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。这意味着它是一种疾病，其中身体的免疫系统，旨在保护我们免受感染，而不是开始攻击身体本身的部分，导致疾病过程。不同的自身免疫性疾病攻击身体的不同部位，并有不同的症状。在APS中，问题在于免疫系统产生称为抗磷脂抗体（aPL）的抗体，这些抗体与各种不同类型的细胞相互作用。受影响的主要细胞在血管或子宫中，因此APS的主要影响是导致血管凝块，脑中风和/或复发性流产。APS是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。目前唯一可用的预防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结的药物。这些药物被称为抗凝剂，包括华法林和肝素。然而，它们有副作用，特别是出血的风险，因为它们反对所有的凝血-甚至是受伤后发生的有助于止血的凝血。我们寻求开发一种全新的APS治疗方法，这种方法不会稀释血液，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其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联合收割机与β 2 GPI结合时，这种结合可以结合到血管或子宫中的细胞表面，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beta2GPI结合，以防止这种有害过程的发生。Beta2GPI由五个部分组成，称为结构域，像一根绳子上的珠子一样首尾相连。我们知道aPL主要附着于末端结构域（结构域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。这可以用纯度超过95%的DI以高产率完成。我们已经证明，这种纯化的DI可以用来阻断塑料板上APS患者的aPL与人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，这使得它不适合用作药物，因为它只能在体内保留几个小时。为了避免这个问题，我们需要修改我们的DI，使其更大。我们通过一种称为聚乙二醇化的过程来实现这一点，在这个过程中，大的聚乙二醇（PEG）分子与小分子结合在一起。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开发了一种技术，可以在小分子表面精确确定的点上使其聚乙二醇化。我们已经实现了PEG化DI的三种不同变体的生产，其具有不同大小的PEG。较大的PEG可能有利于使注射后DI在体内持续更长时间，但也可能阻断DI对aPL的影响。因此，我们需要对所有三种变体进行比较，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对与β 2GPI结合、试管中凝血和小鼠中凝块形成的影响。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最适合阻断aPL的效果，然后将该形式用于动物测试。这些测试将确定它在体内保留多久以及是否有任何毒副作用。假设没有发现毒性，我们将开发这种PEG-DI的大规模生产，其纯度足以进行人体试验。

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.