Synthesis and preclinical evaluation of bifunctional ligands for improved chelation chemistry of 90Y and 177Lu for targeted radioimmunotherapy

Article abstract of DOI:10.1021/bc200696b

We report a practical and high-yield synthesis of a bimodal bifunctional ligand 3p-C-NETA-NCS containing the isothiocyanate group for conjugation to a tumor targeting antibody. 3p-C-NETA-NCS was conjugated to a tumor-targeting antibody, trastuzumab, and the corresponding 3p-C-NETA-trastuzumab conjugate was evaluated and compared to trastuzumab conjugates of the known bifunctional ligands C-DOTA, C-DTPA, and 3p-C-DEPA for radiolabeling kinetics with ⁹⁰Y and ¹⁷⁷Lu. 3p-C-NETA-trastuzumab conjugate exhibited extremely rapid complexation kinetics with ⁹⁰Y and ¹⁷⁷Lu. ⁹⁰Y-3p-C-NETA-trastuzumab and ¹⁷⁷Lu-3p-C-NETA-trastuzumab conjugates were stable in human serum for 2 weeks. A pilot biodistribution study was conducted to evaluate in vivo stability and tumor targeting of ¹⁷⁷Lu-radiolabeled trastuzumab conjugate using nude mice bearing ZR-75-1 human breast cancer. ¹⁷⁷Lu-3p-C-NETA-trastuzumab conjugate displayed low radioactivity level at blood (1.6%), low organ uptake (<2.2%), and high tumor-to-blood ratio (6.4) at 120 h. 3p-C-NETA possesses favorable in vitro and in vivo profiles and is an excellent bifunctional chelator that can be used for targeted RIT applications using ⁹⁰Y and ¹⁷⁷Lu and has the potential to replace DOTA and DTPA analogues in current clinical use.

Full text of DOI:10.1021/bc200696b

Article
pubs.acs.org/bc
Synthesis and Preclinical Evaluation of Bifunctional Ligands for
90
177
Improved Chelation Chemistry of Y and Lu for Targeted
Radioimmunotherapy
Chi Soo Kang,† Xiang Sun,† Fang Jia,‡ Hyun A Song,† Yunwei Chen,† Michael Lewis,‡
and Hyun-Soon Chong*,†
†Chemistry Division, Biological and Chemical Sciences Department, Illinois Institute of Technology, Chicago, Illinois, United States
‡Department of Veterinary Medicine and Surgery, Department of Radiology, Nuclear Science and Engineering Institute, University of
Missouri-Columbia, Columbia, Missouri, United States
S
* Supporting Information
ABSTRACT: We report a practical and high-yield synthesis
of a bimodal bifunctional ligand 3p-C-NETA-NCS containing
the isothiocyanate group for conjugation to a tumor targeting
antibody. 3p-C-NETA-NCS was conjugated to a tumor-
targeting antibody, trastuzumab, and the corresponding 3p-
C-NETA-trastuzumab conjugate was evaluated and compared
to trastuzumab conjugates of the known bifunctional ligands
C-DOTA, C-DTPA, and 3p-C-DEPA for radiolabeling kinetics
with 90Y and 177Lu. 3p-C-NETA-trastuzumab conjugate
exhibited extremely rapid complexation kinetics with 90Y and 177Lu. 90Y-3p-C-NETA-trastuzumab and 177Lu-3p-C-NETA-
trastuzumab conjugates were stable in human serum for 2 weeks. A pilot biodistribution study was conducted to evaluate in vivo
stability and tumor targeting of 177Lu-radiolabeled trastuzumab conjugate using nude mice bearing ZR-75-1 human breast cancer.
177Lu-3p-C-NETA-trastuzumab conjugate displayed low radioactivity level at blood (1.6%), low organ uptake (<2.2%), and high
tumor-to-blood ratio (6.4) at 120 h. 3p-C-NETA possesses favorable in vitro and in vivo profiles and is an excellent bifunctional
chelator that can be used for targeted RIT applications using 90Y and 177Lu and has the potential to replace DOTA and DTPA
analogues in current clinical use.
the slow formation kinetics and low radiolabeling efficiency of
C-DOTA in binding 90Y and 177Lu remain limitations to broad
RIT applications of the macrocyclic chelate.12,13 Rapid
radiolabeling kinetics of a bifunctional ligand conjugated to
an antibody is required to minimize radiolytic damage to
protein conjugates that can occur during radiolabeling of an
antibody conjugate with high specific activity for therapeutic
applications.14 Development of superior chelation chemistry
that allows for scale-up production of stable 90Y- or 177Lu-
radiolabeled radioimmunoconjugates under mild and practical
radiolabeling conditions is necessary for effective and safe RIT
applications of 90Y and 177Lu.
We have synthesized the bimodal ligands in the series of
NETA and DEPA (Figure 1) that possess both macrocyclic and
acyclic moieties for cooperative metal binding.15−18 The acyclic
pendant donor groups in NETA or DEPA are proposed to
rapidly initiate coordination to the metal, as in the case of
DTPA, while the macrocyclic component, a partial NOTA
(Figure 1) or DOTA backbone, is expected to tightly wrap
around the metal ion trapped by the acyclic binding groups and
INTRODUCTION
■
Radioimmunotherapy (RIT) is a potent therapeutic technique
applicable to numerous cancers.1,2 Two RIT drugs, Zevalin and
Bexxar, are clinically available for treatment of B-cell non-
Hodgkin’s lymphoma (NHL).3−5 The RIT system generally
consists of three components, a radionuclide, a tumor-targeting
antibody or peptide, and a bifunctional ligand.2,6 β-emitting
radionuclides, 90Y and 177Lu, have been extensively explored for
RIT. Highly energetic 90Y (t1/2 = 64.1 h, Emax = 2.3 MeV) has
the advantage of relatively long-range tissue penetration and
homogeneous dose distribution.2,6 Less energetic 177Lu (t1/2
=
6.7 d, Emax = 0.5 MeV) has a shorter penetration range relative
to 90Y and is proposed to provide more selective tumor
targeting and resultant lower normal tissue damage and better
tolerance by patients.2 Its longer half-life is sufficient for
preparation and shipment of radioimmunoconjugate and allows
for maximal delivery of dose to tumors.2
C-DOTA (Figure 1) forms a stable complex with lanthanides
including 90Y and 177Lu.7,8 The enhanced stability of the metal
complexes of DOTA compared to acyclic ligand such as C-
DTPA (Figure 1) is attributed to the macrocyclic effect and
increased preorganization.9,10 The therapeutic efficacy of 90Y-
or 177Lu-labeled DOTA−antibody conjugates has been
demonstrated in preclinical and clinical trials.2,11,12 However,
Received: December 31, 2011
Revised: June 7, 2012
Published: August 10, 2012
© 2012 American Chemical Society
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Figure 1. Bifunctional ligands in preclinical and clinical evaluation.
