Quantitative PET of EGFR expression in xenograft-bearing mice using 64Cu-labeled cetuximab, a chimeric anti-EGFR monoclonal antibody

  • Weibo Cai
  • Kai Chen
  • Lina He
  • Qizhen Cao
  • Albert Koong
  • Xiaoyuan ChenEmail author
Molecular imaging



Cetuximab, a chimeric monoclonal antibody targeting epidermal growth factor receptor (EGFR) on the surface of cancer cells, was approved by the FDA to treat patients with metastatic colorectal cancer. It is currently also in advanced-stage development for the treatment of several other solid tumors. Here we report for the first time the quantitative positron emission tomography (PET) imaging of EGFR expression in xenograft-bearing mice using 64Cu-labeled cetuximab.


We conjugated cetuximab with macrocyclic chelating agent 1,4,7,10-tetraazadodecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA), labeled with 64Cu, and tested the resulting 64Cu-DOTA-cetuximab in seven xenograft tumor models. The tracer uptake measured by PET was correlated with the EGFR expression quantified by western blotting. The estimated human dosimetry based on the PET data in Sprague-Dawley rats was also calculated.


MicroPET imaging showed that 64Cu-DOTA-cetuximab had increasing tumor activity accumulation over time in EGFR-positive tumors but relatively low uptake in EGFR-negative tumors at all times examined (<5%ID/g). There was a good correlation (R 2 = 0.80) between the tracer uptake (measured by PET) and the EGFR expression level (measured by western blotting). Human dosimetry estimation indicated that the tracer may be safely administered to human patients for tumor diagnosis, with the dose-limiting organ being the liver.


The success of EGFR-positive tumor imaging using 64Cu-DOTA-cetuximab can be translated into the clinic to characterize the pharmacokinetics, to select the right population of patients for EGFR-targeted therapy, to monitor the therapeutic efficacy of anti-EGFR treatment, and to optimize the dosage of either cetuximab alone or cetuximab in combination with other therapeutic agents.


Cetuximab Epidermal growth factor receptor Micro-positron emission tomography Copper-64 



This project was financially supported by National Institute of Biomedical Imaging and Bioengineering (NIBIB) (R21 EB001785), National Cancer Institute (NCI) (R21 CA102123, P50 CA114747, U54 CA119367, and R24 CA93862), Department of Defense (DOD) (W81XWH-04-1-0697, W81XWH-06-1-0665, W81XWH-06-1-0042, and DAMD17-03-1-0143), and a Benedict Cassen Postdoctoral Fellowship from the Education and Research Foundation of the Society of Nuclear Medicine (to W.Cai).


