Achieving stable, site-­‐selec1ve conjuga1on to THIOMAB™ an1bodies Jack Sadowsky Genentech, Inc. World ADC 2015 San Diego, California ADCs: much more than drug plus anEbody Connec1on Trigger Drug
Linker
•  Target antigen
•  Cleavable vs. non-cleavable
•  Mechanism of action
•  Antigen copy number
•  Cleavage mechanism
•  Standalone potency
•  Nonspecific uptake
•  Hydrophobicity/-philicity
•  Cell permeability
•  Fc receptor binding
•  Stability
•  MDR/PGP sensitivity
Therapeutic index
Need stability in circulaEon, but release in target cell for high efficacy 1
2
ADC
3
Stable in
circulation
4
5a
5b
Release in
target cell
Sievers, et al., 2013
Two flavors of cleavable linkers thiosuccinimide
peptide
O
O
S
N
O
O
H
N
N
H
O
O
O
N
H
HN
O
NH 2
disulfide
S
S
Drug
N
H
N
Drug
O
O
N
N
O
O
O
Thiosuccinimides can become deconjugated in vivo thiosuccinimide
mAb
O
R
N
S
O
maleimide
Deconjugation
O
R
N
mAb-SH
+
O
HS-albumin
Interchain Cys conjugate
Average DAR
~3-4
SH HS
HS
HS
HS
SH
SH
SH
THIOMAB™ approach to stabilizing thiosuccinimides thiosuccinimide
mAb
O
Deconjugation
R
N
S
maleimide
O
O
R
N
+
mAb-SH
O
HS-albumin
THIOMAB™-drug conjugate
engineered
Cys
S
S
Average DAR
1.9-2.0
Thiosuccinimide stability and efficacy are linked and site-­‐dependent thiosuccinimide
hydrolyzed
mAb
S
O
R
N
H
COOH
Hydrolysis
In vivo stability
mAb
S
O
R
N
O
maleimide
Deconjugation
O
R
N
+
mAb-SH
O
In vivo efficacy
Shen, et al., 2012
Linker structure also impacts hydrolysis kineEcs thiosuccinimide
O
mAb
Hydrolysis
R
N
S
hydrolyzed
mAb
S
O
O
R
N
H
COOH
% Hydrolysis (HC A118C)
R=
O
60
N
H
50
Drug
40
30
H
N
20
O
10
0
0
100 200 300 400 500
Time (hours)
Drug
Electron-­‐withdrawing groups speed up thiosuccinimide hydrolysis thiosuccinimide
mAb
S
O
R
N
hydrolyzed
Hydrolysis
O
mAb
S
O
R
N
H
COOH
% Hydrolysis (HC A118C)
R=
NO 2
NO 2
NO 2
VC-PAB-MMAE
O
Time (h)
Conditions: PBS, 37 C
Nitrophenyl succinimide is stable in vivo and gives greater efficacy •  Conjugated to αCD22 Fc S400C
mutant (most unstable in vivo
with mc-vc-PAB-MMAE)
O
O
H
N
N
O
O
O2N
H
N
N
H
O
N
H
N
N
N
O
N
H
O
O
O
O
OH
O
nitrophenyl-mal-VC-PAB-MMAE
HN
O
O
O
NH 2
In vivo stability
•  Fully hydrolyzed post-conjugation
In vivo efficacy (CD22 model)
1200
nitrophenyl/S400C
mc/V205C
80
Tumor volume (mm3)
Normalized Avg DAR
100
60
40
mc/S400C
20
0
mc/S400C
(αHer2) mc/S400C
Vehicle
1000
800
mc/V205C
600
400
nitrophenyl/S400C
200
0
2
4
Day
6
8
0
Dose: 1 mg/kg
0
5
10
Day
15
20
From pepEde to disulfide linkers peptide
O
S
N
O
O
H
N
N
H
thiosuccinimide
O
O
O
O
N
H
N
H
N
Drug
O
O
HN
O
S
NH 2
S
N
N
Drug
disulfide
Disulfide release mechanism distinct from peptide linkers…
…may offer improved therapeutic index
O
O
O
From pepEde to disulfide linkers peptide
O
S
N
O
O
H
N
N
H
thiosuccinimide
O
O
O
O
N
H
N
H
N
Drug
O
O
N
N
O
O
HN
O
S
NH 2
S
Drug
disulfide
For disulfides, mechanism for instability and release are the same…
…stability and release are coupled
O
Decoupling stability and release in disulfide linkers Lysine (heterogeneous)
O
R3 R4
S
N
H
S
Drug
Drug
