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

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-gamma 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.