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

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.

The antiphospholipid syndrome (APS) is an autoimmune disease in which autoantibodies interact with phospholipid-binding proteins, particularly beta-2-glycoprotein I (beta2GPI), leading to thrombosis, strokes and/or pregnancy morbidity. The only evidence-based treatment currently available is long-term non-specific anticoagulation with agents such as warfarin, carrying a significant risk of haemorrhage. No biologic targeted therapies exist.

We, and others, have shown that the key autoantibody-antigen interaction in the pathogenesis of APS is with the N-terminal domain (Domain I or DI) of beta2GPI. Recombinant DI, expressed from bacteria, blocks binding of IgG purified from APS patients (APS-IgG) to beta2GPI in-vitro and inhibits thrombosis caused by these patient-derived APS-IgG in a mouse model. Our proposed therapeutic for APS is a PEGylated form of DI. PEGylation of small molecules improves half-life in-vivo, allowing feasible dosing regimens in patients, and two PEGylated agents are already in clinical use for other rheumatological diseases.

We are collaborating with PolyTherics, a biopharmaceutical company with an innovative proprietary method of PEGylating proteins on disulphide bonds. A shared UCL/PolyTherics MRC CASE PhD student has established high-yield, high-purity expression of PEGylated DI, including three different PEG sizes, which retained the ability to inhibit the activity of IgG from patients with APS in assays of binding and clotting.

In this project we will work with a CMO to optimise and scale-up the manufacture of PEG-DI and produce material to GLP standards. Pharmacokinetics and efficacy studies will show which PEG-DI variant has the optimal balance of half-life and retained activity. Toxicology studies in appropriately selected species will be carried out. The aim is to select a lead PEG-DI candidate for a first-in-man study and to determine from preclinical safety, efficacy and pharmacokinetic studies the dose range for such a study

The main beneficiaries from this research would be people with the antiphospholipid syndrome (APS). The main effects of the syndrome are development of clots in blood vessels, strokes and recurrent miscarriages.

Currently, this research is targeted primarily at the prevention of clots and strokes. Although our proposed therapeutic agent may subsequently play a role in prevention of pregnancy loss in patients with APS this is not one of the current objectives.

APS is one of the commonest causes of strokes in people under 50. A stroke in a young person may have a devastating effect on their capabilities and quality of life as well as ability to work and look after others. Clots may be life-threatening, particularly in the pulmonary circulation. Thus, patients with APS need to take long-term drug treatment in order to prevent clots or strokes.

At present, the treatment of choice for prevention of clots and strokes in patients with APS is long-term anticoagulation with warfarin. This is difficult and inconvenient for patients. Warfarin and similar agents act by blocking all clotting, including the helpful forms of clotting that stop people bleeding when they cut or injure themselves. This means that patients who take these drugs are at risk of bleeding, which may be serious. Regular blood tests to monitor the effects of warfarin on clotting are necessary. In addition, warfarin may interact with a number of other drugs.

Our proposed biologic agent, being specific for the antibody-DI interaction, is designed not to interfere with any other form of clotting apart from that caused by antiphospholipid antibodies and therefore should not increase the risk of bleeding. Our aim is to provide a new form of therapy that can be taken long-term to prevent clotting in APS without causing side-effects. It is important to note that the development of either our biologic agent or any other novel therapeutic agent for APS will ultimately depend on the willingness of patients to enter into clinical trials comparing new agents with warfarin (the current standard-of-care agent). Thus it is crucial to engage patient groups in this research and to elicit their opinions about what sort of new therapies they would wish to see. We work closely with the Hughes Syndrome Foundation (HSF), the UK's leading patient group for APS. Professor Anisur Rahman, the Principal Investigator of this research project, is a Trustee of the HSF and speaks every year at their annual Patients' Day. Over the last few years, he has spoken about the Domain I research project and the challenges of developing new therapies and carrying out clinical trials. A letter of support from HSF has been uploaded as an Attachment.

There are possible economic and industrial benefits as the idea of developing a PEGylated agent to block binding of antigen to antibody in a chronic autoimmune disease is novel. We have used bacterial expression followed by site-specific PEGylation to make our agent. The bacterial expression system allows for high-yield production with relatively low cost compared to eukaryotic systems. If we can establish proof of concept for the utility of our PEGylated antigen as a therapeutic agent in the clinic, this could provide impetus for academics, companies and investors to explore similar drug development strategies in other diseases that are characterised by an identifiable key antigen-antibody interaction. Autoimmune diseases in which such interactions play an important role are systemic lupus erythematosus and rheumatoid arthritis.