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Preclinical Studies and Early Drug Development: In Vitro and In Vivo Assays and Models

In vitro and in vivo interaction of ⁶⁴Cu-DOTA-cetuximab and epidermal growth factor receptor in cervical cancer cell lines

Washington University School of Medicine, Saint Louis, MO

Abstract

B12

Cetuximab (ImClone Systems/Bristol-Myers Squibb), a chimeric monoclonal antibody specific for the extracellular domain of the human epidermal growth factor receptor (EGFR), is approved by the FDA for the treatment of EGFR-expressing, metastatic colorectal carcinoma. Patients treated with cetuximab show an objective response rate of 10.8%, and thus far, no correlation has been found between the degree of EGFR expression and the response to cetuximab treatment. At least 60% of cervical cancers are EGFR positive. Copper-64-labeled cetuximab and cervical cancer cell lines with varying degrees of EGFR expression were used to investigate characteristics of the antibody-EGFR interaction that might correlate with patient response. Five cervical cancer cell lines (C-33A (negative for EGFR), HeLa, DoTc2 4510, ME-180 and CaSki ) were shown by GeneChip microarray analysis to have different EGFR expression. Cetuximab was conjugated with the chelator DOTA and radiolabeled with ⁶⁴Cu (T_1/2 = 12.7 h; ß⁺ (17.4%)). Saturation receptor binding and internalization assays were carried out in the five cell lines using whole cells with ⁶⁴Cu-DOTA-cetuximab and receptor densities (B_max) and binding constants (K_d) were determined. The values for EGFR binding affinity (K_d) of ⁶⁴Cu-DOTA-cetuximab in the five cell lines were very similar (0.1 - 0.7 nM), as was expected for the same substrate-receptor interaction. The EGFR receptor concentration (B_max; fmol/mg total cellular protein) was found to mimic the order of EGFR expression as determined by microarray analysis and was in the order of CaSki (2133) > ME-180 (818) > DoTc2 4510 (610) > HeLa (305) > C-33A (24). The level of internalization of ⁶⁴Cu-DOTA-cetuximab by these cell lines showed the same trend, and studies are currently underway to examine the rate of internalization for each of the cell lines. CaSki cells were implanted into SCID mice and the resulting tumors were imaged by microPET with ⁶⁴Cu-DOTA-cetuximab. High uptake was observed in the CaSki tumors by both microPET imaging and biodistribution (SUV: 1.36; 13.2 2.7 %ID/g). PET imaging of EGFR positive tumors with ⁶⁴Cu-DOTA-cetuximab may give further insight into the actions of cetuximab on EGFR positive tumors.