Identification of Lamprey Antibodies Capable of Noninvasive Brain Drug Delivery

能够无创脑部药物输送的七鳃鳗抗体的鉴定

• 批准号: 9920222
• 负责人: ERIC V SHUSTA
• 金额: $ 33.08万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-15 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9920222
• 关键词: Acquired Immunodeficiency Syndrome; Affinity; Alzheimer' s Disease; American; Antibodies; Antibody Specificity; Antibody-drug conjugates; Antigen Receptors; Binding; Biodistribution; Biological Products; Blood; Blood - brain barrier anatomy; Blood Circulation; Brain; Brain-Derived Neurotrophic Factor; Carbohydrates; Cell membrane; Cerebrum; Characteristics; Clinical; Complementary DNA; Coupling; Data; Development; Diffuse; Disease model; Dose; Drug Delivery Systems; Drug Kinetics; Drug Transport; Endothelium; Exhibits; Family; Fc Receptor; Gene Proteins; Generations; Genes; Goals; Hagfish; Human; Immune; Immune system; Immunization; Immunize; Immunoglobulin G; Immunoprecipitation; Insulin; Lampreys; Lead; Leucine-Rich Repeat; Libraries; Link; Liposomes; Low Density Lipoprotein Receptor; Low-Density Lipoproteins; Lymphocyte; Malignant neoplasm of brain; Mammals; Mediating; Medicine; Mining; Mus; Oryctolagus cuniculus; Parkinson Disease; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacology; Pharmacotherapy; Polysaccharides; Preparation; Proteins; Reagent; Research; Route; Side; Specificity; Stains; Stroke; Structure; Surface; System; Techniques; Technology; Therapeutic; Transferrin; Translating; Translations; Validation; Vertebrates; Yeasts; base; blood-brain barrier permeabilization; brain tissue; cerebral microvasculature; dalton; drug candidate; drug development; high-throughput drug screening; immunogenic; improved; in vivo; innovation; intravenous administration; intravenous injection; lipophilicity; member; nanoparticle; nervous system disorder; novel; novel therapeutics; pharmacokinetic model; programs; receptor; response; screening; small molecule; small molecule therapeutics; stroke model; tandem mass spectrometry; targeted delivery; therapeutic DNA; trafficking; transcytosis; uptake; vector

ABSTRACT Millions of people worldwide suffer from neurological diseases such as Alzheimer's disease, stroke, and brain cancer. Advances in protein/gene profiling techniques and high throughput drug screening technologies have spawned many new drug candidates. However, the blood-brain barrier (BBB) has impeded the development and clinical realization of this new generation of neurotherapeutics by restricting the brain uptake of most small molecule therapeutics, and prohibiting brain uptake of protein- and gene-based medicines. A promising noninvasive brain delivery strategy takes advantage of endogenous BBB transport mechanisms as a means to shuttle drug cargo from the blood to the brain. Such receptor-mediated transport systems can be targeted using the exquisite specificity of antibodies that are in turn linked to a drug payload that can include small molecules, proteins, or DNA therapeutics. After binding to the receptor on the blood side, the antibody- drug conjugate acts as an artificial substrate for the transporter and is transcytosed from the blood, across the BBB, and into the brain. Current approaches have yielded limited brain uptake because the targeted transporters are ubiquitously expressed, and the antibody targeting reagents have a low BBB permeability. Therefore, this proposal is focused on the identification and validation of novel delivery vectors and their cognate BBB transporters that can mediate improved transport efficiency. Rather than deploying traditional mammalian antibody technology, we describe a new strategy that employs lamprey antibodies known as Variable Lymphocyte Receptors (VLRs) to target the BBB. Lampreys and humans last shared a common ancestor >500 million years ago, and due to this tremendous evolutionary divergence, even highly conserved mammalian proteins and carbohydrates are immunogenic in lampreys. By leveraging these unique aspects of the lamprey immune system with innovative screening technologies, we anticipate that the proposed research will provide new BBB-targeting VLRs capable of trafficking into the brain. To achieve these goals, lampreys were immunized with mouse brain microvessel plasma membrane preparations, and staining with the resultant polyclonal antiserum demonstrated that VLRs clearly recognize the in vivo BBB and bind to multiple unique glycan structures. The lymphocyte cDNA of immunized lampreys was then used to create a yeast display library consisting of millions of VLRs that will be screened using an innovative screening approach to select BBB-binding and trafficking monoclonal VLRs. These brain-targeting VLRs will be validated both by pharmacokinetic profiling and their capacity to elicit a pharmacologically- relevant response in a murine stroke model. Finally, the BBB-targeting VLRs will be employed to identify the cognate BBB transporter and any associated glycoforms. Those VLRs exhibiting significant and specific brain uptake would represent new, noninvasive brain drug delivery vectors that could be powerful in the treatment of debilitating neurological disease.

抽象的 全世界有数百万人患有神经系统疾病，例如阿尔茨海默氏病，中风和 脑癌。蛋白质/基因分析技术和高通量药物筛查技术的进步 已经产生了许多新毒品。但是，血脑屏障（BBB）阻碍了 通过限制大脑的吸收，这种新一代神经疗法的开发和临床实现 在大多数小分子疗法中，并禁止脑摄取蛋白质和基因基因药物。一个 有希望的无创脑输送策略利用内源性BBB传输机制作为一种 意味着从血液到大脑的毒品货物。这种受体介导的传输系统可以是 使用抗体的精致特异性针对的，而抗体又与药物有效载荷相关联 小分子，蛋白质或DNA疗法。与血侧的受体结合后，抗体 - 药物共轭物充当转运蛋白的人造底物，并从血液中转移，跨过 BBB，进入大脑。目前的方法产生了有限的脑部吸收 转运蛋白无处不在，靶向试剂的抗体渗透性较低。 因此，该提案的重点是对新型交付向量的识别和验证以及 他们的同源BBB转运蛋白可以介导提高运输效率。而不是部署 传统的哺乳动物抗体技术，我们描述了一种采用七lamp鼠抗体的新策略 称为可变淋巴细胞受体（VLR），以靶向BBB。七lamp和人类最后分享了 共同的祖先> 5亿年前，由于这种巨大的进化差异，甚至很高 保守的哺乳动物蛋白和碳水化合物在七lamp虫中具有免疫原性。通过利用这些独特的 具有创新筛查技术的七lamp带免疫系统的各个方面，我们预计 拟议的研究将为您提供涉及BBB的新型VLR，能够投入到大脑中。 为了实现这些目标，用小鼠脑微血管质膜免疫七lamp鼠 制剂，并用最终的多克隆抗血清染色表明VLR清楚地识别 体内BBB并与多个独特的聚糖结构结合。免疫七lamp鼠的淋巴细胞cDNA 然后被用于创建一个由数百万VLR组成的酵母显示库，该库将使用 创新的筛选方法选择BBB结合和贩运单克隆VLR。这些靶向大脑 VLR将通过药代动力学分析及其引起药理学的能力来验证 在鼠卒中模型中的相关响应。最后，将采用针对BBB的VLR来识别 同源BBB转运蛋白和任何相关的糖型。那些表现出重要大脑的VLR 摄取将代表新的无创脑药物输送媒介 神经系统疾病。