Pharmaceutical Research

, Volume 34, Issue 4, pp 696–703 | Cite as

Detection and Specific Elimination of EGFR+ Ovarian Cancer Cells Using a Near Infrared Photoimmunotheranostic Approach

  • Dirk Bauerschlag
  • Ivo Meinhold-Heerlein
  • Nicolai Maass
  • Andreas Bleilevens
  • Karen Bräutigam
  • Wa’el Al Rawashdeh
  • Stefano Di Fiore
  • Anke Maria Haugg
  • Felix Gremse
  • Julia Steitz
  • Rainer Fischer
  • Elmar Stickeler
  • Stefan Barth
  • Ahmad Fawzi Hussain
Research Paper



Targeted theranostics is an alternative strategy in cancer management that aims to improve cancer detection and treatment simultaneously. This approach combines potent therapeutic and diagnostic agents with the specificity of different cell receptor ligands in one product. The success of antibody drug conjugates (ADCs) in clinical practice has encouraged the development of antibody theranostics conjugates (ATCs). However, the generation of homogeneous and pharmaceutically-acceptable ATCs remains a major challenge. The aim of this study is to detect and eliminate ovarian cancer cells on-demand using an ATC directed to EGFR.


An ATC with a defined drug-to-antibody ratio was generated by the site-directed conjugation of IRDye®700 to a self-labeling protein (SNAP-tag) fused to an EGFR-specific antibody fragment (scFv-425).


In vitro and ex vivo imaging showed that the ATC based on scFv-425 is suitable for the highly specific detection of EGFR+ ovarian cancer cell, human tissues and ascites samples. The construct was also able to eliminate EGFR+ cells and human ascites cells with IC50 values of 45–66 nM and 40–90 nM, respectively.


Our experiments provide a framework to create a versatile technology platform for the development of ATCs for precise detection and treatment of ovarian cancer cells.


antibody theranostic conjugate molecular targeting ovarian cancer photodynamic therapy theranostics 



Antibody drug conjugate


Antibody theranostic conjugate


Epidermal growth factor receptor


Fluorescein isothiocyanate


Indocyanine green




Single-chain variable fragment


Sentinel lymph nodes



The authors would like to thank Dr. Richard M Twyman for editing the manuscript. The authors declare that they have no conflict of interest.


