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The Advent ofAntibody-Drug Conjugates
MacMillan Group Meeting4 June 2015
Tracy Liu
Traditional Cancer TherapyThe Double Edged Sword
Anti-cancer treatments should be as aggressive as possible to fully eradicate the tumor...
...but it is precisely this aggressiveness that often causes severe side effects
N
N N
NN
NH
O
CO2H
CO2H
Me
NH2
H2N
methotrexateanti-folate
HN NH
O
OF
5-fluorouracilthymidylate synthase
inhibitor
PtCl NH3
Cl NH3
cisplatinDNA crosslinking agent
Common Chemotherapeutic Agents
■ lack of tumor selectivity - killing of proliferating normal cells
■ requires administration at near the maximum tolerated dosage
■ 99% of cells in a tumor must be killed to achieve complete remission
Traditional Cancer TherapyMaximizing the Therapeutic Window
Limited clinical efficacy of chemotherapeutics is due to an insufficient therapeutic window -
lack of ability to kill enough cancel cells without causing toxicity to normal cells
Current most critical need: Maximization of the Therapeutic Window
Dosage
Maximum Tolerated Dose (MTD)
Minimum Effective Dose (MED)Therapeutic Window
increase MTD = increase selectivity
decrease MED = increase potency
Angew. Chem. Int. Ed. 2014, 53, 3796.
Targeted Cancer TherapyDirect Approaches
Targeting tumor-associated or specific proteins to directly alter their signaling by:
■ direct binding of monoclonal antibody to ■ binding of small molecule drugs to activeantigen expressed on tumor cell surface site of a protein to disrupt normal function
monoclonal antibody
antigen
tumor cell
HN
NH
O
OF
protein
Nature Rev. 2006, 5, 147.
to induce immune responses
Targeted Cancer TherapyIndirect Approaches
that function as a targeting platform for fusion proteins bearing different effector molecules
■ antibody-directed enzyme prodrug therapy
■ seletive activation of a mildly toxic prodrug to a toxic drug at the tumor site through
Reliance on proteins specifically expressed or overexpressed on tumor cell surfaces
monoclonal antibody (fusion protein)
tumor cell
antigen (overexpressed protein)
prodrug
drug
conjugation of an enzyme to a tumor-specific antibody
= enzyme
Nature Rev. 2006, 5, 147.
Targeted Cancer TherapyIndirect Approaches
that function as a targeting platform for fusion proteins bearing different effector molecules
small molecule drug (antibody-drug conjugate)
Reliance on proteins specifically expressed or overexpressed on tumor cell surfaces
toxin (immunotoxins)radionucleotides (radioimmuno conjugate)
immunoregulatory cytokines (antibody-cytokine fusion protein)
effector molecule
Nature Rev. 2006, 5, 147.
monoclonal antibody (fusion protein)
tumor cell
antigen (overexpressed protein)
effector molecule
500
1000
1500
2000
1900-19
10
1911-19
20
1921-19
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1931-19
40
1941-19
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1951-19
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1971-19
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1981-19
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1991-20
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2001-20
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2011-present
19961686
787464
471020000
Antibody-Drug ConjugatesA Brief Introduction and History
Anatomy of an Antibody-Drug Conjugate
A. Antibody
B. Linker
C. Small-molecule drug warhead
Number of Publications in Antibody-Drug Conjugate Research1900 - present day
A
B C
Antibody-Drug ConjugatesA Brief Introduction and History
Nature Rev. 2006, 5, 147.; Bioconjugate Chem. 2015, 26, 176.
2000199019801970
1975 - advent ofmurine mAB
1987 - first1997 - first therapeutic mAB(unconjugated) - Rituxan
chimeric mAB Genentech/Biogen
2010
2000 - first FDAapproved ADC - MylotargWyeth (Pfizer)
2010 - Pfizerwithdraws Mylotarg
2011 - FDA approvesAdcetrisSeattle Genetics
2010 - FDAapproval ofKadcylaGenentech/Immunogen
mAB = monoclonal antibodyADC = antibody-drug conjugate
Part I.First Generation
Antibody-Drug Conjugates
Part II.Second Generation
Antibody-Drug Conjugates
Part III.Current Challenges
and Overview ofClinical Performanceand Their Improvementsand Lessons Learned
Antibody-Drug ConjugatesA Brief Introduction and History
Nature Rev. 2006, 5, 147.; Bioconjugate Chem. 2015, 26, 176.
2000199019801970
1975 - advent ofmurine mAB
1987 - first1997 - first therapeutic mAB(unconjugated) - Rituxan
chimeric mAB Genentech/Biogen
2010
2000 - first FDAapproved ADC - MylotargWyeth (Pfizer)
2010 - Pfizerwithdraws Mylotarg
2011 - FDA approvesAdcetrisSeattle Genetics
2010 - FDAapproval ofKadcylaGenentech/Immunogen
mAB = monoclonal antibodyADC = antibody-drug conjugate
Part I.First Generation
Antibody-Drug Conjugates
Part II.Second Generation
Antibody-Drug Conjugates
Part III.Current Challenges
and Overview ofClinical Performanceand Their Improvementsand Lessons Learned
Deconstruction of Antibody-Drug ConjugatesKey Domains of the Immunoglobulin G Antibody Scaffold
Angew. Chem. Int. Ed. 2014, 53, 3796.
