Download Methods to Design and Synthesize Antibody

International Journal of
Molecular Sciences
Review
Methods to Design and Synthesize Antibody-Drug
Conjugates (ADCs)
Houzong Yao 1,† , Feng Jiang 1,2,† , Aiping Lu 1, * and Ge Zhang 1, *
1
2
*
†
Institute for Advancing Translational Medicine in Bone & Joint Diseases, School of Chinese Medicine,
Hong Kong Baptist University, Hong Kong, China; [email protected] (H.Y.); [email protected] (F.J.)
Faculty of Materials Science and Chemical Engineering, the State Key Laboratory Base of
Novel Functional Materials and Preparation Science, Ningbo University, Ningbo 315211, Zhejiang, China
Correspondence: [email protected] (A.L.); [email protected] (G.Z.);
Tel.: +852-3411-2456 (A.L.); +852-3411-2958 (G.Z.)
These authors contributed equally to this work.
Academic Editor: Már Másson
Received: 11 January 2016; Accepted: 28 January 2016; Published: 2 February 2016
Abstract: Antibody-drug conjugates (ADCs) have become a promising targeted therapy strategy
that combines the specificity, favorable pharmacokinetics and biodistributions of antibodies with the
destructive potential of highly potent drugs. One of the biggest challenges in the development of
ADCs is the application of suitable linkers for conjugating drugs to antibodies. Recently, the design
and synthesis of linkers are making great progress. In this review, we present the methods that are
currently used to synthesize antibody-drug conjugates by using thiols, amines, alcohols, aldehydes
and azides.
Keywords: antibody-drug conjugates (ADCs); targeted therapy; monoclonal antibodies (mAbs);
drugs; linkers
1. Introduction
Cancer is still one of the major threats to human health. However, cancer therapies used today
always have more or less adverse side effects to normal tissues. Targeted therapy is a promising strategy
to address this challenge. The pioneer of targeted therapy is Paul Ehrlich who introduced the principle
“magic bullet” at the beginning of the 20th century [1]. To avoid side effects, drugs should be specifically
delivered to cancer cells via binding to ligands that can specifically recognize the cancer-associated
biomarkers such as antigens. Among the ligands for targeted therapy, antibodies are excellent
candidates because of their specific recognitions and high affinities. Nowadays, antibody-drug
conjugates (ADCs) are attracting tremendous attention for targeted cancer therapy.
Antibody-drug conjugates are biotherapeutics that consist of monoclonal antibodies, potent
cytotoxic drugs and linkers between them (Figure 1). The monoclonal antibodies lead the drug
precursors to the target cancer cells, in which the prodrugs can be chemically or enzymatically
converted to drugs in their active forms [2]. Conjugating cytotoxins to monoclonal antibodies that
specifically tie to tumor cell surface antigens enables the drugs to be target-delivered to cancer cells and
leaves normal cells unaffected. More important, many of the cytotoxic drugs that are too toxic for use
in traditional chemotherapy can also be used in the construction of antibody-drug conjugates [3,4]. The
linkers are also essential parts of antibody-drug conjugates, which account for stability in circulation,
good pharmacokinetics and efficient release of toxic drugs in the tumor cells.
The selection of antibody, drug, and linker has recently been summarized in a few excellent
reviews [5–11]. In this review, we mainly describe the linking methods to design and synthesize ADCs,
including those that are not discussed in the reviews mentioned above.
Int. J. Mol. Sci. 2016, 17, 194; doi:10.3390/ijms17020194
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The selection of antibody, drug, and linker has recently been summarized in a few excellent
The [5–11].
selection
drug,
and linker
hasthe
recently
been
summarized
in aand
fewsynthesize
excellent
reviews
In of
thisantibody,
review, we
mainly
describe
linking
methods
to design
reviews
[5–11].
In
this
review,
we
mainly
describe
the
linking
methods
to
design
and
synthesize
ADCs,
including
those
Int.
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194 that are not discussed in the reviews mentioned above.
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ADCs, including those that are not discussed in the reviews mentioned above.
Figure 1. Schematic representation of an antibody-drug conjugate (ADC). Reprinted with permission
Figure
1.
from Reference
[2]. representation
Figure
1. Schematic
Schematic
representation of
of an
an antibody-drug
antibody-drug conjugate
conjugate (ADC).
(ADC). Reprinted
Reprinted with
with permission
permission
from
[2].
from Reference
Reference [2].
2. Conjugation via Various Functional Groups to Synthesize Antibody-Drug Conjugates (ADCs)
2.
2. Conjugation
Conjugationvia
viaVarious
VariousFunctional
FunctionalGroups
Groups to
to Synthesize
Synthesize Antibody-Drug
Antibody-Drug Conjugates
Conjugates (ADCs)
(ADCs)
2.1. Conjugation via Thiols
2.1. Conjugation via Thiols
Employing the thiols of interchain cysteine residues in monoclonal antibodies as attachment
the thiols
thiolsisofof
interchain
cysteine
residues
in monoclonal
as attachment
the
interchain
cysteine
residues
in monoclonal
as attachment
sites Employing
for drug molecules
one
of the most
used
conjugation
methods.antibodies
In antibodies
a human
IgG1,
theresites
are
sites
for drug
molecules
isof
one
ofmost
the
used
methods.
In a [12].
human
there
are
for
molecules
is one
the
used
conjugation
methods.
In a sites
human
IgG1,
there
are four
fourdrug
interchain
disulfide
bonds
that
canmost
be used
as conjugation
potential
conjugation
TheIgG1,
four
interchain
four
interchain
disulfide
bonds
be used
potential
conjugation
sites[12].
[12].
The four
four interchain
interchain
disulfide
thatthat
can
be
used
as as
potential
conjugation
sites
The
disulfide
bonds
can bonds
be reduced
bycan
tris(2-carboxyethyl)
phosphine
(TCEP)
or dithiothreitol
(DTT),
disulfide
bonds
can
be
reduced
by
tris(2-carboxyethyl)
phosphine
(TCEP)
or
dithiothreitol
(DTT),
or dithiothreitol
(DTT),
which results in eight thiol groups that are available for phosphine
conjugating(TCEP)
drug molecules.
Through
this
are available
available
for conjugating
conjugating
drug molecules.
molecules.
which
results
in eight
thiol
groupsratio
that are
for
drug
Through
this
method,
different
drug
antibody
(DAR)
conjugates
will be obtained
when targeting
typical
method,
drug
antibody antibody-drug
ratio (DAR) conjugates
be obtained
when targeting
typical
DARs of different
2–4 [13,14].
In addition,
conjugatewill
at each
drug antibody
ratio has several
DARs
of
2–4
[13,14].
In
addition,
antibody-drug
conjugate
at
each
drug
antibody
ratio
has
several
DARs
of Thus,
2–4 [13,14].
In addition,
antibody-drug
each
drug antibody
ratio has
several
isomers.
over a hundred
different
species areconjugate
present inatthe
antibody-drug
conjugate.
Although
isomers.
Thus,
over
a
hundred
different
species
are
present
in
the
antibody-drug
conjugate.
Although
isomers.
Thus,
over
a
hundred
different
species
are
present
in
the
antibody-drug
conjugate.
Although
conventional methods that employ cysteine residues as conjugation sites are highly heterogeneous,
conventional
that
conventional
methods
that
employ
cysteine residues
residues as
as conjugation
conjugation sites
sites are
are highly
highly heterogeneous,
heterogeneous,
Adcetris® wasmethods
approved
by employ
FDA in cysteine
2011.
®
®
Adcetris
was
by
2011.
Adcetris
was approved
approved
by FDA
FDA in
in conjugates
2011.
Homogeneous
antibody-drug
can be produced through cysteine residues when all
Homogeneous
conjugates
can
through
cysteine
residues
when
all
Homogeneous
antibody-drug
conjugates
can be
be produced
produced
through
cysteine
residues
when
all
interchain
cysteines antibody-drug
are
coupled to drugs.
For example,
Senter and
coworkers
[15,16]
developed
such
interchain
cysteines
are
coupled
to
drugs.
For
example,
Senter
and
coworkers
[15,16]
developed
such
interchain
cysteines
coupledof
to cAC10,
drugs. For
Senter
and coworkers
[15,16]
developed
such
a conjugate
which are
consisted
an example,
anti-CD30
monoclonal
antibody,
and
monomethyl
aaauristatin
conjugate
which
consisted
of cAC10,
anconjugate
anti-CD30
monoclonal
antibody,
and monomethyl
conjugate
consisted
of cAC10,
an anti-CD30
monoclonal
antibody,
and per
monomethyl
auristatin
Ewhich
(MMAE).
This cAC10-vcMMAE
contains
eight drugs
antibody,
which is
auristatin
(MMAE).
This ratio
cAC10-vcMMAE
conjugate
contains
eight
drugs
antibody,
is
E
(MMAE).
cAC10-vcMMAE
conjugate
contains
eight
drugs
per antibody,
which
iscysteines
thewhich
highest
the
highestE This
drug
antibody
(DAR)
that can
be obtained
through
using per
interchain
as
the
highest
drug
antibody
ratio
(DAR)
that
can
be
obtained
through
using
interchain
cysteines
as
drug
antibody
ratioHowever,
(DAR) that
can be obtained
through with
usingfour
interchain
as conjugation
sites.
conjugation
sites.
antibody-drug
conjugates
drugs cysteines
per antibody
generally have
conjugation
sites.
However,
antibody-drug
conjugates
with
four drugs
per antibody
generally
However,
antibody-drug
conjugates
with four
per
antibody
improved
inhave
vivo
improved in
vivo performance
[17]. McDonagh
etdrugs
al. [18]
developed
agenerally
method
tohave
control
the conjugate
improved
in
vivo
performance
[17].
McDonagh
et
al.
[18]
developed
a
method
to
control
the
conjugate
performance
[17]. McDonagh
[18]interchain
developedcysteines
a methodtotoserines,
control the
conjugate
sites by
mutating
sites by mutating
four or sixetofal.the
therefore
leaving
four
or two
sites
bysix
mutating
four
or six
of the(Scheme
interchain
cysteines
to serines,
leaving
fourmutated
or two
four
or
of the interchain
cysteines
to
serines,
leaving
four
or
two cysteines
for
cysteines
accessible
for conjugating
1). therefore
After
reduction
of
thetherefore
disulfide
bonds,accessible
the
cysteines
accessible
for
conjugating
(Scheme
1).
After
reduction
of
the
disulfide
bonds,
the
mutated
conjugating
(Scheme
1).
After
reduction
of
the
disulfide
bonds,
the
mutated
monoclonal
antibodies
monoclonal antibodies with the reduced number of interchain cysteines were conjugated with the
monoclonal
antibodies
with
the
reduced
numberwere
ofantibody-drug
interchain
cysteines
conjugated
with the
with
reduced
number
of interchain
cysteines
conjugated
with
thewere
drug
vcMMAE.
Through
drug the
vcMMAE.
Through
this
method,
homogenous
conjugates
with
clear attachment
drug
vcMMAE.
Through this
method, homogenous
conjugates
clear
this
antibody-drug
conjugatesantibody-drug
with clear attachment
siteswith
could
be attachment
produced.
sitesmethod,
could
behomogenous
produced.
sites could be produced.
Scheme 1.
1. Interchain
conjugate to
to the
the remaining
remaining
Scheme
Interchain cysteine
cysteine to
to serine
serine mutagenesis
mutagenesis enables
enables drugs
drugs to
to conjugate
Scheme
1.
Interchain
cysteine
to
serine
mutagenesis
enables
drugs
to
conjugate
to
the
remaining
cysteines.
Adapted
from
reference
[18].
cysteines. Adapted from reference [18].
cysteines. Adapted from reference [18].
