Bispecific CAR-iNKT cells: beyond dual antigen targeting Free

In this issue of Blood, Ren et al¹ report that bispecific chimeric antigen receptor (CAR) invariant natural killer T (iNKT) cells targeting CD19 and CD133 exhibit superior antileukemic activity compared with conventional CAR T cells in preclinical models of KMT2A-rearranged (KMT2Ar) leukemia, acting through both CAR-mediated and NKG2D-dependent mechanisms.

iNKT cells are a rare subset of innate lymphocytes that express a semi-invariant T-cell receptor (TCR) that recognizes glycolipids presented by the monomorphic, MHC-like molecule CD1d. Owing to their unique TCR composition and antigen recognition, iNKT cells do not induce graft-versus-host disease, thus positioning them as an ideal platform for the development of off-the-shelf cellular therapies. Beyond this safety profile, iNKT cells display potent antitumor activity, and several groups have successfully generated CAR-iNKT cells with robust antitumor properties in vitro and in xenograft models,^2,3 efforts that have now progressed into early-phase clinical trials.^4,5 

In the current study, Ren et al engineered iNKT cells to express 2 CARs targeting the pan–B-cell antigen CD19 and CD133 (PROM1), a stem cell marker directly regulated by KMT2A-fusion oncoproteins. Using rigorous in vitro and in vivo models of KMT2Ar leukemia, the authors demonstrated that CAR-iNKT cells mediated more potent antitumor activity than conventional CAR T cells and maintained such activity against antigen-low leukemic cells. Remarkably, bispecific CAR-iNKT cells eradicated leptomeningeal disease, a major clinical hurdle in KMT2Ar leukemia. This finding aligns with previous work from Rotolo et al showing that CAR-iNKT cells outperform CAR T cells in eradicating central nervous system lymphoma, according to their elevated expression of ITGA4 and ITGB1, encoding the integrin α4β1 (VLA-4), which facilitates adhesion and trafficking across the blood-brain and choroid plexus barriers.⁶ 

Mechanistic analyses presented in this study provide an additional explanation for the enhanced efficacy of CAR-iNKT cells. The authors observed robust CAR- and antigen-dependent upregulation of the innate activating receptor NKG2D on CAR-iNKT cells and demonstrated that engagement of NKG2D ligands expressed on KMT2Ar-ALL blasts contributed to CAR-iNKT cell antitumor activity (see figure). By leveraging both CAR antigens and NKG2D ligands, CAR-iNKT cells create a layered targeting platform for KMT2Ar-ALL and potentially other malignancies. Target antigen loss or downregulation is a well-recognized driver of relapse after CAR T-cell therapy. Here, the dual antigen recognition of CAR-iNKT cells, combined with the NKG2D-mediated killing, enabled effective targeting of leukemia cells with low antigen expression. Previous studies have also highlighted advantages of CAR-iNKT cells over conventional CAR T cells, including their ability to recognize CD1d and CAR antigens simultaneously⁶ and to cross-prime host T cells.^7,8 This study identifies NKG2D-dependent cytotoxicity as an additional mechanism favoring CAR-iNKT cells over conventional CAR T cells, further underscoring their expanding therapeutic versatility.

Despite these promising findings, important questions remain. The in vivo work relied on lymphopenic murine models, which preclude assessment of how CAR-iNKT cells interact with a fully competent immune system. In a clinical setting, managing host-versus-graft rejection and ensuring CAR-iNKT cell persistence will be critical. In B-cell acute lymphoblastic leukemia, long-term CAR T persistence and sustained B-cell aplasia correlate with durable remissions. Whether off-the-shelf CAR-iNKT products, expected to have shorter persistence due to rejection by the host immune system, can deliver similar long-term benefit remains unclear. Antirejection strategies, including further cellular engineering⁹ and/or pharmacological immunosuppression, may be needed to optimize the therapeutic window of CAR-iNKT therapy.

In summary, the work by Ren et al provides compelling evidence that bispecific CAR-iNKT cells targeting CD19 and CD133 can represent a powerful new approach for KMT2Ar leukemia. Beyond demonstrating potent preclinical efficacy, the study deepens our understanding of CAR-iNKT biology by uncovering a new NKG2D-dependent mechanism of action. These findings offer strong rationale for advancing iNKT-based cellular therapies in hematologic malignancies and for exploring NKG2D coengagement as a generalizable concept to overcome antigen escape in engineered cell therapies.

Conflict-of-interest disclosure: F.S. received institutional consulting fees from Bristol Myers Squibb (BMS)/Celgene, Incyte, and Kite/Gilead; speaker fees from Kite/Gilead and Incyte; travel support from Kite/Gilead, Novartis, AstraZeneca, Neovii, and Janssen; and research funding from Kite/Gilead, Novartis, and BMS/Celgene.