Development of 170Tm-DOTA-cetuximab for radioimmunotherapy

Article abstract of DOI:10.1002/jlcr.1957

Thulium-170 [T_1/2=128.4days, E_β(max)=968keV, E_γ=84keV (3.26%)] has radionuclidic properties suitable for use in therapy. ¹⁷⁰Tm can be produced by a relatively feasible route involving thermal neutron bombardment on natural Tm(NO₃)₃ (100% ¹⁶⁹Tm) in medium flux research reactors. The combination of beta-particle emission of Tm-170 with therapeutic properties of C225 monoclonal antibody (cetuximab) as well as optimization studies for future Tm-167 labeling was targeted in this study. Conjugated cetuximab was obtained by the addition of 0.5ml of a cetuximab pharmaceutical solution (1mg, in phosphate buffer, pH7.8) to a glass tube pre-coated with in situ prepared 1,4,7,10-tetraazacyclododecane- N,N,N,N-tetraacetic acid mono-(N-hydroxysuccinimidyl) ester (DOTA-NHS) (～5mg) at 25°C. Cetuximab was labeled with ¹⁷⁰Tm-Thulium chloride (100MBq) after conjugation with DOTA-NHS in 2-3h (radiochemical purity >99%, instant thin-layer chromatography, specific activity=77-385TBq/mmol). Biodistribution studies in wild-type rats for free Tm-170 and the radioimmunoconjugate were performed to determine the distribution up to 72h. A comparative time-frame study was performed for critical organs for both radiochemical species. The major organs of accumulation were shown to be the lung, liver, and spleen, respectively.

Full text of DOI:10.1002/jlcr.1957

Research Article
Received 02 November 2011,
Revised 30 November 2011,
Accepted 01 December 2011
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1957
170
Development of Tm-DOTA-cetuximab for
radioimmunotherapy
*
Simindokht Shirvani-Arani, Ali Bahrami-Samani, Amir Reza Jalilian,
Amirsaleh Shirvani-Arani, and Mohammad Ghannadi-Maragheh
Thulium-170 [T1/2 = 128.4 days, Eb(max) = 968 keV, Eg = 84 keV (3.26%)] has radionuclidic properties suitable for use in
therapy. 170Tm can be produced by a relatively feasible route involving thermal neutron bombardment on natural Tm
(NO3)3 (100% 169Tm) in medium flux research reactors. The combination of beta-particle emission of Tm-170 with
therapeutic properties of C225 monoclonal antibody (cetuximab) as well as optimization studies for future Tm-167 labeling
was targeted in this study. Conjugated cetuximab was obtained by the addition of 0.5 ml of a cetuximab pharmaceutical solu-
tion (1 mg, in phosphate buffer, pH 7.8) to a glass tube pre-coated with in situ prepared 1,4,7,10-tetraazacyclododecane-N,N,
N,N-tetraacetic acid mono-(N-hydroxysuccinimidyl) ester (DOTA-NHS) (~5 mg) at 25ꢀC. Cetuximab was labeled with 170Tm-
Thulium chloride (100 MBq) after conjugation with DOTA-NHS in 2–3 h (radiochemical purity >99%, instant thin-layer
chromatography, specific activity = 77–385 TBq/mmol). Biodistribution studies in wild-type rats for free Tm-170 and the
radioimmunoconjugate were performed to determine the distribution up to 72 h. A comparative time-frame study was
performed for critical organs for both radiochemical species. The major organs of accumulation were shown to be the lung,
liver, and spleen, respectively.
Keywords: cetuximab; Thulium-170; biodistribution; radioimmunotherapy
to the authors’ knowledge no reports are available on develop-
ing Tm-170-labeled immunomolecules. Radiopharmaceuticals
prepared by Thulium-170 have the advantage of comparatively
Introduction
Cetuximab (IMC-C225—marketed under the name Erbitux) is a
chimeric (mouse/human) monoclonal antibody, an epidermal
longer half-life of 128.4 days, which makes it possible to supply
growth factor receptor (EGFR) inhibitor, given by intravenous
the radiopharmaceuticals worldwide.
