Transcript Anti-inflammatory effect

Therapies targeting the
immune system:
 Stimulation
 Modulation
 Suppression
IMMUNOMODULATORS
modify the immune system either on a positive or at a negative way
1) Bacterial immunomodulators:
Freund adjuvants (CFA)-mycobacterium tuberculosis
human: BCG (mycobacterium) – melanoma, carcinoma
Stimulation of Mf, T, B, Nk cells, IL-1 production
muramyl dipeptide and derivatives, less toxic
Staphylococcus aureus – superantigen – polyclonal stimuli
Escherichia coli heatlabile enterotoxin (LT), CT - adjuvant effect
2) Cytokines:
rekombinant proteins (IL-1. IL-2, Epo)
cytokine antagonists:
inhibitors of signaling
soluble receptors: TNF, IL-1, IL-4
3) Antibodies:
antibodies specific for cytokines, or cytokine receptors,
or recognizing molecules on cell surface, receptors, co-stimulators
etc.
Applications:
Autoimmune and allergic diseases
Causal treatment:
 Peptides – e.g.: DNA-mimotope peptides, epitópe peptides
corresponding to the autoantigens in SLE
Tolerance induction
Inhibition of pathogenic antibody production (autoreactive, or
IgE)
Regulation of cytokine network
Regulation od signaling
Therapies influencing apoptosis
1. Bacterial immunomodulators
Immunmodulation with cholera
toxin-like enterotoxins
Escherichia coli heatlabile toxin (Etx)
A subunit:
A1 (toxic),
A2 (adaptor)
(ADP ribozilation, cAMP ,-> PKA Cl- egress,
H2O influx)
B subunit
(non toxic) - adjuvant
pentamer ring, stabile non-kovalent
binding
high affinity:
KD= 7-5 x 1010
Receptor: GM1 gangliosid
Efficient immunogen if get into the
body through mucosa or at a
systemic way
(membrane rafts)
Effect: tolerance induction
When and how cholera-like enterotoxin is used for tolerance
induction?
Disease
antigen
prep
immunization
SRBC
SRBC-CtxB
p.o.
BCG
BCG-CtxB
p.o.
EAE (rat)
MBP
MBP-CtxB
p.o.
Diabetes
insulin
ins-XCtxB
p.o.
Arthritis
none
ETXB
s.c.
Diabetes
none
CTxB
i.v., i.p
(Experimental Autoimmune Encephalomyelitis - mice model of multiple sclerosis)
(NOD mice)
(mucosal vaccination)
Human: CTB – non-toxic, good adjuvant, vaccination against cholera
oral vaccine: inactivated vibrio cholera +CTB  IgA, memory
Inducing tolerance in HSP uveitis: mucosal immunization tolerance
(CTB-HSP peptide conjugates) small phase I/II trial in patients with Behcet’s
disease (BD) was undertaken with very encouraging results
Etx
B subunit
Interaction with GM1
receptor
•Polyclonal B cell activation
•Without proliferation
•Increase of synthesis of :
MHCII, B7, CD40, ICAM1,
IL2Ra
•Therapy: local antibody
production
•TH1 –linked diseases,
•Autoimmune diseases
• graft rejection
Endotoxin exposition: effect on allergy and asthma
Gram negative bacteria
outer cell wall – LPS
O-antigen: repeated O-polysacharides
(glycan polimer)
Immunostimulator: Lipid A
conserved in
different bacteria species
Adjuvant effect -T memory
IL-12, IFNg production
In microbe-enriched environment 
Less asthma
LPS induces IL12 production in blood lymphocytes
Similar effect on IFNg production TH1 shift, TH2 cytokines repressed
prevents diseases like athopy
Endotoxin induces TH1 type response,
mitigating TH2 mediated allergy and asthma
Reverse correlation between exposure to microbe infection and
the appearance of allergy and asthma
House dust, animals, non-pasteurized milk  lower number
of children have allergy
Towns >>> farms:
Allergy
Frequent infections in children communities (airways,
intestinal infections
„Hygiene hypothesis”
BUT: endotoxin is a double edged sward, may also cause asthma
endotoxin
endotoxin
Timing, dosage, environment, genetic factors influence endotoxin effect
