CL Antibody Drug Conjugates

The Advent of
Antibody-Drug Conjugates
MacMillan Group Meeting

4 June 2015
Tracy Liu
Traditional Cancer Therapy

The 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
Common Chemotherapeutic Agents
NH 2
O
HN
CO2H
NH
H 2N
N
H
N
Cl
CO2H
Cl
Pt
NH 3
NH 3
Me
5-fluorouracil
methotrexate
cisplatin
thymidylate synthase
inhibitor
anti-folate
DNA crosslinking agent
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 Therapy

Maximizing 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
increase MTD = increase selectivity
Maximum Tolerated Dose (MTD)

Dosage
Therapeutic Window
Minimum Effective Dose (MED)
decrease MED = increase potency
Angew. Chem. Int. Ed. 2014, 53, 3796.
Targeted Cancer Therapy

Direct Approaches
Targeting tumor-associated or specific proteins to directly alter their signaling by:
monoclonal antibody
O
F
HN
O
antigen
tumor cell
direct binding of monoclonal antibody to

antigen expressed on tumor cell surface
N
H
protein
binding of small molecule drugs to active

site of a protein to disrupt normal function
to induce immune responses

Nature Rev. 2006, 5, 147.

Indirect Approaches
Reliance on proteins specifically expressed or overexpressed on tumor cell surfaces
that function as a targeting platform for fusion proteins bearing different effector molecules
monoclonal antibody (fusion protein)
prodrug
antigen (overexpressed protein)
drug
tumor cell
= enzyme
antibody-directed enzyme prodrug therapy

seletive activation of a mildly toxic prodrug to a toxic drug at the tumor site through
conjugation of an enzyme to a tumor-specific antibody
Nature Rev. 2006, 5, 147.

Indirect Approaches
Reliance on proteins specifically expressed or overexpressed on tumor cell surfaces
that function as a targeting platform for fusion proteins bearing different effector molecules
monoclonal antibody (fusion protein)
effector molecule
antigen (overexpressed protein)
tumor cell
effector molecule
small molecule drug (antibody-drug conjugate)
toxin (immunotoxins)
radionucleotides (radioimmuno conjugate)
immunoregulatory cytokines (antibody-cytokine fusion protein)
Nature Rev. 2006, 5, 147.
A Brief Introduction and History
Anatomy of an Antibody-Drug Conjugate
A. Antibody
B
B. Linker
C. Small-molecule drug warhead
Number of Publications in Antibody-Drug Conjugate Research

1900 - present day
1996
2000
1686
1500
1000
787
464
500
en
0
11
20
20
01
-2
-p
re
s
01
0
19
91
-2
00
0
19
81
-1
99
0
19
71
-1
98
0
19
61
-1
97
0
19
51
-1
96
0
-1
41
19
47
95
0
19
31
-1
94
0
93
21
19
-1
92
19
11
-1
91
-1
00
19
0
0
0
0
1987 - first
chimeric mAB
1970
1980
1997 - first therapeutic mAB

(unconjugated) - Rituxan
Genentech/Biogen
1990
2011 - FDA approves

Adcetris
Seattle Genetics
2000
1975 - advent of
murine mAB
2010
2000 - first FDA

approved ADC - Mylotarg
Wyeth (Pfizer)
2010 - FDA
approval of
Kadcyla
Genentech/
Immunogen
2010 - Pfizer
withdraws Mylotarg
mAB = monoclonal antibody

ADC = antibody-drug conjugate
Part I.
Part II.
Part III.
First Generation
Second Generation
Current Challenges
and Overview of
and Lessons Learned
and Their Improvements
Clinical Performance
Nature Rev. 2006, 5, 147.; Bioconjugate Chem. 2015, 26, 176.
1987 - first
chimeric mAB
1970
1980

Genentech/Biogen
1990
2011 - FDA approves

Adcetris
Seattle Genetics
2000
1975 - advent of
murine mAB
2010
2000 - first FDA

Wyeth (Pfizer)
2010 - FDA
approval of
Kadcyla
Genentech/
Immunogen
2010 - Pfizer
withdraws Mylotarg

