Regiospecific radiolabelling of Nanofitin on Ni magnetic beads with [18F]FBEM and in vivo PET studies

Abstract Introduction Nanofitins are low molecular weight, single chain and cysteine-free protein scaffolds able to selectively bind a defined biological target. They derive from Sac7d bacterial protein family and are highly stable over a wide range of pH (0–13) and temperature (Tm ~80°C). Their extreme stability, low cost of production and high tolerability for chemical coupling make Nanofitins a very interesting alternative to antibodies and their fragments. Here, a hexahistidine tagged model Nanofitin (H4) directed against hen egg white lysozyme was radiolabelled and injected in mice to provide a baseline biodistribution and pharmacokinetic profiles to support future Nanofitin development programs. Method A single cysteine residue has been genetically inserted in a model Nanofitin and its regioselective radiolabelling has been performed with 4-[ 18 F]fluorobenzamido-N-ethylamino-maleimide ([ 18 F]FBEM). The synthesis of [ 18 F]FBEM has been completely implemented on a radiosynthesis unit (FastLab) including HPLC purification and formulation. Coupling with the [ 18 F]FBEM has been achieved on a solid support (Ni magnetic beads) allowing rapid purification at room temperature without organic solvent. PET-MRI studies on C57BL/6 mice were conducted after injection of [ 18 F]FBEM-Cys-H4 in order to access the biodistribution of this Nanofitin model. Results Radiochemical yield (decay corrected) of 54±7% (n=4) was obtained after optimization for coupling the [ 18 F]FBEM to Nanofitin. Pharmacokinetics results of [ 18 F]FBEM-Cys-H4 revealed a fast clearance through the liver and the kidneys. Conclusion An efficient new method on Ni magnetic beads was developed to radiolabelled his-tagged biomolecules with [ 18 F]FBEM. This procedure was applied on a Nanofitin model Cys-H4 and biodistribution kinetic studies were achieved to evaluate the potential use of Nanofitin for diagnostic imaging. Fast clearance indicates that Nanofitins represent very interesting tools for diagnostic imaging. Image 1