the sum of its parts? Antibody-drug conjugates (ADCs): Can an ADC be greater than

2 This educational program is prepared by F. Hoffmann-La Roche Ltd The purpose of this educational program is to provide an overview of an investigational mechanism of action and should not be construed as a recommendation for use of any product for unapproved uses. Products under investigational study have not been approved by any Health Authority for the use outside clinical trial setting. This educational program may be implemented only upon receipt of local jurisdiction's regulatory approval and following local affiliate legal/regulatory review. Disclosure

Overview The definition of an ADC Components of an ADC Proposed mechanisms of action of ADCs Potential of ADCs Summary 3

What are antibody-drug conjugates (ADCs)? An ADC is a unique combination of 1-4 –A targeted monoclonal antibody (mAb) –A stable linker –A potent cytotoxic agent ADCs are designed to selectively kill cancer cells while minimising effects on normal tissue Jaracz et al. Bioorg Med Chem. 2005;13: Wu et al. Nat Biotechnol. 2005;23: Ricart et al. Nat Clin Pract Oncol. 2007;4: Junutula et al. Nat Biotechnol. 2008;26: Potent cytotoxic agent Stable linker mAb

Critical parameters for development of an ideal ADC 5 Potent cytotoxic agent Stable linker Targeted mAbEach element of an ADC must fulfill exacting criteria

Overview The definition of an ADC Components of an ADC Proposed mechanisms of action of ADCs Potential of ADCs Summary 6

mAbs are designed to target cancer cells and may possess their own anticancer effects Preclinical studies show that mAbs integrated into an ADC should 1-5 –Target antigens that are preferentially or exclusively expressed on the surface of cancer cells –Display a high binding affinity and high selectivity to the antigen –Persist in circulation to allow prolonged exposure of the cancer cells to the ADC Jaracz et al. Bioorg Med Chem. 2005;13: Oflazoglu et al. Clin Cancer Res. 2008;14: Carter et al. Cancer J. 2008;14: Chari. Acc Chem Res. 2008;41: Ricart et al. Nat Clin Pract Oncol. 2007;4:

8 mAbs are designed to target cancer cells and may possess their own anticancer effects mAb-mediated anticancer activities may include 1-3 : –Prevention of signaling –Ability to initiate a cytotoxic immune response by effector cells (eg, antibody-dependent cellular cytotoxicity [ADCC]) –Induction of apoptosis mAbs may retain these activities when integrated into an ADC 1,2 1. Oflazoglu et al. Clin Cancer Res. 2008;14: Carter et al. Cancer J. 2008;14: Ricart et al. Nat Clin Pract Oncol. 2007;4:

mAbs may be developed to target a variety of cancers *This is not a comprehensive list of antigen targets under investigation in ADCs. 9 Target antigen*Cancer type CanAg 1-4 Gastric cancer, solid tumours CD19 1,4,5 Non-Hodgkin’s lymphoma (NHL) CD20 4 B-cell malignancies CD22 1,4,6 NHL CD30 1,4,7-9 CD30+ malignancies, Hodgkin lymphoma, anaplastic large cell lymphoma CD56 1,4,10 Multiple myeloma, solid tumours CD79b 4,11 NHL EphA2 4,12 Solid tumours HER2 1,4 HER2-positive breast cancer Lewis Y 1,4,13 Epithelial cancers 1. Carter et al. Cancer J. 2008;14: Erickson et al. Cancer Res. 2006;66: Widdison et al. J Med Chem. 2006;49: Teicher. Curr Cancer Drug Targets. 2009;9: Gerber et al. Blood. 2009;113: DiJoseph et al. Clin Cancer Res. 2004;10: Oflazoglu et al. Br J Haematol. 2008;142: Ansell et al. J Clin Oncol. 2007;25: Bartlett et al. Blood. 2008;111: Tassone et al. Cancer Res. 2004;64: Dornan et al. Blood. 2009;114: Jackson et al. Cancer Res. 2008;68: Herbertson et al. Clin Cancer Res. 2009;15:

Stable linkers are designed to prevent the release of the cytotoxic agent into the circulation 1-4 Preclinical studies suggest that selection of a linker depends on 4 –Cancer type and the cytotoxic agent in use –Stability in circulation –Ability to be efficiently cleaved within cancer cells Jaracz et al. Bioorg Med Chem. 2005;13: Wu et al. Nat Biotechnol. 2005;23: Chari. Acc Chem Res. 2008;41: Sanderson et al. Clin Cancer Res. 2005;11:

