Advertisement

Rapid optical imaging of EGF receptor expression with a single-chain antibody SNAP-tag fusion protein

  • Florian Kampmeier
  • Judith Niesen
  • Alexander Koers
  • Markus Ribbert
  • Andreas Brecht
  • Rainer Fischer
  • Fabian Kießling
  • Stefan Barth
  • Theo Thepen
Original Article

Abstract

Purpose

The epidermal growth factor receptor (EGFR) is overexpressed in several types of cancer and its inhibition can effectively inhibit tumour progression. The purpose of this study was to design an EGFR-specific imaging probe that combines efficient tumour targeting with rapid systemic clearance to facilitate non-invasive assessment of EGFR expression.

Methods

Genetic fusion of a single-chain antibody fragment with the SNAP-tag produced a 48-kDa antibody derivative that can be covalently and site-specifically labelled with substrates containing 0 6-benzylguanine. The EGFR-specific single-chain variable fragment (scFv) fusion protein 425(scFv)SNAP was labelled with the near infrared (NIR) dye BG-747, and its accumulation, specificity and kinetics were monitored using NIR fluorescence imaging in a subcutaneous pancreatic carcinoma xenograft model.

Results

The 425(scFv)SNAP fusion protein accumulates rapidly and specifically at the tumour site. Its small size allows efficient renal clearance and a high tumour to background ratio (TBR) of 33.2 ± 6.3 (n = 4) 10 h after injection. Binding of the labelled antibody was efficiently competed with a 20-fold excess of unlabelled probe, resulting in an average TBR of 6 ± 1.35 (n = 4), which is similar to that obtained with a non-tumour-specific probe (5.44 ± 1.92, n = 4). When compared with a full-length antibody against EGFR (cetuximab), 425(scFv)SNAP-747 showed significantly higher TBRs and complete clearance 72 h post-injection.

Conclusion

The 425(scFv)SNAP fusion protein combines rapid and specific targeting of EGFR-positive tumours with a versatile and robust labelling technique that facilitates the attachment of fluorophores for use in optical imaging. The same approach could be used to couple a chelating agent for use in nuclear imaging.

Keywords

Single-chain antibody hAGT (SNAP)-tag NIR optical imaging Site-specific labelling Molecular imaging Tumour targeting 

Abbreviations

hAGT

Human O 6-alkyl-DNA alkyltransferase

NIR

Near infrared

scFv

Single-chain variable fragment

EGFR

Epidermal growth factor receptor 1

BG

Benzylguanine

Notes

Acknowledgments

We would like to thank Dr. Richard Twyman for the critical reading of the manuscript and Agnieszka Dreier (Institute for Neuropathology, University Hospital, RWTH Aachen University) for supplying the cetuximab antibody.

Conflicts of interest

None.

