Identifying, characterising and evaluating antibodies in Guillain-Barré syndrome

Inflammatory neuropathies are conditions in which the immune system mistakenly attacks and damages peripheral nerves. This leads to progressive weakness, numbness, and imbalance. In severe cases, patients become completely paralysed, unable to talk or breathe for themselves. It is critically important that we rapidly improve on the current situation where some patients spend months on intensive care and many more are left with persistent disability.

Recently, several research groups, including our own, have identified disease-causing 'nodal' antibodies which target a specific and important part of the nerve fibre in a proportion of patients with inflammatory neuropathies. Crucially, these patients have more severe illness and respond poorly to standard treatments. However, they can markedly improve if their antibody-producing cells are targeted directly. Following on from this, we established a nodal antibody diagnostic testing service, and led an NHS commissioning review. This means that doctors in the UK now have a simple means of identifying these patients and then accessing a more effective treatment.

Unfortunately, the majority of patients with the most common inflammatory neuropathy, known as Guillain-Barré syndrome (GBS), still do not have any clinically useful blood markers to help with diagnosis or guide treatment, and receive only poorly targeted, incompletely effective and non-specific immune-supressing therapies.

Using live human nerve fibres, generated from stem cells in our laboratory, we have shown that a proportion of GBS patients nevertheless have nerve-reactive antibodies in their blood that are not detected by current clinical tests. At present, the precise target and relevance of these antibodies is unclear.

In this project, we will identify the target of these antibodies, develop efficient tests to detect them in large numbers of samples, and establish their implications for patients.

To do this, we will use the method we successfully employed to detect and identify nodal antibodies. Firstly, we apply blood samples from patients to dishes of human motor or sensory nerves, Schwann cells, or combinations of these cell types. In the 'co-cultures', Schwann cells align with the nerve fibres and wrap them in myelin insulation. This also forms nodes and their surrounding structures, similar to those found in the body itself. We can then identify which cell types or nerve regions are targeted by patient's antibodies by labelling them with a fluorescent marker and looking at the pattern of binding under the microscope. We have already detected new, nerve-targeting antibodies using this technique in over 20 patients with GBS and related neuropathies. To broadly identify the type of molecule attacked by the antibodies, we will use a range of chemical treatments to modify the nerves. We will then check to see whether they reduce antibody binding.

To specifically identify the target, we will grow larger dishes of nerves. We will select patient samples which produce the most intense and widespread nerve binding, and incubate them with the cells. After careful washing, the antibodies will remain stuck to their target on the nerves. By breaking up the fibres and capturing the antibodies using specially coated magnetic beads, we can also purify out the target molecule which they remain bound to. We will then run the purified molecule though a mass spectrometry machine to identify it.

We will then develop specific tests using manufactured chemical or genetically modified cells to confirm these initial results. These more efficient tests will then be used to assess how often these antibodies are found in the neuropathy patients versus healthy controls and to evaluate whether they can help diagnosis, provide information on disease severity, or guide treatment. Blood will be obtained from national/international repositories of neuropathy patient samples linked to detailed clinical information.

The acute inflammatory neuropathy Guillain-Barrè syndrome (GBS) involves devastating immune-mediated damage to peripheral nerves. Affected individuals develop progressive paralysis and sensory loss, frequently necessitating ventilatory and intensive care support. There is an urgent need to improve the current situation where non-specific and incompletely effective immunotherapies leave many patients with persistent disability.

We and others have recently identified disease-causing antibodies targeting specific proteins at the node of Ranvier in a subset of patients with the rarer chronic inflammatory demyelinating polyneuropathy (CIDP). We have shown that patients with these antibodies have distinct features. Crucially, they respond poorly to standard therapies, yet can show marked and sustained improvements following treatment with CD20/B-cell targeting agents such as rituximab.

However, the majority of patients with GBS are seronegative for all currently identified antibodies. Despite GBS undoubtedly encompassing a range of underlying pathologies, patients typically receive standard therapies, to which a significant proportion do not respond. Using myelinating co-cultures of human stem-cell derived sensory neurones and rat Schwann cells as an unbiased screen, we are able to detect peripheral nerve reactive antibodies in 10-20%. However, the target of these antibodies, and whether they indicate a mechanistically distinct disease process and/or influence treatment responses, is currently unclear. In this project, using an established and previously successful immunoprecipitation/mass spectrometry workflow, we will identify the relevant antigens. High throughout immunoassays will then be developed to confirm these findings and assess the frequency and clinical implications of the antibodies using established national/international neuropathy biobanks linked to detailed clinical and outcome data.