Westerlund Kristina, Honarvar Hadis, Norrström Emily, Strand Joanna, Mitran Bogdan, Orlova Anna, Eriksson Karlström Amelie, Tolmachev Vladimir
Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, AlbaNova University Center , 106 91 Stockholm, Sweden.
Institute for Immunology, Genetics and Pathology, Uppsala University , 751 05 Uppsala, Sweden.
Mol Pharm. 2016 May 2;13(5):1668-78. doi: 10.1021/acs.molpharmaceut.6b00089. Epub 2016 Apr 6.
A promising strategy to enable patient stratification for targeted therapies is to monitor the target expression in a tumor by radionuclide molecular imaging. Affibody molecules (7 kDa) are nonimmunoglobulin scaffold proteins with a 25-fold smaller size than intact antibodies. They have shown an apparent potential as molecular imaging probes both in preclinical and clinical studies. Earlier, we found that hepatic uptake can be reduced by the incorporation of negatively charged purification tags at the N-terminus of Affibody molecules. We hypothesized that liver uptake might similarly be reduced by positioning the chelator at the N-terminus, where the chelator-radionuclide complex will provide negative charges. To test this hypothesis, a second generation synthetic anti-HER2 ZHER2:2891 Affibody molecule was synthesized and labeled with (111)In and (68)Ga using DOTAGA and DOTA chelators. The chelators were manually coupled to the N-terminus of ZHER2:2891 forming an amide bond. Labeling DOTAGA-ZHER2:2891 and DOTA-ZHER2:2891 with (68)Ga and (111)In resulted in stable radioconjugates. The tumor-targeting and biodistribution properties of the (111)In- and (68)Ga-labeled conjugates were compared in SKOV-3 tumor-bearing nude mice at 2 h postinjection. The HER2-specific binding of the radioconjugates was verified both in vitro and in vivo. Using the DOTAGA chelator gave significantly lower radioactivity in liver and blood for both radionuclides. The (111)In-labeled conjugates showed more rapid blood clearance than the (68)Ga-labeled conjugates. The most pronounced influence of the chelators was found when they were labeled with (68)Ga. The DOTAGA chelator gave significantly higher tumor-to-blood (61 ± 6 vs 23 ± 5, p < 0.05) and tumor-to-liver (10.4 ± 0.6 vs 4.5 ± 0.5, p < 0.05) ratios than the DOTA chelator. This study demonstrated that chelators may be used to alter the uptake of Affibody molecules, and most likely other scaffold-based imaging probes, for improvement of imaging contrast.
一种实现针对靶向治疗的患者分层的有前景策略是通过放射性核素分子成像监测肿瘤中的靶标表达。亲和体分子（7 kDa）是非免疫球蛋白支架蛋白，其大小比完整抗体小25倍。在临床前和临床研究中，它们已显示出作为分子成像探针的明显潜力。早些时候，我们发现通过在亲和体分子的N端引入带负电荷的纯化标签，可以降低肝脏摄取。我们推测，通过将螯合剂定位在N端，肝脏摄取可能同样会降低，因为螯合剂 - 放射性核素复合物会提供负电荷。为了验证这一假设，合成了第二代合成抗HER2 ZHER2:2891亲和体分子，并使用DOTAGA和DOTA螯合剂用（111）In和（68）Ga进行标记。螯合剂通过手动偶联到ZHER2:2891的N端形成酰胺键。用（68）Ga和（111）In标记DOTAGA-ZHER2:2891和DOTA-ZHER2:2891产生了稳定的放射性缀合物。在注射后2小时，在荷SKOV-3肿瘤的裸鼠中比较了（111）In和（68）Ga标记的缀合物的肿瘤靶向和生物分布特性。在体外和体内都验证了放射性缀合物的HER2特异性结合。对于两种放射性核素，使用DOTAGA螯合剂在肝脏和血液中的放射性显著降低。（111）In标记的缀合物比（68）Ga标记的缀合物显示出更快的血液清除。当螯合剂用（68）Ga标记时，发现其影响最为明显。与DOTA螯合剂相比，DOTAGA螯合剂产生的肿瘤与血液比值（61±6对23±5，p <0.05）和肿瘤与肝脏比值（10.4±0.6对4.5±0.5，p <0.05）显著更高。这项研究表明，螯合剂可用于改变亲和体分子以及很可能其他基于支架的成像探针的摄取，以改善成像对比度。
