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ADC Analyte Diversity and Appropriate PK Assays
Part II: Next Generation ADCs & Challenges
Surinder Kaur, Ph. D.
Director, ADC Programs & MS
BioAnalytical Sciences, Genentech
S. San Francisco, California
European Bioanalysis Forum – ADC Training Day
Bringing ADC into Practice
Defining the Bioanalytical Strategy
20th June, 2017, Lisbon
Overview
Next generation antibody drug conjugates
Novel conjugation formats, payloads and linkers
New bioanalytical challenges and strategies
Case Study highlights
Summary and acknowledgments
S. Kaur, EBF ADC Training, Analyte Diversity Part II, Lisbon, 06.20.17 2
Future Directions of ADCs
S. Kaur, EBF ADC Training, Analyte Diversity Part II, Lisbon, 06.20.17 3
1980sEarly ADCs
2000Mylotarg®
Approval (withdrawn 2010)
2011Adcetris®
Approval
2013Kadcyla®
Approval
More Data
Next Generation
ADCs
Beyond Oncology
Success Required Technology Advances Across Multiple Fields
• Humanized monoclonal antibody production
• Stable chemical linker chemistries
• Cytotoxins with appropriate potency and mechanism of action
• Genomic profiling to identify unique tumor antigens
• Novel hybrid large molecule/small molecule bioanalytical technologies
S. Kaur, EBF ADC Training, Analyte Diversity Part II, Lisbon, 06.20.17 4
Many Players in the ADC Field
Genentech
Three Generations of ADCs
Conjugation Linker Payload Limitations
1st generation
(e.g., Mylotarg®
2000 -2010;
withdrawn)
Random lysines Unstable Low potency; e.g.,
conventional
chemotherapy
Heterogeneity; lack of
efficacy; systemic
toxicity due to
premature drug loss;
highly immunogenic
2nd generation
(e.g., Adcetris®
2011; Kadcyla®
2013)
Random lysines;
reduced interchain
cysteines
Improved
stability;
cleavable vs.
noncleavable
~1000x more potent
than chemo; anti-
microtubule MOA;
only active against
proliferating cells
Heterogeneity; fast
clearance for high
DARs; premature
drug loss; narrow TI;
drug resistance
3rd generation
Site specific
adopted;
engineered cysteins
(e.g., THIOMABTM);
novel constructs
Stable in
circulation; fine-
tuned to match
drug; release
drugs in tumors
Highly potent; also
DNA damaging MOA;
target proliferating &
non-proliferating cells;
against modest target
expression
Possible toxicity due
to highly potent
payloads; catabolism
may be different
across species
Vankemmelbeke, Durrant. Ther. Deliv. (2016) 7, 141; Mack, Ritchie, Sapra. Seminar in Oncology (2014) 41, 637
5
Evolution of ADC Generations
2nd generation ADCs
Panowski et al. mAbs (2014)
~1 kDa ~150 kDa
(MCC) (Herceptin®)
KadcylaTM
AdcetrisTM
M MMAE
maleimide-
derived
C
caproic acid-
derived
mono-methyl auristatin E
vcVal-Cit
PAB
p-amino-
benzyl
3rd generation ADCs
Conjugation
siteLinker Drug
Components: Antibody, conjugation site, linker and payload
S. Kaur, EBF ADC Training, Analyte Diversity Part II, Lisbon, 06.20.17 6
S. Kaur, EBF ADC Training, Analyte Diversity Part II, Lisbon, 06.20.17 7
2nd Gen ADC Challenges in Development:
Conventional Format vc-MMAE ADCs (DAR ~4) Dosing
1Saber, H., Leighton, J.K. An FDA oncology analysis of antibody-drug conjugates. Regul. Toxicol. Pharmacol. (2015), In press.
http:// dx.doi.org/10.1016/j.yrtph.2015.01.0142Junutula, J., Raab, H., Clark, S., et al. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index.
