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Biological therapy for the manipulation of complement system

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1 Biological therapy for the manipulation of complement system
Prohászka Zoltán, IIIrd Department of Medicine, Research Laboratory Semmelweis University

2 Biological therapy Biological therapy refers to the use of medication that is tailored to specifically target an immune mediator of disease or induce an immunological mechanism to cure a disease. Targeted therapy in clinical immunology (or oncology) refers to medications acting through specific molecular targets to achieve immunomodulation or oncolysis, in contrast to less specific treatments, like steroids or cytostatica. Specific form of targeted therapy is the substitutional therapy with purified „factors”, like coagulation factors in haemophilia, or insulin therapy Biological response modifiers (BRMs) are substances influencing biological functions, like interferons, interleukins, growth factors and colony stimulating factors Vaccination

3 Milestones in biological therapy
Serum therapy for diphtheria (1890)

4 The first therapeutic approach, that was created with the understanding of the etiopathogensis of disease Emil von Behring Nobel Prize in Physiology and Medicine, 1901: Orvosi Nobel díj, 1901: "for his work on serum therapy, especially its application against diphtheria, by which he has opened a new road in the domain of medical science and thereby placed in the hands of the physician a victorious weapon against illness and deaths". Diphteria antitoxin, 1890 Johannes Bókay Jr „based on an international mandate, he checked the safity of the diphteria antitoxin” 1894 Edwin Klebs Corynebacterium diphtheriae Klebs-Löffler bacillus (1883) Tom, the Horse (1894, London)

5 Milestones in biological therapy
Serum therapy for diphtheria (1890) Treatment for agammaglobulinemia with purified immunogobulin G (1952) The development of monoclonal antibody (mAb) technology by Köhler and Milstein (1975) leading to the approval of the first therapeutic murine mAb, Muromonab-OKT3 (1986), for the prevention of transplantation rejection.

6 Niels K. Jerne, Georges J.F. Köhler and César Milstein
In 1984, the Nobel Prize in Physiology and Medicine was awarded jointly to Niels K. Jerne, Georges J.F. Köhler and César Milstein "for theories concerning the specificity in development and control of the immune system and the discovery of the principle for production of monoclonal antibodies".

7 Milestones in biological therapy
Serum therapy for diphtheria (1890) Treatment for agammaglobulinemia with purified immunogobulin G (1952) The development of monoclonal antibody (mAb) technology by Köhler and Milstein (1975) leading to the approval of the first therapeutic murine mAb, Muromonab-OKT3 (1986), for the prevention of transplantation rejection. Moreover, the progress of molecular and transgenic technologies has enabled the development of chimeric mAb, Abciximab-ReoPro (Gp IIb-IIIa, 1994) and Rituximab-Rituxan (CD20, 1997), humanized (complementarity-determining region; CDR-grafted) mAb, Trastuzumab-Herceptin (Her2/Neu, 1998) and Infliximab-Remicade (TNFa, 1998) fully human mAb, phage display–derived Adalimumab-Humira (TNFa, 2002) and transgenic mouse-derived Panitumumab-Vectibix (EGFR, 2006) The progress of development of these substances has found a niche in the management of various severe diseases, including cancerous, autoimmune and inflammatory syndromes.

8 Monoclonal antibody product analysis, historical and forecast sales growth ($m)
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9 Monoclonal antibody product trends - companies
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10 Outline of the lecture Overview on monoclonal antibodies as therapeutics Molecular biological technologies to manipulate and produce human antibody based therapeutics Examples to highlight the application of biological therapeutics to manipulate the complement system

11 The structure of human immunoglobulin G
Antigen binding Light chain (L) Variable (V) domains (VL and VH domains) Heavy chain (H) Two light chains/molecule (kappa or lambda) Two heavy chains/molecule (mu, gamma, delta, alpha or epsilon Constant (C) domains (CL, CH1, CH2, CH3)

12 Complementarity determining (CDR) and hypervariable regions
in the heavy and light chains

13 How to produce humanized or human antibodies in large scale?
The sequence of the variable domains (VH, VL) with the 3+3 hypervariable regions are required these sequences are unique, and only present in the mature B cells (after immunization or infectious disease) The sequence of the constant domains are also required Known and available Genetic modification of mouse monoclonal antibodies Chimera production Humanization Production of human antibodies Hybridoma technology Antibody (Phage) libraries Transgenic animals

