Treatment of Transplant Reperfusion with an Anti-CD47 Antibody

描述(申请人提供)：器官移植是现代医学最成功的故事之一。然而，合适的器官供应远远落后于需求，许多患者在等待器官时死亡。目前的做法包括“供体预适应”，以稳定供移植的器官和组织，试图增加可供移植的供体器官的供应。预适应的一个关键目标是减少缺血-再灌注损伤(IRI)造成的损害，IRI是当缺血/缺氧器官连接到受体的循环时发生的。这场由活性氧、炎症介质和原虫因子组成的风暴可能会杀死新的器官，并对受体造成严重破坏。目前的工作表明，提高eNOS和nNOS产生的低水平一氧化氮(NO)可以显著改善IRI和移植结果。Vasculox公司的创始人发现了一种受体CD47，它在所有血管细胞中持续反对有益的NO的作用。在小鼠中敲除CD47或用单抗(MAb)阻断CD47可在许多外科缺血模型中增强组织灌注量，并在肝脏和后肢模型中保护免受IRI。在这一阶段的建议中，我们试图在一个具有良好特征的大鼠肝移植模型中进行概念验证研究，测试抗CD47单抗治疗供体肝脏以保护其免受IRI的有效性。在这里，我们将比较最好的情况，其中抗CD47单抗被注射给供体大鼠，并在整个肝脏采集、冷藏和再灌流过程中保持不变，与最差的情况不阻断CD47。在我们的方案中，心脏跳动的供体大鼠将用抗CD47单抗处理，取肝脏，用抗CD47单抗冲洗，进行冷保存缺血，然后在单抗存在的情况下，在受控的机器再灌流条件下复温。再灌流一段时间后，测定肝组织中释放的可溶性肝酶和炎症介质的水平，取肝组织进行三磷酸腺苷和环核苷酸的测定，并对肝组织进行定量细胞学分析和组织化学染色，以检测抗CD47单抗的分布和细胞凋亡(TUNEL和裂解半胱氨酸天冬氨酸蛋白酶)。当单抗注射和器官采集之间有一段时间的热缺血时，也将进行简化的无心脏跳动供体模型。在这个第一阶段项目中获得的数据将为在这个肝移植模型中进行抗CD47单抗治疗建立概念证明。第二阶段将解决“真实世界”的情况，即将经过治疗或控制的肝脏移植到活体受者体内，然后可以用抗CD47单抗治疗(或不治疗)。虽然我们选择将重点放在肝脏模型上，但我们预计抗CD47治疗将有益于其他器官的移植(心、肾、肺等)以及创伤和癌症的器官切除手术，以及潜在的心肌梗死和中风。与公共健康相关：Vasculox Inc.的创始人发现了一种调节性受体CD47，它可以抑制所有血管组织中的一氧化氮信号。一氧化氮在血管系统中有许多有益的作用，包括限制缺血再灌注损伤。因此，阻断CD47和解除一氧化氮抑制可改善缺血再灌注损伤，有望成为改善移植器官状况和控制缺血再灌注损伤对受体损伤的一种手段。Vasculox正在开发一种针对CD47的单抗，在这个项目中，旨在测试这种抗体在大鼠器官采集、运输和再灌注模型中的有效性。

DESCRIPTION (provided by applicant): Organ transplantation is one of the great success stories of modern medicine. However, the supply of suitable organs lags far behind the need and many patients die while waitlisted for organs. Current practice involves "donor preconditioning" to stabilize organs and tissues for transplant in attempts to increase the supply of donor organs acceptable for transplantation. A key goal of preconditioning is to reduce the damage inflicted by ischemia-reperfusion injury (IRI) that occurs when the ischemic/ hypoxic organ is connected to the recipient's circulation. This storm of reactive oxygen species, inflammatory mediators and prothombotic factors threatens to kill the new organ and wreaks havoc upon the recipient as well. Current work indicates that enhancing the low levels of nitric oxide (NO) produced by eNOS and nNOS can dramatically improve IRI and transplant outcomes. The founders of Vasculox, Inc have discovered a receptor, CD47, that continually opposes the action of beneficial NO in all vascular cells. Knocking out CD47 in mice or blocking CD47 with a monoclonal antibody (mAb) results in enhanced tissue perfusion in a number of surgical ischemia models and protection from IRI in both liver and hindlimb models. In this phase one proposal, we seek to perform proof of concept studies in a well-characterized rat model of liver transplantation, testing the efficacy of anti-CD47 mAb treatment of the donor liver to protect it from IRI. Here we will compare the best case scenario in which anti-CD47 mAb is administered to the donor rat and maintained throughout liver harvest, cold storage and reperfusion, with the worst case of no CD47 blockade. In our protocol, the heart-beating donor rat will be treated with anti-CD47 mAb, the liver harvested and flushed with anti-CD47 mAb, subjected to cold storage ischemia and then rewarmed in the presence of mAb under controlled conditions of machine reperfusion. After a fixed interval of reperfusion, levels of released soluble liver enzymes and inflammatory mediators will be assayed, tissue taken for assays of ATP and cyclic nucleotides and the liver fixed for quantitative cytologic analysis and histochemical staining for the distribution of the anti-CD47 mAb and apoptosis (TUNEL and cleaved caspase). A simplified nonheart-beating donor model will also be performed when a period of warm ischemia is interposed between mAb administration and organ harvest. The data obtained in this phase I project will establish proof of concept for anti-CD47 mAb therapy in this liver transplant model. Phase II will address the "real world" situation of transplantation of treated or control livers into living recipients that can then be treated (or not) with the anti-CD47 mAb. While we have chosen to focus here on the liver model, we anticipate that anti-CD47 therapy will be of benefit in the transplantation of other organs (heart, kidney, lung, etc) as well as in surgeries involving organ resection for trauma and cancer and, potentially, in myocardial infarction and stroke. PUBLIC HEALTH RELEVANCE: The founders of Vasculox Inc, have discovered a regulatory receptor, CD47, that inhibits nitric oxide signaling in all vascular tissues. Nitric oxide provides many beneficial effects in the vascular system, including limiting ischemia-reperfusion injury. Therefore blocking CD47 and removing nitric oxide inhibition improves ischemia-reperfusion injury and holds promise as a means to improve the condition of organs destined for transplant and to control the damage to the recipient caused by ischemia reperfusion injury. Vasculox is developing a monoclonal antibody that targets CD47 and in this project aims to test the efficacy of such an antibody in a rat model of organ harvest, transport and reperfusion.