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Introduction
Human colonic carcinoma is one of the most common
cancers. The 5-year survival rate of patients with
chemotherapy is zero. More than half of the patients with this
tumor experience metastasis or reoccurrence after treatment.
The liver is the most common metastasis
foci[1]. Radiolabeled MoAbs offer the prospect of a localized, highly
targeted radiation treatment for these cancers. The range of
action for radionuclides is defined predominantly by the
nature of the particle and energy of the emission. One of the
earliest radioisotopes to be coupled to antibodies for
therapeutic purposes was Iodine 131
(131I). Its high-energy a particles can penetrate approximately three tumor cells, so it
can be effective even when only deposited near the tumor
cells and has minor toxicology to normal
cells[2]. There are several antibodies for a variety of human tumors that have
been used to localize human tumors in xenograft models as
well as in patients. Several of these antigens have served as
targets for testing whether MoAbs as conjugates with
radionuclides can act as selective therapeutic agents. For
example, antibodies directed against CEA, a-fetoprotein,
ferritin, melanoma, and epithelial-specific antibody have been
radiolabeled with 131I and used in the treatment of human
cancers[3,4].
In histological classifications, colon cancers are over 90%
adenocarcinoma. CEA can be observed in either the cancer
cell surface or patients blood serum in this type of
tumor[5]. Until recently, three products have been approved
worldwide for the treatment of tumors in patients: Bexxar, Zevalin
and ChTNT. The antibody used in this experiment is a new
product awaiting permission for clinical trial, provided by
Beijing Second Pharmaceutical Co, Ltd (Beijing). We
undertook this study to determine the antitumor effect of the
[131I]-labeled anti-CEA MoAbs and its distribution in nude mice
bearing xenografts.
Materials and methods
Mice Athymic nude female BABL/c nu/nu mice, 4-6 weeks
old, were obtained from the Institute of Laboratory Animals,
Chinese Medical Science Academy. Mice were kept under
SPF conditions and were fed with a diet of sterile mice chow
and water. Animals were given 10% LugolĄ¯s (5% Iodine and
10% KI) water from 2 d before the start of the experiment
beginning until the experiment was completed.
Cell lines Three colonic carcinoma derivative cell lines
were used: LS180 (ATCC No: CL-187) with a cell surface CEA
expression rate of 81%; LS174T (ATCC No: CL-188) with a
cell surface CEA expression rate of 66%; SW1116 (ATCC
No: CCL-233) with a cell surface CEA expression rate of 2654
ng/106 cells[6]. LS180 was grown in DMEM/F-12 (Hyclone)
medium, LS174T and SW1116 in MEM (Invitrogen Technologies, Inc, Carlsbad) essential medium, supplemented
with 10% FBS, 2 mmol/L L-glutamine, 100 U/mL penicillin
and 100 U/mL streptomycin.
[131I]-labeled anti-CEA MoAbs
[131I]-labeled anti-CEA humanized chimeric recombinant MoAbs
([131I]-labeled-rch24) were supplied by Beijing SaiKe Pharmaceutical.
Radioactivity was 5 mCi/mg. Radiochemical purity was more
than 98.5%.
Establishing colon tumors in nude mice The three
tumor cells were harvested and suspended in sterile PBS at a
concentration of 25×106 cells/mL. Cell viability was
determined by trypan blue dye exclusion. Cells
(5×106) in sterile PBS were inoculated subcutaneously into the flank of nude
mice[7]. Tumors became apparent in 8-10 d.
Radiolabeled antibody treatment of
tumors Mice bearing tumors were randomly divided into groups outlined in
Table 1. Mice were administered i.v. in the tail vein.
Antibodies were given 2 times with the interval of 10 d. The
positive chemotherapy drug (5-FU) was given 2 weeks, 6
times a week.
Radiolabeled antibody effect The tumor growth rate was
determined by measuring the length (a) and width (b) (mm)
of each tumor using a caliper. Tumor
volume=a×b2/2. The relative tumor volume (RTV),
RTV=Vt/Vo.
Vo is the tumor volume when the experiment started.
Vt is the measured tumor volume at different experiment time. The relative tumor
growth rate was calculated by % of
T/C=TRTV (treated
group)/CRTV (control group)×100%. The effective criterion is T/C
(%) above or equal to 60%. Tumor growth inhibition rate was
calculated by S%=(mean weight of treated group-mean
weight of control group)/(mean weight of control
group)×100%[8].
To evaluate peripheral plasma CEA levels, mice in each
group were bled from the eye using heparinized capillary
tubes. The plasma CEA level was determined by ELISA
(Hoffmann-La Roche Ltd).
Radiolabeled antibody biodistribution Two animals from
each group were bled, killed, and dissected at 24 h, 48 h, or
96 h after treatment, respectively. Tissues and organs were
immediately dissected, rinsed with saline, blotted dry, and
placed in plastic tubes and weighed. The radioactivity of
each sample of blood, liver, heart, lung, kidney, and tumor
tissue was measured using a well-type gamma counter. From
the data, [131I]-labeled anti-CEA MoAbs biodistributions
(%ID/g) were calculated: %ID/g=(tissue or organ cpm)/(total
injected cpm)/ (tissue or organ weight).
Statistical analysis Differences among the groups were
tested using a one-way ANOVA. Results are given as
mean±SD unless indicated otherwise.
