非病毒 TRAC 敲入 CD19(KI)CAR-T 与 gp350(KI)CAR-T 细胞针对 1 型或 2 型 EBV 感染伯基特淋巴瘤的测试：体内细胞动力学与效力

We used CRISPR/Cas9 gene editing methods to knock in (KI) the CD19CAR.CD28z or gp350CAR.CD28z into the T cell receptor (TCR) alpha chain ( TRAC ) locus.

Applying upscaled methods with the ExPERT ATx MaxCyte system, KI efficacy was ~20% of the total ~2 10 8 TCR-knocked-out (KO) generated cells. KO TCR KI CAR-T cells were co-cultured in vitro with the gp350 + CD19 + BL cell lines Daudi (infected with type 1 EBV) or with Jiyoye (harboring a lytic type 2 EBV). Both types of CAR-T cells showed cytotoxic effects against the BL lines in vitro . CD8 + KI CAR-T cells showed higher persistency than CD4 + KI CAR-T cells after in vitro co-culture with BL and upregulation of the activation/exhaustion markers PD-1, LAG-3, and TIM-3. Two preclinical in vivo xenograft models were set up with Nod.Rag.Gamma mice injected intravenously (i.v.) with 2 10 5 Daudi/fLuc-GFP or with Jiyoye/fLuc-GFP cells. Compared with the non-treated controls, mice challenged with BL and treated with CD19 KI CAR-T cells showed delayed lymphoma dissemination with lower EBV DNA load. Notably, for the Jiyoye/fLuc-GFP model, almost exclusively CD4 + CD19 KI CAR-T cells were detectable at the endpoint analyses in the bone marrow, with increased frequencies of regulatory T cells (T regs ) and TIM-3 + CD4 + T cells. Administration of gp350 KI CAR-T cells to mice after Jiyoye/GFP-fLuc challenge did not inhibit BL growth in vivo but reduced the EBV DNA load in the bone marrow and promoted gp350 antigen escape. CD8 + PD-1 + LAG-3 + gp350 KI CAR-T cells were predominant in the bone marrow. DISCUSSION: The two types of KO TCR KI CAR-T cells showed different therapeutic effects and in vivo dynamics. These findings reflect the complexities of the immune escape mechanisms of EBV, which may interfere with the CAR-T cell property and potency and should be taken into account for future clinical translation.