1. European Congress of Pathology: Helsinki, 31 st August 2011 Complement activation and its relation to the kidney Matthew Pickering Centre for Complement and Inflammation Research

2. Overview  Complement biology: lessons from nature complement deficiency states  Complement regulation and the kidney C3 glomerulopathies  Dense deposit disease  CFHR5 nephropathy Atypical haemolytic uraemic syndrome  Complement therapeutics eculizumab

3. C3b C3 Factor B Factor D C3b FOREIGN SURFACE C3b ‘Alternative pathway C3 amplification loop’ Complement activation lectin pathway Bacterial Carbohydrate, ficolins C4b2a classical pathway immune complexes C3bBb alternative pathway ‘always on’ C5 activation MAC C5a

4. C3 Factor B Factor D C3b FOREIGN SURFACE Complement function lectin pathway C3bBb alternative pathway C5 activation C4b2a ‘safe’ disposal of immune complexes and apoptotic cells classical pathway C3b opsonization leucocyte activation augment antibody response anaphylatoxin C5a Membrane damage Cell lysis MAC C3b

5. C3 iC3bC3b Complement regulation lectin pathway C4b2a classical pathway C3bBb alternative pathway C1 inhibitor C4bp C1 inhibitor Factor H Factor I iC3b CR1CD46CD59 MAC Factor I Factor H DAF (CD55) DAF – decay-accelerating factor

6. CFHCFHR3CFHR1CFHR4CFHR5CFHR2 Regulators of complement activation gene cluster (RCA) The factor H family

7. Deletion homozygotes:African American 16% Hageman et al, Ann. Medicine 2006 European Americans4.7%

8. Disorders of complement Activation protein deficiency ‘too little’ complement Tell us what might happen if we therapeutically inhibit complement Regulatory protein deficiency ‘too much’ complement’ Provide diseases in which complement inhibiting therapies ought to be effective

9. Complement activation protein deficiency Terminal pathway Recurrent Neisseria infections C3 Infection Classical pathway SLE-like illness Recurrent infection with encapsulated bacteria e.g. pneumococci, Haemophilus influenzae Vasculitis, glomerulonephritis

10.  Physiological control of complement activation REGULATORS Complement dysregulation and disease: ACTIVATORS Loss of functionGain of function The balance is influenced by mutations (extreme) and and/or polymorphisms (‘fine tuning’)

11. Complement dysregulation and disease: Terminal pathway dysregulation Paroxysmal nocturnal haemoglobinuria C1 inhibitor deficiency [classical pathway dysregulation] Hereditary angioedema Alternative pathway dysregulation Dense deposit disease, Atypical haemolytic uraemic syndrome renal thrombotic microangiopathy

12. C1 inhibitor deficiency - treatment C1 inhibitor deficiency Hereditary angioedema C1 inhibitor concentrates Recombinant C1 inhibitor Berinert™ CSL Behring Viro Pharma Rhucin™ Pharming

13. Paroxysmal nocturnal haemoglobinuria Terminal pathway dysregulation Paroxysmal nocturnal haemoglobinuria Eculizumab - Soliris™ Alexion Pharmaceuticals Humanised anti-C5 antibody

14.  Pathogenesis of haemolysis: CD59 deficiency results in uncontrolled terminal pathway activation and formation of membrane attack complex  cell lysis CD59 DAF Charles Parker, Lancet, 2009, vol. 373, pp759-67. DAF – decay-accelerating factor Paroxysmal nocturnal haemoglobinuria

15. C3b C3 Factor B Factor D C3b PNH-RED CELL SURFACE C3b ‘Alternative pathway C3 amplification loop’ C3bBb alternative pathway ‘always on’ C5 activation MAC C5a eculizumab Humanised monoclonal antibody against complement C5

16. Paroxysmal nocturnal haemoglobinuria  Eculizumab Clinical trials shown to reduce transfusion requirements Drug is safe and well-tolerated Side-effects:  Increased risk of Neisserial infections  patients must be immunised before starting treatment  May reveal additional mechanisms of anaemia  Allows accumulation of C3 on PNH-red cell  In some patients this results in extra-vascular haemolysis and may result in eculizumab resistance Not ideal – indiscriminate C5 blockade rather than targeting to surface of PNH-red cell Expensive!  One year treatment - £252,000 per patient…… Risitano et al, Blood, 2009, vol. 113, pp4094-4100. Hillmen et al, Blood, 2007, vol. 110, pp4123-28.

17. Alternative pathway dysregulation and disease  It is associated with renal disease: Dense deposit disease Atypical haemolytic uraemic syndrome mutations rare ‘protective’ and ‘at risk’ polymorphisms common

18. Dense deposit disease  Electron-dense transformation of the glomerular basement membrane Glomerular C3 staining in DDD DDD retinopathy

19. 12% 18% 45% 25% Dense deposit disease  Heterogeneous light microscopic appearances Patrick Walker et al., Modern Pathology, 20; 605-16, 2007 Mesangial proliferation Membranoproliferative Crescentic Acute proliferative & exudative

20. C3 glomerulopathy  Glomerular pathology associated with deposition of complement C3 in the absence of immunoglobulins Dense deposit disease CFHR5 nephropathy

21.  Impaired plasma regulation REGULATION  Dense deposit disease ACTIVATION  Loss of function FACTOR H Gain of function C3 plasma regulation surface recognition

22. Dense deposit disease C3b C3 C3bBb Factor H B, D C3 nephritic factor Anti-factor H  Impaired plasma regulation plasma regulation surface recognition

