Peptide-based B-cell epitope vaccines targeting HER-2/neu.

摘要

HER-2/neu (ErbB2), a member of the epidermal growth factor family of receptors (EGFR) is overexpressed in a significant fraction of breast cancers. It is an attractive target for receptor-directed antitumor therapy using monoclonal antibodies. Trastuzumab and pertuzumab are growth-inhibitory humanized antibodies targeting the oncogenic protein HER-2/neu. Although passive immunotherapy with trastuzumab is approved for treatment of breast cancer, a number of concerns exist with passive immunotherapy. Treatment is expensive, and has a limited duration of action, necessitating repeated administrations of the monoclonal antibody. Active immunotherapy with conformational B-cell epitopes affords the possibility of generating an enduring immune response, eliciting protein-reactive high-affinity anti-peptide antibodies.The three-dimensional structure of human HER-2 in complex with trastuzumab reveals that the antigen binding region of HER-2 spans residues 563-626 that comprises an extensive disulfide bonding pattern. In order to minimally dissect the interacting binding region of HER-2, we have designed four synthetic peptides with different levels of conformational flexibility. Chimeric peptides incorporating the measles virus fusion 'promiscuous' T cell epitope via a four-residue linker sequence were synthesized, purified, and characterized. All conformationally restricted peptides were recognized by trastuzumab and prevented the function of trastuzumab inhibiting tumor cell proliferation, with 563-598 and 597-626 showing greater reactivity. All epitopes were immunogenic in FVB/n mice with antibodies against 597-626 and 613-626 recognizing HER-2. The 597-626 epitope was immunogenic in outbred rabbits eliciting antibodies which recognized HER-2, competed with trastuzumab for the same epitope, inhibited proliferation of HER-2-expressing breast cancer cells in vitro and caused their antibody-dependent cell-mediated cytotoxicity (ADCC). Moreover, immunization with the 597-626 epitope significantly reduced tumor burden in transgenic BALB-neuT mice.Based on the three-dimensional structure of the HER-2: pertuzumab Fab fragment complex, we have designed three conformational peptide constructs to mimic regions of the dimerization loop of the receptor and to characterize their in vitro and in vivo anti-tumor efficacy. All the constructs elicited high affinity anti-peptide antibodies and all the anti-peptide antibodies showed ADCC to varying degrees with the 266-296 constructs being equally effective as compared to trastuzumab. The 266-296 peptide vaccine statistically reduced tumor onset in both transplantable tumor models (FVB/n and BALB/c) and significant reduction in tumor development in a transgenic mouse tumor model (Balb-neuT).Finally, we report on a phase I clinical trial using the first generation peptide vaccines MVF 316-339 and MVF 628-647 with nor-MDP as adjuvant. The goals of the trial were to determine the safety and toxicity of the vaccine as well as the maximum tolerable dose. The vaccine was well-tolerated and the maximum tolerable dose was identified as the highest dose level, 1.5 mg of each peptide. Additionally, patients produced antibodies of the IgG isotype against the vaccine, and patients receiving the highest dose level had a statistically significant increase in the IgG antibody response compared to patients receiving the lowest dose level.