A Study Of C-Met CAR-T And PD1/CD28 Fusion Receptor CAR-T In Gastric Cancer

Objective:To design a chimeric antigen receptor T cell（CAR-T）targeting c-Met,verify its efficacy and safety in gastric cancer（GC）,and optimize a PD1/CD28 fusion receptor for c-Met CAR to restrain immunosuppression and improve anti-tumor effects.Methods:The expression of c-Met and the relationship between c-Met expression and survival prognosis were analyzed by bioinformatic methods from TCGA stomach adenocarcinoma datasets.Gastric cancer and adjacent tissues from GC patients were collected,and the expression of c-Met in these tissues were detected by immunohistochemistry（IHC）.The expression of c-Met and PD-L1 in different gastric cancer cell lines were detected by PCR,WB,and flow cytometry（FCM）.The c-Met CAR and cMet-PD1/CD28 CAR plasmids were constructed and then infected into T cells by lentivirus,the same empty vector was used for Mock T as negative control.In vitro,the changes of different subgroups and phenotype in CAR-T cells,and the changes of activation and PD-1 expression on CAR-Ts after the stimulation of target cells were measured by FCM.The secretion levels of different cytokines were detected by ELISA,and the killing ability of CAR-T to target cells was tested by lactate dehydrogenase（LDH）release assay,7-AAD staining and CD107a degranulation reaction.In vivo,the effects of different CAR-Ts on subcutaneously（s.c.）transplanted GC models in mice were observed by in vivo small animal imaging and the off-target toxicity to normal organs of mice were observed by HE-staining.Results:The expression of c-Met was higher in GC tissues than normal or adjacent tissues according to both TCGA datasets and IHC results from GC patients.Besides,the expression of c-Met was higher in GC patients with microsatellite instability（MSI）,pathological stageⅢ-Ⅳand intestinal type.The high expression of c-Met was related to poor prognosis in GC patients.MKN-45 was selected as the highest c-Met expression cell line and HGC-27 as the lowest from 6 different GC cell lines by PCR,WB and FCM.HGC27-OE was HGC-27 over-expressed with c-Met which was constructed as a positive control.The CD3⁺CD8⁺subgroup and CD62L⁺CCR7⁺central memory phenotype(T_CM)in c-Met CAR-T were increased compared with Mock T.The PD1/CD28 fusion receptor could increase the proportion of CD62L⁺CCR7⁺T_CMM cells,but did not change the subgroups of CD4⁺and CD8⁺T cells.The stimulation from target cells could increase the expression of PD-1 and increase the activation phenotype as CD69⁺and CD71⁺in c-Met CAR-T.Compared with Mock T,c-Met CAR-T could secrete more cytokines such as IFN-γ,TNF-α,IL-6,IL-10 after co-cultured with target cells.Furthermore,the killing ability to target cells and CD107a degranulation reaction of c-Met CAR-T were significantly improved,and its killing activity depends on the expression of c-Met on target cells.The PD1/CD28 fusion receptor could further enhance the killing ability of c-Met CAR-T on c-Met positive GC cells,in addition,it could reduce the release level of IL-6.The two CAR-Ts could significantly inhibit the growth of tumors in mice with subcutaneously transplanted GC model,and with no obvious off-target toxicity to normal organs such as stomach,small intestine,heart,liver,spleen,lung,and kidney.PD1/CD28 fusion receptor could further enhance the long-term anti-tumor effect of c-Met CAR-T.Conclusions:c-Met CAR-T had a good killing activity on c-Met-positive gastric cancer cells,could significantly inhibit the growth of c-Met-positive gastric cancer tumors,and had no serious off-target toxicity.The design of PD1/CD28 fusion receptor could further enhance the anti-tumor ability of c-Met CAR-T by reversing the PD-1immunosuppressive signal,and could reduce the secretion of IL-6 which may reduce the incidence of cytokine release syndrome and improve security.