This document provides an outline of a presentation and is incomplete without the accompanying oral commentary and discussion. Conclusions and/ or potential strategies contained herein are NOT necessarily endorsed by Pfizer

management. Any implied strategy herein would be subject to management, regulatory and legal review and approval before implementation.

Development and Characterization of

Multi-Platform Antibody Drug Conjugates

Jason A. Starkey

Senior Principal Scientist - BioTherapeutics Pharmaceutical Sciences

Pfizer, Inc.

ADCs Approved and in Clinical

Development

2

2000

2011

2013

Tomorrow...

FDA Approvals

Pfizer

Approved in 2000 for AML

Anti-CD33 conjugated to calicheamicin

Subsequently withdrawn from US in 2010

Available in Japan

Seattle Genetics

Anti-CD30 mAb conjugated with vcMMAE

C&E News 92:3, 13-21(2014)

3

•Antibody:

– Monoclonal antibody (mAb) or related molecule (e.g. Fc fusion protein) specific

to a cell surface tumor antigen/protein

– Abundant target expression and internalization

•Drug:

– Often highly potent small molecule drug/toxin with validated antitumor/cytotoxic

mechanism of action (e.g. microtubule inhibition, DNA damage)

•Linker:

– Tethers the drug/toxin to the antibody

– Stable in plasma, labile upon internalization to release drug

What is an Antibody Drug Conjugate?

3

Clin Cancer Res 17, 6389-6397 (2011)

Antibody-Drug Conjugate Concept

4

Goals

Improve selectivity

Improve efficacy

Decrease systemic toxicity

Improve therapeutic index

Examples of ADC Structures

5

Wu and Senter Nature Biotech. 23:9, 1137-1146 (2005)

(Shaded: bonds lending to drug release)

Common ADC Conjugation Chemistries

6

Cysteine conjugation Lysine conjugation

Site-specific conjugation

Potential impact of new technologies

(e.g site specific conjugation) Controlled drug loading, eliminate mixtures

May improve in pharmaceutical properties

May simplify analytics and development, and

present newer challenges

Junutula, J. R et al Nat Biotechnol, 26, 925-932 (2008)

Strop, P, et al Chem Biol, 20, 161-167 (2013)

Unique Regulatory Space of ADCs

• EMA: EMEA/CHMP/BWP/157653/2007 Guideline on the Development,

Production Characterization and Specifications for Monoclonal Antibodies

and Related Products

• FDA: Points to Consider in the Manufacture and Testing of Monoclonal

Antibody Products for Human Use, 1997

• Unique aspects of ADCs include:

• Characterization of each individual component (mAb, linker, drug)

• Understanding of potency pre- and post-conjugation

• Understanding of product-related impurities pre- and post-conjugation

• Understanding of the amount of free vs conjugated mAb

• Understanding of the amount of free drug and related species

• Number LP attached to mAb and sites of conjugation

7

8

Early Stage Testing Strategy for mAb and ADC

DS/DP - Release/Stability Test Methods

Test Relevant To

Appearance and Description mAb/DS/DP

pH mAb/DS/DP

Protein Content (UV) mAb/DS/DP

Drug loading and drug profile (HIC,

UV, iCE or RP HPLC) DS/DP

Charge profiles1 mAb/DS1/DP1

Identity (peptide mapping) mAb/DS/DP

Purity/Impurity (SEC-HPLC) mAb/DS/DP

Purity/Impurity (CGE or SDS-CE) mAb/DS/DP

Impurity: free drug & related/ free

antibody DS/DP

Biological Activity (Binding ELISA) mAb/DS2/DP2

Biological Activity (Cytotoxicity) DS/DP

Test Relevant To

Safety (Bioburden or Sterility) mAb/DS/DP

Safety (Endotoxin) mAb/DS/DP

Uniformity of Dosage Units DP

Reconstitution Time (for Lyo) DP

Residual Moisture (for Lyo) DP

Particulate Matter (Subvisible

particles) DP

Impurities: Residual Host Cell

Protein, rProtein A (ELISAs) & DNA mAb

N-linked Oligosaccharide profile

(Glycan fingerprint) mAb

Osmolality DS/DP

1 charge profiles may not be as meaningful for Lys conjugates 2 may be replaced by cytotoxicity

