title: Antibody dependent cellular phagocytosis (ADCP) and antibody dependent cellular cytotoxicity (ADCC) of breast cancer cells mediated by bispecific antibody, MDX-210.
abstract: BACKGROUND
  MDX-210 is a bispecific antibody (BsAb) with specificity for both the proto-oncogene product of HER-2/neu (c-erbB-2) and FcgammaRI (CD64). HER-2/neu is overexpressed in malignant tissue of approximately 30% of patients with breast cancer, and FcgammaRI is expressed on human monocytes, macrophages, and IFN-gamma activated granulocytes. We investigated phagocytosis and cytolysis of cultured human breast cancer cells by human monocyte-derived macrophages (MDM) mediated by BsAb MDX-210, its partially humanized derivative (MDX-H210), and its parent MoAb 520C9 (anti-HER-2/neu) under various conditions.
  
  
  METHODS
  Purified monocytes were cultured with GM-CSF, M-CSF, or no cytokine for five or six days. Antibody dependent cellular phagocytosis (ADCP) and cytolysis (ADCC) assays were performed with the MDM and HER-2/neu positive target cells (SK-BR-3). ADCP was measured by two-color fluorescence flow cytometry using PKH2 (green fluorescent dye) and phycoerythrin-conjugated (red) monoclonal antibodies (MoAb) against human CD14 and CD11b. ADCC was measured with a non-radioactive LDH detection kit.
  
  
  RESULTS
  Both BsAb MDX-210 (via FcgammaRI) and MoAb 520C9 (mouse IgG1, via FcgammaRII) mediated similar levels of ADCP and ADCC. ADCP mediated by BsAb MDX-H210 was identical to that mediated by BsAb MDX-210. Confocal microscopy demonstrated that dual-labeled cells represented true phagocytosis. Both ADCP and ADCC were higher when MDM were pre-incubated with GM-CSF than when incubated with M-CSF.
  
  
  CONCLUSIONS
  BsAb MDX-210 is as active in vitro as the parent MoAb 520C9 in inducing both phagocytosis and cytolysis of MDM. MDX-210 and its partially humanized derivative, MDX-H210, mediated similar levels of ADCP. GM-CSF appears to superior to M-CSF in inducing MDM-mediated ADCC and ADCP. These studies support the ongoing clinical investigations of BsAb MDX-210 and its partially humanized derivative.
authors.affiliation: The Breast Cancer Program, Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC 20007, USA.
authors.forename: M
authors.initials: M
authors.lastname: Watanabe
authors.forename: P K
authors.initials: PK
authors.lastname: Wallace
authors.forename: T
authors.initials: T
authors.lastname: Keler
authors.forename: Y M
authors.initials: YM
authors.lastname: Deo
authors.forename: C
authors.initials: C
authors.lastname: Akewanlop
authors.forename: D F
authors.initials: DF
authors.lastname: Hayes
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000378:metabolism / Q000494:pharmacology*; D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000494:pharmacology*; D000918:Antibody Specificity; D000920:Antibody-Dependent Cell Cytotoxicity / Q000187:drug effects* / Q000276:immunology; D001943:Breast Neoplasms / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D002277:Carcinoma / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D016207:Cytokines / Q000494:pharmacology; D003601:Cytotoxicity Tests, Immunologic; D004305:Dose-Response Relationship, Drug; D000939:Epitopes; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D008264:Macrophages / Q000166:cytology / Q000187:drug effects; D051379:Mice; D018613:Microscopy, Confocal; D010587:Phagocytosis / Q000187:drug effects* / Q000276:immunology; D000090063:Proto-Oncogene Mas; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*; D017452:Receptors, IgG / Q000276:immunology*; D014407:Tumor Cells, Cultured; D020298:U937 Cells
chemical_list: D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D016207:Cytokines; D000939:Epitopes; D000090063:Proto-Oncogene Mas; D018719:Erb-b2 Receptor Tyrosine Kinases; D017452:Receptors, IgG; C000715470:MAS1 protein, human