The Role Of Bi-specific CAR-T Cells Targeting B7H3 And IL13Rα2 In The Treatment Of Lung Metastasis Of Triple Negative Breast Cancer

Background:The median survival time after metastasis of triple-negative breast cancer(mTNBC)is about 13.3 months.Chemotherapy remains the most important systemic therapy for mTNBC because mTNBC lacks therapeutic targets such as ER,PR,HER2.However,the median progression-free survival time of first-line chemotherapy is not optimistic,also the incidence of grade 3-5 adverse events related to chemotherapy remains high,which make TNBC in urgent need new and curative treatments.Treatment of solid tumors with chimeric antigen receptor T(CAR-T)cells has made some progress recently,and triple-negative breast cancer is considered to be a tumor exhibiting immunogenic potential and immunotherapy potential in all types of breast cancer.Immune checkpoint inhibitors also achieved good clinical benefits in TNBC,reflecting the prospect of immunotherapy in TNBC.In triple negative breast cancer,while there is a lack of effective treatment,the risk of metastasis and poor prognosis seriously threaten the life and safety of patients.CAR T is a T cell that transferred chimeric antigen receptors,which can help T cells recognize tumor antigen in a way independent of MHC presentation and activate it specifically to kill tumor.And it has recently achieved remarkable efficacy in hematologic tumors,and certain progress has also been made in the treatment of solid tumors.Combined with the previous bioinformatics analysis,we explored the potential therapeutic targets of metastatic triple negative breast cancer,evaluated the therapeutic effect of CAR-T cell immunotherapy in triple negative breast cancer,and made various attempts to improve the benefits of solid tumors including breast cancer.We used CAR-T therapy under high or low tumor burden to explore the mechanism of immune escape,used bi-specific CAR-T to try to overcome immune escape,and optimized the treatment regimen to treat TNBC lung metastasis by injecting CAR-T in advance and increasing the therapeutic dose of CAR-T.Objective:In view of the lack of targets in the current treatment of triple negative breast cancer and its malignant metastasis,the effect of chemotherapy is not ideal,and the prognosis is poor,we explored the immunotherapy targets of negative breast cancer,looking for better targets suitable for CAR-T cell therapy,so as to provide theoretical support and transformation research reference for CAR-T therapy of clinical metastatic triple negative breast cancer.Methods:1.We used bioinformatics research methods to determine the potential therapeutic targets of metastatic triple negative breast cancer by comparing KaplanMeier survival analysis and IHC analysis.2.We constructed lentivirus plasmid by molecular cloning and infected CD3 T to construct CAR-T cells.3.CAR-T was coincubated with tumor cells to detect the cytotoxicity of CAR-T in vitro.4.We verified the therapeutic effect of B7H3 CAR-T under low or high tumor burden by xenografts model in mice,and explored the mechanism of immune escape by flow cytometry and Weston-blot.5.We used a mouse model of metastatic triple negative breast cancer to estimate the therapeutic effect of candidate targeting B7H3 and IL13Rα2 as CAR-T targets.Results:1.Kaplan-Meier survival analysis showed that patients with triple negative breast cancer with high expression of B7H3 and IL13Rα2 had shorter survival time of distant metastasis.At the same time,IHC results of primary tumor and lymph node metastasis in patients with triple negative breast cancer with metastasis showed that B7H3 and IL13Rα2 were highly expressed in primary tumor and lymph node metastasis,which indicated that B7H3 and IL13Rα2 were potential in the treatment of lung metastasis of TNBC.2.Flow cytometry and fluorescence microscopy showed that B7H3 CAR and BI CAR were successfully expressed in CD3 T cells.3.B7H3 CAR-T has a significant killing effect on TNBC cell line in vitro.4.In vivo,B7H3 CAR-T can remove subcutaneous tumors when the tumor burden is low,immune escape occurs when the tumor load is high,and the effect of B7H3 CAR-T in the treatment of lung metastasis of TNBC is not ideal.5.BI CAR-T was constructed and its killing effect in vitro was better than that of B7H3 CAR-T.6.The therapeutic effect of BI CAR-T is better than that of B7H3 CAR-T in the treatment of TNBC lung metastasis.Conclusion:Bi-specific CAR-T cells targeting B7H3 and IL13Rα2(BI CAR-T)overcame the unsatisfactory therapeutic effect of single targeted B7H3 CAR-T on mice with high tumor burden and the occurrence of immune escape,and enhanced the clearance and killing effect on metastatic tumor cell TNBC lung metastasis.because the BI CAR-T has two antigen receptors,it enhances the recognition specificity,expands the CAR-T recognition window and reduces the immune escape.The research results obtained in this project provide a new therapeutic strategy for mTNBC;on the other hand,it is confirmed that Bi-specific CAR-T can mediate the effective anti-tumor activity of mTNBC,and also provide principle proof data for the design of new CAR-T.