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Abstract

Despite the encouraging outcome of anti-CD19 chimeric antigen receptor T (CAR T) cell therapy in patients with B cell malignancies, CAR T cell persistence remains a major clinical challenge. CAR T cells highly express death receptor CD95 (also known as Fas), which might be sensitive to CD95L-mediated apoptosis and thus impair CAR T cell persistence. Therefore, in this thesis, it was investigated whether disruption of CD95-CD95L signaling within CAR T cells would prevent CAR T cells from CD95L-mediated apoptosis and improve their persistence. In this study, third-generation CD19.CAR T cells generated from healthy donors expressed high levels of CD95 and were susceptible to CD95L-mediated cell death. CD95 ligand (CD95L) was also expressed on CAR T cells upon tumor stimulation, resulting in activation-induced cell death in CAR T cells via binding to CD95 receptor. The CD95L inhibitor APG101, a soluble CD95-Fc fusion protein, protected CAR T cells from activation-induced cell death through blockade of the CD95-CD95L pathway. CD95L blockade enhanced the survival of CAR T cells after repetitively being challenged by CD19 expressing target tumor cell lines, and promoted killing of tumor cells in vitro. In addition, Fas knockout CD19.CAR T cells generated via electroporation-based CRISPR-Cas9 displayed high targeted efficiency and were resistant to CD95L-mediated apoptosis. Moreover, Fas knockout CD19.CAR T cells exhibited prolonged persistence upon repeated antigen stimulation and superior killing efficiency against CD19+ tumor cells. In conclusion, disruption of the CD95-CD95L pathway in CD19.CAR T cells through pharmacologic and genetic approaches prevents CAR T cells from CD95L-mediated apoptosis and improves their persistence, resulting in enhanced antitumor efficacy of these CAR T cells. Thus, combining CAR T cell therapy with CD95L inhibitor might improve CAR T cell persistence in vivo and thus enhance the effect of CAR T cell therapy in patients with relapsed or refractory hematologic malignancies.