Optimalization: minimal risk, optimal protection
2. Cytokines:
•recombinant proteins (IL-1. IL-2, Epo)
•cytokine antagonists: IL-1RA signaling inhibitors
•soluble receptors : TNF, IL-1, IL-4
•specific, high affinity binding,
•natural occurence in body fluids
(proteolitic cleavage or alternative splicing)
•do not activate immune response
•neutralise ligands
•relative long life time
•less immunogen
Cytokines:
Immunmodulator
effect
result
IFNα
enhance innate immunity
increased anti-tumor response
IFNβ
„
IFNγ
enhance immune response
„
IL-2
activate killer cells
anti-tumor effect
IL-4
TH2 response increased
increased antibody synthesis
IL-10
TH1 response suppressed
decreased cellular cytotoxicity, autoimmun
IL-12
TH1 response stengthened
cellular cytotoxicity increased, anti-tumor
Fas(CD95)
CD8 cytotoxicity,
deletion in thymus
soluble ligand, inhibits graft rejction
autoimmunity
TGFb
suppresses specific
anti-inflammatory
„
„
, against infections
response
IL-1RA
inhibits IL-1R binding
septic shock, prvent allergy
GM-CSF
increased number of white
neutropenia
blood cells
Anti-inflammatory effects of anti-TNF-α therapy
in rheumatoid arthritis
Anti-TNF in RA:
•
Inhibits inflammatory cytokine /chemokine production
•
inhibits angiogenesis
•
inhibits leukocyte invasion
•
inhibits matrix metalloproteinases
3). Antibody mediated therapies:
 elimination of the pathogen – hyperimmune sera (passive immunization)
(rabies, hepatitis B, CMV, RSV, varicella/zoster)
 prevention of infection : RSV (respiratory syncytial virus)
toxin neutralization e.g. snake toxin, tetanus
 inhibition of blood coagulation
 cell depletion: e.g.. anti-CD20 CD20+ B cell depletion
Targeting targeted therapies
Targets of antibodies:
Cell surface receptors
Cytokines and their receptors:
•Graft versus host disease (GVH)
•Malignus tumor
•Immunosuppression (a-MHCII, a-MHCI)
•inflammation
•Platelets aggregation
Antibodies are applied for:
Diagnosis: detect malignant cells - in metastasis
Prognosis – based on detection of membrane markers
Hyperimmune antibodies : intramuscular, intravenous application
Anti-inflammatory effect (IVIG): autoimmune dieases, allergy
Substitution therapy : immunodefficieny, autoimmune diseases
Polyclonal antibodies :
Non-antigen specific immunosuppression
Suppresssion of cellular immune responses:
 anti-thymocyte serum, -globulin: inhibits T cell responses
 anti-lymphocyte serum, anti-lymphocyte globulin
Transplantation: inhibits graft rejection, GVH
Problems:
standardization,
non-selective
antigenicity –serum disease
Monoclonal antibodies:
 Homogenous
 Selective
 Humanized antibodies, human antibodies -no immune response
Block Graft rejection :
anti CD3: CD3+T cells are transiently depleted  function is
depleted
antibodies against APC and/or T cell receptors  immunosuppression
Non-mitogen anti-CD3
MHC II
MHC I
ICAM-1
APC
B7
CD40
CD4 or
CD8
TCR
CD3
T cell
LFA-1
CD28
CD40L
IL-2 R
Inhibition of costimulation: CTLA4-Fc
T cell depletion : anti-CD52
(Campath)
Antigen specific inhibition: ag/peptide
CD28 superagonist -Treg increase (but:
„cytokin storm” )
TNFa inhibition
Citokin-citokin-R inhibition
Antiinflammatory effect
Nature Reviews Cancer 2; 750-763 (2002); doi:10.1038/nrc903
LIGAND-TARGETED THERAPEUTICS IN ANTICANCER THERAPY
Antibodies and antibody fragments
< previous next >
Bio-similar, bio-better, me-better
• Biosimilar antibodies are “generic” versions of “innovator”
(or “originator”) antibodies with the same amino acid
sequence, but produced from different clones and
manufacturing processes.
