Abstract B054: Identification and functional characterization of CD8 T cells recognizing neoepitopes with low affinity (11,987 nM IC50) for MHCI (Kd)

Abstract Cancer neoepitopes are considered arguably central to cancer immunology. The neoepitopes which elicit CD8 T cell–dependent tumor control in murine models, display a broad range of affinities (between IC50 values of 8 nM and 40,000 nM) to MHCI. Proportionally, most of the neoepitopes active in tumor control have low to very low affinities for MHC I (see Srivastava 2024 J. Clinical Investigation for review). Yet the low-affinity neoepitopes elicit robust CD8 T cell-dependent tumor immunity. Analysis of the CD8 T response to the low affinity MHCI-binding peptides is hindered by our inability to create MHCI tetramers of low affinity peptide-MHCI (pMHCI) complexes. Here, we address this problem by generating tetramers from single chain trimers (SCT) of peptide-MHCI-β2 microglobulin and using these SCT-tetramers to detect and characterize the CD8 T cell response to low affinity pMHCI complexes. We analyze the CD8 T cell response to the ccdc85cmut peptide (TYIRPFETKVK) from Meth A fibrosarcoma of BALB/c mice. Immunization with ccdc85cmut mediates strong rejection of the Meth A sarcoma in a CD8 T cell dependent manner. The predicted affinity of the ccdc85cmut to Kd is 11,987 nM. We have created a SCT for the low-affinity ccdc85cmut -Kd- β2 microglobulin, and have made tetramers using this SCT. Most importantly, the ccdc85cmut- SCT tetramer detects significantly higher number of CD8 T cells in mice immunized with ccdc85cmut-pulsed bone marrow derived dendritic cells (BMDCs), than in mice immunized with un-pulsed BMDCs or un-immunized mice. This is the first identification of CD8 T cells reactive to a peptide with an extraordinarily low affinity for MHC I (11,987 nM). Interestingly, ccdc85cmut -SCT tetramer detects CD8 T cells in mice immunized with un-pulsed BMDCs albeit at a statistically significantly lower level than in mice immunized with ccdc85cmut -pulsed BMDCs, and at a significantly higher level than in un-immunized mice. In order to test the functional activity of CD8 T cells reactive to the ccdc85cmut-SCT tetramer, such cells isolated from ccdc85cmut-pulsed BMDCs-immunized mice or un-pulsed BMDCs-immunized mice were adoptively transferred into mice bearing palpable and progressively growing Meth A tumors of 5-7 mm diameter (day 9 after tumor challenge). The ccdc85cmut reactive CD8 T cells from mice immunized with ccdc85cmut-pulsed BMDCs led to tumor growth suppression, while the ccdc85cmut reactive CD8 T cells from mice immunized with un-pulsed BMDCs, did not control tumor growth. Nevertheless, we have detected un-expected biological activity in the ccdc85cmut reactive CD8 T cells in mice immunized with un-pulsed BMDCs under certain conditions; this finding brings into sharp focus the nexus between autoimmunity and cancer immunity in immunization with cancer neoepitopes and shall be presented and discussed. Citation Format: Stabonia Maji, Joseph Dempsey, Tatiana Shcheglova, Adam Hagymasi, Steven Chou, Pramod Srivastava. Identification and functional characterization of CD8 T cells recognizing neoepitopes with low affinity (11,987 nM IC50) for MHCI (Kd) [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr B054.