Novel car design reversing the PD-1 signal boosts the efficacy of car-T cells with low cytokine release and no off-target effect

Abstract

Background & Aim Purpose The immunosuppressive tumor microenvironment (TIME) and cytokine release syndrome (CRS) hinder the progression of chimeric antigen receptor (CAR) T cell therapy. Strategies that block or reverse PD-1/PD-L1 inhibitory signals through CAR structural redesign to overcome TIME are extensively studied but still need be improved. Targeting the widely expressed PD-L1 without increasing the off-tumor effect while reduce cytokines release are critical challenges. Based on the above considerations, we engineered a novel bispecific CAR T cell type targeting CD19 and PD-L1 by incorporating the PD-1 extracellular domain next to the transmembrane domain (TMD) of CD19-targeting CAR domain, which we named EMD.PD-1 CAR. Methods, Results & Conclusion Experimental design In this study, we firstly engineered a novel CD19/PD-L1 bispecific CARs by incorporating the PD-1 domain into CD19 CAR and constructed second-generation CAR as control. Second, to assess the function of the novel anti-CD19 CAR T cells via antigen-specific stimulation, the antitumor functions, cytokine release, and off-target effects were studied in vitro and in vivo. We demonstrated that PD-L1 stimulation alone is not sufficient to activate EMD.PD-1 CAR T cells for the production of a antigen-specific immune response, including specific lysis, cytokines release, and proliferation. When challenged with CD19+PDL1+ tumor cells, EMD.PD-1 CARs showed a stronger specific immune response than 2nd CAR in vitro. Moreover, EMD.PD-1 CAR T cells exhibited only moderate specific cytokine release compared with 2nd CAR T cells when challenged with target tumor cells. In xenograft NOD/SCID/γc−/−(NSG) mouse model bearing CD19+PD-L1+ tumor cells, our results demonstrated that EMD.PD-1 CAR prolonged the overall survival and decreased the tumor burden compared with 2nd CAR. In addition, EMD.PD-1 CAR T cells showed a higher fraction of stem cell memory (TSCM) and central memory (TCM) T cell phenotype and lower PD-L1, TIM-3, CTLA-4, and intrinsic PD-1 expression in flow cytometry, CAR T cells demonstrated decreased differentiation and exhaustion marker expression. In conclusion, when faced with heterogeneous tumors with high expression of immunosuppressive signals, traditional second-generation CAR T cells are insufficient. The novel CAR, called EMD.PD-1 CAR with a low-avidity affinity to PD-L1 and moderate cytokine release can address this critical challenge.