The Chemistry Behind Antibody-Drug Conjugation
Eran Sella
Baran Lab
Group Meeting
Dominant Investigator
Antibody-Drug Conjugation (ADC)
- Peter D. Senter, Ph.D. - B.A Biotechnology (Berkeley), Ph.D. Chemistry (University of Illinois),
Postdoctoral in the Max-Planck Institute for Experimental Medicine.
Worked at Cytokine Networks, Bristol-Myers-Squibb Research Institute.
Current position: Vice president, Seattle Genetic.
(Development of potent drugs, novel linker systems and conjugation
methodology)
Senior editor of Molecular Cancer Therapeutics
Affilate Professor (University of Washington)
- Taking the advantage of the specificity of mAb (monoclonal antybodies) to deliver
potent cytotoxic drug selectively to antigen-expressing tumor cells.
Chemistry allows to use highly potent drugs that could not be used alone.
- No. of publication with the concept "antibody drug conjugated" over ther years:
Antibody-Drug Conjugates - Clinical Reports (clinicaltrials.gov 2012)
Agensys - 3 ADCs in Phase I (auristatine) - carcinoma and prostate cancer
Antibody-Drug Conjugates - History
H2N
Bayer HealthCare - 2 ADCs in Phase I (Auristatine/maytansinoid)
N
Biogen - 1 ADC in Phase I (DM4) - breast cancer
NH2
Biotest - 1 ADC in Phase II (DM4) - myeloma
N
N
N
Mylotarg withdrown
from market
N
H
N
CO2H
First FDA
approved ADC
Mylotarg
O
CO2H
Paul Erlich
"Magic Bullet"
MTX-Ab
Noncovalent linked
ADC tested in animal Humanized mAbs
reported
models
BMS - 1 ADC in Phase I (duocarmycin) - non-Hodgkin's lymphoma
Celldex - 1 ADC in Phase II (auristatine)
Kadcyla
approved
Immunogen - 2 ADCs in Phase I (DM1 and DM4)
Pfizer - 1 ADC in Phase III - non-Hodgkin's lymphoma
Progenics - 1 ADC in Phase I (auristatine) - prostate cancer
1913
1910
1958
1920
1930
1940
1950
1960
1988 1993
1972
1975
1970
1980
Covalent linked
ADC tested in animal
models
Drug Discov. Today, 2013
1990
Roche - 1 ADC in Phase III and 2 ADCs in Phase I (DM1) - breast cancer
2000
ADC with
calicheamicin
2013
2010
2011
Sanofi - 2 ADCs in Phase II (DM4) - lymphoma and leukemia
Seattle Genetics - 1 ADC in Phase III and 1 ADC in Phase I (auristatine) - non-Hosdgkin's
lymphoma and carcinoma
Adcetris
approved
1
The Chemistry Behind Antibody-Drug Conjugation
Eran Sella
Mode of Action
Baran Lab
Group Meeting
General Linkers
- Use of highly potent drugs
(Especially negative-charged drugs)
- Ab can be also anti-cancer
- Drug is stable and usually inactive
until it reaches the cancer cells
ADC with a specific drug is usually
more potent than the drug alone
O
O
O
NH2
N O
R
N
H
NHS
O
R
amide
S
NH2
S C N R
N
H
isothiocyanate
O
SH
X
N R
H thiourea
O
S
R
R
haloacetyl
thioether
ImmunoGen Inc.
O
Antibody-Drug Conjugates - Components & Demands
N
O
Attachment
Site
Linker
O
SH
S
R
N
maleimide
O
-Stable
-Slow hydrolysis
Drug
R
thioether
Simple Conjugation
Antibody:
1. Targets a well-characterized antigen with high tumor expression
2. Maintain all of it's properties upon conjugation to the drug
3. Minimal non specific binding
Attechment site:
1. Typically through cysteine or lysine residues on the Ab
2. Variable drug:Ab ratio or site-selective conjugation
O
O
O
N
H
O
S
N
O
N
H
H
N
Drug
maleimidocaproyl
H
N
Drug
mercaptoacetamidocaproyl
O
O
S
Drug
O
O
Linker:
1. Cleavable or non cleavable
2. Stable with selective release of the drug
Drug:
1. Highly potent (usually 2-4 drugs per mAb)
2. Non-immunogenic
3. Could be linked to the Ab
4. Defined mechanism of action
H
N
O
O
Current Opinion in Chemical Biology, 2010, 14, 529
2
The Chemistry Behind Antibody-Drug Conjugation
Eran Sella
Canadian J. Org. Chem., 1984, 62(3), 586
Chem. Abstr., 1968, 68
Thiolation Reagents
Chem. Commun., 2013, 49, 8187
O
S
R NH2
R N
H
SH
NH
S
R
NH2
S
O
Br
Br
Toluene
reflux
Cl
S
S
S
N3
-N2
1g = 3.5 $
SH
CN
K2CO3
quant.
