Fluorine-18 labeling of an anti-HER2 VHH using a residualizing prosthetic group via a strain-promoted click reaction: Chemistry and preliminary evaluation

Article abstract of DOI:10.1016/j.bmc.2018.02.040

In a previous study, we evaluated a HER2-specific single domain antibody fragment (sdAb) 2Rs15d labeled with ¹⁸F via conjugation of a residualizing prosthetic agent that was synthesized by copper-catalyzed azide-alkyne cycloaddition (CuAAC). In

Full text of DOI:10.1016/j.bmc.2018.02.040

Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
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Bioorganic & Medicinal Chemistry
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Fluorine-18 labeling of an anti-HER2 VHH using a residualizing
prosthetic group via a strain-promoted click reaction: Chemistry and
preliminary evaluation
Zhengyuan Zhou a, Satish K Chitneni a, Nick Devoogdt b, Michael R. Zalutsky a, Ganesan Vaidyanathan a,
⇑
a Department of Radiology, Duke University Medical Center, Durham, NC 27710, USA
b In Vivo Cellular and Molecular Imaging Laboratory, Vrije Universiteit Brussel, (VUB), 1090 Brussels, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
In a previous study, we evaluated a HER2-specific single domain antibody fragment (sdAb) 2Rs15d
labeled with 18F via conjugation of a residualizing prosthetic agent that was synthesized by copper-cat-
alyzed azide-alkyne cycloaddition (CuAAC). In order to potentially increase overall efficiency and
decrease the time required for labeling, we now investigate the use of a strain-promoted azide-alkyne
cycloaddition (SPAAC) between the 2Rs15d sdAb, which had been pre-derivatized with an azide-contain-
ing residualizing moiety, and an 18F-labeled aza-dibenzocyclooctyne derivative. The HER2-targeted sdAb
2Rs15d and a nonspecific sdAb R3B23 were pre-conjugated with a moiety containing both azide- and
guanidine functionalities. The thus derivatized sdAbs were radiolabeled with 18F using an 18F-labeled
aza-dibenzocyclooctyne derivative ([18F]F-ADIBO) via SPAAC, generating the desired conjugate ([18F]
RL-II-sdAb). For comparison, unmodified 2Rs15d was labeled with N-succinimidyl 4-guanidinomethyl-
3-[125I]iodobenzoate ([125I]SGMIB), the prototypical residualizing agent for radioiodination.
Radiochemical purity (RCP), immunoreactive fraction (IRF), HER2-binding affinity and cellular uptake
of [18F]RL-II-2Rs15d were assessed in vitro. Paired label biodistribution of [18F]RL-II-2Rs15d and [125I]
SGMIB-2Rs15d, and microPET/CT imaging of [18F]RL-II-2Rs15d and the [18F]RL-II-R3B23 control sdAb
were performed in nude mice bearing HER2-expressing SKOV-3 xenografts. A radiochemical yield of
23.9 6.9% (n = 8) was achieved for the SPAAC reaction between [18F]F-ADIBO and azide-modified
2Rs15d and the RCP of the labeled sdAb was >95%. The affinity (Kd) and IRF for the binding of [18F]RL-
II-2Rs15d to HER2 were 5.6 1.3 nM and 73.1 22.5% (n = 3), respectively. The specific uptake of [18F]
RL-II-2Rs15d by HER2-expressing BT474M1 breast carcinoma cells in vitro was 14–17% of the input dose
at 1, 2, and 4 h, slightly higher than seen for co-incubated [125I]SGMIB-2Rs15d. The uptake of [18F]RL-II-
2Rs15d in SKOV-3 xenografts at 1 h and 2 h p.i. were 5.54 0.77% ID/g and 6.42 1.70% ID/g, respectively,
slightly higher than those for co-administered [125I]SGMIB-2Rs15d (4.80 0.78% ID/g and 4.78 1.39%
ID/g). MicroPET/CT imaging with [18F]RL-II-2Rs15d at 1–3 h p.i. clearly delineated SKOV-3 tumors while
no significant accumulation of activity in tumor was seen for [18F]RL-II-R3B23. With the exception of kid-
neys, normal tissue levels for [18F]RL-II-2Rs15d were low and cleared rapidly. To our knowledge, this is
the first time SPAAC method has been used to label an sdAb with 18F, especially with residualizing
functionality.
Received 22 December 2017
Revised 19 February 2018
Accepted 21 February 2018
Available online xxxx
Keywords:
Fluorine-18
Strain-promoted click reaction
VHH
HER2
Residualizing label
Ó 2018 Elsevier Ltd. All rights reserved.
1. Introduction
Single-domain antibody fragments (sdAbs), also known as
Abbreviations: sdAb, single domain antibody fragment; CuAAC, copper-cat-
nanobodies (Nbs) and VHH molecules, are derived from camelid
heavy-chain-only antibodies. Compared with other protein-based
vehicles, advantages of sdAbs include stability, low immunogenic-
ity, and nanomolar to picomolar affinity. To date, a variety of sdAb-
based agents specific to oncological targets such as epidermal
growth factor receptor (EGFR),1–3 prostate-specific membrane
alyzed azide-alkyne cycloaddition; SPAAC, strain-promoted azide-alkyne cycload-
dition;
SGMIB,
N-succinimidyl
4-guanidinomethyl-3-iodobenzoate;
RCP,
radiochemical purity; ADIBO, aza-dibenzocyclooctyne; PET, positron emission
tomography.
⇑
Corresponding author at: 161C, Bryan Research Building, 311 Research Drive,
Durham, NC 27710, USA.
E-mail address: ganesan.v@duke.edu (G. Vaidyanathan).
https://doi.org/10.1016/j.bmc.2018.02.040
0968-0896/Ó 2018 Elsevier Ltd. All rights reserved.
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2
Z. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
antigen (PSMA),4–6 multiple myeloma 5T2 M-protein7,8
,
and
designed for use with an internalizing biomolecule like 2Rs15d
anti-HER2 sdAb.
human epidermal growth factor receptor type 2 (HER2)9–11 have
been developed and evaluated both preclinically and clinically.
The small size (ꢀ15 kDa) of sdAbs facilitates vascular extravasation
and tissue penetration as well as rapid blood clearance. The latter
property makes them excellent vehicles for molecular imaging
with short-lived positron emitters such as 18F and 68Ga.
The aim of this study was to develop a method for labeling
2Rs15d with 18F using a residualizing label via SPAAC. This was
accomplished by first pre-conjugating the sdAb with the
azide- and guanidine-containing prosthetic moiety N-succinimidyl
3-(azidomethyl)-5-(guanidinomethyl)benzoate (1)26 and then
clicking the derivatized 2Rs15d with [18F]F-ADIBO27 (Scheme 1).
This radiolabeled sdAb was evaluated on HER2-expressing cell
lines and by necropsy biodistribution and microPET imaging in
athymic mice bearing HER2-expressing SKOV-3 ovarian carcinoma
xenografts. As a benchmark for comparison, 2Rs15d radiolabeled
with the prototypical radioiodination residualizing label [125I]
SGMIB was evaluated in parallel.
