Dissecting the antigen presentation pathway by mass spectrometry to elucidate HLA neoantigen presentation 9084

Description The HLA-peptide (HLAp) complex facilitates adaptive immune responses by presenting the intra- and extracellular protein content to T cells. Frequent somatic mutations presented as HLAp (shared neoantigens) are attractive targets for cancer immunotherapy due to their exclusive prevalence in disease; however, only a fraction is presented as HLAp. To understand the mechanisms that govern neoantigen presentation, we developed a mass spectrometry-based peptidomic approach to dissect the antigen presentation pathway by characterizing proteasome-associated peptides, cytoplasmic peptides, ER-associated peptides and HLA-presented peptides. Our workflow identifies tens of thousands of HLA-precursors in each compartment revealing the intracellular peptide pool available for HLA loading. We applied these methods to HLA-A*02:01 monoallelic C1R cells that co-express 27 amino acid long sequences of frequent KRAS mutations. Our analyses reveal cleavage sites that preclude the presentation of the KRAS HLA-A*02:01 specific neoepitope, indicating that the HLA precursor peptide is not generated and therefore not available for HLA loading. We expanded our initial observations by mapping cleavage patterns for prevalent cancer neoantigens across cellular compartments informing if and how their detection in the proteasome and ER translate to HLA presentation. These HLA-allotype agnostic datasets can improve neoantigen selection, promising increased clinical efficacy of T-cell-based immunotherapies.