Skip to main content

Advertisement

SpringerLink
Log in

Production of Novel VHH Nanobody Inhibiting Angiogenesis by Targeting Binding Site of VEGF

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Vascular endothelial growth factor (VEGF) is one of the key players in angiogenesis and is considered as one of the major targets in cancer therapy. VEGF is secreted by the cancerous cells to form new vessels that carry oxygen and nutrients to the tumor, allowing it to grow beyond 1–2 mm. Cancerous cells spread using these veins and cause malignancy. Therefore, neutralization of VEGF could prevent tumor growth and malignancy, and nowadays, antibodies are widely used for such purpose. Among antibody fragments, nanobodies possess unique characteristics which make them appropriate tools for cancer therapy. In this study, the receptor-binding region of VEGF was used to produce a nanobody using phage display technology. A camel was immunized with the recombinant VEGF, and VHH fragments were amplified using nested PCR on lymphocyte complementary DNA (cDNA). The highest binding affinity was achieved after three rounds of panning. Twenty-four clones were tested by monoclonal phage ELISA, and the clone with the highest affinity (VA12) was selected for soluble expression of nanobody. VA12 was tested under various physicochemical conditions and showed considerable stability in extreme temperatures, pH, and various urea concentrations. Stability of VA12 under such conditions is considered as an advantage over the prevailing antibodies.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Hicklin, D. J., & Ellis, L. M. (2005). Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. Journal of Clinical Oncology, 23, 1011–1027.

    Article  CAS  Google Scholar 

  2. Schlaeppi, J.-M., & Wood, J. M. (1999). Targeting vascular endothelial growth factor (VEGF) for anti-tumor therapy, by anti-VEGF neutralizing monoclonal antibodies or by VEGF receptor tyrosine-kinase inhibitors. Cancer and Metastasis Reviews, 18, 473–481.

    Article  CAS  Google Scholar 

  3. Ferrara, N. (2002). Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: therapeutic implications. Seminars in Oncology, 29, 10–14.

  4. Awata, T., Inoue, K., Kurihara, S., Ohkubo, T., Watanabe, M., Inukai, K., Inoue, I., & Katayama, S. (2002). A common polymorphism in the 5′-untranslated region of the VEGF gene is associated with diabetic retinopathy in type 2 diabetes. Diabetes, 51, 1635–1639.

    Article  CAS  Google Scholar 

  5. Carmeliet, P. (2005). VEGF as a key mediator of angiogenesis in cancer. Oncology, 69, 4–10.

    Article  CAS  Google Scholar 

  6. Hirakawa, S., Kodama, S., Kunstfeld, R., Kajiya, K., Brown, L. F., & Detmar, M. (2005). VEGF-A induces tumor and sentinel lymph node lymphangiogenesis and promotes lymphatic metastasis. The Journal of Experimental Medicine, 201, 1089–1099.

    Article  CAS  Google Scholar 

  7. Ishigami, S., Arii, S., Furutani, M., Niwano, M., Harada, T., Mizumoto, M., Mori, A., Onodera, H., & Imamura, M. (1998). Predictive value of vascular endothelial growth factor (VEGF) in metastasis and prognosis of human colorectal cancer. British Journal of Cancer, 78, 1379.

    Article  CAS  Google Scholar 

  8. Fuh, G., Wu, P., Liang, W.-C., Ultsch, M., Lee, C. V., Moffat, B., & Wiesmann, C. (2006). Structure-function studies of two synthetic anti-vascular endothelial growth factor Fabs and comparison with the Avastin™ Fab. Journal of Biological Chemistry, 281, 6625–6631.

    Article  CAS  Google Scholar 

  9. Lien, S., & Lowman, H. B. (2008). In therapeutic anti-VEGF antibodies (vol 181 p 6). Berlin Heidelberg: Springer.

  10. Behdani, M., Zeinali, S., Khanahmad, H., Karimipour, M., Asadzadeh, N., Azadmanesh, K., Khabiri, A., Schoonooghe, S., Habibi Anbouhi, M., & Hassanzadeh-Ghassabeh, G. (2012). Generation and characterization of a functional Nanobody against the vascular endothelial growth factor receptor-2; angiogenesis cell receptor. Molecular Immunology, 50, 35–41.

