, consistent with the characteristics of the latter antibody.

[0174] Example 12 - CDC effect of hCD40 antibody

[0175] The disease-causing-disruption (CDC) effect is an important mechanism by which complement and antibodies participate in tumor killing. Specific antibodies bind to corresponding antigens on the cell membrane to form a complex, activating complement in the blood and thus mediating the CDC effect, leading to the lysis of target cells. 293T-hCD40 is a recombinant cell line that highly expresses hCD4, while Ramos cells naturally express hCD40, with the latter showing a significantly lower hCD40 expression level. This experiment used cell lines with different expression levels to detect the CDC effect of hCD40 antibodies.

[0176] The experimental method is briefly described below:

[0177] 293T-CD40 cells and Ramos cells were collected separately and counted using DMEM complete medium (containing 5% FBS) and 1640 complete medium (containing 5% FBS), respectively. Cell density was adjusted, and cells were seeded in 96-well plates at a density of 2.0 × 10^4 cells / 40 μL / well. The corresponding prepared antibodies—hCD40 antibody, control antibody PcAb, and rituximab—were added, all starting at a concentration of 30 μg / mL and serially diluted 1:3 in DMEM complete medium (containing 5% FBS) (for 293T-CD40 cells) and 1640 complete medium (containing 5% FBS) (for Ramos cells), respectively. Negative control groups were prepared using DMEM complete medium (containing 5% FBS) and 1640 complete medium (containing 5% FBS), respectively. Antibody groups, negative control groups, and high-dose control groups were added to 96-well plates at 50 μL / well. Each sample was tested in duplicate. Each well contained a double antibiotic P / S (1:100). Cells were then incubated at 37°C in a 5% CO2 incubator for 0.5 h. After 0.5 h incubation, 10 μL of fresh human serum was added to each well, and the cells were incubated again at 37°C in a 5% CO2 incubator for 3 h. After 2.5 h incubation, the high-control group received 10 μL of Lysis Buffer from the Cytotoxicity LDH Assay Kit per well and was incubated at 37°C in a 5% CO2 incubator for 0.5 h. After 0.5 h incubation, 100 μL of the prepared Working Solution from the Cytotoxicity LDH Assay Kit was added to each well, and the reaction was carried out at room temperature in the dark for 30 min. Finally, 50 μL of the Stop Solution from the Cytotoxicity LDH Assay Kit was added to each well, and the absorbance at 490 nm (OD490) was immediately measured using a microplate reader in absorbance mode. The LDH levels mediated by the negative control group, high reference group, reference control group, and CD40 candidate antibody were analyzed using GraphPad Prism 5 data processing software.

[0178] Experimental results are as follows Figure 11 As shown. By Figure 11 As shown in Figure A, the reference antibody PcAb, the humanized antibodies 36F11-H4L2 and 72F10-H3L3 all exhibited significant CDC-killing effects on 293T-hCD40 cells with high hCD40 expression. Meanwhile, the... Figure 11As shown in Figure B, Ramos cells with low hCD40 expression exhibit a very weak CDC effect. Rituximab, an antibody against CD20 protein and used as a control group, does not express CD20 on the surface of 293T-hCD40 cells, thus failing to produce a CDC effect. However, Ramos cells significantly express CD20 protein, resulting in a very strong CDC effect.

[0179] The experimental results show that the degree of CDC effect of the candidate antibody is closely related to the abundance of hCD40 expression in the target cells. Generally speaking, the endogenous expression level of hCD40 in normal tissue cells is not too high. Therefore, it can be inferred that the CDC killing effect caused by the candidate antibody has little toxic side effect on normal tissue cells.

[0180] Example 13: Affinity of hCD40 antibody to FcγRII A (CD32a) and FcγRII B (CD32b)

[0181] The difference in affinity of hCD40 antibodies for CD32a and CD32b plays a crucial role in the cross-linking reaction of the antibody Fc fragment. Generally, binding of the antibody Fc fragment to CD32a can activate FcγR; while CD32b is an inhibitory receptor, and binding to CD32b can inhibit FcγR-mediated activation. The affinity for CD32a is closely related to antibody-mediated macrophage ADCP, while the affinity for CD32b is closely related to the enhancement of cell signal transduction and cell function activity mediated by antibody cross-linking.

