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

Monoclonal Antibodies Generation of Monoclonal Antibodies G. Köhler and C. Milstein 1975

Characteristics of Monoclonal Antibodies

Mechanisms Operating with Therapeutic Antibodies

Therapeutic Effects of Monoclonal Antibodies

Strategies for Therapeutic Application of Monoclonal Antibodies

Patient Suffering from NHL

Immunocytology of Bone Marrow Smear from a Patient with NHL

Non-Hodgkin‘s lymphomas

Target Antigens on Malignant B-cells HLA-DR TDT CD19 CD10 CD20 CD22 CD21 CD38 Stem cell Pro- B cell Pre- B cells Immature Mature Activated Plasma 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 Patient with Monoclonal Antibody

Monoclonal Antibody Therapy - Problems

Domain Structure of Immunoglobulins Fab Fv Fc Hinge VL VH CL CH CH2 CH3 Domain Structure of Immunoglobulins scFv IgG

Antibodies for Therapeutic Application Human Murine Chimeric Humanized Antibodies for Therapeutic Application scFv Fragment Bispecific

Rituximab

Rituximab

Strategies for Therapeutic Application of Monoclonal Antibodies

Kinase Growth Factor Pathway Cell membrane Ligand binding Activated receptor Y P Proliferation Migration Tumour growth and 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 epidermal growth factor receptor (EGFR) is a transmembrane receptor with an intracellular tyrosine kinase domain, which is activated by growth factors. The main activating ligands are epidermal growth factor (EGF) and transforming growth factor- (TGF- ) [1]. - EGFR is a protooncogene and its activation plays a key role in the development of many cancers. Activation occurs because of gene amplification, gene mutation and/or autocrine stimulation due to the overproduction of the EGFR ligands (eg EGF and TGF- ). Activation leads to the pairing of receptors with either another EGFR (homodimers) or another member of the EGFR family (heterodimers) [1]. - Receptor dimerization initiates an intracellular signaling cascade, gene activation and the stimulation of cell cycle progression [1]. 1. Baselga J. Eur J Cancer 2001; 37 Suppl 4:S16–S22.

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.”* - The EGFR intracellular signaling cascade stimulates not only cell proliferation but also protection from apoptosis, loss of differentiation, angiogenesis, cell migration and metastasis formation (ie all the key processes involved in tumorigenesis) [1]. - EGFR is expressed in a high proportion of solid tumors, in particular head and neck, lung and colorectal cancer [2]. Expression has been correlated with disease progression [3]. Many studies have shown that EGFR expression can be an adverse prognostic factor for cancer treatment outcome [2]. - ‘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 [3]. 1. Baselga J. Eur J Cancer 2001; 37 Suppl 4:S16–S22. 2. Nicholson RI, Gee JMW, Harper ME. Eur J Cancer 2001; 37 Suppl 4:S9–S15. 3. Baselga J. The Oncologist 2002; 7 Suppl 4:2–8. * 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 - Erbitux® (cetuximab) is an IgG1 MAb that targets EGFR with a high specificity and a higher affinity than either of the two main activating growth factors (EGF and TGF-) [1]. - The binding of Erbitux® to EGFR blocks ligand-induced EGFR signaling and results in the inhibition of proliferation, - stimulation of apoptosis, inhibition of angiogenesis, inhibition of de-differentiation and prevention of metastasis formation [1]. Being an IgG1 monoclonal antibody Erbitux® has been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) in vitro [2]. Therefore, in addition to its inhibitory function on receptor signaling, patients with EGFR-expressing tumors may also benefit from this immune stimulatory effect of Erbitux®. - The antibody has a long half-life of around 3-4 days, making it suitable for convenient weekly administration. Erbitux® is administered 400 mg/m2 for the 1st infusion followed by 250 mg/m2 weekly. - Erbitux® is well tolerated, lacks the hematologic toxicity [3] commonly associated with a number of anticancer agents, and does not exacerbate the toxicity of the investigated chemotherapeutic drugs [4]. The main side effect is an acne-like rash which generally improves during treatment and usually does not preclude continued treatment [5]. 1. Baselga J. Eur J Cancer 2001; 37 Suppl 4:S16–S22. 2. Naramura M, Gillies SD, Mendelsohn J, et al. Cancer Immunol Immunother 1993; 37:343-349. 3. Mendelsohn J. J Clin Oncol 2002; 20 Suppl 18:1S–13S. 4. O’Dwyer PJ, Benson AB. Semin Oncol 2002; 29 Suppl 14:10–17. 5. Needle MN. Semin Oncol 2002; 29 Suppl 14:55–60.

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 ?“

Strategies for Therapeutic Application of Monoclonal Antibodies

Inhibition of Angiogenesis

Strategies for Therapeutic Application of Monoclonal Antibodies

Internal Radiation by Radiolabeled mAb

Immunoscintigraphy with 131I-labeled mAb

RIT: Clinical Trials for NHL * myeloablative (autologous stem cell rescue)   Antibody Antigen Nuclide Patients n Response % (CR) Witzig et al. 2002 Ibritumomab (Zevalin®) CD20 90Y 54 74 (15) Kaminski et al. 2001 Tositumomab (BexxarTM) 131I 60 65 (20) De Nardo et al. 1998 Lym-1 HLA-DR 51 75 (20) Juweid et al. 1999 HLL2 CD22 131I, 90Y 22 18 (5) Press et al. 1998 * B1 29 86 (79)

Strategies for Therapeutic Application of Monoclonal Antibodies

Immunotoxin

Recombinant Immunotoxin Fusion Protein tumor cell normal ligand A-chain B-chain immunotoxin facilitates delivery into cytosol binds to many cells, must be blocked or removed inhibits protein synthesis mAb, scFv, cytokine receptor cytotoxic domain translocation binding toxin moiety Recombinant Immunotoxin Fusion Protein Original by Dr. D. Vallera

Strategies for Therapeutic Application of Monoclonal Antibodies

Monoclonal anti-Idiotype Antibodies

Strategies for Therapeutic Application of Monoclonal Antibodies

Cytotoxic T-cell Targeting Kill EGF-R MUC-1 cytotoxic T lymphocyte carcinoma cell CD3 Ep-CAM bispecific antibody HEA125xCD3 Cytotoxic T-cell Targeting

Ovarian Carcinoma Stained for Ep-CAM Expression

Selection of Quadromas X Hybridoma A (HGPRT-) Hybridoma B (Iodoacetamide) HAT Hybrid-Hybridoma

Antibody Mixture Produced by Hybrid-Hybridomas

Ovarian Cancer ¨ 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)

Clinical Trial of Intraperitoneal BsAb Therapy in Patients with Advanced Ovarian Cancer

Changes in Ascites Volume during bsAb Therapy Patient 2 All patients Ascites production lowest value during therapy Patient 1 , 020 263 2 135 3 218 4 190 5 533 6 290 7 571 350 8 000 9 200 10 320 before therapy Ascites volume in ml/day HEA125xOKT3 applications 800 4000 ascites ml/d 700 3500 ascites volume CA125 U/ml 600 3000 CA125 serum level 500 2500 400 2000 300 1500 200 1000 100 500 8 month of follow up 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 Ehrlich who considered antibodies as magic bullets have become reality. Monoclonal antibodies have established themselves as the most important and rapidly expanding class of drugs in oncology. Paul Ehrlich (1854-1915)