Identification of Lamprey Antibodies Capable of Noninvasive Brain Drug Delivery

摘要 全世界数以百万计的人患有阿尔茨海默氏症、中风和 脑癌。蛋白质/基因图谱技术和高通量药物筛选技术的研究进展 催生了许多新的候选药物。然而，血脑屏障(BBB)阻碍了 限制脑摄取这一新一代神经疗法的发展和临床应用 大多数小分子疗法，并禁止大脑摄取基于蛋白质和基因的药物。一个 有希望的无创脑递送策略利用内源性血脑屏障转运机制作为一种 意味着将毒品货物从血液输送到大脑。这种受体介导的运输系统可以是 利用抗体的精致特异性进行靶向，这些抗体又与药物有效载荷相联系，包括 小分子、蛋白质或DNA疗法。在与血侧的受体结合后，抗体- 药物结合物充当转运体的人工底物，从血液中转运，穿过 血脑屏障，进入大脑。目前的方法已经产生了有限的大脑吸收，因为靶向 转运蛋白广泛表达，抗体靶向试剂具有较低的血脑屏障通透性。 因此，这项建议的重点是识别和验证新的递送载体和 他们的同源血脑屏障转运体可以调节运输效率的提高。而非部署 传统的哺乳动物抗体技术，我们描述了一种使用七鳃鳗抗体的新策略 被称为可变淋巴细胞受体(VLR)，以BBB为靶点。七鳃鳗和人类最后一次共享 共同的祖先&gt；5亿年前，由于这种巨大的进化分歧，甚至高度 在七鳃鳗中，保守的哺乳动物蛋白质和碳水化合物是免疫原性。通过利用这些独特的 利用创新的筛选技术研究七鳃鳗免疫系统，我们预计 拟议的研究将提供新的以血脑屏障为靶点的VLRs，能够贩运到大脑中。 为了实现这些目标，用小鼠脑微血管质膜免疫七鳃鳗。 制备的多克隆抗血清和染色结果表明，VLRS清楚地识别 体内的血脑屏障与多种独特的糖链结构结合。免疫七鳃鳗淋巴细胞基因的研究 然后用来创建由数百万个VLR组成的酵母展示文库，该文库将使用 创新筛选方法筛选结合和运输BBB的单抗VLRs。这些大脑靶向 VLRs将通过药代动力学图谱和它们在药理学上的诱导能力进行验证- 在小鼠中风模型中的相关反应。最后，将使用以BBB为目标的VLRS来识别 同源的血脑屏障转运体和任何相关的糖形式。那些显示有意义和特定大脑的VLR 摄取将代表新的、非侵入性的脑部给药载体，可能在治疗糖尿病方面发挥强大的作用。 使人衰弱的神经系统疾病。

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.