Download The Interferons

In The Name Of God
The Most Compassionate
The Most Merciful
Cytokines and Growth Factors
By
Ali Jahanian Najafabadi
Email: Jahanian@pharm.mui.ac.ir
Introduction
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A diverse group of regulatory proteins or glycoproteins
Produced in minute quantities by the body
Act as chemical communicators
Intracellular signal transduction
The major proteins/protein families that constitute the cytokine group of regulatory molecules
Introduction
• Mostly produced by, or act upon leukocytes
IgG FcR
Opsonized antigen
IgER
Hyper sensitivity
IgER
Parasitic worms
Kupffer cells, microglia, and alveolar macrophages
Introduction
• Mostly produced by leukocytes
 Play a central role in:
 Regulating both immune and inflammatory function
 Processes such as haematopoiesis (the production of blood cells from
haematopoietic stem cells in the adult bone marrow)
 Wound healing
Introduction
• Nomenclature:
 Cytokines: Firstly introduced in the mid 1970s for the
polypeptide growth factors controlling the differentiation and
regulation of cells of the immune system
 Additional classification:
 Lymphokines (cytokines such as IL-2 and IFN-γ, produced by lymphocytes)
 Monokines (cytokines such as TNF-α, produced by monocytes)
 On the basis of the specific biological activity by which the
cytokine was first discovered
 TNF exhibited cytotoxic effects on some cancer cell lines
 CSFs promoted the growth in vitro of various leukocytes in clumps or
colonies).
Introduction
• Nomenclature:
 Primary sequence analysis of cytokines coupled to determination of
secondary and tertiary structure  one of six families:
Many are known by more than one name:
IL-1: lymphocyte activating factor (LAF), endogenous pyrogen, catabolin and
mononuclear cell factor
Introduction
• The effects of recombinant DNA technology and monoclonal
antibody technology on the understanding of cytokine biology:
 Genetic engineering allowed production of large quantities of most cytokines.
These could be used for structural and functional studies of the cytokine itself, and its
receptor
 Analysis of cytokine genes established the exact evolutionary relationships between
these molecules
 Detection of cytokine mRNA and cytokine receptor mRNA allowed identification of the
full range of sources and target cells of individual cytokines
 Hybridoma technology facilitated development of immunoassays capable of
detecting and quantifying cytokines
 Inhibition of cytokine activity in vivo by administration of monoclonal antibodies (and,
more recently, by gene knockout studies) continues to elucidate the physiological and
pathophysiological effect of various cytokines.
Introduction
• The following generalizations may be made with regard to most
cytokines:
 Very potent regulatory molecules, nanomolar to picomolar concentrations
 Produced by a variety of cell types, which may be leukocytes or nonleukocytes: e.g. IL-1: leukocytes, non-leukocytes (such as smooth muscle cells,
vascular endothelial cells, fibroblasts cells and collagen fibre, astrocytes and
chondrocytes
 Many cell types can produce more than one cytokine:
 Lymphocytes: wide range of interleukins, CSFs, TNF, IFN-αs and IFN-γ
 Fibroblasts can produce IL-1, -6, -8, and -11, CSFs, IFN-β and TNF
Introduction
• The following generalizations may be made with regard to most
cytokines:
 Most cytokines are pleiotropic, i.e. can affect a variety of cell types:
 IFN-γ stimulates activation and growth of

T- and B lymphocytes,

macrophages,

NK cells,

fibroblasts

endothelial cells

It also displays weak anti-proliferative activity
with some cell types.
Introduction
• The following generalizations may be made with regard to most
cytokines:
 Many cytokines play a regulatory role in processes other than immunity and
inflammation: EPO
 Most cytokines are inducible, and are secreted by their producer cell:
IL1IL2
 potent cytokine inducers : infectious agents, tissue injury and toxic stimuli
 some cytokines appear to be expressed constitutively: EPO, CSFs
 Many cytokines exhibit redundancy: i.e. two or more cytokines can induce a
similar biological effect: TNF-α and -β
Introduction
• The following generalizations may be made with regard to most
cytokines:
 Cascade induction
Introduction
• Not all polypeptide regulatory factors are classified as cytokines:
– Classical polypeptide hormones are not considered members of the cytokine
family: Insulin, FSH and GH
•
•
•
•
•
Hormones
Secreted by one types of
specialized cells
Unique action
Restricted target cells and a
limited spectrum of actions
(excp insulin)
Act at a distant site
•
•
•
•
Cytokines
Made by more than one type of
cells
Overlap actions (redundancy)
Multiple target cells, multiple
actions
Short action radius
EPO is produced in the kidney and liver and acts in an endocrine manner, promoting production of red
blood cells in the bone marrow. EPO could thus also be considered to be a true hormone.
