Emerging and Unexpected Regulatory Roles for Complement in The

10/29/2013

Disclosures - V. Michael Holers, MD

Types of financial relationships and the companies with whom Emerging and Unexpected Regulatory Roles for I have relationships are as follows: Complement in the Rheumatic Diseases Licensed intellectual property rights/patents: Taligen/Alexion Pharmaceuticals, Inc.

ACR Annual Meeting Royalties: Taligen/Alexion Pharmaceuticals, Inc. Basic Science Symposium October 30, 2013 Contracted research: Taligen/Alexion Pharmaceuticals, Inc.

Consulting fees: Alexion Pharmaceuticals, Inc. V. Michael Holers, MD Scoville Professor and Head, Division of Rheumatology University of Colorado Denver School of Medicine

Pivotal Role of Complement in Induction and Topics Maintenance of Experimental Inflammatory Injury Injury/Insult • Complement System Overview Recognition by complement • Predominant Role of the Alternative Pathway in Tissue Injury • Current Status of the Complement Inhibitor Field – Paroxysmal Nocturnal Hemoglobinuria Recruitment of Cells – Atypical Hemolytic Uremic Syndrome (aHUS) – Positive/Supportive Clinical Trials/Pilot Studies • Complement and Rheumatic Disease: Mechanisms/Insights Release of Mediators from Studies of Inflammatory Arthritis Models (enzymes, cytokines) – Effector Pathways – Modulation of Humoral Autoimmunity • New Concepts Tissue Damage – In Vivo Imaging of Complement Activation

Holers MV. Immunological Reviews. 2008; 223: 300–316. 4

Complement Pathway – Central Roles of Complement Pathway “Activators”: Injury, Immunity, Infection C3 and C5 in Activation Steps C3 fragment (C3b/ C3a/C5a iC3b/C3d) receptors MAC • Inflammation (host defense) • B cell response • Inflammation • T cell response • Apoptotic cell/debris clearance • Hemolysis Classical Alternative Lectin • Tissue repair/regeneration • Immune complex clearance • Neisseria control • Immune complexes • “Tickover” • Repeating simple • Opsonization and RES clearance (natural IgM, IgG) sugars C3a C5a • C-reactive protein (CRP) • Amplification pathway • G0 carbohydrate (chromatin, Streptococcus) glycoforms on IgM Classical Classical,Lectin Classical, Lectin • SIGN-R1 • Endotoxin • Acetylated sugars C3 Convertases C5 Convertases Lectin • Apoptotic bodies • IgA I.C. • Cytokeratin-1 C3 C3b C5 C5b MAC • Beta-amyloid fibrils • Polysaccharides Alternative Alternative Alternative C3 Convertases C5 Convertases • Serum amyloid P (SAP) • C3 nephritic factor • Phosphatidylserine Amplific. • Mitochondrial products

Holers MV. Immunological Reviews. 2008; 223: 300–316.

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Complement Pathway “Activators”: Injury, Immunity, Infection, Spontaneous/ The Tickover Mechanism Continuous

Classical Alternative Lectin

• Immune complexes • “Tickover” • Repeating simple C3 Spontaneous conversion (natural IgM, IgG) sugars (~1%/hour) • C-reactive protein (CRP) • Amplification pathway • G0 carbohydrate (chromatin, Streptococcus) glycoforms on IgM C3(H2O) Factor B • SIGN-R1 • Endotoxin • Acetylated sugars • Apoptotic bodies • IgA I.C. • Cytokeratin-1 Factor D • Beta-amyloid fibrils • Polysaccharides C3(H2O) Bb • Serum amyloid P (SAP) • C3 nephritic factor • Phosphatidylserine C3 C3b • Mitochondrial products

Holers MV. Immunological Reviews. 2008; 223: 300–316.

