Cancer-targeting antibody-drug conjugates: Site-specific conjugation of doxorubicin to anti-EGFR 528 Fab′ through a polyethylene glycol linker

Article abstract of DOI:10.1071/CH11071

Antibodydrug conjugates have been prepared to examine the effect that attaching small-molecule drugs to an antibody fragment has on antibody activity. The anticancer drug doxorubicin was covalently attached through a polyethylene glycol linker to a cancer-targeting, anti-epidermal growth factor receptor antibody fragment (Fab′). The reactivity of maleimide was compared with a substituted maleimide derivative (citraconimide) in conjugation reactions with cysteine residues on a Fab′. Introduction of polyethylene glycol increased aqueous solubility of the cytotoxic drug, which led to an improvement in overall yield of the conjugation reaction with the antibody fragment. Antibodydrug conjugates prepared retained activity of the parent antibody, as determined by antigen binding experiments measured by surface plasmon resonance.

Full text of DOI:10.1071/CH11071

RESEARCH FRONT
Full Paper
CSIRO PUBLISHING
www.publish.csiro.au/journals/ajc
Aust. J. Chem. 2011, 64, 779–789
Cancer-targeting Antibody Drug Conjugates: Site-specific
]
Conjugation of Doxorubicin to Anti-EGFR 528 Fab9
through a Polyethylene Glycol Linker
A
A
A
A
Lisa P. T. Hong, Judith A. Scoble, Larissa Doughty, Gregory Coia,
A B
,
and Charlotte C. Williams
A
CSIRO Materials Science and Engineering, 343 Royal Parade, Parkville,
Vic. 3052, Australia.
B
Corresponding author. Email: charlotte.williams@csiro.au
Antibody–drug conjugates have been prepared to examine the effect that attaching small-molecule drugs to an antibody
fragment has on antibody activity. The anticancer drug doxorubicin was covalently attached through a polyethylene glycol
linker to a cancer-targeting, anti-epidermal growth factor receptor antibody fragment (Fab0). The reactivity of maleimide
was compared with a substituted maleimide derivative (citraconimide) in conjugation reactions with cysteine residues on a
Fab0. Introduction of polyethylene glycol increased aqueous solubility of the cytotoxic drug, which led to an improvement
in overall yield of the conjugation reaction with the antibody fragment. Antibody–drug conjugates prepared retained
activity of the parent antibody, as determined by antigen binding experiments measured by surface plasmon resonance.
Manuscript received: 12 February 2011.
Manuscript accepted: 16 March 2011.
Introduction
an antibody approved for treatment of metastatic colorectal
cancer[14] and head and neck cancers.[15] Only approximately
one-third of patients with colorectal cancer respond to therapy
with the antibody alone.[14] Arming the antibody with a cyto-
toxic drug could result in improved efficacy.
Antibodies can be enzymatically digested to form fragments
that retain the antigen binding site of the full antibody. These
fragments (Fabs) are attracting interest for use as therapeutic
agents, several of which have received approval for a range
of clinical applications.[16] Although full antibodies dominate
clinical application, antibody fragments have advantages such
as an increase in tissue penetration, leading to an increased
uptake in tumours,[16,17] as well as their potential for reduced
adverse reactions due to lack of the immunogenic Fc portion,
which is present on full antibodies.
To date, the focus has been on the use of the whole antibody,
with conjugation of the cytotoxic drug through amine groups
on lysine residues; this leads to a heterogeneous mixture as
the drugs are attached to numerous residues that are located at
various sites throughout the antibody.[18] Antibody fragments,
however, are ideally suited for site-specific conjugation reac-
tions as they can possess free sulfhydryl groups (i.e. Fab0), which
Antibody–drug conjugates (ADCs) combine the exquisite tar-
geting properties of anticancer monoclonal antibodies (mAbs)
with small-molecule cytotoxic drugs, increasing the selectivity
of current anticancer agents.[1–5] ADCs aim to increase the
efficacy of therapeutic mAbs as well as provide targeted
delivery of cytotoxic drugs to tumour cells (avoiding healthy
cells), thus ameliorating dose-limiting toxicity of current che-
motherapies.[2] To be clinically valid, an ADC must contain an
antibody against a well-characterized antigen, a potent drug, a
linker system that is stable in the circulation, and perhaps most
importantly, a conjugation strategy that preserves the char-
acteristics of the antibody.[3]
The epidermal growth factor receptor (EGFR) family of
tyrosine kinase receptor proteins are mediators of cell growth
and survival. Consequently, many human cancers overexpress
members of the EGFR family on the cell surface.[6,7] The anti-
EGFR mAb 528[8–11] inhibits proliferation of a variety of
malignant human cell lines that express EGFR, and in combina-
tion therapy with cisplatin, is capable of producing a striking
antitumour effect in well-established tumour xenografts.[12]
Anti-EGFR mAb 528 has a clinical counterpart, Erbitux,[13]
Charlotte Williams was educated at the University of Western Australia, B.Sc. (Hons), where she was awarded a Ph.D. with
distinction under the supervision of Professor M. V. Baker. After postdoctoral research at The University of Oxford with
Professor H. L. Anderson, she undertook a research scientist position with Johnson Matthey (U.K.) before returning to
Australia and taking up a position at Starpharma Pty. Ltd. where she worked for over 5 years as a senior research chemist and
research manager. Since 2009 Charlotte has held a position with CSIRO Materials Science and Engineering where she has
been involved in the development of bioconjugation capabilities within the division. She is a member of both the ACS and RSC
as well as an active committee member of the RACI. Her current research interests include investigations into unique and
interesting methods to conjugate small molecules, peptides or proteins to larger biomolecules such as antibodies.
Ó CSIRO 2011
10.1071/CH11071
0004-9425/11/060779

RESEARCH FRONT
780
L. P. T. Hong et al.
O
OH
O
O
O
OH
OH
O
HO
HO
1
10
HO
2
3
13
14
HO 8
HO
O
7
H
O
OH
OMe
H
6ꢀ
O
5ꢀ 1ꢀ
O
O
OMe
3ꢀ
O
3ꢀ
1
HO
NH
R
N
R1
NH2
O
O
Fig. 1. Structure of doxorubicin – linkers can be attached through the C30
2 R ꢁ CH2, R1 ꢁ H
3 R ꢁ PEG24-NHCO, R1 ꢁ H
4 R ꢁ PEG24-NHCO-(CH2)3, R1 ꢁ CH3
amine, C13 ketone, or C14 hydroxyl functional groups.
can be used for conjugation of drugs. A potential drawback for
therapeutic use of Fab0-conjugates is that given their smaller
size, they will be subject to renal filtration,[19] but the introduc-
tion of polyethylene glycol (PEG) to the Fab0 protein will
increase the circulating serum half-life.
Fig. 2. Structure of DOX-linker constructs 2–4.
Michael addition reaction of a maleimide with cysteine thiol
residues on a Fab0 fragment. Three DOX constructs were
prepared for conjugation to Fab0 fragments (Fig. 2). For all
constructs, the thiol-reactive functional group, maleimide or
citraconimide, was attached through a linker to DOX by the
formation of an amide at the 30-amine of DOX. To study the
effect of linker length and composition, the linker was either a
short hydrocarbon chain (2) or a long PEG chain (3 and 4).