[0182] Flow cytometry (FCM) was used to detect the binding activity of hCD40 antibody, positive control PcAb, Rituximab, and IgG2 antibody against CD32a or CD32b on the surface of Jurkat-NFAT-Luc2-CD32a-R167 cells (from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd., catalog number: KC-1524) or 293F-CD32b cells. The 293F-CD32b construction method was as follows: The full-length CD32b gene sequence was designed and constructed into the pCDH-CMV-PURO blank vector. Sequence synthesis and cloning were performed by Guangzhou Aiji Biotechnology Co., Ltd., obtaining the pCDH-CD32b-PURO target vector. The pCDH-CD32b-PURO vector was transfected into 293F cells using liposomes (jetPRIME, Polysciences), and the cells were cultured for 48 hours. After 48 hours of culture, Puromycin antibiotic was added to the cells to a concentration of 2.5 μg / mL, and the cells were cultured for another 5 days. Monoclonal screening was performed on the resulting drug-resistant cell clones. Flow cytometry was then used to identify cell lines that highly expressed CD32b protein.

[0183] Jurkat-NFAT-Luc2-CD32a-R167 cells (or 293F-CD32b cells) were digested, terminated, centrifuged, resuspended, and counted. 20 × 10⁻⁶ cells were collected. 4 Cells were added to 1.5 mL EP tubes, 50 μL / tube. The antibody was diluted to an initial concentration of 36 μg / mL, and six dilutions were obtained using a 1:3 serial dilution method. 50 μL of each diluted antibody was added to the corresponding tube, gently pipetted to mix, and incubated on ice for 30 min. After 30 min, 100 μL of the prepared goat anti-human fluorescent secondary antibody (diluted 1:500, v / v) was added to each experimental group, for a total of 200 μL / tube. The mixture was gently pipetted to mix, and the tubes were incubated on ice in the dark for 30 min. After 30 min of incubation, the signal was detected using flow cytometry.

[0184] Figure 12 The affinity results for hCD40 humanized antibodies against CD32a and CD32b are shown. Figure 12 As shown in Figure A, the hCD40 antibodies 36F11-H4L2 and 72F10-H3L3 exhibit very low affinity and biological activity for CD32a, significantly lower than those of the IgG1 subtype antibody. Figure 12 As shown in section B, the candidate antibodies 36F11-H4L2 and 72F10-H3L3 of the enhanced IgG1 subtype exhibit slightly stronger affinity for CD32b than antibodies of the ordinary IgG1 subtype, and the reference antibody PcAb also shows stronger binding activity to CD32b than the ordinary IgG1 antibody. Antibodies of the IgG2 subtype show very weak binding activity to CD32b, consistent with the characteristics of this subtype.

[0185] The results show that hCD40 antibodies 36F11-H4L2 and 72F10-H3L3 have a strong ability to enhance cellular functional activity after cross-linking with CD32b, but lack ADCP activity (phagocytic activity) on CD40-expressing cells.

[0186] Example 14 - Side effects of hCD40 antibody on human platelet activation

[0187] hCD40 protein is expressed on the surface of platelets. Some hCD40 antibodies can activate platelets, causing platelet aggregation, which can lead to adverse effects such as vascular thrombosis.

[0188] This embodiment evaluates whether hCD40 antibody can activate platelets.

[0189] The experimental method is briefly described below:

[0190] Platelet-rich supernatant was obtained using the Ficoll lymphocyte separation method. PBS was set as the blank control group, TRAP (a platelet activation reagent) as the positive control group, and individual stimulation with each hCD40 antibody, or stimulation with a mixture of each antibody and CD40L-hFc (at a fixed concentration of 5 μg / mL), were used as experimental groups. All groups were incubated at room temperature for 30 min. Platelet activation and aggregation were detected by flow cytometry. Data processing and graphing were performed using FlowJo and GraphPad Prism 5 software.

[0191] Experimental results are as follows Figure 13 As shown in the figure. The results are converted from flow cytometry analysis results into bar charts. The results show that hCD40 antibodies 36F11-H4L2, 72F10-H3L3, and the PBS negative control group did not show any ability to activate platelets; the positive control group showed significant platelet activation and the ability to initiate aggregation; in addition, CD40L-hFc showed the ability to activate platelets. The hCD40 antibody and CD40L-hFc mixed stimulation group, 36F11-H4L2, and 72F10-H3L3 all inhibited CD40L-hFc-induced platelet activation.

