Discovery of Novel Linker Payloads and Antibody Drug...
Transcript of Discovery of Novel Linker Payloads and Antibody Drug...
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
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• 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
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9 steps
9 steps
13 steps
32 steps total
Longest linear sequence – 19 steps
0.002% Overall yield
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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
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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
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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
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Inactive metabolites
Active metabolite
After 4h in plasma, all active payload equivalents have decomposed
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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
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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
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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 – [email protected]
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