New Strategy Of Antibody Affinity Maturation, Based On High-throughput DNA Synthesis And Sequencing

ErbB2is a member of erbB receptor family, which is frequently over-expressed in epithelial cancers, especially in breast and ovarian cancers. This over-expression is associated with tumor metastasis and poor prognosis, making it an attractive target for caner immunotherapy. In the previous study, we generated an anti-ErbB2mAb A21, which could specifically recognize ErbB2and inhibit the growth of ErbB2over-expressing cancer cells, An engineered single-chain chimeric antibody chA21was constructed. It was proved that chA21can inhibit growth of ErbB2over-expressing cancer cells in vitro and in vivo. However, in order to exploit the engineered antibody for the clinical utility, the affinity of this antibody should be improved.Traditional method for antibody affinity maturation is the stochastic mutagenesis of complementarity determining regions (CDRs) of antibodies by overlap PCR. It is time-consuming because it needs steps of digestion and ligation for each library separatly, and needs to construct several libraries for each CDR. The achieved libraries has low ratio of target antibodies because of there are a lot of redundant sequences, and the epitope of antibody may be changed by stochastic mutation. Herein present a novel strategy to diversify the CDRs of single-chain fragment(scFv) of A21antibody, by simultaneous synthesizing all the six CDRs and construct mutational libraries by one-step PCR reaction without digestion and ligation. The design of mutants was based on a scoring system that selects best degenerate codon to generate proper amino acids distribution, according to the analysis of the CDRs Chothia classification from Kabat dataset. So the range of mutated amino acids can be controlled closing to the spontaneous natural mutagenesis. Then the designed oligonucleotides were synthesized and purified by a programmable PicoArray synthesis method.Accurate mutagenesis was performed by long-chain PCR, using the synthesized CDRs oligonucleotides as primers and SKN3, a phage display recombinant plasmid pCANTAB5E with A21scFv DNA, as template. Six libraries of each CDR were constructed by transforming E.coli. The variety of libraries was confirmed by DNA sequencing. These single or double point mutants were displayed on M13phage, then higher affinity antibodies were screened with adsorbed and soluble antigen. About600clones were picked and analyzed by phage ELISA and soluble ELISA. Positive clones of each CDR were identified and sequenced, Significant mutations were selected for new combinatorial library design.The new combinatorial library of5CDRs was also constructed by one-step PCR and screened with different concentration of Biotin labeled antigen, and positive clones were subcloned and expressed in mammalian cells. Their affinity were compared by ELIS A, some higher affinity clones were identified.Because there is no homologous classification of VH CDR3in Kabat dataset, We constructed VH CDR3library by stochastic mutagenesis, now we are scanning the library for higher affinity antibodies.For exploring optimal selection methods, we also tried and compared different selection conditions, such as selected by adsorbed and soluble antigen, and eluted with different buffer.We have set up a system of rapid library construction and selection. It is the first time that high-throughput synthesis and sequencing were introduced to antibody affinity maturation and phage display technology, and it is also the first time that library be constructed by one-step PCR. Application of this new strategy of libraries construction for antibody affinity maturation will be explored.