Abstract B007: Phenotypic Signatures of Circulating Neoantigen-Reactive CD8+ T Cells in Patients with Metastatic Cancers
Authors: Krishna Sri , Yoseph Rami , Sindiri Sivasish , Lowery Frank J , Robbins Paul F , Rosenberg Steven A 
Published: December 2023

Abstract
Background: Immunotherapies represent some of the most promising approaches to treat metastatic solid epithelial cancers, yet their response rates remain low. Identifying antitumor T cells, their antigenic specificities, and their cognate T cell receptors (TCRs) will provide crucial insights into the design of next-generation engineered cellular immunotherapies. Circulating T cells from the peripheral blood (PBL) can provide a rich and non-invasive source for identifying and studying antitumor T cells as an alternative to tumor-infiltrating lymphocytes (TIL). Yet, pre-immunotherapy pre-surgery antitumor T cell frequencies circulating in PBL of patients with metastatic cancer are often low, limiting the accurate definition of their phenotypic states.
Methods: We employed single-cell phenotypic profiling of 36 experimentally defined neoantigen-specific T cell clones from 6 metastatic epithelial cancer patients to derive the transcriptional and cell surface protein signatures of pre-surgery PBL-resident antitumor CD8+ T cells (NeoTCRPBL). and compared T cell gene expression signatures neoantigen TCR clonotypes between the PBL and TIL compartments. We developed a NeoTCRPBL gene signature to assess its sensitivity and specificity in discovering new antitumor TCRs from PBL of prospective patients with different solid tumor types.
Results: Circulating NeoTCRPBL T cells were clonally expanded, but low in frequency in the PBL (⇐0.001-0.005% per clone) necessitating prior-enrichment for studies. NeoTCRPBL T cells exhibited phenotypes distinct from common blood T cell subsets and bystander viral-reactive T cells displaying transcriptional programs of both dysfunctional as well as tissue-resident memory T cells. Within the same patient, intra-clonotype comparison of 24 TIL-and PBL-neoantigen-specific T cell clones revealed that relative to their TIL counterparts, circulating NeoTCRPBLT cells displayed less-dysfunctional immunotherapy-response associated progenitor phenotypic states. Combined analysis of 100 antitumor T cell clones revealed that circulating NeoTCRPBL T cells largely targeted the same clonal, subclonal neoantigens with comparable avidity as TIL (&gt;79% shared), but their TCR-repertoire was only partially shared with TIL (47% shared). Finally, prediction and testing of 64 clonally expanded TCRs based on NeoTCRPBL gene expression signature-enrichment from prospective PBL samples discovered 20 neoantigen-TCR clonotypes suggesting that the NeoTCRPBL signature can successfully identify antitumor TCRs from very low circulating PBL frequencies(&lt; 0.002%).
Conclusions: Circulating PBL-resident antitumor T cells are low in frequency exhibiting distinct clonotypic repertoire and phenotypic states in patients with metastatic solid tumors. The NeoTCRPBL signature provides an alternative source for identifying antitumor T cells and their TCRs non-invasively from pre-surgery blood samples from cancer patients enabling immune monitoring and immunotherapies.
Citation Format: Sri Krishna, Rami Yoseph, Sivasish Sindiri, Frank J Lowery, Paul F Robbins, Steven A Rosenberg. Phenotypic Signatures of Circulating Neoantigen-Reactive CD8+ T Cells in Patients with Metastatic Cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B007.