Plant-Made Nanobodies for Botulism Treatment

用于肉毒杆菌中毒治疗的植物制造纳米抗体

• 批准号: 8058704
• 负责人: KEITH WYCOFF
• 金额: $ 23.68万
• 依托单位: PLANET BIOTECHNOLOGY, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-25 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8058704
• 关键词: Affinity; Amino Acid Sequence; Antibodies; Antitoxins; Binding; Biological; Biological Assay; Biotechnology; Bontoxilysin; Botulinum Toxins; Botulism; Catalytic Domain; Cell membrane; Cells; Characteristics; Chimera organism; Cleaved cell; Cultured Cells; Cytosol; Dose; Endosomes; Equus caballus; Event; Excision; Genes; Goals; Grant; Human; Immunoglobulin Fragments; Immunotherapy; In Vitro; Injectable; Label; Laboratories; Libraries; Light; Llama; Mechanical Ventilators; Metalloproteases; Monoclonal Antibodies; Motor Neurons; Mus; N-terminal; Nerve; Neurons; Paralysed; Peptides; Phase; Planets; Plants; Poisoning; Proteins; Rattus; Recombinant Proteins; Recombinants; Research; Research Support; S-nitro-N-acetylpenicillamine; Serotyping; Spinal Cord; Supportive care; Symptoms; System; Technology; Testing; Therapeutic; Toxic effect; Toxicity Tests; Toxin; Work; base; botulism immune globulin; effective therapy; fluorophore; in vivo; neurotransmission; protein aminoacid sequence; public health priorities; research study; success; uptake

DESCRIPTION (provided by applicant): The ultimate goal of the research described in this application is to develop an economical, safe and effective therapeutic that can reverse the paralytic symptoms of botulism. Success in achieving this goal could save countless lives in the event of a bioterrorist attack by botulinum toxin. The goal of administering an anti-toxin antibody is rapid and complete removal of the toxin from the body. Currently available immunotherapies for botulism (equine antitoxin, human botulism immune globulin, monoclonal antibodies) can remove only toxin molecules that have not yet reached motor neurons. Once the catalytic light chain of the toxin reaches the cytosol of motor neurons, where it cleaves proteins involved in neurotransmission, it is inaccessible to normal antibodies. This is why victims of botulinum neurotoxin poisoning may need supportive care on a respirator for many weeks. We propose using a new single-chain variable fragment, a nanobody, against the catalytic site of the toxin light chain, along with technology that allows proteins to penetrate into the cytosol of motor neurons, to reverse the paralysis caused by the toxin. We will start with a single-domain variable fragment, isolated from a llama antibody library in the laboratory of our collaborator, Dr James Marks (UCSF), that inhibits the ability of the toxin light chain to cleave it's protein substrate. We will express fusions of this nanobody to various cell-penetrating peptides, using a plant expression system. We will then test the ability of these nanobody fusions to enter primary rat neuron cells and inhibit toxin activity. Finally, we will test the toxicity of the cell-penetrating nanobody in mice. PUBLIC HEALTH RELEVANCE: Botulinum toxin is the deadliest biological substance known, and would be easy for a terrorist to produce and use to cause large numbers of casualties. This makes developing an effective and economical countermeasure to protect the public a public health priority. The research supported by this grant will lay the groundwork for using plants to produce a highly protective, safe and economical antitoxin therapy.

描述（由申请人提供）：本申请中描述的研究的最终目标是开发一种经济，安全和有效的治疗方法，可以逆转肉毒杆菌中毒的麻痹症状。如果成功实现这一目标，就可以在肉毒杆菌毒素生物恐怖袭击中挽救无数生命。施用抗毒素抗体的目的是快速且完全地从体内去除毒素。目前可用的肉毒中毒免疫疗法（马抗毒素，人肉毒中毒免疫球蛋白，单克隆抗体）只能清除尚未到达运动神经元的毒素分子。一旦毒素的催化轻链到达运动神经元的胞质溶胶，在那里它切割参与神经传递的蛋白质，正常抗体就无法接近它。这就是为什么肉毒杆菌神经毒素中毒的受害者可能需要在呼吸器上进行数周的支持性护理。我们建议使用一种新的单链可变片段，一种纳米抗体，对抗毒素轻链的催化位点，沿着技术，使蛋白质渗透到运动神经元的胞质中，以逆转毒素引起的瘫痪。我们将从我们的合作者James Marks博士（UCSF）实验室的美洲驼抗体库中分离出的单域可变片段开始，该片段抑制毒素轻链切割其蛋白质底物的能力。我们将使用植物表达系统表达这种纳米抗体与各种细胞穿透肽的融合体。然后，我们将测试这些纳米抗体融合物进入原代大鼠神经元细胞并抑制毒素活性的能力。最后，我们将在小鼠中测试细胞穿透纳米抗体的毒性。 公共卫生关系：肉毒杆菌毒素是已知的最致命的生物物质，恐怖分子很容易生产和使用，造成大量伤亡。这使得制定有效和经济的对策来保护公众成为公共卫生的优先事项。这项资助支持的研究将为利用植物生产高度保护性，安全和经济的抗毒素疗法奠定基础。