Background As cellular immunotherapies utilizing genetically engineered T cells become a more significant focus of clinical investigation, identification of patient-specific neoantigen-reactive T cell receptors (TCRs) in a practical and efficient manner is a top priority. Using high-dimensional single-cell analysis, we recently identified a common gene expression signature in anti-tumor, neoantigen-reactive tumor-infiltrating lymphocytes (TIL) from patients with metastatic cancer, which included high gene expression of cell-surface markers of exhaustion.1 Furthermore, analyses of intra-tumoral T cell populations have shown that neoantigen-specific TIL are enriched in subsets defined by cell-surface markers of exhaustion, likely secondary to oligoclonal expansion that occurs upon tumor antigen recognition in vivo.2–5 In this study we describe an efficient method to prospectively capture and reconstruct neoantigen-reactive T cell receptors from tumor digest based on co-expression of CD39, PD-1, and TIGIT.
Methods We evaluated the ability of PD-1+, CD39+, and TIGIT+ TIL (TIL-TP) to enrich for neoantigen-reactivity by sorting, sequencing, and reconstructing high-frequency TCR alpha/beta pairs from tumor digest in 5 patients with metastatic epithelial cancers. We then tested the ability of PD-1/CD39/TIGIT co-expressing TILs to sustain reactivity to patient-specific tumor neoantigens following in vitro expansion under similar conditions to our current clinical trial protocols (figure 1).
Results We prospectively reconstructed TIL-TP TCRs to identify additional novel TCRs, with 35% of prospectively screened TCRs being neoantigen- or tumor-reactive. Including both previously known and newly predicted TCRs, TIL-TP demonstrated enrichment for neoantigen-reactivity in 4 of 5 patients, with a median of at least 26.8% of sequenced TIL-TP cells being neoantigen-reactive (range: 11.9 – 88.4%). TIL-TP TCR isolation demonstrated a high degree of correlation with single-cell transcriptomic approaches to identification of neoantigen-reactive TCRs, though TIL-TP TCRs represent a more cost-effective and widely available approach compared to those utilizing more advanced technologies. However, despite their substantial enrichment for neoantigen-reactive TCR clonotypes, the majority of TIL-TP populations failed to demonstrate neoantigen-reactivity following in vitro expansion and exhibited loss of neoantigen-reactive clones as well as functional impairment.
Conclusions TIL-TP serve as a highly efficient and reliable source of tumor-reactive TCRs. While direct utilization of these TIL-TP as a source for cellular therapy presents significant challenges, sorting for TIL-TP offers a streamlined approach using readily available and affordable technology to identify neoantigen-reactive TCRs that may be used to design TCR engineered cellular immunotherapies.
Lowery, FJ, et al. 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.
Duhen, T. et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat Commun 2018;9:2724, doi:10.1038/s41467-018-05072-0.
Gros A, et al. PD-1 identifies the patient-specific CD8(+) tumor-reactive repertoire infiltrating human tumors. J Clin Invest 2014;124:2246–2259, doi:10.1172/JCI73639.
Pasetto, A. et al. Tumor- and Neoantigen-Reactive T-cell Receptors Can Be Identified Based on Their Frequency in Fresh Tumor. Cancer Immunol Res 2016;4:734–743, doi:10.1158/2326-6066.CIR-16-0001.
Yossef, R. et al. Enhanced detection of neoantigen-reactive T cells targeting unique and shared oncogenes for personalized cancer immunotherapy. JCI Insight 2018;3: doi:10.1172/jci.insight.122467.
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