Background

[PubMed]

The epidermal growth factor receptor (EGFR) is a 170-kDa transmembrane protein that promotes cell proliferation by the specific binding of the autocrine epidermal growth factor (EGF) and transforming growth factor α (TGFα). The activity of these factors is believed to contribute to the progression of cancers. The EGFR operates through a receptor-associated tyrosine kinase–mediated signal transduction pathway that triggers cell proliferation. Overexpression of the EGFR is a characteristic feature of a variety of cancers such as squamous cell lung carcinoma and breast, ovarian, head and neck, bladder, and colon cancers (1). Much evidence indicates, in addition to overexpression, that a mutated EGFR gene may also play a role in development of these cancers (2). The most common mutation results in the development of a truncated receptor gene (EGFRvIII) that is deficient in exons 2–7 of the coding sequence. The mutant gene produces a receptor that lacks a part of the extracellular ligand-binding domain that is not activated by ligand binding. However, the intracellular tyrosine kinase portion of the receptor is active constitutively and autophosphorylates the tyrosine residue that initiates the signaling pathway for cell proliferation. In an effort to develop cancer therapies, a variety of EGFR inhibitors have been developed that either inhibit activation of the receptor kinase or the receptor binding of EGF and TGFα, e.g., monoclonal antibodies (MAb) to the receptor (3, 4).

Cetuximab is a human chimeric anti-EGFR MAb that has been successfully used by itself and in combination with chemo- and radiation therapy for the treatment of solid tumors or metastasized cancers (5, 6). Cetuximab is available commercially in the United States and has been approved by the US Food and Drug Administration for the treatment of metastasized colorectal cancer and advanced squamous cell carcinoma of the head and neck. Currently it is being evaluated in several clinical trials for the treatment of a variety of cancers. Because of EGFR heterogeneity in cancers, the imaging of cetuximab binding could assist in the selection and treatment of patients who are likely to respond to treatment and enhance the possibility of a positive outcome of the therapy. Radiolabeled cetuximab has been used to visualize and monitor EGFR tumors in animals and humans (7-10). In an effort to develop a non-radiolabeled imaging agent for EGFR, Aerts et al. developed a fluorescent dye, Oregon Green 488 (OG), and labeled cetuximab with this dye (cetuximab-OG) for the in vitro and in vivo visualization of EGFR in tumor tissue (11).

Synthesis

[PubMed]

The synthesis of cetuximab-OG was described by Aerts et al. (11). Cetuximab was purchased commercially as a solution and dialyzed against Hepes-buffered saline (pH 7.4). The protein concentration of the dialysate was determined, and the solution was incubated with succinimidyl-acetylacetate to obtain cetuximab-(Lysine)-acetylthioacetate (cetuximab-(Lys)-ATA). The duration of this incubation and yield of the product was not mentioned in the publication. For de-acetylation, cetuximab-(Lys)-ATA was treated with hydroxylamine and ethylenediamine tetraacetic acid (pH 7.4) for 1 h to obtain cetuximab-(Lys)-SH (yield not provided in the publication). This compound was incubated with OG 488–maleimide for 2 h, which resulted in 1:1 labeling (on average) of cetuximab with OG 488. All intermediates and the final product generated by the synthesis were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy. The final yield of cetuximab-OG was not provided in the publication.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

In vitro binding of cetuximab-OG was investigated in A431 (of human squamous cell carcinoma origin) and T47D (of human breast tumor origin) cells that have high and low levels of EGFR expression, respectively (11). The cells were incubated with cetuximab-OG for 3 h using an increasing concentration range of the labeled MAb (5.2 fmol to 5.2 µmol). Binding of the labeled MAb was observed under a fluorescent microscope, and only the A431 cells showed clear cetuximab-OG binding with a saturation point of 5.2 nM. The T47D cells had only background levels of cetuximab-OG binding at all concentrations. Subsequently, the T47D cell signals were used as the negative control for all experiments. On average, cetuximab-OG binding in A431 cells was 50-fold higher than the negative controls.

