The Chemistry Behind Antibody-Drug Conjugation
14
The Chemistry Behind Antibody-Drug Conjugation Eran Sella Baran Lab Group Meeting 1 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) Dominant Investigator 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010 1913 1958 1972 1975 1988 1993 Paul Erlich "Magic Bullet" N N N N H 2 N NH 2 N H N O CO 2 H CO 2 H MTX-Ab Noncovalent linked ADC tested in animal models Covalent linked ADC tested in animal models Humanized mAbs reported ADC with calicheamicin First FDA approved ADC Mylotarg Mylotarg withdrown from market Kadcyla approved Adcetris approved 2011 2013 Antibody-Drug Conjugates - History - 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: Drug Discov. Today, 2013 Antibody-Drug Conjugates - Clinical Reports (clinicaltrials.gov 2012) Agensys - 3 ADCs in Phase I (auristatine) - carcinoma and prostate cancer Bayer HealthCare - 2 ADCs in Phase I (Auristatine/maytansinoid) Biogen - 1 ADC in Phase I (DM4) - breast cancer Biotest - 1 ADC in Phase II (DM4) - myeloma BMS - 1 ADC in Phase I (duocarmycin) - non-Hodgkin's lymphoma Celldex - 1 ADC in Phase II (auristatine) 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 Roche - 1 ADC in Phase III and 2 ADCs in Phase I (DM1) - breast cancer 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
Transcript of The Chemistry Behind Antibody-Drug Conjugation
The Chemistry Behind Antibody-Drug ConjugationEran SellaBaran Lab
Group Meeting
1
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)
Dominant Investigator
1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010
1913 1958 1972
1975
1988 1993
Paul Erlich"Magic Bullet"
NN
N
NH2N NH2
NHN
O
CO2H
CO2H
MTX-AbNoncovalent linked
ADC tested in animalmodels
Covalent linkedADC tested in animal
models
Humanized mAbsreported
ADC withcalicheamicin
First FDAapproved ADC
Mylotarg
Mylotarg withdrownfrom market
Kadcylaapproved
Adcetrisapproved
2011
2013
Antibody-Drug Conjugates - History
- 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:
Drug Discov. Today, 2013
Antibody-Drug Conjugates - Clinical Reports (clinicaltrials.gov 2012)
Agensys - 3 ADCs in Phase I (auristatine) - carcinoma and prostate cancer
Bayer HealthCare - 2 ADCs in Phase I (Auristatine/maytansinoid)
Biogen - 1 ADC in Phase I (DM4) - breast cancer
Biotest - 1 ADC in Phase II (DM4) - myeloma
BMS - 1 ADC in Phase I (duocarmycin) - non-Hodgkin's lymphoma
Celldex - 1 ADC in Phase II (auristatine)
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
Roche - 1 ADC in Phase III and 2 ADCs in Phase I (DM1) - breast cancer
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
The Chemistry Behind Antibody-Drug ConjugationEran SellaBaran Lab
Group Meeting
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Mode of Action
ImmunoGen Inc.
DrugLinkerAttachmentSite
Antibody-Drug Conjugates - Components & Demands
Antibody:1. Targets a well-characterized antigen with high tumor expression2. Maintain all of it's properties upon conjugation to the drug3. Minimal non specific binding
Attechment site:1. Typically through cysteine or lysine residues on the Ab2. Variable drug:Ab ratio or site-selective conjugation
Linker:1. Cleavable or non cleavable2. Stable with selective release of the drug
Drug:1. Highly potent (usually 2-4 drugs per mAb)2. Non-immunogenic3. Could be linked to the Ab4. Defined mechanism of action
Current Opinion in Chemical Biology, 2010, 14, 529
General Linkers
N
O
O
O R
O
N
O
OR
S C N R
NH2
NH
R
O
SHS N
O
O
R
NH2
NH
NH
S
R
XR
O SHS
O
R
NHS amide
maleimidethioether
thioureaisothiocyanate
haloacetyl thioether
Simple Conjugation
NH
O
O
OO
O
O
HN Drug
S N
O
O
HN
O
Drug
SNH
O HN
O
Drug
-Stable-Slow hydrolysis
maleimidocaproyl
mercaptoacetamidocaproyl
- 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
The Chemistry Behind Antibody-Drug ConjugationEran SellaBaran Lab
Group Meeting
3
Thiolation Reagents
R NH2 R NH
SH R = mAb or Drug
S NHR NH2 R N
H
NHSH
2-iminothiolane"Traut's Reagent"
S
NCS
S SCl N3
ToluenerefluxNaN3
S NH-N2
35%1g = 3.5 $
BrCN SH
O
K2CO3quant.
