The Study On Anti-prostate Cancer Effect And Mechanism Of The Enhanced NKG2D-CAR T Cells

The advent of CAR T-cell immunotherapy is considered as an important milestone in the field of human cancer treatment.CAR T cells are highly effective in the treatment of hematological malignancies,but there are still many challenges in the treatment of solid tumors.Therefore,how to improve the efficacy of CAR T cells in solid tumors has become the focus of researchers.NKG2D is an activated immune receptor mainly expressed in NK cells.Once binding to the ligands,NK cells are activated and show the killing effect by releasing cytotoxic particles and cytokines.As NKG2D ligands are highly expressed in a variety of solid tumors,introduction of NKG2D into T-cell chimeric antigen receptor has become a promising immunotherapy approach.Prostate cancer is an epithelial malignant solid tumor which has a high incidence,high risk of recurrence and metastasis.The current methods for treating metastatic prostate cancer are very limited,so it is urgent to find a better strategy to treat metastatic prostate cancer.In this study,we found that NKG2D ligands were highly expressed on prostate tumor cells.Therefore,T cells expressing NKG2D-CAR were constructed to detect the killing activity against three prostate tumor cell lines by cytotoxicity experiments.The results indicated that NKG2D-CAR T cells could effectively kill three types of prostate tumor cells,indicating that NKG2D-CAR was a feasible and effective method for prostate cancer with high expression of NKG2D ligands.However,like most solid tumors,prostate tumor,especially metastasis prostate tumor,has a powerful immunosuppressive microenvironment,which can inhibit the proliferation,survival and antitumor effect of CAR T cells.Therefore,we co-expressed IL-7 or down regulation of GPR116 in NKG2D-CAR T cells to enhance the proliferation,survival and antitumor function of CAR T cells,which provided a new idea and potential target for improving the efficacy of solid tumors,and a theoretical basis for clinical application.Part Ⅰ Co-expression of IL-7 enhances anti-prostate cancer of NKG2D-CAR T cellsIL-7 is a kind of growth promoting factor,which can promote the proliferation and survival of T cells and maintain the growth of memory T cells.In order to investigate the regulatory role of IL-7 in CAR T cells,NKG2D-CAR T cells co-expressed of IL-7 were produced.We found that NKG2DIL7-CAR T cells could enhance the killing effect of NKG2D-CAR T cells,which depended on the incubation time.Then we detected the proliferation,apoptosis,and exhaustion of two kinds of CAR T cells.Compared with NKG2D-CAR T cells,the proliferation ability of NKG2DIL7-CAR T cells was enhanced,and the proportion of CD8~+T cells was higher.Moreover,NKG2DIL7-CAR T cells had lower proportion of apoptotic cells,and the expression of anti-apoptotic protein Bcl-2 was higher,which was mainly caused by the increase in CD8~+T cells.Furthermore,we found that NKG2DIL7-CAR T cells expressed lower levels of exhaustion molecules,such as PD-1 and Tim-3,and the proportion of Tscm like cells were higher,all evidences indicated that NKG2D-CAR T cells expressing IL-7 had stronger antitumor function.Finally,we used the prostate tumor model of immunodeficient mice to verify the therapeutic effect of two kinds of CAR T cells in vivo.Compared with the untreated group,both two kind of CAR T cells had obvious antitumor effect,and more remarkable effect was observed in NKG2DIL7-CAR T-cell treatment group,in which there were larger numbers of CAR T cells,especially CD8~+T cells in tumor site.In conclusion,our results showed that co-expression of IL-7could enhance the killing,proliferation and survival,reduce the apoptosis and exhaustion,and eventually improve the ability of NKG2D-CAR T cells in treating prostate cancer.Our study provided a new idea for enhancing the antitumor function of CAR T cells,and also provided a new clue and direction for improving the treatment of solid tumors.Part Ⅱ Downregulation of GPR116 enhances the anti-prostate cancer of NKG2D-CAR T cellsIt is reported that GPR116,an adhesive G protein-coupled receptor,involved in the regulation of the immune system,but its function and mechanism remined limited.In previous experiments,we found that Gpr116 knockout mice showed significantly enlarged spleen compared with wild-type mice,and splenic CD8~+T cells secreted higher levels of IFN-γ,which suggested that GPR116 receptor might affect the cytotoxicity of CD8~+T cells.Therefore,we constructed the mouse-derived NKG2D-CAR.Cytotoxicity experiment results indicated that Gpr116 knockout mNKG2D-CAR T cells had stronger effect on killing prostate cancer cells,expressed higher levels of activating molecules CD25 and CD107a,and secreted more IFN-γand granzyme B.In addition,Gpr116 knockout mNKG2D-CAR T cells showed increased proliferation and antitumor effect in vivo.In order to further verify the effect of GPR116 on the antitumor function of CAR T cells,we constructed and screened the shRNA sequences and vectors,which were mostly significantly knockdown of GPR116 by RNA interference,and further prepared human-derived NKG2D-CAR T cells to detect the antitumor function.Consistent with the results of mouse-derived CAR T,downregulation of GPR116 could significantly enhance the killing effect on prostate cancer.The activation level,cytokine secretion,proliferation and viability were enhanced.These results showed that GPR116 is a negative regulator protein and knockdown of GPR116 could enhance the killing,proliferation and anti-apoptosis functions of NKG2D-CAR T cells.Western blot results revealed that GPR116receptors could inhibit the downstream PI3K-Akt-NF-κB signaling pathway by regulating Gαq protein,which in turn affected T cell killing,proliferation,and survival functions.Our study provided a new target and clue for enhancing the antitumor function of CAR T cells,and also opened up new ideas for improving solid tumor treatment and drug target development.