Radiochemical synthesis and preliminary evaluation of anti-bladder cancer monoclonal antibody BDI-1 labeled with rhenium-188

Article abstract of DOI:10.1002/jlcr.470

The anti-human bladder cancer monoclonal antibody BDI-1 was radio-labeled with rhenium-188 by direct labeling methods using SnCl₂ as reductant and MDP as stannous stabilizer or by indirect method using NHS-ECM ester as bifunctional chelator. Radiochemical yields of 30 ± 7.23% and 87.4 ± 5.67% and radiochemical purity of more than 95% were achieved. The inmmunoreactive fraction was 58.7%. The results showed that radionuclide ¹⁸⁸Re might be employed using the same labeling methodology with ^99mTc labeling of BDI-1 for radiommunoimaging (RII) and radioimmunotherapy (RIT) and that may be useful in radioimmunodetection and RIT of human bladder carcinoma in vivo. Copyright

Full text of DOI:10.1002/jlcr.470

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Labelled Cpd Radiopharm 2001; 44: 437–444.
DOI: 10.1002/jlcr.470
Radiochemical synthesis and preliminary
evaluation of anti-bladder cancer monoclonal
antibody BDI-1 labeled with rhenium-188
Rongfu Wang*, Chungli Zhang, Lizhang Yu, Yifeng Guo, Ying Bai
Department of Nuclear Medicine, Peking University First Hospital,
Beijing 100034, P.R. China
Summary
The anti-human bladder cancer monoclonal antibody BDI-1 was radio-
labeled with rhenium-188 by direct labeling methods using SnCl2 as reductant
and MDP as stannous stabilizer or by indirect method using NHS-ECM
ester as bifunctional chelator. Radiochemical yields of 30 ꢀ 7.23% and
87.4 ꢀ 5.67% and radiochemical purity of more than 95% were achieved.
The inmmunoreactive fraction was 58.7%. The results showed that radio-
nuclide 188Re might be employed using the same labeling methodology
with 99mTclabeling of BDI-1 for radiommunoimaging (RII) and radio-
immunotherapy (RIT) and that may be useful in radioimmunodetection and
RIT of human bladder carcinoma in vivo. Copyright # 2001 John Wiley &
Sons, Ltd.
Key Words: Rhenium-188; Bladder carcinoma; Monoclonal antibody BDI-1;
Radioimmunoimaging (RII); Radioimmunotherapy (RIT)
1. Introduction
Bladder and ureteral cancers are common urological malignancies and
represent the most common urological cancers in the world. The
traditional diagnosis is established by cystoscopy and biopsies of
bladder mucosa and additional examinations such as ultrasonography,
*Correspondence to: R. Wang, Department of Nuclear Medicine, Peking University First Hospital,
Beijing 100034, P.R. China.
Copyright # 2001 John Wiley & Sons, Ltd.
Received 26 Jamuary 2000
Revised 28 December 2000
Accepted 19 February 2001

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R. WANG ET AL.
radiography and computed axial tomography (CT) may also contribute
to the diagnosis. However, these methods are all non-specific and have
variable sensitivity; for flat or in situ carcinoma, small solid lesions, and
bladder cancer metastatic foci, quantitative diagnosis is relatively
difficult. On the other hand, the characteristics of bladder cancer
constitute the superficial features for most primary tumors and the high
recurrent rate (50–70%) after surgery. Currently, there are no ideal
therapeutic means besides chemotherapy and the use of bacillus
Calmette–Guerin (BCG) vaccine administered intravesically due to
whole body toxic reaction and poor efficacy.1,2 For these reasons, we are
evaluating approaches with RII using 188Re–BDI-1 in a template in a
new radioimmunotherapeuticapproach.