  1. 1.
    Arteaga C. Targeting HER1/EGFR: a molecular approach to cancer therapy. Semin Oncol 2003;30:3–14.Google Scholar
  2. 2.
    Blobel CP. ADAMs: key components in EGFR signalling and development. Nat Rev Mol Cell Biol 2005;6:32–43.PubMedCrossRefGoogle Scholar
  3. 3.
    Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell 2000;103:211–25.PubMedCrossRefGoogle Scholar
  4. 4.
    Starling N, Cunningham D. Monoclonal antibodies against vascular endothelial growth factor and epidermal growth factor receptor in advanced colorectal cancers: present and future directions. Curr Opin Oncol 2004;16:385–90.PubMedCrossRefGoogle Scholar
  5. 5.
    Wu AM, Senter PD. Arming antibodies: prospects and challenges for immunoconjugates. Nat Biotechnol 2005;23:1137–46.PubMedCrossRefGoogle Scholar
  6. 6.
    Holliger P, Hudson PJ. Engineered antibody fragments and the rise of single domains. Nat Biotechnol 2005;23:1126–36.PubMedCrossRefGoogle Scholar
  7. 7.
    Ciardiello F, Damiano V, Bianco R, Bianco C, Fontanini G, De Laurentiis M, et al. Antitumor activity of combined blockade of epidermal growth factor receptor and protein kinase A. J Natl Cancer Inst 1996;88:1770–6.PubMedCrossRefGoogle Scholar
  8. 8.
    Mendelsohn J, Baselga J. Status of epidermal growth factor receptor antagonists in the biology and treatment of cancer. J Clin Oncol 2003;21:2787–99.PubMedCrossRefGoogle Scholar
  9. 9.
    Bonner JA, Harari PM, Giralt J, Azarnia N, Shin DM, Cohen RB, et al. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med 2006;354:567–78.PubMedCrossRefGoogle Scholar
  10. 10.
    Thienelt CD, Bunn PA Jr, Hanna N, Rosenberg A, Needle MN, Long ME, et al. Multicenter phase I/II study of cetuximab with paclitaxel and carboplatin in untreated patients with stage IV non-small-cell lung cancer. J Clin Oncol 2005;23:8786–93.PubMedCrossRefGoogle Scholar
  11. 11.
    Xiong HQ, Rosenberg A, LoBuglio A, Schmidt W, Wolff RA, Deutsch J, et al. Cetuximab, a monoclonal antibody targeting the epidermal growth factor receptor, in combination with gemcitabine for advanced pancreatic cancer: a multicenter phase II trial. J Clin Oncol 2004;22:2610–6.PubMedCrossRefGoogle Scholar
  12. 12.
    Frieze DA, McCune JS. Current status of cetuximab for the treatment of patients with solid tumors. Ann Pharmacother 2006;40:241–50.PubMedCrossRefGoogle Scholar
  13. 13.
    Schechter NR, Wendt RE 3rd, Yang DJ, Azhdarinia A, Erwin WD, Stachowiak AM, et al. Radiation dosimetry of 99mTc-labeled C225 in patients with squamous cell carcinoma of the head and neck. J Nucl Med 2004;45:1683–7.PubMedGoogle Scholar
  14. 14.
    Perk LR, Visser GW, Vosjan MJ, Stigter-van Walsum M, Tijink BM, Leemans CR, et al. 89Zr as a PET surrogate radioisotope for scouting biodistribution of the therapeutic radiometals 90Y and 177Lu in tumor-bearing nude mice after coupling to the internalizing antibody cetuximab. J Nucl Med 2005;46:1898–906.PubMedGoogle Scholar
  15. 15.
    Velikyan I, Sundberg AL, Lindhe O, Hoglund AU, Eriksson O, Werner E, et al. Preparation and evaluation of 68Ga-DOTA-hEGF for visualization of EGFR expression in malignant tumors. J Nucl Med 2005;46:1881–8.PubMedGoogle Scholar
  16. 16.
    Wu AM, Williams LE, Zieran L, Padma A, Sherman MA, Bebb GG, et al. Anti-carcinoembryonic antigen (CEA) diabody for rapid tumor targeting and imaging. Tumor Targeting 1999;4:47–58.Google Scholar
  17. 17.
    Chen X, Park R, Hou Y, Tohme M, Shahinian AH, Bading JR, et al. MicroPET and autoradiographic imaging of GRP receptor expression with 64Cu-DOTA-[Lys3]bombesin in human prostate adenocarcinoma xenografts. J Nucl Med 2004;45:1390–7.PubMedGoogle Scholar
  18. 18.
    Zhang X, Xiong Z, Wu X, Cai W, Tseng JR, Gambhir SS, et al. Quantitative PET imaging of tumor integrin αvβ3 expression with 18F-FRGD2. J Nucl Med 2006;47:113–21.PubMedGoogle Scholar
  19. 19.
    Cai W, Shin DW, Chen K, Gheysens O, Cao Q, Wang SX, et al. Peptide-labeled near-infrared quantum dots for imaging tumor vasculature in living subjects. Nano Lett 2006;6:669–76.PubMedCrossRefGoogle Scholar
  20. 20.
    Cai W, Zhang X, Wu Y, Chen X. A thiol-reactive 18F-labeling agent, N-[2-(4-18F-fluorobenzamido)ethyl]maleimide (18F-FBEM), and the synthesis of RGD peptide-based tracer for PET imaging of αvβ3 integrin expression. J Nucl Med 2006;47:1172–80.PubMedGoogle Scholar
