[¹¹¹In]Bz-DTPA-hEGF: Preparation andIn VitroCharacterization of a Potential Anti-Glioblastoma Targeting Agent

Abstract

The overexpression of epidermal growth factor receptors, EGFR, in glioblastomas is well documented. Hence, the EGFR can be used as target structure for a specific targeting of glioblastomas. Both radiolabeled anti-EGFR antibodies and the natural ligand EGF are candidate agents for targeting. However, EGF, which has a rather low molecular weight (6 kDa), might have better tissue penetration properties through both normal tissue and tumors in comparison with anti-EGF antibodies and their fragments. The aim of this study was to prepare and evaluate in vitro an EGF-based antiglioma conjugate with residualizing label. Human recombinant EGF (hEGF) was coupled to isothiocyanate-benzyl-DTPA. The conjugate was purified from unreacted chelator using solid-phase extraction and labeled with (111)In. The labeling yield was 87% +/- 7%. The label was reasonably stable; the transchelation of (111)In to serum proteins was about 5% after incubation at 37 degrees C during 24 hours. The obtained [(111)In]benzyl-DTPA-hEGF conjugate was characterized in vitro using the EGFR expressing glioma cell line U343MGaCl2:6. The binding affinity, internalization, and retention of the conjugate were studied. The conjugate had receptor specific binding and the radioactivity was quickly internalized. The intracellular retention of radioactivity after interrupted incubation with conjugate was 71% +/- 1% and 59% +/- 1.5% at 24 and 45 hours, respectively. The dissociation constant was estimated to 2.0 nM. The results indicate that [(111)In]benzyl-DTPA-hEGF is a potential candidate for targeting glioblastoma cells, possibly using locoregional injection.

Directed Evolution to Low Nanomolar Affinity of a Tumor-Targeting Epidermal Growth Factor Receptor-Binding Affibody Molecule

“…It is known that EGF is very rapidly internalized after binding to EGFR. 50,51 This internalization makes binding to EGFR-expressing cells, including hepatocytes, irreversible. As internalization of Affibody molecules seems to be a slower process, the binding of Affibody molecules to EGFR receptors in the wellperfused liver should be followed by a release of Affibody conjugates back into the blood.…”

Affibody Molecules for Epidermal Growth Factor Receptor Targeting In Vivo: Aspects of Dimerization and Labeling Chemistry

“…Lipophilic radiocatabolites can diffuse through lysosomal and cellular membranes and ''leak'' from the malignant cells, as, for example, in the case of radiohalogen labels (12,35). In the case of a radiometal label, bulky charged radiocatabolites of labeled EGF cannot penetrate membranes and remain trapped in the cell, resulting in favorable retention (12,14,36,37). However, a radiometal label does not offer improved tumor retention if the internalization into malignant cells is a slow clathrin-independent process (generally observed with antagonistic binders).…”

Influence of Labelling Methods on Biodistribution and Imaging Properties of Radiolabelled Peptides for Visualisation of Molecular Therapeutic Targets

“…The use of a residualising radiometal label dramatically improved the cellular retention of the rapidly internalised EGF in comparison with non-residualising radioiodine [153]. The cellular retention of 111 In-Bz-DTPA-EGF [169] was nearly equal to the retention of the 177 Lu-labelled counterpart [170]. There was essentially no difference between the cellular retention of 111 In in the case of monoamide-DTPA and benzyl-DTPA based chelators [169].…”

“…The cellular retention of 111 In-Bz-DTPA-EGF [169] was nearly equal to the retention of the 177 Lu-labelled counterpart [170]. There was essentially no difference between the cellular retention of 111 In in the case of monoamide-DTPA and benzyl-DTPA based chelators [169]. The variation of co-ligands in 99m Tc-HYNIC-EGF conjugate influenced the overall label stability, but the cellular retention of 99m Tc was the same for both tricine and EDDA-co-ligands [171], and similar to the cellular retention of 111 In and 177 Lu.…”