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

描述（由申请人提供）：由于此类药物进入CNS的途径有限，因此目前尚未实现基于大分子的神经治疗剂的临床潜力。长期目标是开发有效和特异性的血管递送方法。血脑屏障上的受体介导的转运机制已显示出用于此目的的前景，但迄今为止靶向的受体系统（例如转铁蛋白或胰岛素受体）不是真正的脑特异性的。该特定应用的目的是在脑血管内皮上建立特异性肽-主要组织相容性（MHC）I类复合物作为药物递送的可行入口。肽-MHC复合物由多种内源性肽片段和MHC I类分子的不同等位基因形成，并且在生理上被T细胞受体（TCR）识别。最近，产生对不同的人肽-MHC组合具有高特异性和亲和力的TCR模拟单克隆抗体成为可能。TCR模拟物具有组织/细胞类型特异性靶向的潜力。虽然它们迄今为止主要被探索为靶向病变细胞的工具，但TCR模拟策略主要适用于靶向由任何细胞类型表达的特异性复合物。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开发以TCR模拟物为载体的高度血脑屏障特异性诊断和治疗药物奠定基础。对TCR模拟物之一的初步研究表明，源自人脑的内皮细胞呈递其相应的靶肽-MHC组合，并且可以结合并内化抗体。本论文的主要工作有两个方面：1）研究TCR模拟抗体与脑源性内皮细胞的相互作用。这将包括体外定量结合和摄取研究和所涉及的亚细胞区室的表征，以及解决跨内皮转运（胞吞作用）程度的实验。将检查表达人MHC I类等位基因的转基因小鼠品系，以确定用于后续项目的合适的体内模型。2)确定TCR模拟物靶向的纳米颗粒药物的药理学活性。TCR模拟抗体将与DNA/聚合物复合物偶联，该复合物此前已证明，当通过转铁蛋白受体抗体靶向脑源性内皮细胞时，具有强效的抗炎药物作用。内皮细胞培养物将接受炎性刺激，并且通过炎性标志物的定量RT-PCR和功能测定（单核细胞粘附）来测量TCR模拟物靶向的药物的作用。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，其潜在应用范围从CNS疾病到其他血管化器官疾病和肿瘤。项目叙述：拟议的研究涉及一个重要的研究领域，即开发更好的药物来治疗大脑疾病。这些发现可能对潜在的基于蛋白质或DNA的治疗方法产生广泛的影响，这些治疗方法通常因无法到达靶组织而受到阻碍。

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.