Dual-targeting Tandem CAR T Cells Targeting UPAR And HER2 Treat Epithelial Ovarian Cancer

[Background]Ovarian cancer is the malignant tumor with the highest mortality rate in gynecology,which seriously affects the physical and mental health of women and imposes a heavy economic burden on society.Traditional treatment methods for ovarian cancer include radical surgical resection,chemotherapy,and targeted therapy.Although these methods prolong the survival time,the recurrence rate is still high,and worldwide data show that the 5-year survival rate is less than 50%.In recent years,the development of immunotherapy techniques has provided a new direction to improve the outcome of ovarian cancer treatment.Chimeric antigen receptor T cell(CAR-T cells)therapy is a novel and promising immunotherapeutic approach to cure malignant tumors.Briefly,CAR-T cells consist of two parts,CAR and T lymphocytes.The role of CAR is to specifically identify and bind tumor cells,while the role of T lymphocytes is to kill tumor cells,and the combination of the two completes the targeted killing of tumor cells.CAR-T-cell therapy is now used clinically in adult patients with relapsed or refractory large B-cell lymphoma.On June 23,2021,the National Regulatory Commission of China approved the marketing of the first CAR-T-cell formulation(Aquilenside Injection).However,the application of CAR-T-cell therapy in ovarian cancer is still in the preclinical research stage because ovarian cancer is a solid tumor that differs from hematological tumors in many ways and still faces many problems,such as tumor antigen escape,difficulties in settlement and colonization,on target and off target,and immunosuppressive tumor microenvironment,which directly affect the therapeutic effect of CAR-T-cells.In addition,there are differences in the type of tumor antigens expressed in different pathological types of ovarian cancer.Therefore,the selection of tumor antigens is critical for the efficacy of CAR-T cells.Urokinase-type plasminogen activator receptor(uPAR)and human epidermal growth factor receptor 2(HER2)are all tumor antigens.Up-regulation of uPAR or HER2 has been found to be associated with ovarian cancer development,progression,and metastasis.However,studies of bispecific CAR-T cells targeting both uPAR and HER2 to kill ovarian tumors are not known.To address the problem of tumor antigen escape and difficulty in homing and colonization of CAR-T cells in ovarian cancer treatment,we constructed a novel,tandem,third-generation CAR-T cell that dual-targets uPAR and HER2 antigens,with the aim of improving the effectiveness of CAR-T cells in targeting and killing epithelial ovarian cancer and elucidating the mechanism to provide a theoretical basis for clinical translation and application.[Objective]1.The first objective of this study is to solve the problems of tumor antigen escape,difficult homing,and colonization in CAR-T cell therapy for ovarian cancer,and we construct a novel dual-targeted tandem CAR-T cell to enhance the killing effect of CAR-T cells on epithelial ovarian tumor cells.2.The second objective of this study was to explore the mechanism of epithelial ovarian tumor killing by dual-targeted tandem CAR-T cells and provide theoretical basis for clinical translation and application.[Method]1.The expression of uPAR and HER2 antigen in four human epithelial ovarian cancer cells(ES-2,SKOV3,OVTOKO,A2780)and one human normal ovarian epithelial cell(Hosepic)was studied by qRT-PCR,flow cytometry,and immunohistochemistry.Ovarian cancer cells with uPAR+/HER2+were selected,and then ovarian cancer cells with low uPAR expression and low HER2 expression were constructed by silencing gene technology,respectively.2.We used the basic framework of third-generation CARs to design tandem CAR structures that dual-targeted uPAR and HER2 antigens and then packaged them with lentiviruses.Subsequently,we extracted human T lymphocytes from peripheral blood by density gradient centrifugation