Stepwise Engineering of Heterodimeric Single Domain Camelid VHH Antibodies That Passively Protect Mice from Ricin Toxin
Stepwise Engineering of Heterodimeric Single Domain Camelid VHH Antibodies That Passively Protect Mice from Ricin Toxin
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DOI：
10.1074/jbc.m113.519207
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发表时间：
2013-12-20
影响因子：
4.8
通讯作者：
Shoemaker, Charles B.
中科院分区：
文献类型：
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作者：
Vance, David J.;Tremblay, Jacqueline M.;Shoemaker, Charles B.
背景：我们试图设计高度有效的药物来中和蓖麻毒素。结果如下：我们鉴定了能够在体外中和蓖麻毒素的单体单链骆驼科动物V-H结构域（V（H）H）和在体内中和蓖麻毒素的工程异二聚体V（H）H。结论：V（H）Hs的逐步工程化产生了高度有效的蓖麻毒素中和抗体。重要性：本研究强调了VHH平台作为针对不同生物毒素的治疗策略的潜在用途。为了设计针对B类毒素蓖麻毒素的对策，我们产生并表征了一系列表位标记的仅重链抗体V-H结构域（V（H）H），其特异于蓖麻毒素酶（RTA）和结合（RT B）亚基。在我们鉴定的20种独特的蓖麻毒素特异性V（H）H中，6种具有毒素中和活性：5种特异于RTA，1种特异于RTB。三个中和RTA特异性V（H）H各自通过短肽间隔子连接至唯一的中和抗RTB VHH以产生VHH异二聚体。与等摩尔浓度的它们各自的单价单体相比，所有三种VHH异二聚体对蓖麻毒素具有更高的亲和力，并且在异二聚体D10/B7的情况下，体外毒素中和活性增加6倍。当以4：1的异源二聚体：毒素比例被动给予小鼠时，D10/B7对10 x LD 50蓖麻毒素攻击的应答赋予100%的存活率，而2：1的异源二聚体：毒素比例赋予20%的存活率。然而，当低剂量的D10/B7与IgG 1抗表位标签单克隆抗体组合时，可以实现完全存活，这可能是因为用多达四个IgG修饰毒素促进血清清除。另外两种蓖麻毒素特异性异源二聚体，当在体内测试时，提供了与D10/B7相等或更大的被动保护，从而避免了对所有三种异源二聚体作为可能的治疗剂的进一步研究。
Background: We sought to engineer highly efficacious agents that neutralize ricin toxin. Results: We identified monomeric single-chain camelid V-H domains (V(H)Hs) capable of neutralizing ricin in vitro and engineered heterodimeric V(H)Hs that neutralized ricin in vivo. Conclusion: Stepwise engineering of V(H)Hs resulted in highly potent ricin toxin-neutralizing antibodies. Significance: This study highlights the potential use of a VHH platform as a strategy for therapeutics against diverse biological toxins.In an effort to engineer countermeasures for the category B toxin ricin, we produced and characterized a collection of epitopic tagged, heavy chain-only antibody V-H domains (V(H)Hs) specific for the ricin enzymatic (RTA) and binding (RTB) subunits. Among the 20 unique ricin-specific V(H)Hs we identified, six had toxin-neutralizing activity: five specific for RTA and one specific for RTB. Three neutralizing RTA-specific V(H)Hs were each linked via a short peptide spacer to the sole neutralizing anti-RTB VHH to create VHH heterodimers. As compared with equimolar concentrations of their respective monovalent monomers, all three VHH heterodimers had higher affinities for ricin and, in the case of heterodimer D10/B7, a 6-fold increase in in vitro toxin-neutralizing activity. When passively administered to mice at a 4:1 heterodimer:toxin ratio, D10/B7 conferred 100% survival in response to a 10 x LD50 ricin challenge, whereas a 2:1 heterodimer:toxin ratio conferred 20% survival. However, complete survival was achievable when the low dose of D10/B7 was combined with an IgG1 anti-epitopic tag monoclonal antibody, possibly because decorating the toxin with up to four IgGs promoted serum clearance. The two additional ricin-specific heterodimers, when tested in vivo, provided equal or greater passive protection than D10/B7, thereby warranting further investigation of all three heterodimers as possible therapeutics.