[0192] Example 15: The therapeutic effect of 6-hCD40 antibody on C57BL / 6-hCD40 transgenic mice bearing colon cancer MC-38 cell line.

[0193] Healthy C57BL / 6J murine colon cancer cell line MC38 was subcutaneously inoculated into 8-12 week old adult C57BL / 6-hCD40 mice. Once tumor volume reached the target level, mice were randomly assigned to nine groups: a control group (PBS), a reference control antibody PcAb group (Sotigalimab), and humanized hCD40 antibody groups (36F11-H4L2 and 72F10-H3L3). The PcAb groups received doses of 0.2 mpk and 6 mpk, while the hCD40 humanized antibody groups received doses of 0.2 mpk, 3 mpk, and 6 mpk. Mice were then administered the drug intraperitoneally. Mouse weight and tumor volume were continuously monitored until the experimental endpoint. After sacrifice, tumors were dissected, photographed, and weighed. Drug efficacy (tumor size)-drug concentration curves, mouse weight change curves, and survival curves were plotted.

[0194] Experimental results are as follows Figure 14 As shown. By Figure 14As shown in A and B, the control group PcAb (0.3 mpk and 6 mpk), the humanized hCD40 antibody group (36F11-H4L2, 0.3 mpk, 2 mpk and 6 mpk, and 72F10-H3L3, 0.3 mpk, 2 mpk and 6 mpk) all inhibited tumor growth. The TGI of the control group was 56.2% and 64%, respectively; the TGI of the 36F11-H4L2 group was 54%, 70.8%, and 46.1%, respectively; and the TGI of the 72F10-H3L3 group was 61.5%, 53.9%, and 71.5%, respectively.

[0195] When the experimental endpoint is reached or the tumor volume exceeds 3000 mm, 3 The mice were euthanized and survival curves were plotted.

[0196] like Figure 14 As shown in C and D, the reference control group (0.3 mpk), the candidate CD40 humanized antibody group (36F11-H4L22 mpk, 72F10-H3L32 mpk, and 6 mpk) significantly delayed tumor growth.

[0197] Statistical analysis of the weight change data of mice in each group, such as Figure 14 As shown in E and F, compared with the blank control group, there was no significant difference in the change of body weight of mice in each drug administration group and the difference in change between the groups was small and there was no statistical difference.

[0198] After the experiment reached the treatment endpoint, the mouse tumor tissue was separated and weighed, such as... Figure 14 As shown in G and H, the results showed that the tumor weight of the reference control group 6mpk, the hCD40 humanized antibody group 36F11-H4L22mpk and 72F10-H3L32mpk showed a decreasing trend (tumor weight TGI was 16.88%, 20.22% and 29.41% respectively, indicating that each therapeutic antibody had a good effect on inhibiting tumor growth).

[0199] Example 16 - Detection of metabolic kinetic parameters of humanized hCD40 antibody

[0200] Eight-week-old Balb / c mice (excluding females) were randomly assigned to five groups: a reference control group (Sotigali mab, 6 mpk), a candidate CD40 humanized antibody group (2 mpk and 6 mpk). A single intravenous dose was administered according to the mouse's body weight and dosage. Blood samples were collected at 30 min, 1 h, 2 h, 4 h, 24 h, 48 h, 4 d, 8 d, and 11 d post-administration. Blood drug concentrations were measured using ELISA to assess the metabolism of each hCD40 antibody in the mice.

[0201] The results are as follows Figure 15As shown, CD40 antibodies at all concentrations exhibited good pharmacokinetic activity in mice, and the drug concentration in mouse serum decreased slowly over time, indicating that the antibodies can be stably present in mouse blood, which helps the antibodies exert their effects in the body.

[0202] The main pharmacokinetic parameters of the reference antibody (Sotigalimab) and candidate hCD40 antibodies (36F11-H4L2, 72F10-H3L3) at each time point were obtained using the data processing software PKS2.0: area under the plasma concentration-time curve (AUC(0-t); area under the plasma concentration-time curve (AUC(0-∞); peak concentration (Cmax); time to peak concentration (Tmax); elimination half-life (T1 / 2); apparent volume of distribution (Vd); and clearance rate (CL).

[0203] like Figure 15 As shown, at the same dosage, 36F11-H4L2 and 72F10-H3L3 are superior to the reference antibody Sotig alimab in all pharmacokinetic parameters such as area under the plasma concentration-time curve, half-life, etc., indicating that the candidate hCD40 antibody has a better drug metabolism effect.