Reducing the disulfide bonds of a monoclonal antibody should not affect its functions [19]. What
Reducing the
disulfide bonds
of
monoclonal antibody
should
not affect
functions [19].
What
Reducing
the disulfide
bonds
of aaare
monoclonal
antibody
should
affect its
itsdisulfide
functions
[19]. What
is more, interchain
disulfide
bonds
easier to be
reduced
than not
intrachain
bonds
[20].
is
more,
interchain
disulfide
bonds
are
easier
to
be
reduced
than
intrachain
disulfide
bonds
[20].
These
is more, interchain disulfide bonds are easier to be reduced than intrachain disulfide bonds [20].
allow free thiol groups to be generated under mild reducing conditions while leaving the antibody
intact at the same time. Liu et al. [21] took advantage of the fact that different disulfide bonds in a
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J. Mol.
Mol. Sci.
Sci. 2016,
2016, 17,
17, 194
194
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These allow
allow free
free thiol
thiol groups
groups to
to be
be generated
generated under
under mild
mild reducing
reducing conditions
conditions while
while leaving
leaving the
the
These
antibody
intact
at
the
same
time.
Liu
et
al.
[21]
took
advantage
of
the
fact
that
different
disulfide
bonds
antibody intact at the same time. Liu et al. [21] took advantage of the fact that different disulfide bonds
in aa monoclonal
monoclonal
antibody
have different
different
susceptibilities
towards
reduction
and developed
developed
another
monoclonal
antibody
have different
susceptibilities
towards
reduction
and developed
another strategy
in
antibody
have
susceptibilities
towards
reduction
and
another
strategy
to
tightly
control
the
site
of
conjugation.
Limited
reduction
with
TCEP
or
DTT
to
tightly
control
the
site
of
conjugation.
Limited
reduction
with
TCEP
or
DTT
predominantly
yielded
strategy to tightly control the site of conjugation. Limited reduction with TCEP or
DTT
predominantly
yielded
conjugates
in
which
drugs
were
attached
to
heavy-light
chain
disulfides;
conjugates
in which
drugs
were attached
to heavy-light
chain
disulfides;
partial re-oxidation
of fully
predominantly
yielded
conjugates
in which
drugs were
attached
to heavy-light
chain disulfides;
1 -dithiobis
partial
re-oxidation
of
fully
reduced
antibodies
with
5,5′-dithiobis
(2-nitrobenzoic
acid)
(DTNB)
reduced
antibodies
with
5,5
(2-nitrobenzoic
acid)
(DTNB)
yielded
conjugates
that
drugs
partial re-oxidation of fully reduced antibodies with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB)
yielded
conjugates
that
drugs
were
mainly
attached
to
by
heavy-heavy
chain
disulfides
[13].
were
mainly
attached
to
by
heavy-heavy
chain
disulfides
[13].
yielded conjugates that drugs were mainly attached to by heavy-heavy chain disulfides [13].
2.1.1.
Addition to
2.1.1. Addition
to Maleimides
Maleimides
Classically,
Classically, cysteine
cysteine residues
residues can
can be
be modified
modified through
through addition
addition of
of thiols
thiols to
Classically,
cysteine
residues
can
be
modified
through
addition
of
thiols
to electrophiles
electrophiles such
such as
as
maleimides
(Scheme
2)
[22–25].
The
conjugate
could
be
achieved
by
reducing
the
disulfide
bonds
maleimides (Scheme 2) [22–25]. The conjugate could be achieved by reducing the disulfide bonds of
of
the
then adding
adding to
to maleimides.
maleimides. Addition
most common
common method
method
the antibody
antibody and
and then
then
adding
to
maleimides.
Addition to
to maleimides
maleimides is
is the
the most
most
common
method
® which was approved by
®
for
for the
attaching drugs
to antibodies.
antibodies. Adcetris
which was
was approved
approved by
by the
the FDA
FDA for
for attaching
attaching
drugs to
to
antibodies.
Adcetris®,,, which
the treatment
treatment of
of
patients
with
Hodgkin’s
lymphoma
after
failed
autologous
stem
cell
transplantation
or
patients
with
patients
with
patients with Hodgkin’s lymphoma after failed autologous stem cell transplantation or patients with
systemic
large-cell
lymphoma
after the
failure
at leastof
systemic anaplastic
anaplastic
large-cell
lymphoma
after
the of
failure
ofoneat
atprior
leastmulti-agent
one prior
priorchemotherapy
multi-agent
systemic
anaplastic
large-cell
lymphoma
after
the
failure
least
one
multi-agent
regimen,
was
produced
by
this
method
in
which
a
maleimide-functionalized
drug
was
conjugated
chemotherapy regimen,
regimen, was
was produced
produced by
by this
this method
method in
in which
which aa maleimide-functionalized
maleimide-functionalized
drug
chemotherapy
drug
to
the
interchain
cysteine
residues
of
an
anti-CD30
antibody
[15].
Maleimide-based
antibody-drug
was
conjugated
to
the
interchain
cysteine
residues
of
an
anti-CD30
antibody
[15].
Maleimide-based
was conjugated to the interchain cysteine residues of an anti-CD30 antibody [15]. Maleimide-based
conjugates
wereconjugates
recently found
have limited
blood circulation
which
would lower
antibody-drug
conjugates
weretorecently
recently
foundstability
to have
haveinlimited
limited
stability in
in[26],
blood
circulation
[26],
antibody-drug
were
found
to
stability
blood
circulation
[26],
the
efficacy
of the
conjugates
and damage
healthy tissue.
Succinimide
or maleimide
hydrolysis or
is
which
would
lower
the efficacy
efficacy
of the
the conjugates
conjugates
and damage
damage
healthy
tissue. Succinimide
Succinimide
or
which
would
lower
the
of
and
healthy
tissue.
amaleimide
promisinghydrolysis
method tois
around
this
problem.
Once
hydrolyzed,
the
antibody-drug
conjugates
maleimide
hydrolysis
isget
a
promising
method
to
get
around
this
problem.
Once
hydrolyzed,
the
a promising method to get around this problem. Once hydrolyzed, the
were
no longer conjugates
subject to elimination
reactions
of maleimides
through
retro-Michael
reactions,
thus
antibody-drug
conjugates
were no
no longer
longer
subject
to elimination
elimination
reactions
of maleimides
maleimides
through
antibody-drug
were
subject
to
reactions
of
through
improving
thereactions,
stabilities thus
and potencies
ADCs
[27–29].
retro-Michael
reactions,
thus
improvingofthe
the
stabilities
and potencies
potencies of
of ADCs
ADCs [27–29].
[27–29].
retro-Michael
improving
stabilities
and
Scheme 2.
2. The
The
synthesis
of
antibody-drug
conjugates
(ADCs)
through
addition of
of thiols
thiols to
to
Scheme
The synthesis
synthesis of
of antibody-drug
antibody-drug conjugates
conjugates (ADCs)
(ADCs) through
through the
the addition
Scheme
2.
maleimides.
Adapted
from
reference
[23].
maleimides.
Adapted
from
reference
[23].
maleimides. Adapted from reference [23].
2.1.2. Disulfide-Thiol
Disulfide-Thiol Exchange
Exchange
2.1.2.
The approach
approach disulfide-thiol
disulfide-thiol exchange
exchange could
could also
also be
be used
used to
to synthesis
synthesis ADCs
ADCs by
by forming
forming aa new
new
The
disulfide
bond
between
drugs
and
antibodies
[30,31].
Ojima
et
al.
[30]
designed
and
synthesized
novel
disulfide bond
bond between
between drugs
drugs and
and antibodies
antibodies [30,31].
[30,31]. Ojima
Ojima et al. [30] designed and synthesized novel
disulfide
antibody-taxoid
conjugates
that
include
highly
cytotoxic
taxoid drug
drug and
and monoclonal
monoclonal antibodies
antibodies that
that
antibody-taxoid conjugates
conjugates that include highly cytotoxic taxoid
antibody-taxoid
could
recognize
the
EGFR
expressed
in
cancer
cells.
In
this
study,
taxoid
bearing
a
free
thiol
group
could recognize the EGFR expressed in cancer cells. In this study, taxoid bearing a free thiol group
was attached
attachedtoto
tothe
thepyridyldithio
pyridyldithiogroups
groups
of
the
modified
anti-EGFR
antibodies
through
disulfidewas
attached
the
pyridyldithio
groups
modified
anti-EGFR
antibodies
through
disulfidewas
of of
thethe
modified
anti-EGFR
antibodies
through
disulfide-thiol
thiol exchange
exchange
(Scheme
3). The
The
resulting
conjugates
possessremarkable
remarkableantitumor
antitumoractivities
activities against
against
thiol
(Scheme
3).
resulting
conjugates
possess
remarkable
antitumor
activities
exchange
(Scheme
3). The
resulting
conjugates
possess
EGFR-expressing
A431
(human
epidermoid)
tumor
xenografts
in
immune
deficient
mice.
(human epidermoid)
epidermoid) tumor
tumor xenografts
xenografts in
in immune
immune deficient
deficientmice.
mice.
EGFR-expressing A431 (human
Scheme 3.
3. Preparation
Preparation of
of antibody-taxoid
antibody-taxoid conjugates
conjugates via
via disulfide-thiol
disulfide-thiol exchange.
exchange. Adapted
Adapted from
from
Scheme
Scheme
3. Preparation
of antibody-taxoid
conjugates via
disulfide-thiol exchange.
Adapted from
reference
[30].
reference [30].
[30].
reference
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2.1.3. Addition to Alkynes
Addition to Alkynes
To 2.1.3.
avoid
the maleimide instability issue, Kolodych et al. [32] developed a heterobifunctional
To avoid
the maleimide instability issue, Kolodych et al. [32] developed a heterobifunctional
reagent, sodium
4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate
(CBTF), for
reagent,
sodium
4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate
(CBTF),
for
amine-to-thiol
coupling
(Scheme 4). This reagent comprises a 3-arylpropionitrile (APN)4 of
group
that
Int. J. Mol. Sci.
2016, 17, 194
16
amine-to-thiol coupling (Scheme 4). This reagent comprises a 3-arylpropionitrile (APN) group that
replaces the maleimide and allows for the preparation of remarkably stable conjugates. Addition
replaces
the maleimide
and allows for the preparation of remarkably stable conjugates. Addition of
to Alkynes
of thiols2.1.3.
in Addition
the
3-arylpropionitriles
predominantly
produced
Z-isomers
thiols
in theantibodies
antibodies totothethe
3-arylpropionitriles
predominantly
produced Z-isomers
of the
addition of the
To
avoid
the
maleimide
instability
issue,
Kolodych
et
al.
[32]
developed
a
heterobifunctional
additionproducts.
products.
reagent, sodium 4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate (CBTF), for
amine-to-thiol coupling (Scheme 4). This reagent comprises a 3-arylpropionitrile (APN) group that
replaces the maleimide and allows for the preparation of remarkably stable conjugates. Addition of
thiols in the antibodies to the 3-arylpropionitriles predominantly produced Z-isomers of the addition
products.
Scheme
4.
The
conjugation
of
amine-drug
to
trastuzumab
by
using
sodium
4-((4-
Scheme 4.
The conjugation of amine-drug to trastuzumab by using sodium
(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate (CBTF). Reprinted with permission
4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate
(CBTF). Reprinted with
from reference [32].
permission from reference [32].
