A novel radiofluorinated agouti-related protein for tumor angiogenesis imaging

Article abstract of DOI:10.1007/s00726-012-1391-y

A novel protein scaffold based on the cystine knot domain of the agouti-related protein (AgRP) has been used to engineer mutants that can bind to the α_vβ₃ integrin receptor with high affinity and specificity. In the current study, an ¹⁸F-labeled AgRP mutant (7C) was prepared and evaluated as a positron emission tomography (PET) probe for imaging tumor angiogenesis. AgRP-7C was synthesized by solid phase peptide synthesis and site-specifically conjugated with 4-nitrophenyl 2- ^18/19F-fluoropropionate (^18/19F-NFP) to produce the fluorinated peptide, ^18/19F-FP-AgRP-7C. Competition binding assays were used to measure the relative affinities of AgRP-7C and ¹⁹F-FP- AgRP-7C to human glioblastoma U87MG cells that overexpress α _vβ₃ integrin. In addition, biodistribution, metabolic stability, and small animal PET imaging studies were conducted with ¹⁸F-FP-AgRP-7C using U87MG tumor-bearing mice. Both AgRP-7C and ¹⁹F-FP-AgRP-7C specifically competed with ¹²⁵I-echistatin for binding to U87MG cells with half maximal inhibitory concentration (IC ₅₀) values of 9.40 and 8.37 nM, respectively. Non-invasive small animal PET imaging revealed that ¹⁸F-FP-AgRP-7C exhibited rapid and good tumor uptake (3.24 percentage injected dose per gram [% ID/g] at 0.5 h post injection [p.i.]). The probe was rapidly cleared from the blood and from most organs, resulting in excellent tumor-to-normal tissue contrasts. Tumor uptake and rapid clearance were further confirmed with biodistribution studies. Furthermore, co-injection of ¹⁸F-FP-AgRP-7C with a large molar excess of blocking peptide c(RGDyK) significantly inhibited tumor uptake in U87MG xenograft models, demonstrating the integrin-targeting specificity of the probe. Metabolite assays showed that the probe had high stability, making it suitable for in vivo applications. ¹⁸F-FP-AgRP-7C exhibits promising in vivo properties such as rapid tumor targeting, good tumor uptake, and excellent tumor-to-normal tissue ratios, and warrants further investigation as a novel PET probe for imaging tumor angiogenesis.

Full text of DOI:10.1007/s00726-012-1391-y

Amino Acids (2013) 44:673–681
DOI 10.1007/s00726-012-1391-y
ORIGINAL ARTICLE
A novel radiofluorinated agouti-related protein for tumor
angiogenesis imaging
•
Han Jiang Sarah J. Moore Shuanglong Liu Hongguang Liu
•
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•
•
•
•
Zheng Miao Frank V. Cochran Yang Liu Mei Tian
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Jennifer R. Cochran Hong Zhang Zhen Cheng
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Received: 20 May 2012 / Accepted: 20 August 2012 / Published online: 4 September 2012
Ó Springer-Verlag 2012
Abstract A novel protein scaffold based on the cystine
knot domain of the agouti-related protein (AgRP) has been
used to engineer mutants that can bind to the avb3 integrin
receptor with high affinity and specificity. In the current
study, an 18F-labeled AgRP mutant (7C) was prepared and
evaluated as a positron emission tomography (PET) probe
for imaging tumor angiogenesis. AgRP-7C was synthesized
by solid phase peptide synthesis and site-specifically
conjugated with 4-nitrophenyl 2-18/19F-fluoropropionate
and 8.37 nM, respectively. Non-invasive small animal PET
imaging revealed that 18F-FP-AgRP-7C exhibited rapid
and good tumor uptake (3.24 percentage injected dose per
gram [% ID/g] at 0.5 h post injection [p.i.]). The probe was
rapidly cleared from the blood and from most organs,
resulting in excellent tumor-to-normal tissue contrasts.
Tumor uptake and rapid clearance were further confirmed
with biodistribution studies. Furthermore, co-injection of
18F-FP-AgRP-7C with a large molar excess of blocking
peptide c(RGDyK) significantly inhibited tumor uptake in
U87MG xenograft models, demonstrating the integrin-
targeting specificity of the probe. Metabolite assays
showed that the probe had high stability, making it suitable
for in vivo applications. 18F-FP-AgRP-7C exhibits prom-
ising in vivo properties such as rapid tumor targeting, good
tumor uptake, and excellent tumor-to-normal tissue ratios,
and warrants further investigation as a novel PET probe for
imaging tumor angiogenesis.
