Comparison of Al18F- and 68Ga-labeled NOTA-PEG4-LLP2A for PET imaging of very late antigen-4 in melanoma

Article abstract of DOI:10.1007/s00775-019-01742-6

Abstract: Malignant melanoma is an aggressive cancer with poor prognosis. Very late antigen-4 (VLA-4) is overexpressed in melanoma and many other tumors, making it an attractive target for developing molecular diagnostic and therapeutic agents. We compare

Full text of DOI:10.1007/s00775-019-01742-6

JBIC Journal of Biological Inorganic Chemistry
https://doi.org/10.1007/s00775-019-01742-6
ORIGINAL PAPER
Comparison of Al18F‑ and 68Ga‑labeled NOTA‑PEG4‑LLP2A for PET
imaging of very late antigen‑4 in melanoma
Yongkang Gai1,2 · Lujie Yuan1,2 · Lingyi Sun3 · Huiling Li1,2 · Mengting Li1,2 · Hanyi Fang1,2 · Bouhari Altine1,2
Qingyao Liu1,2 · Yongxue Zhang1,2 · Dexing Zeng3 · Xiaoli Lan1,2
·
Received: 2 July 2019 / Accepted: 6 November 2019
© Society for Biological Inorganic Chemistry (SBIC) 2019
Abstract
Malignant melanoma is an aggressive cancer with poor prognosis. Very late antigen-4 (VLA-4) is overexpressed in mela-
noma and many other tumors, making it an attractive target for developing molecular diagnostic and therapeutic agents. We
compared Al18F- and 68Ga-labeled LLP2A peptides for PET imaging of VLA-4 expression in melanoma. The peptidomi-
metic ligand LLP2A was modifed with chelator 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid
(p-SCN-Bn-NOTA), and the resulting NOTA-PEG4-LLP2A peptide was then radiolabeled with Al18F or 68Ga. The two labeled
peptides were assayed for in vitro and in vivo VLA-4 targeting efciency. Good Al18F and 68Ga radiolabeling yields were
achieved, and the resulting PET tracers showed good serum stability. In the in vivo evaluation of the B16F10 xenograft mouse
model, both tracers exhibited high accumulation with good contrast in static PET images. Compared with 68Ga-NOTA-PEG4-
LLP2A, Al18F-NOTA-PEG4-LLP2A resulted in relatively higher background, including higher liver uptake (1 h: 20.1 2.6 vs.
15.3 1.7%ID/g, P<0.05; 2 h: 11.0 1.2 vs. 8.0 0.8%ID/g, P<0.05) and lower tumor-to-blood ratios (2.5 0.4 vs. 3.3 0.5
at 1 h, P<0.05; 5.1 0.9 vs. 7.3 0.6 at 2 h, P<0.01) at some time points. The results obtained from the mice blocked with
unlabeled peptides and VLA-4-negative A375 xenografts groups confrmed the high specifcity of the developed tracers. Despite
the relatively high liver uptake, both Al18F-NOTA-PEG4-LLP2A and 68Ga-NOTA-PEG4-LLP2A exhibited high VLA-4 targeting
efcacy with comparable in vivo performance, rendering them promising candidates for imaging tumors that overexpress VLA-4.
Graphic abstract
Yongkang Gai and Lujie Yuan contributed equally to this work.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00775-019-01742-6) contains
supplementary material, which is available to authorized users.
Extended author information available on the last page of the article
Vol.:(0123456789)
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JBIC Journal of Biological Inorganic Chemistry
Keywords VLA-4 · Melanoma · Al18F radiolabeling · 68Ga · PET imaging · LLP2A
and prostate-specifc membrane antigen (PSMA)-targeted
ligands [35–39]. LLP2A-based diagnostic and therapeutic
agents have also shown great potential in the treatment of
VLA-4-overexpressing melanomas.
Introduction
Melanoma is a very aggressive malignant tumor with an
increasing incidence, especially among Caucasians, and
has become one of the major health problems in Western
countries [1]. It is one of the most devastating cancers, and
is resistant to most treatments. Despite recent advances in
the treatment of malignant melanomas, the overall 5-year-
survival rate is <10% [2]. Early diagnosis and accurate stag-
ing are important for the choice of appropriate therapeutic
interventions to improve patient outcomes.
18F is the most commonly used radionuclide for PET with
a favorable half-life (109.78 min), high imaging resolution,
and good scalability to GBq levels via on-demand cyclo-
tron production. It can be labeled to ligands via the attached
1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) chela-
tor by Al18F chelating. In addition to 18F, 68Gallium (half-
life = 67.71 min) has been increasingly considered as a
promising radionuclide for routine clinical PET imaging. It
is conveniently produced from an in-house 68Ge/68Ga gen-
erator, and has a very simple radiolabeling procedure. Both
18F and 68Ga have their own intrinsic advantages and attract
most of the interest in constructing peptide-based tracers.