Part I.
Part II.
Part III.
First Generation
Second Generation
Current Challenges
and Overview of
and Lessons Learned
Deconstruction of Antibody-Drug Conjugates

Key Domains of the Immunoglobulin G Antibody Scaffold
The majority of antibody-drug conjugates are built upon the immunoglobulin G (IgG) scaffold
antigen binding complementarity
determining region (CDR)
hinge
region
represents 75% of serum antibodies in humans
protein complex of 4 peptide chains in a Y shape

2 identical heavy chains (light purple)
2 identical light chains (dark purple)
F ab region
CH 2 domain
F ab = Fragment antigen-binding domain

Consists of variable (V) and constant (C) domains
Antigen binding CDR domains found at termini
F C region
CH 3 domain
F c = Fragment crystallizable/constant domain

Ideal location for drug conjugation - far from CDR
Consists of a CH 2 domain and a CH 3 domain
Domains of a Typical IgG Antibody

Angew. Chem. Int. Ed. 2014, 53, 3796.

Key Domains of the Immunoglobulin G Antibody Scaffold Relevant to Drug Conjugation
Nature of the chemistry between antibody and linker is primarily determined by the naturally occuring
functional groups present on the surface of the antibody
Linking through native cysteine residues
S
S
S
S
S
S
S

Bioconj. Chem. 2015, 26, 176.

Traditional Methods of Linker Conjugation to Antibody
Linking through native Cysteine residues
Pros
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)
4 interchain disulfide bridges - easier to reduce
12 intrachain disulfide bridges - harder to reduce
Cons
No free thiols naturally present - partial reduction required
Selective reduction of the 4 interchain disulfide bridges is most common, but this partial reduction
can result in a destabilized antibody
SS
SH HS
Bioconj. Chem. 2015, 26, 176.

Linking through native Cysteine residues
Common disulfide bridge reducing agents
CO2H
OH
SH
HS
HO 2C
OH
dithiothreitol (DTT)
HO
CO2H
tris(2-carboxyethyl)phosphine (TCEP)
SH
2-mercaptoethanol
O
HO
HO
HO 2C
CO2H
S
CO2H
Corresponding oxidized byproducts
HO
OH


Requires reduction to access free thiol
S
S
S
S
S
S
S

Bioconj. Chem. 2015, 26, 176.


NH 2

NH 2
Linking through native lysine residues

NH 2
NH 2

Bioconj. Chem. 2015, 26, 176.

Linking through native Lysine residues
Pros
Naturally nucleophilic functional handle
No requirement for pre-functionalization prior to conjugation with linker
Cons
Greater natural abundance of lysine - control of drug to antibody ratio significantly more difficult
~86 lysine residues total spanning all domains
~20 accessible for functionalization
Low levels of competitive cysteine and tyrosine conjugation observed in some cases
Lysine
Cysteine
O
H 2N
Tyrosine
O
OH
H 2N
O
H 2N
OH
OH
HS
HO
NH 2
Garbaccio, R. M. Chemistry of Antibody-Small Molecule Drug Conjugates. From Comprehensive Organic Synthesis II, 2014, 9, 438.


NH 2

NH 2

NH 2

NH 2

Bioconj. Chem. 2015, 26, 176.



Asn
H
O
Fucose
GlcNAc
Mannose
Linking through conserved glycans in CH2 domain

Bioconj. Chem. 2015, 26, 176.

Linking through native Glycan residues
Pros
Post-translational glycosylation of N297 (Asparagine) residue provides facile site-specific conjugation
Glycosylation site is in CH2 domain - well removed from antigen binding domain
Cons
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
HO
OH
CO2 OH
O
HO HO
OH
fully elaborated glycan
OH
AcHN
OH
O
HO
O
O
OH
NHAc
OH
OH
O
O
OH
O
HO
HO
OH
CO2 OH
O
AcHN
HO HO
sialic acid
OH
O
O
OH
O
HO
O
O
NHAc
galactose
GlcNAc
OH
OH
O
HO
OH
OH
OH
OH
OH
Me
O
HO
NHAc
GlcNAc
OH
fucose
O
O
H
N
N 297
NHAc
GlcNAc
mannose
Bioconj. Chem., 2015, 26, 176.