(
18/19F-NFP) to produce the fluorinated peptide, 18/19F-FP-
AgRP-7C. Competition binding assays were used to measure
the relative affinities of AgRP-7C and 19F-FP-AgRP-7C to
human glioblastoma U87MG cells that overexpress avb3
integrin. In addition, biodistribution, metabolic stability,
and small animal PET imaging studies were conducted
with 18F-FP-AgRP-7C using U87MG tumor-bearing mice.
Both AgRP-7C and 19F-FP-AgRP-7C specifically com-
peted with 125I-echistatin for binding to U87MG cells with
half maximal inhibitory concentration (IC50) values of 9.40
Keywords Knottin Á avb3 integrin Á PET Á 18F
H. Jiang Á Y. Liu Á M. Tian Á H. Zhang (&)
Department of Nuclear Medicine, Second Affiliated Hospital
of Zhejiang University School of Medicine, Institute of Nuclear
Medicine and Molecular Imaging of Zhejiang University,
Key Laboratory of Medical Molecular Imaging of Zhejiang
Province, Hangzhou 310009, China
Introduction
Small protein scaffolds are constrained polypeptides con-
sisting of structural elements such as a-helix, b-sheet, and
loop structures, with size ranges of 3–20 kDa (Miao et al.
2010). Rational and combinatorial methods have been used
to engineer these scaffolds to bind to tumor-related
receptors with low nanomolar or picomolar affinity. Tumor
targets have included avb3 integrin, epidermal growth
factor receptor, human epidermal growth factor receptor 2,
tumor necrosis factor alpha, and carcinoembryonic antigen
(Miao et al. 2010). Recently, engineered protein scaffolds
have demonstrated much promise in the area of molecular
e-mail: hzhang21@gmail.com
H. Jiang Á S. Liu Á H. Liu Á Z. Miao Á Y. Liu Á Z. Cheng (&)
Department of Radiology, Molecular Imaging Program at
Stanford (MIPS), Bio-X Program and Canary Center at Stanford
for Cancer Early Detection, Stanford University,
Stanford, CA 94305, USA
e-mail: zcheng@stanford.edu
S. J. Moore Á F. V. Cochran Á J. R. Cochran
Department of Bioengineering, Cancer Institute, Bio-X Program,
Stanford University, Stanford, CA 94305, USA
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H. Jiang et al.
probe development. A myriad of protein scaffolds,
including affibodies, cystine knot peptides, and ankyrin
repeat proteins, have been engineered to bind to various
tumor biomarkers and evaluated as in vivo molecular
imaging agents (Cheng et al. 2008; Huang et al. 2008;
Kimura et al. 2009a). These small protein scaffolds are
highly structured and possess favorable properties such as
rapid tumor accumulation, fast blood clearance, high in
vivo stability, excellent biocompatibility, low immunoge-
nicity and toxicity (Cheng et al. 2008, 2010; Miao et al.
2009; Ren et al. 2009).
evaluated in nude mice bearing subcutaneous integrin-
positive human glioblastoma U87MG xenografts.
Materials and methods
General
All 9-fluorenylmethyloxycarbonyl (Fmoc) protected amino
acids were purchased from Novabiochem/EMD Chemi-
cals Inc (La Jolla, CA) or CS Bio (Menlo Park, CA).
125I-labeled echistatin was purchased from GE Healthcare
Life Sciences (Piscataway, NJ). Phosphate buffered saline
(PBS, 0.01 M, pH 7.4) was obtained from Gibco/Invitro-
gen (Carlsbad, CA). All other chemicals were purchased
from Fisher Scientific (Fair Lawn, NJ) unless otherwise
specified. The U87MG human glioblastoma cell line was
obtained from American Type Culture Collection
(Manassas, VA). Female athymic nude mice (nu/nu) were
purchased from Charles River Laboratory (Wilmington,
MA).
Cystine knots (also known as knottins) are small, con-
strained polypeptides that share a common disulfide-bon-
ded framework and triple-stranded b-sheet fold and possess
one or more surface-exposed loops that bind diverse bio-
logical targets (Daly and Craik 2011; Kolmar 2010) The
disulfide-bonded knottin core confers high stability in
various chemical, physical, and biological environments.
Furthermore, their relatively short amino acid sequences
(typically 30–50 residues) allow them to be produced by
chemical means such as solid-phase peptide synthesis.
Several engineered knottins have been reported in the lit-
erature that bind to tumor-associated integrin receptors,
including mutants derived from the Ecballium elaterium
trypsin inhibitor II (EETI II) (Kimura et al. 2009a, b, 2010,
2011), a-amylase inhibitor tendamistat (Li et al. 2003), and
agouti-related protein (AgRP) (Silverman et al. 2009).