Few attempts have been made to develop 68Ga- and 18F-BF3-
labeled LLP2A peptide for melanoma imaging [27–30],
and there has been no direct comparison between 68Ga-
and 18F-labeled LLP2A. We prepared a new Al18F-labeled
LLP2A using a one-step Al18F-chelation method, and
assessed its performance in B16F10 (VLA-4-positive) and
A375 (VLA-4-negative) mouse models. The results were
compared with those obtained from 68Ga-NOTA-PEG4-
LLP2A. Characterization and biological evaluations of the
labeled peptides included LogP values, serum stability,
cell uptake, cell internalization, PET imaging, and ex vivo
biodistribution.
Noninvasive imaging techniques, including 18F-fuorode-
oxyglucose-based positron emission tomography/computed
tomography (PET/CT), play an increasingly critical role in
the staging of melanoma and for assessing the response
to treatment [3–5]. However, limitations of 18F-FDG-PET
have been described in detecting distant small metastatic
lesions, such as sentinel lymph node deposits in stages I
and II of this cancer [6, 7]. In addition, the high accumu-
lation of 18F-FDG in infectious and infammatory lesions
may mimic tumor deposits [8]. With the rapid growth of
molecular imaging and precision medicine in the last dec-
ades, many radiopharmaceuticals that bind receptors have
been developed for more specifc imaging of melanoma [9].
Some, including radiolabeled benzamides that target mela-
nin [10–13], α-melanocyte-stimulating hormone analogues
that bind to the melanocortin-1 receptor (MC1R) [14–16],
and peptides that bind to the integrin family [17–19], have
exhibited promising results.
Very late antigen-4 (VLA-4, also called integrin α4β1) is
a non-covalent, heterodimeric transmembrane receptor. It is
overexpressed in melanoma, and plays a vital role in tumor
progression, angiogenesis and metastasis by promoting
adhesion and migration of tumor cells [20–22]. Enhanced
VLA-4 expression has also been found in lymphomas, leu-
kemias, osteosarcoma, and multiple myeloma [23]. Peptid-
omimetic LLP2A is a recently discovered VLA-4 targeting
ligand with a very high afnity (IC50 = 2 pM) [24]. Many
radionuclides, including 64Cu [25–27], 68Ga [27, 28], 18F
[29, 30], 111In [31], 99mTc [32], and 177Lu [33] have been
successfully attached to this ligand for diagnosis and/or
therapy of VLA-4-abundant tumors. Good images with high
tumor-to-background contrasts have been obtained in difer-
ent animal models [25–33]. In a mouse melanoma model,
the 177Lu-LLP2A bioconjugate showed promising therapeu-
tic efcacy, especially when combined with immune check-
point inhibitor therapy [34]. It also has shown great thera-
peutic potential in the treatment of melanoma metastases
[33]. There has been clinical success with theranostic com-
binations of 68Ga- and 177Lu-labeled somatostatin ligands
Materials and methods
Reagents and instruments
All chemicals were obtained from J&K Chemicals (Beijing,
China), or Sigma-Aldrich (St. Louis, MO, USA) and were
used without further purifcation. The mass spectrometry
spectra were recorded on Bruker SolarX 7.0T (Germany).
Radioactivity was quantifed using a dose calibrator (CRC-
15R, Capintec, Ramsey, NJ, USA) or gamma counter (2470
WIZARD; PerkinElmer, Waltham, MA, USA). For purifca-
tion of peptides and analysis of the radiolabeled conjugates
using two elution bufers [0.1 v% trifuoroacetic acid (TFA)
in deionized water as elution bufer A and 0.1 v% TFA in
acetonitrile as elution bufer B], high-performance liquid
chromatography (HPLC) was performed on an LC-20AT
system (Shimadzu Corporation, Tokyo, Japan) equipped
with an SPD-20A UV/vis detector (Shimadzu) and a fow
count radiation detector (Bioscan, Washington, DC, USA).
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JBIC Journal of Biological Inorganic Chemistry
844.8514; observed, [M+H]2+ =844.8493. The same HPLC
method as NOTA-PEG4-LLP2A was used for this chemical.
For AlF-NOTA-PEG4-LLP2A: AlCl3 (100 µL, 200 mM)
and NaF (100 µL, 200 mM) were mixed and shook for
10 min at room temperature to obtain AlF2+ solution.
NOTA-PEG4-LLP2A (20 μL, 5 mM) and AlF2+ solution
(2 μL, 100 mM) were added to sodium acetate bufer (50 µL,
0.25 M, pH 4.5). The mixture was heated at 100 °C for
10 min and the pH was monitored and kept at 4–5. Then,
HPLC purifcation was performed and the peak at 15.9 min
was collected. HRMS (Bruker SolariX 7.0T): calcd. for
C79H108AlFN15O20S: m/z [M+2H]2+ 833.3786; observed,
[M+H]2+ =833.3889. The same HPLC method as NOTA-
PEG4-LLP2A was used for this chemical.
