Rational library design by functional CDR resampling

Abstract

Successful antibody discovery relies on diversified libraries, where two aspects are implied, namely the absolute number of unique clones and the percentage of functional clones. Instead of pursuing the absolute quantity thresholded by current display technology, we have sought to maximize the effective diversity by improving functional clone percentage. With the combined effort of bioinformatics, structural biology, molecular immunology and phage display technology, we devised a bioinformatic pipeline to construct and validate libraries via combinatorial assembly of sequences from a database of experimentally validated antibodies. Furthermore, we showed that the libraries constructed as such yielded a significantly increased success rate against different antigen types and generated over 20-fold more unique hits per targets compared with libraries based on traditional degenerate nucleotide methods. Our study indicated that predefined CDR sequences with optimized CDR-framework compatibility could be a productive direction of functional library construction for in vitro antibody development.

Derivation of splice junction-specific antibodies using a unique hapten targeting strategy and directed evolution

“…All libraries employed single-chain variable fragment (scFv)-display strategies[ 18 - 20 ] ( Supplementary Tables S1 and S5 ). AXL40 is a naïve single-framework Discovery library (Abcam)[ 18 , 21 ] with predetermined complementarity determining regions (CDRs) including a 5% molar ratio of four phospho-binding CDR-H2 sequences identified in-house and in [ 22 ]. AXL41 is a naïve single-framework Discovery library (Abcam) based on a SEP-specific scFv that has been mutagenized using degenerate nucleotides ( i.e.…”

Advances in the Production and Batch Reformatting of Phage Antibody Libraries

“…An alternative approach to scFv library construction has been recently described by Zhao et al that used the modular cloning of different CDR regions [54]. Antibody sequence databases were mined for heavy and light chain CDR2 and CDR3 sequences contained within a particular scFv framework.…”

A Simple Whole-Plasmid PCR Method to Construct High-Diversity Synthetic Phage Display Libraries

“…Subsequent cloning results in a DNA sequence devoid of any restriction enzyme site and therefore addition of extra amino acids within expressed sequences can be avoided and the process is irreversible. Alternative methods use a variation on Kunkel mutagenesis to introduce diversity into multiple CDRs simultaneously by annealing oligonucleotides (containing diversity) to CDR regions in scFv–phagemid template ssDNA and then synthesising the second strand of the DNA to produce heteroduplex DNA that is used directly for transformation [ 19 ] or is further amplified by rolling circle amplification before transformation [ 20 ].…”

“…Cloning of the CDR diversity was carried out using engineered restriction sites within the scaffolds flanking the CDR3 domains. An alternative approach to scFv library construction used the modular cloning of different CDR2 and CDR3 regions [ 20 ]. Antibody sequence databases were mined for heavy and light chain CDR2 and CDR3 sequences contained within a particular scFv framework.…”