Komplementsystemet ------

ny behandling

Tom Eirik Mollnes

Vårkonferanse i medisinsk immunologi 27. april 2016 Bordet (1895): Complement lysis

1. Bacteria + Antiserum Lysis

o 2. Bacteria + Antiserum (56 C, 30´) No lysis

3. Experiment 2 + Normal serum Lysis

4. Bacteria + Normal serum No lysis

Conclusion: Heat stable (Ab) and heat labile (C) factor Complement in 1985

Meeting in Royal Society, London, by M. Hobart i Imm Today 1985.

”Many immunologists hold that complement is baffling or irrelevant or, most conveniently, both but a recent meeting emphasized that complement is interesting and that it may be important, even only as an elegant model system.

Complement in 2015 30 years later Complement therapeutics is in the clinic!

No way back! Interaction between cascades

Complement

C1-INHIBITOR Coagulation Kallikrein- Fibrinolysis Kinin Cascade Principles “Undetonated bombs” Local vs systemic Biological effects Inhibition E3

E2 P3 P = Proenzyme E = Enzyme P3 P2 ”The point of E3 P1 E1

E3 no return” P2

P3

E2 P3

Amplification E3 The Classical Pathway Alternative Pathway Host Foreign surface surface

SAP Ab CRP PTX3 IgM C3 FH C3 FB

Ficolins MBL Collectins C1qrs Cross-talk

C1-INH C3 MASPs Spontaneous C1-INH hydrolysis C4a C4 C4 C2b C4a C2b FH C4b C2 C4b C2 Regulation FI C3 C4BP FB MCP C4BP H2O

Ba DAF FD

CR1 Anaphylatoxins C3b C4b C2a C3b Bb C3b CR1 C3 H2O FP CRIg C3a iC3b CR3 C3aR amplification CR4 C5a C5 AI phagocytosis C5aR1 C5a C4b C2a C3b C9 desArg C6 C8 CD59 C5b-7 C5aR2 C5b C6 Cl Vn C7 C8 C7 C8 C9 C9 ｓC5b9 MAC TCC Lysis ©Mollnes TE Inflammatory effects mediated by C5a

Chemotaxis Histamin release

Lysosomal enzyme release Smooth muscle contraction Neutrophil aggregation Increased permeability

Cell adhesion Cytokines CR3 (CD11b/18) C5a IL-1, IL-6, IL-8, TNF

CR1 and FcR expression B- and T-cell responses

Reactive oxygen Arachidonic acid metabolites metabolites (LT, PG)

Platelet activating factor (PAF) What is inflammation?

Heat Redness Swelling Pain Reduced function Reasons to analyse complement

• Complement deficiencies – are associated with certain diseases • Complement activation – clinical: reflects ongoing disease processes – experimental: animal and in vitro models • Monitoring complement inhibition – is already in the clinic (eculizumab, C5 inhibition) What is serum and what is plasma? A traditional question I get is:

“We have a unique serum biobank – can you do complement analyses of this material?

My answer is: - Do you have plasma and in case what kind of samples do you have? – how where they obtained, prepared and stored etc. The traditional answer I get back is: “I am not quite sure, but I think is serum obtained from the Central Laboratory after they finished their routine analyses”.

What is serum and what is plasma?

SERUM:

- Coagulated - Fibrinogen converted to fibrin and coagulation activation completed

- Fresh frozen: after 1hr preparation safely stored at -70 degrees.

- Aged: serum from the routine lab – nobody knows how it has been stored (1 day, 1 week; - on bench, in fridge).

- Heat inactivated: 56 degrees, 30 min. Not complement specific. What is serum and what is plasma? PLASMA: Anticoagulated in order to avoid clotting. - Heparin: Affects different coagulation factors. Influence the complement system (low doses activates, high dioses inhibits). Citrate: Calcium blocker, needed for coagulation analyses. Inhibits complement, but not sufficient to block it in vitro. - EDTA: Ca/Mg blocker and one the best complement inhibitors and should be used for sampling to detect complement activation in vivo. Cannot be use to study complement experimentally in vitro. - Lepirudin: blocks thrombin specifically. Keeps all other inflammatory arms open – including complement (“serum-like” or even better). Complement tests

