title: Epitope mapping and functional studies with three monoclonal antibodies to the c-kit receptor tyrosine kinase, YB5.B8, 17F11, and SR-1.
abstract: Three monoclonal antibodies (MAbs) to the human c-kit receptor tyrosine kinase (P145c-kit), derived in independent laboratories, have been extensively used in studies of c-kit expression and the role of its ligand, steel factor (SLF), in hemopoiesis and mast cell differentiation and function. In this study, the relationship between the epitopes they identify, and their effects on SLF binding, receptor internalization, and signal transduction are compared. Epitope mapping studies carried out on the high P145c-kit-expressing cell line HEL-DR showed that SR-1 identifies an epitope independent of those bound by YB5.B8 and 17F11, while the latter two antibodies bound to distinct but interacting epitopes. SR-1 potently blocked the binding of SLF to P145c-kit on these cells and also on cells of the factor-dependent line MO7e. In contrast, YB5.B8 and 17F11 had minimal effects on ligand binding. Conversely, SLF partially blocked the binding of SR-1 and YB5.B8 to cells, while binding of 17F11 was actually enhanced by SLf on some target cells. Preincubation of HEL-DR and MO7e cells with MAbs prior to exposure to SLF revealed that 17F11 itself brought about partial down-regulation of P145c-kit and did not inhibit SLF-mediated down-regulation. SR-1 caused minimal down-regulation and inhibited SLF-mediated receptor internalization. YB5.B8 had minimal effects on either cell line in this assay. To determine whether the antibodies had any agonist activity, they were compared with SLF for their ability to bring about receptor phosphorylation in intact MO7e cells. All three antibodies induced detectable tyrosine phosphorylation with 17F11 being the most effective, while YB5.B8 was the least effective. Finally, the ability of the antibodies to influence the proliferation of the MO7e cells was examined. As expected, SR-1 potently inhibited the proliferative response to SLF, while 17F11 weakly inhibited and YB5.B8 had negligible effect. In the absence of SLF both 17F11 and YB5.B8 displayed very weak but reproducible agonist activity.
authors.affiliation: Leukaemia Research Unit, Hanson Centre for Cancer Research, Adelaide, South Australia.
authors.forename: L K
authors.initials: LK
authors.lastname: Ashman
authors.forename: H J
authors.initials: HJ
authors.lastname: Bühring
authors.forename: G W
authors.initials: GW
authors.lastname: Aylett
authors.forename: V C
authors.initials: VC
authors.lastname: Broudy
authors.forename: C
authors.initials: C
authors.lastname: Müller
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D001853:Bone Marrow / Q000378:metabolism / Q000648:ultrastructure; D001854:Bone Marrow Cells; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D004705:Endocytosis; D000939:Epitopes / Q000276:immunology*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D016298:Hematopoietic Cell Growth Factors / Q000378:metabolism; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism / Q000473:pathology; D007947:Leukemia, Megakaryoblastic, Acute / Q000378:metabolism / Q000473:pathology; D010449:Peptide Mapping; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000276:immunology* / Q000378:metabolism / Q000502:physiology*; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases / Q000276:immunology* / Q000378:metabolism / Q000502:physiology*; D016184:Receptors, Colony-Stimulating Factor / Q000276:immunology* / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D019089:Stem Cell Factor; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D016298:Hematopoietic Cell Growth Factors; D011518:Proto-Oncogene Proteins; D016184:Receptors, Colony-Stimulating Factor; D019089:Stem Cell Factor; D019009:Proto-Oncogene Proteins c-kit; D020794:Receptor Protein-Tyrosine Kinases