lysosomal
degradation
NH 2
O
HOOC
S
S
Drug
Drug
Stability in circulation
conferred by Ab and R groups
S
Drug
Drug
R1 R 2
Stability is retained in
catabolite
Stability in circulation
conferred by R groups
R3 R4
S
N
H
R1 R 2
THIOMAB™(site-specific)
R3 R4
lysosomal
degradation
NH 2
HOOC
R3 R4
S
S
Drug
Drug
Stability conferred by
Ab is removed
We can find sites that stabilize disulfides O
THIOMAB™
O
NH
OMe
OH
O
S
S
N
O
O
N
O
DM1
In#vivo#stability#
OMe
1200
Site 1
V205C
Tumor volume (mm3)
a)#
Normalized Avg DAR
a)#
a)#
O
Cl
CD22#model#(dose:#90#ug/m2)#
1000
Vehicle
800
V205C
600
400
200
Site 1
0
Day
In#vivo#efficacy#
0
5
10
15
Day
20
25
Disulfide linkers that release amines Disulfide linker releases a thiol…
O
O
O
NH
O
OMe
O
S
N
O
O
O
OMe
mAb catabolism
OH
S
NH
glutathione
OH
O
HS
N
N
O
O
N
O
O
Cl
O
Cl
OMe
OMe
DM1
(microtubule disruption)
…self-immolative linker releases amines
self-immolation
immolative spacer
O
HS
S
O
S
HO
H
O
N
N
mAb catabolism
O
O
O
N
N
O
O
H
glutathione
HO
H
O
N
N
O
O
O
O
O
N
H
N
O
O
SG3231
15 Disulfide linkers that release amines Disulfide linker releases a thiol…
O
O
O
NH
O
OMe
O
S
N
O
O
O
OMe
mAb catabolism
OH
S
NH
glutathione
OH
O
HS
N
N
O
O
N
O
O
Cl
O
Cl
OMe
OMe
DM1
(microtubule disruption)
…self-immolative linker releases amines
self-immolation
immolative spacer
CO2
S
S
O
S
HO
H
O
N
N
mAb catabolism
O
O
O
O
O
N
N
O
SG3231
H
glutathione
N
H
N
O
O
O
O
O
N
H
N
O
SG2057
(DNA damage agent)16 Achieving site-­‐dependent stability and efficacy with SG3231 S
O
S
THIOMAB™
HO
H
O
N
N
O
O
O
O
N
N
O
O
SG3231
In#vivo#stability#
In#vivo#efficacy#
Tmab SG3231, Mouse Efficacy Study
120
Normalized
Avg
DAR
Average DAR,
Normalized
80
Site#1#
60
Tumor volume (mm3)
2000
100
V205C#
20
Her2#model#(dose:#150#ug/m2)#
Vehicle
1600
V205C#
G2 LC K149C
G3 LC V205C 1200
G4 LC S121C
G5 HC A118C
G7 HC V211C
40
H
800
Site#1#
400
0
00
1
250
3
4100 5 6150 7
Time (Hour)
Day
8200 9 10250
0
0
10
20
Day
30
Stability with DM1 and SG3231 even on Site 1 is not opEmal a)#
a)#
O
NH
OMe
OH
O
S
S
N
O
O
O
a)#
Normalized Avg DAR
O
In#vivo#stability#
N
O
Cl
OMe
DM1
DM1/Site 1
S
O
S
HO
H
O
N
N
O
O
O
O
O
SG3231
N
H
N
O
Normalized Avg DAR
a)#
b)#
b)#
b)#
SG3231/Site 1
Day
Increasing hindrance next to disulfide to improve stability and/or release O
O
O
NH
O
NH
OMe
OMe
OH
Ab
O
S
S
N
OH
Reduction
O
O
HS
O
N
O
N
O
O
Cl
OMe
OMe
R
Ab
Reduction
S
O
S
HO
H
O
N
N
N
O
O
Cl
DM1
O
O
O
O
O
O
N
N
O
SG3231
H
R'
PBD
O
-S
O
Immolation
N
H
N
O
CO2
S
O
O
O
O
N
H
N
O
ConjugaEon challenges with THIOMAB™-­‐disulfide conjugates S-
S-
may
S
S
PBD
N
O
S
S
N
O
O
PDS leaving
group
LC/MS (Fab portion)
may
S SS
S SS
O
O
S S
S S
N
PBD
O
Avg DAR
1.4
N
Avg DAR
1.2
SH
SH
20 Improving conjugaEon regioselecEvity through leaving group opEmizaEon may
S
S
R'
N
PBD
R
X
DAR
N
S
S
X
O
O
X=
O
1.4
%LG
X=
N
24
N
O2N
DAR
%LG
1.2
31
1.5
15
1.9
2
N
N
NO 2
5-nitro-PDS
1.9
4
NO 2
5-nitro-PDS
21 Increasing sterics next to disulfide improves stability in vivo… O
O
DM3
OMe
OH
O
N
S S
O
NH
O
O
S
O
S
HO
H
N
O
Cl
N
N
OMe
SG3451
O
O
O
O
O
O
DM3
