Improved Synthesis and In Vitro Evaluation of an Aptamer Ribosomal Toxin Conjugate
Improved Synthesis and In Vitro Evaluation of an Aptamer Ribosomal Toxin Conjugate
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DOI：
10.1089/nat.2015.0599
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发表时间：
2016-06-01
影响因子：
4
通讯作者：
Levy, Matthew
中科院分区：
文献类型：
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作者：
Kelly, Linsley;Kratschmer, Christina;Levy, Matthew
使用抗体递送毒素，如蓖麻毒素A链、假单胞菌外毒素和白树毒素，在癌症治疗中诱导特异性毒性方面取得了一些成功。然而，这些称为免疫毒素的抗体-毒素缀合物可能体积庞大，难以表达，并且在体内施用时可能诱导免疫应答。我们先前报道了通过全长前列腺特异性膜抗原（PSMA）结合适体A9递送白树素的重组变体（rGel），以潜在地规避这些问题中的一些。在这里，我们报告了一个流线型的方法来产生适配体-rGel共轭物利用化学合成的最小化形式的A9适配体。与全长A9适体不同，这种最小化的变体可以用5'末端硫醇化学合成。这有利于通过可还原的二硫键连接的适体毒素缀合物的大规模合成和产生。使用这种方法，我们产生了适体-毒素缀合物，并评估了它们的结合特异性和毒性。在PSMA（+）LNCaP前列腺癌细胞上，A9. min-rGel偶联物显示出与60 nM相似的IC 50。此外，我们在小鼠血清中进行了该缀合物的稳定性分析，其中缀合物显示t（1/2）类似于4 h，为将来的体内实验铺平了道路。
Delivery of toxins, such as the ricin A chain, Pseudomonas exotoxin, and gelonin, using antibodies has had some success in inducing specific toxicity in cancer treatments. However, these antibody-toxin conjugates, called immunotoxins, can be bulky, difficult to express, and may induce an immune response upon in vivo administration. We previously reported delivery of a recombinant variant of gelonin (rGel) by the full-length prostate-specific membrane antigen (PSMA) binding aptamer, A9, to potentially circumvent some of these problems. Here, we report a streamlined approach to generating aptamer-rGel conjugates utilizing a chemically synthesized minimized form of the A9 aptamer. Unlike the full-length A9 aptamer, this minimized variant can be chemically synthesized with a 5' terminal thiol. This facilitates the large scale synthesis and generation of aptamer toxin conjugates linked by a reducible disulfide linkage. Using this approach, we generated aptamer-toxin conjugates and evaluated their binding specificity and toxicity. On PSMA(+) LNCaP prostate cancer cells, the A9. min-rGel conjugate demonstrated an IC50 of similar to 60 nM. Additionally, we performed a stability analysis of this conjugate in mouse serum where the conjugate displayed a t(1/2) of similar to 4 h, paving the way for future in vivo experiments.