Augmentation of Antioxidant Defenses by Immunotargeting

描述（由申请人提供）：血管氧化应激是许多肺部疾病的关键组成部分，但其治疗并不充分，部分原因是抗氧化剂向内皮细胞（EC）的输送不理想。先前的研究表明：1）血小板内皮粘附分子（PECAM）抗体允许通过一种不同于网格蛋白和小窝介导的内吞作用的新型内吞途径将药物在细胞内递送至EC； 2）静脉注射后，与抗PECAM结合的抗氧化酶（AOE）过氧化氢酶在肺部积聚，并在某些动物模型中防止氧化性肺损伤。这项转化资助的目的是将这一有前途的策略的保护作用的持续时间和有效性提高到临床显着水平。我们假设： i) 通过优化其设计以及操纵细胞内运输和溶酶体降解，可以延长缀合物的活性； ii) 有针对性地向 EC 输送额外的 AOE 将增强对氧化应激的保护； iii) 优化 AOE 缀合物的肺部靶向将可防止高氧血症。 我们将在以下具体目标中对此进行测试：1）定义 EC 中抗 PECAM 缀合物的代谢机制。 我们将检验以下假设：内吞作用和运输涉及 PECAM 的胞质结构域、Na -H 交换器 (NHE) 和细胞骨架的重排，这些结构可以受到辅助剂的影响以延长缀合物的持续时间； 2）额外AOE的设计瞄准。为了增强保护，将生产串联 SOD/过氧化氢酶（用于解毒 02- 和 H2O2）和 1-CysPrx 过氧化还原蛋白（用于解毒 H2O2 和脂质过氧化物）缀合物。将在细胞培养中研究它们的组成、活性、EC摄取和保护，同时将在幼稚动物和患有氧化性肺损伤的动物中确定它们的药代动力学和肺部靶向； 3)评估缀合物在动物模型中的保护作用。将比较新缀合物的有效性和持续时间，并在小鼠肺血管系统中 H2O2 诱导的急性 EC 损伤模型中研究 AOE 靶向方案和保护机制。 最后，将测试最佳缀合物对高氧肺损伤的保护作用。该提案的总体目标是优化血管 AOE 靶向策略，最终启动该策略向临床领域的转化。

DESCRIPTION (provided by applicant): Vascular oxidative stress is a key component of many lung diseases, but its treatment is inadequate, in part due to sub-optimal delivery of antioxidants to endothelial cells (EC). Previous studies have shown that: 1) antibodies to Platelet Endothelial Adhesion Molecule (PECAM) permit intracellular delivery of drugs to EC via a novel endocytotic pathway distinct from clathrin- and caveoli-mediated endocytosis; and 2) the antioxidant enzyme (AOE) catalase conjugated with anti-PECAM accumulates in the lungs after IV injection and protects against oxidative lung injury in some animal models. The aim of this translational grant is to enhance the duration and effectiveness of protective effects of this promising strategy to clinically significant levels. We hypothesize that: i) the activity of the conjugates can be prolonged by optimization of their design and by manipulating intracellular trafficking and lysosomal degradation; ii) targeted delivery of additional AOEs to EC will permit enhanced protection from oxidative stress; and iii) pulmonary targeting of optimized AOE conjugates will be protective against hyperoxia. We will test this in the following Specific Aims: 1) define the mechanisms of metabolism of anti-PECAM conjugates in EC. We will test the hypothesis that endocytosis and trafficking involve the cytosolic domain of PECAM, Na+-H+ exchangers (NHE) and rearrangements of cytoskeleton, which can be affected by auxiliary agents to prolong duration of conjugates; 2) design targeting of additional AOE. To enhance protection, tandem SOD/catalase (to detoxify 02- and H202) and 1-CysPrx peroxiredoxin (to detoxify H202 and lipid peroxides) conjugates will be produced. Their composition, activities, EC uptake and protection will be studied in cell culture, while their pharmacokinetics and pulmonary targeting will be determined in naive animals and animals with oxidative lung injury; and 3) evaluate protective effects of the conjugates in animal models. The effectiveness and duration of the effects of new conjugates will be compared and regimens of AOE targeting and mechanisms of protection will be studied in a model of acute EC injury induced by H202 in the pulmonary vasculature in mice. Finally, protection against hyperoxic lung injury by best conjugates will be tested. The overall goal of this proposal is to optimize strategies for vascular AOE targeting, to ultimately initiate translation of this strategy into the clinical domain.