Discovery of Novel Linker Payloads and

Antibody Drug Conjugates for the Treatment of

Cancer

Christopher J. O’Donnell, Ph.D.

Executive Director, Oncology Medicinal Chemistry, WWMC

Presentation Outline

• Brief History on recent payload discovery efforts at Pfizer

• Discovery of novel linker technology to enable Site-Specific

Conjugation

• Discovery of novel CXI DNA Damaging Payloads, Linker

Payloads and ADCs

2

Discovery of PF-06380101 and its use as a

Payload for ADC Discovery at Pfizer

3

• Maderna, A. et al. J. Med.

Chem. 2014, 57, 10527.

PF-06380101 is an analog of

dolastatin 10 discovered via a

SBDD approach

“vc-0101” used as LP in

multiple conjugates

undergoing clinical

investigation

• PTK7-vc0101 – Damelin et.

al. 2016 AACR

• Notch3-vc0101 – Ken

Geles – 3:00 pm

• NG-Her2-vc0101 – Puja

Sapra et. al. 2016 AACR

• HP Gerber – 6:00 pm

• Trop2 ADC (AcLys linker)

Discovery of Novel Tubulysin Analogs and their use as

ADC payloads – New Insights on Metabolism

4

• Leverett, C. A. et al. ACS Med. Chem Lett. 2016, in press,

DOI: 10.1021/acsmedchemlett.6b00274

• Tumey, L. N. et al. ACS Med. Chem. Lett. 2016, in press, DOI:

10.1021/acsmedchemlett.6b00195

Discovery of Thialanostatin analogs and use as

ADC payloads

5

Burkholderia sp.

FERM BP-3421

1. Sequence genome

2. ID biosynthetic gene cluster

3. Engineer oxido-reductase genes

4. Bioprocess development

N87 (+++) IC50 = 0.44 nM

BT474 (+++) IC50 = 1.02 nM

DYT2 (++) IC50 = 1.4 nM

MB-468 (-) IC50 >1000 nM

0 20 40 60 80 1000

500

1000

1500

Amide_Spliceo-1389 3 mpk

Amide_Spliceo-1389 1 mpk

Amide_Spliceo-1389 0.3 mpk

Vehicle

mcMMAF 3 mpk

Days post-randomization

Tu

mo

r v

ol

(mm

3)

Vehicle

0.3 mpk Spliceo P4 ADC

1.0 mpk Spliceo P4 ADC

3.0 mpk Spliceo P4 ADC

3.0 mpk Comparitor ADC

Gastric Cancer Xenograft

min10 11 12 13 14 157 8 9

2.5 gram/L

Cheng, Y.-Q., et al. J. Nat. Prod., 2013, 76, 685

He, H., et al. J. Nat. Prod., 2014, 77, 1864

Eustaquio, A.S., et al. PNAS, 2014, 111(33), E3376

Eustaquio, A.S., et al. Metab. Eng. 2016, 33, 67

Puthenveetil, S., et al. Bioconj. Chem. 2016, 27, 1880

Development of Novel Linkers to Enable

Enzyme Mediated Site-Specific Conjugation

6

AcLys-ValCitPABC-Aur0101 (LP used on Trop2 ADC)

Aminocaproyl-ValCitPABC-Aur0101

C16-LC

C16-HC

Payload, Linker and Site all critical in optimizing efficacy and safety of ADCs

Strop, P. et al. Chem. & Biol. 2013, 20, 161

Dorywalska, M. et al. Bioconj. Chem. 2015, 26, 650

Dorywalska, M. et al. Mol. Cancer Ther. 2016, 15, 958

Strop, P. et al. Mol. Cancer Ther. 2016, in press, DOI: 10.1158/1535-7163.MCT-16-0431

Magdalena Dorywalska talk at 2:30

DNA Damaging Agents as ADC Payloads

7

• These payloads effectively kill both proliferating and quiescent cells

• Many are “ultra potent” – picomolar to femtomolar IC50’s

• Clinical ‘success’ has been realized with one drug approval and some ADCs

using these payload classes are in advanced stages of development

CXI History: Extraordinary Potency but Limited

Clinical Efficacy due to Toxicity

8

Natural Products Synthetic Analogs

• Mono alkylators and DNA Cross Linkers

• Alkylate N3 nitrogen of Adenine in AT rich regions

• Limited Clinical Efficacy due to Toxicity

CXI’s: Design Approach

9

Our Approach

Payload SAR: Discovery of Novel CBI Dimers

10

HEL IC50 (nM) MDR+

HL

-60

IC

50

(nM

) M

DR

-

Linker Payload Design: A Controlled Chemical and

Enzymatic Conversion Into The Final Active Payload

11

T1/2:

