Efficient bifunctional decadentate ligand 3p-C-DEPA for targeted α-radioimmunotherapy applications

Article abstract of DOI:10.1021/bc100586y

A new bifunctional ligand 3p-C-DEPA was synthesized and evaluated for use in targeted α-radioimmunotherapy. 3p-C-DEPA was efficiently prepared via regiospecific ring opening of an aziridinium ion and conjugated with trastuzumab. The 3p-C-DEPA-trastuzumab

Full text of DOI:10.1021/bc100586y

ARTICLE
pubs.acs.org/bc
Efficient Bifunctional Decadentate Ligand 3p-C-DEPA for Targeted
r-Radioimmunotherapy Applications
Hyun A Song,† Chi Soo Kang,† Kwamena E. Baidoo,‡ Diane E. Milenic,‡ Yunwei Chen,† Anzhi Dai,†
M. W. Brechbiel,‡ and Hyun-Soon Chong*,†
†Chemistry Division, Biological, Chemical, and Physical Sciences Department, Illinois Institute of Technology, Chicago, Illinois,
United States
‡Radioimmune & Inorganic Chemistry Section, Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute,
NIH, Bethesda, Maryland, United States
S Supporting Information
b
ABSTRACT:
A new bifunctional ligand 3p-C-DEPA was synthesized and evaluated for use in targeted r-radioimmunotherapy. 3p-C-DEPA was
efficiently prepared via regiospecific ring opening of an aziridinium ion and conjugated with trastuzumab. The 3p-C-DEPA-
trastuzumab conjugate was extremely rapid in binding 205/6Bi, and the corresponding 205/6Bi-3p-C-DEPA-trastuzumab complex was
stable in human serum. Biodistribution studies were performed to evaluate in vivo stability and tumor targeting of 205/6Bi-3p-C-
DEPA-trastuzumab conjugate in tumor bearing athymic mice. 205/6Bi-3p-C-DEPA-trastuzumab conjugate displayed excellent in vivo
stability and targeting as evidenced by low organ uptake and high tumor uptake. The results of the in vitro and in vivo studies indicate
that 3p-C-DEPA is a promising chelator for radioimmunotherapy of 212Bi and 213Bi.
’ INTRODUCTION
use in RIT when employing short-lived radionuclides such as
213Bi and 212Bi.9,10 The acyclic bifunctional ligand, C-DTPA,
displayed rapid and high-yield radiolabeling with Bi(III) radio-
isotopes.3 However, C-DTPA produced a less stable Bi(III)-C-
DTPA complex than that of Bi(III)-C-DOTA,3,11,12
The r-emitting radioisotopes, 212Bi (t1/2 = 60.6 m) and 213Bi
(t1/2 = 45.6 m), have proven to be effective for radioimmu-
notherapy (RIT) of cancers.1 The r-emitters with high r-energy
(5ꢀ8 MeV) and a short emission path length (50ꢀ80 μm) can
be closely deposited in the target tumor cells resulting in minimum
damage to normal tissues.2 The radionuclides, 212Bi and 213Bi, with
very short half-lives decay ultimately to stable bismuth nuclides and
are considered to be suitable for convenient outpatient RIT.3 The
therapeutic efficacy of 212Bi and 213Bi has been demonstrated in
numerous preclinical and clinical trials involving cancer patients with
leukemia, melanoma, and glioblastoma.4ꢀ7
Three optimal components, a bifunctional ligand, an antibody,
and a radioisotope, are required for a successful RIT. An effective
bifunctional ligand that can rapidly form a stable complex with
the radionuclide should be employed to minimize toxic side
effects related to biological deposition of the radionuclide if it be-
comes dissociated from the radiolabeled ligandꢀantibody con-
jugate during RIT.8 Research efforts have been directed toward
improving chelation chemistry for RIT. C-DOTA and C-DTPA
(Figure 1) analogues are two bifunctional ligands that are fre-
quently explored for RIT applications.3C-DOTA forms a kine-
tically inert and stable complex with Bi(III); however, the slow
complex formation kinetics render this chelator unacceptable for
We recently reported the synthesis and evaluation of DEPA
(Figure 1, 7-[2-(bis-carboxymethyl-amino)-ethyl]-4,10-bis-
carboxymethyl-1,4,7,10-tetraaza-cyclododec-1-yl-acetic acid).10
DEPA is a decadentate ligand in a hybridized form of the macro-
cyclic DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracar-
boxylic acid) and the acyclic DTPA (diethylenetriamine pentaa-
cetic acid). This novel bimodal ligand DEPA was hypothesized to
rapidly form a stable complex with a metal based on coopera-
tive coordination of the macrocyclic and acyclic binding moieties.
DEPA radiolabeled with 205/6Bi (t1/2 = 15.3 d for 205Bi; t1/2 = 6.24
for 206Bi), aγ-emitting surrogate of 212Bi and 213Bi was indeed found
to be stable in human serum without any loss of the radioactivity for
two weeks and displayed excellent in vivo stability in mice.10
With the promising complexation kinetics and stability data
for DEPA, we sought preparation of an effective bifunctional
Received: December 20, 2010
Revised:
May 4, 2011
Published: May 23, 2011
r
2011 American Chemical Society
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dx.doi.org/10.1021/bc100586y Bioconjugate Chem. 2011, 22, 1128–1135
|

Bioconjugate Chemistry
ARTICLE
Figure 1. Ligands in clinical and preclinical use for RIT.
version of DEPA for RIT. In this paper, we report the synthesis
and evaluation of a bifunctional DEPA analogue, 3p-C-DEPA
(Figure 1, 2-[(carboxymethyl)][5-(4-nitrophenyl-1-[4,7,10-tris-
(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]pentan-2-yl)-
amino]acetic acid), which contains the parent DEPA backbone
and the isothiocyanate (NCS) group for conjugation to an
antibody or a peptide. 3p-C-DEPA was synthesized, character-
ized, and conjugated to trastuzumab. Trastuzumab is a HER2
(human epidermal growth factor receptor 2) targeting antibody
which has been reported to selectively target the HER2 protein
overproduced in various tumors, including 90% of colorectal
carcinomas.13,14 The corresponding 3p-C-DEPA-trastuzumab
conjugate was evaluated for its radiolabeling reaction kinetics
with 205/6Bi. In vitro analysis of the 3p-C-DEPA-trastuzumab
conjugate radiolabeled with 205/6Bi included assessment of its
stability in human serum and retention of reactivity with HER2
using a radioimmunoassay. Finally, the in vivo biodistribution and
tumor uptake of the 205/6Bi-labeled 3p-C-DEPA-trastuzumab
was assessed in mice bearing s.c. tumor (LS-174T) xenografts.
