Preparation Of Human Antibody Fab’ Fragment Against Tumor Necrosis Factor-α By Phage Surface Display Technology

Tumor necrosis factor-alpha （TNF-alpha） is a kind of cytokines playing a keyrole in the pathological changes of rheumatoid arthritis （RA） and other diseases, andclinical trial results confirmed that TNF-alpha is the appropriate therapeutic target ofsuch diseases. The antibodies of TNF-alpha which were used in the treatment of RAhave achieved good results, such as Enbrel, Remicade, and Humira. With thelaunching of clinical trials, new indications of TNF-alpha antibodies were continuallyreported, and in the meantime the market space was expanded. These drugs with highspecificity, good target activities and lower side effects than other similar drugs, havegradually become the first-line treatment in the treatment of RA together withmethotrexate （MTX）. Although existing TNF antibody drugs have many advantages,they also have different drawbacks. First, Remicade is a chimeric antibody containing25%of the murine fragment, and long-term treatment may give rise to humananti-mouse antibodies （HAMA） response. Second, the Fc fragment of these antibodiescan activate complements and ADCC, which will lead to apoptosis of cells thatexpress TNF and a series of side-effects. Therefore, development of a small moleculefully human TNF-α antibody has special significances.Firstly, the antigen of TNF-alpha was expressed and purified. According to thesequence of human TNF-α in GenBank, the TNF-α gene could be created throughchemical synthesis, then the gene was extracted to construct expression vectorpET-23b/TNF-α of TNF-α and was transformed into E. coli BL21（DE3）, and thenpositive clones were obtained. After positive clones were induced at37°C for16h,soluble TNF-α was secreted into the supernatant of the fermentation broth, and thenthe target protein was purified. The purity of TNF-α is greater than95%, the specific activity of4.4×10⁶U/mg, and the yield of4mg/L.Secondly, isolates are screened from antibody phage-display. We usedself-prepared TNF-α as the antigen to screen isolates from antibody phage-display, inorder to obtain the TNF-α antibody clone which has specific affinity. After six timesof solidoid panning, the phage antibody clones that bound TNF-α specifically rosefrom1.1×10³pfu to2×10⁵pfu, and the ratio for input and output increased from3.2×10^-8to1.2×10^-6, and we finally got a200-fold enrichment although the screeningrounds increased and the elution conditions became more strict.200clones wereselected randomly from the sixth round of panning for ELISA screening, and fivepositive clones were obtained. Then the antigen binding specificities of the five cloneswere compared, finally a most TNF-alpha-specific clone was determined as G11.Thirdly, anti-TNF-α Fab antibody was expressed and purified. G11clone wasscreened and sequenced to obtain gene sequences to build Fab antibody expressionvector. When we got positive clones, we compared the influence of the medium andinduction time to optimize the expression conditions. The results showed that usingLB medium at30℃,0.1mM IPTG induction for24h, the expression level of Fabantibody reached the highest. A comparison was made among bacteria disruptionmethods, and finally we chose repeated freezing and thawing method from ultrasound,osmotic shock, lysozyme methods for cell disruption. The purity of purified Fabantibody protein was nearly100%and non-competitive ELISA showed that affinityconstant of the antibody and TNF-α was Ka=4×10-13M-1. In addition, neutralizationtest in vitro showed that in a certain extent, Fab antibody could neutralize the TNF-αkilling ability on L929cells. When the concentration of the Fab antibody was3.2ng/ml, the rate of neutralization of TNF-alpha cytotoxicity was85%. When theconcentration was800pg/ml, the rate was48%.In this study, TNF-α that worked as an antigen was expressed in prokaryotic cells.TNF-α antibody was selected from the phage antibody library screening and its Fabwas transformed and, ultimately, fully human TNF-alpha-derived Fab antibody with acertain biological activity was obtained, which laid a good foundation for thetreatment of TNF-alpha related diseases.