thereby achieve maximum complex stability by saturating the
metal coordination sphere. The NETA- and DEPA-backboned
chelators were shown to display favorable complexation kinetics
and stability in binding 90Y, 177Lu, or 205/6Bi.15−18
hexanes to afford 3 (120 mg, 97%) as a brownish oil. 1H NMR
(CDCl3, 300 MHz) δ 1.45 (s, 18H), 1.71−1.82 (m, 2H), 1.83−
2.05 (m, 2H), 2.66−2.87 (m, 2H), 2.95 (dd, J = 8.6 Hz, J = 14.1
Hz, 1H), 3.29 (dd, J = 8.6 Hz, J = 14.1 Hz, 1H), 3.42 (dd, J =
17.3 Hz, J = 22.4 Hz, 4H), 4.10−4.18 (m, 1H), 7.35 (d, J = 8.6
Hz, 2H), 8.14 (d, J = 8.6 Hz, 2H). 13C NMR (CDCl3, 300
MHz) δ 28.2 (q), 30.8 (t), 34.9 (t), 36.0 (d), 36.3 (t), 56.9 (t),
64.1 (t), 81.2 (s), 123.6 (d), 129.2 (d), 146.4 (s), 149.9 (s),
170.4 (s). HRMS (Positive ion FAB) Calcd for C23H36IN2O6:
Herein, we report an efficient synthesis of bifunctional ligand
3p-C-NETA-NCS containing the isothiocyanate group for
conjugation to a tumor targeting antibody. 3p-C-NETA-NCS
conjugated to trastuzumab was compared to trastuzumab
conjugates of the known bifunctional ligands C-DOTA, C-
DTPA, and 3p-C-DEPA for radiolabeling kinetics with 90Y and
177Lu. 90Y-and 177Lu-radiolabeled trastuzumab conjugates of 3p-
C-NETA, 3p-C-DEPA, and C-DOTA were evaluated for in vitro
serum stability. 177Lu-3p-C-NETA-trastuzumab conjugate was
also evaluated for in vivo biodistribution and tumor uptake
study.
+
[M − I + OH]+ m/z 453.2601. Found: [M − I+ OH] m/z
453.2603.
tert-Butyl-2-[(1-{4,7-bis[2-(tert-butoxy)-2-oxoethyl]-
1,4,7-triazonan-1-yl}-5-[4-(hydroxyl-nitro)phenyl]-
pentan-2-yl)[2-(tert-butoxy)-2-oxoethyl]amino]acetate
(7). To a solution of 3 (50 mg, 0.089 mmol) in CH3CN (1 mL)
at 0 °C was added compound 5 (35.0 mg, 0.098 mmol) and
DIPEA (34.5 mg, 0.267 mmol). The resulting mixture was
stirred for 40 h at room temperature, while monitoring the
reaction progress using TLC. The reaction mixture was
concentrated to dryness. The residue was purified via column
chromatography on silica gel (220−440 mesh) eluting with 3%
CH3OH in CH2Cl2 to provide product 7 (65 mg, 94%). 1H and
13C NMR data of 7 were identical to those as previously
reported.18
tert-Butyl-2-[(1-{4,7-bis[2-(tert-butoxy)-2-oxoethyl]-
1,4,7-triazonan-1-yl}-5-[4-(hydroxyl-nitro)phenyl]-
pentan-2-yl)[2-(tert-butoxy)-2-oxoethyl]amino]acetate
(7). Reaction of 2 with 5 in the Presence of AgClO4. To a
solution of 2 (50 mg, 0.097 mmol) in CH3CN (0.5 mL) at −5
°C was added AgClO4 (20.1 mg, 0.097 mmol) and stirred for
10 min at the same temperature. Then, compound 5 (34.7 mg,
0.097 mmol) and DIPEA (37.6 mg, 0.291 mmol) in CH3CN
(0.5 mL) was sequentially added to the reaction mixture at −5
°C. The resulting mixture was warmed to room temperature
and stirred for 24 h while monitoring the reaction progress
using TLC. The residue was purified via column chromatog-
raphy on silica gel (60−230 mesh) eluting with 3% CH3OH in
CH2Cl2 to provide the crude product 7 containing a tiny
amount of the starting material 5 as an impurity. The crude
product was treated with 0.1 M HCl solution (10 mL) and
extracted with CHCl3 (10 mL × 3). The combined organic
layers were concentrated to dryness. The residue was treated
with 0.1 M NaOH solution (10 mL) and extracted with CHCl3
(10 mL × 3). The combined organic layers were dried over
MgSO4, filtered, and concentrated in vacuo to dryness to
EXPERIMENTAL PROCEDURES
■
1
Instruments and Methods. H, 13C, and DEPT NMR
spectra were obtained using a Bruker 300 instrument, and
chemical shifts are reported in ppm on the δ scale relative to
TMS. Electrospray ionization (ESI) high-resolution mass
spectra (HRMS) were obtained on JEOL double sector JMS-
AX505HA mass spectrometer (University of Notre Dame, IN).
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 250−5 column (Bio-Rad,
Hercules, CA), TSK-G3000PW column (Tosoh bioscience,
King of Prussia, PA), or a BioSep-SEC S 3000 column
(Phenomenex, Torrance, CA).
Reagents. All reagents were purchased from Sigma-Aldrich
(St. Louis, MO) and used as received unless otherwise noted.
Trastuzumab (Herceptin; Genentech, South San Francisco,
CA) was obtained through the Veterinary Resources Program
(National Institutes of Health, Bethesda, MD) and provided by
Dr. Martin Brechbiel (Radioimmune and Inorganic Chemistry
Section, Radiation Oncology Branch, NIH). 90Y (0.05 M HCl)
and 177Lu (0.05 M HCl) were purchased from Perkin-Elmer. C-
DOTA was purchased from Macrocyclics (Dallas, TX).
Caution: 90Y (t1/2 = 64.1 h) is a β-emitting radionuclide.
177Lu (t1/2 = 6.7 days) is a β/γ-emitting radionuclide.
Appropriate shielding and handling protocols should be in
place when using the isotope.
tert-Butyl 2-{[2-(tert-Butoxy)-2-oxoethyl][2-iodo-5-(4-
nitrophenyl)pentyl]amino} acetate (3). To a solution of
118 (100 mg, 0.221 mmol) and PPh3 (69.5 mg, 0.265 mmol) in
CH2Cl2 (2 mL) at 0 °C was added imidazole (18 mg, 0.265
mmol) and iodine (67.3 mg, 0.265 mmol) portionwise over 5
min. The reaction mixture was stirred for 4 h at 0 °C and RT
for 1 h after which the reaction mixture was concentrated to
dryness. The residue was purified by silica gel (60−230 mesh)
column chromatography eluted with 10% ethyl acetate in
provide product 7 (66 mg, 86%) as a yellowish oil. 1H and 13
C
NMR data of 7 were identical to those previously reported.18
tert-Butyl-2-[(1-{4,7-bis[2-(tert-butoxy)-2-oxoethyl]-
1,4,7-triazonan-1-yl}-5-[4-(hydroxyl-nitro)phenyl]-
pentan-2-yl)[2-(tert-butoxy)-2-oxoethyl]amino]acetate
(7). Reaction of 3 with 5 in the Presence of AgClO4. To a
solution of 3 (50 mg, 0.089 mmol) in CH3CN (0.5 mL) at −5
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°C was added AgClO4 (18.4 mg, 0.089 mmol) and stirred for
10 min at the same temperature. Then, compound 5 (31.8 mg,
0.089 mmol) and DIPEA (34.5 mg, 0.267 mmol) in CH3CN
(0.5 mL) was sequentially added to the reaction mixture at −5
°C. The resulting mixture was warmed to room temperature
and stirred for 24 h while monitoring the reaction progress
using TLC. The residue was purified via column chromatog-
raphy on silica gel (60−230 mesh) eluting with 3% CH3OH in
CH2Cl2 to provide the crude product 7 containing a tiny
amount of the starting material 5 as an impurity. The crude
product was treated with 0.1 M HCl solution (10 mL) and
extracted with CHCl3 (10 mL × 3). The combined organic
layers were concentrated to dryness. The residue was treated
with 0.1 M NaOH solution (10 mL) and extracted with CHCl3
(10 mL × 3). The combined organic layers were dried over
MgSO4, filtered, and concentrated in vacuo to dryness to
added dropwise 1 M thiophosgene in CHCl3 (11 μL, 0.011
mmol). The resulting mixture was stirred at room temperature
for 3 h. The aqueous layer was concentrated in vacuo to provide
1
pure 10 (6.0 mg, 95%) as a light yellowish solid. H NMR
(D2O, 300 MHz) δ 1.31 (s, 1H), 1.57 (s, 3H), 2.95−3.28 (m,
5H), 3.29−3.53 (m, 12H), 3.60 (d, J = 17.3 Hz, 2H), 3.85 (s,
4H), 7.13 (s, 4H). HRMS (Positive ion FAB) Calcd for
C26H38N5O8S: [M + H]+ m/z 580.2441. Found: [M + H]+ m/z
580.2439.
Conjugation of 3p-C-NETA-NCS to Trastuzumab. C-
DTPA-NCS, 3p-C-DEPA-NCS, and C-DOTA-NCS were
conjugated to trastuzumab as previously reported.16 All
absorbance measurements were obtained on an Agilent 8453
diode array spectrophotometer equipped with an 8-cell
transport system (designed for 1 cm cells). Metal-free stock
solutions of all buffers were prepared using Chelex-100 resin
(200−400 mesh, Bio-Rad Lab, Hercules, CA, Cat# 142−2842).
Chelex resin (1 g/100 mL) was added into the buffer solution
and the mixture was shaken overnight in a shaker and filtered
through a Corning filter system (Cat# 430513, pore size 0.2
μm). Disposable PD-10 Sephadex G-25 M columns (GE
Healthcare, #17−0851−01) were rinsed with 25 mL of the
conjugation buffer prior to addition of antibody. Amicon
centricon C-50 (50 000 MWCO) centrifugal filter devices
(Millipore, Bedford, MA, Cat# UFC805008) were used for
purification of trastuzumab conjugate. The initial concentration
of trastuzumab was determined by UV/vis spectroscopic
method. Phosphate-buffered saline (PBS, 1×, 11.9 mM
phosphates, 137 mM NaCl, and 2.7 mM KCl, pH 7.4) was
purchased from Fisher Scientific and used as received.