Amplification Loop Begins When Circulating Why Does Tickover Exist? C3 is Acted Upon by Convertases and Fixed as C3b to a Surface

• Provides capability for immediate response to foreign pathogens

• Does not require any other recognition molecules to work – Independent of antibody and other innate immune mechanisms

• Helps to separate “self” from “non-self” – “Self” tissues express regulators that block C3b from engaging factor B and continuing to activate system – “Non-self” does not have same regulatory molecules and thus is susceptible to continued complement activation

• Lack of appropriate control of complement initiation through tickover and subsequent amplification steps underlies fundamental pathogenesis of PNH and aHUS Janssen, Nature 444:213.-216, 2006

Amplification Loop Uses Alternative Pathway Engagement of the Amplification Loop; Components for Next C3 Convertase Exponential Activity Can Generate the Majority Formation of Effector Factors in Vivo

C3 C3b

(On)B C3bB

Factor D Properdin C3bBbP

F. Forneris, D. Ricklin, J. Wu, A. Tzekou, R.S. Wallace, J. Lambris and P. Gros, Science 330, 1816-1820 (2010)

Holers MV. Immunological Reviews. 2008; 223: 300–316. 12

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Alternative Pathway/Amplification Loop – Evidence The Alternative Pathway of Complement is Required Supporting Role as Primary Pathogenic Process in for Many Experimental Models of Human Disease, Human Disease Regardless of Classical/Lectin Pathway Dependence

• Genetically programmed deficiency states that primarily affect the Human Disease Model(s) Pathway Requirements alternative pathway (either rare germ line mutations or Classical/ Alternative/ acquired somatic cell mutations) Lectin Amplification 1. Rheumatoid Arthritis K/BxN serum transfer N Y - Paroxysmal Nocturnal Hemoglobinuria, Atypical Hemolytic Anti-CII Ab transfer N Y Uremic Syndrome 2. Antiphospholipid aPL Ab Transfer Y Y Syndrome • Linkage by Genome Wide Association Studies (GWAS) of 3. Lupus Nephritis MRL/lpr strain ? Y polymorphisms in complement alternative pathway activation C4-/-/B6 lpr strain N ? 4. Asthma Ova, ragweed N Y and regulatory protein encoding genes with human diseases immunization/inhalation (Human Genome Project trailblazing – in both GWAS and 5. Ischemia-Reperfusion Intestinal/Skeletal Y Y Injury Renal N Y dense sequencing projects) 6. Atypical Hemolytic- NA ? Y Uremic Syndrome - Age-Related Macular Degeneration 7. Type II Membrano- fH-/- strain N Y • Informative animal models - Thurman and Holers, J. Immunol. Proliferative GN 2006; Holers Immunology Reviews 2008 8. Macular Degeneration CNV N Y 9. Spontaneous Fetal Crry-/- strain N Y Loss

Thurman and Holers, J. Immunol. 2006; Holers Immunology Reviews 2008

The Alternative Pathway of Complement is Required Effector Mechanisms for Many Experimental Models of Human Disease, Effects on Adaptive Immunity In Complement •Enhance humoral immunity Regardless of Classical/Lectin Pathway Dependence •Modulate natural Ab repertoire Activation •Regulate tolerance •Modify T cell immunity Human Disease Model(s) Pathway Requirements Classical Alternative/ Lectin Amplification Complement 1. Rheumatoid Arthritis K/BxN serum transfer N Y Activation Anti-CII Ab transfer N Y Opsonization by C3b 2. Antiphospholipid aPL Ab Transfer Y Y Direct Effects of C5a and/or Syndrome C3a generation Direct Lytic 3. Lupus Nephritis MRL/lpr strain ? Y Effects C4-/-/B6 lpr strain N ? C5aR/C5L2/C3aR MAC of MAC C3b 4. Asthma Ova, ragweed N Y immunization/inhalation 5. Ischemia-Reperfusion Intestinal/Skeletal Y Y Injury Renal N Y 6. Atypical Hemolytic- NA ? Y Uremic Syndrome 7. Type II Membrano- fH-/- strain N Y Proliferative GN 8. Macular Degeneration CNV N Y Cell Activating Effects 9. Spontaneous Fetal Crry-/- strain N Y •Pro-coagulant state Loss •Adhesion Molecules

Thurman and Holers, J. Immunol. 2006; Holers Immunology Reviews 2008 •Pro-inflammatory mediators

