Synthesis and evaluation of [18F]FP-Lys-GE11 as a new radiolabeled peptide probe for epidermal growth factor receptor (EGFR) imaging

Article abstract of DOI:10.1016/j.nucmedbio.2020.10.004

Introduction: The epidermal growth factor receptor (EGFR) has emerged as an attractive target in the treatment of various cancers. Radiolabeled small molecules, antibodies, and peptides that specifically target EGFR are promising probes for tumor imaging to guide personalized treatment with EGFR-targeted drugs. This study aimed to radiolabel GE11 (an EGFR-specific targeting peptide) with 18-fluorine to develop a new EGFR-targeting positron emission tomography (PET) probe, [¹⁸F]FP-Lys-GE11, for imaging tumors overexpressing EGFR. Methods: [¹⁸F]FP-Lys-GE11 was produced by radiolabeling a GE11 peptide with the prosthetic group 4-nitrophenyl-2-[¹⁸F]fluoropropionate ([¹⁸F]NFP). Stability in PBS and mice serum, affinity for A431 cell line, U87 and PC-3 cells uptake and blocking studies, and biodistribution of [¹⁸F]FP-Lys-GE11 were determined. 2 h dynamic and static PET scans of probe for tumor-bearing mice normal and inhibition uptake were performed. Results: [¹⁸F]FP-Lys-GE11 was stable in PBS and mice serum. The Kd and Bmax values of probe for A431 were 42.43 ± 3.75 nM and 3383 ± 81.73 CPM, respectively. In cell uptake and blocking experiments, a significant reduction in radioactivity accumulation (over 4-fold) was observed by blocking U87 and PC-3 cells with unlabeled peptide. PET imaging of U87 and PC-3 tumor-bearing mice revealed clear tumor imaging (tumor radioactivity accumulation was 3.48 ± 0.44 and 3.68 ± 0.76%ID/g respectively, tumor-to-muscle ratio was 3.45 ± 0.43 and 3.64 ± 0.76 respectively). Blocking imaging revealed that the U87 tumor uptake was significantly inhibited (2.21 ± 0.41%ID/g). The biodistribution and dynamic PET imaging showed that [¹⁸F]FP-Lys-GE11 was mainly excreted by the kidneys and the rest was excreted through the bile and intestines. Conclusion: The current results showed that [¹⁸F]FP-Lys-GE11was a good radiolabeled peptide probe for EGFR overexpression tumor's imaging.

Full text of DOI:10.1016/j.nucmedbio.2020.10.004

Nuclear Medicine and Biology 90–91 (2020) 84–92
Contents lists available at ScienceDirect
Nuclear Medicine and Biology
journal homepage: www.elsevier.com/locate/nucmedbio
18
Synthesis and evaluation of [ F]FP-Lys-GE11 as a new radiolabeled
peptide probe for epidermal growth factor receptor (EGFR) imaging
Xueli Li a,1, Kongzhen Hu , Wenfeng Liu , Yuanfeng Wei , Runhua Sha , Yongxuan Long , Yanjiang Han ,
b,1
c
a
a
a
b
b
b
b
b
a,b,
Penhui Sun , Hubing Wu , Guiping Li , Ganghua Tang , Shun Huang
⁎
a
Medical Imaging Profession, Nanfang Hospital, Southern Medical University, Guangdong 510515, China
Nanfang PET Center, Nanfang Hospital, Southern Medical University, Guangdong 510515, China
School of Biotechnology and Health Sciences, Wuyi University, Jiangmen 529020, China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Introduction: The epidermal growth factor receptor (EGFR) has emerged as an attractive target in the treatment of
various cancers. Radiolabeled small molecules, antibodies, and peptides that specifically target EGFR are promis-
ing probes for tumor imaging to guide personalized treatment with EGFR-targeted drugs. This study aimed to ra-
diolabel GE11 (an EGFR-specific targeting peptide) with 18-fluorine to develop a new EGFR-targeting positron
Received 19 August 2020
Received in revised form 27 September 2020
Accepted 21 October 2020
Available online xxxx
18
emission tomography (PET) probe, [ F]FP-Lys-GE11, for imaging tumors overexpressing EGFR.
1
8
Methods: [ F]FP-Lys-GE11 was produced by radiolabeling a GE11 peptide with the prosthetic group 4-
Keywords:
1
8
18
nitrophenyl-2-[ F]fluoropropionate ([ F]NFP). Stability in PBS and mice serum, affinity for A431 cell line, U87
Epidermal growth factor receptor (EGFR)
Positron emission tomography (PET)
GE11
18
and PC-3 cells uptake and blocking studies, and biodistribution of [ F]FP-Lys-GE11 were determined. 2 h dy-
namic and static PET scans of probe for tumor-bearing mice normal and inhibition uptake were performed.
Results: [ F]FP-Lys-GE11 was stable in PBS and mice serum. The Kd and Bmax values of probe for A431 were
18F]FP-Lys-GE11
Tumor imaging
18
[
42.43 ± 3.75 nM and 3383 ± 81.73 CPM, respectively. In cell uptake and blocking experiments, a significant re-
duction in radioactivity accumulation (over 4-fold) was observed by blocking U87 and PC-3 cells with unlabeled
peptide. PET imaging of U87 and PC-3 tumor-bearing mice revealed clear tumor imaging (tumor radioactivity ac-
cumulation was 3.48 ± 0.44 and 3.68 ± 0.76%ID/g respectively, tumor-to-muscle ratio was 3.45 ± 0.43 and
3
(
.64 ± 0.76 respectively). Blocking imaging revealed that the U87 tumor uptake was significantly inhibited
2.21 ± 0.41%ID/g). The biodistribution and dynamic PET imaging showed that [1 F]FP-Lys-GE11 was mainly ex-
8
creted by the kidneys and the rest was excreted through the bile and intestines.