14 Induction of anti-mouse immune response in humans
Figure 2 | Antibody engineering.   Mouse hybridoma technology generates mouse monoclonal antibodies. Genetic engineering has fostered the generation of chimeric, humanized and human antibodies. Cloning of mouse variable genes into human constant-region genes generates chimeric antibodies. Humanized antibodies are generated by the insertion of mouse complementarity-determining regions (CDRs) onto human constant and variable domain frameworks; however, additional changes in the framework regions have, in several cases, been shown to be crucial in maintaining identical antigen specificity75, 76. Fully human antibodies can be generated by the selection of human antibody fragments from in vitro libraries (see Box 2 and Fig. 3a), by transgenic mice (Fig. 3b) and through selection from human hybridomas. Induction of anti-mouse immune response in humans „HAMA”: human anti-mouse antibodies loss of functional activity of the therapeutics induction of side effects interference in immunoassays

15 Production of human antibodies
Single-chain Ab Figure 3 | In vitro and in vivo human antibody techniques exemplified by phage display and transgenic mouse technologies.   a | The in vitro process is based on panning the library of antibodies against an immobilized target. The non-binding phage antibodies are washed away and the recovered antibodies are amplified by infection in Escherichia coli. The selection rounds are subsequently repeated until the desired specificity is obtained. The antibody format for screening is either Fab or single-chain Fv. The expression of antibodies in E. coli and recent developments in screening technologies77 have made it possible to screen tens of thousands of clones for specificity. The antibody fragments themselves can be used as therapeutic agents as discussed in this review, but they can also be converted into intact immunoglobulins by the cloning of the variable genes into plasmids incorporating the constant-region genes of immunoglobulins. The genes are transfected into cell lines and therefore produce fully human immunoglobulins. b | The in vivo process is based on the immunization of a transgenic mouse. The mouse has been genetically engineered and bred for the expression of human immunoglobulins. The B cells harvested after immunization can be immortalized by fusion with a myeloma cell line, as in traditional hybridoma technology. The hybridomas can then be screened for specific antibodies.

16 Engineering of constant domains
Constant domains determine The biological functions of the antibodies Receptor interactions (Fc receptors) Complement activation (IgG1: ADCC reaction and CDC) Neutralization (IgG4) In vivo half-life and access to storage pools depends on glycosilation, which is determined by expression/production systems Tissue culture: prokaryotes, yeast, insect cells, eukaryote cells Living organisms: transgenic plants, transgenic animals (secretion of antibodies to milk, to serum, etc…) The compartment of its production Bloodstream Milk (secretory component)

17 Targeting the human complement system by biological therapeutics, examples
The complement system is a plasma serine protease system, composed by soluble (zymogen) proteases, proteins, humoral regulators, cell-surface regulators and cellular receptors It is part of the complex plasma serine protease system, including Coagulation Fibrinolysis Contact (kinin-kallikrein) system Complement system These systems have common activators (injury) and common regulators (protease inhibitors)

18 Key biological functions of complement
Tissue macrophages Dendritic cells Mast cells B cells Monocytes T cells Neutrophils Complement system A komplementrendszer híd a veleszületett és az adaptív immunitás között. Bár tradícionálisan a komplementrendszert úgy tekintjük, mint a patogének elleni védekezés ősi szisztémáját, azonban tudtalevőleg a szervezet homeosztázisának fenntartásában is részt vesz, felismeri és eltakarítja az apoptotikus és nekrotikus sejteket, az immunkomplexeke. Kommunikál számos immunsejttel és beindítja, modulálja az immunfolyamatrokat. Innate immunity Clearance Adaptive immunity Opsonisation Lysis of pathogens Chemotaxis Inflammation Activation of target cells Immunecomplexes Apoptotic cells Necrotic cells Augmentation of antibody production T-cell response Depletion of self-reacting B-cells Induction of B-cell memory

19 Schematic presentation of the complement system
Classical pathway (Immunecomplexes) Alternative pathway (Spontaneous C3 activation) Factor B and Factor D Regulators: C1-inhibitor, C4-binding protein, Factor I C3 activation Regulators: MCP, CD59, DAF, Factor H and Factor I Lectin pathway (Carbohydrate structures) Alternative pathway amplification C3b Opsonization Antigen presentation Antibody production C5 activation Regulators: S protein and Clusterin Anaphylatoxins C3a, C5a Inflammation Chemotaxis C5-C9 Terminal Pathway Lysis Cellular damages Induction of apoptosis

20 Complement related human pathologies
Deficiency (genetic or acquired) C1-inhibitor (hereditary angioedema) Alternative pathway regulators (Paroxysmal Nocturnal Hemoglobinuria, atypical Hemolytic Uremic Syndrome, ) Terminal pathway components (meningitis) Pathological activation Autoimmune diseases (immunecomplex diseases) Transplant rejection Ischemia/reperfusion (stroke, myocardial infarction, etc…) Hemodialysis, on-pump cardiac operation Dysregulated activation and consumption Sepsis Pathological pregnancies, preeclampsia, HELLP syndrome, DIC Complement related biological therapies Substitution of deficient factor/protein Non-specific inhibition of pathological activation Targeted inhibition of complement activation

