Uncovering the role of LRG1 in disease mechanisms

After a team of researchers at the University College London (UCL) Institute of Ophthalmology (IoO), London, started to explore the causes of malformed blood vessels in the eye, they discovered that a molecule called LRG1 (leucine-rich alpha-2-glycoprotein 1) was partly responsible. They realised that if they could block the function of this molecule, they might be able to improve the formation of blood vessels so they would not be as leaky.

The team is now collaborating with Moorfields Eye Hospital, London, with the aim of moving towards clinical testing of new therapies that could improve disease outcomes in patients with a range of disorders including wet age-related macular degeneration (AMD), diabetes and cancer. I spoke with professors John Greenwood and Stephen Moss, who led the team at UCL, to find out more.

What prompted your research into LRG1?

Our research began in 2005, when Professor Mark Gillies, a clinical colleague from Sydney, Australia, invited us to join the MacTel Project, a new international consortium set up by the Lowy Medical Research Institute to understand the mechanism behind the rare retinal condition macular telangiectasia type 2, or MacTel. The disease leads to a permanent loss of central vision and at the time, little was known about its causes.

The MacTel Project provided us with almost one million pounds over 5 years, funding which enabled us to see which genes and biological mechanisms were involved in abnormal vessel formation in the retina. This was a hallmark feature of MacTel, and thought to be a cause of the condition, although it was subsequently found to be one of its consequences.

It led to us identifying the gene LRG1, which we found was implicated in the growth of abnormal blood vessels. We realised that the LRG1 molecule might be a factor not just in MacTel, but in other eye conditions and in diseases affecting other parts of the body. At the time, there were fewer than ten publications on it, and almost nothing was known about its function.

What did you do after finding your target?

We developed a function-blocking humanised monoclonal antibody against the LRG1 protein, ‘Magacizumab’. It has already been through preclinical testing and it looks extremely promising.

More recently, we have generated a Fab fragment (an antigen-binding fragment) from our antibody: ‘MagaFab’. Rather like how Lucentis (ranibizumab, Novartis), the Fab fragment of anti-tumour drug Avastin (bevacizumab, Roche), is used for retinal disease, our aim is for Magafab to be used in eye conditions, with Magacizumab having systemic applications.

Importantly, in the context of eye disease, we have learnt that inhibiting LRG1improves clinical outcomes and reduces lesions in animal models of ocular disease. It does this totally independently of vascular endothelial growth factor (VEGF), which is the key target for most ocular treatments, such as Avastin and Eylea (aflibercept, Regeneron Pharmaceuticals). This means we have identified a completely separate pathway that may explain why some patients do not respond to current treatments.

Could you tell me about the process of developing the antibody?

It took us 4 years to generate the antibody and then engineer it so it could be used in patients without causing an immune response. We then had it manufactured, via good manufacturing practices, through a contract research organisation in San Diego, United States.

However, in preclinical studies, the full-length antibody caused an acute inflammatory response in the eye. There is always a risk that a protein may cause some inflammation, but this was a real puzzle for us, because antibodies being injected into the eye have a long history of being very safe and non-toxic.

We thought this would set us back, but then we got some seed-funding through the UCL Technology Fund, which enabled us to produce the Fab fragment, thus removing the unneeded half of the molecule which we thought might be eliciting the inflammatory response.

This time, preclinical studies yielded no adverse events. In addition, since MagaFab is a smaller molecule, more of it can be contained in solution, which means you can inject a larger, longer-lasting dose.

We have also been researching its role in cancer, as well as collaborating with scientists working on lung disease, colitis and vascular inflammation, where LRG1is also emerging as a contributing factor in disease progression. This opens up the possibility of using Magacizumab to treat patients systemically – something we were able to do without problems in our earlier safety studies. In particular, we have shown that our antibody reduces the growth of many solid tumours, and enhances the effectiveness of existing therapies such as cytotoxics and the new immunotherapies.