1g = 7.4 $
CN
S
67%
S
NH2
Cl
PhS
PhS
O
O
N O
SAc R
O
NH2
R
N
H
SAc
R
N
H
O
PNPCl
Pyridine
O
O
H2N
O
O
O
Drug
NO2
O
O
HN
O
PhS
NH2OH
Drug
O
N R
PhS
TCEP
O
O
Br
O
N
PhS
OMe
OH
O
N
HCl/MeOH
S S
O
S
O
N R
S
HS SH
O
SH
S
O
pH=7.4
S
O
OH
N R
S
OH
N
O
N
PhS
PhS
O
N
O
Br
PABA
O
PhS
NH
O
O
Br
PhSH
NEt3
S
O
Br
MeOCOCl
NMM
O
35%
Br
Drug
O
NH
NaN3
O
SH
N
H
2-iminothiolane
"Traut's Reagent"
NCS
O
O
N
R = mAb or Drug
NH
R
Baran Lab
Group Meeting
O
S
S
pH=4.5
O
O
NHR
S
O
H2N
Drug
O
PABA
3
The Chemistry Behind Antibody-Drug Conjugation
Eran Sella
Bifunctional Linkers
O
O
O
O
O
N O
NH2
HS
HO
S
O
N
H
O
N
H
B
Drug
H
O
S
O
HO
O
N
H
H
N
N
H
S
N
H
Can deconjugate
(retro-michael)
H
Drug
S
N
H
S
Drug
Hydrazone (Rapid hydrolysis
in the lysosome)
Disulfide (Reduction in lysosome)
Mainly by Glutathione
O
O
N
H
HO
O
OH
O
NH
H
N
N
OH
O
S
SH
H
N
NH
H2N
S
Drug Linking by Click
N
H
O
N
H
H
N
O
Drug
Val-Cit dipeptide
(site-specific proteolysis)
Sometimes also Phe-Lys
copper-free click - developed
by Bertozzi (2007)
PNAS, 104(43), 16793
bosutinib
Drug
O
Can't deconjugate
Drug
NH2
N
H
O
N
O
O
N
H
J. Controlled Release, 2012,
161, 422
O
S
Drug
O
S
O
-TolSO2-
SH
O
O
O
pH=7.4
O
Piperidine
O
Drug
O
O
Oxone
Misc. Drug Linking with Specific Release Mechanism
Bioconjugate Chem., 2014, 25, 460
O
STol
N
O
NH
Drug
O
Drug
O
HO
O
Drug
HS
O
HO
O
N
O
O
O
O
N
H
N
O
CH2O
Piperidine/HCl
O
Baran Lab
Group Meeting
N3
Drug
N
N
N
N
JACS, 2013, 135, 12994
4
The Chemistry Behind Antibody-Drug Conjugation
Eran Sella
Traceless Linkers
NH2
Precise Controll on Conjugation to Synthesize homogeneous ADCs
Advantages: 1. Known mAb:Drug ratio.
