Design and construction of small perturbation mutagenesis libraries for antibody affinity maturation using massive microchip-synthesized oligonucleotides

Effective Optimization of Antibody Affinity by Phage Display Integrated with High-Throughput DNA Synthesis and Sequencing Technologies

“…However, saturation mutagenesis, which can generate all combinations of twenty natural amino acids in beyond eight positions, typically requires a library of > 1 x 10 12 diverse sequences, making it impractical for the current display systems. Novel strategies to improve CDR diversification efficiency have been reported, such as hot-spot mutagenesis [ 6 ], look-through mutagenesis [ 7 ], simultaneous mutagenesis [ 8 ], and small perturbation mutagenesis (SPM) which was recently developed by our group [ 9 ]. In addition, the structures of antibody-antigen complexes indicate that the majority, if not all, of the six CDRs may contribute to antigen-binding in most cases [ 10 ].…”

“…However, using current microarray-based techniques for the synthesis of a large number of different degenerate oligonucleotides remains challenging. Previously, we demonstrated the capacity of a programmable microfluidic microchip to produce hundreds of degenerate oligonucleotides that are suitable for antibody library construction [ 9 ]. In general, the microchip contains nearly four thousand independent reaction chambers, and each chamber can be assigned to synthesize one degenerate oligonucleotide.…”

Structural insight into a matured humanized monoclonal antibody HuA21 against HER2-overexpressing cancer cells

“…In previous studies, the anti-HER2 chimeric monoclonal antibody chA21 was developed to inhibit the proliferation and metastasis of HER2-overexpressing human breast cancer cells (Zhou et al, 2011;Cheng et al, 2003;Hu et al, 2008;Zhang et al, 2010). To reduce potential human anti-mouse immune response, chA21 was further humanized and affinity-matured to yield HuA21 (Hu et al, 2015;Xu et al, 2015). The antitumor activity and mechanism of HuA21 have also been characterized in vivo and in vitro (Li et al, 2016).…”

“…To reduce potential human anti-mouse antibody (HAMA) response and improve its binding affinity, chA21 was further developed by phage-display and antibody affinity maturation technologies to yield the antibody HuA21. The humanization level of HuA21 was greater than 95% (Hu et al, 2015;Xu et al, 2015). Similar to chA21, HuA21 also significantly inhibited the proliferation and migration of tumor cells (Li et al, 2016).…”

High Affinity Maturated Human Antibodies from Naïve and Synthetic Antibody Repertoires

“…The humanized anti-ErbB2 antibody (HuA21) was targeted to diversify the CDR regions via a small perturbation mutagenesis method and was validated using deep sequencing by the Illumina platform. Finally, the mutant candidates were screened by phage display to select for high affinity binders [115,116].…”