To study the effect of Michael acceptor structure on reactivity
towards protein thiols, maleimide (2 and 3) and methyl-
maleimide (citraconimide 4) functional groups were prepared
(Fig. 2).
Doxorubicin (DOX) is a well-known anthracycline antibiotic
that is used in the clinic as a chemotherapeutic drug. DOX was
chosen as the cytotoxic drug to exemplify this work given its
clinical relevance, that it can be chemically modified for linker
installation, and that it possesses an absorption maximum at
l 482 nm, which facilitates analysis of protein conjugates. The
objectives of this work were to investigate the site-specific
conjugation of DOX or PEG-DOX to an anti-EGFR 528 Fab0,
to understand the effect of conjugation on the solubility of the
Fab0 as well as its ability to retain antigen binding (i.e. retain
activity). We believe this is the first time that an ADC has been
prepared using an anti-EGFR 528 Fab0. This work demonstrates
the need for water-soluble drugs in order to achieve good yields
of ADCs as well as retaining solubility of the antibody after
conjugation, especially if a small Fab0 fragment is used. Surface
plasmon resonance (SPR) binding experiments show that insert-
ing a long PEG linker does not adversely affect the ability of the
antibody fragment to bind its antigen; thus, the ADC retains its
targeting potential.
This work also investigates the use of citraconimide as an
alternative Michael acceptor to maleimide in bioconjugation
reactions to protein sulfhydryls. Although citraconimides are
known, we believe that this is the first time that they have been
studied in detail with respect to hydrolytic stability and Michael-
type addition reactions with thiols in particular proteins, as
compared with maleimides. There are several contradictory
reports regarding the regioselectivity of thiolate addition to an
N-substituted citraconimide, which we felt required further
investigation and clarification.[20–22]
The short hydrocarbon linker was introduced by reaction of
6-maleimidocaproic acid 5 (Scheme 1), prepared by a modifi-
cation of the reported method,[28] with the C30 amine of DOX to
yield 6-maleimidocaproyl-doxorubicin 2 (Fig. 2). Reaction of
DOX with a discrete heterobifunctional PEGylation crosslink-
ing reagent MAL-PEG24-NHS gave 3 (Fig. 2) in high yield.
The production of 4 required a more elaborate approach
(Scheme 1). A 1.1-kDa PEG with a protected t-butyl ester was
introduced at the carboxylic acid of 6-citraconimidocaproic acid
(6, prepared by a modification of the reported method)[30] to
t
give CIT-(CH2)5-PEG24-CO2 Bu (7). The t-butyl protecting
group was removed and the resulting CIT-(CH2)5-PEG24-
CO2H (8) was activated and coupled in situ with the C30 amine
of DOX to give 4.
Chemistry of Citraconimide versus Maleimide
We were interested in the ability of citraconimide to participate
in Michael addition reactions with thiols, as well as its aqueous
stability, compared with maleimide. A facet of Michael addition
to citraconimides for consideration involves determining with
which carbon atom the thiolate will react. There are conflicting
reports regarding this regioselectivity, with support for vicinal
substitution by reaction of the thiolate at the non-substituted
carbon atom[21,22] as well as support for geminal substitution
by thiolate attack of the olefin at the carbon atom that bears
the methyl group.[20] Another factor for consideration is that
Michael addition of thiolates to any maleimide will result in a
mixture of diastereomers. The presence of diastereomers is a
potential complication given that they may display different
biological activity;[31] this could have a negative impact on the
overall biological activity of conjugates and needs to be a con-
sideration for successful ADCs in the future. The presence of
Results and Discussion
Synthesis of Doxorubicin with Reactive Linkers
Doxorubicin (1) contains three functional groups to which lin-
kers can be installed for subsequent attachment to proteins or
polymers (Fig. 1). DOX has been functionalized by formation of
an amide or carbamate[23,24] to the 30-amine of the daunosamine
sugar, formation of a hydrazone by reaction of the C13-ketone
with a hydrazine[25–28] or formation of an ester by reaction of
the C14 hydroxyl.[29] For the present work, we required a stable
bond to DOX so that characterization and antibody binding
assays would not be complicated by cleavage reactions.
The bioconjugation chemistry employed to load functiona-
lized DOX to antigen-binding fragments is the commonly used

RESEARCH FRONT
A Cancer Targeting Antibody–Drug Conjugate
781
O
O
H
N
O
O
H2N
CO2H
R
O
H2N-PEG24-CO2tBu
O
CO2tBu
CO2H
N
N
N
O
23
R
O
O
AcOH, reflux
TSTU, DIPEA
O
O
O
O
7
8
TFA, DCM
5
6
R ꢁ H
R ꢁ CH3
R ꢁ H or CH3
H
N
O
CO2H
O
23
OH
OH
O
O
O
HO
O
HO
TSTU, DIPEA
DOX·HCl
H
O
OMe
HO
O
H
N
NH
O
O
N
23
O
O
O
4
Scheme 1. Synthesis of 4 CIT-(CH2)5-PEG24-DOX.
NH2
EtO
SH
O
O
N
O
O
OH
OH
OH
H2N
EtO
N
H2N
EtO
N
S
S
ꢂ
O
O
O
O
O
O
6
O
O
Cys-CIT
Cys-CIT
R,R diastereomer
R,S diastereomer
9
10
O
OH
H2N
EtO
N
S
O
O
O
Cys-CIT
A
Scheme 2. Reaction of 6-citraconimidocaproic acid (6) with L-Cys-OEt yields a mixture of geminal thioether diastereomers (9 and 10).
diastereoisomers would be difficult to determine when conju-
gated to the target protein; hence, it was important to study the
outcome of such reactions on a model system.
Hydrolysis of Citraconimide versus Maleimide
Hydrolysis of maleimides has been studied previously,[31] but it
was of interest to explore the difference between the rate of
hydrolysis of maleimide and citraconimide. The water solubility
of 6-maleimidocaproic acid (5) and 6-citraconimidocaproic acid
(6) was improved by the introduction of a PEG chain
(Scheme 3).
Hydrolysis of the water-soluble analogues (11 and 7) was
analyzed by 1H NMR, and at room temperature, the hydrolysis
of both analogues was slow. Given that the addition of base is
known to promote the hydrolysis of maleimides,[31,33,34] the rate
at which each analogue was hydrolyzed was expedited first by
incubating the samples at 808C, followed by the addition of an
increasing amount of base. After 6 days of incubation in the
presence of ,0.02 M NaOH, complete hydrolysis of the mal-
eimide ring (11) was observed. In comparison, 25% of the
PEGylated citraconimide construct (7) remained intact after
this time.
