Skip to main content

Advertisement

SpringerLink
Log in

Efficient inhibition of EGFR signalling and of tumour growth by antagonistic anti-EGFR Nanobodies

  • Original Article
  • Published:
Cancer Immunology, Immunotherapy Aims and scope Submit manuscript

Abstract

The development of a number of different solid tumours is associated with over-expression of ErbB1, or the epidermal growth factor receptor (EGFR), and this over-expression is often correlated with poor prognosis of patients. Therefore, this receptor tyrosine kinase is considered to be an attractive target for antibody-based therapy. Indeed, antibodies to the EGFR have already proven their value for the treatment of several solid tumours, especially in combination with chemotherapeutic treatment regimens. Variable domains of camelid heavy chain-only antibodies (called Nanobodies™) have superior properties compared with classical antibodies in that they are small, very stable, easy to produce in large quantities and easy to re-format into multi-valent or multi-specific proteins. Furthermore, they can specifically be selected for a desired function by phage antibody display. In this report, we describe the successful selection and the characterisation of antagonistic anti-EGFR Nanobodies. By using a functional selection strategy, Nanobodies that specifically competed for EGF binding to the EGFR were isolated from ‘immune’ phage Nanobody repertoires. The selected antibody fragments were found to efficiently inhibit EGF binding to the EGFR without acting as receptor agonists themselves. In addition, they blocked EGF-mediated signalling and EGF-induced cell proliferation. In an in vivo murine xenograft model, the Nanobodies were effective in delaying the outgrowth of A431-derived solid tumours. This is the first report describing the successful use of untagged Nanobodies for the in vivo treatment of solid tumours. The results show that functional phage antibody selection, coupled to the rational design of Nanobodies, permits the rapid development of novel anti-cancer antibody-based therapeutics.

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
Fig. 6

Similar content being viewed by others

References

  1. Arbabi Ghahroudi M, Desmyter A, Wyns L, Hamers R, Muyldermans S (1997) Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett 414:521

    Article  PubMed  CAS  Google Scholar 

  2. Bleeker WK, Lammerts van Bueren JJ, van Ojik HH, Gerritsen AF, Pluyter M, Houtkamp M, Halk E, Goldstein J, Schuurman J, van Dijk MA, van de Winkel JG, Parren PW (2004) Dual mode of action of a human anti-epidermal growth factor receptor monoclonal antibody for cancer therapy. J Immunol 173:4699

    PubMed  CAS  Google Scholar 

  3. Bremer E, Samplonius DF, van Genne L, Dijkstra MH, Kroesen BJ, de Leij LF, Helfrich W (2005) Simultaneous inhibition of EGFR signaling and enhanced activation of TRAIL-R-mediated apoptosis induction by an scFv:sTRAIL fusion protein with specificity for human EGFR. J Biol Chem 280(11):10025–10033

    Article  PubMed  CAS  Google Scholar 

  4. Bruell D, Stocker M, Huhn M, Redding N, Kupper M, Schumacher P, Paetz A, Bruns CJ, Haisma HJ, Fischer R, Finnern R, Barth S (2003) The recombinant anti-EGF receptor immunotoxin 425(scFv)-ETA’ suppresses growth of a highly metastatic pancreatic carcinoma cell line. Int J Oncol 23:1179

    PubMed  CAS  Google Scholar 

  5. Buday L, Downward J (1993) Epidermal growth factor regulates p21ras through the formation of a complex of receptor, Grb2 adapter protein, and Sos nucleotide exchange factor. Cell 73:611

    Article  PubMed  CAS  Google Scholar 

  6. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156

    Article  PubMed  CAS  Google Scholar 

  7. Clackson T, Hoogenboom HR, Griffiths AD, Winter G (1991) Making antibody fragments using phage display libraries. Nature 352:624

    Article  PubMed  CAS  Google Scholar 

  8. Cohen S, Ushiro H, Stoscheck C, Chinkers M (1982) A native 170,000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles. J Biol Chem 257:1523

    PubMed  CAS  Google Scholar 

  9. Cortez-Retamozo V, Backmann N, Senter PD, Wernery U, De Baetselier P, Muyldermans S, Revets H (2004) Efficient cancer therapy with a nanobody-based conjugate. Cancer Res 64:2853

    Article  PubMed  CAS  Google Scholar 

  10. Cortez-Retamozo V, Lauwereys M, Hassanzadeh Gh G, Gobert M, Conrath K, Muyldermans S, De Baetselier P, Revets H (2002) Efficient tumor targeting by single-domain antibody fragments of camels. Int J Cancer 98:456