HER2 overexpression occurs in about 20% of breast cancers and
is generally associated with tumor aggressiveness.12 Therefore,
robust assessment of HER2 status is crucial for selecting patients
who might benefit from HER2-targeted treatments. Current meth-
ods for the assessment of HER2 status – immunohistochemistry
and fluorescence in situ hybridization – are not ideal because they
require invasive biopsy and are inappropriate for dealing with the
heterogeneous nature of HER2 expression in the primary tumor as
well as variability in expression between primary and metastatic
tumors.13 Furthermore, these methods do not provide tumor
HER2 status in real-time. Given the risks involved in invasive pro-
cedures and the potential for misleading results from these ex-vivo
methods, non-invasive alternatives that can provide global HER2
status in real time are needed. Positron emission tomography
(PET) is a molecular imaging technique with high quantitative
capability, making PET an attractive approach for achieving this
goal.
Certain biomolecules undergo internalization after binding to
their respective receptors/antigens on the surface of tumor cells,
translocate to the lysosomes wherein they are catabolized. When
these molecules are radiolabeled, the low molecular weight
catabolites bearing the radiolabel often wash out of the cells
decreasing the radioactive signal within the tumor cells. Residual-
izing prosthetic agents, containing charged or polar moieties,
2. Materials and methods
2.1. General
All reagents were purchased from Sigma-Aldrich except where
noted. Sodium [125I]iodide (81.4 TBq (2200 Ci)/mmol) in 0.1 N
NaOH was obtained from Perkin-Elmer Life and Analytical Sciences
(Boston, MA). Synthesis of N-succinimidyl 4-guanidinomethyl-3-
[
125I]iodobenzoate ([125I]SGMIB)14 and N-succinimidyl 3-(azido-
methyl)-5-((1,2-bis(tert-butoxycarbonyl)guanidino)methyl)benzoate26
has been described. The sdAb 2Rs15d was radioiodinated using
[
125I]SGMIB as reported before.16 N-(3-Aminopropionyl)-5,6-dihy-
dro-11,12-didehydrodibenzo[b,f]azocine (ADIBO-amine; com-
pound 2, Scheme 2) was obtained from Click Chemistry Tools
(Scottsdale, AZ). Normal phase column chromatography was per-
formed using the Biotage Isolera chromatography system (Char-
lotte, NC) and their prepacked columns. High performance liquid
carbohydrate residues, and/or D-amino acid peptides generate cell-
and lysosome-membrane-impermeable catabolites, thereby
increasing activity retention in the tumor cells after internaliza-
tion.14,15 In a previous study, we labeled the anti-HER2 sdAb
2Rs15d with 18F using a residualizing label ([18F]RL-I) designed
to entrap the 18F in HER2-expressing cancer cells after receptor-
mediated internalization. The [18F]RL-I prosthetic group was pre-
synthesized employing conventional Cu(I)-assisted [3 + 2] Huisgen
alkyne-azide cycloaddition (CuAAC).16 The thus labeled 2Rs15d
([18F]RL-I-2Rs15d) demonstrated good immunoreactivity and
affinity for HER2 as well as specific tumor targeting in vivo. How-
ever, the labeling procedure required about 3 h and the overall
radiochemical yield was only 3–4%. One approach for streamlining
the labeling procedure and potentially increasing the radiochemi-
cal yield is the use of bioorthogonal chemistry to directly label
the sdAb. Although attempts have been made to label proteins
and other biomolecules using CuAAC,17–19 limitations of CuAAC
include the toxicity of copper as well as its potential for forming
complexes with biomolecules.20 A copper-free, strain-promoted
azide-alkyne cycloaddition (SPAAC) has been developed to sur-
mount these problems21 and SPAAC has been utilized successfully
with both nonradiolabeled and radiolabeled prosthetic agents to
modify peptides, which can tolerate relatively harsh conditions like
higher temperature and organic solvents.20,22 Proteins also have
been modified using SPAAC with nonradiolabeled prosthetic
agents, where one has the luxury of using stoichiometric or excess
amounts of the agent, and running the reaction for an extended
period.23 Of some relevance to this work, SPAAC has been used
to generate dimeric sdAbs and to PEGylate an sdAb.24 To our
knowledge, SPAAC has not been utilized for labeling proteins with
radionuclides, particularly those with short half-lives like 18F (t
=
½
109.8 min). It should be noted that during the final stages of
preparing this manuscript, labeling of an Adnectin protein with
18F by a similar approach was published25; however, this method
did not involve introduction of a residualizing label that was
Scheme 1. Pre-derivatization of sdAb with
1
and subsequent 18F-labeling via
SPAAC with [18F]F-ADIBO.
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Z. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
3
Scheme 2. Scheme for synthesis of ADIBO-OTs and F-ADIBO. a) tert-butyldimethylsilyl chloride, imidazole, DMF b) TritonB, MeOH c) EDC, DMAP, N-hydroxysuccinimide,
DMF d) Tsf, DBU, MeCN e) TBAF, THF
chromatography (HPLC) was performed using the following sys-
tems: 1) An Agilent 1260 MWD system with PrepStar 218 pumps
and controlled by the Agilent ChemStation software (this system
was used for purification of compound 1) 2) an Agilent 1260 Infin-
ity system equipped with a 1260 Infinity Multiple Wavelength
Detector (Santa Clara, CA) connected to a LabLogic Dual Scan-
RAM flow radioactivity detector/TLC scanner (Tampa, FL); the
whole system was controlled by LabLogic LauraÒ software. In addi-
tion, for semi-preparative HPLC purification of compounds 5 and 6,
the Agilent HPLC attached to the LCMS (see below) was used.
When the Agilent system was used, for both radiolabeled and unla-
beled compounds, HPLC was performed using an Agilent Poroshell
EC-120 (9.4 ꢁ 250 mm 2.7 mm) reversed-phase semi-preparative
column. EmporeTM SPE C18 cartridges used for concentrating HPLC
samples were purchased from 3 M (Maplewood, MN). Disposable
PD 10 desalting columns for gel filtration were purchased from
GE Healthcare (Piscataway, NJ). Instant thin layer chromatography
(ITLC) was performed using silica gel impregnated glass fiber
sheets (Pall Corporation, East Hills, NY) using PBS, pH 7.4 as the
mobile phase. Developed sheets were analyzed for activity using
the TLC scanner described above. Activity levels in various samples
were assessed using either an LKB 1282 (Wallac, Finland) or Perkin
Elmer Wizard II (Shelton, CT) automated gamma counter. Proton
NMR spectra were obtained on a 400 MHz spectrometer (Varian/
Agilent, Inova; Palo Alto, CA) and chemical shifts are reported in
d units using the residual solvent peaks as a reference. For com-
pound 1, both proton and 13C NMR spectra were obtained using
a Varian 500 MHz (125.8 MHz) spectrometer. Mass spectra were
recorded using an Agilent LC/MSD Trap for electrospray ionization
(ESI) LC/MS and/or an Agilent LCMS-TOF (ESI); the latter is a high-
resolution mass spectrometer. In addition, mass spectra were also
obtained using an Advion (Ithaca, NY) ExpressionL CMS LC-MS Sys-
tem attached to an Agilent HPLC like the one described above. This
equipment has the capability of determining molecular weights of
compounds directly from TLC plates (Plate Express) and by ESI,
APCI, and ASAP. For the determination of derivatized sdAb molec-
ular weights, either the above Advion system (LCMS) or an Applied
Biosystems DE-PRO Biospectrometry Workstation (for Maldi) were
used. Surface plasmon resonance experiments were performed on
a GE Biacore T200 machine located in the Biacore Facility, Duke
University Human Vaccine Research Institute.
