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 2014 Genentech USA, Inc. All rights reserved. 1 Disclosure/Disclaimer The Molecular Basis of Cancer Educational Series is not intended to promote any.

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Presentation on theme: " 2014 Genentech USA, Inc. All rights reserved. 1 Disclosure/Disclaimer The Molecular Basis of Cancer Educational Series is not intended to promote any."— Presentation transcript:

1  2014 Genentech USA, Inc. All rights reserved. 1 Disclosure/Disclaimer The Molecular Basis of Cancer Educational Series is not intended to promote any cancer agent or class approved by the FDA or currently under clinical development. The contents of these slide presentations are owned solely by Genentech; any unauthorized uses are prohibited. These programs are intended to provide general information about the molecular basis of cancer, not medical advice for any particular patient. The information is presented on behalf of Genentech, and is consistent with FDA guidelines. BIO/092914/0038

2  2014 Genentech USA, Inc. All rights reserved. 2 Biomarkers are used to assess distinct biological characteristics of cancer CTCs=circulating tumor cells; FISH=fluorescence in situ hybridization; IHC=immunohistochemistry; NGS=next-generation sequencing; PCR=polymerase chain reaction; qRT-PCR=quantitative reverse transcriptase polymerase chain reaction; SKY=spectral karyotyping. Courtesy: National Human Genome Research Institute. FISH image Reprinted by permission from Macmillan Publishers Ltd: Modern Pathology 16(7): , copyright CTCs image from Marrinucci D et al. Fluid biopsy in patients with metastatic prostate, pancreatic and breast cancers. Phys. Biol. 2012; 9:1-9.© IOP Publishing. Reproduced with permission. All rights reserved. IHC image ©2009 American Society of Clinical Oncology. All rights reserved. Shaw, A et al: J Clin Oncol Vol. 7(26),2009: Gene ProteinCells DNANucleosomeChromosome RNA SKYBisulfite PCR PCR NGS FISHqRT-PCRIHC proteomics CTCs

3  2014 Genentech USA, Inc. All rights reserved. 3 Biomarkers are used to assess distinct biological characteristics of cancer CTCs=circulating tumor cells; FISH=fluorescence in situ hybridization; IHC=immunohistochemistry; NGS=next-generation sequencing; PCR=polymerase chain reaction; qRT-PCR=quantitative reverse transcriptase polymerase chain reaction; SKY=spectral karyotyping. Courtesy: National Human Genome Research Institute. FISH image Reprinted by permission from Macmillan Publishers Ltd: Modern Pathology 16(7): , copyright CTCs image from Marrinucci D et al. Fluid biopsy in patients with metastatic prostate, pancreatic and breast cancers. Phys. Biol. 2012; 9:1-9.© IOP Publishing. Reproduced with permission. All rights reserved. IHC image ©2009 American Society of Clinical Oncology. All rights reserved. Shaw, A et al: J Clin Oncol Vol. 7(26),2009: Gene ProteinCells DNANucleosomeChromosome RNA SKYBisulfite PCR PCR NGS FISHqRT-PCRIHC proteomics CTCs Precision medicine in oncology depends on the use of biomarkers measured at several structural levels, which reciprocally demands assessment of biological samples using precise molecular assays 1 This slide depicts the hierarchy of biomolecular structures, from the level of the chromosome, down to single DNA base pairs, and spanning outward through the components of gene expression (ie, genes, RNA, and proteins), ultimately facilitating the construction of whole cells ‒ Unique analytical techniques probe each structural level Goals of biomarker identification include 1 : ‒ Discovery of genetic and molecular abnormalities relevant to many or all patients in a particular type of cancer ‒ Identification of optimal targets to develop novel therapies ‒ Personalization of therapy for each patient Technology-enabled insights into the molecular details of cancer biology are influencing treatment decisions and outcomes at an increasing rate 2 Next-generation sequencing (NGS) is capable of identifying base mutations, indels, amplifications, and deletions in 1 test, rapidly generating a wealth of clinically actionable data 3 As proteomic technologies quickly advance, they are informing our understanding of the functional effects of complex genomic abnormalities and better defining patient subpopulations 2 Understanding and utilizing the rapidly expanding, clinically actionable information from genomic, methylomic, transcriptomic, proteomic, and metabolomic techniques will require integrated approaches to interpretation and clinical validation References: 1.Dolsten M, Søgaard M. Precision medicine: an approach to R&D for delivering superior medicines to patients. Clin Transl Med. 2012;1:7. 2.Kalia M. Personalized oncology: recent advances and future challenges. Metabolism. 2013;62(suppl 1):S11-S14. 3.Meldrum C, Doyle MA, Tothill RW. Next-generation sequencing for cancer diagnostics: a practical perspective. Clin Biochem Rev. 2011;32: Notes

