Download Current Approaches for ADME Characterization of

2016 IQ Webinar Series Presents:
Current Approaches for
ADME Characterization of
Antibody-Drug Conjugates
Sponsored by the IQ Drug Metabolism Leadership Group
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Current Approaches for ADME Characterization of
Antibody-Drug Conjugates
IQ- ADC ADME Working Group
Feb 19, 2016
Team members:
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Eugenia Kraynov (Team Lead) – Pfizer
Amrita Kamath – Genentech
Markus Walles – Novartis
Edit Tarcsa – Abbvie
Nagendra Chemuturi – Seattle Genetics
Antoine Deslandes – Sanofi
Ramaswamy Iyer – Bristol-Myers Squibb
Amita Datta-Mannan – Eli Lilly
Dan Rock – Amgen
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Priya Sriraman – Celgene
Michaela Bairlein – Bayer
Johnny Yang – Takeda
Matthew Barfield – GlaxoSmithKline
Guangqing Xiao – Biogen
Enrique Escandon – Merck
Weirong Wang – Jansen
David Moore – Roche
Current Approaches for ADME Characterization of Antibody-Drug Conjugates: An
Industry White Paper. Kraynov et al, Drug Metabolism & Disposition, December 2015
Outline
• Overview of ADC PK
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Nomenclature/Definitions
Mechanisms of disposition
Bioanalytical considerations
Factors impacting PK of ADCs
• Currents approaches to characterize ADC ADME
– In vitro and In vivo studies
– Novel vs. previously used payloads
• Summary and Overall recommendations
Current Approaches for ADME Characterization of Antibody-Drug Conjugates: An
Industry White Paper. Kraynov et al, Drug Metabolism & Disposition, December 2015
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ADC Components
ANTIBODY
• mAbs
• ThioMAbs
LINKER
• Cleavable
• Peptide
• Disulfide
• Acid-labile
• Non-Cleavable
• Thioether
DRUG
‘payload’, ‘warhead’, ‘toxin’
• Tubulin polymerization inhibitors
• maytansines (DM1, DM4)
• auristatins (MMAE, MMAF)
• DNA damaging agents
• calicheamicin, duocarmycin
• doxorubicin
DAR: Drug Antibody Ratio, i.e., number of drugs per antibody
ADC Antibody Formats
ADC: Conjugation through
Lysine residues
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ADC: Conjugation through reduced
inter-chain disulfide bonds
DAR
DAR
Thiomab™; TDC: Conjugation through
engineered cysteine residues
DAR
DAR = Drug-Antibody Ratio
Panowski, et al. mAbs 2014
ADC: Mechanism of Disposition
Receptor-mediated Endocytosis
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Non-Specific
Pinocytosis
Endosome
Endosome
Drug Release in Lysosome
Linker Cleavage
Metabolism of
released drug
Metabolite of released
drug in systemic
circulation or bile
(from hepatocyte)
Lysosome
Drug release by
linker proteolysis
or whole ADC
catabolism
Catabolism
Unconjugated drug in systemic
circulation, extracellular space, or bile
(from hepatocyte)
PK of ADC: What to Measure?
