Many large synthetic antibody libraries have been designed and constructed to successfully generate high-quality antibodies suitable for various demanding applications. An advantage of synthetic antibody libraries is the ability to exert fine control over factors like framework sequences, amino acid and codon usage, and complementary determining region (CDR) diversity. While synthetic antibody libraries have many advantages such as optimized framework sequences and a broader sequence landscape than natural antibodies, their sequence diversities are typically generated by random combinatorial synthetic processes. 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, researchers from Ewha Womans University describe the construction and validation of a human single-chain variable fragment (scFv) library with a novel design approach to synthetic CDR diversification.
The SCIEN (Simulation of CDRs Inspired by and Emulating Nature) principle is based on the simulation of natural rearranged and hypermutated CDRs and the parallel synthesis of thousands of oligonucleotides encoding the predefined CDR sequences. Because the CDR sequence diversity is predefined and synthesized without relying on random combinatorial events, the incorporation of undesired sequences to the library can be prevented in the design stage.
The researchers 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.
Construction of the scFv library with six non-combinatorially diversified CDRs
Pools of oligonucleotides with the designed CDR sequences were array-synthesized and amplified by PCR. A single-CDR library (scFv library with only one of the six CDRs are diversified) was constructed for each CDR using the amplified CDR oligonucleotide mixtures, and panned against anti-HA antibody that bind to HA-tag at the C-terminus of scFv in order to proofread the CDR repertoires for in-frame sequences. The proofread CDRs were consolidated into the final scFv library with six diversified CDRs