Conjugation buffer (50 mM HEPES, 150 mM NaCl, pH 8.6)
was prepared as 1× solutions, chelexed, and filtered through the
Corning filter. Trastuzumab (9.2 mg) was diluted to 2.5 mL
using conjugation buffer, and the resulting solution was added
to a PD-10 column. Conjugation buffer (6.0 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 (7.0 mg) was added a 10-fold excess of 3p-C-
NETA-NCS (45 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-NETA-trastuzumab conjugate followed by centrifugation to
remove unreacted ligand. The volume of purified conjugate
antibody was brought to 1.0 mL. To measure [trastuzumab] in
the conjugates, a UV/vis spectrometer was zeroed against a
cuvette filled with 2 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-NETA-trastuzumab conjugate
solution was added, and absorbance of the solution at 280 nm
was noted. Beer’s Law was used to calculate [trastuzumab] in
the conjugate with molar absorptivity of 1.42. After
centrifugation, 5.9 mg (4.06 × 10−5 M, 84%) of the
trastuzumab remained.
provide product 7 (62 mg, 88%) as a yellowish oil. 1H and 13
C
NMR data of 7 were identical to those as previously reported.18
{ 4 - [ 5 - ( 4 - A m i n o p h e n y l ) - 2 - ( b i s - t e r t -
butoxycarbonylmethylamino)pentyl]-7-tert-butoxycar-
bo-nylmethyl-[1,4,7]triazonan-1-yl}acetic Acid tert-Butyl
Ester (8). To a solution of 7 (14.6 mg, 0.018 mmol) in ethanol
(5 mL) at room temperature was added 10% Pd/C (3 mg)
under Ar (g). The reaction mixture was placed under
hydrogenation apparatus for 14 h. The resulting mixture was
filtered via Celite bed and washed thoroughly with ethanol. The
filtrate was concentrated to provide 8 (13.1 mg, 96%) as a
1
yellowish solid. H NMR (CDCl3, 300 MHz) δ 1.45 (s, 36H),
1.50−1.70 (m, 4H), 1.92−2.05 (m, 1H), 2.18−2.32 (m, 1H),
2.38−2.90 (m, 15H), 3.08 (d, J = 16.6 Hz, 1H), 3.26 (d, J =
16.5 Hz, 1H), 3.34 (s, 2H), 3.44 (s, 4H), 6.60 (d, J = 8.3 Hz,
2H), 6.94 (d, J = 8.2 Hz, 2H); 13C NMR (CDCl3, 300 MHz) δ
27.8(t), 28.0(q), 28.1(t), 28.4(t), 29.6(t), 35.8(t), 51.3(t),
51.7(t), 53.5(t), 54.7(t), 55.9(t), 58.1(t), 58.5(t), 59.4(d),
81.2(s), 81.3(s), 82.2(s), 115.2(d), 119.1(d), 131.2(s),
145.5(s), 170.7(s), 170.9(s), 171.4(s). HRMS (Positive ion
FAB) Calcd for C41H72N5O8: [M + H]+ m/z 762.5381. Found:
[M + H]+ m/z 762.5364.
{4-[5-(4-Amino-phenyl)-2-(bis-carboxymethylamino)-
pentyl]-7-carboxymethyl-[1,4,7]triazo-nan-1-yl}acetic
Acid (9). To a flask containing compound 8 (8.5 mg, 0.011
mmol) at 0−5 °C was added dropwise 4 M HCl (g) in 1,4-
dioxane (3 mL) over 20 min. The resulting mixture was
gradually warmed to room temperature and continuously
stirred for 18 h. Ether (20 mL) was added to the reaction
mixture which was then stirred for 10 min. The resulting
mixture was placed in the freezer for 1 h. The resulting
precipitate was filtered and washed with ether. The solid
product was quickly dissolved in deionized water. The aqueous
solution was concentrated in vacuo to provide 9 (7.1 mg, 88%)
as an off-white solid. 1H NMR (CDCl3, 300 MHz) δ 1.15−1.34
(m, 1H), 1.39−1.55 (m, 3H), 2.55 (t, J = 6.4 Hz, 2H), 3.00−
3.20 (m, 2H), 3.20−3.73 (m, 17H), 3.87 (s, 4H), 7.18 (dd, J =
8.1 Hz, J = 20.3 Hz, 4H); 13C NMR (D2O, 300 MHz) δ 25.7
(t), 26.6 (t), 33.5 (t), 48.2 (t), 49.6 (t), 50.4 (t), 51.8 (t), 52.5
(t), 53.7 (t), 54.0 (t), 56.0 (t), 60.3 (d), 123.0 (d), 127.5 (s),
130.0 (d), 142.2 (s), 168.0 (s), 170.0 (s), 173.6 (s). HRMS
(Positive ion FAB) Calcd for C25H40N5O8: [M + H]+ m/z
538.2877. Found: [M + H]+ m/z 538.2880.
Spectroscopic Determination of Ligand to Protein (L/
P) Ratio. A stock solution of the Lu(III)-AAIII reagent was
prepared in 0.15 M NH4OAc (pH 7.0) by adding an aliquot of
lutetium atomic absorption solution (5.79 × 10−3 M) into a 10
μM solution of AAIII to afford a 5 μM solution of Lu(III). This
solution was stored in the dark to avoid degradation over time.
A UV/vis spectrometer was zeroed against 8 well-dried blank
{ 4 - [ 2 - ( B i s - c a r b o x y m e t h y l a m i n o ) - 5 - ( 4 -
isothiocyanatophenyl)pentyl]-7-carboxymethyl[1,4,7]-
triazonan-1-yl}acetic Acid (3p-C-NETA-NCS, 10). To a
solution of 9 (6.3 mg, 0.009 mmol) in water (0.15 mL) was
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cuvettes with a window open from 190 to 1100 nm. A cuvette
was filled with AAIII solution (2 mL), and the other 7 cuvettes
with the Lu(III)-AAIII (2 mL). Lu(III)-AAIII (50 μL) in the 7
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-NETA (0.1 mM) were added to the 5 cuvettes to give a
series of 5 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. Trastuzumab
conjugate of 3p-C-NETA (50 μL) was added to the eighth
cuvette containing Lu(III)-AAIII reagent (1950 μL). After
addition of trastuzumab conjugate to the Lu(III)-AAIII
solution, the resulting solution was equilibrated for 10 min.
