Abstract 3711: Enriching CD8+ and CD4+ neoantigen-reactive tumor infiltrating lymphocytes by cell surface marker-based sorting

Abstract Introduction: Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL) is FDA-approved for metastatic melanoma and is under investigation for metastatic epithelial cancers. Identifying neoantigen-reactive TIL currently requires time- and resource-intensive testing against patient-specific neoantigens, enabling selection only at the culture level. Cell-surface markers capable of isolating tumor-relevant CD8+ and CD4+ TIL could eliminate the need for personalized sequencing and screening. Methods: Single-cell RNA/TCR sequencing of mixed population of neoantigen-specific and bystander TIL derived from pooled cultures from a metastatic GI cancer lesion with known reactivity identified CD103 as the top marker of tumor-relevant CD8+ TIL and CD31 as a bystander marker. Similar analysis of bulk TIL from another GI malignancy lesion derived from a different patient identified PD-1 and ADGRG1 as CD4+ enrichment markers and CD69 and KLRB1 as depletion markers. Markers were tested for their ability to enrich tumor-relevant CD8+ and CD4+ TIL from pooled epithelial cancer fragments (11 retrospective and 5 prospective CD8+ samples; 10 retrospective CD4+ samples). Following culture in IL-2 in GREX100 flasks for 21 days, TIL were sorted based on expression of CD103 and CD31 for the CD8+ TIL and PD1, ADGRG1, CD69 and KLRB1 for the CD4+ TIL. Sorted populations were expanded using rapid expansion protocol (REP) and evaluated for neoantigen reactivity by upregulation of 4-1BB and/or OX40 expression and IFN-γ release via ELISpot. Single-cell TCR sequencing of populations was done to identify and confirm neoantigen specificity. Group comparisons were performed using non-parametric paired t-tests. Results: Across retrospective and prospective samples, CD103+CD31- CD8+ TIL showed mean neoantigen reactivity of 19.3% and 18.3% and median enrichment of 5.7x (p = 0.0244*) and 8.1x over bulk CD8+ TIL. Single-cell TCR sequencing confirmed enrichment in retrospective samples (p = 0.0312*) and validated 1-3 unique neoantigens per patient. Among 10 patients with CD4+ neoantigen-specific responses, CD4+ bulk TIL showed a mean reactivity of 13.8%, with 1-5 neoantigens validated per patient. CD4+CD69-PD-1+ selection produced a 2.2x median increase in reactivity (p = 0.0195*) and superior capture of neoantigen-specific TCRs vs CD4+ Bulk (p = 0.0039**). Overall 14 of 16 patient samples studied for CD8+ reactivity showed enrichment by CD103+CD31- while 9 of 10 patient samples studied for CD4+ reactivity showed enrichment with CD4+ CD69-PD1+. Conclusion: Selection of CD103+CD31- cells enriches tumor-relevant CD8+ TIL, while CD4+CD69-PD-1+ most effectively enriches CD4+ TIL. Clinical-scale sorting using these markers could generate neoantigen-enriched TIL without patient-specific screening, expanding treatment feasibility for patients with more rapidly progressing disease. Citation Format: Lisa M. Kenney, Nivedita Ratnam, Abraham A. Hakim, Juliette Rault-Wang, Steven A. Rosenberg, Frank J. Lowery, . Enriching CD8+ and CD4+ neoantigen-reactive tumor infiltrating lymphocytes by cell surface marker-based sorting abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3711.