Drug Delivery to CNS Endothelial Cells by Targeting Peptide-MHC Complexes

描述(由申请人提供)：基于大分子的神经疗法的临床潜力目前尚未实现，因为此类药物进入中枢神经系统的机会有限。长期目标是开发有效和特定的血管输送方法。受体介导的血脑屏障转运机制已经显示出实现这一目的的希望，但迄今为止靶向的受体系统(例如转铁蛋白或胰岛素受体)并不是真正的大脑特异性的。这一特殊应用的目的是在脑血管内皮细胞上建立特异性的多肽主要组织相容性(MHC)I类复合体，作为药物输送的可行门户。多肽-MHC复合体是由多种内源性多肽片段和MHC I类分子的不同等位基因形成的，并被T细胞受体(TCR)生理识别。最近，针对不同的人多肽-MHC组合产生高特异性和高亲和力的TCR模拟单抗成为可能。TCR模拟物具有组织/细胞类型特异性靶向的潜力。虽然到目前为止，它们主要是作为靶向疾病细胞的工具进行探索，但TCR模拟策略主要用于靶向由任何细胞类型表达的特定复合体。这项工作的中心假设是TCR模拟抗体进入脑源性内皮细胞，因此可以用作递送载体。该项目的基本原理是为以TCR模拟物为载体的高度血脑屏障特异性诊断和治疗试剂的开发奠定基础。对其中一种TCR模拟物的初步研究表明，人脑来源的内皮细胞呈现其相应的靶肽-MHC结合，并能结合和内化抗体。这项拟议的工作将遵循两个特定的目标：1)研究TCR模拟抗体与脑源性内皮细胞的相互作用。这将包括体外的定量结合和摄取研究以及所涉及的亚细胞间隔的特征，以及解决跨内皮运输(跨细胞作用)的程度的实验。表达人类MHC I类等位基因的转基因小鼠品系将被检测，以确定适合后续项目的体内模型。2)确定TCR模拟物靶向的纳米药物的药理活性。TCR模拟抗体将偶联到DNA/聚合物复合体上，以前通过转铁蛋白受体抗体靶向脑源性内皮细胞时，DNA/聚合物复合体显示出强大的抗炎药物作用。内皮细胞培养将受到炎症刺激，TCR-MIMIC靶向药物的效果将通过炎症标记物的定量RT-PCR和功能分析(单核细胞黏附)来衡量。这种方法是创新的，因为TCR模拟物以前没有被认为是靶向大脑的载体。这项拟议的研究意义重大，因为它可以为一种新的血管靶向策略铺平道路，潜在的应用范围从中枢神经系统疾病到其他血管器官的疾病，以及肿瘤。项目简介：拟议的研究涉及与开发更好的治疗脑部疾病的药物有关的一个重要研究领域。这些发现可能会对潜在的基于蛋白质或DNA的疗法产生广泛的影响，这些疗法往往因无法到达目标组织而受到阻碍。

DESCRIPTION (provided by applicant): The clinical potential of macromolecule-based neurotherapeutics is currently not realized due to the limited access such drugs gain to the CNS. The long term goal is to develop efficient and specific vascular delivery approaches. Receptor-mediated transport mechanisms at the blood-brain barrier have shown promise for this purpose, but the receptor systems targeted to date (e.g. transferrin or insulin receptor) are not truly brain specific. The objective of this particular application is to establish specific peptide- major histocompatibility (MHC) class I complexes on brain vascular endothelium as viable portals for drug delivery. Peptide-MHC complexes are formed from a vast variety of endogenous peptide fragments and different alleles of MHC class I molecules, and are physiologically recognized by T-cell receptors (TCR). Recently it became possible to generate TCR mimic monoclonal antibodies of high specificity and affinity for distinct human peptide-MHC combinations. TCR mimics hold the potential of tissue / cell type specific targeting. While they have to date primarily been explored as tools for targeting diseased cells, the TCR mimic strategy lends itself principally to target specific complexes expressed by any cell type. The central hypothesis of this work is that TCR mimic antibodies enter brain-derived endothelial cells and thus can be used as delivery vectors. The rationale of the project is to lay the base for the development of highly blood-brain barrier specific diagnostic and therapeutic agents using TCR mimics as vectors. Preliminary studies with one of the TCR mimics indicate that endothelial cells derived from human brain present its corresponding target peptide-MHC combination, and can bind and internalize the antibody. The proposed work will follow two specific aims: 1) Investigate the interaction of TCR mimic antibodies with brain-derived endothelial cells. This will include quantitative binding and uptake studies in vitro and characterization of the subcellular compartments involved, as well as experiments addressing the extent of transendothelial transport (transcytosis). Transgenic mouse strains expressing human MHC class I alleles will be examined to identify suitable in vivo models for follow up projects. 2) Determine the pharmacological activity of a nanoparticulate drug targeted by a TCR mimic. The TCR mimic antibody will be coupled to a DNA/polymer complex, which has previously demonstrated potent anti- inflammatory drug effects when targeted to brain-derived endothelial cells by a transferrin receptor antibody. Endothelial cell cultures will receive inflammatory stimuli and the effect of drug targeted by the TCR-mimic will be measured by quantitative RT-PCR of inflammatory markers and by a functional assay (monocyte adhesion). The approach is innovative, because TCR mimics have not been previously considered as vectors for targeting brain. The proposed research is significant, because it can pave the way to a novel vascular targeting strategy, with potential applications reaching from CNS disorders to diseases of other vascularized organs, and tumors. PROJECT NARRATIVE: The proposed studies address an important area of research concerned with the development of better drugs to treat diseases of the brain. The findings can have wide implications for potential protein- or DNA-based therapeutics, which are often hampered by their inability to reach the target tissues.