DM1
Day
H
N
O
a)#
SG3451
Normalized Avg DAR
a)#
Normalized Avg DAR
a)#
a)#
N
SG3231
Day
…and efficacy O
O
DM3
OMe
OH
O
N
S S
NH
O
S
O
HO
H
N
O
O
S
O
Cl
SG3451
O
N
N
OMe
O
O
O
O
N
N
O
Efficacy in CD22 model
Vehicle
1000
DM1
800
600
400
200
O
Efficacy in Her2 model
2000
Tumor volume (mm3)
Tumor volume (mm3)
1200
1600
Vehicle
SG3231
1 3 6 mpk
1200
800
SG3451
1 mpk
400
DM3
0
0
5
10
15
Day
20
25
0
H
0
5
10
15
Day
20
25
What about dimethyl disulfides? O
O
NH
OMe
OH
O
S
O2N
N
S
O
O
N
X
O
N
may
S S
S
O
O
Cl
OMe
DAR = 0.1
0% LG
S
O2N
N
O
S
HO
H
O
N
N
O
O
O
O
O
N
H
N
O
X
S S
S
PBD
O
O
DAR = 0.4
71% LG
24 Reversing reacEon roles to achieve dimethyl disulfide conjugaEon nucleophile
electrophile
X
Ab
S-
S δ+
S
O2N
may
O
electrophile
nucleophile
-S
Ab δ+S
S
N
N
may
O
NO 2
LC/MS (Fab portion)
may
S S
S
O
Avg DAR
1.9
SH
Reversing reacEon roles to achieve dimethyl disulfide conjugaEon nucleophile
electrophile
X
S-
Ab
S δ+
S
electrophile
may
nucleophile
Ab δ+S
S
O
may
-S
O
N
N thiols
This
O2Napproach does not work for immolating
NO 2
CO2
O
O
HS
LC/MS (Fab
portion)
HO
N
H
N
O
S
O
O
O
O
N
H
N
O
N
H
N
O
O
O
O
N
H
N
O
O
may
S S
S
O
Avg DAR
1.9
SH
Exploring novel leaving groups for hindered immolaEng disulfides O
O
H
N
N
O
S
S
X
O
X=
N
NO 2
OH
N
H
O
O
O
O
N
N
N
N
MMAE
H
O
O
S
S
X
O
DAR
%LG
X=
DAR
%LG
0.6
51
LG1
0.7
14
0
96
LG2
1.9
0
NO 2
N
*
N
NO 2
0
100 (SNAr)
NO 2
S S
S
MMAE
O
O
LG1
1.5
0
SH
Summary •  Selecting appropriate conjugation site and linker modifications
in THIOMAB™-drug conjugates can be used in combination to
achieve both high stability and potency in vivo
- Improving thiosuccinimide stability through EWG
- Decoupling stability and release in disulfide conjugates
•  Conjugation challenges with disulfides can be overcome
through reversing reaction roles of antibody and drug (nonimmolating thiols) or improvement of leaving group electronics
(immolating thiols)
ConjugaEon lab at Genentech ADC conjugation lab
Hans Erickson
Dick Vandlen
Craig Blanchette
Jack Sadowsky
Byoung-Chul Lee
Chris Nelson
Martine Darwish
Rachana Ohri
Breanna Vollmar
Pragya Adhikari
Neelie Zacharias
WUXI Aaptec (Jiawei Lu)
and Mr. ADC
Acknowledgements Chemistry: Thomas Pillow, Pete Dragovich, Zhonghua Pei, John Flygare, Leanna Staben, Vishal
Verma, Jinhua Chen, Jingtian Chen, Yihui Chen, Youbing Gao, Changrong He, Jiahui Lu, John
Wai, Jing Wang, Tao Wang, Yanli Wang, Qinglu Yang, Hui Yao, Zijin Xu, JuanJuan Xue, Hao Zhou
(WUXI Aaptec)
Antibody format: Jagath Junutula, Sunil Bhakta
Biology: Jyoti Asundi, Carter Fields, Jun Guo, Ginny Li, Weiguang Mao, Gail Phillips, Andy
Polson, Paul Polakis, Bing Zheng
DMPK: Donglu Zhang
Biochem. & Cell. Pharm.: Phillip Chu, Josefa Chuh, Leanne Goon, Kathy Kozak, Jenny Li, Yichin
Liu, Aimee O Donohue, John Tran, Siao Ping Tsai
BioAnalytical Sciences: Carl Ng, Ola Saad, Dian Su, Keyang Xu
Translational Oncology: Beth Blackwood, Geoff Del Rosario, MaryAnn Go, Rebecca Rowntree,
Susan Spencer, Shang-Fan Yu
Legal: Alex Andrus
Nerviano, Spirogen and University of Auckland