Peptide synthesis
LLP2A was prepared via standard 9‐fuorenylmethoxy-
carbonyl (Fmoc)-based solid phase synthesis as reported
previously [24]. Briefy, Fmoc-Lys(Dde)-OH was frst
attached to Rink amide resin followed by Fmoc-PEG4-
CH2COOH, Fmoc-Ach-OH, Fmoc-Aad(tBu)-OH, N-α-
Fmoc-Lys(Alloc)-OH, 2-(4-3-o-tolylureido)phenylacetic
acid, and Trans-3-(3-pyridyl)acrylic acid. All coupling
was performed in dimethylformamide (DMF) using O-(7-
azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafuorophosphate (HATU) (3 equivalents), 1-hydroxy-
benzotriazole (HOBt) (3 equivalents), and N,N-diisopro-
pylethylamine (DIEA, 9 equivalents) as coupling rea-
gents. After all the coupling, the Dde protecting group
was cleaved with 2% hydrazine in DMF (3 × 20 min),
washed with DMF and dichloromethane (DCM), and
cleaved from the resin by twice treating with TFA/Phenol/
H2O/triisopropylsilane (92.5/2.5/2.5/2.5) for 2 h at room
temperature. The solution was fltered and the peptide
was precipitated in cold diethyl ester. The crude peptide
was purifed on a semi-preparative C18 column (Sepax
GP-C18, 250 × 10 mm) using HPLC. Fractions from 12.5
to 15 min were collected and lyophilized to obtain pure
PEG4-LLP2A. HPLC method: 0–3 min, 25% bufer B;
3–30 min, from 25% bufer B to 40% bufer B; fow rate,
4 mL/min.
Radiolabeling
Al18F labeling: no-carrier-added 18F-fluoride was used,
which was produced by a GE MINItrace cyclotron via the
18O(p,n)18F reaction. Briefy, the target was washed with
4 mL water to collect residual activity after routine clinical
18F-FDG production. The target water was passed through
a Sep-Pak QMA light cartridge (Waters), which was pre-
conditioned with 5 mL of 0.5 M potassium bicarbonate.
The 18F was eluted with 300 μL saline and the fractions
were collected into six tubes (each 50 μL). To the tube con-
taining the highest activity (500–740 MBq), AlCl3 (2 μL,
5 mM in sodium acetate bufer, pH 4, 0.1 M), sodium acetate
bufer (10 μL, pH 4, 1 M), acetonitrile (50 μL) and NOTA-
PEG4-LLP2A (4 μL, 5 mM) were added. The solution was
heated at 100 °C for 15 min, and then diluted with 1 mL
water. The resulting mixture passed through a C18 light
cartridge (Waters), followed by washing the cartridge with
5 mL water to remove unreacted 18F, and then the desired
Al18F-NOTA-PEG4-LLP2A was eluted with 0.5 mL ethanol.
The product was reconstituted in saline and passed through
a 0.22 μm Millipore flter into a sterile vial, and its radio-
chemical purity was determined by radio-HPLC.
Synthesis of NOTA‑PEG4‑LLP2A
p-SCN-Bn-NOTA (1.9 mg) and DIEA (10 μL) were added
to a solution of LLP2A-PEG4 (2.0 mg) in DMF (0.2 mL).
After reaction at room temperature for 4 h, 0.8 mL water was
added, and followed by HPLC purifcation. 2.1 mg prod-
uct was obtained after lyophilization (yield, ~71%). HPLC
method: Sepax GP-C18 (250×4.6 mm), 0–3 min, from 10%
bufer B to 20% bufer B; 3–23 min, from 20% bufer B to
60% bufer B; fow rate 1 mL/min. Retention time: 16.1 min.
HRMS (Bruker SolariX 7.0T): calcd. for C79H111N15O20S:
m/z [M+2H]2+ 811.9003; observed, [M+H]2+ =811.9000.
68Ga labeling: 68Ga3+ in 0.05 M HCl was obtained from
a
68Ge/68Ga generator (Isotope Technologies Graching,
Germany). Briefy, 4 mL 0.05 M HCl was passed through
the column and the fractions were collected into eight tubes
(each 0.5 mL). Sodium acetate bufer (150 µL, 0.25 M, pH
4.5) and NOTA-PEG4-LLP2A (2 μL, 5 mM) were added
to the tube containing the highest activity (370–500 MBq).
The pH of the fnal radiolabeling solution was 4–5. Then,
the mixture was heated at 100 °C for 10 min. The radiola-
beling yield of the product 68Ga-NOTA-PEG4-LLP2A was
determined by radio-HPLC.
Synthesis of nonradioactive Ga‑
and AlF‑NOTA‑PEG4‑LLP2A
For Ga-NOTA-PEG4-LLP2A: NOTA-PEG4-LLP2A (20 μL,
5 mM) and GaCl3 (2 μL, 100 mM) were added to sodium
acetate bufer (50 µL, 0.25 M, pH 4.5). The mixture was
heated at 100 °C for 10 min and the pH was monitored and
kept at 4–5. Then, HPLC purifcation was performed and
the peak at 16.0 min was collected. HRMS (Bruker SolariX
7.0T): calcd. for C79H108GaN15O20S: m/z [M + 2H]2+
Radio-HPLC method: 0–4 min, 5% bufer B; 4–5 min,
from 5% bufer B to 90% bufer B; 5–10 min, 90% bufer B;
10–15 min, 5% bufer B; fow rate, 1.0 mL/min.
