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首页> 外文期刊>BMC Biotechnology >Site-specific modification of ED-B-targeting antibody using intein-fusion technology
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Site-specific modification of ED-B-targeting antibody using intein-fusion technology

机译：使用内含蛋白融合技术对ED-B靶向抗体进行位点特异性修饰

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Background A promising new approach in cancer therapy is the use of tumor specific antibodies coupled to cytotoxic agents. Currently these immunoconjugates are prepared by rather unspecific coupling chemistries, resulting in heterogeneous products. As the drug load is a key parameter for the antitumor activity, site-specific strategies are desired. Expressed protein ligation (EPL) and protein trans-splicing (PTS) are methods for the specific C-terminal modification of a target protein. Both include the expression as an intein fusion protein, followed by the exchange of the intein for a functionalized moiety. Results A full-length IgG specific for fibronectin ED-B was expressed as fusion protein with an intein ( Mxe GyrA or Npu DnaE ) attached to each heavy chain. In vitro protocols were established to site-specifically modify the antibodies in high yields by EPL or PTS, respectively. Although reducing conditions had to be employed during the process, the integrity or affinity of the antibody was not affected. The protocols were used to prepare immunoconjugates containing two biotin molecules per antibody, attached to the C-termini of the heavy chains. Conclusion Full-length antibodies can be efficiently and site-specifically modified at the C-termini of their heavy chains by intein-fusion technologies. The described protocols can be used to prepare immunoconjugates of high homogeneity and with a defined drug load of two. The attachment to the C-termini is expected to retain the affinity and effector functions of the antibodies.
机译：背景技术在癌症治疗中有希望的新方法是使用与细胞毒性剂偶联的肿瘤特异性抗体。目前，这些免疫偶联物是通过相当非特异性的偶联化学制备的，从而产生异质产物。由于载药量是抗肿瘤活性的关键参数，因此需要针对位点的策略。表达的蛋白质连接（EPL）和蛋白质反式剪接（PTS）是对目标蛋白质进行特定C末端修饰的方法。两者都包括表达为内含蛋白融合蛋白，然后将内含蛋白交换为功能化部分。结果对纤连蛋白ED-B特异的全长IgG被表达为融合蛋白，每个重链上均附有内含肽（Mxe GyrA或Npu DnaE）。建立了体外方案以分别通过EPL或PTS以高产量进行位点特异性修饰。尽管在该过程中必须采用还原条件，但是抗体的完整性或亲和力没有受到影响。该方案用于制备每个抗体包含两个生物素分子的免疫偶联物，该抗体与重链的C末端连接。结论全长蛋白可以通过intein融合技术在其重链的C末端进行有效的位点特异性修饰。所描述的方案可用于制备高同质性和确定的药物载量为两个的免疫缀合物。预期与C末端的结合将保留抗体的亲和力和效应子功能。

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