Extensive Survey of Antibody Invariant Positions for Efficient Chemical Conjugation Using Expanded Genetic Codes
Extensive Survey of Antibody Invariant Positions for Efficient Chemical Conjugation Using Expanded Genetic Codes
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DOI：
10.1021/acs.bioconjchem.7b00265
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发表时间：
2017-08-01
影响因子：
4.7
通讯作者：
Shiraishi, Yasuhisa
中科院分区：
文献类型：
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作者：
Kato, Akifumi;Kuratani, Mitsuo;Shiraishi, Yasuhisa
在用扩展的遗传密码合成后，蛋白质的位点特异性化学缀合有望推进基于抗体的技术，包括抗体药物缀合和双特异性 Fab 二聚体的创建。将非天然氨基酸掺入抗体中不仅保证了位点特异性，而且还允许使用生物正交化学。然而，氨基酸掺入效率在不同位点之间波动显着，从而阻碍了有用缀合位点的鉴定。在本研究中，我们应用密码子重新分配技术来实现在指定位置含有 N-ε-(o-azidobenzyloxycarbony1)-L-lysine (o-Az-Z-Lys) 的化学功能化抗体的稳健和高效合成。这种赖氨酸衍生物在长侧链末端具有生物正交反应基团，能够识别 Fab 恒定结构域中的多个新位置，从而实现高效的化学缀合。对具有 o-Az-Z-Lys 的 Fab 变体进行的 X 射线晶体学研究揭示了叠氮基对溶剂的高水平暴露，其中六个已识别的位置随后用于设计“Variabodies”，这是一种允许两个 Fab 分子之间存在各种连接的新型抗体形式。我们的研究结果表明，一些创建的 Variabody 在培养细胞中表现出激动活性，而不是抗体的拮抗性质。这些结果表明，我们的方法极大地提高了抗体化学缀合的可用性，并可能有助于开发新的治疗抗体。
The site-specific chemical conjugation of proteins, following synthesis with an expanded genetic code, promises to advance antibody-based technologies, including antibody drug conjugation and the creation of bispecific Fab dimers. The incorporation of non-natural amino acids into antibodies not only guarantees site specificity but also allows the use of bio-orthogonal chemistry. However, the efficiency of amino acid incorporation fluctuates significantly among different sites, thereby hampering the identification of useful conjugation sites. In this study, we applied the codon reassignment technology to achieve the robust and efficient synthesis of chemically functionalized antibodies containing N-epsilon-(o-azidobenzyloxycarbony1)-L-lysine (o-Az-Z-Lys) at defined positions. This lysine derivative has a bio-orthogonally reactive group at the end of a long side chain, enabling identification of multiple new positions in Fab-constant domains, allowing chemical conjugation with high efficiency. An X-ray crystallographic study of a Fab variant with o-Az-Z-Lys revealed high-level exposure of the azido group to solvent, with six of the identified positions subsequently used to engineer "Variabodies", a novel antibody format allowing various connections between two Fab molecules. Our findings indicated that some of the created Variabodies exhibited agonistic activity in cultured cells as opposed to the antagonistic nature of antibodies. These results showed that our approach greatly enhanced the availability of antibodies for chemical conjugation and might aid in the development of new therapeutic antibodies.