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JBIC Journal of Biological Inorganic Chemistry
Serum stability
Animal model
Al18F-NOTA-PEG4-LLP2A and 68Ga-NOTA-PEG4-LLP2A
(~3.7 MBq) were each mixed with 0.5 mL human serum,
and the two mixtures were incubated at 37 °C for 4 h
(Al18F-tracer) and 2 h (68Ga-tracer), respectively. To remove
plasma protein, 0.5 mL acetonitrile was added to the mix-
tures, followed by 5 min centrifugation at 14,000 rpm. The
supernatants were separated and 90% of the radioactivity
was recovered. Approximately 37 kBq of the supernatants
was taken for radioactive purity determination using radio-
HPLC. Free 64Cu/68Ga came out at ~3.5 min and the radi-
olabeled conjugates came out at 11 min.
Four-week-old female C57BL/6 and BALB/c nude mice
purchased from HFK Bioscience (Beijing, China) were
used in this study. All mice were maintained under specifc
pathogen-free conditions, within facilities approved by the
Laboratory Animal Care of Huazhong University of Science
and Technology, and in compliance with the regulations and
standards of the Institutional Animal Care and Use Com-
mittee of Tongji Medical College of Huazhong University
of Science and Technology. For B16F10 and A375 tumor
xenografts, C57BL/6 and BALB/c nude mice were injected
subcutaneously in the right shoulder with one million cells
in PBS bufer, respectively.
Cell culture
Micro‑PET imaging
Cells were cultured in Dulbecco’s Modifed Eagle Medium
(DMEM), supplemented with 10% fetal bovine serum, peni-
cillin (100 units/mL), streptomycin (100 μg/mL) l-glutamine
(300 μg/mL), sodium pyruvate (100 mg/mL), and glucose
(4.5 g/L) and maintained at 37 °C, 5% CO2.
Mice bearing B16F10/A375 xenografts (4 mice per group)
were injected intravenously with Al18F-NOTA-PEG4-LLP2A
or 68Ga-NOTA-PEG4-LLP2A (5.5–7.4 MBq, 100–200 pmol
per mouse) with or without a blocking dose (100 µg unla-
beled LLP2A-PEG4). At 0.5 h, 1 h and 2 h (and 4 h for 18F)
post injection (p.i.), mice were anesthetized with 2% isofu-
rane and PET images were acquired. All PET scans were
performed with a small-animal PET scanner (BioCaliburn
LH Raycan Technology Co., Ltd.™, Suzhou, China). The
intrinsic spatial resolution was 1.0 mm, the time resolution
was 1.50 ns full-width at half-maximum, the energy resolu-
tion was 13.0% at 511 keV and the axial feld of view was
5.3 cm.
Cell uptake and internalization
B16F10 cells were seeded in 12-well plates (200,000 cells
per well) 24 h prior to the experiment. The cells were then
washed twice with 1 mL Hank’s Balanced Salt Solution
(HBSS) and 1 mL media (DMEM with 0.1% BSA and 1 mM
Mn2+) was added to each well. The cells were then incubated
with Al18F-NOTA-PEG4-LLP2A/68Ga-NOTA-PEG4-LLP2A
(~74 kBq per well) with and without the addition of excess
LLP2A-PEG4 (10 μg per well) to determine nonspecifc
uptake. At diferent time points (0.5, 1, and 2 h for 68Ga-
tracer; 0.5, 1, 2, and 4 h for 18F-tracer), the radioactive
media were aspirated, and the plate was washed twice with
PBS bufer (pH 7.2). The total cell uptakes were measured
by counting the radioactivity on a gamma counter. To col-
lect the surface-bound fraction, each well was treated with
20 mM sodium acetate-HBSS (pH 4.0) and was incubated
at 37 °C for 10 min. After removal of the surface-bound
fraction, cell pellets were dissolved in 0.5% SDS. All of the
fractions were counted in a well counter (2470 Automatic
Gamma Counter WIZARD, PerkinElmer, Norwalk, CT,
USA). The amount internalized was the amount of activity
in the fnal cell pellet, corrected for activity in the blocked
fractions and background activity. The percentages of the
radiotracers internalized into the cell were calculated based
on the equation: (the gamma counts obtained from cell-inter-
nalized radioactivity)/(the gamma counts obtained from the
total cell-associated radioactivity).
Ex vivo biodistribution
Mice bearing B16F10/A375 xenografts (5 mice per group)
were injected intravenously with Al18F-NOTA-PEG4-LLP2A
or 68Ga-NOTA-PEG4-LLP2A (5.5–7.4 MBq, 100–200 pmol
per mouse) with or without a blocking dose (100 µg unla-
beled LLP2A-PEG4). The tumor-bearing mice were killed
at 0.5 h, 1 h, and 2 h (and 4 h for 18F-tracer) p.i.. The organs
and tumors were harvested, weighed, and counted in a
gamma counter. The radioactivity in each tissue was nor-
malized as the percentage injected dose per gram of tissue
(%ID/g).
