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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 prohoz@kut.sote.hu

  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 1854-1917 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 1858-1937 „based on an international mandate, he checked the safity of the diphteria antitoxin” 1894 Edwin Klebs 1834-1913 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 theprevention of transplantation rejection.

  6. César Milstein Niels K Jerne Georges JF Köhler • 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) 8

  9. Monoclonal antibody producttrends - companies 9

  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. Two light chains/molecule (kappa or lambda) Two heavy chains/molecule (mu, gamma, delta, alpha or epsilon The structure of human immunoglobulin G Antigen binding Light chain (L) Variable (V) domains (VL and VH domains) Heavy chain (H) 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 • „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

  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 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 C3 activation Regulators: C1-inhibitor, C4-binding protein, Factor I 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 C3 activation Regulators: C1-inhibitor, C4-binding protein, Factor I 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, 350.000 $/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 C3 activation Regulators: C1-inhibitor, C4-binding protein, Factor I 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. Inhibition of pathological complement activation Classical pathway (Immunecomplexes) Alternative pathway (Spontaneous C3 activation) Factor B and Factor D C3 activation Regulators: C1-inhibitor, C4-binding protein, Factor I Regulators: MCP, CD59, DAF, Factor H and Factor I Lectin pathway (Carbohydrate structures) Alternative pathway amplification C3b Y Opsonization Antigen presentation Antibody production IgG4 Eculizumab (humanized murine anti-C5 Ab) 1st most expensive drug, 409.500 $/year Pexelizumab (scV anti-C5 Ab) C5 activation 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. 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 renal failure, HUS Acute neurological symptoms, TTP TMA as severe complication A simplified overview on the classification of thrombotic microangiopathies (based on Besbas et al., 2006, Kidney Int.)

  26. 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, 9 14-15, 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) Laboratory tests currently used for the work-up of patients with clinical TMA in our laboratory

  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. Haematoma evac. Hysterect. 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 factorsfor 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: 1997+15=2012) • Biosimilarity, in contrast to bioequivalency

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