High-throughput construction of multivalent binders and subsequent screening for biological activity represent a fundamental challenge: A linear increase of monovalent components translates to the square of possible bivalent combinations. Even high-efficiency cloning and expression methods become limiting when thousands of bispecific binders need to be screened for activity. In this study, we present an in vitro method for the efficient production of flexibly linked bispecific binding agents from individually expressed and purified monovalent binders. We established a sortase A–mediated coupling reaction to generate bispecific designed ankyrin repeat proteins (DARPins), with an optimized reaction maximizing the bivalent coupling product with low levels of monovalent side-products. These one-pot reaction mixtures could be used directly, without further purification, in cell-based assays. We generated a matrix of 441 different bispecific DARPins against the extracellular domains of the cancer-associated receptors EGFR, ErbB2, ErbB3, ErbB4, EpCAM, and c-MET and screened on two different ErbB2-positive cancer cells lines for growth-inhibitory effects. We identified not only known but also novel biologically active biparatopic DARPins. Furthermore, we found that the cancer cell lines respond in a highly reproducible and defined manner to the treatment with the 441 different bivalent binding agents. The generated response profiles can thus be used for functional characterization of cell lines because they are strongly related to the cell line–specific surface receptor landscape. Thus, our method not only represents a robust tool for screening and lead identification of bispecific binding agents, but additionally offers an orthogonal approach for the functional characterization of cancer cell lines.

中文翻译：

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通过分选酶 A 介导的偶联高通量生成双特异性结合蛋白，用于细胞培养中的直接功能筛选

多价结合剂的高通量构建和随后的生物活性筛选是一项基本挑战：单价组分的线性增加转化为可能的二价组合的平方。当需要筛选数以千计的双特异性结合物的活性时，即使是高效的克隆和表达方法也会受到限制。在这项研究中，我们提出了一种体外方法，用于从单独表达和纯化的单价结合剂中有效生产灵活连接的双特异性结合剂。我们建立了一种分选酶 A 介导的偶联反应，以产生双特异性设计的锚蛋白重复蛋白 (DARPins)，优化的反应使二价偶联产物最大化，单价副产物含量低。这些一锅反应混合物可以直接使用，无需进一步纯化，在基于细胞的检测中。我们针对癌症相关受体 EGFR、ErbB2、ErbB3、ErbB4、EpCAM 和 c-MET 的细胞外结构域生成了 441 个不同的双特异性 DARPins 矩阵，并在两种不同的 ErbB2 阳性癌细胞系上筛选了生长抑制作用。我们不仅确定了已知的而且还确定了新的具有生物活性的双互补位 DARPins。此外，我们发现癌细胞系以高度可重复和明确的方式对 441 种不同的二价结合剂进行治疗。因此，生成的响应曲线可用于细胞系的功能表征，因为它们与细胞系特异性表面受体景观密切相关。因此，