Cellular immunotherapy has transformed the treatment of hematologic malignancies, yet acute myeloid leukemia (AML) remains stubbornly refractory. Key obstacles include extreme antigen heterogeneity, a paucity of safe targets, an immunosuppressive micro-environment, and on-target toxicity to normal hematopoietic stem cells (HSCs). Current AML-directed CAR-T cells that focus on CD33 or CD123 frequently induce life-threatening pancytopenia.

We therefore engineered allogeneic CAR-T cells that co-target CLL-1 and Siglec-6 two antigens highly expressed on AML blasts and leukemia stem cells but absent from HSCs. Nanobodies against CLL-1 and Siglec-6 were isolated by phage display and integrated into a next-generation construct: UCART-CLL1.BiTE-Siglec6. These “off-the-shelf” CAR-T cells express a CLL-1-directed CAR while continuously secreting a Siglec-6×CD3 bispecific T-cell engager (BiTE).

Compared with CLL-1-only UCAR-T cells, UCART-CLL1.BiTE-Siglec6 proliferate more robustly, display superior persistence, and exert markedly stronger cytotoxicity against AML cells co-expressing CLL-1 and Siglec-6. The autocrine BiTE recruits endogenous T cells to eradicate Siglec-6+ blasts that might otherwise escape through antigen heterogeneity, thereby enhancing overall anti-leukemic efficacy without compromising HSCs. Our dual-target, BiTE-armored allogeneic platform offers a promising new strategy for safer and more effective AML therapy.

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2025
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