Generation Of Anti-TNF-α Multivalent Antibodies And Studies On Their In Vitro And In Vivo Bioactivities

TNF-α, originally identified in 1970s by its ability to effect hemorrhagic necrosis of specific tumors, is now known to play a major role in promoting immunologic, inflammatory and pathobiologic reactions. Antagonists including neutralizing antibodies of TNF-a are considered as useful agents for the treatment of inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis and septic shock. There are three biological agents approved by American FDA, which are Etanercept, Inflixima and Adalimumab. The latter two are monoclonal antibody. The development of therapeutic antibody has undergone the consecutive phase of mouse hybridoma technology, chimeric antibody, humanized antibody and human antibody. The genetic engineering technology developed in 1980s have pushed the generation of small weight antibodies such as Fab, scFv and VH or VL into reality. These small weight antibodies, which have an advantage over monoclonal antibodies because of its great tissue penetration due to their small size, low kidney uptake, ease to generate and functional expression in E. coli, showed very promising in clinical application. In this study, we generated an anti-TNF-α single chain Fv antibody and made a comprehensive research on it bioactivity. In order to increase the functional affinity, we oligomerized the anti-TNF-α single chain Fv antibody and further studied their in vivo and in vitro anti-TNF-α efficacy. 1. Generation and in vitro bioactivity analysis of TNF-scFvSingle chain Fv antibody is a recombinant protein by joining the VH and VL domains of an intact antibody with a flexible linker. In this work, the amino acids sequence of an anti-TNF-α single chain Fv antibody (TNF-scFv) was designed by join the VH and VL with a (GGGGS)3 linker. The whole amino acids sequence was back-translated into the DNA sequence with the prior codons of E. coli. 22 oligonucleotides were synthesized based on the DNA sequence and the PCR was performed to assemble the 22 oligonucleotides to an intact gene fragment coding for...