The native AgRP is a neuropeptide produced in the
human brain that plays an important role in the regulation
of appetite and metabolism (Ollmann et al. 1997; Shutter
et al. 1997; Backberg et al. 2004). A truncated form of
AgRP (AgRP*), a 4-kDa cystine-knot peptide with four
disulfide bonds and four solvent-exposed loops, has been
successfully used as a scaffold for engineering peptides
that bind to tumor angiogenesis biomarkers, such as avb3
integrins, with high affinity and specificity (Silverman et al.
2009). AgRP* has several distinct advantages as a scaffold
for engineering tumor targeting agents (Kolmar 2010). The
protein is of human origin, thus is expected to have low
immunogenicity. In addition, its small size (*4 kDa) will
impart fast blood clearance which is desirable for molec-
ular imaging applications. The rigid AgRP* structure
contains four solvent-accessible loops that could be muta-
ted to engineer novel binding function. Finally, engineered
AgRP-based knottins are amenable to recombinant or
synthetic production, which allows site-specific incorpo-
ration of labels or chemical functionality for various
applications.
Semipreparative reversed-phase high performance
liquid chromatography (RP-HPLC) using a Vydac protein
and peptide column (218TP510; 5 lm, 250 9 10 mm) was
performed on a Dionex 680 chromatography system with a
UVD 170U absorbance detector and model 105S single-
channel radiation detector (Carroll & Ramsey Associates,
Berkley, CA). The recorded data were processed with use
of Chromeleon version 6.50 software (Sunnyvale, CA).
With a flow rate of 5.0 mL/min, the mobile phase was
changed from 95 % solvent A (0.1 % trifluoroacetic acid
[TFA] in water) and 5 % solvent B (0.1 % TFA in aceto-
nitrile [MeCN]) to 35 % solvent A and 65 % solvent B
over 32 min. Analytical HPLC had the same gradient
system except that the flow rate was 1.0 mL/min with a
Vydac protein and peptide column (218TP510; 5 lm,
250 9 4.6 mm). Absorbance was monitored at 218 nm,
and peptide identification was based on the UV spectrum
acquired using a PDA detector. All instruments, including
matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry (MALDI-TOF–MS) instruments, RP-
HPLC equipment including a radioaction detector, and a
PET dose calibrator, were the same as those described
previously (Cheng et al. 2007b).
Chemistry and radiochemistry
Linear AgRP-7C (sequence shown in Fig. 1) was synthe-
sized with a CS BioCS336 instrument using Fmoc-based
solid-phase peptide synthesis, and deprotected and cleaved
from resin as reported previously (Jiang et al. 2010).
Without intermediate purification, precursor peptide was
oxidized and folded in 4 M guanidinium chloride, 10 mM
In this study, for the first time, an AgRP knottin peptide
(AgRP-7C) was site-specifically conjugated with a small
18F prosthetic group, 4-nitrophenyl 2-18F-fluoropropionate
(18F-NFP), through the peptide N-terminal amino group
(Fig. 1). The resulting probe, 18F-FP-AgRP-7C, was
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A novel radiofluorinated AgRP for tumor imaging
675
Fig. 1 Schematic and
sequence of AgRP. a Structure
of a radiofluorinated AgRP
knottin scaffold. The imaging
label 18F-NFP was site-
specifically conjugated to the N
terminus of the knottin peptide.
b Amino acid sequence of
AgRP-7C with disulfide bonds
indicating the connectivity
between Cys1-Cys4, Cys2-Cy5,
Cys3-Cys8, and Cys6-Cys7.
Yellow lines represent the
disulfide bonds. c Synthetic
scheme for 18F-NFP synthon
and knottin radiofluorination
reduced glutathione, 2 mM oxidized glutathione, and 3.5 %
(v/v) dimethylsulfoxide (DMSO) at pH 8.0 in ammonium
bicarbonate buffer at room temperature for 3 days with gentle
mixing. Folded peptide was purified on a Vydac C18 prepa-
ratory scale column to[95 % purity and lyophilized.
18F-NFP was prepared and used for AgRP-7C radiola-
beling according to a previously reported protocol (Liu et al.
2010). 18F-NFP (specific activity of 40–100 GBq/lmol, at
the end of synthesis in 100 lL of DMSO) and 10 lL of
DIPEA were added to 200 lg of AgRP-7C peptide and
reacted for 20 min at 60 °C. After 1 mL of water containing
50 lL of TFA was added to quench the reaction, the radi-
olabeled product was separated by semipreparative HPLC
using the same elution gradient as for 19F-FP-AgRP-7C
purification. The HPLC fractions containing 18F-FP-AgRP-
7C were collected and dried with a rotary evaporator. The
radiolabeled peptide was reconstituted in PBS and passed
through a 0.22–lm millipore (Billerica, MA) filter into a
sterile vial for in vitro and in vivo experiments.
