The Ionic Charge of Copper-64 Complexes Conjugated to an Engineered Antibody Affects Biodistribution

Abstract

The development of biomolecules as imaging probes requires radiolabeling methods that do not significantly influence their biodistribution. Sarcophagine (Sar) chelators form extremely stable complexes with copper and are therefore a promising option for labeling proteins with (64)Cu. However, initial studies using the first-generation sarcophagine bifunctional chelator SarAr to label the engineered antibody fragment ch14.18-ΔCH2 (MW 120 kDa) with (64)Cu showed high tracer retention in the kidneys, presumably because the high local positive charge on the Cu(II)-SarAr moiety resulted in increased binding of the labeled protein to the negatively charged basal cells of the glomerulus. To test this hypothesis, ch14.18-ΔCH2 was conjugated with a series of Sar derivatives of decreasing positive charge and three commonly used macrocyclic polyaza polycarboxylate (PAC) bifunctional chelators (BFC). The immunoconjugates were labeled with (64)Cu and injected into mice, and PET/CT images were obtained at 24 and 48 h postinjection (p.i.). At 48 h p.i., ex vivo biodistribution was assessed. In addition, to demonstrate the potential of metastasis detection using (64)Cu-labeled ch14.18-ΔCH2, a preclinical imaging study of intrahepatic neuroblastoma tumors was performed. Reducing the positive charge on the Sar chelators decreased kidney uptake of Cu-labeled ch14.18-ΔCH2 by more than 6-fold, from >45 to <6% ID/g, whereas the uptake in most other tissues, including liver, was relatively unchanged. However, despite this dramatic decrease, the renal uptake of the PAC BFCs was generally lower than that of the Sar derivatives, as was the liver uptake. Uptake of (64)Cu-labeled ch14.18-ΔCH2 in neuroblastoma hepatic metastases was detected using PET.

Site‐Specific In Vivo Bioorthogonal Ligation via Chemical Modulation

“…[23] This result is in accordance with the previous report by Dearling and Packard where Cu-chelators with different surface charges changed their biodistribution significantly. [24] …”

In vitro and in vivo evaluation of the bifunctional chelator NODIA-Me in combination with a prostate-specific membrane antigen targeting vector

“…Interestingly, the influence of the metal chelate is not limited to radiotracers based on small molecules or peptides. For example, the biodistribution profiles of 64 Cu-labelled antibody fragments as well as full antibody conjugates can also be influenced by the nature of the metal chelate [13][14]. Evidently the important role that chemical structure has on the biological performance of a radiotracer demands continued development and optimization of new BFCs.…”

Positron Emission Tomography Detects In Vivo Expression of Disialoganglioside GD2 in Mouse Models of Primary and Metastatic Osteosarcoma

“…Conjugation of hu14.18K322A (4 mg/mL in 0.1 mol/L sodium bicarbonate, pH 9.0) with 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA; Macrocyclics, catalog no. B-605) was performed at a molar ratio of 10:1 (chelator:antibody) by the method of Vosjan and colleagues (31,38). The conjugation solution was incubated at 37 C for 40 minutes with gentle mixing.…”

“…Z648035) to a final antibody concentration of 4 mg/mL for storage at À80 C until radiolabeling. The number of chelates per antibody molecule available for 64 Cu complexation was estimated by the isotopic dilution method (39) 64 Cu was performed using 5 mCi/mg (185 Bq/mg) of antibody in 0.25 mol/L sodium acetate, pH 5.5 (final antibody concentration 2 mg/mL) with gentle mixing for 40 minutes at 37 C. Labeling efficiency and radiochemical purity were determined using thin-layer chromatography as described previously (31). The final product had >95% radiochemical purity and 4-5 mCi/mg (145-185 Bq/mg) specific activity.…”

“…More recently, studies demonstrated the feasibility of radiolabeling 14G.2a, ch14.18 (15), and hu14.18K322A 14with 64 Cu as well as providing preliminary PET imaging results in strongly GD2-positive mouse models of neuroblastoma and melanoma. A subsequent study with ch14.18-DCH2, examining the effect of chelators on biodistribution, reported that S-2-benzyl-1,4,7-triazacyclononane-1,4,7-triacetic acid (Bn-NOTA) chelator conjugated to the antibody via a thiourea linkage provided the optimum signal-tonoise profile (31).…”