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Background Aggressive T cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis and high rates of relapse and refractory cases, with long-term survival rates of <10%, highlighting a need for alternative therapies. CD5 is an attractive target for CAR-based therapy in T-ALL. However, the high CD5 expression in normal and malignant T cells makes difficult to use CAR T cells as therapy due to the induction of fratricide or product contamination with malignant cells during cell manufacturing. NK cells are innate immune cells with potent anti-tumor properties. NK cells are CD5-negative and have been shown to be safe in clinical trials, making them a suitable therapy for treating T-ALL.Methods In-house developed single-chain variable fragment (scFV) and domain-only (VL) CD5 binders were formatted to into a second generation CAR (CD8hinge-TM, 41BB-CD3zeta) and baboon envelope pseudotyped lentivirus vectors generated to transduce primary NK cells. Anti-CD5 CAR-NK cells were expanded in vitro for up to 2 weeks and their functionality assessed in killing assays. Cytokine production was assessed after coculture with target cells. Safety of anti-CD5 CAR NK cells against T cells was investigated in in vitro cocultures of healthy T:anti-CD5 CAR NK cells. In vivo efficacy of anti-CD5 CAR NK cells was evaluated using a xenograft model in NSG mice.Results CAR expression of most of the CD5 binders was efficient and did not compromise cell expansion. In killing assays, two scFV binders and four VL binders showed the highest killing activity against CD5+ tumor cell lines and primary T-ALL samples. IFN-g, TNF and GM-CSF secretion were minimal in unstimulated NK cells but upregulated after coculture with targets. Granzyme and perforin were also upregulated upon coculture with targets. Despite a binder-specific initial transient drop in T cell expansion, most T cells expanded well in the presence of anti-CD5 CAR NK cells. In contrast to scFV binders, VL did not affect CD5 expression in T cells. When tested in vivo, anti-CD5 CAR NK cells maintained reduced tumor burden compared to untreated mice reaching a 2-Log decrease in bioluminescent imaging signal by day 23 post tumor implantation.Conclusions Anti-CD5 CAR NK cells are effective in vitro and in vivo against T-ALL targets. Anti-CD5 CAR expression did not compromised NK cell expansion and showed good but binder-dependent safety profile against T cells. These data demonstrate that anti-CD5 CAR NK cells have potent anti-tumor activity against T-ALL and warrants further investigation in early clinical trials