Abstract 3604: PSC-derived T cells as a novel source of tumor antigen-specific T cell receptors

Abstract T cell receptor (TCR)-engineered T cells are a promising approach to cell therapy for cancer. TCRs targeting public tumor associated antigens (TAAs) offer the widest potential coverage of patients within and across tumor types. TAA-specific TCRs, however, are rare in the blood and when detected are frequently of low affinity due to negative selection of high-affinity self-antigen reactive T cells in the thymus. Here, we present a TCR discovery platform based on in vitro generation of T cells from human pluripotent stem cells (PSCs) using the previously described artificial thymic organoid (ATO) system. We hypothesized that ATO-derived T cells offer several advantages for TCR discovery including a novel TCR repertoire and the absence of negative selection against TAAs during T cell development. Sequencing of the TCR repertoire of polyclonal CD8+ T cells generated from a HLA-A*02:01+ PSC line confirmed low junctional diversity at both the TCR beta and alpha loci, consistent with our previous finding of low TdT expression during T cell development from PSCs and an enrichment for germline-like TCRs. We then interrogated this novel TCR repertoire for reactivity against two well-described HLA-A2-restricted epitopes of NY-ESO-1 and WT1, reactivities to which are rare in the blood. Following co-culture with artificial APCs, a surprisingly high frequency of antigen-specific T cells was detected by MHC-tetramer staining. From these, high frequency TCR clonotypes were functionally validated by reconstitution in Jurkat TCR-reporter cells. Interestingly, from a single experimental run multiple antigen-specific TCRs were validated (5 out of 10 tested clonotypes for NY-ESO-1, and 2 out of 5 for WT1). This finding was reproducible across independent experimental runs, yielding further validated TCRs against these targets. To benchmark relative TCR affinities, we compared a panel of 5 ATO-derived NY-ESO-1 TCRs with 4 blood-derived TCRs with the same specificity, as well as the affinity-enhanced 1G4 TCR. ATO-derived TCRs showed not only superior performance to their blood derived counterparts in peptide dilution assays, but TCRs were identified with equivalent or superior cytotoxicity to 1G4, including against cell lines with endogenous NY-ESO-1 expression. Comparative in vivo anti-tumor efficacy studies are in process. A similarly high level of specificity and cytotoxicity was observed for ATO-derived WT1-specific TCRs. Finally, we demonstrate proof-of-concept identification of TCRs specific to a prostate-specific antigen for which high quality TCRs have remained elusive. In conclusion, the PSC ATO TCR-discovery platform efficiently identifies high-affinity TAA-specific TCRs, including against previously intractable targets. Considering the potential for MHC modularity of this in vitro system, this approach may theoretically enable rapid discovery of therapeutic TCRs based on any designated peptide/MHC-I combination. Citation Format: Chloe S. Wang, Suwen Li, Zhiyuan Mao, Olivia Zhou, Carlos Botero, William Satyadi, Patrick Chang, Sang Pil Yoo, Ho-Chung Chen, Shawn Lopez, Jami McLaughlin, Amélie Montel-Hagen, Owen N. Witte, Gay M. Crooks, Christopher S. Seet. PSC-derived T cells as a novel source of tumor antigen-specific T cell receptors [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 3604.