SCN
O
S NH2Cl
HCl/MeOH
67%
N
O
O
O
OSAc R NH2
R NH
OSAc
R NH
OSH
NH2OH
1g = 7.4 $
N
O
O
S
S
OO
O Drug
Chem. Commun., 2013, 49, 8187
NH
O
O
Br
Br
MeOCOClNMM
N
O
O
Br
Br
O
OMe
PhSHNEt3
N
O
O
OHBr
Br
PNPClPyridineN
O
O
PhS
PhS
PABA
OH
N
O
O
PhS
PhS
OH2N Drug
N
O
O
PhS
PhS
O
O
O NO2
HN
O
Drug
HS SHS S
TCEP
N
O
O
PhS
PhSR
N
O
O
S
SR
N
O
O
S
SR
pH=7.4O
O
S
S
OH
NHR
pH=4.5O
O
O
S
S
H2N Drug PABA
Canadian J. Org. Chem., 1984, 62(3), 586Chem. Abstr., 1968, 68
The Chemistry Behind Antibody-Drug ConjugationEran SellaBaran Lab
Group Meeting
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Bifunctional Linkers
N
O
O
O
O
N
O
O
NH2
O
N
O
O
NH
HS Drug
O
N
O
O
NH
S Drug
Drug NH
OOSO
O
pH=7.4
BH
Drug NH
OO SH
Drug
NH
OO
S
Bioconjugate Chem., 2014, 25, 460
Drug NH
OO
S
Drug NH
OHO
S
Can deconjugate(retro-michael) Can't deconjugate
O
HO
O CH2OPiperidine/HCl O
HO
ONH
HS
Piperidine
O
HO
OSTol
Oxone
HO
OOSO
O
-TolSO2-
H
Misc. Drug Linking with Specific Release Mechanism
O
NH
O
HN
N DrugHydrazone (Rapid hydrolysis in the lysosome)
O
NH
SS Drug Disulfide (Reduction in lysosome)
Mainly by Glutathione
O
HN
OH
OSH
NHNH2
HO
O O
SN
O
O
HN
O
NH
HN
O
NH
NHH2N
DrugVal-Cit dipeptide(site-specific proteolysis)
Sometimes also Phe-Lys
Drug Linking by Click
O
NH
N
Drug N3
NN
N
Drug
J. Controlled Release, 2012, 161, 422
copper-free click - developed by Bertozzi (2007)PNAS, 104(43), 16793
JACS, 2013, 135, 12994
bosutinib
The Chemistry Behind Antibody-Drug ConjugationEran SellaBaran Lab
Group Meeting
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Traceless Linkers
SH
NH2Drug CHO
S
HN
Drug(Hydrolysis)
thiazolidine
SH S
Drug SH
S Drug (Reduction)
SH
HOOC
O2N S
2
Ellman ReagentS S NO2
COOH
Drug SH
S S DrugNature Protocols, 2013, 8, 2079
Precise Controll on Conjugation to Synthesize homogeneous ADCsAdvantages: 1. Known mAb:Drug ratio. 2. mAb stability remains
OO
Drug O NH2
Oxime Ligation
NNOO
Drug Drug
Schultz, PNAS, 2012, 92(3), 13
Genetically encoded unnatural amino acid with ortogonal chemical reactivity
NH
OO
ONH2Drug
N SS N
SHS S P
COOH
COOHHOOC
HSSH
OH
OHDTT(dithiothreitol)
(Cleland's Reagent)
TCEP
S S
SS
HO
HO
2,2'-dithiopyridine
NH
OH
O
NN
N
O
O
Ph
Drug = auristatine
NH
OH
O
NNH
N
O
OPh
PBS, RT, 30 min
Barbas, JACS, 2010, 132, 1523
H2N
O
OH
1. TFAA2. EDC, HOBt
N3 ONH23
3. K2CO3 / MeOH H2N
O
NH
ON3
3
1. PNPCl, NEt32. EtOCONHNH2
NH
O
NH
ON3
3O
NH
HN
O
EtOK2CO3 / MeOH
N
O
NH
ON3
3
HNHN
O
O
NBS, PyridineN
O
NH
ON3
3
NN
O
O
OH
N NH
NO
O
RGD
Herceptin
Click-like reaction for Tyrosine
The Chemistry Behind Antibody-Drug ConjugationEran SellaBaran Lab
Group Meeting
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Self-Immolative Spacers (Cont.)