    Article  CAS  Google Scholar 

  11. Willett, C. G., Boucher, Y., di Tomaso, E., Duda, D. G., Munn, L. L., Tong, R. T., Chung, D. C., Sahani, D. V., Kalva, S. P., & Kozin, S. V. (2004). Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer. Nature Medicine, 10, 145–147.

    Article  CAS  Google Scholar 

  12. Muller, Y. A., Chen, Y., Christinger, H. W., Li, B., Cunningham, B. C., Lowman, H. B., & de Vos, A. M. (1998). VEGF and the Fab fragment of a humanized neutralizing antibody: crystal structure of the complex at 2.4 Å resolution and mutational analysis of the interface. Structure, 6, 1153–1167.

    Article  CAS  Google Scholar 

  13. Muller, Y. A., Li, B., Christinger, H. W., Wells, J. A., Cunningham, B. C., & De Vos, A. M. (1997). Vascular endothelial growth factor: crystal structure and functional mapping of the kinase domain receptor binding site. Proceedings of the National Academy of Sciences, 94, 7192–7197.

    Article  CAS  Google Scholar 

  14. Deffar, K., Shi, H., Li, L., Wang, X., & Zhu, X. (2009). Nanobodies-the new concept in antibody engineering. African Journal of Biotechnology, 8, 2645–2652.

  15. Ferrari, A., Rodriguez, M., Power, P., Weill, F., De Simone, E., Gutkind, G., & Leoni, J. (2007). Immunobiological role of llama heavy-chain antibodies against a bacterial β-lactamase. Veterinary Immunology and Immunopathology, 117, 173–182.

    Article  CAS  Google Scholar 

  16. Harmsen, M., & De Haard, H. (2007). Properties, production, and applications of camelid single-domain antibody fragments. Applied Microbiology and Biotechnology, 77, 13–22.

    Article  CAS  Google Scholar 

  17. Muyldermans, S., Baral, T., Retamozzo, V. C., De Baetselier, P., De Genst, E., Kinne, J., Leonhardt, H., Magez, S., Nguyen, V., & Revets, H. (2009). Camelid immunoglobulins and nanobody technology. Veterinary Immunology and Immunopathology, 128, 178–183.

    Article  CAS  Google Scholar 

  18. Ebrahimizadeh, W., & Rajabibazl, M. (2014). Bacteriophage vehicles for phage display: biology, mechanism, and application. Current Microbiology, 69, 109–120.

    Article  CAS  Google Scholar 

  19. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.

    Article  CAS  Google Scholar 

  20. Ardekani, L.S., Gargari, S.L.M., Rasooli, I., Bazl, M.R., Mohammadi, M., Ebrahimizadeh, W., Bakherad, H., Zare, H. (2013). A novel nanobody against urease activity of Helicobacter pylori. International Journal of Infectious Diseases,

  21. Bakherad, H., Mousavi Gargari, S. L., Rasooli, I., Rajabibazl, M., Mohammadi, M., Ebrahimizadeh, W., Safaee Ardakani, L., & Zare, H. (2013). In vivo neutralization of botulinum neurotoxins serotype E with heavy-chain camelid antibodies (VHH). Molecular Biotechnology, 55, 159–167.

    Article  CAS  Google Scholar 

  22. Ebrahimizadeh, W., Gargari, S. M., Rajabibazl, M., Ardekani, L. S., Zare, H., & Bakherad, H. (2013). Isolation and characterization of protective anti-LPS nanobody against V. cholerae O1 recognizing Inaba and Ogawa serotypes. Applied Microbiology and Biotechnology, 97, 4457–4466.

    Article  CAS  Google Scholar 

  23. Araste, F., Ebrahimizadeh, W., Rasooli, I., Rajabibazl, M., & Gargari, S. L. M. (2014). A novel VHH nanobody against the active site (the CA domain) of tumor-associated, carbonic anhydrase isoform IX and its usefulness for cancer diagnosis. Biotechnology Letters, 36, 21–28.

    Article  CAS  Google Scholar 

  24. Beatty, J. D., Beatty, B. G., & Vlahos, W. G. (1987). Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. Journal of Immunological Methods, 100, 173–179.

    Article  CAS  Google Scholar 

  25. Kolkman, J. A., & Law, D. A. (2010). Nanobodies—from llamas to therapeutic proteins. Drug Discov Today Tech., 7, e139–e146.

    Article  CAS  Google Scholar 

  26. Ferrara, N., Hillan, K. J., & Novotny, W. (2005). Bevacizumab (Avastin), a humanized anti-VEGF monoclonal antibody for cancer therapy. Biochemical and Biophysical Research Communications, 333, 328–335.

