title: Production of macrophage-activated killer cells for targeting of glioblastoma cells with bispecific antibody to FcgammaRI and the epidermal growth factor receptor.
abstract: OBJECTIVE
  The aim was to determine the ability of macrophage-activated killer cells (MAK cells) obtained from peripheral blood of normal volunteers to kill glioblastoma multiforme (GBM) cell lines. Another goal was to investigate whether a bispecific antibody (bsAb) MDX-447, recognizing the high-affinity Fc receptor for IgG (FcgammaRI) and epidermal growth factor receptor (EGFR), would enhance MAK cell tumoricidal activity.
  
  
  METHODS
  Monocytes, from leukapheresis product, were isolated by countercurrent elutriation and differentiated into MAK cells by culture with granulocyte/macrophage-colony-stimulating factor, vitamin D3 and interferon gamma. Cells were checked for sterility, endotoxin and phenotypic markers. MAK cell functional activity was measured by a flow-cytometric phagocytosis assay. Target cells, a carcinoma cell line and two glioma cell lines expressing EGFR, were stained with PKH-26. MAK cells were labeled with fluorescein-conjugated anti-CD14. Combined effectors, targets and bsAb were incubated and the percentage of MAK cells with phagocytosed targets was determined by flow cytometry.
  
  
  CONCLUSIONS
  We demonstrate that a large number of highly purified monocytes, isolated from peripheral blood, can be differentiated into MAK cells for use as an adjuvant for cancer treatment. After culture these cells are sterile, endotoxin-free and comprise more than 95% MAK cells. Increased amounts of CD14, CD64 and HLA-DR, which are characteristics of macrophage activation, were expressed. MAK cells were extremely phagocytic in comparison to monocytes, even in the absence of bsAb. Moreover, bsAb enhanced the tumoricidal activity of elutriated MAK cells targeted against GBM cell lines. Therefore, intracavity MAK cells armed with MDX-447 could be an effective adoptive immunotherapy for EGFR-positive GBM.
authors.affiliation: Department of Microbiology, Dartmouth Medical School, Lebanon, NH 03756, USA.
authors.forename: P K
authors.initials: PK
authors.lastname: Wallace
authors.forename: J L
authors.initials: JL
authors.lastname: Romet-Lemonne
authors.forename: M
authors.initials: M
authors.lastname: Chokri
authors.forename: L H
authors.initials: LH
authors.lastname: Kasper
authors.forename: M W
authors.initials: MW
authors.lastname: Fanger
authors.forename: C E
authors.initials: CE
authors.lastname: Fadul
mesh_terms: D000276:Adjuvants, Immunologic / Q000627:therapeutic use; D000906:Antibodies / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D002762:Cholecalciferol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D066246:ErbB Receptors / Q000096:biosynthesis / Q000276:immunology*; D005434:Flow Cytometry; D005909:Glioblastoma / Q000276:immunology* / Q000378:metabolism*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D007167:Immunotherapy / Q000379:methods; D007371:Interferon-gamma / Q000494:pharmacology; D007694:Killer Cells, Natural / Q000378:metabolism*; D018950:Lipopolysaccharide Receptors / Q000276:immunology; D008175:Lung Neoplasms / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism*; D018613:Microscopy, Confocal; D009000:Monocytes / Q000378:metabolism; D010587:Phagocytosis; D010641:Phenotype; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis; D017452:Receptors, IgG / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D000276:Adjuvants, Immunologic; D000906:Antibodies; D000911:Antibodies, Monoclonal; D002762:Cholecalciferol; D066246:ErbB Receptors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D007371:Interferon-gamma; D018950:Lipopolysaccharide Receptors; D018719:Erb-b2 Receptor Tyrosine Kinases; D017452:Receptors, IgG