Abstract 6105: iPSC-derived HER2 CAR-iNKT cells enhance the activity of immune cells against cancer cells

Abstract Invariant natural killer T (iNKT) cells directly kill tumor cells through TCR and NKG2D, and also exert anti-tumor activity indirectly by promoting dendritic cell (DC) maturation, activating CD8+T cells and repolarizing macrophages. iNKT cells do not trigger graft versus host disease, making them an attractive cell type for an “off-the-shelf” cell therapy platform. We are developing iPSC-derived iNKT (ipsNKT) cell therapy to overcome manufacturing challenges due to the low abundance of iNKT cells in blood. It is not clear whether ipsNKT cells, especially when the cells are gene edited, still preserve multifunctional properties of native iNKT cells. In this study, we evaluated these intrinsic direct and indirect anti-tumor functions of iNKT cells using HER2-targeting CAR-ipsNKT cells. HER2-CAR-ipsNKT cells were differentiated from HER2 CAR-edited iPS cell line under a feeder cell-free condition and exerted potent cytotoxicity when cultured with HER2 positive HCC1954-CMVpp65 cells. HER2-CAR-ipsNKT cells released cytokines including IL-4, TNF-α and IFN-γ upon CAR-stimulation by HER2 antigen. After co-culturing monocyte derived immature DCs with HER2-CAR-ipsNKT cells, the expression of DC maturation markers like CD80, CD83, CD86 and HLA-DR were markedly upregulated, suggesting that HER2-CAR-ipsNKT cells promote DC maturation. When monocytes were cultured in conditioned medium from HER2-CAR-ipsNKT cells, polarization of M1 macrophages was observed by detecting expression of the M1 macrophages markers using flowcytometry. Intriguingly, conditioned medium of HER2-CAR-T cells did not show the same result. To evaluate whether HER2-CAR-ipsNKT cells can promote cytotoxic T lymphocyte (CTL) activity in vivo, HCC1954-CMVpp65 cells were inoculated subcutaneously into NOG-hIL15Tg mice. After the tumor volumes reached 100∼200 mm3, 1 × 105 PBMCs were injected intratumorally, followed by intravenous doses of HER2-CAR-ipsNKT cells. Initially, all animals showed comparable tumor growth, however, the tumor xenografts in the HER2-CAR-ipsNKT cell treatment group started to regress after 3 to 4 weeks of treatment, suggesting an indirect anti-tumor contribution of HER2-CAR-ipsNKT cell by activating-tumor specific CTLs. This speculation is supported by the observation that the CD8+T cells and CMVpp65 tetramer+ cells were increased in both spleen and tumor of the treatment group. This study demonstrated that HER2-CAR-ipsNKT cells promote DC maturation and M1 macrophage polarization, likely due to cytokine release from HER2-CAR-ipsNKT cells as a result of CAR-stimulation, and also activate tumor antigen-specific CD8+T cells, thereby enhancing anti-tumor effect in vivo. These results suggest that administration of iPSC-derived CAR-iNKT cells to a patient can improve tumor microenvironment and activate the host endogenous tumor-specific CTLs, potentially achieving a durable therapeutic response. Citation Format: Koichiro Shioya, Tomio Matsumura, Akane Urakami, Tomokuni Shigeura, Aki Naito, Yu-Ching Lin, Mio Shiraishi, Yuji Mishima, Haruhiko Koseki, Shinichiro Motohashi, Lilin Zhang. iPSC-derived HER2 CAR-iNKT cells enhance the activity of immune cells against cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6105.