mRNA‐Engineered CD5‐CAR‐γδT<sup>CD5‐</sup> Cells for the Immunotherapy of T‐Cell Acute Lymphoblastic Leukemia
Zhixiong Zhu, Hexian Li, Qizhong Lu, Zongliang Zhang, Li Jia, Zeng Wang, Nian Yang, Zhengyu Yu, Yang Chen, Yongdong Chen, Huaqing Lu, Wei Wang, Ting Niu, Chunlai Nie, Aiping Tong
Abstract
Abstract Clinical trials of Chimeric Antigen Receptor T‐cell (CAR‐T) therapy have demonstrated remarkable success in treating both solid tumors and hematological malignancies. Nanobodies (Nbs) have emerged as promising antigen‐targeting domains for CARs, owing to their high specificity, robust stability, and strong affinity, leading to significant advancements in the field of Nb‐CAR‐T. In the realm of T‐cell acute lymphoblastic leukemia (T‐ALL) targets, CD5 stands out as a potentially excellent candidate for T‐cell‐based CAR therapy, due to its distinct expression on the surface of malignant T‐ALL cells. To mitigate graft‐versus‐host disease associated with allogeneic CAR‐T, γδT cells are selected and stimulated from peripheral blood mononuclear cells, and γδT cells are engineered via CRISPR/Cas9 to eliminate fratricide, enabling the creation of fratricide‐resistant CAR‐γδT CD5− cells. In vitro transcribed (IVT) mRNA is used to construct CAR‐T, presenting a safer, faster, and cost‐effective method compared to traditional viral vector approaches. In this study, a CD5‐VHH library is constructed, and specific CD5‐nanobodies are screened for subsequent use in CD5‐CAR‐γδT CD5− therapy. IVT‐mRNA‐CD5‐CAR‐γδT CD5− cells exhibited favorable functional characteristics and demonstrated antitumor efficacy against malignant T cell lines, underlining the potential for advancing mRNA‐CD5‐CAR‐γδT CD5− therapy.