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

Abstract In a previous study, we evaluated a HER2-specific single domain antibody fragment (sdAb) 2Rs15d labeled with 18 F 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 18 F-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 18 F using an 18 F-labeled aza-dibenzocyclooctyne derivative ([ 18 F]F-ADIBO) via SPAAC, generating the desired conjugate ([ 18 F]RL-II-sdAb). For comparison, unmodified 2Rs15d was labeled with N -succinimidyl 4-guanidinomethyl-3-[ 125 I]iodobenzoate ([ 125 I]SGMIB), the prototypical residualizing agent for radioiodination. Radiochemical purity (RCP), immunoreactive fraction (IRF), HER2-binding affinity and cellular uptake of [ 18 F]RL-II-2Rs15d were assessed in vitro. Paired label biodistribution of [ 18 F]RL-II-2Rs15d and [ 125 I]SGMIB-2Rs15d, and microPET/CT imaging of [ 18 F]RL-II-2Rs15d and the [ 18 F]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 [ 18 F]F-ADIBO and azide-modified 2Rs15d and the RCP of the labeled sdAb was >95%. The affinity (K d ) and IRF for the binding of [ 18 F]RL-II-2Rs15d to HER2 were 5.6 ± 1.3 nM and 73.1 ± 22.5% (n = 3), respectively. The specific uptake of [ 18 F]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 [ 125 I]SGMIB-2Rs15d. The uptake of [ 18 F]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 [ 125 I]SGMIB-2Rs15d (4.80 ± 0.78% ID/g and 4.78 ± 1.39% ID/g). MicroPET/CT imaging with [ 18 F]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 [ 18 F]RL-II-R3B23. With the exception of kidneys, normal tissue levels for [ 18 F]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 18 F, especially with residualizing functionality.