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Therapies targeting the immune system :

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  1. Therapiestargetingtheimmunesystem: • Stimulation • Modulation • Suppression

  2. IMMUNOMODULATORS modifytheimmunesystemeitheron a positiveoratanegativeway 1) Bacterialimmunomodulators: Freund adjuvants (CFA)-mycobacteriumtuberculosis human: BCG (mycobacterium) – melanoma, carcinoma Stimulation of Mf, T, B, Nkcells, IL-1 production muramyldipeptide and derivatives, less toxic Staphylococcusaureus – superantigen – polyclonalstimuli Escherichia coli heatlabileenterotoxin (LT), CT - adjuvanteffect 2) Cytokines:rekombinantproteins (IL-1. IL-2, Epo) cytokineantagonists: inhibitors of signaling solublereceptors: TNF, IL-1, IL-4 3)Antibodies:antibodiesspecificforcytokines, orcytokinereceptors, orrecognizingmoleculesoncellsurface, receptors, co-stimulators etc.

  3. Applications: Autoimmune and allergicdiseases • 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

  4. 1. Bacterialimmunomodulators

  5. Immunmodulationwithcholeratoxin-likeenterotoxins 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 (membrane rafts) Effect: tolerance induction Efficient immunogen if get into the body through mucosa or at a systemic way

  6. When and howcholera-likeenterotoxin is usedfortoleranceinduction? Diseaseantigenprepimmunization SRBC SRBC-CtxBp.o. BCG BCG-CtxBp.o. EAE (rat) MBP MBP-CtxBp.o.(ExperimentalAutoimmuneEncephalomyelitis - micemodel of multiple sclerosis) Diabetes insulinins-XCtxBp.o. (NOD mice) Arthritisnone ETXB s.c. Diabetes noneCTxBi.v., i.p (mucosalvaccination) Human: CTB – non-toxic, goodadjuvant, vaccinationagainstcholeraoralvaccine: inactivatedvibriocholera +CTB  IgA, memory Inducingtolerancein HSP uveitis: mucosalimmunization tolerance (CTB-HSP peptideconjugates) small phase I/II trial in patients with Behcet’sdisease (BD) was undertaken with very encouraging results

  7. 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

  8. Endotoxinexposition: effectonallergy 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

  9. LPS induces IL12 productioninbloodlymphocytes Similar effect on IFNg production TH1 shift, TH2 cytokines repressed prevents diseases like athopy

  10. Endotoxininduces TH1 typeresponse, mitigating TH2 mediatedallergy and asthma

  11. 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”

  12. BUT: endotoxin is a double edged sward, may also cause asthma

  13. endotoxin endotoxin Timing, dosage, environment, geneticfactorsinfluenceendotoxineffect Optimalization: minimalrisk, optimalprotection

  14. 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

  15. Cytokines: Immunmodulator effect result IFNα enhance innate immunity increased anti-tumor response IFNβ „ „ IFNγ enhance immune response „ „ , against infections 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, soluble ligand, inhibits graft rejction deletion in thymus autoimmunity TGFb suppresses specific anti-inflammatory response IL-1RA inhibits IL-1R binding septic shock, prvent allergy GM-CSF increased number of white neutropenia blood cells

  16. Anti-inflammatoryeffects of anti-TNF-αtherapyinrheumatoidarthritis • Anti-TNF in RA: • Inhibitsinflammatorycytokine /chemokine production • inhibits angiogenesis • inhibits leukocyteinvasion • inhibits matrix metalloproteinases

  17. 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

  18. 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

  19. 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

  20. 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 immuno- suppression CD4 or CD8 MHC II MHC I TCR T cell CD3 ICAM-1 B7 LFA-1 CD28 IL-2 R CD40 CD40L APC • 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

  21. Nature Reviews Cancer2; 750-763 (2002); doi:10.1038/nrc903LIGAND-TARGETED THERAPEUTICS IN ANTICANCER THERAPY < previousnext > Antibodies and antibody fragments

  22. 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

  23. B cell receptors, their role in growth and activation: potential therapies for autoimmune disease. Benlista (belimumab) Autoimmun phenotype

  24. 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

  25. Monoclonal antibodies applied in autoimmune diseases

  26. 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

  27. 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 ,

  28. 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

  29. 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

  30. 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)

  31. CH2OH C=O CH2OH C=O CH2OH C=O CH2OH C=O OH OH OH OH 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

  32. cell membrane steroid steroid receptor HSP-90 regulatory gene element nucleus Mechanism of action cytoplasm transcription mRNA translation protein

  33. 10,000- 4,000- 2,000- 400- 300- 100- Effect on cell numbers – transient change Neutrofils Lymphocytes Eosinophils Monocytes Basophils cells/mm3 6 h 12 h 24 h • metabolic effect: , lipid, protein, carbohydrate degradation increased, • toxicity 1% of genes are regulated!

  34. Anti-inflammatory effect of corticosteroids

  35. Mechanismof activation of gene transcription IkBa gene corticosteroids Increased transcription and protein syntesis Cytokin gene X NF-kB IkBa transcription

  36. 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)

  37. 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 Genetranscription No activation of transcription

  38. 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

  39. 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.

  40. Antigenspecificimmunosuppression Rh negative mothers - anti-D IgG prophylaxis

  41. INDUCTIONORAL TOLERANCE • Myelin basic protein (MBP) • Insulin • Collagen II-IV • Local effect on mucosal immunsystem • (Th2 activation, TGF ß productionenhanced)

  42. Antigengivenorallycan lead toprotectionagainstautoimmunedisease Experimental allergic encephalomyelitis= EAE

  43. Signal-therapy – selectivity? (tyrosin kinase inhibitors, JAK inhibitors, PI3-Kg inhibitors….) Survival receptors Death receptors