Abstract 5495: Screens to identify TCR-specific epitopes using engineered antigen-presenting cells

Abstract Despite the key role of T-cell receptors (TCRs) in adaptive immune responses, experimental validation of interactions between activated TCRs and cognate antigens remains a holy grail in developing novel molecular and cell-based therapeutics. To facilitate efficient screening of TCR-antigen interactions, we engineered both reporter Jurkat-NFAT-GFP cells to express candidate-activated TCRs, and antigen-presenting K562-HLA cells (APC) expressing a library of predicted peptide epitopes. To detect antigen-activation of Jurkat cells interacting with cognate epitopes, the K562 cells were engineered to express a single-chain variable fragment (scFv) which could bind IL2 released by the activated Jurkat-TCR cells. Jurkat-TCR reporter cells co-cultured with K562-APC cells induce expression of the HLA cognate epitope, which enables them to be labeled with PE anti-IL2 antibody and purified by FACS. The cognate epitope is characterized using NGS. To validate this TCR-epitope library screening system, we constructed and expressed a library of ∼100 cancer-associated epitopes in K562-HLA_A02:01 cells. The K562-HLA-epitope library was then co-cultured with the Jurkat reporter cells expressing MART1 TCR, NY-ESO-1 TCR or, as a control, CMV TCR. After staining with the IL2 antibody and FACS selection, stained APC cells detected specific epitopes with high specificity for cancer-associated and control TCRs. Citation Format: Debbie Deng, Tianbing Liu, Kitt Paraiso, Paul Diehl, Alex Chenchik. Screens to identify TCR-specific epitopes using engineered antigen-presenting cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5495.