5178
H. Xu et al. / Bioorg. Med. Chem. 17 (2009) 5176–5181
C18 column using a 0–50% CH3CN to 15 mM NH4OAc gradient over
30 min followed by lyophilization of the solvent from the relevant
fraction to yield trifunctional chelate 5 (1.50 mg, 31%). RP-HPLC:
tR = 21.5 min; ES-MS m/z: calcd for C78H100N9O21S2 [MꢂH]ꢂ,
[Mꢂ2H]2ꢂ: 1562.7, 780.9 found 1562.4, 780.7.
appropriate dilution and allowed to attach overnight at 37 °C prior
to treatment.
To demonstrate the binding of trastuzumab-Cy5.5-CHX-A00 (1)
and cetuximab-Cy7-CHX-A00 (2) by fluorescent microscopy both
monoclonal antibodies were used at a concentration of 10 lg/mL
in culture medium. The individual cell lines seeded on chambered
slides with 60–80% confluency were incubated for 2 h at 37 °C in
the dark. After incubation the cell culture medium was removed
and slides were washed three times with PBS (pH 7.4) and imme-
diately fixed in 4% paraformaldehyde prepared in PBS (pH 7.4) for
5 min at room temperature. Fluorescent mounting medium
(S3023, DakoCytomation) was added at the time the slide was cov-
ered with a cover glass to stabilize the fluorophores before and
during confocal microscopy.
2.1.3. Conjugation of 5 to cetuximab (general procedure)
Cetuximab (ꢀ10 mg) was reacted with Traut’s reagent27 (Sigma
Chemical Co., St. Louis, MO) at a 1:15 molar ratio for 1.5 h at room
temperature in 1 mL of PBS plus 10 mM EDTA buffer. Excess Traut’s
reagent was removed by passage of the reaction solution through a
PD-10 column eluted with PBS plus 10 mM EDTA buffer. The num-
ber of thiols introduced was determined using Ellman’s reagent.28
Just prior to protein conjugation, 5 was dissolved in the same buf-
fer and then added drop wise to the mAb solution to achieve a mo-
lar reaction ratio of 10:1 (5: Cetuximab) and gently vortexed. The
solution was then gently agitated in the dark at 25 °C for 1.5 h. Ex-
cess free unreacted SH groups were capped by the addition of iodo-
acetamide solution (2.0 mM). Finally, the reaction mixture was
dialyzed into PBS buffer at 4 °C with 4 buffer changes (4 L total)
over 48 h. Protein concentration was determined by Lowry assay
and the number of dye (and chelate) molecules per antibody was
calculated based on Cy 7 dye UV absorption at 747 nm.
2.1.7. Confocal fluorescence imaging
For confocal images, cells were examined with a Zeiss LSM 510
confocal system (Carl Zeiss Inc., Thornwood, NY, USA) with an
Axiovert 100 M inverted microscope operating with a 5 mW HeNe
laser tuned to 633 nm. Fluorescence (Cy5.5 or Cy7) and DIC images
were collected simultaneously using a 63ꢁ Plan-Apochromat 1.4
NA oil immersion objective and a long pass 650 nm emission filter.
In the acquisitions for all channels the Zeiss AIM software version
3.2 sp2 (Carl Zeiss GmbH, Heidelberg, Germany) was used. All con-
focal images were acquired with a frame size of 512 by 512 with
identical settings.
2.1.4. Radiolabeling
Trastuzumab-Cy5.5-CHX-A00 and cetuximab-Cy7-CHX-A00 were
each labeled with 111In as follows. A solution of 111InCl in HCl
(0.05 M, 1–3
l
L, 0.5–1 mCi) was added to either antibody conju-
L, 0.15 M, pH 7),
3. Results and discussion
gate (50 g) contained in NH4OAc buffer (100
l
l
vortexed immediately and incubated at room temperature for
Synthesis of lysine derivative 3, bearing the radiometal chelate
30 min. The reaction was quenched by the addition of EDTA solu-
tion (0.1 M, 4 l
L). The 111In labeled antibodies were purified by
CHX-A00 and the thiol-reactive maleimide moiety attached to the
a
-COOH and e-NH2 of lysine functional groups, respectively, was
gel filtration chromatography using PD-10 columns (GE Health-
care) eluted with PBS. Radiolabeling yields as determined by the
PD-10 separation ranged from 75% to 94%. The purity of the PD-
10 purified product was ascertained by SE-HPLC as described
above.
recognized as a versatile intermediate that could serve as a nexus
for the modular introduction of fluorescent dyes or other agents.