■ represents 75% of serum antibodies in humans
The majority of antibody-drug conjugates are built upon the immunoglobulin G (IgG) scaffold
■ protein complex of 4 peptide chains in a Y shape2 identical heavy chains (light purple)2 identical light chains (dark purple)
hinge
antigen binding complementaritydetermining region (CDR)
FC region
region
Fab region ■ Fab = Fragment antigen-binding domain
■ Fc = Fragment crystallizable/constant domainIdeal location for drug conjugation - far from CDR
Domains of a Typical IgG Antibody
Consists of variable (V) and constant (C) domains
Antigen binding CDR domains found at termini
C
V
CH2 domain
Consists of a CH2 domain and a CH3 domain
CH3 domain
Deconstruction of Antibody-Drug ConjugatesKey Domains of the Immunoglobulin G Antibody Scaffold Relevant to Drug Conjugation
Bioconj. Chem. 2015, 26, 176.
■ Linking through native cysteine residues
S SS
SS
S
S S
Domains of a Typical IgG Antibody
functional groups present on the surface of the antibodyNature of the chemistry between antibody and linker is primarily determined by the naturally occuring
Deconstruction of Antibody-Drug ConjugatesTraditional Methods of Linker Conjugation to Antibody
Bioconj. Chem. 2015, 26, 176.
■ High nucleophilicity of sulfur - naturally high reactivity for conjugation chemistry■ Low abundance of cysteine in primary sequence - easier control of drug to antibody ratio (DAR)
Linking through native Cysteine residues
Pros
■ No free thiols naturally present - partial reduction required■ Selective reduction of the 4 interchain disulfide bridges is most common, but this partial reduction
Cons
can result in a destabilized antibody
S S SH HS
4 interchain disulfide bridges - easier to reduce12 intrachain disulfide bridges - harder to reduce
Deconstruction of Antibody-Drug ConjugatesTraditional Methods of Linker Conjugation to Antibody
Linking through native Cysteine residues
HSSH
OH
OH
dithiothreitol (DTT)
PCO2HHO2C
CO2H
tris(2-carboxyethyl)phosphine (TCEP)
HOSH
2-mercaptoethanol
Common disulfide bridge reducing agents
SS
HO
HO PO
CO2HHO2C
CO2H
HOS
SOH
Corresponding oxidized byproducts
Deconstruction of Antibody-Drug ConjugatesKey Domains of the Immunoglobulin G Antibody Scaffold Relevant to Drug Conjugation
Bioconj. Chem. 2015, 26, 176.
■ Linking through native cysteine residues
4 interchain disulfide bridges - easier to reduce12 intrachain disulfide bridges - harder to reduce
S SS
SS
S
S S
Domains of a Typical IgG Antibody
functional groups present on the surface of the antibody
Requires reduction to access free thiol
Nature of the chemistry between antibody and linker is primarily determined by the naturally occuring
NH2
NH2
NH2
NH2
Deconstruction of Antibody-Drug ConjugatesKey Domains of the Immunoglobulin G Antibody Scaffold Relevant to Drug Conjugation
Bioconj. Chem. 2015, 26, 176.
■ Linking through native cysteine residues
4 interchain disulfide bridges - easier to reduce12 intrachain disulfide bridges - harder to reduce
■ Linking through native lysine residues
Domains of a Typical IgG Antibody
functional groups present on the surface of the antibody
Requires reduction to access free thiol
Nature of the chemistry between antibody and linker is primarily determined by the naturally occuring
Deconstruction of Antibody-Drug ConjugatesTraditional Methods of Linker Conjugation to Antibody
Garbaccio, R. M. Chemistry of Antibody-Small Molecule Drug Conjugates. From Comprehensive Organic Synthesis II, 2014, 9, 438.
■ Naturally nucleophilic functional handle
Linking through native Lysine residues
Pros
■ Greater natural abundance of lysine - control of drug to antibody ratio significantly more difficult
■ Low levels of competitive cysteine and tyrosine conjugation observed in some cases
Cons
H2N OH
OH2N OH
O
HS
H2N OH
O
HONH2
■ No requirement for pre-functionalization prior to conjugation with linker
Lysine Cysteine Tyrosine
~86 lysine residues total spanning all domains~20 accessible for functionalization
NH2
NH2
NH2
NH2
Deconstruction of Antibody-Drug ConjugatesKey Domains of the Immunoglobulin G Antibody Scaffold Relevant to Drug Conjugation
Bioconj. Chem. 2015, 26, 176.
