Evaluation of Copper-64-Labeled αvβ6-Targeting Peptides: Addition of an Albumin Binding Moiety to Improve Pharmacokinetics.

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Citation 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 [ 18 F]AlF NOTA-K(ABM)-α v β 6 -BP, where α v β 6 -BP is a peptide with high affinity for the cell surface receptor integrin α v β 6 that is overexpressed in several cancers, and the ABM is an iodophenyl-based moiety. [ 18 F]AlF NOTA-K(ABM)-α v β 6 -BP demonstrated prolonged blood circulation compared to the non-ABM parent peptide, resulting in high, α v β 6 -targeted uptake with continuously improving detection of α v β 6 (+) tumors using PET/CT. To further extend the imaging window beyond that of fluorine-18 ( t 1/2 = 110 min) and to investigate the pharmacokinetics at later time points, we radiolabeled the α v β 6 -BP with copper-64 ( t 1/2 = 12.7 h). Two peptides were synthesized without ( 1 ) and with ( 2 ) the ABM and radiolabeled with copper-64 to yield [ 64 Cu] 1 and [ 64 Cu] 2, respectively. The affinity of [ nat Cu] 1 and [ nat Cu] 2 for the integrin α v β 6 was assessed by enzyme-linked immunosorbent assay. [ 64 Cu] 1 and [ 64 Cu] 2 were evaluated in vitro (cell binding and internalization) using DX3puroβ6 (α v β 6 (+)), DX3puro (α v β 6 (-)), and pancreatic BxPC-3 (α v β 6 (+)) 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. [ 64 Cu] 1 and [ 64 Cu] 2 were synthesized in ≥97% radiochemical purity. In vitro, [ nat Cu] 1 and [ nat Cu] 2 maintained low nanomolar affinity for integrin α v β 6 (IC 50 = 28 ± 3 and 19 ± 5 nM, respectively); [ 64 Cu] 1 and [ 64 Cu] 2 showed comparable binding to α v β 6 (+) cells (DX3puroβ6: ≥70%, ≥42% internalized; BxPC-3: ≥19%, ≥12% internalized) and ≤3% to the α v β 6 (-) DX3puro cells. Both radiotracers were ≥98% stable in human serum at 24 h, and [ 64 Cu] 2 showed a 6-fold higher binding to human serum protein than [ 64 Cu] 1. In vivo, selective uptake in the α v β 6 (+) tumors was observed with tumor visualization up to 72 h for [ 64 Cu] 2. A 3-5-fold higher α v β 6 (+) tumor uptake of [ 64 Cu] 2 vs [ 64 Cu] 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 [ 64 Cu] 2 at 48 h. Incorporation of an iodophenyl-based ABM into the α v β 6 -BP ([ 64 Cu] 2 ) prolonged circulation time and resulted in improved pharmacokinetics, including increased uptake in α v β 6 (+) tumors that enabled visualization of α v β 6 (+) tumors up to 72 h by PET/CT imaging.