Proteogenomic Analysis Unveils the HLA Class I-Presented Immunopeptidome in Melanoma and EGFR-Mutant Lung Adenocarcinoma

•Proteogenomics identified ∼35,000 Class I-presented peptides.•Class I-presented variant peptides identified in EGFR mutant low TMB tumor.•Cancer germline antigen and PTM peptides identified in melanoma and lung cancer.•De novo search identified variant and lncRNA-derived peptides.•A new strategy to identify Class I-presented lncRNA-derived peptides developed. Immune checkpoint inhibitors and adoptive lymphocyte transfer-based therapies have shown great therapeutic potential in cancers with high tumor mutational burden (TMB), such as melanoma, but not in cancers with low TMB, such as mutant epidermal growth factor receptor (EGFR)-driven lung adenocarcinoma. Precision immunotherapy is an unmet need for most cancers, particularly for cancers that respond inadequately to immune checkpoint inhibitors. Here, we employed large-scale mass spectrometry (MS)-based proteogenomic profiling to identify potential immunogenic human leukocyte antigen (HLA) Class I-presented peptides in melanoma and EGFR-mutant lung adenocarcinoma. Similar number of peptides were identified from both tumor types. Cell line and patient-specific databases were constructed using variants identified from whole-exome sequencing. A de novo search algorithm was used to interrogate the HLA Class I immunopeptidome mass spectrometry data. We identified 12 variant peptides and several classes of tumor-associated antigen-derived peptides. We constructed a cancer germline (CG) antigen database with 285 antigens. This allowed us to identify 40 Class I-presented CG antigen-derived peptides. The Class I immunopeptidome comprised more than 1000 post-translationally modified (PTM) peptides representing 58 different PTMs, underscoring the critical role PTMs may play in HLA binding. Finally, leveraging de novo search algorithm and an annotated long non-coding RNAs (lncRNAs) database, we developed a novel lncRNA-encoded peptide discovery pipeline to identify 44 lncRNA-derived peptides that are presented by Class I. We validated MS/MS spectra of select variant, CG antigen, and lncRNA-derived peptides using synthetic peptides and performed HLA Class I binding assays to demonstrate binding to Class I proteins. In summary, we provide direct evidence of HLA Class I presentation of a large number of variant and tumor-associated peptides in both low and high TMB cancer. These results can potentially be useful for precision immunotherapies, such as vaccine or adoptive cell therapies in melanoma and EGFR mutant lung cancers. Immune checkpoint inhibitors and adoptive lymphocyte transfer-based therapies have shown great therapeutic potential in cancers with high tumor mutational burden (TMB), such as melanoma, but not in cancers with low TMB, such as mutant epidermal growth factor receptor (EGFR)-driven lung adenocarcinoma. Precision immunotherapy is an unmet need for most cancers, particularly for cancers that respond inadequately to immune checkpoint inhibitors. Here, we employed large-scale mass spectrometry (MS)-based proteogenomic profiling to identify potential immunogenic human leukocyte antigen (HLA) Class I-presented peptides in melanoma and EGFR-mutant lung adenocarcinoma. Similar number of peptides were identified from both tumor types. Cell line and patient-specific databases were constructed using variants identified from whole-exome sequencing. A de novo search algorithm was used to interrogate the HLA Class I immunopeptidome mass spectrometry data. We identified 12 variant peptides and several classes of tumor-associated antigen-derived peptides. We constructed a cancer germline (CG) antigen database with 285 antigens. This allowed us to identify 40 Class I-presented CG antigen-derived peptides. The Class I immunopeptidome comprised more than 1000 post-translationally modified (PTM) peptides representing 58 different PTMs, underscoring the critical role PTMs may play in HLA binding. Finally, leveraging de novo search algorithm and an annotated long non-coding RNAs (lncRNAs) database, we developed a novel lncRNA-encoded peptide discovery pipeline to identify 44 lncRNA-derived peptides that are presented by Class I. We validated MS/MS spectra of select variant, CG antigen, and lncRNA-derived peptides using synthetic peptides and performed HLA Class I binding assays to demonstrate binding to Class I proteins. In summary, we provide direct evidence of HLA Class I presentation of a large number of variant and tumor-associated peptides in both low and high TMB cancer. These results can potentially be useful for precision immunotherapies, such as vaccine or adoptive cell therapies in melanoma and EGFR mutant lung cancers.