Recombinant antibodies against Iranian cobra venom as a new emerging therapy by phage display technology

Abstract Background: The production of antivenom from immunized animals is an established treatment for snakebites; however, antibody phage display technology may have the capacity to delivery results more quickly and with a better match to local need. Naja oxiana, the Iranian cobra, is a medically important species, responsible for a significant number of deaths annually. This study was designed as proof of principle to determine whether recombinant antibodies with the capacity to neutralize cobra venom could be isolated by phage display. Methods: Toxic fractions from cobra venom were prepared by chromatography and used as targets in phage display to isolate recombinant antibodies from a human scFv library. Candidate antibodies were expressed in E. coli HB2151 and purified by IMAC chromatography. The selected clones were analyzed in in vivo and in vitro experiments. Results: Venom toxicity was contained in two fractions. Around a hundred phage clones were isolated against each fraction, those showing the best promise were G12F3 and G1F4. While all chosen clones showed low but detectable neutralizing effect against Naja oxiana venom, clone G12F3 could inhibit PLA2 activity. Conclusion: Therefore, phage display is believed to have a good potential as an approach to the development of snake antivenom.研究背景：从免疫动物体内制备抗蛇毒血清是治疗蛇咬伤的成熟治疗方案，但抗体噬菌体展示（phage display）技术或可实现更快速的研发，并更好地适配本地临床需求。伊朗眼镜蛇（Naja oxiana）是具有重要医学意义的蛇类物种，每年造成大量人员死亡。本研究作为原理验证性实验，旨在探究能否通过噬菌体展示技术分离得到可中和眼镜蛇毒液的重组抗体。 实验方法：通过色谱法分离得到眼镜蛇毒液的毒性组分，并将其作为靶标用于噬菌体展示筛选，从人类单链可变片段（scFv, single-chain variable fragment）噬菌体展示库中分离重组抗体。候选抗体在大肠杆菌HB2151（E. coli HB2151）中进行表达，并通过固定化金属亲和色谱（IMAC, Immobilized Metal Ion Affinity Chromatography）进行纯化。对筛选得到的克隆开展体内及体外实验分析。 实验结果：眼镜蛇毒液的毒性集中于两个组分。针对每个组分分别分离得到约百个噬菌体克隆，其中最具应用潜力的克隆为G12F3与G1F4。所有筛选得到的克隆均对伊朗眼镜蛇毒液表现出低水平但可检测到的中和活性，其中G12F3克隆可抑制磷脂酶A2（PLA2, Phospholipase A2）活性。 研究结论：综上，噬菌体展示技术在抗蛇毒血清研发领域具备良好的应用潜力。