Conclusion: The current results showed that [18F]FP-Lys-GE11was a good radiolabeled peptide probe for EGFR
overexpression tumor's imaging.
©
2020 Elsevier Inc. All rights reserved.
1
. Introduction
[3], erlotinib [4], lapatinib [5], vandetanib [6], afatinib [7], and
osimertinib [8], and the other is monoclonal antibody drugs, such as
cetuximab [9], panitumumab [10], and necitumumab [11]. Assessment
of a patient's EGFR expression levels can guide the screening of targeted
drug-sensitive individuals and specify personalized treatment options
[12]. Immunohistochemistry (IHC) is the traditional method to detect
EGFR status. It requires an invasive puncture but the result is highly var-
iable because of the heterogeneity and branched evolution of EGFR in
both primary and metastatic tumors [13]. Therefore, noninvasive molec-
ular imaging, such as radionuclide imaging for the expression of EGFR in
tumor regions, is needed.
The overexpression of epidermal growth factor receptor (EGFR) has
been demonstrated in malignant tumors, including non-small cell lung
cancer and breast, renal, ovarian, colon, glioma, pancreatic, bladder,
and head and neck cancers [1]. The overexpression and/or aberrant ac-
tivation of EGFR were shown to play major roles in the invasion and me-
tastasis of tumors [2]. Therefore, EGFR is an attractive biomarker for the
diagnosis, prognosis, and therapy of related cancers.
Currently, two types of drugs are used clinically for targeted thera-
pies in patients with high expression of or mutations in EGFR. One
type is small molecule tyrosine kinase inhibitors (TKIs), such as gefitinib
Several radiolabeled EGFR-targeted imaging probes have been re-
ported for possible diagnostic purposes in the clinic [14]. These probes
fall into two classes. One class is radiolabeled tyrosine kinase inhibitors,
⁎
Corresponding author at: Dr. Nanfang PET Center, Department of Nuclear Medicine,
11
18
11
such as [ C]erlotinib [15], [ F]gefitinib [16], and [ C]PD153035 [17].
Nanfang Hospital, Southern Medical University, 1838 Guangzhou Avenue North, Guang-
zhou, Guangdong Province 510515, China.
18
We previously reported that the [ F]FEA-Erlotinib probe, which targets
the intracellular tyrosine kinase (TK) part of EGFR, had the potential to
screen EGFR TKI-sensitive patients with exon 19 deletion mutations in
E-mail address: shunhuang007@163.com (S. Huang).
Xueli Li and Kongzhen Hu contributed equally to this work.
1
https://doi.org/10.1016/j.nucmedbio.2020.10.004
0969-8051/© 2020 Elsevier Inc. All rights reserved.

X. Li, K. Hu, W. Liu et al.
Nuclear Medicine and Biology 90–91 (2020) 84–92
EGFR [18]. However, such small-molecule probes have a common
defect. They are relatively fat-soluble and have a high background in
the abdomen. Another class is biomolecular probes, including protein,
antibodies, affibodies, and nanobodies, which can direct patient's preci-
sion therapy of EGFR-targeted monoclonal antibodies. Representative
The identification of samples was performed by a Bruker DRX
400 MHz instrument (Bruker, Karlsruhe, Germany) for proton
1
nuclear magnetic resonance ( H NMR) studies, a Bruker maXis im-
pact spectrometer (Bruker, Karlsruhe, Germany) for positive-ion
electrospray ionization–mass spectrometry (ESI-MS) studies, and
a Thermo-Scientific™ Obitrap Fusion™ Tribrid™ mass spectrome-
ter (Waltham, MA, USA) for high-resolution mass spectra (HRMS)
studies. PET/CT scans and imaging analyses were performed by an
Inveon MicoPET scanner (Siemens Healthiness, Munich, Germany).
HPLC separation was carried out with a PET-MF-2V-IT-I synthesizer
module (PET Co. Ltd., Beijing, China) built-in the HPLC system with a
semi-preparative reverse-phase C18 column (10 × 250 mm). The
mobile phase was 61% solvent A (0.1% trifluoroacetic acid (TFA) in
water): 39% solvent B (0.1% TFA in acetonitrile (MeCN)) with a flow
rate of 4 mL/min. Analytical HPLC was performed using an LC-20AD
HPLC system (Shimadzu, Kyoto, Japan) equipped with a UV-VIS detector
(Shimadzu Corporation, Kyoto, Japan) set at 220 nm, a γ-radioactivity
detection system (LabLogic Systems Ltd., Sheffield, UK), and a Therom
100-5C18 4.6 × 250 mm 5 μ analytical column. The gradient program
started from 95% solvent A (0.1% TFA in water): 5% solvent B (0.1%
TFA in MeCN) (0–2 min) and ramped to 20% solvent A: 80% solvent B
at 25 min with a flow rate of 1 mL/min. Radioactivity was measured
by a calibrated ion chamber (Capintec CRC-15R) or a gamma counter
(γ-counter) (GC-1200, USTC Chuangxin Co. Ltd. Zonkia Branch, China).
PET scans were performed using an Inveon Micro-PET/CT system (Sie-
mens Healthiness, Munich, Germany). An Inveon Research Workplace
Micro-PET/CT scanner was used for image analyses.