Nat Biotechnol 2008; 26:925-32
Conventional (DAR = 4) vc-MMAE ADCs show dosing challenges1
Relatively narrow therapeutic window: responses and maximum doses close
Next generation ADCs (DAR = 2) have potential to overcome limitations2
S. Kaur, EBF ADC Training, Analyte Diversity Part II, Lisbon, 06.20.17 8
:
Multiple Potential Mechanisms of Toxicity:On-Target and Off-target ADC Toxicity
• Off-target
Toxicity mechanisms not fully
understood
• Both large and small
catabolites could contribute
• Non-specific uptake of small
catabolites?
• Fc or mannose mediated
cellular internalization
• Non-specific antibody mediated
pinocytosis
• On-target
Toxicity can result from
target antigen expression
in normal organs/tissues
and mechanisms better
understood
Okeley et al (2010) Clin Cancer Res;16(3)888-97
Sassoon, Blanc (2013) Methods Mol Biol;1045:1-27
S. Kaur, EBF ADC Training, Analyte Diversity Part II, Lisbon, 06.20.17 9
Opportunities for Next Generation ADCs
Next generation ADCs have lower Drug:Antibody ratio (DAR = 2)
Potential for wider therapeutic window1,2
Drugs with new mechanisms of action (MOAs) for diversity
Combine with other therapeutics for synergistic effects
Combination warheads for dual MOAs
New disease areas: e.g., infectious diseases
1Junutula J, Raab H, Clark S, et al. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index.
Nat Biotechnol 2008; 26:925-322Hamblett KJ, Senter PD, Chace DF, et al. Effects of drug loading on the antitumor activity of a monoclonal antibody drug conjugate.
Clin Cancer Res 2004; 10:7063-70
Genentech
Multiple Changing Parameters for 3rd Generation ADCs
These components influence each other resulting in highly complex 3rd generation ADCs
• Stable in circulation
• Cross species variations?
• Released in tumor
Chemical linker
• Favorable stability
• No impact on target binding
Conjugation Site
• Normal internalization
Antibody
• Good pharmacokinetics
• Favorable biological activity
• Proper target binding
• Cross species reactivity
Cytotoxic drug
• Stable in circulation
• Non-immunogenic
• Highly potent
• Proper MOA
10S. Kaur, EBF ADC Training, Analyte Diversity Part II, Lisbon, 06.20.17
Genentech
Some Current and Emerging ADC Formats
Deonarain et al. Expert Opin Drug Discov. (2015) 10(5), 463
Lysine
Red. disulfide
Engineered cysteine
Glycoengineering
(e.g., sialic acid, alkyne, azide)
Non-natural amino acid
Ab fragments
11
Genentech
Next Generation ADCs Incorporate Novel Technologies
Mack, Ritchie, Sapra. Seminar in Oncology (2014) 41(5), 637
• More homogeneous
• Improved stability &
biologic activity
• Responsive to both
proliferating & non-
proliferating cells
• Targeting multiple
component of tumor
microenvironment
• Highly potent payloads
• Different MOAs; potential
for combination therapy
• Reduce drug resistance
• More selective binding
to tumor cells
• Improved tumor
biodistribution
• Solid tumors
12
Genentech
Example of Novel ADC Constructs : ProbodyTM Drug Conjugate (PDC)
ProbodyTM Drug Conjugate
Desnoyers. 15th Annual PepTalk, 201613
Genentech
• Small in size; fast penetration
in tumors
• “Plug & play” options for novel
multi-specific biologics
• Tunable serum half-life
Examples of Novel Small Drug-Conjugate Constructs
Humabody™ Drug Conjugate (HDC)
http://www.crescendobiologics.com/
Pentarins™ miniaturized biologic
drug conjugate (mBDC)
• Small targeting ligands based
• Quick penetration to solid tumors
• Tunable biodistribution
• Nanoparticle incorporation
http://www.tarveda.com/14
Genentech
Overview
Next (3rd) Generation Antibody drug conjugates
Novel conjugation formats, payloads and linkers
New bioanalytical challenges and strategies
Case Studies
Summary and acknowledgments
15
S. Kaur, EBF ADC Training, Analyte Diversity Part II, Lisbon, 06.20.17 16
Complex Catabolites In Vivo for Some ADCs:Linker cleavage and/or loss of functional groups in drug
Cleavage at labile sites in linker or drug
• Loss of functional group(s) & potency drives what to measure for PK
Partial drug loss
DAR2 or DAR0?
