Improved 18F labeling of peptides with a fluoride-aluminum- chelate complex

Article abstract of DOI:10.1021/bc100137x

We reported previously the feasibility to radiolabel peptides with fluorine-18 (¹⁸F) using a rapid one-pot method that first mixes ¹⁸F^- with Al³⁺ and then binds the (Al ¹⁸F)²⁺ complex to a

Full text of DOI:10.1021/bc100137x

Bioconjugate Chem. 2010, 21, 1331–1340
1331
18
Improved F Labeling of Peptides with a Fluoride-Aluminum-Chelate
Complex
William J. McBride,*,† Christopher A. D’Souza,† Robert M. Sharkey,§ Habibe Karacay,§ Edmund A. Rossi,‡
Chien-Hsing Chang,‡ and David M. Goldenberg§
Immunomedics, Inc., Morris Plains, New Jersey 07950, IBC Pharmaceuticals, Inc., Morris Plains, New Jersey 07950, and
Garden State Cancer Center, Center for Molecular Medicine and Immunology, Belleville, New Jersey 07109. Received
March 17, 2010; Revised Manuscript Received May 10, 2010
We reported previously the feasibility to radiolabel peptides with fluorine-18 (18F) using a rapid one-pot method
that first mixes 18F- with Al3+ and then binds the (Al18F)2+ complex to a NOTA ligand on the peptide. In this
report, we examined several new NOTA ligands and determined how temperature, reaction time, and reagent
concentration affected the radiolabeling yield. Four structural variations of the NOTA ligand had isolated
radiolabeling yields ranging from 5.8% to 87% under similar reaction conditions. All of the Al18F NOTA complexes
were stable in vitro in human serum, and those that were tested in vivo also were stable. The radiolabeling
reactions were performed at 100 °C, and the peptides could be labeled in as little as 5 min. The IMP467 peptide
could be labeled up to 115 GBq/µmol (3100 Ci/mmol), with a total reaction and purification time of 30 min
without chromatographic purification.
prosthetic groups through amide bonds, aldehydes, and “click
chemistry” (8–12).
INTRODUCTION
18F is the most commonly used isotope for positron-emission
tomography (PET), due to its nearly ideal imaging properties
(ꢀ+ 0.635 MeV 97%, T1/2 110 min). Conventionally, 18F is
attached to peptides by binding it to a carbon atom (1–4), but
attachments to silicon (5, 6) and boron (7) also have been
reported. Binding to carbon usually involves multistep syntheses,
in some instances taking several hours to complete, which can
be problematic for an isotope with a 110 min half-life.
The most common amide bond-forming reagent has been
N-succinimidyl 4-18F-fluorobenzoate (18F-SFB), but a number
of other groups have been tested (1–3). In some cases, such as
when 18F-labeled active ester amide-forming groups are used,
it may be necessary to protect certain groups on a peptide during
the coupling reaction, after which they are cleaved. The synthesis
of the 18F-SFB reagent and subsequent conjugation to the peptide
requires many synthetic steps and takes about 1.5-3 h (11, 12).
The most common use of 18F-labeled peptides is to image
receptor sites in vivo (2). The number of receptor sites is usually
limited, so a high specific activity is necessary to avoid blocking
target receptors with the excess unlabeled peptide. In most 18F-
peptide applications, the final peptide must be purified by high-
pressure liquid chromatography (HPLC) to separate the unla-
beled peptide from the radiolabeled peptide in order to obtain
the specific activity needed for imaging. In one case (8), the
radiolabeled peptide was generated with a high specific activity
and an excellent yield (79%) without the need for HPLC
purification. However, most of the 18F-peptide radiolabeling
processes are complicated, take several reaction steps, and
require specialized equipment and highly trained personnel. For
example, Table 1 lists the properties of several of the more
commonly reported fluorination procedures. Peptide labeling
through carbon often involves 18F-binding to a prosthetic group
through nucleophilic substitution, usually in 2 or 3 steps, where
the 18F- is first boiled to dryness in the presence of KHCO3
and Kryptofix 2.2.2 (K222), then mixed with acetonitrile and
dried 2 more times (dry-down step). The prosthetic group is
then labeled with 18F- in the presence of excess precursor. The
18F-prosthetic group is purified, attached to the targeting peptide,
and then purified again. This method has been used to attach
A simpler, more efficient 18F-peptide labeling method was
developed by Poethko et al. (10), where a 4-18F-fluorobenzal-
dehyde reagent was conjugated to a peptide through an oxime
linkage in about 75 min, including the dry-down step. The newer
“click chemistry” method attaches 18F-labeled molecules onto
peptides with an acetylene or azide in the presence of a copper
catalyst (8, 9). The reaction between the azide and acetylene
groups forms a triazole connection, which is quite stable and
forms very efficiently on peptides without the need for protecting
groups. Click chemistry produces the 18F-labeled peptides in
good to excellent yield (∼50-79%) in about 30-90 min,
including the dry-down step.
A more recent method of binding 18F to silicon uses isotopic
exchange to displace 19F with 18F (5). Performed at room
temperature in 10 min, this reaction produces the 18F-prosthetic
aldehyde group with high specific activity (225-680 GBq/µmol;
6100-18 400 Ci/mmol). The 18F-labeled aldehyde is subse-
quently conjugated to a peptide and purified by HPLC, and the
purified labeled peptide is obtained within 40 min (including
dry-down) with ∼55% yield. The 18F-silicon approach was
modified subsequently to a single-step process by incorporating
the silicon into the peptide before the labeling reaction (6).
Biodistribution studies in mice with an 18F-silicon-bombesin
derivative showed increasing bone uptake over time (1.35 (
0.47% injected dose (ID)/g at 0.5 h vs 5.14 ( 2.71% ID/g at
4.0 h), suggesting a release of 18F from the peptide, since
unbound 18F is known to localize in bone. HPLC analysis of
urine showed a substantial amount of 18F activity in the void
volume, which may be due to 18F- released from the peptide.
