Nanobody-Engineered CLL-1 CAR T Cells: Optimizing Tumor-Specific Cytotoxicity and Minimizing Off-Tumor Toxicity

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by the rapid expansion of undifferentiated myeloid progenitors, leading to impaired hematopoiesis and poor patient prognosis. Although chimeric antigen receptor (CAR) T-cell therapy using single-chain variable fragments has revolutionized immunotherapy, clinical application in AML remains limited by on-target, off-tumor toxicities, largely due to shared antigen expression on normal hematopoietic stem and progenitor cells. To address this challenge, we developed a nanobody-based CAR T-cell platform directed against C-type lectin-like molecule-1 (CLL-1), a myeloid-restricted surface antigen minimally expressed on healthy hematopoietic stem cells but consistently enriched on AML blasts and leukemic stem cells. Leveraging the high specificity, solubility, and reduced immunogenicity of llama-derived single-domain variable heavy-chain antibodies, we engineered both CLL-1 and CD33 nanobody CAR constructs and systematically compared their functional activity. Functional validation included real-time cytotoxicity monitoring using IncuCyte imaging of mKate2-labeled AML cells, serial tumor rechallenge assays to assess sustained killing, and NOD/SCID/IL2Rγnull xenograft models to evaluate in vivo efficacy under conditions of high leukemic burden. CLL-1 and CD33 CAR T cells demonstrated rapid and durable cytotoxicity, with significant killing efficiency at low effector-to-target ratios (0.33:1). Unlike CD33 CAR T cells, CLL-1-directed CARs spared normal hematopoietic progenitors, preserving colony-forming capacity. Importantly, CLL-1 CAR T cells retained a favorable memory phenotype with stable proliferation and viability, whereas cytokine release assays confirmed effective yet antigen-specific immune activation. In vivo, treatment with CLL-1 CAR T cells resulted in profound and sustained tumor regression in AML xenografts, accompanied by the persistence of functional CAR T cells. Together, these findings establish CLL-1-targeted nanobody-based CAR T cells as a precision-engineered immunotherapy with potent antileukemic activity, reduced off-target toxicity, and enhanced translational potential. This platform provides a promising therapeutic avenue to overcome current barriers in AML CAR T-cell development and improve patient outcomes. SIGNIFICANCE: Nanobody-based CLL-1 CAR T-cell therapy balances potent antitumor activity with hematopoietic preservation, highlighting the potential of our CLL-1 CAR T-cell platform as a next-generation, safer, and clinically superior strategy for effective AML treatment.