For comparison and as a reference standard, the C-DOTA-
trastuzumab conjugate was evaluated in the same in vitro and
in vivo studies.
noted. Trastuzumab (Herceptin; Genentech, South San
Francisco, California) was obtained through the Veterinary
Resources Program (National Institutes of Health, Bethesda,
MD). 205,6Bi was produced using a CS30 cyclotron (PET
Dept, Clinical Center, NIH) and purified as described
previously.15 C-DOTA was purchased from Macrocyclics
(Dallas, TX).
tert-Butyl-2,20,200-(10-(2-(bis(2-tert-butoxy-2-oxoethyl)-
amino)-5-(4-nitrophenyl)-pentyl)-1,4,7,10-tetraazacyclodo-
decane-1,4,7-triyl)triacetate (4). To a solution of 116 (547.7 mg,
1.06 mmol) and DIPEA (410.9 mg, 3.18 mmol) in CH3CN
(10 mL) was added trisubstituted cyclen 3 (546.6 mg, 1.06 mmol).
The resulting mixture was stirred for 4 weeks at room temperature
while monitoring the reaction progress using TLC. The reaction
mixture containing the starting materials and the product 4 was
concentrated to dryness. The residue was purified via column
chromatography on silica gel (220ꢀ440 mesh) eluted with 3%
CH3OH in CH2Cl2. The fractions containing the product 4 along
with the starting material 3 as impurity were combined. After
evaporating the solvent, ether (20 mL) was added to the residue,
and the starting material 3 formed a slurry which was removed by
filtration. The filtrate containing the product 4 was washed with DI
water (2 ꢁ 10 mL). The ether layer was dried over MgSO4, filtered,
and concentrated in vacuo to provide pure 4 (610 mg, 61%). 1H
NMR (CDCl3, 300 MHz) δ 1.37ꢀ1.45 (m, 45 H), 1.60ꢀ1.78 (m,
2 H), 1.81ꢀ1.95 (m, 1 H), 2.02ꢀ2.19 (m, 1 H), 2.39ꢀ2.50 (m,
3H), 2.56ꢀ2.83 (m, 18 H), 3.21 (s, 4H), 3.26 (s, 2 H), 3.36 (dd, J =
16.9, 21.9 Hz, 4 H), 7.36 (d, J = 8.6 Hz, 2 H), 8.09 (d, J = 8.7 Hz, 2
H); 13C NMR (CDCl3, 300 MHz) δ 27.89 (t), 28.11 (q), 28.22
(q), 30.98 (t), 35.91 (t), 52.04 (t), 52.11 (t), 52.20 (t), 53.09 (t),
53.19 (t), 56.34 (t), 56.44 (t), 58.38 (t), 60.08 (d), 80.41 (s), 80.57
(s), 123.43 (d), 129.30 (d), 146.18 (s), 151.17 (s), 170.98 (s),
171.10 (s), 171.41 (s). HRMS (Positive ion FAB) Calcd for
C49H85N6O12 [M þ H]þ m/z 949.6225. Found: [M þ H]þ m/z
949.6256. Analytical HPLC (tR = 42 min, method 1).
Tri-tert-butyl-10-(2-(tert-butoxycarbonyl)-5-(4-nitrophenyl)-
pentyl)-1,4,7,10-tetraazacyclo-dodecane-1,4,7-tricarboxylate
(7). To a mixture of 5 (1.24 g, 3.85 mmol) and 6 (1.82 g, 3.85
mmol) in 1,2-dichloroethane (30 mL) was added portionwise
sodium triacetoxyborohydride (1.14 g, 5.4 mmol). The mixture
was stirred at room temperature for 14 h. The reaction mixture
was quenched with saturated NaHCO3 (40 mL), and the product
was extracted while washing with CH2Cl2. The combined
organic layers were dried over MgSO4, filtered, and concentrated
in vacuo. The residue was purified by silica gel (60ꢀ230 mesh)
column chromatography eluted with 25% EtOAc in hexanes to
provide 7 (1.90 g, 63%). 1H NMR (CDCl3, 300 MHz) δ 1.03ꢀ
1.51 (m, 36 H), 1.51ꢀ2.01 (m, 4 H), 2.50ꢀ3.99 (m, 21 H), 7.30
(d, J = 8.5 Hz, 2 H), 8.08 (d, J = 8.4 Hz, 2H); 13C NMR (CDCl3,
300 MHz) 24.69 (t), 27.16 (t), 28.57 (q), 28.69 (q), 35.13 (t),
35.68, (t), 36.64 (t), 47.49 (t), 48.46 (d), 50.54 (t), 51.20 (t),
58.18 (t), 58.64 (t), 79.18 (s), 79.28 (s), 79.57 (s), 123.56 (d),
129.19 (d), 146.27 (s), 150.39 (s), 155.14 (s), 155.73 (s),
’ EXPERIMENTAL PROCEDURE
Instruments and Methods. 1H and 13C NMR spectra were
obtained using a Bruker 300 instrument and chemical shifts are
reported in ppm on the δ scale relative to TMS or solvent.
