Receptor-Binding, Biodistribution, and Metabolism Studies of⁶⁴Cu-DOTA-Cetuximab, a PET-Imaging Agent for Epidermal Growth-Factor Receptor-Positive Tumors

Abstract

The epidermal growth-factor receptor (EGFR) and its ligands have been recognized as critical factors in the pathophysiology of tumorigenesis. Overexpression of the EGFR plays a significant role in the tumor progression of a wide variety of solid human cancers. Therefore, the EGFR represents an attractive target for the design of novel diagnostic and therapeutic agents for cancer. Cetuximab (C225, Erbitux) was the first monoclonal antibody targeted against the ligand-binding site of EGFR approved by the Food and Drug Administration for the treatment of patients with EGFR-expressing, metastatic colorectal carcinoma, although clinical trials showed variability in the response to this treatment. The aim of this study involved using cetuximab to design a positron emission tomography (PET) agent to image the overexpression of EGFR in tumors. Cetuximab was conjugated with the chelator, DOTA, for radiolabeling with the positron-emitter, 64Cu (T(1/2) = 12.7 hours). 64Cu-DOTA-cetuximab showed high binding affinity to EGFR-positive A431 cells (K(D) of 0.28 nM). Both biodistribution and microPET imaging studies with 64Cu-DOTA-cetuximab demonstrated greater uptake at 24 hours postinjection in EGFR-positive A431 tumors (18.49% +/- 6.50% injected dose per gram [ID/g]), compared to EGFR-negative MDA-MB-435 tumors (2.60% +/- 0.35% ID/g). A431 tumor uptake at 24 hours was blocked with unlabeled cetuximab (10.69% +/- 2.72% ID/g), suggesting that the tumor uptake was receptor mediated. Metabolism experiments in vivo showed that 64Cu-DOTA-cetuximab was relatively stable in the blood of tumor-bearing mice; however, there was significant metabolism in the liver and tumors. 64Cu-DOTA-cetuximab is a potential agent for imaging EGFR-positive tumors in humans.

Comparison of Conjugation Strategies of Cross-Bridged Macrocyclic Chelators with Cetuximab for Copper-64 Radiolabeling and PET Imaging of EGFR in Colorectal Tumor-Bearing Mice

“…The CB-TE1A1P:cetuximab ratio was 1.5 for CB-TE1A1P–cetuximab synthesized via NHS-aided reactions, compared to 5 chelators/mAb for previously reported DOTA–cetuximab. 10 The low ratio of the chelator to antibody is likely responsible for the lower SA of 64 Cu-CB-TE1A1P–cetuximab compared to the DOTA and CB-TE1K1P-PEG 4 -DBCO conjugates.…”

“…10 Copper-64-labeled cetuximab (∼0.74 MBq, 1.0–6.0 μg in 150 μL) was injected via tail vein into HCT116 tumor-bearing mice (7–9 weeks). At 24 and 48 h after injection, mice were sacrificed and selected organs were removed, weighed, and counted on a γ counter (Beckman 8000; Irvine, CA).…”

“…Cetuximab has been radiolabeled with 99m Tc and 111 In for SPECT imaging 8,9 and 64 Cu, 86 Y, and 89 Zr for PET imaging. 10−12 Copper-64 ( T 1/2 = 12.7 h) is particularly promising because of its suitable half-life and unique decay characteristics: β + (656 keV, 17.4%) and β – (573 keV, 38.5%), and the beta-decay makes this radionuclide attractive for therapy. 13−16 Radiolabeling at 37 °C or below is critical to maintain the biological integrity of antibodies, therefore DOTA has been the most commonly used chelator for 64 Cu radiolabeling of cetuximab and other antibodies.…”

“…However, the in vivo behavior of the 64 Cu-DOTA complex is less than optimal due to dissociation of 64 Cu from the DOTA complex, and this might lead to relatively poor tumor to nontumor ratios. 10,17,18 Cross-bridged macrocyclic chelators (such as CB-TE2A, CB-TE1A1P, and CB-TE1K1P, Figure 1) have shown impressive kinetic inertness for Cu complexes. 19−22 The monocarboxylate monophosphonate chelator CB-TE1A1P can be labeled with 64 Cu at room temperature with comparable in vivo stability to CB-TE2A, 21 and its peptide conjugate 64 Cu-CB-TE1A1P-Y3-TATE demonstrated improved tumor targeting and biodistribution compared to 64 Cu-CB-TE2A-Y3-TATE conjugate in a pancreatic tumor bearing rat model.…”

Development of radioimmunoconjugate for diagnosis and management of head-and-neck subclinical cancer and colorectal carcinoma

“…For therapy, lutetium-177 (Jalilian et al, 2016) and yttrium-90 (Niu et al, 2010) have been used. Ping et al (2008) reported a preclinical study related to the use of cetuximab as a PET agent, directed to tumors overexpression` EGFR. Cetuximab was conjugated with DOTA chelator (1,4,7,10 -tetraazacyclododecane tetraacetic acid), for radiolabeling with copper-64 (T 1/2 = 12.7 hours).…”