NIR dyes Cy5.5 and Cy7 were then selected to demonstrate synthetic
feasibility. Cy 5.5 derivative 4 and its antibody conjugate 1 were re-
cently developed in our laboratory.6 The synthetic approach to 4 was
applied to the synthesis of the corresponding Cy 7 conjugates 5 and 2
using similar procedures with minor modifications. In brief, Cy 7
2.1.5. Radioimmunoassays
The immunoreactivity of the cetuximab-Cy7-CHX-A00 conjugate
was evaluated in a competition radioimmunoassay. Fifty ng of
mono-NHS ester was reacted with the
a-NH2 of 5 in DMSO. The
product was precipitated from diethyl ether and then purified by re-
verse-phase HPLC using a C18 column to isolate 5 in ꢀ31% yield
(Fig. 2). To conjugate 5 to cetuximab, the mAb was first thiolated
with 15 equiv of Traut’s agent using standard procedures.27 In our
hands, ꢀ2–4 –SH groups per mAb were introduced as quantitated
by Ellman’s reagent.28 Thiolated mAb was then reacted with
10 equiv of 5 at rt for 1.5 h to produce antibody conjugate 2. Unre-
acted remaining thiols were capped with iodoacetamide to mini-
mize cross-linking of the antibody conjugate products. Finally, the
reaction mixture was dialyzed into phosphate-buffered saline
(PBS; pH 7.2) at 4 °C with four buffer changes over 48 h. The number
of Cy7 dye moieties per cetuximab was calculated to be ꢀ2 based on
UV absorption at 747 nm, which directly corresponds to the same
number of CHX-A00 chelates on cetuximab according to the 1:1 ratio
of Cy7 and CHX-A00 within 5.
EGFR (Sigma–Aldrich, St. Louis, MO) in 50
Mg2+ and Ca2+ was added to each well of a 96-well plate. Following
an overnight incubation at 4 °C, wells were aspirated and 150 L of
PBS/BSA added to each well and allowed to sit for an additional
hour at ambient temperature. The wells were aspirated and serial
lL of PBS containing
l
dilutions (0.01–500 ng in 50 lL BSA/PBS) of unmodified cetuximab
or cetuximab-Cy7-CHX-A00 conjugate were added to the wells in
triplicate (one set of wells received BSA/PBS without any compet-
itor), along with 125I-cetuximab (ꢀ50,000 cpm in 50
lL BSA/PBS).
The wells were aspirated after 4 h incubation at 37 °C and washed
three times with BSA/PBS. The bound radioactivity was removed
with 100
lL 0.2 M NaOH, adsorbed to cotton filters, placed in
12 ꢁ 75 mm tubes and counted in a
c
-scintillation counter (Wizard
One, PerkinElmer, Shelton, CT). The percent inhibition was calcu-
lated using the buffer control and plotted. HuM195, a mAb that re-
acts with human CD33, served as a negative control.
Both trastuzumab-Cy5.5-CHX-A00 and cetuximab-Cy7-CHX-A00
radiolabeled efficiently (ꢀ75–94%) with the SPECT radionuclide
111In within 30 min at room temperature. HPLC profiles of both 1
and 2 reveal single symmetric peaks of near identical retention time
(see Supplementary data).
2.1.6. Cell culture and treatment
A431, SKOV3, and NIH3T3 cells were obtained from ATCC (Man-
asass, VA). The NIH3T3 cells were transfected with HER2 genes
(3T3/HER2+), in order to over-express HER2 receptors.22 All of the
cell lines were grown at 37 °C in the presence of 5% CO2 in DMEM
medium supplemented with 1% L-glutamine, and 5% FBS. For
the confocal fluorescence microscopy experiments cells were
plated in Lab-Tek chambered slides (#178599 Nalge Nunc) at an
Radioimmunoassay of the cetuximab-Cy7-CHXA00 conjugate
showed that conjugation of 5 to cetuximab resulted in comparable
binding to the EGFR receptor (Fig. 3). Fifty percent inhibition was
achieved with 1.7 nmol cetuximab-Cy7-CHXA00 versus 1 nmol for
the native cetuximab. The fluorescent labeling of HER2 expressing