21 Substitution therapy for HAE with C1-deficiency
Classical pathway (Immuncomplexes) Alternative pathway (Spontaneous C3 activation) Factor B and Factor D Regulators: C1-inhibitor, C4-binding protein, Factor I C3 activation Regulators: MCP, CD59, DAF, Factor H and Factor I Lectin pathway (Carbohydrate strucutures) Life-threatening edematous attacks (Bradikinin overproduction) Acute treatment with C1-inhibitor concentrate Purified human C1-inhibitor Cetor/Sanquin or Berinert P/Behring Nanofiltrated Cinryze/ViroPharma (4th most expensive drug, $/year Recombinant human C1-inhibitor Rhucin/Pharming Alternative pathway amplification C3b Opsonization Antigen presentation Antibody production C5 activation Regulators: S protein and Clusterin Anaphylatoxins C3a, C5a Inflammation Chemotaxis C5-C9 Terminal Pathway Lysis Cellular damages Induction of apoptosis

22 Inhibition of pathological complement activation
Classical pathway (Immunecomplexes) Alternative pathway (Spontaneous C3 activation) Factor B and Factor D Regulators: C1-inhibitor, C4-binding protein, Factor I C3 activation Regulators: MCP, CD59, DAF, Factor H and Factor I Lectin pathway (Carbohydrate structures) Alternative pathway amplification C3b Opsonization Antigen presentation Antibody production C5 activation sCR1 (soluble complement receptor 1) Regulators: S protein and Clusterin Inhibition of complement activation on multiple levels Aimed to be used in I/R injury situation, i.e. by-pass operation Lack of breakthrough results with this drug Anaphylatoxins C3a, C5a Inflammation Chemotaxis C5-C9 Terminal Pathway Lysis Cellular damages Induction of apoptosis

23 Y Inhibition of pathological complement activation IgG4 Eculizumab
Classical pathway (Immunecomplexes) Alternative pathway (Spontaneous C3 activation) Factor B and Factor D Regulators: C1-inhibitor, C4-binding protein, Factor I C3 activation Regulators: MCP, CD59, DAF, Factor H and Factor I Lectin pathway (Carbohydrate structures) Alternative pathway amplification C3b Y Opsonization Antigen presentation Antibody production IgG4 C5 activation Eculizumab (humanized murine anti-C5 Ab) 1st most expensive drug, $/year Pexelizumab (scV anti-C5 Ab) Regulators: S protein and Clusterin Anaphylatoxins C3a, C5a Inflammation Chemotaxis C5-C9 Terminal Pathway Lysis Cellular damages Induction of apoptosis

24 Current on-label indication and off-label applications for Eculizumab
On-label: Paroxysmal Nocturnal Hemoglobinuria (PNH) Disease of hemopoetic stem cells (clonal deletion of GPI-anchor for receptors, including complement regulators CD59 and DAF) Red blood cells are susceptible to episodic hemolysis mediated by complement Chronic, progressive disease with recurrent thrombosis and organ-ischemia Current management: regular transfusions, anticoagulation, bone-marrow transplantation, and since 2007 targeted therapy with Eculizumab Off-label applications: Current clinical trials with Eculizumab Atypical hemolytic uremic syndrome Age-related macular degenration Complement-mediated injury after kidney transplantation Dense-deposit disease, C3-nephropathy Neuromyelitis optica Catastrophic Antiphospholipid syndrome Cold-agglutinin disease ANCA-vasculitis Sickle-cell disease

25 A simplified overview on the classification of thrombotic microangiopathies (based on Besbas et al., 2006, Kidney Int.) Advanced etiology, no underlying disease Infections Shiga-like toxin producing pathogens Neuraminidase producing pathogens Complement dysregulation Alternative pathway dysregultaion Thrombomodulin mutation Failure of von-Willebrand factor processing Acquired ADAMTS13 inhibitory antibodies Congenital defect of ADAMTS13 protease (Upshaw-Schülman sy) Secondary forms, underlying diseases Typical clinical presentation Acute renal failure, HUS Critically ill, HUS Acute neurological symptoms, TTP TMA as severe complication

