Understanding the Properdin Paradox using C3 D1115N (gain-of-function) knock-in mice

We at the National Renal Complement Therapeutic Centre (NRCTC) were one of the first to recognise that eculizumab (Soliris, a drug that blocks part of our immune system known as complement) is a highly effective treatment in many patients with rare blood and kidney diseases, particularly paroxysmal nocturnal haemoglobinuria (PNH) and atypical haemolytic uraemic syndrome (aHUS). However, we also readily accept that eculizumab is incredibly expensive, it does work on all patients with aHUS (or indeed in many patients with a related disease known as C3 glomerulopathy) and because it completely switches off an important component of our immune system, it leaves patients treated with the drug highly susceptible (>1000 fold more so) to infections that can cause meningitis. Therefore, the need for better anti-complement drugs is clear.

Many such drugs are in development that target complement component C3. C3 is at heart of the complement system, an important part of the alternative pathway of complement activation which is a self-activating amplification loop and drives much of the anti-bacterial function of complement. Use of drugs that target this part of the complement system may have significant side effects with respect to susceptibility to infection and poor response to vaccines.

Models systems to test drugs that target C3 in a comprehensive, realistic manner, with respect to aHUS, are generally lacking. Over the last decade we have successfully generated several unique copies of mouse C3 molecules based on changes (or mutations) found in human C3 that associated with aHUS. In doing so, we demonstrated that we could transfer functional changes from patients to a mouse model. In this case, we generated a C3 molecule which was hyperactive. Mice with this change on both gene copies of C3 rapidly develop aHUS.

Our analysis confirms that blockade of the complement system in a manner equivalent to use of eculizumab in man completely protects mice from aHUS and therefore validates the model for testing of other anti-complement drugs.

Recent work by our colleagues has suggested that drugs that block the function of another protein in the complement system, known as properdin, may prevent aspects of aHUS and indeed, may be useful in PNH. However, other studies examining the blockade of properdin in C3 glomerulopathy were catastrophic. This leaves important questions around the suitability of use of anti-properdin drugs in aHUS to be resolved. Furthermore, addressing these questions may have significant impact on our knowledge of properdin function overall and allow better choice of anti-complement drug use in more general conditions such as common autoimmune diseases like rheumatoid arthritis and systemic lupus erythematosus or indeed during organ transplantation.

Therefore, the aim of this study is to provide detailed data regarding the efficacy of using anti-properdin drugs in the treatment of aHUS using the most sophisticated small animal model of aHUS available. To compare these findings to the current front line anti-complement drugs. To assess whether new anti-complement drugs can provide better protection than existing drugs regardless of the complement protein variant that caused the disease, i.e. establish the need or provide the rationale for a detailed framework for personalised medicine in the treatment of aHUS patients for the future.

Finally, as activation of the complement system is involved in most, if not all, inflammatory conditions (such as age-related macular degeneration and control of cancer) and is linked to many autoimmune diseases (such as rheumatoid arthritis and lupus), the further validation of this comparative model, which has hyperactive C3 at its heart, will cement its place as the premier model to analyse the role of complement activation in the progression, and the treatment, of a wide range of inflammatory diseases/conditions.

Recent published data suggests that blockade of properdin (FP), a key positive regulator of the alternative pathway of complement (AP), can prevent thrombotic aHUS. However, previous studies in the related AP driven disease, C3 glomerulopathy, suggested FP blockade exacerbated disease.
We have recently generated a C3 gain-of-function mouse model of aHUS based on the C3 D1115N change found in the NRCTC patient cohort. Homozygous C3 D1115N mice develop aHUS by post-partum day 21 (as assessed by all biochemical and histological criteria) which can be reversed or blocked through either use of BB5.1 or genetic deletion of C5.
Interestingly, our pilot data indicates that FP blockade is ineffective in preventing disease in the C3 D1115N model.
Herein, we wish to establish if FP blockade is a valid treatment for aHUS (as a result of C3 variance) by:
1. Treating homozygous C3 D1115N mice with H4 (anti-FP) either after disease onset (proteinuria and haematuria present) or prophylactically from post partum day 14. Increased mouse survival, improved biochemical and cellular characteristics with reduced kidney pathology would be anticipated if anti-FP works in C3 variant driven aHUS.
2. Backcross onto the FP-/- knockout mice followed by comprehensive analysis as above.
3. Using SPR (Biacore) to assess changes in binding and interaction of FP to recombinant or ex vivo purified and generated C3bBb in the presence of C3 variants. Furthermore, the ability of recombinant Factor H (FH) variants influence FP function and vice versa would also be analysed to gain a much clear picture of why modulation of FP can have disparate effects in murine models of aHUS and C3G.
4. Establish the optimal complement therapeutic regime to treat individuals with C3 variants. We will compare the effectiveness of murine versions of complement therapeutics i.e. Homodimeric minimal FH, anti-FP, anti-Factor B to that of BB5.1 in the model to assess the most efficacious treatment regime.

Who will benefit?
Complement system malfunction is linked to clinical conditions affecting nearly every organ in the body. Hence our first and second outputs - the full understanding of the role of properdin in a novel model of aHUS and the effect of blocking properdin - could impact indirectly on the work of physicians, and potentially on clinical practice and disease management in all renal units. The ability to perform reliable, insightful pre-clinical tests on complement-suppressing molecules, tied to our third output - significant steps towards an optimal complement therapeutic strategy for treating individuals with C3 variants - will expedite the development of anti-complement therapeutics and will directly benefit pharmaceutical companies and of course patients and their relatives and carers.

How will they benefit?
1. A deeper understanding of the relationship between the complement system and disease; aHUS and C3G may be orphan diseases but they devastate the lives of patients who routinely progress to end-stage renal failure. While eculizumab (Soliris) can treat aHUS in most cases, it is extremely expensive, and has had mixed success in C3G, with trials ongoing in MPGN. Extravascular haemolysis is problem in PNH, even when disease is managed by eculizumab, another disease of driven by complement dysregulation. Anti-properdin therapy has been shown experimentally to be of benefit in this disease also. Thus, any additional data on the safety of these drugs will be of significant interest to patients and clinicians
The links discovered between inherited variations in complement genes and disease were key breakthroughs, but the mechanisms linking protein sequences to disease symptoms remain poorly understood, particularly when it comes to the role of properdin. The work proposed here on observations of phenotypic outcomes of a single-nucleotide substitution in complement C3 in conjunction with the role of properdin will help to address fundamental questions such as: why does a change in C3 sometimes result in aHUS while others results in C3G? Can mice (and by extension, humans) have a "bad complotype" and what are the consequences? Can mice like man replicate the other genetic and environmental conditions that contribute to illness?

2. The generation of the mice models in this proposal will allow pre-clinical screening of potentially therapeutic agents designed to correct defects in the complement system in man. This will benefit a range of big and small pharma companies currently competing to get anti-complement therapies to market.

3. Improving quality of life for patients inflicted by deregulated complement activation is a major strategic goal. Complement maintains homeostasis as well as fighting infections. Complement malfunctions increase with age, for example the largest single risk factor of susceptibility to age-related macular degeneration are linked to polymorphisms in complement proteins that control C3 activation; there is the link between Alzhemier's disease and variants in the genes for clusterin and complement receptor-type 1. The mouse models we are developing are based on 'subtle' individual residue substitutions (rather than gene deletions) affecting specific biological functions of multifunctional proteins. These will allow the chronic effects of tiny genetic variations and long-term therapeutic interventions - for example elevating or reducing properdin, FH or FI levels or administering homodimeric minimal FH or TT30 - to be assessed over a lifespan.