Cysteine-selective [188Re]Re(V) radiolabelling of a Nanobody® for targeted radionuclide therapy using a “chelate-then-click” approach.

Abstract

In this study, we present the first reported use of bioorthogonal click chemistry with rhenium-188 (¹⁸⁸Re) for radiolabelling of an anti-c-Met V_HH Nanobody^®. We employed a "chelate-then-click" strategy, wherein a bifunctional chelator was designed in two parts, which were subsequently joined post-labelling and post-conjugation via the strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. Cysteine-selective conjugation of the V_HH was achieved through thiol-Michael addition, forming a V_HH-DBCO construct. Radiolabelling of the azide-functionalised chelator with [¹⁸⁸Re]Re(V) was optimised to achieve a radiochemical conversion of ~70%, despite challenges associated with maintaining the azide functionality under reducing conditions. The final product, [¹⁸⁸Re]Re-V_HH, demonstrated high radiochemical purity and good in vitro stability over 48 h. In vitro cell-binding studies against U87MG and BxPC3 cell lines proved the retention of c-Met binding post-labelling. In vivo biodistribution studies on mice bearing BxPC3 tumour xenografts, however, exhibited suboptimal tumour uptake, likely a result of the low molar activity (1.4 – 3.3 MBq/nmol) of the radioconjugate. This work illustrates the potential of bioorthogonal click chemistry for radiolabelling biomolecules with ¹⁸⁸Re, although further optimisation or alternative radiolabelling strategies to enhance the molar activity are necessary to improve pharmacokinetics.