As a model system, maleimide (5) and citraconimide (6)
were reacted with L-cysteine ethyl ester (L-Cys-OEt, Scheme 2)
in phosphate buffered saline (PBS) at pH 7.2. Michael addition
of L-Cys-OEt to 6 resulted in the formation of L-cysteine-
citraconimidocaproic acid (Cys-CIT), which was isolated as a
pair of diastereomers (9 and 10). Rather than vicinal attack of
the thiolate on the citraconimide olefin to give A, the product
isolated was that for geminal attack of the thiolate at the
substituted olefinic carbon atom (9 and 10). Assignment as a
geminal thioether was established by 1H NMR.
Generally, one would expect that the reactivity of a malei-
mide would decrease if electron-rich substituent groups were
placed on the olefin owing to both steric and electronic effects at
the substituted carbon atom.[32] Thus, it was surprising to us that
thiolate substitution occurred at the methylated olefinic carbon
atom. The mechanism most likely involves a complex set of
equilibria and further work is required to elucidate the reason for
the observed regioselectivity (see Accessory Publication). Anal-
The addition of base and heat increased the rate of hydrolysis
for both analogues. Nonetheless, these studies show that the rate
of hydrolysis of maleimides is faster than that of citraconimides.
These results support the use of citraconimides over maleimides
for aqueous reactions with protein sulfhydryls. Citraconimides
may be more stable than maleimides for long-term storage of
thiol-reactive reagents, although further work under normal
1
ysis of H NMR spectra of the Cys-CIT mixture of diastereo-
mers 9 and 10 and spectra of the diastereomers produced from
the reaction of L-Cys-OEt and 5 to give R,R-Cys-MAL and R,S-
Cys-MAL, are also available in the Accessory Publication.

RESEARCH FRONT
782
L. P. T. Hong et al.
O
O
H
N
O
CO2H
O
CO2tBu
N
R
N
R
H2N-PEG24-CO2tBu
23
O
O
O
PyBOP, DIPEA, DMF
11
7
R ꢁ H
R ꢁ CH3
5 R ꢁ H
6 R ꢁ CH3
Scheme 3. Synthesis of water-soluble maleimide and citraconimide derivatives for use in hydrolysis reactions.
SH
SH
Pepsin
TCEP
IAA
Fab-(SCH2CONH2)2
F(abꢀ)2
Fabꢀ
12a Fab ꢁ α-FLAG
12b Fab ꢁ 528
IgG
2, 3 or 4
O
HO
O
OH
O
O
HO
HO
H
O
OH
OMe
2
Fab
O
N
13a R ꢁ CH2, R1 ꢁ H, Fab ꢁ α-FLAG
13b R ꢁ CH2, R1 ꢁ H, Fab ꢁ 528
NH
R
R1
S
14a R ꢁ PEG24-NHCO, R1 ꢁ H, Fab ꢁ α-FLAG
14b R ꢁ PEG24-NHCO, R1 ꢁ H, Fab ꢁ 528
O
O
15a R ꢁ PEG24-NHCO-(CH2)3, R1 ꢁ CH3, Fab ꢁ α-FLAG
15b R ꢁ PEG24-NHCO-(CH2)3, R1 ꢁ CH3, Fab ꢁ 528
Scheme 4. Synthesis of the immunoconjugates 12a–15a and 12b–15b.
conjugation conditions (PBS, pH 8.0, 48C) is required to confirm
the hypothesis that citraconimides are more stable in aqueous
conditions.
thiol-reactive doxorubicin constructs 2, 3, or 4 (Fig. 2), or a 100-
fold molar excess of iodoacetamide (IAA) in PBS (0.5 mM
EDTA, pH 7.2) for 16 h at 48C (Scheme 4). Reaction with IAA
blocks the free thiols from re-oxidation and is representative of
unconjugated Fab0.
Conjugation Reactions
Model Antibody: a-FLAG Fab0 Conjugations
F(ab0)2 fragments of the anti-EGFR mouse monoclonal antibody
528 were prepared by enzymatic digestion of the immuno-
globulin (IgG) (Scheme 4) using the protease pepsin, which
selectively cleaves disulfides in the hinge region of anti-
bodies.[16,17] Pepsin digestion produced the bivalent antigen-
binding fragment F(ab0)2, which was selectively reduced in the
hinge region using tris(2-carboxyethyl)phosphine (TCEP). Mild
reduction led to cleavage of the hinge disulfides holding
together the heavy chains, yielding two Fab0 fragments, each
containing one antigen-binding site and free cysteine resid-
ues near the C-terminus.[16,17] Reduced Fab0 fragments were
purified by size exclusion to remove TCEP as there is precedent
for TCEP reacting with maleimides.[35–37]
To model ADC conjugation reactions, an anti-FLAG anti-
body that recognizes the FLAG epitope (a short, hydrophilic
peptide, DYKDDDDK) was used.[38] The anti-FLAG antibody
is robust, well understood and available in large quantities and
consequently serves as a good ‘model’ antibody to optimize
conjugation strategies. Freshly prepared Fab0 (Fab-SH) frag-
ments were either treated with a 10-fold molar excess of the
The conjugation reactions were analyzed by gel filtration
chromatography (GFC), which revealed little difference
between the reactivity of N-maleimido-PEG24-DOX (3) and
6-citraconimidocaproyl-PEG24-DOX (4) towards free thiols on
Fab0 (Fig. 3). Citraconimide (Fig. 3d) is equally as good as
maleimide (Fig. 3c) for reaction with reduced thiols on proteins.
The conjugation reactions were analysed by measuring absor-
bance at two wavelengths, l 280 nm (absorption maxima for
proteins) and l 482 nm (absorption maxima for DOX) simul-
taneously (Fig. 3).
The a-FLAG Fab0-IAA conjugate 12a (Fig. 3a) is a single
peak, which elutes at a retention volume (Rv) of 80 mL. Freshly
reduced Fab0 (Fab-SH) also elutes at this retention volume (not
shown). In the reaction of a-FLAG Fab0 with 2, a red precipitate
was observed, which is attributed to a combination of 2 coming
out of solution on dilution into PBS as well as some precipitation
of Fab0-2 conjugate (13a). The hydrophobicity of DOX with a
hydrocarbon linker leads to precipitation from PBS. Conse-
quently, this reaction (Fig. 3b) results in a mixture of unreacted

RESEARCH FRONT
A Cancer Targeting Antibody–Drug Conjugate
783
158 kDa
44 kDa
17 kDa
1.35 kDa
3a
12a λ 280 nm
α-FLAG Fab-IAA
12a λ 482 nm
150
100
50
3b
3c
3d
13a λ 280 nm
13a λ 482 nm
α-FLAG Fab-2
α-FLAG Fab-3
14a λ 280 nm
14a λ 482 nm
15a λ 280 nm
15a λ 482 nm
α-FLAG Fab-4
0
40
50
60
70
80
90
100
110
Elution volume [mL]
Fig. 3. Gel filtration chromatography (GFC) spectra of a-FLAG Fab0 conjugates 12a–15a (3a–3d respectively);
dashed lines indicate absorbance detected at l 482 nm (DOX) and solid lines indicate absorbance detected
at l 280 nm (protein). Vertical lines indicate retention time of protein MW standards, g-globulin (bovine) has
a molecular mass of 158 kDa, ovalbumin (chicken) 44 kDa, myoglobin (horse) 17 kDa, and vitamin B12 has a
molecular mass of 1.35 kDa.