4  2014 Genentech USA, Inc. All rights reserved. 4 Molecular pathways can be targeted by small molecule and biologic agents ADCC=antibody-dependent cell-mediated cytotoxicity; Grb2=growth factor receptor-bound protein 2; MAbs=monoclonal antibodies; PDK1=phosphoinositide- dependent kinase-1; PI3K=phosphatidylinositol 3-kinase; Raf=rapidly accelerating fibrosarcoma; Ras=rat sarcoma; Sos=son of sevenless. Adjei AA, Hidalgo M. J Clin Oncol. 2005;23: Small Molecules Generally, chemical agents (~400 daltons) Varying degrees of specificity Penetrate through the plasma membrane Cannot elicit ADCC immune response, eg, TKIs Large Molecules Generally, large proteins (~150,000 daltons) Highly specific Cannot penetrate through the plasma membrane May elicit immune response: ADCC, eg, monoclonal antibodies AKT PDK1RAS Sos Grb2Shc Raf Cell surface receptors AKT PDK1RAS Sos Grb2Shc Raf PI3K Cell surface receptors PI3K

5  2014 Genentech USA, Inc. All rights reserved. 5 Molecular pathways can be targeted by small molecule and biologic agents ADCC=antibody-dependent cell-mediated cytotoxicity; Grb2=growth factor receptor-bound protein 2; MAbs=monoclonal antibodies; PDK1=phosphoinositide- dependent kinase-1; PI3K=phosphatidylinositol 3-kinase; Raf=rapidly accelerating fibrosarcoma; Ras=rat sarcoma; Sos=son of sevenless. Adjei AA, Hidalgo M. J Clin Oncol. 2005;23: Small Molecules Generally, chemical agents (~400 daltons) Varying degrees of specificity Penetrate through the plasma membrane Cannot elicit ADCC immune response, eg, TKIs Large Molecules Generally, large proteins (~150,000 daltons) Highly specific Cannot penetrate through the plasma membrane May elicit immune response: ADCC, eg, monoclonal antibodies AKT PDK1RAS Sos Grb2Shc Raf Cell surface receptors AKT PDK1RAS Sos Grb2Shc Raf PI3K Cell surface receptors PI3K Notes Molecular targeted agents fall into 2 categories 1 ‒ Small molecules, eg, small molecule inhibitors ‒ Large molecules, eg, monoclonal antibodies Small molecules are generally chemical agents that are capable of targeting the plasma membrane 1 Large molecules are generally large proteins that are highly specific and cannot penetrate the plasma membrane 2 Large molecules, such as monoclonal antibodies, help mediate antibody- dependent cell-mediated cytotoxicity (ADCC), wherein immune effector cells are recruited to—and can attack—the antibody-coated target cell 2,3 References: 1.Adjei AA, Hidalgo M. Intracellular signal transduction pathway proteins as targets for cancer therapy. J Clin Oncol. 2005;23: Mellor JD, Brown MP, Irving HR, Zalcberg JR, Dobrovic A. A critical review of the role of Fc gamma receptor polymorphisms in the response to monoclonal antibodies in cancer. J Hematol Oncol. 2013;6:1. 3.Boross P, Lohse S, Nederend M, et al. IgA EGFR antibodies mediate tumour killing in vivo. EMBO Mol Med. 2013;5:

6  2014 Genentech USA, Inc. All rights reserved. 6 Bispecific monoclonal antibodies may confer a distinct advantage over single-target agents EGFR=endothelial growth factor receptor; Fv=variable fragment; HER2=human epidermal growth factor receptor 2; HGF=hepatocyte growth factor. 1 antibody, 2 epitopes Fv 2 mechanistic classes Limitations of single-target agents Pathway crosstalk Redundancy

