Presentation on theme: "Biological therapy for the manipulation of complement system"— Presentation transcript:
1 Biological therapy for the manipulation of complement system Prohászka Zoltán,IIIrd Department of Medicine, Research LaboratorySemmelweis University
2 Biological therapyBiological 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 therapyBiological response modifiers (BRMs) are substances influencing biological functions, like interferons, interleukins, growth factors and colony stimulating factorsVaccination
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 diseaseEmil von BehringNobel 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, 1890Johannes Bókay Jr„based on an international mandate, he checked the safity of the diphteria antitoxin”1894Edwin KlebsCorynebacterium diphtheriaeKlebs-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 toNiels 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 ofchimeric 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.
10 Outline of the lectureOverview on monoclonal antibodies as therapeuticsMolecular biological technologies to manipulate and produce human antibody based therapeuticsExamples to highlight the application of biological therapeutics to manipulate the complement system
11 The structure of human immunoglobulin G Antigen bindingLight 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 epsilonConstant (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 requiredthese sequences are unique, and only present in the mature B cells (after immunization or infectious disease)The sequence of the constant domains are also requiredKnown and availableGenetic modification of mouse monoclonal antibodiesChimera productionHumanizationProduction of human antibodiesHybridoma technologyAntibody (Phage) librariesTransgenic 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 antibodiesloss of functional activity of the therapeuticsinduction of side effectsinterference in immunoassays
15 Production of human antibodies Single-chain AbFigure 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 determineThe biological functions of the antibodiesReceptor 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 systemsTissue culture: prokaryotes, yeast, insect cells, eukaryote cellsLiving organisms: transgenic plants, transgenic animals (secretion of antibodies to milk, to serum, etc…)The compartment of its productionBloodstreamMilk (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 receptorsIt is part of the complex plasma serine protease system, includingCoagulationFibrinolysisContact (kinin-kallikrein) systemComplement systemThese systems have common activators (injury) and common regulators (protease inhibitors)
18 Key biological functions of complement Tissue macrophagesDendritic cellsMast cellsB cellsMonocytesT cellsNeutrophilsComplement systemA 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 immunityClearanceAdaptive immunityOpsonisationLysis of pathogensChemotaxisInflammationActivation of target cellsImmunecomplexesApoptotic cellsNecrotic cellsAugmentation of antibody productionT-cell responseDepletion of self-reacting B-cellsInduction of B-cell memory
19 Schematic presentation of the complement system Classical pathway (Immunecomplexes)Alternative pathway (Spontaneous C3 activation)Factor B and Factor DRegulators:C1-inhibitor, C4-binding protein, Factor IC3 activationRegulators:MCP, CD59, DAF, Factor H and Factor ILectin pathway (Carbohydrate structures)Alternative pathwayamplificationC3bOpsonizationAntigen presentationAntibody productionC5 activationRegulators:S protein and ClusterinAnaphylatoxins C3a, C5aInflammationChemotaxisC5-C9Terminal PathwayLysisCellular damagesInduction 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 activationAutoimmune diseases (immunecomplex diseases)Transplant rejectionIschemia/reperfusion (stroke, myocardial infarction, etc…)Hemodialysis, on-pump cardiac operationDysregulated activation and consumptionSepsisPathological pregnancies, preeclampsia, HELLP syndrome, DICComplement related biological therapiesSubstitution of deficient factor/proteinNon-specific inhibition of pathological activationTargeted inhibition of complement activation
21 Substitution therapy for HAE with C1-deficiency Classical pathway (Immuncomplexes)Alternative pathway (Spontaneous C3 activation)Factor B and Factor DRegulators:C1-inhibitor, C4-binding protein, Factor IC3 activationRegulators:MCP, CD59, DAF, Factor H and Factor ILectin pathway (Carbohydrate strucutures)Life-threatening edematous attacks (Bradikinin overproduction)Acute treatment with C1-inhibitor concentratePurified human C1-inhibitorCetor/Sanquin or Berinert P/BehringNanofiltrated Cinryze/ViroPharma (4th most expensive drug, $/yearRecombinant human C1-inhibitorRhucin/PharmingAlternative pathwayamplificationC3bOpsonizationAntigen presentationAntibody productionC5 activationRegulators:S protein and ClusterinAnaphylatoxins C3a, C5aInflammationChemotaxisC5-C9Terminal PathwayLysisCellular damagesInduction of apoptosis
22 Inhibition of pathological complement activation Classical pathway (Immunecomplexes)Alternative pathway (Spontaneous C3 activation)Factor B and Factor DRegulators:C1-inhibitor, C4-binding protein, Factor IC3 activationRegulators:MCP, CD59, DAF, Factor H and Factor ILectin pathway (Carbohydrate structures)Alternative pathwayamplificationC3bOpsonizationAntigen presentationAntibody productionC5 activationsCR1(soluble complement receptor 1)Regulators:S protein and ClusterinInhibition of complement activation on multiple levelsAimed to be used in I/R injury situation, i.e. by-pass operationLack of breakthrough results with this drugAnaphylatoxins C3a, C5aInflammationChemotaxisC5-C9Terminal PathwayLysisCellular damagesInduction of apoptosis