The synthesis of 4-nitrophenyl 2-fluoropropionate
(19F-NFP) is briefly described as follows: To a solution of
2-fluoropropionic acid (5.0 mg, 54.3 lmol) in 200 lL of
dimethylformamide, bis(4-nitrophenyl) carbonate (15.2
mg, 50.0 lmol) and diisopropylethylamine (DIPEA;
20 lL) were added. After incubating at 60 °C for 3 h, the
reaction mixture was cooled to room temperature and
diluted with 1 mL of 5 % acetic acid solution. The product
19F-NFP was isolated by semipreparative RP-HPLC. The
collected fractions were combined, and the solvent was
removed using a rotary evaporator. The product obtained
was a white powder (5.9 mg, 56 %). ESI–MS: m/z 214.3
[MH]?; 1H NMR (CDCl3, 300 MHz): d = 8.24 (d,
J = 9.0 Hz, 2H), 7.27 (d, J = 9.0 Hz, 2H), 5.21 (m, 1H),
1.70 (dd, J = 6.8 Hz, 23.4 Hz, 3H). 13C NMR (CDCl3,
75 MHz): d = 18.9 (d, J = 22.5 Hz), 86.0 (d, J =
184.0 Hz), 122.9, 126.0, 146.4, 155.2, 168.5. 19F-NFP
conjugated AgRP-7C (19F-FP-AgRP-7C) was prepared as a
reference compound. Briefly, AgRP-7C (0.5 mg, 0.12 lmol)
was dissolved in 400 lL of dimethyl sulfoxide (DMSO) and
19F-NFP (0.3 mg, 1.5 lmol) was dissolved in 100 lL of
DMSO, and both solutions, along with 10 lL of DIPEA, were
reacted for 1 h at room temperature. The resulting conjugate,
19F-FP-AgRP-7C, was purified by the semipreparative RP-
HPLC, and fractions containing the product were collected and
lyophilized. The identity of 19F-FP-AgRP-7C was confirmed
by MALDI-TOF–MS.
Cell culture and animal model
U87MG cells were cultured in Dulbecco’s modified
Eagle’s high-glucose medium (GIBCO, Carlsbad, CA) and
supplemented with 10 % fetal bovine serum (FBS) and
1 % penicillin–streptomycin in a humidified incubator
containing 5 % CO2 at 37 °C. A 70–80 % confluent
monolayer was detached with 0.25 % trypsin–ethylene-
diaminetetraacetic acid (EDTA) and dissociated into a
single-cell suspension for further cell culture and assays.
All animal studies were carried out in compliance with
federal and local institutional rules for the conduct of
animal experimentation. Approximately 5 9 106 cultured
U87MG cells were suspended in 100 lL of PBS and sub-
cutaneously implanted in the right shoulders of nude mice.
Tumors were grown to a size of 0.5–1 cm in diameter
(2–3 weeks).
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H. Jiang et al.
U87MG cell uptake assay
percent injected radioactive dose per gram of tissue (% ID/g)
values were determined by dividing counts per gram per
minute by injected dose. No attenuation correction was
performed.
Cell uptake studies were performed as previously described
(Cheng et al. 2007a). Briefly, U87MG cells (3 9 105) were
seeded in 12-well tissue culture plates and allowed to
attach overnight. After a wash with 0.01 M PBS, the cells
were incubated with 18F-FP-AgRP-7C (37 kBq, 1 lCi per
well, in culture medium) with or without c(RGDyK)
(2 mg/well) at 37 °C for 1 and 2 h. The cells were then
washed 3 times with PBS and lysed in 1 mL of 1.0 M
NaOH and 0.1 % sodium dodecyl sulfate. Radioactivity
was measured by using a c-counter (PerkinElmer 1470,
Waltham, MA). Cell uptake was expressed as the per-
centage of added radioactivity. Experiments were per-
formed twice with triplicate wells.
Biodistribution studies
For biodistribution studies, nude mice bearing U87MG
xenografts (n = 4 per group) were injected via the tail vein
with approximately 3.7 MBq (100 lCi) of 18F-FP-AgRP-
7C with or without 10 mg of c(RGDyK) per kg of mouse
body weight and euthanized at 2 h p.i. Tumor and normal
tissues of interest were removed and weighed, and their
radioactivity was measured in a c-counter. Radioactivity
uptake was expressed as the % ID/g.
U87MG cell binding assay
In vivo metabolite analysis
The in vivo metabolic stability of 18F-FP-AgRP-7C was
determined from samples recovered from the tumor and
several murine organs using a procedure described previ-
ously (Jiang et al. 2010). Briefly, 18F-FP-AgRP-7C was
injected into U87MG tumor-bearing mice via the tail vein,
and the mice were euthanized at 1 h p.i. Urine was col-
lected, and tumor, liver, and kidneys were homogenized
and extracted with PBS and dimethylformamide (500 lL
each). After centrifugation in Costar nylon filter tubes,
samples were analyzed by reversed-phase HPLC using a
radioactive detector (radio-HPLC), and compared to the
untreated radiolabeled peptide.