infusion for treatment of metastatic colorectal cancer and head
In this work, we report radiolabeling, quality control, and bio-
distribution studies for 170Tm-DOTA-cetuximab.
and neck cancer. It received Food and Drug Administration approval
in 2006 for use in combination with radiation therapy for treating
squamous cell carcinoma of the head and neck or as a single agent
in patients who received prior platinum-based therapy.1
Experimental
Many radiolabeled cetuximab compounds have been developed
Thulium nitrate (spectroscopic grade N99.99% pure) was
to visualize and monitor EGFR tumors in animals and humans using
copper-64,2 technetium-99m,3 and yttrium-86 or for therapeutic
4
obtained from E. Merck (Darmstadt, Germany). Sephadex G-50,
purposes including 177Lu and 90Y in tumor-bearing nude mice 5 as
sodium acetate, phosphate buffer components, and chloroform
well as I-131.6
were purchased from Sigma–Aldrich Company Ltd., Poole, England.
Cetuximab (erbitux) was a pharmaceutical sample purchased from
Thulium-170, having both gamma and beta emissions (Eb =968
Merck UK and was used without further purification. DOTA-NHS
keV and Eg = 84 keV), is a new candidate for labeling of immunocon-
ester was freshly synthesized in our laboratory on the basis of the
jugates, which guarantees both applications (radioimmunotherapy
conventional method and kept under a blanket of dry N2. Radio-
and imaging studies). Thulium-170 can be prepared by neutron
chromatography was performed by counting Whatman No. 2 TLC
paper, using a thin-layer chromatography scanner, Bioscan
AR2000, Paris, France. Animal studies were performed in accor-
irradiation of natural target (natural Tm (NO3)3) using (n, g) reaction
in reasonable amounts.7 The cation 170Tm3+ as a lanthanide has sui-
table chelating properties with 1,4,7,10-tetraazacyclododecane-N,N,
dance with the United Kingdom Biological Council’s Guidelines on
N,N-tetraacetic acid (DOTA); thus, in this work we chose DOTA
the Use of Living Animals in Scientific Investigations, 2nd Ed. All
as the chelating agent, which can be linked to the antibodies
via N-hydroxysuccinimidyl (NHS). Activation of the DOTA molecule,
resulting in 1,4,7,10-tetraazacyclododecane-N,N,N,N-tetraacetic acid
mono-(N-hydroxysuccinimidyl) ester (DOTA-NHS), allows conjugation
to cetuximab antibody.
Very few reports are available on the development of Tm-170-
labeled compounds for therapeutic applications including
170Tm-ethylenediamine tetra(methylene phosphonic acid),8 and
Radiopharmaceutical Research and Development Lab (RRDL), Nuclear Science
and Technology Research Institute (NSTRI), P.O. Box: 14155-1339, Tehran, Iran
*Correspondence to: Simindokht Shirvani-Arani, Radiopharmaceutical Research
and Development Lab (RRDL), Nuclear Science and Technology Research Institute
(NSTRI), P.O. Box: 14155-1339, Tehran, Iran.
E-mail: smshirvani@aeoi.org.ir
J. Label Compd. Radiopharm 2012, 55 103–107
Copyright © 2012 John Wiley & Sons, Ltd.

S. Shirvani-Arani et al.
of the rats were male NMRI purchased from Pasteur Institute of Iran, and mixed gently for 30 s. The resulting solution was incubated
weighing 180–200 g. In each group/interval, five rats were used and at 37–40ꢀC for 2–3 h. After radiochemical purity determination
kept at routine day/night light program and under common rodent using instant thin-layer chromatography (ITLC) (Whatman No. 2
diet pellets.
paper, 1 mM diethylene triamine pentaacetic acid (DTPA), pH 5),
the mixture can be further purified using size-exclusion chroma-
tography in case of low radiochemical purity.
Production and quality control of 170Tm
Briefly, the radiolabeled antibody conjugate was purified from
free 170Tm by size-exclusion chromatography on a Sephadex
G-50 (fine) column (15–20 ml bed volume) and eluted with
phosphate-buffered saline (PBS). Fractions (1 ml) were collected,
and the radioactivity of each fraction was measured by a recently
calibrated radioisotope dose calibrator. The protein presence in
each fraction was determined using a fast protein assay method
described above.12
The fractions containing the proteins (visible blue color by
naked eye) with the maximum radioactivity (fraction No. 7) was
tested for purity by ITLC using a radio-TLC scanner.
