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Background Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL) is an FDA-approved treatment for metastatic melanoma that can mediate durable tumor regression 1–3 and is an investigational therapy for metastatic epithelial cancers in an ongoing trial.4 In this trial, a 24% objective response rate was associated with infusion of higher CD4+ neoantigen-reactive TIL.4 The current treatment paradigm identifies candidate neoepitopes by tumor sequencing followed by TIL screening against corresponding mutation-encoded long peptides and tandem minigenes for culture-level selection of reactive cells (‘Selected TIL’).4 Identification of surrogate surface markers for neoantigen-reactive CD4+ TIL could streamline enrichment of tumor-relevant CD4+ cells, allowing cell-level selection without requiring patient-specific reagents.Methods Single-cell RNA/TCR sequencing of mixed populations of neoantigen-reactive and bystander CD4+ TIL identified candidate enrichment markers including CD69, PD-1, ADGRG1, and KLRB1 and marker combinations. Bulk TIL was grown in IL-2 from pooled tumor fragments from 10 patients with epithelial cancers in GREX100 flasks for 21 days. Cells were sorted by surface markers into CD4+ Bulk, CD69+/–, PD1+/–, ADGRG1+/–, KLRB1+/–, and double-marker groups (e.g., CD4+ CD69-PD1+, CD4+ CD69+PD1–) and expanded via rapid expansion protocol (REP). After 14 days, populations were tested for neoantigen reactivity by co-culture against peptide pools containing possible neo-epitopes, using 4-1BB and OX40 as activation markers and IFN-γ ELISPOT assays. Group comparisons were done by non-parametric paired T tests.Results Among 10 patients with previously known CD4+ neoantigen TIL reactivity, the median frequency of neoantigen reactive cells within CD4+ Bulk was 8.9%, with most samples showing ≥ 2 neoantigenic targets. Among the individual markers, CD4+ CD69– was effective for enriching reactive TIL, increasing median reactivity 2x vs. CD4+ Bulk (p = 0.0039**), while CD4+ CD69+ depleted reactive cells (0.7x, p = 0.0391*). CD4+ PD1+ and PD1- did not significantly enrich or deplete reactive cells (p = 0.56 and 0.0742, respectively). Double-marker CD4+ CD69-PD1+ increased median reactivity 2.2x vs. CD4+ Bulk (p = 0.0195*) ( figure 1). Of 10 evaluated patient samples, 9 demonstrated enrichment with dual selection by CD4+ CD69-PD1+ (figure 2).Conclusions On functional testing, CD69– is the most promising single surface marker for enriching reactivity of CD4+ tumor-infiltrating lymphocytes, while CD69-PD1+ is the most promising combination surface marker. Single-cell TCR sequencing and validation testing are pending to further evaluate enrichment of neoantigen-reactive lymphocytes. Sorting of such cells to clinical scale may help enable neoantigen-enriched TIL therapy without necessity of patient-specific screening.References Seitter SJ, et al. Impact of prior treatment on the efficacy of adoptive transfer of tumor-infiltrating lymphocytes in patients with metastatic melanoma. Clin Cancer Res 2021;27(19):5289-5298.Kwong MLM, Yang JC. Lifileucel: FDA-approved T-cell therapy for melanoma. Oncologist 2024;29(8):648-650.Mullard A. FDA approves first tumour-infiltrating lymphocyte (TIL) therapy, bolstering hopes for cell therapies in solid cancers. Nat Rev Drug Discov 2024;23(4):238.Lowery FJ, et al. Neoantigen-specific tumor-infiltrating lymphocytes in gastrointestinal cancers: a phase 2 trial. Nature Medicine 2025;31(6):1994-2003.Ethics Approval This protocol (03-C-0277) was performed at the Surgery Branch, National Cancer Institute, and obtained ethics approval in compliance with the IRB in accordance with International Council on Harmonization Good Clinical Practice and relevant portions of the USA Code of Federal Regulations. Informed written consent was provided by all participants, and the trial was reviewed annually by the Center for Cancer Research Safety Monitoring Board.Abstract 372 Figure 1Comparing % frequency of neoantigen reactive cells in 10 analyzed patient samples. Scatter dot plot of total neoantigen reactivity by patient. Bars indicate median values per marker populationAbstract 372 Figure 2Comparing neoantigen reactivity of CD4+ Bulk vs. CD4+ CD69-PD1+. Dot plot comparing paired% frequency of neoantigen reactive cells of CD4+ Bulk population vs. CD4+ CD69-PD1+. Bar indicates population median values