Electrospray iodization (ESI) high-resolution mass spectra
(HRMS) were obtained on JEOL double sector JMS-AX505HA
mass spectrometer (University of Notre Dame, IN). Analytical
HPLC was performed on an Agilent 1200 instrument (Agilent,
Santa Clara, CA) equipped with a diode array detector (λ = 254
and 280 nm), a thermostat set at 35 °C, and a Zorbax Eclipse
XDB-C18 column (4.6 ꢁ 150 mm, 80 Å, Agilent, Santa Clara,
CA). The mobile phase of a binary gradient (0ꢀ100% B/40
min; solvent A, 0.05 M AcOH/Et3N, pH 6.0; solvent B,
CH3OH for method 1) at a flow rate of 1 mL/min was used
for analytical HPLC. Semiprep HPLC was performed on an
Agilent 1200 equipped with a diode array detector (λ = 254 and
280 nm), a thermostat set at 35 °C, and a Zorbax Eclipse XDB-
C18 column (9.4 ꢁ 250 mm, 80 Å). The mobile phase of a
binary gradient (0ꢀ100% B/160 min; solvent A = 0.05 M
AcOH/Et3N, pH 6.0; solvent B = MeOH for method 2) at a
flow rate of 2 mL/min was used for semiprep HPLC. Size-
exclusion HPLC (SE-HPLC) chromatograms were obtained on
Agilent 1200 equipped with a diode array detector and an in-line
IN/US γ-Ram model 2 radiodetector (Tampa, FL), fitted
with Bio-Silect SEC 250ꢀ5 column (Biorad, Hercules, CA)
or a TSKgel G3000PW column (Tosoh Biosep, Montgomery-
ville, PA).
Reagents. All reagents were purchased from Sigma-Aldrich
(St. Louis, MO) and used as received unless otherwise
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Bioconjugate Chemistry
ARTICLE
155.95 (s), 156.72 (s). HRMS (Positive ion ESI) Calcd for
C39H67N6O10 [M þ H]þ m/z 779.4913, Found: [M þ H]þ
m/z 779.4890.
H2O (12 mL) was added dry 10% Pd/C (14.0 mg) under argon.
The reaction mixture was subject to hydrogenation in a hydro-
genation apparatus set at the constant pressure (15 psi) of H2 (g)
for 19 h. The resulting mixture was filtered over a Celite bed and
washed thoroughly with DI H2O. The filtrate was concentrated
5-(4-Nitrophenyl)-1-(1,4,7,10-tetraazacyclododecan-1-yl)-
pentan-2-amine (8). Compound 7 (1.84 g, 2.36 mmol) at
0ꢀ5 °C was treated dropwise with 4 M HCl (g) in 1,4-dioxane
(18 mL) over 20 min. The resulting mixture was warmed to room
temperature and stirred for 22 h. Ether (100 mL) was added and
stirred for 10 min. The resulting mixture was capped and placed
in the freezer for 1 h. The slurry formed was filtered, washed with
ether, and quickly dissolved in deionized (DI) water. The aqu-
eous solution was concentrated in vacuo to provide 8 as an acidic
salt (1.17 g, 89%). 1H NMR (D2O, 300 MHz) δ 1.50ꢀ1.71 (m,
4 H), 2.50ꢀ2.80 (m, 6 H), 2.82ꢀ3.40 (m, 15 H), 7.30 (d, J = 8.7
Hz, 2 H), 8.00 (d, J = 8.7 Hz, 2 H); 13C NMR (D2O, 300 MHz) δ
25.65 (t), 30.63 (t), 34.46 (t), 41.30 (t), 42.13 (t), 44.21 (t),
48.56 (t), 48.57 (d), 56.81 (t), 123.66 (d), 129.40 (d), 145.90 (s),
150.21 (s). HRMS (Positive ion ESI) Calcd for C19H35N6O2
[M þ H]þ m/z 379.2816. Found: [M þ H]þ m/z 379.2804.
A solution of the acidic salt 8 (670 mg, 1.7 mmol) in DI water
(5 mL) was neutralized using 0.5 M NaOH. The aqueous layer
was then extracted with CHCl3 (2 ꢁ 25 mL). The aqueous layer
was further adjusted to pH 10 and re-extracted with CHCl3 (2 ꢁ
25 mL). The organic layers extracted from both neutral (pH 7)
and basic (pH 10) solutions were combined, dried over MgSO4,
filtered, and concentrated in vacuo to provide free amine 8 (452
mg, 100%). 1H NMR (CDCl3, 300 MHz) δ 1.20ꢀ1.49 (m, 2 H),
1.60ꢀ1.89(m, 2H), 2.15ꢀ3.00(m, 21H), 7.33(d, J= 8.6 Hz, 2 H),
8.14 (d, J = 8.6 Hz, 2 H); 13C NMR (CDCl3, 300 MHz) δ 27.26 (t),
35.00 (t), 35.64 (t), 45.16 (t), 46.01 (t), 46.95 (t), 48.71 (d), 52.10
(t), 62.56 (t), 123.27 (d), 129.00 (d), 145.94 (s), 150.24 (s).
Synthesis of 4 from Compound 8. To a solution of 8 (170.3
mg, 0.45 mmol) in CH3CN (5 mL) was added tert-butyl
bromoacetate (438.9 mg, 2.25 mmol) and K2CO3 (311.0 mg,
2.25 mmol). The resulting mixture was heated at 65 °C and
stirred for 13 h while monitoring the reaction progress by
analytical HPLC. The reaction mixture was cooled to room
temperature, and the solvent was evaporated. The residue was
purified by semi-prep HPLC (method 2, 138ꢀ143 min) to afford
4 (37 mg, 9%).
