Abstract 6111: Reprogramming ROR1 CAR-T cells with SNIPR logic gating promote safety, while keeping anti-tumor efficacy in a tumor-bearing animal model

Abstract CAR-T cell therapies have shown promise in the treatment of cancers. However, off-target toxicities often occur due to heterogeneous and low-abundance target expression by normal tissues. ROR1 is a good tumor target being expressed by many tumor tissues and cells. But normal tissues also express a lower level of ROR1, which sometimes led to patient fatalities in clinical trials with ROR1 CAR-T. Here we developed and demonstrated a two-antigen recognition platform using SNIPR logic gating to control the cytotoxicity of ROR1 CAR-T cells, limiting them to the tumor site in vivo, and thus mitigating the risk of attacking normal vital tissues. Methods: Using SNIPR (synthetic intramembrane proteolysis receptors) (Zhu, et al. Cell, 185(8):1431-1443.e16, 2022), we engineered T cells with an inducible ROR1 VHH CAR expression that was controlled by an external sensor protein PD1. We then applied the PD1/SNIPR-based ROR1 CAR-T cells to a tumor-bearing animal model, which could recognize PDL1, commonly expressed in many tumor cells, and then turned on the CAR-mediated killings to those cells. Results: PD1-specific reporter induction of the SNIPR-based ROR1 CAR-T cells was confirmed in vitro using flow, IL2 production, and Incucyte’s real-time killing assays when co-culturing separately with PDL1-overexpressing MDA MB231 cells (ROR1+) as a positive control, and other ROR1+ tumor cell lines expressing variable levels of PDL1 at an E:T ratio of 1:1. In the native MDA MB231 and PD1-overexpressing MDA MB231 xenografted animal models, after we infused the CAR-T cells at 4x106 cells/mouse via the tail vein, we found that non-gated ROR1 CAR-T group showing a quicker tumor shrinkage starting D11 at a mean tumor size of 70mm3 when treating the native tumor. However, in the gated group treating the same native tumor we observed a delayed and steady decline of the tumor volume starting D15 (the mean 120mm3). Its shrinkage continued down to a mean tumor volume of 48mm3 D29, almost close to the size (the mean 49mm3) of the non-gated ROR1 CAR-T group D29, whereas the gated group treating the PD1-overexpressing MDA MB231 revealed a quick tumor shrinkage D15 (the mean 100mm3), leveled the tumor curve D22 (the mean 50mm3), and began to show the signs of GHVD on D25. Conclusion: We successfully demonstrated the therapeutic potential of our two-antigen recognition platform using SNIPR logic gating in an xenografted animal model. This platform enables us to reconfigure and develop safer and more effective CAR products suitable for clinical translation. Citation Format: Linyan Deng, Yuhua Wan, Jingyu Li, Jian Hua, Di Zhu, Hua Zhang. Reprogramming ROR1 CAR-T cells with SNIPR logic gating promote safety, while keeping anti-tumor efficacy in a tumor-bearing animal model [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 6111.