6
8
89
probes include [ Ga]-DOTA-hEGF [19], [ Zr] labeled panitumumab
and cetuximab [20,21], [64Cu]-DOTA-cetuximab-F(ab′)
[22], and [ F]
18
2
FBEM-Cys-ZEGFR:1907 [23]. However, these biomolecules, due to the
large size, have low blood clearance and poor stability, which cause
high background and limit the diffusion of the probes in the tumor, lead-
ing to reduced imaging contrast [24]. Thus, using small molecular
weight biomolecules, such as peptides, may overcome these limitations
and obtain better imaging results. Recently, peptides have been widely
studied as radiotracers for cancer imaging because of their small molec-
ular weight, simple structure, high affinity, strong penetrability, and
68
weak immune response induction [25]. Probes such as [ Ga]-PSMA
and [68Ga]-DOTATATE have been widely used in clinical research and
applications worldwide [26–28]. Our team has also previously con-
ducted the preclinical research and development of many peptide PET
probes [29,30].
Two peptide sequences (D4(Leu-Ala-Arg-Leu-Leu-Thr) and GE11
(
Tyr-His-Trp-Tyr-Gly-Tyr-Thr-Pro-Gln-Asn-Val-Ile))
have
been
radiolabeled as probes for imaging EGFR-positive cancers [31–35].
Hosseinimehr et al. used 9 Tc to label a D4 peptide ([
9m
99m
Tc]HYNIC-
Tc]HYNIC-
EDDA/tricine-(Ser)3-D4, [9 Tc]HYNIC-(Ser)3-D4, and [
9m
99m
D4) and evaluated it for targeting and imaging non-small-cell lung can-
cer [31–33]. Compared to PET imaging, SPECT imaging of the tumor
could not be visualized with good contrast, but no further PET studies
have been reported. Another EGFR-specific peptide, GE11, was reported
by Li et al. [36]. It was obtained by phage screening technology with high
affinity (Kd = 22 nM) for EGFR-overexpressing cancer cells (human
hepatoma cell line, SMMC-7721). In 2011, Dissoki et al. first radiolabeled
2.2. Cell culture
U87 (human glioma cell line), PC-3(human prostate cancer cell line)
and A431(human epidermal cancer cell line) were obtained from the
cell bank of the Chinese Academy of Sciences (Shanghai, China). The
cells were cultured in DMEM medium with 10% fetal bovine serum
18
GE11 with some prosthetic-labeled groups, such as [ F]N-succinimidyl
1
8
18
18
4
-fluorobenzoate ([ F]SFB), [ F](2-[1]-ethoxy)-propyne ([ F]F-
1
24
PEG4-propyne), [ I]N-succinimidyl 4-iodobenzoate, and the NOTA-
(FBS) at 37 °C in a humidified 5% CO atmosphere.
2
111
Bn-NCS chelator for
In labeling [37]. The next year, Dejesus reported
64
the synthesis of [ Cu]-NOTA-Bn-GE11 for PET imaging of EGFR-rich tu-
mors. However, neither study conducted biological evaluation and im-
aging studies [34]. In 2017, Rahmanian et al. developed a 9 Tc-
labeled GE11 peptide as a radiotracer for the diagnosis and staging of tu-
mors highly overexpressing EGFR. The biodistribution of the tracer was
analyzed in normal female mice and SKOV3 tumor-bearing mice with a
tumor/muscle ratio of 3.2 after 4 h injection [35,38]. The next year,
Striese et al. synthesized 64Cu-labeled GE11 tracers by the chelating
agent p-SCN-Bn-NOTA and found that it was not suitable for further bi-
ological investigations because they presumably formed aggregates [39]
2.3. Animal models
9m
Tumor-bearing models were developed in 4 to 6-week-old athymic
nude mice (Animal Experiment Center of Nanfang Hospital) by injection
7
with 5 × 10 cells in their right shoulders. Tumor growth was monitored
by caliper measurements of the diameter of the tumor. Micro-PET/CT
imaging and other animal experiments were performed when the
tumor diameter reached 8–12 mm (3–4 weeks after inoculation). All
of the animal experiments were in accordance with the principles and
procedures of the Southern Medical University Institutional Animal
Care and Use Committee.
.
In general, there are no current effective radiolabeled peptide probes
that target EGFR for the precise guidance of EGFR-targeted therapy.
Herein, we introduced the prosthetic group of 4-nitrophenyl-2-[18F]
fluoropropionate ([ F]NFP) to radiolabel a GE11 peptide. Also, the
in vitro stability, cellular affinity and uptake, and PET imaging in EGFR-
overexpressing tumor-bearing mice were analyzed.
2.4. Immunohistochemistry
1
8
Immunohistochemistry staining was performed on the xenografted
tumors to analyze EGFR expression. The primary EGFR antibody (clone
EP38Y) was purchased from Abcam (ab52894, Abcam, Cambridge,
UK). The secondary antibody was rabbit anti-human horseradish perox-
idase (P0214, Dako, Glostrup, Denmark). The procedures were con-
ducted according to the antibody manufacturers' protocols.
2
. Methods
2
.1. General
GE11 and Lys-GE11 peptides were synthesized by GL Biochem
2.5. Chemistry and radiochemistry
Ltd. (Shanghai, China). The QMA, C-18, and Alumina N columns
were purchased from Waters (Milford, MA, USA). All chemicals, un-
less otherwise indicated, were purchased from Fisher Scientific
2.5.1. Synthesis of 4-nitrophenyl-2-fluoropropionate (NFP)
The synthesis of NFP was performed as described previously [29].
