Synthesis of phosphine and antibody-azide probes for in vivo Staudinger ligation in a pretargeted imaging and therapy approach

Bioconjug Chem. 2011 Oct 19;22(10):2072-81. doi: 10.1021/bc200298v. Epub 2011 Sep 9.


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.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / blood
  • Antibodies, Monoclonal / chemistry*
  • Antibodies, Monoclonal / pharmacokinetics
  • Azides / blood
  • Azides / chemistry*
  • Azides / pharmacokinetics
  • Carcinoma, Squamous Cell / diagnosis*
  • Cell Line, Tumor
  • Goats
  • Head and Neck Neoplasms / diagnosis*
  • Humans
  • Immunoconjugates / blood
  • Immunoconjugates / chemistry*
  • Immunoconjugates / pharmacokinetics
  • Mice
  • Phosphines / blood
  • Phosphines / chemistry*
  • Phosphines / pharmacokinetics
  • Rabbits
  • Radiopharmaceuticals / blood
  • Radiopharmaceuticals / chemistry*
  • Radiopharmaceuticals / pharmacokinetics
  • Squamous Cell Carcinoma of Head and Neck
  • Swine


  • (124)I-cMAb U36
  • Antibodies, Monoclonal
  • Azides
  • Immunoconjugates
  • Phosphines
  • Radiopharmaceuticals
  • phosphine