An immunoadhesin for treatment of hypercholesterolemia

描述（由申请人提供）：在本项目中，我们提出生产和体内测试一种重组蛋白，该蛋白应可持续降低血浆低密度脂蛋白胆固醇（LDL-C）。前蛋白转化酶枯草杆菌蛋白酶/kexin 9型（PCSK 9）是一种在肝脏中分泌的蛋白质，通过调节低密度脂蛋白受体（LDLR）（一种从血浆中清除LDL-C颗粒的质膜糖蛋白）的细胞表面水平，在维持胆固醇稳态中发挥关键作用。人群中循环PCSK 9水平的变化范围超过100倍，PCSK 9水平与LDL-C水平（以及PCSK 9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（高胆固醇）。下调PCSK 9的治疗应作为他汀类药物的辅助药物，以维持LDL-C的健康水平，特别是在家族性高胆固醇血症患者中。含有LDLR的表皮生长因子样重复AB（EGF-AB）结构域的蛋白片段与PCSK 9结合，并阻断PCSK 9消耗肝细胞上LDLR的能力。然而，EGF-AB片段在体内不稳定，因此作为治疗剂是不切实际的。在这个项目中，我们建议生产并在体内测试一种稳定的包含EGF-AB的诱饵分子，该分子应该耗尽血浆PCSK 9并可持续地降低血浆LDL-C。 使用植物表达系统，我们将产生一系列重组蛋白，其由融合至人IgG 1的Fc的EGF-AB变体组成。一种变体将并入野生型EGF-AB序列，但其他变体将包含在患有高胆固醇血症的个体的LDLR中发现的单个氨基酸突变（和一些非天然突变）。预计这些变体中的一些将更紧密地结合PCSK 9，从而更好地阻断其消耗LDLR的能力。将首先测定EGF-AB变体干扰标记的LDLR胞外结构域与PCSK 9结合的能力。然后在细胞LDL摄取试验中检测具有最大活性的变体，以确定它们对外源性添加的PCSK 9的LDL降低活性的抑制程度。将检测两种最佳EGF-AB-Fc变体在表达升高水平的人PCSK 9的转基因小鼠中降低LDL-C的能力。 公共卫生相关性：冠状动脉疾病是全球主要的死亡原因。冠状动脉疾病发病机制中的主要致病因素是血浆低密度脂蛋白胆固醇（LDL-C）水平升高。基于融合至IgG Fc的低密度脂蛋白受体的蛋白质片段的受体诱饵预期可显著降低LDL-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.