[0204] Table 1 - Mean values ​​of major pharmacokinetic parameters for each dose group of the humanized hCD40 antibody (mean ± standard error)

[0205]

[0206] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A CD40 antibody or antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, characterized in that: (1) The amino acid sequence of CDR1 of the heavy chain variable region is shown in SEQ ID NO: 2; the amino acid sequence of CDR2 of the heavy chain variable region is shown in SEQ ID NO: 3; the amino acid sequence of CDR3 of the heavy chain variable region is shown in SEQ ID NO: 4; and the amino acid sequence of CDR1 of the light chain variable region is shown in SEQ ID NO: 7; the amino acid sequence of CDR2 of the light chain variable region is shown in SEQ ID NO: 8; the amino acid sequence of CDR3 of the light chain variable region is shown in SEQ ID NO: 9; or (2) The amino acid sequence of CDR1 of the heavy chain variable region is shown in SEQ ID NO: 12; the amino acid sequence of CDR2 of the heavy chain variable region is shown in SEQ ID NO: 13; the amino acid sequence of CDR3 of the heavy chain variable region is shown in SEQ ID NO: 14; and the amino acid sequence of CDR1 of the light chain variable region is shown in SEQ ID NO: 17; the amino acid sequence of CDR2 of the light chain variable region is shown in SEQ ID NO: 18; and the amino acid sequence of CDR3 of the light chain variable region is shown in SEQ ID NO:

19.

2. The CD40 antibody or antigen-binding fragment according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1; The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:

6.

3. The CD40 antibody or antigen-binding fragment according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 11; The amino acid sequence of the light chain variable region is shown in SEQ ID NO:

16.

4. The CD40 antibody or antigen-binding fragment according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 21; The amino acid sequence of the light chain variable region is shown in SEQ ID NO:

22.

5. The CD40 antibody or antigen-binding fragment according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 23; The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:

24.

6. A nucleic acid molecule encoding the CD40 antibody or antigen-binding fragment of any one of claims 1 to 5.

7. A gene expression cassette comprising the nucleic acid molecule of claim 6.

8. A recombinant vector comprising the nucleic acid molecule of claim 6 or the gene expression cassette of claim 7.

9. A transgenic cell line comprising the nucleic acid molecule of claim 6, or the gene expression cassette of claim 7, or the recombinant vector of claim 8; wherein the transgenic cell line does not include plant or animal varieties.

10. A product for detecting CD40, comprising a CD40 antibody or antigen-binding fragment according to any one of claims 1 to 5, or a nucleic acid molecule according to claim 6, or a gene expression cassette according to claim 7, or a recombinant vector according to claim 8, or a transgenic cell line according to claim 9.

11. A pharmaceutical composition comprising a CD40 antibody or antigen-binding fragment according to any one of claims 1 to 5, or a nucleic acid molecule according to claim 6, or a gene expression cassette according to claim 7, or a recombinant vector according to claim 8, or a transgenic cell line according to claim 9.

12. The pharmaceutical composition according to claim 11, characterized in that, It also includes pharmaceutically acceptable carriers.

13. The use of any CD40 antibody or antigen-binding fragment of claim 1 to 5, or the nucleic acid molecule of claim 6, or the gene expression cassette of claim 7, or the recombinant vector of claim 8, or the transgenic cell line of claim 9 in the preparation of a medicament for treating tumors, wherein the tumor is colon cancer.

14. The use of any CD40 antibody or antigen-binding fragment according to any one of claims 1 to 5, or the nucleic acid molecule according to claim 6, or the gene expression cassette according to claim 7, or the recombinant vector according to claim 8, or the transgenic cell line according to claim 9 in the preparation of a reagent for detecting CD40.

15. The use of any CD40 antibody or antigen-binding fragment according to any one of claims 1 to 5, or the nucleic acid molecule according to claim 6, or the gene expression cassette according to claim 7, or the recombinant vector according to claim 8, or the transgenic cell line according to claim 9 in the preparation of diagnostic reagents for diagnosing CD40-related diseases.

16. A method for producing protein, characterized in that... Includes the following steps: (1) Cultivate the transgenic cell line according to claim 9, so that the transgenic cell line expresses the CD40 antibody or antigen-binding fragment according to any one of claims 1 to 5; (2) Recover the expressed protein.

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