Direct technetium-99m labeling of anti-hepatoma monoclonal antibody fragment: a radioimmunoconjugate for hepatocellular carcinoma imaging
technetium-99m labeling of anti-hepatoma monoclonal antibody fragment:
a radioimmunoconjugate for hepatocellular carcinoma imaging
Jie Bian1, Zhi Nan Chen1 and Jing Lan Deng2
Engineering Research Center, Basic Medical Department, Fourth
Military Medical University, Xi’an 710032, Shaanxi Province, China
2Department of Clinical Nuclear Medicine, Xijing
Hospital, Xi’an 710033, Shaanxi Province, China
J Gastroenterol 2000;June6(3):348-352
Supported by National Natural Science Foundation of China,
headings: antibody, monoclonal;
antibody fragments; technetium-99m; hepatocellular
carcinoma; liver neoplasms; radioimmunoimaging
AIM: To directly radiolabel an
anti-hepatoma mAb fragment HAb18 F(ab’)2 with 99mTc
by stannous-reduced method, and assess the stability,
biodistribution and radioimmun oimaging (RⅡ).
METHODS: Immunoreactive fraction was determined according to
Lin dmo’s method. Ellman’s reagent was used to determine the
number of thiols in the reduced F(ab’)2. Labeling
efficiency and homogeneity were measured by paper chromatography,
sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE)
and autora diography. Challenge assay involved the incubation of
aliquots of labeled antibo dy in ethylenediaminetetraacetate (EDTA)
and L-cysteine (L-cys) solutions with different molar ratio at 37℃
for 1h, respectively. Investigations in vivo utilized nude
mice bearing human hepatocellular carcinoma (HHCC) xenografts with
gamma camera imaging and tissue biodistribution studies at regular
RESULTS: The labeling procedure was finished within 1.5h
compared with the “pretinning” method which would take at least
21h. In vitro studies demonstrated that the radiolabeled mAb
fragment was homogen eous and retained its immunoreactivity.
Challenge studies indicated that 99mTc-labeled HAb18 F(ab’)2
in EDTA is more stable than in L-cys. Imaging and biodistribution
showed a significant tumor uptake at 24h post-injection of 99mTc-labeled
HAb18 F(ab’)2. The blood, kidney, liver and tumor
uptakes at 24h were 0.56±0.09, 56.45±11.36, 1.43±0.27 and 6.57±3.01
CONCLUSION: 99mTc-HAb18 F(ab’)2
conjugate prepare d by this direct method appears to be an effective
way to detect hepatoma in nude mice model.
The introduction of mAbs as targeting devices in nuclear
medicine is well develo ped and many different antibodies which
labeled with a variety of isotopes have been reported in cancer
diagnosis. It seemed that 99mTc is the most popular
radionuclide for nuclear medicine imaging because of its favorable
physi cal characteristics, low cost, and ready availability. 99mTc
labeled mAb fragments should be superior to other big molecule
radioimmunoconjugates for u se in tumor RⅡ.
A number of methods have been proposed for 99mTc labe
ling proteins, and mAbs in particular. In general, these
methodologies can be d ivided into two categories: indirect and
direct methods. In indirect method the protein was
modified with a technetium binding ligand and then reacte d with a
technetium complex. Several bifunctional chelating agents have been
syn thesized and used, such as diethylenetriaminepenta acetic acid (DTPA)
, diamide dimercaptide N2S2 ligands, and
hydrazino nicotinamide analog . Although it is said
that the indirect method can lead to loss of immuno reactivity.
Joiris et al. have tested that the derivatization of antibody
or fragment by iminothiolane does not split the protein and keeps
the immunoreact ivity. By direct method, 99mTc
metal ion binds directly to endogenous donor groups on the antibody.
The method is simple to perform and co mpatible with practical
clinical use. However, direct labeling of mAbs with 99mTc
was reported to be unstable due to non-specific binding (low and
some reports suggest an improved labeling of proteins with 99mTc.
In the Schwarz and Steinstrasser procedure, as modified by Mather
and Ellison, disulide bridges in the mAb are reduc ed
with 2-mercaptoethanol (2-ME). After purification, the resulting
reduced an tibody can be stored frozen until required for use.
