Introduction: The epidermal growth factor receptor (EGFR) is one of the most frequently mutated oncogenes in human lung cancer. Although immune checkpoint inhibitors have been therapeutically effective in multiple cancers, patients harboring EGFR mutations have not responded well to this immunotherapy. Adoptive T cell therapy (ACT) has resulted in complete and durable regression of metastatic cancers. ACT requires that neoantigens are presented by major histocompatibility complex (MHC) class I molecules on tumor but not normal cells. The MHC binding algorithms, such as IEDB, most commonly used for antigen prediction on MHC molecules is far from perfect; thus, experimental proof of MHC-bound peptides is needed to provide direct evidence of antigen presentation.

Method: We developed a mass spectrometry (MS)-based platform integrated with a whole exome sequencing (WES)-based protein database to identify tumor-specific mutant peptides presented by MHC class I. Experiments were conducted using EGFR mutant PC9 lung adenocarcinoma cell line that harbors the EGFRDel746-750mutant. MHC class I proteins and their associated peptides were immunoprecipitated, and the peptides separated from MHC proteins. The MHC proteins were subjected to trypsin digestion and HLA typing using MS. Class I-associated peptides underwent C-18 separation and tandem MS analysis. A PC9-specific database was built adding all SNVs and INDELs identified by WES to the normal human database and used to search MS data. Peptides identified by MS were subjected to IEDB algorithm search using PC9 specific HLA types.

Results: The MS HLA typing identified A*02, A*24, and B*39 in PC9 cells. We identified 13,765 MHC bound peptides. 10,711 peptides were 6~15 amino acid residues in length (6~15mer), which are generally considered as class I epitopes. Using the PC9-specific protein database, 11 variant peptides (neo-peptides) were identified. These include plectin isoform 1 (PLEC_H1459R, 9mer), proteasome subunit beta type-4 (PSMB4_I234T, 9mer), prostaglandin reductase 1 (PTGR1_A27S, 8mer), cytochrome b-c1 complex subunit Rieske mitochondrial (UQCRFS1_S6A, 7mer) and obscurin-like protein 1 (OBSL1_E1365D, 10mer). The IEDB results predicted the binding of PLEC_H1459R with HLA-B*39, OBSL1_E1365D with HLA-A*02 and PSMB4_I234T with HLA-A*24; however, 8 identified variant peptides were not predicted by IEDB.

Conclusion: Our MS data provided direct experimental evidence for MHC class I presentation of 11 neopeptides in PC9 cells. Neopeptides discovered by this proteogenomic platform could potentially trigger T cell response, which needs to be validated by immunological assays. Experiments are underway to utilize this workflow to identify variant neopeptides from patient tumors and patient-derived xenografts (PDXs).

Citation Format: Yue Qi, Tapan K. Maity, Xu Zhang, Shaojian Gao, Nitin Roper, Meriam Bahta, Khoadang Nguyen, Constance M. Cultraro, Udayan Guha. Integrated proteogenomics utilizing mass spectrometry to identify MHC-associated neopeptides in EGFR mutant lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1438.