title: Novel engineered trastuzumab conformational epitopes demonstrate in vitro and in vivo antitumor properties against HER-2/neu.
abstract: Trastuzumab is a growth-inhibitory humanized Ab targeting the oncogenic protein HER-2/neu. Although trastuzumab is approved for treatment of advanced 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 mAb. Active immunotherapy with conformational B cell epitopes affords the possibility of generating an enduring immune response, eliciting protein-reactive high-affinity anti-peptide Abs. The three-dimensional structure of human HER-2 in complex with trastuzumab reveals that the Ag-binding region of HER-2 spans residues 563-626 that comprises an extensive disulfide-bonding pattern. To delineate the 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 conformational 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 Abs against 597-626 and 613-626 recognizing HER-2. The 597-626 epitope was immunogenic in outbred rabbits eliciting Abs 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 Ab-dependent cell-mediated cytotoxicity. Moreover, immunization with the 597-626 epitope significantly reduced tumor burden in transgenic BALB-neuT mice. These results suggest the peptide B cell immunogen is appropriate as a vaccine for HER-2-overexpressing cancers because the resulting Abs show analogous biological properties to trastuzumab.
authors.affiliation: Departments of Obstetrics and Gynecology, Arthur G. James Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210, USA.
authors.forename: Joan T
authors.initials: JT
authors.lastname: Garrett
authors.forename: Sharad
authors.initials: S
authors.lastname: Rawale
authors.forename: Stephanie D
authors.initials: SD
authors.lastname: Allen
authors.forename: Gary
authors.initials: G
authors.lastname: Phillips
authors.forename: Guido
authors.initials: G
authors.lastname: Forni
authors.forename: John C
authors.initials: JC
authors.lastname: Morris
authors.forename: Pravin T P
authors.initials: PT
authors.lastname: Kaumaya
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000627:therapeutic use*; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents / Q000138:chemical synthesis* / Q000627:therapeutic use; D019496:Cancer Vaccines / Q000138:chemical synthesis / Q000627:therapeutic use; D045744:Cell Line, Tumor; D000939:Epitopes / Q000737:chemistry* / Q000627:therapeutic use; D005260:Female; D006801:Humans; D008325:Mammary Neoplasms, Experimental / Q000276:immunology* / Q000378:metabolism / Q000517:prevention & control*; D051379:Mice; D008807:Mice, Inbred BALB C; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000627:therapeutic use; D011487:Protein Conformation; D015202:Protein Engineering; D011817:Rabbits; D051381:Rats; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000276:immunology*; D000068878:Trastuzumab
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D019496:Cancer Vaccines; D000939:Epitopes; D010446:Peptide Fragments; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab