Synthesis and Feasibility Evaluation of a new Trastuzumab Conjugate Integrated with Paclitaxel and 89Zr for Theranostic Application Against HER2-Expressing Breast Cancers

Article abstract of DOI:10.1002/open.201900037

The preparation and in vitro evaluation of a theranostic conjugate composed of trastuzumab, paclitaxel (PTX), and deferoxamine (DFO)-chelated ⁸⁹Zr have been reported. These comounds have potential applications against HER2 receptor positive bre

Full text of DOI:10.1002/open.201900037

DOI: 10.1002/open.201900037
Full Papers
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Synthesis and Feasibility Evaluation of a new Trastuzumab
Conjugate Integrated with Paclitaxel and 89Zr for
Theranostic Application Against HER2-Expressing Breast
Cancers
Joo Hee Jang, Sang Jin Han, Jung Young Kim, Kwang Il Kim, Kyo Chul Lee, and
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11 Chi Soo Kang*[a]
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The preparation and in vitro evaluation of
a
theranostic
in vitro stability in human serum. Furthermore, DFO-trastuzu-
mab-PTX displayed comparable cytotoxicity with PTX and 89Zr-
DFO-trastuzumab-PTX exhibited HER2 receptor-mediated bind-
ing on HER2-positive MDA-MB-231 breast cancer cells. The
results of our in vitro study indicate high potential of 89Zr-DFO-
trastuzumab-PTX to be utilized in the theranostic application
against HER2-postive breast cancers.
conjugate composed of trastuzumab, paclitaxel (PTX), and
deferoxamine (DFO)-chelated 89Zr have been reported. These
comounds have potential applications against HER2 receptor
positive breast cancers. We conjugated DFO and PTX to
trastuzumab by exploiting simple conjugation chemistry. The
conjugate (DFO-trastuzumab-PTX) showed excellent radiolabel-
ing efficiency with 89Zr and the labeled conjugate had high
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1. Introduction
mortality rate is also very high.[8,9] Researchers have put
significant effort to understand novel characteristics of breast
cancer and to develop appropriate therapeutic strategies. It was
found that around 25–30% of all breast cancer and approx-
imately 50% of breast cancer with bone metastasis overexpress
HER2 receptor, which is a member of human epidermal growth
factor receptor family, and its expression causes poor
prognosis.[10–12] Trastuzumab (Herceptin®), which is a FDA-
approved humanized monoclonal antibody for the treatment of
metastatic breast cancer, selectively targets the HER2 receptor
and is known to block HER2-related cellular transduction
pathway and cause antibody-dependent cell-mediated
cytotoxicity.[12] Trastuzumab’s clinical efficacy was proven when
used solely as well as in combination with chemotherapeutics,
and its antibody drug conjugate version (Trastuzumab emtan-
sine, T-DM1) has shown improved treatment outcome com-
pared to sole treatment of trastuzumab.[13] Therefore, trastuzu-
mab is a pertinent choice for the development of theranostic
platform when HER2-expressing breast cancer is considered.
Paclitaxel (PTX) is an effective chemotherapeutics used in a
wide range of cancers including breast, ovarian, head and neck
cancers, etc., and it stabilizes microtubules leading to
apoptosis.[14,15] However, the clinical use of PTX is limited by its
poor water-solubility and high toxicity to normal cells neces-
sitating the development of appropriate delivery systems for
improved solubility and target selectivity.[14]
Theranostic agents, defined as a material capable of diagnosing
and treating a disease simultaneously using a single platform
composed of multiple functional units, have withdrawn tremen-
dous interest for past decades from research community. These
agents have potential to provide information about localization
and biodistribution of a drug in a patient for better assessment
of the drug as well as planning of treatment regimen.[1]
Furthermore, theranostic agents are capable of minimizing the
inevitable difference in biodistribution and selectivity existing
between diagnostic and therapeutic materials for a specific
disease.[2,3] Especially for cancer, which is a highly heterogenous
disease, application of theranostic agent is essential for better
assessment of treatment options to different individuals to
achieve improved prognosis.[4,5] There can be variety of
theranostic platforms for simultaneous accommodation of
imaging and therapeutic moieties, and antibody conjugate
would be a great choice since the formulation has been proven
to be effective in several clinical cases.[6,7]
Breast cancer is the most common cancer in women and its
incidence is over 1.5 million cases worldwide every year and the
[a] Dr. J. H. Jang, S. J. Han, Dr. J. Y. Kim, Dr. K. I. Kim, Dr. K. C. Lee,
Dr. C. S. Kang
Division of Applied RI
Positron emission tomography (PET) is a sensitive and
noninvasive molecular imaging technique utilizing positron-
emitting radioisotopes and most frequently used option for
cancer diagnostics. 2-deoxy-2-[18F]-fluoroglucose (18F-FDG),
which exploits increased glucose metabolism of cancer, is the
predominantly used as a PET agent for cancer imaging.
However, it poses a crucial limitation such that 18F-FDG is not
cancer specific, i.e. it accumulates not only at cancer sites but
Korea Institute of Radiological and Medical Sciences
75 Nowon-ro, Nowon-gu, Seoul, Korea 01812
E-mail: cskang0224@kirams.re.kr
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/open.201900037
©2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution Non-Commercial License, which permits use, distribution and
reproduction in any medium, provided the original work is properly cited
and is not used for commercial purposes.
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also at the sites with increased glucose metabolism, such as
infectious and inflammatory sites, leading to false-positive
result.[16] To overcome the limitation of 18F-FDG, development of
monoclonal antibody labeled with a positron emitting radio-
isotope has attracted a lot of attention.[17,18] Accordingly, various
positron emitting radioisotopes including 124I, 86Y, 64Cu and 68Ga
have been explored to be labeled to the antibodies. However,
they have some drawbacks that hamper the optimal construct
of immuno-PET agents. For instance, in vivo dehalogenation of
PET agent using 124I might cause radiation damage to heathy
tissue, and the short half-lives of 64Cu (12.7 h), 68Ga (67.7 min),
and 86Y (14.7 h) do not match with in vivo half-life (2–3 days) of
typical antibodies.[19] On the other hand, 89Zr has optimal
physical characteristics that can be exploited in PET imaging
application of cancer. Its decay half-life (78.4 h) is in line with
physiological half-life of typical antibodies, and the methods for
production with high radiochemical purity and specific activity
are well developed.[18–21] Furthermore, suitable chelator (Defer-
oxamine, DFO) for 89Zr is already commercially available fueling
the investigation of 89Zr as an immuno-PET agent.[22–24]
tion) evaluation using reversed-phase C-18 column validates
the coupling of maleimide group to PTX, and clean single peak
also confirms the purity of the compound. Mass analysis via
MALDI ([M+Na]+ calculated/found: 1027.35/1027.49) further
confirms the synthesis of 2’-maleimido-PTX.
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2.2 Preparation of Trastuzumab Conjugate
Preparation of DFO-trastuzumab-PTX is shown in scheme 2.
Conjugation of DFO with trastuzumab was done via thiourea
linkage between lysine residues from trastuzumab and isothio-
cyanate group from DFO. Solution of p-SCN-DFO was prepared
in DMSO and %v/v of DMSO (<1.0%) in a reaction buffer (0.1 M
NaHCO3, pH 9.0) was carefully controlled to minimize possible
antibody aggregation. It was found between 0.45 to 0.55 DFO
was conjugated (Supporting Information) to a molecule of
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°
trastuzumab after 16 h of reaction at 37 C. Following DFO
conjugation, thiolation of trastuzumab in DFO-trastuzumab was
carried out using 2-iminothiolane (Traut’s reagent), after which
conjugation with 2’-maleimido-PTX was done. The solution of
2’-maleimido-PTX was prepared in DMSO, and conjugation with
thiolated DFO-trastuzumab was progressed in the reaction
buffer for 6 h at RT, and it was found between 3.8 to 4.2 PTX
was conjugated (Supporting Information) to a molecule of DFO-
trastuzumab via thioether linkage. Although more extensive
investigation needs be done to find the best conditions for
conjugation of antibody with DFO and PTX, especially when
considering they are not water soluble, the number of
conjugated DFO or PTX to trastuzumab was good enough to
test in vitro feasibility for the development of theranostic agent.
In the present study, we first synthesized a new antibody
conjugate composed of trastuzumab, PTX, and DFO by
sequential conjugation of DFO and PTX with trastuzumab.
Following characterization of the conjugate, radiolabeling
efficiency using 89Zr and serum stability of 89Zr-labeled complex
were assessed. Furthermore, in vitro cell binding study and
cytotoxicity evaluation were done using HER2-expressing
(HER2-transduced) MDA-MB-231 human breast cancer cell line.
2. Results and Discussion
2.1 Synthesis of 2’-Maleimido-PTX
2.3 Radiolabeling with 89Zr and Serum Stability of
89Zr-Labeled Complex
Synthetic route of 2’-maleimido-PTX is outlined in scheme 1.
Steglich esterification using DIC/DMAP as coupling reagents
The complexation kinetics of the new antibody conjugate (DFO-
trastuzumab-PTX) with 89Zr was highly efficient (100% at 1 min,
supporting information), and the labeling efficiency was
comparable with that of DFO-trastuzumab. As DFO is an
excellent chelator for 89Zr,[25,26] it was expected that DFO-
trastuzumab would have a good radiolabeling efficiency with
89Zr. However, it was not unreasonable to assume that complex-
ation kinetics of DFO-trastuzumab-PTX would be compromised
due to more intricate conjugation status. Nontheless, DFO-
trastuzumab-PTX instantly grabbed 89Zr at RT with specific
activity of 0.457 Ci/μmol. Since rapid chelation of radionuclide
in mild condition is an important prerequisite for the develop-
ment of antibody-based radiopharmaceuticals due to the
concern for antibody’s integrity, the labeling result of DFO-
trastuzumab-PTX with 89Zr indicated it has the potential to be a
theranostic agent utilizing antibody and radionuclide. Stability
of 89Zr-labeled DFO-trastuzumab-PTX and DFO-trastuzumab was
evaluated by incubating the complexes in human serum at
Scheme 1. Synthesis of maleimide coupled PTX by utilization of Steglich
esterification with DMAP/DIC coupling reagents. DMAP, 4-(Dimethylamino)
pyridine; DIC, N,N’-Diisopropylcarbodiimide.
was preferentially done at the 2’-hydroxyl group with 50.2%
yield due to steric hindrance at 7’-hydroxyl group of PTX. NMR
analysis indicated that C-2’ proton peak was shifted from
4.78 ppm in unmodified PTX to 5.47 ppm in maleimide-coupled
PTX, and there was the appearance of proton signal from
ethylene in maleimide at 6.49 ppm. Furthermore, retention time
shift from 6.77 min to 10.99 min on HPLC (Supporting Informa-
°
37 C (Supporting Information). As human serum has variety of
proteins and trace metals, trans-chelation would occur if the
complex is not very stable, thereby incubation of labeled
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Scheme 2. Preparation of a new antibody conjugate composed of DFO, trastuzumab and PTX.
complex in human serum is a good way to evaluate stability of
the labeled complex. Stability of 89Zr-labeled DFO-trastuzumab-
PTX was assessed up to 7 days and it was shown that no
significant amount of 89Zr was dissociated from the labeled
complex, and the result was comparable with that of 89Zr-
labeled DFO-trastuzumab indicating that the addition of PTX
did not affect the stability of labeled complex.
0.0002) showing cell binding was receptor mediated. 89Zr-
labeled DFO-trastuzumab also had similar binding percentage
(51.0�3.3%) indicating additional conjugation of PTX did not
alter biological activity of trastuzumab. Free 89Zr was also
incubated with the cells to ensure %bound of the 89Zr-labeled
conjugates was not due to adsorption of dissociated free 89Zr,
and the uptake of the free 89Zr was only 2.4�0.3% confirming
intact complexes of 89Zr-labeled conjugates were bound to the
cells. 89Zr-labeled DFO-trastuzumab-PTX was also incubated
with HER2-negative MDA-MB-231 cells, and it showed signifi-
cantly lower binding (4.9�2.2%, p-value=0.0002) validating
receptor-mediated uptake of 89Zr-labeled DFO-trastuzumab-
PTX.
2.4 Binding Assay of 89Zr-Labeled Theranostic Agent
To evaluate binding property of 89Zr-labeled DFO-trastuzumab-
PTX, in vitro binding assay was performed on HER2-positive
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MDA-MB-231 cells (Figure 1). After 5 h incubation at 4 C, 49.5�
5.3% of the 89Zr-labeled DFO-trastuzumab-PTX treated were
remained bound to cells, while blocking with excess trastuzu-
mab reduced the cell bound percentage significantly (p-value=
2.5 In Vitro Toxicity Evaluation
Cytotoxicity screening (50 μM, PTX equimolar) of DFO-trastuzu-
mab-PTX was done using MTT assay on HER2-positive and
negative MDA-MB-231 cells and compared with PTX itself
(Figure 2). Approximately half of the HER2-positive MDA-MB-
231 cells were dead after 72 h incubation with DFO-trastuzu-
mab-PTX, which was similar with the effect of PTX treatment.
Exposure of trastuzumab only on the cells did not exert any
cytotoxic effect, as expected, since it is known trastuzumab
requires other effectors to trigger anti-cancer effect and would
not result in cell death in vitro.[27] On the HER2-negative cells,
PTX also had almost the same level of cytotoxic effect (55.5%
vs. 56.4%) as it is not a targeted therapeutics. However, DFO-
trastuzumab-PTX also showed quite similar cytotoxic effect on
the HER2-negative cells compared to HER2-positive cells (62.4%
vs. 55.8%), which was not expected, since less anti-cancer effect
was anticipated due to the lack of HER2 receptor. It was
Figure 1. Binding assay of 89Zr-labeled trastuzumab conjugates on HER2-
positive and negative MDA-MB-231 cells [a: (89Zr)DFO-Tras-PTX; b: Blocking;
c: (89Zr)DFO-Tras; d: 89Zr]. 89Zr-labeled trastuzumab conjugate (1 μg,
6
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~700kcpm) was incubated on the cells (1×10 ) for 5 h at 4 C, after which
the cells were washed with cold PBS (3×1 mL) and radioactivity remained
on the cell pellets was measured using gamma counter. The evaluation was
done in triplicate (mean�standard deviation %).
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Experimental Section
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Instruments and Materials: 1H NMR spectrum was obtained using a
Bruker 300 instrument, and chemical shifts are reported in ppm on
the δ scale relative to TMS. Matrix-assisted laser desorption
ionization mass (MALDI-MS) was obtained using Applied Biosystems
ABI5800 Plus MALDI-TOF/TOF analyser (Korea Drug Development
Platform using Radio-isotope, Korea Institute of Radiological and
Medical Sciences, Seoul, Korea). Analytical HPLC was performed on
a Waters 2998 system (Waters, Milford, MA, USA) equipped with
photodiode array detector (λ=230 nm) using Atlantis® dC18, 5 μm,
3.0×150 mm column with solvent system consisting of 0.1% TFA in
water (solvent A) and 0.1% TFA in acetonitrile (solvent B), where
isocratic eluent profile 0–20 min, 50% A, 50% B (0.5 mL/min) was
applied. All measurements of protein concentration were obtained
using Thermo Scientific Nanodrop 2000 (Waltham, MA, USA).
Radioactive Thin layer chromatography (TLC) measurements was
done using Eckert & Ziegler TLC scanner B-AR2000-1 (Eckert &
Ziegler, Valencia, CA, USA). For measurements of radioactivity in
cell binding assay, PerkinElmer 2480 Automatic Gamma Counter
(Waltham, MA, USA) was used. RT-PCR was performed using 7500
Real Time PCR System (Applied Biosystems, Foster City, CA, USA)
and fluorescence images were obtained using Olympus IX71
(Olympus, Tokyo, Japan). 4-(Dimethylamino)pyridine (Cat# 522805),
N,N’-Diisopropylcarbodiimide (Cat# D125407) and 2-iminothiolane
hydrochloride (Cat# I6256) were obtained from Sigma Aldrich (St.
Louis, MO, USA). 3-maleimidopropionic acid (Cat# M1962), paclitax-
el (Cat# P106869) and p-SCNÀ Bn-Deferoxamine (Cat# B-705) was
obtained from Tokyo Chemical Industry (Tokyo, Japan), Aladdin
Industrial Corporation (City of Industry, CA, USA), and Macrocyclics
(Plano, TX, USA), respectively. hERBB2 inducible lentiviral particle
(Cat# LVP504) was purchased from GenTarget Inc. (San Diego, CA,
USA).
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Figure 2. In vitro toxicity evaluation of DFO-trastuzumab-PTX on HER2-
positive and negative MDA-MB-231 cells [a: DFO-Tras-PTX; b: PTX; c: Tras].
50 μM (PTX equimolar) of the conjugate was incubated for 72 h, and relative
cell viability was measured using MTT assay. The evaluation was done in
triplicate (mean � standard deviation %).
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speculated that the ester linkage between PTX and trastuzumab
was broken due to its susceptibility to hydrolysis even
in vitro,[28,29] and PTX was released during the incubation period
leading to cell death. Therefore, the cytotoxicity exhibited by
DFO-trastuzumab-PTX on MDA-MB-231 breast cancer cells,
whether they express HER2 receptor or not, was indeed the
result of premature release of PTX rather than the receptor-
mediated delivery of PTX. Therefore, smarter conjugate strat-
egies linking PTX and trastuzumab should be exploited for
improved performance of targeted delivery and tumor-selective
action of the drug. Furthermore, the 2’-hydroxyl group of PTX
should be intact after release from the conjugate to properly
interfere with tubulin.[30,31] Consequently, a linker that is stable
enough in human plasma but hydrolyzed within cancer cells
releasing PTX without liker residues should be applied. Recently,
alcohol modification chemistry using acetal pyrophosphate
ester, which satisfies above mentioned requirements, to con-
jugate an anti-cancer drug with an antibody was developed,[32]
and it would be interesting to apply the acetal pyrophosphate
ester into our conjugate system in the future.
Production of 89Zr Chloride: 89Zr was produced as previously
described[33] with little modification. Briefly, nuclear reaction of 89Y
(p,n)89Zr on an enriched yttrium target (89Y, 200×200×0.025 mm,
99.9%) was carried out with a bombardment by the proton beam
(about 20 MeV) of medical cyclotron (MC50, Scanditronix co., 1985).
To obtain 89Zr with high radiochemical purify, the solution of
radioactive yttrium target melted by hydrochloric acid (6.0 N,
10 mL) was passed through a hydroxamate resin column. 89Zr
trapped in the column was recovered by oxalate solution (1.0 N,
2.0 mL), and the 89Zr-oxalate solution was slowly loaded into a QMA
Sep-Pak column (Waters co.). To remove oxalate, the column was
slowly washed using distilled water (100 mL). The product left in
the column was obtained by hydrochloric acid (1.0 N, 1.0 mL), and
the radiochemical yield of 89Zr chloride was found to be 6.5�
1.5 mCi/h with over 99% radionuclide purity.
3. Conclusions
A new antibody conjugate (DFO-trastuzumab-PTX) was pre-
pared via simple modification of PTX and sequential conjuga-
tion of DFO and the modified PTX with trastuzumab. DFO-
trastuzumab-PTX showed efficient labeling with 89Zr, and the
labeled complex was highly stable in human serum. Further-
more, DFO-trastuzumab-PTX exhibited comparable cytotoxicity
with PTX in vitro, and 89Zr-labeled DFO-trastuzumab-PTX had
receptor-specific binding on HER2-positive MDA-MB-231 cells.
Although improved linker chemistry needs to be applied to the
conjugation of PTX with trastuzumab for better selective anti-
cancer effects, our in vitro results present promising potential of
Establishment of HER2-Expressing MDA-MB-231 Breast Cancer
Cell: Lentivirus vectors carrying HER2 and red fluorescent protein
(RFP) fused with blasticidin deaminase (Bsd) genes (200 μL, 1×
107 IFU/mL in DMEM medium) were transduced into MDA-MB-231
cells. HER2-expressing cells were selected by blasticidin for 4 weeks,
and confirmation of HER2 expression was done by RT-PCR and
immunofluorescence imaging (Supporting Information).
Cell Culture: HER2-positive and negative MDA-MB-231 human
breast cancer cell lines were cultured in a humidified atmospheric
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condition with 5% CO2, at 37 C using RPMI-1640 culture medium
containing 10% fetal bovine serum (FBS) and 1% antibiotic-
antimycotic.
89Zr-labeled DFO-trastuzumab-PTX as
against HER2-positive breast cancers.
a theranostic agent
Synthesis of 2’-Maleimido-PTX: 4-(Dimethylamino)pyridine
(0.70 mg, 0.006 mmol), 3-maleimidopropionic acid (10.9 mg,
0.06 mmol) and N,N’-Diisopropylcarbodiimide (7.4 mg, 0.06 mmol)
were sequentially added to PTX (50 mg, 0.06 mmol) in dichloro-
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°
°
methane (5 mL) at 0 C. The temperature was gradually increased to
in a thermomixer at 37 C. 89Zr bound percentage was assessed for
7 consecutive days using radio-TLC (20 mM EDTA in 0.15 M NH4OAC
as an eluent).
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room temperature, and the reaction mixture was reacted for 24 h
under argon atmosphere while the progress was monitored by TLC.
After the reaction was complete, the mixture was washed using
saturated solution of NH4Cl (2×15 mL) and NaHCO3 (2×15 mL),
followed by NH4Cl (3×15 mL). The organic layer was dried over
MgSO4, filtered, and evaporated to obtain a crude product. The
crude product was purified by auto column chromatography (0%
B/3 min, 0–2% B/3–5 min, 2% B/5–10 min; solvent A=dichloro-
methane, solvent B=MeOH) and dried in vacuo to provide pure
product (29.6 mg, 50.2%). The product was characterized by 1H
NMR (300 MHz, CDCl3),28 HPLC (tR =10.99 min and 6.77 min for 2’-
maleimido-PTX and unmodified PTX, respectively, Supporting
Information), and MALDI-MS [M+Na]+ (calculated/found: 1027.35/
1027.49).
In Vitro Binding Assay: 89Zr-labeled DFO-trastuzumab-PTX and
DFO-trastuzumab were prepared as described in the radiolabelling
section. An aliquot of 89Zr-labeled conjugate (1 μg, ~700 kcpm) was
added to HER2-positive or negative MDA-MB-231 breast cancer
cells (1×106) suspended in PBS with 1% BSA (0.5 mL) in a sterile
polystyrene round-bottom tube (BD Falcon, Cat# 352052) and
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incubated at 4 C with vigorous stirring for 5 h. The radioactivity of
each tube was measured using gamma counter, after which the cell
pellets were washed with cold PBS (3×1 mL). The radioactivity after
the wash was measured again, and the percentage bound was
calculated by comparing the radioactivity before and after the
wash. For blocking experiment, 5000-fold excess of trastuzumab
was added to the cells prior to the addition of 89Zr-labeled
conjugate.
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Conjugation of DFO with Trastuzumab: To
trastuzumab (19.5 mg) in 0.1 M NaHCO3 (pH 9.0, 5 mL) was added
20-fold excess p-SCN-DFO in DMSO (44.6 μL). The reaction mixture
was agitated in a thermomixer at 37 C for 16 h, after which
unreacted p-SCN-DFO was removed using Amicon Ultra centrifugal
filters (Millipore, Ultracel-30K, UFC803024) while washing with 0.1 M
NaHCO3 (3×2 mL). DFO-trastuzumab was recovered (18.6 mg,
97.0%) using 0.1 M NaHCO3 (pH 9.0, 1 mL), and the number of DFO
conjugated to a molecule of trastuzumab was found to be 0.45–
0.55 by MALDI-Mass analysis (Supporting Information).
a solution of
Cytotoxicity Evaluation: HER2-positive and negative MDA-MB-231
breast cancer cells (1×104) were seeded on a 96-well plate and
allowed for them to attach for 24 h. PTX (50 μM), DFO-trastuzumab-
PTX (50 μM, PTX equimolar), and trastuzumab (12.5 μM, trastuzu-
mab concentration resulted when PTX to trastuzumab ratio was
considered for 50 μM PTX in DFO-trastuzumab-PTX) were treated
on the cells and incubated for 72 h. After the incubation, relative
cell viability was obtained by comparing the viabilities between
untreated control cells and treated ones by using MTT assay.
°
Thiolation of Trastuzumab: To a solution of DFO-trastuzumab
(18.6 mg) in 0.1 M NaHCO3 (pH 9.0, 1 mL) was added 20-fold excess
2-iminothiolane. The reaction mixture was agitated in a thermo-
mixer at RT for 2 h, after which unreacted 2-iminothiolane was
removed using Amicon Ultra centrifugal filters (Millipore, Ultracel-
30K, UFC803024) while washing with 0.1 M NaHCO3 (3×2 mL).
Thiolated DFO-trastuzumab was recovered (15.2 mg, 82.0%) using
0.1 M NaHCO3 (pH 9.0, 0.9 mL).
Statistical Analysis: Data were analysed by unpaired, two-tailed
Student’s t-test using GraphPad Prism 8.0.2 (GraphPad Software
Inc.), and the difference with p-value of <0.05 (95% confidence
level) was considered as statistically significant.
Conjugation of 2’-Maleimido-PTX with DFO-Trastuzumab: To a
solution of thiolated DFO-trastuzumab (7.0 mg) in 0.1 M NaHCO3
(pH 9.0, 6 mL) was added 30-fold excess 2’-maleimido-PTX in DMSO
(28.8 μL). The reaction mixture was agitated in a thermomixer at RT
for 6 h, after which unreacted 2’-maleimido-PTX was removed using
Amicon Ultra centrifugal filters (Millipore, Ultracel-30K, UFC803024)
while washing with 0.1 M NaHCO3 (3×2 mL). DFO-trastuzumab-PTX
was recovered (5.2 mg, 75.0%) using 0.1 M NaHCO3 (pH 9.0,
0.7 mL), and the number of PTX conjugated to a molecule of
trastuzumab was found to be 3.8-4.2 by MALDI-Mass analysis
(Supporting Information).
Acknowledgements
This study was supported by a grant of the Korea Institute of
Radiological and Medical Sciences (KIRAMS), funded by Ministry of
Science and ICT (MSIT), Korea (No. 50461-2018) and the Nuclear
Research and Development Program of the National Research
Foundation of Korea (NRF) grant funded by the Korean govern-
ment (No. 2017M2A2A6A02019904). We thank Ms. Eunbi Shin for
MALDI-MS measurement and analysis using MALDI-TOF/TOF
analyser (Korea Drug Development Platform using Radio-isotope,
Korea Institute of Radiological and Medical Sciences, Seoul,
Korea).
Radiolabeling of DFO-Linked Trastuzumab Conjugates with 89Zr
Chloride: Radiolabeling reaction buffer (0.25 M NH4OAc, pH 7.0)
was prepared by using 99.999% trace metals basis NH4OAc (Sigma
Aldrich, Cat# 372331) and treated with Chelex-100 resin (Biorad,
Cat# 1422842) to remove any trace metals possibly existing in the
solution. To a buffer solution (17.96–18.32 μL) in a capped micro-
centrifuge tube was sequentially added a solution of either DFO-
Conflict of Interest
trastuzumab-PTX (10 μg) or DFO-trastuzumab (10 μg) and
a
solution of 89Zr chloride (30 μCi, 1.5 μL, 0.1 N HCl). The reaction
mixture was agitated in a thermomixer (1000 rpm) at RT. An aliquot
of the mixture was withdrawn at designated time points (1, 10, 30,
60 min), spotted on a TLC plate and eluted using 20 mM EDTA in
0.15 M NH4OAc for measurement of labelling efficiency by radio-
TLC scanner.
The authors declare no conflict of interest.
Keywords: theranostics · trastuzumab · paclitaxel · 89Zr · breast
cancer
Serum Stability Evaluation of 89Zr-Labeled Complexes: 89Zr-labeled
complexes were prepared as described in the radiolabelling section.
After complete complexation of the DFO-trastuzumab conjugates
was confirmed by radio-TLC, human serum (40 μL) was added to
the 89Zr-labelled conjugate (40 μL), and the mixture was incubated
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Manuscript received: January 24, 2019
Revised manuscript received: March 12, 2019
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