23. Models of ‘dense deposit disease’  Animal models : Spontaneous porcine factor H deficiency and gene-targeted factor H-deficient mice  Profound plasma C3 depletion – 5% of normal C3 levels  Spontaneous renal disease – ‘murine/porcine DDD’ Factor H deficiency Wild-type C3 staining wild-type 0 200 400 600 Cfh -/- Plasma C3 - mg/l

24. Alternative pathway dysregulation and disease  It is associated with renal disease: Dense deposit disease Atypical haemolytic uraemic syndrome mutations rare ‘protective’ and ‘at risk’ polymorphisms common

25.  Impaired surface regulation REGULATION  Atypical Haemolytic uraemic syndrome ACTIVATION  Loss of function FACTOR H FACTOR I CD46 Gain of function C3 FACTOR B plasma regulation surface recognition

26. Atypical Haemolytic uraemic syndrome C3b C3 Factor B Factor D C3b RENAL ENDOTHELIUM C3b ‘Alternative pathway C3 amplification loop’ C3bBb alternative pathway ‘always on’ C5 activation MAC C5a

27. Murine model of factor H-associated atypical haemolytic uraemic syndrome  Gene-targeted factor H-deficient mice transgenically expressing a mutant mouse factor H protein (FH  16-20) Cfh -/-  FH16-20 Cfh -/- 0 25 50 75 100 Plasma C3 - mg/l wild-type mouse CFH Mutated mouse FH 16-20 Renal histology in Cfh -/-.FH16-20

28. Atypical haemolytic uraemic syndrome - therapy  C5 inhibition successful in case reports – examples: Eculizumab for aHUS – N. Engl. J. Med. 2009 360:5 pp542-543 Eculizumab for congenital aHUS – N. Engl. J. Med. 2009 360:5 pp544-6  Open Label Controlled Trial of Eculizumab in Adult Patients With Plasma Therapy-sensitive / -resistant Atypical Hemolytic Uremic Syndrome (aHUS) Successful outcomes announced in ASN 2010 meeting http://clinicaltrials.gov/ct2/results?term=eculizumab

29. Alternative pathway dysregulation and disease  It is associated with renal disease: Dense deposit disease Atypical haemolytic uraemic syndrome mutations rare ‘protective’ and ‘at risk’ polymorphisms common

30. Factor H and Age-related macular degeneration

31. Factor H and susceptibility to meningococcal infection Meningococcal sepsis

32. Alternative pathway dysregulation and disease  It is associated with renal and non-renal disease: Dense deposit diseaseAtypical haemolytic uraemic syndrome mutations rare ‘protective’ and ‘at risk’ polymorphisms common Age-related macular degeneration Meningococcal sepsis

33. The factor H family

34. Why are the factor H-related proteins important?  They are associated with renal disease: mutations rare ‘protective’ and ‘at risk’ polymorphisms common

35. C3 glomerulonephritis

36. CFHR5 nephropathy  Familial C3 glomerulonephritis and mutation in CFH-related protein 5 Aberrantly increased size protein detected in sera CLINICAL FEATURES Autosomal dominant Persistent microscopic haematuria Episodes of synpharyngitic macroscopic haematuria Progression to end stage renal disease mainly in males

37. CFHR5 and renal complement  Does CFHR5 regulate renal complement deposition? IgA Post-infectious MembranousClass IV lupus Am J Kidney Dis, vol 39, N 1, 2002: pp24-27

38. Why are the factor H-related proteins important?  They are associated with renal disease: mutations rare ‘protective’ and ‘at risk’ polymorphisms common PLOS GENETICS May 2011

39. Why are the factor H-related proteins important?  They are associated with renal disease: mutations rare ‘protective’ and ‘at risk’ polymorphisms common Age-related macular degeneration CFHR1-3 deletion allele polymorphism associated with protection against AMD

40. Complement and tissue injury Pathologies in which complement is activated

41. Antibody-mediated injury to allografts  Complement-fixing donor-specific antibodies  Associated with peritubular C4d staining  Can complement inhibition prevent / ameliorate antibody- mediated rejection? Eculizumab Alexion Pharmaceuticals Humanised anti-C5 antibody C1 inhibitor concentrates Berinert™ CSL Behring Viro Pharma

42. Complement therapeutics Examples of the many complement inhibitors in development Eric Wagner and Michael Frank Nature Reviews 2010, vol. 9, 43-56.

43. Summary  Abnormal regulation of complement is associated with disease The kidney appears to be particularly susceptible Renal pathologies resulting directly from complement dysregulation include:  Atypical haemolytic uraemic syndrome  C3 glomerulopathies  Dense deposit disease  CFHR5 nephropathy  Our understanding of these rare pathologies may be very relevant to more common renal pathologies  Eculizumab is the first complement inhibitor and is highly effective in: Preventing red cell lysis in paroxysmal nocturnal haemoglobinuria Atypical haemolytic uraemic syndrome  Eculizumab may be indicated in more common renal pathologies...

44. Thanks  Elena Goicoechea de Jorge  Katherine Vernon  Mitali Patel  Kirsten Rose  Talat Malik  Sharmal Narayan  Marieta Ruseva  Tamara Montes  Lola Sanchez-Nino  Danielle Paixao-Cavalcante  Fadi Fakhouri  Terence Cook  Marina Botto  Santiago Rodriguez de Cordoba  Veronique Fremeaux -Bacchi  Patrick Maxwell  Danny Gale