Complement Therapeutics: Topics Indications

• Complement System Overview • Approved • Predominant Role of the Alternative Pathway in Tissue Injury – Hereditary Angioedema (C1-INH replacement) – Paroxysmal Nocturnal Hemoglobinuria (Anti-C5) • Current Status of the Complement Inhibitor Field – Atypical Hemolytic Uremic Syndrome (aHUS) (Anti-C5) – Paroxysmal Nocturnal Hemoglobinuria – Atypical Hemolytic Uremic Syndrome (aHUS) – Positive/Supportive Clinical Trials/Pilot Studies • Positive/Supportive Human Clinical Trials/Pilot Studies: • Complement and Rheumatic Disease: Mechanisms/Insights – Neuromyelitis Optica from Studies of Inflammatory Arthritis Models – Age-related macular degeneration (AMD) – Effector Pathways – Acute humoral renal transplant rejection – Modulation of Humoral Autoimmunity – Myasthenia gravis • New Concepts – C3 nephropathy – Catastrophic anti-phospholipid antibody syndrome (CAPS) – In Vivo Imaging of Complement Activation

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Overview:Conformational Interaction Change Inhibitors of C3 upon& Monoclonal Cleavage Antibodies to C3a/C3b ConformationalOverview: ChangeSoluble Complementof C3 upon Cleavage Regulators to C3a/C3b

Targets: Numerous, at any level Targets: C3b, Convertases, MAC anti-C1q On Market: Eculizumab On Market: none High specificity & affinity Physiological approach Administration (non-oral) Act at the central level of activation Production costs (proteins/mAb) No oral bioavalability High production costs

sCR1-sLex anti-C5 anti-FD anti-FB anti-C5 aptamer anti-C5 minibody anti-C5a spiegelmer TP-20 Eculizumab/Soliris N/A TA106, Bikaciomab ARC1905 MUBODINA NOX-D15 [Avant] [Alexion] [Genentech] [Alexion], [Novelmed] [Ophthotech] [Adienne] [NOXXON] APT070 CD59- sCR1 Mirococept Prodaptin CDX1135 [MRC] [Inflazyme] [Celldex] MCP-DAF CAB-2 FH-CR2 anti-C3 peptide sCRIg [Millenium] TT30 rFH anti-MASP anti-Properdin anti-C3b Compstatin anti-C5 proteins [Genentech] N/A N/A S77 POT-4 [Potentia/Alcon, Apellis] OmCI (Ticks) [Alexion] [Optherion] [Omeros] [Novelmed] [Genentech] Cp30/Cp40 [Anosos] SSL7 (Staph) Ricklin and Lambris, Nature Biotechnology. 25:1265-75, 2007 (updates /slide courtesy of Dan Ricklin) Ricklin and Lambris, Nature Biotechnology. 25:1265-75, 2007 (updates /slide courtesy of Dan Ricklin)

Paroxysmal Nocturnal Hemoglobinuria (PNH): A Paroxysmal Nocturnal Hemoglobinuria (PNH): An Disease of Unregulated Alternative Pathway Orphan Disease of Unregulated Alternative Activation Pathway Activation

• Rare form of hemolytic anemia • Incidence 1.3 cases per million per year • Prevalence 15.9 cases per million • Acquired genetic deficiency of complement inhibitors that require glycophosphatidyl inositol (GPI) membrane anchor for expression • Associated with aplastic anemia, myelodysplastic syndrome • Mutations are in X-linked phosphatidylinositol glycan-complementation class A gene (PIGA) – diagnosis previously by Ham Test, now flow cytometry

Rother et al, Nat Biotech 25:1256-1264, 2007

Ameliorative Effects Eculizuman on Intravascular Soliris – Direct C5 Inhibition Hemolysis in Cross-Over Extension Study

C3a C5a

Classical Classical,Lectin Classical, Lectin C3 Convertases C5 Convertases Lectin C3 C3b C5 C5b MAC Alternative Alternative Alternative C3 Convertases C5 Convertases

Amplific. SOLIRIS

Rother et al, Nat Biotech 25:1256-1264, 2007

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Thrombotic Microangiopathy Eculizumab – Adverse Events Definition and Clinical Overlaps

• Microangiopathic hemolytic anemia • Thrombocytopenia • Neisseria Infections – N meningitidis • Fever • Renal Failure • Rate constant: ~1/100 patient-years • Mental status changes • CNS changes • Renal failure • Managed variably by: TTP HUS – Vaccination of all patients prior to initiation of treatment (or antibiotic coverage for initial period) – Patient education, physician availability Typical, or Atypical, or D+ HUS (90%) D- HUS (10%) – Antibiotic coverage – prophylactic or at first infection • Diarrhea with STEC- • No antecedent • Improving vaccine coverage HUS [Shiga toxin infection with producing E. coli STEC-HUS E. Coli (O157:H7)] • Familial 2% of cases

