Construction And Selection Of Synthetic Human And Humanization Antibody Library

Phage display technique is the combination of combinatorial technology.1 and engineering antibody technique. It provides a convenient and robust tool for the selection of high-quality antibodies, enabling the identification of antibodies in a fast, high-throughput mode. Here in the dissertation, we applied the fully synthetic phage display antibody library technology in construction and selection of human library and humanization antibody library.Non-immunized human antibody libraries play an important role in isolating antibody for any antigen., While it is an important and hard work to construct large antibody libraries which .contain antibodies with affinities to an unlimited array of antigens. Considering the shortcomings exist in the antibody libraries nowadays, we design to construct a fully synthetic human library. Fourteen reported antibody framework sequences were adopted and synthesized by spliced overlap extension PCR since they are representative of the antibody repertoire in human and were designed to be fold well with good expression behavior. After acquiring fourteen frameworks identical to the reported sequences, we combined them and got 49 frameworks totally. A frequently used framework K3H3 was selected to construct a model library by appending partially randomized CDR3 regions. A novel split-mix-split method was adopted to synthesize the CDR3 sequences. It is validated that the construction of fully synthetic library is successful by sequencing, expression analysis and selection.During the past thirty years, thousands of murine monoclonal antibodies have been made and some of them can be used therapeutically. However, the property of provoking strong Human anti-Murine Antibody (HAMA) immune responses restricts their application clinically. Thus, as one of the strategies for antibody humanization,antibody reshaping has been a 'hotspot' in antibody engineering. We designed a new strategy of molecular directed evolution for antibody reshaping which is different from the structure-based strategy. It can be used to explore large mutant pool simultaneously to select reshaped antibodies.CD28 is an important co-stimulatory molecule for T cell activation which is cooperated with signal I provided by the interaction of the MHC-antigen complex with T cell receptor (TCR)i Anti-CD28 antibody can be used for T cell activation as the alternative of natural ligand of CD28 (B7). Here we successfully reshaped an mouse anti-CD28 VL single chain antibody by phage display 'and strategy of molecular directed evolution. We grafted the CDR region of light-chain of mouse anti-CD28 monoclonal antibody 9.3 to the framework region of light-chain of human antibody gi/598166 and determined the VL amino acid sequence of reshaping anti-CD28 antibody by comparing the conserved sequences of human and mouse frameworks. In order to keep the binding activity of original McAb and increase the diversity of sequence, some amino acid residues were substituted or retained by code degeneracy. According to preferential codons for E. coli, the oligonucleotides fragments from amino acids were synthesized and the ?full-length VL gene were amplified by overlap PCR. This VL gene was ligated to previously constructed reshaped anti-CD28 VH gene and then the complete VL-VH was transformed to E. coli, yielding a reshaped anti-CD28 primary antibody library. Further ELISA experiments showed the selected single-chain antibody could specifically bind CD28 antigen with higher avidity than recombinant single chain Fv.