* Corresponding author. William J. McBride. E-mail bmcbride@
immunomedics.com. Immunomedics, Inc., 300 American Road, Morris
Plains, NJ 07950; Phone 973-605-8200ext. 233. Fax 973-605-1340.
† Immunomedics, Inc.
‡ IBC Pharmaceuticals, Inc.
§ Garden State Cancer Center.
10.1021/bc100137x  2010 American Chemical Society
Published on Web 06/14/2010

1332 Bioconjugate Chem., Vol. 21, No. 7, 2010
McBride et al.
Table 1. Summary of Selected 18F-Peptide Labeling Methods
author/ref
Schirrmacher 5 Ho¨hne 6
Marik 8
click
2
Glaser 9
Poethko 10
Wester/Ma¨ding Becaud 13
(11, 12)
McBride
attachment
silicon
silicon
1
click
aldehyde/oxime amide
direct-
substitution
aluminum
complex
1
Rx steps
process
2
2
2
75
many
60+
1
35
40
115-155 30
65b
30c
time (min)a
yield (%)d
HPLC-purification
steps
55
1
13
1
79
50
40
1
10
2
57
1
51
SPE
distillation + 1 + distillation
Sep-Pak
specific activity
(GBq/µmol)
225-680
62
>35
high
high
high
74
115
a Includes dry-down step. b Estimated. c Dry-down step is not required. d Decay-corrected.
Substantial hepatobiliary excretion also was reported, attributed
to the lipophilic nature of the 18F-silicon-binding substrate, and
requiring future derivatives to be more hydrophilic. Methods
of attaching 18F to boron also have been explored; however,
the current process produces conjugates with low specific
activity (7).
suggested that only two carboxyl groups of the NOTA were
needed for the Al18F-NOTA complex (21); and the C-NETA-
containing peptide, IMP467, because the literature had indicated
that this NOTA ligand could have increased binding kinetics
for metals (31). As shown herein, IMP467 proved to be the
best of the 4 NOTA ligands we have evaluated to date, and
thus, additional studies were performed to assess optimal
labeling conditions for IMP467.
A direct 18F-labeling method has also been explored (13).
Here, the peptide contains a trimethylammonium-leaving group
attached to an aromatic ring containing an electron-withdrawing
group. The 18F- is dried down (10 min) with K222/K2CO3 or
Cs2CO3 and heated (50-90 °C) with the peptide for 15 min to
substitute the 18F for the trimethylammonium group. The
reaction mixture is then purified by HPLC (∼10 min) to produce
the high-specific-activity 18F-labeled peptide (74 GBq/µmol) in
20-57% isolated yield. The radiolabeled peptide was stable in
vitro in mouse plasma.
EXPERIMENTAL PROCEDURES
Materials. The p-SCN-Bn-NOTA and TACN were purchased
from Macrocyclics, Inc. (Dallas, TX). The DiBocTACN,
NODA-GA(tBu)3, and NO2AtBu were obtained from CheMat-
ech (Dijon, France). Protected amino acids, other peptide
synthesis reagents, and resins were procured from Creosalus
(Louisville, KY), Chem Impex (Wood Dale, IL), Bachem
(Torrance, CA), and EMD Biosciences (San Diego, CA). The
peptides were synthesized by the Fmoc method either manually
or on a Protein Technologies, Inc. (Tucson, AZ) peptide
synthesizer. The aluminum chloride hexahydrate was acquired
from Sigma-Aldrich (Milwaukee, WI). The remaining solvents
and reagents were obtained from Fisher Scientific (Pittsburgh,
PA) or Sigma-Aldrich. The analytical and preparative reverse-
phase HPLC (RP-HPLC) columns were bought from Phenom-
enex (Torrance, CA) and Waters Corp. (Milford, MA). The Sep-
Pak Light Waters Accell Plus QMA and CM cartridges used to
purify the 18F- and Waters Oasis HLB 1 cm3 flangeless
cartridges, which were used to purify the radiolabeled peptides,
were purchased from Waters Corp. The size exclusion HPLC
(SE-HPLC) column and the AG 1-X8 resin were from Bio-
Rad (Hercules, CA). The Tricorn 5/20 Column was acquired
from GE Healthcare (Piscataway, NJ). 18F- was supplied by
IBA Molecular (Somerset, NJ). Female nude mice (NCr nu-
m), 23.1 ( 2.3 g, were obtained from Taconic Farms, German-
town, NY.
In contrast to these multistep, time-consuming procedures,
antibodies and peptides are radiolabeled routinely with radio-
metals typically in 15 min and in quantitative yields (14, 15).
For PET imaging, copper-64 and, more recently, gallium-68
have been bound to peptides via a chelate, and have shown
reasonably good PET-imaging properties (16). Since fluoride
binds to most metals (17), we sought to determine if an 18F-
metal complex could be bound to a chelate on a targeting
molecule. We focused on the binding of an (Al18F)2+ complex,
since the aluminum-fluoride bond is one of the strongest
fluoride-metal bonds and the (AlF)2+ complex was known to
bind to ligands (18). The AlFn complex is stable in vivo, since
this is part of the mechanism that the body uses to incorporate
fluoride into tooth enamel, and thus, low doses of AlFn should
be compatible for human use (19, 20). We reported previously
initial studies that showed the feasibility of this approach, using
an 18F-labeled peptide for in vivo targeting of cancer with a
bispecific antibody (bsMAb) pretargeting system (21), a pro-
cedure that was shown to be a highly sensitive and specific
technique for localizing cancer, in some cases better than 18F-
FDG (fluorodeoxyglucose) (22–30). In this initial report, we
found that an (Al18F)2+ complex could bind stably to a 1,4,7-
triazacyclononane-1,4,7-triacetic acid (NOTA) ligand, but the
yields were low and the labeled peptide had to be purified by
HPLC to obtain the specific activity needed for the imaging
study.