Cell binding assays were performed as previously descri-
bed (Miao et al. 2009). Briefly, U87MG cells (2 9 105)
were incubated with 0.06 nM 125I-echistatin and varying
concentrations of peptides (AgRP-7C and 19F-FP-AgRP-
7C) in integrin-binding buffer (25 mM Tris pH 7.4,
150 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2,
and 0.1 % bovine serum albumin) at room temperature for
1 h. The cell-bound radioactivity remaining after washing
was determined by c-counting. The data were analyzed
with use of GraphPad Prism (GraphPad Software, Inc. La
Jolla, CA), and the concentration of unlabeled peptide
required to inhibit 50 % of the 125I-echistatin binding (IC50
value) was calculated. The experiment was performed with
quadruplicate samples.
Statistical methods
Small-animal PET imaging
Statistical analysis was performed using the Student’s t test
for unpaired data. A 95 % confidence level was chosen to
determine the significance between groups, with P values
of less than 0.05 being designated as significantly different.
Small-animal PET imaging was performed on a micro-PET
R4 rodent model scanner (Concorde Microsystems Inc.
Knoxville, TN). Mice bearing U87MG (n = 4 for each
group) xenografts were injected via the tail vein with
approximately 3.7 MBq (100 lCi) of 18F-FP-AgRP-7C
with or without 10 mg of c(RGDyK) per kg of mouse body
weight. At 0.5, 1, and 2 h post-injection (p.i.), mice were
anesthetized with 2 % isoflurane and placed in the prone
position near the center of the field of view of the scanner.
Five-minute static scans were obtained, and the images
were reconstructed with use of a 2-dimensional ordered-
subsets expectation maximization (OSEM) algorithm. No
background correction was performed. Regions of interest
(ROIs: 5 pixels for coronal and transaxial slices) were
drawn over the tumor on decay-corrected whole-body
coronal images. Maximum counts per pixel per minute
were obtained from the ROIs and converted to counts per
milliliter per minute by using a calibration constant. The
Results
Chemistry and radiochemistry
AgRP-7C was synthesized by solid phase peptide
synthesis, folded in vitro, and purified by RP-HPLC.
19F-FP-AgRP-7C was prepared by site-specific conjugation
of 19F-NFP to the N terminus of AgRP-7C with a 92 % yield.
This non-radioactive analog was used as a standard for ra-
diosynthesis and integrin receptor binding assays. The
product purities were more than 95 %, as determined by
analytical HPLC, and the molecular masses were character-
ized by MALDI-TOF–MS. Folded peptide AgRP-7C:
m/z = 4222.5 for [MH]? (C171H259N55O55S8, calculated
123

A novel radiofluorinated AgRP for tumor imaging
677
[MH]? = 4221.8 after accounting for the loss of 8 protons
upon formation of 4 disulfide bonds). 19F-FP-AgRP-7C:
m/z = 4297.3 for [MH]? (C174H262FN55O56S8, calculated
[MH]? = 4295.8). The retention times on analytical HPLC
for folded AgRP-7C and 19F-FP-AgRP-7C were 14.3 and
18.3 min, respectively.
For radiolabeling, the synthesis time for 18F-NFP was
approximately 100 min, and the decay corrected yield was
67 11 % using a modified GE synthetic module
(TRACERlab FXFN) (Liu et al. 2010; Haubner et al.
2001). Counted from 18F-NFP, the radiochemical yield of
18F-FP-AgRP-7C (decay corrected) was generally greater
than 70 %. Because the unlabeled AgRP-7C could be
easily separated from the 18F-FP-AgRP-7C, the radio-
chemical purity of 18F-FP-AgRP-7C was[95 % according
to analytic HPLC, and the specific radioactivity of the
probe was estimated to be 20–40 GBq/lmol on the basis of
the prosthetic labeling agent 18F-NFP.
In vitro cell uptake
U87MG cell uptake of 18F-FP-AgRP-7C was performed to
evaluate integrin binding and specificity. Levels of cell
uptake of 18F-FP-AgRP-7C were observed after 1 h and
2 h incubation at 37 °C (Fig. 2a). 18F-FP-AgRP-7C
exhibited high accumulation in U87MG cells, with uptake
values reaching about 3 % of applied activity at 1 h. Fur-
thermore, probe accumulation was efficiently inhibited by
the addition of unlabeled c(RGDyK) (P \ 0.05), an inte-
grin-binding peptidomimetic, indicating that 18F-FP-
AgRP-7C specifically targets integrin receptors.