Fab0 (Rv 80 mL) and 13a (Rv 77.5 mL); the latter exhibits
absorbance at l 482 nm, confirming the presence of DOX
(i.e. 2). Re-oxidized F(ab0)2 is also present in Fig. 3b (at Rv
69 mL), characterized by a peak with absorbance at only 280 nm,
whichcorrespondstotheexpectedMWforF(ab0)2. Re-oxidation
is to be expected given the poor solubility of 2 in PBS; thus the
conjugation reaction is competing with Fab0 re-oxidation.
Conjugation reactions to prepare the PEGylated conjugates
14a (Fig. 3c) and 15a (Fig. 3d) were more successful than
conjugation using the non-PEG-linked 2 (13a, Fig. 3b). This
confirms the hypothesis that a PEG linker offers superior
aqueous solubility to DOX and conjugation to the Fab0 is more
efficient. The chromatograms of 14a (Fig. 3c) and 15a (Fig. 3d)
show a major peak at Rv 75 mL, indicating a much higher MW
than Fab0 alone – PEG contributes to a large hydrodynamic
radius, which leads to the appearance of a higher MW.[39] The
peaks at Rv 75 mL also have a significant contribution absorbing
at 482 nm, indicating the presence of DOX (i.e. 3 and 4). The
chromatograms of both reactions 14a (Fig. 3c) and 15a (Fig. 3d)
also have a later-running shoulder at approximately Rv 80 mL,
which has little to no absorbance at 482 nm, likely to be due to
unreacted Fab0. Unreacted 3 and 4 (MW ,1800) elute from the
column at Rv 105 mL.
)
2
ꢀ
MW [kDaF] (ab
12a
100
50
40
20
Fig. 4. SDS-PAGE gel of anti-FLAG Fab0 conjugates 12a–15a. Invitrogen
NuPAGE 4–12% Bis-Tris precast gel was used with Invitrogen Bench-
MarkTM MW markers.

RESEARCH FRONT
784
L. P. T. Hong et al.
Table 1. Data from kinetic evaluation of FLAG binding to a-FLAG Fab9 conjugates
Values given as ꢀ s.d.
Ligand
Ka [Mꢁ1 sꢁ1] ꢂ 105
Kd [sꢁ1] ꢂ 10ꢁ4
KD [nM]
a-FLAG IgG
6.3 ꢀ 0.6
5.0 ꢀ 0.3
6.4 ꢀ 0.7
4.7 ꢀ 0.2
4.4 ꢀ 0.4
1.1 ꢀ 0.1
1.6 ꢀ 0.2
2.1 ꢀ 0.1
1.9 ꢀ 0.2
1.7 ꢀ 0.1
180 ꢀ 12
325 ꢀ 41
334 ꢀ 37
402 ꢀ 38
389 ꢀ 8
a-FLAG Fab0-IAA (12a)
a-FLAG Fab0-2 (13a)
a-FLAG Fab0-3 (14a)
a-FLAG Fab0-4 (15a)
Table 2. Data from kinetic evaluation of sEGFR-501 binding to 528 Fab9 conjugates
Ligand
Ka [Mꢁ1 sꢁ1] ꢂ 105
Kd [sꢁ1] ꢂ 10ꢁ4
KD [nM]
528 IgG
4.20 ꢀ 0.10
3.42 ꢀ 0.12
3.42 ꢀ 0.05
3.46 ꢀ 0.08
8.80 ꢀ 0.40
2.10 ꢀ 0.10
2.62 ꢀ 0.09
2.45 ꢀ 0.05
2.51 ꢀ 0.05
528 Fab0-IAA (12b)
528 Fab0-2 (13b)
528 Fab0-3 (14b)
8.93 ꢀ (7.6 ꢂ 10ꢁ6
8.40 ꢀ (4.2 ꢂ 10ꢁ6
8.70 ꢀ (4.0 ꢂ 10ꢁ6
)
)
)
Each conjugate was isolated by pooling fractions containing
12a at Rv 80 mL, 13a at Rv 77.5 mL, and 14a and 15a at
Rv 75 mL, excluding fractions contaminated with unreacted
Fab0. See the Accessory Publication for analytical GFC of
isolated conjugates. The pooled fractions were concentrated
and analyzed by sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE) (Fig. 4) and SPR (Table 1).
Analysis of conjugation reactions 12a–15a by SDS-PAGE
(Fig. 4) augments conclusions drawn from GFC. The a-FLAG
Fab0-IAA conjugate 12a runs just short of 50 kDa (compared
with the MW markers), which is the expected MW for Fab. The
conjugate 13a runs at a molecular mass slightly larger than
that of Fab0 alone. In this lane, there is also evidence for some
re-oxidation to F(ab0)2 at 100 kDa as well as some breakdown
of the Fab0 to fragments at 20 kDa. This reflects previous
observations that conjugation of poorly soluble 2 competes with
Fab0 re-oxidation and Fab0 degradation. The SDS-PAGE gel of
both conjugates 14a and 15a, which result from reaction of the
PEGylated-DOX constructs 3 and 4 respectively, clearly shows
a major new band at an appropriate molecular mass for the
addition of ,3.6 kDa (two PEGylated-DOX molecules per
Fab0). Both reactions also contain small amounts of unreacted
Fab0 as well as some mono-PEG-DOX Fab0.
that conjugation of 2, 3, and 4 to anti-FLAG Fab0 did not
adversely affect the activity of the antibody fragment with
respect to antigen binding.
Anti-EGFR Antibody 528 Fab0 Conjugations
An anti-EGFR antibody 528 Fab0 was prepared by digestion
of the 528 IgG with pepsin followed by reduction with TCEP,
as outlined in Scheme 4. Freshly prepared Fab0 (Fab-SH) was
treated with a 20-fold molar excess of the thiol-reactive DOX
constructs 2, 3, or 4, or a 100-fold molar excess of IAA in PBS at
pH 7.2 (Scheme 4).
Each of the 528 Fab0 conjugation reactions was analysed and
purified by GFC (see Accessory Publication). The major peak in
the trace for 12b can be attributed to Fab0-IAA with a smaller
contribution from F(ab0)2. Reaction of 2 with 528 Fab0 encoun-
tered similar problems to the anti-FLAG Fab0, in that the
hydrophobic construct 2 and the conjugate 13b both displayed
insolubility in PBS. Despite this, conjugation of 2 to 528 Fab0
was successful and the major peak in the GFC trace is attributed
to the conjugate 13b. The reaction of PEGylated-DOX 3 with
528 Fab0 to yield the conjugate 14b proceeded well. The major
peak represents a considerable increase in MW with respect to
12b, due to installation of PEGylated-DOX 3.
SPR Analysis of a-FLAG Fab0 Conjugates
Each conjugate was isolated by pooling fractions containing
12b, 13b, and 14b, strictly excluding fractions on either side of
the major peak. The pooled fractions were concentrated before
analysis by SPR. Unfortunately, the reaction of 6-citraconimi-
docaproyl-PEG24-DOX 4 with 528 Fab0 (to give 15b) was
difficult to interpret by GFC and there was insufficient material
recovered for further analysis.