The absorbance of the resulting solution at 652 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 Lu(III)-AAIII solution containing 6 different
concentrations were used to construct a calibration plot of A652
versus [3p-C-NETA] by the equation, Y = 0.1202 − (1.2138 ×
104)[3p-C-NETA] (R2 = 0.9967) wherein Y = A652 nm. The
concentration of 3p-C-NETA in the trastuzumab conjugate was
calculated (1.27 × 10−4). The L/P ratio of 3p-C-NETA-
trastuzumab conjugate ([1.27 × 10−4 M]/[4.06 × 10−5 M])
was measured to be 3.1.
Radiolabeling of Bifunctional Ligand−Trastuzumab
Conjugates with 90Y or 177Lu. C-DTPA-trastuzumab, 3p-C-
DEPA-trastuzumab, and C-DOTA-trastuzumab conjugates
were prepared and purified as previously reported.16 All HCl
solutions were prepared from ultrapure HCl (Fisher Scientific,
Cat# A466−500). 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 used to prepare all NH4OAc buffer solutions
(0.25 M, pH 5.5). The buffer solutions were treated with
Chelex-100 resin (Biorad, #142−2842, 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 (30 μL, pH 5.5) in a capped microcentrifuge
tube (1.5 mL, Fisher Scientific #05−408−129) was sequentially
added a solution of bifunctional ligand−trastuzumab conjugate
(30 μg) in PBS (5−10 μL) and 177Lu (0.05 M HCl, 60 μCi, 2−
3 μL). To a conjugate solution (15−27 μL, 30 μg) in a capped
microcentrifuge tube (1.5 mL) was sequentially added a
solution of 0.05 M HCl (6−15 μL) and 90Y (0.05 M HCl,
60 μCi, 2−4 μL). The final volume of the resulting solution was
around 40 μL, and the pH of the resulting reaction mixture was
5.5. The reaction mixture was agitated on the thermomixer
(Eppendorf, #022670549) set at 1000 rpm at room temper-
ature for 1 h. The labeling efficiency was determined by SE-
HPLC (Bio-Silect SEC 250−5 column, PBS (pH 7.4) at a flow
rate of 1 mL/min for method 1; BioSep-SEC S 3000, a solution
of 0.05 M NaSO4/0.02 M NaH2PO4/0.05% NaN3 (pH 6.8) at
a flow rate of 1 mL/min for method 2; or TSK-G3000 PW
column, PBS (pH 7.4) at a flow rate of 1 mL/min for method
3) and ITLC (solvent: 20 mM EDTA/0.15 M NH4OAc).
A solution of radiolabeled mixture (6 μL) 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, 0.6 μL) was
added to the mixture, and the resulting mixture was left for at
least 20 min at RT. In ITLC analysis, peaks for bound and
unbound radioisotope appeared around 30 mm and 50 mm
from the origin, respectively. Bound and unbound radioisotope
peaks appeared around 7.8 and 10.7 min (Method 1), 9.0 and
12.0 min (method 2), and 6.5 and 8.5 min (method 3),
respectively.
In Vitro Stability of 90Y- and 177Lu-Radiolabeled
Trastuzumab Conjugate. The radiolabeled trastuzumab
conjugates were evaluated for stability in human serum (pH
7, 37 °C). 177Lu-3p-C-NETA-trastuzumab, 177Lu-C-DOTA-
trastuzumab, or 177Lu-3p-C-DEPA-trastuzumab was radio-
labeled with 177Lu at RT or 37 °C (0.25 M NH4OAc, pH
5.5). The complex formed was purified by gel filtration
chromatography using Biospin 6 column (Biorad, Cat# 732−
6002) eluted with PBS (pH 7.4). The fraction containing 177Lu-
3p-C-NETA-trastuzumab (150 μL, ∼200 μCi), 177Lu-C-
DOTA-trastuzumab (30 μL, ∼100 μCi), or 177Lu-3p-C-
DEPA-trastuzumab (30 μL, ∼100 μCi) was added into
human serum (1 mL, Gemini Bioproducts, #100110) in a
microcentrifuge tube. The stability of the purified 177Lu-3p-C-
NETA-trastuzumab or 177Lu-C-DOTA-trastuzumab in human
serum (37 °C, pH 7) was evaluated for 14 days, and 177Lu-3p-
C-DEPA-trastuzumab in human serum (37 °C, pH 7) was
evaluated for 2 days.
90Y-3p-C-NETA-trastuzumab, 90Y-C-DOTA-trastuzumab, or
90Y-3p-C-DEPA-trastuzumab was radiolabeled with 90Y at RT
or 37 °C (0.25 M NH4OAc, pH 5.5). The complex formed was
purified by gel filtration chromatography using Biospin 6
column or PD-10 Sephadex G-25 M columns (GE Healthcare,
#17−0851−01) eluted with PBS (pH 7.4). The fraction
containing 90Y-3p-C-NETA-trastuzumab (20 μL, ∼110 μCi),
90Y-C-DOTA-trastuzumab (30 μL, ∼110 μCi), or 90Y-3p-C-
DEPA-trastuzumab (560 μL, 128 μCi) was added into human
serum (1 mL, Gemini Bioproducts, #100110) in a micro-
centrifuge tube. The stability of the purified 90Y-3p-C-NETA-
trastuzumab or 90Y-C-DOTA-trastuzumab in human serum (37
°C, pH 7) was evaluated for 14 days. The stability of 90Y-3p-C-
DEPA-trastuzumab in human serum (37 °C, pH 7) was
evaluated for 7 days.
