Labeling single domain antibody fragments with ¹⁸F using a novel residualizing prosthetic agent — N-succinimidyl 3-(1-(2-(2-(2-(2-[¹⁸F]fluoroethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-5-(guanidinomethyl)benzoate

Introduction: Labeling single domain antibody fragments (sdAbs) with ¹⁸F is an attractive strategy for immunoPET. Earlier, we developed a residualizing label, N-succinimidyl 3-((4-(4-fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([¹⁸F]RL-I), synthesized via a click reaction for labeling sdAbs with ¹⁸F, that has attractive features but suffered from modest radiochemical yields and suboptimal hydrophobicity. Herein, we have evaluated the potential utility of an analogous agent, N-succinimidyl 3-(1-(2-(2-(2-(2-[¹⁸F]fluoroethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-5-(guanidinomethyl)benzoate ([¹⁸F]SFETGMB; [¹⁸F]RL-III) designed to address these limitations. Methods: [¹⁸F]RL-III was synthesized by the click reaction between 3-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)-5-ethynylbenzoate and 1-azido-2-(2-(2-(2-[¹⁸F]fluoroethoxy)ethoxy)ethoxy)ethane and subsequent deprotection. The anti-HER2 sdAbs 5F7 and 2Rs15d were labeled by conjugation with [¹⁸F]RL-III and compared in a paired-label fashion to the sdAbs labeled using N-succinimidyl 4-guanidinomethyl-3-[¹²⁵I]iodobenzoate ([¹²⁵I]SGMIB) or N-succinimidyl 3-guanidinomethyl-5-[¹²⁵I]iodobenzoate (iso-[¹²⁵I]SGMIB). The ¹⁸F-labeled sdAbs were evaluated in vitro using HER2-expressing breast and ovarian carcinoma cells (BT474/BT474M1 and SKOV-3) and in vivo in athymic mice bearing subcutaneous SKOV-3 or BT474 xenografts. PET imaging of athymic mice bearing either subcutaneous BT474 or intracranial BT474M1Br-Fluc xenografts after administration of [¹⁸F]RL-III-5F7 also was performed. Results: Radiochemical yields for the synthesis of Boc₂-[¹⁸F]RL-III (21.5 ± 3.4%) were significantly higher than reported for Boc₂-[¹⁸F]RL-I. The overall radiochemical yields for the synthesis of [¹⁸F]RL-III-2Rs15d and [¹⁸F]RL-III-5F7 from aqueous [¹⁸F]fluoride were 1.7 ± 0.7% and 3.8 ± 2.3%, respectively. Both sdAbs, labeled using [¹⁸F]RL-III, retained affinity and immunoreactivity to HER2. Uptake and internalization of [¹⁸F]RL-III-5F7 in HER2-expressing cells was higher than that seen for [¹⁸F]RL-III-2Rs15d. Although different xenograft models were used, [¹⁸F]RL-III-2Rs15d showed relatively high uptake in a number of normal tissues, while uptake of [¹⁸F]RL-III-5F7 was mainly in tumor and kidneys with minimal background activity. Concordant with the necropsy experiments, microPET imaging with [¹⁸F]RL-III-5F7 in the BT474 subcutaneous model demonstrated clear delineation of the tumor (12.2 ± 5.1% ID/g) with minimal background activity except in kidneys. A tumor uptake (max) of 0.98%ID/g and a tumor-to-normal brain ratio of 9.8:1 were observed for [¹⁸F]RL-III-5F7 in the intracranial model. Conclusions: Although higher radiochemical yields than that reported for [¹⁸F]RL-I were obtained, considerable improvements are needed for this method to be of practical utility. Despite clear tumor delineation with [¹⁸F]RL-III-5F7 as early as 1 h, high activity levels in the kidneys remain a concern.