Introduction
• Autocrine,Paracrine and Endocrine
Introduction
• Cytokine receptors
Introduction
• Cytokine receptors
 Based upon amino acid sequence homology, receptors are usually classified as
belonging to one of 5 known superfamilies:
 Immunoglobulin superfamily receptors
 Class I cytokine receptor family (also known as the hematopoietin receptor family)
 Class II cytokine receptor family (also known as the interferon receptor family)
 TNF receptor family
 Chemokine receptor family
Introduction
• Cytokine receptors
 Based upon amino acid sequence homology, receptors are usually classified as
belonging to one of 5 known superfamilies:
 Immunoglobulin superfamily receptors
Introduction
• Cytokine receptors
 Based upon amino acid sequence homology, receptors are usually classified as
belonging to one of 5 known superfamilies:
 Class I cytokine receptor family (also known as the hematopoietin receptor family)
Introduction
• Cytokine receptors
 Based upon amino acid sequence homology, receptors are usually classified as
belonging to one of 5 known superfamilies:
 Class II cytokine receptor family (also known as the interferon receptor family)
Introduction
• Cytokine receptors
 Based upon amino acid sequence homology, receptors are usually classified as
belonging to one of 5 known superfamilies:
 TNF receptor family
Introduction
• Cytokine receptors
 Based upon amino acid sequence homology, receptors are usually classified as
belonging to one of 5 known superfamilies:
 Chemokine receptor family
Introduction
• Cytokine receptors
Introduction
• Cytokine receptors
 Individual members of any one superfamily characteristically display 20–50 per cent
homology
 In some cases a single receptor may contain domains characteristic of two or
more superfamilies. e.g. IL-6: both the haematopoietic and immunoglobulin
superfamilies
 Some cytokine receptors are composed of a single transmembrane polypeptide
(e.g. receptors for IL-8, -9 and -10). Many contain two polypeptide components
(including the IL-3, -4, and -5 receptors), and a few contain three or more polypeptide
components (e.g. the IL-2 receptor contains three polypeptide chains)
 In some instances a single cytokine may be capable of initiating signal transduction by
binding two or more distinct receptors (e.g. IL-1 has two distinct receptors (types
I and II), both of which are transmembrane glycoproteins).
Introduction
• Cytokine receptors
– In many cases where a receptor consists of multiple polypeptides, one of those polypeptides (which
will be unique to that receptor) will interact directly with the ligand. The additional polypeptide(s),
responsible for initiation of signal transduction, may be shared by a number of receptors.
Leukaemia inhibitory factor (LIF)
Introduction
• Cytokines as biopharmaceuticals
As coordinators of the immune and inflammatory response
EPO in anemic person
Growth factors and wound healing
Neurotrophic factors in the abatement of certain neurodegenerative diseases
Defeating certain viral pathogens:
 Cowpox: IL1 binding protein
 EBV: IL10 homologous protein
 Overproduction of certain cytokines
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 Pro-inflammatory cytokines: IL6, IL8 and TNF: septic shock and
rheumatoid arthritis
 Administration of mABs or soluble receptors
The Interferons
The Interferons
 The first family of cytokines to be discovered
 History: In 1957, researchers observed that susceptible animal cells, if they were
exposed to a colonizing virus, immediately became resistant to attack by other
viruses. This resistance was induced by a substance secreted by virally infected
cells which was named interferon
 It has been shown that most species actually produce a whole range of interferons.
Humans produce at least three distinct classes, IFN-α, IFN-β and IFN-γ.
