title: Specific localization, gamma camera imaging, and intracellular trafficking of radiolabelled chimeric anti-G(D3) ganglioside monoclonal antibody KM871 in SK-MEL-28 melanoma xenografts.
abstract: 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 (125)I via tyrosine residues and with (111)In 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 (111)In or (125)I, 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. (111)In-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 (111)In-labeled conjugate was found in normal tissues. In contrast, there was little accumulation of (125)I-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 (111)In-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(D3)-expressing 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.
authors.affiliation: Ludwig Institute For Cancer Research, Melbourne Branch, Austin and Repatriation Medical Centre, Studley Road, Heidelberg, Victoria 3084, Australia.
authors.forename: F T
authors.initials: FT
authors.lastname: Lee
authors.forename: A
authors.initials: A
authors.lastname: Rigopoulos
authors.forename: C
authors.initials: C
authors.lastname: Hall
authors.forename: K
authors.initials: K
authors.lastname: Clarke
authors.forename: S H
authors.initials: SH
authors.lastname: Cody
authors.forename: F E
authors.initials: FE
authors.lastname: Smyth
authors.forename: Z
authors.initials: Z
authors.lastname: Liu
authors.forename: M W
authors.initials: MW
authors.lastname: Brechbiel
authors.forename: N
authors.initials: N
authors.lastname: Hanai
authors.forename: E C
authors.initials: EC
authors.lastname: Nice
authors.forename: B
authors.initials: B
authors.lastname: Catimel
authors.forename: A W
authors.initials: AW
authors.lastname: Burgess
authors.forename: S
authors.initials: S
authors.lastname: Welt
authors.forename: G
authors.initials: G
authors.lastname: Ritter
authors.forename: L J
authors.initials: LJ
authors.lastname: Old
authors.forename: A M
authors.initials: AM
authors.lastname: Scott
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000493:pharmacokinetics*; D002614:Chelating Agents / Q000737:chemistry / Q000493:pharmacokinetics; D005260:Female; D015902:Gamma Cameras; D005732:Gangliosides / Q000096:biosynthesis / Q000276:immunology*; D006801:Humans; D018796:Immunoconjugates / Q000493:pharmacokinetics*; D007150:Immunohistochemistry; D007205:Indium Radioisotopes / Q000737:chemistry; D007457:Iodine Radioisotopes / Q000737:chemistry; D017879:Isothiocyanates / Q000737:chemistry / Q000493:pharmacokinetics; D007553:Isotope Labeling; D008545:Melanoma / Q000000981:diagnostic imaging* / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D004369:Pentetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000493:pharmacokinetics; D011877:Radionuclide Imaging; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics*; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000493:pharmacokinetics; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
chemical_list: D000911:Antibodies, Monoclonal; D002614:Chelating Agents; D005732:Gangliosides; D018796:Immunoconjugates; D007205:Indium Radioisotopes; D007457:Iodine Radioisotopes; D017879:Isothiocyanates; D019275:Radiopharmaceuticals; D011993:Recombinant Fusion Proteins; C085678:N-(2-amino-3-(4-isothiocyanatophenyl)propyl)cyclohexane-1,2-diamine-N,N',N',N'',N''-pentaacetic acid; C026226:ganglioside, GD3; D004369:Pentetic Acid