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

对于未接种疫苗的中毒性疾病(毒素感染和蛇咬伤等)高危人群的治疗和被动预防来说，抗毒素是必不可少的。目前，供应来源有限的同源人免疫球蛋白抗毒素制剂(高度免疫的人)和可能存在的病毒感染风险仅适用于破伤风，以及所有其他有不良反应(如血清病)的毒性疾病的异种马抗毒素制剂。因此，从培养的杂交瘤细胞中制备人型单抗和在非动物细胞中生产更经济的重组抗毒素是可取的。在这项研究中，我们试图开发重组抗破伤风抗毒素作为更好的抗毒素制剂的模型，因为破伤风抗毒素已经被很好地描述了。我们克隆了一株人型抗破伤风超强毒素中和单抗-G6的重链可变区(H)和轻链可变区(κ链)的基因(V_H和V_K)在杂交瘤细胞株的基础上，利用噬菌体p Cantab5e，建立并构建了由类似物(L)[(Gly)4-Ser]n，(n：1~3)以H-Ln-K或K-Ln-H形式结合的单链抗体片段(ScFv)的表达系统。在优化的条件下，用我们改进的毒素中和试验方法首次证明了C端带有E标签的H-L_1-K基因产物能够完全中和毒性(解救中毒小鼠)。为了获得更多自然形式的ScFv-G6，我们利用载体pPJC9建立了毕赤酵母表达系统，首先利用ScFv-G6H-L_1-K(His)_6标签，然后不使用标签。为了更好地生产，我们用含有卡那霉素抗性基因的载体pPIC9K构建了毕赤酵母系统，通过选择带有多拷贝scFv-G6基因的G418抗性克隆，使scFv-G6的产量是E.col系统的300多倍。较少

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