0.5 h (buffer pH 7)

10 h (buffer pH 5)

T1/2 > 4h (buffer), < 1-60 min (mouse plasma)

Not observed in mouse plasma

-CO2

Total Synthesis of the Linker Payload

12

9 steps

9 steps

13 steps

32 steps total

Longest linear sequence – 19 steps

0.002% Overall yield

13

R = acetate, cSFLogD=3.89

CBI payloads more lipophilic than Auristatins

R = phosphate, cSFLogD=1.10

110374 1389 1001:10_UV1_280nm 110374 1389 1001:10_Logbook

0

500

1000

1500

2000

2500

3000

3500

mAU

0.0 5.0 10.0 15.0 20.0 25.0 ml

Aggregate:Poorly loaded ADC

Monomer:Unmodified Ab

Monomer:loaded ADC

Conventional Cysteine Conjugations of CBI LPs & The Importance of Linker Payload Properties

No isolation of desired well-loaded and monomeric ADC

mAb = Anti-CD33

Analytical SEC

Isolation of desired well-loaded and monomeric ADC in 65-70% overall yield )

Precipitate observed (even w/20% organic)

CBI Thiophene Phosphate ADC – Potent,

Selective & Active in MDR+ and SOC Res Models

14

ID mAb HL-60 NB4 HEL

(MDR+)

TF-1

(MDR+)

Raji (neg.

control)

CBI thiophene

ADC

11A1-WT 0.23 0.27 0.09 0.40 1341.34

AcBut Calich

ADC

11-A1-WT 2.68 4.25 >1000 >1000 392.28

IC50 [ng/ml]

Mylotarg

0.1 1 10 100 1000

50

100

NB4-Parental NB4-CYTAR-R

ng/ml

Perc

en

t C

on

tro

l

ADC 3030 (KC183-ValAla)

0.1 1 10 100 1000

50

100

NB4-Parental NB4-CYTAR-R

ng/ml

Perc

en

t C

on

tro

l

SOC-Resistant Models (not MDR1)

CBI Thiophene ADC

DAR ~ 4

AcBut-Calich ADC

Vehicle

CBI Thiophene ADC, 1 mpk

CBI Thiophene ADC, 1 mpk

CBI Thiophene ADC, 3 mpk

Mylotarg, 2 mpk

Mylotarg, 2 mpk

CBI Thiophene DAR 4 Conventional: Active in

both MDR- and MDR+ in vivo models

15

H L 6 0 (M D R -)

0 1 0 2 0 3 0

0

1 0 0 0

2 0 0 0

3 0 0 0

D a y s

Tu

mo

r v

olu

me

(m

m3

)

V e h ic le

A D C 3 2 2 1 ( th io p h e n e C B I) , 1 m p k

A D C 3 2 2 1 ( th io p h e n e C B I) , 3 m p k

M y lo ta r g (2 m p k )

But why does it

require ‘higher’ then

expected doses for

activity?

Vehicle

CBI Thiophene ADC, 1 mpk

CBI Thiophene ADC, 3 mpk

Mylotarg, 2 mpk

Payload Instability in Mouse Plasma

16

Inactive metabolites

Active metabolite

After 4h in plasma, all active payload equivalents have decomposed

17

ADC Model For In Vivo Efficacy: Three Confirmed

MOA’s To Deliver Payloads To The Tumor Tissue

MOA 1: Drug remains in cell: Only receptor expressing cells are killed

MOA 2: Drug escapes into extracellular space: Initial selective cell kill

followed by collateral damage

MOA 3: Drug cleaves extracellulary, collateral damage in tumor tissue

Standard cell proliferation assay only represents MOA 1.

But: In vivo efficacy is sum of all three MOA’s (ADC’s with cleavable linkers).

1

2

3

1

2

1

Significant hydrolase activity in

extracellular space.

Use plasma stability assay as a

surrogate to screen for payload

Instability.

AACR 2014 #4837: Extracellular proteolytic cleavage of peptide-linked

antibody-drug conjugates promotes bystander killing of cancer cells

The design objective: stabilize the amide bonds

between the two warheads

18

Replacing the aromatic amides with various carbocyclic spacers without compromising potency.

The increase in sterical bulk around the amide bonds was sought to reduce affinity of the

compounds to the active site of the plasma-hydrolases.

Bicyclo[1.1.1]pentane as

a phenyl ring isostere:

Stepan, A.F. et. al. J. Med.

