Moutel Sandrine, Bery Nicolas, Bernard Virginie, Keller Laura, Lemesre Emilie, de Marco Ario, Ligat Laetitia, Rain Jean-Christophe, Favre Gilles, Olichon Aurélien, Perez Franck
Institut Curie, PSL Research University, Paris, France.
CNRS UMR144, Paris, France.
Elife. 2016 Jul 19;5:e16228. doi: 10.7554/eLife.16228.
In vitro selection of antibodies allows to obtain highly functional binders, rapidly and at lower cost. Here, we describe the first fully synthetic phage display library of humanized llama single domain antibody (NaLi-H1: Nanobody Library Humanized 1). Based on a humanized synthetic single domain antibody (hs2dAb) scaffold optimized for intracellular stability, the highly diverse library provides high affinity binders without animal immunization. NaLi-H1 was screened following several selection schemes against various targets (Fluorescent proteins, actin, tubulin, p53, HP1). Conformation antibodies against active RHO GTPase were also obtained. Selected hs2dAb were used in various immunoassays and were often found to be functional intrabodies, enabling tracking or inhibition of endogenous targets. Functionalization of intrabodies allowed specific protein knockdown in living cells. Finally, direct selection against the surface of tumor cells produced hs2dAb directed against tumor-specific antigens further highlighting the potential use of this library for therapeutic applications.
体外抗体筛选能够迅速且低成本地获得高功能结合物。在此，我们描述了首个完全合成的人源化羊驼单域抗体噬菌体展示文库（NaLi-H1：人源化单域抗体文库1）。基于针对细胞内稳定性优化的人源化合成单域抗体（hs2dAb）支架，这个高度多样化的文库无需动物免疫就能提供高亲和力结合物。按照几种筛选方案，利用NaLi-H1针对各种靶标（荧光蛋白、肌动蛋白、微管蛋白、p53、HP1）进行筛选。还获得了针对活性RHO GTP酶的构象抗体。筛选出的hs2dAb用于各种免疫测定，并且常常被发现是有功能的细胞内抗体，能够追踪或抑制内源性靶标。细胞内抗体的功能化能够在活细胞中实现特定蛋白质的敲低。最后，针对肿瘤细胞表面进行直接筛选产生了针对肿瘤特异性抗原的hs2dAb，进一步凸显了该文库在治疗应用方面的潜在用途。