The Interferons
• Biological Effects
 Induction of cellular resistance to viral attack
 Regulation of most aspects of immune function
 Regulation of growth and differentiation of many cell types
 Sustenance of early phases of pregnancy in some animal species
No one interferon will display all of these biological activities
Effects are initiated by the binding of the interferon to its specific cell surface receptor
present in the plasma membrane of sensitive cells
The Interferons
• IFN-α and INF-β
 Significant amino acid sequence homology (30 per cent)
 Bind to the same receptor
 Induce similar biological activities
 Acid stable
 IFN-α and IFN-β are sometimes collectively referred to as type I interferons, or acid-stable
interferons.
 IFN-γ  Type II interferon
The Interferons
• Producer cells
The Interferons
• Medical conditions in which IFNs could be used:
 Augmentation of the immune response against infectious agents (viral, bacterial,
protozoal, etc.)
 Treatment of some autoimmune conditions
 Treatment of certain cancer types
The Interferons
• The biochemistry of interferon-α
 In human, 24 related genes or pseudo-genes, code for the production of at least 16
distinct mature IFN-αs
 15 type I: 166 aa,
 1 type II: 172 aa
 23 aa Signal peptide
 Predicted mass of 19-20 kD, but observed up to 27 kD (why?):
 70% aa homology, Rich in leucine and glutamic acid
 Conserved cysteines (usually at positions 1, 29, 99 and 139), two disulfide bonds
 Each have an identifying name: In most cases the names were assigned by placing a
letter after the ‘α’ (i.e. IFN-αA, IFN-αB, etc.)
 Some exceptions exist which contain a number or a number and letter, e.g. IFN-α7,
IFN-α8, IFN-α2B.
 To ensure total confusion, several are known by two different names, e.g. IFN-α7 is also
known as IFN-αJ1
The Interferons
• The biochemistry of interferon-β
 Normally produced by fibroblasts
 The first interferon to be purified.
 In human, a single IFN-β molecule
 166 aa
 30 per cent sequence homology to IFN-αs
 A single disulfide bond
 Glycoprotein of molecular mass in excess of 20 kDa: N-gly, Asn 80
The Interferons
• The biochemistry of interferon-γ
 “Immune” interferon
 Initially purified from human peripheral blood lymphocytes
 Is produced predominantly by lymphocytes
 IL-2 and -12, can induce IFN-γ production under certain circumstances
 143 aa
 No significant sequence homology to type I family
 Predicted mass of 17 kD, but observed three bands: 16-17, 20 and 25 (Why?): N-gly, Asn 25,
97
 Its biologically active form appears to be a homodimer in which the two subunits are
associated in an antiparallel manner
The Interferons
• The interferon receptors: type I IFN receptors
 Two type I interferon receptor polypeptides
 Belonging to the class II cytokine receptor family
 Both are transmembrane N-linked glycoproteins
 α/β: capable of binding all type I interferons
 αβ: specific for IFN-α-B (a specific member of the IFN-α family)
 Present on most cell types
The Interferons
• The interferon receptors: type II IFN receptors
 The IFN-γ receptor
 More limited cellular distribution
 a transmembrane glycoprotein of molecular mass 50 kDa
 Function as a homodimer
 The extracellular IFN-γ binding region consists of approximately 200 amino acid
residues
 AF-1 (accessory factor 1, a transmembrane glycoprotein) associates with the
extracellular region of the receptor
The Interferons
• The interferon receptors: type II IFN receptors
 Cell types which display an IFN-γ receptor on their surface
The Interferons
• Interferon signal transduction
 The intracellular events triggered upon binding of type I or II interferons to their
respective receptors are quite similar
 The JAK–STAT pathway
 signal transducers and activators of transcription (STAT1-STAT6)
 Janus kinases: Janus, a Roman god with two faces: two potential active sites
The Interferons
• The JAK–STAT pathway
Ligands which, upon binding to their
cell surface receptors, are known to
promote activation of one or more
STATs. (The STATs activated are also
shown.)
The Interferons
• The JAK–STAT pathway
The Interferons
• The biological effects of interferons:
 Type I IFNs:
 Antiviral activity
 Anti tumor effects
 Anti-proliferative effect
 by increasing NK and T-cytotoxic cell activity
The Interferons
• The biological effects of interferons:
 Type I IFNs: Antiviral activity
 2–5 oligoadenylate synthetase (2,5-An synthetase) and the eIF-2α protein kinase.
