Therapies targeting the immune system:

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Therapies targeting the immune system

• Stimulation

• Modulation

• Suppression

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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 Stimulati
on 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.
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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

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1. Bacterial immunomodulators
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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.(Exper
imental Autoimmune Encephalomyelitis - mice model
of multiple sclerosis) Diabetes
insulin ins-XCtxB p.o. (NOD
mice) Arthritis none ETXB s.c. Diabetes none C
TxB i.v., i.p (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 Behcets disease (BD) was undertaken with
very encouraging results
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• 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

Etx B subunit
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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
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LPS induces IL12 production in blood lymphocytes

Similar effect on IFNg production? TH1 shift, TH2
cytokines repressed prevents diseases like
athopy
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Endotoxin induces TH1 type response, mitigating
TH2 mediated allergy and asthma
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• 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 gtgtgt farms
• Allergy
• Frequent infections in children communities
  (airways, intestinal infections
• Hygiene hypothesis

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BUT endotoxin is a double edged sward, may also
cause asthma
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endotoxin
endotoxin
Timing, dosage, environment, genetic factors
influence endotoxin effect Optimalization
minimal risk, optimal protection
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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

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Cytokines
Immunmodulator effect result IFNa enhance
innate immunity increased anti-tumor
response IFNß IFN? enhance immune response
, against infections IL-2 activ
ate 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, soluble ligand, inhibits graft
rejction deletion in thymus autoimmunity TGFb
suppresses specific anti-inflammatory respon
se IL-1RA inhibits IL-1R binding septic shock,
prvent allergy GM-CSF increased
number of white neutropenia blood cells
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Anti-inflammatory effects of anti-TNF-a therapy
in rheumatoid arthritis

• Anti-TNF in RA
• Inhibits inflammatory cytokine /chemokine
  production
• inhibits angiogenesis
• inhibits leukocyte invasion
• inhibits matrix metalloproteinases

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• 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

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• 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

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• 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

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• Monoclonal antibodies
• Homogenous
• Selective
• Humanized antibodies, human antibodies -no immune
  response
•  
• Block Graft rejection
•   anti CD3 CD3T cells are transiently depleted
  ? function is depleted
• antibodies against APC and/or T cell receptors
  ? immuno- suppression
•  

• Non-mitogen anti-CD3
• 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

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Antibodies and antibody fragments
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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

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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 mutation
SHP-1
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depleting antibodies against molecules expressed
on B cells (CD20, CD22, and CD52).
Monoclonal antibodies
Inhibition of factors, pathways necessary for B
cell survival (CD40/CD40L és BLyS/BR3 )

• AIM
• 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

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Monoclonal antibodies applied in autoimmune
diseases
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Antibody therapies substitution of
antibodies Plasmapheresis 50 removed (IgG
20 , IgM 50 ) Elimination of immunecomplexes
autoimmune diseases self-specific IgG
Goodpastures syndrom lung, kidney,
(antibodies against glomerulal basal
membrane) myasthenia gravis (anti-acetilcholinre
ceptor) Antibody overproduction Waldenström
macroglobulinemia IgM cold agglutinin
haemolitic anemia -IgM
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Intravenous Ig therapy, (IVIG) Immunmodulatory,
anti-inflammatory effect
IVIG therapy - examples

• Neuroimmunological diseases
• diseases with demyelination - inhibiting
  complement effect
• MS (?)
• Primairy immunodefficiencies Ig lt 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 ,

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a2,6 SA containing IVIG antiinflammatory effect -
model
a2,6 sialyc acid recognizing receptor SIGN-R,
humanDC-SIGN
Anthony and Ravetch, J CLin Immunol. 2010.30
suppl. S9-S14
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• 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

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• 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)
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The structure of the anti-inflammatory
corticosteroid drug prednisone
structure
synthetic products
OH
cortisol prednisolon
cortison prednison (4x more efficient)
O
O
O
O
O
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
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Mechanism of action
cytoplasm
transcription mRNA
translation protein
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Effect on cell numbers transient change
Neutrofils Lymphocytes Eosinophils Monocytes Ba
sophils
cells/mm3
6 h 12 h
24 h

• metabolic effect , lipid, protein,
  carbohydrate degradation increased,

• toxicity

1 of genes are regulated!
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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)
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Mechanism of activation of gene transcription
IkBa gene
corticosteroids
Increased transcription and protein syntesis
Cytokin gene
X
NF-kB
IkBa
transcription
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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)

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Cyclosporin A and tacrolimus inhibit T-cell
activation by interfering with the
serine/threonine-specific phosphatase
calcineurin Blocks T cell response, decreases B
cell response
Gene transcription No activation of
transcription
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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)
antibody producing cells
100 - 10 - 1 - 0.1 -
dose
24 h before antigen 24 h after antigen
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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.
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Antigen specific immunosuppression
Rh negative mothers - anti-D IgG prophylaxis
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INDUCTION ORAL TOLERANCE

• Myelin basic protein (MBP)
• Insulin
• Collagen II-IV
• Local effect on mucosal immunsystem
• (Th2 activation, TGF ß production enhanced)

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Antigen given orally can lead to protection
against autoimmune disease
Experimental allergic encephalomyelitis EAE
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Signal-therapy selectivity? (tyrosin kinase
inhibitors, JAK inhibitors, PI3-Kg inhibitors.)
Survival receptors
Death receptors