GASTROENTEROLOGY 1995;108:172-182

Inhibition of Hepatic Metastases of Human Colon Cancer in
Nude Mice by a Chimeric SF-25 Monoclonal Antibody
HIROSHI TAKAHASHI,*'* TETSUYA NAKADA,* MOTOE NAKAKI,* and JACK R. WANDS**Gastrointestinal Unit, Massachusetts General Hospital, and Department of Medicine, Harvard Medical School, Boston; and ~MolecularHepatology Laboratory, Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts

Background~Aims: Hepatic metastasis is one of themost serious complications of human colon cancer. A
murine-human chimeric SF-25 monocional antibody
was prepared, and this construct recognizes a cell surface antigen highly present in human colon adenocarcinoma. Methods: This study determined if the chimeric
SF-25 monoclonal antibody inhibits the outgrowth ofhepatic metastases of human colon adenocarcinoma
using an athymic nude mouse model. Results: A single
intravenous injection of chimeric SF-25 monoclonal antibody significantly inhibited the outgrowth of 5- and 7day hepatic micrometastases (P = 0.0001 and 0.004,
respectively, vs. untreated) and improved the survivalof the animals. No detectable tumor was found in theliver when mice were treated by multiple injections of
the antibody immediately after tumor cell grafting into
the portal vein. In contrast, F(ab')a fragments did notshow antitumor effects, and the administration of natural killer cell or macrophage depleting agents (anti-asialo GM1 antibody and carrageenan, respectively) substantially inhibited the antitumor effects of chimeric
SF-25 monoclonal antibody in vivo. Conclusions: Chimeric SF-25 monoclonal antibody inhibits growth of hepatic metastasis of human colon cancer, and cell-mediated host immune mechanisms seem to be importantfor its in vivo antitumor activity.
espite major advances in general patient care andsurgical therapy, the mortality rate associated withcolon adenocarcinoma has not changed significantly during the last 40 years, principally because of metastatic
spread and tumor recurrence. Although 7 0 % - 8 0 % ofpatients will present with operable primary tumors at
the time of diagnosis, surgery alone is often unable topermit a long-term survival even after complete surgicalresection because of metachronous metastatic spread andtumor recurrence. The liver is the primary site of metastasis in patients with colon cancer. 1 At autopsy, liver
involvement is frequently found in patients with coloncancer and is the sole site of metastasis in one third ofpatients. 2 Those patients who develop hepatic involvement, either solitary or multiple, are incurable in thevast majority of cases, and it is generally accepted thatD

the presence of hepatic metastases is associated with apoor prognosis.
Metachronous hepatic metastases arise from the tumor
cells that are seeded into the portal vein system from theprimary site or micrometastases that are already present
in the liver at the time of surgery. The prevention ofthese hepatic metastases has been the subject of greatinterest in the treatment of human colon cancer. In thisregard, inhibition of tumor cell spread via the vascularsystem as well as the treatment of synchronous hepatic
micrometastases are important. Numerous trials of postoperative adjuvant treatment regimens have been performed in an attempt to protect from metastatic spreadand tumor recurrence, but most have not shown a reduction in the incidence of hepatic metastasis. 3-6 The death
rate will undoubtedly remain the same until improved
methods for treatment of hepatic involvement become
available. Thus, it is of general interest to develop and
explore new experimental approaches that may eventually
be translated into useful clinical treatment regimens forthis disease.
A murine SF-25 monoclonal antibody (MAb) has beenfound to recognize a 125-kilodalton cell surface antigen
highly expressed in transformed human cells/Metabolicand cell surface labeling studies have shown that SF-25
antigen was a disulfide bond-linked heterodimer composed of 35-kilodalton and 90-kilodalton glycosylated
subunits termed o~ and ~, and the epitope was located on
the core protein of ~ subunit (unpublished data, January1992). The SF-25 antigen was expressed in all cases ofsurgically obtained human colon adenocarcinoma, andthe distribution of antigen in the tissues was uniform.
A number of normal human tissues were found to be
negative by immunohistological staining, including thestomach, colon, small intestine, liver, lung, adrenal
gland, and other tissues. 7'8 Exceptions were the kidney
Abbreviations used in this paper: ADCC, antibody-dependent ceilmediated cytotoxicity; CGN, carrageenan; MAb, monoclonal antibody; NK, natural killer.
© 1995 by the Amedcan Gastroenterological Association
0016-5085/95/$3.00

