Clinical-scale radiolabeling of a humanized anticarcinoembryonic antigen monoclonal antibody, hMN-14, with residualizing 131I for use in radioimmunotherapy.
Clinical-scale radiolabeling of a humanized anticarcinoembryonic antigen monoclonal antibody, hMN-14, with residualizing 131I for use in radioimmunotherapy.
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发表时间：
2005
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通讯作者：
S. Govindan;G. Griffiths;R. Stein;Philip M. Andrews;R. Sharkey;H. Hansen;I. Horak;D. Goldenberg
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作者：
S. Govindan;G. Griffiths;R. Stein;Philip M. Andrews;R. Sharkey;H. Hansen;I. Horak;D. Goldenberg
用胞内捕获形式的（131）I（残基化（131）I）对单克隆抗体（mAb）进行未标记的放射性标记，包括放射性碘标记小分子实体，将其与mAb偶联，并纯化。在涉及多千兆克放射性水平的程序中，柱纯化是不切实际的。本研究的目的是开发一种简单、远程、“一锅法”的放射性标记和纯化方法，用于人源化抗癌胚抗原（CEA）mAb（人源化MN-14（hMN-14;拉贝珠单抗））的放大放射性碘标记，并使用优化的残留（131）I部分（131）I-IMP-R4）。IMP-R4为MCC-Lys（MCC）-Lys（X）-d-Tyr-d-Lys（X）-OH，其中MCC为4-（N-马来酰亚胺基甲基）-环己烷-1-羰基，X为1-（（4-硫代羰基氨基）苄基）-二亚乙基三胺五乙酸。方法采用基于IODO-GEN的远程标记系统。在pH 7.0-7.4下对IMP-R4进行放射性碘标记（0.13 μ mol/3.7 GBq的（131）I），并在未使用的反应性（131）I淬灭后与二硫键还原的hMN-14偶联。产物通过与20%（w/v）阴离子交换树脂悬浮液搅拌5分钟来纯化，并无菌过滤到密封小瓶中。加入终浓度为1%-2.5%的人血清白蛋白。通过与CEA混合并通过尺寸排阻高压液相色谱法测定络合水平来测定免疫反应性。还进行了两种对照放射性标记，使用未还原的hMN-14或省略IMP-R4。结果在18个131 I标记的放射性核素中，131 I的放射性活度范围为2.04- 4.81GBq（55-130 mCi），产率59.9% +/- 7.9%（平均值+/- SD），比活度为200 +/- 26 MBq/mg（5.4 +/- 0.7 mCi/mg），其中>或=95%的放射性与hMN-14有关，3.7 GBq的（131）I。制备物的免疫反应性通常> 95%。在不存在IMP-R4的情况下的非特异性掺入为0.5%，而用未还原的IgG获得的非特异性掺入约为8%，这可能是因为IMP-R4在赖氨酸位点的缀合。该方法还去除了>99%的所用猝灭剂。用新鲜制备的溶液或冻干制剂进行放射性标记产生相似的产率，结果表明可选择一次性试剂盒设计。结论阴离子交换树脂搅拌5 min可有效去除（131）I-IMP-R4和淬灭（131）I，使（131）I-IMP-R4-hMN-14的制备达到数千兆贝克勒尔水平，安全、方便、实用。
UNLABELLED Radiolabeling of monoclonal antibodies (mAbs) with an intracellularly trapped form of (131)I (residualizing (131)I) involves radioiodinating a small molecular entity, conjugating it to the mAb, and purification. Column purifications are impractical during procedures involving multi-gigabecquerel levels of radioactivity. The goal of this study was to develop a simple, remote, "1-pot" method of radiolabeling and purification for the scaled-up radioiodination of a humanized anti-carcinoembryonic antigen (CEA) mAb, humanized MN-14 (hMN-14; labetuzumab), with an optimized residualizing (131)I moiety, (131)I-IMP-R4. IMP-R4 is MCC-Lys(MCC)-Lys(X)-d-Tyr-d-Lys(X)-OH, where MCC is 4-(N-maleimidomethyl)-cyclohexane-1-carbonyl and X is 1-((4-thiocarbonylamino)benzyl)-diethylenetriaminepentaacetic acid. METHODS An IODO-GEN-based remote labeling system was used. IMP-R4 was radioiodinated (0.13 mumol per 3.7 GBq of (131)I) at a pH of 7.0-7.4 and conjugated to disulfide-reduced hMN-14 after quenching of unused reactive (131)I. The product was purified by stirring for 5 min with a 20% (w/v) suspension of an anion-exchange resin and sterilely filtered into a sealed vial. Human serum albumin was added at a final concentration of 1%-2.5%. Immunoreactivity was determined by mixing with CEA and determining the complexation level by size-exclusion high-pressure liquid chromatography. Two control radiolabelings, either with unreduced hMN-14 or with IMP-R4 omitted, also were performed. RESULTS In 18 radiolabelings with (131)I in the range of 2.04-4.81 GBq (55-130 mCi), yields of 59.9% +/- 7.9% (mean +/- SD) at specific activities of 200 +/- 26 MBq/mg (5.4 +/- 0.7 mCi/mg) were obtained, with > or =95% of the radioactivity being associated with hMN-14 and with 3.7 GBq of (131)I. The immunoreactivities of the preparations were typically >95%. Nonspecific incorporation in the absence of IMP-R4 was 0.5%, whereas that obtained with unreduced IgG was approximately 8%, possibly because of conjugation of IMP-R4 at lysine sites. The process also removed >99% of the quenching reagent used. Radiolabelings performed with freshly prepared solutions or lyophilized preparations produced similar yields, a result that suggested the option for a single-use kit design. CONCLUSION Efficient removal of (131)I-IMP-R4 and quenched (131)I by 5 min of stirring with anion-exchange resin renders a multi-gigabecquerel-level preparation of (131)I-IMP-R4-hMN-14 safe, convenient, and practical.