Download A humanized monoclonal antibody to carcinoembryonic antigen

Molecular Cancer Therapeutics
A humanized monoclonal antibody to carcinoembryonic
antigen, labetuzumab, inhibits tumor growth and
sensitizes human medullary thyroid cancer
xenografts to dacarbazine chemotherapy
Rhona Stein and David M. Goldenberg
Garden State Cancer Center, Center for Molecular Medicine and
Immunology, Belleville, New Jersey
for MTC management and possibly other CEA-expressing
neoplasms. [Mol Cancer Ther 2004;3(12):1559 – 64]
Introduction
Abstract
A variety of observations have shown that carcinoembryonic antigen (CEA) is associated with growth and
metastasis of cancers, including correlation of CEA serum
levels with poor clinical outcome, mediation of cell-cell
adhesion by CEA, and involvement of CEA in the immune
recognition of tumors and apoptotic pathways. The
purpose of this study was to investigate the effect that
an anti-CEA monoclonal antibody (MAb) may have on the
growth of medullary thyroid cancer (MTC), a CEAexpressing tumor, alone and in combination with chemotherapy. Antitumor effects were evaluated in a nude
mouse-human MTC xenograft model. Using the TT MTC
cell line grown s.c., we compared tumor growth in
untreated mice with that of mice given the humanized
anti-CEA MAb labetuzumab or an isotype-matched control
MAb. The effects of time of administration post-tumor
injection, MAb dose response, specificity of response, and
combination with dacarbazine (DTIC) chemotherapy were
studied. The humanized anti-CEA MAb, labetuzumab, has
direct, specific, antitumor effects in this model, without
conjugation to a cytotoxic agent. In addition, labetuzumab
sensitizes these tumor cells to chemotherapy with an
effective drug in this model, DTIC, without increased
toxicity. Significant delays in tumor growth were caused
by the MAb therapy or chemotherapy alone; however, the
combination of these agents was significantly more
effective than either agent given as a monotherapy or
use of an irrelevant MAb in this model. The superiority of
the combined modality treatment argues for the integration of CEA MAb therapy into chemotherapeutic regimens
Received 7/29/04; revised 9/30/04; accepted 10/7/04.
Grant support: Immunomedics, Inc., Morris Plains, New Jersey.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
Requests for reprints: Rhona Stein, Garden State Cancer Center, Center for
Molecular Medicine and Immunology, 520 Belleville Avenue, Belleville,
NJ 07109. Phone: 973-844-7012; Fax: 943-844-7020.
E-mail: [email protected]
Copyright C 2004 American Association for Cancer Research.
Although medullary thyroid cancer (MTC) confined to the
thyroid gland is potentially curable by total thyroidectomy
and central lymph node dissection, disease recurs in f30%
to 50% of these patients. The 5-year survival rate for all
types of MTC is between 78% and 91%, and the 10-year
survival rate is between 61% and 75% (1). These facts, as
well as the limited value shown by chemotherapy and
radiation therapy in disseminated disease, have led to the
search for novel treatment modalities. Alternative strategies have been explored in recent years including gene
therapy approaches (2), dendritic cell vaccination (3), and
monoclonal antibody (MAb) therapies (4 – 6).
The expression of carcinoembryonic antigen (CEA) in
MTC has been well documented for over 25 years and this
has led to the exploitation of radiolabeled anti-CEA MAbs
for therapy of this disease. Numerous reports on the
association of CEA with MTC have been published,
including detection of CEA in serum, immunohistologic
detection in paraffin sections of MTC biopsies, and
scintigraphic detection of MTC lesions using radiolabeled
anti-CEA antibodies (7 – 10). The availability of a human
MTC cell line, TT (11), has allowed examination of
treatment options in a preclinical setting. TT cells express
a high level of CEA, and 131I- and 90Y-labeled anti-CEA
MAbs specifically target TT tumors and cause significant
antitumor effects in nude mice bearing these xenografts (4).
In addition, the combination of radioimmunotherapy and
chemotherapy augments the antitumor effects of either
treatment alone in the xenograft model, without a
significant increase in toxicity (12, 13). Clinically, excellent
targeting of MTC has been found with radiolabeled antiCEA antibodies (5, 14), and antitumor effects have been
achieved with 131I-labeled anti-CEA antibodies (6).
