title: Novel anti-HER2 monoclonal antibodies: synergy and antagonism with tumor necrosis factor-α.
abstract: BACKGROUND
  One-third of breast cancers display amplifications of the ERBB2 gene encoding the HER2 kinase receptor. Trastuzumab, a humanized antibody directed against an epitope on subdomain IV of the extracellular domain of HER2 is used for therapy of HER2-overexpressing mammary tumors. However, many tumors are either natively resistant or acquire resistance against Trastuzumab. Antibodies directed to different epitopes on the extracellular domain of HER2 are promising candidates for replacement or combinatorial therapy. For example, Pertuzumab that binds to subdomain II of HER2 extracellular domain and inhibits receptor dimerization is under clinical trial. Alternative antibodies directed to novel HER2 epitopes may serve as additional tools for breast cancer therapy. Our aim was to generate novel anti-HER2 monoclonal antibodies inhibiting the growth of breast cancer cells, either alone or in combination with tumor necrosis factor-α (TNF-α).
  
  
  METHODS
  Mice were immunized against SK-BR-3 cells and recombinant HER2 extracellular domain protein to produce monoclonal antibodies. Anti-HER2 antibodies were characterized with breast cancer cell lines using immunofluorescence, flow cytometry, immunoprecipitation, western blot techniques. Antibody epitopes were localized using plasmids encoding recombinant HER2 protein variants. Antibodies, either alone or in combination with TNF-α, were tested for their effects on breast cancer cell proliferation.
  
  
  RESULTS
  We produced five new anti-HER2 monoclonal antibodies, all directed against conformational epitope or epitopes restricted to the native form of the extracellular domain. When tested alone, some antibodies inhibited modestly but significantly the growth of SK-BR-3, BT-474 and MDA-MB-361 cells displaying ERBB2 amplification. They had no detectable effect on MCF-7 and T47D cells lacking ERBB2 amplification. When tested in combination with TNF-α, antibodies acted synergistically on SK-BR-3 cells, but antagonistically on BT-474 cells. A representative anti-HER2 antibody inhibited Akt and ERK1/2 phosphorylation leading to cyclin D1 accumulation and growth arrest in SK-BR-3 cells, independently from TNF-α.
  
  
  CONCLUSIONS
  Novel antibodies against extracellular domain of HER2 may serve as potent anti-cancer bioactive molecules. Cell-dependent synergy and antagonism between anti-HER2 antibodies and TNF-α provide evidence for a complex interplay between HER2 and TNF-α signaling pathways. Such complexity may drastically affect the outcome of HER2-directed therapeutic interventions.
authors.affiliation: BilGen Genetics and Biotechnology Research Center, Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey.
authors.forename: Ceyhan
authors.initials: C
authors.lastname: Ceran
authors.forename: Murat
authors.initials: M
authors.lastname: Cokol
authors.forename: Sultan
authors.initials: S
authors.lastname: Cingoz
authors.forename: Ipek
authors.initials: I
authors.lastname: Tasan
authors.forename: Mehmet
authors.initials: M
authors.lastname: Ozturk
authors.forename: Tamer
authors.initials: T
authors.lastname: Yagci
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology*; D000915:Antibody Affinity / Q000276:immunology; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D019938:Cyclin D1 / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004336:Drug Antagonism; D004357:Drug Synergism; D000939:Epitopes / Q000276:immunology / Q000378:metabolism; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D061986:MCF-7 Cells; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000276:immunology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D000911:Antibodies, Monoclonal; D019938:Cyclin D1; D000939:Epitopes; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D051057:Proto-Oncogene Proteins c-akt; D018719:Erb-b2 Receptor Tyrosine Kinases; D014409:Tumor Necrosis Factor-alpha; C535150:MAPK1 protein, human