Department of Otolaryngology/Head and Neck Surgery, VU University Medical Center, Amsterdam, The Netherlands.
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.
完整单克隆抗体（mAbs）作为靶向剂在核医学成像和放射免疫治疗中的应用受到这些分子药代动力学缓慢的阻碍。利用 mAbs 进行预靶向可以降低患者的辐射负担，同时利用 mAbs 出色的靶向能力。在这项研究中，我们评估了使用抗体-叠氮化物缀合物作为肿瘤靶向分子与含有小膦的成像/治疗探针结合的作为预靶向策略的 Staudinger 连接的适用性。多达 8 个三氮化物分子连接到抗体上，而不会严重影响其免疫反应性、药代动力学和荷瘤裸鼠中的肿瘤摄取。此外，两种（89）Zr 和（67/68）Ga 标记的去铁胺（DFO）-膦、（177）Lu-1、4、7、10-四氮杂环十二烷-1、4、7、10-四乙酸（DOTA）-膦和（123）I-立方基膦探针被合成并对其在裸鼠中的药代动力学行为进行了表征。对于膦探针，注射后 30 分钟的血液水平<5%注射剂量/克组织，表明血液清除迅速。在水性溶液中，用 1 当量放射性标记的膦与 3.33 μM 抗体-叠氮化物缀合物进行体外 Staudinger 连接，2 小时后得到 20-25%的效率。在存在 37%人血清的情况下，连接效率降低（在 2 小时时最大降低 30%），而膦仍保持>80%的完整性。在注射 500μg 抗体-叠氮化物后，在 2 小时时注射 68μg DFO-膦，并在 7 小时时在血液中进行评估，在小鼠模型中未观察到体内 Staudinger 连接。为了解释在小鼠中观察到的阴性结果，在小鼠血清中进行了体外 Staudinger 连接。在这些条件下，形成了带有膦的副产物，并且连接效率严重降低。结论是，由于该连接反应不是生物正交的，并且效率不够高，因此在小鼠中应用 Staudinger 连接进行预靶向方法在体内是不可行的。缓慢的反应动力学也将严重限制 Staudinger 连接在人类中的适用性。