2. mAb stability remains
H
N
CHO
Drug
Drug
SH
Baran Lab
Group Meeting
O
(Hydrolysis)
S
O
O NH2
Drug
Drug
Drug
O
O
N
N
thiazolidine
Oxime Ligation
SH
Drug
S S
SH
(Reduction)
Drug
Genetically encoded unnatural amino acid with ortogonal chemical reactivity
HOOC
Schultz, PNAS, 2012, 92(3), 13
O2N
S
COOH
O
2
SH
S S
Ellman Reagent
NO2
N
N
OH
= auristatine
Drug
Ph
N Ph
S S
N
N
S
O
N
N
H
OH
O
N3
O
H2N
O
NH2
3
N
H
O
3
N3
1. PNPCl, NEt3
2. EtOCONHNH2
H2N
3. K2CO3 / MeOH
2,2'-dithiopyridine
O
O
COOH
S S
P
HOOC
H
N
EtO
TCEP
SH
O
N
H
O
N
H
O
SH
HS
OH
S S
HO
HO
N
H
O
N
HN
HN
O
N3
3
O
O
COOH
N
H
O
3
N3
N
N
S
S
K2CO3 / MeOH
N
H
NBS, Pyridine
OH
N3
3
O
DTT
(dithiothreitol)
(Cleland's Reagent)
N
1. TFAA
2. EDC, HOBt
OH
N
O
OH
Herceptin
O
S
NH2
RGD
NH
O
Nature Protocols, 2013, 8, 2079
O
O
Click-like reaction for Tyrosine
O
O
N
H
Drug
O
PBS, RT, 30 min
N
H
SH
N
H
N
O
Drug
Drug
N
O
Barbas, JACS, 2010, 132, 1523
5
The Chemistry Behind Antibody-Drug Conjugation
Eran Sella
Spacers
Baran Lab
Group Meeting
Self-Immolative Spacers (Cont.)
Drug
n
X
O
Drug
O
O
O
Drug
n
X
O
Self-Immolative Spacers
X
-CO2
Drug
Drug
R
O
X
Examples
O
N
H
O
X
O
N
H
OH
X
-CO2
O
Drug
H2N
O
O
Drug
O
Drug
HO
Drug
HO
Drug
Drug
O
H2O
OH
O
O
X
O
Drug
O
OH
O
X
Y
O
Drug
O
S
S
O
O
N
N
S
O
O
Drug
O
O
X
Y
O
O
O
Drug
Y
X
HO
Drug
-CO2
S
O
O
N
N
HO
Drug
6
The Chemistry Behind Antibody-Drug Conjugation
Eran Sella
Baran Lab
Group Meeting
Adcetris (Brentuximab Vedotin)
mAb - Brentuximab, directs to protein CD30 (expressed in Hodgkin's lymphoma and large-cell lymphoma)
Attachment Site - thiomaleimido caproyl
Linker - Cathepsin (protease) cleavable linker (Val-Cit)
Spacer - PABA
Drug - Monomethyl auristatine E (MMAE)
O
O
O
N
S
O
N
H
H
N
O
O
N
O
H
N
N
N
O
N
H
O
H
N
O
O
O
OH
O
NH
H2N
NH
MMAE - antimitotic drug (inhibits mitosis, cell division), from the marine shell less Dolabella Auricularia
- Blocking the polymerisation of tubulin
- 200 times more potent than vinblastine
H
N
N
O
O
H
N
N
N
O
O
O
N
S
O
Dolastatine
7
The Chemistry Behind Antibody-Drug Conjugation
Eran Sella
Adcetris (Brentuximab Vedotin)
O
H
N
N
O
O
O
O
Pettit, JACS, 1989, 111(14), 5463
N
H
N
N
N
Baran Lab
Group Meeting
S
O
Dolastatine
O
NaH, CH3I
THF
OH
NHCbz
O
O
BH3, THF
OH
NCbz
83%
OH
NCbz
95%
SO3-Pyridine, DMSO
H
78%
NCbz
O
O
N
Cbz
LDA, THF
-78o
87% total
33% separated
H
N
Boc
CH3N2, BF3 etherate
O
OH
N
Cbz
67%
O
H2, Pd/C
O
N
H
63%
OMe O
O
OMe O
(3R,4S, 5S)
1. BH3 - THF
2. modified Swern
H
OH
N
Boc
75% (2 steps)
O
O
H
N
Boc
Ph
O
O
Ph
H
LDA, MgBr2
Ph
O
OH O
H
Me3O BF4
Ph
N
Boc
DCM
Ph
OMe O
Ph
O
Ph
Ph
Ph
47%
after separation
-20o
57%
Ph
H
KOtBu
N
Boc
O
OMe O
Ph
Ph
H
1. H2/Pd
2. TFA
TFA
N
H2
OMe O
1.
