A novel nanobody-based TCR-like CAR-T therapy targeting PRAME for the treatment of acute myeloid leukemia

PURPOSE: Chimeric antigen receptor (CAR) T-cell immunotherapy in acute myeloid leukemia (AML) remains challenging due to the lack of specific cell surface antigens that are highly expressed on leukemic blasts but largely absent in hematopoietic stem/progenitor cells (HSPCs) and healthy tissues. Targeting intracellular antigen via TCR-like CAR-T cells offers a promising alternative. This study aimed to develop nanobodies targeting the intracellular antigen PRAME and develop a novel nanobody-based TCR-like CAR-T cell therapy. EXPERIMENTAL DESIGN: We investigated PRAME expression level by analyzing the RNA sequencing data from 1,007 AML patients and healthy donor samples. We explored the relationships between PRAME expression and the prognosis of AML. Novel nanobodies targeting PRAME425-433/HLA-A2 were generated via alpaca immunization and yeast surface display, then used to construct TCR-like CAR-T cells. The antileukemia potency of the PRAME-targeted TCR-like CAR-T cells and their on-target/off-tumor toxicity against normal HSPCs were evaluated. RESULTS: PRAME was highly expressed in AML cells but largely absent in normal hematopoietic cells and healthy tissues, and is correlated with poor clinical outcomes in AML. The CAR-T cells based on the nanobody targeting PRAME425-433/HLA-A2 exhibited specific and potent anti-leukemic cytotoxicity against PRAME+HLA-A2+ AML cells in vitro and in vivo, whereas they showed negligible effects on the viability and function of normal HSPCs. CONCLUSIONS: This study demonstrates that PRAME is a promising target for the immunotherapy of AML, and nanobody-based TCR-like CAR-T cells targeting PRAME425-433HLA-A2 exhibit potent anti-leukemic activity with a favorable off-target safety profile.