DAR2 or DAR1?
17
Linker cleavage
Complex In Vivo Biotransformation:Need to understand Active Analytes for PK
DAR 1.6
Max. 586.2 cps.
1.450e5 1.455e5 1.460e5 1.465e5 1.470e5 1.475e5 1.480e5 1.485e5 1.490e5Mass, Da
0
50
100
150
200
250
300
350
400
450
500
550
586
Inte
nsity
, cp
s
146580145206145182 145231 145308 148291146595145422 146766 148372146825145503 148456146554145596 148555145674 148639145761 146293145993 148776 148801 148905146022145962 146270
DAR2Linker
deconj.
Linker deconjugation
N
O
O
S
Improvement in stability needed
to enhance drug potency
Linker deconj.
DAR 0.8
1.445e5 1.450e5 1.455e5 1.460e5 1.465e5 1.470e5 1.475e5 1.480e5 1.485e5 1.490e5 1.495e5 1.500e5Mass, Da
144100
Linker deconjugation
N
O
O
S
Linker deconj. +
Partial drug cleavage
partial drug cleavage
Linker cleavage
+ partial drug cleavage
S. Kaur, EBF ADC Training, Analyte Diversity Part II, Lisbon, 06.20.17
18
DAR 2 - ether
DAR 2 – 2 ethersDAR 1 - ether
1.440e5 1.445e5 1.450e5 1.455e5 1.460e5 1.465e5 1.470e5 1.475e5 1.480e5
Mass, Da
5.0
10.0
15.0
20.0
25.0
Inte
nsity,
cps
DAR 2
1.440e5 1.445e5 1.450e5 1.455e5 1.460e5 1.465e5 1.470e5 1.475e5 1.485e5 1.490e5
10
20
30
40
50
Inte
nsity,
cps
DAR 2
1.480e5
DAR 1
In Vitro
(Cyno, 48 h)
In Vivo
(Cyno, Day 1)
Linker – R1
OR2
R1 – Linker
OR2
R1 – Linker
OR2
Linker – R1
OR2
R1 – Linker
OR2 Ether cleavage
Catabolites In Vivo Can be More
Complex than In Vitro
S. Kaur, EBF ADC Training, Analyte Diversity Part II, Lisbon, 06.20.17
Cross Species Differences Can Occur in CatabolitesExample of a TDC More Stable in Cyno than Mouse
Day 0
Day 1
Day 3
Day 0
Day 1
Day 3
Stability: cyno > mouse
19S. Kaur, EBF ADC Training, Analyte Diversity Part II, Lisbon, 06.20.17
Overview
Next (3rd) Generation Antibody drug conjugates
Novel conjugation formats, payloads and linkers
New bioanalytical challenges and strategies
Case Studies
Summary and acknowledgments
20
Integrated Bioanalytical Strategies Needed
for ADC Drug Development
• ADC biotransformation in vivo by affinity capture LC-MS
• Three key pharmacokinetic assays
1) Total antibody by LBA or immunoaffinity LC-MS/MS
2) Antibody-conjugated drug (conjugate) by immunoaffinity LC-MS/MS
or Conjugated antibody by LBA
3) Unconjugated drug by LC-MS/MS
• Catabolite assays in circulation & tissues, as needed
• Immunogenicity assays
Multi-disciplinary bioanalytical team to enable innovation:
S. Kaur, EBF ADC Training, Analyte Diversity Part I, Lisbon, 06.20.17
Kaur et al., Bioanalysis 5 (2) 201-26 (2013)
Gorovits et al., Bioanalysis 5 (9) 997-1006 (2013)
22
1.43e5 1.44e5 1.45e5 1.46e5 1.47e5
Mass
0
50
100
150
200
250
300
350
400
450
Reco
nst
r uct
ed
I nt e
nsi
t y
145935.95