Labeling is accomplished by addition of stannous ion from a bone-
scanning kit and pertechnetate. In addit ion to using regular
reducing agents, such as 2-ME, stannous ions，b
orohydride, ascorbic acid, dithionite,
or gl utathioneto generate sulphydryl groups, other
peculiar approaches also appeared recently. Direct 99mTc
labeling of mAbs were finis hed by reduction of antibodies using
photoactivation and insoluble macromolecula r Sn(Ⅱ)
complex[13,14]. With the development of direct method,
there ha ve been a few reports of successful use of this technique
in colorectal, breast, and ovarian cancer imaging[15-17].
this report, we describe a direct method for radiolabeling anti-hepatoma
mon oclonal antibody fragment HAb 18F (ab’)2 with 99mTc.
The stability and homogeneity of 99mＴc-HAb18
F(ab’)2 were evaluated. The biodis tribution and tumor
localization in nude mice bearing a HHCC xenograft were studied.
Materials and Methods
The mAb HAb18 is of murine IgG1 isotype and was
developed by our laboratory. F(ab’)2
fragment of HAb18 was generated by papain digestion with a molecular
weight of ～96000
Hepatocellular carcinoma grown in Balb/c mice was used as a
prototype tumo r model. Approximately 107 HHCC cells
obtained from Shanghai Cell Institute of Chinese Academy of Sciences
were implanted in the left thigh of the animals and the tumors were
allowed to grow for 8-10 days to approximately 1cm in diameter.
The antibody concentrated to 8g/L in neutral PBS was reduced
by reaction with a molar excess of stannous/glucoheptonate (Sn/GH)
ranging from 10∶1
(Sn/GH: MAb) at 37℃
for 15min-30min. The Sn/GH with a mass ratio of 1∶100
was dissolved in 50mM acetate-buffered saline (ABS), pH 5.3 purged
with nitrogen. The reduced antibody was isolated from reductant t
hro ugh a PD-10 column (Pharmacia) equilibrated with 0.05mol/L ABS.
The number of resulting free sulphydryl groups was assayed with
Ellman’s reagent 5，5’dithio-bis
(2-nitrobenzoic acid), (DTNB, Sigma Chemical Co., USA) .
One hundred μL of sample was mixed with 20μL of 0.01mol/L-DTNB
and diluted to 3mL with 0.05mol/L Tris-HCl buffer pH 8.4. The
mixture was incubated at room te mperature for 15min and coloration
measured with an UV/VIS spectrophotome ter at 412nm. The number of
thiols was obtained by comparison with a seri es of L-cysteine (L-cys)
standards ranging from 0.312mg/L to 10mg/L.
integrity of the reduced F(ab’)2 was determined by
non-reduced SDS-PAGE with 100g/L gel using Vertical Gel
Electrophoresis System (Bio-Rad). The gel was stained with Coomassie
brilliant blue R250.Control experiments were run using unreduced mAb
For labeling, 160μg of reduced HAb18 F(ab’)2
was mixed with a 10μL-20μL of diluted Sn/GH solution
(0.2g/L), and pertechnetium solution (0.2mL , 74MBq), (Chinese
Academy of Atomic Energy) was injected into the mixture . The Sn/GH
solution was freshly prepared each time by dissolving 100mg GH and
1mg SnCl2·2H2O in 5mL of saline purged with
nitrogen. The reaction mixture was incubated for 0.5h-1h at 37℃
before it was analyzed by Whatman 3MM paper chromatography which was
then developed in acetone or 100g/L trichloroacetic acid (TCA). R-f
values for acetone are: mAb 0.0, 99mTc-GH 0.0, and 99mTcO4-0.9-1.0.
R-f values for 100g/L TCA are: mAb 0.0, 99mTcGH 0, and 99mTcO4-
0.7. Labeled mAb was differentiated from 99mTc colloid by
the method of Thrall et al. The same strips
impregnated with 10g/L-20g/L human serum albumin before development
NH4OH. Colloid remained on the bottom of the strip while
mAb-bound isotope migrated with the solvent front.
integrity of the labeled F(ab’)2 was assayed using the
same non-reduced SDS-PAGE as described above. The gel was
autoradiograghied on x-ray film before stained with Coomassie
brilliant blue R250.
The in vitro immunoreactivity of the radiolabeled
HAb18 F(ab’)2 was evalu ated by a live cell assay.
