Transcript Monovalent mAbs are distinguished by a single antigen

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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
 2014 Genentech USA, Inc. All rights reserved.
1
Biomarkers are used to assess distinct biological
characteristics
of cancer
• Precision medicine in oncology depends on the use of biomarkers measured at several structural levels,
Notes
which reciprocally demands assessment of biological samples using precise molecular assays1
• 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 include1:
‒ 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 rate2
• Next-generation sequencing (NGS) is capable of identifying base mutations, indels, amplifications, and
deletions in 1 test, rapidly generating a wealth of clinically actionable data3
Chromosome
Nucleosome
DNA they Gene
Protein
Cells
• As proteomic technologies
quickly advance,
are informing RNA
our understanding
of the functional
2
effects of complex genomic abnormalities and better defining patient subpopulations
• 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
SKYTransl Med. 2012;1:7.
Bisulfite
PCR
FISH
qRT-PCR
IHC
2. Kalia M. Personalized
oncology: recent NGS
advances and future challenges. Metabolism. 2013;62(suppl
1):S11-S14.
PCR
proteomics
3. Meldrum C, Doyle MA, Tothill RW. Next-generation sequencing for cancer diagnostics: a practical perspective.
Clin Biochem Rev. 2011;32:177-195.
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):708-715, copyright 2003.
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.
 2014 Genentech USA, Inc.
All rights reserved. IHC image ©2009 American Society of Clinical Oncology. All rights reserved. Shaw, A et al: J Clin Oncol Vol. 7(26),2009:4247-4253.
CTCs
All rights reserved.
3
Molecular pathways can be targeted by small
molecule and biologic agents
Notes
• Molecular targeted agents fall into 2 categories1
Large Molecules
Small Molecules
•
‒
‒
Generally,
Small molecules, eg, small molecule inhibitors
Large molecules,
• Generally,
chemical
agentseg, monoclonal antibodies
large proteins
• Small
molecules are generally chemical agents that
are capable
of targeting the
(~150,000
daltons)
(~400
daltons)
plasma membrane1
• Highly specific
• Varying degrees of specificity
• Large molecules are generally large proteins that are highly specific and cannot
penetrate
the plasma
membrane2
• Cannot penetrate through the
• Penetrate
through
the plasma
membrane
membrane
• Large molecules, such as monoclonal antibodies,plasma
help mediate
antibodydependent cell-mediated cytotoxicity (ADCC), wherein immune effector cells are
• May
elicit
response:
• Cannot
elicit ADCC
2,3
recruited
to—andimmune
can attack—the antibody-coated
target
cellimmune
ADCC, eg, monoclonal antibodies
response, eg, TKIs
References:
1. Adjei AA, Hidalgo M. Intracellular signal transduction pathway proteins as targets for cancer therapy.
J Clin Oncol. 2005;23:5386-5403.
2. Mellor JD, Brown MP, Irving
HR,surface
Zalcberg JR, Dobrovic A. A critical review of the role of Fc gamma
Cell surface
Cell
receptor polymorphisms in the response to monoclonal antibodies in cancer. J Hematol Oncol.
receptors
receptors
2013;6:1.
3. Boross P, Lohse S, Nederend M, et al. IgA EGFR antibodies mediate tumour killing in vivo.
EMBO Mol Med. 2013;5:1213-1226.
Sos
Sos
PDK1
Grb2 Shc
RAS
PI3K
PDK1
AKT
Raf
PI3K
AKT
RAS
Grb2 Shc
Raf
ADCC=antibody-dependent cell-mediated cytotoxicity; Grb2=growth factor receptor-bound protein 2; MAbs=monoclonal antibodies; PDK1=phosphoinositidedependent kinase-1; PI3K=phosphatidylinositol 3-kinase; Raf=rapidly accelerating fibrosarcoma; Ras=rat sarcoma; Sos=son of sevenless.
 2014
Adjei AA, Hidalgo M. J Clin Oncol. 2005;23:5386-5403.
Genentech USA, Inc. All rights reserved.
5
Bispecific monoclonal antibodies may confer a
Key takeaway
distinct
advantage
over
single-target
agents
• Bispecific antibodies provide a distinct advantage over typical monospecific antibodies by binding 2 epitopes with 1
Notes
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 ligands1,2
‒ 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 compound3
‒ Either multiple sites on a single antigen or single sites on multiple antigens2
1 antibody, 2 epitopes
Fv
2 mechanistic
classes
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 classes2
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
Limitations of single-target agents
References:
• Pathway
crosstalk
1. Chames P, Baty D. Bispecific antibodies
for cancer therapy:
the light at the end of the tunnel? MAbs. 2009;1:539-547.
2. Kontermann R. Dual targeting strategies
with bispecific antibodies. MAbs. 2014;4:182-197.
• Redundancy
3. Holmes D. Buy buy bispecific antibodies. Nat Rev Drug Discov. 2011;10:798-800.
EGFR=endothelial growth factor receptor; Fv=variable fragment; HER2=human epidermal growth
factor receptor 2; HGF=hepatocyte growth factor.