One of the aims of this work was to investigate the effect
of installing a cytotoxic drug on a Fab0 on the activity and
integrity of the antibody. Structural integrity of an antibody
can be assessed by determination of the binding interaction
between the antibody and its antigen. Surface plasmon reso-
nance binding experiments permit real-time monitoring of
biomolecular interactions and estimations of kinetic binding as
well as affinity parameters.[40] The anti-FLAG Fab0 conjugates
12a–15a as well as parental anti-FLAG IgG were covalently
coupled onto the surface of a sensor chip. A FLAG-epitope-
containing peptide (GGGDYKDDDDK) was injected over the
immobilized antibody fragments and the binding interactions
were monitored in real time. Sensorgrams resulting from these
binding experiments can be found in the Accessory Publica-
tion. Estimated rate parameters (ka and kd) and overall affini-
ties (KD ¼ kd/ka) are summarized in Table 1. Similar binding
parameters were obtained for all Fab0 conjugates, indicating
SPR Analysis of Anti-EGFR Antibody 528 Fab0
Conjugates
The 528 Fab0 conjugates (12b, 13b, and 14b) and the parental
528 IgG (full antibody) were immobilized onto the surface
of a sensor chip. A soluble form of the EGF receptor
(sEGFR501)[41,42] was injected over the immobilized con-
jugates and immobilized parental 528 IgG. Gratifyingly, our
estimated KD value for the interaction of EGFR with parental
528 IgG (Table 2, entry 1, 2.1 ꢀ 0.1 nM) is in excellent agree-
ment with reported values (2.5 nM).[10,43] The binding

RESEARCH FRONT
A Cancer Targeting Antibody–Drug Conjugate
785
(a)
(c)
200
800
700
600
500
400
300
200
100
0
528 IgG
528 Fabꢀ-2 (13b)
150
100
50
0
0
100
200
300
400
500
600
0
100
200
300
400
500
600
Time
Time
(b)
(d)
400
350
300
250
200
150
100
50
800
700
600
500
400
300
200
100
0
528 Fabꢀ-3 (14b)
528 Fabꢀ-IAA (12b)
0
0
100
200
300
400
500
600
0
100
200
300
400
500
600
Time
Time
Fig. 5. Surface plasmon resonance (SPR) binding data for the sEGFR501 protein binding to various 528 anti-EGFR Fab0 conjugates immobilized on the
surface of a GLC chip docked in a ProteOn XPR36 biosensor: (a) 528 IgG; (b) 528 Fab0-IAA conjugate 12b; (c) 528 Fab0-2 conjugate 13b; and (d) 528 Fab0-3
conjugate 14b. sEGFR501 protein samples were tested at 45, 15, 5, 1.7, and 0.6 nM. Overlayed triplicate binding responses are shown (black lines). Binding
data were globally fit to a simple 1:1 interaction model (orange lines).
sensorgrams of Fab0 conjugates (12b, 13b, and 14b; Fig. 5)
yielded near-identical association and dissociation rate para-
meters (Table 2, entries 2–4). These binding data clearly dem-
onstrate that conjugation of a small-molecule drug to 528 Fab0
(13b, Fig. 5c) or introduction of a PEGylated small-molecule
drug (14b, Fig. 5d) do not impede the ability of 528 Fab0 to bind
its antigen. This work also confirms that preparation of 528 Fab0
(12b, Fig. 5b) does not diminish antibody activity compared
with the full 528 IgG (Fig. 5a).
drugs are hydrophobic. Binding studies by SPR demonstrated
that ADCs prepared in this work retained the ability to robustly
bind their antigens, even with the introduction of long PEG
linkers. In principle, the incorporation of a PEG linker between
any cytotoxic drug and an antibody will improve the efficiency
of conjugation reactions without compromising activity of the
antibody. The ability to do so is only limited by the availability
of a functional group on the cytotoxic drug of interest for linker
attachment. Another benefit of PEG group installation is an
increase in circulation time in vivo and potentially an improved
uptake in tumour cells.
Retention of antibody activity is pivotal to the success of
ADCs. This work demonstrates for the first time site-specific
conjugation of a cytotoxic drug to a clinically relevant anti-
EGFR 528 Fab0, with retention of antigen binding. This work
will guide our future studies towards using pH-sensitive (e.g.
hydrazone)[25–27,44] or enzymatically cleavable linkers,[2,45,46]
given that the synthetic chemistry used to conjugate the cyto-
toxic drug to the antibody is crucial for the success of armed
antibodies.
Conclusions
ADCs comprising an anti-EGFR 528 Fab0 and DOX-linked
derivatives were prepared and isolated. These derivatives con-
tained either a short hydrocarbon linker or a longer PEG linker
between a thiol-reactive functional group and DOX. Citraco-
nimide was introduced as an alternative thiol-reactive functional
group for bioconjugation reactions with Fab0 free sulfhydryls;
citraconimide represents a more hydrolytically stable analogue
to maleimide. The best ADC yields were obtained using
PEGylated-DOX constructs in conjugation reactions rather than
constructs with the short hydrocarbon linker. The resulting Fab0-
PEG-DOX conjugates also displayed much better solubility
in PBS than conjugates containing the short hydrocarbon
linker. The improvement in aqueous solubility of PEGylated
drugs leads to an increase in conjugation reaction yields. It is
likely that the benefits of improved aqueous solubility are not
restricted to anthracyclines, as many cytotoxic small-molecule
Experimental
General
All chemicals were used as received (Aldrich) and PEG reagents
were purchased from Quanta Biodesign Ltd (Ohio, USA). NMR
spectra were recorded with a Bruker ARX-400 spectrometer
at ambient temperature and were referenced with respect to

RESEARCH FRONT
786
L. P. T. Hong et al.
residual solvent peaks in deuterated solvents. Electrospray
ionization (ESI) mass spectra were recorded on an Applied
Biosystems API 150EX mass spectrometer. Surface plasmon
resonance binding experiments were performed on the Bio-Rad
ProteOnTM XPR36 protein interaction array system and kinetic
analyses were performed using ScrubberPro software. GFC was
performed on GE instruments using preparative HiLoad 16/60
Superdex 200 pg and analytical Superdex 200 10/300 GL col-
umns. GFC traces were analyzed using Unicorn software. Pro-
tein molecular weight standards were purchased from Bio-Rad.