As shown in this report, we have improved the radiolabeling
yields and specific activity of this NOTA ligand, but have also
prepared several new NOTA ligands to determine if structural
changes would yield further improvements. Our goal is to
develop a one-pot radiolabeling method that provides the
radiolabeled peptide with a specific activity greater than 37 GBq/
µmol with no more than a simple filtration-type solid-phase
extraction (SPE) purification needed before formulation for
injection. Three new NOTA ligands were chosen: the NODA-
GA ligand (IMP460), because it was commercially available
in a form suitable for peptide synthesis; a simple NOT2A
derivative (IMP461) because our experience with IMP449
The recombinant, humanized, tri-Fab bsMAb, TF2, was
provided by IBC Pharmaceuticals, Inc. (Morris Plains, NJ). TF2
binds divalently to carcinoembryonic antigen (CEACAM5 or
CD66e) and monovalently to the synthetic hapten, HSG
(histamine-succinyl-glycine) (30). The bsMAb was >95% im-
munoreactive against CEACAM5 and the divalent-HSG NOTA-
peptide, IMP449, using a SE-HPLC method described previ-
ously (30).
1H and 13C NMR spectra were obtained using a Varian Inova
NMR Spectrometer (Varian Inc., Palo Alto, CA) at 500 MHz
for 1H and 125.7 MHz for 13C at Rutgers University Chemistry
Department (Newark, NJ). Chemical shifts were referenced to
tetramethyl silane. High-resolution mass spectra (HRMS) were
obtained on an Agilent ESI-TOF instrument at the Scripps
Center for Mass Spectrometry (La Jolla, CA) or at Immuno-
medics, Inc. (Morris Plains, NJ).
Methods. Synthesis of IMP460: NODA-GA-D-Ala-D-Lys(HSG)-
D-Tyr-D-Lys(HSG)-NH2. The peptide was synthesized on Sieber

Radiolabeling Peptides with Chelated Al18F
Bioconjugate Chem., Vol. 21, No. 7, 2010 1333
Scheme 2. Synthesis of Succinyl C-NETA Ligand (4)
Scheme 1. Synthesis of Bis-t-butyl NOTA
amide resin with the amino acids and other agents added in the
following order: Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, Aloc
removal, Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH, Trt-
HSG-OH, Aloc removal, Fmoc-D-Ala-OH, and NODA-GA(t-
Bu)3. The peptide was then cleaved and purified by HPLC.
HRMS C61H92N18O18 MH+ calcd 1365.6909, found 1365.6912.
Synthesis of IMP461: NOT2A-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys-
(HSG)-NH2 2-{4,7-bis-tert-butoxycarbonylmethyl-[1,4,7] triazocy-
clononan-1-yl }-acetic acid (Bis-t-butyl-NOTA). The NO2AtBu
(Scheme 1) (0.501 g 1.4 × 10-3 mol) was dissolved in 5 mL
anhydrous acetonitrile. The benzyl-2-bromoacetate (0.222 mL,
1.4 × 10-3 mol) was added to the solution followed by 0.387
g of anhydrous K2CO3. The reaction was stirred at room
temperature overnight, then filtered and concentrated to obtain
0.605 g (86% yield) of the benzyl ester conjugate (1). The crude
product was dissolved in 50 mL of isopropanol, mixed with
0.2 g of 10% Pd/C (under Ar), and placed under 50 psi H2 for
3 days. The product was then filtered and concentrated under
vacuum to obtain 0.462 g of the desired product (2). HRMS
Preparation of Aluminum Acetate Stock Solution. An alumi-
num acetate buffer solution was prepared by dissolving
AlCl3 ·6H2O in a 0.1 M, pH 4, sodium acetate solution to
provide a 2 mM Al-stock solution.
Concentration and Purification of 18F-. Radiochemical-grade
18F- needs to be purified and concentrated before use. We
examined four different SPE purification procedures to process
the 18F- prior to its use.
1
C20H37N3O6 MH+ calcd 416.2755, found 416.2759. H NMR
(500 MHz, CDCl3, 25 °C, TMS) δ 1.45 (s, 18 H), 2.8-3.3 (m,
12 H), 3.44 (s, 4 H), 3.67 (s, 2 H); 13C (125.7 MHz, CDCl3) δ
28.15, 49.75, 52.43, 53.54, 57.25, 58.99, 81.56, 168.77, 170.68.
Synthesis of IMP461. The peptide was synthesized as described
above with Bis-t-butyl-NOTA-OH added last. The peptide was
then cleaved and purified by HPLC to obtain the product. HRMS
C58H88N18O16 MH+ calcd 1293.6698, found 1293.6707.
Most of the radiolabeling procedures were performed using
18F- prepared by a conventional process (32). The 18F- in 2
mL of water was loaded onto a Sep-Pak Light, Waters Accell
QMA Plus Cartridge that was prewashed with 10 mL of 0.4 M
KHCO3, followed by 10 mL water. After loading the 18F- onto
the cartridge, it was washed with 5 mL water to remove any
dissolved metal and radiometal impurities. The isotope was then
eluted with ∼1 mL of 0.4 M KHCO3 in several fractions to
isolate the fraction with the highest concentration of activity.
The eluted fractions were neutralized with 5 µL of glacial acetic
acid per 100 µL of solution to adjust the eluent to pH 4-5.
In the second process, the QMA cartridge was washed with
10 mL pH 8.4, 0.5 M NaOAc followed by 10 mL DI H2O.
18F- was loaded onto the column as described above and eluted
with 1 mL, pH 6, 0.05 M KNO3 in 200 µL fractions with
60-70% of the activity in one of the fractions. No pH
adjustment of this solution was needed.
In the third process, the QMA cartridge was washed with 10
mL pH 8.4, 0.5 M NaOAc followed by 10 mL DI H2O. The
18F- was loaded onto the column as described above and eluted
with 1 mL, pH 5-7, 0.154 M commercial normal saline in 200
µL fractions with 80% of the activity in one of the fractions.
No pH adjustment of this solution was needed.
Finally, we devised a method to prepare a more concentrated
and high-activity 18F- solution, using tandem ion exchange.