NH 2
S
S
NH 2
S
S
S
S
S

NH 2
Asn
H
O
NH 2
Fucose
GlcNAc
Mannose
Linking through conserved glycans in CH2 domain

Post translational modification glycosylates N297
Oxidation of terminal sugar furnishes aldehyde

Bioconj. Chem. 2015, 26, 176.

More on the Antibody
Advances in recombinant DNA technology have enabled the generation of engineered antibodies
mouse CDR
murine mAB
human CDR
chimeric mAB
humanized mAB
F c region of murine mAB

replaced with corresponding
human constant domain
only the essential

murine CDR regions
are retained
human mAB
increasing humanization
replacement of protein sequences of a mouse antibody with naturally occuring
sequences in humans significantly reduces undesired immune responses
Angew. Chem. Int. Ed. 2014, 53, 3796.
First Generation Antibody-Drug Conjugates

Transition from Chemotherapeutics
In an attempt to achieve greater selectivity for chemotherapy drugs, first generation antibody-drug
conjugates took clinically established cancer drugs as warheads
NH 2
H 2N
OH
N
H
OH
CO2H
OH
O
OH
N
Me
OMe O
OH
methotrexate
NH 2
O
anti-folate
OH
Me
OH
N
N
H
MeO
doxorubicin
Me
R = Me
R = CHO
Survey of 4 clinically evaluated first generation

antibody-drug conjugates
CO2Me
N
Me
H
N
R
OAc
HO
antibiotic
CO2Me
vinca alkaloids
anti-mitotic/microtubule agent
KS1/4 - methotrexate
KS1/4 - DAVLB
KS1/4 - DAVLBHYD
BR96 - doxorubicin
Adv. Drug Delivery Rev., 1998, 31, 89.

KS1/4S2 - Methotextrate Conjugate
CO2H
O
NH 2
N
N
H 2N
OH
N
H
N
Me
methotrexate
KS1/4S2 murine mAB
non-cleavable amide linker formed

through non-selective EDC coupling
KS1/4-methotrexate
NH 2
N
H 2N
N
H
N
Me
significant localization to tumor
CO2H
H 2N Lys
H
N Lys
O
Phase I clinical trials revealed

little therapeutic benefit potentially
due to non-cleavable linker
murine mAB illicited a human
anti-murine antibody (HAMA)
response in patients
Am. J. Respir. Crit. Care Med., 1994, 150 , 1114.

KS1/4 4-Desacetylvinblastine Conjugates
OH
N
N
H
MeO
Me
CO2Me
N
Me
H
N
Me
H 2N Lys
OH
HO
CO2Me
4-desacetylvinblastine
succinic anhydride
KS1/4S2 murine mAB
esterase-labile hemisuccinate linker
KS1/4-DAVLB
highly potent in vivo activity with

greater efficacy than unconjugated drug
Phase I clinical trials using radiolabeled
conjugate indicates localization of
drug to tumor cells
N
MeO
H
N
Me
Et
H
N Lys
O
HO
CO2Me
no increased therapeutic effect

patients developed immune responses
to both the antibody and vinca alkaloid
Clin. Pharmacol. Ther., 1990, 47, 36.
Cancer Res., 1991, 51, 2286.

KS1/4 4-Desacetylvinblastine Conjugates
OH
N
Me
CO2Me
N
H
MeO
N
Me
H
N
Me
H
O
OH
HO
Fucose
GlcNAc
Mannose
Asn
NH
O
NH 2
KS1/4S2 murine mAB

treated with NaIO 4
4-desacetylvinblastine derivative
cleavable acid-labile hydrazone linker
KS1/4-DAVLBHYD

N
Phase I clinical trials indicates localization

drug to tumor cells
MeO
H
N
Me
Et
Asn
OH
H
N
HO
O
no increased therapeutic effect - premature

cleavage of hydrazone
patients developed immune responses
to both the antibody and vinca alkaloid
Cancer Res., 1991, 51, 2286.

