| Popis: |
Clinically effective CAR-T cell therapy for solid tumors, such as clear cell renal cell carcinoma (ccRCC), will require substantial T cell engineering to increase their specificity and potency. We have developed an Integrated Circuit T cell (ICT) that encodes multiple synthetic “modules” in order to overcome diverse barriers to efficacy in ccRCC; ICT cells are generated via CRISPR-mediated, targeted knock-in of a single large transgene into the novel GS94 safe-harbor locus. Both primary and metastatic sites of ccRCC are highly vascularized, with the majority of tumor cells expressing elevated levels of carbonic anhydrase IX (CA9), suggesting CA9 may be an excellent CAR target. However, CA9 is also expressed in healthy bile ducts and stomach tissue which has led to on-target, off-tumor toxicities in patients treated with constitutive CA9 CAR T cells. To improve the therapeutic index of CA9 CAR T cells, we developed an “AND” logic gated ICT cell that requires the presence of two antigens to trigger tumor cell killing, thereby enhancing tumor specificity. Induction of the CA9 CAR is gated on the expression of PSMA found on the tumor neovasculature of ccRCC. Importantly, PSMA and CA9 are not co-expressed in normal tissues. When the anti-PSMA priming receptor (PrimeRTM) binds PSMA, PrimeRTM engagement triggers proteolytic release of a chimeric, fully human transcription factor that induces expression of a CA9 CAR. We confirmed the feasibility of vascular priming using a transwell assay where ICTs were primed by a PSMA expressing endothelial cell line and then migrated across the transwell membrane to kill CA9 expressing RCC cells. In addition, a dual flank xenograft model was used to show logic gated circuits selectively kill tumors that express both CA9 and PSMA, and not tumors that express CA9 alone. Transforming growth factor beta (TGFb) is an immunosuppressive cytokine known to be highly expressed in ccRCC. To further increase the potency and persistence of the ICT cells an shRNA cassette was developed targeting both FAS and TGFBR2, a receptor required for TGFB signaling in T cells. Addition of FAS/TGFBR2 shRNA enhanced antitumor activity of PSMAxCA9 logic gate expressing T cells during in vitro chronic stimulation assays conducted in the presence of exogenous TGFb. Furthermore, FAS/TGFBR shRNA containing ICTs demonstrated enhanced antitumor activity in multiple xenograft RCC models. Collectively, these results demonstrate that PSMAxCA9 ICT cells can (i) selectively target antigens that cannot be safely targeted by conventional CARs and (ii) overcome multiple suppressive mechanisms in the tumor microenvironment. Citation Format: Angela C. Boroughs, Irene Scarfo, Nickolas Attanasio, Thomas Gardner, Jenessa B. Smith, Jennifer McDevitt, Laura Lim, Nishant Mehta, Suchismita Mohanty, James Zhang, Eric Cui, Vibhavari Sail, Amanda Fearon, Samuel Williams, Stephen Santoro, W. Nicholas Haining, Levi Gray-Rupp. A neovasculature-inducible CA9 CAR resistant to FASL and TGFb mediated suppression for the treatment of ccRCC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4088. |
