Skip to main content
SpringerLink
Log in

Synthesis and preclinical investigation of 99mTc-p-SCN-Bzl-DTPA-cetuximab for targeting EGFR using head and neck squamous cell carcinoma (HNSCC) xenografts

  • Original Article
  • Published:
Molecular Biology Reports Aims and scope Submit manuscript

Abstract

To assess the preclinical potential of technetium-99m labelled conjugated para-isothiocyanato-benzyl diethylene triamine penta-acetic acid cetuximab (99mTc-p-SCN-Bzl-DTPA cetuximab) for imaging EGFR in HNSCC mice and rabbits xenografts. Cetuximab, a chimeric monoclonal antibody targeting EGFR, was conjugated with p-SCN-Bzl-DTPA followed by labelling with 99mTc. The labelled conjugate was evaluated for in vitro stability in L−cysteine at 37 °C. The 99mTc-p-SCN-Bzl-DTPA cetuximab was also investigated for immunoreactivity, internationalization kinetics, dose escalation (up to 300 µg) and biodistribution in HNSCC mice xenograft. The suitability of labelled moiety as a specific EGFR radio-tracer was assessed in HNSCC rabbit xenograft. 99mTc-p-SCN-Bzl-DTPA cetuximab exhibited more than 98% radiochemical purity at room temperature. In excess L−cysteine, it showed a stable behaviour at 37 °C up to 4 h p.l. The labelled conjugate was internalized in vitro in FaDu tumor cells up to 19.55%. Significantly higher uptake in tumor (at 10 µg; 34.75 ± 0.38% ID/g: pi) was seen in HNSCC mice xenograft with dose escalation assay from 1 to 300 µg/mouse. Blocking of EGFR with excess cetuximab consequently decreased the uptake of tumor up to 6.80 ± 1.25%. SPECT images of rabbit xenograft confirmed increase in tumor to background ratio after 4 h pi and validated its potential in preclinical trial as a specific FaDu tumor tracer. Our in vitro and in vivo preclinical findings indicate that the 99mTc-p-SCN-Bzl-DTPA cetuximab prepared at optimal dose of cetuximab could become a useful tool for EGFR imaging in HNSCC using SPECT.

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. Chong CR, Janne PA (2013) The quest to overcome resistance to EGFR-targeted therapies in cancer. Nat Med 19:1389–1400

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Scaltriti M, Baselga J (2006) The epidermal growth factor receptor pathway: a model for targeted therapy. Clin Cancer Res 12:5268–5272

    Article  CAS  PubMed  Google Scholar 

  3. Nicholson RI, Gee JM, Harper ME (2001) EGFR and cancer prognosis. Eur J Cancer 37:S9–S15

    Article  CAS  PubMed  Google Scholar 

  4. Schlomm T, Kirstein P, Iwers L et al (2007) Clinical significance of epidermal growth factor receptor protein overexpression and gene copy number gains in prostate cancer. Clin Cancer Res 13:6579–6584

    Article  CAS  PubMed  Google Scholar 

  5. Parra HS, Cavina R, Latteri F et al (2004) Analysis of epidermal growth factor receptor expression as a predictive factor for response to gefitinib (‘Iressa’, ZD1839) in non-small-cell lung cancer. Br J Cancer 91:208–212

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Zlobec I, Vuong T, Hayashi S et al (2007) A simple and reproducible scoring system for EGFR in colorectal cancer: application to prognosis and prediction of response to preoperative brachytherapy. Br J Cancer 96:793–800

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Garousi J, Andersson KG, Mitran B et al (2016) PET imaging of epidermal growth factor receptor expression in tumours using 89Zr-labelled ZEGFR:2377 affibody molecules. Int J Oncol 48:1325–1332

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Menke-van der Houven van Oordt CW, Gootjes EC, Huisman MC et al. 89Zr-cetuximab PET imaging in patients with advanced colorectal cancer. Oncotarget. 2015;6:30384–30393

    PubMed  PubMed Central  Google Scholar 

  9. Nayak TK, Regino CA, Wong KJ et al (2010) PET imaging of HER1-expressing xenografts in mice with 86Y-CHX-A’’-DTPA-cetuximab. Eur J Nucl Med Mol Imaging 37:1368–1376