BR96 - Doxorubicin Conjugate
OH
O
OH
O
OH
OMe O
OH
NH 2
H
N
H 2N
HS
O
O
OH
Me
doxorubicin
linker with hydrazide and maleimide
acid-labile hydrazone linker
BR96 - doxorubicin

O
S
O
OH
OH
OH
advanced to Phase II clinical trials
N
NH
O
OH
OMe O
BR96 chimeric mAB
significant gastrointestinal toxicity target antigen expression on normal

tissue cells
50% of patients developed immune
responses despite chimeric mAB
J. Clin. Oncol., 1999, 17 , 478.

Universal Shortcomings and Lessons Learned
All four case studies successfully demonstrated localization of drug payload to tumor sites,
but in all cases no significant improvement in therapeutic activity was observed...
I. Low in vitro potency - conjugation results in decreased cytotoxicity compared to free drug
Different mechanisms of cellular uptake
Free drugs can diffuse through cell membrane
Conjugated drugs require efficient internalization after binding to antigen
Need 10 6 molecules/cell of a moderately potent cytotoxic drug to effect cell kill

Limited expression of antigen - tumor cells typically express 1 x 10 5 receptors/cell
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
Acc. Chem. Res., 2008, 41, 98.
Improving Antibody-Drug Conjugates

Ideal Characteristics of an Antibody-Drug Conjugate
A. Antibody
B. Linker
B
C. Small-molecule drug
selective for antigens with high

copy numbers (>10 5/cell) on
target cell
selective for antigens uniquely
expressed on tumor cell
homogeneous expression of
antigen on tumor cell
Stable to circulation in vivo

Selectively cleaved only once
internalized inside target cell
disulfide linkers
protease labile linkers
Designed to release drug in
its active form (without linkers)
self immolative linkers

induces minimal immunogenic
response
Stable to long-term storage

in aqueous environments
highly potent in vitro - potency

in picomolar range required
sensitive to the ideal mechanism
of action for specific tumor types
amenable to introduction of
functional groups for linking
water soluble
Acc. Chem. Res., 2008, 41, 98.
1987 - first
chimeric mAB
1970
1980

Genentech/Biogen
1990
2011 - FDA approves

Adcetris
Seattle Genetics
2000
1975 - advent of
murine mAB
2010
2000 - first FDA

Wyeth (Pfizer)
2010 - FDA
approval of
Kadcyla
Genentech/
Immunogen
2010 - Pfizer
withdraws Mylotarg

Part I.
Part II.
Part III.
First Generation
Second Generation
Current Challenges
and Overview of
and Lessons Learned
Second Generation Antibody-Drug Conjugates

Most Commonly Used Drug Payloads
O
O
Cl
MeO
HN
Me
Me
OH
Me
iPr
Me
Me
Me
O Me
Me
N
H
H
N
N
Me
R1
Me
Me
O
N
N
iPr
Me
OMe O
R2
OMe O
maytansanoids
auristatins
anti-mitotic agent
anti-mitotic agent
O
HO
Me
X
Me
S
O
Me
O
HO
MeO
OH
OMe
OMe
OH
MeSSS
O
Me
O N
H
HO
calicheamicins
O
O
DNA damaging agent
H
O
N
R
MeO
Angew. Chem. Int. Ed., 2014, 53, 3796.

Maytansanoid Antibody-Drug Conjugates
O
O
Cl
HN
MeO
Me
O Me
OH
Me
1000 fold more cytotoxic than first generation payloads
Me
Binds tubulin to suppress microtubule dynamics,

resulting in cell arrest in G2/M phase
Me
Me
Me
N
H
Good aqueous solubility
SAR activity indicates that the ester at C3 can be derivatized

for linker conjugation without impacting drug activity
maytansine
anti-mitotic agent
1.
O
OH
O
Me
N
HO
Me
S
O
Me
O
O
O Me
3
Me
2. DTT reduction
Me
SH
O
Me
maytansinol
Angew. Chem. Int. Ed., 2014, 53, 3796.

O
O
Cl
MeO
HN
Me
N
O Me
OH
Me
R1R 2
Me
Me
Me
SH
N
H
R3 R4
H 2N Lys
O
maytansine
heterobifunctional linker
O
O
Me
R3 R4
O
O Me
3
S
O
R1 R 2
humanized C242 mAB
H
N
S
O
Me
Angew. Chem. Int. Ed., 2014, 53, 3796.

