Rational Structure-Based Design of Broad Neutralizing Humanized svMP mAbs

项目摘要 在这个R21奖项中，我们的目标是开发一种新的和广泛的中和人单克隆抗体，用于治疗 毒蛇咬伤毒液通过合理的结构设计，以产生更有效，更安全的下 代抗蛇毒血清。 蛇毒中毒是一个严重的全球性公共卫生问题，并被列为世界卫生组织（WHO） LD 世界卫生组织的被忽视的热带病名单， 平均每年有12.5万人死亡， 造成另外40万人终身残疾。在美国，大多数的蛇毒液中毒是由 毒蛇科的成员，引起局部组织损伤（如肌坏死、水泡和局部 炎症和疼痛）和全身效应，包括出血和凝血病，这可能导致 休克、肾衰竭和死亡。蛇毒金属蛋白酶是自发性 全身出血和凝血功能障碍。目前的抗蛇毒血清是通过对家畜进行免疫接种而生产的， 在预防蝰蛇科毒液蛰入的局部和全身效应方面的效力有限， 超敏反应的相关风险。我们的长期目标是开发新颖、有效的人性化 毒蛇科毒液蛰入的抗蛇毒血清疗法。这个项目的目的是检验假设， 骆驼启发的抑制性互补位合成人抗体靶向医学相关的活性位点 蝰蛇毒金属蛋白酶（svMPs）可以提供广泛的抗蛇毒保护，而不与 人金属蛋白酶，而没有超敏反应的风险。这一目标将通过我们的 建立了蛇毒毒理学团队之间的互补专业知识的合作， 天然毒素研究中心（NNTRC）和加州大学的抗体发现小组 滨江（UCR）.为了验证我们的假设，我们将讨论以下三个具体目标。目标1：定性 蝰蛇svMP（Galan）的出血活性的定量表征，目的2：发现 广泛中和svMP特异性人mAb（Ge）。目的3：评价svMP抗蛇毒血清的效力 体外（Ge）和体内（Sanchez）的抑制性mAb。这项研究意义重大，因为它将 在生化/细胞水平上推进我们对蛇毒液蛰入引起的出血方面的理解 水平，并开发有效的人源化mAb抗蛇毒血清，这将是直接翻译用于治疗用途。 本课题的创新点是：（1）开发和应用一种新的出血评分系统， 表征svMP;（2）从携带新型抗蛇毒血清的文库中分离人源化svMP特异性抗蛇毒mAb， 凸互补位;（3）开发突破性的功能（而不是基于绑定的）HTS， 发现单克隆抗体抑制出血性蛇毒;（4）潜在地改变传统的抗蛇毒血清 制备成特异性中和人源化mAb治疗剂。

PROJECT SUMMARY In this R21 award, we aim to develop a novel and broad neutralizing human monoclonal antibody for treating snakebite envenoming by rational structure-based design in order to produce a more effective and safer next generation antivenom. Snake envenomation is a serious global public health concern and ranked on the Wor ld Health Organization’s list of neglected tropical diseases, killing on average 125,000 people per year and leaving another 400,000 permanently disabled. The majority of snake envenomation in the US, inflicted by members of the snake family Viperidae, causes local tissue damage (such as myonecrosis, blisters, and local inflammation and pain) and systemic effects, including hemorrhage and coagulopathies which can lead to shock, renal failure and death. Snake venom metalloproteinases are major causative agents for spontaneous systemic bleeding and coagulopathies. Current antivenoms, produced by immunization of domestic animals, have limited efficacy in the prevention of both local and systemic effects of Viperidae envenomation as well as an associated risk of hypersensitivity reactions. Our long-term goal is to develop novel, effective humanized antivenom therapeutics for Viperidae envenomation. The objective of this project is to test the hypothesis that camelid-inspired inhibitory paratope synthetic human antibodies targeted to the active site of medically-relevant viperid venom metalloproteinases (svMPs) can provide broad antivenom protection without cross-reaction with human metalloproteinases and without the risk of hypersensitivity. This objective will be addressed through our established collaboration of complementary expertise between the snake venom toxinology team at National Natural Toxins Research Center (NNTRC) and the antibody discovery team at University of California Riverside (UCR). To test our hypothesis, we will address the following three Specific Aims. Aim 1: Qualitative and Quantitative Characterization of the hemorrhagic activity of viperid svMPs (Galan), Aim 2: Discovery of Broadly Neutralizing svMP-Specific Human mAbs (Ge). Aim 3: Evaluation of the antivenom efficacy of svMP inhibitory mAbs in vitro (Ge) and in vivo (Sanchez). The proposed research is significant because it will advance our understanding of the hemorrhagic aspects caused by snake envenomation at biochemical/cellular levels and develop effective humanized mAb antivenoms, which will be directly translatable for therapeutic use. The novelties of our project are (1) development and application of a novel Hemorrhage Score system to characterize svMPs; (2) isolation of humanized svMP-specific antivenom mAbs from libraries carrying novel convex paratopes; (3) development groundbreaking functional (rather than binding-based) HTS for facile discovery of mAbs inhibiting hemorrhagic snake toxins; and (4) potentially shifting the conventional antivenom production into specific neutralizing humanized mAb therapeutics.