Development Considerations - Unconjugated Antibody

• Unconjugated antibody is a recognized attribute that reflects process

consistency capabilities

• Analytical methods for unconjugated antibody have evolved

significantly since the first ADC licensure

– Capillary electrophoresis for charge distribution of lysine-based

conjugates

– HIC for cysteine-based conjugates

– Mass Spectrometry as an orthogonal characterization technique

9

0%

5%

10%

15%

20%

25%

0 1 2 3 4 5 6 7 8

Drug Load

0%

5%

10%

15%

20%

25%

0 1 2 3 4 5 6 7 8

Drug Load

10

Drug Load Profile by HIC

- Cysteine Conjugates

DL0

DL1

DL2

Cysteine Chemistry

Site Specific Conjugation

HIC is a powerful tool for

determining drug load profiles in

ADCs

AU

0.00

0.20

0.40

0.60

0.80

Minutes

6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

DL0

DL2

DL4a DL4b

DL6

Drug Load Profile by HIC

- Lysine Conjugates

11

low

co

nju

ga

ted

fracti

on

s

Lysine Chemistry – Heterogenous conjugation at various

sites and not in sets, like cysteine conjugation

Absorb

ance

0.00

0.05

0.10

0.15

pI

6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80

Absorb

ance

0.00

0.02

0.04

0.06

0.08

0.10

0.12

pI

6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80

Characterization of Drug Load Distribution

- Lysine Conjugation HIC and iCE

Abs

orba

nce

0.00

0.05

0.10

0.15

0.20

0.25

pI

6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80

Absorb

ance

0.000

0.010

0.020

0.030

0.040

0.050

pI

6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80

Absorb

ance

0.000

0.010

0.020

0.030

0.040

0.050

pI

6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80

D5

D7

D8

D4

iCE ADC + mAb 7.2

77

7.3

70

7.4

55

7.5

58

7.6

52

7.7

70

7.8

92

8.0

19

8.1

55

8.3

28

8.4

72

Absorb

ance

-0.010

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

0.080

0.090

0.100

0.110

0.120

pI

6.80 6.90 7.00 7.10 7.20 7.30 7.40 7.50 7.60 7.70 7.80 7.90 8.00 8.10 8.20 8.30 8.40 8.50 8.60 8.70 8.80

D7 iCE D5

D4

6

D0

D4

D5

D8

D7HIC

Cytotoxicity Assay for ADC Drug Substance and

Drug Product

13

Assay format

mAb target antigen

ADC Tumor

cell

tubulin

Payload inhibits tubulin

polymerization, inhibiting

cell division and results

in decreased ATP levels.

payload

Plate tumor cells in assay plate

Serially dilute ADC in dilution plate

Transfer from dilution plate to assay plate

Incubate ~72 hrs in 37 C in 5% CO2 incubator

Add Cell Titer-Glo®, and lyse cells.

Cell Titer-Glo® generates a luminescent response

directly proportional to the amount of ATP present.

ADC concentration

Cell number

ATP

Luminescence

ADC concentration

Cell number

ATP

Luminescence

Representative Standard Curve

Qualification parameters evaluated:

Plate bias

Precision

Accuracy

Range

Linearity

Specificity/selectivity

14

Specification ranges are initially established based on bioassay

variability and not process variability.

Cytotoxicity assay is one of many analytical release and/or

characterization tools used to define a product.

Understand clinical dosing escalation plans in order to determine what

range cytotoxicity specification needs to be within.

• Initially set assay specification wide with tighter limits to allow

investigation into out-of-limit results and further understand

assay variability

• Based on assay and process variability, design assay to

support tighter specification if able

• Clinical development timelines and assay development

timelines are challenging to align.

Requires early discussions on clinical plan, early start of cytotoxicity

assay development & more resources to develop and characterize

assay.

