KV 8.3 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:http://www.iib.unibe.ch/wiki/?p=teaching/medical_students&l=de

Protein Pharmaceutika 2011

• 134 FDA-zugelassene

biotechnologisch hergestellte

(Proteine/Peptide) Medikamente

• Protein Pharmaceutika kosten

derzeit ca. $50 Milliarden/Jahr

2

3

Top Ten Monoclonal Antibodies2009/2010

Generic Name Brands® IndicationsSales $ billion2009 / 2010

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 1.1 1.0

Thearapeutische Antikörper auch zu teuer für uns?

KV 8.3 5

Übersicht / Lernziele

• Antikörper Produktion einst• Passive Immuntherapie einst • Monoklonale /

rekombinante Antikörper• Antikörper als Therapie für:

• Immunsuppression• Krebs Therapie• Immunmodulation• Anti-Adhäsionstherapien

• Rückblick und ZukunftPaul Ehrlich vor 100 Jahren

KV 8.3 6

Passive Immuntherapie einst

KV 8.3 7

Passive Immuntherapie mit polyklonalen Antikörpern

Multispender human Gammaglobulin oder Pferd Antitoxin

Tetanus

Pferd AntiveninSchlangenbiss

Multispender human GammaglobulinTollwut

Multispender human GammaglobulinMasern

Multispender human GammaglobulinHepatitis A and B

Pferd AntitoxinDiphtherie

Pferd AntitoxinBotulismus

Pferd AntiveninSchwarze Spinnenbiss

TherapeutikumKrankheit

KV 8.3 8

Monoklonale AntikörperHybridoma Technologie

KV 8.3 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)

Human anti-mouse Ab

Human/mouse chimeric Ab (Infliximab)

• 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

KV 8.3 11

Diversity of Antibodies is Generated by Gene RearrangementDank Wissen um Grundlagen

rekombinante Antikörper

Die Domänenstruktur hilft zum Umbau von Antikörpern

KV 8.3 12

Fab

Fv

Fc

Hinge

VL

VH

CL

CH

CH2

CH3

(a) Immunoglobulin G

VHVL

scFv IgG

KV 8.3 13

Ezzell, Scientific American

Humanisierung von Maus Antikörpern

HumanMaus Chimäre Humanisiert

Therapeutische Antikörper

KV 8.3 14

Generic name Trade name Target Type

Muromonab Orthoclone OKT3 CD3 Murine

Abciximab ReoPro Platelet GP IIb/IIIa Murine

Rituximab Rituxan CD20 Chimeric

Daclizumab Zenapax IL2Ra Humanized

Basiliximab Simulect IL2R Chimeric

Palivizumab Synagis RSV Humanized

Infliximab Remicade TNFa Chimeric

Trastuzumab Herceptin Her2/neu/ErbB2 Humanized

Gemtuzumab Mylotarg CD33 Humanized

Alemtuzumab Campath-1H CD52 Humanized

Endrecolomab Panorex 17A-1 Humanized

Ibritumomab tiuxetan Zevalin CD20 Chimeric

Adalimumab Humira TNFa Human

Omalizumab Xolair IgE Humanized

Centuximab Erbitux EGFR/ErbB1/Her1 Humanized

Bevacisumab Avastin VEGF Humanized

Efalizumab Raptiva CD11a Humanized

KV 8.3 15

VHVL VHVLVHVL

CH1CL

VLVH

Fv scFvFab VHdsFv

(a) Monovalent antibody fragments

Fab scFvFv dsFv VH

Mono- & plurivalente Ak-Fragmente

scFv Diabody orTriabody Tetrabody

VH VL

L

Ag

L

VH VL

L

Ag

Ag

L

L

Ag

Ag

Ag

Ag

Ag

Ag

Ag

VH VL

(b) Single chain two-domain constructs

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

mRNA von B Zellen

mRNA cDNA

PCRofH&Lchaingenes

PCRofH&Lchaingenes

BlutAntikörper (Fab)

