Skip to main content

Advertisement

SpringerLink
Log in

Efficient growth inhibition of EGFR over-expressing tumor cells by an anti-EGFR nanobody

  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

Epidermal growth factor receptor (EGFR) is deemed to be one of the main molecular targets for diagnosis and treatment of cancer. It has been identified that EGFR involves in pathogenesis of some forms of human cancers. Monoclonal antibodies targeting EGFR could control the tumor cell growth, proliferation, and apoptosis by suppressing the signal transduction pathways. Nanobodies can be regarded as the smallest intact antigen binding fragments, derived from heavy chain-only antibodies existing in camelids. Here, we describe the identification of an EGFR-specific nanobody, referred to as OA-cb6, obtained from immunized camel with a cell line expressing high levels of EGFR. Utilizing flow cytometry (FACS) and blotting methods, we demonstrated that OA-cb6 nanobody binds specifically to EGFR expressing on the surface of A431 cells. In addition, OA-cb6 nanobody potently causes the inhibition of EGFR over expression, cell growth and proliferation. The antibody fragments can probably be regarded as worthwhile binding block for further rational design of anti-cancer therapy.

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. Alroy I, Yarden Y (1997) The ErbB signaling network in embryogenesis and oncogenesis: signal diversification through combinatorial ligand/receptor interactions. FEBS Lett 410(1):83–86

    Google Scholar 

  2. Citri A, Yarden Y (2006) EGF-ERBB signaling: towards the systems level. Nat Rev Mol Cell Biol 7:505–516

    Article  PubMed  CAS  Google Scholar 

  3. Baselga J (2002) Why the epidermal growth factor receptor? The rationale for cancer therapy. Oncologist 7(suppl 4):2–8

    Article  PubMed  CAS  Google Scholar 

  4. Aerts HJ, Dubois L, Hackeng TM, Straathof R, Chiu RK, Lieuwes NG, Jutten B, Weppler SA, Lammering G, Wouters BG, Lambin P (2007) Development and evaluation of a cetuximab-based imaging probe to target EGFR and EGFRvIII. Radiother Oncol 83:326–332

    Article  PubMed  CAS  Google Scholar 

  5. Boland W, Bebb G (2009) Nimotuzumab: a novel anti- EGFR monoclonal antibody that retains anti-EGFR activity while minimizing skin toxicity. Expert Opin Biol 9:1199–1206

    Article  CAS  Google Scholar 

  6. Cohenuram M, Saif MW (2007) Panitumumab the first fully human monoclonal antibody: from the bench to the clinic. Anticancer Drugs 18(1):7–15

    Article  PubMed  CAS  Google Scholar 

  7. Ciardiello F, Tortora G (2008) EGFR antagonists in cancer treatment. N Engl J Med 358:1160–1174

    Article  PubMed  CAS  Google Scholar 

  8. Imai K, Takaoka A (2006) Comparing antibody and small molecule therapies for cancer. Nat Rev Cancer 6:714–727

    Article  PubMed  CAS  Google Scholar 

  9. Altintas I, Kok RJ, Schiffelers RM (2012) Targeting epidermal growth factor receptor in tumors: from conventional monoclonal antibodies via heavy chain-only antibodies to nanobodies. Eur J Pharm Sci 45:399–407

    Article  PubMed  CAS  Google Scholar 

  10. Dienstmann R, Tabernero J (2010) Necitumumab, a fully human IgG1 mAb directed against the EGFR for the potential treatment of cancer. Curr Opin Invest Drugs 11:1434–1441

    CAS  Google Scholar 

  11. Graeven U, Kremer B, Sudhoff T, Killing B, Rojo F, Weber D, Tillner J, Unal C, Schmiege W (2006) Phase I study of the humanized anti-EGFR monoclonal antibody matuzumab (EMD 72000) combined with gemcitabine in advanced pancreatic cancer. Br J Cancer 94:1293–1299

    Article  PubMed  CAS  Google Scholar 

  12. Martinelli E, Palma RD, Orditura M, Vita FD, Ciardiello F (2009) Anti-epidermal growth factor receptor monoclonal antibodies in cancer therapy. Clin Exp Immunol 158(1):1–9

    Article  PubMed  CAS  Google Scholar 

  13. Omidfar K, Shirvani Z (2012) Single domain antibodies: a new concept for epidermal growth factor receptor and EGFRvIII targeting. DNA Cell Biol 31(6):1015–1026

    Article  PubMed  CAS  Google Scholar 

  14. Roovers RC, Laeremans T, Huang L, De Taeye S, Verkleij AJ, Revets H, de Haard HJ, van Bergen en Henegouwen PMP (2007) Efficient inhibition of EGFR signaling and of tumour growth by antagonistic anti-EFGR nanobodies. Cancer Immunol Immunother 56:303–317

    Article  PubMed  CAS  Google Scholar 

  15. Deffar K, Shi H, Li L, Wang X, Zhu X (2009) Nanobodies the new concept in antibody engineering. Afr J Biotechnol 8:2645–2652

    CAS  Google Scholar 

  16. Dumoulin M, Conrath K, Van Meirhaeghe A, Meersman F, Heremans K, Frenken LG, Muyldermans S, Wyns L, Matagne A (2002) Single-domain antibody fragments with high conformational stability. Protein Sci 11:500–515

    Article  PubMed  CAS  Google Scholar 

  17. Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hammers C, Songa EB, Bendahman N, Hammers R (1993) Naturally occurring antibodies devoid of light chains. Nature 363:446–448

