Summary

该生物耦合和重新布置<sup> 89</sup>锆DFO标记的抗体

Published: February 12, 2015
doi:

Summary

Due to its multi-day radioactive half-life and favorable decay properties, the positron-emitting radiometal 89Zr is extremely well-suited for use in antibody-based radiopharmaceuticals for PET imaging. In this protocol, the bioconjugation, radiosynthesis, and preclinical application of 89Zr-labeled antibodies will be described.

Abstract

特殊的亲和力,特异性和选择性的抗体使其成为肿瘤靶向PET放射性药物非常有吸引力的载体。由于其多天的生物半衰期,抗体必须贴有正电子发射放射性核素具有相对长的物理衰变半衰期。传统上,正电子发射同位素124 I(叔1/2 = 4.18 d)中,86 Y(吨1/2 = 14.7小时),和64的Cu(叔1/2 = 12.7小时),已被用于标记抗体PET成像。最近,然而,该领域已经见证了在基于抗体的PET成像剂的使用的正电子发射放射性金属89 Zr的显着增加。89锆是一种近乎理想的放射性同位素用于PET成像的免疫,因为它具有一个物理半-life(吨1/2 = 78.4小时),它与抗体的体内药动学相容并发射相对低的烯产生高分辨率的图像红绿黄正电子。此外,抗体可直接地使用铁载体衍生的螯合剂去铁胺(DFO)标记89锆。在这个协议中,前列腺特异性膜抗原靶向抗体J591将被用作模型系统,以说明(1)的双功能螯合剂的生物缀DFO-异硫氰酸酯的抗体,(2)的放射和89 ZR-纯化DFO-单抗放射免疫偶联,和(3) 在体内 PET成像与89锆DFO-单抗放射免疫偶联中的癌症的小鼠模型。

Introduction

由于其显着的敏感性,亲和力和选择性,抗体长期以来被认为是有前途的载体用于递送放射性同位素至肿瘤细胞。但是,它们在正电子发射断层扫描(PET)成像中的应用受到阻碍,缺乏适当的正电子发射放射性同位素为其标签1-3之一在放射性免疫的设计中最关键的考虑因素是匹配的物理衰变半寿命放射性同位素与抗体的体内药动学。更具体地说,抗体通常具有相对长的,多天的生物半衰期,因此必须用放射性同位素标记具有可比的物理半衰期。用于PET成像的应用中,抗体历来放射性标记的64铜(叔1/2 = 12.7小时),86 Y(吨1/2 = 14.7小时),或124 I(叔1/2 = 4.18 d)所示。4, 5然而,每个这些放射性同位素拥有妨碍其适用于临床影像显著的局限性。而放射免疫标记86 Y和64铜已被证明有希望的临床前调查,既同位素具有物理半衰期太短是有效用于成像在人类。124 I,与此相反,具有接近理想的物理半衰期为成像用的抗体,但它是昂贵的,并且具有导致相对较低的分辨率临床图像欠佳衰减特性。此外,124 I标记的放射性免疫能受到脱卤体内 ,一种能降低肿瘤-背景活动比率的过程。6,7

该驱动器找到一个正电子发射放射性同位素来取代64,86 Y,124我放射免疫起到了推波助澜的最近激增的研究89锆标记的抗体。8-12牛逼他原因89锆问世是直截了当:放射性金属具有近乎理想的化学和物理性质用于诊断的PET放射性免疫使用13 89锆经由89 Y(P,N)上使用一个回旋加速器89 Zr的反应而产生。市售和100%自然丰富89ÿ目标。14,15的放射性金属具有23%的正电子产率,衰变78.4小时的半衰期,并且发射正电子与395.5千电子伏( 图1)的相对低的能量。 13,16,17要注意的是89锆还发射较高能量是很重要的,909千电子伏γ射线具有99%的效率。虽然这种发射使劲与发射的511keV光子干扰,但是它对于运输,处理,和剂量需要额外考虑。尽管这样的警告,这些衰减特性最终意味着89锆不仅拥有更有利的ħ用于成像ALF-寿命的抗体比86 Y和64铜,但也可以比124 I产生更高分辨率的图像,其发射正电子与687和975 keV的更高的能量,以及一个数的光子能量与内100-150千电子伏的在511千电子伏的正电子创建的光子。13此外,89的Zr也更安全地处理,以产生更便宜,并且residualizes肿瘤更有效地比其对应物的放射性碘。18,19的一个潜在的89 Zr的限制是,它不具有治疗isotopologue, 比如 ,86 Y(PET)与90 Y(治疗)。这就排除了可以用作剂量球探对他们的治疗对口化学性质相同,替代显像剂的建设。尽管如此,调查结果表明,89锆标记的抗体是有潜力的成像代理人90 Y-和177路标记免疫。20,21