O
O
Me
R3 R4
O
O Me
3
S
O
R1 R 2
H
N Lys
S
O
Me
Improved mechanism of selective drug release
O
HO 2C
NH 2
glutathione naturally present in high concentration inside tumor
SH
N
H
H
N
O
glutathione
disulfide bond reducing agent
cells (millimolar range), but exceptionally low (micromolar range)
CO2H
in blood stream - selective cleavage upon internalization

disulfide linkage is stable under physiological pH
stability of the antibody-drug conjugate can be tuned by
varying the sterics of the R groups flanking the disulfide bond
Angew. Chem. Int. Ed., 2014, 53, 3796.

Study on effect of linker stability to therapeutic efficacy
O
O
HuC242-DM1
Me
Me
O
O Me
H
N Lys
O
Me
O
HuC242-DM3
Me
Me
O
O Me
O
S
N Lys
H
increased
stability in
blood stream
increasing
therapeutic
efficacy
Me
O
HuC242-DM4
Me
N
O
O Me
3
Me Me
S
O
N Lys
H
Me
Extreme case - synthesis of a non-cleavable conjugate?
Acc. Chem. Res., 2008, 41, 98.

O
O
Cl
MeO
HN
Me
N
O Me
OH
Me
O
O
Me
Me
Me
SH
N
H
O
O
H 2N Lys
O
N
O
maytansine
SMCC (non-cleavable) linker
humanized C242 mAB
succinimidyl 4-(N-maleimidomethyl)
cyclohexane-1-carboxylate
HuC242-MCC-DM1
O
O
Me
N
O
O Me
3
S
O
H
N
O
O
Me
Angew. Chem. Int. Ed., 2014, 53, 3796.
tumor size (mm 3 )
% injected dose
plasma concentration ( g/mL)
time (hrs) post administration
days post tumor inoculation
HuC242-MCC-DM1 shows greatest stability
HuC242-DM4 shows greatest cytotoxicity
(in vivo half life of 134 hrs)
What is the mechanism of action of these maytansanoid antibody-drug conjugates?
Acc. Chem. Res., 2008, 41, 98.

Cellular Processing of Disulfide Linked Maytansanoids

O
Lys
R1 R 2
S
N
H
May
Lys
Me
N
H
May
N
O
lysosomal
processing
CO2
lysosomal
processing
H 3N
R1 R 2
S
N
H
May
CO2
Me
O
H 3N
N
H
disulfide
reduction
May
N
O
R1 R 2
HS
good
May
bystander killing
poor bystander killing

charged nature prevents diffusion into
neighboring cells
S-methylation
excellent
R1 R 2
Me
May
X
bystander killing
Angew. Chem. Int. Ed., 2014, 53, 3796.

Enhanced Therapeutic Efficacy of Cleavable Disulfide Linkers is Due to Bystander Cell Killing
Angew. Chem. Int. Ed., 2014, 53, 3796.

Auristatin Antibody-Drug Conjugates
inhibits tubulin-dependent GTP binding and
microtubule dynamics
Me
iPr
Me
N
Me
H
N
Me
O
Me
iPr
H
N
N
Me
OMe O
OMe O
fully synthetic series of highly potent

anti-mitotic agents based on SAR studies
S
N
on dolastatin 10
Ph
inhibits tubulin-dependent GTP binding and
dolastatin 10
SAR indicates terminal 3 amine can be
derivatized for conjugation AND terminal
anti-mitotic agent
phenethyl amine can be changed without

loss of efficacy
Me
iPr
Me
N
H
H
N
Me
O
Me
N
N
iPr
Me
OMe O
OMe O
H
N
R1
R2
Ph
monomethyl auristatin E (MMAE) (R1 = Me, R 2 = OH)

monomethyl auristatin F (MMAF) (R1 = CO2H, R 2 = H)
Angew. Chem. Int. Ed., 2014, 53, 3796.

