Passive Immunisierung und Antikörpertherapien

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1 Passive Immunisierung und Antikörpertherapien
Konteptvorlesung 3 Themenblock 8 Beda M. Stadler Institut für Immunologie Diese Folien stehen auch als PPT Files zum download bereit unter: KV 8.3

2 Protein Pharmaceutika 2011
134 FDA-zugelassene biotechnologisch hergestellte (Proteine/Peptide) Medikamente Protein Pharmaceutika kosten derzeit ca. $50 Milliarden/Jahr

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4 Top Ten Monoclonal Antibodies 2009/2010
Generic Name Brands® Indications Sales $ billion / Infliximab Remicade RA,UC,CD,Ps,PsA,AS 6.9 8.0 Bevacizumab Avastin Colon cancer 5.9 6.8 Rituximab Rituxan NHL,RA 5.8 6.7 Adalimumab Humira RA,Ps,JIA,PsA,AS,CD 5.4 6.5 Trastuzumab Herceptin Breast Cancer 5.0 5.5 Cetuximab Erbitux Colon, head and neck cancer 2.5 3.2 Ranibizumab Lucentis Wet macular degeneration 2.4 3.1 Natalizumab Tysabri Multiple sclerosis 1.0 1.7 Omalizumab Xolair Allergic Asthma 0.9 1.1 Palivizumab Synagis RSV Thearapeutische Antikörper auch zu teuer für uns?

5 Übersicht / Lernziele Antikörper Produktion einst
Passive Immuntherapie einst Monoklonale / rekombinante Antikörper Antikörper als Therapie für: Immunsuppression Krebs Therapie Immunmodulation Anti-Adhäsionstherapien Rückblick und Zukunft Paul Ehrlich vor 100 Jahren KV 8.3

6 Passive Immuntherapie einst
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7 Passive Immuntherapie mit polyklonalen Antikörpern
Multispender human Gammaglobulin oder Pferd Antitoxin Tetanus Pferd Antivenin Schlangenbiss Multispender human Gammaglobulin Tollwut Masern Hepatitis A and B Pferd Antitoxin Diphtherie Botulismus Schwarze Spinnenbiss Therapeutikum Krankheit KV 8.3

8 Monoklonale Antikörper Hybridoma Technologie
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9 Therapeutische Maus Antikörper
Probleme Immunogenizität (humane anti-Maus AK, HAMA) keine Effektor Mechanismen (Maus Fc) Unerwartete Toxizität (Reaktivitiät mit normalem Gewebe, Kreuzreaktion mit anderen Antigenen) Verlust von Tumorantigen (z.B. anti-idiotyp Behandlung von Lymphomen) KV 8.3

10 Immunogenicität von Infliximab (chimärer monokl. Ak gegen TNFα)
61% of patients had detectable antibodies after the fifth infusion of Infliximab Incidence of the formation of antibodies to Infliximab is reduced with immuno-suppressive therapy Human anti-mouse Ab Human/mouse chimeric Ab (Infliximab) KV 8.3

11 Dank Wissen um Grundlagen rekombinante Antikörper
Diversity of Antibodies is Generated by Gene Rearrangement Dank Wissen um Grundlagen rekombinante Antikörper KV 8.3

12 Die Domänenstruktur hilft zum Umbau von Antikörpern
Fab Fv Fc Hinge VL VH CL CH CH2 CH3 scFv IgG KV 8.3

13 Humanisierung von Maus Antikörpern
Chimäre Humanisiert Human KV 8.3 Ezzell, Scientific American

14 Therapeutische Antikörper
Generic name Trade name Target Type Muromonab Orthoclone OKT3 CD3 Murine Abciximab ReoPro Platelet GP IIb/IIIa Rituximab Rituxan CD20 Chimeric Daclizumab Zenapax IL2Ra Humanized Basiliximab Simulect IL2R Palivizumab Synagis RSV Infliximab Remicade TNFa Trastuzumab Herceptin Her2/neu/ErbB2 Gemtuzumab Mylotarg CD33 Alemtuzumab Campath-1H CD52 Endrecolomab Panorex 17A-1 Ibritumomab tiuxetan Zevalin Adalimumab Humira Human Omalizumab Xolair IgE Centuximab Erbitux EGFR/ErbB1/Her1 Bevacisumab Avastin VEGF Efalizumab Raptiva CD11a KV 8.3