2.2. Single domain antibodies, cells culture conditions and animal
model
Details of the production, purification and characterization of
2Rs15d sdAb have been reported.11,28 An anti-paraprotein sdAb
R3B23,7 which doesn’t bind to HER2, was used for evaluating speci-
ficity of uptake. Both 2Rs15d and R3B23 are devoid of any tags
such as a His-tag. Cell culture reagents were purchased from
Thermo Fisher Scientific (Waltham, MA) except where noted.
BT474M1 human breast carcinoma cells,29 a more tumorigenic
version of the original BT474 line, were grown in RPMI1640 med-
ium (Sigma-Aldrich, St. Louis, MO) containing 10% fetal bovine
serum, 1% penicillin-streptomycin, 1% non-essential amino acids,
1% sodium pyruvate, 1% 4-(2-hydroxyethyl)-1-piperazineethane-
sulfonic acid (HEPES) and 10 mg/mL insulin (Sigma-Aldrich, St.
Louis, MO). SKOV-3 human ovarian carcinoma cells, obtained from
the Duke University Cell Culture Facility, were grown in McCoy’s
5A medium containing 10% fetal bovine serum and 1% penicillin-
streptomycin. Cells were cultured at 37 °C in a 5% CO2 humidified
incubator. All experiments involving animals were performed
using a protocol approved by the Duke University IACUC. Subcuta-
neous SKOV-3 xenografts were established by inoculating 10-week
old female athymic mice, obtained from the Duke University, Divi-
sion of Laboratory Animal Resources, with 5 ꢁ 106 SKOV-3 cells in
50% Matrigel (Corning Inc. NY) in the above medium (100 mL). The
tumors were allowed to grow until they reached a volume of 350–
500 mm3 (ꢀ6–8 weeks).
2.3. N-Succinimidyl 3-azidomethyl-5-guanidinomethylbenzoate (1)
A 95:2.5:2.5 (v/v/v) mixture of TFA:water:tri-isopropyl silane
(0.25 mL) was added to N-succinimidyl 3-(azidomethyl)-5-((1,2-
bis(tert-butoxycarbonyl)guanidino)methyl)benzoate26
(29 mg;
0.05 mmol) and the mixture stirred at 20 °C for 30 min. Solvents
were evaporated and the residue re-dissolved in 0.25 mL of ace-
tonitrile and subjected to semi-preparative HPLC purification. For
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4
Z. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
this, the Poroshell EC-120 reversed-phase semi-preparative col-
umn was eluted at a flow rate of 4 mL/min with a gradient consist-
ing of water (solvent A) and acetonitrile (solvent B); the proportion
of B was increased from 5% to 15% in 20 min. Under these condi-
tions the product eluted with a retention time of 13.9 min. Solvents
from the pooled HPLC fractions were evaporated to get 19.5 mg
(80% based on trifluoroacetate salt) of a clear oil. Analytical HPLC
of the isolated product was performed using the analytical Poro-
shell column with flow rate of 2 mL/min with a gradient consisting
of 0.1% formic acid in both water (solvent A) and acetonitrile (sol-
vent B); the proportion of B was increased from 5% to 25% in 5 min.
The product eluted with a retention time of 2.9 min and its purity
was ꢀ 99%. 1H NMR (CD3CN) d 2.85 (s, 4H), 4.43 (s, 2H), 4.52 (s,
2H), 7.67 (s, 1H), 8.00 (s, 1H), 8.01 (s, 1H). LRMS (LCMS-ESI) m/z:
346.1 (M+H)+. 13C NMR (CD3CN) d 26.87, 45.09, 54.56, 127.16,
129.61, 130.26, 134.58, 139.62, 140.10, 158.94, 162.89, 171.53.
Poroshell EC-120 reversed-phase semi-preparative column was
eluted with a gradient mobile phase consisting of 0.1% formic acid
in both water (solvent A) and acetonitrile (solvent B) at a flow rate
of 3 mL/min; the proportion of B was linearly increased from 40%
to 70% over a period of 30 min. HPLC fractions containing the pro-
duct (tR = 25 min) were pooled, and solvents evaporated to obtain
50 mg (0.09 mmol, 83%) of compound 5 as a colorless oil: 1H
NMR (CDCl3) d 1.22–1.32 (m, 2H), 1.40–1.47 (m, 2H), 1.58–1.65
(m, 2H), 1.90–1.97 (m, 3H), 2.41–2.47 (m, 1H), 2.45 (s, 3H), 3.18–
3.37 (m, 2H), 3.68 (d, 1H), 4.01 (t, 2H), 5.12 (d, 1H), 5.97 (t, 1H),
7.25–7.40 (m,7H), 7.78 (d, 1H). LRMS (LCMS-ESI) m/z: 545.1 (M
+H)+.
2.7. Synthesis of F-ADIBO (6)27
Tetrabutyl ammonium fluoride (27 lL of 1 M THF solution, 27
HRMS (ESI) calcd for
346.1254 0.0001 (n = 4).
C
14H15N7O4 (M+H)+ 346.1583, found
mmol) was added to a solution of 5 (5 mg, 9 mmol) in 1 mL of
THF. The reaction mixture was refluxed for 30 min, the solvent
removed under vacuum, and the residue purified by RP-HPLC using
the conditions described above to give 2.5 mg (6.4 mmol, 69%) of 6
(tR = 19 min) as a colorless oil: 1H NMR (CD3CN): 1.25–1.50 (m,
2H), 1.55–1.63 (m, 2H), 1.65–1.72 (m, 2H), 1.90–2.00 (m, 3H),
3.69 (d, 1H), 4.35 (t, 1H), 4.47 (t, 1H), 5.10 (d, 1H), 6.17 (t, 1H),
7.25–7.50 (m,7H), 7.66 (d, 1H). LRMS (LCMS-ESI) m/z: 393.2 (M
+H)+.
2.4. 6-((tert-Butyldimethylsilyl)oxy)hexanoic acid (3)
This was synthesized essentially following the procedure
reported previously.30 Briefly, a mixture of tert-butyldimethylsi-
lylchloride (1.84 g, 12.21 mmol), imidazole (816 mg, 11.99 mmol)
and ethyl 6-hydroxyhexanoate (640 mg, 3.99 mmol) in DMF (10
mL) was stirred under nitrogen at 20 °C. After 24 h, the reaction
mixture was diluted with ether (50 mL) and the ethereal solution
washed with brine (150 mL ꢁ 3). The ether solution was separated,
dried over Na2SO4, and the ether removed by rotary evaporation to
obtain a yellow oil. A methanolic solution of Triton B (40% w/w, 10
mL) was added to this and the mixture stirred at 20 °C for an hour.
Methanol was removed under vacuum, water (20 mL) was added
to the residue and the pH adjusted to 4 using 1 M HCl. The aqueous
phase was then extracted with ether (100 mL ꢁ 3) and the com-
bined ether solution was dried over Na2SO4. Ether was evaporated
to yield 609 mg (2.47 mmol, 61.9%) of compound 3 as a yellow oil:
1H NMR (CDCl3) d 0.04 (s, 6H), 0.89 (s, 9H), 1.40 (dd, 2H), 1.53 (m,
2H), 1.65 (m, 2H), 3.61 (t, 2H).