Briefly, diisopropylethylamine (DIPEA, 200 μL) and bis(4-nitrophenyl)
carbonate (518 mg, 1.65 mmol) were added to a solution of 2-
fluoropropionic acid (150 mg, 1.65 mmol) in dry dimethylformamide
(DMF, 2 mL). After stirring for 3 h at 60 °C, the reaction mixture was
cooled to room temperature and quenched with a 5% acetic acid solution
(
Pittsburgh, PA, USA), ABX GmbH (Radeberg, Germany), J&K Scien-
tific (Shanghai China), or Sigma-Aldrich (Tel Aviv, Israel), without
1
8
further purification. The no carrier-added F-fluoride was prepared
18
by the PETtrace Cyclotron (GE Medical Systems, Uppsala) via an
O
1
8
(
p, n) F reaction.
85

X. Li, K. Hu, W. Liu et al.
Nuclear Medicine and Biology 90–91 (2020) 84–92
(
2 mL). The crude product was purified by silica-gel column chromatog-
The in vivo metabolic analysis of [18F]FP-Lys-GE11 in plasma was
measured using BALB/c blood samples (0.4 mL) collected at 30, 60,
and 120 min after tracer injection. The blood supernatant after centri-
fuged (12,000g, 10 min) was filtered through a 30 KDa Millipore filter
and analyzed by HPLC. Samples were collected in 0.5-min fractions for
30 min, then analyzed in a γ-counter for 20 s. Counts were decay-
corrected to the injection time and added together. The counts were
as the plotted intensity (cpm) versus fractions to show the profile of
the sample. The data were plotted to reconstruct the HPLC spectrum.
raphy with ethyl acetate and hexanes (EtOAc:hexanes = 1:1) to afford
the title compound as a red solid (160 mg, 0.75 mmol, 46% yield). NMR
Spectroscopy: H NMR (400 MHz, CDCl
1
3
) δ 8.30 (d, J = 8.8 Hz, 2H), 7.36
(
d, J = 8.8 Hz, 2H), 5.30 (dd, J = 48.1, 6.9 Hz, 1H), 1.77 (dd, J = 23.4,
1
3
6
2
2
.9 Hz, 3H). C NMR (100 MHz, (CD3)2SO, 25 °C, δ): 168.1 (d, J =
4 Hz), 154.6, 145.8, 125.3, 122.3, 85.4 (d, J = 183 Hz), 18.3 (d, J =
2 Hz).
2
.5.2. Synthesis of FP- Lys-GE11
NFP (0.64 mg, 3.0 μmol in 0.1 mL of DMSO) was slowly added to a so-
2.9. Cellular uptake and blocking studies
lution of Lys-GE11 (5 mg, 3.0 μmol in 0.2 mL of DMSO and 40 μL of N,N-
Diisopropylethylamine (DIPEA)) at room temperature. The reaction
mixture was stirred for 3 h at room temperature. The desired product
was purified by semi-preparative HPLC and lyophilized to afford FP-
Lys-GE11 as a white powder (3.5 mg, 2.0 μmol, 66.7% yield). Analytical
Cell uptake and blocking studies of [18F]FP-Lys-GE11 were evaluated
in U87 and PC-3 tumor cells (EGFR-positive expression). For the cell up-
take experiment, tumor cells were seeded into 24-well plates at a den-
sity of 2 × 105 cells/well and incubated overnight. Then, the culture
HPLC (Rt = 17.5 min) and HRMS (m/z: calculated for C84
[M − H]), 1740.8191, found, 1740.8193) analyses confirmed the prod-
uct identification (Fig. S1).
H112FN19O
21
18
medium was discarded and medium containing [ F]FP-Lys-GE11 was
(
added in quadruplicate wells (37 kBq/well). After incubation at 37 °C
for 5, 15, 30, 60, 90, and 120 min, the cells were rinsed thrice with cooled
PBS and lysed with NaOH-SDS (0.2 M NaOH, 1% SDS). The cell lysate was
collected and measured in a well-type γ counter. For the blocking exper-
iments, Lys-GE11 (10 μmol/L) was added to the culture medium con-
taining the radiotracer for incubation with the tumor cells. The other
procedures were as above.
1
8
2
.5.3. Radiosynthesis of [ F]FP-Lys-GE11
[
18
F]NFP was prepared and used for peptide radiolabeling based on a
previously reported procedure [40]. In brief, about 350 mCi of [ F]NFP
18
was obtained after a substitution, hydrolysis, and esterification reaction
1
8
from 1000 mCi [ F]fluoride. Then, a solution of Lys-GE11 (50 μg) in
18
DMSO (200 μL) and DIPEA (40 μL) was added to anhydrous [ F]NFP.
The reaction mixture was reacted 10 min at room temperature and
was diluted with water (10 mL). Then, the dilution was passed through
a Plus C18 cartridge and the cartridge was washed with water (10 mL)
twice. About 35 mCi desired radiolabeled compound, [18F]FP-Lys-
GE11, was eluted with 1 mL of ethanol. For the in vitro and in vivo exper-
iments, the solvent was evaporated in vacuo and [18F]FP-Lys-GE11 was
formulated with 0.9% saline.
2
.10. PET imaging
PET/CT scans and imaging analyses were performed by an Inveon
MicoPET scanner (Siemens Healthiness, Munich, Germany). About
5
MBq of [18F]FP-Lys-GE11 was injected in U87 xenograft nude mice
via the tail vein and imaged under anesthetization with pentobarbital.