X O
O
Drug
X O
O
Drug X
RDrug
-CO2
Examples
OH
O
O Drug
O OHO Drug
OH
O
O DrugO O
HO Drug
SS
O N
ON
O
SO O
O
Drug
OS
O
NN
O-CO2
HO Drug
Self-Immolative Spacers
Spacers
Drug
OO Drug
n
n
X
O NH
O
Drug
X
O NH
O
Drug
X
H2N Drug
H2O
X
OH
-CO2
XY O
O
Drug
XY O
O
Drug XY
OHO Drug
The Chemistry Behind Antibody-Drug ConjugationEran SellaBaran Lab
Group Meeting
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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)
SN
O
O
O
NH O
HN
O
NH
O
O
N
NH
NHH2N
O
HN
O
NO
N
O
HN
OH
OO
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
NO
HN
O
NO
N
O
HN
OOS
N
Dolastatine
The Chemistry Behind Antibody-Drug ConjugationEran SellaBaran Lab
Group Meeting
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Adcetris (Brentuximab Vedotin)
NO
HN
O
NO
N
O
HN
OOS
N
Dolastatine
OH
O
NHCbz
NaH, CH3ITHF
83%
Pettit, JACS, 1989, 111(14), 5463
OH
O
NCbz
BH3, THF
95% OHNCbz
SO3-Pyridine, DMSO
78%NCbz
O
H
O
O
LDA, THF-78o
87% total33% separated
O
ONCbz OH
(3R,4S, 5S)
CH3N2, BF3 etherate
67%O
ONCbz OMe
H2, Pd/C
63%O
ONH OMe
NBoc
OH
O
H1. BH3 - THF2. modified Swern
75% (2 steps) NBoc
HH
OO
OPh
PhPh
LDA, MgBr2
NBoc
H
OH O
OPh
PhPh
47%after separation
Me3O BF4
DCM NBoc
H
OMe O
OPh
PhPh
KOtBu
-20o
57%
NBoc
H
OMe O
OPh
PhPh
1. H2/Pd2. TFA
NH2
H
OMe O
OH
TFA
O
OHNHBoc
O
HNHBoc
H2NSH
1.
2. MnO2
N
SNHBoc
The Chemistry Behind Antibody-Drug ConjugationEran SellaBaran Lab
Group Meeting
9
Adcetris (Brentuximab Vedotin)
NO
HN
O
NO
N
O
HN
OOS
N
Dolastatine
Tetrahedron, 2007, 63, 6115
COOHBocHN
1. CDI2. Mg(O2CCH2COOEt)2
62% BocHNO O
OEt
RuBr2[(S)-SYNPHOS)H2, 12bar, 50o, 24h
100% conversion BocHNO
OEt
OH92:8
O
OO
O
PPh2PPh2
1. LHMDS. HMPA, MeOTf2. aq. NaOH / EtOH
60% (2 steps) BocHNO
OH
OMe
NBoc O
OEt
ONBoc O
OHH
NBoc
OH
OOMe
(S)-Boc-Isoleucine
(S)-Boc-Proline
Cl
O
HN OMeHCl
Pyridine77%
N
O
OMeS
NLi
56% ON
S 1. Ipc2BCl2. NaOH3. H2O2
60%99% ee
N
S
OH
PO
PhOPhO
N3
PPh3, DEAD
N
S
N3
1. PPh3, NH4OH2. DIBOC
44%>99%ee
N
S
NHBoc
The Chemistry Behind Antibody-Drug ConjugationEran SellaBaran Lab
Group Meeting
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Kadcyla (Trastuzumab emtansine)
mAb - Trastuzumab, targets HER2 receptor, also stop cell groth alone
Attachment Site - SMCC (succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate)
Drug - Mertansine (3.5 units/mAb in avarage)
Mertansine (DM1)- Thiol derivative of Maytansine- Antimitotic
HN
O
N
O
O
S
O
NO
O
O
O
O
HN
OCl
N
O
HOHMeO
ClNHMe
COOEt
MeO LiAlH4THF
88%
ClNHMeMeO
OH
1. MeOCOCl, K2CO32. NaOH/MeOH
90%
ClNMeO
OH
O
OMe 1. MsCl, NEt32. NaI
94%
ClNMeO
I
O
OMe
OHBr2, CCl4
OH
Br
Br
LDA
50%
Br
OH
1.DHP, pTsOH2. Buli
3.OMe
Cu
OMeCu
OTHP
Li
ClNMeO
I
O
OMe
OMeCu
OTHP
Li
90%
ClNMeO
O
OMe
OTHP
1. pTsOH/MeOH2. MnO2
95%
ClNMeO
O
OMeONH2
N LDA, TMSCl
73% TMS
N 1. sec-BuLi
2.
