384 Features of neoantigen-reactive T cells with <i>ex vivo</i> expansion capacity

Background A high number of neoantigen-reactive tumor-infiltrating T lymphocytes (TIL) for adoptive transfer immunotherapy is a key mediator of clinical responses in metastatic melanoma and gastrointestinal cancers. 1 2 However, the baseline molecular features defining neoantigen-reactive TIL with ex vivo proliferative capacity remain poorly defined, which in turn limits the development of novel manufacturing methods and patient-selection criteria.Methods We analyzed 21 tumors from patients with colorectal, lung, ovarian, and melanoma cancers. Primary expansion of TIL from tumors was obtained with IL-2, followed by a standard bulk rapid expansion protocol (bulkREP) with irradiated allogeneic PBMC feeders, OKT3 and IL-2, and in parallel, a REP of TIL sorted based on their reactivity to predicted neoantigen peptides pulsed on autologous APC (neoREP). Single-cell CITEseq, RNA and TCR-seq ( n = 13) were performed at baseline in TIL from fresh tumors combined with flow cytometry and bulk TCR-seq, and ex vivo expansion was tracked with bulk TCR sequencing after primary expansion and REP.Results Flow cytometry and bulk TCR-seq revealed substantial clonotype attrition during REP, especially among CD8 + T cells, regardless of enrichment for neoantigen-reactivity. From the baseline single-cell analysis of 41,966 TIL, the starting frequency of predicted neoantigen-reactive TIL inferred from the most validated published gene signatures 3 was not predictive of neoantigen reactivity after primary expansion, bulkREP, or neoREP. However, neoantigen-reactive and ex vivo proliferative TIL (neoTILprolif) arose from specific cell clusters. In CD4+ T cells, follicular-like T cells expressing CXCL13 represented the main source, while in CD8+ T cells, clusters of already proliferative T cells and of GZMK-expressing effector memory cells sourced most cells, contrasting with a low input from tissue-resident memory T cells. Genes most highly expressed in neoTILprolif had little overlap with gene signatures that only predicted neoantigen reactivity. By differential protein expression analysis, the integrin α2 (CD49b) and PD-1 significantly distinguished both CD4+ and CD8+ neoTILprolif from neoantigen-reactive TIL that contracted in cultures, as well as from proliferative, naïve-like, bystanders.Conclusions Neoantigen-reactive TIL with ex vivo proliferative potential constitute a minority of T cells in tumors and have a unique transcriptomic profile and cell surface protein expression to guide antigen-agnostic manufacturing of T cells more likely to mediate clinical response.Acknowledgements This work was supported by preclinical research funding from Turnstone Biologics.References Kristensen NP, Heeke C, Tvingsholm SA, et al. Neoantigen-reactive CD8+ T cells affect clinical outcome of adoptive cell therapy with tumor-infiltrating lymphocytes in melanoma. J Clin Investig 2022;132:e150535.Lowery FJ, Goff SL, Gasmi B, et al. Neoantigen-specific tumor-infiltrating lymphocytes in gastrointestinal cancers: a phase 2 trial. Nat Med 2025;1–10.Lowery FJ, Krishna S, Yossef R, et al. Molecular signatures of antitumor neoantigen-reactive T cells from metastatic human cancers. Science 2022;375:877–84.Ethics Approval This study was approved by the Centre hospitalier de l’Université de Montréal Institutional Review Board, approval number 21.299.

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