  1. 1.
    Vargas AN. Natural history of ovarian cancer. Ecancermedicalscience. 2014;8:465–75.PubMedPubMedCentralGoogle Scholar
  2. 2.
    Coleman RL, Monk BJ, Sood AK, Herzog TJ. Latest research and clinical treatment of advanced-stage epithelial ovarian cancer. Nat Rev Clin Oncol. 2013;10:211–24.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Oliver KE, McGuire WP. Ovarian cancer and antiangiogenic therapy: Caveat Emptor. J Clin Oncol. 2014;32:3353–6.CrossRefPubMedGoogle Scholar
  4. 4.
    Burger RA, Brady MF, Bookman MA, Fleming GF, Monk BJ, Huang H, et al. Incorporation of Bevacizumab in the primary treatment of ovarian cancer. N Engl J Med. 2011;365:2473–83.CrossRefPubMedGoogle Scholar
  5. 5.
    Perren TJ, Swart AM, Pfisterer J, Ledermann JA, Pujade-Lauraine E, Kristensen G, et al. A Phase 3 trial of Bevacizumab in ovarian cancer. N Engl J Med. 2011;365:2484–96.CrossRefPubMedGoogle Scholar
  6. 6.
    Lengyel E. Ovarian cancer development and metastasis. Am J Pathol. 2010;177:1053–64.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    du Bois A, Reuss A, Pujade-Lauraine E, Harter P, Ray-Coquard I, Pfisterer J. Role of surgical outcome as prognostic factor in advanced epithelial ovarian cancer: a combined exploratory analysis of 3 prospectively randomized phase 3 multicenter trials. Cancer. 2009;115:1234–44.CrossRefPubMedGoogle Scholar
  8. 8.
    Sato K, Hanaoka H, Watanabe R, Nakajima T, Choyke PL, Kobayashi H. Near infrared photoimmunotherapy in the treatment of disseminated peritoneal ovarian cancer. Mol Cancer Ther. 2015;14:141–50.CrossRefPubMedGoogle Scholar
  9. 9.
    Spring BQ, Abu-Yousif AO, Palanisami A, Rizvi I, Zheng X, Mai Z, et al. Selective treatment and monitoring of disseminated cancer micrometastases in vivo using dual-function, activatable immunoconjugates. Proc Natl Acad Sci U S A. 2014;111:E933–42.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Strop P, Liu SH, Dorywalska M, Delaria K, Dushin RG, Tran TT, et al. Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates. Chem Biol. 2013;20:161–7.CrossRefPubMedGoogle Scholar
  11. 11.
    Gronemeyer T, Chidley C, Juillerat A, Heinis C, Johnsson K. Directed evolution of O6-alkylguanine-DNA alkyltransferase for applications in protein labeling. Protein Engineering. Design Select. 2006;19:309–16.CrossRefGoogle Scholar
  12. 12.
    Hussain AF, Amoury M, Barth S. SNAP-tag technology: a powerful tool for site specific conjugation of therapeutic and imaging agents. Curr Pharm Des. 2013;19:5437–42.CrossRefPubMedGoogle Scholar
  13. 13.
    Hussain AF, Kampmeier F, von Felbert V, Merk HF, Tur MK, Barth S. SNAP-tag technology mediates site specific conjugation of antibody fragments with a photosensitizer and improves target specific phototoxicity in tumor cells. Bioconjug Chem. 2011;22:2487–95.CrossRefPubMedGoogle Scholar
  14. 14.
    Hussain AF, Kruger HR, Kampmeier F, Weissbach T, Licha K, Kratz F, et al. Targeted delivery of dendritic polyglycerol-doxorubicin conjugates by scFv-SNAP fusion protein suppresses EGFR+ cancer cell growth. Biomacromolecules. 2013;14:2510–20.CrossRefPubMedGoogle Scholar
  15. 15.
    Amoury M, Bauerschlag D, Zeppernick F, von Felbert V, Berges N, Di Fiore S et al. Photoimmunotheranostic agents for triple-negative breast cancer diagnosis and therapy that can be activated on demand. Oncotarget. 2016; 54925–36.Google Scholar
  16. 16.
    von Felbert V, Bauerschlag D, Maass N, Brautigam K, Meinhold-Heerlein I, Woitok M, et al. A specific photoimmunotheranostics agent to detect and eliminate skin cancer cells expressing EGFR. J Cancer Res Clin Oncol. 2016;142:1003–11.CrossRefGoogle Scholar
  17. 17.
    Mitsunaga M, Nakajima T, Sano K, Choyke PL, Kobayashi H. Near-infrared theranostic photoimmunotherapy (PIT): repeated exposure of light enhances the effect of immunoconjugate. Bioconjug Chem. 2012;23:604–9.CrossRefPubMedGoogle Scholar
  18. 18.
    Knutson S, Raja E, Bomgarden R, Nlend M, Chen A, Kalyanasundaram R. Development and evaluation of a fluorescent antibody-drug conjugate for molecular imaging and targeted therapy of pancreatic cancer. PLoS One. 2016;11:e0157762.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Sharma SK, Pourat J, Carlin S, Abdel-Atti D, Bankovich A, Sisodiya V, et al. A DLL3-targeted theranostic for small cell lung cancer: Imaging a low density target with a site-specifically modified radioimmunoconjugate. J Nucl Med. 2016;57:50.Google Scholar
  20. 20.
    Mitsunaga M, Nakajima T, Sano K, Kramer-Marek G, Choyke PL, Kobayashi H. Immediate in vivo target-specific cancer cell death after near infrared photoimmunotherapy. BMC Cancer. 2012;12:345.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Mitsunaga M, Ogawa M, Kosaka N, Rosenblum LT, Choyke PL, Kobayashi H. Cancer cell-selective in vivo near infrared photoimmunotherapy targeting specific membrane molecules. Nat Med. 2011;17:1685–91.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Kipps E, Tan DS, Kaye SB. Meeting the challenge of ascites in ovarian cancer: new avenues for therapy and research. Nat Rev Cancer. 2013;13:273–82.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    van Dam GM, Themelis G, Crane LM, Harlaar NJ, Pleijhuis RG, Kelder W, et al. Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-alpha targeting: first in-human results. Nat Med. 2011;17:1315–9.CrossRefPubMedGoogle Scholar
  24. 24.
    Tummers QR, Hoogstins CE, Gaarenstroom KN, de Kroon CD, van Poelgeest MI, Vuyk J et al. Intraoperative imaging of folate receptor alpha positive ovarian and breast cancer using the tumor specific agent EC17. Oncotarget. 2016: 32144–55.Google Scholar
  25. 25.
    Tummers QR, Hoogstins CE, Peters AA, de Kroon CD, Trimbos JB, van de Velde CJ, et al. The value of intraoperative near-infrared fluorescence imaging based on enhanced permeability and retention of indocyanine green: feasibility and false-positives in ovarian cancer. PLoS One. 2015;10:e0129766.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Cohen R, Stammes MA, de Roos IH, Stigter-van Walsum M, Visser GW, van Dongen GA. Inert coupling of IRDye800CW to monoclonal antibodies for clinical optical imaging of tumor targets. EJNMMI Res. 2011;1:31.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Dirk Bauerschlag
    • 1
  • Ivo Meinhold-Heerlein
    • 2
  • Nicolai Maass
    • 1
  • Andreas Bleilevens
    • 2
  • Karen Bräutigam
    • 3
  • Wa’el Al Rawashdeh
    • 4
  • Stefano Di Fiore
    • 5
  • Anke Maria Haugg
    • 2
  • Felix Gremse
    • 4
  • Julia Steitz
    • 6
  • Rainer Fischer
    • 5
    • 7
  • Elmar Stickeler
    • 2
  • Stefan Barth
    • 8
    • 9
  • Ahmad Fawzi Hussain
    • 2
  1. 1.Department of Gynecology and ObstetricsUniversity Medical Center Schleswig-HolsteinKielGermany
  2. 2.Department of Gynecology and ObstetricsUniversity Hospital RWTH AachenAachenGermany
  3. 3.Department of Gynecology and ObstetricsUniversity Hospital Schleswig-HolsteinLübeckGermany
  4. 4.Department of Experimental Molecular ImagingRWTH Aachen UniversityAachenGermany
  5. 5.Fraunhofer Institute for Molecular Biology and Applied Ecology IMEAachenGermany
  6. 6.Institute for Laboratory Animal ScienceUniversity Hospital RWTH AachenAachenGermany
  7. 7.Institute of Molecular BiotechnologyRWTH Aachen UniversityAachenGermany
  8. 8.Department of Pharmaceutical Product DevelopmentFraunhofer Institute for Molecular Biology and Applied Ecology IMEAachenGermany
  9. 9.South African Research Chair in Cancer Biotechnology, Institute of Infectious Disease and Molecular Medicine (IDM), Department of Integrative Biomedical Sciences, Faculty of Health SciencesUniversity of Cape TownObservatorySouth Africa

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