Biological therapy for the manipulation of complement system

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Biological therapy for the manipulation of
complement system
Prohászka Zoltán, IIIrd Department of Medicine,
Research Laboratory Semmelweis University prohoz
_at_kut.sote.hu
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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

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Milestones in biological therapy

• Serum therapy for diphtheria (1890)

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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)
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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.

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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".
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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 displayderived
  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.

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Monoclonal antibody product analysis, historical
and forecast sales growth (m)
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Monoclonal antibody product trends - companies
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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

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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)
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Complementarity determining (CDR) and
hypervariable regions in the heavy and light
chains
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How to produce humanized or human antibodies in
large scale?

• The sequence of the variable domains (VH, VL)
  with the 33 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

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• 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

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Production of human antibodies
Single-chain Ab
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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)

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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)

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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

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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
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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

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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
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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
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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
C5 activation
Eculizumab (humanized murine anti-C5 Ab) 1st
most expensive drug, 409.500 /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
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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

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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 renal failure, HUS
• Acute neurological symptoms, TTP
• TMA as severe complication

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Laboratory tests currently used for the work-up
of patients with clinical TMA in our laboratory

• 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)

• 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

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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
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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

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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

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CD20-positive B-cell depletion in autoimmune
diseases

• Rheumatological diseases
• Rheumatoid arthritis
• Systemic lupus erythematosus (SLE)
• Sjögrens 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
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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
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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 idiotypicanti-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 cellproliferative responses
• Expansion, activation, or both of a population of
  Treg cells
• Inhibition of the differentiation and maturation
  of dendritic cells

Ballow M, JACI, 2011
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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.
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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 1997152012)
• Biosimilarity, in contrast to bioequivalency

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