Control labeling experiments were also performed using
170TmCl3, and DOTA with 170TmCl3. Both reaction mixtures were
passed through separate size-exclusion chromatography columns
and eluted with PBS. The fraction number 5-7 showed the pre-
sence of protein. Fraction 6 was used in the other experiments
(n = 3).
170Tm was produced by thermal neutron bombardment on
natural Tm (NO3)3 (100% 169Tm) at Tehran Research Reactor
(TRR) for a period of 7 days at a flux of 3–4 Â 1013 neutrons/cm2 s.
In a typical procedure, the amount of target that was equal to
1 mg of Tm was dissolved in HNO3 and transferred to a quartz
ampoule followed by evaporating the liquid. The ampoule was
sealed and put in an aluminum can and sent for irradiation.
The irradiated powder was dissolved in 100 ml of 0.1 M HCl, and
this radiochemical form was used for radionuclidic purity deter-
mination using beta and gamma spectroscopy for the detection
of various interfering beta and gamma emitting radionuclides.
Preparation of 1,4,7,10-tetraazacyclododecane-N,N,N,
N-tetraacetic acid mono-(N-hydroxysuccinimidyl) ester
This compound was prepared according to methods previously
given in the literature with slight modifications.9,10 For a single
run, a mixture of DOTA in acidic form (4.04 mg, 0.01 mmol),
N-hydroxysuccinicimide (1.15 mg, 0.01 mmol), and dicyclohexyl
carbodiimide (DCC) (2 mg, 0.01 mmol) were dissolved in anhydrous
CH2Cl2 (300–500 ml) under a blanket of N2 in a glass vial. The
mixture was vortexed for 30 s and stirred at 25ꢀC for 15 h. Thin-layer
chromatography using ethyl acetate : hexane (1:1) mixture as mobile
phase and silica gel as stationary phase was performed to monitor
the reaction progress.
Stability testing of the radiolabeled compound in final
formulation
Stability of 170Tm-DOTA-cetuximab in PBS was determined by
storing the final solution at 4ꢀC for 14 days and performing fre-
quent ITLC analysis to determine radiochemical purity. The stabi-
lity of the conjugated DOTA-cetuximab stored at 20ꢀC for more
than 1 month was also investigated. ITLC analysis of the conju-
gated product was performed to monitor for degradation pro-
ducts or other impurities. After subsequent 170Tm-labeling of
the stored conjugated product, both labeling efficiency and
radiochemical purity were determined.
Conjugation of DOTA-NHS with the cetuximab
The DOTA-NHS prepared above was conjugated to the antibody
using a small modification of the DOTA-NHS method.11 The resi-
due mixture was evaporated under a flow of N2 gas in a glass
tube producing a thin film coat of DOTA-NHS. The commercially
available cetuximab (2 mg/ml pH 7.8) was added to the coated
glass tube while gently mixing at room temperature for 30 s.
The mixture was then incubated in a water bath at 25ꢀC for
15–18 h. The conjugation mixture was then passed through a
Sephadex G-50 (fine) column (2 Â 15 cm, prepared by soaking
3 g of the resin in 50 ml milli-Q water for 4 h), and 1-ml fractions
were collected and checked for the presence of protein using
visible Lowry colorimetric assay by mixing freshly prepared
Folin–Colciteau reagent (prepared by mixing 5 ml of Folin and
25 ml of fresh Cu-tartrate solution) and 10 ml of the eluted frac-
tions using UV absorbance at 280 nm or visible Folin-phenol
reagent.12 The fraction containing the highest concentration of
immonoconjugate was chosen and kept at 4ꢀC for radiolabeling.
Stability testing of the radiolabeled compound in presence
of human serum
Briefly, a 1:3 mixture of radiolabeled antibody to freshly prepared
human serum was incubated at 37ꢀC while it was gently stirred.
