Background Glioblastoma (GBM) is an aggressive form of adult brain cancer with poor prognosis. Checkpoint inhibitors and other immunotherapies have shown limited efficacy in GBM, largely due to the immunosuppressive tumor microenvironment (TME). The GBM TME is highly infiltrated with tumor associated macrophages (TAMs) that promote tumor aggression and impede cytotoxic T cell activities. Immunopeptidomics, the profiling of MHC-I associated peptides presented on cell surfaces using liquid chromatograph tandem mass spectrometry (LC-MS/MS) enables high throughput antigen discovery for the development of new targeted immunotherapies. Profiling the immunopeptidome of TAMs and GBM cells during their interaction will uncover potential targets against TAMs and GBM cells to revert the immunosuppressive TME in GBM.

Methods We used direct co-culture of murine bone marrow derived macrophages (BMDMs) and two mouse syngeneic GBM cell lines CT2A and GL261 to model macrophage:GBM interaction. Phenotypic alterations of BMDMs and GBM cells were profiled using flow cytometry. Post co-culture, TAMs and tumor cells were separated by CD45 antibody labeled magnetic beads. We performed LC-MS/MS with tandem mass tag (TMT) labeling to quantify the MHC-I associated peptide repertoire presented on co-cultured TAMs and tumor cells in comparison to their naïve state. We identified co-culture induced very significantly altered (CIVS) peptides from TAMs and adapted tumor cells, and performed absolute quantification of selected peptides (figure 1). To validate the therapeutic efficacy of these tumor associated peptide, we encoded 6 CIVS peptides into mRNA vaccines and treated GL261 subcutaneous tumors.

Results Co-culture of BMDMs with CT2A or GL261 cells induced phenotypic alteration of BMDMs into TAM like state. We identified dozens of CIVS peptides on TAMs and co-cultured CT2A and GL261 cells. CIVS peptides from TAMs enrich for proteins associated with cytokine signaling pathways, whereas CIVS of adapted tumor cells enrich for RhoGTPase signaling pathway. Surequant analysis revealed that CIVS peptides are presented at 10s-1000s copies per cell and are upregulated 2–5 times in TAMs. CIVS peptides from tumor cells are presented at 100s-1000s copies per cell and can be upregulated >20 times on adapted tumor cells. Despite being self-peptides, a CIVS peptide encoded mRNA vaccine controlled tumor growth in 40% mice bearing GL261 tumor (1 cured).

Conclusions We quantitatively profiled alteration of the immunopeptide repertoire on TAMs and adapted GBM tumor cells to identify targetable MHC-I peptides and developed targeted immunotherapy that showed some efficacy against GBM, highlighting the translational potential of CIVS peptides as GBM vaccines.

Ethics Approval All animal procedures were approved by the Committee on Animal Care at MIT under protocol 2204000324.

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Abstract 897 Figure 1

CIVS identification workflow