2.1.4. Disulfide Re-Bridging
Scheme
4. The conjugation of amine-drug to trastuzumab by using sodium 4-((42.1.4. Disulfide
Re-Bridging
Recently,
various novel cysteine-relied conjugation methods have been developed [33]. Godwin
(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate (CBTF). Reprinted with permission
and coworkers [34–36] developed a thiol conjugation approach in which interchain disulfide bonds
from
referencenovel
[32]. cysteine-relied conjugation methods have been developed [33]. Godwin
Recently,
various
of the cysteines were partially reduced, followed by bis-alkylation (including Michael addition and
and coworkers
[34–36]
developed
a thiol
conjugation
approach
in5).
which
interchain
elimination)
to introduce
thiols of
two cysteines
to the drug
(Scheme
Depending
on the disulfide
reduction bonds
2.1.4.
Disulfide
Re-Bridging
of the cysteines
partially
reduced,
followedcan
bybe
bis-alkylation
Michael
and
degree, thewere
numbers
of cysteines
for conjugation
eight or four to (including
generate drug
antibodyaddition
ratios
Recently,
novel
cysteine-relied
methods
havethe
been
developed
[33].
Godwin
(DARs)
fourvarious
and two,
respectively.
Theyconjugation
also
that
thiobridge
ADCs
are
more
elimination)
toofintroduce
thiols
of
two cysteines
todemonstrated
the drug
(Scheme
5).
Depending
on
the
reduction
and coworkers
[34–36]ADCs
developed
ahuman
thiol conjugation
approach in which interchain disulfide bonds
than maleimide
in
serum.
degree, stable
the numbers
of cysteines
fortheconjugation
can be eight or four to generate drug antibody ratios
of theBehrens
cysteines
were
partially
reduced,
followed
by
bis-alkylation
(including
Michaelresidues,
additionthen
and
et al. [37] reduced all the disulfide bonds, exposing
eight cysteine
(DARs)elimination)
of four and
two, respectively.
They
alsotodemonstrated
that
the thiobridge
ADCs are more
to introduce
thiols of two
cysteines
the
drug
5). groups
Depending
on antibody
the reduction
similarly used
dibromomaleimide
(DBM)
to react
with
the(Scheme
free thiol
of the
and
stable than
maleimide
ADCs
in the
human
serum. cytotoxic
degree,
theanumbers
of cysteines
for conjugation
be eightdrugs
or fourwith
to generate
drug antibody
produced
dithiomaleimide
(DTM)
ADC. Fourcan
this functional
linker ratios
were
Behrens
et
al.
[37]
reduced
all
the
disulfide
bonds,
eight
cysteine
residues,
then
similarly
(DARs)
and two, respectively.
They also
demonstrated
that
thecysteine
thiobridge
ADCs are
more
attachedof
tofour
the monoclonal
antibodies conveniently
byexposing
linking with
the
residues.
stable
than
maleimide
ADCs
in
the
human
serum.
used dibromomaleimide
to [27,38–40]
react with
the freea thiol
groups
of the
antibody
and produced a
Chudasama and(DBM)
coworkers
presented
significant
method
towards
next-generation
Behrens
et therapeutics
al. [37]
reduced
all disulfide
the disulfide
bonds,
exposing
eight
residues,
then to the
antibody-based
through
re-bridging.
their
works,
thecysteine
reduction
of disulfides
dithiomaleimide
(DTM)
ADC.
Four
cytotoxic
drugs
with In
this
functional
linker
were
attached
similarly
used
dibromomaleimide
(DBM)
to
react
with
the
free
thiol
groups
of
the
antibody
and
and disulfide
re-bridging
could by
belinking
achieved
in the
one cysteine
step by residues.
the use of a single reagent:
monoclonal
antibodies
conveniently
with
produced
a
dithiomaleimide
(DTM)
ADC.
Four
cytotoxic
drugs
with
this
functional
linker
were
dithioaryl(TCEP)pyridazinedione
[38].
Disulfide
re-bridging
through
the
use
of
Chudasama
andmonoclonal
coworkers
[27,38–40]
presented
significant
method
towards
next-generation
attached
to the
antibodies
conveniently
by alinking
with
the
cysteine
residues.
dibromopyridazinedione
derivatives
after
disulfide reduction
by
TCEP
was
another
strategy for the
antibody-based
therapeutics
through
disulfide
re-bridging.
InThe
their
works,
the reduction
of
Chudasama
and coworkers
[27,38–40]
presented
a significant
method
towards
next-generation
construction
of antibody-based
therapeutics
in their
studies
[39,40].
resulting
conjugates
were
antibody-based
therapeutics
through
disulfide
re-bridging.
In their
the reduction
disulfides
disulfides
and
disulfide
re-bridging
could
be
achieved
in works,
one step
by the ofuse
of a single
highly
stable
and
had potent
cytotoxicites
against
tumor
cells.
disulfide re-bridging could be achieved
step by re-bridging
the use of a through
single reagent:
reagent:anddithioaryl(TCEP)pyridazinedione
[38].in one
Disulfide
the use of
dithioaryl(TCEP)pyridazinedione
[38].
Disulfide
re-bridging
through
the
use
of
dibromopyridazinedione derivatives after disulfide reduction by TCEP was another strategy
for
dibromopyridazinedione derivatives after disulfide reduction by TCEP was another strategy for the
the construction
of
antibody-based
therapeutics
in
their
studies
[39,40].
The
resulting
conjugates
were
construction of antibody-based therapeutics in their studies [39,40]. The resulting conjugates were
highly stable
cytotoxicites
cells.
highly and
stablehad
andpotent
had potent
cytotoxicitesagainst
against tumor
tumor cells.
Scheme 5. Three approaches to make ADCs through disulfide re-bridging: thiobridge,
dibromomaleimide and pyridazinedione. Adapted from reference [35,37,40].
Scheme 5. Three approaches to make ADCs through disulfide re-bridging: thiobridge,
Scheme 5.
Three approaches to make ADCs through disulfide re-bridging:
dibromomaleimide and pyridazinedione. Adapted from reference [35,37,40].
dibromomaleimide and pyridazinedione. Adapted from reference [35,37,40].
thiobridge,
Int. J. Mol. Sci. 2016, 17, 194
Int. J. Mol. Sci. 2016, 17, 194
Int. J. Mol. Sci. 2016, 17, 194
5 of 16
5 of 16
5 of 16
Ligation (EPL)
(EPL) and Alkylguanine-DNA-Alkyl
Alkylguanine-DNA-Alkyl Transferase(AGT)
(AGT) Reaction
2.1.5.
2.1.5. Expressed
Expressed Protein
Protein Ligation
Ligation (EPL) and
and Alkylguanine-DNA-Alkyl Transferase
Transferase (AGT)Reaction
Reaction
Another
Another strategy
strategy for
for making
making homogeneous
homogeneous ADCs
ADCs was
was inserting
inserting entire
entire domains
domains or
or proteins
proteins into
into
The
most
well
known
such
method
isisexpressed
protein
ligation
(EPL),
which
relied
a
antibodies.
The
most
well
known
such
method
expressed
protein
ligation
(EPL),
which
reliedon
antibodies. The most well known such method is expressed protein ligation (EPL), which relied
onona
self-splicing
intein
to
activate
the
C-terminal
of
the
target
protein
and
thus
formed
a
new
amide
bond
aself-splicing
self-splicing
intein
activate
C-terminal
of the
target
protein
formed
new amide
intein
to to
activate
thethe
C-terminal
of the
target
protein
and and
thusthus
formed
a newa amide
bond
with with
a small
molecule,
peptide
or or
protein.
EPL
followed
the
mechanism:
(1)
C-terminal thioester
thioester
bond
a
small
molecule,
peptide
protein.
EPL
followed
the
mechanism:
(1)
with a small molecule, peptide or protein. EPL followed the mechanism: (1) C-terminal thioester
through
rearrangement
an
selected
thiol
displacement
of
formation
through the
the spontaneous
spontaneousN
Nto
toS
rearrangementof
anintein;
intein;(2)
selected
thiol
displacement
formation through
the
spontaneous
N
to
SSrearrangement
ofofan
intein;
(2)(2)selected
thiol
displacement
of
the
intein
sequence
to
give
an
activated
thioester;
(3)
thiol
exchange
of
the
thioester
with
a
β-amino
of
the
intein
sequence
to
give
an
activated
thioester;
(3)
thiol
exchange
of
the
thioester
a
β-amino
the intein sequence to give an activated thioester; (3) thiol exchange of the thioester with a β-amino
ligand
oror
protein);
andand
(4) spontaneous
N toNCto
rearrangement
to form
mercapto
ligand(small
(smallmolecule,
molecule,peptide
peptide
protein);
spontaneous
C rearrangement
to
mercapto ligand
(small
molecule,
peptide
or protein);
and (4) (4)
spontaneous
N to C rearrangement
to form
an stable
amideamide
bond that
links
the
antibody
of interest
to
the drug
with
a inserted
cysteine
(Scheme
6)
form
an
stable
bond
that
links
the
antibody
of
interest
to
the
drug
with
a
inserted
cysteine
an stable amide bond that links the antibody of interest to the drug with a inserted cysteine (Scheme 6)
[34].
(Scheme
6) [34].
[34].
Scheme 6.
6. Amino mercapto-derivitized
mercapto-derivitized drug can
be
to
Scheme
be attached
attached
to an
an antibody
antibody through
through intein
intein splicing.
splicing.
Scheme 6. Amino
Amino mercapto-derivitized drug
drug can
can be
attached to
an
antibody
through
intein
splicing.
Reprinted
with
permission
from
reference
[34].
Reprinted
with
permission
from
reference
[34].
Reprinted with permission from reference [34].
Proteins that do not interfere with the function of an insertion can take advantage of the human
interfere with
with the function of an insertion can take advantage of the human
Proteins that do not interfere
O666-alkylguanine-DNA alkyltransferase (hAGT) reaction in which the guanine attached to the O666
O -alkylguanine-DNA
alkyltransferase
to the
the O
O
-alkylguanine-DNA alkyltransferase (hAGT)
(hAGT) reaction
reaction in
in which the guanine attached to
benzyl group is attacked by the cysteine of hAGT and thus transferred to the drug-AGT conjugate
attacked by the
the cysteine
cysteine of
of hAGT
hAGT and
and thus
thus transferred
transferred to
to the
the drug-AGT
drug-AGT conjugate
conjugate
benzyl group is attacked
(Scheme 7). To realize this reaction, hAGT was directly evolved to possess comparable kinetics to the
(Scheme 7).
7). To
To realize
realize this
this reaction,
reaction, hAGT
hAGT was
was directly
directly evolved
evolved to
to possess
possess comparable
comparable kinetics
kinetics to the
(Scheme
wild type hAGT, while retaining the substrate tolerance for the O-benzyl moiety [41].
wild type
type hAGT,
hAGT,while
whileretaining
retainingthe
thesubstrate
substratetolerance
tolerancefor
forthe
theO-benzyl
O-benzylmoiety
moiety[41].
[41].
wild
Scheme 7. Human O66-alkylguanine-DNA alkyltransferase (hAGT) used guanine as a leaving group,
Scheme 7.
7. Human
Human O
O6-alkylguanine-DNA
Scheme
-alkylguanine-DNA alkyltransferase
alkyltransferase (hAGT)
(hAGT) used
used guanine
guanine as
as aa leaving
leaving group,
group,
forming a thioether bond to a benzyl-derivitized drug. Adapted from Reference [41].
forming
a
thioether
bond
to
a
benzyl-derivitized
drug.
Adapted
from
Reference
[41].
forming a thioether bond to a benzyl-derivitized drug. Adapted from Reference [41].
2.2. Conjugation via Amines
2.2. Conjugation
Conjugation via
via Amines
Amines
2.2.
2.2.1. Formation of Amides
2.2.1. Formation
Formation of
of Amides
Amides
2.2.1.
