Abstract 1783: High-throughput arrayed screening of logic-gated CARs enables the selection of candidates for ccRCC with optimal potency and fidelity traits
Authors: Mehta Nishant , Thomas Jamie , Yashin Edward , Fua Andrea , Li Jonathan , Chen Jonathan , Lim Laura , Chua Je , Cardozo Andrew , Sandoval Marian , Nguyen Duy P. , Hong Ziyan , Wu Jimmy , Sue Catherine , Guzman Gustavo , Wang Li , Panagiotopoulou Sofia K. , Xu Sophie , Boroughs Angela C. , Haining W. Nicholas 
Published: April 2023

Abstract
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 &lt;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.