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

以具有高毒素中和活性的抗破伤风人单克隆抗体衍生的单链重组抗体片段作为模型的功效评价

• 批准号: 14570249
• 负责人: MATSUDA Morihiro
• 金额: $ 2.56万
• 依托单位: Koshien University, College of Nutition
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570249/
• 关键词: single-chain antibody fragments; anti-tetanus antitoxin antibody; human monoclonal antibody; neutralizing antibody

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

抗毒素是治疗和被动预防未接种疫苗的人的高风险的毒性疾病（蜱虫感染和蛇咬伤等）所必需的。目前同源人IgG抗毒素制剂的供应来源有限（超免疫人），可能存在病毒感染的风险，仅可用于破伤风，而对于所有其他毒性疾病，异源马抗毒素制剂具有不良副反应，如血清病。因此，需要从培养的杂交瘤细胞中获得人型单克隆抗毒素抗体（MAb）和在非动物细胞中产生的更经济的重组抗毒素。在这项研究中，我们试图开发重组抗破伤风抗毒素作为一个更好的抗毒素制剂的模型，因为破伤风抗毒素已被最充分的特点。我们克隆了人型抗破伤风极高毒力中和单抗G6的重链可变区（H）和轻链可变区（K）的基因（V_H和V_K），并将其克隆到大肠杆菌BL 21（DE 3）中。 ...更多信息 我们以杂交瘤细胞系为基础，利用噬菌粒p CANTAB 5 E，通过噬菌体展示技术，建立并构建了由H和K通过连接物（L）：[（Gly）4-Ser]n（n：1〜3）以H-Ln-K或K-Ln-H形式组成的MAb-G6单链抗体片段（ScFv）的大肠杆菌表达系统。艾德。在优化的条件下，用我们改进的毒素中和试验方法，首次证明了基因产物（C末端带有E标签的H-L_1-K，用抗E标签的ELISA检测）能完全中和毒素（拯救中毒小鼠）。为了提高ScFv-G6的产量，我们利用毕赤酵母表达载体pPJC 9，首先将ScFv-G6的H-L_1-K（His）_6标签转化到毕赤酵母中，然后将其去除。为了提高ScFv-G6的产量，我们利用卡那霉素抗性载体pPIC 9 K构建了毕赤酵母表达系统，筛选出多拷贝ScFv-G6基因的G418抗性克隆，使ScFv-G6的产量比大肠杆菌表达系统提高了300倍以上。少

项目成果

Matsuda, M., Nonaka, K., Horiguchi, Y.: "Expression in E.coli of anti-tetanus human single-chain antibody fragments with toxin-neutralizing activity"Jpn.J.Bacteriol. (Nihon Saikingaku Zasshi) (in Japanese). 57(1). 96 (2002)

Matsuda，M.，Nonaka，K.，Horiguchi，Y.：“具有毒素中和活性的抗破伤风人单链抗体片段在大肠杆菌中的表达”Jpn.J.Bacteriol。

Sugimoto, N., Matsuda, M.: "9.Motor neurons, In Site-selective neurotoxicity(D.Lester, W.Slikker Jr. & P.Lazarovici eds.), Cellular & Molecular Mechanisms of Toxin Action vol.3"Taylor & Francis Books Ltd, London & New York. 13/308 (2002)

Sugimoto, N., Matsuda, M.：“9.运动神经元，位点选择性神经毒性（D.Lester，W.Slikker Jr.）

松田守弘: "広川タンパク質化学 第1巻「毒素タンパクII」破傷風毒素(林恭三 編)"広川書店. 21/201 (2003)

松田盛弘：“广川蛋白质化学第 1 卷‘毒素蛋白质 II’破伤风毒素（林恭三编辑）”广川书店 21/201（2003 年）。

松田守弘: "Clostridium tetani,「細菌学」(竹田美文・林英生 編)"朝倉書店. 706(13) (2002)

Morihiro Matsuda：“破伤风梭菌，‘细菌学’（由竹田义文和林秀夫编辑）”朝仓书店 706(13) (2002)。

Okada, Y., Miyake, M., Oka, K., Horiguchi, Y., Matsuda, M.: "Establishment of an expression system in Pichia pastries for anti single-chain antibody fragments (ScFv-G6) an anti-tetanus monoclonal antibody with high toxin-neutralizing activity"Jpn.J.Bacter

Okada, Y.、Miyake, M.、Oka, K.、Horiguchi, Y.、Matsuda, M.：“在毕赤酵母糕点中建立抗单链抗体片段 (ScFv-G6) 和抗破伤风表达系统