and constructed uPAR/HER2-CAR-T cells by lentiviral transduction.Finally,the expression of CD3,CD4,and CD8 in T lymphocytes was detected by flow cytometry.3.Five effector cells(T cells,Control-CAR-T cells,uPAR-CAR-T cells,HER2-CAR-T cells,and uPAR/HER2-CAR-T cells)were compared with four target cells(uPAR+/HER2+,uPAR+/HER2-,uPAR-/HER2+,and uPAR-/HER2ovarian cancer cells),and the killing effect was monitored by lactate dehydrogenase release assay and the optimal effect and target ratio was determined.Then,under the condition of the optimal effect and target ratio,the ability to secrete Th cytokines and granzyme B in killing assay was detected by CBA kit and ELISA,respectively.4.We constructed a tumor-bearing mouse model by implanting ES-2 cells subcutaneously into immunodeficient BALB/c-nu mice.We injected effector cells into the mice through the tail vein after observing successful implantation of tumors.Five experimental groups were divided according to the type of effector cells:namely,T cell group,Control-CAR-T cell group,uPAR-CAR-T cell group,HER2-CAR-T cell group,and uPAR/HER2-CAR-T cell group.The tumor tissues were removed after performing the fractions,and then the mean volume and mean mass of the tumors in each group of mice were recorded.The CAR copy number in the tumor tissue was determined by qRT-PCR,the number of CD3+cells in the tumor tissue was observed by HE staining and immunofluorescence,and the types and levels of cytokines in the blood of the five groups of mice were detected by Luminex technology.[Results]1.The qRT-PCR results showed that the relative quantification of uPAR and HER2 on the surface of ES-2 cells was 8.69 and 7.70 times higher than that of Hosepic cells,respectively.The flow cytometry results showed that the expression levels of uPAR and HER2 on the surface of ES-2 cells were 94.80%and 98.48%,respectively.The results of immunohistochemistry showed that both uPAR and HER2 were positively expressed on the surface of ES-2 cells.The results of qRT-PCR and flow cytometry showed that sh-uPAR-ES2 cells with low expression of uPAR and sh-HER2-ES2 cells with low expression of HER2 were successfully constructed.2.We constructed tandem uPAR/HER2 CAR-T cells with CD8α in the transmembrane region and CD28-CD137-CD247 in the intracellular signaling region.Flow cytometric identification of T lymphocyte bodies showed that the percentage of CD3-positive cells was 98.20%.Compared with normal cells,the percentage of CD8 cells in each CAR-T cell was higher than that of CD4 cells.Flow cytometry results showed that the transduction efficiency of dual-target tandem CAR lentivirus for T lymphocytes was 73.70%.3.The killing effect of uPAR/HER2-CAR-T cells on ES-2(uPAR+/HER2+),A2780(uPAR-/HER2-),sh-uPAR-ES2(uPAR-/HER2+),and sh-HER2-ES2(uPAR+/HER2-)cells was variable,with the strongest killing effect on ES-2 cells,whose lysis rate was 86.29±0.23%.The optimal effective target ratio of uPAR/HER2-CAR-T cells against ES-2 cells was 10:1.In the same type of target cells,uPAR/HER2-CAR-T cells killed tumor cells better than both uPAR-CAR-T cells and HER2-CAR-T cells and secreted more IFN-γ,TNF-α,IL-2,and granulocyte enzyme B.4.Compared with the T cell group,the control CAR-T cell group,the uPAR-CAR-T cell group,and the HER2-CAR-T cell group,implanted tumors in the uPAR/HER2-CAR-T cell group had a smaller mean volume,a smaller mean mass,more CAR-copies and a higher number of CD3+cells in the tumor tissue,as well a higher concentrations of tumor-killing cytokines(such as IFN-γ,TNF,IL-2)in the blood.[Conclusions]1.uPAR/HER2-CAR-T cells can overcome the problems of tumor antigen escape,difficult homing and colonization in the treatment of epithelial ovarian cancer and improve the therapeutic effect of ovarian cancer.2.The mechanism by which uPAR/HER2-CAR-T exerts efficient anti-tumor effects is their enhanced ability to recognize and bind target antigens and secrete large amounts of cytokines such as granzyme B,IFN-γ,TNF and IL-2.