Rational Structure-Based Design of Broad Neutralizing Humanized svMP mAbs

项目概要 在这个 R21 奖项中，我们的目标是开发一种新型且广泛的中和性人单克隆抗体，用于治疗 通过基于合理结构的设计来毒蛇咬伤，以产生更有效、更安全的下一步 一代抗蛇毒血清。 蛇毒中毒是一个严重的全球公共卫生问题，并被列为世界严重问题 LD 卫生组织列出的被忽视的热带病， 平均每年造成 125,000 人死亡 另有 40 万人永久残疾。在美国，大部分蛇中毒事件都是由 蛇科蝰蛇科成员，引起局部组织损伤（如肌坏死、水泡和局部组织损伤） 炎症和疼痛）和全身影响，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蛇毒的主要致病因子 全身出血和凝血病。目前的抗蛇毒血清是通过家畜免疫产生的， 在预防蝰蛇毒液的局部和全身影响以及预防方面效果有限 过敏反应的相关风险。我们的长期目标是开发新颖、有效的人性化 针对蝰蛇科毒液的抗蛇毒血清疗法。该项目的目的是检验以下假设： 受骆驼启发的抑制性互补位合成人类抗体，靶向医学相关的活性位点 蝰蛇毒液金属蛋白酶 (svMP) 可提供广泛的抗蛇毒血清保护，且不会与其他毒液发生交叉反应 人类金属蛋白酶并且没有过敏的风险。这一目标将通过我们的 国家蛇毒毒理学团队之间建立了互补专业知识合作 天然毒素研究中心 (NNTRC) 和加州大学抗体发现团队 河滨（UCR）。为了检验我们的假设，我们将实现以下三个具体目标。目标 1：定性 蝰蛇 svMP（Galan）出血活性的定量表征，目标 2：发现 广泛中和 svMP 特异性人单克隆抗体 (Ge)。目标 3：评估 svMP 的抗蛇毒血清功效 体外 (Ge) 和体内 (Sanchez) 抑制性单克隆抗体。拟议的研究意义重大，因为它将 增进我们对生化/细胞领域蛇毒中毒引起的出血方面的理解 水平并开发有效的人源化单克隆抗体抗蛇毒血清，该抗体将可直接用于治疗用途。 我们项目的新颖之处在于（1）开发和应用新型出血评分系统 描述 svMP 的特征； (2) 从携带新型抗体的文库中分离人源化 svMP 特异性抗蛇毒血清单克隆抗体 凸互补位； (3) 开发突破性的功能性（而不是基于结合的）HTS，以方便 发现抑制出血蛇毒素的单克隆抗体； (4) 可能改变传统的抗蛇毒血清 生产成特定的中和人源化单克隆抗体疗法。

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.