tumor therapy with monoclonal antibodies gerhard moldenhauer

Tumor Therapy with Monoclonal Antibodies

Gerhard Moldenhauer

Innate and Adaptive Immunity

Lymphocyte Activation

Effector Functions of Antibodies

Model of Secreted IgG

Structure of an IgG Antibody

Original by Dr. Mike Clark

Generation ofMonoclonal Antibodies

G. Köhler and C. Milstein1975

Monoclonal antibodies are

♦ monospecific

=> recognize onl y one epitope (antigenic determinan)t

♦ homogenous

=> identica l immunoglobulin molecules=> display identica l binding strengths (affinity)

♦ produced in unlimited quantities

♦ usually deri vedfrom mouse

Characteristics of Monoclonal Antibodies

Mechanisms Operating with Therapeutic Antibodies

Direct effects

♦ / Blockade of growth factors growth factor receptors

♦ Induction of apoptosis

♦ Inhibition of angiogenesis

Indirect effects

♦ - Complement dependent ( )cytotoxicity CDC

♦ - Antibody dependent cellular ( )cytotoxicity ADCC

♦ , Vehicel for toxins radionuclides and cytostatic drugs

♦ -Anti idiotype antibody formation

♦ Effector cell targeting using bispecific antibodies

Therapeutic Effects of Monoclonal Antibodies

in vivo:♦ mAb against differentia tionantigens (AD ,CC compleme ntfixation)♦ mAb with direc tanti-proliferative effects (grow threceptors, apoptosis)♦ mAb interfering w ithangiogenesis♦ mAb as carrier sfo r radioisotope s(radioimmunotherapy)♦ mAb as carrier sfo r toxins(immunotoxins)♦ Ant -i idiotype mAb♦ Bispecific mAb tofoc useffector cell ac tivity(effector cell targeting)

e xvivo:♦ Autologous / BM SC transplanta :tion pur ging of th eautograf twith mAb♦ Allogeneic /BM SC transplanta : tion prevention of GVHD by T ce ll depletion

Strategies for Therapeutic Application of Monoclonal Antibodies

Patient Suffering from NHL

Immunocytology of Bone Marrow Smear from a Patient with NHL

♦ Group of malignancies that affect primarily the lymphatic system.

♦ Prevalent forms derive from malignant B lymphocytes (80% of cases).

=> Incidence 6-17 cases per 100,00 habitants per year.

=> Increase world-wide at about 4% a year.

=> Mortality 9,000 patients in Western Europe (330 Million population).

♦ Early and intermediate stages of NHL can be cured by chemotherapy.

♦ The disease-free survival at advanced stage-NHL is 25% at 5 years and less

than 10% at 10 years.

Non-Hodgkin‘s lymphomas

Target Antigens on Malignant B-cellsTarget Antigens on Malignant B-cells

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

First Report on Treatment of a Cancer Patientwith Monoclonal Antibody

♦ Tumor cell heterogeneity (escape)

♦ Antigen modulation (loss of surface antigens)

♦ Blocking effects of circulating antigens

♦ Insufficient penetration into the tumor

♦ Insufficient activation of human effector mechanisms

♦ Cross reactivity with normal tissue antigens

♦ Antibodies against mouse immunoglobulin (anti-isotype,

anti-allotype, anti-idiotype)

Monoclonal Antibody Therapy - Problems

Fab

Fv

Fc

Hinge

VL

VH

CL

CH

CH2

CH3

(a) Immunoglobulin G

VHVL

Domain Structure of Immunoglobulins

scFv IgG

Human

Murine Chimeric Humanized

Antibodies for Therapeutic Application

scFvFragment

Bispecific

Rituximab

Kinase Growth Factor Pathway

Cell membrane

Ligand binding

Activated receptor

Y Y

YYP

P

P

P

Proliferation Migration

Tumour growthand metastases

Survival

Signal transduction

Tyrosine kinase receptor

Tyrosine kinase domain

Original by Dr. Axel Ullrich

Effects of Trastuzumab (Herceptin) on Breast Cancer Cells

EGFR signaling

The EGFR is activated by growth factors (e.g. epidermal growth factor (EGF) and transforming growth factor- (TGF-)).

EGFR-activation leads to the building of either receptor homo- or hetero-dimers.

Receptor dimerization initiates an intracellular signaling cascade, gene activation and the stimulation of cell cycle progression.

The importance of EGFR as a target

“Evidence for a role for the EGFR in the inhibition and pathogenesis of various cancers has led to the rational design and development of agents that selectively target this receptor.”*

* Baselga 2002

Erbitux® (cetuximab)

• Erbitux® (cetuximab) is an IgG1 MAb targeting the EGFR

• Binding blocks EGFR signaling and inhibits proliferation, angio-genesis and metastasis, and stimulates apoptosis and differentiation

• The main toxicity is an acne-like rash that generally improves during treatment, and usually does not preclude continued treatment

Deborah Schrag : The Price Tag on Progress N Engl J Med, July 22, 2004

“Just how much are we as a society willing to spend on the treatment of advanced

cancer ?“

Inhibition of Angiogenesis

Internal Radiation by Radiolabeled mAb

Immunoscintigraphy with 131I-labeled mAb

RIT: Clinical Trials for NHL

* myeloablative (autologous stem cell rescue)