26 Laboratory tests currently used for the work-up of patients with clinical TMA in our laboratory
Advanced etiology, no underlying disease Infections Shiga-like toxin producing pathogens Neuraminidase producing pathogens Complement dysregulation Alternative pathway dysregultaion Thrombomodulin mutation Failure of von-Willebrand factor processing Acquired ADAMTS13 inhibitory antibodies Congenital defect of ADAMTS13 protease (Upshaw-Schülman sy) Secondary forms Functional complement measurements CH50 and WIELISA-ALT Complement protein determination C3, C4, FH, FB, FI Mutation screening CFH exons 2, 4, 6, , 17, 18, 20-23 CFI exons 3, 5-6, 9-10, 12-13 CD46 exons 5-6 C3 exons 14, 20, 26-27, 37 CFB exons 6-7 THBD in progress Haplotype analysis CFH tag SNPs MCP tag SNPs Copy number determination on 1q32 (MLPA) Screening for autoimmune form of aHUS (anti-Factor H IgG)

27 Current and future therapeutic options for patients with aHUS
Episodic occurence of disease shub (hemolysis with fragmented erythrocytes, LDH increase , low platelet count) Therapy: Plasma exchange Immunosuppression Cytostatica ESRD, dialysis, tx Eculizumab 900 mg/week for 4 weeks, thereafter 1200 mg/two weeks

28 The autoimmune form of atypical HUS (Biologicals for the treatment of autoimmune disease)
Presence of pathogenic autoantibodies against factor H Linked to CFHR1-3 deletion Binding to the functionally active N-terminal part of the molcule Inhibition of the complement regulating activity of FH Specific therapeutic approach: inhibition of autoantibody production by the depletion of B-cells

29 Rituximab (Rituxan, MabThera)
Anti-CD20 monclonal antibody (human-mouse chimera) developed to deplete B-cells (treatment of lymphomas and leukemias) The ligand of CD20 is unknown, the molecule is involved in the regulation of calcium flux The mechanisms of action are: induction of ADCC reaction, of complement dependent cytotoxicity, and of apoptosis; and saturation of Fc receptors Recently, the drug found its way to treat diseases characterized by hyperactive B-cells, producing autoantibodies One treatment cycle (4 doses of 375 mg/m2, 1 each week) depletes CD20-pos B cells from the periphery for ~2 years

30 CD20-positive B-cell depletion in autoimmune diseases
Rheumatological diseases Rheumatoid arthritis Systemic lupus erythematosus (SLE) Sjögren’s syndrome Dermatomyositis and polymyositis Vasculitides Non-rheumatological autoimmune diseases Idiopathic thrombocytopenic purpura (ITP) Thrombotic thrombocytopenic purpura (TTP) Autoimmune hemolytic anaemia (AIHA) Pemphigus vulgaris and foliaceus Perosa et al, J Intern Med, 2010

31 IVIG Diagnosis of TTP Hysterect. 109 /l Curettage Sectio Hgmm g/l
Haematoma evac. 109 /l Curettage Sectio Hgmm IVIG g/l Diagnosis of TTP Feresis Madách K és mtsai: Aneszteziológia és Intenzív Terápia, 2008; 38(1): 34-38

32 Mechanisms of action of IVIG in autoimmune and inflammatory diseases
Blockade of Fc receptors on macrophages of the reticuloendothelial system of liver and spleen Restoration of the idiotypic–anti-idiotypic network Suppression or neutralization of cytokines by specific antibodies in the IVIG Blockage of binding of adhesion molecules on leukocytes to vascular endothelium Inhibition of complement uptake on target tissues Neutralization of microbial toxins Saturation of the FcRn receptors to enhance the clearance of autoantibodies Induction of inhibitory FcgRIIb receptors on effector macrophages Neutralization of growth factors for B cells, such as B-cell activating factor Inhibition of T cell–proliferative responses Expansion, activation, or both of a population of Treg cells Inhibition of the differentiation and maturation of dendritic cells Ballow M, JACI, 2011

33 Mechanisms of action of intravenous immune globulin (IgIV) on the immune modulation of various components of the innate and adaptive immune systems. (Adapted from Tha-In et al. Trend Immunol, 2008) DC, Dendritic cell; Mo, monocyte; NK, natural killer.

34 Take home messages Biological therapy, 2011: 29 companies, 52 products, several hundreds of indications, 40 milliard US dollars annual turnover Several diseases, that were untreatable or treatable but only in non-specific manner, are now efficiently cured or treated Based on continuous product development, there is increased efficacy (engineering of biological effects) decreased side-effects of novel products (100% human antibodies) Drugs, currently in clinical practice are increasingly used off-label, and this will soon result in broadening of the field of indications rituximab for autoimmune diseases Alternative applications of different preparations for substitution therapies is also spreading IVIG for modulation of autoimmunity and inflammation The appearance of generic drugs will also arrive soon (for rituximab: =2012) Biosimilarity, in contrast to bioequivalency

35 Thank you for your attenetion! www.kutlab.hu


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