# Isolation and Characterization of the Vascular Endothelial Growth Factor Receptor Targeting ScFv Antibody Fragments Derived from Phage Display Technology

## TL;DR

This study isolates and characterizes antibody fragments targeting VEGFR-2, a key protein in tumor angiogenesis, using phage display technology.

## Contribution

The novel contribution is the isolation of four unique anti-VEGFR-2 scFv clones that can block the VEGFR-2/VEGF-A interaction.

## Key findings

- Four anti-VEGFR-2 scFv clones (D3, E1, H1, E9) were successfully isolated and shown to bind VEGFR-2 domains.
- ScFvs H1 and D3 effectively blocked the interaction between VEGFR-2 and VEGF-A.
- Molecular docking and competition assays confirmed the receptor-blocking properties of the scFvs.

## Abstract

Angiogenesis, as a tumor hallmark, plays an important
role in the
growth and development of the tumor vasculature system. There is a
huge amount of evidence suggesting that the vascular endothelial growth
factor receptor (VEGFR-2)/VEGF-A axis is one of the main contributors
to tumor angiogenesis and metastasis. Thus, inhibition of the VEGFR-2
signaling pathway by anti-VEGFR-2 mAb can retard tumor growth. In
this study, we employ phage display technology and solution-phase
biopanning (SPB) to isolate specific single-chain variable fragments
(scFvs) against VEGFR-2 and report on the receptor binding characteristics
of the candidate scFvs A semisynthetic phage antibody library to isolate
anti-VEGFR-2 scFvs through an SPB performed with decreasing concentrations
of the VEGFR-2-His tag and VEGFR-2-biotin. After successful expression
and purification, the specificity of the selected scFv clones was
further analyzed by enzyme-linked immunosorbent assay (ELISA), flow
cytometry, and immunoblotting. The competition assay was undertaken
to identify the VEGFR-2 receptor-blocking properties of the scFvs.
Furthermore, the molecular binding characteristics of candidate scFvs
were extensively studied by peptide–protein docking. Polyclonal
ELISA analysis subsequent to four rounds of biopanning showed a significant
enrichment of VEGFR-2-specific phage clones by increasing positive
signals from the first round toward the fourth round of selection.
The individual VEGFR-2-reactive scFv phage clones were identified
by monoclonal phage ELISA. The sequence analysis and complementarity-determining
region alignment identified the four unique anti-VEGFR-2-scFv clones.
The soluble and purified scFvs displayed binding activity against
soluble and cell-associated forms of VEGFR-2 protein in the ELISA
and flow cytometry assays. Based on the inference from the molecular
docking results, scFvs D3, E1, H1, and E9 recognized domains 2 and
3 on the VEGFR-2 protein and displayed competition with VEGF-A for
binding to VEGFR-2. The competition assay confirmed that scFvs H1
and D3 can block the VEGFR-2/VEGF-A interaction. In conclusion, we
identified novel VEGFR-2-blocking scFvs that perhaps exhibit the potential
for angiogenesis inhibition in VEGFR-2-overexpressed tumor cells.