1
in vacuo to provide a light yellow solid 10 (44 mg, 100%). H
NMR (D2O, 300 MHz) δ 1.22ꢀ1.38 (m, 1H), 1.43ꢀ1.68 (m,
3H), 2.45ꢀ2.63 (m, 2H), 2.81ꢀ3.70 (m, 27H), 3.80ꢀ3.98 (m,
2H), 7.17 (d, J = 8.5 Hz, 2H), 7.24 (d, J = 8.5 Hz, 2H); 13C NMR
(D2O, 300 MHz) δ 27.04 (t), 27.46 (t), 34.18 (t), 48.50 (t),
48.98 (t), 50.28 (t), 51.12 (t), 52.50 (t), 53.69 (t), 54.50 (t),
55.49 (t), 59.40 (d), 123.01 (d), 127.55 (s), 130.10 (d), 143.28
(s), 169.50 (s), 173.13 (s), 174.22 (s); HRMS (positive ion ESI)
Calcd for C29H47N6O10 [M þ H]þ m/z 639.3348. Found: [M þ
H]þ m/z 639.3342.
2,20,200-(10-(2-(Bis(carboxymethyl)amino)-5-(4-isothiocya-
natophenyl)pentyl)-1,4,7,10-tetraazacyclododecane-1,4,7-
triyl)triacetic Acid (11). To a solution of 10 (7.0 mg, 8.2 μmol)
in water (0.1 mL) was added CSCl2 (7.0 μL, 7.0 μmol) in CHCl3.
The resulting mixture was stirred at room temperature for 2 h.
The aqueous layer was isolated and concentrated in vacuo to give
pure 3p-C-DEPA-NCS 11 as a solid (8.0 mg, 100%). 1H NMR
(D2O, 300 MHz) δ 1.23ꢀ1.72 (m, 4H), 2.40ꢀ2.58 (m, 2H),
2.80ꢀ3.98 (m, 29H), 7.13 (s, 4H). HRMS (Positive ion FAB)
Calcd for C30H43N6O12S [M þ H]þ m/z 679.2761. Found: [M þ
H]þ m/z 679.2747.
Conjugation of 3p-C-DEPA-NCS to Trastuzumab. All ab-
sorbance measurements were obtained on an Agilent 8453 diode
array spectrophotometer equipped with a 8-cell transport system
(designed for 1 cm cells). Metal-free stock solutions of all buffers
were prepared using Chelex-100 resin (200ꢀ400 mesh, Bio-Rad
Lab, Hercules, CA, Cat no 142ꢀ2842). Chelex resin (2.5 g) was
added into the buffer solution (250 mL) and the mixture was
shaken overnight in a shaker and filtered through a Corning filter
system (Cat no 430513, pore size 0.2 μm). Disposable PD-10
Sephadex G-25 M columns (GE Healthcare, #17ꢀ0851ꢀ01)
were rinsed with 25 mL of the appropriate buffer prior to
addition of antibody or its ligand conjugates. Amicon centricon
C-50 (50 000 MWCO) centrifugal filter devices (Millipore, Cat
no UFC805008) were used for purification of trastuzumab
conjugate (Bedford, MA). The initial concentration of trastuzu-
mab was determined by the UV/vis spectroscopic method.
Phosphate buffered saline (PBS, 1ꢁ, 11.9 mM Phosphates,
137 mM NaCl, and 2.7 mM KCl, pH 7.4) was purchased from
Fisher and used as received. Conjugation buffer (50 mM HEPES,
150 mM NaCl, pH 8.6) were prepared as 1ꢁ solutions, chelexed,
and filtered through the Corning filter. Trastuzumab (6.67 mg)
was diluted to 1.6 mL using conjugation buffer (1:0.6), and the
resulting solution was added to a PD-10 column. Conjugation
buffer (10 mL) was added to the PD-10 column to exchange the
buffer solution of the antibody and collected in a sterile test tube
and checked for the presence of trastuzumab via analysis of the
UV/vis spectrum at 280 nm. To a sterile test tube containing the
recovered trastuzumab (6.14 mg) was added a 10-fold excess of
3p-C-DEPA-NCS (39.6 μL, 10 mM). The resulting solution was
gently agitated for 16 h at room temperature and placed on a
Centricon C-50 membrane and spun down to reduce volume.
PBS (3 ꢁ 2 mL) was added to the remaining solution of the 3p-
C-DEPA-trastuzumab conjugate, followed by centrifugation in
order to remove unreacted ligand. The volume of purified
conjugate antibody was brought to 1.0 mL with PBS. To measure
[trastuzumab] in the 3p-C-DEPA-trastuzumab conjugate, a
2-[(Carboxymethyl)][5-(4-nitrophenyl-1-[4,7,10-tris(carboxy-
methyl)-1,4,7,10-tetraazacyclo-dodecan-1-yl]pentan-2-yl)-
amino]acetic Acid (9). Compound 4 (77.0 mg, 0.08 mmol) at
0ꢀ5 °C was treated dropwise with 4 M HCl (g) in 1,4-dioxane
(15 mL) over 20 min. The resulting mixture was allowed to warm
to room temperature for 22 h. Ether (∼20 mL) was added and
continuously stirred for 10 min. The resulting mixture was
capped and placed in the freezer for 1 h. The solid formed was
filtered, washed with ether, and quickly dissolved in DI water.
Evaporation of the aqueous solution gave 9 (68.0 mg, 97%) as an
1
off-white solid. H NMR (D2O, 300 MHz) δ 1.26ꢀ1.40 (m,
1H), 1.48ꢀ1.70 (m, 3H), 2.52ꢀ2.78 (m, 2H), 2.90ꢀ3.70 (m,
27H), 3.75ꢀ3.98 (m, 2H), 7.35 (d, J = 8.3 Hz, 2H), 8.06 (d, J =
8.2 Hz, 2H); 13C NMR (D2O, 300 MHz) δ 27.08 (t), 34.64 (t),
48.45 (t), 48.82 (t), 50.22 (t), 51.09 (t), 52.40 (t), 53.58 (t),
54.28 (t), 55.46 (t), 59.43 (d), 123.55 (d), 129.40 (d), 145.71 (s),
150.54 (s), 169.54 (s), 172.97 (s), 173.93 (s); HRMS (Positive
ion FAB) Calcd for C29H45N6O12 [M þ H]þ m/z 669.3095.
