Abstract
CD117 (c-Kit) is a tyrosine kinase receptor that is overexpressed in multiple dog tumors. There is 100% homology between the juxtamembrane domain of human and canine CD117, and many cancer-causing mutations occur in this region in both species. Thus, CD117 is an important target for cancer treatment in dogs and for comparative oncology studies. Currently, there is no monoclonal antibody (mAb) specifically designed to target the exposed region of canine CD117, although there exist some with species cross-reactivity. We panned a naïve phage display library to isolate antibodies against recombinant CD117 on whole cells. Several mAbs were isolated and were shown to bind recombinant canine CD117 at low- to sub-nanomolar affinity. Additionally, binding to native canine CD117 was confirmed by immunohistochemistry and by flow cytometry. Competitive binding assays also identified mAbs that competed with the CD117 receptor-specific ligand, the stem cell factor (SCF). These results show the ability of our cell-based biopanning strategy to isolate a panel of antibodies that have varied characteristics when used in different binding assays. These in vitro/ex vivo assessments suggest that some of the isolated mAbs might be promising candidates for targeting overexpressed CD117 in canine cancers for different useful applications.
Highlights
Membrane proteins represent 20% to 30% of all proteins in most living entities and have a major role in transportation and signal transduction [1,2]
The desirable properties of an monoclonal antibody (mAb) depend upon its final application, whether that be for diagnosis or for therapy
Alternating the transfected cell line in each round of cell-based panning reduced the isolation of irrelevant binders and increased the yield of scFv-phage specific to canine CD117
Summary
Membrane proteins represent 20% to 30% of all proteins in most living entities and have a major role in transportation and signal transduction [1,2]. They account for 44% of human drug targets, which make them the biggest group of drug targets and one of the most attractive targets for drug. Monoclonal antibodies (mAbs) against membrane proteins are highly desirable as research reagents and as diagnostic and therapeutic agents. MAbs are created specific to a cell surface antigen, and a large amount of research and development is focused on the utility of mAbs as standalone drugs, antibody–drug conjugates, or antibody-targeted, drug-loaded nanoparticles for cancer therapy [5,6,7,8,9]. Immunohistochemistry is commonly used for testing formalin-fixed tissue sections, which requires mAbs that can bind denatured or linear epitopes
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