References

  1. 1.
    Mendelsohn J. Targeting the epidermal growth factor receptor for cancer therapy. J Clin Oncol 2002;20:1S–13.PubMedGoogle Scholar
  2. 2.
    Nicholson RI, Gee JM, Harper ME. EGFR and cancer prognosis. Eur J Cancer 2001;37 Suppl 4:S9–15.CrossRefPubMedGoogle Scholar
  3. 3.
    Harandi A, Zaidi AS, Stocker AM, Laber DA. Clinical efficacy and toxicity of anti-EGFR therapy in common cancers. J Oncol 2009;2009:567486.PubMedGoogle Scholar
  4. 4.
    Ang KK, Berkey BA, Tu X, Zhang HZ, Katz R, Hammond EH, et al. Impact of epidermal growth factor receptor expression on survival and pattern of relapse in patients with advanced head and neck carcinoma. Cancer Res 2002;62:7350–6.PubMedGoogle Scholar
  5. 5.
    Hirsch FR, Herbst RS, Olsen C, Chansky K, Crowley J, Kelly K, et al. Increased EGFR gene copy number detected by fluorescent in situ hybridization predicts outcome in non-small-cell lung cancer patients treated with cetuximab and chemotherapy. J Clin Oncol 2008;26:3351–7.CrossRefPubMedGoogle Scholar
  6. 6.
    Schlomm T, Kirstein P, Iwers L, Daniel B, Steuber T, Walz J, et al. Clinical significance of epidermal growth factor receptor protein overexpression and gene copy number gains in prostate cancer. Clin Cancer Res 2007;13:6579–84.CrossRefPubMedGoogle Scholar
  7. 7.
    Weissleder R, Ntziachristos V. Shedding light onto live molecular targets. Nat Med 2003;9:123–8.CrossRefPubMedGoogle Scholar
  8. 8.
    Ke S, Wen X, Gurfinkel M, Charnsangavej C, Wallace S, Sevick-Muraca EM, et al. Near-infrared optical imaging of epidermal growth factor receptor in breast cancer xenografts. Cancer Res 2003;63:7870–5.PubMedGoogle Scholar
  9. 9.
    Diagaradjane P, Orenstein-Cardona JM, Colón-Casasnovas NE, Deorukhkar A, Shentu S, Kuno N, et al. Imaging epidermal growth factor receptor expression in vivo: pharmacokinetic and biodistribution characterization of a bioconjugated quantum dot nanoprobe. Clin Cancer Res 2008;14:731–41.CrossRefPubMedGoogle Scholar
  10. 10.
    Koyama Y, Barrett T, Hama Y, Ravizzini G, Choyke PL, Kobayashi H. In vivo molecular imaging to diagnose and subtype tumors through receptor-targeted optically labeled monoclonal antibodies. Neoplasia 2007;9:1021–9.CrossRefPubMedGoogle Scholar
  11. 11.
    Kampmeier F, Ribbert M, Nachreiner T, Dembski S, Beaufils F, Brecht A, et al. Site-specific, covalent labeling of recombinant antibody fragments via fusion to an engineered version of 6-O-alkylguanine DNA alkyltransferase. Bioconjug Chem 2009.Google Scholar
  12. 12.
    Bruns CJ, Harbison MT, Kuniyasu H, Eue I, Fidler IJ. In vivo selection and characterization of metastatic variants from human pancreatic adenocarcinoma by using orthotopic implantation in nude mice. Neoplasia 1999;1:50–62.CrossRefPubMedGoogle Scholar
  13. 13.
    Kamat V, Donaldson JM, Kari C, Quadros MR, Lelkes PI, Chaiken I, et al. Enhanced EGFR inhibition and distinct epitope recognition by EGFR antagonistic mAbs C225 and 425. Cancer Biol Ther 2008;7:726–33.PubMedGoogle Scholar
  14. 14.
    Bruell D, Bruns CJ, Yezhelyev M, Huhn M, Müller J, Ischenko I, et al. Recombinant anti-EGFR immunotoxin 425(scFv)-ETA' demonstrates anti-tumor activity against disseminated human pancreatic cancer in nude mice. Int J Mol Med 2005;15:305–13.PubMedGoogle Scholar
  15. 15.
    Kindermann M, George N, Johnsson N, Johnsson K. Covalent and selective immobilization of fusion proteins. J Am Chem Soc 2003;125:7810–1.CrossRefPubMedGoogle Scholar
  16. 16.
    Keppler A, Kindermann M, Gendreizig S, Pick H, Vogel H, Johnsson K. Labeling of fusion proteins of O6-alkylguanine-DNA alkyltransferase with small molecules in vivo and in vitro. Methods 2004;32:437–44.CrossRefPubMedGoogle Scholar
  17. 17.
