The Effect Of CRISPR/Cas9-mediated SHP-1 Or PD-1 Editing On The Anti-tumor Activity Of CAR T Cells

Tumor immunotherapy,which takes use of the ability of immune system to identify and remove alien constituents or abnormal cells,is a new strategy to fight against cancer.In order to maintain the balance of the immune system and prevent the excessive activation of T cells,T cells themselves have evolved a set of immunosuppressive regulation mechanisms.Activated T cells overexpress immune checkpoint molecules,such as CTLA-4 and PD-1,on the cell surface.Once receptors combined with corresponding ligands,inhibitory signals are transmitted to weak the activation of T cells.The inhibition of signals from the inhibitory receptors expressed on the cell surface can be blocked by the synthesized corresponding antibody.In rencent years,PD-1 and CTLA-4 antibodies have been extensively used in the treatment of multiple solid tumors and hematologic malignancies.However,the antibody treatment also has its disadvantages such as the cumbersome preparation and the side effects.Meanwhile,another immunotherapy approach,CAR T therapy,has also been successfully used in the treatment of tumors.Nevertheless,the breakthrough by the CAR T cell therapy is limited in the treatment of blood tumors,but not the solid tumors,mainly because the solid tumors have more complex immunosuppression microenvironment.Interestingly,specifically knockout PD-1 by a rencent developed CRISPR/Cas9 technology in CD 19 CAR T cells has shown improved antitumor effect,suggesting the potential of the combination of gene editing and CAR T therapy in tumor immunotherapy.In order to explore the effect of PD-1 knockout on CAR T cell therapy against solid tumors,we used PiggyBac-transposase system to mediate the stable expression of EGFRVIII CAR,and CRISPR/Cas9 system to specifically knockout of PD-1 in human primary T cells.Flow cytometry assay showed that more than 90%of T cells express CAR gene.And the PD-1 knockout efficiency(nearly 60%)was quantified by T7EN1 assay,Sanger sequencing and flow cytometry.Then,we detected the composition of CAR T cell subsets,including the compositions of CD4+and CD8+ T cells,and the proportion of T cells expressing the activated molecule CD28,because the subset of T cells might affect the efficacy of T cell therapy.The ratio of CD4/CD8 and the proporation of activated T cells showed no differences between EGFRVIII CAR T cells and PD-1 deficient EGFRVIII CAR T cells.Next,we evaluated the cytotoxic function of these gene-edited CAR T cells in vitro,no obvious differences were observed in cytokine secration or proliferation.But in immunodeficient NPG mouse engrafted with U251-EGFRVIII tumor cells,PD-1 disruption CAR T cells significantly improved the antitumor effect.Besides PD-1,there is also a large number of inhibitory molecules expressed within T cells,which can not be amenable to antibody-mediated therapy.Otherwise,the small molecule inhibitors are difficult to be applied to the clinical tries due to poor specificity,strong side effects and other characteristics.However,the emergence of CRISPR/Cas9 system provides an opportunity to study the effects of intracellular molecules on immunotherapy.Taken the advantage of established CD133 CAR system,we achieved both expression of CD 133 CAR and knockout of SHP-1,an intracellular inhibitory molecule,in human T cells.After two rounds of amplification,efficiencies of CAR expression and SHP-1 knockout were confirmed by flow cytometry,T7EN1 assay and Sanger sequencing,which were both higher than 80%.Then,we detected the composition of CAR T cell subsets,including the compositions of CD4+and CD8+naive and memory T cells,the proportion of T cells expressing the activated molecule CD27 and CD28.Flow cytometry assays showed no differences between CD 133 CAR T cells and SHP-1 deficient CD133 CAR T cells.In vitro cytotoxic assay shown that the disruption of SHP-1 in CAR T cells could significantly improve the cytolysis effect on glioma cell lines,which was about 40%higher compared with CAR T cells when the ratio of effect to target is 16:1.After further study,we found that the enhanced antitumor efficacy of SHP-1 deletion might due to the increased release of TNF-α and IFN-y.Next,we deleted both PD-1 and SHP-1 in CD 133 CAR T cells to explore the antitumor effect on CAR T therapy.Finally,we tested the biosafety of Cas9 genome editing but didn’t find any insertions of Cas9,nor obvious editing in off-target sites.In conclusion,we used the CRISPR/Cas9 system and the PiggyBac/transposase system to achieve both PD-1 or SHP-1 gene knockout and CAR gene expression in human T cells.Further Cytotoxic assay shown PD-1 or SHP-1 disruption in CAR T cells could significantly improve antitumor effect.