2.8. Synthesis of [18F]F-ADIBO ([18F]6)
[
18F]F-ADIBO was synthesized by slight modifications of a pre-
viously reported method.27 Fluorine-18 was obtained either by in
house cyclotron irradiation of [18O]H2O as described before26 or
from PET-NET solutions (Durham, NC). For labeling reactions, 18F
activity trapped in a QMA cartridge was eluted with 0.5 mL of a
solution of tetraethylammonium bicarbonate in 80% acetonitrile
(6 mg/mL) and dried by azeotroping with acetonitrile (3 ꢁ 1 mL).
A solution of 5 (2 mg, 3.57 mmol) in 300 mL acetonitrile was added
to the dried 18F activity (1.9–3.7 GBq; 50–100 mCi), and heated at
100 °C for 15 min. Acetonitrile was evaporated, and the residual
activity dissolved in 100 mL of 40% acetonitrile and injected onto
a Poroshell EC-120 column that was eluted under the conditions
described above for 6. The HPLC fractions containing [18F]6 were
pooled and concentrated by solid-phase extraction using an
EmporeTM SPE cartridge (3M, St. Paul, MN) with acetonitrile (3 ꢁ
150 mL) as the eluent and acetonitrile from the pooled fractions
was evaporated.
2.5. Synthesis of compound 427
Compound 3 (187 mg, 0.76 mmol), EDC (58.3 mg, 0.30 mmol),
and DMAP (3.1 mg, 0.03 mmol) were added to a solution of
ADIBO-amine (70 mg, 0.25 mmol) in 5 mL DMF. The reaction mix-
ture was stirred overnight at 20 °C. The solvent was removed under
vacuum and the residue dissolved in 50 mL dichloromethane. The
resultant solution was washed with brine (3 ꢁ 50 mL), dried over
Na2SO4, and dichloromethane removed using a rotary evaporator.
The residue was purified by chromatography using 2% methanol
in dichloromethane to obtain 100 mg (0.20 mmol, 78.2%) of com-
pound 4 as a yellow oil: 1H NMR (CDCl3) d 0.05 (s, 6H), 0.90 (s,
9H), 0.90 (s, 9H), 1.25–1.35 (m, 2H), 1.45–1.55 (m, 4H), 1.95–2.05
(m, 3H), 2.40–2.50 (m, 1H), 3.27–3.37 (m, 2H), 3.60 (t, 2H), 3.70
(d, 1H), 5.15 (d, 1H), 6.00 (t, 1H), 7.26–7.42 (m, 7H), 7.68 (d, 1H).
LRMS (LCMS-ESI) m/z: 505.2 (M+H)+.
2.9. Derivatization of sdAbs with N-succinimidyl 3-azidomethyl-5-
guanidinomethylbenzoate
A solution of the sdAb (2Rs15d or R3B23; 1.0 mg, ꢀ0.08
in 0.1 M borate buffer, pH 8.5 (300 L) was added to 1 (850
2.46 mol) and the mixture stirred at 30 °C for 2 h. The derivatized
lmol)
l
l
g,
l
and non-derivatized sdAbs were isolated from unreacted reagent
and borate salts by ultrafiltration using a VivaspinÒ protein con-
centrator (5 kDa MWCO; GE Healthcare) and lyophilized to dry-
ness. The molecular weight of sdAb conjugates was determined
by MALDI-TOF mass spectrometry. The affinity for binding of the
2Rs15d conjugate to HER2 extracellular domain was determined
by surface plasmon resonance (SPR).
2.6. Synthesis of ADIBO-OTs (5)27
A mixture of compound 4 (100 mg, 0.20 mmol), tosyl fluoride
(104 mg, 0.60 mmol) and DBU (15 lL, 0.10 mmol) in 3 mL of ace-
tonitrile was stirred at 90 °C for 2 h. The reaction mixture was
cooled to 20 °C, and partitioned between dichloromethane and
brine (50 mL ꢁ 3). The pooled dichloromethane solution was dried
with Na2SO4, and concentrated to dryness. The residue was taken
up in acetonitrile and subjected to semi-preparative HPLC
using the Agilent 1260 Infinity HPLC system. For this, an Agilent
2.10. Labeling derivatized sdAbs with 18F via SPAAC using [18F]F-
ADIBO
A solution of each sdAb (100 mL of 1 mg mLꢂ1) in PBS, pH 7.4
was added to a vial containing dried [18F]F-ADIBO activity, and
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Z. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
5
the mixture incubated at 20 °C for 20 min. The entire mixture was
2.14. Biodistribution
loaded onto a PD-10 gel filtration column and eluted with PBS.
Paired-label biodistribution of
[
18F]RL-II-2Rs15d and
[
125I]
SGMIB-2Rs15d was evaluated in SKOV-3 tumor bearing mice. Each
mouse weighing about 25 g received 259 kBq (7 mCi, 1.7 mg) of [18F]
RL-II-2Rs15d and 185 kBq (5 mCi, 0.8 mg) of [125I]SGMIB-2Rs15d in
2.11. Determination of radiochemical purity
Three methods were used to determine the radiochemical pur-
ity/integrity of radiolabeled sdAbs: 1) To determine protein-associ-
ated activity, trichloroacetic acid precipitability was performed by
a total of 100 lL of PBS by bolus injection via the tail vein. Blood
and urine were collected from groups of five mice, which were
then killed and their tissues harvested at 1 and 2 h post injection.
The isolated solid tissues were blot-dried, weighed, and the activity
in them as well as in blood and urine was counted along with input
standards. From these, the percentage of the injected dose (ID) per
organ and per gram of tissue (%ID/g) were calculated. Mouse thy-
roid being a very small organ, it is harvested along with surround-
ing muscle tissue. The following formula was used to calculate the
%ID/organ in the case of thyroid.
incubating ꢀ5 ng of the labeled sdAb with 300
lL of 1% human
serum albumin (HSA) and 500 L of 10% TCA in triplicate at 20 °C
l
for 1 min. 2) ITLC was done on silica gel-impregnated glass fiber
sheets eluted with PBS (pH 7.4). The sheets were scanned by
LabLogic Scan-RAM described above. The sdAbs remained at the
origin and lower molecular weight species eluted with a retention
factor of 0.47. 3) Non-reducing SDS-PAGE and subsequent phos-
phor imaging using a Storage Phosphor System Cyclone Plus phos-
phor imager (Perkin-Elmer Life and Analytical Sciences, Downers
Grove, IL) was used to assess the integrity of labeled proteins as
previously described.16
ꢀꢀꢀ
ꢁ
ꢁ
ꢁ
n
N
%ID ¼
ꢃ 100 ꢂ A ꢃ W
Where %ID is the percentage of injected dose in thyroid, n is the
counts per minute obtained for thyroid and associated muscle
combined, N is the counts per minute in the injected dose, A is
the %ID/g in muscle and W is the weight of thyroid and associated
muscle.
2.12. Determination of immunoreactivity and Kd of [18F]RL-II-2Rs15d
The immunoreactive fraction of [18F]RL-II-2Rs15d was deter-
mined using the Lindmo method.31 Briefly, streptavidin-coated
magnetic beads (PureBiotech, Middlesex, NJ) were conjugated with
the extracellular domain of HER2 (Acrobiosystems, Newark, DE), or
to evaluate nonspecific binding, human serum albumin. Aliquots of
Statistical significance of the difference in uptake between the
two radioisotopes was determined by a paired Student t test using
Microsoft Excel program; a P value of < 0.05 was considered to be
significant.