With the development of hybridoma technology the use of
radiolabeled monoclonal antibodies (MoAbs) for radioimmunodetec-
tion and RIT has undergone extensive investigation. For example, there
are several approaches to the diagnosis of bladder cancer using
radiolabeled monoclonal antibodies which have demonstrated that
RII may be valuable for the diagnosis of bladder cancer.3–5 The BDI-1
is an anti-bladder carcinoma cell line BIU-87 monoclonal antibody
which has been prepared in our hospital. In our previous work, 131I-
BDI-1 and 99mTc-BDI-1 have been shown to have good affinity to
human bladder carcinoma cell line BIU-87 and have shown excellent
localization in nude mice bearing human bladder cancer xenograft.6–8
Recently, the therapeutic radioisotopes of rhenium, rhenium-186 and
rhenium-188, have been proposed for use in RII, especially in RIT due
to their excellent therapeutic physical characteristics and chemical
similarities to technetium. This is due to the hypothesis that, if an
acceptable 99mTclabeling of MoAbs is available, 188Re might be used
with the same labeling methodology for RII and RIT.
In this paper, we describe the preparation of anti-bladder tumor
monoclonal antibody (BDI-1) labeled with radionuclide rhenium-188
(
188Re–BDI-1) for use in radioimmuno-guided treatment of bladder
cancer and have evaluated its property of locating bladder cancer.
Experimental
Preparation of monoclonal antibody BDI-1
The BDI-1 monoclonal antibody was obtained from a hybridoma
produced by fusing spleen cells from mice immunized against the
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bladder carcinoma cell line BIU-87 with SP 2/0 mouse myeloma.
It was purified from mouse ascitic fluid by protein A Sepharose
CL-4B affinity chromatography. The monoclonal antibody was
characterized by indirect immunofluoresence assay and ABC–
ELISA immunohistochemical staining which demonstrated that
BDI-1 has a strong binding reaction with bladder transitional cell
carcinoma tissue and BIU-87 and E-J bladder carcinoma cell lines but
there was no reaction with normal bladder tissue and other normal
human tissues.9
Immunohistochemistry was used to study the expression of the
antigen in tumors with different grades and stages in 45 specimens
of TCC (transitional cellular carcinoma) by virtue of mono-
clonal antibody BDI-1 bindings. The bindings of BDI-1 was strong,
when the tumor grading was high, but no significant differences were
observed between the different stages (p > 0:05). The expression of
antigen, which was related to MoAb BDI-1, was correlated with the
grade of tumor. The higher the tumor grading, the higher was the
expression of antigen.
Radiochemical synthesis of 188Re–BDI-1
[
188Re]-perrhenate was obtained from 188W/188Re generator
which was purchased from Syncor Company. 188Re isotope has a
155 keV g-emission suitable for RII, and has 2.21 MeV
a
b-emission suitable for RIT. MDP kit was purchased from
Shanghai Hongqi Paramaceutical Factory and 2-mercaptoethanol
(2-ME) was purchased from Merck Company. IR was recorded on
Perkin Elmer.
BDI-1 reduced by 2-mercaptoethanol was labeled with Re-188 either
by indirect method with 188Re-NHS-ECM ester (Scheme 1) or by direct
labeling with 188ReO4ꢁ using SnCl2 as reductant and MDP as stannous
stabilizer (Scheme 2).
Formaldehyde (HCHO) was added in l-cysteine dissolved in aqueous
HCl reacted overnight at r.t., and then purified to produce l-
thiazolidine-4-carboxylic acid. EC was obtained by reducing the l-
thiazolidine-carboxylic acid with sodium in liquid ammonia and then
reacted with ethanol under reflux for 24 h to obtain a mixture which was
then isolated by HPLC to obtain ECM, IR (CHCl3): 2977 cmꢁ1 (CH3),
1230 cmꢁ1 (CO), 1076 cmꢁ1 (C–N), 940 cmꢁ1 (O–H). ECM was added
to a solution of 188Re sodium perrhenate 37–740 MBq, followed by the
Copyright # 2001 John Wiley & Sons, Ltd.
