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CAR T cell therapies for solid tumors are limited by a paucity of tumor-specific single target antigens and insufficient potency due to limitations of CAR T biology. Logic gated (LG) CARs increase control of cell therapy activation and target cell killing by requiring recognition of two or more antigens through AND or AND-NOT Boolean logic. They represent an attractive strategy for cell therapy to improve the therapeutic index by increasing specificity for tumor antigens. We created an AND logic gate cassette deliverable by CRISPR-mediated, non-viral, site-specific integration into human T cells. This logic gate activates expression of a MSLN-targeting CAR upon ALPP/G binding of a priming receptor (PrimeR) within high-grade serous carcinoma ovarian tumors. The PrimeR triggers proteolytic release of a chimeric, fully human transcription factor, which then translocates to the nucleus to induce expression of a minigene encoding a CAR. The PrimeR is built only from human protein sequences, reducing theoretical risk of cell therapy immunogenicity and rejection within a patient. In order to test the specificity of our LG CARs, we integrated LG cassettes into a defined site in the T cell genome, and then co-cultured T cells with K562 cells engineered with only one target antigen (K562-ALPG or K562-MSLN), both target antigens (K562-ALPG/MSLN), or neither target antigen (K562). LG CAR T cells only killed K562-ALPG/MSLN cells. The specificity of this LG was also demonstrated in vivo in a dual-flank K562 model, in which only the ALPG/MSLN tumor was inhibited by LG CAR T cells. CAR expression was not observed on LG CAR T cells recovered outside the ALPG/MSLN tumor, and no growth inhibition was observed in K562-MSLN tumors, unlike a constitutive CAR T control. To model priming antigen heterogeneity expected in tumors, we mixed K562-MSLN cells with varying proportions of K562-ALPG/MSLN cells. We found that the presence of only 5-15% ALPG/MSLN cells was sufficient to effect killing of all target cells by the induced MSLN CAR. The ALPG/MSLN LG CAR will be tested as a component of AB-1015, a novel therapy for indications including ovarian, fallopian tube, or primary peritoneal cancer. Citation Format: Jasper Williams, Kevin Martinez, Angela Boroughs, Laura Lim, Marian Sandoval, Cate Sue, Matthew Drever, Anzhi Yao, Joseph Choe, Maxim Sidorov, Sophia Phillips, Dina Polyak, Suchismita Mohanty, Stephen Santoro, Aaron Cooper, W. Nicholas Haining. Logic gates controlled by priming receptors increase specificity and potency of CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2854. |