■ Linking through native cysteine residues
4 interchain disulfide bridges - easier to reduce12 intrachain disulfide bridges - harder to reduce
■ Linking through native lysine residues~86 lysine residues total spanning all domains
Domains of a Typical IgG Antibody
functional groups present on the surface of the antibody
Requires reduction to access free thiol
~20 accessible for functionalization
Nature of the chemistry between antibody and linker is primarily determined by the naturally occuring
Deconstruction of Antibody-Drug ConjugatesKey Domains of the Immunoglobulin G Antibody Scaffold Relevant to Drug Conjugation
Bioconj. Chem. 2015, 26, 176.
■ Linking through native cysteine residues
4 interchain disulfide bridges - easier to reduce12 intrachain disulfide bridges - harder to reduce
■ Linking through native lysine residues
■ Linking through conserved glycans in CH2 domain
~86 lysine residues total spanning all domains
Domains of a Typical IgG Antibody
functional groups present on the surface of the antibody
Requires reduction to access free thiol
~20 accessible for functionalizationAsn
OH
FucoseGlcNAcMannose
Nature of the chemistry between antibody and linker is primarily determined by the naturally occuring
Deconstruction of Antibody-Drug ConjugatesTraditional Methods of Linker Conjugation to Antibody
Bioconj. Chem., 2015, 26, 176.
■ Post-translational glycosylation of N297 (Asparagine) residue provides facile site-specific conjugation
Linking through native Glycan residues
Pros
■ Glycosylation is a heterogenous post-translational modification - difficult to control drug to antibody ratio■ Requires pre-oxidation of vicinal diol moiety on glycan to access the bioorthogonal aldehyde handle
Cons
■ Glycosylation site is in CH2 domain - well removed from antigen binding domain
O HN
ON297NHAc
O
O
OH
MeOH
OH
OHO
O
NHAc
OHOHO
OOH
OHO
O
O
OHOH
OH
O OHOH
OHO
OO
O
NHAc
OHOHO
OH
NHAcHOO
O
O
OH
OHOH
O
OH
OHOH
OO
HO
AcHN
HO OH
HO
CO2
O
HO
AcHN
HO OH
HO
CO2 fully elaborated glycan
fucose
GlcNAcGlcNAc
mannose
GlcNAcgalactosesialic acid
NH2
NH2
NH2
NH2
Deconstruction of Antibody-Drug ConjugatesKey Domains of the Immunoglobulin G Antibody Scaffold Relevant to Drug Conjugation
Bioconj. Chem. 2015, 26, 176.
■ Linking through native cysteine residues
4 interchain disulfide bridges - easier to reduce12 intrachain disulfide bridges - harder to reduce
S SS
S
■ Linking through native lysine residues
■ Linking through conserved glycans in CH2 domainPost translational modification glycosylates N297
SS
S S
~86 lysine residues total spanning all domains
Domains of a Typical IgG Antibody
functional groups present on the surface of the antibody
Requires reduction to access free thiol
~20 accessible for functionalization
Oxidation of terminal sugar furnishes aldehyde
Asn
OH
FucoseGlcNAcMannose
Nature of the chemistry between antibody and linker is primarily determined by the naturally occuring
Deconstruction of Antibody-Drug ConjugatesMore on the Antibody
Angew. Chem. Int. Ed. 2014, 53, 3796.
replacement of protein sequences of a mouse antibody with naturally occuring
Advances in recombinant DNA technology have enabled the generation of engineered antibodies
murine mAB chimeric mAB humanized mAB human mAB
mouse CDR human CDR
sequences in humans significantly reduces undesired immune responses
increasing humanization
Fc region of murine mABreplaced with corresponding
human constant domain
only the essentialmurine CDR regions
are retained
First Generation Antibody-Drug ConjugatesTransition from Chemotherapeutics
Adv. Drug Delivery Rev., 1998, 31, 89.
In an attempt to achieve greater selectivity for chemotherapy drugs, first generation antibody-drug conjugates took clinically established cancer drugs as warheads
N
N N
NN
NH
O CO2H
Me
NH2
H2N
methotrexateanti-folate
N
N
OAc
Me
HO CO2MeR
H
MeO
NH
NOH
Me
CO2Me
R = MeR = CHO
vinca alkaloidsanti-mitotic/microtubule agent
KS1/4 - methotrexateKS1/4 - DAVLB
KS1/4 - DAVLBHYDBR96 - doxorubicin
antibody-drug conjugates
O
OH
Survey of 4 clinically evaluated first generation
O
O
O
OOMe OH
OH
MeOH
NH2
OHO
OH
doxorubicinantibiotic
First Generation Antibody-Drug ConjugatesKS1/4S2 - Methotextrate Conjugate
Am. J. Respir. Crit. Care Med., 1994, 150 , 1114.
N
N N
NN
NH
O CO2H
Me
NH2
H2N
O
OH
Lys
KS1/4S2 murine mAB
H2N
methotrexate
LysHN
ONH
O CO2H
NMe
N
N
N
N
NH2
H2N
KS1/4-methotrexate through non-selective EDC coupling
■ significant localization to tumor
■ Phase I clinical trials revealed
■ murine mAB illicited a humananti-murine antibody (HAMA)response in patients
little therapeutic benefit potentiallydue to non-cleavable linker
■ non-cleavable amide linker formed
First Generation Antibody-Drug ConjugatesKS1/4 – 4-Desacetylvinblastine Conjugates
Cancer Res., 1991, 51, 2286.