Ten-minute static PET scans were acquired 2 h post-injection (p.i;
n = 3/group). For the blocking studies, 100 μg of unlabeled GE11 pep-
tide was conjugated with approximately 5 MBq of [18F]FP-Lys-GE11
and PET/CT scans were performed. The PET scan was immediately
followed by a low-dose CT scan. The CT scan was used for attenuation
correction and localization of the lesion site. Two-hour dynamic PET
scans were also performed immediately after the injection of about
2
.6. In vitro receptor binding assay
The cellular affinity of [18F]FP-Lys-GE11 for the A431 cell line was
determined by a saturation binding experiment to calculate the dissoci-
5
ation constant (Kd). Briefly, the A431 cells were seeded at 1.3 × 10
5
MBq of [18F]FP-Lys-GE11.
The images were reconstructed using a three-dimensional ordered-
density in 48-well plates and incubated overnight. Then, the culture me-
dium was changed to DMEM (without FBS) containing different concen-
trations (0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10.0, 50.0, 100.0 μCi/mL) of
radiolabeled peptide. A block well was included which contained
subset expectation maximum (3D OSEM) algorithm. For every scan,
the regions of interest (ROIs) were drawn using vendor software
(
Inveon Research Workplace). The concentration of radioactivity accu-
5
3
0
00 μM unlabeled Lys-GE11peptide. After incubation of the cells at
7 °C for 60 min, the cells were rinsed three times with cold PBS (with
.5% bull serum albumin) and lysed with NaOH-sodium dodecyl sulfate
mulated within the tumors, brain, hearts, lungs, liver, kidneys, gall blad-
ders, muscle, and bone was obtained from the mean pixel values of
multiple ROI volumes. Then, these values were converted to MBq/mL
to obtain the image-ROI-derived percent injected dose per gram (%ID/
g). For the dynamic scans, the frame rates of the histogram for the imag-
ing were 10 × 3, 3 × 10, 4 × 60, 5 × 300, 9 × 600 (frames × seconds).
(
SDS) (0.2 M NaOH, 1% SDS). The cell lysates were collected and mea-
sured using a well-type γ counter. The Kd value was calculated by
GraphPad software with nonlinear regression.
2
.7. Octanol/water partition coefficient
2.11. Biodistribution
The [18F]FP-Lys-GE11 radiotracer (10 μL, ≈100 kBq) was added to
tubes containing octanol (1000 μL) and PBS (pH = 7.4, 990 μL) in tripli-
Approximately 1 MBq of [18F]FP-Lys-GE11 was directly injected intra-
venously into 5 to 6-week-old BALB/c 5 female mice via the tail vein. The
mice were sacrificed and dissected 2 h post-injection. The brain, heart,
lungs, liver, kidneys, gall bladder, muscle, and bone were collected,
weighed, and counted for radioactivity in a γ counter (n = 3). All mea-
surements were background-subtracted and decay-corrected to the
time of injection. The results were calculated as the percentage injected
dose per gram organ or tissue (%ID/g).
cate. After the tubes were gently and continuously mixed for over
3
0 min at room temperature, the aqueous and organic phases were iso-
lated. The radioactivity of each of the samples (300 μL) was measured
using a γ-counter and the logP values were calculated.
2
.8. In vitro stability and in vivo metabolism
For the in vitro stability, the [18F]FP-Lys-GE11 radiotracer (10 μL,
≈2 MBq) was added to PBS (200 μL) or mice serum (MS, 200 μL) and in-
2.12. Statistical analyses
cubated at 37 °C for 2 h. For the PBS study, the solution was directly
injected onto the HPLC for analysis. For the mice serum sample, the so-
lution was filtered through a 0.22 μm membrane for HPLC analysis.
All data are expressed as means ± SD. One-way analysis of
variance (ANOVA) was used for the statistical analyses. A p-value
86

X. Li, K. Hu, W. Liu et al.
Nuclear Medicine and Biology 90–91 (2020) 84–92
less than 0.05 was considered statistically significant (*p > 0.05,
found that the probe was mainly metabolized to three radioactive prod-
ucts (peak 1, peak 2, and peak 3), and the retention time in HPLC spec-
trogram was 4 to 5 min, 6 to 7.5 min, and 11 to 12 min, respectively. The
probe first metabolized to peak 1 (about 40% at 30 min). Then all the
probe prototypes of [18F]FP-Lys-GE11 metabolized to peak 1, while an-
other new metabolite peak 2 (the ratio of peak 1 to peak 2 at 60 min was
*
*p > 0.01, ***p > 0.001).
3
. Results
3
.1. Chemistry and radiochemistry
3
:1) was produced, and a very small amount of metabolite peak 3 was
The radiosynthesis of [18F]FP-Lys-GE11 followed our previously
also present. The ratio of these three metabolites (peak 1, peak 2, and
peak 3) was about 2:4:1 by 120 min.
published four-step two-pot methodology [40]. For the first pot
1
8
radiosynthesis, an [ F]fluorination reaction was carried out using
ethyl 2-bromopropionate as a precursor, then potassium hydroxide
was directly added for hydrolysis. Finally, 2-[18F]fluoropropanoate salt
3.3. Cell-specific binding
18
The cellular binding affinity of [18F]FP-Lys-GE11 for the EGFR high-
was activated with bis(4-nitrophenyl) carbonate to yield [ F]NFP. For
the second pot radiosynthesis, the [18F]NFP was coupled with Lys-
GE11 at 40 °C for 10 min (Scheme 1). The product, [18F]FP-Lys-GE11,
was isolated by solid-phase extraction with an overall yield (from the
expressing A431 cell line was determined by saturation-binding assays.
The specific binding curve of [ F]FP-Lys-GE11 to the A431 cells is
shown in Fig. 2. The Kd (dissociation constant) and Bmax (maximum
number of binding sites) values for the radiolabeled peptide were ob-
tained as 42.43 ± 3.75 nM and 3383 ± 81.73 CPM, respectively.