Found: [M þ H]þ m/z 669.3086.
2,20,200-(10-(5-(4-Aminophenyl)-2-(bis(carboxymethyl)-
amino)pentyl)-1,4,7,10-tetraaza-cyclododecane-1,4,7-triyl)-
triacetic acid (10). To a solution of 9 (46.0 mg, 68.8 μmol) in DI
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ARTICLE
UV/vis spectrometer was zeroed against a cuvette filled with
2.0 mL of PBS with a window open from 190 to 1100 nm. A
50 μL portion of PBS was removed and discarded, 50 μL of the
3p-C-DEPA-trastuzumab conjugate solution was added, and
absorbance at 280 nm was noted. Beer’s Law was used to
calculate [trastuzumab] in the conjugate with molar absorptivity
of 1.42. After centrifugation, 2.98 mg (2.02 ꢁ 10ꢀ5 M, 49.0%) of
the trastuzumab remained.
SE-HPLC (Biorad, Bio-Silect SEC 250ꢀ5 column, 7.8 ꢁ 30 cm,
eluent, PBS; flow rate, 1 mL/min). A solution of radiolabeled
mixture (10 μL for C-DOTA-trastuzumab and 7 μL for 3p-C-
DEPA-trastuzumab) was withdrawn at the designated time
points (1 min, 5 min, 10 min, 20 min, 30 min, and 60 min),
and DTPA solution (10 mM, 1 or 1.4 μL) was added to the
mixture, and the resulting mixture was left for at least 20 min.
HPLC samples were prepared and evaluated by SE-HPLC. Peaks
for bound and unbound radioisotope appeared around 8.3 and
11 min, respectively.
Spectroscopic Determination of Ligand to Protein (L/P)
Ratio. A stock solution of the Cu(II)-AAIII reagent was prepared
in 0.15 M NH4OAc, pH 7.0 by adding an aliquot of copper
atomic absorption solution (1.55 ꢁ 10ꢀ2 M) into a 10 μM
solution of AAIII to afford a 5 μM solution of Cu(II).17 This
solution was stored in the dark to avoid degradation over time. A
UV/vis spectrometer was zeroed against eight well-dried blank
cuvettes with a window open from 190 to 1100 nm. A cuvette was
filled with AAIII solution (2 mL), and the other seven cuvettes
with the Cu(II)-AAIII solution (2 mL). Cu(II)-AAIII solution
(50 μL) in the seven cuvettes was removed and discarded. Milli-
Q water (50 μL) was added to the second cuvette, and one to five
10 μL additions of 3p-C-DEPA (0.1 mM) were added to the five
cuvettes to give a series of five different concentrations (2 mL
total volume). The solutions in the third to the seventh cuvette
were diluted to 2.0 mL by adding an aliquot of Milli-Q water. 3p-
C-DEPA-trastuzumab conjugate (50 μL) was added to the eighth
cuvette containing Cu(II)-AAIII reagent (1950 μL). After addi-
tion of 3p-C-DEPA-trastuzumab conjugate to the Cu(II)-AAIII
solution, the resulting solution was equilibrated for 10 min. The
absorbance of the resulting solution at 610 nm was monitored
every 30 s over 6 min. The average of the absorbance of each
solution was calculated, and the absorbance data from the
Cu(II)-AAIII solutions containing six different concentrations
were used to construct a calibration plot of A610 versus [3p-C-
DEPA] by the equation, Y = 0.0663 ꢀ (1.5284 ꢁ 104)[3p-C-
DEPA] (R2 = 0.9969), wherein Y = A610 nm. The concentration of
3p-C-DEPA in the 3p-C-DEPA-trastuzumab conjugate was
calculated (3.98 ꢁ 10ꢀ5 M). The L/P ratio of the 3p-C-DEPA-
trastuzumab conjugate ([3.98 ꢁ 10ꢀ5 M]/[2.02 ꢁ 10ꢀ5 M]) was
measured to be 1.97.
Radiolabeling of 3p-C-DEPA-trastuzumab or C-DOTA-
Trastuzumab Conjugates with 205/6Bi. All HCl solutions were
prepared from ultrapure HCl (Fisher, Cat no A466ꢀ500). For
metal-free radiolabeling, plasticware including pipet tips, tubes,
and caps was soaked in 0.1 M HCl overnight, washed thoroughly
with Milli-Q (18.2 MΩ) water, and air-dried overnight. Ultra-
pure NH4OAc (Aldrich, #372331) was purchased from Aldrich
and used to prepare all NH4OAc buffer solutions (0.1 M, pH 7).
