Abstract 127: Single cell mapping of tumor infiltrating lymphocytes enables neoantigen-reactive T cell identification in metastatic human cancer
Authors: Lowery Frank J. , Krishna Sri , Yossef Rami , Chatani Praveen D. , Parikh Neilesh B. , Lu Yong-Chen , Zacharakis Nikolaos , Levin Noam , Hanada Ken-ichi , Yang James C. , Robbins Paul F. , Parkhurst Maria R. , Rosenberg Steven A. 
Published: July 2021

Abstract
The adoptive transfer of tumor-infiltrating lymphocytes (TILs) expanded in vitro can mediate regressions of established tumors, yet only a minority of intratumoral T cells are reactive to cancer antigens while the majority represent bystander cells. This complexity underpins the need to phenotypically characterize T cells infiltrating the tumor microenvironment. In this study, we performed single-cell RNA and T cell receptor (TCR) sequencing (scRNA/TCR-seq) on over 45,000 T cells isolated from ten archival metastatic tumor samples whose primary histologies included colon, rectal, breast, and melanoma. By transcriptomic clustering of the T cells and mapping TCR identity data onto these blueprints, we observed a diversity of T cell phenotypes spanning from memory-like to dysfunctional effector-like, with clonal expansion occurring more widely in the effector-like cells. Through sequencing of peripheral blood lymphocytes (PBL) obtained at the time of tumor resection, we found T cell clusters that were either tumor enriched or distinctly PBL enriched, potentially representing recent tumor immigrants. Prior knowledge of 30 CD4 and CD8 tumor neoantigen-reactive TCRs (NeoTCRs) from eight patients allowed us to project these NeoTCRs onto the TIL transcriptomic map where we observed 380 total T cells bearing these NeoTCRs. The majority (&gt;85%) of NeoTCRs were expressed by T cells within 2 clusters, one CD4+ and one CD8+, marked by expression of CXCL13, ENTPD1 (CD39), TOX, and PDCD1 (PD-1), indicating a dysfunctional state. We reasoned that other dominant T cell clonotypes within the NeoTCR clusters could contain additional novel NeoTCR clonotypes. We expressed these predicted NeoTCRs in donor PBL and screened them in vitro with antigen presenting cells (APCs) pulsed with peptides or tandem minigenes encoded by patient-specific tumor mutations, yielding 24 new NeoTCR clones (251 cells), which included neoantigen reactivities undetected in the same patients' fragment TIL screens. Using a NeoTCR signature score derived from the archival samples, three prospective patients' TIL were analyzed by scRNA/TCRseq and clusters scored according to their NeoTCR signature. TCRs reconstructed from these clusters and tested against patients' tumor-specific reagents yielded novel CD4 and CD8 NeoTCRs. Altogether, this study enabled successful enrichment and detection of tumor-specific NeoTCRs in the sequenced TIL of 12/12 patients for whom reactivity was either previously assessed or prospectively identified. Deconvolution of NeoTCRs from bystander TCRs within the tumor-immune microenvironment represents an important step in the development of personalized immunotherapy, as prospective NeoTCR isolation based on transcriptional phenotype of TIL will allow for rapid therapeutic development in the form of T cells expressing these tumor-specific TCRs.
Citation Format: Frank J. Lowery, Sri Krishna, Rami Yossef, Praveen D. Chatani, Neilesh B. Parikh, Yong-Chen Lu, Nikolaos Zacharakis, Noam Levin, Ken-ichi Hanada, James C. Yang, Paul F. Robbins, Maria R. Parkhurst, Steven A. Rosenberg. Single cell mapping of tumor infiltrating lymphocytes enables neoantigen-reactive T cell identification in metastatic human cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 127.