Expression aufp3 des M13

Phagen

Klonierungund Rekombination

Humane Antikörper mittels Repertoire Klonierung

KV 8.3 18

Expression von Antikörpern

AntikörperDNA

AntikörperVektor

Transfektion

Zellkultur

Expression

KV 8.3 19

Industrielle Antikörper Produktion

Flask SpinnerBioreaktor

(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 IgGdurch rekombinante anti-RhD

RhD+

Erythrozytenrekombinante Antikörper

mittels phage display

KV 8.3 21

Neue Therapeutische Antikörper

KV 8.3 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

OKT3

Campath-1H

CD3

CD52

Polyclonal Anti-Thymocyte Globulin

Lymphocyte Depleting Antibody Therapies

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

DRCD19 CD20

CD22

KV 8.3 25

Side effects

• fever • chills • weakness • headache • nausea • vomiting • diarrhea • low blood pressure • rashes

KV 8.3 26

Complement pathway Tumor cell

ADCC

Anti-Idiotyp IL-1

TNF

Mac

NK

-Tox

-Tox

-Tox

-Tox

-Y-90

-Y-90-Y-90

-Y-90

-Y-9

0-Drug

-Drug

-Drug

1 2

3 4

AntikörperKrebs Therapie Strategien

5Anti

Angiogenese

KV 8.3 27

Antikörper-abhängige Zytotoxizität

(ADCC)

Apoptosis viaInduktion von intrazellulären

Signal-Pathways

Complement-abhängigeZytotoxizität

(CDC)

Ziel Zelle

Ziel ZelleZiel Zelle

NK Zelle

Strategie 1Antikörper vermittelte Zytotoxizität

KV 8.3 28

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

z.B. Herceptin® (Trastuzumab)

normal cell tumor cell treatment

KV 8.3 29

HLA-DR

TDT

CD19

CD10

CD20

CD22

CD21

CD38

Stem cell

Pro-B cell

Pre-B cells

ImmatureB cell

MatureB cell

ActivatedB cell

Plasmacell

Neoplasias: Leukemias from B-cell Precursors (B-ALL)

B-cell Lymphomas (NHL, CLL)

Multiple Myeloma

Antigen independent Antigen dependent

Strategie 1: maligne B-Zellen

KV 8.3 30

Strategie 1anti idiotypische Antikörper

(B-Z

ell L

ymp

ho

me)

KV 8.3 31

Strategie 2: Verschiedene Isotope

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

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.

Strategie 3: Mylotarg® (anti CD-33)

KV 8.3 34

Strategie 4: Enzym markierte Antikörper

Antibody directed enzyme prodrug therapy

(ADEPT)

Tumorantigen

Tumorzelle

ZytotoxischerWirkstoff

Prodrug132

4

Enzym

Antikörper

KV 8.3 35

Strategie 5: Anti-Angiogenes

KV 8.3 36

• Recombinant humanized monoclonal antibody against VEGF; prevents binding with its receptor.

• Blocks stimulation of angiogenesis stops tumor growth.

Anti-VEGF: Avastin ®

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

• 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

Chimeric monoclonal antibody

KV 8.3 39

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

TNFR2

Nucleus

NFB

transcriptionNo Signal

TNF

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 CellPhagocytosis

KV 8.3 41

EtanerceptInfliximab

TNF-a: Rezeptor oder Antikörper

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

Omalizumab (Xolair®)

BMC

↓ free IgE ↓ FcεRI ↓ Degranulation↓ Inflammation↓ Symptoms

IgE

FcεRI

MediatorRelease

Allergic Inflammation

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

anti-IgE

OmalizumabReduces IgE and FcεRI Levels

KV 8.3 44

Anti-IgE: Omalizumab (Xolair®)

KV 8.3 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

Anti-GpIIb/IIIa Inhibits the clumping of platelets by binding the receptors on their surface that normally are linked by fibrinogen.

Abciximab (Reopro®)

Helpful in preventing reclogging of the coronaryarteries 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…