Statistics analysis
Signifcance analysis of the data was performed by unpaired
t-testing using Prism software (GraphPad Software Inc., San
Diego, CA, USA). Multiple comparisons were performed by
multiple t-tests together with the Holm–Sidak method for
correction. A value of P<0.05 was considered signifcant
and P<0.01 was considered highly signifcant.
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JBIC Journal of Biological Inorganic Chemistry
Results
Al18F-NOTA-PEG4-LLP2A/68Ga-NOTA-PEG4-LLP2A
were −1.80 0.03 and −1.92 0.05, respectively, indicat-
ing that both tracers were hydrophilic.
Synthesis and radiolabeling
In vitro cell study
A good isolated yield (~ 50%) of NH2-PEG4-LLP2A was
obtained from the traditional Fmoc-based solid phase syn-
thesis. p-SCN-Bn-NOTA was attached to the prepared
NH2-PEG4-LLP2A with a 71% yield; after HPLC purif-
cation, the purity of the NOTA-PEG4-LLP2A product was
>95% (see Fig. 1a for the structures).
To determine the specifc binding and internalization pro-
fles of Al18F-NOTA-PEG4-LLP2A and 68Ga-NOTA-PEG4-
LLP2A, cell uptake studies were performed using the VLA-4
overexpressing mouse B16F10 melanoma cells. 4 h and 2 h
cell uptake studies were conducted for 18F- and 68Ga-tracers,
respectively, because of their diferent half-life times. As
shown in Fig. 1c, e, the uptake of Al18F-NOTA-PEG4-LLP2A
increased within 1 h and then remained at a plateau up to
4 h, while the uptake of 68Ga-NOTA-PEG4-LLP2A gradu-
ally increased over 2 h. The uptake values of 68Ga-NOTA-
PEG4-LLP2A were significantly higher than those of
Al18F-NOTA-PEG4-LLP2A at the same time point (P<0.01).
A total of 32.0–35.5% (Al18F-tracer) and 26.9–36.5% (68Ga-
tracer) of the cell-bonded tracers (1–1.4% and 2.2–2.4%
of total added dose, respectively) were internalized into
the cells, while most of them remained on the cell surface.
In the groups receiving the blocking dose, the uptake of
Al18F-NOTA-PEG4-LLP2A and 68Ga-NOTA-PEG4-LLP2A
were reduced signifcantly by 61.7 and 79.2%, respectively,
validating their high specifcity for VLA-4.
The Al18F radiolabeling of the conjugate was performed
at 100 °C in 0.1 M sodium acetate bufer (pH 4) for 15 min.
Radiochemical yields ranging from 38 to 42% were obtained
with high radiochemical purity (>95%). The 68Ga radiolabe-
ling of the conjugate was performed at 100 °C in a solution of
0.5 mL 68GaCl3 in HCl (0.05 M) and 0.15 mL sodium acetate
(0.25 M) for 15 min. A greater than 95% radiolabeling yield
was achieved. Nonradioactive Ga-NOTA-PEG4-LLP2A and
AlF-NOTA-PEG4-LLP2A standards were prepared to con-
frm the successful 68Ga and Al18F radiolabeling. The molar
activity of 68Ga-NOTA-PEG4-LLP2A (37–74 MBq/nmol)
was slightly higher than that of Al18F-NOTA-PEG4-LLP2A
(20–37 MBq/nmol). Both Al18F2+ and 68Ga3+ labeled trac-
ers exhibited good stability, and less than 5% disassocia-
tion of 18F and 68Ga were observed after being incubated
in human serum at 37 °C (Fig. 1b). The LogP values of
Fig. 1 a Structures of Al18F-NOTA-PEG4-LLP2A and 68Ga-NOTA-
ered as the membrane-bound fraction. The radioactivity measured
in the cell lysates was considered as the internalized fraction. Total
cell-associated activity equals the activity of internalized fraction plus
membrane-bound fraction. d, f B16F10 cell uptake of the two trac-
ers and after blocking with co-incubation of 10 µg unlabeled LLP2A-
PEG4 (***P<0.001; n=3)
PEG4-LLP2A.
b
Stability
of
Al18F-NOTA-PEG4-LLP2A
and 68Ga-NOTA-PEG4-LLP2A after incubation in serum at
37 °C for 4 h and 2 h, respectively. c, e Cell internalization of
Al18F-NOTA-PEG4-LLP2A and 68Ga-NOTA-PEG4-LLP2A in
B16F10 cells. The radioactivity that was removed from the cells by
treatment with 20 mM sodium acetate-HBSS (pH 4.0) was consid-
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JBIC Journal of Biological Inorganic Chemistry
Fig. 2 Representative static PET
images of the B16F10 xenograft
at 0.5 h, 1 h, 2 h, and 4 h p.i. of
5.5–7.4 MBq (100–200 pmol)
Al18F-NOTA-PEG4-LLP2A (a)
and 68Ga-NOTA-PEG4-LLP2A
(b), and the blocked group co-
injected with 100 µg unlabeled
LLP2A-PEG4 (c and d, respec-
tively). Arrows indicate tumors
To validate in vivo specificity, blocking doses were
applied to the mice injected with Al18F-NOTA-PEG4-LLP2A
and 68Ga-NOTA-PEG4-LLP2A. As shown in Fig. 2c, d, the
activity accumulation in B16F10 xenografts was signif-
cantly reduced by the blocking agent, showing good speci-
fcity. To further confrm their VLA-4 targeting specifc-
ity, mice bearing VLA-4-negative A375 xenografts were
injected with 68Ga-NOTA-PEG4-LLP2A and PET imaging
was conducted at the same time points. The resulting PET
images did not show specifc accumulation in the A375
xenografts (Fig. 3).