Based on these observations and the large body of
literature indicating that CEA is associated with growth
and metastasis of cancers (15 – 17), we investigated the
effect that an unlabeled anti-CEA MAb may have on the
growth of a CEA-expressing MTC tumor alone and in
combination with chemotherapy with dacarbazine (DTIC).
Here, we show that the unlabeled humanized anti-CEA
MAb, labetuzumab, has antitumor efficacy in MTC,
without conjugation to a cytotoxic agent. However,
combined therapy of the anti-CEA MAb with DTIC
augments the antitumor effects of antibody or chemotherapy alone, without increased toxicity to the host.
Mol Cancer Ther 2004;3(12). December 2004
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1560 Anti-CEA Immunotherapy and Chemoimmunotherapy of MTC
Materials and Methods
Monoclonal Antibodies, Cell Lines, and Chemotherapy
TT, a human MTC cell line, was purchased from
the American Type Culture Collection (Rockville, MD).
The cells were grown as monolayers in DMEM (Life
Technologies, Gaithersburg, MD) supplemented with 10%
fetal bovine serum, penicillin (100 units/mL), streptomycin
(100 Ag/mL), and L-glutamine (2 mmol/L). The cells
were routinely passaged after detachment with trypsin0.2% EDTA.
MN-14 is a class III anti-CEA (CD66e, CEACAM-5) MAb,
reacting with CEA and unreactive with the normal crossreactive antigen, biliary antigen, and meconium antigen
(16). The construction and characterization of the humanized forms of MN-14 (labetuzumab) and LL2 (epratuzumab), the anti-CD22 MAb used here as a negative
isotype-matched control, have been described previously
(15, 18). Both antibodies were provided by Immunomedics,
Inc. (Morris Plains, NJ). The antibodies were purified by
protein A chromatography.
The choice of DTIC as the chemotherapeutic agent for
these studies and the dose schedule used were based on
results of our earlier work (12). DTIC was purchased from
Florida Infusion, Inc. (Palm Harbor, FL). In our previous
study, the maximum given dose of DTIC was selected
based on the dose of the drug given clinically to humans on
a mg/m2 basis (300 mg/m2, days 0, 1, and 2; equivalent
to 1.08 mg per dose in a 0.0036 m2 mouse). The present
study was designed using submaximal doses of the
drug, so that any putative advantage due to the combination of agents would not be masked by the general
sensitivity of this tumor model to DTIC. DTIC was given
at 75 Ag per dose, on days 2 to 4 post-tumor cell injection,
by i.p. injection.
In vivo Studies
Tumors were propagated in female nu/nu mice (Taconic
Farms, Germantown, NY) at 6 to 8 weeks of age by s.c.
injection of 2 108 washed TT cells, which had been
propagated in tissue culture. Antibodies were injected i.v.,
via the lateral tail vein, into the tumor-bearing animals.
Details on the quantities of antibodies injected and the time
of administration are indicated in the Results section for
each study. Results are given as the mean F SE. Tumor
size was monitored by weekly measurements of the
length, width, and depth of the tumor using a caliper.
Tumor volume was calculated as the product of the three
measurements. Statistical comparisons were made using
Student’s t test to compare tumor volumes and area under
the growth curves. Toxicity was evaluated by weekly measurement of body weights. Animal studies were done
under protocols approved by the Institutional Animal
Care and Use Committee.
Results
To study the effect of labetuzumab on the growth of TT
tumors in nude mice, the MAb was given either as a single
i.v. injection (0.5 mg labetuzumab per dose) given 1 or
Figure 1. Labetuzumab treatment of nude mice bearing TT xenografts.
Animals were given s.c. injections of TT cells and then (A) given an i.v.
injection of 0.5 mg labetuzumab 1 or 11 days later ( , untreated; n, day 1
treated; E, day 11 treated) or (B) given weekly i.v. injections (0.25 mg
labetuzumab/dose) initiated either 1, 3, or 7 days after inoculation of TT
cells ( , untreated; ., day 1 treated; E, day 3 treated; n, day 7 treated).