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Scheme 1. Synthetic route of indirect labeling BDI-1 with rhenium-188
Scheme 2. Synthetic route of direct labeling BDI-1 with rhenium-188
addition of 0.2 ml of freshly prepared solution of stannous chloride
(0.1 mg) in 0.1 N HCl of pH=4.0. 188Re-ECM was determined by paper
chromatography using two developing systems. System 1 used
CHCl3 : CH3OH=9 : 1 (v/v) as eluent, Rf (ECM, ECD)=0.9–1.0, Rf
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(
99mTcOꢁ4 , EC)=0–0.1. System 2 used acetone as eluent, Rf (ECM,
ECD)=0–0.1, Rf (ECD, 99mTcOꢁ4 )=0.9–1.0. 188Re-ECM reacted with
N-hydroxysuccinimide using N-(3-dimethylaminopropyl)-N0-ethyl car-
bodiimide as condensing agent to produce 188Re–NHS–ECM ester.
After purification with RP18-TCL, the pre-ester was coupled to BDI-1
under pH 8.0–8.5 to obtain 188Re–ECM–BDI-1. The radiolabeling
yields of the antibody were determined on silica gel using an eluent of
0.9% NaCl. Rf (MoAbs)=0–0.1, Rf (188ReO4ꢁ)=0.9–1.0.
The direct labeling approach was based on the method
previously described by Griffiths et al.10 with some modi-
fications. Briefly, 188Re–BDI-1 was prepared by 2-mercaptoethanol
(2-ME) direct reducing method. Five ml 2-ME was added to 100 ml
BDI-1, then reacted for 30 min at 30–348C to reduce the disulfide
bonds of the antibody to mercapto groups. After the reduced
antibody was purified by Sephadex G-50, 148–296 MBq (4–8 mCi)
188ReOꢁ4 , 40 ml dissolved MDP kit by 2 ml saline and 135 mg
SnCl2 in HCl aqueous were added and shaken for 15 min at
room temperature. The labeled antibody was purified by
Sephadex G-50 and passed through a 0.22 microfilter to obtain sterile
product. The radiochemical purity was controlled by paper chromato-
graphy
Immunoreactivity studies
Immunoreactivity studies were performed using the method described in
the literature11. Briefly, six serial 1 : 2 dilutions of two aliquots of human
bladder cancer cell line BIU-87 starting at 5 ꢂ 106 cell/ml were made.
Then, the cell suspensions were centrifuged and resolved in 100 ml 1%
BSA. To 1 of 2 aliquots of cells, unlabled antibody was added to a final
concentration of 50 mg/ml and the cells were incubated for 2 h to
saturate the binding sites for the subsequent measurement of nonspecific
binding of radiolabled antibody. Radiolabeled antibody at a concen-
tration of 40 mg/ml in 100 ml 1% BSA was added to each cell suspension.
After 2 h of incubation, the radioactivity (total applied) was measured.
The cells were centrifuged, washed three times with 1% BSA, then the
radioactivity (specific binding) was measured. The data were plotted
by total applied radioactivity over specific binding as a function of
the inverse of cell concentration. The immunoreactive fraction
was determined by means of linear extrapolation to infinite cell
concentration.
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Radioimmunoimaging
Radioimmunoimaging of three nude mice bearing human
bladder cancer xenografts was performed at 24 h after i.v. injection of
11.1 MBq of 188Re–BDI-1 using a General Electric STARCAM 300AC
g camera equipped with a low-energy high-resolution collimator.
Analog and digital images were acquired from the dorsa view with
the collimator 10 cm from the animal. Images were acquired with an
energy peak of 150 keV and matrix of 128 ꢂ 128 from approximately
50000 accumulation counts resulting in imaging times ranging from 5 to
15 min. Digital images were normalized with a General Electric
STARCAM computer to produce visually similar levels of activity in
the central torso. Tumor and whole body regions of interest were
identified on each mouse image.
