Biological therapy for the manipulation of complement system Biological therapy


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Biological therapy for the manipulation of complement system


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



Milestones in biological therapy

  • Serum therapy for diphtheria (1890)



The first therapeutic approach, that was created with the understanding of the etiopathogensis of disease



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.





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.







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



The structure of human immunoglobulin G

  • Two light chains/molecule

    • (kappa or lambda)
  • Two heavy chains/molecule

    • (mu, gamma, delta, alpha or epsilon




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

  • Production of human antibodies

    • Hybridoma technology
    • Antibody (Phage) libraries
    • Transgenic animals






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)


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)



Key biological functions of complement





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

  • Complement related biological therapies

    • Substitution of deficient factor/protein
    • Non-specific inhibition of pathological activation
    • Targeted inhibition of complement activation








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


A simplified overview on the classification of thrombotic microangiopathies (based on Besbas et al., 2006, Kidney Int.)

  • Advanced etiology, no underlying disease

    • Infections
    • 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



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



Current and future therapeutic options for patients with aHUS



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



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



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




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





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


Thank you for your attenetion! www.kutlab.hu





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