Points, means of respective treatment groups; bars, SE.
y
y
11 days post-tumor cell injection or as weekly i.v. injections
(0.25 mg labetuzumab per dose) initiated either 1, 3, or 7
days after inoculation of TT cells. Figure 1A shows the
tumor growth curves of animals treated with a single
0.5 mg dose of labetuzumab per mouse compared with
untreated controls. The untreated group contained 16
animals; the two treatment groups contained 10 animals
each. A significant growth delay was observed between
the untreated group and the group treated 1 day posttumor injection. Significant differences in the mean tumor
sizes (P < 0.05) were observed from days 32 to 93. Between
days 32 and 60, there was a 64% to 70% inhibition of tumor
size in the labetuzumab-treated group compared with the
untreated animals. There were no significant differences
between the mean tumor sizes in the day 11 group and
untreated animals. Significant delay in tumor growth was
also seen by t test analysis of the area under the growth
curves: P < 0.05 for the untreated group compared with
the group treated 1 day following tumor injection but not
for the group treated 11 days following tumor injection.
Similar results were observed when labetuzumab was
given as 0.25 mg weekly doses, as seen in Fig. 1B. Groups
Mol Cancer Ther 2004;3(12). December 2004
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Molecular Cancer Therapeutics
of seven to eight animals were studied. Significant
differences in mean tumor sizes (P < 0.05) between the
untreated group and all three treatment groups were
observed. However, the difference in mean tumor size
between the untreated mice and the day 7 treatment group
was only significant at one time point, day 28. Day 1 –
treated mice yielded significant differences from 21 to 77
days, and day 3 – treated mice yielded significant differences from 21 to 70 days. t Test analysis of the area under
the growth curves indicated significant growth inhibition
for the groups treated with labetuzumab either 1 or 3 days
after TT cell administration compared with the untreated
group. However, this analysis did not reach the 95%
confidence limit for a difference between the untreated
group and the group treated on day 7 (P = 0.057 at 5
weeks). No significant differences in body weights were
caused by any of the treatments.
Figure 2 summarizes the results of a study on the
specificity of the antitumor response. The effect of
unlabeled labetuzumab on the growth of TT tumors in
nude mice was compared with that of an isotype-matched
control humanized MAb, epratuzumab (anti-CD22 IgG1),
and the murine anti-CEA MAb MN-14. MAbs (0.5 mg per
mouse) were given i.v. 1 day after inoculation of the TT
cells followed by three additional weekly doses of 0.5 mg
per mouse. Groups of 15 animals were studied. The growth
inhibition, without effect on body weight, shown in the
experiment summarized in Fig. 1 was confirmed in this
study. Significant differences in mean tumor sizes (P <
0.05) between the labetuzumab and the untreated groups
were observed starting at day 23. At day 37, the mean
tumor volume in the group treated with labetuzumab was
42.7% of the untreated control animals. Treatment with
murine MN-14 yielded results similar to the labetuzumab.
Treatment with epratuzumab (anti-CD22 humanized MAb)
did not slow tumor growth; instead, there was a small
Figure 2. Specificity of treatment. Animals were given s.c. injections of
TT cells and either left untreated or given an i.v. injection of MAb (0.5 mg)
1 day later. Points, means of respective treatment groups ( , untreated;
n, labetuzumab; E, murine MN-14; , human LL2); bars, SE.
y
Figure 3. Effect of labetuzumab dose. Animals were given i.v. injections
of increasing doses of labetuzumab 1 day after s.c. injection of TT cells.
Points, means of respective treatment groups ( , untreated; ., 0.125 mg;
o, 0.25 mg; n, 0.50 mg; , 1.0 mg; E, 2.0 mg); bars, SE for the untreated group and the group that received 0.50 mg labetuzumab per
mouse.
y
(insignificant) increase in growth rate. For example, at
day 37, 87% of the tumors treated with labetuzumab
were <0.5 cm3 compared with 40% of the untreated group
and 29% of the epratuzumab-treated group. t Test analysis
of the area under the growth curves showed significant
differences (P < 0.05) between the untreated group and the
groups treated with either labetuzumab or murine MN-14
but not the group treated with epratuzumab. In addition,
the labetuzumab group was significantly different from the
epratuzumab group but not from the murine MN-14treated animals.
