CRISPR-Cas9 Mediated Disruption Of PD-1 On Human T-cells And Its Anti-tumor Efficacy In The Treatment Of EBV Positive Gastric Cancer

Purpose:Immunotherapy is one of the most promising therapeutic methods for tumor treatment.In recent years,immunetherapy targeting PD-1/PD-L1 blocking in the treatment of malignant melanoma and lung cancer has been approved for clinical use and achieved gratifying effect,also showing good therapeutic effect in blood system malignancies and liver cancer,breast cancer,colon cancer and gastric cancer.In addition,adoptive cell therapy strategies such as CAR-T,TCR-T and DC-CTL technologies are also showing promising effectiveness in clinic.These two significant devenlopements bring a new revelation.to the cancer treatment.The treatment of gastric cancer is a worldwide difficult problem.The selection of specific antigen for gastric cancer has been one of the difficult problems.Studies show that a part of gastric cancer is closely related with EBV infection and this subtype of gastric cancer is with a high degree of lymphocyte infiltration and PD-L1 gene amplification.In this study,we used the third generation gene knockout system CRISPR-Cas9 to establish the method of PD-1 gene disruption on human peripheral blood T cells,and to evaluate its function.More over,we further evaluate the possibility of PD-1 disruption by CRISPR-Cas9 in the generation of EBV-LMP2A-CTL in vitro or in vivo through an immune-deficient mice tumor xenograft model and evaluate the synergy effect when combined with small dose radiotherapy,providing new strategies and methods for the treatment of gastric cancer.Methords:We established PD-1 gene disrupted T cells by electroporation of the plasmis pGL3-hPD-1sgRNA and pST1374-Cas9 on human primary T cells.The transfection efficacy was observed by fluorescence microscope and further comfirmed by the T7EN1 assay and sequencing.The proliferation capcity and the phenotype of the gene modified T cells was evaluated and expression of PD-1 was monitored through the prolonged culturing with cytokine cocktail.IFN-y production was measured through Elispot by stimulation of gene modified T cells with DCs loaded with tumor associated antigen.The cytotoxicity was then evaluated by CFSE/PI assay through flow cytometry.In addition,PD-1 disrupted EBV-CTLs were established by co-culturing these gene modified T cells with LMP2A-loaded autologous DCs.Similaly,the proliferation and phenotype of the gene modified CTLs were evaluated and the cytokine production was measured by Elispot as well as multi-cytokine flow cytometry.The cytotoxicity of the PD-1 disrupted EBV-CTLs was compared between EBV positive gastric cancer cell line and EBV negtive counter part.In addition,the expression of PD-1/PD-L1 on the tumor tissue of EBV associated gastric cancer and EBV non-associated gastric cancer patiens were detected by immunohistochemistry.PD-1 disrupted EBV-CTL derived from EBVaGC patient was also characterized as described above.For the in vivo experiment,a tumor xenograft model of EBV associated gastric cancer was established by inoculation of SNU-719 cell line derived from an EBVaGC patient into the immune deficient mouse.The gene modified T cells was administered intravenously post radiotherapy at a dose of 2 Gy for 2 circles,7 days apart,followed by intraperitoneal injection of IL-2.The antitumor effect including tumor volume and survival was monitored followed by the treatment.The tumor was excised and used for the detection of CD3+T cells infiltration by immunohistochemistry and flow cytometry.The blood serum was collected and analysed for cytokine production.Results:We described for the first time a non-viral mediated approach to reprogram primary human T cells by disruption of PD-1.We showed that:1.The selected sgRNA worked effectively with Cas9 on human genomes.2.The gene disruption of PD-1 by electroporation of plasmids encoding sgRNA and Cas9 was technically feasible and the higest efficacy achieved was 66.67%.The condition of nucleotransfection and the ratio of plasmids concertration is of great importance.3.The disruption of gene PD-1 resulted in significant reduction of PD-1 protein expression of primary human T cells during the prolonged in vitro culture and didn't affect the viability and subsets of transfected T cells 4.Cellular immune response of the gene modified T cells was characterized by up-regulated IFN-? production by Elispot and enhanced cytotoxicity.More over,we further demonstrated that:1.PD-L1 expressed on the tumor cells of 64%EBV associated gastric cancer patients,while it only expressed on 15%of EBV non associated gastric cancer patients;PD-1 is up-regulated on EBV-LMP2A-specific CD8+T cells and associated with impaired function of CTLs towards EBV positice gastric cancer cells.2.PD-1 disruption by CRISPR-Cas9 in the generation of EBV-LMP2A-CTL derived from healthy donors or EBVaGC patients is technically feasible.3.Cellular immune response of these gene modified CTL cells was characterized by enhanced cytokine production of IFN-?,IL-2 and TNF-?.4.Enhanced cytotoxicity was shown of these PD-1 disrupted EBV-LMP2A-CTL towards EBV positive gastric cancer cells rather than the negtive cell.4.The in vivo injection of these gene modified T cells was safe and improved the survival of tumor bearing mice.5.These PD-1 disrupted CTL cells mediates efficient antitumor efficacy when co-administered with low-dose radiotherapy in a EBVaGC xenograft mouse model and the enhanced antitumor effect may be correlated with enhanced infiltration with transferred T cells.Conclusion:These results suggest that electroporation of plasmids encoding sgRNA-Cas9 DNA is technically feasible and it is efficient to established PD-1 disrupted T cells.This easy handling technique has great potential to achieve nice effect.And this electroporation mediated approach provides an alternative to labor-intensive and time-consuming viral-mediated gene transfer methods.More importantly,we presumed that PD1/PD-L1 pathway plays an important role in the immune tolerance of EBVaGC.We achieved PD-1 disruption by CRISPR-Cas9 on EBV-CTLs for adoptive cell therapy.The gene edited CTLs performed superior in immune responses and cytotoxicity.Importantly,low-dose radiotherapy can be safely and effectively combined with adoptive cell transfer and orchestrated best with PD-1 disrupted CTLs in an xenograft model of EBVaGC.Collectively,these results provide validation for the utilization of CRISPR-Cas9 system to the gene editing of human primary T cells for the "super CTLs" and provide recommendation for combining radiotherapy with immune checkpoint modified CTLs for the clinical tumor treatment.