Antibodies anti-FLAG F(ab0)2 and anti-EGFR 528 IgG were
supplied by CSIRO.
up into MeOH (2 mL) and filtered through a 0.45-mm Acrodisc
for purification by HPLC. HPLC conditions: 0.1% TFA, 20–
80% MeCN from 2 to 18 min, major peak (retention time Rt
16.0 min) gave N-maleimido-PEG24-DOX 3 as a pale red resi-
due (24.6 mg, 94%). dH (400 MHz, CD3OD) 7.81–7.69 (m, 2H,
DOX–ArCH C1 and C2), 7.46 (m, 1H, DOX–ArCH C3), 6.81 (s,
2H, 2 ꢂ MAL–CH), 5.38 (m, 1H, DOX–CH C10), 5.03 (m, 1H,
DOX–CH C7), 4.73 (s, 2H, DOX–COCH2OH C14), 4.25
(q, J 6.6, 1H, DOX–CHCH3 C50), 3.98 (s, 3H, DOX–OCH3
C4), 3.75 (t, J 7.0, 2H, PEG O–CH2CH2–CONH–DOX), 3.66
(t, J 6.0, 2H, PEG NH–CH2CH2–O), 3.64–3.52 (m, 92H, PEG,
O–CH2CH2–O and 1H, DOX–CHOH C40 and t, 2H, MAL–
NCH2CH2CONH), 3.49 (t, J 5.6, 2H, PEG NH–CH2CH2–O),
2.99 and 2.83 (2 ꢂ d, J 18.5, 2H, DOX–CHH C10), 2.80–2.72
(m, 1H, DOX–CH(NH) C30), 2.46 (t, J 7.0, 2H, PEG O–
Synthesis of Doxorubicin with Reactive Linkers
6-Maleimidocaproyl-doxorubicin 2
CH2CH2CONH–DOX),
2.44–2.39
(m,
2H,
MAL–
6-Maleimidocaproic Acid 5: A solution of 6-aminocaproic
acid (100 mg, 0.76 mmol) and maleic anhydride (74.8 mg,
0.76 mmol) in acetic acid (10 mL) was refluxed for 4 h. The
reaction mixture was concentrated under reduced pressure and
the residue triturated with diethyl ether to yield 6-maleimido-
caproic acid as a waxy solid, which was used without further
purification. HPLC (0.1% TFA) indicates 61% purity. dH
(400 MHz, CD3OD) 6.81 (s, 2H, 2 ꢂ MAL–CH), 3.48 (t, J 7.1,
2H, N–CH2), 2.27 (t, J 7.3, 2H, CH2–CO2H), 1.66–1.53 (m,
4H, N–CH2CH2 and CH2CH2CO2H), 1.36–1.26 (m, 2H, N–
CH2CH2CH2).
NCH2CH2CONH), 2.34, 2.12 (2 ꢂ m, 2H, DOX–CHH C8),
1.96, 1.70 (2 ꢂ m, 2H, DOX–CHH C20), 1.26 (d, J 6.6, 3H,
DOX–CHCH3 C60). m/z (ESI) calc.: 1822.98. Found: 1822.8
(Mþ), 912.1 (M2þ).
Towards 6-Citraconimidocaproyl-PEG24-doxorubicin 4
6-Citraconimidocaproic Acid 6: To a stirred solution of 6-
aminocaproic acid (100 mg, 0.76 mmol) in acetic acid (10 mL)
was added citraconic anhydride (68.5 mL, 0.76 mmol); the col-
ourless solution was refluxed for 4 h. The reaction mixture was
concentrated under reduced pressure and the residue triturated
with diethyl ether to yield 6-citraconimidocaproic acid 6 as a
waxy solid, which was used without further purification. HPLC
(0.1% TFA) indicates 73% purity. dH (400 MHz, CD3OD) 6.40
(q, J 1.8, 1H, CIT–CH), 3.46 (t, J 7.0, 2H, N–CH2), 2.28 (m, 2H,
CH2–CO2H), 2.03 (d, J 1.8, 3H, CIT–CH3), 1.59 (m, 4H, N–
CH2CH2 and CH2CH2CO2H), 1.31 (m, 2H, N–CH2CH2CH2).
6-Citraconimidocaproyl-PEG24-CO2H 8: To a stirred solu-
tion of 6-citraconimidocaproic acid 6 (9.1 mg, 0.04 mmol) in
DMF (1.5 mL) was added DIPEA (35 mL, 0.202 mmol) followed
by a solution of TSTU (15.3 mg, 0.051 mmol) in DMF (1.0 mL);
after stirring at room temperature under an atmosphere of
6-Maleimidocaproyl-doxorubicin 2: To a solution of 6-mal-
eimidocaproic acid 5 (20.6 mg, 0.097 mmol) in DMF (1.5 mL)
was added N,N-diisopropylethylamine (DIPEA) (85 mL,
0.487 mmol) followed by a solution of N,N,N0,N0-tetramethyl-
O-(N-succinimidyl)uronium tetrafluoroborate (TSTU) (37 mg,
0.123 mmol) in DMF (1.0 mL); after stirring under nitrogen
at room temperature for 1 h, a solution of doxorubicinꢃHCl
(56.6 mg, 0.097 mmol) in DMF (2.0 mL) was added and the
red solution was stirred at room temperature under nitrogen and
protected from light for 14 h. The reaction mixture was concen-
trated under reduced pressure and the solid residue was triturated
with MeCN. 6-Maleimidocaproyl-DOX 2 was isolated as a red
solid after recrystallization from MeOH/MeCN (30.9 mg, 43%).
dH (400 MHz, [D6]DMSO) 7.89–7.74 (m, 2H, DOX-ArCH C1
and C2), 7.52–7.40 (m, 1H, DOX–ArCH C3), 6.95 (s, 2H,
2 ꢂ MAL–CH), 5.20 (m, 1H, DOX–CH C10), 4.85 (m, 1H,
DOX–CH C7), 4.57 (s, 2H, DOX–COCH2OH C14), 4.15 (q,
J 6.6, 1H, DOX–CHCH3 C50), 3.95 (s, 3H, DOX–OCH3 C4),
3.94–3.87 (m, 2H, MAL–NCH2), 3.47–3.27 (residual H2O and
1H, DOX–CHOH C40), 3.01–2.70 (2 ꢂ m, 2H, DOX–CHH C10
and m, 1H, DOX–CH(NH) C30), 2.24–1.70 (2 ꢂ m, 2 ꢂ 2H,
2 ꢂ DOX–CHH C8 and C20 and m, 2H, caproyl CH2CH2–
CONHDOX), 1.48–1.27 (m, 4H, MAL–NCH2CH2 and cap-
royl CH2CH2CONHDOX), 1.18–1.00 (m, 2H, MAL–
NCH2CH2CH2), 1.11 (d, J 6.6, 3H, DOX–CHCH3 C60). m/z
(ESI) calc.: 736.72. Found: 737.2 (Mþ), 759.4 (M þ Naþ), 783.0
(M þ 2Naþ).
t
nitrogen for 1 h, a solution of H2N–PEG24–CO2 Bu (Quanta
Biodesign, 48.5 mg, 0.04 mmol) in DMF (2.0 mL) was added in
one portion. After stirring under nitrogen at room temperature
for 14 h, the reaction mixture was concentrated under reduced
pressure and the residue was taken up in MeOH (2 mL) and
filtered through a 0.45-mm Acrodisc for purification by HPLC.