Antibody Antigen Nuclide Patientsn

Response% (CR)

Witzig et al. 2002

Ibritumomab

(Zevalin®)

CD20 90Y 54 74 (15)

Kaminski et al. 2001

Tositumomab(BexxarTM)

CD20 131I 60 65 (20)

De Nardo et al. 1998

Lym-1 HLA-DR 131I 51 75 (20)

Juweid et al. 1999

HLL2 CD22 131I, 90Y 22 18 (5)

Press et al. 1998 *

B1 CD20 131I 29 86 (79)

in vivo: ♦ mAb against differentiation antigens ( ,ADCC complement fixatio )n ♦ mAb with direct anti-proliferative effects (growth ,receptors apop )tosis ♦ mAb interfering with angiogenesis ♦ mAb as carriers for radioisotopes (radioimmunotherap )y ♦ mAb as ca rriers for toxins (immunotox )ins ♦ Anti-idiotype mAb ♦ Bispecific mAb to focus effector cell activity (effector cell targeti )ng

ex vivo: ♦ Autologous BM/SC transplantation: purging of the autograft with mAb ♦ Allogeneic BM/SC transplantation: preventio n o f GVHDby T cell depletion


Immunotoxin

tumorcell

normalcellligand A-chain B-chain

immunotoxin

facilitatesdeliveryinto cytosol

binds to many cells,must be blockedor removed

inhibitsproteinsynthesis

mAb,scFv,cytokine

receptor

cytotoxicdomain

translocationdomain

bindingdomain

toxin moiety

Recombinant Immunotoxin Fusion Protein

Original by Dr. D. Vallera

in vivo:♦ mAb against leukocyte differentiation antige ns(ADCC, complement fixation)♦ mAb with direc tanti-proliferative effects (grow threceptors, apoptosis)♦ mAb as carrier sfo r radioisotope s(radioimmunotherapy)♦ mAb as carrier sfo r toxins(immunotoxins)♦ Ant -i idiotype mAb♦ Bispecific mAb tofoc useffector cell ac tivity(effector cell targeting)



Monoclonal anti-Idiotype Antibodies

Kill

EGF-R

MUC-1

cytotoxicT lymphocyte

carcinoma cell

CD3Ep-CAM

bispecific antibodyHEA125xCD3

Cytotoxic T-cell Targeting

Ovarian Carcinoma Stained for Ep-CAM Expression

Selection of Quadromas

X

Hybridoma A (HGPRT-)

Hybridoma B (Iodoacetamide)

HAT

Hybrid-Hybridoma

Zur Anzeige wird der QuickTime™ Dekompressor „TIFF (Unkomprimiert)“ benötigt.

Antibody Mixture Produced by Hybrid-Hybridomas

♦ Leading cause of death among gynecological malignancies

♦ Therapy consists of cytoreductive surgery and combination chemotherapy

♦ Prognosis of advanced disease is very poor (5 year survival at stages III and IV

less than 20%)

=> Early spread of carcinoma cells from ovaries into the peritoneal cavity

giving rise to multiple metastases

=> Formation of malignant ascites leading to severe symptoms (anorexia,

dyspnea, obstruction of the gastrointestinal tract)

Ovarian Cancer

Patient inclusion criteria

- Histologically confirmed ovarian carcinoma

- Recurrent ascites

- Resistance to second- or third line chemotherapy

- Life expectancy > 6 month

- Written informed consent

S tud y p ro toc ol

- Removal of malignant ascites by ultrasound-guided puncture

- Weekly intraperitoneal application of 1 mg bsAb in 500 ml saline

- Evaluation after 4 courses of therapy

- In case of ascites regression continuation of therapy until progression

Obje ctives

- Toxicity (cytokine syndrome?)

- Reduction of ascites volume

- Cytokine profile in ascites

Clinical Trial of Intraperitoneal BsAb Therapyin Patients with Advanced Ovarian Cancer

Ascites production lowest value

during therapyPatient

1 1,020 263

2 135 03 218 04 190 05 533 06 290 07 571 3508 1,000 09 200 010 320 0

Ascites production before therapy

Ascites volume in ml/day

asc

ites

vo

lum

e

Changes in Ascites Volume during bsAb Therapy

Patient 2

0

100

200

300

400

500

600

700

800

0

500

1000

1500

2000

2500

3000

3500

4000

CA

125

ser

um

lev

el

ascites ml/d

CA125 U/ml

HEA125xOKT3 applications

8 month of follow up

All patients

Collaboration with the Department of Obstetrics and Gynecology at the University of Heidelberg(Dr. A. Marmé, Prof. G. Bastert)

Eighteen Therapeutic Antibodies

Approved by the FDA

Paul Ehrlich(1854-1915)

Finally, the dreams of Paul Ehrlichwho considered antibodiesas magic bullets have become reality.

Monoclonal antibodies haveestablished themselves as the mostimportant and rapidly expandingclass of drugs in oncology.

tumor therapy with monoclonal antibodies gerhard moldenhauer

Documents

nhl slide

rituximab slide

monoclonal antibody

continued treatment

radiolabeled mab slide

lymphocyte activation

mike clark slide

adaptive immunity slide