Abstract
The present work describes the construction and validation of a human scFv library with a novel design approach to synthetic complementarity determining region (CDR) diversification. The advantage of synthetic antibody libraries includes the possibility of exerting fine control over factors like framework sequences, amino acid and codon usage, and CDR diversity. However, random combinatorial synthesis of oligonucleotides for CDR sequence diversity also produces many clones with unnatural sequences and/or undesirable modification motifs. To alleviate these issues, we designed and constructed a novel semi-synthetic human scFv library with non-combinatorial, pre-designed CDR diversity and a single native human framework each for heavy, kappa, and lambda chain variable domains. Next-generation sequencing analysis indicated that the library consists of antibody clones with highly nature-like CDR sequences and the occurrence of the post-translational modification motifs is minimized. Multiple unique clones with nanomolar affinity could be isolated from the library against a number of target antigens, validating the library design strategy. The results demonstrate that it is possible to construct a functional antibody library using low, non-combinatorial synthetic CDR diversity, and provides a new strategy for the design of antibody libraries suitable for demanding applications.
Highlights
Target-specific antibodies can be rapidly isolated from a large antibody library by in vitro display technologies, such as phage or yeast display
Those sequences for which the end of FR1 and the beginning of FR4 could be identified were selected, and redundant variable region sequences were removed
complementarity determining region (CDR) sequences were extracted from these variable region sequences [13], and the somatic mutations of the CDRs were identified by comparing their sequences with the closest human germline CDR sequence
Summary
Target-specific antibodies can be rapidly isolated from a large antibody library by in vitro display technologies, such as phage or yeast display. The size and quality of the antibody library is a major determinant of the success of in vitro antibody generation, and many different strategies have been employed to design and construct large, highly functional antibody libraries [1]. While the size of an antibody library is mostly determined by the transformation efficiency of bacteria or yeast, multiple different factors can influence the functionality of a library and need to be considered in the library design. One important factor in library design is the source and nature of the sequence diversity, which can originate from natural (animal B-cells), synthetic, or semi-synthetic sources.
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