The application of intact
monoclonal antibodies (mAbs) as targeting agents in nuclear imaging and
radioimmunotherapy is hampered by the slow pharmacokinetics of these molecules. Pretargeting with mAbs could be beneficial to reduce the radiation burden to the patient, while using the excellent targeting capacity of the mAbs. In this study, we evaluated the applicability of the Staudinger
ligation as pretargeting strategy using an antibody-
azide conjugate as
tumor-targeting molecule in combination with a small
phosphine-containing imaging/therapeutic probe. Up to 8
triazide molecules were attached to the antibody without seriously affecting its immunoreactivity, pharmacokinetics, and
tumor uptake in
tumor bearing nude mice. In addition, two (89)Zr- and (67/68)Ga-labeled
desferrioxamine (DFO)-
phosphines, a (177)Lu-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid (
DOTA)-
phosphine and a (123)I-cubyl
phosphine probe were synthesized and characterized for their pharmacokinetic behavior in nude mice. With respect to the
phosphine probes, blood levels at 30 min after injection were <5% injected dose per gram tissue, indicating rapid blood clearance. In vitro Staudinger
ligation of 3.33 μM antibody-
azide conjugate with 1 equiv of radiolabeled
phosphine, relative to the
azide, in aqueous
solution resulted in 20-25% efficiency after 2 h. The presence of 37% human serum resulted in a reduced
ligation efficiency (reduction max. 30% at 2 h), while the
phosphines were still >80% intact. No in vivo Staudinger
ligation was observed in a mouse model after injection of 500 μg antibody-
azide, followed by 68 μg DFO-
phosphine at t = 2 h, and evaluation in blood at t = 7 h. To explain negative results in mice, Staudinger
ligation was performed in vitro in mouse serum. Under these conditions, a side product with the
phosphine was formed and
ligation efficiency was severely reduced. It is concluded that in vivo application of the Staudinger
ligation in a pretargeting approach in mice is not feasible, since this
ligation reaction is not bioorthogonal and efficient enough. Slow reaction kinetics will also severely restrict the applicability of Staudinger
ligation in humans.