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Background Chimeric antigen receptor (CAR)-modified invariant natural killer T (iNKT) cells have shown encouraging results in clinical trials for cancer and autoimmune diseases. Their unique tumor-targeting properties, strong infiltration into solid tumors, and ability to modulate the tumor microenvironment (TME) make them a promising therapeutic approach for solid tumor treatment. In addition, their inability to induce graft-versus-host disease (GvHD) makes them well-suited for allogeneic, off-the-shelf cell therapy applications.Methods Two CAR constructs targeting CD19 and GPC3 were independently developed to assess their therapeutic efficacy. To evaluate the ability of CD19 CAR-iNKT cells in eliminating malignant B cells, 1×10 6 Nalm6 cells per mouse were intravenously injected into NSG mice, allowed to engraft for two days, and subsequently treated with one or three injections of 1×106 CAR+ iNKT cells per mouse on days 2, 4, and 6, respectively. In contrast, the efficacy of GPC3 CAR-iNKT modified with PD-1 co-deletion was tested in an IL-15-producing NSG mouse xenograft model by implanting 1×106 Hep3B cells per mouse three days prior to treatment with three tail vein injections of 18×106 GPC3 CAR-iNKT cells (50% CAR+) or untransduced iNKT cells on days 3, 6, and 9.Results Compared to unmodified iNKT cells and single injections of CD19 CAR-iNKT, three doses led to significant clearance of CD19-positive Nalm6 cells in bioluminescent imaging ( figure 1), highlighting its potential for treating B-cell malignancies and autoimmune diseases such as ALL, CLL, and SLE, etc. Whereas in the Hep3B xenograft model, only GPC3 CAR-iNKT with PD-1 deletion, but not conventional GPC3 CAR-iNKT, induced tumor shrinkage starting day 10 and achieved complete tumor elimination by day 24 and 31 without producing GvHD (figure 2).Conclusions These results demonstrate that iNKT cells offer a highly effective allogeneic platform for CAR-based immunotherapy in both cancer and autoimmune diseases, providing a strong foundation for further clinical evaluation.Ethics Approval The study was approved by Medicilon’s Ethics Board, approval number SWSH(YF)2025-034.Abstract 300 Figure 1Nalm6 tumor growth, directly correlated with color intensity, was in different treatment groups and was monitored by BLI as days indicatedAbstract 300 Figure 2Hep 3B cells were inoculated subcutaneously at 1×106 into the NSG mice. On the day 3, 6 and 9 after the inoculation, the mice were dosed with 18x106/mouse of the cells (CAR+≈50%) via the tail vein each time