    Article  CAS  Google Scholar 

  27. Klement, G., Baruchel, S., Rak, J., Man, S., Clark, K., Hicklin, D. J., Bohlen, P., & Kerbel, R. S. (2000). Continuous low-dose therapy with vinblastine and VEGF receptor-2 antibody induces sustained tumor regression without overt toxicity. Journal of Clinical Investigation, 105, R15.

    Article  CAS  Google Scholar 

  28. Folkman, J. (2002). Role of angiogenesis in tumor growth and metastasis. Seminar in Oncology, 29, 15–18.

    Article  CAS  Google Scholar 

  29. Duda, D. G., Batchelor, T. T., Willett, C. G., & Jain, R. K. (2007). VEGF-targeted cancer therapy strategies: current progress, hurdles and future prospects. Trends in Molecular Medicine, 13, 223–230.

    Article  CAS  Google Scholar 

  30. Shojaei, F. (2012). Anti-angiogenesis therapy in cancer: current challenges and future perspectives. Cancer Letters, 320, 130–137.

    Article  CAS  Google Scholar 

  31. Samant, R. S., & Shevde, L. A. (2011). Recent advances in anti-angiogenic therapy of cancer. Oncotarget, 2, 122.

    Google Scholar 

  32. Eichelberg, C., Heuer, R., Chun, F. K., Hinrichs, K., Zacharias, M., Huland, H., & Heinzer, H. (2008). Sequential use of the tyrosine kinase inhibitors sorafenib and sunitinib in metastatic renal cell carcinoma: a retrospective outcome analysis. European Urology, 54, 1373–1378.

    Article  CAS  Google Scholar 

  33. Demetri, G. D., van Oosterom, A. T., Garrett, C. R., Blackstein, M. E., Shah, M. H., Verweij, J., McArthur, G., Judson, I. R., Heinrich, M. C., & Morgan, J. A. (2006). Efficacy and safety of sunitinib in patients with advanced gastrointestinal stromal tumour after failure of imatinib: a randomised controlled trial. The Lancet, 368, 1329–1338.

    Article  CAS  Google Scholar 

  34. Ellis, L. M., & Hicklin, D. J. (2008). VEGF-targeted therapy: mechanisms of anti-tumour activity. Nature Reviews Cancer, 8, 579–591.

    Article  CAS  Google Scholar 

  35. Ferreira, L. M. R. (2010). Cancer metabolism: the Warburg effect today. Experimental and Molecular Pathology, 89, 372–380.

    Article  CAS  Google Scholar 

  36. Berg, J. M., T.J., Stryer, L. (2002). Biochemistry, 5th edn. In section 33.3, antibodies bind specific molecules through their hypervariable loops. New York: W H Freeman.

  37. Wesolowski, J., Alzogaray, V., Reyelt, J., Unger, M., Juarez, K., Urrutia, M., Cauerhff, A., Danquah, W., Rissiek, B., & Scheuplein, F. (2009). Single domain antibodies: promising experimental and therapeutic tools in infection and immunity. Medical Microbiology and Immunology, 198, 157–174.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors wish to thank Shahed University and Biotechnology Development Council of I. R. Iran for their financial support.

Author information

Authors and Affiliations

  1. Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran

    Walead Ebrahimizadeh

  2. Department of Biology, Faculty of Basic Science, Shahed University, Opposite Imam Khomeini’s Shrine, Tehran-Qom Express Way, Tehran, 3319118651, Iran

    Walead Ebrahimizadeh, Seyed Latif Mousavi Mousavi Gargari & Zahra Javidan

  3. Department of Clinical Biochemistry, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Masoumeh Rajabibazl

Authors
  1. Walead Ebrahimizadeh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Seyed Latif Mousavi Mousavi Gargari
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zahra Javidan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Masoumeh Rajabibazl
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Seyed Latif Mousavi Mousavi Gargari.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOCX 176 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ebrahimizadeh, W., Mousavi Gargari, S.L.M., Javidan, Z. et al. Production of Novel VHH Nanobody Inhibiting Angiogenesis by Targeting Binding Site of VEGF. Appl Biochem Biotechnol 176, 1985–1995 (2015). https://doi.org/10.1007/s12010-015-1695-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-015-1695-y

Keywords

Search

Navigation