    Article  PubMed  CAS  Google Scholar 

  11. Daub H, Weiss FU, Wallasch C, Ullrich A (1996) Role of transactivation of the EGF receptor in signalling by G-protein-coupled receptors. Nature 379:557

    Article  PubMed  CAS  Google Scholar 

  12. De Haard HJ, Bezemer S, Ledeboer AM, Muller WH, Boender PJ, Moineau S, Coppelmans MC, Verkleij AJ, Frenken LG, Verrips CT (2005) Llama antibodies against a lactococcal protein located at the tip of the phage tail prevent phage infection. J Bacteriol 187:4531

    Article  PubMed  Google Scholar 

  13. Delgado C, Pedley RB, Herraez A, Boden R, Boden JA, Keep PA, Chester KA, Fisher D, Begent RH, Francis GE (1996) Enhanced tumour specificity of an anti-carcinoembrionic antigen Fab’ fragment by poly(ethylene glycol) (PEG) modification. Br J Cancer 73:175

    PubMed  CAS  Google Scholar 

  14. Dolk E, van Vliet C, Perez JM, Vriend G, Darbon H, Ferrat G, Cambillau C, Frenken LG, Verrips T (2005) Induced refolding of a temperature denatured llama heavy-chain antibody fragment by its antigen. Proteins 59:555

    Article  PubMed  CAS  Google Scholar 

  15. Els Conrath K, Lauwereys M, Wyns L, Muyldermans S (2001) Camel single-domain antibodies as modular building units in bispecific and bivalent antibody constructs. J Biol Chem 276:7346

    Article  PubMed  CAS  Google Scholar 

  16. Ewert S, Cambillau C, Conrath K, Pluckthun A (2002) Biophysical properties of camelid V(HH) domains compared to those of human V(H)3 domains. Biochemistry 41:3628

    Article  PubMed  CAS  Google Scholar 

  17. Foon KA, Yang XD, Weiner LM, Belldegrun AS, Figlin RA, Crawford J, Rowinsky EK, Dutcher JP, Vogelzang NJ, Gollub J, Thompson JA, Schwartz G, Bukowski RM, Roskos LK, Schwab GM (2004) Preclinical and clinical evaluations of ABX-EGF, a fully human anti-epidermal growth factor receptor antibody. Int J Radiat Oncol Biol Phys 58:984

    Article  PubMed  CAS  Google Scholar 

  18. Frenken LG, van der Linden RH, Hermans PW, Bos JW, Ruuls RC, de Geus B, Verrips CT (2000) Isolation of antigen specific llama VHH antibody fragments and their high level secretion by Saccharomyces cerevisiae. J Biotechnol 78:11

    Article  PubMed  CAS  Google Scholar 

  19. Giard DJ, Aaronson SA, Todaro GJ, Arnstein P, Kersey JH, Dosik H, Parks WP (1973) In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J Natl Cancer Inst 51:1417

    PubMed  CAS  Google Scholar 

  20. Haisma HJ, Grill J, Curiel DT, Hoogeland S, van Beusechem VW, Pinedo HM, Gerritsen WR (2000) Targeting of adenoviral vectors through a bispecific single-chain antibody. Cancer Gene Ther 7:901

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  22. Hayashi H, Asano R, Tsumoto K, Katayose Y, Suzuki M, Unno M, Kodama H, Takemura S, Yoshida H, Makabe K, Imai K, Matsuno S, Kumagai I, Kudo T (2004) A highly effective and stable bispecific diabody for cancer immunotherapy: cure of xenografted tumors by bispecific diabody and T-LAK cells. Cancer Immunol Immunother 53:497

    Article  PubMed  CAS  Google Scholar 

  23. Heitner T, Moor A, Garrison JL, Marks C, Hasan T, Marks JD (2001) Selection of cell binding and internalizing epidermal growth factor receptor antibodies from a phage display library. J Immunol Methods 248:17

    Article  PubMed  CAS  Google Scholar 

  24. Herbst RS, Arquette M, Shin DM, Dicke K, Vokes EE, Azarnia N, Hong WK, Kies MS (2005) Phase II multicenter study of the epidermal growth factor receptor antibody Cetuximab and Cisplatin for recurrent and refractory squamous cell carcinoma of the head and neck. J Clin Oncol 23:5578

    Article  PubMed  CAS  Google Scholar 

  25. Herbst RS, Langer CJ (2002) Epidermal growth factor receptors as a target for cancer treatment: the emerging role of IMC-C225 in the treatment of lung and head and neck cancers. Semin Oncol 29:27