Chem. 2012, 55, 3414

The bicyclo[1.1.1]pentane spacer has improved

plasma stability – but ADC is less potent

19

Payload Mouse Plasma Stability

• 50x less potent in vitro

• But only 5x less potent

in vivo

RT: 3.96 - 7.06

4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0

Time (min)

0

50000

100000

150000

200000

uA

U

0

50000

100000

150000

uA

U

0

50000

100000

uA

U

0

50000

100000

150000

uA

U

0

50000

100000

150000

200000

uA

U

6.15

6.62

6.28

6.62

6.72

6.29

5.61

6.62

5.72 6.485.49

5.59

6.31

6.625.715.48 6.444.53

5.58

6.614.52 6.344.86 5.484.17

NL:2.36E5

Channel A UV 2013-11-6_PF-06757725_30uM_Stock

NL:1.85E5

Channel A UV 2013-11-7_pf-06757725_phosphateb_30um_t1min

NL:1.41E5

Channel A UV 2013-11-7_pf-06757725_phosphateb_30um_t30min

NL:1.59E5

Channel A UV 2013-11-7_pf-06757725_phosphateb_30um_t1h

NL:2.10E5

Channel A UV 2013-11-7_pf-06757725_phosphateb_30um_t4h

Parent30 uM DMSO Stock: Parent

T = 1 min

T = 1h

T = 4h

T = 30 min

Parent

RT: 3.96 - 7.06

4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0

Time (min)

0

50000

100000

150000

200000

uA

U

0

50000

100000

150000

uA

U

0

50000

100000

uA

U

0

50000

100000

150000

uA

U

0

50000

100000

150000

200000

uA

U

6.15

6.62

6.28

6.62

6.72

6.29

5.61

6.62

5.72 6.485.49

5.59

6.31

6.625.715.48 6.444.53

5.58

6.614.52 6.344.86 5.484.17

NL:2.36E5

Channel A UV 2013-11-6_PF-06757725_30uM_Stock

NL:1.85E5

Channel A UV 2013-11-7_pf-06757725_phosphateb_30um_t1min

NL:1.41E5

Channel A UV 2013-11-7_pf-06757725_phosphateb_30um_t30min

NL:1.59E5

Channel A UV 2013-11-7_pf-06757725_phosphateb_30um_t1h

NL:2.10E5

Channel A UV 2013-11-7_pf-06757725_phosphateb_30um_t4h

Parent30 uM DMSO Stock: Parent

T = 1 min

T = 1h

T = 4h

T = 30 min

Parent

Swap CBI core for CPI to improve potency

20

Inspired by:

CPI Dimers Induce Inter-strand DNA

Crosslinking

0 60 180 540 nM Active form

Double

Strand DNA

Single

Strand DNA

ds

DNA

Linearized Plasmid

DNA

DNA CROSSLINKING AGENT

+

21

*By analogy with selectivities of bizelesin bis-alkylation of DNAThompson, A.S. and Hurley, L. H. J. Mol. Biol. 1995, 252, 86.

Demonstration of crosslinking ability in vitro:

CPI Dimer DNA Binding Sites and Sequence

Selectivity

5’ –TTTTAAATTAA

Primarily binding to A

Preferential binding sequence

for the CPI dimer

5’ -TTTAAATCAA

Less preferential binding sequence

C C T T T T A A A T T A A A A A T G

22

Binding Mode – DNA Minor Groove

23Thompson, A.S.; Hurley, L.H. J. Mol. Biol. 1995, 252, 86

PDB ID = 226D

Recap on the Evolution of the CPI-1.1.1 Dimer

ADC

24

CBI-Thiophene SeriesPoor payload plasma stability

(labile amide bonds)PK also indicates loss of LP from ADC

CBI-1.1.1-bicyclopentaneImproved payload plasma stability

(improved amide stability with 1.1.1-spacer)

But significantly less potent (~50-60x in vitro, ~5x in vivo)

CPI-1.1.1-bicyclopentaneRetained payload plasma stability(with CPI series and 1.1.1-spacer)

Comparable potency in vivo vs. thiophene

But…..

Conventional CPI

DAR 3.4 PK

0 4 8 9 6 1 4 4 1 9 2 2 4 0 2 8 8 3 3 6

0 .1

1

1 0

1 0 0

T im e (h r )

Co

nc

en

tra

tio

n (

ug

/mL

)

cNOAEL 3 mpk

Switch Linker to enable Site-Specific Conjugate

to Improve ADC PK

25

Certain site specifics displayed vastly improved stability in vitro (plasma stability)

This also resulted in improved in vivo stability (and tolerability)

But did this translate to good efficacy?