(A) The OAS/RNase L system, an innate immunity pathway that acts against viral infections.
Silverman R H J. Virol. 2007;81:12720-12729
The Interferons
• The biological effects of interferons:
 IFN-γ:
 Weak antiviral and anti-proliferative activity
 Potentates the IFN-α/β activities
 Regulating most aspects of the immune and inflammatory responses
 main macrophage-activating factor
 Destruction of invading microorganisms;
 Destruction of intracellular pathogens;
 Tumour cell cytotoxicity;
 Increased major histocompatibility complex (MHC) antigen expression,
leading to enhanced activation of lymphocytes via antigen presentation
The Interferons
• The biological effects of interferons:
 IFN-γ:
 Binding to its receptor on Neutrofils  increasing in expression of a surface
protein capable of binding to Fc portion of IgG
 Direct modulation of immune responses by affecting growth, differentiation and
function of both T- and B-cells
The Interferons
• Interferon Biotechnology
 The antiviral and anti-proliferative activity of interferons, as well as their ability to
modulate the immune and inflammatory response renders obvious their potential
medical application
 Large-scale purification from sources such as blood was non-viable.
 Interferons exhibit species preference and, in some cases, strict species specificity.
 Transfused blood supplies: only 1% pure
 Mammalian cell culture: cancer cell lines; the namalwa cell line (8000 L) +sendai virus
 Recombinant DNA technology
 E. coli
 Fungi
 Yeast
 Some mammalian cell lines, such as CHO cell lines and monkey kidney cell
lines
The Interferons
• Interferon Biotechnology
The Interferons
• Medical uses of interferon-α
 Anti cancer agent


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
Breast cancer
Certain lymphomas (malignant tumour of the lymph nodes)
Multiple myeloma (malignant disease of the bone marrow)
Delay recurrence of tumour growth after surgery in osteogenic sarcoma (cancer of connective
tissue involved in bone formation)
 Approved indications of different INF-αs:
 rhIFN-α2a and -α2b (Intron A): Hairy cell leukemia
 More than 16 medical conditions
The Interferons
• Medical uses of interferon-α
 Antiviral agent: Mostly approved for viral hepatitis
 PEGylated interferons
 PEG IntronA (Pegylated rhIFN-α2b): HCV
 Viraferon Peg (Pegylated rhIFN-α2b): HCV
 PEGasys (Pegylated rhIFN-α2a): HCV
Synthetic Interferon:
 Infergen (interferon alfacon-1 or consensus interferon):
 The most frequently occurring amino acid residue in each corresponding position of the
native interferons
 Cloning and expression of the synthesized coding sequenceby E. coli
 Approved for Hepatitis C
 When compared on a mass basis, the synthetic interferon displays higher antiviral,
antiproliferative and cytokine-inducing activity than do native type I interferons.
The Interferons
• Medical uses of interferon-β
 Relapsing–remitting multiple sclerosis (MS)
 Betaferon (rIFN-β-1b, mutated form: C17S): E. coli
 Betaseron (rIFN-β-1b, mutated form: C17S): E. coli
 Avonex (rhIFN-β-1a): CHO
 Rebif (rhIFN-β-1a): CHO
 Cinnovex (Iran, (rhIFN-β-1a))
 Mechanism of action:
 Perhaps by inhibiting the production of IFN-γ and TNF-α and hence mediating downregulation of the pro-inflammatory responses against the myelin sheath
The Interferons
• Medical uses of interferon-γ
 Chronic granulomatous disease (CGD)
 Phagocytic cells are poorly capable/incapable of ingesting or destroying infectious agents such
as bacteria or protozoa
 Healthy phagocytes produce highly reactive oxidative substances, such as hydrogen peroxide
and hypochlorous acid, via a multicomponent NADPH oxidase system, which are lethal to
pathogens
 CGD: a genetic defect in any component of this oxidase system
NADPH oxidase system
The Interferons
• Medical uses of interferon-γ
 The recombinant human IFN-γ produced in E. coli: termed IFN-γ1b (Actimmune)
 A potent activator of phagocytes
 Potentiates their ability to generate toxic oxidative products
 As long as the genetic defect has not totally inactivated a component of the system,
it promotes increased synthesis of these oxidative substances
 Promotes increased expression of IgG Fc receptors on the surface of phagocytes
 Destroy opsonized infectious agents via phagocytes
The Interferons
Increased expression of IgG Fc receptors on
phagocytes results in enhanced phagocytosis.
