Basic Study On The Therapeutic Function Of Tri-specific Antibodies Targeting CD3-TROP2-PDL1 On Triple-negative Breast Cancer

Objective: Breast cancer is a serious threat to women’s health.Tumor immunotherapy has become a hot spot.Immunotherapy has become a potential treatment due to high immune cell infiltration and PDL1 expression in triple-negative breast cancer.Therefore,we designed a trispecific antibody CD3-TROP2-PDL1 with targeting function and tested its therapeutic effect on triple-negative breast cancer in vitro in order to provide a reference for the development of immunotherapy methods for triple-negative breast cancer.Methods: 1.We connected the TROP2-sc Fv fragments to the heavy chains of CD3 and PDL1 monoclonal antibodies,and then expressed them in mammalian cells,and finally purified the trispecific antibody.2.After obtaining the antibody,we used the Coomassie brilliant blue staining method to detect the molecular weight of the antibody and the size of the monomer structure.3.Using flow cytometry,cells expressing only one target antigen were chosen to detect the binding ability of the antibody to its target antigenpositive cells.4.Using the fluorescent reporter gene system,we tested the ability of antibodies to activate Jurkat cells in the presence or absence of tumor cells.5.Pan-T cells,tumor cells and antibody were co-cultured to detect the ability of antibodymediated T cells to kill tumor cells by using CCK8 method.6.The amount of IL-2 secreted in the supernatant of Pan-T co-cultured with tumor cells was detected by ELISA.Results: We successfully obtained the trispecific antibody with the correct configuration.The molecular weight of the antibody was about 190 k Da by Coomassie brilliant blue staining.After opening the disulfide bond,it was found that the antibody actually consists of a pair of light chains and a pair of linked heavy chains of TROP2-sc Fv.From the results of flow cytometry,the antibody can bind well to the target antigen.The results of the fluorescent reporter gene confirmed that the antibody can effectively activate Jurkat cells in the presence of tumor cells.The results of CCK8 and ELISA showed that the trispecific antibody can not only effectively guide T cells to clear tumor cells,but also stimulate T cells to secrete the cytokine IL-2.Conclusion: The trispecific antibody CD3-TROP2-PDL1 can bind to its target antigen,it can effectively activate T cells and mediate T cells to kill tumor cells.Meanwhile it stimulated T cells to secrete cytokine IL-2.This antibody may provide new ideas for the development of therapeutic antibodies for triple-negative breast cancer.