Academic Trainees Meeting – 5 th May, 2011

1. Academic Trainees Meeting – 5th May, 2011 Interesting aspects of complement regulation…… Matthew Pickering Wellcome Trust Senior Fellow in Clinical Science Consultant Rheumatologist

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

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

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

5. C3bBb Complement activation Bacterial Carbohydrate, ficolins ‘always on’ immune complexes classical pathway lectin pathway alternative pathway C3 C5a C4b2a C5 activation C3b ‘C3b amplification loop’ C3b C3b C3b C3b MAC FOREIGN SURFACE MAC = membrane attack complex

6. C3bBb C1 inhibitor C4bp Complement regulation Factor H C1 inhibitor classical pathway lectin pathway alternative pathway C3 Factor H C4b2a C3b Factor I iC3b C3b Factor I MAC CD59 CD46 CR1 DAF (CD55) Factor H iC3b MAC = membrane attack complex

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

8. What does factor H do? • Critical negative regulator of the alternative pathway and C3b amplification loop • What happens to C3 levels in individuals with complete genetic deficiency of CFH? • Uncontrolled spontaneous activation of the alternative pathway and secondary consumption of C3

9. Why is factor H important? • It is associated with human disease: ‘protective’ and ‘at risk’ polymorphisms common mutations rare Dense deposit disease

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

11. C3 nephritic factor C3bBb Anti-factor H Dense deposit disease • Associated with plasma C3 activation: Factor H C3 B, D C3b

12. 600 400 Plasma C3 - mg/l 200 0 wild-type Cfh-/- 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

13. Dense deposit disease • What have the animal models taught us? • The renal disease does not develop if activation of C3 is blocked • The renal disease does develop if C5 activation is blocked • Dense deposits still develop • Glomerular inflammation reduced but not absent • Murine dense deposit disease is dependent on the ability to activate C3 but not C5 Glomerular basement membrane deposits in mice with combined deficiency of factor H and C5 Pickering MC, et al. PNAS 2006 103(25):9649-54.

14. C3 Factor I Factor H absent low low C3b iC3b, C3d Human complement deficiency Deficiency State: Plasma C3: Recurrent infection Associations: immune complex-mediated renal disease e.g. MPGN type I Dense deposit disease Pickering MC, Cook HT. Clin Exp Immunol. 2008 51(2):210-30.

15. Factor H C3bBb Plasma C3 regulation • Continuous activation of C3 occurs in plasma through the C3 ‘tick-over’pathway C3c C3d C3 iC3b Factor B Factor D Factor I C3b C3b

16. injections 200 150 100 0 24 48 72 Plasma C3 levels (mg/l) 50 0 hours Dense deposit disease • Administration of factor I to mice with combined deficiency of H and I restores GBM C3 staining Rose KL et al. J Clin Invest. 2008 118(2):608-18.

17. Why is factor H important? • It is associated with human disease: ‘protective’ and ‘at risk’ polymorphisms common mutations rare Atypical haemolytic uraemic syndrome Dense deposit disease

18. Atypical Haemolytic uraemic syndrome Alternative pathway dysregulation • Associated with: • COMPLEMENT MUTATIONS • Loss of function mutations in regulators • Factor H • Mutations • Hybrid gene (copy number variation) • Factor I • MCP (CD46) • Gain of function mutations in activation proteins • C3 • Factor B • ACQUIRED COMPLEMENT DYSREGULATION • Anti-factor H autoantibodies renal thrombotic microangiopathy Atypical haemolytic uraemic syndrome

19. C3 MAC C5a C3bBb B, D C5 activation C3b C3b C3b C3b C3b iC3b Atypical Haemolytic uraemic syndrome – factor H mutations C3 regulation Surface recognition Factor I C3b CD46 HOST SURFACE RENAL ENDOTHELIUM

20. 100 75 Plasma C3 - mg/l 50 25 0 Cfh-/- Cfh-/-FH16-20 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) Mutated mouse FH16-20 wild-type mouse CFH Renal histology in Cfh-/-.FH16-20

21. Murine model of factor H-associated atypical haemolytic uraemic syndrome • Use this model to determine contribution of C5 activation to renal injury • Spontaneous renal disease does not occur in C5-deficient Cfh-/-FH16-20 animals

22. Murine model of factor H-associated atypical haemolytic uraemic syndrome • Cfh-/-FH16-20 animals are hypersensitive to experimentally triggered renal injury – this injurious response is C5 dependent C3 C9

23. 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

24. Why is factor H important? • It is associated with human disease: ‘protective’ and ‘at risk’ polymorphisms common mutations rare Atypical haemolytic uraemic syndrome Dense deposit disease

25. Factor H and Age-related macular degeneration

26. Factor H and AMD – the ‘Y402H’ polymorphism From Sofat et al., Atherosclerosis 213 (2010) 184-90

27. Factor H and Age-related macular degeneration Alternative pathway dysregulation • Associated with: • Polymorphic variants in: • Regulators • Factor H Y402H ‘at risk’ • V62I ‘protective’ • activationproteins • C3 C3FF ‘at risk’ • Factor B Bf32Q ‘protective’ Ocular drusen Age-related macular degeneration

28. Factor H and Age-related macular degeneration 62Valine 62Isoleucine Age-related macular degeneration Functional differences in the Valine62Isoleucine CFH polymorphism 62Isoleucine more efficient at preventing red cell lysis 14nM vs. 22.6nM at 50% lysis

29. Complement dysregulation and eye disease – age-related macular degeneration Factor H 402Y* Factor H 62I Factor B 32Q C3S CFHR1/3 deletion* Factor H 402H* Factor H 62V Factor B 32R C3F Factor H null alleles C3 3923∆DG ‘protective’ polymorphisms ‘at risk’ polymorphisms mutations alternative pathway activation DDD retinopathy Ocular drusen Age-related macular degeneration Dense deposit disease *functional consequences not understood

30. Why is factor H important? • It is associated with human disease: ‘protective’ and ‘at risk’ polymorphisms common Age-related macular degeneration Meningococcal sepsis mutations rare Atypical haemolytic uraemic syndrome Dense deposit disease

31. Factor H and susceptibility to meningococcal infection Meningococcal sepsis

32. The factor H family

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

34. The factor H family: copy number variation CFH CFHR4 CFHR2 CFHR5 Most frequent CFH-CFHR allele CFH CFHR3 CFHR1 CFHR4 CFHR2 CFHR5 CFHR1-3 deletion allele polymorphism (common) Deletion homozygotes: African American 16% Hageman et al, Ann. Medicine 2006 European Americans 4.7% Others (uncommon - <1%) CFH CFHR1 CFHR4 CFHR2 CFHR5 CFH CFHR3 CFHR2 CFHR5 CFH CFH CFHR3 CFHR3 CFHR1 CFHR3 CFHR4 CFHR1 CFHR2 CFHR4 CFHR5 CFHR2 CFHR5 CFH CFHR3 CFHR1 CFHR1 CFHR4 CFHR2 CFHR5

35. Why are the factor H-related proteins important? • They are associated with human disease: ‘protective’ and ‘at risk’ polymorphisms CFHR1-3 deletion allele polymorphism associated with protection against AMD Mol Immunology 44 (2007):3921. common Age-related macular degeneration

36. Complement therapeutics Pathologies in which complement is activated

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

38. 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