Molecular conjugation to antibodies has emerged as a growing strategy to combine the mechanistic activities of the attached molecule with the specificity of antibodies. A variety of technologies have been applied for molecular conjugation; however, these approaches face several limitations, including disruption of antibody structure, destabilization of the antibody, and/or heterogeneous conjugation patterns. Collectively, these challenges lead to reduced yield, purity, and function of conjugated antibodies. While glycoengineering strategies have largely been applied to study protein glycosylation and manipulate cellular metabolism, these approaches also harbor great potential to enhance the production and performance of protein therapeutics. Here, we devise a novel glycoengineering workflow for the development of site-specific antibody conjugates. This approach combines metabolic glycoengineering using azido-sugar analogs with newly installed N-linked glycosylation sites in the antibody constant domain to achieve specific conjugation to the antibody via the introduced N-glycans. Our technique allows facile and efficient manufacturing of well-defined antibody conjugates without need for complex or destructive chemistries. Moreover, introduction of conjugation sites in the antibody fragment crystallizable (Fc) domain renders this approach widely applicable and target agnostic. Our platform can accommodate up to 3 conjugation sites in tandem, and the extent of conjugation can be tuned through use of different sugar analogs or production in different cell lines. We demonstrated that our platform is compatible with various use-cases, including fluorescent labeling, antibody-drug conjugation, and targeted gene delivery. Overall, this study introduces a versatile and effective yet strikingly simple approach to produce antibody conjugates for research, industrial, and medical applications.

中文翻译：

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使用代谢糖工程和新型 Fc 糖变体生产位点特异性抗体偶联物。


分子偶联抗体已成为一种日益增长的策略，它将附着分子的机械活性与抗体的特异性相结合。分子偶联已应用了多种技术;然而，这些方法面临一些限制，包括抗体结构的破坏、抗体的不稳定和/或异质偶联模式。总的来说，这些挑战会导致偶联抗体的产量、纯度和功能降低。虽然糖工程策略已广泛用于研究蛋白质糖基化和操纵细胞代谢，但这些方法在提高蛋白质治疗药物的生产和性能方面也具有巨大潜力。在这里，我们设计了一种新的糖工程工作流程，用于开发位点特异性抗体偶联物。该方法将使用叠氮基糖类似物的代谢糖工程与抗体恒定域中新安装的 N-连接糖基化位点相结合，以通过引入的 N-糖实现与抗体的特异性偶联。我们的技术可以简单高效地生产成分明确的抗体偶联物，而无需复杂或破坏性的化学试剂。此外，在抗体片段可结晶 （Fc） 结构域中引入偶联位点使该方法广泛适用且与靶标无关。我们的平台可以同时容纳多达 3 个偶联位点，并且可以通过使用不同的糖类似物或在不同细胞系中生产来调整偶联程度。我们证明，我们的平台与各种用例兼容，包括荧光标记、抗体-药物偶联和靶向基因递送。 总体而言，本研究介绍了一种多功能且有效但非常简单的方法来生产用于研究、工业和医学应用的抗体偶联物。