167 High-efficiency capture of anti-tumor neoantigen-reactive T cell receptors from tumor digest (2021)

<h3>Background</h3> 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.<sup>1</sup> 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.<sup>2–5</sup> 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. <h3>Methods</h3> 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). <h3>Results</h3> 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. <h3>Conclusions</h3> 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. <h3>References</h3> 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. <i>Nat Commun</i> 2018;<b>9</b>: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. <i>J Clin Invest</i> 2014;<b>124</b>: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. <i>Cancer Immunol Res</i> 2016;<b>4</b>: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. <i>JCI Insight</i> 2018;<b>3</b>: doi:10.1172/jci.insight.122467.