Forming amide is one of the most important reactions for the nucleophilic amines. Amines could
Forming amide
of theofmost
reactions reactions
for the nucleophilic
Amines
could
Forming
amideis one
is one
the important
most important
for the amines.
nucleophilic
amines.
typically be acylated by carboxyl via some familiar activating reagents, such as N-hydroxysuccinimide
typically
be
acylated
by
carboxyl
via
some
familiar
activating
reagents,
such
as
N-hydroxysuccinimide
Amines could typically be acylated by carboxyl via some familiar activating reagents, such as
(NHS), 2-Succinimido-1,1,3,3-tetra-methyluronium tetrafluoroborate (TSTU), and Benzotriazol-1-yl(NHS), 2-Succinimido-1,1,3,3-tetra-methyluronium
tetrafluoroborate (TSTU),
and Benzotriazol-1-ylN-hydroxysuccinimide
(NHS), 2-Succinimido-1,1,3,3-tetra-methyluronium
tetrafluoroborate
(TSTU),
oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP).
oxytripyrrolidinophosphonium
hexafluorophosphate hexafluorophosphate
(PyBOP).
and
Benzotriazol-1-yl-oxytripyrrolidinophosphonium
(PyBOP).
Amines of the antibodies can react with the carboxyls that derived from the drugs in the effect
Amines of
of the
theantibodies
antibodiescan
canreact
reactwith
withthe
thecarboxyls
carboxylsthat
thatderived
derived
from
drugs
effect
Amines
from
thethe
drugs
in in
thethe
effect
of
of the NHS to give antibody-drug conjugates (Scheme 8) [22,23,42]. Amines of lysines are commonly
of the
NHS
to give
antibody-drug
conjugates
(Scheme
8) [22,23,42].
Amines
of lysines
are commonly
the
NHS
to give
antibody-drug
conjugates
(Scheme
8) [22,23,42].
Amines
of lysines
are commonly
used
used for linking drugs to antibodies because lysines are usually exposed on the surface of the
used
for linking
drugs
to antibodies
lysines
areexposed
usually on
exposed
on the
surface
of the
for
linking
drugs to
antibodies
becausebecause
lysines are
usually
the surface
of the
antibodies
antibodies and therefore easily accessible. Antibodies contain up to 80 lysines [43] and, as a result,
antibodies
and
therefore
easily
accessible.
Antibodies
contain
up
to
80
lysines
[43]
and,
as
a
result,
and therefore easily accessible. Antibodies contain up to 80 lysines [43] and, as a result, conjugation
conjugation through lysine residues inevitably leads to twofold heterogeneity: (1) different number
conjugation
through
lysine
residues
inevitably
leads
to twofold heterogeneity:
(1) different
number
through
lysine
residues
inevitably
leads
to twofold
heterogeneity:
(1) different number
of drugs
per
of drugs per antibody; and (2) antibodies with the same number of drugs attached at different sites
of
drugs
per
antibody;
and
(2)
antibodies
with
the
same
number
of
drugs
attached
at
different
sites
antibody; and (2) antibodies with the same number of drugs attached at different sites [31,44]. The
[31,44]. The heterogeneity with respect to DARs can be restricted to a certain extent by adjusting the
[31,44]. The heterogeneity
respect
can to
bearestricted
to a certain
extentthe
bystoichiometry
adjusting the
heterogeneity
with respect with
to DARs
can to
be DARs
restricted
certain extent
by adjusting
stoichiometry of drug and antibody used in the reaction; and with respect to site-specificity, the
stoichiometry
of drugused
andinantibody
usedand
in with
the reaction;
with respect
site-specificity,
of
drug and antibody
the reaction;
respect toand
site-specificity,
thetoheterogeneity
canthe
be
heterogeneity can be limited by the chemical accessibility of reactive groups
[45,46]. Mylotarg®® was
®
heterogeneity
can
be
limited
by
the
chemical
accessibility
of
reactive
groups
[45,46].
Mylotarg
was
limited by the chemical accessibility of reactive groups [45,46]. Mylotarg was the first antibody-drug
the first antibody-drug conjugate on the market by lysine-coupling. In the conjugate, a semi-synthetic
the first antibody-drug conjugate on the market by lysine-coupling. In the conjugate, a semi-synthetic
calicheamicin derivative was activated with NHS, and then attached to the lysines of a humanized
calicheamicin derivative was activated with NHS, and then attached to the lysines of a humanized
Int. J. Mol. Sci. 2016, 17, 194
6 of 16
conjugate
on the market by lysine-coupling. In the conjugate, a semi-synthetic calicheamicin derivative
Int. J. Mol. Sci. 2016, 17, 194
6 of 16
was
with
and then attached to the lysines of a humanized IgG4 [47]. However,
Int.activated
J. Mol. Sci. 2016,
17, NHS,
194
6 of 16
®
®
Mylotarg
was
withdrawn
from the
in 2010
duethe
to the
lack in
of 2010
benefit
improvement
patients.
Int.
J. Mol.
Sci.
2016,
17, 194Mylotarg
6 of 16
IgG4
[47].
However,
wasmarket
withdrawn
from
market
due
to the lack oftobenefit
® was withdrawn from the market in 2010 due to the lack of benefit
IgG4 [47].
However,
Mylotarg
Recently,
there
was
new
clinically
relevant
conjugate
generated
by lysine modification:
improvement
to apatients.
Recently,
there antibody-drug
was a new clinically
relevant
antibody-drug
conjugate
® was withdrawn
improvement
to patients.
Recently,
there® was
newthe
clinically
antibody-drug
conjugate
® [48].
IgG4
[47].
However,
Mylotarg
market relevant
in 2010 due
to the lack of
benefit
Kadcyla
generated
by
lysine
modification:
Kadcyla
[48]. afrom
generated by lysine
modification:
Kadcyla
[48].a new clinically relevant antibody-drug conjugate
improvement
to patients.
Recently,
there ®was
generated by lysine modification: Kadcyla® [48].
Scheme
8. Amines
of the
reacted
with the
carboxyls
that derived
from thefrom
drugsthe
in the
Scheme
8. Amines
of antibodies
the antibodies
reacted
with
the carboxyls
that derived
Scheme
8.
Amines
of
the
antibodies
reacted
with
the
carboxyls
that
derived
from
the
effect
of
the
N-hydroxysuccinimide
(NHS).
Adapted
from
reference
[23].
drugs in the effect of the N-hydroxysuccinimide (NHS). Adapted from reference [23].
drugs in8.the
effect of the N-hydroxysuccinimide
Adaptedthat
fromderived
reference
[23].
Scheme
Amines
antibodies reacted with(NHS).
the carboxyls
from
the
drugs
in
the
of the N-hydroxysuccinimide
(NHS).
Adapted
from
reference
[23].
Hong
al.[49]
[49]effect
developed
an approach
attach
thethe
anticancer
drugdrug
doxorubicin
to
Hong
etetal.
developed
approachtotocovalently
covalently
attach
anticancer
doxorubicin
Hong et al.
[49] developed
an(Fab’)
approach
to covalently
attach the
anticancer
drug doxorubicin
to
an
anti-EGFR
antibody
fragment
through
a
polyethylene
glycol
(PEG)
linker.
In
this
work,
to an anti-EGFR antibody fragment (Fab’) through a polyethylene glycol (PEG) linker. In this work,
an anti-EGFR
fragment
through
a polyethylene
glycol
(PEG) drug
linker.doxorubicin
In this work,
Hong
et al.antibody
[49] developed
an (Fab’)
approach
to covalently
attach the
anticancer
to
CIT–(CH
2)5–PEG24–CO2H was activated by TSTU or PyBOP and then coupled in situ with the
CIT–(CH
2 )5 –PEG24 –CO2 H was activated by TSTU or PyBOP and then coupled in situ with the
CIT–(CH
2)5–PEG
24–CO2H
was
activated
by
TSTU
or
PyBOP
and
then
coupled
in
situ
with
the
an
anti-EGFR
antibody
fragment
(Fab’)
through
a
polyethylene
glycol
(PEG)
linker.
In
this
work,
1 -amine ofof
DOXtotogive
giveCIT–(CH
CIT–(CH)2)5–PEG
–PEG24–DOX,
which was then conjugated with the cysteine
C3C3′-amine
DOX
2 5
24 –DOX, which was then conjugated with the cysteine
C3′-amine
of
DOX
to 2give
CIT–(CH
2)5–PEG
24
–DOX,
which
wasand
then
conjugated
thewith
cysteine
CIT–(CH
2)5–PEG
24–CO
H Fab’.
was
activated
by of
TSTU
or
PyBOP
then
coupled with
in
situ
the
residues
of
an
anti-EGFR
Introduction
PEG
increased
the
drug,
which
residues of an anti-EGFR Fab’. Introduction of PEG increasedaqueous
aqueoussolubility
solubilityofof
the
drug,
which
residues
of
an
anti-EGFR
Fab’.
Introduction
of
PEG
increased
aqueous
solubility
of
the
drug,
which
C3′-amine
of
DOX
to
give
CIT–(CH
2)5–PEG24
–DOX,
which
was
then
conjugated
with
the
cysteine
a yield
improvementofofthe
theconjugation
conjugation reaction
reaction with
with the
ledled
to to
a yield
improvement
the Fab’.
Fab’.
led
to
a yield
improvement
of the
conjugation
withwith
the
Fab’.
residues
of anfamiliar
anti-EGFR
Fab’.
Introduction
of reaction
PEG
increased
aqueous
solubility
of their
the drug,
which
Besides
activating
reagent,
amines
could
react
carboxyl
acids and
derivatives
Besides
familiar
activating
reagent,
amines
could
react
with
carboxyl
acids
and
their
derivatives
to
Besides
familiar
activating
reagent,
amines
could
react
with
carboxyl
acids
and
their
derivatives
led
to
a
yield
improvement
of
the
conjugation
reaction
with
the
Fab’.
to form the amides under the influence of the BTG, SrtA and BirA. For example, Jeger et al. [50]
form
the
amides
under
the
influence
of
the
BTG,
SrtA
and
BirA.
For
example,
Jeger
et
al.
[50]
observed
to form
the
amides
under
the reagent,
influence
ofbythe
BTG,
SrtA with
and
BirA.
Foracids
example,
Jeger
et al. [50]
Besides
activating
amines
could
react
carboxyl
andflexible
their
derivatives
observed
thefamiliar
selective
acylation
of amines
the
glutamines
in the
heavy
chain’s
regions
of
theto
selective
acylation
ofacylation
amines
byamines
the glutamines
inSrtA
the and
heavy
chain’s
flexibleflexible
regions
of
IgG
observed
the
selective
of
by
the
glutamines
in
the
heavy
chain’s
regions
of
form
the
amides
under
the
influence
of
the
BTG,
BirA.
For
example,
Jeger
et
al. an
[50]
an IgG where the asparagines (N) were mutated to glutamines (Q). By using bacterial
where
(N)
were mutated
towere
glutamines
(Q).toByin
using
bacterial
(BTG)
an the
IgGasparagines
where
the
asparagines
(N)they
glutamines
(Q).transglutaminase
Byflexible
using
bacterial
observed
the
selective
acylation
amines
by
themutated
glutamines
the heavy
chain’s
regions
of
transglutaminase
(BTG)
at theseofsites,
synthesized
homogeneous
conjugates
that
were
tumorat an
these
sites,
they
synthesized
homogeneous
conjugates
that
were
tumor-uptake
selective
in
vivo
transglutaminase
(BTG)
at
these
sites,
they
synthesized
homogeneous
conjugates
that
were
tumorIgGselective
where inthe
uptake
vivoasparagines
(Scheme 9). (N) were mutated to glutamines (Q). By using bacterial
(Scheme
uptake9).
selective in(BTG)
vivo (Scheme
transglutaminase
at these 9).
sites, they synthesized homogeneous conjugates that were tumoruptake selective in vivo (Scheme 9).