3. AcOH/NaOAc
ClNMeO
O
OMe
O
O
ClNMeO
O
OMe
OCoreyJACS, 1978, 100(9), 2916Tel. Lett. 1978, 12, 1051JACS, 1980, 102(4), 1439
The Chemistry Behind Antibody-Drug ConjugationEran SellaBaran Lab
Group Meeting
11
Kadcyla (Trastuzumab emtansine)
HN
O
N
O
O
S
O
NO
O
O
O
O
HN
OCl
N
O
HOHMeO
ClNMeO
O
OMe
O
O
OAcAcO
AcO1. NaOMe/MeOH2. HgOAc
75%O
OH
H
OMe
HOHO
AcOHg 1. NaCl2. NaBH43. HCl
99%
OOH
H
OMe
HOHO
1. TrtCl, Pyridine2. NaH (4 eq.)3. isopropylbenzene sulfonyl imidazole
75%
OOTrt
H
OMeO
MeLi, CuI OOTrt
H
OMeOH
SH
SH
CHCl3/HCl96%
HO
S S
OH
HO OEt
BF3 Et2O86%
O
S S
OH
O MEMCl
99%
O
S S
OMEM
O
1. nBuLi2. NaH, MeI
MeOCl
NO
O
O
OMe
OMe
OMEMSS
1. CH3SLi2. HClO43. Pb(OAc)4
MeOCl
NHO
OMe
OMEMSS
TMSLi N
then Pyridinium chloride82%
MeOCl
NH
OMe
OMEMSS
O
PO O
OMeMeOMeO
Li
1.
2. Bu4NOH3. MesSO2Cl4. 10% H2SO4
MeOCl
N
OMe
OHSS
O
1. PNPCl, Pyridine2. NH4OH / t-butanol3. HgCl2, CaCO3
76%
MeOCl
N
OMe
O
O
NHOH
O
(-)-N-Methylmeysenine
O
S S
OMEM
O
CoreyJACS, 1978, 100(9), 2916Tel. Lett. 1978, 12, 1051JACS, 1980, 102(4), 1439
The Chemistry Behind Antibody-Drug ConjugationEran SellaBaran Lab
Group Meeting
12
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
HO
NHCO2Me
OSS Sugar
O
HO
NHCO2Me
OSugar
S
BergmanCycloaromatization
O
HO
NHCO2Me
OSugar
S
DNA
O
HO
NHCO2Me
OSugar
S
DNA diradicalO2
DNA scission
initiation
The Chemistry Behind Antibody-Drug ConjugationEran SellaBaran Lab
Group Meeting
13
O
NHCO2Me
OHBzO
Et3Si
OO
Pd C-C
acetyilide-aldehydecondensation
O O
HOtetronic acid
ethylene glycolpTsOH
60%
O O
OO
DIBAL
84%
O
OO
OH
MEMO s-BuLi
MEMO LiIpc2BOMe
OMEM
BIpc2
(Z)
H2O2, NaOHHO
OO OH
OMEM
1. TBSCl, Im2. PhCOCl, Pyr
TBSOOO OBz
OMEM 1. TBAF2. Swern3. NH2OH N
OO OBz
OMEMHO NaOCl
NO
BzOOMEM
OO
ON
HOMEM
BzO
O O
isoxazoline51% after separation
1. NaOMe2. CrO3, H2SO4
3.
TMS Li4. Ac2O5. ZnBr2
ON
HOH
O O
AcO
TMS
1. Swern2. PPh3=CHCO2Me
ON
O O
AcO
TMS CO2Me
Nicolaou, JACS, 1992, 114, 10082
The Chemistry Behind Antibody-Drug ConjugationEran SellaBaran Lab
Group Meeting
14
O
NHCO2Me
OHBzO
Et3Si
OO
Pd C-C
acetyilide-aldehydecondensation
ON
O O
AcO
TMS CO2Me
1. K2CO3/MeOH2. TES triflate
3. Cl
TMSPd(PPh3)4, CuI
91%
OO
ON
OEt3Si
TMS
CO2Me
1. Mo(CO)62. K2CO3
OO
CHO
NH2O
Et3Si
H
CO2Me
82%
O
O
ClCl
PyridineMeNO2
OON
OEt3Si
H
CO2Me
O O
O
1. SiO22. Ac2O O
NPhth
CHO
CO2Me
Et3Si
OO
O
NPhth
OHCO2Me
Et3Si
OO
H
KHMDS-90o
48%
1. MsCl2. SiO2, Pyr O
NPhth
Et3Si
OO
OO
1. MeNHNH2 (99%)2. Triphosgene, Pyr/MeOH (82%)3. DIBAL, NaBH4 (82%)4. PhCOCl, Pyr (84%)
O
NHCO2Me
OHBzO
Et3Si
OO
Nicolaou, JACS, 1992, 114, 10082