 2014 Genentech USA, Inc. All rights reserved.
7
Notes
Engineering next-generation antibody-drug conjugates
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 Mab1
‒ Optimal stoichiometry of antibody to drug is 1 to 42
• Does not sterically hinder antibody-binding
• Minimizes unconjugated antibody
• MAbs: high specificity1,3
‒ High potency in low quantity due to localization
• Drug conjugate: potent cytotoxicity (IC50=10-9 to 10-11 M [vs first generation ADCs at 10-7 M])4
‒ Toxins that are generally too toxic for administration as a stand-alone agent are well-suited for ADCs3
• 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
Carrier
•
•
Linker
•
Stability in circulation vs
selective
payload release
Linker stability
•
•
•
High specificity
Potent at relatively low
concentrations due to localization
Effective ADCs must balance the delicate interplay between immunoconjugate stability in the plasma during delivery and subsequent selective
payload release2,4,6
Unique chemical properties of the linker direct selective release (chemically or enzymatically labile) 3
Linker must be stable in circulation5
Chemical linker motifs
• Disulfides
‒ Alkylation of reduced interchain disulfides2
• Hydrazones7
‒ 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 stability5
• Valine-citrulline and phenylalanine-lysine have shown to possess high serum stability4
‒ Improved antitumor effects
‒ Offer enhanced stability over hydrazones5
Cargo
Chemical linker motifs
•
Potent cytotoxic agent
Second-generation ADCs
Disulfides
Hydrazones
• Refinements in linker
technologies and innovations in Peptides
recombinant MAb design define a new generation of improved ADCs8
References:
R1
S
1.
2.
3.
4.
5.
6.
7.
8.
S
NH
O
Rn+1
Kovtun YV, Goldmacher VS. Cell killing by2antibody-drug conjugates.
Cancer Lett. 2007;255:232-240.
H
Sievers2EL, Senter PD. Antibody-drug conjugates in cancerN
therapy. Annu Rev Med. 2013;64:15-29.
Ducry L, Stump B. Antibody-drug conjugates: linking cytotoxic payloads toNmonoclonal antibodies. Bioconjug Chem. 2010;21:5-13.
Kitson SL, Quinn DJ, Moody TS, Speed D, Watters W, Rozzell D. Antibody-drug conjugates (ADCs)—biotherapeutic bullets. Chem Today. 2013;31:30-36.
Jaracz S, Chen J, Kuznetsova LV, Ojima I. Recent advances in R
tumor-targeting
H anticancer drug conjugates. Bioorg Med Chem. 2005;13:5043-5054.
n
1 drug conjugates
2
Senter PD. Potent antibody
for cancer therapy. Curr Opin Chem Biol. 2009;13:235-244.
Hamann PR. Monoclonal antibody-drug conjugates. Expert Opin Ther Patents. 2005;15:1087-1103.
Zolot RS, Basu S, Million RP. Antibody-drug conjugates. Nat Rev Drug Discov. 2013;12:259-260.
N
R
R
C
R
 2014 Genentech USA, Inc. All rights reserved.
9
Notes
ADC
activation is dependent on internalization
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 uptake
agent into the cytoplasm1,2
Modes
of antibody
• The rate and extent of internalization influence drug uptake3
1. Clathrin-mediated
endocytosis
4
Internalization
ADC binds target
2.• Caveolae-mediated
uptake uptake
There are 3 modes of antibody
‒ Clathrin-mediated endocytosis (antigen-dependent)
3. Macropinocytosis
‒ Caveolae-mediated uptake (antigen-dependent)
‒ Macropinocytosis (independent)
Receptor-ADC
Payload release
Potent cytotoxic
complex is
• Disulfides
agent is released
internalized
‒ Cleaved inside tumor cells through disulfide exchange with an intracellular thiol 1
inside the cell
by cell
‒ Hypoxic state of tumor cells results in upregulation of reductive enzymes and boosts intracellular
glutathione concentrations (micromolar) relative to normal cells2
• 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
cytosol3
• Peptides
Selective payload release
‒ Rapid enzymatic proteolysis in lysosome3,5
Disulfides
Hydrazones
Peptides
References:
Disulfide
exchange
pH-dependent
intracellular
1. Jaracz S, Chen J, Kuznetsova LV, Ojima I. Recent
advances in
tumor-targeting anticancer
drug conjugates. Bioorg MedRapid
Chem. 2005;13:5043-5054.
2. Ducry L, Stump B. Antibody-drug conjugates: linking
payloads
to monoclonal
antibodies. Bioconjug
Chem. 2010;21:5-13.
withcytotoxic
cytosolic
thiols
hydrolysis
in acidic
proteolysis
3. Sievers EL, Senter PD. Antibody-drug conjugates in cancer therapy. Annu Rev Med.lumen
2013;64:15-29.
of lysosome
4. Kovtun YV, Goldmacher VS. Cell killing by antibody-drug conjugates. Cancer Lett. 2007;255:232-240.
5. Hamann PR. Monoclonal antibody-drug conjugates. Expert Opin Ther Patents. 2005;15:1087-1103.
ADCs=antibody-drug conjugates.
 2014 Genentech USA, Inc. All rights reserved. 11