Scheme 9. The reaction of the amine-drugs and antibodies under the influence of bacterial
Scheme
The reaction
reaction
of the
the from
amine-drugs
transglutaminase
(BTG). Adapted
reference and
[50]. antibodies
Scheme
9. 9. The
of
amine-drugs
and
antibodies under
underthe
theinfluence
influenceofofbacterial
bacterial
transglutaminase
(BTG).
Adapted
from
reference
[50].
Scheme
9.
The
reaction
of
the
amine-drugs
and
antibodies
under
the
influence
of bacterial
transglutaminase (BTG). Adapted from reference [50].
transglutaminase
Adapted
fromsortases
referencefound
[50]. in Gram-positive bacteria, is a transpeptidase
Sortase A (SrtA),(BTG).
one of
the many
Sortase
A
(SrtA), one
of the many
sortases
found
in Gram-positive
bacteria,
is a transpeptidase
[51]
that
can
recognize
sequence,
break
theinTG
bond and facilitate
theisformation
of a new
Sortase
A (SrtA),
oneaaofLPXTG
the many
sortases
found
Gram-positive
bacteria,
a transpeptidase
[51]
[51]
that
can
recognize
LPXTG
sequence,
break
the
TG
bond
and
facilitate
the
formation
of a new
Sortase
A
(SrtA),
one
of
the
many
sortases
found
in
Gram-positive
bacteria,
is
a transpeptidase
amide bond with the amine of glycine-derivitized drug. This was demonstrated through
conjugating
that
can
recognize
a
LPXTG
sequence,
break
the
TG
bond
and
facilitate
the
formation
of
a
new
amide
amide
bond
the amine
of glycine-derivitized
drug.
was
demonstrated
[51]
that
can with
recognize
a LPXTG
sequence,
break
the
TGThis
bond
and
facilitate
thethrough
formation
of a new
folate,
biotin,
rhodamine
and
so
on [52,53].
The
leaving
glycine
could return
as a conjugating
competing
bond
with
the
amine
of
glycine-derivitized
drug.
This
was
demonstrated
through
conjugating
folate,
folate,
biotin,
rhodamine
and
so
on
[52,53].
The
leaving
glycine
could
return
as
a
competing
amide
bond
with
the
amine
of
glycine-derivitized
drug.
This
was
demonstrated
through
conjugating
nucleophile, which reversed the reaction, unless more than 10 equivalents of glycine-derivitized
biotin,
rhodamine
and
so
on
[52,53].
The
leaving
glycine
could
return
as
a
competing
nucleophile,
nucleophile,
which
reversed
the
unless
than
10 equivalents
ofsolution
glycine-derivitized
folate,
biotin,
rhodamine
and[54].
so reaction,
on [52,53].
The
leaving
glycine
return
as atocompeting
drugs
were
used
(Scheme
10)
Williamson
et
al.more
[55]
put
forward
acould
creative
solve this
which
reversed
the (Scheme
reaction,
unless
more
than
10
equivalents
of
glycine-derivitized
drugs
werethis
used
drugs
were
used
10)
[54].
Williamson
et
al.
[55]
put
forward
a
creative
solution
to solve
nucleophile,
which
reversed
the
reaction,
unless
more
than
10
equivalents
of
glycine-derivitized
problem: they used threonine esters as substrates and the product glycolic acids were
far less
(Scheme
10)they
[54].
Williamson
al.
[55] as
putsubstrates
forward
aand
creative
solution
to solve
thiswere
problem:
they
problem:
used
threonine
esters
the
product
glycolic
acids
far this
less
drugs
were
used
(Scheme
10) et
[54].
Williamson
et al. [55]
put
forward
a creative
solution
to solve
nucleophilic,
enabling
conjugation
with a 1:1
stoichiometry.
used
threonine
esters
as
substrates
and
the
product
glycolic
acids
were
far
less
nucleophilic,
enabling
nucleophilic,
enabling
conjugation
with
a
1:1
stoichiometry.
problem: they used threonine esters as substrates and the product glycolic acids were far less
conjugation
with
a 1:1 stoichiometry.
nucleophilic,
enabling
conjugation with a 1:1 stoichiometry.
Scheme 10. The sortase-mediated conjugation of glycine-derivitized drugs and the antibodies.
Scheme
10.
sortase-mediated
conjugation
of glycine-derivitized
and
the antibodies.
Scheme
10. from
TheThe
sortase-mediated
conjugation
of glycine-derivitized
drugsdrugs
and the
antibodies.
Adapted
Adapted
reference
[54].
Adapted
from
reference
[54].
Scheme
10. The
from
reference
[54]. sortase-mediated conjugation of glycine-derivitized drugs and the antibodies.
Adapted
from
reference
[54].could recognize and acylate the Avi-tag, which is a 15-amino acid
The biotin
ligase
(BirA)
The biotin ligase (BirA)
could
recognize
acylate
the Avi-tag,
which
is analogs
a 15-amino
acid
GLNDIFEAQKIEWHE
sequence.
Chen
et al. [56]and
found
that BirA
could accept
keto
of biotin
GLNDIFEAQKIEWHE
sequence.
Chen
et
al.
[56]
found
that
BirA
could
accept
keto
analogs
of
biotin
The biotin ligase (BirA) could recognize and acylate the Avi-tag, which is a 15-amino acid
GLNDIFEAQKIEWHE sequence. Chen et al. [56] found that BirA could accept keto analogs of biotin
Int. J. Mol. Sci. 2016, 17, 194
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J. Mol.
Mol. Sci.
Sci. 2016,
2016, 17,
17, 194
194
Int.
of 16
16
77 of
The biotin ligase (BirA) could recognize and acylate the Avi-tag, which is a 15-amino acid
GLNDIFEAQKIEWHE
sequence.
et al. [56]
found thatthat
BirAcould
couldconjugate
accept keto
analogs
of biotin
as substrates
substrates to
to react
react with
with
Avi-tagChen
and form
form
intermediates
that
could
conjugate
with
the oxyamines
oxyamines
as
Avi-tag
and
intermediates
with
the
as
to react
and form
intermediates
that could conjugate with
the oxyamines
of
of substrates
drugs (Scheme
(Scheme
11).with
BirAAvi-tag
has aa similar
similar
function
as formylglycine-generating
formylglycine-generating
enzyme
(FGE), which
which
of
drugs
11).
BirA
has
function
as
enzyme
(FGE),
drugs
(Scheme
11).
BirA
has
a
similar
function
as
formylglycine-generating
enzyme
(FGE),
which
can
can site-specifically
site-specifically insert
insert an
an aldehyde
aldehyde group
group into
into an
an antibody.
antibody. In
In this
this case,
case, aa keto
keto group
group was
was
can
site-specifically
an aldehyde
groupBirA
into requires
an
antibody.
In this
case, ait
group
was introduced
introduced into
intoinsert
the antibody.
antibody.
Although
BirA
requires
long
sequence,
itketo
is far
far
less restricted
restricted
in the
the
introduced
the
Although
aa long
sequence,
is
less
in
into
the
antibody.
Although
BirA
requires
a
long
sequence,
it
is
far
less
restricted
in
the
location
along
location
along
the
antibody.
They
demonstrated
the
reaction
with
the
installed
carbonyl
using
location along the antibody. They demonstrated the reaction with the installed carbonyl using
the
antibody.
They demonstrated the reaction with the installed carbonyl using fluorescein hydrazide.
fluorescein
hydrazide.
fluorescein
hydrazide.
Scheme 11.
11. The
The BirA-mediated
BirA-mediated conjugation
conjugation
of biotin-like
biotin-like
ketones,
oxyamine-drugs
and
antibody.
Scheme
BirA-mediated
conjugation of
biotin-like ketones,
ketones, oxyamine-drugs
oxyamine-drugs and
and antibody.
antibody.
11.
Adapted from
from
Reference
[56].
Adapted
Reference
[56].
from Reference [56].
2.2.2. Formation
Formation of
of Carbamates
Carbamates
2.2.2.
Amines of
of the
the
drugs
could
react
with
the
hydroxyls
that
derived
from
the
linkers
in in
thethe
effect
of
the
linkers
in
the
effect
of
Amines
the drugs
drugs could
couldreact
reactwith
withthe
thehydroxyls
hydroxylsthat
thatderived
derivedfrom
from
the
linkers
effect
phosgene,
4-nitrophenyl
chloroformate,
etc.
and
form
the
carbamate
containing
drug-linkers
phosgene,
4-nitrophenyl
chloroformate,
drug-linkers
of
phosgene,
4-nitrophenyl
chloroformate,etc.
etc.and
andform
formthe
the carbamate
carbamate containing
containing drug-linkers
(Scheme
12),
which
was
then
coupled
to
antibodies
[57,58].
For
instance,
Jeffrey
et
al.
[57]
prepared
(Scheme 12), which was then coupled to antibodies [57,58]. For instance, Jeffrey et al. [57] prepared
antibody-drug conjugates
conjugates in
in which
which the
the amino-CBI
amino-CBI drug,
drug, aa DNA
DNA minor
minor groove
groove binder
binder drug
drug (MGBs),
(MGBs),
antibody-drug
was attached
attached to
to
monoclonal
antibodies
through
peptide
linkers
that
designed
to
release
drugs
in the
the
was
monoclonal
antibodies
through
peptide
linkers
that
designed
to
release
drugs
in
to monoclonal antibodies through peptide linkers that designed to release drugs
in
lysosomes
of
target
cells.
In
this
study,
the
amino-CBI
drug
reacted
with
phosgene
to
form
the
lysosomes
of target
cells.
In In
thisthis
study,
the
the
lysosomes
of target
cells.
study,
theamino-CBI
amino-CBIdrug
drugreacted
reactedwith
withphosgene
phosgene to
to form the
corresponding isocyanate
isocyanate and
and then
then the
the linker
linker with
with aa hydroxyl
hydroxyl was
was added
added to
to form
form the
the carbamate.
carbamate.
corresponding
carbamate.
Dubowchik
et
al.
[59]
linked
the
anticancer
drug
doxorubicin
to
chimeric
BR96,
an
internalizing
linked the
the anticancer
anticancer drug
drug doxorubicin
doxorubicin to
to chimeric
chimeric BR96,
BR96, an
an internalizing
internalizing
Dubowchik et al. [59] linked
monoclonal antibody,
antibody, through
throughlysosomally
lysosomallycleavable
cleavable dipeptides.
dipeptides. In
In this
this case,
case, the
the carbamate
carbamate between
between
antibody,
through
lysosomally
cleavable
dipeptides.
In
monoclonal
drug and
and antibody
antibody was
was prepared
prepared with
with 4-nitrophenyl
4-nitrophenyl chloroformate.
chloroformate. These
These antibody-drug
antibody-drug conjugates
conjugates
chloroformate.
drug
usually
insert
a
spacer
such
as
para-aminobenzyl
carbamate
(PABC)
between
the
peptide
linkers
and
usually insert a spacer such as para-aminobenzyl carbamate (PABC) between the peptide linkers and
the drugs
drugs to
to minimize
minimize the
the steric
steric interaction
interaction effects.
effects. This
This approach
approach has
has previously
previously been
been used
used to
to release
release
the
doxorubicin [60],
[60], MMAE
MMAE [17]
[17] and
and camptothecin
camptothecin [61]
[61] from
from antibody-drug
antibody-drugconjugates.
conjugates.
doxorubicin
camptothecin
[61]
from
antibody-drug
conjugates.
Scheme 12.
12. The
The reaction
reaction of
of amines
amines of
of the
the drug
drug and
and hydroxyls
hydroxyls of
of the
the linkers
linkers in the
the effect
effect of
of phosgene
phosgene
Scheme
Scheme
12. The
reaction of
amines of
the drug
and hydroxyls
of the
linkers inin
the effect
of phosgene
or
or 4-nitrophenyl
4-nitrophenyl chloroformate.
chloroformate. Adapted
Adapted from
from Reference
Reference [57,58].