The effect of dose of labetuzumab on the growth of TT
tumors in nude mice was also evaluated. Antibody doses
were given 1 day after the TT cells, then weekly until the
termination of the study. Weekly doses ranged from 0.125
to 2.0 mg labetuzumab per mouse in groups of six mice.
Significant differences in mean tumor sizes and area under
the growth curves between the untreated group and all
treatment groups were observed (Fig. 3), again without
significant changes in body weight. For example, between
days 21 and 49, mean tumor volume in the two lowest
labetuzumab treatment groups were 27% to 40% of the size
of tumors in the untreated animals. Treatment with the
lower doses, 0.125 and 0.25 mg, seemed to be more effective
than treatment with the higher doses, although the
difference did not reach statistical significance. Thus,
dose-dependent results were not observed in this study.
Indeed, dose-dependent results have not been commonly
observed in antibody treatment studies, even clinically, so
this should not be unexpected.
Combining naked anti-CEA MAb therapy and chemotherapy was evaluated by comparing the growth of TT in
untreated mice with those treated with DTIC alone,
labetuzumab alone, or a combined treatment of DTIC with
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labetuzumab. Animals were given i.p. injections of labetuzumab at 100 Ag per dose on days 2 to 5, 7 to 11, and 15 and
then every 7 days. Groups of 10 animals were studied.
DTIC was given at 75 Ag per dose on days 2 to 4. In the
combined modality group, treatment with the anti-CEA
MAb was initiated on the same day as the first dose of
DTIC. Growth curves of TT tumors in mice given these
treatment regimens are shown in Fig. 4. All of the treatment groups had significant improvement in efficacy
compared with the untreated animals, without increased
toxicity. At 7 weeks, P values for t test of area under
curve are 0.0017, 0.0374, and 0.0002 for DTIC-only,
labetuzumab-only, and DTIC + labetuzumab, respectively,
compared with untreated animals. Combined therapy of
the naked anti-CEA MAb with DTIC augments the
antitumor effects of either antibody or chemotherapy
alone. At 7 weeks, P values for t test of area under curve
of the combined treatment were 0.0026 and 0.0002
compared with the DTIC-only and labetuzumab-only
treatment groups, respectively. Eight of 10 mice treated
with DTIC plus labetuzumab exhibited no palpable tumor
at 7 weeks post-treatment compared with only 1 of 10 in the
DTIC-only group and none in the labetuzumab-only and
untreated groups. Mean tumor volumes at 7 weeks were
0.018 F 0.012, 0.284 F 0.062, 0.899 F 0.172, and 1.578
F 0.303 cm3 for the DTIC plus labetuzumab, DTIC,
labetuzumab, and untreated groups, respectively. No
significant differences in body weights were caused by
any of the treatments. Thus, labetuzumab yields antitumor
efficacy in MTC, without conjugation to a cytotoxic
agent. However, combined therapy of the naked antiCEA MAb with DTIC augments the antitumor effects of
antibody or chemotherapy alone, without increased
host toxicity.
Figure 4.
Effect of combination treatment, DTIC + labetuzumab.
Animals were given s.c. injections of TT cells and either left untreated
( ) or given an i.p. injection of labetuzumab at 100 Ag per dose on days
2 – 5, 7 – 11, and 15 and then every 7 days (o); DTIC given at 75 Ag per
dose on days 2 – 4 (E); or combined modality treatment consisting of the
same labetuzumab and DTIC schedules with the labetuzumab treatment
initiated on the same day as the first dose of DTIC (4). Points, means of
respective treatment groups; bars, SE.