[
18F]RL-II-2Rs15d (ꢀ5 ng) were incubated with increasing concen-
trations of both positive (HER2) and negative (HSA) beads. The
reciprocal of the percentage of specific binding was plotted against
the reciprocal of bead concentration and data were fit to a straight
line by linear regression. The immunoreactive fraction was calcu-
lated as the reciprocal of the y-intercept value (binding at infinite
antigen concentration).
2.15. MicroPET/CT imaging
Imaging of SKOV-3 subcutaneous xenograft-bearing mice was
performed on a Siemens Inveon microPET/CT system (Knoxville,
TN). Three mice were imaged 1 h, 2 h and 3 h after administration
of 0.6–1.3 MBq (16–36 mCi; 3.7–8.3 mg) [18F]RL-II-2Rs15d. Another
two mice were imaged 1 h and 2 h after administration of 1.7–2.0
For the saturation binding assay, BT474M1 cells were seeded in
24-well plates at a density of 8 ꢁ 104 cells/well/mL and incubated
at 37 °C overnight. Prior to the addition of [18F]RL-II-2Rs15d, the
cells were washed and replenished with fresh cold medium. Cells
were then incubated with [18F]RL-II-2Rs15d (0.1 to 300 nM; 0.6
mL/well) in triplicate at 4 °C for 2 h, the medium containing
unbound activity was removed, and the cells were washed twice
with cold medium. Finally, the cells were lysed with 0.1% SDS
and the activity in the cell lysate was counted using an automated
gamma counter. To determine non-specific binding, parallel assays
were performed as above but cells were co-incubated with a 100-
fold excess of unlabeled 2Rs15d. The data were fit using GraphPad
Prism software to determine Kd values. The entire experiment was
repeated thrice.
MBq (46–54 mCi; ꢀ11.5–13.5 mg)
[
18F]RL-II-R3B23. Mice were
anesthetized using 2–3% isoflurane in oxygen and placed prone
in the scanner gantry for a 5-min static PET acquisition followed
by a 5 min CT scan. List mode PET data were histogram-processed,
and the images reconstructed using the standard OSEM3D/MAP
algorithm—2 OSEM3D iterations, and 18 MAP iterations—with a
cutoff (Nyquist) of 0.5. Images were corrected for attenuation
(CT-based) and radioactive decay. Image analysis was performed
using Inveon Research Workplace software (Siemens).
3. Results and discussion
HER2-targeted vectors such as diabodies, affibodies and sdAbs
labeled with 18F have been investigated for the determination of
HER2 status by PET imaging.32–34 Because of the short half-life,
widespread availability and favorable radiation dosimetry proper-
ties of 18F, there is great interest in developing improved methods
for labeling fast-clearing biomolecules with 18F that can be per-
formed efficiently under physiological conditions. Of particular
interest is the development of residualizing prosthetic moieties
for labeling internalizing biomolecules with 18F because these
can potentially augment tumor retention of activity after recep-
tor-mediated internalization of the targeting vector occurs. In this
regard, we developed a first-generation residualizing label via a
click chemistry approach ([18F]RL-I) and evaluated its potential
utility for labeling two anti-HER2 sdAbs 5F7 and 2Rs15d.16,34
While excellent tumor targeting was seen with these tracers in
HER2-expressing cells and xenografts, the labeling procedure was
too long and labeling yields were too low for clinical translation.
In an attempt to simplify the labeling procedure and potentially
2.13. In vitro uptake assays
The cell uptake assay was performed in a paired-label format.
BT474M1 cells (8 ꢁ 105 cells per well/3 mL) were seeded in 6-well
plates overnight at 37 °C. The next day, the medium was replaced
with 2 mL fresh medium containing 5 nM each of [18F]RL-II-
2Rs15d and [125I]SGMIB-2Rs15d and the cells were incubated at
37 °C for 1, 2, and 4 h. Then, the cell culture supernatants were
removed, and the cells washed with medium (2 ꢁ 1 mL) and lysed
with SDS treatment. Activity in the cell culture supernatants and
cell-associated fractions was counted in an automated gamma
counter. To determine nonspecific uptake, a parallel experiment
was performed as above but with the addition of a 100-fold molar
excess of unlabeled 2Rs15d in the incubation media. The experi-
ment was performed in triplicate and the entire experiment was
repeated thrice.
Please cite this article in press as: Zhou Z., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.02.040

6
Z. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
increase labeling efficiency, strain-promoted alkyne azide cycload-
dition (SPAAC) was evaluated in the current study.
prosthetic agent 1 did not affect its HER2 binding affinity (1.59
nM) measured by SPR (Fig. 1A), which indicated a similar binding
affinity as unmodified 2Rs15d (1.71 nM).
The azadibenzocyclooctyne (ADIBO)-based SPAAC partner
reagent has been widely investigated in the past few years due
to its good reactivity, stability and commercial availability. With
a reduced activation barrier for SPAAC, azide-containing biomole-
cules such as peptides and proteins can be conveniently ‘‘clicked”
with an ADIBO-containing reagent under physiological conditions.
SPAAC employing ADIBO-azide has been used for site-specific con-
jugation of drugs to antibodies35 and in live cell imaging36 as well
as in pre-targeting strategies.37 Relevant to the work presented
herein, ADIBO-azide SPAAC has been used for 18F-labeling of pep-
tides and small molecules38,39 and recently, for labeling a protein.25
Compound 1 (Scheme 1) was synthesized from its protected
precursor26 in about 80% yield. Its NMR and mass spectrometry
characteristics were consistent with its structure. The precursor 5
and standard 6 (Scheme 2) were synthesized starting with com-
mercially available 2 and known compound 330 in two and three
steps, respectively, following reported procedures.27 The yields
were similar to those reported with 78% for intermediate 4 (versus
87%), 83% for 5 (versus 83%) and 69% for 6 (versus 78%). NMR data
for these compounds (as well as 3) were consistent with their
structures.
Conjugation of 2Rs15d with the azide- and residualizing guani-
dine-containing prosthetic agent (1) (Scheme 1) was accomplished
in 70% yield when about 30 equivalents of the prosthetic agent
were used; higher amounts of the prosthetic agent were precluded
due to its poor water solubility. Although the addition of 3–5%
DMSO was investigated, it failed to further improve the conjuga-
tion yield. MALDI-TOF mass spectrometry analysis revealed that
about 70% of the 2Rs15d molecules were substituted with the
prosthetic group with approximately 60%, 30% and 10% of these
conjugated to one, two and three prosthetic groups, respectively.
In the conjugation of 1 to control sdAb R3B23, about 35% of sdAb
molecules were modified with one prosthetic group and another
15% with two prosthetic groups. Conjugating 2Rs15d with the
The decay-corrected radiochemical yield for the synthesis of
[
18F]F-ADIBO ([18F]6) was 55.9 14.5% (n = 8), and 23.9 6.9% (n
= 8) for SPAAC between derivatized 2Rs15d and [18F]F-ADIBO.
