Unravelling the pathophysiology of HUS in light of recent discoveries on complement activation Marina Noris November 13, 2018 1
THROMBOTIC MICROANGIOPATHY Histology lesions: Swelling and detachment of endothelial cells, accumulation of fluffy material in the subendothelium, thrombi and obstruction of the vessel lumina. Ischemia Stenosis Endothelial injury subendothelial espansion Stenosis Organ dysfunction Thrombosis Thrombocytopenia 2 Shear stress RBC fragmentation Ruggenenti and Remuzzi, Kidney Int, 2001
dhus Stx Adhesion molecules and P-selectin up-regulation ahus Gene mutation Defective complement regulation TTP ADAMTS13 UL-VWF multimers stma Autoimmune or systemic diseases, glomerulopathies, tx, infections Complement activating conditions Microthrombi 3
ATYPICAL HEMOLYTIC UREMIC SYNDROME Ryan ahus Incidence: 0.5-2/1,000,000/year Sex: no difference 4 Patients free of events Death or ESRD (%) 100 80 60 40 CFH mutation 20 0 0 2 4 6 8 Follow up (years) 10 12
100 80 Prevalence of low C3 P < 0.001 Prevalence of low C4 60 (%) 40 P = 0.005 20 20 (% %) 10 0 Cases Controls Cases Controls 0 Cases Controls Cases Controls Relatives Relatives Within families, subjects with lower than normal C3 serum levels had a relative risk of HUS of 16.5 as compared to subjects with normal C3 levels 5 Noris et al., J Am Soc Nephrol, 1999
COMPLEMENT ACTIVATION PATHWAYS Classical pathway IgM, IgG Immune complexes Lectin pathway Mannose residues Alternative pathway bacteria, bacterial toxin, LPS, tick-over C1q,C1r,C1s MBL, MASP C3 C3a C1-est inhib C4,C2 Factor B Factor H C4bp C4bC2a Bb C3 convertase C3 convertase C3 C3a CR1 Factor I DAF MCP C3 C3a C4bC2a () 2 Bb C5 convertase C5 C5 convertase 6 C5a C5b-9 (MAC) CD59
Factor H plays a pivotal role in the regulation of the alternative pathway of complement activation Produced mainly in the liver as a single peptide glycoprotein, factor H circulates in plasma at a concentration of 50 mg/dl Regulatory domain Recognition doman proteoglycans proteoglycans 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Y118Ifs*4 L98F R53C S58A R60G C66Y c.155delagaa Y235C C309W Q400K c.1494dela C597stop C630W C673Y S714stop c.2303insa E850K M823T C853Y - >100 different genetic abnormalities in about 30% of patients - 90% are heterozygous, most in the recognition domain Q950H Y951H T956M I970V W978C S890I H893R Y899D Y899stop C915S Q924stop C926F c.22686del15bp Y1021F C1043R W1037stop Q1076E D1119G D1119N P1130L V1134G E1135 Q1137L Y1142D Y1142C W1157R C1163W c.3486dela c.3493+1g>a P1166L V1168stop I1169L E1172stop Y1197C W1183L, W1183R W1183C T1184R L1189R L1189F S1191L S1191W G1194D E1195stop V1197A E1198A, E1198K F1199S V1200L R1203W G1204E R1206C R1210C, S1211P R1215G,R1215Q T1216stop C1218R P1226S c.3675_3699del c.3695_3698del Warwicker et al., Kidney Int, 1998 Caprioli et al., JASN, 2001 Richards et al., Am J Hum Gen, 2001 Perez-Caballero et al., Am J Hum Gen, 2001 Dragon-Durey et al., J Am Soc Nephrol, 2004 Caprioli et al., Blood, 2006 Noris et al, CJASN 2010
Fluid phase C3 convertase domain Alternative pathway activation (spontaneous hydrolysis, bacteria, viruses) recognition C- FH -N regulatory CFB FD Bb CFI CFH via regulatory domain dissociates Bb from and favors inactivation i C3 CFH via recognition domain binds on endothelium and regulates complement on cell surface C3a CFB FD CFI i CFI Bb Glycosaminoglycans Endothelial cell 8
SINGLE AMINO ACID CHANGES IN SCR 20 OF FACTOR H AFFECT ENDOTHELIAL CELL BINDING co HUVEC incubated with recombinant wild type or mutated factor H stained with fluorescinated anti-factor H antibody and analyzed by FACS Factor H R1210C Factor H Flow cytometry 9 Factor H R1210C WT Factor H Manuelian et al, J Clin Invest, 2003
CONSEQUENCES OF CFH MUTATIONS C- 20 CFH Fluid phase C3 convertase Alternative pathway activation (spontaneous hydrolysis, bacteria, viruses) -N CFB C3 Bb CFI i C3a 20 CFB CFI i Surface bound C3 convertase Bb C5 C5a C5 convertase 10 Glycosaminoglycans Endothelial cell
