Preparation And Functional Study Of Specific An-Ti CD25 Antibodies

Cancer is one of the most complex diseases in human history,characterized not only by the overgrowth of malignant tumor cells,but usually accompanied by an altered immune response.The suppression and re-editing of the immune system plays a key role in tumorigenesis and progression,and immunotherapeutic approaches aim to reactivate anti-tumor immune cells and overcome immune escape from tumors,with the advantages of high accuracy and specificity.However,the overall clinical response rate of tumor immunotherapy still needs to be improved,and in addition to the combination with multiple immunotherapeutic approaches that can improve clinical outcomes,new biomarkers should be further explored and new therapeutic approaches should be investigated and discovered.CD25,the alpha subunit of interleukin-2(IL-2)receptor,is mainly expressed on the surface of regulatory T cells(Treg)and effector T cells(Teff),which play an important role in maintaining immune homeostasis and reducing the development of autoimmune diseases.Patients with lung cancer,ovarian cancer,intestinal tumors,and acute lymphoblastic leukemia have a significant increase in the number of Treg cells in the peripheral blood or tumor localization.IL-2 binds to CD25 on the surface of Teff cells,which effectively activates the cells and promotes cell proliferation,but inhibits the proliferation of Teff cells when competitive binding of CD25 on the surface of Treg cells occurs.Given that CD25 is present on both Teff and Treg cell surfaces,it makes it somewhat limited in clinical applications.The development of therapeutic antibody drugs targeting CD25 on the surface of Treg cells has great potential in the field of future antitumor immunotherapy.The main research of the paper is as follows:The paper investigated the immune response of BALB/c mice.The results of the study showed that good humoral immune responses were obtained in mice with different injection methods.Using spleen cells from mice electrofused with myeloma cells,and using enzyme-linked immunosorbent assay(ELISA)and flow cytometry(FACS)screening techniques,38 polyclonal hybridoma cells with cell supernatants specifically bound to CD25 were identified.34 monoclonal hybridoma cells that could secrete monoclonal antibodies were successfully screened by dilution seeding to plates from the polyclone hybridoma cells.7 murine-derived monoclonal antibodies that bind well to CD25 specifically were further purified.In order to reduce the immunogenicity of the antibodies,the murine-derived monoclonal antibodies were transformed into human-mouse chimeric antibodies,and the two most effective GC-01 and GC-02 antibodies were selected and further transformed into humanized antibodies GHu-01 and GHu-02 based on their specific binding to primary cells and the results of ADCC mediated by the reporter cell lines.The modified humanized antibody can mediate targeted killing of Treg cells by natural killer(NK)cells in peripheral blood cells(PBMC)and does not affect the function of IL-2 in promoting cell proliferation and differentiation during co-culture with PBMC.The antibody did not bind non-specifically to HUVEC cells,erythrocytes,granulocytes,or human-derived serum.Pharmacokinetic assays of the antibody in mice showed that the 10 mg/kg dose of antibody was stable in mice for a longer period of time.The validation results of the mouse-MC38 tumor model showed that the humanized antibody GHu-01 had better tumor suppression effect.In this study,two new epitopes of CD25-specific humanized antibodies were screened using hybridoma technology and humanization modification,and showed good ability to target binding Treg cells,which have promising clinical applications.