Reagents and Methods for PET Using Bispecific Antibody Pretargeting and 68Ga-Radiolabeled Bivalent Hapten-Peptide-Chelate Conjugates

摘要

id="p-1">The aim of this work was to develop reagents and methods potentially useful in PET, using 68Ga in a 2-step pretargeting protocol. >Methods: 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 Faba€2 ?— Faba€2 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,Na€2,Na€3,Na€′-tetraacetic acid (DOTA) was designed to be readily radiolabeled with 68Ga taken directly from a 68Ge/68Ga generator system. Reagents were tested in vitro and, then, for their targeting properties in a preclinical animal model of human cancer. >Results: A chemically cross-linked hMN-14 ?— 679 F(aba€2)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)- class="sc">d-Tyr-Lys(HSG)-NH₂] and labeled it with 68Ga and 67Ga at temperatures from 45?°C to 100?°C, over times of 15 min to 1 h, establishing 15 min at 95?°C as a useful condition for 68Ga labeling. When we formulated the IMP 241 bivalent hapten-peptide with ammonium acetate buffer at pH 4-5 and eluted the 68Ga from the generator directly into the peptide solution, we achieved an almost quantitative incorporation of the 68Ga 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 67Ga-IMP 241 as a surrogate for 68Ga-IMP 241, in view of the short, 68-min half-life of the 68Ga nuclide. The 67Ga-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 67Ga uptake were found for all tissues at 1 to 6 h after injection of 67Ga-IMP 241. When using the BS1.5H diabody for pretargeting, tumor-to-blood, tumor-to-liver, and tumor-to-lung ratios of 67Ga-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. >Conclusion: The general approach described, along with the new compositions and the labeling methods we have developed, may eventually allow for use of 68Ga-labeled specific targeting agents in a routine clinical PET application.