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Glypican-3 (GPC3) is overexpressed in human hepatocellular carcinoma (HCC)but not expressed in normal tissues except for placenta and fetal liver and thereforeis an ideal target for cancer immunotherapy. In this study, we identified an H2-Kbor H2-Db restricted and murine GPC3 (mGPC3)-derived cytotoxic T-lymphocyte (CTL)epitope peptide in C57BL/6 (B6) mice, which can be used in the design of preclinicalstudies of various therapies with GPC3-target immunotherapy in vivo. First, 11types of 9- to 10-mer peptides predicted to bind with H2-Kb or H2-Db were selectedfrom the mGPC3 amino acid sequence based on the binding score as calculated bythe BIMAS software. We evaluated the peptide-binding affinity and confirmed thatall peptides were able to bind to H2-Kb or H2-Db by in vitro cellular bindingassay. Subsequently, a mixed peptide vaccine and single peptide vaccine were givento B6 mice to evaluate immunogenic potential of the 11 selected peptides. Usingthe splenocytes from peptide-vaccinated mice, interferon (IFN)-γ enzyme-linkedimmunospot (ELISPOT) assays showed that mGPC3-1127-136 (AMFKNNYPSL) peptide wasthe most efficient for inducing CTLs among the 11 peptides. Next, we demonstratedthat the mGPC3-1 peptide-specific CTL line could recognize mGPC3-expressing cancercells, suggesting that mGPC3-1 peptide was an endogenously presented peptide.In conclusion, we identified mGPC3-1 as an H2-Kb or H2-Db restricted, mGPC3-derivedCTL epitope peptide.

Identification of an H2-Kb or H2-Db restricted and glypican-3-derived cytotoxic T-lymphocyte epitope peptide