Control Mechanisms that Turn Off the Factor H Mutations in aHUS Patients Alternative Pathway and Amplification Loop

Regulatory Recognition C3 C3b domain doman Factor H, MCP (Cofactors) C3b proteoglycans C3b proteoglycans proteoglycans C3b (On)B (Off) Factor I

1 2 3 4 5 6 7 8 9 1 1 1 1 1 1 1 1 1 1 2 0 1 2 3 4 5 6 7 8 9 0 C3bB iC3b E1172stop (n=2) 1494delA (n=3) Q950H W1183R (n=2) Y951H N1050Y S1191L (n=10) I970V G1194D (n=2) V1197A (n=3) E1198A

Factor D N498K R1210C (n=9) C1163W R78G (n=2) R1215Q (n=4) S890I (n=5) Q1137L del24bp (n=11) Y899stop Dup(3546- W1183stop 279del15bp(n=2) 3103delG 3581) Properdin W920R V1200L C3bBbP E1198K • Patients screened: 267 (Off) • 29 different mutations identified in 70 patients (33 familial, 37 sporadic) Factor H, DAF • Mutation frequency: 26.2% Bb • 27 of 29 are heterozygous (Decay Acceleration) • Most clustering in the C-terminus domain C3b + Caprioli et al., J Am Soc Nephrol, 2001; Caprioli et al., Hum Mol Gen, 2003; Caprioli et al., Blood, 2006; Unpublished data from the International Registry of Non-Stx-HUS. Adapted from: Marina Noris.

Autoantibodies to Factor H in aHUS Patients Mutations of Factor I, MCP, C3 and Factor B Common in aHUS

Complement Ligand regulation recognition • Mutations of factor I in aHUS cause either altered secretion or altered function (Nilsson et al, Eur. J. Immunol. 40:172-185, 2010) 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 1 17 18 1 20 0 6 9 • Atypical hemolytic uremic syndrome in children: additional complement alternative pathway mutations and association of clinical characteristics (Geerdink et al, Peditr. Nephrol. 27:1283-1291, 2012) HUS associated factor H autoantibodies • Combined complement gene mutations in aHUS worsen clinical (described mainly in children) mimic the phenotype (Breslin et al, J. Am. Soc. Nephrol. 24:475-486, 2013) effect of C-terminal factor H mutations, as Anti-factor H • Mutations overlap with those also found in age-related macular they inhibit the regulatory function of factor H IgG antibodies degeneration (AMD) at cell surfaces by blocking its C-terminal recognition region

Dragon-Durey et al, JASN, 2005; Jozsi et al., Blood, 2007. Adapted from: Marina Noris.

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aHUS Model: Primary Endpoint in aHUS Trial Met Study C08-002A/B Complement Regulatory Protein Mutations Using Eculizumab: Change in Platelet Count Complement Activation – by Trigger Mutated Factor H Patients with 9 Mutated Factor I + Plts ≥150x10 /L C3b Proteoglycans Primary endpoint Mutated MCP Sialic acids FI + FI 100 13/15 + 14/17 Change in platelet count through 90 87% C3b iC3b C3b week 26 (ANOVA) 82%

Endothelial cell nucleus C3b 70 Subendothelial matrix Complement 9 60 Activation through C3 and C5 • Point estimate: 73 x 10 /L alternative pathway convertases 50

• P-value= 0.0001 PercentPatients 40 Platelet activation 30 Proteinases C3a C5a Oxygen radicals 20 C5b9 10 0 All Pts Baseline Plts endothelial damage and retraction <150x10^9/L Adapted from: Marina Noris. Slide courtesy of Steve Squinto

Is Complement Activation The Common Antiphospholipid Antibody Syndrome Pathway in TMA?

