Abstract B166: Mass spectrometric characterization of MHC peptides on therapeutic EBV transformed primary lymphoblastoid cells from HLA A*11 patients
Authors: Jain Amit 
Published: February 2019

Abstract
EBV transformed lymphoblastoid cell lines (LCL) derived from patients with Epstein Barr virus (EBV)-related cancer have been used as "feeder cells" after terminally irradiation in multiple cell therapy protocols targeting EBV-related cancers. These cells are subsequently co-cultured with patient peripheral blood mononuclear cells (PBMC) and are sufficient expansion of autologous cell product, given therapeutically back to patients. LCL are known to present both host genome derived cancer-associated antigens relevant for cancer, alongside EBV antigens. PBMC that are co-cultured with LCL have been used in multiple small studies across multiple EBV related cancers. This study was performed in the context of LCL used as feeder cells in the context of EBV-related nasopharyngeal cancer that was treated with autologous cell product after co-culture with LCL as part of a phase II clinical trial in Singapore. The aims of this study were, firstly, to perform unbiased identification of MHC peptides using mass spectrometry; secondly, to map the peptides to respective HLA alleles using publicly available HLA peptide binding prediction algorithms; and thirdly, to look specifically at HLA A*11 allele specific EBV-derived peptides. 6 patients with HLA A*11 were identified and LCL from these patients were grown in cell culture for 1-3 months each, until 5E8 cells were available. Cells were treated in suspension with acidification in order to enable acid elution of MHC peptides. Mass spectrometric analysis was performed for unbiased evaluation of MHC peptides. Based on the consensus peptide sequences derived from 8-mer and 9-mer peptides, HLA A*11:01 appeared to be a dominant allele with at least 20% of peptides having basic end residues that are typical for binding to HLA allele. Between 3,000 to 5,000 distinct peptide sequences across lengths characteristic for both class I and II MHC alleles were identified for each sample, including multiple peptides derived host genome DNA, and some from EBV proteins including LMP1, BARF1, EBNA6, and TTK. There was some overlap of peptides seen across the samples. For example, 2 patient samples, with 3,636 and 4,033 distinct peptides identified respectively, had 1,025 overlapping peptides. 2 patient samples with HLA A*11:01 and HLA A*11:02 respectively, had 1 overlapping EBV protein derived set of peptides that demonstrated differences in trimming of peptides derived from the same epitope across these 2 HLA classes. Only 1 EBV protein was found to be consistently represented in the MHC peptidome across the patient samples, with the same epitope represented across all the samples within the scope of this study. This small study demonstrates that mass spectrometric analysis of large numbers of cells is able to directly deconvolute the MHC peptide, and this is relevant in the context of adoptive cell therapies that utilize "feeder cells" that are required to reliably present clinically relevant antigens to generate autologous cell product. Furthermore, in the absence of reliable peptide prediction algorithms for HLA alleles that are more prevalent in Asia, such as HLA A*11:02, this approach is useful for direct characterization of peptides that bind.
Citation Format: Amit Jain. Mass spectrometric characterization of MHC peptides on therapeutic EBV transformed primary lymphoblastoid cells from HLA A*11 patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B166.