Cell Internalization Studies of Gadofullerene-(ZME-018) Immunoconjugates into A375m Melanoma Cells

Fullerene (C(60))-monoclonal antibody (mAb) immunoconjugates have been determined to internalize into target cells using water-soluble Gd(3+) ion-filled metallofullerenes (Gd@C(60)[OH](x)). Two separate conjugations of Gd@C(60)(OH)(x) with the antibody ZME-018 and a murine antibody mixture (MuIgG) were performed in a 1:5 mAb/Gd@C(60) ratio. Characterization of the immunoconjugates was established using inductively coupled plasma mass spectrometry (ICP-MS) for Gd(3+) and UV-Vis spectrometry (for Gd@C(60) + C(60)). Once conjugated, enzyme-linked immunosorbent assays showed little change in the specific binding of ZME-018. Each immunoconjugate was exposed to two cancer cell lines, A375m (antigen positive), and T24, bladder carcinoma (antigen negative). Internalization levels of the immunoconjugate were determined at various time points during 24 hours by harvesting and digesting the cells with 70% HNO(3) for Gd(3+) ion analysis by ICP-MS. These results are the first to demonstrate the practicality of a targeted cancer therapy based on fullerene immunotherapy.