Bispecific antibodies comprise extremely diverse architectures enabling complex modes of action, such as effector cell recruitment or conditional target modulation via dual targeting, not conveyed by monospecific antibodies. In recent years, research on bispecific therapeutics has substantially grown. However, evaluation of binding moiety combinations often leads to undesired prolonged development times. While high throughput screening for small molecules and classical antibodies has evolved into a mature discipline in the pharmaceutical industry, dual-targeting antibody screening methodologies lack the ability to fully evaluate the tremendous number of possible combinations and cover only a limited portion of the combinatorial screening space. Here, we propose a novel combinatorial screening approach for bispecific IgG-like antibodies to extenuate screening limitations in industrial scale, expanding the limiting screening space. Harnessing the ability of a protein trans-splicing reaction by the split intein Npu DnaE, antibody fragments were reconstituted within the hinge region in vitro. This method allows for fully automated, rapid one-pot antibody reconstitution, providing biological activity in several biochemical and functional assays. The technology presented here is suitable for automated functional and combinatorial high throughput screening of bispecific antibodies.

中文翻译：

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内含子介导的高通量筛选双特异性抗体。

双特异性抗体包含极其多样的结构，能够实现复杂的作用模式，例如效应细胞募集或通过双重靶向进行条件性靶调节，而这些不是单特异性抗体所传递的。近年来，对双特异性疗法的研究已大大增加。但是，结合部分组合的评估通常会导致不希望的延长的开发时间。尽管小分子和经典抗体的高通量筛选已发展成为制药行业的成熟学科，但是双靶抗体筛选方法仍无法全面评估大量可能的组合，并且仅覆盖组合筛选空间的一小部分。这里，我们提出了一种针对双特异性IgG样抗体的新型组合筛选方法，以减轻工业规模的筛选限制，扩大了限制筛选空间。利用分裂的内含蛋白Npu DnaE利用蛋白质反式分裂反应的能力，在体外在铰链区内重建抗体片段。这种方法可实现全自动，快速一锅法抗体重组，从而在几种生化和功能测定中提供生物学活性。本文介绍的技术适用于双特异性抗体的自动化功能和组合高通量筛选。在体外将抗体片段重建在铰链区内。这种方法可实现全自动，快速一锅法抗体重组，从而在几种生化和功能测定中提供生物学活性。本文介绍的技术适用于双特异性抗体的自动化功能和组合高通量筛选。在体外将抗体片段重建在铰链区内。这种方法可实现全自动，快速一锅法抗体重组，从而在几种生化和功能测定中提供生物学活性。本文介绍的技术适用于双特异性抗体的自动化功能和组合高通量筛选。