The buffer solutions were treated with Chelex-100 resin (Biorad,
#142ꢀ2842, 1 g/100 mL buffer solution), shaken overnight at
room temperature, and filtered through 0.22 μm filter (Corning,
#430320) prior to use. To a buffer solution (120 μL, pH 7) in a
capped microcentrifuge tube (1.5 mL, Fisher Scientific #05ꢀ
408ꢀ129) was sequentially added a solution of C-DOTA-
trastuzumab (30 μg) in PBS (13.5 μL) and 205/6Bi (0.1 M HI,
60.5 μCi, 12.7 μL) or 3p-C-DEPA-trastuzumab (30 μg) in PBS
(9.6 μL) and 205/6Bi (0.1 M HI, 60.5 μCi, 12.7 μL). The final
volume of the resulting solution was 146.2 and 142.3 μL for C-
DOTA-trastuzumab and 3p-C-DEPA-trastuzumab, respectively,
and the pH of the resulting reaction mixture was 5.5 for both
conjugates. The reaction mixture was agitated on the thermo-
mixer (Eppendorf, #022670549) set at 1000 rpm at room
temperature for 1 h. The labeling efficiency was determined by
In Vitro Stability of 205/6Bi-3p-C-DEPA-Trastuzumab Con-
jugate. 205/6Bi-3p-C-DEPA-trastuzumab was prepared by reac-
tion of 3p-C-DEPA-trastuzumab with 205/6Bi at either RT or
37 °C (5.0 M NH4OAc, pH 5.5). The complex formed was
purified from 205/6Bi by gel filtration chromatography using PD-
10 column (Disposable PD-10 Sephadex G-25 M columns, GE
Healthcare, Cat no 17ꢀ0851ꢀ01) eluted with PBS (pH 7.4,
Fisher Scientific, Cat no BP2438ꢀ4). The fractions containing
205/6Bi-3p-C-DEPA-trastuzumab (700 μL, 22 μCi) were com-
bined and added into human serum (700 μL, Gemini Biopro-
ducts, #100110) in a microcentrifuge tube (Fisher Scientific,
#05ꢀ408ꢀ129). The stability of the purified 205/6Bi-3p-C-
DEPA-trastuzumab was evaluated for 4 days. The serum stability
of the radiolabeled complexes was assessed by measuring the
transfer of the radionuclide from each complex to serum proteins
using SE-HPLC (TSKgel G3000PW column, 7.5 ꢁ 30 cm,
eluent, PBS; flow rate, 1 mL/min). A solution of the radiolabeled
complex in serum (20ꢀ80 μL) was withdrawn at the designated
time point, treated with DTPA (5 mM, 1.0 μL), incubated at
room temperature for 20 min, and then diluted with PBS (pH
7.4, 50ꢀ100 μL) prior to SE-HPLC.
Preparation of 205/6Bi-3p-C-DEPA-trastuzumab and
205/6Bi-C-DOTA-trastuzumab for Immunoreactivity and In
Vivo Studies. For use in the in vitro binding and in vivo
biodistribution and tumor targeting studies, 205/6Bi-3p-C-
DEPA-trastuzumab and 205/6Bi-C-DOTA-trastuzumab with high
specific activity were prepared. In this instance, two aliquots of
205/6Bi solution (100 μL in 0.1 M HI, 1.1 mCi each) were each
neutralized to pH 5.5 with NH4OAc solution (10 μL, 5 M, pH 7).
An aliquot of each immunoconjugate containing 50 μg of protein
in PBS was added to one or the other 205/6Bi solution. The
mixtures were vortexed briefly and incubated at 37 °C. After
30 min, EDTA solution (5 μL, 0.1M) was added to each reaction,
vortexed briefly, and further incubated for 5 min. The products
were purified by gel filtration chromatography using disposable
PD-10 columns. The labeling efficiencies were 85.8% and 67.3%
for 205/6Bi-3p-C-DEPA-trastuzumab and 205/6Bi-C-DOTA-tras-
tuzumab, respectively, as determined by the labeled products
collected from the PD-10 columns. The purity of the labeled
products as determined by SE-HPLC (TSKgel G3000PW col-
umn, PBS eluate at 0.5 mL/min) were >98% for each product.
The specific activities were 16 mCi/mg and 13.6 mCi/mg
for205/6Bi-3p-C-DEPA-trastuzumab and 205/6Bi-C-DOTA-tras-
tuzumab, respectively.
Radioimmunoassay. The immunoreactivity of the 205/6Bi-
labeled trastuzumab conjugates was assessed in a radioimmu-
noassay using purified recombinant human Erb2/Fc chimera
(rhErb2/Fc). Briefly, 50 ng aliquots of the rhErb2/Fc in PBS
with Mg2þ and Ca2þ(50 μL) were added to each well of a 96-well
plate and allowed to adsorb to the well at 4 °C. Following an
overnight incubation, the solution was removed and 100 μL of
1% BSA in PBS (BSA/PBS) was added to each well and
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Scheme 1. Synthesis of Precursor Molecule 4 to 3p-C-DEPA
incubated at room temperature for 30ꢀ60 min. Serial dilutions
of 205/6Bi-trastuzumab conjugate (∼300 000 to ∼12 500 cpm in
50 μL of BSA/PBS) were added to the wells in duplicate,
covered, and incubated at 37 °C for 4 h. The radiolabeled
antibody was then removed and the wells were washed three
times with PBS. The radioactivity was removed from the wells
with a solution of 0.2 M NaOH with 1 mM EDTA (100 μL),
adsorbing the solution to cotton filters, and transferring the filters
to 12 ꢁ 75 mm polypropylene tubes. The radioactivity was
measured in a γ-scintillation counter (WizardOne, Perkin-Elmer,
Shelton, CT), and the percentage binding calculated for each
dilution. The values presented are an average of the percent
bound for the serial dilutions. The specificity of the radiolabeled
trastuzumab was confirmed by incubating one set of wells with
radiolabeled trastuzumab and 10 μg of unlabeled trastuzumab.
In Vivo Biodistribution and Tumor Targeting Studies. In
vivo studies were conducted using the human colon carcinoma cell
line, LS-174T (kindly provided by Dr. J. Greiner, NCI). The LS-
174T cell linewas grown in Dulbecco’s minimum essential medium
(DMEM) supplemented with 10 mM glutamine along with 10%
FetalPlex (Gemini Bio-Products, Woodland, CA) and 1 mM
nonessential amino acids. Media and supplements were obtained
from Lonza (Walkersville, MD). All in vivo studies were performed
using 4ꢀ6 week old female athymic (nu/nu) mice (Charles River
Laboratories, Wilmington, MA). All animal protocols were ap-
proved by the National Cancer Institute Animal Care and Use
Committee. Mice were injected subcutaneously (s.c.) in the right
rear leg with 2 ꢁ 106 LS-174T cells in media (200 μL) with 20%
Matrigel (BD Biosciences, San Jose, CA). Mice were utilized in
studies when the tumor xenografts maximal diameter measured
0.4ꢀ0.6 cm. Mice (n = 4 per time point) were injected intrave-
nously (i.v.) with the 205/6Bi-labeled trastuzumab conjugates (∼7.5
μCi on 0.6 μg) and sacrificed by exsanguination at 2, 6, and 24 h.