• quantification (Serum) – C3, C4, C1-inhibitor (and others)

• Complement function (Fresh serum) – Screening for • Total complement system Screen (Wieslab®) (“CH50”) • Monitoring clinical inhibition

• Activation products (EDTA-plasma) – E.g. sC5b-9 (soluble TCC) – Detects activation of the whole cascade

Manifestations of Complement Deficiency • Hereditary angioedema – C1-inhibitor • Infections – Neisseria: Alternative and terminal components – Recurrent infections early in life: MBL, others • Autoimmune diseases (SLE) – C1q, other classical and terminal components • PNH – GPI-anchor defect (DAF, CD59) • Kidney: aHUS, MPGN II, Tx-rejection – , I, MCP (or gain of function: C3, fB) • Eye: Adult macula degeneration Hereditary Angioedema

• Etiology: C1-inhibitor deficiency – Genetic low protein conc. (Type 1) or dysfunction (Type 2) – Acquired (malignancy, autoimmunity) • Pathophysiology: – C4, C2 and kallikrein activation – Increased vascular permeability and EDEMA (bradykinin) • Clinical features: Attacks of local edema in any organ – Duration: 2-5 days. No effect of anti-allergic treatment – Edema of larynx may be lethal • Diagnosis: low C1-inhibitor antigen or function (and C4) • Treatment: Danazol, C1-inhibitor, Bradykinin receptor antagonist Total Complement System Activity

Serum added to microtiter wells (Wieslab®)

C9 C9 C9 C8 C8 C8 C7 C7 C7 C6 C6 C6 C5 C5 C5 C3 C3 C3 P C4 C2 C4 C2 MBL FB FD C1qrs MASP-2 IgM Mannan LPS Classical pathway Lectin pathway Alternative pathway Antigenic changes during complement activation mAbs to Activation Native neoepitopes: products component • Detection • Manipulation

Neoepitopes

Native-restricted epitope Activation independent epitope Enzyme immunoassay (EIA) for quantification of TCC (neoepitope)

anti-C6 Av-Px Biotin

SC5b-9

anti-C9neo mAb aE11 Complement activation products

Classical Lectin Alternative pathway pathway pathway C1rs-C1-INH ? Ba, Bb C4a, C4bc, C4d C3bPBb C3 C3a, C3bc, C3dg Terminal pathway C5a TCC/sC5b-9 TCC as complement activation marker

• TCC = sC5b-9 – Reflects activation through all pathways and release of C5a • In vitro stability – Relatively resistant to spontaneous activation – Relatively resistant to freezing and thawing • In vivo – Relatively long half-life (60 min) compared with C5a (1 min) – Low physiological concentration Complement activation and prognosis in patients with meningococcal disease

Activation products measured in samples obtained on admission to hospital

Terminal Complement Complex (TCC)

< 12.7 AU/mL: 1 of 32 died > 12.7 AU/mL: 6 of 7 died

p < 0.0001

Brandtzaeg et al. J Infec Dis 1989 Spontaneous in vitro complement activation

Stability of TCC in EDTA-plasma at room temperature

Increase (fold) 25 20 C3dg 15 10 5 TCC 1 0 1 2 Days Spontaneous in vitro activation in serum Consequences for different assays

% 100 C3 antigen conc.

75 C3 activation (C3bc) 50

25 C3 hemolytic activity

0 0 20 40 60 Hours Factors influencing the amount of native components (e.g. C3 and C4)

• Synthesis – Reduced amounts in liver failure • Acute phase reaction – Most components increase • In vivo activation – Consumption leads to decreased amounts • Hemodilution

The sum of these factors determines the level Native components and activation products in two patients

Patient 1 (F24) Patient 2 (F35) Reference range

C3 0.20 0.30 0.50 - 1.00 g/L

C4 0.09 0.06 0.10 - 0.50 g/L

C3dg 25 126 20 - 45 AU/mL

TCC 3.9 15 2.2 - 6.6 AU/mL

Diagnosis: Liver Chronic active failure hepatitis Complement activation products: Treatment of samples

Due to rapid in vitro activation it is crucial that the samples are obtained and stored properly

• EDTA tubes (10-20 mM final conc.) – turn tube gently x 3 to ensure good mixing • Store on crushed ice or at +4oC – immediately (< 10 min) • Separate plasma cool and store at -70oC – within a few hours (4-6) Effect of eculizumab

