EF-1α 驱动的纳米抗体基 CD19 重定向 CAR-T 细胞保留 CMV 驱动对应细胞的抗肿瘤疗效、抗原驱动扩增与细胞因子分泌

Chimeric antigen receptor (CAR) T-cell efficacy requires sustained transgene expression. Cytomegalovirus (CMV) promoters drive strong initial expression but undergo silencing, while elongation factor-1 (EF-1 ) promoters resist inactivation.

This study evaluated whether transitioning a previously established VHH-based CD19-redirected CAR construct to EF-1 promoter control compromises antitumor functionality. Lentiviral particles encoding identical second-generation CAR constructs (CD19-specific VHH, CD8 transmembrane, 4-1BB costimulatory, CD3 signaling domains) under CMV (HCAR-T) or EF-1 (ECAR-T) promoters transduced primary human T cells. Cytotoxicity (effector-to-target ratios 1:1-10:1), antigen-driven proliferation, and cytokine secretion against CD19 /CD19 targets were quantified. Promoter substitution from CMV to EF-1 in the matched VHH-based CAR construct did not alter CAR-T cell functionality.

ECAR-T and HCAR-T cells exhibited statistically comparable cytotoxicity against CD19 Raji (81. 6% 2. 4 versus 81. 8% 2. 5 at 10:1, respectively) and NALM6 cells (82. 2% 2. 3 versus 80. 7% 3. 5 at 10:1, respectively). Proliferation against Raji (ECAR-T: 94. 2% 7. 54 versus HCAR-T: 91. 1% 6. 03) and NALM6 (ECAR-T: 94. 9% 4. 35 versus HCAR-T: 92. 5% 6.

34) was comparable. IFN- , IL-2, and TNF- secretion against CD19 targets was statistically comparable between ECAR-T and HCAR-T groups and strictly CD19-dependent. EF-1 preserved the antitumor potency of nanobody-based CD19-redirected CAR-T cells, supporting its use as a clinically relevant promoter for further development of this VHH-based CAR-T cell platform.