At each time interval, 1 ml sample of the mixture was assessed
by size-exclusion chromatography on a Sepharose column
(1 Â 30 cm), which was previously equilibrated with PBS and
eluted at a low rate of 0.5 ml/min at room temperature; 1-ml frac-
tions were collected. Each eluted fraction was checked for the
presence of protein using Lowry method and radioactivity con-
tent. Control experiments were also performed separately for
human serum sample, antibody, and free Tm-170 cation.
Biodistribution of 170Tm-acetate and 170Tm-DOTA-Cetuximab
to wild-type rats
Radiolabeling of the antibody conjugate with 170Tm
To perform the biodistribution studies, 170Tm-acetate and 170Tm-
The antibody conjugate was labeled using an optimization pro- DOTA-cetuximab were administered to wild-type rats. A volume
tocol according to the literature.13 Typically, 80–100 MBq of (100 ml) of final 170Tm-DOTA-cetuximab solution containing
170Tm-chloride (in 0.1 N HCl) was added to a conical vial and 100 mCi (3.7 GBq) of radioactivity was injected intravenously into
dried under a flow of nitrogen. Double-distilled water was added rats via their tail vein. The animals were euthanized at the exact
to the vial containing 170Tm activity (2.32 mCi, 85.8 MBq) fol- time intervals (2, 4, 24, and 48 h, 1 and 2 months) for 170Tm-acetate
lowed by drying the vial using nitrogen flow (two times). The and 2, 4, 24, and 168h for the radioimmunoconjugate. The activity
protein-containing fraction with the maximum protein content in different organs was calculated as a percentage of injected dose
in 1 ml of phosphate buffer (0.1 M, pH 8) was added to the vial per gram using a dose calibrator.
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Copyright © 2012 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2012, 55 103–107

S. Shirvani-Arani et al.
(1:1) mixture as mobile phase and silica gel as stationary phase
was performed to monitor the reaction progress. The formation
of mono-succinimidyl ester at Rf of 0.36 was observed. In case
of longer reaction times, another species at Rf = 0.56–0.6 can be
observed and is related to di-succinimidyl ester. All reactants
remain at the Rf of 0.0–0.1.
Result and discussion
Production and quality control of 170Tm
Irradiation of natural Tm(NO3)3 was performed at a thermal neu-
tron flux of 3–4Â1013 neutrons/cm2 s for 5 days at TRR, and the
radionuclide was prepared according to standard methods with
a range of specific activity 10–15 mCi/mg for radiolabeling use.
However, because of long physical half-life and presence of
carrier Tm-169, the specific activity is rather low. Gamma ray
spectrum of the appropriately diluted 170TmCl3 solution showed
a major peak at 84 keV, which is the photopeak of 170Tm, as well
as two minor peaks at 52 and 59 keV, which are the X-ray peaks
associated with the 170Tm decay. The radioisotope was dissolved
in acidic media as a starting sample and was further diluted and
evaporated to obtain the desired pH and volume followed by
sterile filtration. The absence of any other photo-peaks in the
gamma ray spectrum indicated that the 170Tm was produced
with a radionuclidic purity of >99.99%. The radiochemical purity
of the 170Tm solution was checked in a solvent system contain-
ing DTPA 1 mM and Whatman No. 2 TLC paper, as stationary
phase; Tm3+ cation is complexed to more lipophilic 170Tm-DTPA
ligand and migrates to higher Rf.
Conjugation of cetuximab with DOTA-NHS and radiolabeling
of cetuximab with 170Tm
The conjugated DOTA-cetuximab fractions containing the maxi-
mum protein content were mixed with 170Tm-TmCl3 solution,
vortexed, and kept at room temperature. Small fractions were
taken from this mixture and tested by Radio-thin layer chromatog-
raphy (RTLC) to find the best time scale for labeling. After 2–3 h,
the radiochemical purity of reaction reached almost 100%. The
mixture was then passed through another Sephadex G-50 size-
exclusion chromatography column to remove unbound 170Tm
cation and/or other low molecular weight impurities.