HIGH HOMOLOGY IN THE REGULATORS OF COMPLEMENT ACTIVATION GENE CLUSTER CFH/CFHR1 hybrid gene High degree of sequence identity between the gene for factor H and the genes for the five factor H-related proteins (CFHR1 to 5) which favors non-allelic homologous recombinations giving rise to hybrid genes. Copy number variation assays are required to detect hybrid genes 11 Venables et al., Plos Medicine 2006 Valoti et al, JASN 2015
CFH/CFHR HYBRID PROTEINS FOUND IN PATIENTS WITH ahus CFH/CFHR1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 4 5 Venables et al., PLoS Med 2006 CFH/CFHR1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 5 19 Valoti et al., JASN, 2015 CFH/CFHR3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 1 2 3 4 5 Francis et al., Blood, 2012 CFH/CFHR3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1 2 3 4 5 Challis et al., Blood, 2015 In the CFH/CFHR hybrid molecules the C-terminal SCRs of CFH are substituted with those of CFHR1 or with the entire CFHR3, resulting in decreased complement regulatory activity on endothelial cell surface
HOMOZYGOUS DELETION OF CFHR1-CFHR3 GENES IS ASSOCIATED WITH FORMATION OF ANTI-CFH AUTOANTIBODIES CFH 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 CFHR1 1 2 3 4 5 Blanc et al., J Immunol, 2012 Eight percent of patients (mostly children) develop anti-cfh autoantibodies Most patients with ahus and CFH autoantibodies are homozygous for a deletion of genes encoding CFH related proteins 1 and 3 Jozsi et al, Blood, 2008 Moore et al, Blood 2010 Failure of central and/or peripheral tolerance to FH related 1 in subjects completely lacking CFHR1? 13
MCP VARIANTS IN ahus spontaneous hydrolysis, bacteria, viruses 1 C35X C35Y (n=3) E36X P50T R59X (n=4) C64P K65D C3 SCRs 2 192T>C+193-198del C99R R103W G130V (96-129)del+G130I+Y132T+L133X STP 3 4 Y189D G204R T267fs270X (858-872)del+D277N+P278S F242C C3a TM A353V Y328X CFB CT MCP 10% CFI MCP i Endothelial cell Loss of function heterozygous variants: low expression or reduced binding and cofactor activity 14 Noris et al, Lancet 2003 Richards et al, PNAS 2003 Caprioli et al, Blood 2006
C3 IN ahus β-chain α-chain MG1 MG2 MG3 MG4 MG5 MG6β LNK ANA α NT MG6α MG7 CUB g TED CUB f MG8 Anchor C345C T140R T140K Q163E C3 7-10% R456L R570W K1029M R1041S D1093N I1135T T1361M C3 C3a CFB CFB C3 convertase CFI C5 MCP i Bb C5 convertase Glycosaminoglycans Gain of function C3 mutations: reduced binding to MCP and CFH, increased affinity to CFB Endothelial cell Goicoechea et al., PNAS, 2007 Roumenina et al., Blood, 2009 Fremeaux-Bacchi et al, Blood 2008
GENETICS OF AHUS: AN EUROPEAN DISCOVERY CFH 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 CFH/CFHR1 hybrid MCP Newcastle 1 Jena C3 2 3 anti FH Ab Paris ~60% Del CFHR3/CFHR13 THBD CFI Bergamo Madrid CFB 1 16 4 2 3 17 18 19 20
COMBINED COMPLEMENT GENE ABNORMALITIES IN ahus 795 patients from 4 European cohorts 8.7% 8.2% 10% 22.6% 27% Combined complement gene abnormalities were found in about one quarter of patients with MCP or CFI mutations, and in around 10% of patients with CFH or C3 or CFB mutations 17 Bresin et al, JASN 2013
THREE CFH COMMON POLYMORPHISMS TAGGING THE H3 HAPLOTYPE ARE STRONGLY ASSOCIATED WITH AHUS Genotypes Odds Ratios (95 % C.I.) P value -332C/T Promoter c.2016a/g (p.q672q, SCR11) c.2808g/t (p.e936d,scr16) -332T+c.2016G+p.936D High risk 1 2 3 4 5 6 7 8 9 0.0015 < 0.0001 < 0.0001 < 0.0001 Caprioli et al., Hum Mol Genet, 2003 18 Pickering et al., J Exp Med, 200
TRIGGERING /UNDERLYING CONDITIONS % of HUS patients 40 30 20 10 URT infection Diarrhea Pregnancy Others (malignant hypertension, glomerulopathy, de novo post-tx, systemic diseases) 0 CFH MCP CFI C3 Gene mutations 19 Noris et al, CJASN 2010
Trigger C3 C3a Eculizumab A humanized monoclonal antibody that binds to C5 PMP CD40L CFI CFB C3 convertase C3 C5 convertase C5a Coagulation Platelet aggregation MCP Bb Bb C5 C5b C5b-9 VWF Endothelial cell Endothelial cell 20 Modified from Noris and Remuzzi, NEJM 2009