7  2014 Genentech USA, Inc. All rights reserved. 7 Bispecific monoclonal antibodies may confer a distinct advantage over single-target agents EGFR=endothelial growth factor receptor; Fv=variable fragment; HER2=human epidermal growth factor receptor 2; HGF=hepatocyte growth factor. 1 antibody, 2 epitopes Fv 2 mechanistic classes Limitations of single-target agents Pathway crosstalk Redundancy Key takeaway Bispecific antibodies provide a distinct advantage over typical monospecific antibodies by binding 2 epitopes with 1 antibody (or fused/recombinant Fab). Supporting information The multifactorial nature of complex diseases, such as cancer, involves crosstalk between signaling pathways and redundancy of mediating receptors and ligands 1,2 ‒ Upregulation of alternative receptors and pathway switching provide mechanisms of therapeutic resistance and underscore the limitations of single-target agents Typical (monospecific) bivalent antibodies are characterized by 2 identical antigen binding sites 2,3 Bispecific antibodies offer a means of targeting 2 individual paratopes with 1 therapeutic compound 3 ‒ Either multiple sites on a single antigen or single sites on multiple antigens 2 2-for-1 Combination therapies with bivalent monospecific antibodies necessitate a substantial investment of resources for development, manufacturing, clinical studies, and approval of the individual components 2 2 mechanistic classes 2 1. Direct: exert effect directly through epitope binding Dual-receptor inhibition Receptor-ligand dual targeting 2. Indirect (retargeting): utilize dual targeting to coordinate delivery of a therapeutically active moiety Exploiting immune effector mechanisms (ADCC, complement-dependent cytotoxicity [CDC]) ‒ Achieved by directly targeting effectors or by targeting cytokines Drug-loaded nanoparticles Prodrug-converting enzymes References: 1. Chames P, Baty D. Bispecific antibodies for cancer therapy: the light at the end of the tunnel? MAbs. 2009;1: Kontermann R. Dual targeting strategies with bispecific antibodies. MAbs. 2014;4: Holmes D. Buy buy bispecific antibodies. Nat Rev Drug Discov. 2011;10: Notes

8  2014 Genentech USA, Inc. All rights reserved. 8 Engineering next-generation antibody-drug conjugates Carrier High specificity Potent at relatively low concentrations due to localization Cargo Potent cytotoxic agent Linker Stability in circulation vs selective payload release Chemical linker motifs R1R1 R2R2 S S R1R1 R2R2 NH 2 N C O N H HNHN R n+1 RnRn DisulfidesHydrazonesPeptides

9  2014 Genentech USA, Inc. All rights reserved. 9 Engineering next-generation antibody-drug conjugates Carrier High specificity Potent at relatively low concentrations due to localization Cargo Potent cytotoxic agent Linker Stability in circulation vs selective payload release Chemical linker motifs R1R1 R2R2 S S R1R1 R2R2 NH 2 N C O N H HNHN R n+1 RnRn DisulfidesHydrazonesPeptides Key takeaway Antibody-drug conjugates (ADCs) optimize target specificity and drug potency through a 3-component molecular assembly, refined through advances in linker chemistry and recombinant antibody design. Supporting information Engineering ADCs Composed of 1 or several drug molecules covalently linked to a Mab 1 ‒ Optimal stoichiometry of antibody to drug is 1 to 4 2 Does not sterically hinder antibody-binding Minimizes unconjugated antibody MAbs: high specificity 1,3 ‒ High potency in low quantity due to localization Drug conjugate: potent cytotoxicity (IC50=10 -9 to M [vs first generation ADCs at M]) 4 ‒ Toxins that are generally too toxic for administration as a stand-alone agent are well-suited for ADCs 3 Can be 100 to 1000 times more cytotoxic than traditional chemotherapeutics ‒ DNA-targeted agents Minor groove disruption Strand breakage Alkylation ‒ Microtubule polymerization inhibitors ‒ Topo II inhibitors Agents must be internalized by target cells to exert effect 5 Linker stability Effective ADCs must balance the delicate interplay between immunoconjugate stability in the plasma during delivery and subsequent selective payload release 2,4,6 Unique chemical properties of the linker direct selective release (chemically or enzymatically labile) 3 Linker must be stable in circulation 5 Chemical linker motifs Disulfides ‒ Alkylation of reduced interchain disulfides 2 Hydrazones 7 ‒ Naturally occurring carbohydrates in the Fc region of mAbs are oxidized to generate aldehyde groups ‒ Enables nucleophilic addition of hydrazine to yield hydrazone Peptides ‒ Optimally designed for high serum stability 5 Valine-citrulline and phenylalanine-lysine have shown to possess high serum stability 4 ‒ Improved antitumor effects ‒ Offer enhanced stability over hydrazones 5 Second-generation ADCs Refinements in linker technologies and innovations in recombinant MAb design define a new generation of improved ADCs 8 References: 1. Kovtun YV, Goldmacher VS. Cell killing by antibody-drug conjugates. Cancer Lett. 2007;255: Sievers EL, Senter PD. Antibody-drug conjugates in cancer therapy. Annu Rev Med. 2013;64: Ducry L, Stump B. Antibody-drug conjugates: linking cytotoxic payloads to monoclonal antibodies. Bioconjug Chem. 2010;21: Kitson SL, Quinn DJ, Moody TS, Speed D, Watters W, Rozzell D. Antibody-drug conjugates (ADCs)—biotherapeutic bullets. Chem Today. 2013;31: Jaracz S, Chen J, Kuznetsova LV, Ojima I. Recent advances in tumor-targeting anticancer drug conjugates. Bioorg Med Chem. 2005;13: Senter PD. Potent antibody drug conjugates for cancer therapy. Curr Opin Chem Biol. 2009;13: Hamann PR. Monoclonal antibody-drug conjugates. Expert Opin Ther Patents. 2005;15: Zolot RS, Basu S, Million RP. Antibody-drug conjugates. Nat Rev Drug Discov. 2013;12: Notes