Results and discussion
A
new anti-bladder cancer monoclonal antibody BDI-1 pre-
pared by ourselves was reduced by 2-mercaptoethanol. Rhenium-188
labeling with reduced antibody was carried out either by indirect
method with 188Re–NHS-ECM or by direct labeling with 188ReOꢁ4 using
SnCl2 as reductant and MDP as stannous stabilizer. Labeling yields at
different SnCl2 concentration, reaction time and pH values were
studied.
Both the direct labeling method10 and indirect labeling method12 can
be used for labeling antibodies with radioactive rhenium. In our study,
radiolabeling yields of 30 ꢀ 7.23% by the direct method and
87.4 ꢀ 5.76% by the indirect method were demonstrated and more
than 95% radiochemical purity of both the means was achieved. Direct
labeling is a simple method and widely used, but it is not very effective in
labeling BDI-1 with Re-188. The indirect method with NHS–ECM was
developed in our study and higher radiolabeling yield was achieved as
ECM could be labeled under high acidity. The immunoreactive fraction
of 188Re–BDI-1 was more than 58.7%. The optimum radiolabeling
conditions considering both yields and immunoreactivity require further
investigation
RII of nude mice bearing human bladder cancer xenografts showed
significant radioactive accumulation on the tumor at 24 h postinjection
(Figure 1). The result demonstrated the excellent immunoreactivity of
188Re–BDI-1 in vivo.
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PREPARATION OF ANTI-BLADDER CANCER
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Figure 1. RII of nude mice bearing human bladder cancer xenografts. Radio-
immunoimaging in nude mice bearing human bladder human bladder cancer
xenografts was performed at 24 h after intravenous injection of 11.1 MBq of
188Re–BDI-1 using a General Electric STARCAM 300AC g camera equipped
with a low-energy high resolution collimated. It demonstrated the selected
radioactive accumulation on tumor (in arrowhead). The radioactivity in the liver
can also be seen
Bladder cancer is one of the most common urological tumors and
postoperative recurrence is common, which necessitates the examination
of methods in the early diagnosis of bladder cancer for treatment
planning and prognosis. RII may be used to diagnose bladder tumor,
which is associated with the following two factors. The first factor
includes the expression of tumor antigen, the immunoactivity and
specificity of monoclonal antibody (MoAbs). The second factor includes
the availability and cost effectiveness of the selected radioisotopes. The
physical properties of Rhenium-188 (155 keV g-emission suitable for
RII, and 2.12 MeV b-emission suitable for RIT) make it an excellent
candidate as a radionuclide for radioimmunotherapy when it is
covalently chelated to MoAbs. In comparison with 131I, 188Re has
many favorable nuclear properties such as suitable g-energy for
scintigraphic imaging and strong b-energy for intraradiation therapy
of tumors and is more and more widely used in therapeuticnulcear
medicine.13 Furthermore, 188Re can be easily obtained from the
generator of 188W/188Re,14 which is more available in routine clinical
practice.
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Conclusions
We developed a new potential radiopharmaceutial rhenium-188–BDI-1,
for radioimmunotherapy, permitting its successful labeling with radio-
active rhenium. The labeling compound showed higher radiolabeling
yields of 87 ꢀ 5.76% from indirect labeling method with 188Re–NHS–
ECM than that of 30 ꢀ 7.23% from direct labeling method using 2-
mercaptoethanol reduction, and has an available immunoreactivity of
57.8%. RII in animals bearing human bladder cancer xenograft has
demonstrated significant radioactive accumulation in the tumor. The
preliminary results shown by our study also justify the development of
radioimmunoconjugates for RIT, especially to provide a novel means
for radionuclide intraradiation therapy of bladder cancer by intravesical
perfusion of 188Re–BDI-1.
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Copyright # 2001 John Wiley & Sons, Ltd.
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