    Article  PubMed  CAS  Google Scholar 

  26. Holbro T, Civenni G, Hynes NE (2003) The ErbB receptors and their role in cancer progression. Exp Cell Res 284:99

    Article  PubMed  CAS  Google Scholar 

  27. Honegger AM, Dull TJ, Felder S, Van Obberghen E, Bellot F, Szapary D, Schmidt A, Ullrich A, Schlessinger J (1987) Point mutation at the ATP binding site of EGF receptor abolishes protein-tyrosine kinase activity and alters cellular routing. Cell 51:199

    Article  PubMed  CAS  Google Scholar 

  28. Hoogenboom HR (2005) Selecting and screening recombinant antibody libraries. Nat Biotechnol 23:1105

    Article  PubMed  CAS  Google Scholar 

  29. Hoogenboom HR, de Bruine AP, Hufton SE, Hoet RM, Arends JW, Roovers RC (1998) Antibody phage display technology and its applications. Immunotechnology 4:1

    Article  PubMed  CAS  Google Scholar 

  30. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680

    Article  PubMed  CAS  Google Scholar 

  31. Lange IG, Daxenberger A, Meyer HH (2001) Studies on the antibody response of Lama glama—evaluation of the binding capacity of different IgG subtypes in ELISAs for clenbuterol and BSA. Vet Immunol Immunopathol 83:1

    Article  PubMed  CAS  Google Scholar 

  32. Lauwereys M, Arbabi Ghahroudi M, Desmyter A, Kinne J, Holzer W, De Genst E, Wyns L, Muyldermans S (1998) Potent enzyme inhibitors derived from dromedary heavy-chain antibodies. Embo J 17:3512

    Article  PubMed  CAS  Google Scholar 

  33. Mamot C, Drummond DC, Greiser U, Hong K, Kirpotin DB, Marks JD, Park JW (2003) Epidermal growth factor receptor (EGFR)-targeted immunoliposomes mediate specific and efficient drug delivery to EGFR- and EGFRvIII-overexpressing tumor cells. Cancer Res 63:3154

    PubMed  CAS  Google Scholar 

  34. Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G (1991) By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol 222:581

    Article  PubMed  CAS  Google Scholar 

  35. McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552

    Article  PubMed  CAS  Google Scholar 

  36. Merlino GT, Xu YH, Ishii S, Clark AJ, Semba K, Toyoshima K, Yamamoto T, Pastan I (1984) Amplification and enhanced expression of the epidermal growth factor receptor gene in A431 human carcinoma cells. Science 224:417

    Article  PubMed  CAS  Google Scholar 

  37. Meulemans EV, Slobbe R, Wasterval P, Ramaekers FC, van Eys GJ (1994) Selection of phage-displayed antibodies specific for a cytoskeletal antigen by competitive elution with a monoclonal antibody. J Mol Biol 244:353

    Article  PubMed  CAS  Google Scholar 

  38. Mosesson Y, Yarden Y (2004) Oncogenic growth factor receptors: implications for signal transduction therapy. Semin Cancer Biol 14:262

    Article  PubMed  CAS  Google Scholar 

  39. Muyldermans S (2001) Single domain camel antibodies: current status. J Biotechnol 74:277

    PubMed  CAS  Google Scholar 

  40. Nguyen VK, Hamers R, Wyns L, Muyldermans S (2000) Camel heavy-chain antibodies: diverse germline V(H)H and specific mechanisms enlarge the antigen-binding repertoire. Embo J 19:921

    Article  PubMed  CAS  Google Scholar 

  41. Raben D, Helfrich B, Chan DC, Ciardiello F, Zhao L, Franklin W, Baron AE, Zeng C, Johnson TK, Bunn PA Jr (2005) The effects of cetuximab alone and in combination with radiation and/or chemotherapy in lung cancer. Clin Cancer Res 11:795

    PubMed  CAS  Google Scholar 

  42. Revets H, De Baetselier P, Muyldermans S (2005) Nanobodies as novel agents for cancer therapy. Expert Opin Biol Ther 5:111

    Article  PubMed  CAS  Google Scholar 

  43. Roovers RC, Henderikx P, Helfrich W, van der Linden E, Reurs A, de Bruine AP, Arends JW, de Leij L, Hoogenboom HR (1998) High-affinity recombinant phage antibodies to the pan-carcinoma marker epithelial glycoprotein-2 for tumour targeting. Br J Cancer 78:1407

    PubMed  CAS  Google Scholar 

  44. Roovers RC, van der Linden E, de Bruine AP, Arends JW, Hoogenboom HR (2001) In vitro characterisation of a monovalent and bivalent form of a fully human anti Ep-CAM phage antibody. Cancer Immunol Immunother 50:51

