Fluorine-18 labeling of an anti-HER2 VHH using a residualizing prosthetic group via a strain-promoted click reaction: Chemistry and preliminary evaluation.

In a previous study, we evaluated a HER2-specific single domain antibody fragment (sdAb) 2Rs15d labeled with 18F via conjugation of a residualizing prosthetic agent that was synthesized by copper-catalyzed azide-alkyne cycloaddition (CuAAC). In order to potentially increase overall efficiency and decrease the time required for labeling, we now investigate the use of a strain-promoted azide-alkyne cycloaddition (SPAAC) between the 2Rs15d sdAb, which had been pre-derivatized with an azide-containing residualizing moiety, and an 18F-labeled aza-dibenzocyclooctyne derivative. The HER2-targeted sdAb 2Rs15d and a nonspecific sdAb R3B23 were pre-conjugated with a moiety containing both azide- and guanidine functionalities. The thus derivatized sdAbs were radiolabeled with 18F using an 18F-labeled aza-dibenzocyclooctyne derivative ([18F]F-ADIBO) via SPAAC, generating the desired conjugate ([18F]RL-II-sdAb). For comparison, unmodified 2Rs15d was labeled with N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB), the prototypical residualizing agent for radioiodination. Radiochemical purity (RCP), immunoreactive fraction (IRF), HER2-binding affinity and cellular uptake of [18F]RL-II-2Rs15d were assessed in vitro. Paired label biodistribution of [18F]RL-II-2Rs15d and [125I]SGMIB-2Rs15d, and microPET/CT imaging of [18F]RL-II-2Rs15d and the [18F]RL-II-R3B23 control sdAb were performed in nude mice bearing HER2-expressing SKOV-3 xenografts. A radiochemical yield of 23.9 ± 6.9% (n = 8) was achieved for the SPAAC reaction between [18F]F-ADIBO and azide-modified 2Rs15d and the RCP of the labeled sdAb was >95%. The affinity (Kd) and IRF for the binding of [18F]RL-II-2Rs15d to HER2 were 5.6 ± 1.3 nM and 73.1 ± 22.5% (n = 3), respectively. The specific uptake of [18F]RL-II-2Rs15d by HER2-expressing BT474M1 breast carcinoma cells in vitro was 14-17% of the input dose at 1, 2, and 4 h, slightly higher than seen for co-incubated [125I]SGMIB-2Rs15d. The uptake of [18F]RL-II-2Rs15d in SKOV-3 xenografts at 1 h and 2 h p.i. were 5.54 ± 0.77% ID/g and 6.42 ± 1.70% ID/g, respectively, slightly higher than those for co-administered [125I]SGMIB-2Rs15d (4.80 ± 0.78% ID/g and 4.78 ± 1.39% ID/g). MicroPET/CT imaging with [18F]RL-II-2Rs15d at 1-3 h p.i. clearly delineated SKOV-3 tumors while no significant accumulation of activity in tumor was seen for [18F]RL-II-R3B23. With the exception of kidneys, normal tissue levels for [18F]RL-II-2Rs15d were low and cleared rapidly. To our knowledge, this is the first time SPAAC method has been used to label an sdAb with 18F, especially with residualizing functionality.

Duke Scholars

Author Michael Rod Zalutsky Radiology

Author Ganesan Vaidyanathan Radiology