Fig. 2 a In vitro U87MG cell uptake assay. U87MG cells were
incubated with 37 kBq (1 lCi) of 18F-FP-AgRP-7C for 1 and 2 h at
37 °C, with or without c(RGDyK) as a blocking agent. Data represent
the mean percentage of total radioactivity added. The experiment was
performed in triplicate, and the error bars represent standard
deviation. b Competition binding of 125I-echistatin binding to integrin
receptors on U87MG cells by AgRP-7C and 19F-FP-AgRP-7C
(IC50 = 9.4 1.8 and 8.4 2.3 nM, respectively). Results are
expressed as the mean of quadruplicate measurements standard
deviation
The relative receptor binding affinities of AgRP-7C and
19F-FP-AgRP-7C were determined by competitive binding
assays with 125I-echistatin. Both peptides inhibited the
binding of 125I-echistatin to U87MG cells in a concentra-
tion-dependent manner (Fig. 2b), with IC50 values of
9.4 1.8 and 8.4 2.3 nM for AgRP-7C and 19F-FP-
AgRP-7C, respectively. Therefore, the N-terminal fluoro-
propionate modification had no effect on the integrin-
binding affinity of AgRP-7C.
Co-injection of excess c(RGDyK) as a blocking agent
significantly reduced tumor uptake, which confirmed the in
vivo tumor-targeting specificity of the probe. In addition,
no visible bone uptake was observed in any PET images,
suggesting no defluorination of the probe. Tumor and
major organ activity accumulation was quantified by
measuring the ROIs encompassing the entire organ on the
coronal images (Fig. 3b). 18F-FP-AgRP-7C displayed good
tumor uptake and retention, with values of 3.24 0.33,
2.39 0.15, and 1.51 0.19 % ID/g observed at 0.5, 1,
and 2 h, respectively. For the blocking group, the tumor
uptake value was only 0.97 0.27 % ID/g at 1 h p.i.
(P \ 0.05), indicating 59.4 % inhibition. 18F-FP-AgRP-7C
was also rapidly cleared from the blood, with the highest
uptake value of 1.16 0.11 % ID/g observed at 0.5 h.
Small-animal PET imaging
Decay-corrected coronal PET images of a U87MG tumor-
bearing mouse at 0.5, 1, and 2 h p.i., of 18F-FP-AgRP-7C
were obtained by micro PET imaging (Fig. 3a). The tumor
was clearly visible at 0.5 h p.i., and enhanced tumor-to-
background contrast was achieved at 2 h. The probe
showed rapid clearance from blood and most normal
organs, and low accumulation in lung, liver, and abdominal
regions were observed, resulting in excellent tumor imag-
ing quality. Relatively high levels of radioactivity accu-
mulation and retention were observed in the kidneys.
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H. Jiang et al.
Fig. 3 PET imaging of 18F-FP-AgRP-7C in nude mice bearing
U87MG tumor xenografts (n = 4 per group). a Coronal PET images
of U87MG tumor-bearing mice at 0.5, 1, and 2 h post injection of
approximately 3.7 MBq (100 lCi) of 18F-FP-AgRP-7C with or
without 10 mg c(RGDyK) per kg of mouse body weight at 1 h after
injection. Arrows indicate the location of tumors. b Small animal PET
quantification results, expressed as % ID/g, in various organs and
tumors after injection of 18F-FP-AgRP-7C. c Tumor-to-normal tissue
ratios at 0.5, 1, and 2 h p.i. (T/Li, T/M, T/B and T/Lu represent
tumor-to-liver, tumor-to-muscle, tumor-to-blood and tumor-to-lung
ratios, respectively)
In vivo metabolite analysis
Muscle uptake was also very low (\0.5 % ID/g) at all
times tested. The tumor-to-muscle, tumor-to-blood, and
tumor-to-liver ratios were 7.0, 2.8 and 5.2, respectively, at
0.5 h (Fig. 3c).
18F-FP-AgRP-7C showed high stability in PBS with no free
18F or other catabolic products as assayed by radio-HPLC
(Fig. 5a). In vivo metabolite analyses were also conducted
in tumor, liver, kidney, and urine samples, which were
obtained from the U87MG tumor-bearing mice at 1 h after
injection of the probe (Fig. 5b–e). Only minor peaks with
retention times between 5 and 10 min were observed in
tested samples. These results suggest that 18F-FP-AgRP-7C
is highly metabolically stable in vivo.
Biodistribution studies
Biodistribution studies confirmed that the probe had good
uptake in tumors and low accumulation in most normal
organs (Fig. 4). Tumor uptake of 18F-FP-AgRP-7C was
1.49 0.20 % ID/g at 2 h p.i., whereas for most of normal
organs, the uptake of the probe was lower than 1 % ID/g.