N
N
OH
Me
HO CO2MeMe
H
MeO
NH
NOH
Me
CO2Me
4-desacetylvinblastine
OO O
Lys
succinic anhydride KS1/4S2 murine mAB
N
N
OEt
HO CO2MeMe
H
MeOO
HN
O
Lys
H2N
KS1/4-DAVLB ■ highly potent in vivo activity with
■ Phase I clinical trials using radiolabeled
■ no increased therapeutic effect
conjugate indicates localization ofdrug to tumor cells
■ patients developed immune responsesto both the antibody and vinca alkaloid
Clin. Pharmacol. Ther., 1990, 47, 36.
■ esterase-labile hemisuccinate linker
greater efficacy than unconjugated drug
First Generation Antibody-Drug ConjugatesKS1/4 – 4-Desacetylvinblastine Conjugates
Cancer Res., 1991, 51, 2286.
N
N
OH
Me
HOMe
H
MeO
NH
NOH
Me
CO2Me
4-desacetylvinblastine derivative
Asn
KS1/4S2 murine mABO
NHNH2
OH
treated with NaIO4
FucoseGlcNAcMannose
N
N
OHEt
HOMe
H
MeO
O
HN
Asn
NH
KS1/4-DAVLBHYD ■ cleavable acid-labile hydrazone linker
■ Phase I clinical trials indicates localization
■ no increased therapeutic effect - premature
drug to tumor cells
■ patients developed immune responsesto both the antibody and vinca alkaloid
■ highly potent in vivo activity withgreater efficacy than unconjugated drug
cleavage of hydrazone
SN
O
O
O
NHN
OHOH
OMe
O
O
OH
OH O
HS
First Generation Antibody-Drug ConjugatesBR96 - Doxorubicin Conjugate
J. Clin. Oncol., 1999, 17 , 478.
O
O
O
OOMe OH
OH
MeOH
NH2
OHO
OH
doxorubicin BR96 chimeric mAB
HN
ON
O
O
H2N
linker with hydrazide and maleimide
BR96 - doxorubicin■ highly potent in vivo activity with
■ advanced to Phase II clinical trials
■ significant gastrointestinal toxicity -
■ 50% of patients developed immune
■ acid-labile hydrazone linker
greater efficacy than unconjugated drug
responses despite chimeric mAB
target antigen expression on normaltissue cells
First Generation Antibody-Drug ConjugatesUniversal Shortcomings and Lessons Learned
Acc. Chem. Res., 2008, 41, 98.
I. Low in vitro potency - conjugation results in decreased cytotoxicity compared to free drug
All four case studies successfully demonstrated localization of drug payload to tumor sites,
■ Limited expression of antigen - tumor cells typically express 1 x 105 receptors/cell
■ Different mechanisms of cellular uptake
■ Need 106 molecules/cell of a moderately potent cytotoxic drug to effect cell kill
■ Conjugated drugs require efficient internalization after binding to antigen
■ Free drugs can diffuse through cell membrane
but in all cases no significant improvement in therapeutic activity was observed...
II. Stability of the linker was inadequately tuned■ Hydrazone linkers were too labile - prone to cleavage prior to cellular uptake■ Amide linker not labile enough - no cleavage to release drug after internalization
III. Antibodies of murine or chimeric origin illicited undesired immune response■ Generation of human anti-murine antibodies■ Rapid clearance of antibody-drug conjugate upon repeat dosing
Improving Antibody-Drug ConjugatesIdeal Characteristics of an Antibody-Drug Conjugate
Acc. Chem. Res., 2008, 41, 98.
A
B C
A. Antibody
B. Linker
C. Small-molecule drug
■ selective for antigens with highcopy numbers (>105/cell) on
■ induces minimal immunogenicresponse
■ homogeneous expression of
■ Stable to circulation in vivo
■ Selectively cleaved only onceinternalized inside target cell
■ Stable to long-term storagein aqueous environments
■ highly potent in vitro - potencyin picomolar range required
■ amenable to introduction offunctional groups for linking
■ water soluble
target cell
■ selective for antigens uniquelyexpressed on tumor cell
antigen on tumor cell
disulfide linkersprotease labile linkers
■ Designed to release drug inits active form (without linkers)
self immolative linkers
■ sensitive to the ideal mechanismof action for specific tumor types
Antibody-Drug ConjugatesA Brief Introduction and History
Nature Rev. 2006, 5, 147.; Bioconjugate Chem. 2015, 26, 176.
