An immunoadhesin for treatment of hypercholesterolemia

描述(申请人提供)：在这个项目中，我们建议生产并在体内测试一种可持续降低血浆低密度脂蛋白胆固醇(LDL-C)的重组蛋白。枯草杆菌前蛋白转换酶9(PCSK9)是一种在肝脏中分泌的蛋白质，通过调节细胞表面低密度脂蛋白受体(LDLR)的水平，在维持胆固醇平衡方面发挥关键作用。低密度脂蛋白受体是一种质膜糖蛋白，可以从血浆中清除低密度脂蛋白颗粒。人类循环中的PCSK9水平变化幅度超过100倍，并且PCSK9水平与低密度脂蛋白-C水平(以及PCSK9与冠状动脉疾病事件之间)呈正相关。他汀类药物被广泛用于治疗高胆固醇血症。下调PCSK9的治疗应该作为他汀类药物的辅助药物，以维持健康的低密度脂蛋白-C水平，特别是在患有家族性高胆固醇血症的患者中。一个含有LDLR的表皮生长因子样重复AB(EGF-AB)结构域的蛋白质片段与PCSK9结合，并阻断PCSK9耗尽肝细胞上LDLR的能力。然而，EGF-AB片段在体内不稳定，因此作为治疗药物不切实际。在这个项目中，我们建议生产并在体内测试一种稳定的含有EGF-AB的诱饵分子，它应该会耗尽血浆PCSK9并可持续地降低血浆低密度脂蛋白-C。利用植物表达系统，我们将生产一系列由EGF-AB变异体与人IgG1 Fc融合而成的重组蛋白。一个变体将包含野生型EGF-AB序列，但其他变体将包含在高胆固醇血症患者LDLR中发现的单一氨基酸突变(以及一些非自然突变)。预计其中一些变体将与PCSK9结合得更紧密，从而更好地阻断其耗尽LDLR的能力。首先将检测EGF-AB变异体干扰标记的LDLR胞外区与PCSK9结合的能力。然后，活性最高的变异体将在细胞低密度脂蛋白摄取试验中进行测试，以确定它们对外源添加的PCSK9降低低密度脂蛋白受体活性的抑制程度。两个最好的EGF-AB-Fc变种将在表达高水平人PCSK9的转基因小鼠中测试它们降低低密度脂蛋白-C的能力。 公共卫生相关性：冠状动脉疾病是世界范围内的主要死亡原因。冠状动脉疾病发病的主要原因是血浆低密度脂蛋白胆固醇(LDL-C)水平升高。一种基于低密度脂蛋白受体与免疫球蛋白Fc融合的蛋白片段的受体诱饵有望显著降低低密度脂蛋白-C并降低冠状动脉疾病的风险。

DESCRIPTION (provided by applicant): In this project we propose producing, and testing in vivo, a recombinant protein that should sustainably reduce plasma low-density lipoprotein cholesterol (LDL-C). Proprotein convertase subtilisin/kexin type 9 (PCSK9), a protein secreted in the liver, plays a key role in maintaining cholesterol homeostasis by regulating cell-surface levels of the low-density lipoprotein receptor (LDLR), a plasma membrane glycoprotein that removes LDL-C particles from the plasma. Levels of circulating PCSK9 in human populations vary over a >100- fold range, and there is a positive correlation between PCSK9 levels and levels of LDL-C (and between PCSK9 and coronary artery disease events). Statins are widely prescribed as therapy for hypercholesterolemia (high cholesterol). A therapy that would down-regulate PCSK9 should act as an adjunct to statins in maintaining healthy levels of LDL-C, especially in individuals with familial hypercholesterolemia. A protein fragment containing the epidermal growth factor-like repeat AB (EGF-AB) domains of the LDLR binds to PCSK9 and blocks the ability of PCSK9 to deplete LDLR on hepatocytes. The EGF-AB fragment, however, is unstable in vivo, and thus impractical as a therapeutic. In this project we propose to produce, and test in vivo, a stable decoy molecule incorporating EGF-AB, which should deplete plasma PCSK9 and sustainably reduce plasma LDL-C. Using a plant expression system we will produce a series of recombinant proteins comprised of EGF- AB variants fused to the Fc of human IgG1. One variant will incorporate the wild-type EGF-AB sequence, but the others will contain single amino acid mutations found in the LDLR of individuals with hypercholesterolemia (and some non-natural mutations). It is expected that some of these variants will bind more tightly to PCSK9 and thus better block its ability to deplete LDLR. The EGF-AB variants will first be assayed for their ability to interfere with the binding of labeled LDLR extracellular domain to PCSK9. The variants with the greatest activity will then be tested in a cellular LDL uptake assay to determine how well they inhibit the LDLR-lowering activity of exogenously added PCSK9. The two best EGF-AB-Fc variants will be tested for their ability to lower LDL-C in transgenic mice expressing elevated levels of human PCSK9. PUBLIC HEALTH RELEVANCE: Coronary artery disease is the leading cause of death worldwide. The primary causal factor in the pathogenesis of coronary artery disease is an elevated plasma level of low-density lipoprotein cholesterol (LDL-C). A receptor decoy based on a protein fragment of the low-density lipoprotein receptor fused to IgG Fc is expected to dramatically lower LDL-C and reduce the risk of coronary artery disease.