Despite the significant advances of antibodies as therapeutic agents, there is still much room for improvement concerning the discovery of these macromolecules. Here, we present a new synthetic cell-based strategy that takes advantage of eukaryotic cell biology to produce highly diverse antibody libraries and, simultaneously, link them to a high-throughput selection mechanism, replicating B cell diversification mechanisms. The interference of site-specific recognition by CRISPR/Cas9 with error-prone DNA repair mechanisms was explored for the generation of diversity, in a cell population containing a gene for a light chain antibody fragment. We achieved up to 93% of cells containing a mutated antibody gene after diversification mechanisms, specifically inside one of the antigen-binding sites. This targeted variability strategy was then integrated into an intracellular selection mechanism. By fusing the antibody with a KDEL retention signal, the interaction of antibodies and native membrane antigens occurs inside the endoplasmic reticulum during the process of protein secretion, enabling the detection of high-quality leads for expression and affinity by flow cytometry. We successfully obtained antibody lead candidates against CD3 as proof of concept. In summary, we developed a novel antibody discovery platform against native antigens by endoplasmic synthetic library generation using CRISPR/Cas9, which will contribute to a faster discovery of new biotherapeutic molecules, reducing the time-to-market.

中文翻译：

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通过CRISPR / Cas9库生成和内质网筛选发现针对天然抗原的合成抗体。

尽管抗体作为治疗剂取得了显着进步，但是关于这些大分子的发现仍然有很大的改进空间。在这里，我们提出了一种新的基于合成细胞的策略，该策略利用了真核细胞生物学来产生高度多样化的抗体库，并同时将它们链接至高通量选择机制，从而复制了B细胞的多样化机制。在含有轻链抗体片段基因的细胞群中，探索了CRISPR / Cas9对位点特异性识别的干扰以及易于出错的DNA修复机制的产生多样性。经过多样化机制，特别是在一个抗原结合位点内部，我们获得了高达93％的包含突变抗体基因的细胞。然后将这种有针对性的可变性策略整合到细胞内选择机制中。通过将抗体与KDEL保留信号融合，可以在蛋白质分泌过程中在内质网内部发生抗体与天然膜抗原之间的相互作用，从而可以通过流式细胞仪检测高质量的表达和亲和力。我们成功获得了针对CD3的抗体前导候选物作为概念证明。总而言之，我们通过使用CRISPR / Cas9生成内质合成文库，开发了针对天然抗原的新型抗体发现平台，这将有助于更快地发现新的生物治疗分子，从而缩短上市时间。抗体和天然膜抗原之间的相互作用发生在蛋白质分泌过程中的内质网内部，从而可以通过流式细胞仪检测高质量的表达和亲和力线索。我们成功获得了针对CD3的抗体前导候选物作为概念证明。总而言之，我们通过使用CRISPR / Cas9生成内质合成文库，开发了针对天然抗原的新型抗体发现平台，这将有助于更快地发现新的生物治疗分子，从而缩短上市时间。抗体和天然膜抗原之间的相互作用发生在蛋白质分泌过程中的内质网内部，从而可以通过流式细胞仪检测高质量的表达和亲和力线索。我们成功获得了针对CD3的抗体前导候选物作为概念证明。总而言之，我们通过使用CRISPR / Cas9生成内质合成文库，开发了针对天然抗原的新型抗体发现平台，这将有助于更快地发现新的生物治疗分子，从而缩短上市时间。