N-Succinimidyl 3-((4-(4-[¹⁸F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([¹⁸F]SFBTMGMB): a residualizing label for ¹⁸F-labeling of internalizing biomolecules

Abstract

Residualizing labeling methods for internalizing peptides and proteins are designed to trap the radionuclide inside the cell after intracellular degradation of the biomolecule. The goal of this work was to develop a residualizing label for the 18F-labeling of internalizing biomolecules based on a template used successfully for radioiodination. N-succinimidyl 3-((4-(4-[18F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(bis-Boc-guanidinomethyl)benzoate (Boc2-[18F]SFBTMGMB) was synthesized by click reaction of an azide precursor and [18F]fluorohexyne in 8.5 ± 2.8% average decay-corrected radiochemical yield (n =15). An anti-HER2 nanobody 5F7 was labeled with 18F using [18F]SFBTMGMB ([18F]RL-I), obtained by the deprotection of Boc2-[18F]SFBTMGMB, in 31.2 ± 6.7% (n =5) conjugation efficiency. Thus labeled nanobody had a radiochemical purity of >95%, bound to the HER2-expressing BT474M1 breast cancer cells with an affinity of 4.7 ± 0.9 nM, and had an immunoreactive fraction of 62–80%. In summary, a novel residualizing prosthetic agent for labeling biomolecules with 18F has been developed. An anti-HER2 nanobody was labeled using this prosthetic group with retention of affinity and immunoreactivity to HER2.

Fluorine-18 Labeling of the HER2-Targeting Single-Domain Antibody 2Rs15d Using a Residualizing Label and Preclinical Evaluation

“…The methods for radiolabeling sdAb 2Rs15d with [ 125 I]SGMIB and [ 18 F]RL-I were essentially identical to those reported previously for labeling 5F7 with these prosthetic agents [25, 31]. Briefly, a solution of 2Rs15d in borate buffer, pH 8.5 (100 μg in 50 μl), was added to the dried prosthetic agent (0.37–0.56 GBq for [ 18 F]RL-I and 0.04–0.11 GBq for [ 125 I]SGMIB), and the mixture was incubated at 20 °C for 20 min.…”

“…Sodium [ 125 I]iodide (81.4 TBq/mmol) in 0.1 N NaOH was obtained from PerkinElmer Life and Analytical Sciences (Boston, MA). N -succinimidyl 4-guanidinomethyl-3-[ 125 I]iodobenzoate ([ 125 I]SGMIB) [26] and the F-18 residualizing prosthetic agent [ 18 F]RL-I [25] were synthesized as reported previously. High-pressure liquid chromatography (HPLC) was performed using a Beckman Gold HPLC system equipped with a Model 126 programmable solvent module, a Model 168 diode array detector, and Beckman System Gold remote interface module SS420X; data were acquired using 32 Karat® software.…”

“…R3B23 was labeled using [ 18 F]RL-I in a similar fashion. The purity of labeled sdAbs was determined by trichloroacetic acid (TCA) precipitation, ITLC, and SDS-PAGE/phosphor imaging as described previously [25, 27, 31]. In some cases, size-exclusion HPLC was also performed.…”

“…The immunoreactive fraction of radiolabeled sdAb conjugates was determined by the Lindmo method [32] using magnetic beads coated with the extracellular domain of HER2 or, as a negative control, with HSA, prepared as described [25, 27, 31]. Briefly, aliquots of labeled sdAb (~5 ng) were incubated in duplicate with three doubling concentrations of both positive (HER2) and negative (HSA) beads.…”

“…1) [19, 25] and evaluate the potential utility of this radioconjugate for imaging HER2-expressing tumors by PET. Studies were performed in BT474M1 breast carcinoma cells as well as in subcutaneous and intracranial xenografts derived from this line and included comparisons to 2Rs15d radioiodinated with the prototypical residualizing prosthetic agent SGMIB [26].…”