These receptors will retain opsonized (i.e.
antibody-coated) infectious agents at their surface
by binding the Fc portion of the antibody. This
facilitates subsequent phagocytosis
The Interferons
• Interferon toxicity
 Like most drugs, administration of interferons can elicit a number of unwanted side
effects
 In some instances the severity of such effects can limit the maximum recommended
therapeutic dose to a level below that which might have maximum therapeutic effect
 The common adverse effect between the different IFNs is Flu-like symptoms
 Fever
 Headache
 Chills
The Interleukins
The Interleukins
• General aspects
 The interleukins represent another large family of cytokines
 at least 36 different constituent members
 Most of these polypeptide regulatory factors are glycosylated (a notable
exception being IL-1) and display a molecular mass ranging from 15 to 30 kDa
(IL-9: heavily glycosylated, 40 kD)
 Most of the interleukins are produced by a number of different cell types
 IL-1  17 different cell types, IL-8  at least 10 different cell types
 IL-2, IL-9 and IL-13  produced only by T- lymphocytes
The Interleukins
• General aspects
 Most cells capable of synthesizing one interleukin are capable of synthesizing
several, and many prominent producers of interleukins are non-immune
system cells
 In most instances, induction or repression of any one interleukin is prompted by
numerous regulators (mostly additional cytokines)
 IL-1 promotes increased synthesis and release of IL-2 from activated Tlymphocytes
 It is highly unlikely that cells capable of synthesizing multiple interleukins
concurrently synthesize them all at high levels
The Interleukins
• General aspects
 Nearly all of the interleukins are soluble molecules (one form of IL-1 is cell
associated)
 Their biological response is induced by binding to specific receptors on the
surface of target cells
Most interleukins exhibit paracrine activity
Some display autocrine activity : IL-2
Some interleukins display more systematic endocrine effects: IL-1
The Interleukins
 Signal Transduction:
 Tyrosine phosphorylation
 Serine and threonine residues of specific intracellular substrates are also
phosphorylated
 Promoting an increase in intracellular calcium concentration
 Inducing hydrolysis of phosphatidylethanolamine with release of diacyl
glycerol
The Interleukins
• General aspects
Many cell types are capable of producing a whole range of
interleukins
T-lymphocytes are capable of producing all the interleukins, with
the possible exception of IL-7 and IL-15.
Many cell types producing multiple interleukins can also produce
additional cytokines
For example, both macrophages and fibroblasts are capable of
producing several interleukins, CSFs and PDGF.
The Interleukins
• Biological effects
 Interleukins regulate a variety of physiological and pathological conditions, including:
 Normal and malignant cell growth;
 All aspects of the immune response;
 Regulation of inflammation.
 6 interleukin-based products have gained approval for general medical use
 Proleukin: an IL-2
 Neumega: an IL-11
 Kineret: anti IL-1
 Ustekinumab: anti IL-12 and IL23
 Reslizumab: Anti IL-5
 Secukinumab: anti IL-17A
 Ontak: DT-IL2 immunotoxin
The Interleukins
• Interleukin-2
 known as T-cell growth factor
 The first T-cell growth factor to be identified
 It is produced exclusively by T-lymphocytes (especially T-helper cells), in response to
activation by antigen and mitogens
 A single-chain glycoprotein containing 133 amino acids
 The mature molecule displays a molecular mass ranging from 15 to 20 kDa
 The carbohydrate moiety is not required for biological activity
 O-gly to 3rd Thr
The Interleukins
• Interleukin-2 Receptor
 The high-affinity receptor complex consists of three membrane-spanning polypeptide
chains (α, β and γ)
 The α chain binds IL-2 with low affinity
 The γ subunit does not interact directly with IL-2: γc (common): IL-4, 7, 9, 13 and 15
 αγ or βγ can bind IL-2 with intermediate affinity
 The heterotrimeric αβγ complex represents the cytokine’s true high-affinity receptor
Prolonged elevated levels of IL-2
promote the shedding of the IL-2
receptor α subunit from the cell
surface
The Interleukins
• Interleukin-2 Receptor
 The IL-2 receptor is associated with a number of cell types, mainly cells playing a central
role in the immune response
 Binding of IL-2 to its receptor induces growth and differentiation of these cells
Lymphokine Activated Killer
The Interleukins
• Interleukin-2 Receptor
 Quiescent T-lymphocytes are stimulated largely by direct binding to an antigen fragment
presented on the surface of a macrophage in the context of MHC complex:
 This results in the induction of expression of at least 70 genes whose products are
collectively important in immune stimulation
The Interleukins
• Interleukin-2 Receptor
 Several cytoplasmic proteins capable of inducing T-cell growth (i.e. several cellular
protooncogenes, including C-fos and C-myc).
