1. 1Kitasato-Harvard Symposium 10/03/2002 New Monoclonal Antibody Approved for Advanced Breast Cancer Shin-ichi Nihira, Ph.D. Dept. Clinical Research 3 Chugai Pharmaceutical Co., Ltd.

2. 2Kitasato-Harvard Symposium 10/03/2002 Tailor-Made Drug Therapy for Cancer Identification of genetic abnormality and molecular mechanism responsible for generation and exacerbation of cancer Selection of particular patient population, in which higher efficacy and safety of the drug therapy is expected. ex.) Anti-HER2 humanized monoclonal antibody; Trastuzumab (Herceptin ® )

3. 3Kitasato-Harvard Symposium 10/03/2002 Trastuzumab (Herceptin ® ) : Humanized Anti-HER2 Antibody Targets HER2 oncoprotein High affinity (K d = 0.1 nM) and specificity 95% human, 5% murine - Decrease potential for immunogenicity - Increase potential for recruiting immune-effector mechanisms - ADCC - CDC Trastuzumab is a humanized monoclonal antibody that binds to the extracellular domain of the human epidermal growth factor 2 gene (HER2)

4. 4Kitasato-Harvard Symposium 10/03/2002 Trastuzumab Therapy Offering the alternative option to therapeutic algorithms for MBC Hormone therapy Chemotherapy Antibody therapy Molecular target drug for HER2 based on genetic abnormality “Tailor-Made” cancer therapy for solid tumors Survival benefit for HER2 overexpressing metastatic breast cancer (MBC) patients

5. 5Kitasato-Harvard Symposium 10/03/2002 HER2 Overexpression A Key Strategy for Clinical Development of Trastuzumab Patient population for Trastuzumab was focused on MBC patients diagnosed as HER2 amplification/overexpression in the clinical development (Phase I - III).  HER2 detection assay had been developed prior to the clinical studies. Clinical Trial Assay (CTA) by Genentech  Development of HercepTest ® by DAKO Know-how and expertise of CTA transferred to DAKO by GNE  FISH test Confirmation of the concordance have been done by GNE using clinical trials samples, retrospectively.

6. 6Kitasato-Harvard Symposium 10/03/2002 HER2 Receptor Provides an Extracellular Therapeutic Target Signal transduction to nucleus Nucleus Binding site Tyrosine kinase activity Cytoplasm Plasma membrane Gene activation CELL DIVISION

7. 7Kitasato-Harvard Symposium 10/03/2002 HER2 Overexpression 1 =  gene copy number 2 =  mRNA transcription 3 =  cell surface receptor protein expression 4 =  release of receptor extracellular domain A = HER2 DNA B = HER2 mRNA C = HER2 receptor protein Normal Amplification / Overexpression Nucleus Cytoplasm Cytoplasmic membrane 1 2 3 4 C B A

8. 8Kitasato-Harvard Symposium 10/03/2002 HER2 in Breast Cancer Slamon et al. 1987 HER2 oncoprotein overexpression HER2 oncogene amplification HER2 overexpressing 3 years HER2 normal 6–7 years Women whose breast cancers are HER2 positive have a shorter overall survival

9. 9Kitasato-Harvard Symposium 10/03/2002 Methods of Assessing HER2 Status Gene amplification  Fluorescence in-situ hybridisation (FISH)  Southern hybridisation  Polymerase chain reaction (PCR)  in-situ hybridisation (ISH; non-fluorescence) Protein overexpression  Immunohistochemistry (IHC)  Western blot  ELISA (serum) for circulating protein

10. 10Kitasato-Harvard Symposium 10/03/2002 Detection of Gene Amplification by FISH

11. 11Kitasato-Harvard Symposium 10/03/2002 HER2 Status in IHC & FISH IHC Images courtesy of MJ Kornstein, MD, Medical College of Virginia Abnormal 2+Abnormal 3+Normal 0Normal 1+ Normal Abnormal low amplification Abnormal high amplification

12. 12Kitasato-Harvard Symposium 10/03/2002 The New Biology Comes of Age Diagnosis of Breast Cancer Tumour genotype HER2/neu gene amplification Tumour phenotype Aggressive Selection of therapy Trastuzumab

13. 13Kitasato-Harvard Symposium 10/03/2002 Pivotal Trastuzumab Combination Multinational Study No prior anthracyclinesPrior anthracyclines Paclitaxel (n=96) Trastuzumab + paclitaxel (n=92) AC (n=138) Trastuzumab + AC (n=143) Metastatic breast cancer HER2 overexpression (HER2 2+&3+) No prior CT for MBC Measurable disease KPS  60% Eligible patients (n=469) AC = doxorubicin/epirubicin + cyclophosphamide, CT = chemotherapy, MBC = metastatic breast cancer AC = doxorubicin/epirubicin + cyclophosphamide, CT = chemotherapy, MBC = metastatic breast cancer D.J Slamon, et al. N Engl J Med 2001; 344: 783-792

14. 14Kitasato-Harvard Symposium 10/03/2002 Increasing Efficacy by Level of HER2 Overexpression Trastuzumab + CT CT alone p<0.05 1.0 0.8 0.6 0.4 0.2 0.0 2029 Time (months) Probability of survival Mass R et al. Proc ASCO 2000;19:Abstract 291 0 10 20 30 40 50 1.0 0.8 0.6 0.4 0.2 0.0 Probability of survival 2025 0 10 20 30 40 50 Time (months) HER2 3+HER2 2+ & 3+ - Overall Survival -

15. 15Kitasato-Harvard Symposium 10/03/2002 -- all patients (HER2 2+&3+) and HER2 3+ patients -- *p<0.05 All (HER2 2+&3+): n=469, HER2 3+ : n=349 Increasing Efficacy by Level of HER2 Overexpression

16. 16Kitasato-Harvard Symposium 10/03/2002 Trastuzumab Efficacy by Level of HER2 Overexpression Percentage responding (%) HHH+PH+ACPAC Salvage1st line IHC Response Rate (CR+PR) Combination Therapy H0649gH0650g Monotherapy H0648g

17. 17Kitasato-Harvard Symposium 10/03/2002 HER2 Testing Algorithm Being Applied for Breast Cancer Clinical Practice in US Patient tumour sample IHCFISH 2+3++– Retest with FISH Trastuzumab therapy – +

18. 18Kitasato-Harvard Symposium 10/03/2002 Conclusions Trastuzumab is a HER2 specific humanised monoclonal antibody, providing survival benefit for metastatic breast cancer patients with HER2/neu gene amplification/HER2 overexpression. Patient population for Trastuzumab should be selected based on diagnosis of HER2 status, where the efficacy of trastuzumab correlated with the level of HER2 status. Development of standardised diagnostic methods for HER2 status was indispensable for the clinical development of Trastuzumab. HER2 diagnosis algorithm needs to be implemented in the clinical practice for breast cancer patients.