15 Mono- & plurivalente Ak-Fragmente
Fab scFv Fv dsFv VH scFv Diabody Triabody Tetrabody KV 8.3

16 Transgene Humanisation
humane Antikörper in human Ig transgenenen Mäusen KV 8.3

17 Der Wunsch nach humanen AK
Humane Antikörper mittels Repertoire Klonierung mRNA von B Zellen Antikörper (Fab) Expression auf p3 des M13 Phagen Blut PCR of H&L chain genes Klonierung und Rekombination mRNA  cDNA KV 8.3

18 Expression von Antikörpern
DNA Transfektion Antikörper Vektor Zellkultur Expression KV 8.3

19 Industrielle Antikörper Produktion
Flask Spinner Bioreaktor (Bench scale) Bio Reactor (Plant) Subkultur Produktion 1mL → 100mL → 1L → 1Lx3 → 12L → 100L → 300L → 2’000L→ 10’000L KV 8.3

20 Ersatz von passivem IgG durch rekombinante anti-RhD
Erythrozyten rekombinante Antikörper mittels phage display KV 8.3

21 Neue Therapeutische Antikörper
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22 Immunsuppressive Anikörper (anti-T Zell Antikörper)
Lymphocyte immune globulin (Atgam®) lytic to human thymic lymphocytes; blocks T-cell responding Anti-CD3: (OKT3®, muromonab-CD3) binds CD3, blocks antigen binding; depletes T-cells Anti-CD3: huOKT3 Humanized version of OKT3 Anti-CD4: OKT4 less infusion-related reaction Anti-Tac monoclonal antibody (Zenopax®) binds IL-2 receptor of activated T-cells causing their inactivation KV 8.3

23 Lymphocyte Depleting Antibody Therapies
Polyclonal Anti-Thymocyte Globulin Campath-1H OKT3 CD52 CD3 Currently available induction antibodies include antibodies that cause varying degrees lymphocyte depletion. These include both monoclonal agents (OKT3, Campath-1H) and a variety of polyclonal agents. KV 8.3

24 Immunsuppressive Anikörper (anti B-Zell Antikörper)
Anti-CD20: Rituxan® (Rituximab) results in mature B-cell depletion and induces apoptosis (B-cell mediated vascular rejection) for B-cell Non Hodgkin’s Lymphoma anti-CD52: Campath-1H (Alemtuzumab) depletes T and B lymphocytes, NK cells, monocytes and macrophages slg DR CD19 CD20 CD22 KV 8.3

25 Side effects fever chills weakness headache nausea vomiting diarrhea
low blood pressure rashes KV 8.3

26 Antikörper Krebs Therapie Strategien
1 2 5 ADCC -Y-90 -Y-90 NK Anti Angiogenese Complement pathway -Y-90 Tumor cell TNF -Y-90 Anti-Idiotyp IL-1 -Y-90 Mac -Drug . Early studies of non-specific immunotherapy using agents such as alpha interferon and Interleukin-2 demonstrated that lymphomas were susceptible to immune modulation. Development of newer specific immunotherapy approaches, such as monoclonal antibodies, have largely focused on lymphomas Monoclonal antibody therapy is revolutionizing the way we treat lymphoma May allow for selective cytotoxicity of tumor cells and relative sparing of normal tissues The following are types of antibody-based cancer therapy1,2: 1. Unmodified antibodies may kill cancer cells by complement-mediated lysis, antibody-dependent cell-mediated cytotoxicity (ADCC), opsonization, cytokine release from macrophages or natural killer (NK) cells, or interruption of anti-idiotypic networks, and induction of apoptosis. 2. RICs emit cytotoxic radioactive particles possibly cytocidal over many cell diameters. Antigen-negative tumor cells and malignant cells with poor antibody access may be killed by cross fire from labeled neighboring cells. 3. Antibody-toxin conjugates (immunotoxins) must be internalized to permit enzymatic inactivation of ribosomes (ricin or abrin-A chain) or elongation factor-2 (diphtheria toxin and Pseudomonas endotoxin). 4. Antibody-drug conjugates also must be internalized for maximal cytotoxicity, which is often mediated by disruption of DNA replication. 1. Press et al. Biologic Therapy of Cancer Updates. JB Lippincott; 1994;4:1-13. 2. Weiner. Semin Oncol. 1999;26(No. 5, suppl 14):43-51. -Tox 3 4 -Tox -Drug -Tox -Tox -Drug KV 8.3

