Bispecific CD37–Siglec6 CAR‑T cells exhibit enhanced anti‑leukemic activity and reduced exhaustion in acute myeloid leukemia xenograft models

Relapsed/refractory pediatric acute myeloid leukemia (AML) remains a significant therapeutic challenge, largely due to the persistence of leukemia stem cells (LSCs), antigen heterogeneity and off-tumor toxicity. Siglec6 and CD37 are highly expressed in AML LSC subpopulations. To enhance specificity and reduce relapse risk, we explored the efficacy of a bispecific CD37–Siglec6 chimeric antigen receptor (CAR) T-cell strategy, where both receptors are enriched on AML LSCs. Single-cell RNA sequencing data from the GEO dataset and Bulk RNA-sequencing were subsequently obtained from newly diagnosed, remission, and relapsed patients’ AML bone marrow samples. Meanwhile, Siglec6 CAR, CD37 CAR, and CD37–Siglec6 bispecific CAR constructs were prepared. CAR genes were synthesized and cloned into lentiviral vectors, which were then packaged in 293T cells. Lentivirus was transduced to generate Siglec6 CAR-T, CD37 CAR-T, and CD37–Siglec6 CAR-T cells. In vitro assays were used to assess CAR expression, immunophenotype, cytotoxicity against U-937 cells, and cytokine secretion. In vivo efficacy was evaluated in an NSG mouse model engrafted with luciferase‑U-937 cells. Siglec6 was highly expressed in erythroid progenitors and malignant blasts, which was correlated with relapse and showed potential interaction with CD37 via protein‑network analysis. Compared with single‑target CAR constructs, the bispecific CD37–Siglec6 CAR‑T cells exhibited higher transduction efficiency (52.8%) and expansion (145‑fold). Furthermore, these bispecific CAR-T cells displayed an enriched central and effector memory phenotype, reduced TIM‑3 expression, and superior cytotoxicity in vitro (87.8% specific killing at 48 h), accompanied by elevated IFN‑γ, TNF‑α and IL‑6 secretion. In vivo, CD37‑Siglec6 CAR‑T cells achieved the most potent tumor control, prolonged median survival (60 days), and they demonstrated enhanced persistence in the bone marrow, spleen and peripheral blood, while showing few signs of acute toxicity. Bispecific CD37–Siglec6 CAR‑T cells exhibit enhanced anti‑AML activity, improved persistence, and a favorable safety profile in preclinical models. Importantly, these bispecific CD37–Siglec6 CAR-T cells demonstrated enhanced preclinical efficacy against AML cell lines without significantly affecting normal hematopoiesis, providing a promising therapeutic strategy for pediatric AML.