Design and Evaluation of Novel Albumin-Binding Folate Radioconjugates: Systematic Approach of Varying the Linker Entities

Tumor targeting using folate radioconjugates is a promising strategy for theragnostics of folate receptor-positive tumors. The aim of this study was to investigate the impact of structural modifications of folate radioconjugates on their pharmacokinetic properties. Four novel folate radioconjugates ([¹⁷⁷Lu]Lu-OxFol-2, [¹⁷⁷Lu]Lu-OxFol-3, [¹⁷⁷Lu]Lu-OxFol-4, and [¹⁷⁷Lu]Lu-OxFol-5), modified with a lipophilic or hydrophilic linker entity in close proximity to the albumin-binding 4-(p-iodophenyl)butanoate entity or the DOTA chelator, respectively, were designed and evaluated for comparison with the previously developed [¹⁷⁷Lu]Lu-OxFol-1. A hydrophobic 4-(aminomethyl)benzoic acid linker, incorporated in close proximity to the 4-(p-iodophenyl)butanoate entity, enhanced the albumin-binding properties (relative affinity 7.3) of [¹⁷⁷Lu]Lu-OxFol-3 as compared to those of [¹⁷⁷Lu]Lu-OxFol-1 (relative affinity set as 1.0). On the other hand, a hydrophilic d-glutamic acid (d-Glu) linker entity used in [¹⁷⁷Lu]Lu-OxFol-2 compromised the albumin-binding properties. [¹⁷⁷Lu]Lu-OxFol-4 and [¹⁷⁷Lu]Lu-OxFol-5, in which the respective linker entities were incorporated adjacent to the DOTA chelator, showed similar albumin-binding properties (0.6 and 1.0, respectively) as [¹⁷⁷Lu]Lu-OxFol-1. Biodistribution studies in KB tumor-bearing nude mice revealed twofold higher tumor-to-kidney ratios at 4 h and 24 h after injection of [¹⁷⁷Lu]Lu-OxFol-3 (∼1.2) than after injection of [¹⁷⁷Lu]Lu-OxFol-1 (∼0.6). The tumor-to-kidney ratios of [¹⁷⁷Lu]Lu-OxFol-2 were, however, much lower (∼0.2) due to the high kidney retention of this radioconjugate. The tumor-to-kidney ratios of [¹⁷⁷Lu]Lu-OxFol-5 were only slightly increased (∼0.9), and the ratios for [¹⁷⁷Lu]Lu-OxFol-4 (∼0.7) were in the same range as for [¹⁷⁷Lu]Lu-OxFol-1. SPECT/CT imaging studies demonstrated similar tumor uptake of all radioconjugates but a clearly improved tumor-to-kidney ratio for [¹⁷⁷Lu]Lu-OxFol-3 as compared to that for [¹⁷⁷Lu]Lu-OxFol-1. Based on these data, it can be concluded that the linker entity in close proximity to the 4-(p-iodophenyl)butanoate entity affects the radioconjugate's pharmacokinetic profile considerably due to the altered affinity to albumin. Changes in the linker entity, which connects the DOTA chelator with the folate molecule, do not have a major impact on the radioconjugate's tissue distribution profile, however. As a result of these findings, [¹⁷⁷Lu]Lu-OxFol-3 had a comparable therapeutic effect to that of [¹⁷⁷Lu]Lu-OxFol-1 but appeared advantageous in preventing kidney damage. Provided that the kidneys will present the dose-limiting organs in patients, [¹⁷⁷Lu]Lu-OxFol-3 would be the preferred candidate for a clinical translation.