| Triple negative breast cancer is a subtype of breast cancer with the worst clinical prognosis.Due to the lack of specific targets,patients with triple negative breast cancer are not sensitive to the hormone or antibody therapy.The successful application of adoptive cell therapy,especially as the adoptive of chimeric antigen receptor T cells in the treatment of hematological malignancies provides a new trend for the treatment of solid tumors.However,the immunosuppression and low T cell immigration of solid tumor micro-environment limit the clinical efficacy of CAR-T therapy.Conventional CAR-T therapy is an individualized treatment,which poses great challenges to the quality control,poor CAR-T consistency and long preparation period.To improve the target migration ability of CAR-T cells,regulated the tumor micro-environment artificially and explore the feasibility of universal CAR-T,in the part 1,the chemokine CCL19 and activating cytokine IL-7 were introduced to the new generation of chimeric antigen receptors targeting FRα.7x19FRα-CAR-γδT cells were generated by using lentivirus vector.In the part2,the cytotoxicity of 7x19FRα-CAR-γδT against breast cancer and triple negative breast cancer cell lines in vitro and their chemotactic function of various autoimmune cells were investigated and determined.In the part 3,NOD/SCID tumor-bearing models of breast cancer and triple-negative breast cancer were established and the 7x19FRα-CAR-γδT or FRα-CAR-γδT cells were infused through tail vein injection respectively.PBMC was administered through tail vein to induce the synergistic antitumor effect of immune cells in vivo.In vivo antitumor effects of 7x19FRα-CAR-γδT and FRα-CAR-γδT were evaluated based on tumor volume and pathology results.Part 1.The construction of 7x19FRα-CAR and the generation of7x19FRα-CAR-γδT cellsObjective:To construct a chimeric antigen receptor FRαsc Fv-CD28-CD3ζ-F2A-IL7-F2A-CCL19(7x19FRα-CAR)with secreting type of IL-7 and CCL19.The gene encoding 7x19FRα-CAR was delivered intoγδT cells by lentiviral vectors to produce 7x19FRα-CAR-γδT.Methods:The gene sequences encoding 7x19FRα-CAR and FRα-CAR were synthesized by overlapping PCR and cloned into p LVX vectors respectively.Western blot was used to detect the expression of FRα-CAR in HEK-293T cells.The lentiviral vectors were produced by packaging in HEK-293T cells.7x19 FRα-CAR-γδT and CAR-γδT cells were transfected with the lentiviral vectors.The expression of FRα-CAR onγδT cells was detected by flow cytometry.Results:The gene sequence encoding 7x19FRα-CAR was synthesized and cloned into p LVX vector and there is no mutation was detected.1 Western blot showed that the FRα-CAR protein of the target gene fragment can fully expressed in HEK-293T cells and the self-shearing peptides can be cleaved efficiently.1.HEK-293T cells were co-transfected with helper plasmids and packaging the lentiviral vectors.The 7x19FRα-CAR lentivirus vector titer was5E7 TU/m L.2.The concentration ofγδT cells from peripheral blood of healthy donors can be increased to 99.7%after magnetic bead sorted.5.The transfection efficiency of 7x19FRα-CAR-γδT was 40.7%.Part 2.Biological function evaluation of 7x19FRα-CAR-γδT cells in vitroObjective:To study the biological functions of 7x19FRα-CAR-γδT cells and evaluate its antitumor activity in vitro.Methods:The expression levels of FRαantigen on the surface of MCF-7,MDA-MB-231,MDA-MB-436,MDA-MB-468,and C30 cells were detected by flow cytometry.The expression levels of CCL19 ligand CCR7 on the surface of DC,T and PBMC cells were determined by flow cytometry.The7x19FRα-CAR-γδT cells were co-cultured with tumor cell lines with different expression levels of FRαantigen.The specific cytotoxicity of7x19FRα-CAR-γδT cells was detected by CCK8 assay;The secretion levels of cytokines IFN-γ,CCL19 and IL-7 were detected by ELISA.Transwell Assay was used to evaluate the chemotaxis of 7x19FRα-CAR-γδT cells to DC,T,and PBMC migration.Results:1.Flow