280 Novel AND-gated LINK CAR-T cells demonstrate improved safety compared to 2nd-generation CAIX CAR-T cells for the treatment of clear cell renal cell carcinoma

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Background Safety of chimeric antigen receptor (CAR) targets, such as CAIX, in solid tumors can be compromised by target expression in normal tissues.1 High-fidelity AND-gated CAR-T cells (LINK CAR-T) can overcome this issue. Here we de-risk CAIX as a target for clear cell renal cell carcinoma (ccRCC). We identified a second target antigen (Target B) on ccRCC which displays high tumor-specific expression, with a different subset of healthy tissue liabilities. Combined, co-expression of these antigens is limited to ccRCC tumor. We hypothesized concomitantly targeting both antigens with LINK CAR-T cells could eliminate on-target, off-tumor toxicity as a safe and efficacious therapeutic. Methods LINK CAR design co-opts the combined downstream T cell signaling proteins, SLP76 and LAT.2 Potential target pairs were rigorously evaluated using a proprietary combination of in silico and functional cell and tissue screening tools. For ccRCC, the SLP76 arm is directed against CAIX, and the LAT arm binder recognizes Target B. Subsequently, in vitro functional experiments targeted RCC cell lines expressing both targets and cell lines expressing each single antigen to mimic expression on healthy tissue. Xenograft mouse models of RCC with dual-positive and single-positive antigen-expressing tumors were utilized to evaluate efficacy and safety within the same animal. In addition, we employed an AAV vector to induce mouse tissue expression of CAIX prior to engrafting dual-antigen positive tumor to demonstrate anti-tumor efficacy and safety of single-antigen positive healthy tissues. Results Against dual-positive targets, LINK CAR-T cells show substantial cytokine secretion and cytotoxicity in vitro. However, when only one antigen was expressed, LINK CARs did not display cytotoxicity. In contrast, 2nd-generation CAIX CAR-Ts showed activity in both settings. In xenograft mouse models, LINK CARs showed efficacy against dual-positive tumors but not single-antigen expressing tumors whereas both were controlled with 2nd-generation CARs. In the AAV xenograft model with healthy tissue expression of CAIX, LINK CAR-T cells shrank subcutaneous tumor without targeting normal tissues, whereas 2nd-generation CAR-T cells mirrored clinical toxicity profiles and caused severe liver toxicity. Conclusions Rigorous selection of potential CAR target pairs, coupled with high fidelity [AND]-logic gated LINK CAR-T cells, can result in safe and effective tumor control in in vitro and in vivo models of ccRCC with a target that previously has shown clinical on-target, off-tumor toxicity. We demonstrate the non-clinical utility of novel AND-gated CAR-T cells as a potential therapeutic for ccRCC. References Lamers CH, Sleijfer S, van Steenbergen S, van Elzakker P, van Krimpen B, Groot C, Vulto A, den Bakker M, Oosterwijk E, Debets R, Gratama JW. Treatment of metastatic renal cell carcinoma with CAIX CAR-engineered T cells: clinical evaluation and management of on-target toxicity. Mol Ther. 2013 Apr;21(4):904–12. doi: 10.1038/mt.2013.17. Epub 2013 Feb 19. PMID: 23423337; PMCID: PMC5189272. Tousley AM, Rotiroti MC, Labanieh L, Rysavy LW, Kim WJ, Lareau C, Sotillo E, Weber EW, Rietberg SP, Dalton GN, Yin Y, Klysz D, Xu P, de la Serna EL, Dunn AR, Satpathy AT, Mackall CL, Majzner RG. Co-opting signalling molecules enables logic-gated control of CAR T cells. Nature. 2023 Mar;615(7952):507–516. doi: 10.1038/s41586-023-05778-2. Epub 2023 Mar 8. PMID: 36890224; PMCID: PMC10564584.

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