O
OH
NHBoc
O
H
NHBoc
OH
H2N
2. MnO2
SH
N
S
NHBoc
8
The Chemistry Behind Antibody-Drug Conjugation
Eran Sella
Adcetris (Brentuximab Vedotin)
O
H
N
N
H
N
N
N
O
O
O
O
Baran Lab
Group Meeting
Tetrahedron, 2007, 63, 6115
N
S
O
Dolastatine
1. CDI
2. Mg(O2CCH2COOEt)2
BocHN
62%
COOH
RuBr2[(S)-SYNPHOS)
H2, 12bar, 50o, 24h
OEt
BocHN
O
(S)-Boc-Isoleucine
O
1. LHMDS. HMPA, MeOTf
2. aq. NaOH / EtOH
100% conversion
92:8
OEt
BocHN
60% (2 steps)
OH O
O
OH
BocHN
OMe O
PPh2
PPh2
O
O
O
H
N
Boc O
OH
N
Boc O
OEt
N
Boc OMe O
O
OH
(S)-Boc-Proline
HN OMe
Cl
O
HCl
Pyridine
77%
O
O
PhO
P N3
PhO
PPh3, DEAD
OMe
N
S
S
N
N
56%
O
1. Ipc2BCl
2. NaOH
3. H2O2
60%
99% ee
S
N
OH
S
N
N3
S
Li
1. PPh3, NH4OH
2. DIBOC
44%
>99%ee
N
NHBoc
9
The Chemistry Behind Antibody-Drug Conjugation
Eran Sella
Baran Lab
Group Meeting
O
Kadcyla (Trastuzumab emtansine)
HN
O
MeO
mAb - Trastuzumab, targets HER2 receptor, also stop cell groth alone
H
OH
Attachment Site - SMCC (succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate)
Drug - Mertansine (3.5 units/mAb in avarage)
O
Mertansine (DM1)
- Thiol derivative of Maytansine
- Antimitotic
O
O
N
O
N
S
N
O
Cl
H
N
O
O
O
O
Cl
Cl
LiAlH4
THF
NHMe
MeO
Cl
NHMe
MeO
1. MeOCOCl, K2CO3
2. NaOH/MeOH
COOEt
OH
OH
OH
LDA
1.DHP, pTsOH
2. Buli
50%
3.
Br
I
Li
OMe
Cu
OTHP
Cu
N
MeO
OTHP
OMe
Cl
Cl
Cu
Cl
N
O
94%
OMe
OH
OMe
Li
OMe
MeO
N
MeO
1. MsCl, NEt3
2. NaI
OH
Br
Br
OMe
O
90%
88%
Br2, CCl4
Cl
N
MeO
O
90%
O
1. pTsOH/MeOH
2. MnO2
OMe
N
MeO
OMe
O
95%
O
OTHP
I
O
NH2
Cl
N
N
LDA, TMSCl
73%
TMS
MeO
1. sec-BuLi
2.
OMe
O
Cl
MeO
N
N
OMe
O
O
Corey
JACS, 1978, 100(9), 2916
Tel. Lett. 1978, 12, 1051
JACS, 1980, 102(4), 1439
O
3. AcOH/NaOAc
10
The Chemistry Behind Antibody-Drug Conjugation
Eran Sella
Baran Lab
Group Meeting
O
Kadcyla (Trastuzumab emtansine)
HN
O
MeO
AcO
1. NaOMe/MeOH
2. HgOAc
O
75%
AcO
AcOHg
O
H
HO
HO
O
H
HO
HO
99%
OTrt
O
H
MeLi, CuI
OH
HO
SH
OEt
OH
CHCl3/HCl
OMe
96%
75%
O
O
HO
SH
S
O
O
H
N
OMe
Cl
O
O
O
N
S
O
N
H
N
O
O
O
O
O
O
O
MEMCl
OH
BF3 Et2O
86%
S
H
OTrt
OMe
OMe
OAc
OH
1. TrtCl, Pyridine
2. NaH (4 eq.)
3. isopropylbenzene
sulfonyl imidazole
OH
1. NaCl
2. NaBH4
3. HCl
OH
S
99%
OMEM
S
S
S
O
O
MeO
O
N
1. nBuLi
2. NaH, MeI
OMe
O
OMe
N
MeO
S
S
O
S
Cl
1. CH3SLi
2. HClO4
3. Pb(OAc)4
O
O
OMEM
S
Cl
Cl
OMEM
O
MeO P
MeO
NH
OMe
OMEM
then Pyridinium chloride
82%
O
O
Cl
OMe
MeO
Li
S
S
OMEM
N
OMe
1.
