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Nanobodies (Nbs) have emerged as promising antigen\u2010targeting domains for CARs, owing to their high specificity, robust stability, and strong affinity, leading to significant advancements in the field of Nb\u2010CAR\u2010T. In the realm of T\u2010cell acute lymphoblastic leukemia (T\u2010ALL) targets, CD5 stands out as a potentially excellent candidate for T\u2010cell\u2010based CAR therapy, due to its distinct expression on the surface of malignant T\u2010ALL cells. To mitigate graft\u2010versus\u2010host disease associated with allogeneic CAR\u2010T, \u03b3\u03b4T cells are selected and stimulated from peripheral blood mononuclear cells, and \u03b3\u03b4T cells are engineered via CRISPR\/Cas9 to eliminate fratricide, enabling the creation of fratricide\u2010resistant CAR\u2010\u03b3\u03b4T<jats:sup>CD5\u2212<\/jats:sup> cells. In vitro transcribed (IVT) mRNA is used to construct CAR\u2010T, presenting a safer, faster, and cost\u2010effective method compared to traditional viral vector approaches. In this study, a CD5\u2010VHH library is constructed, and specific CD5\u2010nanobodies are screened for subsequent use in CD5\u2010CAR\u2010\u03b3\u03b4T<jats:sup>CD5\u2212<\/jats:sup> therapy. IVT\u2010mRNA\u2010CD5\u2010CAR\u2010\u03b3\u03b4T<jats:sup>CD5\u2212<\/jats:sup> cells exhibited favorable functional characteristics and demonstrated antitumor efficacy against malignant T cell lines, underlining the potential for advancing mRNA\u2010CD5\u2010CAR\u2010\u03b3\u03b4T<jats:sup>CD5\u2212<\/jats:sup> therapy.<\/jats:p>","DOI":"10.1002\/advs.202400024","type":"journal-article","created":{"date-parts":[[2024,7,16]],"date-time":"2024-07-16T15:15:46Z","timestamp":1721142946000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":18,"title":["mRNA\u2010Engineered CD5\u2010CAR\u2010\u03b3\u03b4T<sup>CD5\u2010<\/sup> Cells for the Immunotherapy of T\u2010Cell Acute Lymphoblastic Leukemia"],"prefix":"10.1002","volume":"11","author":[{"given":"Zhixiong","family":"Zhu","sequence":"first","affiliation":[{"name":"State Key Laboratory of Biotherapy and Cancer Center Collaborative Innovation Center of Biotherapy West China 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