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Eiblmaier M, Meyer LA, Watson MA, Fracasso PM, Pike LJ, Anderson CJ (2008) Correlating EGFR expression with receptor-binding properties and internalization of 64Cu-DOTA-cetuximab in 5 cervical cancer cell lines. J Nucl Med 49:1472–1479

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Yun M, Kim DY, Lee JJ et al (2017) A high-affinity repebody for molecular imaging of EGFRexpressing malignant tumors. Theranostics 7:2620–2633

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hwang DE, Ryou JH, Oh JR, Han JW, Park TK, Kim HS (2016) Anti-human VEGF repebody effectively suppresses choroidal neovascularization and vascular leakage. PLoS ONE 11:e0152522

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Lee JJ, Choi HJ, Yun M et al (2015) Enzymatic prenylation and oxime ligation for the synthesis of stable and homogeneous protein-drug conjugates for targeted therapy. Angew Chem Int Ed Engl 54:12020–12024

    Article  CAS  PubMed  Google Scholar 

  14. Lee JJ, Kim HJ, Yang C-S et al (2014) A high-affinity protein binder that blocks the IL-6/STAT3 signaling pathway effectively suppresses non–small cell lung cancer. Mol Ther 22:1254-

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Kim JY, Park H, Lee JC et al (2009) A simple Cu-64 production and its application of Cu-64 ATSM. Appl Radiat Isot 67:1190–1194

    Article  CAS  PubMed  Google Scholar 

  16. Cai W, Chen K, Mohamedali KA et al (2006) PET of vascular endothelial growth factor receptor expression. J Nucl Med 47:2048–2056

    CAS  PubMed  Google Scholar 

  17. Yang CH, Chou HC, Fu YN et al (2015) EGFR over-expression in non-small cell lung cancers harboring EGFR mutations is associated with marked down-regulation of CD82. Biochim Biophys Acta 1852:1540–1549

    Article  CAS  PubMed  Google Scholar 

  18. Cross DA, Ashton SE, Ghiorghiu S et al (2014) AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer. Cancer Discov 4:1046–1061

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Petrulli JR, Sullivan JM, Zheng MQ et al (2013) Quantitative analysis of [11C]-erlotinib PET demonstrates specific binding for activating mutations of the EGFR kinase domain. Neoplasia 15:1347–1353

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Cooper MS, Ma MT, Sunassee K et al (2012) Comparison of 64Cu-complexing bifunctional chelators for radioimmunoconjugation: labeling efficiency, specific activity, and in vitro/in vivo stability. Bioconjug Chem 23:1029–1039

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Schechter NR, Wendt RE, Yang DJ et al (2004) Radiation dosimetry of 99mTc-labeled C225 in patients with squamous cell carcinoma of the head and neck. J Nucl Med 45:1683–1687

    CAS  PubMed  Google Scholar 

  22. Wen X, Wu QP, Ke S et al (2001) Conjugation with (111)InDTPA-poly(ethylene glycol) improves imaging of anti-EGF receptor antibody C225. J Nucl Med 42:1530–1537

    CAS  PubMed  Google Scholar 

  23. Li WP, Meyer LA, Capretto DA et al (2008) Receptor-binding, biodistribution, and metabolism studies of 64Cu-DOTA-cetuximab, a PET-imaging agent for epidermal growth-factor receptor-positive tumors. Cancer Biother Radiopharm 23:158–171

    Article  Google Scholar 

  24. Perk LR, Visser GW, Vosjan MJ et al (2005) (89)Zr as a PET surrogate radioisotope for scouting biodistribution of the therapeutic radiometals (90)Y and (177)Lu in tumor-bearing nude mice after coupling to the internalizing antibody cetuximab. J Nucl Med 46:1898–1906

    CAS  PubMed  Google Scholar 

  25. Nayak TK, Regino CA, Wong KJ et al (2010) PET imaging of HER1-expressing xenografts in mice with 86Y-CHX-A″- DTPA-cetuximab. Eur J Nucl Med Mol Imaging 37:1368–1376

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Shah SQ, Gul-E-Raana, Uddin G (2018) Imaging prostate cancer (PCa) with [99mTc(CO)3finasteride dithiocarbamate. J Labelled Comp Radiopharm 61:550–556

