adc analyte diversity and appropriate pk assays part ii ...€¦ · dar 2 1.480e5 dar 1 in vitro...

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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


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Approval (withdrawn 2010)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


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withdrawn)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



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


:

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


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



Case Studies


15


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



Case Studies


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


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

adc analyte diversity and appropriate pk assays part ii ...€¦ · dar 2 1.480e5 dar 1 in vitro...

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