A Vascularized Blood-Brain Barrier Model for In Vitro Testing of Drug and Immunotherapy Delivery

项目总结 大脑是一个高度专业化、调节精细的器官，其功能依赖于维持动态平衡。 在狭义的范围内。这在很大程度上是通过血脑屏障(BBB)实现的，它紧密地 调节什么能和什么不能进入大脑。从功能上讲，血脑屏障是内皮细胞共同作用的结果 (EC)、周细胞和星形胶质细胞，后两者作用于EC以维持广泛的紧密连接网络。 连接，促进专门的转运体的表达，并限制跨细胞吞饮速率。这 非亲脂性物种缺乏血液和大脑之间的自由转移，严重限制了许多物种的进入， 否则就是有用的小分子药物。例如，顺铂(3%脑/血浆比率)对非脑组织有效 这种药物对肿瘤有效，但对脑肿瘤(如胶质瘤)的透视性差，疗效低。同样，交付 贝伐单抗(0.2%)和双特异性抗体等免疫疗法也受到限制，仍有许多工作要做 了解免疫细胞向大脑的传递，包括CART-T细胞、NK细胞、γδT细胞和 病人自己的单核细胞和淋巴细胞。最后，鼠标和鼠标之间存在显著差异 包括免疫系统在内的人类生理学。因此，许多药物开发人员正在转向人类细胞-- 基于系统而不是鼠标模型。此第一阶段SBIR应用程序的主要目标是演示 阿拉卡里生物科学公司的专利技术用于模拟合适的药物和免疫疗法的可行性 通过人体血脑屏障传递。Aracari的专利核心技术是可灌流的人体血管网络 在符合方便的96孔板格式的微流控装置内。这项技术已经成功地 开发成商品化产品，包括带血管的微型器官(VMOTM)和带血管的 微肿瘤(VMTTM)。重要的是，白细胞可以通过血管灌流，在刺激下，它们将 黏附和渗出，就像他们在体内做的那样。我们还开发了一种血管微脑(VMB)，这是 这个应用程序的重点。这包含了一个由人血脑屏障组成的灌流神经血管单位(NVU) EC、周细胞和星形胶质细胞，它们都嵌入了一种模仿大脑的细胞外基质中。这些船只显示 血脑屏障转运蛋白和结合蛋白上调，通透性显著降低 北威州大学的船只。肿瘤细胞(胶质瘤)可添加到模型脑肿瘤(VMB-T)和潜在的折衷 是BBB的。这一模型将为研究神经胶质瘤和其他疾病的药物反应提供前所未有的机会。 脑瘤在更自然的环境中。我们的初步数据显示了我们目标的可行性，这些目标是： 具体目标1：确定一组脑靶向药物在VMB和VMB-T中的渗透性 具体目标2：确定一小批免疫治疗药物在VMB和VMB-T中的传递特性 具体目标3：确定免疫细胞在VMB和VMB-T中的传递 这些目标的完成将为我们提供一个独特的工具，我们可以提供给制药公司进行研究 关于血脑屏障的特性，以及神经活性药物和免疫肿瘤药物和细胞的输送。

PROJECT SUMMARY The brain is a highly-specialized, finely-tuned organ that depends for its function on maintaining homeostasis within narrowly-defined limits. This is achieved in large part by the Blood-Brain Barrier (BBB), which tightly regulates what can and can’t get into the brain. Functionally, the BBB is the combined work of endothelial cells (EC), pericytes and astrocytes, the latter two of which act on the EC to maintain an extensive network of tight junctions, promote the expression of specialized transporters, and limit the rate of transcellular pinocytosis. This lack of free transfer between blood and brain for non-lipophilic species severely limits the entry of many, otherwise useful, small molecule drugs. For example, cisplatin (<3% Brain/Plasma ratio), is effective in non-brain tumors, but has poor penetrance and low efficacy against brain tumors such as glioma. Similarly, delivery of immunotherapeutics such as bevacizumab (0.2%) and bi-specific antibodies is also limited, and much is still to be understood regarding the delivery of immune cells to the brain, including CAR-Ts, NK cells, γδ T cells and the patient’s own monocytes and lymphocytes. Finally, there are significant differences between mouse and human physiology including in the immune system. Many drug developers are therefore turning to human cell- based systems rather than mouse models. The primary goal of this Phase I SBIR application is to demonstrate the feasibility of Aracari Biosciences’ proprietary technology to model appropriate drug and immunotherapeutic delivery across the human BBB. Aracari’s patented core technology is a perfusable human vascular network within a microfluidic device that fits in a convenient 96-well plate format. This technology has been successfully developed into commercialized products including the vascularized micro-organ (VMOTM) and the vascularized micro-tumor (VMTTM). Importantly, leukocytes can be perfused through the vessels and on stimulation they will adhere and extravasate as they do in vivo. We have also developed a vascularized micro-brain (VMB), which is the focus of this application. This incorporates a perfused neurovascular unit (NVU) comprised of human BBB EC, pericytes and astrocytes, all embedded in a brain-mimicking extracellular matrix. The vessels show upregulation of BBB transporters and junctional proteins, and greatly-reduced permeability compared to non- NVU vessels. Tumor cells (glioma) can be added to model brain tumors (VMB-T) and the potential compromise of the BBB. This model will provide unprecedented opportunities to study drug responses of glioma and other brain tumors in a more natural environment. Our preliminary data show the feasibility of our goals which are: Specific Aim 1: Characterize permeability of a small panel of brain-targeted drugs in the VMB and VMB-T Specific Aim 2: Characterize delivery of a small panel of immunotherapeutic drugs in the VMB and VMB-T Specific Aim 3: Characterize delivery of immune cells in the VMB and VMB-T Completion of these aims will provide us with a unique tool we can offer to pharmaceutical companies for studies on BBB properties, and delivery of neuroactive drugs and immuno-oncology drugs and cells.