cNOAEL 3 mpk cNOAEL >10 mpk

Conventional Cys CPI

DAR 3.4TGase-AcLys CPI

DAR 1.8

0 4 8 9 6 1 4 4 1 9 2 2 4 0 2 8 8 3 3 6

0 .1

1

1 0

1 0 0

T im e (h r )

Co

nc

en

tra

tio

n (

ug

/mL

)

0 4 8 9 6 1 4 4 1 9 2 2 4 0 2 8 8 3 3 6

0 .1

1

1 0

1 0 0

T im e (h r)

Co

nc

en

tra

tio

n (

ug

/mL

)

Amine Handle for

SS TG based conj

DAR 2 Site-specific Conjugates are efficacious

in both MDR- and MDR+ in vivo models

26

H L 6 0 (M D R - )

C D 3 3 -H 1 6 -C P I

0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

D a y s

Tu

mo

r v

olu

me

(m

m3)

M y lo ta rg A D C

C D 3 3 -H 1 6 A D C

C o n tro l A D C

0 .3

0 .6

1 m p k

1 m p k

T F -1 (M D R + )

C D 3 3 -H 1 6 C P I

0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

D a y s

Tu

mo

r v

olu

me

(m

m3)

1m pk

0 .3

0 .6 , 1m pk

M y lo ta rg A D C

C D 3 3 -H 1 6 A D C

C o n tro l A D C

Site Specific DAR 2 CPI ADC efficacious in both MDR - and MDR +

models – at lower doses than DAR 4 Conventional conjugate

More potent than conventional DAR 4 conjugate bearing the same LP

used in Mylotarg in MDR+ model

TI = [NOAEL] / [TSC] = >7 (single dose rat study)

Pfizer Payloads/Linker Payloads

27

• Multiple LP with both cleavable and non-cleavable linkers with each payload class

• Allows us to match the right LP with the right antibody depending on target

Overview of Pfizer’s ADC Technology Licensing

Strategy

• Pfizer has invested heavily in ADC technology for the past 10

years to build up world class ADC technology and expertise

• We are now in a position to offer this technology and

expertise to select external partners on a target exclusive

basis

– Biology of the partner’s targets/mAbs will be a critical factor for

licensing consideration

– Partner’s development capabilities, preclinical and clinical, will

also be a critical factor for licensing consideration

• Pfizer’s preferred partnering structure

– We wish to participate in successful program development at

some predetermined option point

– Pfizer has worldwide regulatory, clinical development and

commercial capabilities that can drive broad commercial success

• Contact Denis Patrick – denis.patrick@pfizer.com28

Pfizer’s Cross-Functional Team Enables

Scientific Innovation in ADC Development

Oncology Research Unit

World Wide Medicinal Chemistry

Global Biotherapeutics Technology

Pharmacokinetics Disposition and Metabolism

Drug Safety

Rinat

Pharmaceutical Sciences

Center of Therapeutic Innovation

External Collaborators

29

TEAMWORK

AcknowledgmentsMany thanks to all of the Dedicated Scientists that Contributed

Global Biotherapeutics

Technology

Lioudmila Tchistiakova

Kim Marquette

Madan Katragadda

Will Somers

Eric Bennett

Pharmacokinetics Drug

Metabolism

Steve Hansel

Frank Barletta

Mauricio Leal

Xiaogang Han

Rinat

Pavel Strop

Drug Safety

Martin Finkelstein

Bob Veneziale

Hadi Falahatpisheh

Magali Guffroy

Legal

David Rubin

Medicinal Chemistry

Russell Dushin

Edmund Graziani

Frank Koehn

Andreas Maderna

Chakrapani Subramanyam

Sai Chetan Sukuru

Jeff Casavant

Sujiet Puthenveetil

Nathan Tumey

Anokha Ratnayake

Zecheng Chen

Matt Doroski

Alex Porte

Hud Risley

Dahui Zhou

Ken DiRico

Carolyn Leverett

Jesse Teske

Beth Vetelino

Li-Ping Chang

Haiyin He

Alessandra Eustaquio

Kathy Jones

Melissa Wagenaar

Xidong Feng

Rob Maguire

Dave Bernhardson

30

Oncology Research Unit

Hans Peter Gerber

Puja Sapra

Frank Loganzo

Jen Kahler

Brian Kennedy

Sylvia Musto

Xingzhi Tran

My-Hahn Lam

Judy Lucas

Ed Rosfjord

Christine Hosselet

Stephane Thibault

Manoj Charati

Robert Abraham

Pharmaceutical Sciences

Cheryl Hayward

Jeff Sperry

Carlos Martinex

Jarad Van Haitsma

Anne Akin

Mari Stephan

Yingxin Zhang

John Ragan

Huijun Dong

Jennifer Thorn

Steve Max