    Article  PubMed  CAS  Google Scholar 

  45. Rowinsky EK (2004) The erbB family: targets for therapeutic development against cancer and therapeutic strategies using monoclonal antibodies and tyrosine kinase inhibitors. Annu Rev Med 55:433

    Article  PubMed  CAS  Google Scholar 

  46. Rowinsky EK, Schwartz GH, Gollob JA, Thompson JA, Vogelzang NJ, Figlin R, Bukowski R, Haas N, Lockbaum P, Li YP, Arends R, Foon KA, Schwab G, Dutcher J (2004) Safety, pharmacokinetics, and activity of ABX-EGF, a fully human anti-epidermal growth factor receptor monoclonal antibody in patients with metastatic renal cell cancer. J Clin Oncol 22:3003

    Article  PubMed  CAS  Google Scholar 

  47. Saltz LB, Meropol NJ, Loehrer PJ Sr, Needle MN, Kopit J, Mayer RJ (2004) Phase II trial of cetuximab in patients with refractory colorectal cancer that expresses the epidermal growth factor receptor. J Clin Oncol 22:1201

    Article  PubMed  CAS  Google Scholar 

  48. Savoca KV, Abuchowski A, van Es T, Davis FF, Palczuk NC (1979) Preparation of a non-immunogenic arginase by the covalent attachment of polyethylene glycol. Biochim Biophys Acta 578:47

    PubMed  CAS  Google Scholar 

  49. Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 82:1107

    PubMed  CAS  Google Scholar 

  50. Smith GP (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228:1315

    Article  PubMed  CAS  Google Scholar 

  51. Souriau C, Rothacker J, Hoogenboom HR, Nice E (2004) Human antibody fragments specific for the epidermal growth factor receptor selected from large non-immunised phage display libraries. Growth Factors 22:185

    Article  PubMed  CAS  Google Scholar 

  52. Spano JP, Lagorce C, Atlan D, Milano G, Domont J, Benamouzig R, Attar A, Benichou J, Martin A, Morere JF, Raphael M, Penault-Llorca F, Breau JL, Fagard R, Khayat D, Wind P (2005) Impact of EGFR expression on colorectal cancer patient prognosis and survival. Ann Oncol 16:102

    Article  PubMed  Google Scholar 

  53. Todorovska A, Roovers RC, Dolezal O, Kortt AA, Hoogenboom HR, Hudson PJ (2001) Design and application of diabodies, triabodies and tetrabodies for cancer targeting. J Immunol Methods 248:47

    Article  PubMed  CAS  Google Scholar 

  54. Verheesen P, ten Haaft MR, Lindner N, Verrips CT, de Haard JJ (2003) Beneficial properties of single-domain antibody fragments for application in immunoaffinity purification and immuno-perfusion chromatography. Biochim Biophys Acta 1624:21

    PubMed  CAS  Google Scholar 

  55. Yarden Y, Sliwkowski MX (2001) Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2:127

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank prof. Dr. Guus van Dongen (VUMC, Amsterdam, The Netherlands) for pharmacokinetic data of trivalent, bispecific Nanobody fragments and for critically reading the manuscript. The term Nanobody™ was used with the kind permission of Ablynx N.V.

Author information

Authors and Affiliations

  1. Department of Molecular Cell Biology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584, Utrecht, CH, The Netherlands

    Rob C. Roovers, Arie J. Verkleij & Paul M. P. van Bergen en Henegouwen

  2. Ablynx N.V., Technologiepark 4, 9052, Zwijnaarde, Belgium

    Toon Laeremans, Severine De Taeye & Hans J. de Haard

  3. Laboratory of Cellular and Molecular Immunology, Department of Molecular and Cellular Interactions, Flanders Interuniversity Institute for Biotechnology (VIB), Free University of Brussels, Pleinlaan 2, 1050, Brussels, Belgium

    Lieven Huang & Hilde Revets

Authors
  1. Rob C. Roovers
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Toon Laeremans
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lieven Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Severine De Taeye
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Arie J. Verkleij
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hilde Revets
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hans J. de Haard
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Paul M. P. van Bergen en Henegouwen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Paul M. P. van Bergen en Henegouwen.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Roovers, R.C., Laeremans, T., Huang, L. et al. Efficient inhibition of EGFR signalling and of tumour growth by antagonistic anti-EGFR Nanobodies. Cancer Immunol Immunother 56, 303–317 (2007). https://doi.org/10.1007/s00262-006-0180-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00262-006-0180-4

Keywords

Search

Navigation