O
O
O
N
5
SH
N
H
H
N
O
HN
LG
N
H
iPr
Me
H 2N
chimeric cAC10 mAB
mal-caproyl-val-cit-PAB linker
H
N
O
N
iPr
Me
monomethyl auristatin
Me
iPr
O
O
O
S
N
O
N
H
H
N
O
HN
O
N
H
N
Me
H
N
Me
O
Me
N
iPr
H
N
Me
OMe O
HO
Ph
Me
OMe O
3
O
Mal-caproyl-val-cit-PAB-MMAE
H 2N
Angew. Chem. Int. Ed., 2014, 53, 3796.

Me
valine-citrulline dipeptide
iPr
O
O
O
S
N
O
mal-caproyl
N
H
H
N
O
HN
N
Me
N
H
Me
Me
Me
OMe O
HO
Ph
N
iPr
H
N
OMe O
3
O
H 2N
H
N
Me
O
p-aminobenzyl

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
Angew. Chem. Int. Ed., 2014, 53, 3796.

Mechanism of cellular processing of mal-caproyl-val-cit-MMAE conjugate

Me
valine-citrulline dipeptide
iPr
O
O
O
S
N
O
H
N
N
H
Me
N
H
O
HN
Me
Me
OMe O
HO
Ph
N
iPr
H
N
Me
OMe O
3
O
mal-caproyl
H
N
Me
O
p-aminobenzyl
H 2N
cathepsin B
mediated peptide
cleavage
iPr
O
O
H 2N
N
Me
H
N
Me
N
iPr
iPr
self immolation
Me
-PAB
-CO2
N
H
H
N
O
N
iPr
Me
free MMAE
good bystander killing
Angew. Chem. Int. Ed., 2014, 53, 3796.

O
O
SH
iPr
N
chimeric cAC10 mAB
Me
O
N
H
mal-caproyl (non-cleavable) linker
H
N
N
iPr
Me
monomethyl auristatin
Me
O
iPr
O
N
Cys S
O
N
Me
Me
O
H
N
Me
N
N
iPr
Me
OMe O
OMe O
H
N
O
OH
Ph
Mal-caproyl-MMAF
Angew. Chem. Int. Ed., 2014, 53, 3796.

Cellular Processing of Non-Cleavable Mal-caproyl-MMAF Conjugates
Me
O
iPr
O
N
Cys S
H
N
Me
Me
O
Me
N
N
iPr
Me
OMe O
H
N
OMe O
O
OH
Ph
lysosomal
processing
Me
O
iPr
O
H 3N
S
CO2
N
Me
H
N
Me
O
Me
N
N
iPr
Me
OMe O
OMe O
H
N
O
OH
Ph
poor bystander killing

charged nature prevents diffusion into neighboring cells
Angew. Chem. Int. Ed., 2014, 53, 3796.

Calicheamicin Antibody-Drug Conjugates
O
HO
Me
X
Me
OMe
NHCO 2Me
MeSSS
O
O
Me
O
HO
MeO
OH
OH
OMe
Me
O N
H
HO
O
O
O
calicheamicins
DNA damaging agent
H
O
N
R
MeO
insanely cytotoxic class of anti-tumor
antibiotics (0.15g/kg dose)
calicheamicin 1 Br - X = Br, R = iPr
aryl tetrasaccharide moiety binds in
calicheamicin 1 Br - X = Br, R = Et
minor groove of DNA, placing ene-
calicheamicin 1 I - X = I, R = Et
diyne warhead within double helix

too toxic for use as drug warhead -
N-acetyl calicheamicin 1 I - X = I
20 fold less potent N-acetyl analogue
Me
Me
O
N
MeO
developed for applications to ADCs

trisulfide converted to disulfide provides a handle for conjugation
Angew. Chem. Int. Ed., 2014, 53, 3796.

Mechanism of Action of the Calicheamicins
O
HO
Me
Me
OH
OMe
Me
O
HO
MeO
OH
Me
NHCO 2Me
MeSSS
OMe
Me
glutathione
mediated
O N
H
HO
Me
O
HO
NHCO 2Me
O
thiol reduction
N
MeO
sugar
Michael addn
O
HO
DNA
NHCO 2Me
S
sugar
O
HO
NHCO 2Me
S
O
double
stranded DNA
diradical
O2
sugar
O
HO
cycloaromatization
NHCO 2Me
S
(relief of ring strain)

O
double
stranded DNA
cleavage
sugar
cell death
Angew. Chem. Int. Ed., 2014, 53, 3796.