Briefly, Tygon tubing (1.27 cm long, 0.64 cm OD) was inserted
into a Tricorn 5/20 column and filled with ∼200 µL of AG
1-X8 resin, 100-200 mesh. The resin was washed with 6 mL
0.4 M K2CO3 followed by 6 mL H2O. A Sep-Pak light Waters
Synthesis of IMP467: tert-Butyl {4-[2-(Bis-tert-butyoxycarbon-
ylmethylamino-3-(4-succinylamidophenyl)propyl]-7-tert-butyoxy-
carbonylmethyl-[1,4,7]triazacyclononan-1-yl}acetate (Succinyl-C-
NETA) (4). To a solution of 3 (31) (Scheme 2) (148.1 mg, 0.202
mmol) in anhydrous CH3CN (3 mL) was added (21.1 mg, 0.210
mmol) succinic anhydride. After 3 h, the solvent was evaporated
and the reaction was purified by preparative RP-HPLC to yield
pure 4 (137.7 mg, 82%) as dark brown oil. HRMS C43H71N5O11
1
MH+ calcd 834.5223, found 834.5221. H NMR (500 MHz,
CDCl3, 25 °C, TMS) δ 1.43 (s, 18 H), 1.44 (s, 18 H), 2.65-2.66
(m, 4 H), 2.85-2.88 (m, 5 H), 3.18-3.32 (m, 10 H), 3.43-3.46
(m, 10 H), 6.99 (d, 2H), 7.44 (d, 2H); 13C (125.7 MHz, CDCl3)
δ 27.95, 28.05, 29.96,31.58, 33.36, 58.10, 58.74, 58.77, 60.69,
81.93, 82.40, 120.76, 129.20, 132.21, 137.26, 170.19, 170.21,
171.61, 171.75, 175.10.
Synthesis of IMP467: C-NETA-succinyl-D-Lys(HSG)-D-Tyr-D-
Lys(HSG)-NH2. IMP467 was made on a Sieber amide resin, as
described above, except when the last Aloc was cleaved, the
tert-butyl{4-[bis-(tert-butoxycarbonylmethyl)amino)-3-(4-suc-
cinylamidophenyl)propyl]-7-tert-butoxycarbonylmethyl[1,4,7]-
triazanonan-1-yl}acetate (4) was added. The peptide was then
cleaved from the resin and purified by RP-HPLC to yield 6.3
mg of IMP 467. HRMS C70H101N19O20 MH+ calcd 1528.7543,
found 1528.7565.

1334 Bioconjugate Chem., Vol. 21, No. 7, 2010
McBride et al.
Accell Plus CM cartridge was washed with DI H2O. Using a
syringe pump, the crude 18F- that was received in a 5 mL syringe
in 2 mL DI H2O flowed slowly through the CM cartridge and
the Tricorn column over ∼5 min followed by a 6 mL wash
with DI H2O through both ion-binding columns. Finally, 0.4 M
K2CO3 was pushed through only the Tricorn column in 50 µL
fractions. Typically, 40-60% of the eluted activity was in one
50 µL fraction. The fractions were collected in 2.0 mL free-
standing screw-cap microcentrifuge tubes containing 5 µL
glacial acetic acid to neutralize the carbonate solution. The
elution vial with the most activity was then used as the reaction
vial.
Comparison of Al18F Radiolabeling Yields of IMP449, IMP460,
IMP461, and IMP467. Three microliters of 2 mM Al3+ stock
solution was added to 60 µL of 18F- (44 MBq) followed by the
addition of 10 µL of 0.05 M peptide solution in pH 4.1, 0.5 M
NaOAc. The four reaction mixtures were formulated and placed
in a 103 °C heating block for 19 min. The reaction mixtures
were purified by an HLB column, as described above, to
determine the radiochemical reaction yield. All of the reaction
yields noted in this work are isolated radiochemical yields.
(AlCl3 ·6H2O) in 0.1 M NaOAc (pH 4), followed by 2 µL 0.01
M IMP467 (20 nmol) in 0.1 M NaOAc (pH 4), and incubated
in a 105 °C heating block for 17 min. The solution was cooled
briefly and diluted with 1 mL H2O, and then placed in an HLB
1-cc (30 mg) cartridge. The solution was eluted under vacuum
into an empty crimp-sealed vial as follows. The reaction vessel
and the column were rinsed with 2 × 1 mL portions of H2O.
The column was moved to a vial containing lyophilized ascorbic
acid (15 mg, buffered at pH 5.5) and eluted with 2 × 200 µL
of 1:1 ethanol/water.
Radiolabeling of IMP467 Using Saline-Eluted 18F-Fluoride
Ion. 18F-fluoride ion (70.3 MBq) in 50 µL saline was mixed
with 2 µL 0.01 M (AlCl3 ·6H2O) in 0.1 M NaOAc (pH 4)
followed by 20 µL IMP467 (2 mM, 40 nmol) in 0.1 M NaOAc
(pH 4), and incubated in a 105 °C heating block for 15 min.
The solution was cooled briefly, diluted with 1 mL PBS (pH
7.4), and then placed in a HLB cartridge. The solution was
purified as described above to obtain 48.3 MBq of the
radiolabeled product in 85% isolated yield. The total reaction
and purification time was 30 min.
Synthesis and Characterization of 19F-IMP467. The 19F-IMP467
was prepared by mixing 20 µL of 2 mM IMP467 in 2 mM
NaOAc buffer, pH 4.18, with 50 µL of 2 mM AlCl3 and 100
µL of 2 mM NaF in the same acetate buffer in a 2 mL screwcap
microcentrifuge vial and heated (sealed) for 5 min at 101 °C.
The sample was then examined by HPLC on a Waters 2695
HPLC system equipped with a Phenomenex Gemini C18 reverse-
phase column (250 × 4.6 mm, 5 µm, 110 Å), using a linear
gradient of 90% A (0.1% TFA) to 20% B (90% acetonitrile,
10% water, 0.1% TFA) over 20 min at a flow rate of 1 mL/
min, absorbance was detected at 254 nm using Waters PDA
2996 detector. The 19F-IMP467 forms two complexes (12.92
and 14.84 min), which were isolated by HPLC. The isolates
were stored in the eluent buffer and reinjected 0.5, 1, 1.5, and
2 h after isolation. The complexes were examined by HPLC on
the Agilent ESI-TOF.
