Screening Human Antibodies Against HCC With Guided-selection And Phage Antibody Library Techniques

Primary hepatic cell cancer (HCC) is one of the malignant tumors threatening the human. About 130 thousands patients dead of HCC every year in China,. The development of gene-engineering antibody provides a new method for the diagnosis and treatment of HCC. Now about 80 McAbs have enter the clinic trials. HAb18, the mouse anti-HCC McAb, have been complete the clinic trial with better prospect for the diagnosis and treatment. But as mouse McAb, HAMA(human anti-mouse antibody reaction) can be induced. Chimeric HAbl8-Fab with high humanization level still can induce significant HAMA.The humanization of McAb can be completed by a set of techniques. Antibody library displayed on phage surface was a new technique developed in the last ten years. The technology of phage displaying antibody library allows much more rapid selection of specific antibodies from large libraries. The phage display antibody library provides a powerful methods for the production of gene-engineering McAb. Here the complete human anti-HAb18G Fabs were selected with guided-selectionand phage displaying antibody library techniques.1.The optimization of the primers for the construction of human Fabantibody library.The construction of a large human phage antibody library with a good diversity is based on the amplification of all the human antibody genes.According to the human germ-line V-genes in the V-BASE, the germ-line V-genes are divided into different groups. Based on these, the specific 5' primers are designed and optimized. All the germ-line V-genes are aligned with the 5' primers. With the match rate, the amplification efficiency is analyzed. The human Fd genes were amplified by RT-PCR from PBMC of hepatoma patients. Then with the primers, we amplified the genes of Fd and light chains, and the genes of PCR products were sequenced. In the study, we designed a set of specific 5 primers, including 5 primers for the Fd genes, 6 primers for the kappa chain and 10 primers for lamdda chain. All the primers have high match rate with the germ-line V-genes with a limited primer number, specially in the 3'regions of the primers. The results of PCR showed all primer-pairs could be used to amplify the interested antibody genes. Genes sequencing confirmed most interested genes could be amplified. The set of optimized family-specific primers can amplify the human genes of the Fd and light chains with a high efficiency. These will be helpful for the construction of human Fab antibody libraries. 2. The optimized strategy for the amplifying and screening of human-mouse phage Fab antibody library.Based on the chimeric cFab-HAb18, the PCR products were cloned in the vectors pComb3 and pComb3X with the chimeric light chain anti-FLAb18G to construct the human-mouse Fab phage antibody libraries. In the study, the chimeric CL genes were paired as template with a repertoire of Fd chains, forming phage antibody library of human-mouse heterozygosis. With the GST fusion and no-fusion extracellular region of HAb18G, a cancer associated antigen, the target antibodies were selected. During the course, the strategy were optimized to increase the efficiency. When the host bacteria XLl-Blu was cultivated at 37 C, the clones could not cut out the correct fragments with the restriction endonucleases. It indicates the recombinant-deletion occurs. At 30C, about 17% of the clones had the phenomenon. GST-fusion HAb18GE had low efficacy because of the cross-action. In the study, the phage ELISA cannot be used the positiveclones selection because the serious cross-reaction. But the lyses supernatant after induced with IPTG could identify positive clones with ELISA. Through the construction of the HuMFab phage antibody library and the optimized screening strategy, we get the available procedures for subsequent research.3. The selection of human anti-hepatoma Fd fragments guided with the chimeric light chain.With the constructed human-mouse Fab phage library, HAbl8GE, extracellular region of HAb18G, were used as antigen to screening. The positive clones were ident...