2000199019801970
1975 - advent ofmurine mAB
1987 - first1997 - first therapeutic mAB(unconjugated) - Rituxan
chimeric mAB Genentech/Biogen
2010
2000 - first FDAapproved ADC - MylotargWyeth (Pfizer)
2010 - Pfizerwithdraws Mylotarg
2011 - FDA approvesAdcetrisSeattle Genetics
2010 - FDAapproval ofKadcylaGenentech/Immunogen
mAB = monoclonal antibodyADC = antibody-drug conjugate
Part I.First Generation
Antibody-Drug Conjugates
Part II.Second Generation
Antibody-Drug Conjugates
Part III.Current Challenges
and Overview ofClinical Performanceand Their Improvementsand Lessons Learned
Second Generation Antibody-Drug ConjugatesMost Commonly Used Drug Payloads
Angew. Chem. Int. Ed., 2014, 53, 3796.
Me ONH
O
O
Me
OH
ClMeO HN
O
OO
ON
MeR
OMe
Me
MeH
maytansanoidsanti-mitotic agent
MeNMe
iPrHN
O iPr
O
NMe
MeMe
OMe O
N
OMe
Me
OR2
R1
auristatinsanti-mitotic agent
MeX
OMeOMe
O
S
O
O
OMe
OH
O
OHMeOHO
Me
NH
OMe
OO
HOO
MeO
HNR
OHO
MeSSScalicheamicins
DNA damaging agent
Second Generation Antibody-Drug ConjugatesMaytansanoid Antibody-Drug Conjugates
Angew. Chem. Int. Ed., 2014, 53, 3796.
Me ONH
O
O
Me
OH
ClMeO HN
O
OO
ON
MeMe
OMe
Me
MeH
maytansineanti-mitotic agent
■ 1000 fold more cytotoxic than first generation payloads
resulting in cell arrest in G2/M phase
■ Good aqueous solubility
■ SAR activity indicates that the ester at C3 can be derivatizedfor linker conjugation without impacting drug activity
3
OOH
3
Me
O
maytansinol
OO
3
Me
O
ON
Me
Me
O
SHHO
ONMe
O
SS
Me
Me
1.
2. DTT reduction
■ Binds tubulin to suppress microtubule dynamics,
H2N Lys
Second Generation Antibody-Drug ConjugatesMaytansanoid Antibody-Drug Conjugates
Angew. Chem. Int. Ed., 2014, 53, 3796.
Me ONH
O
O
Me
OH
ClMeO HN
O
OO
ON
Me
OMe
Me
MeH
maytansine
3
SH
O
ONS
SNR4R3
O
O
humanized C242 mAB
R1R2
heterobifunctional linker
OO
3
Me
O
ON
Me
Me
O
SS
R3 R4
R1 R2 O
HN
OO
3
Me
O
ON
Me
Me
O
SS
R3 R4
R1 R2 O
HN Lys
Second Generation Antibody-Drug ConjugatesMaytansanoid Antibody-Drug Conjugates
Angew. Chem. Int. Ed., 2014, 53, 3796.
Improved mechanism of selective drug release
NH
OSH
O
HN CO2HHO2C
NH2
glutathione
■ glutathione naturally present in high concentration inside tumorcells (millimolar range), but exceptionally low (micromolar range)
disulfide bond reducing agent
in blood stream - selective cleavage upon internalization
■ disulfide linkage is stable under physiological pH
■ stability of the antibody-drug conjugate can be tuned byvarying the sterics of the R groups flanking the disulfide bond
OO
3
Me
O
ON
Me
Me
O
SS
O
HN Lys
Second Generation Antibody-Drug ConjugatesMaytansanoid Antibody-Drug Conjugates
Acc. Chem. Res., 2008, 41, 98.
Study on effect of linker stability to therapeutic efficacy
Me
OO
3
Me
O
ON
Me
Me
OS
SNH
O
Lys
OO
3
Me
O
ON
Me
Me
OS
SMe Me
NH
O
Lys
Meincreasingtherapeutic
efficacy
HuC242-DM1
HuC242-DM3
HuC242-DM4
increasedstability in
blood stream
Extreme case - synthesis of a non-cleavable conjugate?
Lys
Second Generation Antibody-Drug ConjugatesMaytansanoid Antibody-Drug Conjugates
Angew. Chem. Int. Ed., 2014, 53, 3796.
Me ONH
O
O
Me
OH
ClMeO HN
O
OO
ON
Me
OMe
Me
MeH
maytansine
3
SH
O
ON
O
O
humanized C242 mABSMCC (non-cleavable) linker
OO
3
Me
O
ON
Me
Me
OO
HN
N
O
O
N
O
O
S
H2N
cyclohexane-1-carboxylatesuccinimidyl 4-(N-maleimidomethyl)
HuC242-MCC-DM1
Second Generation Antibody-Drug ConjugatesMaytansanoid Antibody-Drug Conjugates
Acc. Chem. Res., 2008, 41, 98.
time (hrs) post administration
% injected dose
plas
ma
conc
entra
tion
(µg/
mL)
tum
or s
ize
(mm
3 )
days post tumor inoculation
■ HuC242-DM4 shows greatest cytotoxicity■ HuC242-MCC-DM1 shows greatest stability(in vivo half life of 134 hrs)
What is the mechanism of action of these maytansanoid antibody-drug conjugates?