  21. 21.
    Wu Y, Zhang X, Xiong Z, Cheng Z, Fisher DR, Liu S, et al. MicroPET imaging of glioma αv-integrin expression using 64Cu-labeled tetrameric RGD peptide. J Nucl Med 2005;46:1707–18.PubMedGoogle Scholar
  22. 22.
    Cai W, Wu Y, Chen K, Cao Q, Tice DA, Chen X. In vitro and in vivo characterization of 64Cu-labeled Abegrin™, a humanized monoclonal antibody against integrin αvβ3. Cancer Res 2006;66:9673–81.PubMedCrossRefGoogle Scholar
  23. 23.
    Meares CF, McCall MJ, Reardan DT, Goodwin DA, Diamanti CI, McTigue M. Conjugation of antibodies with bifunctional chelating agents: isothiocyanate and bromoacetamide reagents, methods of analysis, and subsequent addition of metal ions. Anal Biochem 1984;142:68–78.PubMedCrossRefGoogle Scholar
  24. 24.
    Cai W, Chen K, Mohamedali KA, Cao Q, Gambhir SS, Rosenblum MG, et al. PET of vascular endothelial growth factor receptor expression. J Nucl Med 2006;47:2048–56.PubMedGoogle Scholar
  25. 25.
    Lindmo T, Boven E, Cuttitta F, Fedorko J, Bunn PA Jr. Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess. J Immunol Methods 1984;72:77–89.PubMedCrossRefGoogle Scholar
  26. 26.
    Chen X, Sievers E, Hou Y, Park R, Tohme M, Bart R, et al. Integrin αvβ3-targeted imaging of lung cancer. Neoplasia 2005;7:271–9.PubMedCrossRefGoogle Scholar
  27. 27.
    Xiong Z, Cheng Z, Zhang X, Patel M, Wu JC, Gambhir SS, et al. Imaging chemically modified adenovirus for targeting tumors expressing integrin αvβ3 in living mice with mutant herpes simplex virus type 1 thymidine kinase PET reporter gene. J Nucl Med 2006;47:130–9.PubMedGoogle Scholar
  28. 28.
    Visvikis D, Cheze-LeRest C, Costa DC, Bomanji J, Gacinovic S, Ell PJ. Influence of OSEM and segmented attenuation correction in the calculation of standardised uptake values for [18F]FDG PET. Eur J Nucl Med 2001;28:1326–35.PubMedCrossRefGoogle Scholar
  29. 29.
    Anderson CJ, Jones LA, Bass LA, Sherman EL, McCarthy DW, Cutler PD, et al. Radiotherapy, toxicity and dosimetry of copper-64-TETA-octreotide in tumor-bearing rats. J Nucl Med 1998;39:1944–51.PubMedGoogle Scholar
  30. 30.
    Sgouros G. Dosimetry of internal emitters. J Nucl Med 2005;46 Suppl 1:18S–27S.PubMedGoogle Scholar
  31. 31.
    Cutler PD, Schwarz SW, Anderson CJ, Connett JM, Welch MJ, Philpott GW, et al. Dosimetry of copper-64-labeled monoclonal antibody 1A3 as determined by PET imaging of the torso. J Nucl Med 1995;36:2363–71.PubMedGoogle Scholar
  32. 32.
    Cai W, Rao J, Gambhir SS, Chen X. How molecular imaging is speeding up anti-angiogenic drug development. Mol Cancer Ther 2006;5:2624–33.PubMedCrossRefGoogle Scholar
  33. 33.
    Cai W, Olafsen T, Zhang X, Cao Q, Gambhir SS, Williams LE, et al. PET imaging of colorectal cancer in xenograft-bearing mice by use of an 18F-labeled T84.66 anti-carcinoembryonic diabody. J Nucl Med 2007:in press.Google Scholar
  34. 34.
    International Commission on Radiological Protection. 1990 Recommendations of the International Commission on Radiological Protection: ICRP publication 60. Ann ICRP 1991;21:6–10.Google Scholar
  35. 35.
    Philpott GW, Schwarz SW, Anderson CJ, Dehdashti F, Connett JM, Zinn KR, et al. RadioimmunoPET: detection of colorectal carcinoma with positron-emitting copper-64-labeled monoclonal antibody. J Nucl Med 1995;36:1818–24.PubMedGoogle Scholar
  36. 36.
    Verel I, Visser GW, Boellaard R, Boerman OC, van Eerd J, Snow GB, et al. Quantitative 89Zr immuno-PET for in vivo scouting of 90Y-labeled monoclonal antibodies in xenograft-bearing nude mice. J Nucl Med 2003;44:1663–70.PubMedGoogle Scholar
  37. 37.
    Maeda H, Wu J, Sawa T, Matsumura Y, Hori K. Tumor vascular permeability and the EPR effect in macromolecular therapeutics. A review. J Control Release 2000;65:271–84.PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Weibo Cai
    • 1
  • Kai Chen
    • 1
  • Lina He
    • 1
  • Qizhen Cao
    • 1
  • Albert Koong
    • 2
  • Xiaoyuan Chen
    • 1
    Email author
  1. 1.The Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X ProgramStanford University School of MedicineStanfordUSA
  2. 2.Department of Radiation OncologyStanford University School of MedicineStanfordUSA

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