    Article  CAS  PubMed  Google Scholar 

  27. Brady ED, Chong HS, Milenic DE, Brechbiel MW (2004) Development of a spectroscopic assay for bifunctional ligand-protein conjugates based on copper. Nucl Med Boil 31:795–802

    Article  CAS  Google Scholar 

  28. Shah SQ, Mahmood S (2018) Evaluation of 99mTc-labeled bevacizumab-N-HYNIC conjugate in human ovarian tumor xenografts. Cancer Biother Radiopharm 33:96–102

    Article  CAS  Google Scholar 

  29. Koenig JA, Kaur R, Dodgeon I, Edwardson JM, Humphrey PP (1998) Fates of endocytosed somatostatin sst2 receptors and associated agonists. Biochem J 336:291–298

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Cai W, Chen K, He L, Cao Q, Koong A, Chen X (2007) Quantitative PET of EGFR expression in xenograft-bearing mice using 64Cu-labeled cetuximab, a chimeric anti-EGFR monoclonal antibody. Eur J Nucl Med Mol Imaging 34:850–858

    Article  CAS  PubMed  Google Scholar 

  31. Niu G, Sun X, Cao Q et al (2010) Cetuximab-based immunotherapy and radioimmunotherapy of head and neck squamous cell carcinoma. Clin Cancer Res 16:2095–2105

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Goldenberg A, Masui H, Divgi C et al (1989) Imaging of human tumor xenografts with an indium-111-labeled anti-epidermal growth factor receptor monoclonal antibody. J Natl Cancer Inst 81:1616–1625

    Article  CAS  PubMed  Google Scholar 

  33. Niu G, Li Z, Xie J, Le QT, Chen X (2009) PET of EGFR antibody distribution in head andneck squamous cell carcinoma models. J Nucl Med 50:1116–1123

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Kareem H, Sandstrom K, Elia R et al (2010) Blocking EGFR in the liver improves the tumor-to-liver uptake ratio of radiolabeled EGF. Tumor Biol 31:79–87

    Article  CAS  Google Scholar 

  35. Divgi CR, Welt S, Kris M et al (1991) Phase I and imaging trial of indium 111-labeledanti-epidermal growth factor receptor monoclonal antibody 225 in patients withsquamous cell lung carcinoma. J Natl Cancer Inst 83:97–104

    Article  CAS  PubMed  Google Scholar 

  36. Milenic DE, Wong KJ, Baidoo KE et al (2008) Cetuximab: preclinical evaluation of a monoclonal antibody targeting EGFR for radioimmunodiagnostic and radioimmunotherapeutic applications. Cancer Biother Radiopharm 23:619–631

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank the Higher Education Commission (HEC), Islamabad, Pakistan for providing all sorts of financial assistance under National Research Program for Universities. The instant study is part of the HEC funded research grant No. 3122.

Author information

Authors and Affiliations

  1. Institute of Chemical Sciences, University of Peshawar, Peshawar, K.P.K, 25120, Pakistan

    Syed Qaiser Shah &  Gul-e-Raana

Authors
  1. Syed Qaiser Shah
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gul-e-Raana
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

GR performed conjugation of cetuximab with p-SCN-Bzl-DTPA, labeling with radionuclide, in vitro studies and biodistribution in animal model mice. SQS performed the imaging studies using rabbit xenograft. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Syed Qaiser Shah.

Ethics declarations

Conflict of interest

No conflict of interests.

Ethics approval and consent to participate

No human was involved in this study. Experiments on mice and rabbits models were executed in compliance with Nuclear Medicine Research Laboratory (NMRL) and approval of ethics committee at Institute of Chemical Sciences (ICS), University of Peshawar.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shah, S.Q., Gul-e-Raana Synthesis and preclinical investigation of 99mTc-p-SCN-Bzl-DTPA-cetuximab for targeting EGFR using head and neck squamous cell carcinoma (HNSCC) xenografts. Mol Biol Rep 46, 1675–1682 (2019). https://doi.org/10.1007/s11033-019-04616-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11033-019-04616-x

Keywords

Search

Navigation