Cl
MeO
TMS
S
S
OMe
O
Li
O
NH
MeO
1. PNPCl, Pyridine
2. NH4OH / t-butanol
3. HgCl2, CaCO3
N
2. Bu4NOH
3. MesSO2Cl
4. 10% H2SO4
S
S
OH
76%
OMe
O
Cl
MeO
N
(-)-N-Methylmeysenine
Corey
JACS, 1978, 100(9), 2916
Tel. Lett. 1978, 12, 1051
JACS, 1980, 102(4), 1439
O
N
H
OMe OH
O
11
The Chemistry Behind Antibody-Drug Conjugation
Eran Sella
Baran Lab
Group Meeting
Mylotarg (Gentuzumab ozogamicin)
mAb - Gentuzumab (antibody to CD33 expressed in leukemia cells)
Linker - includes Hydrazone and stable S-S linkage
Drug - Calicheamicin
Calicheamicin
- from Micromonospora echinospora, Texas, 1980
- Causes DNA strand scission
O
initiation
HO
S S
O
NHCO2Me
O
Bergman
Cycloaromatization
HO
S
Sugar
O
O
NHCO2Me
O
O
Sugar
NHCO2Me
HO
DNA diradical
S
DNA
HO
S
Sugar
NHCO2Me
O
O2
DNA scission
Sugar
12
The Chemistry Behind Antibody-Drug Conjugation
Eran Sella
Baran Lab
Group Meeting
O O NHCO Me
2
Et3Si O
Pd C-C
OH
acetyilide-aldehyde
condensation
BzO
O
O
O
ethylene glycol
pTsOH
O
60%
HO
O
O
DIBAL
O
84%
O
OH
O
tetronic acid
MEMO
1. TBSCl, Im
2. PhCOCl, Pyr
H2O2, NaOH
Ipc2BOMe
MEMO
Li
OMEM
TBSO
OMEM
BIpc2
s-BuLi
O
OMEM
HO
(Z)
O
OMEM
1. TBAF
2. Swern
3. NH2OH
HO
O OBz
N
O OH
O
O
NaOCl
O
N
O OBz
BzO
O
OMEM
O
N
O
BzO
O
1. NaOMe
2. CrO3, H2SO4
O
H
OMEM
N
AcO
3.
TMS
4. Ac2O
5. ZnBr2
O
Li
TMS
O
H
OH
1. Swern
2. PPh3=CHCO2Me
O
O
N
AcO
TMS
O
CO2Me
isoxazoline
51% after separation
Nicolaou, JACS, 1992, 114, 10082
13
The Chemistry Behind Antibody-Drug Conjugation
Eran Sella
Baran Lab
Group Meeting
O O NHCO Me
2
Et3Si O
Pd C-C
OH
acetyilide-aldehyde
condensation
O
BzO
O
O
O
1. K2CO3/MeOH
2. TES triflate
N
AcO
TMS
O
3.
O
O
N
Et3Si
O
O
1. Mo(CO)6
2. K2CO3
Et3Si
NH2
O
CHO
82%
Cl
O
O
Et3Si
O
O
N
O
O
Pyridine
MeNO2
CO2Me
CO2Me
CO2Me
Cl
Cl
O
O
CO2Me
TMS
Pd(PPh3)4, CuI
H
TMS
H
91%
O O NPhth
O O NPhth
1. SiO2
2. Ac2O
KHMDS
-90o
Et3Si O
1. MsCl
2. SiO2, Pyr
Et3Si O
Et3Si O
48%
CHO
CO2Me
1. MeNHNH2 (99%)
2. Triphosgene, Pyr/MeOH (82%)
3. DIBAL, NaBH4 (82%)
4. PhCOCl, Pyr (84%)
O O NPhth
CO2Me
H
OH
O
O
O O NHCO Me
2
Et3Si O
OH
BzO
Nicolaou, JACS, 1992, 114, 10082
14