Abstract 563: Circulating tumor-specific T cells preferentially recognize patient-specific mutational neoantigens and infrequently recognize shared cancer driver mutations
Authors: Sennino Barbara , Mathur Jyoti , Huang Eva , Conroy Andrew , Ting Marc , Yuen Benjamin , Pan Zheng , Stawiski Eric , Mandl Stefanie 
Published: June 2022

Abstract
Background: Due to a lack of prior technologies able to generate a landscape analysis of mutational neoantigen-HLA class I complex T cell recognition, it is unclear how the frequency of T cell receptors (TCR) against patient-specific mutations compares to shared oncogenic mutations.
Methods: We used the imPACT Isolation Technology®, which allows the selective capture of cancer-specific CD8 T cells from the blood of cancer patients at frequencies as low as 1 in 300K CD8 T cells. This TCR isolation platform is based on whole exome sequencing of a tumor and paired normal tissue control for the identification of non-synonymous mutations, including mutations in cancer driver genes (e.g., p53, KRAS, PI3KCA or HRAS). Bioinformatic algorithms were used to predict potential HLA-binding neoantigens (neoAg) in context of the patient’s HLA class I haplotype. A library of multimerized and barcoded predicted neoAg peptide-HLA molecules were generated to interrogate patient CD8 T cells. The paired αβ-TCR sequences were derived from the single cell sorted T cells. TCRs from antigen experienced CD8 T cell were functionally validated by generating T cells expressing neoTCRs, using non-viral precision gene editing to insert the transgenic TCR chains into the endogenous TRAC locus. We previously showed that all (40/40) TCRs isolated using this approach, reacted to patient-matched cancer cell lines (Puig Saus et al. AACR 2020).
Results: Neoantigen-specific T cells isolated from the peripheral blood of 243 patients with melanoma, bladder, endometrial, ovarian, colorectal, head and neck, urothelial and breast cancers were analyzed For each patient, an average of 352 neoAg-HLA capture complexes were predicted. Across all patients 58,058 neoAg-HLA complexes were manufactured, spanning 8,804 unique mutations. Among these, only about 2% represented known oncogene driver mutations. The predicted peptide-HLA binding affinity for cancer driver mutations was significantly lower (P&lt;0.001) compared to patient-specific neoAg. Remarkably, functional characterization of 206 TCRs showed that most TCRs (93.7%) recognized patient-specific neoAg. Despite the low percentage of neoAg-HLA capture complexes covering oncogene driver mutations (~ 2%), 6.3% of TCRs (13) targeted known cancer driver proteins. Primary human T cells were then engineered to express each TCR. TCR T cells became functionally activated only when exposed to the cognate antigen, irrespective of whether they target neoAg or cancer driver mutations, confirming the high degree of specificity of the isolated TCRs.
Conclusion: A landscape analysis of neoAg-specific TCRs recognizing mutations in cancer has revealed that most T cells recognizing neoantigen-HLA complexes are specific for private mutations in each cancer, though low frequencies of TCR targeting driver mutations can still be identified.
Citation Format: Barbara Sennino, Jyoti Mathur, Eva Huang, Andrew Conroy, Marc Ting, Benjamin Yuen, Zheng Pan, Eric Stawiski, Stefanie Mandl. Circulating tumor-specific T cells preferentially recognize patient-specific mutational neoantigens and infrequently recognize shared cancer driver mutations [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 563.