Role for complement: Thrombosis • C3 and C5 activation is required for fetal loss murine model of APS disease manifestations Holers et al, J Exp Med 2002; Girardi et al., JCI 2003 • Complement regulatory gene mutations identified in patients with APS and pre-eclampsia Salmon et al, PLOS Med 2011 Large VWF Anti-Phospholipid • Hypocomplementemia (C3, C4) in over half of primary multimers Complement STEC-E coli APS patients in large prospective study found to be associated Alternative Antibodies with hemolytic anemia Pathway Ramos-Casals et al, Lupus 2004 Thrombomodulin • Case reports of successful Soliris use in CAPS Lonze et al., NEJM (2010)

Endothelial cells Goldberg and Thurman, AJKD 2011

Complement Therapeutics: Neuromyelitis Optica (NMO) and Approved Indications Pathogenic Complement Activation

• Disease associated with pathogenic IgG autoantibodies to Aquaporin 4 (AQP4) • Paroxysmal Nocturnal Hemoglobinuria (Anti-C5) – Major driver of inflammation and astrocytic injury in the central nervous system • Hereditary Angioedema (C1-INH replacement) • Patients exhibit complement activation fragments in lesions • Atypical Hemolytic Uremic Syndrome (aHUS) (Anti-C5) • Animal studies have supported that complement is pathogenic • Seropositive patients with NMO and ongoing disease despite optimal therapy were treated with eculizumab; primary efficacy endpoints met • Positive/Supportive Human Clinical Trials/Pilot Studies:

– Neuromyelitis Optica

– Age-related macular degeneration (AMD) – Acute humoral renal transplant rejection – Myasthenia gravis – C3 nephropathy – Catastrophic anti-phospholipid antibody syndrome (CAPS)

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Alternative Pathway and Amplification “Complotype”: Disease-Associated Polymorphic Variations in Loop: A Matter of Balance C3, FB, and FH Collaborate to Control Relative Alternative Pathway Functional Activity

C3 C3b Factor H, MCP (Cofactors) (On)B (Off) Factor I

C3bB iC3b Factor D Properdin C3bBbP (Off) Factor H, DAF Bb (Decay Acceleration) C3b + Heurich M et al. PNAS 2011;108:8761-8766

Collagen Antibody-Induced Topics Arthritis (CAIA) Model of Rheumatoid Arthritis

• Complement System Overview • Predominant Role of the Alternative Pathway in Tissue Injury • An in vivo immune complex (IC)-mediated model of rheumatoid arthritis • Current Status of the Complement Inhibitor Field – Paroxysmal Nocturnal Hemoglobinuria • Transfer of four IgG monoclonal anti-type II collagen (CII) – Atypical Hemolytic Uremic Syndrome (aHUS) antibodies (Arthrogen) into C57BL/6 WT and complement- – Positive/Supportive Clinical Trials/Pilot Studies deficient mice • Complement and Rheumatic Disease: Mechanisms/Insights from Studies of Inflammatory Arthritis Models • Confirmed previously (and internally) to be C3- and C5- – Effector Pathways dependent model – Modulation of Humoral Autoimmunity • New Concepts – In Vivo Imaging of Complement Activation

Collagen Antibody-Induced Arthritis (CAIA): Assessment of C3 and Factor H Deposition on Anti-CII mAb Passive Transfer Model of Human Cartilage and Synovium During Rheumatoid Arthritis Evolution of CAIA

Overall inflammation

35 C3 in synovium

30 C3 on cartilage

CB6 CB12 CB11 CB8 CB10 CB5 CB9.7 25 fH in synovium 1 41 124 403 552 898 1017 fH on cartilage 20

15 LPS LyC1 LyC2 LyC3 10

124-290 291-374 375-402 Mean/Baseline 5

0 D1 F10 A2 D8 0.5 1 2 4 8 24 72 96 120 144 hr hr hr hr hr D1 D3 D4 D5 D6 Time (Hours or Days)

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Critical Pathways: Bf-/- Mice Are Resistant to Bf-/- Mice Demonstrate Decreased Joint Damage by Arthritis Induced By Anti-Collagen mAbs Histologic Analysis Compared to WT and C4-/- Mice While C4-/- Mice Are Susceptible

4 4

12 10 *P<0.05 WT (n=6) WT (n=3)

WT (n=6) WT (n=3) -/- -/- 10 Bf (n=6) C4 (n=3) 3 3 Bf-/- (n=6) * 8 C4-/- (n=3) 8 *P <0.001 * * 6 6 2 2 4 4 LPS * LPS 2 2 1 1

Clinical Activity Clinical Disease * 0 0 * 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 * 0 * 0 Days After Antibody Injection Days After Antibody Injection Cartilage Bone Inflammation Pannus Cartilage Bone Inflammation Pannus Damage Damage Damage Damage