27 Strategie 1 Antikörper vermittelte Zytotoxizität
NK Zelle Ziel Zelle Ziel Zelle Apoptosis via Induktion von intrazellulären Signal-Pathways Ziel Zelle The means by which monoclonal antibodies mediate cytotoxic events continue to be defined, though it is clear that multiple mechanisms are likely involved. Binding of monoclonal antibodies to tumor cell antigens can initiate: - Recruitment and activation of immune effector cells, such as natural killer (NK) cells and macrophages, thereby facilitating antibody-dependent cellular cytotoxicity (ADCC) - Cross-linking of membrane receptors, which can initiate intracellular signaling pathways that result in apoptosis or cell growth arrest - Complement-dependent cytotoxicity (CDC) via opsonisation and lytic pathways. Cragg MS, French RR, Glennie MJ. Signaling antibodies in cancer therapy. Curr Opinion Immunol. 1999;11: Antikörper-abhängige Zytotoxizität (ADCC) Complement-abhängige Zytotoxizität (CDC) KV 8.3

28 z.B. Herceptin® (Trastuzumab)
normal cell tumor cell treatment Binds HER-2 (human epidermal growth factor receptor 2), a growth factor receptor found on some tumor cells (some breast cancers, lymphomas). Over-expression of HER2 causes increased cell growth and reproduction. HER2 protein over-expression affects approximately 25% to 30% of breast cancer patients KV 8.3

29 Strategie 1: maligne B-Zellen
Stem cell Pro- B cell Pre- B cells Immature B cell Mature B cell Activated B cell Plasma cell Antigen independent Antigen dependent HLA-DR TDT CD19 CD10 CD20 CD22 CD21 CD38 Leukemias from B-cell Precursors (B-ALL) B-cell Lymphomas (NHL, CLL) Neoplasias: Multiple Myeloma KV 8.3

30 Strategie 1 anti idiotypische Antikörper
(B-Zell Lymphome) KV 8.3

31 Strategie 2: Verschiedene Isotope
Radiolabeled monoclonal antibodies mediate immune defenses, as unlabeled antibodies do, while also delivering targeted radiation to lymphoma cells.1,2 Unlabeled MAbs destroy tumor cells via immune mechanisms initiated by antibody binding to targeted antigen (eg, CD20). Radiolabeled MAbs destroy tumor cells via immune mechanisms and also by delivering radiation directly to the tumor cells. Beta particles (electrons) emitted from conjugated radionuclides can penetrate several cell layers to reach more lymphoma cells. Even antigen-negative lymphoma cells can be hit by radioactive “crossfire” from antigen-positive cells coated with radiolabeled MAbs. Press O, Early JP, Appelbaum PR. Radiolabeled antibody therapy of lymphomas. In: DeVita VT, Hellman S, Rosenberg SA, eds. Biologic Therapy of Cancer Updates, vol 5. Philadelphia, PA: JB Lippincott, 1994;1–13. Buske C, Feuring-Buske M, Unterhalt M, Hiddemann W. Monoclonal antibody therapy for non-Hodgkin’s lymphomas: emerging concepts of a tumor-targeted strategy. Eur J Cancer. 1999;35:549–557. Nackter Antikörper Radiomarkierter Antikörper KV 8.3

32 Isotopen markierte Antikörper
LymphoCide (anti CD22, found on some B-cell leukemias. Tositumomab (Anti-CD20) Lym-1 (Oncolym). Anti- HLA-DR expressed at high levels on lymphoma cells. KV 8.3

33 Strategie 3: Mylotarg® (anti CD-33)
A conjugate of a monoclonal antibody that binds CD33 (cell-surface molecule expressed by the cancerous cells in acute myelogenous leukemia (AML) but not found on the normal stem cells needed to repopulate the bone marrow) and calicheamicin, an oligosaccharide that blocks the binding of transcription factors (proteins) to DNA and thus inhibits transcription. KV 8.3

34 Strategie 4: Enzym markierte Antikörper
Antibody directed enzyme prodrug therapy (ADEPT) Tumor antigen Tumorzelle Zytotoxischer Wirkstoff Prodrug 1 3 2 4 Enzym Antikörper KV 8.3

