A Comparison of Evans Blue and 4-(p-Iodophenyl)butyryl Albumin Binding Moieties on an Integrin α_v β₆ Binding Peptide

Serum albumin binding moieties (ABMs) such as the Evans blue (EB) dye fragment and the 4-(p-iodophenyl)butyryl (IP) have been used to improve the pharmacokinetic profile of many radiopharmaceuticals. The goal of this work was to directly compare these two ABMs when conjugated to an integrin α_v β₆ binding peptide (α_v β₆-BP); a peptide that is currently being used for positron emission tomography (PET) imaging in patients with metastatic cancer. The ABM-modified α_v β₆-BP peptides were synthesized with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid (DOTA) chelator for radiolabeling with copper-64 to yield [⁶⁴ Cu]Cu DOTA-EB-α_v β₆-BP ([⁶⁴ Cu]1) and [⁶⁴ Cu]Cu DOTA-IP-α_v β₆-BP ([⁶⁴ Cu]2). Both peptides were evaluated in vitro for serum albumin binding, serum stability, and cell binding and internalization in the paired engineered melanoma cells DX3puroβ6 (α_v β₆ +) and DX3puro (α_v β₆ −), and pancreatic BxPC-3 (α_v β₆ +) cells and in vivo in a BxPC-3 xenograft mouse model. Serum albumin binding for [⁶⁴ Cu]1 and [⁶⁴ Cu]2 was 53–63% and 42–44%, respectively, with good human serum stability (24 h: [⁶⁴ Cu]1 76%, [⁶⁴ Cu]2 90%). Selective α_v β₆ cell binding was observed for both [⁶⁴ Cu]1 and [⁶⁴ Cu]2 (α_v β₆ (+) cells: 30.3–55.8% and 48.5–60.2%, respectively, vs. α_v β₆ (−) cells <3.1% for both). In vivo BxPC-3 tumor uptake for both peptides at 4 h was 5.29 ± 0.59 and 7.60 ± 0.43% ID/g ([⁶⁴ Cu]1 and [⁶⁴ Cu]2, respectively), and remained at 3.32 ± 0.46 and 4.91 ± 1.19% ID/g, respectively, at 72 h, representing a >3-fold improve-ment over the non-ABM parent peptide and thereby providing improved PET images. Comparing [⁶⁴ Cu]1 and [⁶⁴ Cu]2, the IP-ABM-α_v β₆-BP [⁶⁴ Cu]2 displayed higher serum stability, higher tumor accumulation, and lower kidney and liver accumulation, resulting in better tumor-to-organ ratios for high contrast visualization of the α_v β₆ (+) tumor by PET imaging.