y
Discussion
CEA, a member of the immunoglobulin supergene family
(19), was first described as a tumor marker by Gold and
Freedman (20). CEA was originally thought to be a tumorspecific antigen of colorectal cancer. However, it was later
found to be present in a diverse number of carcinomas,
benign tumors, and diseased tissues as well as in the
normal human colon (21). CEA has been shown to mediate
cell-cell adhesion through homotypic and heterotypic
interactions, which in turn have implicated a role for
CEA in various aspects of tumorigenesis (22). In one report,
CEA levels have been indicated to have prognostic
significance with a more favorable prognosis in CEApositive tumors compared with that of CEA-negative
undifferentiated tumors (23). However, other articles report
the opposite, a more aggressive phenotype with increased
metastases was associated with high levels of CEA in
preclinical models (24, 25). The role of CEA in hepatic
metastasis may be based on the ability of circulating CEA to
induce Kupffer cells to release cytokines. These cytokines in
turn may activate liver endothelial cells, enabling them to
bind to circulating CEA-positive tumor cells (26). CEA has
also been implicated in the immune recognition of tumors,
possibly by interfering in natural killer cell-tumor cell
adhesion (27), as well as having an effect on cellular
differentiation, probably by interfering with normal intercellular adhesion forces (28).
The many roles attributed to CEA in neoplasia suggest
that targeting CEA with an anti-CEA MAb may affect
the growth of CEA-expressing tumors by interfering with
its function(s). Indeed, antibody binding to receptor molecules on tumor cells has been shown to affect the behavior and proliferation of these tumor cells. For example,
antitumor efficacy of unlabeled MAbs reacting with
tumor-associated antigens has been reported in nonHodgkin’s lymphoma with anti-CD20 (rituximab; ref. 29)
and anti-CD22 (epratuzumab; ref. 30) MAbs as well as in
colon cancer using the anti-epithelial glycoprotein-2 MAb,
17-1A (31), and A33 (32), in breast cancer using anti-HER-2
(33), and in colonic and other tumors with anti – epidermal
growth factor receptor (34) MAbs. These unconjugated
MAbs may inhibit tumor growth by blocking intracellular
signaling or other biological activities of their respective
antigens or by stimulating natural immunologic functions,
such as antibody-dependent cell-mediated cytotoxicity
(ADCC) or complement-mediated lysis. It is also interesting that most of these antibodies seem to have their greatest
effect in combination with anticancer drugs (32 – 35).
Common mechanisms of action among the various antibodies given with drugs include cell cycle arrest, potentiation of apoptosis, and inhibition of angiogenesis, resulting
in augmentation of the antitumor effects of chemotherapy
and, in some cases, radiation therapy (34). In the case of
CEA antibodies, such as labetuzumab studied here, a contribution in terms of apoptosis enhancement needs to be
considered because of the observation that CEA plays a
regulatory role in apoptosis (36). Using a CEA-targeted
ribozyme in human colon cancer cells to regulate CEA
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Molecular Cancer Therapeutics
levels, it has been reported that CEA does not affect cell
cycle or proliferation but does protect the cells from
undergoing apoptosis under various conditions, including
confluent growth, UV light, IFN therapy, and treatment
with 5-fluorouracil (36).
In this study, we show that labetuzumab, an antibody
specific to CEA, has antitumor efficacy in MTC without
conjugation to a cytotoxic agent and can sensitize these
tumor cells to chemotherapy with an effective drug in this
model. The current experiments show that CEA-binding
antibodies have direct antitumor effects and can also
chemosensitize human tumor cells in vivo. In nude mice
bearing human MTC xenografts, labetuzumab was shown
to significantly delay tumor growth. Differences in mean
tumor sizes between the anti-CEA MAb – treated and the
untreated groups were observed beginning at 3 weeks
and lasting at least 2 months. The effect of delaying the
treatment with naked anti-CEA MAb was studied up to
11 days after tumor cell implantation. The antibody
yielded statistically significant inhibition of tumor growth
when given up to 3 days post-tumor cell inoculation but
not when given 7 or 11 days after the tumor cells were
transplanted. Treatment with an isotype-matched non –
CEA-binding control MAb, epratuzumab (anti-CD22 humanized MAb), did not slow tumor growth, demonstrating
that the effect of labetuzumab on tumor growth is antigen
specific. The observation that the effect is greatest when
treatment is given at an early time point after injection of
tumor suggests that the antibody is most active during
early spread of the tumor cells. Although this remains to
be proven, we find this possibility intriguing and of
possible significance in terms of blocking the establishment of new metastatic sites. In addition, the anti-CEA
MAb was able to enhance the effect of chemotherapy; the
combination of chemotherapy with DTIC and anti-CEA
MAb treatment was significantly more effective than either
treatment alone. Of the mice given the combined modality
treatment, 80% had complete responses compared with
10% in the chemotherapy-only group and none in the
untreated and MAb-only groups. Thus, unlabeled labetuzumab has shown antitumor efficacy in MTC without
conjugation to a cytotoxic agent, and combined therapy of
the naked anti-CEA MAb with DTIC augments the antitumor effects of antibody or chemotherapy alone, without
increased host toxicity.
