van Gog F B, Visser G W, Klok R, van der Schors R, Snow G B, van Dongen G A
Department of Otolaryngology/Head and Neck Surgery, Free University Hospital, Amsterdam, The Netherlands.
J Nucl Med. 1996 Feb;37(2):352-62.
Our previous studies on the preparation of 186Re-MAb conjugates for clinical radioimmunotherapy (RIT) were extended with the aim to derive conjugates which have a high Re:MAb molar ratio, are stable in vitro and in vivo, have favorable biodistribution characteristics and can be used together with 99mTc-MAb conjugates as a matched pair in combined radioimmunoscintigraphy/RIT studies.
Rhenium and 99mTc-conjugates of intact MAb E48 were prepared according to our previously described multistep procedure using the MAG3 chelate and analyzed by protein mass spectrometry for the number of chelate molecules coupled to the MAb. For biodistribution analysis, tumor-free nude mice were simultaneously injected with 186Re-, 99mTc/99Tc- and/or 125I-labeled E48 IgG and dissected 1-48 hr postinjection.
Rhenium-186-MAb conjugates with up to 20 Re-MAG3 groups per MAb molecule were prepared with an overall radiochemical yield of 40%-60%. The conjugates did not contain empty MAG3 groups and no aggregates were formed. Only conjugates with a 186Re-MAG3:MAb molar ratio higher than 12 demonstrated slightly impaired immunoreactivity to a maximum of 15% decrease at the 20:1 molar ratio. Biodistribution experiments revealed that a proportion of the conjugate became rapidly eliminated from the blood for conjugates with a Re-MAG3:MAb molar ratio higher than 8. In this case, an increased uptake of activity was observed in the liver and intestines. The 99mTc/99Tc-MAb conjugates showed a similar enhanced blood clearance when containing more than eight Tc-MAG3 groups, while dual labeling of MAbs revealed that the in vivo stability of the conjugated Re-MAG3 complex itself does not differ from the corresponding Tc-MAG3 complex.
With the method described in this study, it is possible to prepare 186Re-MAG3-MAb conjugates that fulfil all the aforementioned criteria for use in clinical RIT. Coupling of too many metal-MAG3 groups to MAbs results in rapid blood clearance. At the same metal-MAG3:MAb molar ratio, 99mTc/99Tc-MAb conjugates show a similar pharmacokinetic behavior as 186Re-MAb conjugates and can thus be used to predict the localization of 186Re-labeled MAbs and make dosimetric predictions in individual patients.
我们之前关于制备用于临床放射免疫治疗（RIT）的186Re-单克隆抗体缀合物的研究得到了扩展，目的是获得具有高铼：单克隆抗体摩尔比、在体外和体内稳定、具有良好的生物分布特征并且可以与99mTc-单克隆抗体缀合物作为联合放射免疫闪烁显像/RIT研究中的匹配对一起使用的缀合物。
使用MAG3螯合物按照我们之前描述的多步程序制备完整单克隆抗体E48的铼和99mTc缀合物，并通过蛋白质质谱分析与单克隆抗体偶联的螯合物分子数量。对于生物分布分析，将无肿瘤的裸鼠同时注射186Re-、99mTc/99Tc-和/或125I标记的E48 IgG，并在注射后1-48小时进行解剖。
制备出每个单克隆抗体分子含有多达20个Re-MAG3基团的186Re-单克隆抗体缀合物，总放射化学产率为40%-60%。缀合物不包含空的MAG3基团，也未形成聚集体。只有186Re-MAG3:单克隆抗体摩尔比高于12的缀合物显示免疫反应性略有受损，在20:1摩尔比时最大降低15%。生物分布实验表明，对于Re-MAG3:单克隆抗体摩尔比高于8的缀合物，一部分缀合物会从血液中迅速清除。在这种情况下，观察到肝脏和肠道中的活性摄取增加。当99mTc/99Tc-单克隆抗体缀合物含有超过八个Tc-MAG3基团时，显示出类似的血液清除增强，而单克隆抗体的双重标记表明，缀合的Re-MAG3复合物本身在体内的稳定性与相应的Tc-MAG3复合物没有差异。
使用本研究中描述的方法，可以制备出满足上述所有用于临床RIT标准的186Re-MAG3-单克隆抗体缀合物。过多的金属-MAG3基团与单克隆抗体偶联会导致血液迅速清除。在相同的金属-MAG3:单克隆抗体摩尔比下，99mTc/99Tc-单克隆抗体缀合物显示出与186Re-单克隆抗体缀合物相似的药代动力学行为，因此可用于预测186Re标记的单克隆抗体的定位并在个体患者中进行剂量学预测。
