Construction And Functional Evaluation Of Integrated SOCS5 Chimeric Antigen Receptor T(CAR-T) Cells

The incidence and mortality rate of cancer in China are increasing year by year,however,the efficacy of traditional treatments such as surgery and radiotherapy is limited,so there is a need for more effective cancer treatments.In recent years,chimeric antigen receptor T(CART)cell therapy has achieved breakthrough efficacy in targeting B-cell malignancies,but the efficacy in patients with solid tumors is still unsatisfactory,thus there is a need to further enhance the anti-tumor activity of CAR-T cells in the field of solid tumors.T cells are the key backbone of precise immuno-therapy,and their cell phenotype,ability to further differentiate,and lifespan can affect the efficacy of the final treatment.Among them,although CD4+ T cells do not have the direct killing ability like CD8+ T cells,the differentiation of CD4+ T cells into helper T(Th)cells after activation can enhance cellular immune efficacy.Under different cytokines culture environments,Th can polarize to subtypes like Th1,Th2,Th17,and Treg,respectively,with Th1 cells being an important CD4+ cell subtype that can assist in the development of tumor resistance.SOCS(Suppressor of cytokine signaling,SOCS)family members include SOCS1-7 and CIS composition,and it was found that SOCS proteins play an important regulatory role in T cell differentiation,maturation,and function.Regulation of SOCS family members has potential for enhancing tumor immuno-therapy by endogenously targeting downstream signaling molecules,prompting CD4+ T cells to polarize toward Th1,Th2,Th17 and other fractions,and excluding the influence of paracrine signaling networks in the immune microenvironment.For example,studies have shown that over-expression of SOCS5 gene upregulates Th1-specific genes and pro-inflammatory pathways,as well as promotes the secretion levels of Th1-type cytokines IL-12,IFN-γ,and TNF-α.Therefore,the scientific question posed in this study was to investigate whether overexpression of SOCS5 on top of second-generation CAR molecules could enhance the antitumor activity of CAR-T cells in the complex tumor micro-environment in solid tumors.In this study,we first validated in vitro that Jurkat cells over-expressing SOCS5(Jurkat-OES5)can secrete higher levels of Th1 type cytokines when co-cultured with target cells in vitro;and then constructed chimeric antigen receptor T cells(MSLN CAR-T OES5)integrating the SOCS5 gene,using second-generation CAR-T cells as a control to explore their in vitro functions.In in vivo experiments,we constructed an immunodeficient(NSI)mouse model of gastric cancer cell line MKN-28;the anti-tumor activity and anti-depletion levels of MSLN CAR-T OES5 cells were evaluated by tumor growth curves,peripheral blood assays,and intra-tumoral cell assays in mice,and our results showed that the MSLN CAR-T OES5 cell group could more significantly inhibit tumor The level of CAR-T cell infiltration was increased in mice tumors,and the proportion of predominantly CD4+ T cells and the expression level of PD-1 molecules was lower compared to the control group.In summary,this study was conducted to validate the effect of CAR-T cells integrated with SOCS5,and the results showed that SOCS5 could enhance the anti-tumor effect of CART cells targeting MSLN in vivo.It provides a pavement for further use of SOCS family proteins to optimize the problems encountered when treating solid tumors with immunosuppressive micro-environment by CAR-T cells.