Off-the-shelf dual CAR-iNKT cell immunotherapy eradicates medullary and leptomeningeal high-risk KMT2A-rearranged leukemia.

Current therapies, including autologous CAR-T immunotherapy, fail to cure half of infants with KMT2A-rearranged acute lymphoblastic leukemia (KMT2Ar-ALL), a disease characterized by frequent central nervous system involvement, poor treatment response, early relapse and lineage switching. More effective treatment strategies, including the availability of 'off-the-shelf' immunotherapies is particularly relevant in infants. PROM1/CD133 is a direct target of KMT2A-fusion oncoproteins and is expressed on leukemic cells. Allogeneic iNKT cells, 'innately' more powerful effectors than T cells can be deployed 'off-the-shelf' without risk of acute graft-versus-host disease. Here, we equip iNKT with CD19- and/or CD133-targeting CARs and investigate their anti-leukaemia activity against KMT2Ar-ALL in relevant in vitro and in vivo models. Compared to mono-specific counterparts and dual, bi-specific CAR-T, bi-specific CD19-CD133 CAR-iNKT have a more potent anti-leukemia activity, effectively targeting both CAR antigen-high and -low leukemia. Bi-specific CAR-iNKT eradicate medullary and, notably, leptomeningeal leukemia and induce sustained remissions without discernible hematologic toxicity. Mechanistically, the more potent anti-leukemia effect of CAR-iNKT over CAR-T cells is mediated by a pronounced CAR- and CAR antigen-dependent upregulation of the innate activating receptor NKG2D on CAR-iNKT and its engagement by its corresponding ligands on KMT2Ar-ALL cells. This ensures effective leukemia targeting even with downregulation of CD133 or CD19. Thus, by engaging with two different types of leukemia-associated antigens i.e., CAR antigens and NKG2D ligands, CAR-iNKT provide a powerful platform for the treatment of KMT2Ar-ALL. This approach can be readily adapted for other high-risk malignancies, including those with otherwise difficult to target leptomeningeal involvement.