Acute myeloid leukemia (AML) faces challenges due to the disease heterogeneity and a lack of tumor-specific antigens. CLEC12A (C-type lectin-like molecule-1) is a promising target for chimeric antigen receptor (CAR)-based immunotherapy, as it is expressed on leukemic blasts and leukemia-initiating cells in up to 92% of AML patients while being absent on hematopoietic stem cells. Furthermore, CAR-engineered natural killer (NK) cells offer an “off-the-shelf” therapeutic option, leveraging CAR-mediated cytotoxicity and innate receptor activity.

This study aimed to improve the anti-leukemic activity of CLEC12A-CAR-NK cells through the selection and implementation of a novel VꮋH binding domain. Llama immunization using VLPs followed by yeast surface display (YSD) screening successfully identified AC12VHH4, a high-affinity CLEC12A-specific VꮋH domain. Following implementation into the CAR and NK cell transduction, VꮋH-based CLEC12A-CAR NK cells exhibited superior tumor cell lysis compared to their scFv-based counterparts and non-transduced NK cells. Enhanced anti-leukemic activity was particularly prominent at lower effector-to-target (E:T) cell ratios. In the OCI-AML2 in vivo xenograft model treatment with VꮋH-based CLEC12A-CAR NK cells inhibited leukemia progression and improved survival. Use of the novel VꮋH domain AC12VHH4 enables potent tumor cell elimination by CLEC12A-CAR NK cells, particularly at low E:T ratios. Thereby, the VꮋH identified in this study constitutes a more effective binding domain for the generation of CLEC12A-CAR NK cells than a scFv domain derived from therapeutic antibody Tepoditamab. Importantly, VꮋH-CLEC12A CAR-NK cells also demonstrated potent in vivo efficacy in an OCI-AML2 xenograft model, highlighting the potential impact of this new CAR-based therapeutic concept for AML immunotherapy.