Abstract A074: On Treatment Identification of Mutant KRAS Specific CD8 T Cells in Cancer Patients Unravels Novel Bespoke Diagnostic and Therapeutic Opportunities

Abstract In Stage 4 mutant KRAS oncogene-driven lung cancer patients with prior treatment response to PD1/PD-L1 immune checkpoint inhibitor (ICI) treatment alone, we hypothesized that patients on treatment have circulating mutant KRAS-specific CD8 T cell immune cells. If so, each patient's KRAS-specific CD8 T cells would be HLA-restricted targeting personal mutant KRAS neo-epitopes. Therefore, we designed a comprehensive and unbiased peripheral blood draw-based neo-epitope screening methodology: 1) To qualitatively identify patients with detectable mutant KRAS neoepitope-specific CD8 T cells, then if positive, 2) to identify specific neo-epitopes that each patient had reactive CD8 T cells against. T-cell reactivity was tested with interferon-gamma ELISpot, flow cytometry, and peptide-MHC tetramer staining. Nine patients who harbored KRAS mutations were recruited for this study (5 KRAS G12C, 3 KRAS G12D, and 1 KRAS G12V). 0/9 patients demonstrated T cell reactivity on ELISpot with a fresh blood draw and overnight incubation (24-hour ELISpot) with all theoretical possible neo-epitopes. We then performed ELISpot after extended peptide incubation (up to 21-day ELISpot). 5/9 patients demonstrated T cell reactivity to their respective mutant KRAS neo-epitopes. Individual peptide ELISpot unraveled each patient's specific set of mutant KRAS CD8 neo-epitopes. Five patients had 1 to 7 distinct neo-epitopes. When only personal neo-epitopes were used, 1/5 patients demonstrated T cell reactivity on 24-hour ELISpot, 5/5 had ELISpot positivity after 8-11 days of extended peptide incubation, and had 10 to 500 fold expansion of neo-epitope specific T cells. Serial blood draws were performed for 5 patients (2-14 blood draws). All 5 patients had persistence of epitope-specific T cells over at least 3 serial time points corresponding with disease control. One patient had a disappearance of T cells corresponding with radiological disease progression, and subsequent reappearance of T cells after a change in treatment and corresponding radiological response. Neo-epitopes were then mapped to specific HLA using in silico prediction and validated with peptide MHC binding assays. Novel neo-epitopes (12 over 3 HLA alleles) with corresponding demonstrable neo-epitope reactive T cells led to the discovery of novel patient-derived neo-epitope-specific paired alpha/beta T cell receptor sequences (20 HLA restricted TCRs). Thus, we have optimized methods for diagnostic detection of pre-existing neo-epitope specific T cells using comprehensive then bespoke peptide pools leading to the identification and potential use of bespoke neo-epitope peptides for vaccines in under 14 days. We have unraveled bespoke diagnostic (selection for cancer vaccination, serial immune-monitoring), theranostic (ex vivo and in vivo vaccination) and immunotherapeutic (vaccine, autologous expanded T cells, TCR based therapy) opportunities. The next steps are to deploy these to sustain anti-cancer responses in participating patients. Citation Format: Amit Jain, Andrea Budiman, Nan Jiang. On Treatment Identification of Mutant KRAS Specific CD8 T Cells in Cancer Patients Unravels Novel Bespoke Diagnostic and Therapeutic Opportunities [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A074.