Furthermore, for the blocking group, tumor uptake
(0.57 0.14 % ID/g at 2 h p.i.) was significantly lower
than that of the control group (P \ 0.05), demonstrating
integrin receptor-mediated tumor targeting of the probe.
Finally, the corresponding organ uptakes obtained from
biodistribution were consistent with the PET quantification
results, and no significant differences were observed
between these two studies.
Discussion
Knottins have generated substantial interest in recent
years in the development of molecular imaging probes
because of their high stability, facile synthesis, and
potential for site-specific labeling. In the current study, we
first demonstrated that AgRP-7C can be site-specifically
123

A novel radiofluorinated AgRP for tumor imaging
679
AgRP-7C was performed in tumor, liver, kidney and urine.
The result displayed high in vivo stability, more stable than
18F-Galacto-RGD (Haubner et al. 2004).
Compared with large protein scaffold, AgRP* has small
molecular weight. Small size allows it to clear through the
kidney rather than the liver, allowing for visualization of
tumors located in the abdominal area in molecular imaging
applications. It enables fast targeting to the tumor and
quick clearance from the other organs. In a previous
study, we used a conventional radiofluorination synthon,
N-succinimidyl-4-18F-fluorobenzoate (18F-SFB) to radio-
fluorinate an EETI-II based knottin peptide (2.5D) that
binds with integrins with low nanomolar affinity. PET
imaging showed that 18F-SFB-labeled 2.5D (18F-FB-2.5D)
had a relatively fast tumor washout rate and high uptake in
gallbladder and liver, indicating the suboptimal pharma-
cokinetics of the probe and low in vivo stability of 18F-FB-
2.5D, which limits the further clinical application for tumor
imaging (Miao et al. 2009). In the current study, 18F-FP-
AgRP-7C showed much better tumor-imaging quality
because of higher tumor uptake (for example, 3.24 0.33
vs. *2 0.72 % ID/g at 0.5 h p.i.), better tumor reten-
tion, and lower uptake in many normal organs. Further-
more, the in vivo receptor targeting specificity of the probe
was demonstrated by blocking experiments, in which
unlabeled c(RGDyK) peptide significantly inhibited tumor
uptake. Of note, kidney uptake of 18F-FP-AgRP-7C was
18.9 2.75 % ID/g at 2 h p.i., which was much lower
than that of 64Cu-DOTA-AgRP-7C (60.22 17.52 %
ID/g) (Jiang et al. 2010), suggesting an advantage to using
18F as a PET label. Moreover, compared with 64Cu-DOTA-
AgRP-7C, 18F-FP-AgRP-7C also displayed reduced accu-
mulation in liver (0.31 0.05 vs. 1.74 0.72 % ID/g). In
contrast to the high-activity concentration found in the
kidneys due to the renal elimination, no increase activity in
the gut was showed, indicating that there is no significant
biliary excretion of the tracer into the intestine during the
observation period of 2 h. Low radioactivity accumulation
in liver of 18F-FP-AgRP-7C results in its potential clinical
application for tumor detection in liver. The background
activity in muscle tissue and blood pool was very low. The
absence of radioactivity accumulation in the skeletal sys-
tem in PET images revealed no defluorination of 18F-FP-
AgRP-7C. The background radioactivity of other organs
was low and decreased with time, creating good contrasts.
In vivo evaluation revealed that the probe exhibits fast
tumor targeting and high tumor retention, rapid clearance
from most normal tissues, and excellent tumor discrimi-
nation. These properties indicate promise for further
development of 18F-labeled peptides based on the AgRP*
scaffold for tumor imaging applications.
Fig. 4 Biodistribution and tumor-to-normal tissue ratios of 18F-FP-
AgRP-7C in U87MG tumor-bearing nude mice 2 h after injection of
approximately 3.7 MBq (100 lCi) of 18F-FP-AgRP-7C with or
without co-injection of 10 mg c(RGDyK) per kg mouse body weight.
Data are expressed as % ID/g SD (n = 4 per group). T/Li, T/M, T/B
and T/Lu represent tumor-to-liver, tumor-to-muscle, tumor-to-blood
and tumor-to-lung ratios, respectively
radiofluorinated and used for in vivo imaging of integrin-
positive tumors. The whole study highlights the high
potential of AgRP knottins for probe development.
