Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
J Mol Biol. 2011 Nov 4;413(4):826-43. doi: 10.1016/j.jmb.2011.09.016. Epub 2011 Sep 21.
Designed Ankyrin Repeat Proteins (DARPins) represent a novel class of binding molecules. Their favorable biophysical properties such as high affinity, stability and expression yields make them ideal candidates for tumor targeting. Here, we describe the selection of DARPins specific for the tumor-associated antigen epithelial cell adhesion molecule (EpCAM), an approved therapeutic target on solid tumors. We selected DARPins from combinatorial libraries by both phage display and ribosome display and compared their binding on tumor cells. By further rounds of random mutagenesis and ribosome display selection, binders with picomolar affinity were obtained that were entirely monomeric and could be expressed at high yields in the cytoplasm of Escherichia coli. One of the binders, denoted Ec1, bound to EpCAM with picomolar affinity (K(d)=68 pM), and another selected DARPin (Ac2) recognized a different epitope on EpCAM. Through the use of a variety of bivalent and tetravalent arrangements with these DARPins, the off-rate on cells was further improved by up to 47-fold. All EpCAM-specific DARPins were efficiently internalized by receptor-mediated endocytosis, which is essential for intracellular delivery of anticancer agents to tumor cells. Thus, using EpCAM as a target, we provide evidence that DARPins can be conveniently selected and rationally engineered to high-affinity binders of various formats for tumor targeting.
设计的锚重复蛋白（DARPins）代表了一类新型的结合分子。它们具有高亲和力、稳定性和表达产量等有利的生物物理特性，使其成为肿瘤靶向的理想候选物。在这里，我们描述了针对肿瘤相关抗原上皮细胞黏附分子（EpCAM）的 DARPins 的选择，EpCAM 是实体瘤上已批准的治疗靶点。我们通过噬菌体展示和核糖体展示从组合文库中选择 DARPins，并比较它们在肿瘤细胞上的结合。通过进一步的随机诱变和核糖体展示选择，获得了具有皮摩尔亲和力的结合物，它们完全是单体的，可以在大肠杆菌的细胞质中以高产量表达。其中一种结合物，称为 Ec1，与 EpCAM 的结合具有皮摩尔亲和力（K(d)=68 pM），另一种选择的 DARPin（Ac2）识别 EpCAM 上的不同表位。通过使用这些 DARPins 的各种二价和四价排列，细胞上的离解速率提高了多达 47 倍。所有 EpCAM 特异性 DARPins 都通过受体介导的内吞作用有效内化，这对于抗癌药物向肿瘤细胞的细胞内递药至关重要。因此，以 EpCAM 为靶点，我们提供了证据表明，DARPins 可以方便地选择和合理设计成各种格式的高亲和力结合物，用于肿瘤靶向。
