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)-α_vβ₆-BP, where α_vβ₆-BP is a peptide with high affinity for the cell surface receptor integrin α_vβ₆ that is overexpressed in several cancers, and the ABM is an iodophenyl-based moiety. [¹⁸F]AlF NOTA-K(ABM)-α_vβ₆-BP demonstrated prolonged blood circulation compared to the non-ABM parent peptide, resulting in high, α_vβ₆-targeted uptake with continuously improving detection of α_vβ₆(+) tumors using PET/CT. To further extend the imaging window beyond that of fluorine-18 (<i>t</i>_1/2 = 110 min) and to investigate the pharmacokinetics at later time points, we radiolabeled the α_vβ₆-BP with copper-64 (<i>t</i>_1/2 = 12.7 h). Two peptides were synthesized without (<b>1</b>) and with (<b>2</b>) the ABM and radiolabeled with copper-64 to yield [⁶⁴Cu]<b>1</b> and [⁶⁴Cu]<b>2</b>, respectively. The affinity of [^natCu]<b>1</b> and [^natCu]<b>2</b> for the integrin α_vβ₆ was assessed by enzyme-linked immunosorbent assay. [⁶⁴Cu]<b>1</b> and [⁶⁴Cu]<b>2</b> were evaluated <i>in vitro</i> (cell binding and internalization) using DX3puroβ6 (α_vβ₆(+)), DX3puro (α_vβ₆(-)), and pancreatic BxPC-3 (α_vβ₆(+)) cells, in an albumin binding assay, and for stability in both mouse and human serum. <i>In vivo</i> (PET/CT imaging) and biodistribution studies were done in mouse models bearing either the paired DX3puroβ6/DX3puro or BxPC-3 xenograft tumors. [⁶⁴Cu]<b>1</b> and [⁶⁴Cu]<b>2</b> were synthesized in ≥97% radiochemical purity. <i>In vitro</i>, [^natCu]<b>1</b> and [^natCu]<b>2</b> maintained low nanomolar affinity for integrin α_vβ₆ (IC₅₀ = 28 ± 3 and 19 ± 5 nM, respectively); [⁶⁴Cu]<b>1</b> and [⁶⁴Cu]<b>2</b> showed comparable binding to α_vβ₆(+) cells (DX3puroβ6: ≥70%, ≥42% internalized; BxPC-3: ≥19%, ≥12% internalized) and ≤3% to the α_vβ₆(-) DX3puro cells. Both radiotracers were ≥98% stable in human serum at 24 h, and [⁶⁴Cu]<b>2</b> showed a 6-fold higher binding to human serum protein than [⁶⁴Cu]<b>1</b>. <i>In vivo</i>, selective uptake in the α_vβ₆(+) tumors was observed with tumor visualization up to 72 h for [⁶⁴Cu]<b>2</b>. A 3-5-fold higher α_vβ₆(+) tumor uptake of [⁶⁴Cu]<b>2</b> vs [⁶⁴Cu]<b>1</b> 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]<b>2</b> at 48 h. Incorporation of an iodophenyl-based ABM into the α_vβ₆-BP ([⁶⁴Cu]<b>2</b>) prolonged circulation time and resulted in improved pharmacokinetics, including increased uptake in α_vβ₆(+) tumors that enabled visualization of α_vβ₆(+) tumors up to 72 h by PET/CT imaging.