Evaluation of Copper-64-Labeled αβ-Targeting Peptides: Addition of an Albumin Binding Moiety to Improve Pharmacokinetics.

The incorporation of non-covalent albumin binding moieties (ABMs) into radiotracers results in increased circulation time, leading to a higher uptake in the target tissues such as the tumor, and, in some cases, reduced kidney retention. We previously developed [F]AlF NOTA-K(ABM)-αβ-BP, where αβ-BP is a peptide with high affinity for the cell surface receptor integrin αβ that is overexpressed in several cancers, and the ABM is an iodophenyl-based moiety. [F]AlF NOTA-K(ABM)-αβ-BP demonstrated prolonged blood circulation compared to the non-ABM parent peptide, resulting in high, αβ-targeted uptake with continuously improving detection of αβ(+) tumors using PET/CT. To further extend the imaging window beyond that of fluorine-18 ( = 110 min) and to investigate the pharmacokinetics at later time points, we radiolabeled the αβ-BP with copper-64 ( = 12.7 h). Two peptides were synthesized without () and with () the ABM and radiolabeled with copper-64 to yield [Cu] and [Cu], respectively. The affinity of [Cu] and [Cu] for the integrin αβ was assessed by enzyme-linked immunosorbent assay. [Cu] and [Cu] were evaluated (cell binding and internalization) using DX3puroβ6 (αβ(+)), DX3puro (αβ(-)), and pancreatic BxPC-3 (αβ(+)) cells, in an albumin binding assay, and for stability in both mouse and human serum. (PET/CT imaging) and biodistribution studies were done in mouse models bearing either the paired DX3puroβ6/DX3puro or BxPC-3 xenograft tumors. [Cu] and [Cu] were synthesized in ≥97% radiochemical purity. , [Cu] and [Cu] maintained low nanomolar affinity for integrin αβ (IC = 28 ± 3 and 19 ± 5 nM, respectively); [Cu] and [Cu] showed comparable binding to αβ(+) cells (DX3puroβ6: ≥70%, ≥42% internalized; BxPC-3: ≥19%, ≥12% internalized) and ≤3% to the αβ(-) DX3puro cells. Both radiotracers were ≥98% stable in human serum at 24 h, and [Cu] showed a 6-fold higher binding to human serum protein than [Cu]. , selective uptake in the αβ(+) tumors was observed with tumor visualization up to 72 h for [Cu]. A 3-5-fold higher αβ(+) tumor uptake of [Cu] vs [Cu] was observed throughout, at least 2.7-fold improved BxPC-3-to-kidney and BxPC-3-to-blood ratios, and 2-fold improved BxPC-3-to-stomach ratios were noted for [Cu] at 48 h. Incorporation of an iodophenyl-based ABM into the αβ-BP ([Cu]) prolonged circulation time and resulted in improved pharmacokinetics, including increased uptake in αβ(+) tumors that enabled visualization of αβ(+) tumors up to 72 h by PET/CT imaging.