Banda NK et al. J Immunol. 2006;177:1904-1912 Banda NK et al. J Immunol. 2006;177:1904-1912

Complement C3 Deposition on Cartilage and in Neither MBL A/C-/- Nor C1q-/- Mice Are Resistant Synovium is Greatly Decreased in Factor B Deficient to Arthritis Induced By Anti-Collagen mAbs

as Compared to Wild Type or C4 Deficient Mice

100 WT (n=7) 12 WT (n=7) -/- 80 MBL A/C-/- 10 MBL A/C (n=7) (n=7) 25 25 60 8 6 *P<0.05 WT (n=6) *P<0.05 WT (n=3) 40 LPS 4 LPS 20 Bf-/- (n=6) 20 C4-/- (n=3)

20 2 Disease Incidence Incidence Disease (%) 0 Activity Clinical Disease 0 15 15 * 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10

100 12

WT (n=4) WT (n=4) 10 10 -/- 10 C1q-/- (n=4) 75 C1q (n=4) 8

6 5 * 5 50 * 4 * LPS LPS 25

0 0 2 Disease Incidence Incidence Disease (%) Synovium Cartilage Total score Synovium Cartilage Total Score 0 Activity Clinical Disease 0 0 1 2 3 4 5 6 7 8 9 10 Banda NK et al. J Immunol. 2006;177:1904-1912 0 1 2 3 4 5 6 7 8 9 10 Days After Antibody Injection Days After Antibody Injection

AP-Only Mice Alone are Capable of Fully Important Structural Features of Factor H Supporting Development of CAIA and Alternative Pathway Regulation CP only LP only AP only 12

12 12 WT WT WT 10 10 10 • FH binds to C3b at 2 sites: SCR1-4 & -/- -/- MBL /Df -/- -/- -/- -/- 8 8 C1q /Df 8 C1q /MBL SCR19-20.

6 6 6 • FH also binds to self-surface biomarkers 4 4 4 such as glycosaminoglcyans (GAGS)

2 2 2 and sialic acid residues: SCR6-8 and Clinical Clinical disease activity 0 0 0 SCR20. 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 Days after antibody injection Days after antibody injection Days after antibody Injection

2 4 3 WT (n=4) WT (n=4) WT (n=4) -/- -/- C1q/MBL (n=4) MBL/Df (n=4) C1q/Df-/- (n=4) Kajander et al., Proc. Natl. Acad. Sci. 1.5 3 USA (2011) 108, 2897-2902 2

1 2

0.5 1 All ScoreAll Joint Mean 0 Morgan, Schmidt et al., Nature Struct. Biol. (2011) 0 0 InflammationPannus Cartilage Bone InflammationPannus Cartilage Bone InflammationPannus Cartilage Bone Damage Damage 18, 463-470 Damage Damage Damage Damage

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CR2-Mediated Delivery of Tools in Mice to Study Alternative Pathway Regulation Complement Inhibitors in Development of Tissue Injury CR2 Heparin Heparin Heparin Covalent Chimera C3d Native Factor H SCR1 1 5 SCR20 attachment Regulatory Polyanion C3b/C3d Binding of C3d to Complement Domain sites of Inhibitor (DAF, MCP, Chimera inflammation Factor H) Native CR2 SCR1 1 4 15 C3d Binding

4 Targeted Fusion Protein CR2-fH 1 1 5 C3d Binding Regulatory Domain Complement Factor H SCR19-20 Heparin and C3b/C3d binding domain Activation Inhibitor “homes” to CR2 1 4 C3d coated Systemic C3d Binding Inflamed Tissue Administration Factor H SCR1-5 1 5 Regulatory Domain

Targeted CR2-fH Demonstrates Substantial Conversely, Disabling Factor H Function Inhibition of Tissue Injury in CAIA Model with fH SCR19-20 Amplifies Injury in

CAIA 12 PBS * * * * 10 rfH19-20 * (300µg) 8 rfH3-5 6 (300µg) * A B 4 100 12 WT PBS 2 * * * * * 10 WT rCR2-fH (250ug) 75 WT rCR2-fH (500ug) 0 8 * * 0 1 2 3 4 5 6 7 8 9 10 * Clinicaldiseaseactivity * 50 6 * 100 PBS 4 rfH19-20 25 * * * 75 (300µg) 2 * *p<0.01

