EPITOPE MAPPING AND FUNCTIONAL-STUDIES WITH 3 MONOCLONAL-ANTIBODIES TO THE C-KIT RECEPTOR TYROSINE KINASE, YB5.B8, 17F11, AND SR-1

Three monoclonal antibodies (MAbs) to the human c-kit receptor tyrosin e kinase (P145(c-kit)), derived in independent laboratories, have been extensively used in studies of c-kit expression and the role of its l igand, steel factor (SLF), in hemopoiesis and mast cell differentiatio n and function. In this study, the relationship between the epitopes t hey identify, and their effects on SLF binding, receptor internalizati on, and signal transduction are compared. Epitope mapping studies carr ied out on the high P145(c-kit)-expressing cell line HEL-DR showed tha t SR-1 identifies an epitope independent of those bound by YB5.B8 and 17F11, while the latter two antibodies bound to distinct but interacti ng epitopes. SR-1 potently blocked the binding of SLF to P145(c-kit) o n these cells and also on cells of the factor-dependent line MO7e. In contrast, YB5.B8 and 17F11 had minimal effects on ligand binding. Conv ersely, 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 ce lls. Preincubation of HEL-DR and MO7e cells with MAbs prior to exposur e to SLF revealed that 17F11 itself brought about partial down-regulat ion of P145(c-kit) and did not inhibit SLF-mediated down-regulation. S R-1 caused minimal down-regulation and inhibited SLF-mediated receptor internalization. YB5.B8 bad minimal effects on either cell line in th is assay. To determine whether the antibodies had any agonist activity , they were compared with SLF for their ability to bring about recepto r phosphorylation in intact MO7e cells. All three antibodies induced d etectable tyrosine phosphorylation with 17F11 being the most effective , while YB5.B8 was the least effective. Finally, the ability of the an tibodies to influence the proliferation of the MO7e cells was examined . As expected, SR-1 potently inhibited the proliferative response to S LF, while 17F11 weakly inhibited and YB5.B8 had negligible effect. In the absence of SLF both 17F11 and YB5.B8 displayed very weak but repro ducible agonist activity. (C) 1994 Wiley-Liss, Inc.