    Klimka A, Barth S, Matthey B, Roovers RC, Lemke H, Hansen H, et al. An anti-CD30 single-chain Fv selected by phage display and fused to Pseudomonas exotoxin A (Ki-4(scFv)-ETA') is a potent immunotoxin against a Hodgkin-derived cell line. Br J Cancer 1999;80:1214–22.CrossRefPubMedGoogle Scholar
  18. 18.
    Maeda H. The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting. Adv Enzyme Regul. 2001;41:189–207.Google Scholar
  19. 19.
    Yokota T, Milenic DE, Whitlow M, Schlom J. Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin forms. Cancer Res 1992;52:3402–8.PubMedGoogle Scholar
  20. 20.
    Choi HS, Liu W, Misra P, Tanaka E, Zimmer JP, Itty Ipe B, et al. Renal clearance of quantum dots. Nat Biotechnol 2007;25:1165–70.CrossRefPubMedGoogle Scholar
  21. 21.
    Goel A, Colcher D, Baranowska-Kortylewicz J, Augustine S, Booth BJ, Pavlinkova G, et al. Genetically engineered tetravalent single-chain Fv of the pancarcinoma monoclonal antibody CC49: improved biodistribution and potential for therapeutic application. Cancer Res 2000;60:6964–71.PubMedGoogle Scholar
  22. 22.
    Adams GP, Tai MS, McCartney JE, Marks JD, Stafford 3rd WF, Houston LL, et al. Avidity-mediated enhancement of in vivo tumor targeting by single-chain Fv dimers. Clin Cancer Res 2006;12:1599–605.CrossRefPubMedGoogle Scholar
  23. 23.
    Holliger P, Hudson PJ. Engineered antibody fragments and the rise of single domains. Nat Biotechnol 2005;23:1126–36.CrossRefPubMedGoogle Scholar
  24. 24.
    Batra SK, Jain M, Wittel UA, Chauhan SC, Colcher D. Pharmacokinetics and biodistribution of genetically engineered antibodies. Curr Opin Biotechnol 2002;13:603–8.CrossRefPubMedGoogle Scholar
  25. 25.
    Schneider DW, Heitner T, Alicke B, Light DR, McLean K, Satozawa N, et al. In vivo biodistribution, PET imaging, and tumor accumulation of 86Y- and 111In-antimindin/RG-1, engineered antibody fragments in LNCaP tumor-bearing nude mice. J Nucl Med 2009;50:435–43.CrossRefPubMedGoogle Scholar
  26. 26.
    Withrow KP, Gleysteen JP, Safavy A, Skipper J, Desmond RA, Zinn K, et al. Assessment of indocyanine green-labeled cetuximab to detect xenografted head and neck cancer cell lines. Otolaryngol Head Neck Surg 2007;137:729–34.CrossRefPubMedGoogle Scholar
  27. 27.
    Cuesta AM, Sánchez-Martín D, Sanz L, Bonet J, Compte M, Kremer L, et al. In vivo tumor targeting and imaging with engineered trivalent antibody fragments containing collagen-derived sequences. PLoS One 2009;4:e5381.CrossRefPubMedGoogle Scholar
  28. 28.
    Yazaki PJ, Kassa T, Cheung CW, Crow DM, Sherman MA, Bading JR, et al. Biodistribution and tumor imaging of an anti-CEA single-chain antibody-albumin fusion protein. Nucl Med Biol 2008;35:151–8.CrossRefPubMedGoogle Scholar
  29. 29.
    Tolmachev V, Friedman M, Sandström M, Eriksson TL, Rosik D, Hodik M, et al. Affibody molecules for epidermal growth factor receptor targeting in vivo: aspects of dimerization and labeling chemistry. J Nucl Med 2009;50:274–83.CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Florian Kampmeier
    • 1
  • Judith Niesen
    • 1
  • Alexander Koers
    • 1
  • Markus Ribbert
    • 1
  • Andreas Brecht
    • 2
  • Rainer Fischer
    • 1
    • 3
  • Fabian Kießling
    • 4
  • Stefan Barth
    • 1
    • 5
  • Theo Thepen
    • 1
  1. 1.Fraunhofer Institute for Molecular Biology and Applied EcologyAachenGermany
  2. 2.Covalys Biosciences AGWitterswilSwitzerland
  3. 3.Institute for Molecular BiotechnologyRWTH Aachen UniversityAachenGermany
  4. 4.Department of Experimental Molecular Imaging, Medical FacultyRWTH Aachen UniversityAachenGermany
  5. 5.Department of Experimental Medicine and Immunotherapy, Helmholtz Institute for Biomedical EngineeringRWTH Aachen UniversityAachenGermany

Personalised recommendations