Abstract 578: A functional approach to identifying and engineering TCRs results in highly-potent and specific TCRs for TCR-T cell therapy

Abstract Peptide-HLA (pHLA)- targeting therapeutics, such as T cell receptor-engineered T cells (TCR-T), have had clinical success in treating solid tumors. However, challenges related to safety exist: major concerns remain surrounding the cross-reactivity of T cell receptors (TCRs) as well as the ability of therapeutics to discriminate between on-target vs off-target pHLAs while maintaining high potency. Therefore, approaches that survey the diversity of the T cell repertoire to discover optimal TCRs, as well as platforms to comprehensively identify potential off-target liabilities, are critical to de-risking and accelerating the development of this promising class of pHLA-targeting therapeutics. We have developed a strategy that (1) queries the TCR repertoire to enrich and identify multiple active, sequence-distinct endogenous TCRs; (2) uses 3T-TRACE, a high-diversity pHLA library, to screen for cross-reactivity; and (3) exploits functional selections to simultaneously optimize for TCR potency and specificity. We applied this approach to identify TCRs of optimal specificity and potency targeting a peptide derived from the cancer-testis antigen NY-ESO-1 (SLLMWITQC) displayed by HLA-A2. We profiled 6 sequence-distinct TCRs using 3T-TRACE and validated potential endogenous off-target cross-reactivities. Many of the identified off-targets exhibited little to no sequence homology to the NY-ESO-1 epitope, highlighting the importance of diverse combinatorial libraries in identifying unexpected cross-reactivities. Leveraging the off-target liabilities identified by 3T-TRACE, we designed a functional library and selection scheme that enabled the identification of TCRs with increased potency and specificity. Optimized TCRs exhibited enhanced killing activity and improved safety against an NY-ESO-1-expressing melanoma cell line compared to benchmark TCRs, indicating that this approach has potential to improve clinical safety and efficacy. Using this multi-faceted and comprehensive approach we rapidly identified highly potent and specific TCRs against NY-ESO-1. Identifying cross-reactivities using 3T-TRACE proved to be critical in selecting TCRs suitable for engineering and functional selections. This approach can be extended to any pHLA target to create safe and effective TCR-Ts for clinical development. Citation Format: John D. Leonard, Alejandro Ramirez, Jason Romero, Jake Kleiner, Joanna Dreux, Bindu Hegde, Bryan Xie, Akshay Sharathchandra, Nathan Katz, Venita I. DeAlmeida, Hans-Peter Gerber, Marvin H. Gee, Leah V. Sibener. A functional approach to identifying and engineering TCRs results in highly-potent and specific TCRs for TCR-T cell therapy [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 578.