 Various cytokines, most notably IL-2.
 Cytokine receptors, most notably the IL-2 receptor α subunit. (The T-lymphocytes
appear to constitutively express the β and γ IL-2 receptor polypeptides. Induction of the α
gene leads to formation of a high-affinity αβγ receptor complex, thereby rendering the
activated T cell highly sensitive to IL-2)
The Interleukins
• Interleukin-2 Biological Activities
 IL-2 acts as a critical autocrine growth factor for T-cells, and the magnitude of the T-cell
response is largely dependent upon the level of IL-2 produced
 IL-2 serves as a growth factor for activated B-lymphocytes. In addition to promoting
proliferation of these cells, IL-2 (as well as some other interleukins) stimulates enhanced
antibody production and secretion  humoral immune response
 IL-2 promotes the growth and differentiation of NK cells: lymphokine activated killer
(LAK) cells (with an enhanced ability to kill tumour cells or virally infected cells
directly). NK cells express the β and γ IL-2 receptor subunits only: elevated
concentrations of IL-2 is required
The Interleukins
• Interleukin-2 Clinical Applications
 Cancers
 T-cell and other forms of immunodeficiency
 Infectious diseases
The Interleukins
• Interleukin-2 production
 Sources of production
Jurkat leukemia cell line
Proleukin:
 E. coli
 Non-gly
 Lacks N-terminal Ala
 C125S
The Interleukins
Whatever the exact nature of tumour escape, it has been demonstrated, both in
vitro and in vivo, that immunostimulation can lead to enhanced tumor detection
and destruction.
• Interleukin-2 and cancer treatment
 Cancer Specific antigens:
Novel surface antigens not expressed by normal cells
Greatly elevated levels of certain antigens present normally on the cell at
extremely low levels.  so low that they have escaped immunological
tolerance
 In this regard, both a humoral and cell-mediated response can be induced,
although the T-cell response appears to be the most significant
The Interleukins
Whatever the exact nature of tumour escape, it has been demonstrated, both in
vitro and in vivo, that immunostimulation can lead to enhanced tumour detection
and destruction.
• Interleukin-2 and cancer treatment
 Some transformed cells obviously display characteristics that allow them
to evade this immune surveillance:
Most transformed cells do not express class II MHC molecules and express
lower than normal levels of class I MHC molecules
Some tumor-specific surface antigens resemble normal surface antigens
or were expressed previously during the neonatal period and considered as
“self”
Some tumors secrete cytokines and additional regulatory molecules
that can suppress local immunological activity: TGF-β
The glycocalyx
The Interleukins
• Interleukin-2 and infectious diseases
 Numerous pathogens exist for which no effective treatment could be find
 Most of these pathogens are non-bacterial (e.g. viral, fungal and parasitic,
including protozoal) capable of survival within macrophages:
 Mycobacteria (e.g. M. tuberculosis and M. leprae)
 Listeria monocytogenes (causes listeriosis which is characterized by flu-like
symptoms, but can cause swelling of the brain and induce abortions)
 Legionella pneumophila (the bacterium that causes legionnaire’s disease)
 The immunological response raised against intracellular pathogens is largely a
