Evaluation of Copper-64-Labeled α_vβ₆-Targeting Peptides: Addition of an Albumin Binding Moiety to Improve Pharmacokinetics

Abstract

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 [18F]AlF NOTA-K(ABM)-αvβ6-BP, where αvβ6-BP is a peptide with high affinity for the cell surface receptor integrin αvβ6 that is overexpressed in several cancers, and the ABM is an iodophenyl-based moiety. [18F]AlF NOTA-K(ABM)-αvβ6-BP demonstrated prolonged blood circulation compared to the non-ABM parent peptide, resulting in high, αvβ6-targeted uptake with continuously improving detection of αvβ6(+) tumors using PET/CT. To further extend the imaging window beyond that of fluorine-18 (t1/2 = 110 min) and to investigate the pharmacokinetics at later time points, we radiolabeled the αvβ6-BP with copper-64 (t1/2 = 12.7 h). Two peptides were synthesized without (1) and with (2) the ABM and radiolabeled with copper-64 to yield [64Cu]1 and [64Cu]2, respectively. The affinity of [natCu]1 and [natCu]2 for the integrin αvβ6 was assessed by enzyme-linked immunosorbent assay. [64Cu]1 and [64Cu]2 were evaluated in vitro (cell binding and internalization) using DX3puroβ6 (αvβ6(+)), DX3puro (αvβ6(−)), and pancreatic BxPC-3 (αvβ6(+)) cells, in an albumin binding assay, and for stability in both mouse and human serum. In vivo (PET/CT imaging) and biodistribution studies were done in mouse models bearing either the paired DX3puroβ6/DX3puro or BxPC-3 xenograft tumors. [64Cu]1 and [64Cu]2 were synthesized in ≥97% radiochemical purity. In vitro, [natCu]1 and [natCu]2 maintained low nanomolar affinity for integrin αvβ6 (IC50 = 28 ± 3 and 19 ± 5 nM, respectively); [64Cu]1 and [64Cu]2 showed comparable binding to αvβ6(+) cells (DX3puroβ6: ≥70%, ≥42% internalized; BxPC-3: ≥19%, ≥12% internalized) and ≤3% to the αvβ6(−) DX3puro cells. Both radiotracers were ≥98% stable in human serum at 24 h, and [64Cu]2 showed a 6-fold higher binding to human serum protein than [64Cu]1. In vivo, selective uptake in the αvβ6(+) tumors was observed with tumor visualization up to 72 h for [64Cu]2. A 3–5-fold higher αvβ6(+) tumor uptake of [64Cu]2 vs [64Cu]1 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 [64Cu]2 at 48 h. Incorporation of an iodophenyl-based ABM into the αvβ6-BP ([64Cu]2) prolonged circulation time and resulted in improved pharmacokinetics, including increased uptake in αvβ6(+) tumors that enabled visualization of αvβ6(+) tumors up to 72 h by PET/CT imaging.

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