* rfH3-5

Disease Disease incidence (%) Clinical Clinical disease activity 0 0 50 (300µg) 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 Days after antibody injection Days after antibody injection

25 Prevalence(%) 0 0 1 2 3 4 5 6 7 8 9 10 Days after antibody injection

Banda et al, JI, 2009 Banda et al, JI, 2012

Complement Factor H Related Protein Family: Factor H & Factor H-related proteins Member of RCA Family

• CFHR locus linked to age-related macular degeneration (AMD), Systemic Lupus Erythematosus, and other inflammatory disorders • RCA gene family is located within 1q32.

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Modulation of Complement In Vivo by “Two Compartment” Model of Joint Inflammation CFHR1, CFHR2, and CFHR5 And Damage as an Experimental Framework

Inadequate Factor H; TNFalpha De-regulation IL-1 Synoviocyte Injury& Adipocyte Pro-Factor D Synthesis* C3a MASP-1/3& C5a Mediated MAC Cleavage Amplification Loop Factor D

C3b Inadequate Factor H; Alternative Pathway De-regulation by CFHR C3 Convertases C3a, C5a Cartilage & Initiation MAC Synovium (FLS , Engagement of Macrophages, Distal Amplification Adipocytes*) Process and Natural Goicoechea de Jorge E et al. PNAS 2013;110:4685-4690 Antibodies Anti-CII Ab

The Traditional Model: CR2 Links Through CD19 Topics Association Antigen:C3d Complexes to Amplify B Cell Receptor Signals

• Complement System Overview C3d C3d antigen • Predominant Role of the Alternative Pathway in Tissue Injury CD21 • Current Status of the Complement Inhibitor Field – Paroxysmal Nocturnal Hemoglobinuria CD19 – Atypical Hemolytic Uremic Syndrome (aHUS) IgM – Positive/Supportive Clinical Trials/Pilot Studies CD81 • Complement and Rheumatic Disease: Mechanisms/Insights from Studies of Inflammatory Arthritis Models Signal 1 – Effector Pathways

– Modulation of Humoral Autoimmunity Signal 2 • New Concepts Signal 2 amplifies BCR signal 10,000-fold, enhances Ag presentation – In Vivo Imaging of Complement Activation

Creation of Highly Inhibitory Mouse Anti-Mouse Structural Determinants of CR2-C3d CR2 and C3d mAbs That Reciprocally Impair Interaction Are Well Understood Ligand-Receptor Interactions

-/- Cr2-/- C3

smCR2(1-4) hC3d i.p. x 4 times i.p. x 4 times

spleen spleen

fusion with 653 cells fusion with Sp2/0 cells

screening screening

1. Direct ELISA using smCR2(1-4) 1. Direct ELISA with hCd 2. Flow cytometry of K562 cells transfected with mCR2 2. Indirect ELISA with hC3dg-biotin van den Elsen and Isenman, Science 332:608-611, 2011 3. w.b. with cell lysates from K562 mCR2+ cells.

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Minimal Modulation Of Receptor Levels Following Type II Collagen-Induced Arthritis Treatment With Highly Inhibitory Mouse Anti- (CIA) in DBA/1J Strain Mouse CR2 mAb 4B2

Treatment: Control IgG1 4B2

Immunize with CII PE

- in CFA 4B2 21 days

Score for APC

- arthritis,

7E9 measure anti- CII Abs B220 Boost with CII/IFA

Treatment With mAb 4B2 Decreases Treatment With mAb 4B2 Decreases Pathogenic Anti-CII Ab Levels in CIA Model Arthritis in CIA Model

IgG3 mouse IgG2A mouse IgG1 mouse 1.5 2.0 p<0.05 2.5 p<0.03

p<0.05 p<0.03 1.5 ) 2.0

m 1.0 n

5 1.5

0 1.0

4 (

D 0.5 1.0 O 0.5 0.5 4.5

0.0 0.0 0.0 4 3.5 3 4B2 2.5 control IgG3 bovine IgG2A bovine IgG1 bovine 1.5 2.5 p<0.02 2.5 2 wt p<0.02 2.0 2.0 ) 1.5

m 1.0

n 1.5 1.5

0 4

( 1

1.0 1.0 D

0.5 DiseaseActivity Clinical O 0.5 0.5 0.5

0.0 0.0 0.0 0 1 2 3 4B2 33 37 42 Ova 30 unmanipulated days

Activation, Durable Covalent Attachment and Topics Proteolytic Processing of Complement C3 On Self and Foreign Antigens

