Human platelet lysate enhances in vivo activity of CAR-Vd2 T cells by reducing cellular senescence and apoptosis

V?9Vd2 T cells are an attractive cell platform for the off-the-shelf cancer immunotherapy due to their lack of alloreactivity and inherent multi-pronged cytotoxicity, which could be further amplified with chimeric antigen receptors (CARs). In this study, we sought to enhance the in vivo longevity of CAR-Vd2 T cells by modulating ex vivo manufacturing conditions and selecting an optimal CAR costimulatory domain. Specifically, we compared the anti-tumor activity of Vd2 T cells expressing anti-CD19 CARs with costimulatory endodomains derived from CD28, 4-1BB or CD27 and generated in either standard fetal bovine serum (FBS)- or human platelet lysate (HPL)-supplemented medium. We found that HPL supported greater expansion of CAR-Vd2 T cells with comparable in vitro cytotoxicity and cytokine secretion to FBS-expanded CAR-Vd2 T cells. HPL-expanded CAR-Vd2 T cells showed enhanced in vivo anti-tumor activity with longer T cell persistence compared to FBS counterparts, with 4-1BB costimulated CAR showing the greatest activity. Mechanistically, HPL-expanded CAR Vd2 T cells exhibited reduced apoptosis and senescence transcriptional pathways compared to FBS-expanded CAR-Vd2 T cells and increased telomerase activity. This study supports enhancement of therapeutic potency of CAR-Vd2 T cells through a manufacturing improvement. Overall design: To investigate the transcriptomic differences of CAR-Vd2 T cells manufactured in FBS and HPL, we generated CD19.CAR(41BBz)-Vd2 T cells in either FBS- or HPL-supplemented media from four donors.