18
1
8
[
F]fluoride) of 7 ± 2% (decay corrected), high radiochemical purity
over 99%), and high specific activity (over 35 GBq/μmol, n = 6) in
(
1
05 min (Fig. S2).
Nonradioactive FP-Lys-GE11 was used as a reference compound for
3.4. Cellular uptake
the radiosynthesis and inhibition experiment. It was prepared by the di-
rect conjugation of NFP to FP-Lys-GE11 peptide with a yield of 66.7%
The cellular uptake and inhibition of [18F]FP-Lys-GE11 were evalu-
ated in EGFR-positive U87 human glioma cells and PC-3 human prostate
cancer cells. The results are shown in Fig. 3. For the uptake experiment,
uptake of the tracer was rapidly increased after the incubation and
reached a peak (approximately 0.4%) at 15 min. For the inhibition ex-
periment, incubation with 10 μmol/L of the unlabeled peptide (Lys-
GE11) significantly decreased cellular uptake (<0.1%).
(
Scheme 1). FP-Lys-GE11 was purified by semipreparative HPLC to
achieve greater than 96% purity, which was confirmed by orbitrap fu-
sion mass spectrometry. In the HPLC spectrogram, the retention time
18
of [ F]FP-Lys-GE11 was 17.39 min in the γ-radioactivity detection sys-
tem, which was matched with FP-Lys-GE11 under the UV absorbance
peak at 254 nm (17.23 min) under similar conditions (Fig. 1). The
octanol/water partition coefficient (Log P = −2.09 ± 0.05) was esti-
mated by a γ-counter method.
3.5. PET imaging
3
.2. In vitro stability and in vivo metabolism
Dynamic microPET studies on nude mice bearing U87 tumor xeno-
grafts were performed with [18F]FP-Lys-GE11. Representative coronal
images of the U87 tumor-bearing mice 5, 15, 30, 60, 90, and 120 min
after the injection of [18F]FP-Lys-GE11 are presented in Fig. 4a. As the
images demonstrate, the tumors of the U87 mice could be clearly visu-
alized. PET images were selected by manually selecting the ROIs to
obtain the time-activity curves (TACs) for analyzing the metabolic char-
acteristics of [18F]FP-Lys-GE11 (Fig. 4). The TACs showed that the tumor
uptake of the probe after injection rapidly increased and reached a pla-
teau (1.81%ID/g) at 3 min. After that, the accumulation of the probe in
the tumor slowly increased with time, and the uptake value reached
2.55% ID/g at 2 h. The tumor accumulation was higher than other tissues
To determine the in vitro characteristics of [18F]FP-Lys-GE11, the sta-
bility was assessed by incubating the radiotracer in PBS and mouse
serum (MS) at 37 °C for 2 h. The stability results were determined as a
percentage of the intact radiotracer according to the radio-HPLC analysis
1
8
(Fig. 1a). [ F]FP-Lys-GE11 showed high in vitro stability for up to 2 h.
The percentage of unchanged tracer in PBS and MS was 100% and
7
7
also studied the metabolic stability of [ F]FP-Lys-GE11 in the blood of
BALB/c mouse at 30, 60, and 120 min after injection (Fig. 1b). We
2.2% respectively. There was a radioactive decomposition peak at
.13 min in MS, which accounted for about 27.8% of the tracer. We
18
Scheme 1. Synthesis of [18F]FP-Lys-GE11 and FP-Lys-GE11.
87

X. Li, K. Hu, W. Liu et al.
Nuclear Medicine and Biology 90–91 (2020) 84–92
Fig. 1. (a) HPLC analysis of the in vitro stability of [18F]FP-Lys-GE11 by incubating [18F]FP-Lys-GE11 in PBS and mouse serum (MS) for 2 h, the quality control radioactive chromatography of
1
8
[
F]FP-Lys-GE11, and the UV chromatography of FP-Lys-GE11. (b) Representative HPLC profiles for in vivo stability studies (blood supernatants) in BALB/c mice after intravenous injection
18
of [ F]FP-Lys-GE11.
or organs, except the kidneys, gallbladder, and intestines at 2 h. Mean-
while, the TACs of the kidneys, liver, gall bladder, and intestine showed
that the probe was mainly excreted by the kidneys and a small part was
excreted by the bile and intestine. Then, the ratios of tumor-to-muscle,
tumor-to-liver, and tumor-to-kidneys at different time points were de-
termined (Fig. 4c). All of the ratios increased with time, which demon-
strated that tumor uptake of the probe was specific. The tumor-to-
muscle ratio reached a plateau at 2 h (2.80), which suggested that the
optimal static imaging time should be 2 h after intravenous injection.
The representative whole-body uptake and inhibition images (CT
images and coronal images of PET) of [18F]FP-Lys-GE11 in U87 and PC-
Fig. 5a. The radioactivity that accumulated in the tumor, liver, and mus-
cle was evaluated by measuring the ROIs of the images (Fig. 5b). The
tumor-to-muscle (U87/M or PC-3/M) and liver-to-muscle (Liver/M)
values are shown in Fig. 5c. For the normal uptake of [18F]FP-Lys-
GE11, the tumor was clearly visible and the radioactivity accumulation
in the tumor (U87: 3.48 ± 0.44, PC-3: 3.68 ± 0.76) was higher than in
most tissues and organs (including the liver, 2.24 ± 0.31(p > 0.01)).