Results
Distribution studies Tables 2, 3 and 4 summarize the
tumor/non-tumor ratios found with either
[131I]-labeled anti-CEA MoAbs or
[131I]-labeled-IgG in mice with tumors. The
results confirmed the tumor-specific targeting and retention
of [131I]-labeled anti-CEA MoAbs in tumor tissues in
contrast to [131I]-labeled-IgG. While the percentage of injected
dose per gram (%ID/g) in the normal tissues continued to
decrease over time for both [131I]-labeled anti-CEA MoAbs
and [131I]-labeled-IgG, the percentage of
[131I]-labeled anti-CEA MoAbs increased in the tumor between d 1 and 4. This
caused the T/NT ratios continue to increase in this period.
T/NT ratios for [131I]-labeled anti-CEA MoAbs were 2-2.5
times higher than [131I]-labeled-IgG on d 1 and continued to
increase so that T/NT ratios were 10-20 times higher than
[131I]-labeled-IgG by day 4.
Inhibition of tumor growth The biological effect of
[131I]-labeled anti-CEA MoAbs in mice bearing three tumor types
was assessed. The tumor growth curves are summarized in
Figures 1, 2 and 3. The volume of both
[131I]-labeled anti-CEA MoAbs groups was less than the control group. As
the administrative dosage increased, the tumor volume
increment rate became slow or was not obvious.
The relative tumor growth rate of three tumor types was
calculated. The growth of tumors were inhibited significantly
at the dosage groups of 3.1 mCi/kg, 6.25 mCi/kg, and
12.5 mCi/kg in nude mice bearing LS180 or LS174T (T/C%<60%).
For SW1116, only the 6.25 mCi/kg and 12.5
mCi/kg dosages were effective. With the increasing dosage, more obvious
inhibition of the tumor growth was observed.
Tumor weight and tumor growth inhibition rate (TIR) were
calculated. The data is shown in Table 5. The tumor weights
of three dosage [131I]-labeled anti-CEA MoAbs groups were
all less than that of the control. With the increase in dosage,
the tumor growth inhibition rate was more obvious. The
tumor growth inhibition rate of the 3.1 mCi/kg CEA MoAbs
group (LS180, LS174T, SW1116) was 47.8%-64.0%. This
was 69.6%-78.6% in the 6.25 mCi/kg CEA MoAbs group,
and 81.8%-86.2% in the 12.5 mCi/kg [131I]-labeled anti-CEA
MoAbs group.
Plasma CEA level The plasma CEA level is shown in
Table 6. Three groupsĄ¯ CEA levels were lower than the
control group. This shows a relationship between the CEA level
and dosage. Compared with the "nude" antibody and
[131I]-labeled-IgG, [131I]-labeled anti-CEA MoAbs was more
effective in lowering the CEA level.
Discussion
A new approach in radiation therapy for cancer involves
the use of radiolabeled MoAbs raised against
tumor-associated antigens[9]. The approach adopted in this study was
the use of [131I]-labeled anti-CEA MoAbs at different doses
to produce tumor growth inhibition in groups of athymic
nude mice bearing human colon adenocarcinoma xenografts.
The two principal objectives of this study were to examine
the biodistribution and antitumor activity of the
[131I]-labeled anti-CEA MoAbs.
Our data show that [131I]-labeled anti-CEA MoAbs at
different dosages can significantly inhibit the growth rate of
tumors (LS180, LS174T, SW1116) in a dose-dependent
manner. We are encouraged by the finding that the
destruction of tumors was apparent in approximately 50% of tumors
in the animals given 3.1 mCi/kg of radiolabeled rch24 antibody.
This suggests that we may be able to use a low dosage to
produce slight toxicity.
With one exception, most therapeutic studies with
[131I]-labeled antibodies in experimental animals have failed to
inhibit completely the growth of well-established
tumors[10-12]. However, Cheung et
al were able to ablate 0.5-2.0 cm3
neuroblastoma xenografts in nude mice with a single injection of
1 mCi of [131I]-labeled 3F8
MoAbs[13]. Whether these results are a result of a property of the antibody, radiosensitivity of
the tumor or some other factor, is unclear, but all current
experimental evidence indicates that radiolabeled
antibodies can be effectively used to inhibit tumor growth.
In this report we examined the distribution of
[131I]-labeled anti-CEA MoAbs. Targeting was observed 24 h after
the drug was administered. It was more obvious 96 h after
administration. The blood and liver have the main uptake
and the kidney has a low uptake. Toxicity was measured by
the change in bodyweight and by determination of the total
peripheral white blood cells (WBC). There was no
significant difference in the bodyweight and peripheral WBC counts
between the treated groups and model control (data not
shown).
Because [131I] is not an as effective radionuclide as other
isotopes, other radioconjugates are being
pursued[14,15]. One of the best candidates for convenient coupling to
antibodies is Yttrium-90 [90Y]. But there are difficulties in the
application. These include high uptake in normal tissue,
especially the liver, and problems associated with obtaining
high specific activity [90Y]. In addition,
[90Y] are known to
concentrate in the bone[16]. This may cause severe problems.
Each radionuclide antibody tumor system has advantages
and disadvantages, but [131I] label is the most promising
method at present.
Overall, the results of the present study indicate that
tumor growth inhibition using radiolabeled antibodies can
be confirmed. Using selectively localizing antitumor
antibodies conjugated with suitably cytotoxic radionuclides may
provide a useful new approach to the treatment of
disseminated cancers.
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