Purified gangliosides (Calbiochem, NORTH PARK, CA) were resuspended in ethanol or DMSO. both for PET imaging (+) and therapy (+ and ?) (5, 7C9). However, a major challenge to developing 64Cu2+-based imaging agents has been identifying bifunctional chelating agents that stably complex 64Cu2+ under physiological conditions (5, 10, 11). Such copper chelators must form complexes with high thermodynamic and kinetic stability and be resistant to processes such as transchelation to endogenous copper transport and binding proteins, and reduction to Cu1+. Furthermore, the chemical conditions for conjugation and radiolabeling must be optimized to account for the biological and physical half-lives of the radioimmunoconjugate and to ensure that the specificity of the Agnuside targeting agent is not impaired (5, 12). A new class of bifunctional chelators has recently been synthesized (13) based on the hexaazamacrobicyclic sarcophagine cage Sar (Fig. 1) (14, 15). These compounds coordinate the Cu2+ ion within the multiple macrocyclic rings comprising the sarcophagine cage structure, yielding extraordinarily stable complexes that are inert to dissociation of the metal ion (5, 16). The Cu2+ cannot be removed from the cage under physiological conditions and thus resists transfer to copper-binding proteins such as ceruloplasmin or superoxide dismutase. In fact, the Sar chelator can inhibit incorporation of copper into endogenous copper-binding proteins present in liver extracts (17). The Cu2+ ion within the Sar complex is also unusually resistant to reduction; in contrast, more facile reduction has compromised the utility of other copper radiopharmaceuticals (5, 18). Open in a separate window Fig. 1. Structure of SarAr. SarAr is based on the macrobicyclic cage diamsar and was modified to contain the reactive aminobenzyl group. Smith (13) have recently developed a derivative of the diamsar ligand, SarAr (Fig. 1), which incorporates an aromatic amine into the cage periphery. This allows SarAr to be readily cross-linked to carboxyl residues on peptides and antibody molecules via carbodiimide-mediated amide bonds. This cross-linking reaction can be carried out in neutral or slightly acidic pH conditions using standard aqueous buffers. The resulting SarAr immunoconjugates are stable, allowing for advance preparation and storage for future labeling with 64Cu2+. The data presented here extend earlier results characterizing the SarAr compound by demonstrating the feasibility of using this chelator to produce tumor-targeted immunoconjugates that can be readily labeled with 64Cu2+ and used for imaging of neuroblastoma and melanoma. The procedures developed for this 64Cu-SarAr-mAb system should also be applicable to the preparation of a broad range of 64Cu-labeled protein-based PET imaging agents. Results Preparation and Characterization of SarAr-Conjugated 64Cu-Labeled anti-GD2 Antibody Constructs. The SarAr ligand was DXS1692E successfully Agnuside conjugated via its aromatic amine functional group to the ch14.18 antibody by using the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) reagent, forming a stable amide bond between the mAb and the chelator molecule. The optimal molar ratios of reagents were found to be a 500-fold excess of EDC to antibody, and a SarAr:IgG molar ratio of 250:1 in acetate buffer, pH 5.0 at 37C for 30 min, consistent with earlier results (19). Unbound SarAr was separated from the immunoconjugate by semipreparative HPLC, resulting in the purified immunoconjugate, SarAr-ch14.18. By using this procedure, up to 1 1.0 mg of immunoconjugate could be prepared in a single reaction, with no Agnuside significant intramolecular IgG cross-linking detectable by HPLC or SDS/PAGE (data not shown), confirming earlier observations (19). Similar results were obtained with murine 14.G2a mAb and other immunoglobulins (data not shown), demonstrating the general applicability of this conjugation technique. Radiolabeling of the SarAr-ch14.18 immunoconjugate was performed with carrier-free 64Cu2+. The incorporation of copper into the immunoconjugate was complete within 10C30 min. By using a SarAr/IgG ratio of 250:1 and 10 Ci 64Cu/g of IgG, 95C99% labeling efficiency was routinely obtained (data not shown). The immunoreactivity of 64Cu-labeled ch14.18 was confirmed by both RIA and direct cell binding studies. Under conditions of antigen excess, solid-phase RIA results showed that the SarAr-64Cu labeling process did not adversely impact antibody immunoreactivity. There was 70% retention of control immunoreactivity (Fig. 2), consistent with results for other types of radioimmunoconjugates (20C22). Open in a separate window Fig. 2. Lindmo plot of GD2 binding data, confirming retention of immunoreactivity. Immunoreactive fraction of 70% was obtained [inverse intercept (1/b = 1/1.4356)] by using a fixed concentration of labeled ch14.18 and increasing concentrations.
Purified gangliosides (Calbiochem, NORTH PARK, CA) were resuspended in ethanol or DMSO