Heterogeneous mixture of different DARs
Additional complexity generated in vivo
Unconjugated
drug
DAR 2
DAR 1
DAR 0
Conjugated antibody/ Antibody-conjugated Drug
Total Antibody
 Multiple bioanalytical assays (ELISA, LC/MS)
Kaur et al, Bioanalysis, 2013; Kamath & Iyer, Pharm Res, 2015; Xu et al, Anal Biochem 2011
Affinity-Capture LC/MS (Exploratory):
Stability & Biotransformation
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Impact of ADC components on PK
Antibody
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Linker
Solid line = Total antibody
Dotted Line = Conjugated antibody
Naked antibody
Total antibody
after dosing ADC
Mouse
Lin & Tibbitts, Pharm Res (2012)
Closed symbols= Total antibody
Open symbols = Conjugated antibody
Drug Load (DAR)
LC, HC, Fc Tabs
LC-TDC
HC-TDC
Mouse
Erickson et al, Mol Cancer Ther (2012)
Fc-TDC
Tab Concs (μg/mL)
Conjugation Site
T-SMCC-DM1 had
better efficacy &
toleratibility than
T-SPP-DM1
Total antibody analyte
Unconjugated Ab
DAR 2
DAR 4
Mouse
DAR 8
Time (Days)
Higher DAR species associated with
faster clearance & increased toxicity
Efficacy: LC-V205C > HC-A114C > Fc-S396C
Shen et al, Nat Biotech (2012)
Hamblett et al, Clin Can Res (2004)
Example of Impact of Linker Type on Catabolite Profile
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• Different intracellular processing of toxin/linker constructs and different metabolites result in
different bystander killing activity
Reducible/Cleavable
Non reducible/Non-cleavable
No bystander effect
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bystander effect
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(100,000X lysine–Ne-MCC-DM1)
SPDB linker was processed into
lipophilic metabolites (which had
bystander effects)
Erickson et al. Cancer Res (2006)
Erickson et al, Bioconj Chem (2010)
ADC PK/ADME: Key Assessments
Types of Assessments
• Linker stability
– What is optimal stability for efficacy/toxicity?
– Should be stable in circulation, but promptly
release the drug in the target cells
ADC Stability
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In Vitro Study
– Plasma or Serum from human and efficacy & tox species
– Incubation conditions
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What is released? Is it active?
Does it accumulate in tissues/tumor?
How it is eliminated?
Is it clinically relevant?
In Vivo PK
Choice of animal species for ADC, same general principles as mAb
• Ideal if cross-reactive in animal species (i.e., binding species)
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• In vivo exposure (efficacy & tox)
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Exposure-response analysis
What is the driver of efficacy/toxicity?
Which analyte correlates with activity?
Plasma conc? Or tissue concs?
• Other assessments
– Drug-drug interactions
– Immunogenicity
37ºC at pH 7.4 for 96 hours
ADC concentration around observed/predicted Cmax in
animal species or human
– Typical analytes: Tab, conjugate, released drug, DAR
– Can help optimize the combination of mAb, linker & drug
• Catabolite/Metabolite ID
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If not cross-reactive, PK & toxicity may not be reflective of humans.
May still provide some information on non-specific disposition of
ADCs and on potential drug-related metabolites
• Important to choose species that has similar in vivo fate/
deconjugation mechanism as in humans
• PK characterization at doses low enough to evaluate target
mediated clearance and high enough to understand toxicokinetics
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Typical PK analytes: Tab, conjugate, released drug, DAR
Is there a single in vitro system that can be used for
characterization of both ADC and drug?
Hepatocytes
Liver microsomes
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Contain all relevant microsomal and cytosolic
enzymes
Target protein is not expressed
Drug may have limited permeability.
Cancer cells
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Selection of cell line would depends on target
expression,
Limited by drug permeability
Drug metabolizing enzymes expressed by
cancer cells are found in the live
Have been shown to up-regulate Phase II
enzymes and down regulate Phase I enzymes
as compared to the liver
Lysosomes
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Mimic ADC degradation in the cell
Artificial system which does not contain drugmetabolizing enzymes
Uptake of ADC might be limited
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Contain most relevant drug metabolizing enzymes
Not confounded by drug permeability or uptake
Lack the lysosomal enzymes responsible for
release of drug from ADC molecule
Plasma
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Contains proteases
Liver S9 fraction
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Contains the same drug metabolizing enzymes
as hepatocytes.
Does not rely on drug permeability
Transporter independent
Can be used at either pH 7.4 (to study
metabolism of the drug) or acidified to mimic
lysosomal degradation of an ADC.
Is there a single in vitro system that can be used for
characterization of both ADC and drug?
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Liver microsomes
Drug’s metabolic
stability, reaction
phenotyping, CYP/UGT
inhibition
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Assessment of linker stability in
the systemic circulation. PPB of
released drug
Plasma
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Contain most relevant drug metabolizing enzymes
Not confounded by drug permeability or uptake
Lack the lysosomal enzymes responsible for
release of drug from ADC molecule
Contains proteases
Liver S9 fraction
Characterization of drugcontaining species released from
an ADC. Identification of
metabolites formed from the drug
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Contains the same drug metabolizing enzymes
as hepatocytes.
