Protease Activated Bi-Specific Antibody Prodrugs for Efficient Transportation Across Blood-Brain Barrier

项目总结 血脑屏障(Bbb)是开发有效的神经系统治疗方法的最大挑战。 疾病。受受体介导的细胞转运的启发，抗转铁蛋白受体的双特异性抗体(BsAbs) (TFR)已经显示出CNS交付的显著改进。然而，总体的大脑渗透率是 仍然很温和，绝大多数接种的bsAb仍然留在血液中。因为血脑屏障的跨细胞反应是双向的 过程中，不可避免地导致抗TFR bsAb在血液和大脑之间达到浓度平衡 此外，我们假设，通过最大限度地减少从腔到腔的流出，浓度平衡可以改变。 朝向血脑屏障渗透。我们的设计原则是将抗TfR的可变区(Fv)融合到N-端 通过大脑中存在的疾病相关蛋白酶的特异性可切割接头(S)，获得一种治疗性免疫球蛋白。一次 通过转铁蛋白受体介导的跨细胞转运，治疗性免疫球蛋白将被激活并停留在脑部。 因为它失去了与TFR的结合能力。释放的抗TFR Fv将被输送回血液侧，然后被 通过肾脏清除而消除。与转铁蛋白受体结合的前体药物将进一步跨细胞转运，从而形成 治疗性抗体从血液渗透到大脑。我们的长期目标是开发一种高效的BBB交付 一种能够有效治疗神经疾病的方法，如脑癌和 神经退行性疾病。这个MPI R21项目的目的是证明这个新的BBB的概念 基于蛋白酶激活的前药设计的给药技术。我们将使用组织蛋白酶S激活的抗淀粉样蛋白 本研究以阿尔茨海默病(AD)的β(Aβ)为模型系统。基于我们在以下方面的集体专业知识 蛋白质工程，酶生物化学，血脑屏障运输和AD，我们将，目标1：设计，建造和 优化蛋白酶激活的双特异性抗体前药；目标2：验证血脑屏障渗透性和治疗性 抗体前药在小鼠脑淀粉样血管病(CAA)模型上的疗效。这些方法包括 创新的是，因为可酶裂解的前药设计可以防止反向跨细胞反应，使 浓度平衡，从而促进治疗药物从血液到大脑的渗透。拟议的研究 具有重要意义，因为它开发了一种平台技术，能够(1)提高生物制品的血脑屏障渗透率 包括单抗(MAbbs)和抗体-药物结合物(ADC)，(2)大大减少了异地对 通过脑内原位激活产生的靶向副作用，以及(3)治疗目前尚未发现的多种神经疾病 有针对性。

PROJECT SUMMARY The blood-brain barrier (BBB) poses the greatest challenge for developing effective therapies for neurological diseases. Inspired by receptor-mediated transcytosis, bi-specific antibodies (bsAbs) against transferrin receptor (TfR) have demonstrated significant improvements of CNS delivery. However, the overall brain penetration was still modest, with large majority of administrated bsAbs remain in blood. As transcytosis at BBB is a bi-directional process and inevitably leads anti-TfR bsAb reaching a concentration equilibrium between the blood and the brain sides, we hypothesize that by minimizing abluminal-to-luminal efflux, the concentration equilibrium can be shifted toward BBB penetration. Our design principle is to fuse the variable fragment (Fv) of anti-TfR to the N-terminal of a therapeutic IgG, via cleavable linker(s) specific to disease-associated protease present in the brain. Once delivered to the brain by TfR-mediated transcytosis, therapeutic IgG will be activated and stays at the brain side because it loses binding ability to TfR. Released anti-TfR Fv will transport back to the blood side then be eliminated by renal clearance. TfR-bound prodrugs will be further transcytosed and thus forming a net flow of therapeutic Ab penetration from blood to brain. Our long-term goal is to develop a highly efficient BBB delivery approach that enables effective treatments of neurological disorders such as brain cancer and neurodegenerative diseases. The objective of this MPI R21 project is to prove the concept of this novel BBB delivery technology based on protease-activated prodrug designs. We will use cathepsin S activated anti-amyloid β (Aβ) for Alzheimer’s disease (AD) as the model system in this study. Building on our collective expertise on protein engineering, protease biochemistry, BBB transportation and AD, we will, Aim 1: design, construct and optimize protease-activated bi-specific antibody prodrugs; and Aim 2: validate BBB penetration and therapeutic efficacy of antibody prodrugs using mouse models of cerebral amyloid angiopathy (CAA). The approaches are innovative, because the protease-cleavable prodrug designs can prevent the reverse transcytosis, shift the concentration equilibrium, and thus promote therapeutics penetration from blood to brain. The proposed research is significant because it develops a platform technology enabling to (1) improve BBB penetration of biologics including monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs), (2) greatly reduce off-site on- target side effects by in situ activation in brain, and (3) treat a variety of neurological disorders currently non- targetable.