Ebaluation of the efficacy of single chain recombinant antibody fragments derived from an anti-tetanus human monoclonal antibody with high toxin-neutralizing activity as a model

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Antitoxins are essential for the treatments and passive prophylaxis for the unvaccinated humans of high risk for toxic disorders(toxi-infections and snake-bites etc.). Currently homologous human IgG antitoxin preparations with limited source of supply(hyperimmunized humans) and possible risk of viral infections are available only for tetanus, and for all other toxic disorders heterologous horse antitoxin preparations with adverse side reactions such as serum sickness. Thus human type monoclonal antitoxin antibody(MAb) from cultured hybridoma cells and more economically recombinant antitoxins produced in non-animal cells are desirable. In this study we attempted to develop recombinant anti-tetanus antitoxin as a model of a better antitoxin preparation, since tetanus antitoxins have been most well-characterized. We cloned the genes(V_H and V_K) of variable regions(H and K) of heavy chain and light chain(κ chain) of a human type anti-tetanus extremely high toxin-neutralizing MAb-G6 produc … More ed by a hybridoma cell line we established and constructed an E.coli expression system for a single-chain antibody fragment(ScFv) of MAb-G6 composed of H and K united by a liker(L): [(Gly)4-Ser]n,(n: 1〜3) in the form of H-Ln-K or K-Ln-H using a phagemid p CANTAB 5E by phage display. Under the optimal conditions the gene-products(H-L_1-K with E tag at the C-terminus for detecting by ELISA using anti-E tag) were shown for the first time to neutralize toxicity completely(rescue intoxicated mice) using our improved method of toxin-neutralization test. For higher production of more natural forms of ScFv-G6, we then established a Pichia pastoris expression system using a vector pPJC9, first for ScFv-G6 H-L_1-K (His)_6 tag and then without the tag. For better production we then constructed a Pichia pastoris system with a vector pPIC9K having a kanamycin-resistance gene by selecting G418 resistant clones with multiple copies of ScFv-G6 gene, resulting production of ScFv-G6 more than 300 times as that in E.coil system. Less