Second Generation Antibody-Drug ConjugatesMaytansanoid Antibody-Drug Conjugates
Angew. Chem. Int. Ed., 2014, 53, 3796.
Cellular Processing of Disulfide Linked Maytansanoids
NH
LysO
Me
SS
MayR1 R2
X
lysosomalprocessing
NH
O
Me
SS
MayR1 R2
XH3N
CO2
disulfidereduction
HSMay
R1 R2
X
S-methylation
SMay
R1 R2
X
bystander killing
bystander killingMe
NH
LysO
N
O
O
S May
lysosomalprocessing
NH
O
N
O
O
S MayCO2
H3N
poor bystander killingcharged nature prevents diffusion into
neighboring cells
good
excellent
Second Generation Antibody-Drug ConjugatesMaytansanoid Antibody-Drug Conjugates
Angew. Chem. Int. Ed., 2014, 53, 3796.
Enhanced Therapeutic Efficacy of Cleavable Disulfide Linkers is Due to Bystander Cell Killing
Second Generation Antibody-Drug ConjugatesAuristatin Antibody-Drug Conjugates
Angew. Chem. Int. Ed., 2014, 53, 3796.
MeNMe
iPrHN
O iPr
O
NMe
MeMe
OMe O
N
OMe
Me
O
dolastatin 10anti-mitotic agent
■ fully synthetic series of highly potent
derivatized for conjugation AND terminal
■ inhibits tubulin-dependent GTP binding andmicrotubule dynamics
■ SAR indicates terminal 3º amine can be
anti-mitotic agents based on SAR studiesHN N
S
Ph
phenethyl amine can be changed without
on dolastatin 10
■ inhibits tubulin-dependent GTP binding and
loss of efficacy
MeNH
iPrHN
O iPr
O
NMe
MeMe
OMe O
N
OMe
Me
O
HN R1
PhR2
monomethyl auristatin E (MMAE) (R1 = Me, R2 = OH)monomethyl auristatin F (MMAF) (R1 = CO2H, R2 = H)
SH
Second Generation Antibody-Drug ConjugatesAuristatin Antibody-Drug Conjugates
Angew. Chem. Int. Ed., 2014, 53, 3796.
MeNH
iPrHN
O iPr
O
NMe
monomethyl auristatin
HN
HN
H2NO
ONH
ON
O
ONH
O O LG
O
53
mal-caproyl-val-cit-PAB linkerchimeric cAC10 mAB
HN
HN
H2NO
ONH
ON
O
ONH
O O
O
53
N
iPrHN
O iPr
O
NMe
MeMe
OMe O
N
OMe
Me
O
HN Me
PhHOMeS
Mal-caproyl-val-cit-PAB-MMAE
Second Generation Antibody-Drug ConjugatesAuristatin Antibody-Drug Conjugates
Angew. Chem. Int. Ed., 2014, 53, 3796.
Improved mechanism of selective drug release
■ Valine-citrulline dipeptide moiety is known to be selectively cleaved by the protease cathepsin B
■ p-aminobenzyl group is self immolating - fragments to release the MMAE drug without any residual groups
HN
HN
H2NO
ONH
ON
O
ONH
O O
O
53
N
iPrHN
O iPr
O
NMe
MeMe
OMe O
N
OMe
Me
O
HN Me
PhHOMeS
Mal-caproyl-val-cit-PAB-MMAEmal-caproyl
valine-citrulline dipeptide
p-aminobenzyl
Second Generation Antibody-Drug ConjugatesAuristatin Antibody-Drug Conjugates
Angew. Chem. Int. Ed., 2014, 53, 3796.
HN
HN
H2NO
ONH
ON
O
ONH
O O
O
53
N
iPrHN
O iPr
O
NMe
MeMe
OMe O
N
OMe
Me
O
HN Me
PhHOMeS
Mal-caproyl-val-cit-PAB-MMAEmal-caproyl
valine-citrulline dipeptide
p-aminobenzyl
Mechanism of cellular processing of mal-caproyl-val-cit-MMAE conjugate
cathepsin Bmediated peptide
cleavage
H2N
O
O
N
iPrHN
O iPr
O
NMeMe
NH
iPrHN
O iPr
O
NMe
Meself immolation
-PAB-CO2
free MMAEgood bystander killing
SH
Second Generation Antibody-Drug ConjugatesAuristatin Antibody-Drug Conjugates
Angew. Chem. Int. Ed., 2014, 53, 3796.
MeNH
iPrHN
O iPr
O
NMe
monomethyl auristatin
N
O
O
mal-caproyl (non-cleavable) linkerchimeric cAC10 mAB
N
iPrHN
O iPr
O
NMe
MeMe
OMe O
N
OMe
Me
O
HN
PhMe
Mal-caproyl-MMAF
O
ON
O
O
ON
O
O
SCys OH
O
Second Generation Antibody-Drug ConjugatesAuristatin Antibody-Drug Conjugates
Angew. Chem. Int. Ed., 2014, 53, 3796.