18F-NFP was selected as a prosthetic group for site-
specific labeling of AgRP-7C because of its small size,
high in vivo stability, and well-established production
method in our program (Liu et al. 2010). The nonradio-
active probe, 19F-FP-AgRP-7C, was prepared as a refer-
ence compound and possessed similar relative binding
affinity compared to that of the unlabeled peptide, indi-
cating that AgRP-7C can preserve its bioactivity when
modified with a small tag such as NFP. These modifica-
tions didn’t influence the binding affinity with avb3 inte-
grin, which was confirmed by the binding assay, raveling
the high affinity and selective binding. Cell uptake studies
performed in the presence and absence of blocking peptide
also demonstrated that the probe can specifically bind to
integrin receptors. The metabolic stability of 18F-FP-
Recently, cancer treatment targeting integrin avb3, such
as MEDI-522 (a humanized anti-human integrin avb3
123

680
H. Jiang et al.
Fig. 5 In vivo metabolite assay. a 18F-FP-AgRP-7C after incubation with PBS for 1 h. b–e Urine or homogenized tissues obtained from mice
1 h post injection of 18F-FP-AgRP-7C. Probe stability was analyzed by radio-HPLC and c-counting
monoclonal antibody) (Hersey et al. 2010) are in clinical
trials for antiangiogenic therapy. Numerous patents have
been administrated with avb3 antagonists for the prevention
and/or treatment of cancer in the past several years.
Meanwhile, clinical trials for evaluating the antagonists
antiangiogenic and anti-tumor results in patients have been
initiated, especially using PET imaging as a powerful tool
to examine the effect of antiangiogenic therapies directly.
A range of radionuclides (e.g., 18F, 64Cu, and 99mTc) can be
carried into the RGD peptides as a targeting biomarker to
the avb3 integrin in various malignant tumors (Beer et al.
2008; Haubner et al. 2003). Radiolabeled RGD peptide
used for detection of avb3 integrin-receptor positive tumors
before and/or after the administration of the antagonists,
permits the selection of the patients and optimization of
the treatments strategies. As demonstrated in this study,
18F-FP-AgRP-7C will be a potential candidate for pre-
dicting and evaluating the antiangiogenic treatment by PET
imaging. Furthermore, AgRP-7C can be radiolabeled with
different therapeutic isotopes to generate a series of agents
for integrin-targeted internal radiotherapy. Additionally,
the important issue in clinical application might be that the
use of 18F-FP-AgRP-7C in human is not expected to have
immunogenicity and toxicity because of its human origin
and low plasma concentration.
the tracer similar to protocols used for imaging with
18F-FDG also can reduce the activity concentration in the
kidney. Although the data in brain was encouraging, future
studies on brain primary and metastatic tumor models
require to be done for further demonstrating the hypothesis
that 18F-FP-AgRP-7C could cross BBB. Moreover, it will
require tracer kinetic analysis, traditional Western blots, in
vitro receptor autoradiography and immunohistochemistry
to detect the relationship between in vivo imaging and
expression avb3 integrin of tumors. The in vivo data was
obtained from mice models and can’t accurately mimic the
complex human situation. Thus, we need better animal
models to demonstrate the potential clinical application of
18F-FP-AgRP-7C.
Conclusion
In this study, 18F-FP-AgRP-7C shows high avb3 integrin-
binding affinity and specificity in vitro. Biodistribution and
small animal PET imaging studies further demonstrated
that 18F-FP-AgRP-7C is a promising PET probe for
imaging avb3 integrin-positive tumors in living subjects.
18F-FP-AgRP-7C exhibits desirable in vivo properties such
as rapid tumor targeting, high tumor uptake and retention,
and good tumor-to-normal tissue ratios. The promising
properties of 18F-FP-AgRP-7C reveal that this probe can be
a powerful tool to evaluate the avb3 integrin overexpression
tumors in basic researches as well as clinical application. In
The current study has some limitations. A major draw-
back was high renal uptake. The probe needs further
modification to reduce the kidney accumulation. Admin-
istration of a diuretic agent (e.g., furosemide) together with
123

A novel radiofluorinated AgRP for tumor imaging
681
metabolic stability, and radiation dose estimates. Bioconjug Chem
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AgRP*-based peptides for molecular imaging applications.
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Acknowledgments This work was supported, in part, by National
Cancer Institute (NCI) 5R01 CA119053, NCI In Vivo Cellular
Molecular Imaging Center (ICMIC) grant P50 CA114747, NCI 5K01
CA104706, and a Stanford Molecular Imaging Scholars postdoctoral
fellowship R25 CA118681. SJM has been supported by an NSF
Graduate Research Fellowship, the Medtronic Stanford Graduate
Fellowship, and a Siebel Scholars Fellowship. This work is also partly
sponsored by Grants from the Zhejiang Provincial Natural Science
Foundation of China (Z2110230), Health Bureau of Zhejiang Prov-
ince (2010ZA075, 2011ZDA013), National Science Foundation of
China (NSFC) (no. 81101023, 81170306, 81173468), and Ministry of
Science and Technology of China (2012BAI13B06). The authors
acknowledge Tamara Locke for help on editing the manuscript.
Conflict of interest The authors declare that they have no conflict
of interest.
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