| Chimeric antigen receptor T cell(CAR-T)technology is regarded as a revolutionary tumor immunotherapy technology by the anticancer field.However,the clinical safety risks caused by severe cytokine release syndrome(sCRS)and severe CAR-T related encephalopathy(sRES)after CAR-T reinfusion and short-term relapse after CAR-T treatment are the two major clinical pain points that seriously hinder the current CAR-T clinical application and promotion.Aimed at these two clinical issues,this project is composed of two research proposals to investigate and develop the new strategies and technologies for providing the urgent solution to the CAR-T therapy.The first part of project is aimed at CAR-T clinical safety problems caused by sCRS and sCRES during the treatment of acute B lymphocyte leukemia by CAR-T.In this study,a new generation CAR vector(ssCAR)was successfully designed and constructed,by which both targeting CD19 antigen CAR gene expression cassette and IL-6shRNA gene silencing element were genetically engineered on the same CAR lentivirus vector,respectively.At the cellular level,the effect of ssCART-19 with IL-6shRNA silencing element on the inhibition of expression and secretion of endogenous IL-6 and on the inhibition of release of IL-6 by monocytes were investigated,followed by study of anti-tumor activity both in vitro and in vivo.The experimental results showed that the expression of endogenous IL-6 gene of traditional CART-19 cells was significantly increased with increased 4-fold and20-fold at mRNA and protein level,respectively,after activated by CD19+ target cells.IL-6 factor is recognized as the most important cytokine that significantly stimulates the immune amplification reaction of monocytes.Compared with traditional CART-19,ssCART-19 carrying IL-6shRNA elements can significantly down-regulate the IL-6 gene expression up to 70% at both mRNA and protein levels,and showedcontinuously and effectively reducing the release of IL-6 cytokine from monocytes.Further research found that IL-6shRNA mediated IL-6 gene silencing did not affect the basic properties and functions of ssCART-19,such as its proliferation and anticancer ability both in vitro and in vivo.The new generation CAR-T designed in this project is expected to reduce the incidence of sCRS and sCRES caused by rapidly overexpression and secretion of IL-6,and provide a new idea and novel strategy for effectively reducing the safety risks caused by CART therapy.The second part of this project is about the short-term relapse of multiple myeloma(r/r MM)treated with CAR-T.Antigen escape and tumor stem cells are the main causes of relapse after CAR-T treatment of r/r MM.In this study,two single chain antibodies(scFv)of anti CD19 antigen(scFv-CD19)and anti BCMA antigen(scFv-BCMA)were linked to the same CAR molecule(tan-CAR)in tandem on lentiviral vector.The transduced tan-CAR T cell(tan-CART)can be activated by either antigen of CD19 or BCMA,as well as both CD19/BCMA antigens,on the target cells.Further in vivo studies showed that the activated tan-CART could significantly kill tumor cells carrying the two corresponding antigens,and effectively inhibit the disease progress of animal models constructed by two tumor cells with the corresponding antigens,while in the same animal model,CAR-T cells with only a single antigen targeting property could not inhibit the disease progress.This dual scFv CAR structure,which has two single chain antibodies on the single CAR molecule,endows T cells with the ability to recognize and kill MM cells or MM tumor stem cells no matter a single or dual antigens expressed on.It provides a unique solution of CAR-T for improving the therapeutic efficacy prolonging the remission period and improving the long-term survival rate of r/r MM patients,To sum up,aiming at the two major clinical issues of CAR-T,in terms of safety risks and short duration of remission,two novel CAR-T technologies were designedand successfully developed: it is demonstrated that ssCART-19 cell with IL-6shRNA technology could significantly improve the CAR-T safety profiles without damaging the therapeutic efficacy,while tan-CART cell with two scFv molecules by tandemly linking two scFv to a single CAR molecule and demonstrated that it has ability to significantly kill the tumor cells with dual antigens.The results from these two research proposals may provide a unique and important strategies to help develop the novel CAR-T technologies to improve the safety risk of CART in the treatment of acute B-lymphocyte leukemia and improve the therapeutic efficacy and prolong the remission rate of multiple myeloma. |
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