LONG TERM OUTCOME OF ahus PATIENTS 100 Patients fre ee of events Death or ESRD (%) 80 60 40 20 MCP mutation THBD mutation C3 mutation No mutation CFI mutation CFH mutation 0 0 2 4 6 8 10 12 Follow up (years) Noris et al CJASN 2010
Platelet count mean change from baseline Estimated GFR mean change from baseline 140 60 120 45 100 (x10-9 /liter) 80 60 (ml/min) 30 15 40 20 0 0 0 2 4 6 8 10 12 14 16 18 20 22 24 Months of Eculizumab treatment 26-15 0 2 4 6 8 10 12 14 16 18 20 22 24 Months of Eculizumab treatment 26 Treatment effect was sustained for up to 26 months Licht et al., Kidney Int, 2015
Both during the acute phase of the disease and at remission about half of ahus patients had normal serum C3 and plasma sc5b-9 levels 23
SOLID-PHASE RESTRICTED COMPLEMENT ACTIVATION IN ahus 20 CFH Alternative pathway activation (spontaneous hydrolysis, bacteria, viruses) CFB CFI C3 Bb i Solid-phase restricted ahus C3a 20 CFB CFI i Surface bound C3 convertase Bb C5 C5a C5 convertase 24 Glycosaminoglycans Endothelial cell
SERUM FROM ahus PATIENTS CAUSES C5b-9 FORMATION ON ACTIVATED HMEC-1 REGARDLESS ON MUTATION STATUS HMEC-1 ADP 10µM 10 min -Control or ahus serum (n=29, in remission no eculizumab) (n=7 in acute phase) Static incubation 4 hours - anti-c5b-9 Ab staining - Confocal microscopy C5b-9 deposition 10,000 CFH CFI C3 CFB anti CFH Ab no mut 8,000 C5b-9 formed (pixel 2 ) 6,000 4,000 2,000 Ctr 0 scr1 - + - + - + - + - + - + Excessive C5b-9 deposits were observed with serum from all ahus patients (with or without identified complement abnormalities) during acute or remission phase. Noris et al., Blood, 2014
330.000 euro per patient per year (in child) 460.000 Every 15 days forever? 26
RISK OF ATYPICAL HEMOLYTIC UREMIC SYNDROME RELAPSE AFTER ECULIZUMAB DISCONTINUATION 38 patients (24 women - 9 children and 29 adults) % of ahus relapse-free patients 100 80 60 40 20 0 MCP variant No variant detected CFH variant 0 6 12 18 24 months Fakhouri et al, CJASN, 2017 Among 52 case reports discontinuing eculizumab 16 (31%) had a subsequent TMA. In eculizumab clinical trials 12/61 patients who discontinued eculizumab experienced 15 TMA complications. TMA relapse occurred irrespective of identified genetic mutations, high risk polymorphism or auto-antibodies. Macia et al, Clin Kidney J, 2017
How to monitor and possibly tapering? 28
Terminal complement inhibitors Preclinical Phase I Eculizumab: ALXN1210: anti-c5 Ab blocks C5 cleavage (Alexion) Anti-C5 antibody (Alexion) Coversin RA101495 Tesidolumab Zimura Phase II Phase III Approved ABP959 Eculizumab ALXN1210 SKY59 CCX-168 REGN3918 ALN-CC5 ALN-CC5: sirna Targeting C5 (Alnylam) CCX-168: C5aR antagonist (Chemocentrix) Coversin: Small C5 inhibitor (Akari) RA101495: cyclic peptide, C5 inhibitor (RaPharma) Tesidolumab: Anti-C5 antibody (Novartis) Zimura: aptamer-based C5 inhibitor (Ophthotech) 11 complement-targeted drug candidates in preclinical 29 ABP959: REGN3918: SKY59: Anti-C5 antibody (Amgen) Anti-C5 antibody (Regeneron) Anti-C5 antibody (Roche)
Multiple choice questions 1) Which of the following statements is correct? Atypical hemolytic uremic syndrome is caused by: a) fluid phase complement activation b) activation of the complement classical pathway on cell surface c) activation of the alternative complement pathway on cell surface d) impaired complement activation 2) Which among the following genes is not altered in atypical hemolytic uremic syndrome? a) Complement factor H (CFH) b) ADAMTS13 c) Complement C3 (C3) d) Membrane cofactor protein (MCP)
Multiple choice questions 3) Which among the following approaches is correct to diagnose atypical hemolytic uremic syndrome? a) Exclude an ADAMTS13 deficit b) Exclude an ADAMTS13 deficit and shiga-toxin producing bacteria c) Wait for the results of genetic screening d) Measure serum C3 levels
Next webinar: Membranous Nephropathy Jack Wetzels (Nijmegen) November 27, 2018 32
These slides belong to Marina Noris, Ph.D. Mario Negri Institute for Pharmacological Research, Bergamo, Italy. Using these slides is only authorized when mentioning the source 33