These studies corroborate a recent report of Blumenthal
et al. (17), in which in vivo antiproliferative and antimetastatic effects of labetuzumab were observed in CEAexpressing human colonic carcinoma models. Significant
survival increases were obtained when the animals were
given granulocyte-macrophage colony-stimulating factor to
increase their peripheral WBC counts or when the tumors
were treated with IFN to up-regulate CEA expression. In
addition, labetuzumab potentiated the effect of two common drugs used in colorectal cancer, 5-fluorouracil and
CPT-11. Blumenthal et al. (17) did not observe induction
of apoptosis or complement-mediated cytotoxicity by
labetuzumab in in vitro studies in colon cancer cell lines.
However, similar to anti-HER-2 and anti – epidermal
growth factor receptor MAbs (37, 38), labetuzumab was
shown to inhibit tumor cell proliferation by ADCC (17). The
lack of complement-mediated cytotoxicity against tumor
cells in vitro may be explained by the high levels of
complement regulatory factors, CD55 and CD59, in these
colon cancer cell lines (17), which are known to inhibit
complement-mediated cytotoxicity (39, 40). Thus, the work
of Blumenthal et al. (17) suggests that labetuzumab induces
effector-cell function against CEA-positive human colonic
tumor cells as shown by the in vitro ADCC results and
the importance of WBCs for in vivo activity.
In contrast, other anti-CEA MAbs have been reported to
induce complement-mediated cytotoxicity as well as ADCC
activity against a CEA-expressing gastric cancer cell line
(41). However, CD55 and CD59 levels were not measured
in this cell line. These authors also found that their fully
human anti-CEA MAb inhibited tumor growth in vivo in
human gastric cancer xenografts grown in severe combined
immunodeficient mice (41), thus agreeing with the results
reported here and by Blumenthal et al. (17).
An important role of CEA in tumorigenic potential was
also shown by transfection of human colon cancer cells
with a CEA antisense-expressing vector (42). Parental cells
with high CEA expression were highly tumorigenic,
whereas clones with decreased CEA expression showed
considerably lower tumorigenicity. It seems likely, therefore, that in addition to functioning by stimulating natural
immunologic functions (ADCC and/or complement-mediated cytotoxicity), anti-CEA MAbs may have antitumorigenic effects because they reduce or block CEA and thus
may overcome the putative role of CEA in protecting tumor
cells from apoptotic stimuli, as experienced with antisense
oligonucleotides (42). It is possible that the chemosensitization we observed for the anti-CEA antibody may also be
caused by overcoming inhibition of apoptosis by CEA,
including apoptosis induced by certain cytotoxic drugs.
Thus, the observations of the antiproliferative effects of
CEA MAb and its potentiation of chemotherapy has been
shown in MTC as well as in other cancer model systems
with various levels of CEA expression and with anticancer
drugs with diverse mechanisms of action. These effects on
tumor growth and metastasis are supportive of the general
view of the role of CEA in tumor biology, despite the need
for additional studies to fully understand the mechanisms
underlying these observations. The superiority of the combined modality treatment argues for the integration of
CEA MAb therapy into chemotherapeutic regimens for
MTC management. Clinical trials are needed to assess these
principles in patients with MTC and other CEA-expressing
cancers.
Acknowledgments
We thank Susan Chen for her excellent technical assistance.
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Mol Cancer Ther 2004;3(12). December 2004
Downloaded from mct.aacrjournals.org on April 30, 2017. © 2004 American Association for Cancer Research.
A humanized monoclonal antibody to carcinoembryonic
antigen, labetuzumab, inhibits tumor growth and sensitizes
human medullary thyroid cancer xenografts to dacarbazine
chemotherapy
Rhona Stein and David M. Goldenberg
Mol Cancer Ther 2004;3:1559-1564.
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