Identification of an H2-K(b) or H2-D(b) restricted and glypican-3-derived cytotoxic T-lymphocyte epitope peptide

Glypican-3 (GPC3) is overexpressed in human hepatocellular carcinoma (HCC) but not expressed in normal tissues except for placenta and fetal liver and therefore is an ideal target for cancer immunotherapy. In this study, we identified an H2-K(b) or H2-D(b) 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 preclinical studies of various therapies with GPC3-target immunotherapy in vivo. First, 11 types of 9- to 10-mer peptides predicted to bind with H2-K(b) or H2-D(b) were selected from the mGPC3 amino acid sequence based on the binding score as calculated by the BIMAS software. We evaluated the peptide-binding affinity and confirmed that all peptides were able to bind to H2-K(b) or H2-D(b) by in vitro cellular binding assay. Subsequently, a mixed peptide vaccine and single peptide vaccine were given to B6 mice to evaluate immunogenic potential of the 11 selected peptides. Using the splenocytes from peptide-vaccinated mice, interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assays showed that mGPC3-1(127–136) (AMFKNNYPSL) peptide was the most efficient for inducing CTLs among the 11 peptides. Next, we demonstrated that the mGPC3-1 peptide-specific CTL line could recognize mGPC3-expressing cancer cells, suggesting that mGPC3-1 peptide was an endogenously presented peptide. In conclusion, we identified mGPC3-1 as an H2-K(b) or H2-D(b) restricted, mGPC3-derived CTL epitope peptide.