Characterization of a Novel Monoclonal Antibody to Human Stem Cell Factor and its Determination Effect on Ex Vivo Stem Cell Expansion

Background: Hematopoietic stem cell (HSC) transplantation can be used to treat blood and immune system disorders. Fresh umbilical cord blood (UCB), a major source of HSC for potential clinical applications, contains a limited number of HSCs. Stem cell factor (SCF) activates HSC self-renewal and is being used to stimulate ex vivo expansion of HSCs for treating various hematologic diseases in clinic. Yet, the mechanism by which SCF stimulates and supports HSCs expansion remains poorly understood. Thus, the purpose of the study is to obtain novel monoclonal antibodies for structural and functional SCF characterizations, as well as for the optimization of HSCs ex vivo expansion. Methods: Recombinant human stem cell factor (rhSCF) was used for producing monoclonal antibody (mAb). High-titer mAb specific to rhSCF was selected by following immunochemical screening to various mAb cell lines. HSCs with CD34(+) epitope were isolated from UCB using affinity chromatography. SCF activity was tested in an ex vivo HSC expansion assay, with use of flow cytometry for detection of CD34(+) cell and total mononuclear cells. Part of rhSCF that contained the antibody-binding site was identified via immunoblot analysis of rhSCF tryptic peptides, rhSCF-specific mAb, and subsequent NH2-terminal amino acid sequence analysis of the detected peptides. Results: The mAb cell line 23C8 with a high titer was found to be specific for rhSCF. In ex vivo cord blood expansion assays, the ability of rhSCF to stimulate the expansion of CD34(+) cells was significantly inhibited by 23C8 in a dose-dependet fashion(?). Through peptide mapping, the binding site of 23C8 on rhSCF was mapped to the first 104 amino acids.. Conclusion: The mAb cell line 23C8 produces specific and inhibitory anti-rhSCF mAb. The mAb appears to bind directly to a part of rhSCF that is critical for biological activity. This functionally active site of rhSCF is located in the first 104 amino acids from the NH2-terminus. The novel anti-rhSCF mAb will be valuable for further dissection of SCF functional domains and optimization of HSCs ex vivo expansion.