The radiochemical yield for the synthesis of [18F]F-ADIBO was
somewhat lower than reported (65%),27 which might reflect the
use of a lower amount of precursor in our experiments (2 mg ver-
sus 7–9 mg). The total duration for synthesis of the [18F]RL-II-
2Rs15d conjugate was about 2 h and the overall decay-corrected
radiochemical yield (based on the initial [18F]fluoride activity)
was 3.2 0.9% (n = 8). With [18F]RL-II-R3B23 control sdAb, the
radiochemical yield for the conjugation reaction was 20.6% and
the radiochemical purity was 97.2% by (ITLC).
It is known that the reaction rate constant for SPAAC is much
lower than that for CuAAC (k2 for CuAAC 10–100 Mꢂ1Sꢂ1 versus
0.001–0.96 Mꢂ1Sꢂ1 for SPAAC).40 Although higher 18F-labeling
yields for SPAAC have been reported using similar systems, these
studies involved low molecular weight azide-containing com-
pounds including peptides for which the use of organic solvents
and higher temperatures could be tolerated.27,38 It has been
demonstrated that addition of surfactants or organic solvents such
as ethanol or acetonitrile, which may enhance the solubility of
agents such as [18F]F-ADIBO, can greatly improve the SPAAC reac-
tion yield.40,41 However, use of organic solvents has been shown to
be counterproductive in some cases.42 In the current work, the use
of higher incubation temperature (40 °C), longer incubation time
(up to 40 min), and addition of up to 5% DMSO were explored,
but none of these tactics significantly increased the conjugation
yield. Moreover, 2Rs15d sdAb precipitated at temperatures above
50 °C or when more than 10% DMSO was added. It is highly likely
that this may be due to derivatization with ADIBO as sdAbs includ-
ing 2Rs15d11 are known to be resistant to such conditions than
intact antibodies. The utilization of SPAAC greatly simplified the
labeling procedure, shortening the total radiosynthesis time to 2
Fig. 1. In vitro quality control data for preconjugated 2Rs15d and [18F]RL-II-2Rs15d. A) Surface Plasmon Resonance analysis. B) SDS-PAGE/phosphor imaging shows only one
band corresponding to the molecular weight of sdAb—left lane molecular weight markers and right lane [18F]RL-I-2Rs15d. C) Immunoreactivity assay data. D) Saturation
binding assay data (Mean SD) obtained using BT474M1 cells.
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Z. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
7
h from 3 h required for labeling 2Rs15d with [18F]RL-I via CuAAC.26
However, the overall radiochemical yield was compromised due to
low yield for the SPAAC reaction; thus there was no significant
advantage compared with the [18F]RL-I approach reported previ-
ously,26 at least with this particular combination of sdAb and
ADIBO reagent. Comparison of labeling 2Rs15d with [18F]RL-I and
[
18F]RL-II is presented in Table 1.
As noted above, a recent publication has described 18F labeling
of an Adnectin protein derivatized with a cyclooctyne moiety via
SPAAC using an 18F-labeled azido compound.25 While the authors
did not give the radiochemical yield for SPAAC per se, the overall
yield for the synthesis of 18F-labeled azido compound and its
SPAAC reaction with cyclooctyne-derivatized Adnectin was about
3–4%, which was similar to the overall yield obtained in the current
study. Given that the yield we obtained for [18F]6 was considerably
lower than they obtained for their 18F-labeled azido compound
(<50% vs >70%), the yield we obtained for the SPAAC reaction
should be at least equal or perhaps better even though we were
constrained to use a lower temperature for this reaction (20 °C vs
45 °C).
Fig. 2. Paired-label cellular uptake studies of [125I]SGMIB-2Rs15d and [18F]RL-II-
2Rs15d. BT474M1 cells were incubated with [125I]SGMIB-2Rs15d (red) and [18F] RL-
II-2Rs15d (blue) at 37 °C and processed at 1, 2 and 4 h as described in the text. Data
(Mean SD) shown are percent of initial added radioactivity that was specifically
(total minus nonspecific) cell-associated.
The specific activity of [18F]RL-II-2Rs15d was in the range of 93–
[
18F]RL-II-2Rs15d
activity determined and expressed as percent of initially added
activity. On the other hand, in the previous study, the cells were
incubated initially at 4 °C and after removing the medium contain-
ing unbound activity, cells were incubated with fresh medium at
37 °C, with surface-bound and internalized activity determined
and expressed as the percent of initially bound activity in these
compartments. However, the 14–17% specific uptake observed on
BT474M1 cells for [18F]RL-II-2Rs15d was similar to total uptake
reported for 2Rs15d radiolabeled using a sortase-based approach
and assayed under similar conditions.43 For example, after 1 h
incubation, the percent of input dose bound to cells was about
16–17% for [111In]In-CHX-A”-DTPA-2Rs15d and about 11–14% for
583 kBq/mg. SDS-PAGE/phosphor imaging of
indicated a single radioactive band, which corresponded to the
molecular weight of 2Rs15d (Fig. 1B). The radiochemical purity of
[
18F]RL-II-2Rs15d was further demonstrated by ITLC (95.5 1.8%)
and TCA precipitability (98.4 0.1%). The immunoreactive fraction
determined by Lindmo assay for [18F]RL-II-2Rs15d was 70.4% (Fig.
1C) and a Kd value of 5.6 1.3 nM was measured by saturation
binding assay on HER2-expressing BT474M1 cells (Fig. 1D). These
data indicate that labeling 2Rs15d using [18F]RL-II-2Rs15d did
not significantly affect its binding to HER2 and this is consistent
with the fact that none of the lysines in 2Rs15d is in the HER2
binding region.9
[
68Ga]Ga-NOTA-2Rs15d. It is worth pointing out that in that study,
The uptake of [18F]RL-II-2Rs15d and [125I]SGMIB-2Rs15d by
BT474M1 human breast carcinoma cells in vitro was determined
in a paired-label format. As shown in Fig. 2, about 14–17% of the
added [18F]RL-II-2Rs15d was specifically bound to the cells over
1–4 h. The uptake was similar to that observed for co-incubated
2Rs15d was labeled with radiometal-containing chelates, which
are generally considered to be residualizing prosthetic agents, fur-
ther supporting the residualizing character of the [18F]RL-II pros-
thetic agent.