11 Current linkers under investigation in ADCs 1. Carter et al. Cancer J. 2008;14: Sanderson et al. Clin Cancer Res. 2005;11: Chari. Acc Chem Res. 2008;41: Oflazoglu et al. Clin Cancer Res. 2008;14: LinkerRelease mechanism HydrazoneDesigned for serum stability and degradation in acidic compartments within the cytoplasm 1 PeptideDesigned to be enzymatically hydrolysed by lysosomal proteases such as cathepsin B 1,2 DisulfideDesigned to be cleaved through disulfide exchange with an intracellular thiol, such as glutathione 1,3 ThioetherNonreducible and designed for intracellular proteolytic degradation 1,4

Potent cytotoxic agent: Killing cancer cells from within Chari. Acc Chem Res. 2008;41: Ricart et al. Nat Clin Pract Oncol. 2007;4: Hamann. Expert Opin Ther Patents. 2005;15: Oflazoglu et al. Br J Haematol. 2008;142: Based on preclinical models, criteria for ideal cytotoxic agents include 1-4 : –High potency in vitro –Sensitivity of the cancer type to the cytotoxic agent –Potential to induce cell death from within the cancer cell –Must be stable while bound to antibody 3 Cannot lose potency while in the blood Cannot be deactivated by lysosomal proteolysis, hydrolysis, or acidity

13 Cytotoxic agents investigated for ADCs may be up to 1000-fold more potent than agents initially investigated Chari. Acc Chem Res. 2008;41: Wu et al. Nat Biotechnol. 2005;23: Carter et al. Cancer J. 2008;14: Minotti et al. Pharmacol Rev. 2004;56: Ricart et al. Nat Clin Pract Oncol. 2007;4: NameTargetMode of action Doxorubicin derivatives 4 Topoisomerase II DNA complexes Inhibit DNA religation, leading to DNA double- strand breaks Maytansinoids 5 α-tubulinPrevent tubulin polymerisation Auristatins 5 α-tubulinPrevent tubulin polymerisation Calicheamicin 5 Sequence-specific minor groove of DNA Causes double-strand DNA breaks CC Sequence-specific minor groove of DNA Induces adenine alkylation

Overview The definition of an ADC Components of an ADC Proposed mechanisms of action of ADCs Potential of ADCs Summary 14

Proposed mechanisms of action of ADCs: Binding to the target antigen Based on preclinical models The ADC binds to the target antigen on the cell surface 1,2 Proposed mechanisms of action of this binding may include mAb-mediated anticancer effects, such as prevention of signaling, ADCC, and induction of apoptosis Jaracz et al. Bioorg Med Chem. 2005;13: Chari. Acc Chem Res. 2008;41: Ricart et al. Nat Clin Pract Oncol. 2007;4: Carter et al. Cancer J. 2008;14: Oflazoglu et al. Clin Cancer Res. 2008;14:

Proposed mechanisms of action of ADCs: Endocytosis Based on preclinical models Upon binding to the target antigen, the ADC-antigen complex is internalised 1,2 This initial process may be required for activation of the ADC Jaracz et al. Bioorg Med Chem. 2005;13: Chari. Acc Chem Res. 2008;41: Erickson et al. Cancer Res. 2006;66:

Proposed mechanisms of action of ADCs: Lysosomal degradation Based on preclinical models Internalisation of the ADC­antigen complex into endosomes is followed by delivery of the complex to the lysosomal compartment 1 The lysosomes are both acidic and rich in proteolytic enzymes Erickson et al. Cancer Res. 2006;66: Carter et al. Cancer J. 2008;14:

Proposed mechanisms of action of ADCs: Cell death Based on preclinical models This lysosomal degradation results in release of the cytotoxic agent 1 The cytotoxic agent then interacts with critical cellular machinery to elicit cell death Carter et al. Cancer J. 2008;14: Alley et al. J Pharmacol Exp Ther. 2009;330:

Proposed mechanisms of action of ADCs 19

Overview The definition of an ADC Components of an ADC Proposed mechanisms of action of ADCs Potential of ADCs Summary 20