The ratios of U87/M, PC-3/M and liver/M were 3.45 ± 0.43, 3.64 ±
0.76, and 2.22 ± 0.31, respectively. The ratios of tumor-to-muscle
were significantly different from liver-to-muscle (p > 0.01). For inhibi-
tion imaging (with 100 μg of unlabeled Lys-GE11 peptide), the uptake
by the U87 tumors and liver were much lower than that of the normal
3
xenografted tumors 2 h after intravenous injection are presented in
88

X. Li, K. Hu, W. Liu et al.
Nuclear Medicine and Biology 90–91 (2020) 84–92
mary excretory route of the probe. At the same time, the liver did not
have a particularly high radioactive uptake, and the gall bladder and in-
testine had high uptakes, suggesting that the remaining probes were
mainly excreted by bile. Other background organs, such as the brain,
lungs, stomach, muscles, and bones, had low levels of radioactivity.
The data are consistent with the PET imaging results, and prove that
the probe [18F]FP-Lys-GE11 was hydrophilic, which is consistent with
its octanol/water partition coefficient (log P = −2.09 ± 0.05).
4
. Discussion
In clinical diagnosis and treatment, EGFR protein overexpression and
gene status are considered very important biomarkers for multiple tu-
mors. Tumors overexpressing EGFR or bearing EGFR mutations are asso-
ciated with high patient morbidity and mortality. At present, targeted
therapies to EGFR, including small-molecule tyrosine kinase inhibitors
(TKIs) that target the intracellular domain of EGFR and antibody drugs
that target the intracellular domain, have benefited many patients
Fig. 2. Affinity assessments of [18F]FP-Lys-GE11 in the A431 cell line by saturation binding
analysis. The errors presented in the insert are standard errors of the curve fit.
[
3–11]. Nuclear medicine imaging (especially PET imaging) studies
uptake group. The quantitative analysis values of the U87-inhibition and
liver-inhibition were 2.21 ± 0.41, and 1.38 ± 0.24, respectively, which
were significantly different from the normal uptake group (p = 0.021
and p = 0.020, respectively). The U87-inhibition/M (2.28 ± 0.42) and
liver-inhibition/M (1.43 ± 0.25) ratios were significantly different
from the normal uptake group (p = 0.028 and p = 0.026, respectively).
Immunohistochemical staining showed high EGFR expression in U87
and PC-3 tumors (Fig. 5d and e). There was no significant difference in
probe uptake between U87 and PC-3 tumors, and no significant differ-
ence in EGFR expression between U87 and PC-3 tumors in immunohis-
tochemical staining.
with emerging widely-used targeting peptides can determine the over-
expression of EGFR to guide costly monoclonal antibody targeted ther-
apy and monitor its efficacy [14]. In previous studies, we developed
several peptide PET probes, such as [ F]FP-D-Lys6-GnRH [29] and Al
[
mulated extensive preclinical research experience on peptide PET
probes. In the present study, we chose 18F-fluoride (with a suitable
half-life and high imaging resolution) to radiolabel the EGFR-targeting
peptide GE11 and conducted relevant in vivo and in vitro biological eval-
uation studies to provide precise guidance for EGFR-targeted monoclo-
nal antibody therapy.
18
18
F]F-GP2633 [30], and conducted preclinical studies. We have accu-
For the radiochemistry, we radiolabeled a GE11 peptide with the
1
8
18
3
.6. Biodistribution
prosthetic group of 4-nitrophenyl-2-[ F]fluoropropionate ([ F]NFP),
which was the smallest 18F-radiolabeling group for peptides and has
less effect on the characteristics of peptides [41]. Furthermore, some
peptide PET probes labeled with [18F]NFP have been successfully used
The ex vivo biodistribution studies of [18F]FP-Lys-GE11 were carried
out in normal BALB/c mice 30, 60, and 120 min after intravenous injec-
tion to evaluate the pharmacokinetics of the probe. The data are shown
in Fig. 6, which are expressed as the percent of the total injected dose
per gram of tissue (%ID/g). The radioactivity level in most organs and tis-
sues, including the blood, brain, heart, lung, liver, kidney, gall bladder,
spleen, stomach, muscle, and bone, decreased steadily over a 120 min
period following injection. For the major metabolic and excretory or-
gans, the kidney had the highest uptake, suggesting that it was the pri-
18
in clinical trials [42]. Then, the cellular binding affinity of [ F]FP-Lys-
GE11 was determined in A431 cells with EGFR-overexpression by
saturation-binding assays. The Kd and Bmax of [ F]FP-Lys-GE11 were
42.43 ± 3.75 nM and 3383 ± 81.73 CPM, respectively. The Kd value
was lower than that of GE11 (22 nM) but higher than that reported
18
9
9m
for some radiolabeled probes, such as [
Tc]-tricine-HYNICSSS-GE11
(73 ± 17 nM) and [9 Tc]EDTA/tricine HYNIC (Ser)
GE11 (114 ±
8 nM). Although the cell lines used in the experiment were different,
9m
2
2
their Kd values were on the same order of magnitude as the parent pep-
tide, indicating that the labeling method we introduced had a small ef-
fect on the affinity of the peptide. Cell uptake and inhibition results also
indicated that the probe specifically bound to the EGFR-overexpressing
cells. Those results suggested that [18F]FP-Lys-GE11 was highly and spe-
cifically bound to the EGF receptor.
