PPT

Passive Immunisierung und Antikörpertherapien Konteptvorlesung 3 Themenblock 8

Beda M. Stadler Institut für Immunologie

KV 8.3

Diese Folien stehen auch als PPT Files zum download bereit unter: http://www.iib.unibe.ch/wiki/?p=teaching/medical_students&l=de

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

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Top Ten Monoclonal Antibodies 2009/2010 Sales $ billion 2009 / 2010

Generic Name

Brands®

Indications

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

Omalizumab

Xolair

Allergic Asthma

0.9

1.1

Palivizumab

Synagis

RSV

1.1

1.0

1.0

1.7

Thearapeutische Antikörper auch zu teuer für uns?

Übersicht / Lernziele • Antikörper Produktion einst • Passive Immuntherapie einst • Monoklonale / rekombinante Antikörper • Antikörper als Therapie für: • Immunsuppression • Krebs Therapie • Immunmodulation

• Anti-Adhäsionstherapien

Paul Ehrlich vor 100 Jahren

• Rückblick und Zukunft

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Passive Immuntherapie einst

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Passive Immuntherapie mit polyklonalen Antikörpern Krankheit

Therapeutikum

Schwarze Spinnenbiss

Pferd Antivenin

Botulismus

Pferd Antitoxin

Diphtherie

Pferd Antitoxin

Hepatitis A and B

Multispender human Gammaglobulin

Masern

Multispender human Gammaglobulin

Tollwut

Multispender human Gammaglobulin

Schlangenbiss

Pferd Antivenin

Tetanus

Multispender human Gammaglobulin oder Pferd Antitoxin

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Monoklonale Antikörper Hybridoma Technologie

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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

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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 immunosuppressive therapy

Human anti-mouse Ab

Human/mouse chimeric Ab (Infliximab) KV 8.3

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Diversity of Antibodies is Generated by Gene Rearrangement

Dank Wissen um Grundlagen rekombinante Antikörper

KV 8.3

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Die Domänenstruktur hilft zum Umbau von Antikörpern scFv

IgG

(a) Immunoglobulin G

Fab CL VL

VH

Hinge CH

Fv VL

KV 8.3

VH

CH2 CH3

Fc

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Humanisierung von Maus Antikörpern

Maus

KV 8.3

Chimäre

Humanisiert

Human

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Ezzell, Scientific American

Therapeutische Antikörper Generic name

Trade name

Target

Type

Muromonab

Orthoclone OKT3

CD3

Murine

Abciximab

ReoPro

Platelet GP IIb/IIIa

Murine

Rituximab Daclizumab

Rituxan Zenapax

CD20

Chimeric Humanized

Basiliximab Palivizumab Infliximab Trastuzumab Gemtuzumab Alemtuzumab

Simulect Synagis Remicade Herceptin Mylotarg Campath-1H

Endrecolomab

IL2Ra IL2R RSV TNFa Her2/neu/ErbB2 CD33 CD52

Chimeric Humanized Chimeric Humanized Humanized Humanized

Panorex

17A-1

Humanized

Ibritumomab tiuxetan

Zevalin

CD20

Chimeric

Adalimumab

Humira

Human

Omalizumab

Xolair

TNFa IgE

Centuximab

Erbitux

EGFR/ErbB1/Her1

Humanized

Bevacisumab

Avastin

VEGF

Humanized

Efalizumab

Raptiva

CD11a

Humanized

KV 8.3

Humanized

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Mono- & plurivalente Ak-Fragmente (a) Monovalent antibody fragments

Fab Fab

Fv Fv

scFv scFv

dsFv dsFv

VL VH

VL VH

VL VH

VH VL

CL

V

VHH

C H1

(b) Single chain two-domain constructs

scFv scFv Ag

Diabody Diabody Ag

Triabody Triabody

L

Tetrabody Tetrabody

or Ag

Ag

Ag

Ag

L

L Ag Ag

Ag VH

VL L

KV 8.3

VH

VL

VH

Ag

VL

L

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Transgene Humanisation

humane Antikörper in human Ig transgenenen Mäusen KV 8.3

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Der Wunsch nach humanen AK Humane Antikörper mittels Repertoire Klonierung mRNA von B Zellen

Blut

PCR of H&L chain genes mRNA  cDNA

KV 8.3

Antikörper (Fab) Expression auf p3 des M13 Phagen

Klonierung und Rekombination

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Expression von Antikörpern Antikörper DNA

Transfektion Antikörper Vektor

Zellkultur

Expression KV 8.3

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Industrielle Antikörper Produktion