Small‑animal PET imaging
The static images of Al18F-NOTA-PEG4-LLP2A in mice
bearing B16F10 tumors aforded excellent tumor visuali-
zation with high tumor retention and tumor-to-background
contrast at 0.5 h, 1 h, 2 h and 4 h after intravenous injec-
tion. The activity accumulation in liver was high and mini-
mal activity accumulations were observed in other normal
organs, except bladder.
PET images obtained from the group injected with
68Ga-NOTA-PEG4-LLP2A tracers were similar to those
injected with Al18F-NOTA-PEG4-LLP2A, and all the
tumors were clearly visualized with highest tumor uptake
achieved at an early p.i. time point (0.5 h). Compared to
Al18F-NOTA-PEG4-LLP2A, 68Ga-NOTA-PEG4-LLP2A
showed faster clearance and also slightly less liver uptake,
with higher bladder uptake due to the renal excretion of
injected radioactivity.
Ex vivo biodistribution
Biodistribution data are summarized in Figs. 4 and 5 and
Tables S1, S2 (see electronic supplementary material).
Both Al18F-NOTA-PEG4-LLP2A and 68Ga-NOTA-PEG4-
LLP2A exhibited similar in vivo distribution and excretion
Fig. 3 Representative static
PET images of C57BL/6
mice bearing B16F10 tumor
xenografts (a) and BALB/c
nude mice bearing A375 tumor
xenografts (b) at 0.5 h, 1 h,
and 2 h p.i. of 5.5–7.4 MBq
(100–200 pmol) 68Ga-NOTA-
PEG4-LLP2A. Arrows indicate
tumors
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JBIC Journal of Biological Inorganic Chemistry
Fig. 4 Biodistribution in mice bearing B16F10 tumors at 0.5 h, 1 h,
2 h and 4 h p.i. of Al18F-NOTA-PEG4-LLP2A (5.5–7.4 MBq, 100–
200 pmol) and 4 h after blocking by coinjection of 100 µg unlabeled
LLP2A-PEG4 with the Al18F-NOTA-PEG4-LLP2A) (a); tumor-to-
blood ratios (b); tumor-to-muscle radios (c) (n=5, **P<0.001)
Fig. 5 Biodistribution of 68Ga-NOTA-PEG4-LLP2A (5.5–7.4 MBq,
100–200 pmol) in mice bearing B16F10 tumors at 0.5 h, 1 h and 2 h
p.i., 2 h blocked (co-injected with 100 µg unlabeled LLP2A-PEG4
with the 68Ga-NOTA-PEG4-LLP2A), and in mice bearing A375
tumors at 2 h p.i. (a); tumor-to-blood ratios (b) and tumor-to-mus-
cle ratios (c) of 68Ga-NOTA-PEG4-LLP2A in mice bearing B16F10
tumors at 0.5, 1 and 2 h post-injection (n=5, **P<0.001)
profles, such as high uptake in tumor, liver, spleen, and
kidneys at the same time point p.i.. Both tracers cleared
from the kidneys and liver; however, the liver clearance
was signifcantly higher for Al18F-NOTA-PEG4-LLP2A
than for 68Ga-NOTA-PEG4-LLP2A at 1 h and 2 h p.i. (1 h:
20.1 2.6 vs. 15.3 1.7%ID/g, P < 0.05; 2 h: 11.0 1.2
vs. 8.0 0.8%ID/g, P < 0.05). Signifcantly lower uptake
in the colon was observed for 68Ga-NOTA-PEG4-LLP2A
than for Al18F-NOTA-PEG4-LLP2A at 1 h and 2 h p.i.
(1 h: 4.8 0.9 vs. 2.4 0.3%ID/g, P < 0.01; 2 h: 2.8 0.2
vs. 2.1 0.2%ID/g, P < 0.01). The kidney uptake was also
signifcantly lower for 68Ga-NOTA-PEG4-LLP2A than
1 3

JBIC Journal of Biological Inorganic Chemistry
for Al18F-NOTA-PEG4-LLP2A at 2 h p.i. (5.9 0.5 vs.
4.5 0.3%ID/g, P < 0.01). The biodistribution compari-
sons of Al18F-NOTA-PEG4-LLP2A and 68Ga-NOTA-
PEG4-LLP2A are shown in Figure S1.