[57,58].
or
4-nitrophenyl chloroformate. Adapted from Reference [57,58].
Another carbamate
carbamate was
was designed
designed for
for the
the hydroxy
hydroxy containing
containing drug.
drug. For
For instance,
instance, antibody-drug
antibody-drug
Another
Another
carbamate
was
designed
for
the
hydroxy
containing
drug.
For
instance,
antibody-drug
conjugate SYD985
SYD985 consists
consists of
of seco-DUBA
seco-DUBA drug,
drug, self-elimination
self-elimination spacer,
spacer, cleavable
cleavable peptides
peptides linker
linker and
and
conjugate
conjugate
SYD985
consists of seco-DUBA
drug, to
self-elimination
spacer,spacer
cleavable
peptides linker
trastuzumab.
The seco-DUBA
seco-DUBA
drug was
was linked
linked
the self-elimination
self-elimination
via carbamate
carbamate
bond and
that
trastuzumab.
The
drug
to the
spacer via
bond
that
trastuzumab.
seco-DUBATreatment
drug was linked
to the self-elimination
spacer via carbamate
bond that
derived from
fromThecarbonate.
carbonate.
of MOM
MOM
protected duocarmycin
duocarmycin
with 4-nitrophenyl
4-nitrophenyl
derived
Treatment of
protected
with
derived
from carbonate.
of MOMcarbonate.
protected duocarmycin
4-nitrophenyl
chloroformate
chloroformate
gave the
theTreatment
corresponding
Commerciallywith
available
tert-butyl
methyl(2chloroformate
gave
corresponding
carbonate. Commercially
available
tert-butyl
methyl(2gave
the
corresponding
carbonate.
Commercially
available
tert-butyl
methyl(2-(methylamino)
(methylamino)
ethyl)carbamate
was
then
used
to
synthesize
the
carbamate.
Removal
of
the
Boc and
and
(methylamino) ethyl)carbamate was then used to synthesize the carbamate. Removal of the Boc
ethyl)carbamate
was
then used toacid
synthesize
the carbamate.
Removal
of the Boc and MOM
groups
MOM
groups
with
trifluoroacetic
(TFA)
provided
cyclization
spacer-duocarmycin
as
a
TFA
salt.
MOM groups with trifluoroacetic acid (TFA) provided cyclization spacer-duocarmycin as a TFA salt.
with
trifluoroacetic
acid (TFA) provided
cyclization
TFA salt. Cyclization
Cyclization
spacer-duocarmycin
was reacted
reacted
with spacer-duocarmycin
the activated
activated linker
linker as
toa synthesis
synthesis
drug-linker
Cyclization
spacer-duocarmycin
was
with
the
to
drug-linker
spacer-duocarmycin
was
reacted
with
the
activated
linker
to
synthesis
drug-linker
module under
module
under
slightly
basic
conditions
[62].
module under slightly basic conditions [62].
slightly basic conditions [62].
Int. J. Mol. Sci. 2016, 17, 194
Int. J. Mol. Sci. 2016, 17, 194
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2.3. Conjugation
2.3.
Conjugation via
via Alcohols
Alcohols
2.3.1. Formation of Carbonates
Similar with amines, alcohols can react with chloroformates to form carbonates. For example,
et al.
al. [63]
[63]conjugated
conjugated7-ethyl-10-hydroxycamptothecin
7-ethyl-10-hydroxycamptothecin(SN-38)
(SN-38)derivatives
derivativesto tohMN-14,
hMN-14,
Moon et
a
a humanizedanti-CEACAM5
anti-CEACAM5mAb,
mAb,via
viaa acarbonate
carbonatebond.
bond. To construct the carbonate bond,
humanized
BOC-SN-38 [64] was firstly converted to its 20-O-chloroformate, and then reacted in situ with the
linker, MC-Phe-Lys(MMT)-PABOH
MC-Phe-Lys(MMT)-PABOH[60].
[60].
known linker,
2.3.2. Formation of Ether Bonds
Jeffrey et al. [57] described
described a method
method to
to conjugate
conjugate amino-CBI drug to the monoclonal antibody
by formation of carbamates (see Section 2.2.2). Another approach for attaching a DNA minor groove
binder drug (MGB) derivative to mAb involved O-alkylation of the hydroxy in aza-CBI to form ether
bond. In this approach,
approach, a para-aminobenzyl ether (PABE)
(PABE) group
group was used as a self- elimination spacer
between the drug and the peptides
[57].
An
important
step
in the synthesis of this antibody-MGB
peptides
antibody-MGB
conjugate was the formation of an ether bond through the O-alkylation of aza-CBI by bromide and
potassium carbonate (Scheme 13). This work also showed that the conjugate could cleave via amide
bond hydrolysis and lead to the release of free phenolic drug [65]. This approach should be broadly
applied to drugs that have a phenolic
phenolic hydroxyl
hydroxyl group
group as
as the
the conjugate
conjugate site
site [66].
[66].
Scheme 13.
13. The
through the
the O-alkylation
O-alkylation of
aza-CBI by
by bromide
bromide and
and
Scheme
The formation
formation of
of an
an ether
ether bond
bond through
of aza-CBI
potassium
carbonate.
Adapted
from
reference
[57].
potassium carbonate. Adapted from reference [57].
2.3.3. Formation of Ester Bond
2.3.3. Formation of Ester Bond
In 2010, Quiles et al. [67] developed paclitaxel-monoclonal antibody (PTX-mAb) conjugates that
In 2010, Quiles et al. [67] developed paclitaxel-monoclonal antibody (PTX-mAb) conjugates that
could deliver significant doses of drugs to the tumor cells using the ester bond. The conjugates used
could deliver significant doses of drugs to the tumor cells using the ester bond. The conjugates used
PEG as linker, and the paclitaxel was attached to the linker with glutarate (GL) or succinate (SX)
PEG as linker, and the paclitaxel was attached to the linker with glutarate (GL) or succinate (SX)
through the ester bond, the resulting PTX–L–Lys[(PEG12)3–PEG4]–PEG6–CO2NHS (L = GL or SX) was
through the ester bond, the resulting PTX–L–Lys[(PEG12 )3 –PEG4 ]–PEG6 –CO2 NHS (L = GL or SX) was
then conjugated to C225, an antiepidermal growth factor receptor (anti-EGFR) monoclonal antibody,
then conjugated to C225, an antiepidermal growth factor receptor (anti-EGFR) monoclonal antibody,
producing completely soluble conjugates.
producing completely soluble conjugates.
2.4. Conjugation
2.4.
Conjugation via
via Aldehydes
Aldehydes
2.4.1. Conjugation via FGE
is another
method method
for linkingfor
drugs
to antibodies.
Conjugation via
viaaldehydes
aldehydes
is another
linking
drugs Formylglycineto antibodies.
generating enzymes (FGEs),enzymes
which recognize
modify
a shortand
CXPXR
(where
X is any
amino
acid)
Formylglycine-generating
(FGEs), and
which
recognize
modify
a short
CXPXR
(where
sequence,
can beacid)
usedsequence,
to modify
of antibodies
to aldehyde-containing
X
is any amino
canthe
be cysteine
used to residues
modify the
cysteine residues
of antibodies to
formylglycine (FGly)formylglycine
residues. This
method
was applied
to generate
site-specific
antibody-drug
aldehyde-containing
(FGly)
residues.
This method
was applied
to generate
site-specific
conjugates via incorporating
cytotoxic
drugs into
monoclonal
with a formylglycine
[68].
antibody-drug
conjugates via
incorporating
cytotoxic
drugs antibodies
into monoclonal
antibodies with
a
Following the [68].
production
of modified
antibody,
a chemical
methoda can
be used
to conjugate
a drug
formylglycine
Following
the production
of modified
antibody,
chemical
method
can be used
to
to the aldehyde
formylglycine.
or hydrazide
drugs
were attached
the modified
conjugate
a druggroup
to the of
aldehyde
group ofOxyamine
formylglycine.
Oxyamine
or hydrazide
drugstowere
attached
antibodies
successfully
(Scheme
14) [69].
Recently,
this kind
of aldehyde
was
to
the modified
antibodies
successfully
(Scheme
14) [69].
Recently,
this kindconjugation
of aldehydestrategy
conjugation
further developed
Agarwal by
et al.
[70], who
used
(HIPS) chemistry
strategy
was furtherbydeveloped
Agarwal
et al.
[70],hydrazino-iso-Pictet-Spengler
who used hydrazino-iso-Pictet-Spengler
(HIPS)
to attach maytansine
to the aldehyde-containing
trastuzumab.
The HIPS
resulted
in the
chemistry
to attach maytansine
to the aldehyde-containing
trastuzumab.
Thechemistry
HIPS chemistry
resulted
formation of a covalent C–C bond, which was more stable than oxime or hydrazone ligation products
in physiological condition. What is more, this study showed that the aldehyde group can be
Int. J. Mol. Sci. 2016, 17, 194
9 of 16
in
formation
of194
a covalent C–C bond, which was more stable than oxime or hydrazone ligation
Int.the
J. Mol.
Mol.
Sci. 2016,
2016, 17,
17,
of 16
16
Int.
J.
Sci.
194
99 of
products in physiological condition. What is more, this study showed that the aldehyde group can
be
introduced
many
locations
theantibody
antibodywithout
withoutaffecting
affectingthe
thestability
stability and
and activity
activity of the
introduced
in in
many
locations
of of
the
introduced
in
many
locations
of
the
antibody without
affecting the
stability and
activity of
the
obtained antibody-drug
conjugates.
antibody-drug conjugates.
conjugates.
obtained antibody-drug
O
O
N
N
H2N
N
H
2
O
O
SH
SH
CTPHSR
CTPHSR
FGE
FGE
fGTPHSR
fGTPHSR
CHO
CHO
fGTPHSR
fGTPHSR
H
H
N
N
O
O
N
N
O
O
N
N
N
N
fGTPHSR
fGTPHSR
Scheme 14.
14. The
The conversion
conversion of
of the
the cysteine
cysteine thiol
thiol to
to an
an aldehyde
aldehyde group
group by
by formylglycine-generating
formylglycine-generating
Scheme
14.
cysteine
thiol
to
an
aldehyde
group
by
formylglycine-generating
enzymes (FGE)
(FGE) enables
enables reactions
reactions with
with oxyamine
oxyamine drugs.
drugs. Adapted
Adapted from
from reference
reference [70].
[70].
enzymes
reactions
with
oxyamine
drugs.
Adapted
from
reference
[70].
2.4.2. Conjugation
Conjugation via
via aaRS
aaRS
2.4.2.
The tRNA/aminoacyl-tRNA
tRNA/aminoacyl-tRNA synthetase
synthetase
(aaRS) pair
pair can
can
site-specifically
incorporate an
an unnatural
unnatural
The
synthetase (aaRS)
can site-specifically
site-specifically incorporate
amino
acid
(e.g.,
p-acetylphenylalanine,
pAcPhe)
into
antibody
[71].
Recently,
the
genetic
amino acid
acid(e.g.,
(e.g.,
p-acetylphenylalanine,
pAcPhe)
into antibody
[71]. the
Recently,
the genetic
amino
p-acetylphenylalanine,
pAcPhe)
into antibody
[71]. Recently,
genetic incorporation
incorporation
of unnatural
unnatural
amino
acids into
into
antibodies
had
become
useful
tooldesign
in the
the [72,73].
ADC design
design
incorporation
of
amino
acids
antibodies
become
aa useful
tool
in
ADC
of
unnatural amino
acids into
antibodies
had
become ahad
useful
tool in
the ADC
This
[72,73].