O Me Me
O
Me
Lys NH 2
H 2N
N
H
HO
NHCO 2Me
O
O
sugar
hP67.6 humanized mAB
AcBut linker
N-acetyl calicheamicin
(4-(4'acetylphenyl)butanoic acid)
Me
N
H
Lys N
O
O
H
N
HO
S
NHCO 2Me
O Me Me
sugar
hP67.6 N-Ac- -calicheamicin DMH AcBut

Angew. Chem. Int. Ed., 2014, 53, 3796.

Me
N
H
Lys N
H
N
HO
S
NHCO 2Me
O Me Me
sugar
hP67.6 N-Ac- -calicheamicin DMH AcBut

Linker specifically designed to provide high stability prior to internalization into tumor cells, but is
readily cleaved once inside the lysosome - hydrazone formed from ketone rather than aldehyde
only 6% hydrolysis observed at pH = 7.4
97% hydrolysis observed at pH = 4.5 at 37C over 24 hrs
Inclusion of a hindered disulfide moiety in the linker provides a second handle for selective drug
cleavage via glutathione reduction upon internalization inside cell
Bioconj. Chem., 2002, 13 , 47.
1987 - first
chimeric mAB
1970
1980

Genentech/Biogen
1990
2011 - FDA approves

Adcetris
Seattle Genetics
2000
1975 - advent of
murine mAB
2010
2000 - first FDA

Wyeth (Pfizer)
2010 - FDA
approval of
Kadcyla
Genentech/
Immunogen
2010 - Pfizer
withdraws Mylotarg

Part I.
Part II.
Part III.
First Generation
Second Generation
Current Challenges
and Overview of
and Lessons Learned
Current Trends in Research in Antibody-Drug Conjugates

Controlling the Drug to Antibody Ratio (DAR) and Achieving Site-Specific Conjugation
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
Bioconj. Chem., 2015, 26, 176.

Methods of Homogeneous Conjugation Using Natural Antibodies
N-terminal conjugation leveraging differences in pK a between terminal and internal amino acids
O
R
H
N
HO
OPO32-
NH 2
O
Me
H
N
R
O
37 - 50 C
phosphate buffer pH 6.5
Though conjugation via this method is in the antigen binding domain, drug conjugation here does
not seem to impact antigen recognition and binding
Resulting ketone product can be easily further functionalized through reaction with oximes bearing
a linker or drug
Limitation: Reaction sensitive to nature of terminal amino acid - works best for alanine, glycine, aspartate
glutamate, and asparagine
Limitation: Some antibodies may not be able to tolerate elevated temperatures required for transamination

Methods of Homogeneous Conjugation Using Natural Antibodies
Site-specific functionalization of glutamines through enzymatic conjugation
O
Gln
NH 2
1. Deglycosylation
2. H 2NR, transaminase
O
Gln
HN R
Selectively functionalizes only Q295 residue (flanked by a consensus recognition sequence for a
bacterial transaminase)
Q295 residue is distant from antigen binding domain
Limitation: Requires deglycosylation in the CH2 domain prior to functionalization, which may impact
function and properties of the antibody-drug conjugate

Methods of Homogeneous Conjugation Using Engineered Antibodies
SH
HSe
SeH
OHC
CHO
THIOMABs
C-terminal selenocysteine
aldehyde tagging
engineered Cys at positions
engineered selenocysteines
engineered recognition
with minimal interference
(more nucleophilic)
sequence for formylglycine
to antibody function
selectively at C-terminus
generating enzyme (FGE)

Methods of Homogeneous Conjugation Using Engineered Antibodies
Protein Engineering to Incorporate Unnatural Amino Acids
O
N3
OH
H 2N
Me
p-azido Phe
OH
H 2N
p-acetyl Phe
OH
H 2N
propynyl Tyr
O
N3
O
Me
Clinically Successful Antibody-Drug Conjugates