Cytotoxicity Assay Specifications Strategy

Characterization Strategy for mAb and ADC DS/DP “Roadmap” for ADCs in Early Development

15

"initial characterization

and process

development support:

MS analyses:

Intact protein mass

Subunit/domain isoforms

Peptide map sites

Biochemical analyses:

Drug loading profile (HIC)

Drug-mAb ratio (DAR)

Free drug

Size heterogeneity

Charge heterogeneity

N-glycan profiling (for

mAb DSI only)

Reference Material

Characterization

MS analyses:

Intact protein mass

Subunit/domain isoforms

Peptide map sites

Biochemical analyses:

Drug loading profile (HIC)

Drug-mAb ratio (DAR)

Free drug

Size heterogeneity

Charge heterogeneity

Free sulfhydryl analysis

Biophysical analyses:

2º and 3º structure

Summary of LC/MS – Subunit Analyses for ADCs Comparison of Characterization Methods

AU

0.00

0.50

1.00

AU

0.00

0.20

AU

0.00

0.20

AU

0.00

0.20

AU

0.00

0.20

AU

0.00

0.20

Minutes

10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00

AU

0.0

0.1

0.2

0.3

0.4

AU

0.0

0.1

0.2

0.3

0.4

Minutes

15.0 20.0 25.0 30.0 35.0 40.0

scFc L Chain Fd’

scFc

L Chain

L Chain(1)

Fd’

Fd’(1)

Fd

’(1

)

Fd

’(2

)

Fd

’(2

)

Fd

’(3

)

mAb

ADC

L Chain

H Chain

XXXX

4/5

3/4

3 2/3

2

1

0

ADC

Site-specific Chemistry:

1. ADC is IdeS digested & reduced

2. Fc region is essentially fully

conjugated via quantitative

chromatographic shift

Cys Chemistry:

1. ADC is IdeS digested & reduced

2. Chromatographic profile shows

extent of conjugation in L chain

and Fd’

Lys Chemistry:

1. ADC is reduced & alkylated

2. Acidic pH quantitatively cleaves

hydrazone bond in linker

3. H chain is fully conjugated at known

Fc sites; trace amts. in L chain

pQ-Fd’LC

scFc D/P+vc0101

scFc+vc0101

scFc LCpQ-Fd’

scFc D/P

Fd’

Fd’

mAb

ADC

LC- H20

0

100

200

300

Intens.

[mAU]

0

100

200

300

Intens.

[mAU]

10 12 14 16 18 20 22 24 26 Time [min]

mAb

ADC

17

Identify the sites of conjugation

Qualitatively determine the relative abundance of conjugation at each site

mAb-Calicheamicin

mAb

= conjugated peptide

LC/MS – Peptide Mapping (Reduced / Alkylated / Lys-C)

Identify Lys Conjugation Sites, Molecular Integrity & Conjugation Extent

LC/MS – Peptide Mapping (Reduced / Alkylated / Lys-C)

Identify Cys Conjugation Sites, Molecular Integrity & Conjugation Extent

18

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

Minutes

60.0 70.0 80.0 90.0 100.0 110.0

H12 (

1)

L15

(1)

H13H

14 (

2)

H13H

14 (

1)

H13H

14 (

1)

mAb

Cys Chemistry ADC

H2

7

H1

5

H2

8

L7

H17H18 (N-

glycan), H2

L8

H1

H13H

14

H4

L

5

H7

H8

H7

H2

7D

H1

+V

HS

H1

5O

x

H1

3H

14

G2

38

R

H13H

14

L15: SFNRGEC H12: SCDK H13H14: THTCPPCPAPELLGGPSVFLFPPKPK

All peptide mass measurements are less than 2 ppm.

Numbers in ( ) indicate number of linker-payloads conjugated

Summary

• Pfizer has significant experience with various Antibody Drug

Conjugate (ADC) technologies, and a portfolio that has evolved

over the years

• Analytics are a key component to the advancement of an ADC

development program. The evolution over time of analytical

procedures, approaches, and technologies has greatly

increased our capabilities

• Our ability to better characterize ADCs enhances our capability

to effectively design compounds and develop robust

manufacturing processes

19

Acknowledgements

Pfizer BioTherapeutics Pharm Sci ARD

• Rob Morrison, Heyi Li, April Xu, Debbie Meyer, Jeff Borgmeyer, Jim Mo,

Meg Ruesch, Jason Rouse, Tom Porter, Marta Czupryn, Ned Mozier,

Steve Max, Olga Friese, Laura Bass

Pfizer GCMC

• Leslie Bloom, Jackie Moxham, Kristin Murray, Stephanie Pluschkell,

Mark Tardie, Ann Subashi, Tish Webber

Pfizer PPMT

• Steve Max

Pfizer Oncology Research Unit

• Puja Supra

20