Epitope Mapping and Immunoneutralization of Recombinant Human Stem‐Cell Factor

Abstract

The epitope regions of three anti-[stem-cell factor (SCF)] Ig have been mapped by characterization of immunoreactivities against truncated forms of SCF in imrnunoblots and against synthetic peptides in solution-phase competition ELISA. Two of the antibodies, mAb 7H6 and mAb 8H7A, were raised against Escherichia coli-derived human SCF-(1-164) while the third, polyclonal antibody (pAb) 1337, was raised against a peptide corresponding to residues 3-31 of human SCF. The epitopes of mAbs 7H6 and 8H7A have been mapped to residues 61 -95 and 95-110, respectively. The epitope of pAb 1337 has been mapped to residues 21-31. The ability of the anti-SCF lg to recognize E. coli-derived human SCF presented in various formats, i.e. partially denatured (fixed in standard ELISA or on a western blot) or native (in solution), was studied. mAb 7H6 recognized its epitope in partially denatured or native SCF with equally high affinity, while mAb 8H7A and pAb 1337 recognized their epitopes only when SCF was at least partially denatured. mAb 7H6 was found to neutralize in vitro SCF-mediated cell proliferation and SCF binding to its receptor, when present in equimolar concentrations relative to the ligand, suggesting that the epitope region is functionally significant. Evidence that the mAb 7H6 epitope is represented by discontinuous regions (residues within sequences 61 -65 and 91 -95 are critically involved) is presented. The observation that the mAb 7H6 epitope is discontinuous has implicatioiis for the structure of S CF.

Structural basis for stem cell factor–KIT signaling and activation of class III receptor tyrosine kinases

“…The SCF/KIT structure can now be used to rationalize previous biochemical and functional data mapping SCF/KIT interaction. Mutagenesis and antibody mapping data on SCF implicate the first few N‐terminal residues before Cys4 (Langley et al , 1994), the regions flanked by residues 61–65 and 91–95 (Mendiaz et al , 1996) or the region of residues 79–97 (Matous et al , 1996), being important for KIT binding. These regions, to some extent, differ from the SCF/KIT interfaces in the complex.…”

Structure of the active core of human stem cell factor and analysis of binding to its receptor Kit

“…Arg121 and Asp124 are adjacent to the main N‐linked glycosylation site, which is not involved in binding (see below), and Asp128 is absent in the 1‐127 truncation mutant that retains full receptor‐binding activity (Langley et al ., 1994). Moreover, a study of human‐murine SCF chimeras narrowed the important receptor recognition epitopes to within residues 1‐35 and 79‐97 (Matous et al ., 1996), and the epitope of a neutralizing monoclonal antibody was mapped to the region of residues 60‐95 (Mendiaz et al ., 1996) and 79‐97 (Matous et al ., 1996).…”

Crystal structure of human stem cell factor: Implication for stem cell factor receptor dimerization and activation

“…Moreover, by using antibodies that neutralize different epitopes on SCF, it was demonstrated that the regions flanked by amino acids 61-65 and 91-95 are also essential for SCF activity (31). In general, the regions mapped by biochemical methods are located in close proximity at the tail region of each SCF protomer.…”