The blood, tumor, and major organs were collected, wet-weighed,
and counted in a γ-scintillation counter. The percent injected dose
per gram (%ID/g) and standard deviation were calculated.
N,N-bisubstituted β-amino bromide 1 produced aziridinium ion 2
which was then reacted with trisubstituted cyclen 3 (1,4,7,10-
tetraazacyclododecane). Regiospecific ring-opening of 2 by nucleo-
philic attack of bulky cyclen analogue 310 occurred at the less
hindered methylene carbon in 2 to provide 4 as the exclusive
product (61%). The reaction was carried out at room temperature,
although it proceeds extremely slowly at this mild condition
(4 weeks). An intramolecular rearrangement product of 1 was
obtained when the reaction was under reflux. The desired coupling
product 4 was isolated via column chromatography and character-
1
ized by H and 13C NMR, analytical HPLC, and HRMS. To
confirm the regiochemistry observed in the nucelophilic ring-
opening of the aziridinium ion 2 at the methylene carbon,
compound 4 was separately prepared via another synthetic route
starting from 5 as shown in Scheme 1. Reductive amination of 5
with 6 using sodium triacetoxyborohydride provided 7 which was
subsequently treated with HCl (g) in 1,4-dioxane to afford 8. A
base-promoted reaction of 8 with tert-butyl bromoacetate pro-
duced compound 4, and comparison of the spectroscopic data of 4
that was separately produced via the two synthetic routes con-
firmed regiochemistry in the ring-opening of 2. Synthesis of the
bifunctional ligands 3p-C-DEPA (9) and 3p-C-DEPA-NCS (11)
is shown in Scheme 2. The tert-butyl groups in 4 was removed by
the reaction of 4 with 4 M HCl (g) in 1,4-dioxane to provide 3p-C-
DEPA (9). The nitro group in 9 was transformed into the amino
group by hydrogenolysis of 9 on Pd/C to provide 10 which was
subsequently reacted with thiophosgene (g) in CHCl3 to the
desired bifunctional ligand 3p-C-DEPA-NCS (11) containing the
isothiocyanate group for conjugation to an antibody.
3p-C-DEPA-NCS was conjugated to trastuzumab, and the
concentration of trastuzumab in 3p-C-DEPA-trastuzumab con-
jugate was quantified by UV spectroscopic assay.18 The Cu(II)-
AAIII based UVꢀvis spectrophotometric assay was used for the
determination of the number of 3p-C-DEPA ligand linked to
trastuzumab (L/P ratio).17 The ligand to protein (L/P) ratio for
3p-C-DEPA-trastuzumab conjugate was measured to be 1.97.
For comparison, C-DOTA-NCS (Macrocyclics, TX) was con-
jugated to trastuzumab as reported previously.19
’ RESULTS AND DISCUSSION
The synthesis of the new bifunctional ligand 3p-C-DEPA is
outlined in Scheme 1. An intramolecular substitution reaction of
The purified 3p-C-DEPA trastuzumab conjugates (30ꢀ50 μg)
in 0.25 M NH4OAc buffer solution at pH 5.5 was radiolabeled
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Bioconjugate Chemistry
ARTICLE
Scheme 2. Synthesis of 3p-C-DEPA (9) and 3p-C-DEPA-NCS (11)
Table 1. Radiolabeling Efficiency (%) of Bifunctional Ligand-
Trastuzumab Conjugates with 205/6Bi (0.25 M NH4OAc,
pH 5.5, RT)*
time (min)
3p-C-DEPA
C-DOTA
1
93.6 ( 0.4
94.7 ( 0.9
95.0 ( 0.4
94.1 ( 0.6
94.4 ( 1.6
94.5 ( 0.5
8.4 ( 1.9
17.2 ( 4.3
28.7 ( 4.5
38.0 ( 4.9
49.7 ( 9.0
60.2 ( 8.0
5
10
20
30
60
* Radiolabeling efficiency (mean ( standard deviation) was measured in
triplicate.
Figure 2. Biodistribution of 205/6Bi-3p-C-DEPA-trastuzumab conju-
gate in athymic mice bearing s.c. LS-174T tumors.
with 205/6Bi (0.1 M HI) (60 μCi) at room temperature (RT).
During the reaction time (1 h), the radiolabeling kinetics was
determined by taking aliquots of the reaction mixture at 6 time
points (1 min, 5 min, 10 min, 20 min, 30 min, and 60 min). The
components were analyzed using SE-HPLC after challenging the
reaction mixture with 10 mM DTPA, and the radiolabeling
efficiency (%) was determined (Table 1 and the Supporting
Information). The data indicate that 205/6Bi labeling of 3p-C-
DEPA-trastuzumab conjugate were extremely rapid (1 min,
>93%) at RT. As expected, radiolabeling of C-DOTA-trastuzu-
mab conjugate with 205/6Bi was slow and incomplete at 24 h
(60.2 ( 8.0%). The 205/6Bi-3p-C-DEPA conjugate was further
evaluated for in vitro serum stability. The 205/6Bi-3p-C-DEPA
conjugate was freshly prepared, purified, and incubated in human
serum at 37 °C. At each time point (0 h, 1, 2, 3, 4 d), aliquots of the
reaction mixture were analyzed using SE-HPLC after challenging
the reaction mixture with 5 mM DTPA. 205/6Bi-3p-C-DEPA-
trastuzumab was found to be stable in human serum without release
of the radioactivity for at least 4 days (the Supporting Information).
