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Phage Display Selection of Antibody Fragments targeted to VEGFR-3 Dimerization Domain

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Title: Phage Display Selection of Antibody Fragments targeted to VEGFR-3 Dimerization Domain
Author(s): Lee, Yoke Seng
Contributor: University of Helsinki, Faculty of Medicine, Haartman Institute
Discipline:
Language: English
Acceptance year: 2014
Abstract:
The inhibition of vascular endothelial growth factor receptor-3 (VEGFR-3) signaling has important implications in circumventing tumor angiogenesis and lymphangiogenesis. Monoclonal antibodies inhibiting ligand binding to its receptor are already in clinical trials, and a murine antibody targeted towards the dimerization domain of the receptor has recently been described. This paves the way for new targeting modalities for counteracting receptor dimerization and activation, but human antibodies have thus far not been reported. We sought to generate novel human Fab antibody fragments targeted towards the dimerization domain of VEGFR-3, by surveying the artificial human Fab phage display library (size ~ 3 x 10^10) through affinity selections with the antigens b-R3D45 (biotinylated domains 4 and 5 of VEGFR-3) and R3D17 (full length extracellular domain of VEGFR-3) in mobile and solid phase panning respectively. After 4 to 5 rounds of affinity selection, significant enrichment of VEGFR-3-specific clones was achieved, and subsequent characterization revealed at least 11 promising novel human Fab candidates. These Fabs were expressed in bacterial expression systems and validated by immunoblotting. Future work may be directed towards their purification and assessment in functional assays, such as the inhibition of ligand-mediated cell survival. In conclusion, this study has demonstrated the robustness of the antibody library in generating novel human Fabs against the VEGFR-3 dimerization interface that could be translated towards the clinical inhibition of pathological angiogenesis and lymphangiogenesis in tumors.


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