HPLC Separation of 19F-IMP467 from Unreacted Peptide.
The unreacted peptide could be separated from the 19F or 18F-
peptide by using a Phenomenex Kinetex C-18 column, 50 ×
4.60 mm, 2.6 µ, 100A and eluting at 0.6 mL/min using 0.01%
formic acid in 5% acetonitrile/water as Buffer A and 0.01%
formic acid in 90% acetonitrile as Buffer B. The gradient ran
for 1 min with 100% Buffer A, then to 80:20 A/B over 6 min.
Under these conditions, the Al-peptide and the unreacted peptide
were eluted before the fluoride-bound peptides. These were the
HPLC conditions used to examine the peptide reaction mixture
on the Agilent ESI-TOF.
Stability of 18F-IMP467 in Phosphate-Buffered Saline (PBS)
and Human Serum. The purified radiolabeled peptide in 50 µL
1:1 EtOH/H2O was mixed with 150 µL of human serum and
placed in the HPLC autosampler heated to 37 °C. Another
sample of 18F-IMP467 was diluted in the same manner with
PBS and heated to 37 °C. The samples were analyzed by RP-
HPLC.
Biodistribution and in ViVo Stability of 18F-IMP467. All animal
studies were approved in advance by the Institutional Animal
Care and Use Committee of the Center for Molecular Medicine
and Immunology.
Kinetics of 18F-IMP467 Radiolabeling. Three microliters of 2
mM Al3+ stock solution was mixed with 40 µL of 18F- followed
by the addition of 20 µL of 2 mM IMP467 in 0.1 M, pH 4,
acetate buffer. Four reaction mixtures were formulated and
placed in a 107 °C heating block for 5, 10, 15, and 30 min.
The crude products were each purified on a Waters Oasis HLB
1 cm3 (30 mg) Flangeless Cartridge. Briefly, 200 µL of water
was added to the reaction solution, which was then removed
via pipet and drawn into the HLB column. The reaction solution
was drawn into the column. The reaction vessel was rinsed with
1 mL of H2O, which was then transferred and drawn into the
column. The column was eluted with 2 × 200 µL portions of
1:1 EtOH/H2O, with the product being isolated in a 3 mL vial
that contained 15 mg of ascorbic acid that was adjusted to pH
6 and previously lyophilized. The yield was determined by
measuring the activity left in the reaction vessel, remaining on
the HLB column, in the water wash, and in the 1:1 EtOH/H2O.
The amount of activity in the 1:1 EtOH/H2O fraction divided
by the total activity from the other fractions gave the isolated
radiochemical yield. The radiolabeled peptides were then
analyzed by RP-HPLC, which showed that the unbound 18F-
was removed in all cases.
Comparison of 18F-IMP467 Reaction Yield Vs Moles of Pep-
tide Used. Three microliters of 2 mM Al3+ stock solution was
added to 40 µL of 18F- (24 MBq) followed by the addition of
5, 10, 15, or 20 µL of 2 mM IMP467 in 0.1 M, pH 4.1 acetate
buffer and 15, 10, 5, or 0 µL of water, respectively. The reaction
solutions were heated to 99 °C for 15 min, then purified by
HLB column as described above to determine the isolated
radiochemical yield.
Comparison of 18F-IMP467 Reaction Yield Vs Reaction pH. A
CM cartridge (to remove metals) was washed with H2O and
placed upstream from the QMA cartridge. The 18F- was purified
by the nitrate method described above. The peptide and AlCl3
were both dissolved in pH 4, 0.1 M NaOAc. The peptide was
radiolabeled with the purified 18F- in duplicate over the pH range
4-7.2. Each sample was labeled by mixing 20 µL of 18F-, 2
µL of 0.01 M AlCl3 in 0.05 M NaOAc pH 4.9, 20 µL 2 mM
IMP467 in 0.1 M NaOAc pH 6, and 158 µL of 0.1 M reaction
buffer. One sample was purified and the duplicate was allowed
to decay while sealed. The pH of the decayed sample was
measured with a calibrated pH meter equipped with a Calomel
glass microcombination pH electrode.
The stability of 18F-IMP467 was examined by comparing
HPLC elution profiles of the peptide prior to injection to that
found in the urine at 0.5 and 1.5 h after the intravenous injection
18F-IMP467 (18.5 MBq (500 µCi), 2.67 × 10-10 mol in 1%
human serum albumin) in nude mice. The materials were
analyzed by RP-HPLC and by SE-HPLC alone and in the
presence of TF2 anti-CEACAM5 × anti-HSG tri-Fab bsMAb,
Radiolabeling of IMP467 with Carbonate-Eluted 18F-Fluoride.
18F-fluoride (5.39 GBq) in 50 µL 0.4 M K2CO3 was neutralized
with 5 µL glacial acetic acid and then mixed with 1 µL 0.01 M

Radiolabeling Peptides with Chelated Al18F
Bioconjugate Chem., Vol. 21, No. 7, 2010 1335
Table 2. NOTA Ligands and Maximum Isolated Yields after
Radiolabeling with 500 nmol Peptide
(R ) D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2)
Table 3. 18F-IMP467 Yield as a Function of pH
reaction buffer pH
final reaction pH
isolated yield (%)
2.88
3.99
3.96
4.27
4.25
5.05
4.25
6.04
7.23
54
70
77
70
69
41
3.0
5.00
6.00
7.30
found to be stable in human serum, further studies focused on
IMP467 to assess the impact that other aspects of the radiola-
beling procedure might have on the yield and specific activity.
Decreasing the amount IMP467 in the reaction mixture from
500 nmol to 40 nmol reduced the labeling yield to 65-75%,
which was still better than the 44% yield achieved with 500
nmol of IMP449. When the amount of peptide added to the
reaction was varied from 40 to 20 nmol (at concentrations from
0.63 to 0.32 mM), the yields remained fairly constant at around
75-82%, but decreased to 49% when only 10 nmol was used
at a concentration of 0.16 mM. Other studies found that binding
was nearly complete within 5 min at 107 °C (5 min, 68%; 10
min, 61%; 15 min, 71%; and 30 min, 75%), with only moderate
increases in isolated yield with reaction times as long as 30
min, but no binding was achieved with labeling at 50 °C.