The results obtained from the biodistribution of [18F]RL-II-
2Rs15d and [125I]SGMIB-2Rs15d at 1, and 2 h post injection are
summarized in Table 2. The tumor uptake of [18F]RL-II-2Rs15d
was 5.54 0.77 %ID/g at 1 h and 6.42 1.70 %ID/g at 2 h (P >
0.05). Although these values were higher than those observed for
co-injected [125I]SGMIB-2Rs15d, the differences between the two
radioconjugates were not statistically significant (P > 0.05). The
tumor uptake of [18F]RL-II-2Rs15d was significantly lower than
observed previously with [18F]RL-I-2Rs15d (15–20 %ID/g)16; how-
ever, it should be noted that BT474M1 xenografts were used in
the earlier study. Although there are conflicting reports concerning
the relative receptor number per cell for these two lines,44,45 it
appears that the BT474 cell line expresses HER2 to a higher
degree.28,45 Concordant with this, we have consistently seen high
tumor uptake (>10% ID/g) for various radiolabeled sdAbs in the
BT474M1 xenograft model.34,46,47 Other radiolabeled 2Rs15d con-
jugates have been evaluated in mice with SKOV-3 xeno-
grafts,11,28,48–50 providing more relevant benchmarks for
comparison. The tumor uptake observed for [18F]RL-II-2Rs15d in
SKOV-3 xenografts compared favorably with the values reported
for these other radiolabeled sdAb conjugates both in terms of peak
tumor uptake and retention of activity in the xenograft. In most of
the studies mentioned above, 2Rs15d was labeled with residualiz-
ing, radiometal-bearing prosthetic agents. On the other hand,
when 2Rs15d was labeled with the non-residualizing [18F]SFB
agent, tumor uptake decreased significantly from 1 h (5.94 1.17
%IA/g) to 3 h (3.74 0.52).50 These comparisons to literature data
performed with the same sdAb and the same tumor model suggest
[
125I]SGMIB-2Rs15d (P > 0.05) at 1 and 2 h and slightly higher (P
< 0.01) at 4 h. These results confirm that substitution of the pros-
thetic moiety onto 2Rs15d and the reaction conditions used for
labeling this sdAb using the [18F]RL-II approach did not affect its
binding and internalization to HER2-positive cancer cells. Further-
more, the results suggest that the degree of residualization of
activity in tumor cells in vitro achieved with [18F]RL-II was similar
to that obtained with the prototypical radioiodination residualiza-
tion agent SGMIB. These results cannot be directly compared with
those reported earlier for [18F]RL-I-2Rs15d16 because the assay for-
mat was different in the two cases. In the current study, cells
were incubated at 37 °C with the labeled sdAb and cell-associated
Table 1
Comparison of the two methods—[18F]RL-I and [18F]RL-II—for 18F-labeling of 2Rs15d.
Parameter
[
18F]RL-I
[
18F]RL-II
Total time for synthesis
Heating cycles
3 h
3
2 h
1
Total time for heating
Evaporation cycles
HPLC
Extraction required
Cartridge other than QMA
TFA cleavage required
RCYa for intermediate
Conjugation yield
50 min
6
Normal phase
Yes
Na2SO4
Yes
8.5 2.8%
40.8 9.1%
3.5 1.0%
15 min
4
Reversed-phase
No
C18
No
55.9 14.5%
23.9 6.9%
3.2 0.9%
Overall decay-corrected yield
a
RCY is radiochemical yield.
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8
Z. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
Table 2
Paired-label biodistribution of [18F]RL-II-2Rs15d and [125I]SGMIB-2Rs15d in athymic mice bearing subcutaneous SKOV-3 ovarian cancer xenograft.
Tissues
Percent injected dose per grama
1 h
2 h
I-125
F-18
I-125
F-18
Liver
Spleen
Lungs
0.36 0.04
0.19 0.03
0.64 0.23
0.23 0.05
43.37 7.54
0.44 0.15
0.52 0.17
0.14 0.03
0.04 0.20
0.36 0.30
0.32 0.12
0.29 0.17
0.05 0.03
4.80 0.78
2.22 0.56
0.78 0.19
1.53 0.37
0.85 0.23
131.13 10.73
0.69 0.24c
1.49 0.22
0.51 0.17
0.20 0.11c
0.61 0.23c
0.91 0.12
1.38 0.28
0.29 0.09
5.54 0.77c
0.55 0.06
0.28 0.06
0.45 0.10
0.18 0.03
24.66 6.54
1.35 0.50
0.30 0.06
0.35 0.07
0.70 0.20
0.12 0.06
0.35 0.03
0.18 0.04
0.03 0.01
4.78 1.39
2.48 0.32
0.84 0.08
1.39 0.43
0.58 0.11
78.81 11.72
0.54 0.15
1.73 0.45
1.45 0.42
0.22 0.06
0.45 0.10
0.62 0.13
2.49 0.76
0.30 0.06
6.42 1.70c
Heart
Kidneys
Stomach
Sm. Int.
Lg. Int.
Thyroidb
Muscle
Blood
Bone
Brain
Tumor
a
b
c
Mean SD (n = 5 except for bone at 1 h).
Percent injected dose per organ.
Difference in the uptake between the two agents statistically not significant.
a potential tumor delivery advantage for using the residualizing
18F]RL-II prosthetic agent for labeling sdAbs that target internaliz-
ing receptors such as HER2.
On the other hand, a triazole and the bulky cyclooctyne moieties
are present only in [18F]RL-II-2Rs15d, and thus might have con-
tributed to higher renal uptake of [18F]RL-II-2Rs15d. While there
are conflicting reports on the influence of triazoles on the uptake
of activity in kidneys and other normal tissues, a direct comparison
of folic acid conjugates with and without a triazole moiety showed
significantly higher renal uptake for the conjugate with the tria-
[
Both radiolabeled sdAbs demonstrated rapid clearance, with
<1% ID/g in the blood even at 1 h. Clearance of both radioconju-
gates occurred mainly through the kidneys, which was expected
because the ꢀ15 kDa size of sdAb is below the cutoff for renal fil-
tration of proteins (ꢀ60 kDa).51 There was a slight advantage in
tumor uptake for 2Rs15d labeled using [18F]RL-II; however, its
uptake in normal tissues such as liver, spleen and kidney was sub-
stantially higher than that seen for [125I]SGMIB-2Rs15d. As a result
of the higher uptake in normal tissues for [18F]RL-II-2Rs15d,
tumor-to-tissue ratios were considerably lower for the 18F-labeled
construct (Table 3).
zole.53 Finally, kidney uptake of
mented,54,55 and [18F]fluoride generated by the catabolism of
18F]RL-II-2Rs15d (see below) also might have contributed to high
[
18F]fluoride is well docu-
[
kidney uptake.
The uptake of [18F]RL-II-2Rs15d in the bone at 1 h was more
than 1.4-fold higher than that observed with [18F]RL-I-2Rs15d in
the BT474M1 xenograft model16 and about 7-fold higher than that
reported for 2Rs15d labeled using [18F]SFB in SKOV-3 xenograft
model.50 Furthermore, bone uptake increased from 1 h to 2 h sug-
gesting that 2Rs15d labeled using [18F]RL-II may be more suscepti-
ble to defluorination than when labeled using the other two
prosthetic moieties. When an sdAb that targets the complement
receptor of the Ig super family was labeled with 18F via the [18F]
[AlF]2+ method, bone uptake increased with time, which was
attributed to degradation of the labeled sdAb in kidney lysosomes
and recycling of [18F]fluoride and [18F][AlF]2+ [54]. We speculate
that [18F]RL-II-2Rs15d also may be subjected to this phenomenon
generating more [18F]fluoride; however, it appears that the [18F]
RL-II method is more inert to defluorination than [18F][AlF]2+
method. It has been reported that the internalization of two
The relatively bulky and hydrophobic cyclooctyne moiety pre-
sent in RL-II has been reported to significantly influence the biodis-
tribution properties of its bioconjugates.52 This may be
a
contributing factor for the higher normal tissue uptake observed
for [18F]RL-II-2Rs15d compared with [125I]SGMIB-2Rs15d. Of note,
kidney uptake of [18F]RL-II-2Rs15d was more than 3-fold higher
than that seen for [125I]SGMIB-2Rs15d, which was unexpected
given the hydrophobic nature of the cyclooctyne moiety. Although
the high renal uptake observed with [18F]RL-II-2Rs15d is consistent
with the molecular weight of an sdAb, the reasons that its kidney
uptake was considerably higher than co-injected [125I]SGMIB-
2Rs15d are unclear. The highly basic guanidine moiety is present
in both [18F]RL-II and [125I]SGMIB, so it is probably not a factor.