How does LRG1affect vasculature and thus lead to disease?

LRG1seems to affect the capacity of a blood vessel to mature and stabilise. When blood vessels grow during development, pericytes crawl along the endothelial cells which line the vessel wall.

Through interactions between these two cell types, the new vessels stabilise and the junctions between them start to become impermeable. If you disrupt this interaction, the vessels fail to stabilise and instead retain an immature and abnormal, leaky characteristic.

We think LRG1 affects the pericytes by preventing crosstalk, so blood vessels remain fragile and haemorrhagic; indeed, if we inject the molecule into the eye as the vasculature is developing, it disrupts the vessels and prevents them from growing normally. In diseases we have looked at, ranging from diabetic eye disease to cancer, LRG1 is switched on in the affected blood vessels.

This might explain why, in nearly every disease you look at, vessels do not grow normally. This opens up the possibility that LRG1 may be involved in the early stages of diabetes, where pericytes are one of the first cell types to be affected.

We know that the gene gets switched on very early in diabetes – you suddenly see levels go shooting up, even before you start to see the pathology, which is very interesting. And our studies show that LRG1 is certainly involved in the later, proliferative stage of diabetes. In fact, in both prolific diabetic retinopathy and wet AMD, when we look in the eyes of patients, we find high levels of LRG1.

What is next with your research?

We would like to see MagaFab succeed in the early-stage trials by showing that it is safe and can be tolerated in patients. Ultimately, however, we would like to see some clinical benefit for the large cohort of patients with wet AMD and diabetic eye disease who either do not respond to current treatments or respond poorly. Given how common these diseases are, if our therapy is able to meet the needs of a significant number of those patients, it has the potential to impact hundreds of thousands of people globally.

The fact that so many patients do not respond to anti-VEGF treatment indicates that processes other than the VEGF pathway are important. The same is true for cancer, which can bypass the VEGF pathway.

However, we think that because of what we know about the mode of action of the LRG1 molecule, diabetic patients may be more likely to benefit. This is because more of them fail to respond to anti-VEGFs than for wet AMD: around 50% non/poor responders in the early, diabetic macular oedema stage. We have evidence that our therapy might be good at that point, as well as later on, when you have new vessel formation.

This also raises the possibility—and this is speculation—that MagaFab would be beneficial in other eye diseases where abnormal vasculature is a problem, such as retinopathy of prematurity and inherited diseases such as Coats disease and familial exudative vitreoretinopathy.

If we can prove it is safe in the eye, then these can all be investigated. Outside of ophthalmology, we are hoping to take the full-length antibody (which is safe in the rest of the body) to cancer trials.

We have created PanAngium Therapeutics, a spin-out company, which is about to seek its first series A funding from external investors for the ophthalmic work. That would enable us to scale-up production of MagaFab and then design and embark on appropriate early-stage clinical trials, probably starting two years from now.

We will be relying on our Moorfields colleagues and the information they have about patients to identify the inclusion and exclusion criteria for potential participants into the trial. A visual acuity test will be our primary outcome measure – this is the gold standard, although we will also be measuring a number of secondary outcomes. These include optical coherence tomography scans to look at retinal thickness and, potentially, adaptive optics scanning laser ophthalmoscopy to detect vessel structure.

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Prof. Stephen Moss, PhD
E: s.moss@ucl.ac.uk
Prof. Moss holds the Norman Ashton Chair of Biomedical Research at the UCL Institute of Ophthalmology. He is a named inventor on several patents relating to annexins and to LRG1 and is a founder and hold equity in PanAngium Therapeutics.

Prof. John Greenwood
E: j.greenwood@ucl.ac.uk
Prof. Greenwood is the Hugh Davson Professor of Biomedical Research at the UCL Institute of Ophthalmology, University College London. He is an inventor on several patents relating to LRG1, and is a founder and holds equity in PanAngium Therapeutics.