Identification of Lamprey Antibodies Capable of Noninvasive Brain Drug Delivery

摘要 全球数百万人患有神经系统疾病，如阿尔茨海默病，中风， 脑癌蛋白质/基因谱分析技术与高通量药物筛选技术研究进展 催生了许多新的候选药物然而，血脑屏障（BBB）阻碍了 通过限制大脑摄取， 大多数小分子治疗药物，并禁止大脑摄取蛋白质和基因为基础的药物。一 有前途的非侵入性脑递送策略利用内源性血脑屏障转运机制作为一种有效的治疗方法。 将毒品从血液运送到大脑这种受体介导的转运系统可以是 使用抗体的精确特异性进行靶向，这些抗体又与药物有效载荷连接， 小分子、蛋白质或DNA治疗剂。在与血液侧的受体结合后，抗体- 药物偶联物作为转运蛋白的人工底物，从血液中跨膜转运， BBB，进入大脑。目前的方法产生了有限的大脑摄取，因为目标 转运蛋白普遍表达，并且抗体靶向试剂具有低的BBB渗透性。 因此，该提案的重点是鉴定和验证新的递送载体， 它们的同源BBB转运蛋白可以介导提高的转运效率。而不是部署 传统的哺乳动物抗体技术，我们描述了一种新的策略，采用七鳃鳗抗体 称为可变淋巴细胞受体（VLR），以靶向BBB。七鳃鳗和人类最后一次分享 共同祖先> 5亿年前，由于这种巨大的进化分歧，甚至高度 保守的哺乳动物蛋白质和碳水化合物在七鳃鳗中具有免疫原性。通过利用这些独特的 方面的七鳃鳗免疫系统与创新的筛选技术，我们预计， 拟议的研究将提供新的BBB靶向VLRs能够贩运到大脑。 为了达到这些目标，用小鼠脑微血管质膜免疫七鳃鳗 制备，并用所得的多克隆抗血清染色，证明VLRs清楚地识别 体内BBB并结合多种独特的聚糖结构。免疫七鳃鳗淋巴细胞cDNA 然后用于创建由数百万个VLR组成的酵母展示文库， 创新的筛选方法来选择BBB结合和运输单克隆VLRs。这些大脑定位 VLR将通过药代动力学特征及其引发药物代谢的能力进行验证。 在鼠中风模型中的相关反应。最后，将使用靶向BBB的VLRs来识别 同源BBB转运蛋白和任何相关的糖型。这些VLRs表现出显著的和特定的大脑 吸收将代表新的，非侵入性的大脑药物输送载体，可能是强大的治疗， 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.