P.059 Identification of T-cell responses to the hepatitis B spliced protein (HBSP) of hepatitis B virus

Abstract

Point mutations in therasproto-oncogenes, notably at codon 12, are found in a high frequency of human malignancies and, thus, may be appropriate targets for the induction of tumor-specific T cell responses in cancer immunotherapy. In this study, we examined the mutantrasprotein sequence reflecting the substitution of Gly to Val at position 12 as a putative point-mutated determinant for potential induction of an HLA-A2-reactive, CD8+cytotoxic T lymphocyte (CTL) response. We identified theras4-12(Val12) sequence as a minimal 9-mer peptide, which displayed specific binding to HLA-A2 by T2 bioassays. Peptide binding to HLA-A2 on T2 cells was weak and required coincubation with exogenous β2-microglobulin to facilitate and enhance complex formation. In contrast, the wild-typeras4-12(Gly12) peptide failed to bind to HLA-A2 even in the presence of β2-microglobulin, consistent with the hypothesis that the point mutation creates a C-terminus anchor residue. A CD8+CTL line against theras4-12(Val12) peptide was derivedin vitrofrom a normal HLA-A2+donor using a model culture system consisting of T2 cells as antigen presenting cells pulsed with exogenous mutantraspeptide and β2-microglobulin plus cytokines (interleukin-2 and 12). Functional characterization of the CD8+CTL line revealed: (1) peptide-specific and HLA-A2-restricted cytotoxicity against a panel of peptide-pulsed targets; (2) no specific lysis using the normalraspeptide sequence; (3) half-maximal lysis with exogenous peptide of ∼0.3 μM; (4) lysis of HLA-A2+B cell lines infected with a recombinant vaccinia virus construct encoding the point-mutated human K-rasgene; and (5) specific lysis of the HLA-A2+SW480 colon carcinoma cell line expressing the naturally occurring K-rasVal12 mutation. Maximal lysis of SW480 cells occurred following interferon (IFN)-γ pretreatment, which correlated with enhanced HLA-A2 and ICAM-1 (CD54) expression. Specificity of lysis was revealed by the absence of lysis against a HLA-A2+melanoma cell line (±IFN-γ), which lacked the mutant Val12 mutation, and the inability of an irrelevant CD8+CTL line to lyse SW480 (±IFN-γ) unless the appropriate exogenous peptide was added. These findings demonstrated that tumor cells may endogenously process and express mutantrasepitopes, such as the 4-12(Val12) sequence, albeit in limiting amounts that may be potentiated by IFN-γ treatment. These data support the biological relevance of this sequence and, thus, may have important implications for the generation ofrasoncogene-specific CTL responses in clinical situations.