Abstract 6319: Characterization of AB-1015 logic-gated CAR induction (ON kinetics), receptor turnover (OFF kinetics), and logic gate sensitivity to ALPG/P and MSLN

Abstract

Abstract CAR T cell activity in solid tumors is limited, in part, by off-tumor toxicity. To address this obstacle, AB-1015 contains an “AND” logic gate, consisting of a priming receptor (PrimeR) and an inducible chimeric antigen receptor (CAR) that is upregulated by PrimeR activation. The AB-1015 logic gate targets ALPG/P and MSLN, which are coexpressed in ovarian tumors but not normal tissues. AB-1015 CAR induction within the tumor is expected to be a dynamic process influenced by multiple factors, including antigen density, target cell abundance, as well as tumor microenvironmental factors. To characterize CAR induction and receptor turnover, we developed an in vitro assay with plate-bound ALPG to induce CAR. CAR induction occurred quickly in response to ALPG priming, with most ICT cells expressing CAR within 24 hrs, and plateaued by 72 hrs. Once removed from ALPG, CAR expression returned to baseline within 96 hrs. Similar to conventional CAR T cells, the presence of the cytolytic target antigen (MSLN) was expected to increase the rate of CAR turnover on AB-1015. To assess this, we measured the CAR functional half-life of AB-1015 cells once removed from ALPG priming. The cytotoxic activity of AB-1015 cells rapidly decreased in the absence of ALPG, reaching baseline within 24 hrs. Of note, our in vitro assay did not result in 100% CAR induction from all PrimeR+ AB-1015 cells. To understand whether this reflected biology versus a limitation of our in vitro assay, we conducted a CAR induction, sorted the CAR- cells, then performed a second induction. ICT cells that did not express CAR after the first induction were shown to be able to induce CAR during the second induction assay, thereby demonstrating that all PrimeR+ ICT cells have the potential to induce CAR. To assess the sensitivity of AB-1015 for low density ALPG/P and MSLN, isogenic cell lines were engineered to express each antigen at variable levels. In co-culture, AB-1015 was able to prime and kill targets expressing ALPG and MSLN at or below the limit of detection using immunohistochemistry. In light of these observations, the specificity of AB-1015 was verified using a dual flank in vivo model where one tumor expressed both ALPG and MSLN, and the contralateral tumor expressed MSLN alone. While robust anti-tumor activity was observed against the ALPG+/MSLN+ tumor, no activity was detected against the MSLN+ tumor. In summary, AB-1015 CAR induction and receptor turnover kinetics is a dynamic process. A reductionist in vitro model system was developed, enabling analysis of CAR induction and receptor turnover. Importantly, we demonstrated that all PrimeR+ ICT cells have the potential to induce CAR. Finally, the in vivo specificity of AB-1015 for ALPG+/MSLN+ tumors was established using a dual flank specificity model. Together, these data support the continued development of AB-1015 in a phase I clinical trial (NCT05617755). Citation Format: Xinyan Tang, Marvin Chew, Rakesh Sudhakar, Hongruo Yun, Doreen Sakamoto, Vibhavari Sail, Amanda Fearon, Jasper Williams, Jun Feng, Stephen Santoro. Characterization of AB-1015 logic-gated CAR induction (ON kinetics), receptor turnover (OFF kinetics), and logic gate sensitivity to ALPG/P and MSLN [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6319.