期刊
JOURNAL OF BIOTECHNOLOGY
卷 191, 期 -, 页码 236-245出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jbiotec.2014.04.023
关键词
Shark vNAR antibody; Semi-synthetic library; Stepwise in vitro affinity maturation; Yeast surface display
资金
- Merck'sche Gesellschaft fur Kunst und Wissenschaft
- Federal Ministry of Education and Research (BMBF) in frame of the consortium Nanokat
A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold. (C) 2014 Elsevier B.V. All rights reserved.
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