School of Environmental Sciences, University of Guelph, Guelph, Ontario, Canada.
PLoS One. 2013 Jul 22;8(7):e69495. doi: 10.1371/journal.pone.0069495. Print 2013.
Small recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab')2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α-Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α-Cbtx. Mouse α-Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α-Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy.
小分子重组抗体片段（例如 scFv 和 VHH）具有高度的组织通透性，正被研究用于抗蛇毒血清的生产，因为由 IgG 或 F(ab')2 抗体片段组成的传统抗蛇毒血清不能有效中和位于深部组织的毒液毒素。然而，完全由小分子抗体片段组成的抗蛇毒血清可能由于其血清半衰期短而疗效不佳。为了增加血清半衰期并保持组织穿透性，我们制备了低和高分子质量的抗蛇毒血清抗体。从免疫 VHH 展示噬菌体文库中分离出 4 种骆驼科 VHH，它们对α-眼镜蛇毒素（α-Cbtx）具有高亲和力，亲和力在低 nM 范围内，α-Cbtx 是眼镜蛇属（Naja kaouthia）毒液中最致命的成分。随后，我们将最高亲和力的 VHH（C2）与人 Fc 片段融合，创造了一种 VHH2-Fc 抗体，可提供延长的血清半衰期。在植物体内（Nicotiana benthamiana）表达和纯化后，我们表明我们的 VHH2-Fc 抗体保留了对α-Cbtx 的高亲和力结合。对小鼠α-Cbtx 攻击的研究表明，我们的最高亲和力 VHH（C2 和 C20）和 VHH2-Fc 抗体在抗体：毒素摩尔比低至约 0.75×：1 时，有效地中和了由α-Cbtx 引起的致死性。进一步研究开发涉及这些抗α-Cbtx VHH 和 VHH2-Fc 抗体分子的抗蛇毒血清治疗方法应涉及将它们作为组合进行测试，以确定它们是否保持组织穿透能力和低免疫原性，以及它们是否表现出改善的血清半衰期和治疗效果。