The serum stability of 90Y- or 177Lu-radiolabeled complex (37
°C, pH 7) was assessed by measuring the transfer of the
radionuclide from the complex to serum proteins using SE-
HPLC (Bio-Silect SEC 250-5 column, PBS (pH 7.4) at a flow
rate of 1 mL/min for method 1 BioSep-SEC S 3000 column at
a flow rate of 1 mL/min, eluent: 0.05 M NaSO4/0.02 M
NaH2PO4/0.05% NaN3, pH 6.8 for method 2 or TSK-
G3000PW column at a flow rate of 1 mL/min, eluent: PBS
for method 3). A solution of the radiolabeled complex in serum
(1−100 μL) was withdrawn at the designated time point,
treated with DTPA (10 mM, 1−10 μL or 5 mM, 0.6 μL),
incubated at room temperature for 20 min and then diluted
with the HPLC eluent (160−200 μL) prior to SE-HPLC.
Radiolabeling with 177Lu of Trastuzumab Conjugate
for in Vivo Studies. 3p-C-NETA-trastuzumab was radio-
labeled with 177Lu for a trial biodistribution study. An aliquot of
0.25 M NH4OAc (65.8 μL, pH = 5.0) was added to a solution
of 177LuCl3 (424 μCi, 2.3 μL) in 0.05 M HCl followed by 100
μg of 3p-C-NETA-trastuzumab in a solution of 0.25 M
NH4OAc (15.9 μL, pH = 7.0). The reaction mixture was
incubated at room temperature for 15 min with mixing at 1000
rpm and quenched with 1 mM diethylenetriaminepentaacetic
acid (DTPA) solution (pH = 6.0). The reaction mixture was
allowed to stand for 15 min at room temperature, and then
radiochemical purity and yield were determined using TLC
(silica gel, Methanol/10% NH4OAC = 1:1). The dose was then
prepared for injection to mice without further purification.
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Scheme 1. Optimized Synthesis of the Key Precursor Molecule 7
Scheme 2. Synthesis of Bifunctional Ligand 3p-C-NETA-NCS (10)
Biodistribution Study. All experiments were conducted in
compliance with the guidelines established by the Animal Care
and Use Committee of the University of Missouri-Columbia
Animal Care Quality Assurance Office. Athymic nude mice
were implanted subcutaneously (s.c.) in the hind flank with a
suspension of 5 × 106 ZR-75−1 human breast cancer cells
(0.15 mL) in Hank’s Balanced Salt Solution. When tumors had
grown to 100−400 mg (∼30 days after tumor implantation),
mice were injected intravenously (i.v.) via the tail vein with
177Lu-3p-C-NETA-trastuzumab (27 μCi, 100 μL). The
biodistributions were obtained at 24, 72, and 120 h post-
injection. Tissues harvested included blood, liver, bone, and
tumor. Tissues were drained of blood, weighed, and counted in
a gamma counter with a standard of the injected dose, such that
decay-corrected uptakes were calculated as percent injected
dose per gram of tissue (% ID/g) and percent injected dose per
organ (% ID/organ).
Inspired by high efficiency and regiospecificity of the ring-
opening reaction of aziridinium ion, we sought optimization of
the reaction conditions for the synthesis of 7. We envisioned
using secondary β-amino iodide 3 as the starting material based
on the rationale that 3 containing iodine as an excellent leaving
group would react significantly faster with the nucleophilic
TACN analogue 5 and can be converted to 7 in higher isolated
yield. We also investigated the use of a halo-sequestering agent
AgClO4 to see if the prompt formation of the azridinium ion
can facilitate the nucleophilic attack of 5. The optimization
results are outlined in Scheme 1. Secondary β-amino iodide 3
was prepared using the efficient synthetic method that we
developed. Iodination of 118 using I2, imidazole, and PPh3
provided 3 in 97% isolated yield. Formation of aziridinium salt
4 from intramolecular rearrangement of 3 and subsequent
regiospecific ring-opening of 4 by nucleophilic bisubstituted
TACN 5 at the less hindered carbon provided the desired
coupling product 7 in excellent isolated yield (94%). The effect
of silver perchlorate on the formation of 7 was explored using
secondary β-amino halides 2 and 3. Treatment of 2 or 3 with
silver perchlorate provided aziridinium perchlorate salt 6 which
was further treated with 5 to provide the product 7 in 86% and
88% isolated yield, respectively. Our optimization strategy to
prepare the key precursor 7 in an improved yield and at a
shorter reaction time was found to be successful. The best
result was obtained from the reaction of the secondary β-amino
iodide 3 with 5 in the absence of silver perchlorate. It is
noteworthy that both of the silver-promoted ring-opening
reactions produced a small portion of the intramolecular
rearrangement byproduct. Synthesis of the bifunctional ligand
3p-C-NETA-NCS (10) was accomplished as shown in Scheme
2. Compound 7 was hydrogenated using 10% Pd/C to afford
compound 8 in 96% isolated yield. tert-butyl groups in 8 were
RESULTS AND DISCUSSION
■
The key step in the synthesis of 3p-C-NETA-NCS is the
regiospecific ring-opening reaction of N,N-bisubstituted azir-
idinium ions 4 or 6 by bisubstituted 1,4,7-triazacyclononane
(5) to generate the key precursor molecule 7 (Schemes 1 and
2). We recently reported the construction of 7 from the
reaction of N,N-bisubstituted secondary β-amino bromide 2
with 5 based on the ring-opening strategy.18 The intermediate
compound 7 was efficiently prepared in 94% isolated yield, and
no byproduct was formed during the ring-opening reaction.