cytometry showed that CCR7 was highly expressed in all overαβT lymphocytes,DC and PBMC cells.The“double peaks”of CCR7’s expression onαβT lymphocytes may be related to CD4 and CD8 phenotypes.2.ELISA results showed that no IL-7 and CCL19 were detected inγδT and FRα-CAR-γδT cells.The 7x19FRα-CAR-γδT cells could secrete 1600±150pg/ml of IL-7 and 503.3±100.2pg/ml of CCL19.Transwell assay showed that 7x19FRα-CAR-γδT chemotaxis of T lymphocytes,B lymphocytes,and DC cells to the tumor site.3.7x19FRα-CAR-γδT,FRα-CAR-γδT had high killing efficiency against FRα-positive expressed MCF-7,MDA-MB-231,MDA-MB-436 and MDA-MB-468 cells.The cytotoxic activity of 7x19FRα-CAR-γδT and FRα-CAR-γδT was significant different from that ofγδT cells.However,there was no significant difference in killing efficiency of C-30 cells with negative FRαexpression.4.MDA-MB-231 and MCF-7 cells with positive FRαexpression can significantly stimulated the production of IFN-γin 7x19FRα-CAR-γδT and FRα-CAR-γδT cells,and the 7x19FRα-CAR-γδT cells showed significant differences compared with FRα-CAR-γδT.It suggested that IL-7 can activateγδT.However,IFN-γsecretion was very low after co-cultured with C30 cells with FRαnegative expression,and the difference was significant statistically.Part 3.The study of 7x19FRα-CAR-γδT against tumor in vivoObjective:To evaluate the antitumor effect of 7x19FRα-CAR-γδT cells on breast cancer and triple negative breast cancer xenograft tumor model in mice.Methods:Xenograft models of breast cancer or triple negative breast cancer were established in NOD/SCID mice by subcutaneous injection of either MCF-7 cells or MDA-MB-231 cells.The breast cancer xenograft models were treated withγδT,PBMC,FRα-CAR-γδT,7x19 FRα-CAR-γδT cell therapy.The triple negative breast cancer xenograft models were treated withγδT,PBMC,FRα-CAR-γδT,7x19FRα-CAR-γδT,FRα-CAR-γδT+PBMC and 7x19FRα-CAR-γδT+PBMC cells,respectively.The tumor volume was observed and recorded.Tumor tissue resection were performed,T cells and DC cells were marked by immunofluorescence staining to observe the infiltration of immune cells.Results:1.Subcutaneous xenograft tumor models of breast cancer and triple negative breast cancer were established successfully.2.In the breast cancer xenograft tumor model,there was a statistically significant difference in tumor volume in the FRα-CAR-γδT,7x19FRα-CAR-γδT treated groups compared with theγδT and PBMC treated groups.3.In the triple negative breast cancer xenograft tumor models,significant tumor volume reduction was observed only in the 7x19FRα-CAR-γδT+PBMC treated group,and the difference was statistically significant compared with the other groups.4.In HE staining of triple negative breast cancer xenograft tumor tissues,the remaining tumor tissue of the 7x19FRα-CAR-γδT+PBMC group showed a large number of necrotizing vacuol-like structures,and a large number of T lymphocytes and DC cells were detected by IF staining.There weren’t large number of necrotic structures or immune cell infiltration were observed in any other groups.Conclusion:1.γδT cells can be amplified a 10~2 levels in vitro by using serum-free culture system containing zoledronic acid,IL-2 and IL-15.2.Lentiviral vectors can be used to deliver the gene encoding 7x19FRα-CAR toγδT cells and achieve high expression of 7x19FRα-CAR on theγδT cell surface.3.7x19FRα-CAR-γδT effectively recognizes and kills FRα-positive triple negative breast cancer cells in vitro and chemotactic DC cells and T lymphocytes to migrate to tumor cell sites by secreted CCL19.3.In breast cancer or triple negative breast cancer xenograft models,7x19FRα-CAR-γδT cells can effectively inhibited the growth of xenograft breast cancer.7x19FRα-CAR-γδT cells can also inhibited the growth of triple negative breast cancer xenografts in mice by chemotactic and recruited DC cells and T lymphocytes to play a synergistic antitumor effect. |
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