Characterization of the Structure and Immunogenicity of HN654-662 and Variant Peptides Bound to HLA-A2.1

摘要

We propose to establish an approach by which tumor cells are eradicated through selective induction of CD8(+) cells specific for a cellular protein, HER-2/neu, that is expressed in many breast and ovarian cancers. Our model system is the class I MHC molecule HLA-A2.1 and the HER-2/neu protein. HLA-A2.1 is present in approximately 50% of Caucasian and African-American women, and HER-2/neu is over-expressed in approximately 30% of breast tumors. A peptide derived from HER-2/neu (HN654-662) has been shown to bind HLA-A2.1 and stimulate cytotoxic T lymphocytes (CTL) that lyse primary tumors from breast or ovarian cancer. The peptide has poor immunogenicity due to poor binding to HLA- A2.1 with low melting temperature (Tm = 36.6 deg C). We are trying to improve the binding affinity by making substitution in the peptide sequence to make it effective therapeutic agent. The crystallographic structure of HN654-662 co- crystallized with HLA-A2.1 shows-that the center of the peptide does not assume one specific conformation and does not make stabilizing contacts with the peptide binding cleft. Altering the primary anchor residues did not improve the binding significantly. Surprisingly, the double variants 12L/V5L are less stable than single substitutions. Also the substitutions at three positions, 12L/V5L/ l9V did not improved the stability. The crystallographic studies of HLA-A2.1 plus HN654-662 variant (12L/V5L/L9V) (Tm = 39.5 deg C) shows - that substituted Leu at fifth position point away from the MHC molecule. It seems that substitution of either of the anchor residues makes the position S Leu point away from class I MHC. The crystallographic studies of another variant HLA-A2.1 HN654-662 (12L/V5L) (Tm = 39.0 deg C) shows that the orientation of substituted Leu at fifth position is same as that of Val 5 in wild type.