This
method
was
successfully
realized
by
Axup
et
al.
[74],
who
developed
site-specific
[72,73]. was
Thissuccessfully
method was
successfully
realized
bywho
Axup
et al. [74],
who developed
method
realized
by Axup
et al. [74],
developed
site-specific
auristatinsite-specific
conjugates
auristatin
conjugates
of trastuzumab.
trastuzumab. A
A was
p-acetylphenylalanine
was codon
loadedofonto
onto
thebyamber
amber
codon
of
auristatin
conjugates
of
p-acetylphenylalanine
was
loaded
the
of
of
trastuzumab.
A p-acetylphenylalanine
loaded onto the amber
tRNA
aaRS codon
and then
tRNA by
by aaRS
aaRS
and then
then specifically
specifically
incorporated
into
the amber
amber site
site
of the
the
trastuzumab
heavy chain.
chain.
tRNA
and
incorporated
into
the
of
trastuzumab
heavy
specifically
incorporated
into the amber
site of the
trastuzumab
heavy
chain.
After purification,
the
After
purification,
the
antibody
was
coupled
to
the
monomethyl
auristatin
F
(MMAF)
derivative
that
After purification,
the antibody
was coupled
to the monomethyl
auristatinthat
F (MMAF)
derivative
that
antibody
was coupled
to the monomethyl
auristatin
F (MMAF) derivative
contains
an oxyamine
contains
an
oxyamine
groupwith
by an
an
oxime
ligation
with(Scheme
the pAcPhe
pAcPhe
residues
(Scheme
15). The
The analysis
analysis
contains
group
by
oxime
ligation
with
the
(Scheme
15).
group
byan
anoxyamine
oxime ligation
the
pAcPhe
residues
15). residues
The analysis
of antitumor
activity
of antitumor
antitumor activity
activity and pharmacokinetics
pharmacokinetics of
of this
this site-specific
site-specific antibody-drug
antibody-drug conjugate confirmed
confirmed
of
and
pharmacokineticsand
of this site-specific antibody-drug
conjugate confirmed its conjugate
efficacy and stability
its efficacy
efficacy and stability in serum.
its
in
serum. and stability in serum.
Scheme 15.
15. Incorporation
Incorporation of
of p-acetylphenylalanine
p-acetylphenylalanine (pAcPhe)
(pAcPhe) allows
allows for
for site-specific
site-specific conjugation
conjugation of
of
Scheme
Scheme
15. Incorporation
of p-acetylphenylalanine
(pAcPhe) allows
for site-specific
conjugation of
drugs to
to the
the modified
modified antibodies.
antibodies. Adapted
Adapted from
from reference
reference [74].
[74].
drugs
drugs
to the
modified antibodies.
Adapted from
reference [74].
2.4.3. Oxidation
Oxidation of
of Sialic
Sialic Acids
Acids
2.4.3.
2.4.3.
Oxidation of
Sialic Acids
Glycoengineering has
has been
been employed
employed to
to synthesize
synthesize site-specific
site-specific antibody-drug
antibody-drug conjugates,
conjugates, in
in
Glycoengineering
Glycoengineering
has been employed
to synthesize
site-specific antibody-drug
conjugates, in
which
sialic
acids
were
used
as
chemical
handles
for
selective
conjugations.
This
was
achieved
by
which sialic
sialic acids
acids were
were used
used as
as chemical
chemical handles
handles for
for selective
selective conjugations. This
was achieved
achieved by
by
which
This was
incorporating
sialic acids
acids into
into the
the native
native glycans
glycans of
of trastuzumab
trastuzumabconjugations.
through β-1,4-galactosyltransferase
β-1,4-galactosyltransferase
incorporating
sialic
through
incorporating
sialic acids into the native
through
β-1,4-galactosyltransferase
(Gal T)
T) and
and α-2,6-sialyltransferase
α-2,6-sialyltransferase
(Sial glycans
T). Prior
Priorofto
totrastuzumab
reaction with
with
the oxyamine
oxyamine
drugs, the
the alcohol
alcohol
(Gal
(Sial
T).
reaction
the
(Gal
T)
and
α-2,6-sialyltransferase
(Sial
T).
Prior
to
reaction
with
the
oxyamine
drugs,drugs,
the
alcohol
groups
groups
of
sialic
acids
were
oxidated
to
aldehyde
groups.
The
resulting
antibodies
could
react
with
groups
of sialic
acids
were to
oxidated
to groups.
aldehyde
groups.
The
resultingcould
antibodies
could
react
with
of
sialic
acids
were
oxidated
aldehyde
The
resulting
antibodies
react
with
the
cytotoxic
the cytotoxic
cytotoxic drugs
drugs via
via an
an oxime
oxime ligation
ligation (Scheme
(Scheme 16).
16). This
This method
method was
was evaluated
evaluated by
by conjugating
conjugating
the
drugs
via an oxime
ligation
(Scheme
16). This method
was evaluated
by conjugating
trastuzumab
trastuzumab
with
two
drugs,
monomethyl
auristatin
E
(MMAE)
and
dolastatin
10. with
The
trastuzumab with two drugs, monomethyl auristatin E (MMAE) and dolastatin 10.
The
glycoengineered
antibody-drug
conjugates
exhibited
comparable
antitumor
activities
to
the
glycoengineered antibody-drug conjugates exhibited comparable antitumor activities to the
conventional analogs
analogs [75].
[75].
conventional
Int. J. Mol. Sci. 2016, 17, 194
10 of 16
two drugs, monomethyl auristatin E (MMAE) and dolastatin 10. The glycoengineered antibody-drug
conjugates
Int.
J. Mol. Sci.exhibited
2016, 17, 194comparable antitumor activities to the conventional analogs [75].
10 of 16
Int. J. Mol. Sci. 2016, 17, 194
10 of 16
Scheme
Scheme 16.
16. The
The modification
modification of
of glycans
glycans of
of trastuzumab
trastuzumab by
by Gal
Gal T
T and
and Sial
Sial T
T leads
leads to
to the
the incorporation
incorporation
Scheme
16.
The
modification
of
glycans
of
trastuzumab
by
Gal
T
and
Sial
T
leads
to
the
incorporation
of
sialic
acids,
which
were
oxidated
and
coupled
to
the
drugs
through
oxime
ligation.
Adapted
of sialic acids, which were oxidated and coupled to the drugs through oxime ligation. Adapted from
from
reference
reference [75].
[75].
2.4.4.
Reagent
2.4.4. Conjugation
Conjugation via
via Transamination
Transamination Reagent
Witus
[76,77]
introduced
carbonyl
groups
by
reagent
pyridoxal
[76,77]
introduced
carbonyl
groups by
transamination
reagent pyridoxal
51 -phosphate
Witus etet
etal. al.
al.
[76,77]
introduced
carbonyl
groups
by transamination
transamination
reagent
pyridoxal
5′-phosphate
(PLP)
at
the
N-terminus
of
antibodies,
which
could
be
used
as
unique
attachment
(PLP)
at the (PLP)
N-terminus
of antibodies,
which could
be used
sites forsites
the
5′-phosphate
at the N-terminus
of antibodies,
which
couldas
beunique
used asattachment
unique attachment
sites
for
the
conjugation
formation
(Scheme
17).
However,
the
reaction
yields
were
not
very
high
and
high
conjugation
formation
(Scheme
17).
However,
the
reaction
yields
were
not
very
high
and
high
for the conjugation formation (Scheme 17). However, the reaction yields were not very high and
temperatures
application of
of this
this method.
method. To
To solve
solve these
these problems,
problems,
temperatures were
were required,
required, which
which limited
limited the
the application
application
of
this
method.
To
solve
these
problems,
they
developed
a
combinatorial
peptide
library
screening
platform
and
found
a
new
they developed a combinatorial peptide library screening platform and found a new transamination
transamination
reagent,
N-methylpyridinium-4-carboxaldehyde
[78]. Antibodies
benzenesulfonate salt
salt (RS)
Antibodies with
reagent, N-methylpyridinium-4-carboxaldehyde
N-methylpyridinium-4-carboxaldehyde benzenesulfonate
benzenesulfonate
salt
(RS) [78].
Antibodies
with
glutamate-rich
sequences
were
particularly
reactive
substrates
for
this
reagent
[79].
glutamate-rich sequences were particularly reactive
reactive substrates
substrates for
for this
this reagent
reagent [79].
[79].
R
R
PLP or RS
PLP or RS
NH2
NH2
R1ONH2
R1ONH2
R
R
R1
R
N O 1
N O
O
O
O
O
OH O
OH O
N
N
PLP
PLP
R
R
H
H
OPO322
OPO3
N
N
H
H
O3S
O3S
RS
RS
Scheme
oxidation of
amines to
aldehyde groups
which could
react with
oxyaminedrugs using
Scheme 17.
17. The
The
The oxidation
oxidation of
of amines
amines to
toaldehyde
aldehyde groups
groups which
which could
could react
react with
with oxyaminedrugs
oxyaminedrugs using
transamination
reagents
pyridoxal
5′-phosphate
(PLP)
or
N-methylpyridinium-4-carboxaldehyde
1
transamination
or N-methylpyridinium-4-carboxaldehyde
N-methylpyridinium-4-carboxaldehyde
transamination reagents pyridoxal 55′-phosphate
-phosphate (PLP) or
benzenesulfonate
salt (RS).
(RS). Adapted
Adapted from
from reference
reference
[79].
benzenesulfonate salt
reference [79].
[79].
2.5.
Conjugation via
Azides
2.5.
2.5. Conjugation
Conjugation via
via Azides
Azides
2.5.1.
2.5.1. Click
Click Reactions
Reactions with
with DBCO
DBCO
Azides
can
react
with
alkynes
to
triazoles
through
click
such
alkynes
to form
triazoles
through
click chemistries,
such as copper-catalyzed
Azides can
canreact
reactwith
with
alkynes
to form
form
triazoles
through
click chemistries,
chemistries,
such as
as coppercoppercatalyzed
azide-alkyne
cycloaddition
(CuAAC)
and
strain-promoted
azide-alkyne
cycloaddition
azide-alkyne
cycloaddition
(CuAAC) (CuAAC)
and strain-promoted
azide-alkyne
cycloaddition
(SPAAC)
catalyzed
azide-alkyne
cycloaddition
and strain-promoted
azide-alkyne
cycloaddition
(SPAAC)
(Scheme
This
approach
used
to
antibody-drug
(Scheme
[80,81]. 18)
This[80,81].
approach
was
recently was
used recently
to construct
antibody-drug
For
(SPAAC) 18)
(Scheme
18)
[80,81].
This
approach
was
recently
used
to construct
construct conjugates.
antibody-drug
conjugates.
For
et
conjugated
drugs
antibodies
using
In
example, Zhou
et al. [81]Zhou
conjugated
drugs
to antibodies
this method.
Inmethod.
this study,
an
conjugates.
For example,
example,
Zhou
et al.
al. [81]
[81]
conjugated
drugs to
tousing
antibodies
using this
this
method.
In this
this
study,
an
azide-containing
reagent,
sodium
(difluoroalkylazido)sulfinate
(DAAS-Na),
allowed
azide
azide-containing
reagent, sodium
(DAAS-Na),
allowedallowed
azide groups
study,
an azide-containing
reagent,(difluoroalkylazido)sulfinate
sodium (difluoroalkylazido)sulfinate
(DAAS-Na),
azide
groups
to
to
and
products
then
to
to be linked
heteroaromatics,
and the products
thencould
be attached
monoclonal
antibodies
groups
to be
betolinked
linked
to heteroaromatics,
heteroaromatics,
and the
the could
products
could
then be
betoattached
attached
to monoclonal
monoclonal
antibodies
by
reactions.