35 Strategie 5: Anti-Angiogenes
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36 Anti-VEGF: Avastin ® Recombinant humanized monoclonal antibody against VEGF; prevents binding with its receptor. Blocks stimulation of angiogenesis  stops tumor growth. KV 8.3

37 Immunmodulatorische Antikörper
Anti-TNF Therapie Infliximab Etanercept Humira etc (Biosimilarika) Anti-IgE Therapie Xolair                                                                                           KV 8.3

38 Anti-TNF-a: Infliximab
Chimeric monoclonal antibody Binds specifically to TNF-a, neutralizing its activity Used for Crohn’s disease and rheumatoid arthritis Side-effect: can convert a latent case of tuberculosis into active disease KV 8.3

39 Infliximab: Mechanism of Action
Binds and neutralizes soluble and membrane bound TNF TNFR2 Nucleus NFB transcription No Signal TNF promotes the healing of fistulae in Crohn’s Diseasae for which few therapeutic options are available. As multiple mechanisms are involved in producing the inflammation in Crohn’s Disease, new targeted biological therapies are being tried. The first of these to gain widespread use in Infliximab. A single infusion can induce remission in about two-thirds of patients with active Crohn’s disease. Three infusions over six weeks can also lead to closure of fistulae in 50% of patients. Repeated infusions at eight-weekly intervals will maintain remisison in over 60% of patients who respond initially KV 8.3

40 Other Postulated Mechanisms of Action…
Antibody dependent cell-mediated cytotoxicity (ADCC) Lysis of TNF-expressing cells through complement (C’) activation Complement Activation C’ Complement Receptor Phagocyte T Cell Phagocytosis KV 8.3

41 TNF-a: Rezeptor oder Antikörper
Infliximab Etanercept KV 8.3

42 Biosimilarika: z.B. anti-TNF
Generic Name Brand name (source) Molecular Derivation Development status Infliximab Remicade® Human/mouse chimeric anti-human TNF monoclonal Ab Approved in US & Europe for CD & RA Etanercept Enbrel® Human recombinant p75 (TNF receptor fusion protein) Good for RA CDP-571 Humicade™ Humanised anti-human TNF mAb Trials ongoing for CD & ulcerative colitis CDP-870 N/A(Celltech) Humanised anti-TNF mAb fragment Phase III trials ongoing Adalimumab Humira® Human anti-human TNF mAb Approved for RA - Focus on inhibition of the TNFalpha molecule itself. KV 8.3

43 Omalizumab Reduces IgE and FcεRI Levels
(Xolair®) ↓ Inflammation ↓ Symptoms ↓ free IgE ↓ FcεRI ↓ Degranulation anti-IgE FcεRI Mediator Release Allergic Inflammation B MC IgE Holgate S. et al., J Allergy Clin Immunol 2005 vol. 115 (3) pp

44 Anti-IgE: Omalizumab (Xolair®)
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45 Anti Adhäsionstherapie
Efalizumab (Raptiva®) Anti-CD11a (subunit of LFA-1) inhibits activation of T cells  Alefacept (Amevive®) binds to CD2 on memory effector T lymphocytes, inhibiting their activation Abciximab (Reopro®) Anti-Gp IIb/IIIa KV 8.3

46 Efalizumab (Raptiva) Interactions between leukocyte-function associated antigen type 1 (LFA-1) and intercellular adhesion molecules are important in the pathogenesis of psoriasis. Efalizumab, a humanized monoclonal antibody, binds to the subunit (CD11a) of LFA-1 and inhibits the activation of T cells KV 8.3

47 Alefacept (Amevive®) CD2 Anti-CD2 KV 8.3

48 Abciximab (Reopro®) Anti-GpIIb/IIIa Inhibits the clumping of platelets by binding the receptors on their surface that normally are linked by fibrinogen. Helpful in preventing reclogging of the coronary arteries in patients who have undergone angioplasty. KV 8.3

49 Ausblick: mehr Antikörper für…
Transplantation Knochenmark, Niere, Leber und Pancreas Insel Zellen. Krebs Metastatic Brust Krebs, CLL, non-Hodgkin’s Lymphome. Autoimmunkrankheiten Rheumatoide Arthritis, Psoriasis, Multiple Sclerosis, Crohn’s Krankheit. Infektionskrankheiten Respiratory syncytial virus, Cytomegalovirus, Septikämien. Allergien: Mehr als 150 weitere Ak in der Pipeline… KV 8.3