Harnessing allogeneic CAR‑iNKT cells for next‑generation treatments in oncology and autoimmunity

Background: Chimeric antigen receptor (CAR)-engineered invariant natural killer T (iNKT) cells have demonstrated promising outcomes in clinical studies for both cancer and autoimmune disorders. Their distinctive ability to recognize and eliminate tumor cells, coupled with efficient infiltration into solid tumors and modulation of the tumor microenvironment (TME), positions them as a compelling therapeutic strategy for solid malignancies. Moreover, because iNKT cells do not trigger graft-versus-host disease (GvHD), they are particularly well-suited for allogeneic, off‑the‑shelf cell therapy approaches. Methods: Two CAR constructs targeting CD19 and GPC3 were independently generated to evaluate therapeutic efficacy. For CD19 CAR‑iNKT cells, NSG mice were intravenously inoculated with 1×10⁶ Nalm6 cells per mouse, allowed to engraft for two days, and subsequently treated with either a single dose or three sequential doses of 1×10⁶ CAR⁺ iNKT cells per mouse administered on days 2, 4, and 6. In parallel, the activity of GPC3 CAR‑iNKT cells engineered with PD‑1 deletion was assessed in NOG‑hIL15Tg mice. Each mouse was implanted with 1×10⁶ Hep3B cells three days prior to treatment and then received three tail‑vein injections of 18×10⁶ GPC3 CAR‑iNKT cells (50% CAR⁺) or untransduced iNKT cells on days 3, 6, and 9. Results: In bioluminescent imaging, three sequential doses of CD19 CAR‑iNKT cells achieved marked clearance of CD19‑positive Nalm6 cells compared with unmodified iNKT cells or a single CD19 CAR‑iNKT injection, underscoring their therapeutic potential in B‑cell malignancies and autoimmune disorders such as ALL, CLL, and SLE. In contrast, within the Hep3B xenograft model, only GPC3 CAR‑iNKT cells engineered with PD‑1 deletion,rather than conventional GPC3 CAR‑iNKT cells induced tumor regression beginning on day 10, ultimately achieving complete elimination by days 24 and 31 without evidence of graft‑versus‑host disease. Conclusions: These findings highlight iNKT cells as a potent allogeneic platform for CAR-based immunotherapy, with broad applicability across cancer and autoimmune diseases, thereby establishing a robust basis for continued clinical investigation. Ethics Approval: The study was approved by Medicilon's Ethics Board, approval number SWSH(YF)2025-034.