    Article  PubMed  CAS  Google Scholar 

  18. Muyldermans S (2001) Single domain camel antibodies: current status. Rev Mol Biotechnol 74:277–302

    Article  CAS  Google Scholar 

  19. Omidfar K, Rasaee MJ, Kashanian S, Paknejad M, Bathaie Z (2007) Studies of thermostability in camelusbactrianus (Bactrian camel) single-domain antibody specific for the mutant epidermal-growth-factor receptor expressed by Pichia. Biotechnol Appl Biochem 46:41–49

    Article  PubMed  CAS  Google Scholar 

  20. Kolkman JA, Law DA (2010) Nanobodies—from llamas to therapeutic proteins. Drug Discov Today Technol 7:139–146

    Article  Google Scholar 

  21. Omidfar K, Moinfar Z, Naderi Sohi A, Tavangar SM, Haghpanah V, Heshmat R, Larijani B (2009) Expression of EGFRvIII in thyroid carcinoma: immunohistochemical study by camel antibodies. Immunol Invest 38:165–180

    Article  PubMed  CAS  Google Scholar 

  22. Stijlemans B, Conrath K, Cortez-Retamozo V, Van Xong H, Wyns L, Senter P, Tillib SV (2011) Camel nanoantibody is an efficient tool for research, diagnostics and therapy. Mol Biol 45:77–85

    Google Scholar 

  23. Evazalipour M, SoltaniTehrani B, Abolhassani M, Morovvati H, Omidfar K (2012) Camel heavy chain antibodies against prostate-specific membrane antigen. DNA Cell Biol 31:424–429

    CAS  Google Scholar 

  24. Omidfar K, Rasaee MJ, Modjtahedi H, Forouzandeh M, Taghikhani M, Bakhtiari A, Paknejad M, Kashanian S (2004) Production and chacterization of a new antibody specific for the mutant EGF receptor, EGFRvIII, in Camelus bactrianus. Tumour Biol 25:179–187

    Article  PubMed  CAS  Google Scholar 

  25. Omidfar K, Rasaee MJ, Modjtahedi H, Forouzandeh M, Taghikhani M, Golmakani N (2004) Production of a novel camel single domain antibody specific for the type III mutant EGFR. Tumour Biol 25:296–305

    Article  PubMed  CAS  Google Scholar 

  26. Tege H, Tourle S, Ottosson J, Persson A (2010) Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta (DE3). Protein Expr Purif 69:159–167

    Article  Google Scholar 

  27. Beatty JD, Beatty BG, Vlahos WG (1987) Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J Immunol Methods 100:173–179

    Article  PubMed  CAS  Google Scholar 

  28. Cunningham MP, Thomas H, Fan Z, Modjtahed H (2006) Responses of human colorectal tumor cells to treatment with the anti–epidermal growth factor receptor monoclonal antibody ICR62 used alone and in combination with the EGFR tyrosine kinase inhibitor gefitinib. Cancer Res 66(15):7708–7715

    Article  PubMed  CAS  Google Scholar 

  29. Wykosky J, Fenton T, Furnari F, Cavenee WK (2011) Therapeutic targeting of epidermal growth factor receptor in human cancer: successes and limitations. Chin J Cancer 30:5–12

    Article  PubMed  CAS  Google Scholar 

  30. Beckman RA, Weiner LM, Davis HM (2007) Antibody constructs in cancer therapy: protein engineering strategies to improve exposure in solid tumors. Cancer 109:170–179

    Article  PubMed  CAS  Google Scholar 

  31. Elbakri A, Nelson PN, Abu Odeh RO (2010) The state of antibody therapy. Hum Immunol 71:1243–1250

    Article  PubMed  CAS  Google Scholar 

  32. Omidfar K, Khorsand F, Darziani Azizi M (2013) New analytical applications of gold nanoparticles as label in antibody based sensors. Biosensor and Bioelectronic 43:336–347

    Article  CAS  Google Scholar 

  33. Behdani M, Zeinali S, Khanahmad H, Karimipour M, Asadzadeh N, Azadmanesh K, Khabiri A, Schoonooghed S, HabibiAnbouhi M, Hassanzadeh-Ghassabehd G, Muyldermans S (2012) Generation and characterization of a functional nanobody against the vascular endothelial growth factor receptor-2; angiogenesis cell receptor. Mol Immunol 50:35–41

    Article  PubMed  CAS  Google Scholar 

  34. Oliveira S, Schiffelers RM, van der Veeken J, van der Meel R, Vongpromek R, van Bergen en Henegouwen PMP, Storm G, Roovers RC (2010) Down regulation of EGFR by a novel multivalent nanobody-liposome platform. J Controlled Release 145:165–175

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This research has been supported by the Endocrinology Metabolism Research Center of Tehran University of Medical Sciences, Tehran, and I.R. Iran.

Author information

Authors and Affiliations

  1. Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, P.O. Box 14395/1179, Tehran, Iran

    Kobra Omidfar, Fatemeh Sadat Amjad Zanjani, Arghavan Golbaz Hagh & Maedeh Darziani Azizi

  2. Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Research Institute, Tehran University of Medical Sciences, Tehran, Iran

    Kobra Omidfar, Seyed Javad Rasouli & Susan Kashanian

Authors
  1. Kobra Omidfar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fatemeh Sadat Amjad Zanjani
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Arghavan Golbaz Hagh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Maedeh Darziani Azizi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Seyed Javad Rasouli
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Susan Kashanian
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kobra Omidfar.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Omidfar, K., Amjad Zanjani, F.S., Hagh, A.G. et al. Efficient growth inhibition of EGFR over-expressing tumor cells by an anti-EGFR nanobody. Mol Biol Rep 40, 6737–6745 (2013). https://doi.org/10.1007/s11033-013-2790-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-013-2790-1

Keywords

Search

Navigation