146108.18
146524.21145348.55
146693.36
146858.96
High Resolution MS May Identify Additional Catabolites
0
20
40
60
80
100
Re
l ati
ve
Inte
ns
i ty 145360.09
146534.27146104.95
145521.94
146128.23 146695.31145704.83 146285.59
145945.49
145500 146000 146500
Mass
145548.20
-2LD+Cys+GSH - LD + GSH
- 2LD + 2Cys + Hex
0
20
40
60
80
100R
ela
tive
Inte
nsit
y 145360.09
146534.27
146695.31
145945.49
142000 143000 144000 145000 146000 147000 148000
Mass
- LD + Cys + Hex
Conventional TOF-MS
HR/AM MS
Q-Exactive Plus
DAR2
J. He, D. Su, C. Ng, L. Liu, S. Yu, T. H. Pillow, G. Del Rosario, M. Darwish, B. Lee, R. Ohri, H. Zhou, X. Wang, J. Lu, S. Kaur and K. Xu.
“High-Resolution Accurate-Mass Mass Spectrometry Enabling In-Depth Characterization of in Vivo Biotransformations for Intact
Antibody-Drug Conjugates,” Anal. Chem. 2017, in press
Genentech
Linker-Drug
Enhanced Mass Resolution Needed for Understanding
Biotransformation for 3rd Generation ADCs & PK Analytes
o
o- Ac (i.e., de-activation)
Mass (kDa)
50
100
148.6 148.8 149.0 149.2 149.4 149.6
Glycated
DAR1
DAR0
Intact
HRAM
50
100
DAR0, DAR1
mixtureIntact
Q-TOF
148.6 148.8 149.0 149.2 149.4 149.6
TDC2
(reduced de-acetylation)
TDC1
(de-acetylation)
Vehicle
© 2017, Genentech
• Nonclinical “Plug-and-Play” Hybrid IA-LC-MS/MS
• Generic capture & hu Fc peptide-based analyte
• Clinical Specific Hybrid IA-LC-MS/MS as needed
• Specific capture & CDR peptide-based analyte
• Orthogonal platforms are complementary & help
troubleshoot assay performance issues
y = 1,1404x + 4,7145R² = 0,9829
0
500
1000
1500
2000
2500
3000
3500
0 1000 2000 3000
LC/M
S/M
S, C
on
c. (
ug
/mL)
ELISA, Conc. (ug/mL)
ELISA vs Hybrid LC-MS/MS
ADC Total Antibody PK Assay:Generic Framework Peptide Hybrid LC-MS/MS & ELISA Comparable
Kaur, S.; Xu, K; Saad, O.M; Patent US2013/0315645
Hybrid Binding LC-MS/MS
Magnet
Trypsin
Signature peptide(s)
from Fc region/CDR
FNWYVDGVEVHNAK
y9
Protein A, anti-HuIgG, anti-ID
Resin/Bead
Total Ab ELISA
(DAR insensitive)
Kaur et al., Bioanalysis, (2016), 8 (15), 1565–1577
Jenkins et al., AAPS J. 17(1):1550–7416 (2015)
Genentech
1. Captured by protein A
or xhuIgG Ab
ADC and
endogenous IgGs
2. Linker cleavage/
ADC digestion
3. LC-MS/MS detection of
released drug
Antibody-conjugated drugTotal antibody
Total Ab
Free Drug
acDrug
Co
nce
ntr
atio
n (
nM
)
Time
2. Hu Fc peptides released
1. Captured by protein A
or xhuIgG Ab
3. LC-MS/MS detection
of hu Fc peptides
Unconjugated drug
Inte
ns
ity
Time
Drug
SIL-IS
Bioanalytical Assays in ADC Development for
Nonclinical Studies (LBA or IA LC-MS/MS)
S. Kaur, EBF ADC Training, Analyte Diversity Part II, Lisbon,
06.20.17
25
Genentech