(37–74 MBq/nmol) was slightly higher than that of
Al18F-NOTA-PEG4-LLP2A (20–37 MBq/nmol), which
might also contribute to the superior in vivo performance of
68Ga-NOTA-PEG4-LLP2A. The tumor uptake of 68Ga-NOTA-
PEG4-LLP2A at 1 h was comparable to that of 68Ga-DOTA-
PEG4-LLP2A [33] (9.1 0.9%ID/g) and 68Ga-NODAGA-
PEG4-LLP2A [27] (8.7 1.3%ID/g) without statical
signifcant. The tumor uptake of Al18F-NOTA-PEG4-LLP2A
at 1 h was comparable to that of 18F-DOTA-AMBF3-PEG2-
LLP2A (9.46 2.19%ID/g) [30], but signifcantly higher than
Discussion
The aim of this study was to develop LLP2A-based PET
tracers using same peptidomimetic precursor, with diferent
positron emitters (18F and 68Ga), and compare their in vivo
performance for PET imaging of melanoma. A commonly
used chelator, p-SCN-Bn-NOTA, served as the key coordi-
nating group to form stable complexes with Al18F and 68Ga.
To our knowledge, this is the frst report of an Al18F-labeled
LLP2A tracer. Previously, there have been only a few reports
of 18F-labeled LLP2A using the 18F-organotrifuoroborate
method [29, 30]. In their study, PEG2-LLP2A-trifuoroborate
conjugates were synthesized and labeled with 18F by the
isotope exchange method with sufcient molar activity but
relatively lower radiochemical yields of 3.7% and 11.3%,
respectively.
−
that of 18F-N-Pyr-p-BF3 -PEG2-LLP2A (4.41 0.65%ID/g,
P=0.001) and 18F-AMBF3-PEG2-LLP2A (2.84 0.34%ID/g,
P < 0.0001) [29]. 68Ga-NOTA-PEG4-LLP2A exhib-
ited relatively quicker clearance from nonspecifc organs,
which might be due to slightly lower lipophilicity than
Al18F-NOTA-PEG4-LLP2A. It is consistent with their LogP
profles. The specifcity of Al18F-NOTA-PEG4-LLP2A and
68Ga-NOTA-PEG4-LLP2A was validated via blocking study,
and further confrmed using a VLA-4-negative A375 tumor
model.
Liver and bladder showed the most intense uptake, and
the activity accumulation in liver decreased over time, indi-
cating both tracers cleared predominantly via the hepa-
tobiliary pathway and partly via renal clearance. Signifi-
cantly higher liver uptake was observed at 1 h and 2 h p.i.
of Al18F-NOTA-PEG4-LLP2A compared to those of
68Ga-NOTA-PEG4-LLP2A (P < 0.01). The overall liver
uptake of Al18F-NOTA-PEG4-LLP2A and 68Ga-NOTA-
PEG4-LLP2A was signifcantly higher than those of 68Ga-
NODAGA-PEG4-LLP2A [27] and our previously reported
68Ga-NE2P1A-PEG4-LLP2A [28], which might due to the
slightly higher lipophilicity of the chelator p-SCN-Bn-NOTA
than those of NODAGA and p-SCN-PhPr-NE2P1A. Com-
pared to the reported 18F-R-BF3–PEG2-LLP2A tracers, the
liver uptake of Al18F-NOTA-PEG4-LLP2A was relatively
higher, but the colon uptake was much lower [29]. The high
colonic uptake of 18F-R-BF3–PEG2-LLP2A was signifcantly
reduced by introducing an appended DOTA moiety, which
usually serves as a metal chelator, but acted as a hydrophilic
appendant in their study [30]. The relatively high liver uptake
of Al18F-NOTA-PEG4-LLP2A and 68Ga-NOTA-PEG4-LLP2A
may hinder their applications in the detection of liver meta-
static lesions, which might also be solved by introducing a
hydrophilic appendant.
Stability is extremely important for radiotracers, espe-
cially for 18F radiolabeled tracers, since free 18F− binds
to bone with high afnity. Thus, we conducted an in vitro
stability test, and both prepared tracers remained intact
after several hours’ incubation in serum and PBS. For
Al18F-NOTA-PEG4-LLP2A, relatively high bone uptake
(4.47 0.37%ID/g at 0.5 h) was determined by an ex vivo
biodistribution study. The high bone uptake usually indi-
cates defuorination and low in vivo stability of 18F-based
tracers. In this case, defuorination might also occur for
Al18F-NOTA-PEG4-LLP2A, however, the majority was most
likely contributed by marrow uptake because of the natural
expression of VLA-4 in the bone marrow [40]. Moreover,
higher bone uptake (5.75 2.04%ID/g at 0.5 h) was also
observed in mice receiving 68Ga-NOTA-PEG4-LLP2A,
which confrmed specifc uptake in bone marrow. The bone
uptake was signifcantly reduced for both tracers when cold
PEG4-LLP2A was co-injected, which further validated the
stability.