DAAS-Na
used
heteroarene
reaction,
by click reactions.
used inwas
the heteroarene
reaction, in which
ZnClin
antibodies
by click
clickDAAS-Na
reactions.was
DAAS-Na
was
used in
in the
the functionalization
heteroarene functionalization
functionalization
reaction,
in2
which
ZnCl
2 and TsOH·H2O were acid additives and tBuOOH was an oxidant. The resulting azideand TsOH¨
acid2O
additives
andadditives
tBuOOHand
wastBuOOH
an oxidant.
resultingThe
azide-linked
drugs
which
ZnClH
2 and
TsOH·H
were acid
wasThe
an oxidant.
resulting azide2 O were
linked
linked drugs
drugs could
could react
react with
with aa dibenzylcyclooctyne
dibenzylcyclooctyne (DBCO)
(DBCO) containing
containing antibody
antibody through
through aa CuAAC
CuAAC
reaction.
This
strategy
expands
the
extent
of
bioactive
drugs
that
can
be
linked
to
monoclonal
reaction. This strategy expands the extent of bioactive drugs that can be linked to monoclonal
antibodies.
antibodies.
Int. J. Mol. Sci. 2016, 17, 194
11 of 16
could react with a dibenzylcyclooctyne (DBCO) containing antibody through a CuAAC reaction. This
Int. J. Mol. Sci. 2016, 17, 194
11 of 16
strategy
expands
the
Int. J. Mol. Sci.
2016, 17,
194extent of bioactive drugs that can be linked to monoclonal antibodies.
11 of 16
Scheme 18. The synthesis of ADCs through copper-catalyzed azide-alkyne cycloaddition (CuAAC)
Scheme
18. The
The synthesis
synthesis
of ADCs
ADCs cycloaddition
through copper-catalyzed
copper-catalyzed
azide-alkyne
cycloaddition
Scheme
18.
of
through
azide-alkyne
cycloaddition
(CuAAC)
and strain-promoted
azide-alkyne
(SPAAC) click
reactions. Adapted
from (CuAAC)
reference
and
strain-promoted
azide-alkyne
cycloaddition
(SPAAC)
click
reactions.
Adapted
from
reference
[81].
[81].strain-promoted azide-alkyne cycloaddition (SPAAC) click reactions. Adapted from reference
[81].
Microbial transglutaminase
cancan
catalyze
the formation
of an isopeptide
bond between
Microbial
transglutaminase(MTGase)
(MTGase)
catalyze
the formation
of an isopeptide
bond
Microbial
transglutaminase
(MTGase)
can
catalyze
the
formation
of
an
isopeptide
bond
between
the aminethe
group
of glutamine
and the primary
amine
of lysine
while
simultaneously
releasing
an
between
amine
group of glutamine
and the
primary
amine
of lysine
while simultaneously
the
amine gas
group
of glutamine
and activity
the primary
amine ofwas
lysine
whiletosimultaneously
releasing an
ammonia
[82].
The
coupling
of
MTGase
applied
synthesize
antibody-drug
releasing an ammonia gas [82]. The coupling activity of MTGase was applied to synthesize
ammonia
[82]. For
Theexample,
couplingDennler
activity etofal.MTGase
was applied
tohomogeneous
synthesize antibody-drug
conjugatesgas
[83–85].
[83]
afforded
highly
trastuzumabantibody-drug
conjugates
[83–85]. For example,
Dennler
et al.a[83]
afforded
a highly homogeneous
conjugates
[83–85].
For
example,
Dennler
et
al.
[83]
afforded
a
highly
homogeneous
trastuzumabMMAE
conjugate
with
DAR
of
2
using
this
enzymatic
conjugation
strategy.
In
this
work,
azidetrastuzumab-MMAE conjugate with DAR of 2 using this enzymatic conjugation strategy.anIn
this
MMAE
conjugate
with
DAR
of 2 using
this enzymatic
conjugation
strategy.
In
this
work,
an azidecontaining
linker,
which
involves
a
primary
amine,
was
coupled
to
Q295
of
the
deglycosylated
work, an azide-containing linker, which involves a primary amine, was coupled to Q295 of the
containing
which
involves
a primary
amine,
was coupled
to Q295
of thewith
deglycosylated
antibody bylinker,
MTGase.
This
enzymatic
reaction
was followed
by afollowed
SPAAC
reaction
the DBCOdeglycosylated
antibody
by MTGase.
This
enzymatic
reaction was
by
a SPAAC
reaction
with
antibody
by
MTGase.
This
enzymatic
reaction
was
followed
by
a
SPAAC
reaction
with
the DBCOcontaining
auristatin
drug.
the DBCO-containing auristatin drug.
containing
auristatin
Cell-free
proteindrug.
synthesis (CFPS)
(CFPS) system
system was
was also
also efficiently
efficiently applied
applied to
to produce
produce monoclonal
monoclonal
Cell-free
protein
synthesis
Cell-free
protein
synthesis
(CFPS)
system
was
also
efficiently
applied
to
produce
monoclonal
antibodies that
that contain
contain unnatural
unnatural amino
amino acids
acids for
antibodies
for antibody-drug
antibody-drug conjugate
conjugate generations.
generations. For
For example,
example,
antibodies
that
contain
unnaturala amino
acids antibody-drug
for antibody-drug
conjugate
generations.
For
example,
Zimmerman
et
al.
[86]
prepared
site-specific
conjugate
via
CFPS
system
using
new
Zimmerman et al. [86] prepared a site-specific antibody-drug conjugate via CFPS system using aa new
Zimmerman
et
al.
[86]
prepared
a
site-specific
antibody-drug
conjugate
via
CFPS
system
using
a
new
synthetase totoincorporate
incorporate
a para-azidomethylphenylalanine
(pAMF)
the monoclonal
antibody,
synthetase
a para-azidomethylphenylalanine
(pAMF)
to thetomonoclonal
antibody,
which
synthetase
to incorporate
aDBCO-functionalized
para-azidomethylphenylalanine
(pAMF)
to the
monoclonal antibody,
which
was
then
linked
to
a
MMAF
drug
by
SPAAC
reaction.
was then linked to a DBCO-functionalized MMAF drug by SPAAC reaction.
which was then linked to a DBCO-functionalized MMAF drug by SPAAC reaction.
Terminal Alkynes
Alkynes
2.5.2. Click Reactions with Terminal
2.5.2. Click Reactions with Terminal Alkynes
In 2014, Bryden
Bryden et
et al.
al. [87]
[87] described
described the
theattachment
attachmentofofazide-functionalized
azide-functionalizedporphyrins
porphyrinstotoa
In
2014,
Bryden
et
al.
[87]
described
the
attachment
of
azide-functionalized
porphyrins
to in
a
atratuzumab
tratuzumabvia
viaa anovel
novelconjugation
conjugationmethod.
method.InInthis
thisstudy,
study,Trastuzumab
Trastuzumab was
was treated with TCEP
tratuzumab
via
a
novel
conjugation
method.
In
this
study,
Trastuzumab
was
treated
with
TCEP
in
to reduce
reduce the
the interchain
interchain disulfide
disulfide bond.
bond. Treatment
Treatment with
with N-propargyl-3,4-dibromomaleimide
N-propargyl-3,4-dibromomaleimide
order to
order toalkyne-containing
reduce the interchain
disulfide which
bond. then
Treatment
with N-propargyl-3,4-dibromomaleimide
yielded
trastuzumab,
successfully
reacted with porphyrin derivatives
yielded
alkyne-containing
trastuzumab,
which
then
successfully
reacted with
porphyrin
derivatives
afford trastuzumab-porphyrin
trastuzumab-porphyrin conjugates
conjugates
(Scheme
19). This
through the CuAAC reaction to afford
(Scheme
19).
method
through
the
CuAAC
reaction
to
afford
trastuzumab-porphyrin
conjugates
(Scheme
19).
This
method
realized using
using SPAAC
SPAACreaction
reaction[39].
[39].
could also be realized
could also be realized using SPAAC reaction [39].
Scheme 19.
of trastuzumab-porphyrin
conjugates through
N-propargyl-3,4Scheme
19. The synthesis
The
synthesis
of
trastuzumab-porphyrin
conjugates
through
Scheme
19. The and
synthesis
of trastuzumab-porphyrin
conjugates
through
N-propargyl-3,4dibromomaleimide
a
click
chemistry.
Reprinted
with
permission
from
Reference
[87].
N-propargyl-3,4-dibromomaleimide and a click chemistry. Reprinted with permission from
dibromomaleimide and a click chemistry. Reprinted with permission from Reference [87].
Reference [87].
3. Conclusions
3. Conclusions
3. Conclusions
A promising approach to improve the potency of drugs is to be conjugated to monoclonal
A promising
approach
to
improve the potency
of drugs is to be conjugated
to monoclonal
antibodies
that enable
these to
cytotoxic
to be site-specifically
to tumor
cells while
A promising
approach
improvedrugs
the potency
of drugs is todelivered
be conjugated
to monoclonal
antibodies
that
enable
these
cytotoxic
drugs
to
be
site-specifically
delivered
to
tumor
cells
while
avoiding the
toxicity
ofthese
drugscytotoxic
on normal
cells.toThe
of antibody-drug
antibodies
that
enable
drugs
be linkers
site-specifically
deliveredconjugates
to tumor profoundly
cells while
avoiding
the
toxicity
of
drugs
on
normal
cells.
The
linkers
of
antibody-drug
conjugates
profoundly
impact their potency and safety. Recently, a variety of methods have been developed to conjugate
impact
potency In
and
safety.
Recently,
a variety of
developed
to conjugate
drugs totheir
antibodies.
this
review,
we summarized
themethods
methodshave
that been
are currently
used
to design
drugs
to
antibodies.
In
this
review,
we
summarized
the
methods
that
are
currently
used
to design
and synthesize antibody-drug conjugates, including heterogeneous ADCs and homogeneous
ADCs,
and
synthesize
antibody-drug
conjugates,
including
ADCsand
andazides.
homogeneous
ADCs,
via various
functional
groups such
as thiols,
amines, heterogeneous
alcohols, aldehydes
Heterogeneous
via various functional groups such as thiols, amines, alcohols, aldehydes and azides. Heterogeneous
Int. J. Mol. Sci. 2016, 17, 194
12 of 16
avoiding the toxicity of drugs on normal cells. The linkers of antibody-drug conjugates profoundly
impact their potency and safety. Recently, a variety of methods have been developed to conjugate
drugs to antibodies. In this review, we summarized the methods that are currently used to design
and synthesize antibody-drug conjugates, including heterogeneous ADCs and homogeneous ADCs,
via various functional groups such as thiols, amines, alcohols, aldehydes and azides. Heterogeneous
ADCs were usually synthesized through the thiols of cysteine residues and the amines of lysines,
however, the heterogeneity diminished their activities and promoted antibody aggregations, and
increased toxicities in the circulation. Homogeneous ADCs made through the catalysis of site-specific
conjugation enzymes such as AGT, BTG, aaRS and Sial T are more stable and have comparable or
even better activities than those conventional analogs in vivo. We believe that a growing number
of methods will be developed to synthesize ADCs in the near future, and more and more ADCs,
especially site-specifically modified ADCs, will be produced.
Acknowledgments: We thank the other academic staff members in Aiping Lu and Ge Zhang’s group at
Hong Kong Baptist University (Hong Kong, China). We also thank Hong Kong Baptist University (Hong Kong,
China) for providing critical comments and technical support. This study was supported by the Hong Kong
General Research Fund (HKBU12102914 to Ge Zhang) and the Faculty Research Grant of Hong Kong Baptist
University (FRG2/12-13/027 to Ge Zhang).
Author Contributions: Houzong Yao and Feng Jiang wrote the manuscript; and Aiping Lu and Ge Zhang revised
and approved the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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