Reagents for astatination of biomolecules. 2. Conjugation of anionic boron cage pendant groups to a protein provides a method for direct labeling that is stable to in vivo deastatination
Reagents for astatination of biomolecules. 2. Conjugation of anionic boron cage pendant groups to a protein provides a method for direct labeling that is stable to in vivo deastatination
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DOI：
10.1021/bc060345s
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发表时间：
2007-07-01
影响因子：
4.7
通讯作者：
Hawthorne, M. Frederick
中科院分区：
文献类型：
--
作者：
Wilbur, D. Scott;Chyan, Ming-Kuan;Hawthorne, M. Frederick
用At-211标记的靶向癌症的生物分子必须对体内脱嘌呤稳定，因为由于α粒子发射的高毒性性质，At-211分布的控制是至关重要的。不幸的是，当标记（相对）快速代谢的蛋白质，如单克隆抗体Fab'片段时，还没有发现具有去嘌呤化作用的体内稳定性。作为增加At-211标记蛋白的体内稳定性的手段，我们一直在研究硼笼部分的抗体缀合物。在该研究中，制备了含有巢式碳硼烷（2）、双巢式碳硼烷衍生物（Venus Flytrap Complex，3）和四种2-九氢-闭合-十硼酸酯（2-）衍生物（4-7）的蛋白质反应性衍生物，并将其与抗体Fab ′片段缀合，从而可以获得随后的astatination和体内组织分布。为了帮助测定体内脱嘌呤的稳定性，Fab ′-硼烷缀合物也用I-125标记，并将该材料与At-211标记的Fab ′共注射。为了比较，用I-125和At-211直接标记Fab ′。用Na[I-125]I和氯胺-T直接标记得到89%的放化产率。然而，用Na[At-211]At和氯胺-T直接标记Fab'导致在用NaS 2 O 5淬灭后产率< 1%。作为另一个比较，将相同的Fab'与对-[At-211]阿司他苯甲酸NHS酯，[At-211] 1c-Fab'，和（分别）与对-[I-125]碘苯甲酸NHS酯，[I-125] 1b-Fab '缀合。在无胸腺小鼠中的评价表明，[At-211]1c-Fab'经历脱蛋白。相比之下，[I-125]1b-Fab'的高体内稳定性使其可用作Fab'自然分布的示踪对照。尽管发现在体内比[At-211] 1c-Fab '稳定得多，但巢式碳硼烷缀合的Fab'（[I-125]2-Fab '/[At-211]2-Fab'）和双巢式碳硼烷（VFC）（[I-125]3-Fab '/[At-211]3-Fab'）的生物分布与对照[I-125] 1b-Fab '具有非常不同的体内分布。[I-125]4-Fab '/[At-211]4-Fab'、[I-125]6-Fab '/[At-211]6-Fab'和[I-125]7-Fab '/[At-211]7-Fab'的生物分布表明，它们对体内脱蛋白稳定，并且具有与对照[I-125] 1b-Fab '相似的分布。相比之下，体内评价的苄基修饰的紧密-癸硼酸酯（2-）衍生物（[I-125]5-Fab '/[At-211]5-Fab'）具有与对照非常不同的组织分布。本研究表明，邻十硼酸酯（2-）的蛋白质结合物对体内脱嘌呤相当稳定，某些衍生物对Fab ′的分布几乎没有影响。另外，与近十硼酸（2-）衍生物缀合的Fab'的直接At-211标记提供非常高的（例如，58-75%）放射化学产率。然而，体内数据也表明，近十硼酸盐（2-）可能导致肝脏中的放射性保留。优化用于蛋白质标记的紧密十硼酸酯（2-）缀合物的研究正在进行中。
Cancer-targeting biomolecules labeled with At-211 must be stable to in vivo deastatination, as control of the At-211 distribution is critical due to the highly toxic nature of alpha-particle emission. Unfortunately, no astatinated aryl conjugates have shown in vivo stability toward deastatination when (relatively) rapidly metabolized proteins, such as monoclonal antibody Fab' fragments, are labeled. As a means of increasing the in vivo stability of At-211-labeled proteins, we have been investigating antibody conjugates of boron cage moieties. In this investigation, protein-reactive derivatives containing a nido-carborane (2), a bis-nido-carborane derivative (Venus Flytrap Complex, 3), and four 2-nonahydro-closo-decaborate(2-) derivatives (4-7) were prepared and conjugated with an antibody Fab' fragment such that subsequent astatination and in vivo tissue distributions could be obtained. To aid in determination of stability toward in vivo deastatination, the Fab'-borane conjugates were also labeled with I-125, and that material was coinjected with the At-211-labeled Fab'. For comparison, direct labeling of the Fab' with I-125 and At-211 was conducted. Direct labeling with Na[I-125]I and Chloramine-T gave an 89% radiochemical yield. However, direct labeling of the Fab' with Na[At-211]At and Chloramine-T resulted in a yield of < 1% after quenching with NaS2O5. As another comparison, the same Fab' was conjugated with p-[At-211]astatobenzoate NHS ester, [At-211]1c-Fab', and (separately) with p-[I-125]iodobenzoate NHS ester, [I-125]1b-Fab'. An evaluation in athymic mice demonstrated that [At-211]1c-Fab' underwent deastatination. In contrast, the high in vivo stability of [I-125]1b-Fab' allowed it to be used as a tracer control for the natural distribution of Fab'. Although found to be much more stable in vivo than [At-211]1c-Fab', the biodistributions of nido-carborane conjugated Fab' ([I-125]2-Fab'/ [At-211]2-Fab') and the bis-nido-carborane (VFC) ([I-125]3-Fab'/[At-211]3-Fab') had very different in vivo distributions than the control [I-125]1b-Fab'. Biodistributions of closo-decaborate(2-) conjugates ([I-125]4-Fab'/[At-211]4-Fab', [I-125]6-Fab'/[At-211]6-Fab', and [I-125]7-Fab'/[At-211]7-Fab') demonstrated that they were stable to in vivo deastatination and had distributions similar to that of the control [I-125]1b-Fab'. In contrast, a benzyl-modified closo-decaborate(2-) derivative evaluated in vivo ([I-125]5-Fab'/[At-211]5-Fab') had a very different tissue distribution from the control. This study has shown that astatinated protein conjugates of closo-decaborate(2-) are quite stable to in vivo deastatination and that some derivatives have little effect on the distribution of Fab'. Additionally, direct At-211 labeling of Fab' conjugated with closo-decaborate(2-) derivatives provide very high (e.g., 58-75%) radiochemical yields. However, in vivo data also indicate that the closo-decaborate(2-) may cause some retention of radioactivity in the liver. Studies to optimize the closo-decaborate(2-) conjugates for protein labeling are underway.