All tumors could be clearly visualized in the PET images
with high retention up to 4 h and 2 h p.i. of 18F- or 68Ga-
labeled tracers, respectively. The tumor uptake values of
the two tracers were comparable at the same time points
p.i. post injection, except a slightly higher tumor uptake of
Al18F-NOTA-PEG4-LLP2A (9.04 2.30%ID/g) at 0.5 h
than 68Ga-NOTA-PEG4-LLP2A (6.77 1.77%ID/g) without
statical signifcance. 68Ga-NOTA-PEG4-LLP2A had signif-
cantly higher tumor-to-blood ratios at 1 h (P<0.05) and 2 h
(P<0.01), and signifcantly higher tumor-to-muscle ratio at
2 h (P<0.05) than Al18F-NOTA-PEG4-LLP2A. It should be
noticed that the molar activity of 68Ga-NOTA-PEG4-LLP2A
Overall, 68Ga-NOTA-PEG4-LLP2A displayed clear supe-
riority, compared with Al18F-NOTA-PEG4-LLP2A, due
to signifcantly lower liver uptake, and signifcantly higher
tumor-to-muscle and tumor-to-blood ratios at some time
points. Considering the wide availability of 18F−, develop-
ment of 18F-labeled VLA-4 tracers may also be in demand
in situations where the supply of 68Ga (and/or 68Ge/68Ga
generators) is limited. In addition, the residual activity of
18F after routine 18F-FDG production was collected by
1 3

JBIC Journal of Biological Inorganic Chemistry
6. Strobel K, Bode B, Dummer R, Veit-Haibach P, Fischer D,
Imhof L, Goldinger S, Steinert HC, Von Schulthess G (2009)
Limited value of 18 F-FDG PET/CT and S-100B tumour marker
in the detection of liver metastases from uveal melanoma com-
pared to liver metastases from cutaneous melanoma. Eur J Nucl
Med Mol Imaging 36(11):1774
washing the target of the cyclotron and used for the produc-
tion of Al18F-NOTA-PEG4-LLP2A in this study. Much larger
amounts of Al18F-NOTA-PEG4-LLP2A can be produced with
higher specifc activity when the entire activity produced by
the cyclotron is used. Thus, Al18F-NOTA-PEG4-LLP2A is a
good alternative VLA-4 imaging tracer for research and clini-
cal translation.
7. Servois V, Mariani P, Malhaire C, Petras S, Piperno-Neumann
S, Plancher C, Levy-Gabriel C, Lumbroso-le Rouic L, Desjar-
dins L, Salmon R (2010) Preoperative staging of liver metas-
tases from uveal melanoma by magnetic resonance imaging
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Conclusion
8. Choi EA, Gershenwald JE (2007) Imaging studies in patients
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In summary, two VLA-4 targeted radiotracers
(Al18F-NOTA-PEG4-LLP2A and 68Ga-NOTA-PEG4-
LLP2A) have been successfully constructed for PET
imaging of melanoma, and both tracers showed high
in vivo VLA-4 targeting efciency and good specifcity.
The favorable biodistribution and PET images make them
promising candidates for noninvasively imaging VLA-4
overexpressing tumors.
10. Xu X, Yuan L, Yin L, Jiang Y, Gai Y, Liu Q, Wang Y, Zhang Y,
Lan X (2017) Synthesis and preclinical evaluation of 18F-PEG3-
FPN for the detection of metastatic pigmented melanoma. Mol
Pharm 14(11):3896–3905
11. Feng H, Xia X, Li C, Song Y, Qin C, Liu Q, Zhang Y, Lan X
(2016) Imaging malignant melanoma with 18F-5-FPN. Eur J
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12. Ma X, Wang S, Wang S, Liu D, Zhao X, Chen H, Kang F, Yang
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Acknowledgements This work was supported, in part, by the National
Natural Science Foundation of China (Nos. 81801738 and 81630049),
the Fundamental Research Fund for the Chinese Central Universities of
Huazhong University of Science and Technology (2017KFYXJJ235),
opening foundation of Hubei key laboratory of molecular imaging
(02.03.2017-187) and research foundation of Wuhan Union Hos-
pital (02.03.2017-12). We also acknowledge the support from the
National Institute of Biomedical Imaging and Bioengineering grant
R21-EB020737, and the American Cancer Society Research Scholar
ACS-RSG-17-004-01-CCE.
13. Xu X, Yuan L, Gai Y, Liu Q, Yin L, Jiang Y, Wang Y, Zhang Y,
Lan X (2018) Targeted radiotherapy of pigmented melanoma
with 131I-5-IPN. J Exp Clin Cancer Res 37(1):306
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Compliance with ethical standards
Conflict of interest The authors declare that they have no conficts of
interest.
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Afliations
Yongkang Gai1,2 · Lujie Yuan1,2 · Lingyi Sun3 · Huiling Li1,2 · Mengting Li1,2 · Hanyi Fang1,2 · Bouhari Altine1,2
·
Qingyao Liu1,2 · Yongxue Zhang1,2 · Dexing Zeng3 · Xiaoli Lan1,2
2
* Dexing Zeng
Hubei Province Key Laboratory of Molecular Imaging,
Wuhan 430022, China
zengd@ohsu.edu
3
* Xiaoli Lan
Center for Radiochemistry Research, Department
of Diagnostic Radiology, Oregon Health and Science
University, Portland, OR 97239, USA
LXL730724@hotmail.com
1
Department of Nuclear Medicine, Union Hospital,
Tongji Medical College, Huazhong University of Science
and Technology, Wuhan 430022, China
1 3