Eculizumab Indications for use of eculizumab (Soliris®)

– Paroxysmal nocturnal hemoglobiumuria (PNH) – Clonal defect in bone marrow cells (PIG-A) – Red cells lack CD55 and CD59 – Spontaneous complement- mediated lysis

– aHUS Normal PNH – Atypical hemolytic uremic syndrome – Others: MPGN II? aHUS

– Thrombotic microangipathy – No pathogens or known external “danger signals” – Caused by internal complement dysfunction – Severe disease affecting children – kidney failure

– Imbalance in complement activation – Mutations in complement and regulators – Reduced efficacy of complement regulators – Factor H, I, MCP, DAF – “Gain on function” of ordinary components – C3, factor B Complement deposition in kidney tissue

MPGN II (DDD) in Acute Ab-mediated factor H dysfunction rejection “C3G” C4d in peritubular capillaries TCC (C5b-9) in glomeruli Complement in the future Makrides SC. Pharmacol Rev 1998 Therapeutic aspects PNH treated COMPLEMENT DISEASES References Acute with eculizumab Adult respiratory distress syndrome Zilow et al., 1992; Rinaldo and Christman 1990; Langlois et al., 1989; Meade et al., 1994 Ischemia-reperfusion injury: Hill and Ward, 1971; Earis et al., 1985; Rubin et al., 1989;Fox, 1990; Entman et al., 1991; Kilgore et al., 1994; Myocardial infarct Homeister and Lucchesi, 1994 Skeletal muscle Rubin et al., 1989; Weiser et al., 1996 Lung inflammation Ward, 1996, 1997; Eppinger et al., 1997 Hyperacute rejection (transplantation) Bach et al., 1995; Baldwin et al., 1995; Sanfilippo, 1996; White, 1996; Lawson and Platt, 1996 Sepsis Hack et al., 1989; Gardinali et al., 1992 Cardiopulmonary bypass Kirklin et al., 1983; Homeister et al., 1992 Burns, wound healing Ward and Till, 1990; Oldham et al., 1988; Davis et al., 1987; Ljunghusen et al., 1996 Normal erytrocyte Asthma Regal et al., 1993; Regal and Fraser, 1996 protected against complement Restenosis Niculescu et al., 1987 Multiple organ dysfunction syndrome Miller et al., 1996 Trauma, hemorrhagic shock Gallinaro et al., 1992;Kaczorowski et al., 1995 Guillain-Barré syndrome Hartung et al., 1987; Sanders et al., 1986; Koski et al., 1987; Koski, 1990

Chronic Paroxysmal nocturnal hemoglobinuria Yomtovian et al., 1993; Shichishima, 1995; Rosse, 1997 Glomerulonephritis Couser, 1993; Couser et al., 1985, 1995;Spitzer et al., 1969 Systemic erythematosus Belmont et al., 1986; Hopkins et al., 1988; Negoro et al., 1989; Gatenby, 1991 Hemolysis of a PNH erytocyte Rheumatoid arthritis Kemp et al., 1992; Satsuma et al., 1993;Abbink et al., 1992 Infertility D’Cruz et al., 1990, 1991;Anderson et al., 1993

Johnson et al., 1992; Rogers et al., 1992; Pasinetti, 1996; Eikelenboom et al., 1994;Velazquez et al., 1997; Alzheimer’s disease Jiang et al., 1994; McGeer et al., 1997; Chen et al., 1996; Morgan et al., 1997

Organ rejection (transplantation) Platt, 1996; Baldwin et al., 1995; Marsh and Ryan, 1997; Dalmasso, 1997 Myasthenia gravis Lennon et al., 1978;Piddlesden et al., 1996 Multiple sclerosis Piddlesden et al., 1994

Biomaterials incompatibility PNH erytovyte protected by Platelet storage Gyongyossy-Issa et al., 1994 eculizumab Hemodialysis Cheung et al., 1994; Himmelfarb et al., 1995; Mollnes, 1997

Cardiopulmonary bypass equipment Craddock et al., 1977; Haslam et al., 1980;Gillinov et al., 1993; Mollnes, 1997; te Velthuis et al., 1996