T-cell response. IL-2’s ability to stimulate T-cells may render it useful in the
treatment of a wide range of such conditions
The Interleukins
• Inhibition of interleukin-2 activity
 A variety of medical conditions exist that are caused or exacerbated by the
immune system itself:
Autoimmune diseases
Tissue/organ transplantation
The Interleukins
• Inhibition of interleukin-2 activity
 Selective immunosupression by preventing the synthesis or functioning of IL2:
Cyclosporin A, one of the foremost immunosuppressive agents currently
in use, functions by preventing IL-2 synthesis
Administration of soluble forms of the IL-2 receptor
Administration of monoclonal antibodies capable of binding the IL-2
receptor (without signal transduction activity)
Administration of IL-2 variants that retain the ability to bind the
receptor, but which fail to initiate signal transduction
Administration of IL-2 coupled to bacterial or other toxins: Immunotoxins
The Interleukins
• Immunotoxins
• Ontak (denileukin diftitox)
The Interleukins
• Immunotoxins
The Interleukins
• Interleukin-1
 It is also known as lymphocyte-activating factor (LAF), endogenous pyrogen and
catabolin
 Two distinct forms: IL-1α (membrane anchored) and IL-1β (soluble)
 20% amino acid homology, different genes, but bind the same receptor
 Different cell types produce the different IL-1s in varying ratios
• Fibroblasts and endothelial cells: similar ratios
• IL-1β is produced in larger quantities than IL-1α in monocytes
• Activated macrophages appear to represent the major cellular source for IL-1
The Interleukins
• Interleukin-1
 Two distinct receptors:
 Type I: fibroblasts, keratinocytes, hepatocytes and endothelial cells
 Type II: B-lymphocytes, bone marrow cells and polymorphonuclear leukocytes
Both IL-1s could bind both receptor types
The Interleukins
• Interleukin-1 Biological Effects
 It is a pro-inflammatory cytokine: major biological function of IL-1
 It plays a role in activating B-lymphocytes
 It acts as a co-stimulator of haematopoietic cell growth/differentiation
 Along with IL-6, it induces synthesis of acute-phase proteins in hepatocytes
• These various biological activities depends largely upon the quantities of IL-1
produced in any given situation
 At low concentrations: paracrine, induction of local inflammation.
 At elevated concentrations: endocrine, inducing systematic effects, such as the
hepatic synthesis of acute-phase proteins, induction of fever (hence the name,
endogenous pyrogen) and general body wasting, such as that associated with some
cancers
The Interleukins
• IL-1-like protein or IL-1 receptor antagonist (IL-1Rα)
 This molecule appears to be capable of binding to the IL-1 receptors without
triggering an intracellular response
 Clinical application: Because of the IL-1 role in mediating acute/chronic
inflammation, (downward) modulation of IL-1 levels may prove effective in
ameliorating the clinical severity of these conditions
anti-IL-1 antibodies
soluble forms of the IL-1 receptor
Native IL-1 receptor antagonist
The Interleukins
• IL-1-like protein or IL-1 receptor antagonist (IL-1Rα)
 Kineret: an approved product based on the latter strategy. Indicated in the
treatment of rheumatoid arthritis
 rhIL-1 receptor antagonist
 17.3 kD, E. coli, Non-gly, add. Met.