21 Potential of ADCs: Targeting an unmet need Despite recent oncology breakthroughs, conventional anticancer treatments may have limitations 1,2 These limitations highlight the need and provide the rationale for the development of ADCs 1. Jaracz et al. Bioorg Med Chem. 2005;13: Wu et al. Nat Biotechnol. 2005;23:

22 Potential clinical application of ADCs in oncology Many cancers are known to either preferentially or exclusively express antigens that can be targeted 1-3 Potential methods for targeting these cancers with ADCs involve 3 –Creating new mAbs specific for the preferentially expressed antigen and linking them with appropriate potent cytotoxic agents 4 –Using active existing mAbs already in clinical use to deliver a potent cytotoxic agent that would be intolerable if administered alone 2 1. Jaracz et al. Bioorg Med Chem. 2005;13: Carter et al. Cancer J. 2008;14: Junutula et al. Nat Biotechnol. 2008;26: Kovtun et al. Cancer Lett. 2007;255:

Overview The definition of an ADC Components of an ADC Proposed mechanisms of action of ADCs Potential of ADCs Summary 23

Summary ADCs are a unique combination of a targeted mAb, a stable linker, and a potent cytotoxic agent ADCs have multiple proposed mechanisms of action, including mAb­mediated anticancer activities and targeted intracellular delivery of a potent cytotoxic agent 1-3 –mAbs are designed to target cancer cells and may possess their own anticancer effects 1-5 –Stable linkers are designed to allow ADCs to remain inactive while in circulation 1,4,6,7 –Potent cytotoxic agents kill cancer cells from within 1,5,8,9 Target antigen expression, selectivity of the mAb, linker stability, and potency of the cytotoxic agent are all key considerations when optimising and developing an ADC Chari. Acc Chem Res. 2008;41: Carter et al. Cancer J. 2008;14: Oflazoglu et al. Clin Cancer Res. 2008;14: Jaracz et al. Bioorg Med Chem. 2005;13: Ricart et al. Nat Clin Pract Oncol. 2007;4: Wu et al. Nat Biotechnol. 2005;23: Sanderson et al. Clin Cancer Res. 2005;11: Hamann. Expert Opin Ther Patents. 2005;15: Oflazoglu et al. Br J Haematol. 2008;142:69-73.

Suggested reading for further information on ADCs Bodey B, Bodey B Jr, Siegel SE, Kaiser HE. Genetically engineered monoclonal antibodies for direct anti-neoplastic treatment and cancer cell specific delivery of chemotherapeutic agents. Curr Pharm Des. 2000;6: Carter PJ, Senter PD. Antibody-drug conjugates for cancer therapy. Cancer J. 2008;14: Chari RVJ. Targeted cancer therapy: conferring specificity to cytotoxic drugs. Acc Chem Res. 2008;41: Erickson HK, Park PU, Widdison WC, et al. Antibody-maytansinoid conjugates are activated in targeted cancer cells by lysosomal degradation and linker-dependent intracellular processing. Cancer Res. 2006;66: Jaracz S, Chen J, Kuznetsova LV, Ojima I. Recent advances in tumor-targeting anticancer drug conjugates. Bioorg Med Chem. 2005;13: Kovtun YV, Goldmacher VS. Cell killing by antibody-drug conjugates. Cancer Lett. 2007;255: Reichert JM, Valge-Archer VE. Development trends for monoclonal antibody cancer therapeutics. Nat Rev Drug Discov. 2007;6: Ricart AD, Tolcher AW. Technology insight: cytotoxic drug immunoconjugates for cancer therapy. Nat Clin Pract Oncol. 2007;4: Wu AM, Senter PD. Arming antibodies: prospects and challenges for immunoconjugates. Nat Biotechnol. 2005;23:

APPENDIX 26

History of ADCs 1. Ghose et al. J Natl Cancer Inst. 1978;61: Ranson et al. Oncology. 2002;63: Ricart et al. Nat Clin Pract Oncol. 2007;4: Jaracz et al. Bioorg Med Chem. 2005;13: Carter et al. Cancer J. 2008;14: s Research into ADCs begins s The concept of a targeted therapy to treat malignant diseases is conceived s/1980s Advances in antibody technology and intensive investigation of linkers to bind cytotoxic agents progress the development of ADCs 2, and beyond Decades of dedicated research have resulted in the capacity to create a new generation of ADCs 1, The first ADC for clinical use in oncology becomes available (anti­CD33) 4 27

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