Briefly, HHCC cells 5×109/L were centrifuged (1000r/min)
for 5min and washed twice with 1% bovine serum albumin (BSA) in PBS,
then 5 serial 1∶2
dilutions were made in 10g/L BSA in Eppendorf tubes precoated with
BSA. Radiolabeled HAb18 F(ab’)2 at a concentration of
40ng/mL in 10g/L BSA was added using a volume equal to half the
volume of cell suspension. The total volume of cell-binding assay
solution was 0.3mL. After incubation for 2h at 37℃,
the total as well as the cell-bound radioactivity were counted in a
In Vitro stability studies
The stability was analyzed by using two different
challenging agents, EDTA and L-cys. An aliquot of 50μL 99mTc-HAb
18 F(ab’)2 solution was incubated with EDTA or L-cys at
for 1h. The molar ratio of mAb to challenging agent was at a maximum
Dissociation ratio was analyzed on paper chromatography.
Biodistribution and imaging
Balb/c mice bearing HHCC were divided into three groups.
Each group consisted of three animals and each animal received
approximately 15μ g antibody with about 7.4MBq through a
lateral tail vein. At time i ntervals of 4, 10 and 24h postinjection,
three groups of mice were killed , and imaged on a SPECT (Starcam
3000, UK). Data were collected 100000 co unts per image and peak
energy settings at the 140ke V (20%) window for 99mTc.
The blood and other organs of interest were collected. Tissues were
w ashed, blotted, weighed and counted in a gamma counter. For each
mouse, data are expressed as percent of injected dose per gram of
tissue (%ID/g) after physical decay corrected.
Figure 1 represents the calibration curve for the determination
of sulphydryl groups using L-cys standards over a range of 0.312 to
10mg/L, by plotting optical density at 412nm versus L-cys standard
concentrations after subtraction of the background due to Ellman’s
reagent. Linear regression was used and correlation coefficient
0.999 was obtained. Table 1 shows the influence of the reduction
conditions on the number of free sulphydryl groups detected by this
thiol assay. As expected, increasing the molar ratio of Sn/GH to
antibody in the reaction mixture does increase the number of
apparent -SH groups per antibody, and increase the labeling
efficiency correspondingly, which results in the labeling efficiency
at a maximum of 84.2%. The free 99mTcO4- and
colloid amounts determined by Whatman 3MM paper using different
developing systems were also showed in Table 1. In control expe ri
ments, labeling efficiency was 2% when unreduced HAb18 F(ab’)2
was used. SD S-PAGE by both staining and autoradiography showed that
the radioactivity co-m igrated with the proteins and that there were
almost no protein fragments prese nt within the 60∶1
of molar ratio of Sn/GH to mAb (Figure 2). However , another SDS-PAGE
in Figure 3 illustrates that fragmentation occurred during t he
reduction procedure when the molar ratio of Sn/GH to mAb was at 500∶
shown in Figure 4, the immunoreactive fraction, 0.84 was determined
by plott ing the inverse of the bound fraction compared with the
inverse of the cell conc entration, which is based on the assumption
that the total antigen concentration (cell concentration) is a good
enough approximation for the free antig en concentration.
with EDTA did not remove 99mTc from the labeling conjugat
e remarkably, while L-cys at a molar ratio of 625∶1
remove approximately one-tenth of the label (Figure 5).
of radioactivity in blood and excised tissues are displayed in Table
2. The preparation localized at the tumors was more than at any
organ examined at both 10h and 24h after injection, except the
kidneys. Th e lower radioactivity in blood at 24h suggested fast
blood clearance. The imaging results in Figure 6 showed significant
tumor uptake at 24h post-i njection.
Great efforts have been made to develop a method that can be
used for the direct labeling of mAbs with 99mTc.
Earlier studies involved the incubation of mAbs with stannous
phthalate tartrate solution for up to 21h at room temperature, which
was named “pretinning” method. Clinical success with this method
has been claimed by the author.
aim of our study was to further evaluate the role of stannous as a
reducing agent in the direct labeling of mAb F(ab’) 2 with 99mTc.
The difference between the “pretinning” method and this method
is that we use GH instead of phthalate-tartrate as transfer ligand
and stabilizer to avoid Sn or Tc-c olloid formation. To do this, we
investigated the effect of the quantity of Sn/ GH on the labeling
time and efficiency. When the molar ratio of Sn/GH to mA b F(ab’)2
was constant, we found that there was no obvious difference on the
number of-SH between the reduction time of 20min and 30min or even
longer[ 24]. The whole labeling process can be
accomplished within 1.5h. Hnatowich et al. reported that
labeling efficiency in the case of the stannous ion-reduced a
ntibodies was generally in excess of 70%, however, in
our method mol ar ratio of Sn/GH to mAb was an important parameter
to obtain good labeling re sults, and molar ratio of 40∶1
or higher were needed to get labeling effici ency of more than 80%
(Table 1). The low percentage of free 99mTcO4
and radiocolloid in each sample implied that pH 5.3 and GH are the
opti mal pH value and transfer ligand. Under this condition, the
labeled mAb HAb18 F( ab’)2 keeps its immunoreactivity.