Does not rely on drug permeability
Transporter independent
Can be used at either pH 7.4 (to study
metabolism of the drug) or acidified to mimic
lysosomal degradation of an ADC.
In general, it is recommended that understanding of the linker and drug chemical structures
and potential reactions that they can undergo, be taken into consideration when selecting
the in vitro test system and the most straightforward (or simplest) system is used.
Assessment of DDI potential.
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In most cases, systemic concentrations of released drug are extremely low, therefore, risk of ADC being a
DDI perpetrator can be considered minimal.
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In a clinical DDI study, ADCETRIS® (vc-MMAE ADC) did not affect the PK of midazolam.
Probability of released drug to be a DDI victim exists and impact can be high due to the drug’s narrow
therapeutic margin.
Han & Zhao, DMD , 2014
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No profound changes in clinical PK of ADCETRIS® was observed when coadministered with rifampicin or
ketoconazole. However, exposure of released MMAE was reduced by ~46 % and increased by ~34 % by
coadministration of rifampicin and ketoconazole, respectively.
Kadcyla® (DM1-containing ADC) label contains a caution that coadministration with strong CYP3A4
inhibitors should be avoided due to the potential for an increase in DM1 exposure and toxicity.
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DDI risk assessment for a novel drug used in an ADC needs to be performed during development to
determine if formal clinical studies should be conducted in accordance with the FDA and EMA
guidelines.
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Studies to assess transporter-mediated DDI may be valuable at later stages of the development.
ADC tissue distribution.
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 Typically conducted in rodents (rats and/or tumor bearing mice) to evaluate distribution
to normal tissues (or tumor).
 Radiolabel is applied on the drug (usually C-14 or H-3), or simultaneously on both the
antibody and drug using a dual-labeled ADC with C-14 and H-3.
* Indicates location of the 14C radiolabels
antibody backbone is 3H-radiolabeled.
Alley et. al., JPET, 2009
Tissue distribution study may challenging and may not be appropriate if there no crossreactivity to rodent targets
ADME (mass balance) evaluation.
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 Currently, a human ADME study using radiolabeled material is not recommended.
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For the cytotoxic/genotoxic drugs typically used in oncology ADCs, dosing of ADCs in
healthy volunteers is not appropriate. Evaluation would have to be conducted in cancer
patients.
Due to typically long ADC half-life, patients would have to be sequestered for prolonged
periods of time (3-4 weeks) with little to no benefit to the patient, which would not be ethical.
An ADME study of shorter duration may not be adequate and can result in incomplete mass
balance data.
Identification of the circulating products of further metabolism of the drug may be
challenging due to typically very low concentrations of those products.
 An animal (rodent) ADME study using an ADC with radiolabel on the drug may be
considered.
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Matrices to be collected: serum/plasma, bile, urine, and feces.
Since most of the ADCs do not cross react with rodent targets, this evaluation would
primarily address nonspecific uptake and degradation pathways and may not necessarily
represent the disposition of ADC in humans.
Due to the long half-life of ADCs the study duration would need to be extended in order to
achieve good recovery of radioactivity and mass balance.
In vitro and in vivo studies for characterization of
ADC ADME
Molecule
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ADME data
ADC*
In vitro stability in plasma or serum from animals and humans.
ADC*
PK in pharmacology and toxicology species
ADC**
Animal (rodent) ADME: PK, excretion, and metabolism
ADC
Identification of circulating metabolites formed from the released drug in patients
Drug
Rodent PK
Drug
Plasma protein binding across species
Drug
In vitro characterization of metabolites formed from the released drug (safety species and human)
Drug
Reaction phenotyping
Drug
Passive/active (uptake or efflux) transport (as substrate)
Drug
CYP inhibition and induction
* Analytes that could be measured as appropriate include Tab, ADC, unconjugated drug
** This evaluation is recommended to be conducted with an ADC bearing a radiolabel on the drug
What should be done for novel ADCs with previously
characterized drugs?
