Targeting novel biotherapeutics to endothelium

 描述：急性肺损伤(ALI)的病理生理特征是血液与内皮血管衬里的界面严重紊乱。内皮细胞(ECs)发挥保护作用的机制包括血栓调节蛋白(TM)介导的保护机制。血栓调节蛋白(TM)通过TM、凝血酶、蛋白C、活化蛋白C与内皮细胞膜上紧邻血栓调节蛋白(TM)的内皮蛋白C受体(EPCR)相互作用，部分抑制凝血酶的病理活动。在ALI中，TM和EPCR在功能上被抑制。这种内皮机制的补充是一个重要的治疗目标，但输注重组APC和TM并不能改善ALI的结果，因为问题递送和无法与自然调节分子配对限制了疗效并导致不良反应。为了解决这些问题，我们将TM与抗PECAM抗体的单链可变区(ScFv)融合，获得了在ALI动物模型中提供优于非靶向TM和APC的保护作用的scFv/TM。ScFv/TM锚定在EPCR附近的内皮细胞上可增强保护作用。为了避免ALI典型的内源性EPCR的丢失，我们使用了scFv/TM和scFv/EPCR的双重传递，这是基于我们最近偶然发现的针对不同PECAM表位的成对单抗对内皮结合的“协同增强”。在这里，我们将研究这些融合的传递、机制和受益/风险比，并开始它们的临床翻译：目的1：以内皮为靶向的双链抗体/TM和单链抗体/EPCR。我们将采用遗传学和药理学相结合的方法来选择性地调节融合的分子调控，以量化它们对ALI的影响。 目的2：动物试验的临床前疗效和安全性。抗PECAMscFv/TM和scFv/EPCR的有利和不利影响将被描述(血管渗漏、肺泡炎性反应、形态和生理结果)。目的3：人肺内皮靶向ScFv/TM。为了实现临床翻译，我们将：A)使用适用于人类的分子元件重新设计融合；以及B)研究它们在分离的人肺中的靶向。

 DESCRIPTION: The patho-physiological hallmark of acute lung injury (ALI) is severe derangement the interface of blood with the endothelial vascular lining. Endothelial cells (ECs) exert protective mechanisms including those mediated by thrombomodulin (TM), which inhibits thrombin's pathological activities in part through the production of activated protein C (APC) from its plasma precursor, via interactions between TM, thrombin, protein C, APC and the endothelial protein C receptor (EPCR) which is located in close proximity to TM in the endothelial plasmalemma. In ALI, TM and EPCR are functionally suppressed. Replenishment of this endothelial mechanism is an important therapeutic goal, but infusion of recombinant APC and TM did not improve the outcome of ALI, because problematic delivery and inability to partner with natural regulatory molecules limited effectiveness and lead to adverse effects. To solve these problems we fused TM to a single-chain variable fragment (scFv) of antibodies to PECAM, obtaining scFv/TM that provides protection superior to untargeted TM and APC in animal models of ALI. scFv/TM anchoring on endothelial cells in proximity to EPCR boosts protection. To circumvent the loss of endogenous EPCR typical of ALI, we use dual delivery of scFv/TM and scFv/EPCR, based on our recent serendipitous discovery of "collaborative enhancement" of endothelial binding of paired monoclonal antibodies directed to distinct PECAM epitopes. Here we will study the delivery, mechanisms and benefit/risk ratio of these fusions and embark on their clinical translation: Aim 1: Dual scFv/TM and scFv/EPCR targeting to endothelium. We will employ a combination of genetic and pharmacologic approaches to selectively modulate molecular regulation of the fusions in order to quantify their effects on ALI. Aim 2: Pre-clinical efficacy and safety in animals. Beneficial and adverse effects of anti-PECAM scFv/TM and scFv/EPCR will be characterized (vascular leakage, alveolar inflammatory response, morphologic and physiologic outcomes). Aim 3: Endothelial targeting scFv/TM in human lungs. To enable the clinical translation, we shall: A) Re-engineer the fusions using molecular elements applicable in humans; and, B) Study their targeting in isolated human lungs.