In an effort to enhance the specific activity further, we devised
a procedure for purifying and concentrating 18F-, starting with
substantially higher levels of 18F- than were employed in all
previous studies. When using only 20 nmol of IMP467 with
5.39 GBq (145.6 mCi) of 18F-, the labeling yield was 52%
(isolated radiochemical yield, without correcting for decay, 51%
decay-corrected). Thus, this process further improved the
specific activity of the preparation [i.e., the isolated peptide
contained 2.29 GBq (61.9 mCi) of the purified peptide, with an
effective specific activity of 115 GBq/µmol (3100 Ci/mmol)],
albeit at the expense of somewhat reduced, but acceptable,
yields. This radiolabeled product was prepared in 28 min (18F-
post purification delay ) 6 min; reaction time ) 17 min;
purification time ) 5 min). A second experiment using 7 min
heating afforded a similar yield and specific activity in only 14
min. Overall, these studies confirmed that increasing the
concentration of reagents improves the reaction kinetics and the
specific activity of the labeled product, with some reduction in
labeling efficiency.
which was used to illustrate the continued association of the
HSG-hapten with the 18F-IMP467 as an indication of product
stability.
Biodistribution studies were performed in nude mice bearing
subcutaneous LS174T human colon cancer xenografts. Mice in
the pretargeting group received 163 µg (1 nmol) of TF2
intravenously, followed with 18F-IMP467 (3.91 MBq (105 µCi),
0.05 nmol in 1% human serum albumin) 16 h later. Mice were
necropsied 1 and 3 h post-peptide injection, and the tissues were
weighed and co-counted in a gamma scintillation counter with
standards prepared from the injected product. The data are
expressed as the percent-injected dose per gram (% ID/g).
RESULTS
IMP449, a benzyl-NOTA derivative (Table 2) of a peptide
used for pretargeting, was the first chelate that formed a stable
complex with (Al18F)2+ suitable for in vivo targeting (21).
IMP449 was labeled previously by mixing 6 nmol of Al3+ with
18F- in a pH 4 sodium acetate buffer to form (Al18F)2+, then
mixed with 521 nmol of IMP449 (total volume 213 µL) and
heated for 15 min at ∼100 °C. Labeling yields were between
5% and 20% after HPLC purification. We found subsequently
that radiolabeling yields with this derivative could be improved
to as much as 44% by reducing the reaction volume (73 µL)
by one-third. Several products also were observed by RP-HPLC
after radiolabeling the IMP449 peptide. Adding ascorbic acid
to the reaction mixture markedly reduced these side products
(not shown).
Three new NOTA and NOT2A derivatives were synthesized
as part of a pretargeting peptide in an attempt to find a ligand
that would improve labeling yields and maintain stability of
the Al18F-NOTA complex. Table 2 shows the maximum
radiolabeling yields obtained with the three new ligands using
the same conditions that enabled 44% yields with IMP449. One
of the derivatives, IMP460, had only 5.8%, while another,
IMP461, had a 31% yield. However, radiolabeling yields nearly
doubled to 87% with IMP467. The reported improved binding
properties of the ligand may be responsible for the increased
labeling yield (33). While all of these products were tested and
We next assessed how the concentration of Al3+ might impact
the labeling yields. When IMP467 (40 nmol) was labeled in
the presence of increasing amounts of Al3+ (0, 5, 10, 15, 20 µL
of 2 mM Al in pH 4 acetate buffer and keeping the total volume
constant), yields of 3.5%, 80%, 77%, 78%, and 74%, respec-
tively, were achieved. These results indicated that (a) nonspecific
binding of 18F- to this peptide in the absence of Al3+ was low,
(b) 10 nmol of Al3+ was sufficient to allow for maximum 18F-
binding, and (c) higher amounts of Al3+ did not reduce binding
substantially, indicating that there was sufficient chelation
capacity at this peptide concentration.
The optimal pH for labeling was between 4.3 and 5.5 (Table
3). The process could be expedited by eluting the 18F- from
the anion exchange column with nitrate or chloride ion instead
of carbonate ion, which eliminates the need to adjust the eluent
to pH 4 with glacial acetic acid before mixing with the AlCl3.
RP-HPLC showed unlabeled IMP467 elutes as a single peak
at 13.454 min (Figure 1A), but when radiolabeled, 18F-IMP467
elutes as two peaks 13.399 and 15.388 min (Figure 1B),
suggesting that the aluminum fluoride forms two complexes with
the peptide. To examine this further, we prepared and character-
ized cold 19F-IMP467 by RP-HPLC (UV) and HPLC-mass
spectroscopy (ESI-TOF). The HPLC of the 19F-IMP467 (Figure
1C) confirmed that the 19F-IMP467 peaks 12.927 and 14.887

1336 Bioconjugate Chem., Vol. 21, No. 7, 2010
McBride et al.
Figure 1. RP-HPLC analysis of IMP467, 18F-IMP467, and 19F-IMP467. The unlabeled peptide elutes as a single peak (A), but when radiolabeled
with Al18F (18F-IMP467), two peaks are observed (B). 19F-IMP467 also showed two main peaks (C) [13.4 min peak is unlabeled peptide as seen
in (A)]. The 14.9 min peak was isolated and evaluated over time (D-F), showing the equilibration of the isomeric counterpart (∼13.0 min).
min corresponded to the same peaks seen with the 18F-IMP467
with the retention times of the radiometric detector being slightly
later since it is downstream from the UV detector. When this
reaction mixture was analyzed by HPLC-mass spectrometry
(high resolution), the results confirmed that each of the two
peaks contained the peptide, one aluminum, and one fluorine
(ESI-TOF C70H99FN19O20Al calcd 1571.7113 found 786.8635
[M+2H]2+).
The individual 18F and 19F peaks could be isolated by HPLC.