The eluted fractions were checked by Folin–ColciteauW
reagent and for the presence of radioactivity to determine the
170Tm-DOTA-Cetuximab containing fractions. Fraction number
7 was chosen as the suitable final product with appropriate spe-
cific activity for animal tests. Instant thin-layer chromatography
using 1 mM DTPA as mobile phase and Whatman No. 2 paper
strips as stationary phase was performed to ensure the existence
of only the desired radiolabeled antibody. Radiolabeled antibody
stayed at the origin, whereas free Tm-170 ions migrated to
higher Rfs (Table. 1).
Preparation and structure conformation of DOTA-NHS
Various solvents can be used in the conjugation reaction of
DOTA and NHS, such as dimethylformamide, dimethylsulfoxide,
etc. Because of the high boiling point of such solvents and the
possibility of protein denaturing caused by these solvents, we
tried to use dichloromethane on the basis of the previous reports
for DCC-mediated conjugation reactions. This reaction is per-
formed at room temperature and can be done overnight as
reported.9
Most of the reaction takes place in the first few hours. Consid-
ering the size-exclusion chromatography performed in the next
step of the process, all the starting materials can be separated.
A more lipophilic species in the reaction mixture (monitored by
TLC) is related to the formation of the ester 3 (Figure 1). The
exact stoichiometry of NHS : DOTA (1:1) is mandatory in order
to avoid the formation of the di-succinimidyl ester. As men-
tioned, thin-layer chromatography using ethyl acetate : hexane
Stability of radiolabeled protein in presence of human serum
After incubation of 170Tm-DOTA-cetuximab (3.7 MBq) with
freshly prepared human serum at 37ꢀC for up to 72 h, 96–98%
of all of the radioactivity eluted in the same position as 170Tm-
DOTA-cetuximab, using size-exclusion chromatography. Thus,
there was no evidence for either degradation or trans-chelation
of 170Tm to other serum proteins over a time period consistent
with the normal blood clearance time of cetuximab.
Biodistribution studies in rats
The animals were sacrificed by CO2 asphyxiation at selected
times post injection (2, 4, 24, and 48 h, 1 and 2 months). Dissec-
tion began by drawing blood from the aorta followed by remov-
ing the heart, spleen, muscle, brain, bone, kidneys, liver,
intestine, stomach, lungs, and skin samples. The tissue uptakes
were calculated as the percent of area under the curve of the
related photo peak per gram of tissue (% ID/g). The biodistribu-
tion of 170Tm cation was determined in wild-type animals for
2–48 h and 1–2 months post injection (Figure 2). The blood con-
tent is low at all time intervals, and this shows the rapid removal
of activity in the circulation. The heart, lung, muscle, intestine,
COOH
HOOC
N
N
N
N
1
COOH
HOOC
O
HON
O
2
O
Table 1. Rf values for various radiochemical species using
1 mM DTPA, pH 5 eluent on Whatman No. 2 paper
N
O
COO
HOOC
N
N
N
Radiochemical species
Rf
N
170Tm3+
0.8
0.3
0.0
COOH
HOOC
170Tm-DOTA
3
170Tm-DOTA-cetuximab
Figure 1. Synthesis of DOTA-NHS active ester for conjugation.
J. Label Compd. Radiopharm 2012, 55 103–107
Copyright © 2012 John Wiley & Sons, Ltd.
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S. Shirvani-Arani et al.
Figure 5. Comparative %ID/g in the liver for 170Tm-DOTA-rituximab (blue) and
170TmCl3 (red) in wild-type rats (n = 3).
Figure 2. Percentage of injected dose per gram (ID/g %) of 170Tm-acetate in wild-
type rat tissues at 2, 4, 24, and 48 h, and 1 and 2 months post injection (n = 3).
Figure 6. Comparative %ID/g in the intestine for 170Tm-DOTA-rituximab (blue)
and 170TmCl3 (red) in wild-type rats (n = 3).
Figure 3. Percentage of injected dose per gram (ID/g %) of 170Tm-DOTA-cetuximab
in wild-type rat tissues at 2, 4, 24, and 168 h post injection (n = 3).
Figure 7. Comparative %ID/g in the spleen for 170Tm-DOTA-rituximab (blue) and
170TmCl3 (red) in wild-type rats (n = 3).
