Hanes J, Schaffitzel C, Knappik A, Plückthun A
Biochemisches Institut, Universität Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland.
Nat Biotechnol. 2000 Dec;18(12):1287-92. doi: 10.1038/82407.
Here we applied ribosome display to in vitro selection and evolution of single-chain antibody fragments (scFvs) from a large synthetic library (Human Combinatorial Antibody Library; HuCAL) against bovine insulin. In three independent ribosome display experiments different clusters of closely related scFvs were selected, all of which bound the antigen with high affinity and specificity. All selected scFvs had affinity-matured up to 40-fold compared to their HuCAL progenitors, by accumulating point mutations during the ribosome display cycles. The dissociation constants of the isolated scFvs were as low as 82 pM, which validates the design of the naïve library and the power of this evolutionary method. We have thus mimicked the process of antibody generation and affinity maturation with a synthetic library in a cell-free system in just a few days, obtaining molecules with higher affinities than most natural antibodies.
在此，我们应用核糖体展示技术从一个大型合成文库（人组合抗体文库；HuCAL）中针对牛胰岛素进行单链抗体片段（scFv）的体外筛选和进化。在三个独立的核糖体展示实验中，筛选出了不同的紧密相关scFv簇，所有这些scFv都以高亲和力和特异性结合抗原。与它们的HuCAL祖先相比，所有筛选出的scFv通过在核糖体展示循环中积累点突变，亲和力成熟了高达40倍。分离出的scFv的解离常数低至82 pM，这验证了原始文库的设计以及这种进化方法的威力。因此，我们仅在几天内就在无细胞系统中用合成文库模拟了抗体产生和亲和力成熟的过程，获得了亲和力高于大多数天然抗体的分子。