N
iPrHN
O iPr
O
NMe
MeMe
OMe O
N
OMe
Me
O
HN
PhMe
ON
O
O
SCys OH
O
Cellular Processing of Non-Cleavable Mal-caproyl-MMAF Conjugates
lysosomalprocessing
N
iPrHN
O iPr
O
NMe
MeMe
OMe O
N
OMe
Me
O
HN
PhMe
ON
O
O
S OH
OH3N
CO2
poor bystander killingcharged nature prevents diffusion into neighboring cells
Second Generation Antibody-Drug ConjugatesCalicheamicin Antibody-Drug Conjugates
Angew. Chem. Int. Ed., 2014, 53, 3796.
MeX
OMeOMe
O
S
O
O
OMe
OH
O
OHMeOHO
Me
NH
OMe
OO
HOO
MeO
HNR
OHO
MeSSS
calicheamicinsDNA damaging agent
■ insanely cytotoxic class of anti-tumor
minor groove of DNA, placing ene-
■ too toxic for use as drug warhead -
■ trisulfide converted to disulfide -provides a handle for conjugation
■ aryl tetrasaccharide moiety binds in
antibiotics (0.15µg/kg dose)
diyne warhead within double helix
20 fold less potent N-acetyl analoguedeveloped for applications to ADCs
calicheamicin β1Br - X = Br, R = iPrcalicheamicin γ1Br - X = Br, R = Etcalicheamicin γ1I - X = I, R = Et
N-acetyl calicheamicin γ1I - X = I
O
MeON
Me
O
Me
NHCO2Me
Second Generation Antibody-Drug ConjugatesCalicheamicin Antibody-Drug Conjugates
Angew. Chem. Int. Ed., 2014, 53, 3796.
MeI
OMeOMe
O
S
O
O
OMe
OH
O
OHMeOHO
Me
NH
OMe
OO
HOO
MeON
OHO
MeSSS
Me
O
Me
Mechanism of Action of the Calicheamicins
glutathionemediated
sugar O
OHO
S
Michael addn
sugar O
OHO
S
NHCO2Me
NHCO2Me
thiol reduction
NHCO2Mecycloaromatization
(relief of ring strain)
OHO
S
NHCO2Me
Osugar
OHO
S
NHCO2Me
Osugar
DNA
doublestranded DNA
diradical
O2 doublestranded DNA
cleavagecell death
Lys
Second Generation Antibody-Drug ConjugatesCalicheamicin Antibody-Drug Conjugates
Angew. Chem. Int. Ed., 2014, 53, 3796.
sugar O
OHO
NHCO2MeSSN
H
O MeMeH2N
hP67.6 humanized mAB
NH2Me
O
OO
ON
O
O
AcBut linker N-acetyl calicheamicin
LysHN
OO
Me
NHN
O Me Me
SS
sugar O
OHO
NHCO2Me
(4-(4'acetylphenyl)butanoic acid)
hP67.6 N-Ac- γ-calicheamicin DMH AcBut
Second Generation Antibody-Drug ConjugatesCalicheamicin Antibody-Drug Conjugates
Bioconj. Chem., 2002, 13 , 47.
LysHN
OO
Me
NHN
O Me Me
SS
sugar O
OHO
NHCO2Me
hP67.6 N-Ac- γ-calicheamicin DMH AcBut
Improved mechanism of selective drug release
■ Linker specifically designed to provide high stability prior to internalization into tumor cells, but is
cleavage via glutathione reduction upon internalization inside cell
only 6% hydrolysis observed at pH = 7.4
readily cleaved once inside the lysosome - hydrazone formed from ketone rather than aldehyde
97% hydrolysis observed at pH = 4.5 at 37ºC over 24 hrs
■ Inclusion of a hindered disulfide moiety in the linker provides a second handle for selective drug
Antibody-Drug ConjugatesA Brief Introduction and History
Nature Rev. 2006, 5, 147.; Bioconjugate Chem. 2015, 26, 176.
2000199019801970
1975 - advent ofmurine mAB
1987 - first1997 - first therapeutic mAB(unconjugated) - Rituxan
chimeric mAB Genentech/Biogen
2010
2000 - first FDAapproved ADC - MylotargWyeth (Pfizer)
2010 - Pfizerwithdraws Mylotarg
2011 - FDA approvesAdcetrisSeattle Genetics
2010 - FDAapproval ofKadcylaGenentech/Immunogen
mAB = monoclonal antibodyADC = antibody-drug conjugate
Part I.First Generation
Antibody-Drug Conjugates
Part II.Second Generation
Antibody-Drug Conjugates
Part III.Current Challenges
and Overview ofClinical Performanceand Their Improvementsand Lessons Learned
Current Trends in Research in Antibody-Drug ConjugatesControlling the Drug to Antibody Ratio (DAR) and Achieving Site-Specific Conjugation
Bioconj. Chem., 2015, 26, 176.
