The development of clinically effective CAR-T cell products for solid tumors will require substantial cell engineering to confer sufficient specificity, potency, and persistence. Advances in genome engineering and synthetic biology have provided an increasingly complex set of features that can be introduced into CAR-T cells to augment their function. However, combining multiple features may result in unpredictable negative interactions between components. Here, we report the use of high-throughput screening to optimize the design of a highly-engineered Integrated Circuit T Cell (ICT) product for the treatment of clear cell renal cell carcinoma (ccRCC). ICT cells are CAR-T cells that contain an AND logic gate requiring two antigens to be present to trigger tumor cell killing together with multiple enhancement modules. First, to create the logic gate we generated hundreds of novel scFv and VH/VHH binders targeting PSMA (as a priming target) and CA9 (as a cytolytic target) via two parallel de novo binder discovery efforts: 1) transgenic mice immunizations and 2) internally-developed phage display panning campaigns. Two independent arrayed screens with 500 PSMA prime receptors (PrimeRTM) and 750 CA9 CARs were conducted to find PrimeRs with high inducibility and CARs with strong on-target potency. From these screens, the top 25 PSMA PrimeRs and 20 CA9 CARs were combined with an shRNA cassette for targeted knockdowns along with two variations of a persistence module. We used a fully-automated workcell to perform end-to-end arrayed screening of the resulting 1,000 member library in T cells engineered from four human donors. Non-viral editing techniques were used to electroporate primary CD4/CD8 cells and robotic handlers were used to set up co-cultures. Circuit specificity and potency were assessed by flow and cytokine secretion and resistance to exhaustion was assessed in a seven day killing assay. Although the library was built from components that functioned well independently, we found that when combined, many of the circuits displayed suboptimal function. Integrated screening identified 20 variants that each far exceeded the performance of a small set of initial prototypes built from “best-guess” selections of individual components. The final candidates are significantly superior to constitutive CAR-T cells in a long term killing assay, show potent cytotoxicity of low expressing antigen lines, and display background levels of cytotoxicity against single antigen targets. Engineering multiple features into T cell products is limited by unpredictable negative interactions between components. We have overcome this limitation by using high-throughput screening which generated development-ready candidates for ccRCC with finely tuned desirability criteria in <18 months. Citation Format: Nishant Mehta, Jamie Thomas, Edward Yashin, Andrea Fua, Jonathan Li, Jonathan Chen, Laura Lim, Je Chua, Andrew Cardozo, Marian Sandoval, Duy P. Nguyen, Ziyan Hong, Jimmy Wu, Catherine Sue, Gustavo Guzman, Li Wang, Sofia K. Panagiotopoulou, Sophie Xu, Angela C. Boroughs, W. Nicholas Haining. High-throughput arrayed screening of logic-gated CARs enables the selection of candidates for ccRCC with optimal potency and fidelity traits [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 1783.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.