In another study, U-373 cells (of human glioblastoma-astrocytoma origin) were transfected with a phβAc vector (not described in the publication) containing the EGFRvIII gene (11). A stable clone with the highest expression of EGFRvIII, as determined by immunofluorescence, was selected for further experiments. It was denoted as U373-vIII(+). Another clone that expressed no EGFRvIII was also selected for use as a control and it was denoted as U373-vIII(-). Compared to the U373-vIII(-) cells, the U373-vIII(+) cells had a 40-fold higher binding of cetuximab-OG. This indicated that the labeled MAb could also bind to, and detect, the mutant EGFR.

No competing studies, to determine specificity of the labeled MAb, were reported in the publication (11).

Animal Studies

Human Studies

[PubMed]

No publications are currently available.

References

1.

Gullick W.J. Prevalence of aberrant expression of the epidermal growth factor receptor in human cancers. Br Med Bull. 1991; 47 (1):87–98. [PubMed: 1863851]

2.

Sok J.C. , Coppelli F.M. , Thomas S.M. , Lango M.N. , Xi S. , Hunt J.L. , Freilino M.L. , Graner M.W. , Wikstrand C.J. , Bigner D.D. , Gooding W.E. , Furnari F.B. , Grandis J.R. Mutant epidermal growth factor receptor (EGFRvIII) contributes to head and neck cancer growth and resistance to EGFR targeting. Clin Cancer Res. 2006; 12 (17):5064–73. [PubMed: 16951222]

3.

Dancey J.E. Recent advances of molecular targeted agents: opportunities for imaging. Cancer Biol Ther. 2003; 2 (6):601–9. [PubMed: 14688462]

4.

Dancey J.E. , Freidlin B. Targeting epidermal growth factor receptor--are we missing the mark? Lancet. 2003; 362 (9377):62–4. [PubMed: 12853203]

5.

Bonner J.A. , Harari P.M. , Giralt J. , Azarnia N. , Shin D.M. , Cohen R.B. , Jones C.U. , Sur R. , Raben D. , Jassem J. , Ove R. , Kies M.S. , Baselga J. , Youssoufian H. , Amellal N. , Rowinsky E.K. , Ang K.K. Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med. 2006; 354 (6):567–78. [PubMed: 16467544]

6.

Xiong H.Q. , Rosenberg A. , LoBuglio A. , Schmidt W. , Wolff R.A. , Deutsch J. , Needle M. , Abbruzzese J.L. 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 (13):2610–6. [PubMed: 15226328]

7.

Cai W. , Chen K. , He L. , Cao Q. , Koong A. , Chen X. Quantitative PET of EGFR expression in xenograft-bearing mice using (64)Cu-labeled cetuximab, a chimeric anti-EGFR monoclonal antibody. Eur J Nucl Med Mol Imaging. 2007; 34 (6):850–8. [PubMed: 17262214]

8. Pantaleo, M.A., S. Fanti, P.L. Lollini, S. Boschi, and G. Biasco, PET detection of epidermal growth factor receptor in colorectal cancer: a real predictor of response to cetuximab treatment? Eur J Nucl Med Mol Imaging, 2007. [PubMed: 17447062]

9.

Schechter N.R. , Wendt R.E. , Yang D.J. , Azhdarinia A. , Erwin W.D. , Stachowiak A.M. , Broemeling L.D. , Kim E.E. , Cox J.D. , Podoloff D.A. , Ang K.K. Radiation dosimetry of 99mTc-labeled C225 in patients with squamous cell carcinoma of the head and neck. J Nucl Med. 2004; 45 (10):1683–7. [PubMed: 15471833]

10.

Schechter N.R. , Yang D.J. , Azhdarinia A. , Kohanim S. , Wendt R. , Oh C.S. , Hu M. , Yu D.F. , Bryant J. , Ang K.K. , Forster K.M. , Kim E.E. , Podoloff D.A. Assessment of epidermal growth factor receptor with 99mTc-ethylenedicysteine-C225 monoclonal antibody. Anticancer Drugs. 2003; 14 (1):49–56. [PubMed: 12544258]

11.

Aerts H.J. , Dubois L. , Hackeng T.M. , Straathof R. , Chiu R.K. , Lieuwes N.G. , Jutten B. , Weppler S.A. , Lammering G. , Wouters B.G. , Lambin P. Development and evaluation of a cetuximab-based imaging probe to target EGFR and EGFRvIII. Radiother Oncol. 2007; 83 (3):326–32. [PubMed: 17531336]