Table 3
Tumor-to-tissue ratios calculated for selected tissues from the biodistribution data presented in Table 1.
Tumor-to-tissue ratiosa
1 h
2 h
I-125
F-18
I-125
F-18
Liver
13.53 2.91
25.22 6.01
8.46 3.58
21.32 4.91
0.11 0.03
17.89 8.07
16.57 6.00
15.11 9.60
2.70 1.07
7.57 2.36
3.84 1.19
6.93 2.10
0.04 0.01
9.92 3.53b
6.23 1.46
3.61 1.90
8.61 1.75
2.60 0.59
7.61 1.42
4.74 1.06
11.16 2.72
0.08 0.02
14.81 4.57
10.47 2.28b
2.70 0.76
Spleen
Lungs
Heart
Kidneys
Muscle
Blood
Bone
17.66 5.05
10.64 1.73
26.10 6.07
0.20 0.07
46.79 23.43
13.63 3.54
29.02 14.66
a
Mean SD (n = 5).
Difference in ratio between the two agents statistically not significant.
b
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Z. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
9
Table 4
18F to 125I uptake ratios obtained from the biodistribution of [18F]RL-II-2Rs15d/[125I]SGMIB-2Rs15d in SKOV-3 model in the current study compared with the same ratios obtained
from the data for [18F]RL-I-2Rs15d/[125I]SGMIB-2Rs15d in BT474M1 model reported (Zhou et al., 2017).
Tissues
18F/125I Ratio
1 h
2 h
RL-I
RL-II
RL-I
RL-II
Liver
10.82 1.87
5.18 1.46
5.02 0.53
2.31 0.36
1.19 0.11
1.67 0.27
1.39 0.22
1.59 0.19
1.83 0.36
1.71 0.73
0.84 0.03
6.21 1.60
4.05 1.00
2.49 0.55
3.91 1.58
3.06 0.29
1.60 0.34
2.07 0.76
3.18 1.11
6.05 3.33
7.88 5.01
1.16 0.11
12.15 1.42
4.92 0.82
4.87 0.80
2.12 0.74
0.88 0.02
1.64 0.15
1.23 0.35
2.07 0.28
2.18 1.15
1.22 0.18
0.80 0.02
4.49 0.26
3.11 0.57
3.09 0.51
3.18 0.46
3.28 0.43
0.42 0.12
4.25 1.77
1.77 0.30
14.90 6.53
11.56 4.57
1.35 0.04
Spleen
Lungs
Heart
Kidneys
Stomach
Muscle
Blood
Bone
Brain
Tumor
anti-HER2 scFv-HSA fusion proteins was different in BT474 and
SKOV-3 cells.56 Given this, it is also possible that internalization,
trapping, and in turn the metabolism of the labeled sdAbs may
be different for BT474M1 and SKOV-3 cells, which also may have
contributed to differential bone uptake.
compare the biodistributions of [18F]RL-II-2Rs15d and [18F]RL-I-
2Rs15d, by normalizing to co-injected [125I]SGMIB-2Rs15d through
calculation of 18F/125I tissue uptake ratios. As shown in Table 4,
[
18F]RL-II is favored over [18F]RL-I providing relatively higher
tumor uptake, as well as lower uptake in liver, spleen and lungs.
On the other hand, compared with [18F]RL-I, [18F]RL-II labeling
results in relatively higher uptake in other normal tissues including
brain, bone and kidneys. Because breast cancers are known to
metastasize to liver, lungs, brain and bone, [18F]RL-II may be more
useful approach for evaluating metastatic spread in the liver and
lungs, but not brain and bone. Ideally, next generation [18F]RL-II
derivatives can be designed with structures that decrease defluori-
nation and minimize uptake in the bone and other normal tissues.
Coronal PET/CT images obtained 1, 2 and 3 h after administra-
tion of [18F]RL-II-2Rs15d in a representative athymic mouse bear-
ing a subcutaneous SKOV-3 xenograft are depicted in Fig. 3A.
Concordant with the data from the necropsy experiment, clear
delineation of tumor was observed with minimal background
activity in normal tissues except in kidneys, which exhibited
prominent and prolonged uptake. That the tumor uptake was
related to specific binding to HER2 was demonstrated by the
absence of activity accumulation in the tumor after injection of
nonspecific [18F]RL-II-R3B23 (Fig. 3B). The tumor-to-muscle ratios
for [18F]RL-II-2Rs15d (n = 3) were 9.6 1.7, 11.5 2.3, and 20.9
2.1 at 1 h, 2 h, and 3 h, respectively. The values at 1 h and 2 h were
similar to that obtained from the necropsy experiment. For [18F]RL-
II-R3B23 (n = 2) tumor-to-muscle ratios were 1.8 0.4 and 1.8
0.1 at 1 h and 2 h, respectively.
Although two different xenograft models were used in the cur-
rent and the previous16 studies evaluating 18F-labeled residualizing
prosthetic agents, in both cases, the biodistribution of co-injected
[
125I]SGMIB-2Rs15d also was determined. Thus, it is possible to
4. Conclusion
Although SPAAC has been used to label peptides, other small
molecules, and in one case the protein Adnectin, to our knowledge,
this is the first study evaluating its utility for labeling an sdAb with
18F. When labeled using [18F]RL-II, sdAb 2Rs15d demonstrated
excellent affinity and immunoreactivity for HER2, and uptake com-
parable to
[
125I]SGMIB-2Rs15d in vitro in HER2-expressing
BT474M1 cells. Moreover, in athymic mice with HER2-expressing
SKOV-3 xenografts, tumor uptake was virtually identical to [125I]
SGMIB-2Rs15d and compared favorably to those reported in the
literature for 2Rs15d radioimmunoconjugates including those
labeled with radiometals. To make this labeling strategy more
attractive, it will be necessary to improve SPAAC yields or adapt
other biorthogonal chemistry approaches that provide higher
radiochemical yields, and to develop labeled sdAb with greater
in vivo stability and lower normal tissue accumulation.
Fig. 3. MicroPET/CT images (coronal) obtained at 1 h, 2 h and 3 h after injection of
18F]RL-II-2Rs15d (A) and 1 h and 2 h after injection of [18F]RL-II-R3B23 (B) into
athymic mice bearing SKOV-3 xenografts. Tumor location, which was different for
the two studies, is indicated by arrows.
[
Please cite this article in press as: Zhou Z., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.02.040

10
Z. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
24. Rashidian M, Wang L, Edens JG, et al. Enzyme-mediated modification of single-
Acknowledgements
domain antibodies for imaging modalities with different characteristics. Angew
Chem Int Ed Engl. 2016;55:528–533.
This work was supported in part by National Institutes of
Health Grants CA188177 and CA42324. The excellent technical
assistance of Elzbieta Krol (in vitro studies) and Xiao-Guang Zhao
(in vivo studies) is greatly appreciated. We also thank Thomas
Hawk, and Simone Degan for their excellent support with micro-
PET/CT imaging studies.
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