Abstract 2762: Single-cell analysis of circulating neoantigen-reactive CD8+ T cells allows isolation of novel tumor reactive TCRs
Authors: Yoseph Rami , Krishna Sri , Lowery Frank J. , Copeland Amy R. , Parikh Neilesh B. , Hitscherich Kyle , Robbins Paul F. , Rosenberg Steven A. 
Published: June 2022

Abstract
Adoptive cell transfer of gene-engineered lymphocytes expressing T cell receptors (TCRs) targeting tumor antigens has shown promising clinical efficacy for the treatment of advanced cancer patients. For personalized treatment and to minimize “off-target” effects, neoantigens have become an attractive class of targets. While Tumor-Infiltrating Lymphocytes (TILs) have served as the main source of neoantigen-reactive TCRs (NeoTCRs), this strategy requires the patient to undergo surgery to obtain tumor samples. Circulating T cells are an alternative source for neoantigen-reactive T cells; however, the frequency of NeoTCRs in the blood is extremely low. Several approaches have been developed aiming to enrich these cells or isolate their TCRs by cell-sorting of cells expressing activation or exhaustion markers. Yet, the expression of these nonspecific markers by bystander T cells hampers the efficiency of these methods. Here, we sought to study the transcriptional and protein markers of circulating neoantigen-reactive CD8+ T cells by Single-Cell RNA Sequencing (scRNA-Seq) coupled with TCR-Seq and Cellular Indexing of Transcriptome and Epitope by Sequencing (CITE-Seq) from apheresis. To this end, we FACS-enriched neoantigen-reactive T cells from blood samples from 3 patients using pHLA tetramers of neoantigens previously discovered in autologous TIL samples. To be able to study the differences in the phenotype of these cells and other subsets of CD8+ cells in the blood, including viral-targeting T cells, we mixed tetramerpos and tetramerneg cells prior to sequencing. Projecting NeoTCRs onto phenotypic clusters showed a distinct population of these clones with upregulation of CD45RO, HLA-DR and -DP, and exhaustion markers (CD39, CD103, PD-1, etc.). To circumvent the limitation of tetramers (HLA tetramer availability, defined neoantigens and minimal epitopes) we FACS-sorted CD45RO+HLA-DRAhi-CD39+, -CD103+ single-positive, and -CD39+CD103+ double-positive cells, based on our CITE-Seq results, to enrich for neoantigen-reactive cells from 3 additional patients. Transcriptional analysis of all 6 patient samples allowed us to define a distinct gene-signature of circulating neoantigen-reactive CD8+ cells that showed an activated resident-memory-like phenotype. Moreover, using the surface markers that were identified in our analysis we FACS-sorted CD45RO+HLA-DRAhiCD39+CD103+ cells from blood samples into 96-well plates and performed single-cell TCR sequencing. We were able to detect NeoTCRs in 6 out of 8 patients, with frequencies ranging from 4-50% of the sorted cells. In summary, we were able to phenotype low-frequency neoantigen-reactive CD8+ T cells from blood samples and develop a simple sorting method that can bypass the need for invasive surgery, allowing isolation of TCRs that can be engineered into autologous lymphocytes to treat patients with metastatic cancer
Citation Format: Rami Yoseph, Sri Krishna, Frank J. Lowery, Amy R. Copeland, Neilesh B. Parikh, Kyle Hitscherich, Paul F. Robbins, Steven A. Rosenberg. Single-cell analysis of circulating neoantigen-reactive CD8+ T cells allows isolation of novel tumor reactive TCRs [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 2762.