A conformation-based phage-display panning to screen neutralizing anti-VEGF VHHs with VEGFR2 mimicry behavior.
The potency of VEGF-based anti-angiogenic strategies in cancer therapy and the brilliant characteristics of VHHs motivated us to directly block VEGF binding to its receptor with neutralizing single domain antibodies, thereby fading away the VEGF signaling pathway. Considering with high resolution crystal structure of VEGF-RBD/VEGFR2 complex, we could adopt a combinatorial screening strategy: stringent panning and competition ELISA, to direct the panning procedure to dominantly screen the favorable binders that bind and block the key functional regions of VEGF. Based on competition assay, the majority of the screened clones (82%) showed the VEGFR2 mimicry behavior for binding to VEGF molecule. The phage pool gets enriched in favor of sequences that bind the receptor binding sites of VEGF. Different immunoassays and molecular docking simulation verified that all selected VHHs could bind and cover the receptor binding sites of VEGF. Consequently, some modifications in panning procedure with considering the structural features and detailed information of functional regions of a protein antigen, led us to successfully trap the high-affinity specific binders against its hot functional regions. Since the selected VHHs could cover the receptor binding site of VEGF and block VEGF binding to the receptor, they might be promising candidates for anti-angiogenic therapies.
Department of Biology, Faculty of Sciences, University of Guilan, Rasht, Iran.
S Shirin
SS
Shahangian
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran. Electronic address: sajedi_r@modares.ac.ir.
Reza
R
H Sajedi
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Sadegh
S
Hasannia
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Shirin
S
Jalili
Department of Biology, Faculty of Basic Science, Shahid Chamran University, Ahvaz, Iran.
Mohammad
M
Mohammadi
Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Majid
M
Taghdir
National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
Abbas
A
Shali
Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran.
Kamran
K
Mansouri
Department of Biology, Faculty of Sciences, University of Guilan, Rasht, Iran.
Reyhaneh
R
Sariri
D000595:Amino Acid Sequence; D057134:Antibodies, Neutralizing / Q000737:chemistry / Q000276:immunology*; D017951:Antigen Presentation; D001665:Binding Sites; D061307:Human Umbilical Vein Endothelial Cells / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007114:Immunization; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology*; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D019151:Peptide Library; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism*
D057134:Antibodies, Neutralizing; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D040301:Vascular Endothelial Growth Factor Receptor-2
Phage display; VEGF; VHH