HPLC conditions 0.1% TFA, 20–80% MeCN from 2 to 18 min,
major peak, Rt 16.8 min, gave 6-citraconimidocaproyl-PEG24-
t
CO2 Bu 7 (38 mg, 67%). m/z (ESI) calc.: 1409.69. Found:
1409.7 (Mþ), 705.6 (M2þ). 6-Citraconimidocaproyl-PEG24-
t
CO2 Bu 7 was taken up in anhydrous DCM (2 mL) and TFA
(0.4 mL) was added dropwise; after stirring for 2 h, the mixture
was concentrated under reduced pressure. The residue was taken
up in 1:1 MeCN/H2O and then the volatiles removed under
vacuum. This was repeated twice to ensure complete removal of
TFA, to yield 6-citraconimidocaproyl-PEG24-CO2H 8 as an oily
residue (32.3 mg, 88%). dH (400 MHz, CD3OD) 6.41 (q, J 1.8,
1H, CIT–CH), 3.72 (t, J 6.3, 2H, CIT–N–CH2), 3.62 (m, 92H,
PEG, O–CH2CH2–O), 3.52 (t, J 5.5, 2H, PEG, NH–CH2CH2–
O), 3.46 (t, J 7, 2H, PEG, O–CH2CH2–CO2H), 3.34 (t, J 5.5, 2H,
PEG, NH–CH2CH2–O), 2.54 (t, J 6.3, 2H, caproyl CH2CH2–
CONH), 2.18 (t, J 7.4, 2H, PEG, O–CH2CH2–CO2H), 2.03
(d, J 1.8, 3H, CIT–CH3), 1.59 (m, 4H, CIT–N–CH2CH2
and caproyl CH2CH2CONH), 1.28 (m, 2H, caproyl
N-Maleimido-PEG24-doxorubicin 3
To
a
stirred solution of doxorubicinꢃHCl (8.3 mg,
0.014 mmol) in DMF (1.5 mL) was added triethylamine (5 mL,
0.036 mmol) followed by a solution of MAL-PEG24-NHS
(Quanta Biodesign, 20 mg, 0.014 mmol) in DMF (1.5 mL); the
orange solution was stirred at room temperature under nitrogen
and protected from light for 14 h. The reaction mixture was
concentrated under reduced pressure and the residue was taken

RESEARCH FRONT
A Cancer Targeting Antibody–Drug Conjugate
787
CH2CH2CH2CONH). m/z (ESI) calc.: 1353.58. Found: 677.7
(M2þ), 688.4 (M2þ þ Na), 1353.5 (Mþ), 1375.7 (M þ Na).
(13.3 mg, 0.063 mmol) and L-cysteine ethyl ester hydrochloride
(14.4 mg, 0.078 mmol). Two major peaks were isolated by
HPLC at Rt 15.2 min (5.3 mg, 23%) and Rt 15.4 min (8.5 mg,
37%) both as colourless oils (overall yield 60%). dH (400 MHz,
CD3OD) 4.53–4.38 (m, 1H, Cys–CH(NH2)), 4.36–4.31 (q, J 7.1,
2H, OCH2CH3), 4.03–3.99 (m, 1H, MAL–CH2(CH)S), 3.71–
3.40 (dd, J 14.9, 4.5, 1H, MAL–CHaHb(CH)S), 3.53–3.50 (t,
J 7.1, 2H, N–CH2), 3.36–3.14 (m, 2H, Cys–CH2S), 2.56–2.47
(dd, J 18.6, 4.3, 1H, MAL–CHaHb(CH)S), 2.29 (t, J 7.4,
2H, CH2CO2H), 1.66–1.59 (m, 4H, N–CH2CH2 and
CH2CH2CO2H), 1.38–1.30 (m, 2H, N–CH2CH2CH2), 1.35 (t,
J 7.1, 3H, OCH2CH3).
6-Citraconimidocaproyl-PEG24-CONH-doxorubicin 4
To a stirred solution of 6-citraconimidocaproyl-PEG24-CO2H 8
(18.7 mg, 0.014 mmol) in DMF (1.5 mL) was added DIPEA
(12 mL, 0.07 mmol), followed by a solution of TSTU (5.3 mg,
0.017 mmol) in DMF (1.0 mL); after stirring under nitrogen
at room temperature for 1 h, a solution of doxorubicinꢃHCl
(8 mg, 0.014 mmol) in DMF (1.5 mL) was added and the
orange solution was stirred at room temperature under nitrogen
and protected from light for 14 h. The reaction mixture was
concentrated under reduced pressure and the residue was taken
up in MeOH (2 mL) and filtered through a 0.45-mm Acrodisc for
purification by HPLC. HPLC conditions: 0.1% TFA, 20–80%
MeCN from 2 to 18min, major peak, Rt 17.3min, gave 6-citra-
conimidocaproyl-PEG24-DOX 4 as a pale red residue (10 mg,
38%). dH (400 MHz, CD3OD) 7.87 (m, 1H, DOX–ArCH C2),
7.78 (m, 1H, DOX–ArCH C1), 7.52 (m, 1H, DOX–ArCH C3),
6.40 (q, J 1.84, 1H, CIT–CH), 5.40 (m, 1H, DOX–CH C10), 5.09
(m, 1H, DOX–CH C7), 4.73 (s, 2H, DOX–COCH2OH C14),
4.26 (q, J 6.8, 1H, DOX–CHCH3 C50), 4.00 (s, 3H, DOX–OCH3
C4), 3.85–3.54 (m, 2H, CIT–N–CH2 and 92H, PEG, O–
CH2CH2–O and 1H, DOX–CHOH C40), 3.52 (t, J 5.6, 2H, PEG
NH–CH2CH2–O), 3.45 (t, J 7.2, 2H, PEG O–CH2CH2–CONH–
DOX), 3.34 (t, J 5.6, 2H, PEG NH–CH2CH2–O), 3.14–
2.80 (2 ꢂ d, J 18.6, 2H, DOX–CHH C10 and m, 1H, DOX–CH
(NH) C30), 2.46–2.26 (m, 2H caproyl CH2CH2–CONH and 2H,
DOX–CHH C8), 2.17 (t, J 7.4, 2H, PEG O–CH2CH2–CONH–
DOX), 2.03 (d, J 1.86, 3H, CIT–CH3), 1.96, 1.69 (2 ꢂ m,
2H, DOX–CHH C20), 1.65–1.51 (m, 4H, CIT–N–CH2CH2
and caproyl CH2CH2CONH), 1.33–1.22 (m, 2H, caproyl
CH2CH2CH2CONH and d, J 6.6, 3H, DOX–CHCH3 C60). m/z
(ESI) calc.: 1879.08. Found: 940.3 (M2þ), 1878.9 (Mþ).
Preparation of Antibody Fab0 Fragments
a-FLAG Fab0
Reduction of a-FLAG F(ab0)2 (1.63 mg mLꢁ1) was per-
formed in PBS, pH 6.0, 0.5 mM EDTA, by treatment with
1 mM TCEP at 08C for 30 min. Complete reduction was
confirmed by analytical GFC on Superdex S200 1030 (using
degassed PBS, pH 6.0, 0.5 mM EDTA). TCEP was removed
using a HiPrep 2610 desalting column and the protein was
exchanged into freshly degassed PBS (pH 6.0, 0.5 mM EDTA)
to yield a-FLAG Fab0 (6 mL, 0.79 mg mLꢁ1, 4.8 mg), which was
used immediately in conjugation reactions.
