P12.06.A DESCRIPTIVE AND FUNCTIONAL ASSESSMENT OF MHC I-BOUND PEPTIDES PRESENTED ON EXTRACELLULAR VESICLES OF GLIOBLASTOMA CANCER CELLS IN RESPONSE TO ONCOLYTIC VIRUS IMMUNOTHERAPY

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Abstract BACKGROUND Immunopeptide-based therapies offer the potential for cancer care breakthroughs, necessitating effective mass spectrometry utilization in preclinical research. Despite advancements in identifying cancer-specific antigens, personalized medicine faces challenges due to antigen scarcity, demanding heightened detection sensitivity. Additionally, limited patient tissue availability requires expanding antigen-presenting material through cell culture techniques. MATERIAL AND METHODS Disparate treatment responses are challenging for heterogeneous glioblastoma. Integrating next-gen sequencing and ex vivo patient stem-like cell (GSC) culture, our model mirrors disease heterogeneity and patient-specific responses to HSV1 oncolytic virus (OV) immunotherapy currently under clinical trials for glioblastoma patients. We developed a high-throughput assay using GSCs and immune cells to identify MHC I receptor-bound immunopeptides as response effectors utilizing 27 immune response markers-based flow cytometry. RESULTS GSCs (n=6), infected ex vivo with OV (after 12h at MOI=0.1), yielded EVs via ultracentrifugation. Global proteomic profiling mass spectrometry of EV detected 5,203 proteins, with TAP1, a pivotal player in loading viral peptide antigens onto MHC class I, as the most EV-enriched protein post-OV infection. MHC I was significantly enriched in EV, covering up to 0.1% of all EV proteins, allowing the detection of 2,276 MHC I-bound immunopeptides (out of a total of 45,963 peptides detected). Functional analysis of EVs’ immunological properties via ex vivo PBMC activation assays revealed OV-deregulated EV secretome, with upregulated proteins associated with immune response activation leading to increased activity of NK cells, DCs, NKTs, and CD8+ T cells subpopulations. The effect was diminished by MHC I antibody neutralization. CONCLUSIONS Designed a high-throughput immune phenotype assay, employing a training cohort of GSCs and immune cells as donors and recipients, respectively, pinpointed MHC I receptor-bound immunopeptides and TAP1 overexpressed in response to OV infection as mechanistic effectors of the immune response. Methodological innovation of zooming on EV fraction instead of whole cell material allowed us to bypass the limitations of antigen and patient material scarcity by offering a higher MHC I presenting surface in the material devoid of bulky cellular protein complexes. EV-MHC immunoprecipitation proteomic approach thus correlates personalized clinical interventions with prognostic therapy-outcome assays, stratifying patients for new immunotherapeutic regimens. Supported by National Science Centre Poland grants: 2021/41/N/NZ6/02682 (K.K.), 2022/45/B/NZ5/04226 (A.B.), 2020/39/B/NZ5/02893, 2021/41/B/NZ6/02436 (J.G.), and the National Agency for Academic Exchange grant: PPN/PPO/2019/1/00001 (J.G.).

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