Though the underlying protein scaffold is constant in a heterogeneous population of
antibody-drug conjugates, each conjugate has its own set of pharmacokinetic, toxicity,
aggregation, antigen affinity, and drug release properties
Forefront of research in this field currently lies in achieving:
1. Populations of antibody-drug conjugates with a homogenous DAR
2. Site-specific conjugation of drugs on a given antibody
Current Trends in Research in Antibody-Drug ConjugatesMethods of Homogeneous Conjugation Using Natural Antibodies
Garbaccio, R. M. Chemistry of Antibody-Small Molecule Drug Conjugates. From Comprehensive Organic Synthesis II, 2014, 9, 438.
■ N-terminal conjugation leveraging differences in pKa between terminal and internal amino acids
HN
O
R
NH2N
HO
HO
Me
OPO32-
37 - 50 ºCphosphate buffer pH 6.5
HN
O
O
R
■ Though conjugation via this method is in the antigen binding domain, drug conjugation here doesnot seem to impact antigen recognition and binding
■ Limitation: Reaction sensitive to nature of terminal amino acid - works best for alanine, glycine, aspartateglutamate, and asparagine
■ Resulting ketone product can be easily further functionalized through reaction with oximes bearinga linker or drug
■ Limitation: Some antibodies may not be able to tolerate elevated temperatures required for transamination
Current Trends in Research in Antibody-Drug ConjugatesMethods of Homogeneous Conjugation Using Natural Antibodies
Garbaccio, R. M. Chemistry of Antibody-Small Molecule Drug Conjugates. From Comprehensive Organic Synthesis II, 2014, 9, 438.
■ Site-specific functionalization of glutamines through enzymatic conjugation
2. H2N–R, transaminase
■ Selectively functionalizes only Q295 residue (flanked by a consensus recognition sequence for abacterial transaminase)
■ Limitation: Requires deglycosylation in the CH2 domain prior to functionalization, which may impact
■ Q295 residue is distant from antigen binding domain
GlnO
NH2
1. Deglycosylation
GlnO
HN R
function and properties of the antibody-drug conjugate
Current Trends in Research in Antibody-Drug ConjugatesMethods of Homogeneous Conjugation Using Engineered Antibodies
Garbaccio, R. M. Chemistry of Antibody-Small Molecule Drug Conjugates. From Comprehensive Organic Synthesis II, 2014, 9, 438.
engineered Cys at positions
SH
engineered selenocysteines
SeHHSe
(more nucleophilic)with minimal interferenceto antibody function
CHOOHC
aldehyde taggingengineered recognition
sequence for formylglycineselectively at C-terminus
THIOMABs
generating enzyme (FGE)
C-terminal selenocysteine
OO
MeN3
Current Trends in Research in Antibody-Drug ConjugatesMethods of Homogeneous Conjugation Using Engineered Antibodies
Garbaccio, R. M. Chemistry of Antibody-Small Molecule Drug Conjugates. From Comprehensive Organic Synthesis II, 2014, 9, 438.
N3
H2NOH
Op-azido Phe
O
H2NOH
Opropynyl Tyr
Me
O
H2NOH
Op-acetyl Phe
Protein Engineering to Incorporate Unnatural Amino Acids
Clinically Successful Antibody-Drug ConjugatesFDA Approved Antibody-Drug Conjugates
Angew. Chem. Int. Ed., 2014, 53, 3796.
HN
HN
H2NO
ONH
ON
O
ONH
O O
O
53
N
iPrHN
O iPr
O
NMe
MeMe
OMe O
N
OMe
Me
O
HN Me
PhHOMeS
brentuximab vedotin (Adcetris)
Roche/Genentech/ImmunoGen
FDA approved in 2011 for Hodgkin lymphoma andanaplastic large cell lymphoma (ALCL)
OO
3
Me
OO
N
Me
Me
O
O
HN
N
O
O
S
Me
NH
O
OOH
OMe
Me
HNCl
MeO
ado-trastuzumab emtansine (Kadycla)
Seattle Genetics
FDA approved in 2013 for metastatic breast cancer
Clinically Successful Antibody-Drug ConjugatesFDA Approved Antibody-Drug Conjugates
Angew. Chem. Int. Ed., 2014, 53, 3796.
Wyeth/PfizerGemtuzumab ozogamicin (Mylotarg)
FDA approved in 2000 for acute lymphoblastic lukemia
MeI
OMeOMe
O
S
O
O
OMe
OH
O
OHMeOHO
Me
NH
OMe
OO
HOO
MeON
OHO
Me
O
Me
NHCO2Me
Lys NH
OO
Me
NNH
O Me Me
SS
Withdrawn in 2010 due to toxicity concernsand lack of improvement in patient survival time
Clinically Successful Antibody-Drug ConjugatesAntibody-Drug Conjugates Currently in Clinical Evaluations
Angew. Chem. Int. Ed., 2014, 53, 3796.
Candidate Drug Antigen Lead Indicator Developer/Partner