528 Fab0
A solution of the 528 mAb in PBS was purified to remove
high-molecular-weight aggregate by GFC using Sephadex S200
2660. Fractions containing clean IgG in PBS were pooled and
concentrated to 1.5 mL (2.5 mg mLꢁ1, 3.8 mg) using a Millipore
3K MWCO Ultracel centrifugal filter. The concentrated protein
was then exchanged into 0.1 M citrate buffer using a HiPrep
26/10 desalting column (6 mL, 0.53 mg mLꢁ1, 3.2 mg). The IgG
in citrate buffer (6 mL, 0.53 mg mLꢁ1, 3.2 mg) was treated with
pepsin (21 mL, 1.5 mg mLꢁ1 in citrate buffer, 32 mg pepsin) at
378C for 90 min. After cooling to room temperature, the diges-
tion was stopped by neutralization with 3 M TRIS-HCl buffer
(600 mL, pH 8). The F(ab0)2 fragment was isolated from digested
fragments by GFC on HiLoad 26/60 Superdex 200 pg. Pooled
fractions containing F(ab0)2 were concentrated using a Millipore
Reaction of Citraconimide and Maleimide with L-Cysteine
L-Cysteine-citraconimidocaproic Acid
(Diastereomers 9 and 10)
A solution of 6-citraconimidocaproic acid 6 (12 mg,
0.054 mmol) in 1:1 MeOH/H2O (1 mL) was added to a stirred
solution of L-cysteine ethyl ester hydrochloride (12.6 mg,
0.068 mmol) in PBS (pH 7.2, 10 mL). After stirring at room
temperature for 24 h, the reaction mixture was concentrated
under reduced pressure. The residue was triturated with MeOH
(2 mL) and filtered through a 0.45-mm Acrodisc for purification
by HPLC. HPLC conditions: 0.1% TFA, 10–50% MeCN from
2 to 20 min. The main peak collected at Rt 16 min gave the
diastereomeric mixture of L-cysteine-citraconimidocaproic acid
(9 and 10) as a colourless oil (10.9 mg, 54%). dH (400 MHz,
CD3OD) 4.40–4.26 (m, 1H, Cys–CH(NH2)), 4.32 (q, J 7.1, 2H,
OCH2CH3), 3.51 (t, J 7.1, 2H, N–CH2), 3.49–3.38 (m, 2H, Cys–
CH2S), 3.00 (d, J 18.6, 1H, CIT–CHaHb(C–CH3)S), 2.71 (2 ꢂ d,
J 18.6, 1H, CIT–CHaHb(C–CH3)S), 2.27–2.31 (2 ꢂ t, J 7.4, 2H,
CH2CO2H), 1.69 (2 ꢂ s, 3H, CIT–CHaHb(C–CH3)S), 1.66–1.57
(m, 4H, N–CH2CH2 and CH2CH2CO2H), 1.36–1.31 (m, 5H,
OCH2CH3 and N–CH2CH2CH2).
3K MWCO Ultracel centrifugal filter (0.65 mL, 1.25 mg mLꢁ1
,
0.825 mg) and reduced with 1 mM TCEP (13.2 mL of a 50 mM
TCEP solution in H2O) at 08C for 30 min. TCEP was removed on
a 5-mL HiTrap desalting column with PBS (0.5 mM EDTA) to
yield the reduced 528 Fab0 (1.25 mL, 0.40 mg mLꢁ1, 0.5 mg),
which was used immediately in conjugation reactions.
Conjugation Reactions
a-FLAG Fab0 Conjugations
To separate solutions ofa-FLAG Fab0 (1.27 mL, 0.79 mg mLꢁ1
,
1 mg) in PBS (0.5 mM EDTA, pH 7.2) was added 100 equiva-
lents of IAA (11.9 mL, 168 mM solution in PBS) to yield
a-FLAG Fab0-IAA (12a); 10 equivalents of 2 (18.4 mL,
10.9 mM solution in DMSO) to yield a-FLAG Fab0-2 (13a); 3
(45.6 mL, 4.4 mM solution in DMSO) to yield a-FLAG Fab0-3
(14a); or 4 (41.7 mL, 4.8 mM solution in DMSO) to yield
a-FLAG Fab0-4 (15a). Conjugations were allowed to proceed
at 48C for 16 h before analysis and purification by GFC (HiLoad
16/60 Superdex 200 pg). SDS-PAGE analyses were performed
using Invitrogen NuPAGE 4–12% Bis-Tris precast gels with
3-(N-morpholino)propanesulfonic acid (MOPS) running buffer
at 240 V for 45 min.
L-Cysteine-maleimidocaproic Acid (Diastereomers
S11 and S12)
L-Cysteine-maleimidocaproic acid was prepared and
isolated by the same procedure as that for L-cysteine-citra-
conimidocaproic acid, using 6-maleimidocaproic acid
5

RESEARCH FRONT
788
L. P. T. Hong et al.
528 Fab0 Conjugations
To separate solutions of 528 Fab0 (300 mL, 0.40 mg mLꢁ1
Kenkare-Mitra, S. D. Spencer, M. X. Sliwkowski, Cancer Res. 2008,
68, 9280. doi:10.1158/0008-5472.CAN-08-1776
[7] A. Rossi, P. Maione, C. Gridelli, Crit. Rev. Oncol. Hematol. 2006, 59,
,
120 mg) in PBS (0.5 mM EDTA, pH 7.2) was added 100
equivalents of IAA (1.4 mL, 168 mM solution in PBS) to yield
528 Fab0-IAA (12b); 20 equivalents of 2 (4.4 mL, 10.9 mM
solution in DMSO) to yield 528 Fab0-2 (13b); 3 (10.9 mL,
4.4 mM solution in DMSO) to yield 528 Fab0-3 (14b); or
4 (10.0 mL, 4.8 mM solution in DMSO) to yield 528 Fab0-4
(15b). Conjugations were allowed to proceed at 48C for
16 h before analysis and purification by GFC (Superdex 200
10/300 GL).
139. doi:10.1016/J.CRITREVONC.2006.02.006
[8] G. N. Gill, T. Kawamoto, C. Cochet, A. Le, J. D. Sato, H. Masui,
C. McLeod, J. Mendelsohn, J. Biol. Chem. 1984, 259, 7755.
[9] T. Kawamoto, J. Mendelsohn, A. Le, G. H. Sato, C. S. Lazar, G. N.
Gill, J. Biol. Chem. 1984, 259, 7761.
[10] T. Kawamoto, J. D. Sato, A. Le, J. Polikoff, G. H. Sato, J. Mendelsohn,
Proc. Natl. Acad. Sci. USA 1983, 80, 1337. doi:10.1073/PNAS.80.
5.1337
[11] J. D. Sato, T. Kawamoto, A. D. Le, J. Mendelsohn, J. Polikoff, G. H.
Sato, Mol. Biol. Med. 1983, 1, 511.
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