Back to basics; the role of complement C3 and its degradation fragments in immuno-inflammatory disease

We will bring together experts in complement-mediated disease from around the UK and focus our attention on diseases of kidney, eye and brain. Complement is part of the innate immune system which reacts immediately to kill infectious agents, or pathogens, when they enter the body. Complement is known to attack these organs in a large number of diseases, including multiple sclerosis, C3 glomerulopathy, the common ageing disease macular degeneration, and many others. Anti-complement drugs being developed for these diseases work by directly turning complement off, this can leave patients vulnerable to infection. We investigate a new hypothesis that cross-talk of complement with cells of the immune system might actually be responsible for some of these diseases. If this is true, then other kinds of treatments might be more effective for therapy.

Complement activates very swiftly and coats pathogens in proteins that marks them for 'engulfment' by white blood cells, resulting in death and 'clearance'. To enable this quick reaction, complement is always active at a low level in the body, thus our own cells can accidentally become coated with complement proteins. In health, this coating is minimal and causes no harm; however, in some circumstances these deposits accumulate in tissues and lead to disease. This can be due to mutations in our genes that prevent correct functioning of 'safe-guarding' mechanisms, or abnormal triggers, such as auto-antibodies which bind cells, tricking the immune system into thinking the cells are foreign or infectious.

We have a lot of knowledge around mechanisms leading to different complement-mediated diseases. To date we have focussed on the 'activating' mechanisms that inappropriately drive complement deposition on our own cells. However, recent data from animal models, and some interesting genetic linkages discovered in humans, have led to the realisation that the downstream inactivation or clearance processes might actually be causing disease.

We will generate and share new research tools, called antibodies, which will enable us to take a close look at complement in human tissues. We will work together to produce preliminary, yet critical, data to validate our hypothesis and build the groundwork for a substantial follow-up proposal.

We will use these new research tools:

-To develop highly specific assays to measure the complement activation fragments, known as 'biomarkers', in samples of blood from healthy people and from patients. In many diseases, the complement system leaves a diagnostic 'signature' in blood, a readily accessible tissue. The novel antibodies will provide more detailed information and give new insight into mechanisms and drivers of disease. The output of this part of the study will be numerical and quantitative.

-To provide an accurate picture of exactly how and where complement is attacking the different organs. We will use a technique called immunohistochemistry to detect the kind of fragment present in tissues and determine which parts of the immune system, including cells, are being recruited to those tissue sites where they cause damage. The output of this part of the study will be scientific images or pictures of the tissues.

-To model and understand the disease situation in the laboratory ('in vitro' studies). We will use samples of blood modified to resemble blood from patients with complement-mediated disease. We will use the assays validated in the first aim to understand the link between disease mechanism and complement biomarkers and sow the seeds for our follow-up proposal which will link together the relevant in vitro functional assays with wider studies of blood biomarkers and tissue imaging.

This detailed examination of the mechanisms behind complement-mediated inflammatory diseases will guide our pursuit of drugs suitable to treat both rare and common diseases and will potentially bring benefit to a huge number of patients.

We will establish a collaborative network of experts in complement dysregulation, each focussed on different diseases and organs. Traditionally, the role of complement in inflammatory disease has been assigned to the central activation fragment, C3b, which is the driving force behind the complement amplification mechanism. However, recent data in genetically-modified animals, and genetic associations in man, have implicated downstream C3 degradation products in pathogenic processes.

In this proposal, novel recombinant antibodies will be used in well-characterised cohorts of patients with diseases of different organs: the eye, brain and kidney. The antibodies will be used for three purposes:

1. To measure precisely levels of different C3 fragments in plasma and extend the biomarker dataset associated with established biobanks of samples and patient cohorts. Findings will be correlated with the extensive genetic and clinical phenotypic data already available for these collections.

2. To stain sections from the three organs for different C3 fragments, obtaining information regarding cellular location and leukocyte engagement. The diseases of focus in this pilot study are C3 glomerulopathy (kidney), age-related macular degeneration (eye) and multiple sclerosis (brain).

3. To carry out in vitro functional assays to correlate the biomarker data with genotypic and phenotypic data observed in patients with specific complement gene mutations.

We aim to identify common mechanisms across a number of inflammatory diseases in diverse organs involving dysregulation of the complement system. The unique research tools described here will be used to validate a mechanism in sufficient depth to warrant a large scale follow-up proposal to confirm and explore common pathogenic processes utilising well-validated cohorts of patients.

The proposal will lead to the following socio-economic benefits.

1. For patients:

Patients will benefit directly from the research due to increased power in diagnosis, patient stratification for clinical trials, patient monitoring and development or repurposing of new drugs.

The NRCTC at Newcastle is a hub for clinical trials in anti-complement therapy, with two current investigator-led trials (eculizumab withdrawal and eculizumab in STEC HUS) and participation in numerous company trials (drugs in development). Clinicians at the NRCTC are directly involved with developing and applying NHS England policy with regards to aHUS and C3G (National aHUS service; National C3G/MPGN Service). The NRCTC is also firmly positioned at the interface of academic research and patient treatment (see Pathways to Impact) with strengths in complement diagnostics/biomarkers and patient treatment. Thus we are perfectly positioned at a local and national level to translate our research to the clinic. Better treatments for patients will lead to improved health and quality of life with less frequent trips to hospital and disruption to schooling or employment (for example, intravenous infusion of eculizumab is required every two weeks for aHUS patients).

Age-related macular degeneration AMD is the leading cause of blindness in Caucasians over 75 years (present in 1.7% of all people aged over 50, with incidence rising with age: 0.7-1.4% in people aged 65-75, 11.0-18.5% in people aged over 85). Currently there is no therapy for the Geographic Atrophy, the dry form of the disease. The applicant, Dr Clark, and his colleagues at the Manchester Eye Hospital, similarly engage with clinical trials of new drugs. Of note, anti-complement therapies developed to date for AMD, are injected directly into the eye every month and no phase 3 clinical trial has yet succeeded. Identification of disease mechanisms and development of effective therapies will have a huge impact with improved quality of life for a significant swathe of the UK population.

We expect to reveal common mechanisms underlying numerous complement-mediated diseases, not just those of the eye, kidney and brain. Thus we will benefit many different patient populations, with potential impact in patients with diseases including those of the blood (paroxysmal nocturnal hemoglobinuria), joints (arthritis), skin (bullous pemphigoid) and many other organs.

2. For the NHS / Policy makers.

The NHS will benefit in multiple ways: i) Patient burden; targeted and more effective therapies for patients will lower burden on the NHS and improve patient well-being and quality of life. Common diseases such as AMD, are a huge burden to the NHS due to the large patient populations and impact on the elderly. ii) Decreased cost; the only approved anti-complement drug, eculizumab (anti-complement C5), costs £327,600 per person per year. By defining downstream pathways involved in disease pathogenesis and understanding the role of other immune effectors in the disease process, the door may be opened for trial of other classes of drugs, including immune-modulatory therapeutics. iii) Improved diagnostics; this incorporates not only at disease presentation, but also for patient monitoring and stratification of patient populations for clinical trial.


3. For the industrial sector; Pharma and Biotech companies.

Commercial companies developing drugs and/or diagnostics will benefit from public dissemination of knowledge, which will strengthen internal research and drug discovery programmes. Increased power to detect disease biomarkers will aid patient stratification and monitoring of response to therapies. All applicants are engaged with industry in various capacities, including consultancy, research collaboration and clinical trials. These interactions facilitate bidirectional sharing of non-confidential knowledge, ensuring informed decisions, cutting-edge research and optimised patient treatment.