Efficient Brain Delivery of Neuroprotective TrkB Agonist Antibodies in Alzheimer’s Disease Using CD98hc Bispecific Shuttles

阿尔茨海默病（Alzheimer's disease，AD）是最常见的与年龄相关的神经退行性疾病。尽管大 生物制剂的潜力（例如，治疗性抗体）来影响AD病理生理学，这些药剂通常是 不能穿透血脑屏障（BBB），这是它们临床疗效的最大障碍。一 一种有前景的策略是使用双特异性抗体BBB穿梭，其涉及将IgG融合到第二亲和配体 其与脑血管内皮靶点结合并诱导跨血脑屏障的转运。虽然大多数以前 针对转铁蛋白受体（TfR-1）的BBB穿梭蛋白，我的实验室开发了一种BBB穿梭蛋白， 其与大中性氨基酸转运蛋白（LAT-1）的重链CD 98 hc接合。值得注意的是，我们 初步数据表明，通过CD 98 hc穿梭的IgG的上级脑实质递送和保留， 与那些通过TfR-1穿梭的相比。重要的是，这种第一代CD 98 hc穿梭使大脑能够传递 通过将单链CD 98 hc抗体连接到两种IgG之一的C末端， 沉重的锁链该提议的目标是双重的：i）定义最佳的CD 98 hc穿梭特性， （CD 98 hc单链亲和力、价态和表位），用于最大脑实质递送和保留， 指导第二代CD 98 hc穿梭机的设计;以及ii）在一个 使用诱导神经保护性信号传导的TrkB激动剂抗体的AD小鼠模型（tau，PS19）。第一 目的，我假设血脑屏障穿梭与单价CD 98 hc单链抗体（如第一次- 降低的CD 98 hc单链抗体亲和力（相对于第一代穿梭）将 通过改善抗体在近腔处的解离， 在脑内皮细胞侧，如对于TfR-1穿梭所观察到的。对于第二个目标，我假设， 将TrkB激动剂IgG递送至tau转基因小鼠的脑实质的CD 98 hc穿梭将减少 神经元和突触损失以及认知缺陷。这一假设得到了非- 穿梭TrkB抗体全身递送和TrkB配体在AD中的病毒脑递送的高功效 小鼠模型。因此，我在目标1中提出通过以下方法开发优化的第二代CD 98 hc穿梭体： 评价CD 98 hc单链抗体亲和力、效价和表位对抗体浓度的影响 在小鼠脑实质中作为时间的函数。其次，在目标2中，我建议评估第一个- 产生CD 98 hc穿梭以将TrkB激动剂IgG递送至脑实质，以激活野生型中的TrkB。 和tau（PS19）转基因小鼠，并在tau转基因小鼠中介导神经保护。一个关键的预期成果 是开发一种优化的CD 98 hc穿梭机，可用于递送广泛的现有的 治疗性抗体第二个关键的预期结果是TrkB的临床前数据 AD小鼠模型中的激动作用。这些发现有望使未来的研究，单一和组合 使用拮抗剂和激动剂抗体靶向机制上独特的途径的疗法。

Alzheimer’s disease (AD) is the most common age-related neurodegenerative disease. Despite the great potential of biologics (e.g., therapeutic antibodies) to affect AD pathophysiology, these agents are generally unable to penetrate the blood-brain barrier (BBB), which represents their biggest hurdle to clinical efficacy. One promising strategy is to use bispecific antibody BBB shuttles, which involve fusing IgGs to a second affinity ligand that engages a cerebrovascular endothelial target and induces transport across the BBB. While most previous work has focused on BBB shuttles that target transferrin receptor (TfR-1), my lab has developed a BBB shuttle that engages CD98hc, the heavy chain of the large neutral amino acid transporter (LAT-1). Notably, our preliminary data demonstrate superior brain parenchymal delivery and retention of IgGs shuttled via CD98hc as compared to those shuttled via TfR-1. Importantly, this first-generation CD98hc shuttle enables brain delivery of off-the-shelf IgGs simply by attaching a single-chain CD98hc antibody to the C-terminus of one of the two IgG heavy chains. The objectives of this proposal are twofold: i) to define the optimal CD98hc shuttle characteristics (CD98hc single-chain affinity, valency, and epitope) for maximal brain parenchymal delivery and retention to guide the design of second-generation CD98hc shuttles; and ii) to generate proof-of-concept efficacy data in an AD mouse model (tau, PS19) using a TrkB agonist antibody that induces neuroprotective signaling. For the first objective, I hypothesize that BBB shuttles with monovalent CD98hc single-chain antibodies (as used in the first- generation shuttle) and reduced CD98hc single-chain antibody affinity (relative to the first-generation shuttle) will maximize antibody concentrations in the brain parenchyma by improving antibody dissociation at the abluminal side of the brain endothelium, as observed for TfR-1 shuttles. For the second objective, I hypothesize that CD98hc shuttles that deliver TrkB agonist IgGs to the brain parenchyma of tau transgenic mice will reduce neuronal and synaptic loss and cognitive deficits. This hypothesis is supported by the partial efficacy of non- shuttled TrkB antibodies delivered systemically and high efficacy of viral brain delivery of the TrkB ligand in AD mouse models. Therefore, I propose in Aim 1 to develop an optimized second-generation CD98hc shuttle by evaluating the impact of CD98hc single-chain antibody affinity, valency, and epitope on antibody concentrations in the mouse brain parenchyma as a function of time. Next, in Aim 2, I propose to evaluate the ability of a first- generation CD98hc shuttle to deliver a TrkB agonist IgG to the brain parenchyma, to activate TrkB in wild-type and tau (PS19) transgenic mice, and to mediate neuroprotection in tau transgenic mice. A key expected outcome is the development of an optimized CD98hc shuttle that can be used to deliver a wide range of existing therapeutic antibodies to the brain parenchyma. A second key expected outcome is preclinical data for TrkB agonism in AD mouse models. These findings are expected to enable future studies of single and combination therapies using antagonist and agonist antibodies that target mechanistically unique pathways.