Nonclinical PK Profiles of Total Ab and Ab-conjugated Drug
by IA-LC/MS/MS for a Next Generation ADC
IA LC-MS/MS in studying next gen ADCs
• Generic ac-drug LC-MS/MS does not rely on customized capture reagents
and is more sensitive to measuring drug changes
• Generic total Ab assay (TAB) is readily applicable across ADCs targeting
various antigens and allows better PK comparison
26
Genentech
Hybrid Binding LC-MS Large Molecule Assay Formats
Mixture of TDC DARs in vivo:
(2) Total Antibody (Ab)
(3) Unconjugated Drug
Co
nc
en
tra
tio
n (
nM
)
Time
Total Ab
Unconjugated
Drug acDrug
(1) Antibody-conjugated
Drug (acDrug)
Trypsin Digestion
ADC and
Endogenous IgG
Capture by
Protein A (resin)
LC-MS/MS detection of
signature human Fc peptides
LC-MS/MS detection of released
drug AND human Fc peptidesLinker cleavage
AND Trypsin Digestion
(1) acDrug Assay AND
(2) Total Antibody Assay
2 Analytes –
1 LC-MS/MS assay
Patent Application No. 62/313,608. Multiplexed
Total Antibody and Antibody-Conjugated Drug
Quantification Assay.
Multiplexing 2 of the 3 Key PK Assays : Total Antibody & acDrug
Genentech
Case Study: Multiplexing acDrug and total Ab
Assays in a single assay for disulfide linked TDC
• Linear concentration
dependent reduction/
release of drug
• high r2, % accuracies
within ± 20%
M.V.Lee
ADC (ug/mL) Drug Calc conc (nM)
AbCalc Conc(nM) Calc Avg DAR
1 13.0 6.7 1.9
50 666.0 362.2 1.8
80 1110.0 535.5 2.1
100 1240.0 676.4 1.8
200 2720.0 1184.8 2.3
Avg: 2.0
• Calculated Avg DAR=2.0 & reported Avg DAR (HIC): 1.9
• Expect constant DAR regardless of ADC conc.
Genentech
Summary
• Next generation ADCs employ a variety of novel conjugation platforms, linkers
and payloads to address limitations of earlier platforms
• Increasing diversity and complexity in next generation ADCs present additional
bioanalytical challenges
• Multiple assay formats including LBA and hybrid IA-LCMS/MS and multiplexed
assays are appropriate to assess ADC biotransformations and PK
• The need for innovative bioanalytical strategies continues with the added
challenges of next generation ADCs
29
S. Kaur, EBF ADC Training, Analyte Diversity Part II, Lisbon, 06.20.17 30
BioAnalytical Sciences/ ADC Group
Mass Spectrometry:
Keyang Xu, Luna Liu, Carl Ng, Jintang He,
Dian Su, Ola Saad, Neelima Koppada,
Violet Lee, Sukjoon Hyung
Immunoassays:
Randy Dere, Montse Carrasco,
Helen Davis, Connie Mahood,
Kyu Hong,
Collaborator Groups
Joo-Hee Yi, Kathy Kozak, Sandhya Girish,
Kelly Flagella, Ben Shen, Jagath Junutula,
Amrita Kamath, Flavia Brunstein
Administrative Assistant
George Heckert
Development Sciences Management
An Song, Patty Siguenza, Sara Kenkare
ADC Teams
Acknowledgements