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Identification and in vitro characterization of phage-displayed VHHs targeting VEGF
Zahra Farajpour1, Fatemeh Rahbarizadeh, Bahram Kazemi
11Department of Pharmaceutical Biotechnology, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Abstract:
Vascular endothelial growth factor (VEGF) is a potential target for cancer treatment because of its role in angiogenesis and its overexpression in most human cancers. Currently, anti-VEGF antibodies have been shown to be promising tools for therapeutic applications. However, large size, poor tumor penetration, immunogenicity, and production in cost- and labor-intensive conditions are major drawbacks of such agents. The antigen-binding regions of camelid single-chain antibodies (VHHs), due to their unique biophysical characteristics, offer an alternative to conventional antibodies for tumor-targeting purposes. The present study was undertaken to generate and characterize anti-VEGF VHHs from an immune VHH library using phage display. Four rounds of panning were performed, and selected VHHs were characterized using various immunological techniques. Assessment of the antigenic profile of VHHs was done using competition enzyme-linked immunosorbent assay (ELISA). Selected VHHs reacted strongly to VEGF in indirect ELISA and cross-reactivity ELISA tests. The binding affinity of three VHHs, ZFR-1, ZFR-2, and ZFR-5, ranged from 2.5 to 80 nM, and among them, ZFR-5, which was selected for proliferation assay, significantly inhibited the endothelial cell growth in a dose-dependent manner. Taken together, our results indicate that ZFR-5 and other VHHs may be promising tools in cancer research and treatment.
Insights
Camelid single-chain antibodies (VHHs) targeting vascular endothelial growth factor (VEGF) show promise for cancer therapy. These VHHs effectively inhibit endothelial cell growth, offering a potential alternative to conventional antibodies.
Area of Science:
- Biotechnology
- Cancer Research
- Immunology
Background:
- Vascular endothelial growth factor (VEGF) is crucial for angiogenesis and overexpressed in many cancers, making it a therapeutic target.
- Conventional anti-VEGF antibodies face limitations including large size, poor tumor penetration, immunogenicity, and costly production.
- Camelid single-chain variable fragments (VHHs) offer unique biophysical properties advantageous for tumor-targeting applications.
Purpose of the Study:
- To generate and characterize novel anti-VEGF VHHs using phage display technology.
- To evaluate the efficacy of these VHHs as potential therapeutic agents against cancer.
Main Methods:
- Generation of an immune VHH library from camelids.
- Phage display panning for selection of VEGF-binding VHHs over four rounds.
- Characterization using immunological techniques including indirect ELISA, cross-reactivity ELISA, and competition ELISA.
- Assessment of binding affinity and functional inhibition of endothelial cell proliferation.
Main Results:
- Selected VHHs demonstrated strong reactivity to VEGF.
- Three VHHs (ZFR-1, ZFR-2, ZFR-5) exhibited binding affinities in the nanomolar range (2.5–80 nM).
- VHH ZFR-5 significantly inhibited endothelial cell growth in a dose-dependent manner.
Conclusions:
- The generated anti-VEGF VHHs, particularly ZFR-5, are effective inhibitors of endothelial cell proliferation.
- These VHHs represent promising alternatives to conventional antibodies for cancer research and treatment.
- Further investigation into VHHs holds potential for developing novel anti-angiogenic cancer therapies.