Over time, the isolated isomers interconvert, as shown in Figure
1C-F, where the ∼14.9 min 19F-IMP467 product was isolated
and then re-evaluated over 3 h. This interconversion also was
seen previously with the simple NOT2A ligand (34). Under the
HPLC conditions shown in Figure 1, the unlabeled peptide is
eluted between the two 19F-IMP467 complexes (∼13.4 min).
This peak also was seen to develop over the 3 h evaluation of

Radiolabeling Peptides with Chelated Al18F
Bioconjugate Chem., Vol. 21, No. 7, 2010 1337
Figure 3. Binding of 18F-IMP467 to TF2 by SE-HPLC: (A) 18F-IMP467
after HLB purification, alone, or (B) mixed with TF2 anti-CEA × anti-
HSG bsMAb.
and 3 h, respectively (21). Tumor uptake in the pretargeted
animals 1 h after injection of the 18F-IMP467 was nearly 50×
higher than that observed with the 18F-IMP467 alone, providing
tumor/nontumor ratios of ∼100:1 for the blood, liver, and other
tissues, and even 5:1 for the kidney, indicating the suitability
of this method to prepare functionally active and stable 18F-
labeled peptides for immunoPET imaging.
Figure 2. Serum stability of IMP467 as analyzed by RP-HPLC: (A)
18F-IMP467 in serum at time-zero, (B) 18F-IMP467 in serum after 1 h,
(C) 18F-IMP467 in serum after 4 h.
DISCUSSION
the isolated 14.9 min 19F-IMP467 peak, suggesting some loss
of Al19F from the product over time when held in 0.1% TFA
buffer. Al-peptide complexes are eluted before these peaks (i.e.,
between 9.8 and 12.2 min). If a different C-18 column is used
(Phenomenex Kinetex C-18), the Al peptide complexes and the
unreacted peptide are eluted first followed by the 18F- or 19F-
complexed peptide. The purification on the Kinetex column
would permit isolation of the labeled peptide if further increases
in effective specific activity were desired (data not shown).
Stability studies with 18F-IMP467 were performed in PBS
and initially, in previously frozen human serum. At time zero,
both samples showed no detectable 18F- above background at
the void volume of the column. At 5.5 h, the PBS sample had
2.3% of the 18F- activity in the void volume. At 1 and 4 h,
there was no detectable activity in the void volume, but the
ratio of the two radiolabeled peaks changed over time, with a
higher portion found in the second peak (Figure 2). The serum
stability study was later repeated in fresh human serum at 37
°C for 5 h and the RP-HPLC indicated that 0.5% of the injected
activity was present at the void volume of the column (not
shown). 18F-IMP467 analyzed by SE-HPLC showed that nearly
all of the activity shifted to a shorter retention time on addition
of TF2, indicating that the binding of the Al18F complex did
not compromise the binding of the HSG hapten (Figure 3).
In order to confirm the suitability of 18F-IMP467 for in vivo
use, biodistribution studies were performed in nude mice using
the peptide alone or in a pretargeting setting (21). The peptide
eliminated in the urine had an identical RP-HPLC elution profile
as the 18F-IMP467 that was injected, and SE-HPLC further
showed the labeled peptide contained the HSG-hapten used to
bind the bsMAb (Figure 4). These results indicate that the
peptide was excreted intact, indicating no dissociation of the
radionuclide. As shown in Table 4, the peptide cleared quickly
from the blood (∼0.1% ID/g at 1 h), with little residual activity
in the tissues and low renal retention (∼2% ID/g). Bone uptake
was also low, averaging 0.4-0.5%, as compared to 6% and
9% ID/g that were reported previously for 18F and Al18F at 1
Molecular imaging has become an important priority, with
far-reaching implications in many fields of biomedical research
and clinical practice. PET imaging represents one of the more
sensitive imaging modalities, with the ability to view the whole-
body distribution of a radiotagged compound. A number of
different positron-emitting radionuclides have been used for PET
imaging, but 18F is preferred because of its ideal imaging
properties, and it is also widely available and relatively
inexpensive because of the extensive use of 18F-fluorodexoy-
glucose in oncology and neurobiology. However, coupling 18
F
to compounds has been a challenge. Currently, there are a
number of groups exploring 68Ga, because products can be
prepared at high specific activities, and also because this
radionuclide can be readily coupled to compounds through
simple chelation chemistry that has been widely used for
radiometal labeling over the past 25 years (14–16). We
envisioned that a similar process could be applied to metal-
fluorine complex that would permit more facile radiolabeling
of compounds with 18F and reported previously the initial
success of this approach applied to a NOTA-containing peptide
used in a pretargeting procedure (21). However, radiolabeling
yields were low (5-20%), and HPLC purification was required
to isolate the 18F-peptide from the unlabeled peptide.
We now report improvements to this radiolabeling procedure
by changing the structure of the core NOTA compound and
the reaction conditions. This method can be performed by simply
mixing Al3+(6-40 nmol) with 18F- in pH 4-5.5 acetate buffer
with a 5 min incubation, followed by an HLB cartridge
purification to remove unbound 18F- and (AlFn18F). Using these
improvements, we have been able to label IMP467 with a
specific activity as high as 115 GBq/µmol (3100 Ci/mmol) in
e30 min without HPLC purification, but this required a more
concentrated and high-activity 18F- than what is commonly
supplied by the commercial vendor and resulted in somewhat
lower yields. Purification of the 18F- to remove trace metals is
important here, as well as in other methods. Importantly, the

1338 Bioconjugate Chem., Vol. 21, No. 7, 2010
McBride et al.
Figure 4. In vivo stability of 18F-IMP467. SE- and RP-HPLC analysis of 18F-IMP467 before injection and in urine samples taken from mice 0.5
and 1.5 h postinjection. RP-HPLC shows the same elution profile in the original product and in the urine, while SE-HPLC was performed to show
18F-IMP467 eliminated in urine continued to retain binding to the TF2 anti-CEACAM5 × anti-HSG bsMAb.