• Drugs or linker-drugs previously tested in the clinic
– Now conjugated to different mAbs to form new ADCs
– Using novel linker or novel conjugation chemistry
• Existing ADME information usually available
– Published reports or filings
– Internal unpublished data
– Need to only generate key data specific to the novel ADC
• Additional ADME evaluation
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Plasma stability of the ADC
Major released drug-containing species
Major ADC clearance mechanisms
Confirm that projected human PK properties support intended dose and frequency of
administration.
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Conclusions
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 ADME characterization for an ADC is a complex process as it needs
to take into account both the mAb and small molecule components of
this modality.
 No standard “one size fits all” approach can be applied to all ADCs.
 ADC ADME working group has evaluated advantages and
disadvantages of the currently used experimental systems and
strategies, and published white paper which provides guidance that
should help investigators to develop successful novel ADCs with
desirable ADME properties.
 Since ADC technology is still evolving, the working group has
proposed that this area of science is continuously monitored as it
matures over the next several years and, if needed, currently used
approaches are re-evaluated.
Acknowledgements
 Colleagues from IQ member companies for their input
and review of the white paper
 IQ DMLG for recognizing the importance of this topic
and for their guidance and support
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Marcel Hop (Genentech)
Volker Fischer (AbbVie)
Thomayant Prueksaritanont (Merck)
Sekhar Surapaneni (Celgene)
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IQ Webinar Series: Current
Approaches for ADME
Characterization of AntibodyDrug Conjugates
Q&A Session
Audience Q&A submissions
• When testing metabolic pathways of an ADC with non cleavable
linker, what should be tested? Drug or linker-drug? Or both?
• When testing metabolic pathways of an ADC with non cleavable
linker, what should be tested? Drug or linker-drug? Or both?
• what about using Cyno PK for translation into the Clinic?
• what are the advantages of having a smaller DAR ratio
• Amrita, The data you showed for the difference in clearance of
DAR2,4,&8 were only for cysteine conjugated mAbs correct? This
my not bee the same for lysine or other amino acid conjugated ADCs
(with cysteine conjugated mAbs the mAb will have reduced covelent
bonds between LC & HC which may impact the PK in a very differnt
manner than ADCs conjugated through other amino acids.
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Audience Q&A submissions
• In the table, CYP inhibition studies were recommended, although
circulatory concentrations are expected to be very low. How about
transporter inhibition studies?
• Is there any study looked at the impact of different PL on the profile
of ADC?
• Have PBPK modelling been used by industries and accepted by
regulatory to address ADC-DDIs
• Has ADME studies been conducted in tumor bearing animals?
• For the rodent ADME study, would you consider doing them in
transgenic mice expressing the relevant DMEs for the toxin (e.g.,
CYP3A4) also bearing orthotopic human cell-based tumors that
express the relevant target?
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Audience Q&A submissions
• Would you still run DAR assay for Thiomab ADC with 2 bound
payloads?
• Generally at whats stage of development the DDI risk need to be
evaluated?
• is there any update for ADC combinational therapy in immunooncology field?
• For in vitro assays, do you have any additional literature except for
apporved two ADCS?
• For approved ADCs, how have other companies determined DAR?
Specifically, did they perform DAR investigations in the preclinical or
clinical phase? If in clinical phase, were those investigations GLP or
non-GLP?
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Audience Q&A submissions
• Can you comment on the status of LC/MS/MS vs. immunoassay for
measuring ADC and TAb, and what are your recommendations?
• Can you comment on special considerations for ADME assessment
both preclinically and clinically for ADCs targeted for CNS
malignancies? There are now ADCs being evaluated for CNS
malignancies. What are the key attributes that maximize likelihood
of success?
• What is the role of FCgamma receptor binding on toxicity of ADCs?
• What species (ADC only or Total Antibody as well?) would you looks
at as the the predictor variable in clinical exposure-response
analyses of ADCs (for efficacy)?
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Audience Q&A submissions
• Can you comment on the role/ utility of clinical imaging approaches
for assessing biodistribution/ target access in tumors as a
component of POM ahead of Phase 2?
• What approach do you recommend for human PK predictions for
ADCs?
• Is it necessary to repeat clinical CYP3A inhibitor or inducer DDI
studies for new ADCs that have MMAE as a payload or can the
results from brentuximab vedotin be extrapolated
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