Flask

Spinner

Bioreaktor (Bench scale)

Bio Reactor (Plant)

1mL → 100mL → 1L → 1Lx3 → 12L → 100L → 300L → 2’000L→ 10’000L Subkultur KV 8.3

Produktion 19

Ersatz von passivem IgG durch rekombinante anti-RhD

RhD+

Erythrozyten

rekombinante Antikörper mittels phage display

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Neue Therapeutische Antikörper

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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

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Lymphocyte Depleting Antibody Therapies Polyclonal Anti-Thymocyte Globulin

Campath-1H

OKT3

CD52 CD3

KV 8.3

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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 Bcell Non Hodgkin’s Lymphoma

CD19 DR

slg

CD20 CD22

• anti-CD52: Campath-1H (Alemtuzumab) • depletes T and B lymphocytes, NK cells, monocytes and macrophages KV 8.3

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Side effects • • • • • • • • •

fever chills weakness headache nausea vomiting diarrhea low blood pressure rashes

KV 8.3

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Antikörper Krebs Therapie Strategien

2

NK

Complement pathway

Tumor cell

5

-Y-90 -Y-90

Anti Angiogenese

-Y-90

1

ADCC

TNF Anti-Idiotyp

-Y-90

IL-1

-Y-90

Mac

3

-Drug

-Tox -Tox

4

-Drug

-Tox -Tox KV 8.3

Drug 26

Strategie 1 Antikörper vermittelte Zytotoxizität NK Zelle Ziel Zelle

Ziel Zelle

Apoptosis via Induktion von intrazellulären Signal-Pathways

Antikörper-abhängige Zytotoxizität (ADCC) KV 8.3

Ziel Zelle

Complement-abhängige Zytotoxizität (CDC) 27

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

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Strategie 1: maligne B-Zellen Stem cell

ProB cell

PreB cells

Immature B cell

Mature Activated B cell B cell

Antigen independent

Plasma cell

Antigen dependent

HLA-DR

TDT CD19 CD10 CD20 CD22

CD21 CD38 Neoplasias: KV 8.3

Leukemias from B-cell Precursors (B-ALL)

B-cell Lymphomas (NHL, CLL)

Multiple Myeloma 29

(B-Zell Lymphome)

Strategie 1 anti idiotypische Antikörper

KV 8.3

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Strategie 2: Verschiedene Isotope

Nackter Antikörper KV 8.3

Radiomarkierter Antikörper 31

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

KV 8.3

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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

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Strategie 4: Enzym markierte Antikörper Antibody directed enzyme prodrug therapy

(ADEPT) 4

Antikörper

1

2

3

Prodrug

Enzym

Tumor antigen

Zytotoxischer Wirkstoff Tumorzelle

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Strategie 5: Anti-Angiogenes

KV 8.3

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Anti-VEGF: Avastin ® • Recombinant humanized monoclonal antibody against VEGF; prevents binding with its receptor. • Blocks stimulation of angiogenesis  stops tumor growth.

KV 8.3

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Immunmodulatorische Antikörper • Anti-TNF Therapie • • • •

Infliximab Etanercept Humira etc (Biosimilarika)

• Anti-IgE Therapie • Xolair

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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

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Infliximab: Mechanism of Action Binds and neutralizes soluble and membrane bound TNFa

TNFa

TNFR2

Nucleus

No Signal

transcription NFB

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Other Postulated Mechanisms of Action… Antibody dependent cell-mediated cytotoxicity (ADCC) Lysis of TNFa-expressing cells through complement (C’) activation

Complement Activation

C’

Complement Receptor

Phagocyte

T Cell

KV 8.3

Phagocytosis

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TNF-a: Rezeptor oder Antikörper

Infliximab KV 8.3

Etanercept 41

Biosimilarika: z.B. anti-TNF Generic Name

Brand name (source)

Molecular Derivation

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 antihuman 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

Infliximab

KV 8.3

Development status

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Omalizumab Reduces IgE and FcεRI Levels Omalizumab (Xolair®) ↓ free IgE

↓ FcεRI

↓ Degranulation

↓ Inflammation ↓ Symptoms

anti-IgE FcεRI

B MC IgE Holgate S. et al., J Allergy Clin Immunol 2005 vol. 115 (3) pp. 459-65

Mediator Release

Allergic Inflammation

Anti-IgE: Omalizumab (Xolair®)

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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

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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

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Alefacept (Amevive®)

Anti-CD2

CD2

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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

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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

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