Table 4. Biodistribution of 18F-IMP467 Alone or Pretargeted by
provided highly favorable tumor localization with the pretar-
geting method (21). Some of the new peptides developed for
TF2 Anti-CEACAM5 × Anti-HSG bsMAba
this work had a much greater separation between the Al-peptide
and the Al18F peptide, making the HPLC purification to obtain
high-specific-activity 18F-peptides somewhat easier. More re-
cently, NOT2A-octreotide (34) and NOT2A-bombesin (35)
derivatives bearing the same simple NOT2A ligand as IMP461
were prepared and labeled with (Al18F)2+. As with IMP461,
radiolabeling yields were in the same range, and in both of these
situations, HPLC purification was able to separate radiolabeled
from unlabeled peptide to improve the specific activity, but we
suspect that higher specific activities may be possible by
modifying the NOTA ligand. As indicated herein, radiolabeling
yields vary from as low as 6% to as high as 87%, based on the
NOTA ligand. The higher labeling yield (87%) observed for
IMP467 may be due to the increased binding kinetics of the
ligand (33).
percent-injected dose per gram tissue (mean ( SD; N ) 5)
TF2-pretargeted TF2-pretargeted 18F-IMP467 18F-IMP467
tissue
LS174T
liver
spleen
kidney
lung
blood
stomach
small intestine
large intestine
scapula
muscle
brain
18F-IMP467 1 h 18F-IMP467 3 h
1 h alone
3 h alone
11.8 ( 2.97
0.29 ( 0.32
0.26 ( 0.19
2.28 ( 0.47
0.29 ( 0.18
0.12 ( 0.06
0.13 ( 0.06
0.46 ( 0.12
0.11 ( 0.09
0.57 ( 0.13
0.54 ( 0.88
0.02 ( 0.00
8.16 ( 4.83
0.09 ( 0.02
0.11 ( 0.04
1.98 ( 0.35
0.08 ( 0.02
0.02 ( 0.01
0.05 ( 0.04
0.12 ( 0.03
0.27 ( 0.11
0.41 ( 0.08
0.03 ( 0.04
0.02 ( 0.02
0.23 ( 0.11 0.09 ( 0.05
0.07 ( 0.01 0.06 ( 0.01
0.05 ( 0.01 0.03 ( 0.01
2.47 ( 0.56 1.96 ( 0.55
0.12 ( 0.02 0.06 ( 0.01
0.08 ( 0.05 0.01 ( 0.00
0.03 ( 0.02 0.03 ( 0.01
0.21 ( 0.05 0.13 ( 0.04
0.07 ( 0.02 0.20 ( 0.09
0.40 ( 0.11 0.44 ( 0.14
0.03 ( 0.01 0.02 ( 0.01
0.01 ( 0.00 0.01 ( 0.00
a Nude mice bearing LS174T human colonic cancer xenografts were
necropsied 1 and 3 h after 18F-IMP467 injection (TF2 given 16 h
earlier).
In our initial studies, many known metal-binding ligands were
examined for their suitability for complexing (Al18F)2+ (21).
Diethylenetriaminepenatacetic acid-(DTPA-Al18F) complexes
were formed in >90% yield, but they were not stable in vitro.
Other ligands known to bind Al3+ were tested but also were
unsuitable. Andre´ et al. reported that the Al-NOTA complex
was stable (36), and in our testing, all the NOTA-Al18F
compounds tested to date showed a high level of stability in
serum, and both IMP449 and IMP467 were stable in vivo. A
recent study also found the targeting of 18F-IMP449 was similar
to the same peptide radiolabeled with 68Ga, attesting to the
stability of the 18F-product (29). While the HPLC analysis of
the 14.9 min peak from the 19F-IMP467 showed some formation
of the unlabeled peptide (peak ∼13.4 min) over time, this
product was isolated in 0.1% TFA buffer, which is certainly
not the condition in which 18F-IMP467 would be held for in
labeling process could be performed by eluting 18F-fluoride with
commercial sterile saline, with optimal yields occurring when
mixing 71 MBq of purified 18F- with 20 nmol Al and 40 nmol
IMP467 in a pH 4.3-5.5 acetate buffer in a total volume of
100 µL, heating to 90 to 110 °C for 15 min, and performing
SPE separation to remove unbound 18F- from the radiolabeled
peptide.
As mentioned, the specific activity and yields are contingent
on the concentration of the reaction mixture, but HPLC
purification also could be used to isolate the Al18F-labeled
peptide from the Al-peptide/peptide if higher specific activities
are necessary. In the case of IMP449, Al-IMP449 had about
the same HPLC retention time as the 18F-IMP449, but it could
be purified to a specific activity of about 1300 Ci/mmol, which

Radiolabeling Peptides with Chelated Al18F
Bioconjugate Chem., Vol. 21, No. 7, 2010 1339
vivo use. Indeed, in PBS, only ∼2% loss of 18F occurred, and
when held in serum over 4 h, no detectable loss of 18F was
observed, but there was a noticeable change in the proportion
of the product in the early eluting peak to the later-eluting peak.
The in vivo studies also clearly support the suitability of the
product’s stability, with very low uptake in bone.
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P., Overall, C. M., Adam, M. J., Ruth, T. J., and Perrin, D. M.
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two peaks changed over time, but this isomerization did not
result in the loss of 18F. Al3+ forms octahedral complexes with
four binding sites in a plane and two axial binding sites. Many
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CONCLUSION
In conclusion, C-NETA-containing peptides, as exemplified
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in high yield (up to 85%) via Al-bound 18F at a specific activity
of up to 115 GBq/µmol, without requiring HPLC purification.
The resulting 18F-IMP467 is stable in human serum and suitable
for in vivo pretargeting applications. The use of the 18F- saline
as a source of purified 18F-fluoride simplifies the labeling
process. We anticipate that similar results can be achieved with
a broad spectrum of peptides derivatized with C-NETA. This
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specific-activity 18F-labeled peptides using conventional equip-
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requiring only the addition of 18F- to a peptide-aluminum
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ACKNOWLEDGMENT
This work was funded in part by NIH grants 9R44 RR028018
and 2R44CA123985. Disclaimer: WJM, CAD, EAR, C-HC, and
DMG are employed or have financial interest in Immunomedics,
Inc., or IBC Pharmaceuticals, Inc. RMS and HK have disclosed
no financial conflicts.
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