¹¹¹In-DTPA-Poly(ethylene glycol)-anti-epidermal growth factor receptor antibody C225

[¹¹¹In-DTPA-PEG-C225]

Background

[PubMed]

The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein that mediates biological activity through an intracellular tyrosine kinase signaling pathway. This receptor is known to be overexpressed in a variety of human malignancies, and the degree of expression often indicates the clinical outcome for the patient (1). Blocking EGFR activity appears to be an effective approach for the treatment of cancers, and a variety of agents, including monoclonal antibodies (MAb), have been developed for this purpose (2). The human-mouse chimeric MAb C225 was developed to target the EGFR and was demonstrated to inhibit the tyrosine kinase signaling pathway of this receptor. C225 was also shown to inhibit the proliferation of tumor cell lines expressing EGFR (3). This MAb is currently being evaluated in several clinical trials for the treatment of human breast, colon, head and neck, lung, prostate, and cervical cancers (4, 5). However, to determine the possible response of a patient, it is necessary to evaluate the degree of EGFR expression in the tumors by immunohistology before and after the MAb treatment. This is an invasive procedure and, because of tumor heterogeneity and metastasis, some tumors may be inaccessible and may be overlooked during patient evaluation (5). Therefore, a radiochemical targeted to the EGFR would be a useful tool to determine the level of EGFR expression in the tumor cells and could help select patients who are most likely to benefit from MAb therapy (5).

Using diethylene triamine pentaacetic acid (DTPA), the radioactive metal chelator indium (¹¹¹In) was linked to C225 and the radiolabeled MAb (¹¹¹In-DTPA-C225) was used in a phase 1 clinical trial to image human squamous cell carcinomas expressing high levels of EGFR (6). As a result of nonspecific uptake of radioactivity, a considerable amount of the radiolabel was detected in the liver of patients undergoing the treatment. A similar observation was made when another MAb against EGFR, MAb 528, was labeled with ¹¹¹In using DTPA. This radiolabeled MAb accumulated in the kidneys as well as the liver (7). These studies indicated a limited usefulness of the two radiolabeled MAbs for the detection of tumors and metastasis in the thorax.

In an attempt to improve the specificity of radiolabeled C225 uptake, Wen et al. (8) synthesized the MAb conjugated to polyethylene glycol (PEG), along with DTPA, to obtain DTPA-PEG-C225. The conjugated MAb was then radiolabeled with ¹¹¹In and evaluated for the detection of xenograft tumors expressing EGFR in nude mice (5).

Synthesis

[PubMed]

The synthesis of ¹¹¹In-DTPA-PEG-C225 has been described in detail by Wen et al. (8). Briefly, DTPA-PEG-amine (DTPA-PEG-NH₂) was prepared by stirring a mixture of DTPA-dianhydride, triethylamine (TEA), and t-Boc-NH-PEG-NH₂ in chloroform at room temperature for 2 h. After the reaction was complete, the chloroform and TEA were removed under vacuum. The t-Boc protecting group was removed without purification by adding TFA to the resulting residue and stirring at room temperature for 4 h. DTPA-PEG-NH₂ was purified by dialysis against phosphate-buffered saline (PBS) and deionized water. The product yield of this reaction was 95%.

The purified DTPA-PEG-NH₂ was reacted with S-acetylthioacetate (SATA) in chloroform for 1 h, purified by dialysis, and passed through a gel filtration column to obtain DTPA-PEG-acetylthioacetate. The yield from this reaction and purification was 92%.

Maleimide-activated C225 was prepared by adding N-γ-maleimidobutyryloxysuccinimide (GMBS) in dimethylformamide (DMF) to an aqueous solution of C225 and stirring the mixture at room temperature for 1 h. The product was purified on a PD-10 column. To obtain DTPA-PEG-SH, which contains free sulfhydral groups, for conjugation with activated C225, the protecting groups on DTPA-PEG-SATA were removed by the addition of hydroxylamine and incubation at room temperature for 30 min. The DTPA-PEG-SH was then mixed with maleimide-activated C225 in a 2:1 molar ratio and the mixture was incubated at 4ºC overnight. Unreacted DTPA-PEG-SH was removed by gel filtration on a Sephacryl S-200 HR column with PBS as the elution buffer.

Four DTPA-PEG-C225 conjugates containing 1:10, 1:20, 1:30, and 1:40 molar ratios of MAb to GMBS were synthesized and the physiochemical properties of these conjugates are detailed by Wen et al. (8). Approximately 20–25% (in the 1:10 ratio conjugate), 40% (in the 1:20 ratio conjugate), 60% (in the 1:30 ratio conjugate), and 70% (in the 1:40 ratio conjugate) of the amino groups in the four conjugates were substituted, respectively.

For radiolabeling, the antibody conjugate was incubated with ¹¹¹InCl₃ in sodium acetate buffer at room temperature for 15 min. Free ¹¹¹In was removed by gel filtration on a PD-10 column using PBS as the elution buffer. The radiochemical yield was >70% and the purity was >97 to 99%. The authors did not provide specific activity of the labeled MAb in the publication.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

The binding and biological activity of ¹¹¹In-DTPA-PEG-C225 was evaluated with in vitro assays using cells that express high levels of EGFR (8). Human breast cancer MDA-MB-468 cells were shown to bind increasing radioactivity with increasing concentrations of ¹¹¹In-DTPA-PEG-C225 that plateaued at 2 µg/mL of the labeled antibody. The binding of ¹¹¹In-DTPA-PEG-C225 was observed to be displaced by unlabeled C225 in a dose-dependent manner, indicating that the radiochemical had a specificity for EGFR similar to that of the parent antibody.

The receptor binding affinity of ¹¹¹In-DTPA-PEG-C225 with different degrees of pegylation was investigated using A431 cells. The cells were exposed to the radiochemical MAb conjugated to different amounts of PEG and subsequently lysed (8). The antibody-bound EGFR was immunoprecipitated from the lysates and visualized on a Western blot. It was observed that the amount of EGFR immunoprecipitated by the C225 conjugates (with C225:GMBS molar ratios of 1:10, 1:20, and 1:40, respectively) decreased with an increase in the degree of pegylation, indicating that the PEG modification altered the binding affinity of the antibody.

The biological activity of unlabeled DTPA-PEG-C225 was also assessed with an antiproliferation assay (8). For this, human colon cancer DiFi cells, which undergo apoptosis when the EGFR tyrosine kinase activity is blocked, were exposed to unlabeled DTPA-PEG-C225. It was observed that the modified antibody inhibited DiFi cell proliferation, indicating that the agent induced apoptosis in the cells similar to the C225 antibody.

Pharmacokinetic studies showed that PEG modification of the C225 MAb did not increase the blood circulating half-life of the antibody (8).

Animal Studies

Human Studies

[PubMed]

No publications are currently available.