The distribution of 170Tm-DOTA-cetuximab among tissues was
determined for normal rats. A volume (100 ml) of final [170Tm]-
DOTA-cetuximab solution containing 3.7 MBq (100 mCi) radioac-
tivity was injected into the dorsal tail vein. The total amount of
radioactivity injected into each rat was measured by counting
Figure 4. Comparative %ID/g in the blood for 170Tm-DOTA-rituximab (blue) and
170TmCl3 (red) in wild-type rats (n = 3).
and also skin do not demonstrate significant uptake, which is in the 1-ml syringe before and after injection in a dose calibrator
accordance with accumulation of other cations. Bone uptake for with a fixed geometry. The animals were sacrificed by ether
the cation goes up to 6% up to 1month and then is reduced to asphyxiation at selected times post injection (2, 4, 24, and 168 h).
1% in the second month. Spleen also has significant uptake possibly
Dissection began by drawing blood from the aorta, followed
related to reticuluendothelial uptake. Tm3+ is a water soluble cation; by collecting heart, spleen, kidneys, liver, intestine, stomach,
therefore kidney plays an important role in excretion via urine.
lung, brain, femur, muscle, and skin samples. The samples were
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J. Label Compd. Radiopharm 2012, 55 103–107

S. Shirvani-Arani et al.
>99%, radiochemical purity >99%). One of the most important
advantages of thulium over some other lanthanides is that a
relatively high radionuclidic purity could be produced from
mono-isotopic Tm-169 target. Cetuximab was conjugated with
DOTA chelating agent, followed by 170Tm-radiolabeling. The
radiochemical purity of the 170Tm-DOTA-cetuximab was deter-
mined by the use of ITLC (>99%). The biodistributions of
radiopharmaceutically acceptable 170TmCl3 and 170Tm-DOTA-
cetuximab formulations were checked in wild-type rats up to
72 h post injection. 170Tm-DOTA-cetuximab accumulated mostly
in the liver and spleen as shown for other radiolabeled cetuxi-
mab immunoconjugates. The distinct accumulation of the
activity in lungs for the radiolabeled antibody and liver and
spleen for the free cation was observed. The development of
other 170Tm-labeled monoclonal antibodies for ultimate radioim-
munotherapy based on DOTA conjugates is possible.
Figure 8. Comparative %ID/g in the lung for 170Tm-DOTA-rituximab (blue) and
170TmCl3 (red) in wild-type rats (n = 3).
weighed and their specific activities determined with a gamma
spectrometer using an HPGe detector counting the area under
the curve of the 84-keV peak. The tissue uptakes were calculated
as the percent of area under the curve of the related photo peak
per gram of tissue (% ID/g) (Figure 3).
Acknowledgements
The authors wish to express their sincere gratitude to the
following people without whose assistance this study would
not have been feasible: Mr. Mazidi, Mr. Mirfallah, Mr. Yaghoubi,
and Mr. Meftahi.
The highest activity concentrations were visualized in the
lungs, spleen, and the liver (~15% ID/g).2 Comparison of vital
organs uptake for 170Tm-DOTA-rituximab and 170Tm-acetate
demonstrates kinetic pattern difference between both species.
Both species are removed from the blood circulation rapidly
via different mechanisms, but in case of free Tm cation, the blood
amount is much lower and reaches minimum after 48 h (Figure 4).
In case of liver, a different pattern is observed for free cation.
The uptake decreased after 48 h possibly because of the billiary
excretion, whereas for the conjugate the liver uptake increases
in 24 h followed by the retention of the activity in the liver
(Figure 5). This has been already shown for other radiolabeled
cetuximab conjugates in the literature with 10–18% liver uptake.5
The intestine activity content is comparable with the liver
because the intestine is an organ receiving hepatobilliary excretion
contents (Figure 6). The liver activity reaches its maximum after
24 h and stays constant, whereas the major part of the free Tm3+
activity is not excreted from the liver and possibly from the kidneys
especially before 24 h.
Conflict of Interest
The authors did not report any conflict of interest.
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Tm-170 was prepared by irradiation of natural thulium nitrate
sample by 4 Â 1013 neutrons/cm2 s neutrons (radionuclide purity
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