For tumor targeting, in vivo 2 h dynamic PET scans of [18F]FP-Lys-
GE11were performed in xenografted U87 tumor-bearing mice. The
TACs showed that the tumor uptake of [18F]FP-Lys-GE11 rapidly in-
creased after injection and the tumor site was clearly visible. Radioactive
accumulation in the tumor and the ratio of tumor to muscle both
reached higher values (2.55%ID/g, and 2.80 respectively) at 2 h, so we
determined that 2 h was the optimal imaging time. Then, static imaging
of the uptake and inhibition 2 h post-injection was performed. We
found that the radioactive accumulation in the tumor (U87 and PC-3 tu-
mors was 3.48 ± 0.44 and 3.68 ± 0.76%ID/g respectively) and the
tumor-to-muscle ratios (U87 and PC-3 tumors was 3.45 ± 0.43 and
3
.64 ± 0.76 respectively) were higher than those of dynamic imaging.
This may be due to the long anesthesia time in the dynamic imaging
process, which slowed down the uptake of the probe. The blocking re-
sults showed that the radioactive accumulation in the tumor (2.21 ±
0.41) was obviously inhibited. We found that the uptake by the liver
in inhibition imaging was lower than the normal uptake experiment
Fig. 3. Cell uptake and inhibition studies of [18F]FP-Lys-GE11 of U87 and PC-3 tumor cells
(EGFR-positive, n = 3, mean ± SD). Blocking studies with 10 μM FP-Lys-GE11 confirmed
the receptor-specific uptake.
89

X. Li, K. Hu, W. Liu et al.
Nuclear Medicine and Biology 90–91 (2020) 84–92
Fig. 4. (a) Dynamic microPET/CT images of [18F]FP-Lys-GE11 in nude mice bearing U87 tumors 5, 15, 30, 60, 90, and 120 min after injection (red arrow indicates the location of the tumor).
b) Time-activity curves of the selected regions of interest (ROIs) from the dynamic PET images of U87 tumor-bearing mice. (c) Comparison of tumor-to-organ (U87/muscle, U87/liver, and
(
18
U87/kidneys) ratios of dynamic microPET/CT images of [ F]FP-Lys-GE11 in U87 tumor-bearing mice from 0 to 120 min after injection. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
both in imaging and quantitative analysis (1.38 ± 0.24 vs 2.24 ± 0.31,
p = 0.020). This was because normal liver tissues express EGFR, and
the addition of blocking agents significantly reduced the uptake of
liver [43]. The TAC curves showed that the main excretion of [18F]FP-
Lys-GE11 was by the kidneys and the rest was excreted through the
bile and intestines. These results were consistent with the biological dis-
PET imaging in EGFR-overexpressing tumors. The preclinical data demon-
strated that [18F]FP-Lys-GE11 is a promising PET probe for the precise
guidance of EGFR-targeted monoclonal antibody therapy.
Acknowledgments
1
8
tribution of [ F]FP-Lys-GE11 in normal mice, probably due to the hy-
drophilicity and negative charge of the peptide sequence, which was
consistent with other studies. At the same time, the selection of [18F]
NFP as a prosthetic group and lysine as a linker did not significantly af-
fect the hydrophilicity (log P = −2.09 ± 0.05) of the peptide. This fea-
ture ensures that the imaging can be used for EGFR positive expression
tumors detection in a variety of locations, except for several excretory
organs, such as the kidney, gallbladder, and intestine.
This research was supported by National Natural Science Foundation
of China (No. 81901781), Natural Science Foundation of Guangdong-
Guangzhou Joint Fund (No. 2019A1515110941), President Foundation
Project of Nanfang Hospital of Southern Medical University, China (No.
2
018Z003). Guangdong College Students Innovation and Entrepreneur-
ship Training Program (No. S201912121086).
Overall, we successfully radiolabeled and tested a novel fluoride-18-
Compliance with ethical standards
1
8
labeled peptide ([ F]FP-Lys-GE11) as a PET probe for imaging EGFR
18
overexpressing tumors. According to our data, [ F]FP-Lys-GE11 had a
high affinity for EGFR-overexpressing cells and good imaging contrast
in PET imaging in EGFR-overexpressing tumors. The preclinical data
All animal studies were approved by the Nanfang Hospital Animal
Ethics Committee at the Southern Medical University, and in accordance
with its guidelines.
1
8
demonstrated that [ F]FP-Lys-GE11 is a promising PET probe for future
clinical translation.
Data availability
5
. Conclusion
The data used to support the findings of this study are included
within the article and supplementary information file.
In this study, we successfully radiolabeled and tested a novel fluoride-
8-labeled peptide ([1 F]FP-Lys-GE11) as a PET probe for imaging EGFR
8
Declaration of competing interest
1
18
overexpressing tumors. According to our data, [ F]FP-Lys-GE11 had a
high affinity for EGFR-overexpressing cells and good imaging contrast in
The authors declare that they have no conflict of interest.
90

X. Li, K. Hu, W. Liu et al.
Nuclear Medicine and Biology 90–91 (2020) 84–92
91

X. Li, K. Hu, W. Liu et al.
Nuclear Medicine and Biology 90–91 (2020) 84–92
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Fig. 5. (a) Representative whole-body CT images, uptake and inhibition PET images of [1 F]FP-Lys-GE11 in PC-3 and U87 tumor-bearing mice 2 h after intravenous injection. The arrows
8
1
8
indicate the tumor location. (b) Quantification of the uptake and inhibition images of [ F]FP-Lys-GE11 in tumors, liver, and muscle. The quantitative data were displayed as the mean
values of %ID/g ± SD after decay correction. (c) Comparison of tumor-to-muscle and liver-to-muscle ratios in xenografted tumors. (d) Immunohistochemical staining of EGFR
expression in PC-3 and U87 xenografted tumors. Images depicted at 200× magnification. (e) The images (three sections per animal) of immunohistochemical staining were captured
and quantified using the Image-Pro Plus software package.
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