Specific localization, gamma camera imaging, and intracellular traffickingof radiolabelled chimeric anti-G(D3) ganglioside monoclonal antibody KM871in 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 th e in vivo evaluation of biodistribution properties and measurement of pharm acokinetics. KM871 was radiolabeled with I-125 via tyrosine residues and wi th In-111 via the bifunctional metal ion chelator C-functionalized hairs-cy clohexyl diethylenetriaminepentaacetic acid (CHX-A " -DTPA) to lysine resid ues, Using antigen-positive SK-MEL-28 melanoma cells, immunoreactivities of 42 and 40% cell binding were obtained, respectively, for the two radioconj ugates, Binding was enhanced in the presence of added unlabeled antibody, A humanized A33 antibody was similarly labeled with the two isotopes and use d as a control. To determine and compare in vivo biodistribution characteri stics of KM871 radiolabeled with In-111 or I-125, mixtures of the radioconj ugates were injected i.v. into BALB/c nude mice bearing GD(3)-positive-SK-M EL-28 melanoma xenografts, Gamma camera images were acquired; groups of fiv e mice were sacrificed at various time intervals, and tumors, blood, and ti ssues were analyzed. In-111-labeIed CHX-A " -DTPA-KM871 showed a maximum tu mor uptake of 41.9 +/- 7.0% injected dose/g at 72 h with prolonged retentio n over a 15-day period, The tumor:blood ratio was 3:1 by 72 h, and higher r atios were observed at later time points. No abnormal accumulation of In-11 1-Iabeled conjugate was found in normal tissues, In contrast, there was lit tle accumulation of I-125-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 In-111-labeled CHX-A " -DTPA-KM871 in the xenografts, Chromatogra phic analyses of xenograft cytosolic extracts demonstrated tumor internaliz ation and catabolism of radiolabeled KM871 with the formation of small mole cular weight metabolites, Laser scanning confocal microscopy demonstrated t hat the majority of intracellular KM871 is localized to lysosomes, Despite the catabolism of the radioconjugate, a dose-dependent increase in KM871 tu mor localization was shown through immunohistochemical examination of xenog raft biopsies. This study demonstrates for the first time the in vivo local ization of a radiolabeled anti-G(D3), monoclonal antibody to G(D3) expressi ng xenografts using gamma camera scanning techniques and tumor cell interna lization of KM871 tagged with a green fluorescent dye, Alexa Fluor 488, thr ough confocal microscopy, KM871 has potential for targeting tumors in patie nts with melanoma.