从化学的观点来看,作为一种Ⅳ族金属,89 Zr的存在是为4阳离子在水溶液。所述的Zr 4+离子被高度带电的,相对大的(有效离子半径= 0.84埃),并且可以被分类为一“硬”阳离子。因此,它表现出偏爱配体承载多达八个硬的,阴离子的氧供体。容易在89锆标记的放射性免疫中使用的最常见的螯合剂是去铁胺(DFO),一铁载体衍生的,无环螯合剂轴承3异羟肟酸基团。配位体稳定地协调的Zr 4+阳离子迅速干净在RT在生物相关的pH水平,并且将所得的Zr-DFO络合物保持稳定的在盐水中,血清多天的过程中,和全血。22计算研究强烈提示该DFO形成六配位配合物的Zr 4+,其中金属中心配位的三NEUT拉尔和配体的3阴离子氧供体,以及两个外源性配体的水 2)。23,24放射免疫采用89锆DFO结合支架的体内行为已普遍出色。然而,在一些情况下,成像和急性生物分布研究显示升高的活性水平在注射89锆标记的抗体的小鼠的骨,该数据表明,osteophilic 89的Zr 4+阳离子选自螯合剂释放在体内并且随后矿化在骨。25最近,一些调查到的新的894+螯合剂的发展尤其是配体与八氧捐助者已经出现在文献中。24,26,27不过,目前,DFO是最广泛采用的螯合剂在89大幅锆标记的放射免疫。各种不同生物结合策略已被用来DFO附加到抗体,包括生物正交点击化学,硫醇反应性DFO构建体与抗体的半胱氨酸反应,和活化酯承载DFO构建体与抗体赖氨酸反应。4,28- 30轻松最常见的策略,然而,已使用DFO,DFO-NCS的异硫氰酸酯轴承衍生物( 2)。22本市售双功能螯合剂鲁棒且可靠地形成稳定的,共价的硫脲用的赖氨酸键抗体( 图3)。

在过去的几年中,各种各样的89锆DFO标记放射性免疫已在文献中报道。临床前研究已经特别丰富,具有抗体不等的较为知名的西妥昔单抗,贝伐单抗,曲妥珠单抗和更深奥的抗体,如CD105靶向牛逼RC105和游离PSA靶向5A10 30-36最近,少数使用89锆DFO标记抗体早期临床试验已经出现在文献中。具体而言,在荷兰团体已经发表的试验采用89锆DFO-政制及内地事务U36,89锆DFO-替伊莫单抗,和89锆DFO -曲妥单抗。21,32,37此外,一系列的89其他临床试验锆标记的放射免疫目前正在进行,其中包括使用PSMA靶向89锆DFO-J591前列腺癌成像和HER2靶向89锆DFO,曲妥珠单抗对乳腺癌影像学检查在这里纪念Sloan Kettering癌症中心23, 30此外,虽然放射性标记的抗体仍然是最常用的89锆标记的放射性药物,放射性金属,也越来越多地被使用与其他载体,包括肽,蛋白质,和纳米材料。38-43 </sup>

89锆DFO标签方法的模块化是 ​​一笔巨大的财富。生物标志物靶向抗体的剧目是不断扩展的,并且在使用这些结构进行体内 PET成像的兴趣日益高涨。因此,我们认为,更加规范的做法和协议的制定可能受益的领域。优异的书面实验方案为DFO-NCS的共轭和89 Zr的放射性标记已经公布由Vosjan 等人 22,我们认为,通过这项工作所提供的视觉演示可进一步帮助调查新到这些技术。在协议中,在另一方面,前列腺特异性膜抗原靶向抗体J591将被用作模型系统,以说明(1)的双功能螯合剂的生物缀DFO-异硫氰酸酯的抗体,89(2)的放射合成和纯化ZR-DFO-单克隆抗体放射免疫,及(3) 在体内 PET成像与89锆DFO-单抗放射免疫偶联在癌症的小鼠模型。23,44,45

Protocol

所有上述的体内动物实验是根据经批准的协议,并在纪念Sloan Kettering癌症中心的机构动物护理和使用委员会(IACUC)的道德准则进行。 1.共轭DFO-NCS来J591 在1.7 ml离心管,准备一个2-5毫克/毫升的溶液J591在1ml任1×磷酸盐缓冲盐水(pH 7.4)中或0.5M的HEPES缓冲液(pH 7.4)中的。 溶解DFO-NCS在无水DMSO 5-10毫米(3.8-7.6毫克/毫升)间的浓度。声处理或涡流,以促进完?…

Representative Results

在这个协议中DFO-NCS的与抗体缀合的第一个步骤通常是相当坚固和可靠的。一般地,纯化的,螯合剂改性的免疫可以在> 90%的产率获得,并且使用DFO-NCS的3摩尔当量在最初的缀合反应将产生一定程度的-的标记的约1.0-1.5螯合剂DFO的/单克隆抗体。 89锆放射性标记和纯化步骤的过程中也同样简单。在上述协议中列出的浓度> 80%的放射性标记的产量和> 2.0毫居里/毫克因此具体活动是在室温…

Discussion

而施工,放射性标记,成像和89锆DFO-标有放射免疫通常是一个相当简单的过程,它保持了几个重要的考虑因素记在该过程的每个步骤是重要的。例如,也许在该过程的缀合步骤关注最可能的原因是该抗体的缀合反应期间的聚合。此问题是最经常在加入DFO-NCS原液后的缀合反应的混合不良的产品。22当这种情况发生时,DFO-NCS的非均匀分布可能导致过高的水平与当地反应的抗体,其可?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者感谢教授托马斯·莱纳,雅各布博士霍顿,和Serge Lyaschenko博士有益的对话。

Materials

Name of the Material/Equipment Company Catalog Number Comments
p-SCN-Bn-DFO Macrocyclics B-705 Store at -80 °C
[89Zr]Zr-oxalate Various, including Perkin-Elmer Caution: Radioactive material
PD-10 Desalting Columns GE Healthcare 17-0851-01  Store at room temperature
Amicon Ultra-4 Centrifugal Filter Units EMD Millipore UFC805024 Store at room temperature
Silica Gel Impregnanted RadioTLC Paper Agilent Technologies SGI0001 Cut into strips 0.5 cm wide

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Cite This Article
Zeglis, B. M., Lewis, J. S. The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies. J. Vis. Exp. (96), e52521, doi:10.3791/52521 (2015).

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