However, the reaction was very slow at room temperature and
remained incomplete after 5 days. The mild reaction condition
was required because an elevated temperature promoted the
formation of an unwanted byproduct from intramolecular
rearrangement of 2.18
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a
Table 1. Evaluation of Radiolabling Efficiency (%) of Bifunctional Ligand−Trastuzumab Conjugates with 90Y (RT, 0.25 M
b
c
NH4OAC, pH 5.5) Using ITLC and HPLC (parentheses)
time
3p-C-NETA-trastuzumab
3p-C-DEPA-trastuzumab
C-DTPA-trastuzumab
C-DOTA-trastuzumab
1 min
91.6 3.1
(86.2 3.5)
99.6 0.2
30.6 1.9
(29.3 2.2)
58.6 1.6
98.4 0.4
(90.1 1.2)
98.7 0.7
(89.9 1.3)
98.6 0.8
(89.5 0.8)
98.7 0.4
(89.3 0.8)
98.9 0.3
(90.3 0.6)
99.2 0.4
23.1 7.7
(24.1 6.6)
43.0 9.9
5 min
(94.4 0.2)
99.6 0.3
(50.3 1.8)
66.3 10.4
(58.2 6.8)
67.9 7.2
(39.8 8.3)
56.5 12.5
(50.9 13.3)
67.4 12.2
(60.1 15.2)
69.7 10.8
(62.7 12.4)
71.7 8.9
10 min
20 min
30 min
60 min
(94.9 1.4)
99.6 0.5
(93.1 1.1)
99.9 0.1
(58.3 7.1)
67.50 7.5
(57.4 7.3)
67.4 9.0
(93.6 0.9)
99.8 0.1
(93.4 0.5)
(56.9 5.3)
(90.0 0.5)
(63.4 11.3)
a
b
Radiolabeling efficiency (mean standard deviation %) was measured in triplicate (n = 3). ITLC eluent (20 mM EDTA/0.15 M NH4OAc).
c
HPLC mobile phase (0.05 M NaSO4/0.02 M NaH2PO4/0.05% NaN3, pH 6.8 or PBS, pH 7.4).
a
Table 2. Evaluation of Radiolabling Efficiency (%) of Bifunctional Ligand−Trastuzumab Conjugates with 177Lu (RT, 0.25 M
b
c
NH4OAC, pH 5.5) Using ITLC and HPLC (parentheses)
time
3p-C-NETA-trastuzumab
3p-C-DEPA-trastuzumab
C-DTPA-trastuzumab
C-DOTA-trastuzumab
1 min
99.2 0.2
(96.5 1.2)
99.9 0.1
35.9 1.8
(29.8 2.1)
71.1 2.2
99.5 0.3
(98.3 0.6)
99.4 0.2
(98.3 0.3)
99.4 0.4
(98.4 0.7)
99.6 0.1
(98.3 0.3)
99.6 0.2
(98.3 0.3)
99.4 0.3
29.7 1.5
(28.1 3.0)
58.6 1.3
5 min
(97.7 0.8)
99.8 0.1
(60.0 1.9)
85.3 1.1
(54.6 1.5)
75.5 1.7
10 min
20 min
30 min
60 min
(97.6 1.3)
99.9 0.1
(75.2 0.4)
94.8 0.4
(70.1 1.6)
89.3 0.1
(97.6 0.8)
99.8 0.1
(85.4 1.0)
97.9 0.4
(84.6 1.4)
94.2 0.4
(97.5 0.3)
99.9 0.1
(87.3 1.4)
98.4 0.8
(90.1 1.5)
97.8 0.3
(97.4 0.4)
(87.6 1.3)
(98.1 0.3)
(94.8 1.3)
a
b
Radiolabeling efficiency (mean standard deviation %) was measured in triplicate (n = 3). ITLC eluent (20 mM EDTA/0.15 M NH4OAc).
c
HPLC mobile phase (0.05 M NaSO4/0.02 M NaH2PO4/0.05% NaN3, pH 6.8 or PBS, pH 7.4).
removed by the treatment of 8 with 4 M HCl (g) in 1,4-
dioxane. Reaction of 9 with thiophosgene in CHCl3/H2O
provided the desired 3p-C-NETA-NCS. The synthesis of 3p-C-
NETA-NCS was achieved in 71% overall yield starting from the
readily available compound 1. The efficient synthetic method to
7 can be readily scaled up and applied to preparation of many
other bifunctional chelates.
kinetics data indicate that 3p-C-NETA-trastuzumab conjugates
were extremely rapid in binding 90Y and 177Lu and complex-
ation was nearly complete in 1 min as determined by ITLC
(91.6% for 90Y and 99.2% for 177Lu). Complexation kinetics of
3p-C-NETA-trastuzumab conjugate with 90Y and 177Lu was
comparable to that of C-DTPA-trastuzumab conjugate. C-
DOTA-trastuzumab displayed significantly slower complexation
kinetics with 90Y (71.7% at 1 h, Table 1), while the conjugate
more rapidly bound to 177Lu (>90% at 30 min, Table 2). 3p-C-
DEPA-trastuzumab conjugate exhibited similar 90Y- and 177Lu-
radiolabeling pattern to C-DOTA-trastuzumab, achieving the
respective radiolabeling efficiency of ∼67% (1 h, Table 1) and
>87% (30 min, Table 2) with 90Y and 177Lu. It appears that
decadentate 3p-C-DEPA has too many donor groups to form a
stable Y(III) or Lu(III) complex, and this may allow the
formation of the Lu(III)- or Y(III)-3p-C-DEPA complex to be a
reversible process. The results indicates that the tridentate
pendant acyclic donors in 3p-C-NETA are essential in rapidly
binding Y(III) and Lu(III) and improved complexation kinetics
of NETA with the metals relative to DOTA is ascribed to
cooperative and bimodal binding of acyclic and macrocyclic
donors.
