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Background Developing effective CAR-T cell therapies for solid tumors requires sophisticated engineering to ensure sufficient targeting selectivity to minimize on-target off-tumor toxicities. Additionally, there is a need for long-term persistence against suppressive signals from the tumor microenvironment. Here, we developed a sequential ‘AND’ logic-gated T cell therapy for the intended treatment of metastatic castration-resistant prostate cancer (mCRPC) with a previously described synthetic pathway activator. In this logic-gate design, a priming receptor (PrimeR) induces CAR expression upon detection of a priming antigen, which can then target the cytolytic antigen. This two-step activation is intended to drive T cell killing that is restricted to cells expressing both antigens, thereby minimizing on-target off-tumor toxicity in healthy tissues that express only cytolytic antigen. To overcome immunosuppression in the tumor microenvironment, we incorporated a multiplexed Fas and TGFβR2 targeting shRNA cassette that silences key suppressive genes.Methods We designed an shRNA cassette targeting Fas and TGFβR2 and validated knockdown of Fas and reduced signaling of TGF-β by pSMAD2/3. Functional resistance was assessed using a Fas crosslinking assay and long-term cytotoxicity assay with repeated challenge of target cells with TGF-β. Screens for the logic-gate were performed including testing of 50 CARs, 40 PrimeRs and additional logic-gate architecture variants. Co-cultures were set up with target cell lines expressing either both antigens or cytolytic antigen in short-term cytotoxicity assays. Priming antigen-dependent CAR induction and long-term cytotoxicity assays were utilized to downselect to the top logic-gates which were tested in vivo in both a dual flank specificity model (CAR target alone and both targets), and on-target efficacy model for mCRPC.Results The shRNA cassette significantly reduced Fas expression and TGFb-mediated signaling via pSMAD2/3, and showed improved survival and resistance to TGF-β suppression. The CAR and PrimeR screens demonstrated a range in cytotoxicity and selectivity. The top 20 candidates were further characterized by a priming antigen-dependent CAR induction assay and top 5 candidates were tested in a long-term cytotoxicity assay before in vivo testing. In the dual-flank xenograft model, some candidates showed selectivity at a 1M dose. And several candidates drove complete tumor regression in the mCRPC PC3 xenograft model, outperforming a conventional CAR to the target.Conclusions We demonstrate a multiplexed shRNA knockdown of Fas and TGFβR2 in a logic-gated T cell therapy enhanced performance in vitro. Candidates from the logic-gate screens demonstrate robust selectivity and activity in vivo, supporting potential development as a treatment for mCRPC.