Specific localization, gamma camera imaging, and intracellular trafficking of radiolabelled chimeric anti-G_D3 ganglioside monoclonal antibody KM871 in SK-MEL-28 melanoma xenografts

The chimeric monoclonal antibody KM871, directed against the G_D3 antigen, is under evaluation for its potential to target melanoma. To facilitate the in vivo evaluation of biodistribution properties and measurement of pharmacokinetics, KM871 was radiolabeled with ¹²⁵I via tyrosine residues and with ¹¹¹In via the bifunctional metal ion chelator C-functionalized trans-cyclohexyl diethylenetriaminepentaacetic acid (CHX-A″-DTPA) to lysine residues. Using antigen-positive SK-MEL-28 melanoma cells, immunoreactivities of 42 and 40% cell binding were obtained, respectively, for the two radioconjugates. Binding was enhanced in the presence of added unlabeled antibody. A humanized A33 antibody was similarly labeled with the two isotopes and used as a control. To determine and compare in vivo biodistribution characteristics of KM871 radiolabeled with ¹¹¹In or ¹²⁵I, mixtures of the radioconjugates were injected i.v. into BALB/c nude mice bearing G_D3-positive-SK-MEL-28 melanoma xenografts. Gamma camera images were acquired; groups of five mice were sacrificed at various time intervals, and tumors, blood, and tissues were analyzed, ¹¹¹In-labeled CHX-A″-DTPA-KM871 showed a maximum tumor uptake of 41.9 ± 7.0% injected dose/g at 72 h with prolonged retention over a 15-day period. The tumor:blood ratio was 3:1 by 72 h, and higher ratios were observed at later time points. No abnormal accumulation of ¹¹¹In-labeled conjugate was found in normal tissues. In contrast, there was little accumulation of ¹²⁵I-labeled KM871 in the same tumors. The specificity of antibody localization was confirmed by the low tumor uptake values for radiolabeled control antibody. Gamma camera imaging demonstrated excellent uptake of ¹¹¹In-labeled CHX-A″-DTPA-KM871 in the xenografts. Chromatographic analyses of xenograft cytosolic extracts demonstrated tumor internalization and catabolism of radiolabeled KM871 with the formation of small molecular weight metabolites. Laser scanning confocal microscopy demonstrated that the majority of intracellular KM871 is localized to lysosomes. Despite the catabolism of the radioconjugate, a dose-dependent increase in KM871 tumor localization was shown through immunohistochemical examination of xenograft biopsies. This study demonstrates for the first time the in vivo localization of a radiolabeled anti-G_D3 monoclonal antibody to G_D3expressing xenografts using gamma camera scanning techniques and tumor cell internalization of KM871 tagged with a green fluorescent dye, Alexa Fluor 488, through confocal microscopy. KM871 has potential for targeting tumors in patients with melanoma.