• Bio-better antibodies are antibodies that target the same
validated epitope as a marketed antibody, but have been
engineered to have improved properties, e.g., optimized
glycosylation profiles to enhance effector functions or an
engineered Fc domain to increase the serum half-life
• “Me better” antibodies with controlled and optimized
glycosylation have been obtained in glyco-engineered
CHO cells or yeast strains
B cell receptors, their role in growth and activation:
potential therapies for autoimmune disease.
Benlista (belimumab)
Autoimmun
phenotype
protein
change
CD19
CD22
Bcl-2
Fas
mutation
BAFF
Lyn
SHP-1
mutation
Monoclonal antibodies:
depleting antibodies against molecules
expressed on B cells
(CD20, CD22, and CD52).
AIM:
Inhibition of factors, pathways necessary for
B cell survival (CD40/CD40L és BLyS/BR3 )
 maximal specificity:
targeting
neutralisation
signalisation
 minimal immunogenicity:
humanized/human antibodies
 optimal effector functions:
engineered antibodies :
effector function :
ADCC - FcR
CDC - C1q binding complement
activation
phagocytosis – FcR, CR
halflife - Fcn
Monoclonal antibodies applied in autoimmune diseases
Antibody therapies: substitution of antibodies
Plasmapheresis: ~50 % removed (IgG 20 %, IgM ~50 %)
Elimination of immunecomplexes
autoimmune diseases: self-specific IgG:
Goodpasture’s syndrom: lung, kidney,
(antibodies against glomerulal basal membrane)
myasthenia gravis (anti-acetilcholinreceptor)
Antibody overproduction: Waldenström macroglobulinemia –IgM
cold agglutinin haemolitic anemia -IgM
Intravenous Ig therapy, (IVIG)
Immunmodulatory, anti-inflammatory effect
IVIG therapy - examples
• Neuroimmunological diseases :
diseases with demyelination - inhibiting complement effect
MS (?)
• Primairy immunodefficiencies:
Ig < 400 mg/dl
• Idiopathic trombocytopenia purpura: low platelet number - IVIG inhibits
phagocytosis
• CLL: against bacterial infections
• infectious diseases, toxic shock (100 000/year) –sepsis
• Kawasaki disease: chronic vasculitis - IVIG – neutralization effect ,
α2,6 SA containing IVIG antiinflammatory effect - model
α2,6 sialyc acid recognizing receptor : SIGN-R, human:DC-SIGN
Anthony and Ravetch, J CLin Immunol. 2010.30 suppl. S9-S14
IMMUNSUPPRESSION
Block unwanted immunoresponse:
- Allergy
- Autoimmune diseases
- transplantation: rejection, GVH
Antigen specific immunsuppression – aim: to induce specific tolerance
a, Antigen-specific (pl. oral tolerance)
b, Non-antigen specific
•corticosteroids
•CY-A, FK 506, Rapamycin,
•irradiation
•Cytostatic agents
Antigen non-specific immunosuppression:
Corticosteroids
Inhibit inflammation
Mechanisms: they act via hormon receptors
Naturally occuring 21 C atoms steroid hormon:
Corticosteroid  product of cholesterin metabolism
1948: hydrocortison (Reumatoid arthritis)
The structure of the anti-inflammatory corticosteroid drug prednisone
structure:
synthetic products:
OH
cortisol
O
CH2OH
CH2OH
C=O
C=O
CH2OH
C=O
prednisolon
O
OH
O
O
cortison
CH2OH
O
OH
OH
C=O
OH
prednison
(4x more efficient)
Prednisone is a synthetic analogue of the natural adrenocorticosteroid cortisol. Introduction of the
1,2 double bond into the A ring increases anti-inflammatory potency approximately fourfold
compared with cortisol, without modifying the sodium-retaining activity of the compound
Mechanism of action
cell membrane
steroid
cytoplasm
steroid
receptor
HSP-90
nucleus
regulatory
gene
element
transcription
mRNA
translation
protein
Effect on cell numbers – transient change
cells/mm3
10,000-
4,000-
Neutrofils
2,000-
Lymphocytes
400-
Eosinophils
300-
Monocytes
100-
Basophils
6h
12 h
24 h
 metabolic effect: , lipid, protein, carbohydrate degradation increased,
 toxicity
1% of genes are regulated!