• Complement System Overview • Predominant Role of the Alternative Pathway in Tissue Injury • Current Status of the Complement Inhibitor Field – Paroxysmal Nocturnal Hemoglobinuria – Atypical Hemolytic Uremic Syndrome (aHUS) – Positive/Supportive Clinical Trials/Pilot Studies • Complement and Rheumatic Disease: Mechanisms/Insights from Studies of Inflammatory Arthritis Models C3a C3f C3c – Effector Pathways C3 C3b iC3b C3dg – Modulation of Humoral Autoimmunity • New Concepts C3 Factor I Factor I convertase + + – In Vivo Imaging of Complement Activation Cofactor CR1

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Summary of Key Characteristics of Immunoprecipitation by Group 1 Antibodies Candidate Monoclonal Antibodies –Primary of Specific C3 iC3b and C3dg Fragments in Group 1 Candidates Zymosan-Activated Plasma

Designation Isotype hC3d Inhibition of Zymosan Mouse Cyno binding by hC3d-hCR2 activated serum C3d C3d Western Blot Immunoprecipitation ELISA binding binding binding binding   1 3d3 IgG1 + + 100- ’ ’ 75- b 2 3d10 IgG1 + + ’1 ’1 3 3d11 IgG1 + + + + 50- IgG Heavy chain 4 3d16 IgG1 + +/- + +/-

5 3d9a IgG2a/c + + + + + C3dg C3dg 37- 6 3d15 IgG2a/c + + C3d 7 3d29 IgG2a/c + + + + + 8 3d31 IgG2a/c + + + + 9 3d8b IgG2b + + + + +

Thurman et al, J Clin Invest 123:2218-2230, 2013 Thurman et al, J Clin Invest 123:2218-2230, 2013

Novel mAbs to C3d – Targeting Tissue Novel mAbs to C3d – Imaging Retinal C3d in FH-/- Mice for Imaging Purposes C3d in Live Animals (CNV Model)

Brightfield HB5 Polyclonal anti-C3

3d9a 3d29 3d8b

Brightfield 3d29

Thurman et al, J Clin Invest 123:2218-2230, 2013 Thurman et al, J Clin Invest 123:2218-2230, 2013

Potential Uses of In Vivo Complement Topics Imaging in Rheumatic Diseases

• Lupus Nephritis/Target Organ Dysfunction • Complement System Overview – Assess response to therapy or inform need to advance therapeutics • Predominant Role of the Alternative Pathway in Tissue Injury – Biomarker of response to complement inhibitors • Current Status of the Complement Inhibitor Field

– Paroxysmal Nocturnal Hemoglobinuria • Neuromyelitis Optica – Atypical Hemolytic Uremic Syndrome (aHUS) – Disease activity – Positive/Supportive Clinical Trials/Pilot Studies – Response to complement inhibition • Complement and Rheumatic Disease: Mechanisms/Insights from Studies of Inflammatory Arthritis Models • CNS Vasculitis – Effector Pathways – Modulation of Humoral Autoimmunity • Early Arthritis/Synovitis • New Concepts – In Vivo Imaging of Complement Activation

12 10/29/2013

Acknowledgements Acknowledgements

Academic Barb Rohrer Investigators: William Arend Josh Thurman Michael Pangburn Research Nirmal Banda Anne Lynch Therapeutics Slides: Funding: Liudmila Kulik Viviana Ferreira Daniel Ricklin NIH/NIAMS Rosa Rodriguez Stephanie Hyatt John Lambris NIH/NIAID Alexandra Antonioli Maggie Glogowska Sandler Program in Asthma Research/AAF Steve Tomlinson Kazue Takahashi Models Slides: Lupus Research Institute Carl Atkinson Gregory L. Stahl Marina Noris Beckman Initiative for Macular Research Gary Gilkeson Bao Lu Josh Thurman Taligen/Alexion Jane Salmon Craig Gerard Antonio Risitano Stacey Muller-Ortiz Erwin Gelfand Rick Wetsel Johnny Hannan James Kovacs