Construction And Functional Study Of GITRL Co-Expressing Chimeric Antigen Receptor T (CAR-T) Cells

CAR-T cell therapy has made remarkable achievements in the treatment of tumors,especially in the treatment of hematological malignant tumors,but the efficacy of CAR-T in solid tumors is not yet ideal.The introduction of 4-1BB costimulatory molecule has greatly improved the anti-tumor effect of CAR-T cells,but there is still no substantial breakthrough in the targeted treatment of solid tumors.Through integration of a variety of signal molecules,synergistic improvement of CAR-T cell effect function has gradually become an important breakthrough and research direction in the field.4-1BB,as a member of tumor necrosis factor receptor superfamily(TNFRSF),promotes T cell proliferation and survival by recruiting TRAF1 and TRAF2,and activating NF-κB signal pathway.With continuous study,the important regulatory role of other TNFRSF members and their recruited TRAF molecules on T cells have been gradually revealed.GITR(Glucocorticoid-Induced Tumor necrosis factor Receptor,also known as TNFRSF18)is a new type of costimulatory molecule found in recent years,which is closely related to the regulation of T cell function.Its ligand is the membrane protein GITRL(TNFSFL18).After GITRL/GITR binding,TRAF2,TRAF5 and TRAF6molecules can be recruited,which play an important role in T cell activation,differentiation and memory formation.In the study of a variety of infection and tumor animal models,4-1BB and GITR and their different TRAF molecules have been proved to have unique and highly complementary regulatory effects in T cell-mediated antigen-specific immune responses.Studies have shown that 4-1BB costimulatory signal can promote the differentiation of T_H1 cells in CAR-T,while GITR/GITRL signal can not only enhance the survival and proliferation of effector T cells,inhibit the activity of Treg cells,but also induce the differentiation of T_H9 cells.T_H1 and T_H9 cells are considered to be highly effective anti-tumor immune cells.Even in some solid tumor models and clinical studies,T_H9 cells show better anti-tumor effects than T_H1 cells.Based on the above research,we speculate that the integration of 4-1BB and GITR costimulatory signals in CAR molecules is expected to improve the effector function of CAR-T cells and the therapeutic effect of tumor.Because the expression of GITR is up-regulated in activated T cells,and the costimulatory ligand can transmit costimulatory signals through"cis"self-activation and"trans"bystander activation,this study made CAR-T cells co-express GITR ligand GITRL,to explore the effect of GITR signal on the anti-tumor effect of CAR-T cells on the basis of the second generation CAR-T design of 4-1BB targeting CD19 and PSMA.Firstly,we successfully constructed the corresponding 4-1BB-GITRL CAR-T lentivirus expression vectors by molecular cloning,based on the existing 4-1BB CAR-T lentivirus expression vectors targeting PSMA and CD19 in the laboratory and verified by double enzyme digestion and sequencing.Then,the lentivirus three-plasmid packaging system was used to transfect 293T cells,and the virus solution was collected.After concentration and purification,a high titer virus was produced and used to infect human primary T cells.CAR-T cells with high positive rate of CAR and stable expression were successfully prepared.Co-culture experiments in vitro showed that co-expression of GITRL could enhance the expansion level of CAR-T cells,increase the expression of T cell activation molecules CD25 and CD69,and reduce the expression of T cell depletion molecules PD-1 and Tim3 under the stimulation of tumor antigen.It has been confirmed that co-expression of GITRL can promote the formation of T_H9cells and the secretion of IL-9.In addition,we also found that co-expression of GITRL could enhance the killing effect of CAR-T cells on target cells,promote the secretion of IFN-γ and TNF-α,and enhance the degranulation of CAR-T cells.In order to further evaluate the effect of co-expression of GITRL on the anti-tumor effect of CAR-T cells,a mouse prostate cancer model was established and then treated with our CAR-T cells.The results showed that co-expression of GITRL enhanced the anti-tumor effect of CAR-T cells.To sum up,an enhanced CAR-T cell co-expressing GITRL was designed and prepared in this study.In vitro cell experiments showed that the new CAR-T cell had stronger expansion ability and anti-tumor effect.Co-expression of GITRL slowed down the depletion of CAR-T cells,enhanced the functional persistence of CAR-T cells,and the anti-tumor effect was verified in the prostate cancer mouse model.Our research reveals the application potential of GITR/GITRL,an important costimulatory signal,in CAR-T technology,which provides a new idea for improving the therapeutic effect of CAR-T cells on malignant tumors,especially solid tumors,and shows important research and transformation significance.