Trastuzumab is known to target HER2 (human epidermal
growth factor receptor 2) overproduced in cancer cells and is a
clinically available antibody for treatment of metastatic breast
cancer.19,20 3p-C-NETA-NCS was conjugated with trastuzu-
mab, and concentration of trastuzumab in the conjugate was
quantified by the spectroscopic assay.16 Ligand to protein ratio
(L/P = 3.1) of 3p-C-NETA-trastuzumab conjugate was
determined using Lu(III)-ArsenazoIII based UV−vis spectro-
photometric assay.16 Trastuzumab conjugates of other bifunc-
tional ligands (C-DOTA, C-DTPA, and 3p-C-DEPA) were also
prepared and evaluated for comparison. The purified
trastuzumab conjugate (0.25 M NH4OAc, pH 5.5) was labeled
with 90Y or 177Lu at room temperature (RT).18 During the
reaction time (1 h), the components were analyzed using SE-
HPLC after challenging the reaction mixture with 10 mM
DTPA, and the radiolabeling efficiency (%) was determined
using both ITLC and size exclusion (SE)-HPLC (Tables 1 and
2 and Supporting Information). The radiolabeling formation
90Y- or 177Lu-radiolabeled trastuzumab conjugates of 3p-C-
NETA, 3p-C-DEPA, and C-DOTA were further evaluated for in
vitro serum stability (Supporting Information). Both 90Y-3p-C-
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Bioconjugate Chemistry
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NETA-trastuzumab and 177Lu-3p-C-NETA-trastuzumab re-
mained stable in human serum for 2 weeks as confirmed by
radio-SE-HPLC analysis. However, a significant amount of the
radioactivity was dissociated from 90Y-3p-C-DEPA-trastuzumab
(32%, 2 days) and 177Lu-3p-C-DEPA trastuzumab (45%, 2
days). 90Y-C-DOTA-trastuzumab and 177Lu-C-DOTA-trastuzu-
mab were quite stable releasing a minimal level of radioactivity
over 2 weeks.
3p-C-NETA and 3p-C-DEPA, and other known bifunctional
ligands, C-DOTA and C-DTPA, were evaluated for a
comparative 90Y and 177Lu-radiolabeleing kinetics study. 3p-C-
NETA-trastuzumab conjugate instantly bound to 90Y or 177Lu
with comparable radiolabeling kinetics to C-DTPA-trastuzumab
conjugate, while 3p-C-DEPA- and C-DOTA-trastuzumab
conjugates displayed similar and slow radiolabeling pattern in
binding 90Y and 177Lu. Both 90Y-3p-C-NETA-trastuzumab and
177Lu-3p-C-NETA-trastuzumab were stable in human serum
over 2 weeks. A significant amount of the radioactivity was
released from both 90Y-3p-C-DEPA-trastuzumab and 177Lu-3p-
C-DEPA-trastuzumab in serum over 24 h. 177Lu-3p-C-NETA-
trastuzumab conjugate produced a favorable biodistribution
profile and exhibited low radioactivity level in organs and high
tumor uptake. The results of the in vitro and in vivo studies
indicate that bimodal and cooperative binding of the acyclic and
macrocyclic moieties property in 3p-C-NETA led to fast
complexation kinetics and high complex stability of the
octadentate chelate in binding the lanthanides Y(III) and
Lu(III). The bimodal ligand 3p-C-NETA has broad applica-
tions for RIT of various cancers using 90Y and 177Lu. On the
basis of the favorable in vitro complexation and in vivo
biodistribution data, 3p-C-NETA-NCS radiolabeled with 90Y or
177Lu will be further evaluated for comprehensive in vivo
biodistribution, pharmacokinetics, and dosimetry using differ-
ent antibodies and tumor models.
The in vitro result suggests that 3p-C-NETA was favorably
compared to C-DOTA and displayed excellent complexation
kinetics and stability with 90Y and 177Lu. 1B4M-DTPA20 has
been used as the chelator of 90Y in Zevalin and was reported to
exhibit rapid complexation kinetics with 90Y. However, it should
be noted that 90Y-1B4M-DTPA-antibody conjugate was less
stable than 90Y-DOTA-antibody conjugate both in vitro and in
vivo.21
The in vivo stability and tumor targeting of 177Lu-3p-C-
NETA-trastuzumab was evaluated by performing a pilot
biodistribution study in ZR-75-1-bearing nude mice (Figure
2). The 3p-C-NETA-trastuzumab conjugate was radiolabeled
ASSOCIATED CONTENT
■
S
* Supporting Information
HPLC and ITLC chromatograms for assessment of radio-
labeling 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
■
Figure 2. In vivo biodistribution of 177Lu-3p-C-NETA-trastuzumab
conjugate in ZR-75-1-bearing mice (% ID/g, n = 3, intravenous
injection).
Corresponding Author
*E-mail: Chong@iit.edu, Phone: 312-567-3235, Fax: 312-567-
3494. Mailing address: 3101 S. Dearborn St, LS 182, Chemistry
Division, Biological and Chemical Sciences Department, Illinois
Institute of Technology, Chicago, IL, 60616.
with 177Lu at specific activity of 640 Ci/mmol and in 92.2%
yield and radiochemical purity. The highest tumor uptake
(10.55
Notes
7.93%) of 177Lu-3p-C-NETA-trastuzumab was
The authors declare no competing financial interest.
observed at 72 h. 177Lu-3p-C-NETA-trastuzumab exhibited
the highest radioactivity level in the blood at 24 h (14.57
10.63%), which was not significantly different than 72 h (12.09
1.12%), but significantly decreased at 120 h (1.58 1.30%).
The 177Lu-3p-C-NETA-trastuzumab conjugate resulted in a
very low radioactivity level in the liver (2.20 0.06%) and the
kidneys (1.09 0.12%) at 120 h. The radioimmunoconjugate
displayed low bone uptake and a higher radioactivity level in
the tumor, compared to the normal organs, over the course of
the study and the highest tumor-to-blood ratio (6.39) at 120 h.
The biodistribution data indicate that 177Lu-3p-C-NETA
remains stable in vivo and tumor targeting of 177Lu-3p-C-
NETA-trastuzumab can be improved by selecting an adequate
HER2-positive tumor model.
ACKNOWLEDGMENTS
■
We acknowledge the financial support from the National
Institutes of Health (R01CA112503 to H. S. Chong). We also
acknowledge the Department of Veterans Affairs for providing
resources and use of facilities at the Harry S. Truman Memorial
Veterans’ Hospital in Columbia, MO.
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