10  2014 Genentech USA, Inc. All rights reserved. 10 ADC activation is dependent on internalization ADCs=antibody-drug conjugates. Receptor-ADC complex is internalized by cell Potent cytotoxic agent is released inside the cell ADC binds target Selective payload release DisulfidesHydrazonesPeptides Disulfide exchange with cytosolic thiols pH-dependent hydrolysis in acidic lumen of lysosome Rapid intracellular proteolysis Modes of antibody uptake 1.Clathrin-mediated endocytosis 2.Caveolae-mediated uptake 3.Macropinocytosis

11  2014 Genentech USA, Inc. All rights reserved. 11 ADC activation is dependent on internalization ADCs=antibody-drug conjugates. Receptor-ADC complex is internalized by cell Potent cytotoxic agent is released inside the cell ADC binds target Selective payload release DisulfidesHydrazonesPeptides Disulfide exchange with cytosolic thiols pH-dependent hydrolysis in acidic lumen of lysosome Rapid intracellular proteolysis Modes of antibody uptake 1.Clathrin-mediated endocytosis 2.Caveolae-mediated uptake 3.Macropinocytosis Key takeaway ADCs represent a truly innovative proof-of-concept that relies on specialized linker chemistries. Supporting information Following internalization, ADCs are selectively cleaved along their flexible linker, activating the complex by releasing the cytotoxic agent into the cytoplasm 1,2 The rate and extent of internalization influence drug uptake 3 Internalization 4 There are 3 modes of antibody uptake ‒ Clathrin-mediated endocytosis (antigen-dependent) ‒ Caveolae-mediated uptake (antigen-dependent) ‒ Macropinocytosis (independent) Payload release Disulfides ‒ Cleaved inside tumor cells through disulfide exchange with an intracellular thiol 1 ‒ Hypoxic state of tumor cells results in upregulation of reductive enzymes and boosts intracellular glutathione concentrations (micromolar) relative to normal cells 2 Glutathione is not abundant (nanomolar scale) in circulation, thus establishing selectivity Hydrazones ‒ Undergo pH-dependent hydrolysis in the acidic environment of lysosomes or reductive environment of cytosol 3 Peptides ‒ Rapid enzymatic proteolysis in lysosome 3,5 References: 1.Jaracz S, Chen J, Kuznetsova LV, Ojima I. Recent advances in tumor-targeting anticancer drug conjugates. Bioorg Med Chem. 2005;13: Ducry L, Stump B. Antibody-drug conjugates: linking cytotoxic payloads to monoclonal antibodies. Bioconjug Chem. 2010;21: Sievers EL, Senter PD. Antibody-drug conjugates in cancer therapy. Annu Rev Med. 2013;64: Kovtun YV, Goldmacher VS. Cell killing by antibody-drug conjugates. Cancer Lett. 2007;255: Hamann PR. Monoclonal antibody-drug conjugates. Expert Opin Ther Patents. 2005;15: Notes


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