The Interleukins
• IL-11
 Produced largely by IL-1-activated bone marrow stromal cells and
fibroblasts
 functions as a haematopoietic growth factor
 It stimulates:
 Thrombopoiesis (the production of platelets formed by the shedding of fragments
of cytoplasm from large bone marrow cells called megakaryocytes
 Growth/differentiation of bone marrow cells, derived from stem cells that have
become committed to differentiate into macrophages
The Interleukins
 The rationale for assessing IL-11 as a potential therapeutic agent relates mainly
to its ability to induce platelet synthesis
 Neumega is the tradename given to the IL-11-based product approved for the
prevention of thrombocytopenia
Growth Factors: Haematopoietic growth factors
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Oprelvekin
Trade name: Neumega™
Approval Date: 1997
Description:The protein has a molecular mass of approximately 19kD, and is
non-glycosylated. The polypeptide is 177 amino acids in length and differs from
the 178 amino acid length of native IL-11 only in lacking the amino-terminal
proline residue. This alteration has not resulted in measurable differences in
bioactivity either in vitro or in vivo
Production system: E. coli
Dosage form: Single-use vials containing 5 mg of oprelvekin
Therapeutic Indications: Is indicated for the prevention of severe
thrombocytopenia and the reduction of the need for platelet transfusions
following myelo-suppressive chemotherapy in adult patients with nonmyeloid
malignancies who are at high risk of severe thrombocytopenia
The Tumor Necrosis
Factors
The Interleukins
• Tumour necrosis factor (TNF)
 Two types: α and β
 Bind to same receptor
 Induce very similar biological activities
 Display limited sequece homology
 Chromosome 6, adjacent, 1100bp distance
The Interleukins
• Tumour necrosis factor (TNF)
 TNF-α : macrophage cytotoxic factor, macrophage cytotoxin and necrosin
 As some of these names suggest, activated macrophages appear to represent
the most significant cellular source of TNF-α
 Producer cells do not store TNF-α, but synthesize it de novo following
activation
 Macrophages appear to express TNF-α mRNA constitutively, which is
translated only upon their activation
 Monomeric TNF is biologically inactive; the active form is a homotrimer in
which the three monomers associate non-covalently
The Interleukins
• Tumour necrosis factor (TNF)
 TNF-α: mostly studied, simply as TNF
 Sources of TNF-α:
 Inducers of TNF-α:
The Interleukins
• Biological Activities of TNF- α
 Activation of certain elements of both non-specific and specific immunity, in
particular in response to Gram-negative bacteria
 Induction/regulation of inflammation
 Selective cytotoxic activity against a range of tumor cells
 Mediation of various pathological conditions, including septic shock, cachexia
and anorexia
 The extent of the activity depends on the released amount:
 Low amount: locally, affecting WBCs and ECs
 High amount: Endocrine, is largely detrimental
The Interleukins
• TNF receptors
 2 types:
 Type I; 55kDa (TNF-R55): more distributed, almost all cells
 Type II; 75 kDa (TNF-R75): restricted, most prominent on hematopoietic cells
 Both binds to both TNF types
TNF-R55
and
TNF-R75
Trimerisation following binding of the ligand
to the receptor
The Interleukins
• Clinical Applications of TNF- α
 The initial interest in utilizing TNF as a general anti-cancer agent has
diminished, largely due to the realization that:
• Many tumours are not susceptible to destruction mediated by TNF (indeed, some
tumours produce TNF as an autocrine growth factor)
• Tumour cell necrosis is not TNF’s major biological activity
• Severe side effects usually accompany systemic administration of therapeutically
relevant doses of this cytokine
 Beromun™ (tasonermin): TNF-α-1a
 Soft Tissue Sarcoma (STS) in the limbs to prevent or delay amputation;
 Used in addition to melphalan
The Interleukins
• Clinical Applications of TNF- α
 Most clinical interest in TNF centers around neutralizing its biological effects
in situations where overexpression of TNF induces negative clinical effects
 TNF has been firmly implicated in mediating many of the adverse effects
associated with dozens of diseases
The Interleukins
• Clinical Applications of TNF- α
 Enbrel: an engineered hybrid protein consisting of the extracellular domain of
the TNF p75 receptor fused directly to the Fc (constant) region of human IgG
 Enbrel functions as a competitive inhibitor of TNF, a major pro-inflammatory
cytokine
 Binding of TNF to Enbrel prevents it from binding to its true cell surface
receptors
 The antibody Fc component of the hybrid protein confers an extended serum
half-life on the product, increasing it by fivefold relative to the soluble TNF
receptor portion alone
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Tumor Necrosis Factors
TNF dependent drugs
Etanercept
Trade name: Enbrel™
Approval Date: 1998
Description: Enbrel (etanercept) is a dimeric fusion protein consisting of the
extracellular ligand-binding portion of the human 75 kilodalton (p75) tumor
necrosis factor receptor (TNFR) linked to the Fc portion of human IgG1. It consists
of 934 amino acids and has an apparent molecular weight of approximately 150
kilodaltons
Production system: E. coli
Therapeutic Indications: Enbrel is indicated for reducing signs and symptoms,
inducing major clinical response, inhibiting the progression of structural damage,
and improving physical function in patients with moderately to severely active
rheumatoid arthritis (RA). Enbrel can be initiated in combination with
methotrexate (MTX) or used alone