Generation of novel complete HLA class I monoallelic cell lines used in an MHC stabilization assay for neoantigen evaluation

Iizuka,Akira; Akiyama,Yasuto; Sakura,Naoki; Kanematsu,Akari; Kikuchi,Yasufumi; Nagashima,Takeshi; Urakami,Kenichi; Shimoda,Yuji; Ohshima,Keiichi; Shiomi,Akio; Ohde,Yasuhisa; Terashima,Masanori; Uesaka,Katsuhiko; Mukaigawa,Takashi; Hirashima,Yasuyuki; Yoshikawa,Shusuke; Katagiri,Hirohisa; Sugino,Takashi; Takahashi,Mitsuru; Kenmotsu,Hirotsugu; Yamaguchi,Ken

doi:10.3892/ol.2023.13910

Generation of novel complete HLA class I monoallelic cell lines used in an MHC stabilization assay for neoantigen evaluation

Authors:
- Akira Iizuka
- Yasuto Akiyama
- Naoki Sakura
- Akari Kanematsu
- Yasufumi Kikuchi
- Takeshi Nagashima
- Kenichi Urakami
- Yuji Shimoda
- Keiichi Ohshima
- Akio Shiomi
- Yasuhisa Ohde
- Masanori Terashima
- Katsuhiko Uesaka
- Takashi Mukaigawa
- Yasuyuki Hirashima
- Shusuke Yoshikawa
- Hirohisa Katagiri
- Takashi Sugino
- Mitsuru Takahashi
- Hirotsugu Kenmotsu
- Ken Yamaguchi
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Affiliations: Immunotherapy Division, Shizuoka Cancer Center Research Institute, Shizuoka 411‑8777, Japan, Medical Genetics Division, Shizuoka Cancer Center Research Institute, Shizuoka 411‑8777, Japan, Cancer Diagnostics Research Division, Shizuoka Cancer Center Research Institute, Shizuoka 411‑8777, Japan, Cancer Diagnostics Research Division, Shizuoka Cancer Center Research Institute, Shizuoka 411‑8777, Japan, Division of Colon and Rectal Surgery, Shizuoka Cancer Center Hospital, Shizuoka 411‑8777, Japan, Division of Thoracic Surgery, Shizuoka Cancer Center Hospital, Shizuoka 411‑8777, Japan, Division of Gastric Surgery, Shizuoka Cancer Center Hospital, Shizuoka 411‑8777, Japan, Division of Hepato‑Biliary‑Pancreatic Surgery, Shizuoka Cancer Center Hospital, Shizuoka 411‑8777, Japan, Division of Head and Neck Surgery, Shizuoka Cancer Center Hospital, Shizuoka 411‑8777, Japan, Division of Gynecology, Shizuoka Cancer Center Hospital, Shizuoka 411‑8777, Japan, Division of Dermatology, Shizuoka Cancer Center Hospital, Shizuoka 411‑8777, Japan, Division of Orthopedic Oncology, Shizuoka Cancer Center Hospital, Shizuoka 411‑8777, Japan, Division of Pathology, Shizuoka Cancer Center Hospital, Shizuoka 411‑8777, Japan, Divison of Thoracic Oncology, Shizuoka Cancer Center Hospital, Shizuoka 411‑8777, Japan, Office of The President, Shizuoka Cancer Center, Shizuoka 411‑8777, Japan
Published online on: June 13, 2023 https://doi.org/10.3892/ol.2023.13910
Article Number: 324
Copyright: © Iizuka et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Immunogenic neoantigens derived from somatic mutations in cancer have been identified through clinical studies with the cloning of tumor‑infiltrating T cells, and cancer driver gene mutation‑derived epitopes have been reported; however, these are rare. At present, the validation of epitopes predicted in silico is difficult as human T‑cell clonal diversity cannot be reproduced in vitro or in experimental animal models. To confirm the epitope peptides presented by human leukocyte antigen (HLA) class I molecules predicted in silico, biochemical methods such as major histocompatibility complex (MHC) stabilization assays and mass spectrometry‑mediated identification have been developed based on HLA‑A*02:01 monoallelic T2 cells and HLA‑C*01:02 monoallelic LCL721.221 cells. Therefore, in the present study, to prevent confusion due to peptide cross‑presentation among HLA molecules, HLA class I monoallelic B‑cell clones were generated from the TISI cell line by knocking out HLA‑ABC and TAP2, and knocking in HLA alleles. To explore cancer driver mutations as potential targets for immunotherapy, exome sequencing data from 5,143 patients with cancer enrolled in a comprehensive genome analysis project at the Shizuoka Cancer Center were used to identify somatic amino acid substituted mutations and the 50 most frequent mutations in five genes, TP53, EGFR, PIK3CA, KRAS and BRAF, were identified. Using NetMHC4.1, the present study predicted whether epitopes derived from these mutations are presented on major HLA‑ABC alleles in Japanese individuals and synthesized 138 peptides for MHC stabilization assays. The authors also attempted to examine the candidate epitopes at physiological temperatures by using antibody clone G46‑2.6, which can detect HLA‑ABC, independent of β2‑microglobulin association. In the assays, although the peptide‑induced HLA expression levels were associated with the predicted affinities, the respective HLA alleles exhibited varying degrees of responsiveness, and unexpectedly, p53‑mutant epitopes with predicted weak affinities exhibited strong responses. These results suggested that MHC stabilization assays using completely monoallelic HLA‑expressing B‑cell lines are useful for evaluating the presentation of neoantigen epitopes.

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