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Reagents and methods for PET using bispecific antibody pretargeting and 68Ga-radiolabeled bivalent hapten-peptide-chelate conjugates.

2004; National Institutes of Health; Volume: 45; Issue: 1 Linguagem: Inglês

Gary L. Griffiths, Chien‐Hsing Chang, William J. McBride, Edmund A. Rossi, Agatha Sheerin, German R. Tejada, Habibe Karacay, Robert M. Sharkey, Ivan D. Horak, Hans J. Hansen, David M. Goldenberg,

HER2/EGFR in Cancer Research

The aim of this work was to develop reagents and methods potentially useful in PET, using (68)Ga in a 2-step pretargeting protocol.We prepared bispecific antibodies (bsAbs) for disease-specific targeting of carcinoembryonic antigen-positive cells and recognition of later-administered bivalent hapten-peptide conjugates. The secondary antibody arm (antibody 679) recognizes a histaminyl-succinyl-glycine (HSG) structural subunit. The bsAbs were prepared as Fab' x Fab' conjugates using chemical cross-linking methods and as bispecific diabodies using recombinant DNA technologies. A HSG-bivalent hapten conjugate bearing the macrocyclic ring chelating agent 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) was designed to be readily radiolabeled with (68)Ga taken directly from a (68)Ge/(68)Ga generator system. Reagents were tested in vitro and, then, for their targeting properties in a preclinical animal model of human cancer.A chemically cross-linked hMN-14 x 679 F(ab')(2) and a fully humanized bispecific diabody construct (BS1.5H), expressed in Escherichia coli, were prepared for this work. We synthesized the bivalent peptide termed IMP 241 [DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-NH(2)] and labeled it with (68)Ga and (67)Ga at temperatures from 45 degrees C to 100 degrees C, over times of 15 min to 1 h, establishing 15 min at 95 degrees C as a useful condition for (68)Ga labeling. When we formulated the IMP 241 bivalent hapten-peptide with ammonium acetate buffer at pH 4-5 and eluted the (68)Ga from the generator directly into the peptide solution, we achieved an almost quantitative incorporation of the (68)Ga into IMP 241, as analyzed by size-exclusion high-performance liquid chromatography, after mixing the complex with the 679 antibody. For in vivo studies we used (67)Ga-IMP 241 as a surrogate for (68)Ga-IMP 241, in view of the short, 68-min half-life of the (68)Ga nuclide. The (67)Ga-IMP 241 was successfully pretargeted to human colon tumor xenografts in athymic mice with both the chemical and the diabody bispecific proteins. High tumor-to-normal tissue ratios for (67)Ga uptake were found for all tissues at 1 to 6 h after injection of (67)Ga-IMP 241. When using the BS1.5H diabody for pretargeting, tumor-to-blood, tumor-to-liver, and tumor-to-lung ratios of (67)Ga-IMP 241 at 1 and 3 h after injection were 41:1 and 137:1, 51:1 and 106:1, and 16:1 and 46:1, respectively.The general approach described, along with the new compositions and the labeling methods we have developed, may eventually allow for use of (68)Ga-labeled specific targeting agents in a routine clinical PET application.