MICA: Development of PEGylated Domain I of beta-2-glycoprotein I as a new therapeutic agent for the antiphospholipid syndrome

抗磷脂综合征（APS）是一种自身免疫性疾病。这意味着这是一种疾病，身体的免疫系统，旨在保护我们免受感染，而不是开始攻击身体本身导致疾病过程的部分。不同的自身免疫性疾病攻击身体的不同部位，有不同的症状。在APS中，问题在于免疫系统会产生一种叫做抗磷脂抗体（aPL）的抗体，这种抗体会与各种不同类型的细胞相互作用。受影响的主要细胞是血管或子宫内的细胞，因此APS的主要影响是导致血管凝块、脑中风和/或反复流产。APS是造成这些问题的主要原因之一；例如，它是50岁以下人群中风的最重要原因之一。目前唯一能预防APS患者血栓、中风或流产的治疗方法是稀释血液并阻止其凝血的药物。这些药物被称为抗凝血剂，包括华法林和肝素。然而，它们也有副作用，特别是有出血的风险，因为它们反对所有的凝血——即使是在受伤后为止血而进行的有益凝血。我们寻求开发一种全新的APS治疗方法，它不稀释血液，而是直接针对aPL本身。aPL在APS中造成有害影响的主要方式是将自身附着在血液中称为β -2糖蛋白I （beta2GPI）的蛋白质上。Beta2GPI存在于每个人体内，在没有aPL的情况下是无害的。然而，当aPL与β 2gpi结合时，这种结合可以结合到血管或子宫的细胞表面，改变这些细胞的行为，从而促进凝血或流产。我们正在开发一种药物，旨在阻止aPL与β 2gpi结合，以防止这种有害过程的发生。Beta2GPI由五个称为结构域的部分组成，它们像串珠一样首尾相连。我们知道api主要连接到末端域（域I或DI）。在过去的10年里，我们的研究小组开发了世界上唯一一个在细菌中制造DI的系统。我们现在能够大量培养这些细菌，并从细菌培养物中纯化DI。这可以在高产量下完成，DI纯度超过95%。我们已经证明，这种纯化的DI可以用来阻断APS患者的aPL与塑料板上的人β 2gpi的结合，也可以阻止人aPL在小鼠中引起血栓。然而，DI是一种小分子，这使得它不适合作为药物使用，因为它只会在体内保留几个小时。为了避免这个问题，我们需要修改DI，使其更大。我们通过一个叫做聚乙二醇化的过程来做到这一点，在这个过程中，大的聚乙二醇（PEG）分子与较小的分子连接在一起。我们一直在与一家名为PolyTherics的生物技术公司合作，以实现这一目标。PolyTherics已经开发出一种技术，可以在其表面精确确定的点上对小分子进行聚乙二醇化。我们已经生产了三种不同的聚乙二醇化DI，它们具有不同尺寸的聚乙二醇。较大的peg可能有利于延长注射后DI在体内的停留时间，但也可能阻断DI对aPL的作用。因此，我们需要进行测试，比较所有三种变体，看看哪种是最好的。我们已经证明，我们的PEG-DI阻断了APS患者的aPL与β 2gpi结合、试管凝血和小鼠凝块形成的作用。在这个项目中，我们将进行进一步的测试，以找出哪种形式的PEG-DI对aPL的阻断作用最好，然后将这种形式的PEG-DI进行动物试验。这些测试将决定它在体内停留的时间以及它是否有任何毒副作用。假设没有发现毒性，我们将以足够纯净的形式大规模生产这种PEG-DI，用于人体试验。

Antiphospholipid syndrome (APS) is an autoimmune disease. This means that it is a disease in which the immune system of the body, which is designed to protect us against infections, instead starts to attack parts of the body itself causing the disease process. Different autoimmune diseases attack different parts of the body and have different symptoms. In APS, the problem is that the immune system makes antibodies called antiphospholipid antibodies (aPL) which interact with various different types of cells. The main cells affected are in blood vessels or in the womb, so the main effects of APS are to cause clots in blood vessels, strokes in the brain and/or recurrent miscarriages. APS is one of the main causes of these problems; for example it is one of the most important causes of stroke in people under 50.The only treatments currently available to prevent clots, strokes or miscarriages in patients with APS are drugs that thin the blood and stop it from clotting. These drugs are called anticoagulants, and include warfarin and heparin. However, they have side-effects, notably a risk of bleeding, because they oppose all clotting - even the helpful clotting that occurs after an injury to stop bleeding from a wound. We seek to develop an entirely new form of treatment for APS, which does not thin the blood but which directly targets the aPL themselves.The main way in which aPL cause their harmful effects in APS is to attach themselves to a protein in the blood called beta-2-glycoprotein I (beta2GPI). Beta2GPI is present in everyone and is harmless in the absence of aPL. When aPL combine with beta2GPI, however, this combination can bind to the surfaces of cells in the blood vessels or womb, change the behaviour of these cells and thus promote clotting or miscarriage. We are developing a drug that will be designed to stop aPL binding to beta2GPI to prevent this harmful process from occurring. Beta2GPI is composed of five parts, called domains, arranged end to end like beads on a string. We know that aPL primarily attach to the end domain (Domain I or DI). Over the last 10 years our research group has developed the only system in the world for making DI in bacteria. We are now able to grow these bacteria in large quantities and purify DI from the bacterial cultures. This can be done in high-yield with the DI at over 95% purity. We have shown that this purified DI can be used to block binding of aPL from patients with APS to human beta2GPI on plastic plates and also to stop human aPL from causing clots in mice.However, DI is a small molecule, which makes it unsuitable for use as a drug because it would only be retained in the body for a few hours. To circumvent this problem we need to modify our DI to make it larger. We are doing this by a process called PEGylation, in which large polyethylene glycol (PEG) molecules are joined to smaller molecules. We have been working with a biotechnology company called PolyTherics to achieve this. PolyTherics have developed technology to PEGylate small molecules at precisely determined points on their surface. We have achieved production of three different variants of PEGylated DI, which have PEG of different sizes. Larger PEGs could be good to make the DI last longer in the body after injection but could also block the effects of DI on aPL. Therefore we need to do tests comparing all three variants to see which is best. We have already proved that our PEG-DI blocks effects of aPL from patients with APS on binding to beta2GPI, on clotting in a test tube and on formation of clots in mice. In this project we will carry out further tests to find out which form of PEG-DI is best at blocking effects of aPL then take that form forward to tests in animals. These tests will determine how long it is retained in the body and whether it has any toxic side-effects. Assuming no toxicity is found we will develop production of this PEG-DI at large scale in a form pure enough for human trials.