23 Y Inhibition of pathological complement activation IgG4 Eculizumab Classical pathway (Immunecomplexes)Alternative pathway (Spontaneous C3 activation)Factor B and Factor DRegulators:C1-inhibitor, C4-binding protein, Factor IC3 activationRegulators:MCP, CD59, DAF, Factor H and Factor ILectin pathway (Carbohydrate structures)Alternative pathwayamplificationC3bYOpsonizationAntigen presentationAntibody productionIgG4C5 activationEculizumab(humanized murine anti-C5 Ab)1st most expensive drug, $/yearPexelizumab(scV anti-C5 Ab)Regulators:S protein and ClusterinAnaphylatoxins C3a, C5aInflammationChemotaxisC5-C9Terminal PathwayLysisCellular damagesInduction 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 complementChronic, progressive disease with recurrent thrombosis and organ-ischemiaCurrent management: regular transfusions, anticoagulation, bone-marrow transplantation, and since 2007 targeted therapy with EculizumabOff-label applications: Current clinical trials with EculizumabAtypical hemolytic uremic syndromeAge-related macular degenrationComplement-mediated injury after kidney transplantationDense-deposit disease, C3-nephropathyNeuromyelitis opticaCatastrophic Antiphospholipid syndromeCold-agglutinin diseaseANCA-vasculitisSickle-cell disease
25 A simplified overview on the classification of thrombotic microangiopathies (based on Besbas et al., 2006, Kidney Int.)Advanced etiology, no underlying diseaseInfectionsShiga-like toxin producing pathogensNeuraminidase producing pathogensComplement dysregulationAlternative pathway dysregultaionThrombomodulin mutationFailure of von-Willebrand factor processingAcquired ADAMTS13 inhibitory antibodiesCongenital defect of ADAMTS13 protease(Upshaw-Schülman sy)Secondary forms, underlying diseasesTypical clinical presentationAcute renal failure, HUSCritically ill, HUSAcute neurological symptoms, TTPTMA as severe complication
26 Laboratory tests currently used for the work-up of patients with clinical TMA in our laboratory Advanced etiology, no underlying diseaseInfectionsShiga-like toxin producing pathogensNeuraminidase producing pathogensComplement dysregulationAlternative pathway dysregultaionThrombomodulin mutationFailure of von-Willebrand factor processingAcquired ADAMTS13 inhibitory antibodiesCongenital defect of ADAMTS13 protease(Upshaw-Schülman sy)Secondary formsFunctional complement measurementsCH50 and WIELISA-ALTComplement protein determinationC3, C4, FH, FB, FIMutation screeningCFH exons 2, 4, 6, , 17, 18, 20-23CFI exons 3, 5-6, 9-10, 12-13CD46 exons 5-6C3 exons 14, 20, 26-27, 37CFB exons 6-7THBD in progressHaplotype analysisCFH tag SNPsMCP tag SNPsCopy 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 exchangeImmunosuppressionCytostaticaESRD, dialysis, txEculizumab900 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 HLinked to CFHR1-3 deletionBinding to the functionally active N-terminal part of the molculeInhibition of the complement regulating activity of FHSpecific 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 fluxThe mechanisms of action are: induction of ADCC reaction, of complement dependent cytotoxicity, and of apoptosis; and saturation of Fc receptorsRecently, the drug found its way to treat diseases characterized by hyperactive B-cells, producing autoantibodiesOne 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 diseasesRheumatoid arthritisSystemic lupus erythematosus (SLE)Sjögren’s syndromeDermatomyositis and polymyositisVasculitidesNon-rheumatological autoimmune diseasesIdiopathic thrombocytopenic purpura (ITP)Thrombotic thrombocytopenic purpura (TTP)Autoimmune hemolytic anaemia (AIHA)Pemphigus vulgaris and foliaceusPerosa et al, J Intern Med, 2010
31 IVIG Diagnosis of TTP Hysterect. 109 /l Curettage Sectio Hgmm g/l Haematomaevac.109 /lCurettageSectioHgmmIVIGg/lDiagnosis of TTPFeresisMadách 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 spleenRestoration of the idiotypic–anti-idiotypic networkSuppression or neutralization of cytokines by specific antibodies in the IVIGBlockage of binding of adhesion molecules on leukocytes to vascular endotheliumInhibition of complement uptake on target tissuesNeutralization of microbial toxinsSaturation of the FcRn receptors to enhance the clearance of autoantibodiesInduction of inhibitory FcgRIIb receptors on effector macrophagesNeutralization of growth factors for B cells, such as B-cell activating factorInhibition of T cell–proliferative responsesExpansion, activation, or both of a population of Treg cellsInhibition of the differentiation and maturation of dendritic cellsBallow 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 messagesBiological therapy, 2011: 29 companies, 52 products, several hundreds of indications, 40 milliard US dollars annual turnoverSeveral diseases, that were untreatable or treatable but only in non-specific manner, are now efficiently cured or treatedBased on continuous product development, there isincreased 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 indicationsrituximab for autoimmune diseasesAlternative applications of different preparations for substitution therapies is also spreadingIVIG for modulation of autoimmunity and inflammationThe appearance of generic drugs will also arrive soon (for rituximab: =2012)Biosimilarity, in contrast to bioequivalency