Anti-inflammatory effect of corticosteroids
Activity
Effect
IL-1, TNF, GM-CSF,
IL-3, IL-4. IL-5, IL-8
inflammation
NOS
NO
Phospholipase A2
Cyclooxigenase 2
Lipocortin
Prostaglandins, leukotriens
Adhesion molecules
decreased migration
Endonuclease induction
Apoptosis induction
(lymphocytes, eosinofils)
Mechanism of activation of gene transcription
IkBa gene
corticosteroids
Increased
transcription
and protein
syntesis
X
Cytokin gene
NF-kB
IkBa
transcription
Non-steroid anti-inflammatory agents
400 BC
aszpirin
(Salix alba)- Hippokrates
synthetic production : 19. century
today USA –15x106 kg / year
mechanism:
cyclooxigenase inhibitionprostaglandin production
inhibited
• active site : serine acetilation (irreversible)
• arachidonic acid binding inhibition (reversible)
Cyclosporin A and
tacrolimus inhibit
T-cell activation
by interfering with
the
serine/threoninespecific
phosphatase
calcineurin
Blocks T cell response,
decreases B cell response
Gene transcription
No activation of transcription
Cytotoxic agents:
Kill deviding cells
-during DNS synthesis ( azathioprine, metotrexate), or
-in any phases (cyclophosphamide)
Non-specific for
cell cycle (UV,
irradiation)
S-phase- specific
(azathioprine,
metotrexate)
Cel cycle specific
(cyclophosphamide,
chlorambucil)
100 10 -
10.1 -
24 h before antigen
24 h after antigen
dose
The structure and metabolism of the cytotoxic immunosuppressive
drugs azathioprine and cyclophosphamide
Inhibit purin
biosynthesis
(S phase)
DNA alkylating
agent, unstabile
(every phase)
The structure and metabolism of the cytotoxic immunosuppressive drugs azathioprine and
cyclophosphamide. Azathioprine was developed as a modification of the anti-cancer drug 6-mercaptopurine;
by blocking the reactive thiol group, the metabolism of this drug is slowed down. It is slowly converted in
vivo to 6-mercaptopurine, which is then metabolized to 6-thio-inosinic acid, which blocks the pathway of
purine bio-synthesis. Cyclopho-sphamide was similarly developed as a stable pro-drug, which is activated
enzymatically in the body to phosphoramide mustard, a powerful and unstable DNA-alkylating agent.
Antigen specific immunosuppression
Rh negative mothers - anti-D IgG prophylaxis
INDUCTION ORAL TOLERANCE
•Myelin basic protein (MBP)
•Insulin
•Collagen II-IV
Local effect on mucosal immunsystem
(Th2 activation, TGF ß production enhanced)
Antigen given orally can lead to protection against
autoimmune disease
Experimental allergic encephalomyelitis= EAE
Signal-therapy – selectivity?
(tyrosin kinase inhibitors, JAK inhibitors, PI3-Kg inhibitors….)
Death
receptors
Survival
receptors