Autoradiography of SDS-PAGE had only one m igration of component
identical to that of native HAb18 F(ab’)2 determination
by staini ng with Coomassie brilliant blue R250 (Figure 2), which
demonstrated that S n/GH reduction is mild and does not destroy
interchain bridges in mAbs. Labeli ng efficiency of 2% in control
experiments using unreduced HAb18 F(ab’)2 indicated
that there was no exchange with the low affinity sites and also
demonstrated tha t reduction of disulfides is a necessary initial
step in 99mＴc
direct labeling of antibodies. The bond between SH and Tc is
stronger than that of N -Tc or O-Tc which was verified by the
challenge assay of 99mTc-HAb18 F(ab’)2 in
the presence of EDTA. We found that EDTA even at a molar rat io of
failed to remove a significant amount of 99mTc, this is
in agreement with the results of Rhodes et al.
But L-cys at 625∶1
remove one-tenth of the label (Figure 5). Despite such insta bility
of the label, there was no in vivo evidence of release of
pertechneta te due to no thyroid imaging observed in the whole
imaging process (Figure 7) . Tumor localization of 99mTc-HAb18
F(ab’)2 was successfully dem onstrated in a human
tumor/nude mouse xenograft model. Biodistribution and ima ging
results showed the highest tumor uptake at 24h post-injection. Where
as kidney levels were foun d to be higher in the whole process.
Accumulation of radioactivity in the kidney may be the result of
retention of this metallic radionuclide by the kidney pro ximal
tubule, the possible release of 99mTc-labeled
cysteine and gluta thionestemming from the
radioimmunoconjugate catabolism, and the r elative amount of 99mTc-GH.
A technique has been used in patients to block renal tubule uptake
of 99mTc-anti-CEA Fab’ fragments by amin o acid
conclusion, a radioimmunoimaging conjugate for hepatoma detection
was prepare d by direct labeling mAb HAb18 F(ab’)2-with 99mTc
using stannous/g lucoheptonate as reducing agent. Although the
labeling efficiency is not satisf actory to some degree, it has
several advantapes: simple, easy and quick, besid es, the labeled
mAb fragment retains its immunoreactivity. Biodistribution and
imaging studies reveal that this conjugate is useful for the
detection of hepato ma.
ACKNOWLEGDGMENTS The authors are grateful to Dr. Wang Jing
for mice imaging and the Department of Nuclear Medicine of Shaanxi
People’s Hospital for their support of this project.
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Table 1 Effect of molar ratio (Sn/GH: mAb) on quantity of-SH,
fre e 99mTcO4- and colloid and labeling
efficiency (%, n=3)
2 Biodistribution of 99mTc-HAb
18F(ab’)2 in nude mice bearing hepatoma (x-±s, %ID/g)
after injection (h)
L-cysteine standard curve for
sulphydryl d etermination using Ellman reaction.
(Click image to enlarge)
of reduction on integrity of 99mTc-labeled HAb18 F(ab’)2
as monitored by SDS-PAGE. Vertical lanes re present molar ratios of
Sn/GH to HAb18 F(ab’)2: 1, 10∶1;
1; 4, 40∶1;
(A)Coomassie brilliant blue R250 staining. Molecular weight (kD) is
indicated at the left. (B) autoradiography.
(Click image to enlarge)
of reduction on integrity of HAb18 F(ab’)2 as monitored
by SDS*PAGE. Molecular weights (kD) are indicated at the left.
Vertical lanes represent molar ratios of Sn/GH to HAb18 F(ab’)2:
5, unreduced F(ab’)2.
(Click image to enlarge)
Binding assay for the determination of the immunoreactive
fraction of 99mTc-labeled HAb18 F(ab’)2.
(Click image to enlarge)
Dissociation of 99mTc-labeled HAb18 F(ab’)2
with increasing molar ratio of EDTA to mAb (●)
and L-cys to mAb (○).
(Click image to enlarge)
of nude mice bearing human hepatocel lular carcinoma with 99mTc-HAb18
F(ab’)2 at 24h.
(Click image to enlarge)