The 205/6Bi-3p-C-DEPA-trastuzumab and 205/6Bi-C-DOTA-
trastuzumab conjugates were independently prepared at 37 °C
and pH 5.5 for specific activity and immunoreactivity evaluations
prior to performing comparative in vivo biodistribution studies.
The respective specific activities of 16.0 mCi/mg and 13.6 mCi/
mg were measured for 205/6Bi-3p-C-DEPA and 205/6Bi-C-DOTA.
The radiolabeling yields of 85.8% and 67.3% were determined for 205/
6Bi-3p-C-DEPA and 205/6Bi-C-DOTA, respectively. Immunoreactiv-
ity of 205/6Bi-C-DOTA-trastuzumab and 205/6Bi-3p-C-DEPA-trastu-
zumab was assessed in a radioimmunoassay using recombinant
human ErbB2/Fc Chimera (R&D Systems, Minneapolis,
NM). The respective specific binding of 82.6% and 83.2% was
measured for 205/6Bi-3p-C-DEPA-trastuzumab and 205/6Bi-C-
DOTA-trastuzumab. In the presence of excess trastuzumab, 205/
6Bi-3p-C-DEPA-trastuzumab and 205/6Bi-C-DOTA-trastuzu-
mab exhibited the same nonspecific binding (7.6%).
The in vivo stability and tumor targeting of 205/6Bi-3p-C-
DEPAꢀtrastuzumabwas evaluated by performingbiodistribution
studies in LS174T-bearing athymic mice (i.v. injection, n = 4).
205/6Bi-C-DOTA-trastuzumab conjugate was evaluated for com-
parison. The mice were euthanized at 2 h, 6 h, and 24 h. Tumors,
selected organs, and the blood were harvested, wet-weighed, and
the radioactivity measured in a γ-counter (Figures 2 and 3). The
highest tumor uptake was observed at 24 h for 205/6Bi-3p-C-
DEPA-trastuzumab and 205/6Bi-C-DOTA-trastuzumab (27.3%
and 25.8%, respectively). Among the organs, the highest accre-
tion of radioactivity was observed in the liver: 7.73% for 205/6Bi-
3p-C-DEPA-trastuzumab (6 h) and 8.02% for 205/6Bi-C-DOTA-
trastuzumab (24 h). Both of the 205/6Bi-labeled conjugates
exhibited the highest radioactivity level in the blood at 2 h which
decreased over time. Renal and liver uptake of 205/6Bi-3p-C-
DEPA-trastuzumab peaked at 6 h and decreased by 24 h. 205/6Bi-
3p-C-DEPA-trastuzumab and 205/6Bi-C-DOTA-trastuzumab re-
sulted in the respective radioactivity level of 6.27% and 5.52% in
the kidney at 24 h. 205/6Bi-C-DOTA-trastuzumab exhibited an
upward trend in the accumulation of radioactivity in the liver
over the study period. Femur uptake of 205/6Bi-3p-C-DEPA-
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Bioconjugate Chemistry
ARTICLE
’ ASSOCIATED CONTENT
S
Supporting Information. HPLC chromatograms for as-
b
sessment of radiolabeling reaction kinetics and serum stability
and in vivo biodistribution data. This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: Chong@iit.edu, Fax: 312-567-3494. Mailing address:
3101 S. Dearborn St, LS 182, Chemistry Division, Biological,
Chemical, and Physical Science Department, Illinois Institute of
Technology, Chicago, IL, 60616.
Figure 3. Biodistribution of 205/6Bi-C-DOTA-trastuzumab conjugate in
athymic mice bearing s.c. LS-174T tumors.
’ ACKNOWLEDGMENT
We acknowledge financial support from the National Insti-
tutes of Health (R01CA112503). This work was partly sup-
ported by intramural research program of NIH. Hyun A Song is
the recipient of 2009 Illinois Institute of Technology Kilpatrick
Fellowship.
trastuzumab peaked at 2 h (∼3%) and decreased over the rest of
the time points, while 205/6Bi-C-DOTA-trastuzumab displayed
increased femur uptake from 2.00% at 6 h to 2.49% at 24 h. The
respective tumor-to-blood ratio of 205/6Bi-3p-C-DEPA and 205/
6Bi-C-DOTA at 24 h was 2.26 and 2.15. Both radioimmuno-
conjugates exhibited low blood to tissue ratios (<0.7) in all
tissues and higher radioactivity level in tumor as compared to the
normal organs over the time points.
In summary, the in vitro and in vivo experimental results
indicate that the new ligand 3p-C-DEPA possesses great poten-
tial for RIT of 212Bi and 213Bi. Given complete and ongoing
numerous preclinical and clinical studies of C-DOTA for alpha
RIT, this new ligand deserves more extensive evaluations as a
chelator of other potent r-emitters with a longer half-life
including 212Pb (t1/2 = 10.64 h) and 225Ac (t1/2 = 10 d). 212Pb
is investigated as an in vivo generator of 212Bi to abrogate the
short half-life of the daughter isotope.20 225Ac with the advantage
of multiple r emissions is a potent r emitter and known to
induce apoptosis even with a single particle traversal of the cell.21
Although encouraging results on the therapeutic efficacy of C-
DOTA-antibody conjugates radiolabeled with 212Pb or 225Ac as
demonstrated in the preclinical studies using tumor-bearing mice
was reported,22ꢀ25 C-DOTA-antibody conjugates radiolabeled
with 203Pb or 225Ac were less stable in serum.21,26,27 The
decadentate chelator 3p-C-DEPA possesses a larger macrocyclic
backbone than C-DOTA and may be a suitable ligand for
effectively sequestering 212Pb and 225Ac. 3p-C-DEPA will be
further evaluated for various alpha RIT applications.
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step, regiospecific ring-opening of aziridinium ion by nucleophi-
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