FDA Approved Antibody-Drug Conjugates
Me
iPr
O
O
O
S
N
5
H
N
N
H
O
HN
N
Me
N
H
H
N
Me
O
Me
N
iPr
Me
OMe O
brentuximab vedotin (Adcetris)
Seattle Genetics
H 2N
H
N
Me
OMe O
HO
Ph
FDA approved in 2011 for Hodgkin lymphoma and

anaplastic large cell lymphoma (ALCL)
O
O
Cl
MeO
O
O Me
HN
3
Me
Me
Me
Me
S
O
Me
O
OH
N
H
H
N
O
O
ado-trastuzumab emtansine (Kadycla)

O
Roche/Genentech/ImmunoGen
FDA approved in 2013 for metastatic breast cancer
Angew. Chem. Int. Ed., 2014, 53, 3796.

FDA Approved Antibody-Drug Conjugates
O
O
Lys N
H
O Me Me
N
Me
Me
I
Me
O
HO
MeO
OH
O
HO
NHCO 2Me
Me
OMe
Me
N
H
OH
OMe
O N
H
HO
Me
Me
O
N
MeO
O
O
O
Gemtuzumab ozogamicin (Mylotarg)

Wyeth/Pfizer
FDA approved in 2000 for acute lymphoblastic lukemia
Withdrawn in 2010 due to toxicity concerns
and lack of improvement in patient survival time
Angew. Chem. Int. Ed., 2014, 53, 3796.

Antibody-Drug Conjugates Currently in Clinical Evaluations
Candidate
Drug
Antigen
Lead Indicator
Developer/Partner
Angew. Chem. Int. Ed., 2014, 53, 3796.

CL Antibody Drug Conjugates

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The Advent of

MacMillan Group Meeting

Traditional Cancer Therapy

Anti-cancer treatments should be as aggressive as possible to fully eradicate the tumor...

Common Chemotherapeutic Agents

DNA crosslinking agent

lack of tumor selectivity - killing of proliferating normal cells

Traditional Cancer Therapy

Current most critical need: Maximization of the Therapeutic Window

increase MTD = increase selectivity

Maximum Tolerated Dose (MTD)

decrease MED = increase potency

Angew. Chem. Int. Ed. 2014, 53, 3796.

Targeted Cancer Therapy

Targeting tumor-associated or specific proteins to directly alter their signaling by:

direct binding of monoclonal antibody to

binding of small molecule drugs to active

to induce immune responses

Targeted Cancer Therapy

monoclonal antibody (fusion protein)

antibody-directed enzyme prodrug therapy

Targeted Cancer Therapy

monoclonal antibody (fusion protein)

antigen (overexpressed protein)

C. Small-molecule drug warhead

Number of Publications in Antibody-Drug Conjugate Research

1997 - first therapeutic mAB

2011 - FDA approves

2000 - first FDA

mAB = monoclonal antibody

and Lessons Learned

and Their Improvements

Nature Rev. 2006, 5, 147.; Bioconjugate Chem. 2015, 26, 176.

1997 - first therapeutic mAB

2011 - FDA approves

2000 - first FDA

mAB = monoclonal antibody

and Lessons Learned

and Their Improvements

Nature Rev. 2006, 5, 147.; Bioconjugate Chem. 2015, 26, 176.

Deconstruction of Antibody-Drug Conjugates

represents 75% of serum antibodies in humans

protein complex of 4 peptide chains in a Y shape

F ab = Fragment antigen-binding domain

F c = Fragment crystallizable/constant domain

Domains of a Typical IgG Antibody

Deconstruction of Antibody-Drug Conjugates

Linking through native cysteine residues

Domains of a Typical IgG Antibody

Deconstruction of Antibody-Drug Conjugates

Bioconj. Chem. 2015, 26, 176.

Deconstruction of Antibody-Drug Conjugates

Corresponding oxidized byproducts

Deconstruction of Antibody-Drug Conjugates

Linking through native cysteine residues

12 intrachain disulfide bridges - harder to reduce

Domains of a Typical IgG Antibody

Deconstruction of Antibody-Drug Conjugates

Linking through native cysteine residues

4 interchain disulfide bridges - easier to reduce

12 intrachain disulfide bridges - harder to reduce

Linking through native lysine residues

Domains of a Typical IgG Antibody

Deconstruction of Antibody-Drug Conjugates