Research on allogeneic CAR-T.BiTE cells targeting CLL-1/siglec-6 for the treatment of Acute Myeloid Leukemia

Abstract

Abstract 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.