January1995

and pancreas. Positive staining was present in a subpopulation of proximal tubular cells of kidney, and very weakstaining was also observed in islet cells of the pancreas.
Furthermore, SF-25 antibody was shown to be i m m u n o localized by nuclear imaging studies to hepatic metastasisof human colon adenocarcinoma established in athymic
nude mice. 8
It is well recognized that repeated injections o f m u r i n e
MAb result in the production of human anti-mouse antibody in patients with colorectal carcinoma. 9'1° The human anti-mouse antibody binds to murine MAb andaccelerates clearance from the circulation. In addition,
the anti-mouse i m m u n e response causes some degree of
toxicity and may be responsible for allergic reaction. 9 To
potentially achieve a more clinically useful reagent, wehave investigated a chimeric SF-25 MAb in which theconstant regions of human immunoglobulin G1 werelinked to the variable regions of murine SF-25 MAb byrecombinant D N A techniques. This chimeric antibody
has been shown to be useful for targeting of humaneffector cells to the human colon adenocarcinomas established in the liver of severe combined immunodeficientmice. 11 In the present investigation, we have studied theproperties of this mouse-human chimeric SF-25 MAb
and examined if unmodified chimeric antibody has aninherent antitumor activity and prevents the outgrowthof hepatic metastases of human colon cancer in an experimental metastasis model established in nude mice. W ereport here that chimeric SF-25 MAb was able to inhibit
the growth of human colon cancer cells that are injected
into the portal vein as well as micrometastases establishedin the liver of the animals.

Materials and Methods

INHIBITION OF COLONCANCERMETASTASES 173

Production of the Chimeric MAb
A chimeric SF-25 MAb has been produced by the same
methods as previously reported. 1~'13 In brief, human-mousechimeric immunoglobulin genes were constructed by joining
L and H chain variable region genes isolated from SF-25 M A b secreting hybridoma cells to human K and T 1 constant region
genes, respectively. These constructs were then transfected into
Sp2/0 myeloma cells to produce the chimeric MAb.

Purification and Radiolabeling of
Antibodies
Murine SF-25 MAb (isotype, murine immunoglobulin
G1) and chimeric SF-25 MAb (isotype, human immunoglobulin G1) were purified by Sepharose CL-4B Staphylococcal Protein A-affinity column (Pharmacia LKB Biotechnology Inc.,
Piscataway, NJ). Radiolabeling with 125Iwas performed by the
Iodogen method. 14 Radiolabeled MAb was separated from free
125I by Sephadex G-25 column. Specific activities of iodinatedMAbs were calculated as a ratio of counts per minute of radionuclide attached per mole of antibody.

Association Constant
A constant number of LS 180 cells (105/well) was incubated with 100 gL of 125I-labeled SF-25 MAb serially diluted
with phosphate-buffered saline (PBS) containing 20% bovine
serum for 4 hours at 4°C using 96-well filter-bottomed plates(V & P Scientific Inc., San Diego, CA). Cells were then washedfive times with PBS to remove unbound antibody, and boundradioactivity was counted in the gamma well counter. Concentration of MAbs and the amount of bound MAbs were calculated from radioactivity (cpm) and specific activity (cpm/mol)of I25I-labeled MAbs. The association constant of MAb andthe number of antibody binding sites per tumor cell were
determined by the methods and equation described by Frankel
and Gerhard.15

Cell Lines
All cell lines were maintained in Dulbecco's modified
Eagle medium (M.A. Bioproducts, Walkersville, MD) supplemented with 10% fetal calf serum inactivated at 56°C for
30 minutes, 10 gmol/L nonessential amino acids, 100 U/mL
penicillin, and 100 gg/mL streptomycin. In some experiments,
cells were maintained in RPMI 1640 (Hazleton Biologics, Inc.,
St. Lenexa, KS) supplemented with 10% heat-inactivated fetal
calf serum, 10 ~mol/L nonessential amino acids, 2 mmol/L Lglutamine, 100 U/mL penicillin, and 100 gtg/mL streptomycin.
Ceils used for in vitro testing were harvested from the monolayer cultures by treatment with 0.04% ethylenediaminetetraacetic acid (EDTA) solution in Hank's balanced salts in theabsence of trypsin for 5 minutes at 37°C because SF-25 antigen
was sensitive to trypsin treatment. All of the cell lines usedwere shown to be free of mycoplasma contamination by anucleic acid hybridization technique using Mycoplasma T.C.
11 (Gen-Probe Inc., San Diego, CA).

Flow Cytometric Analysis of SF-25 MAb
Binding to LS 180 Tumor Cell Surface
One million LS 180 cells were incubated for 1 hour at
4°C with 200 ~tL of murine SF-25 or B3/25 MAb (Boehringer
Mannheim, Indianapolis, IN) adjusted to 10 ~g/mL in 0.01
mol/L PBS (pH 7.2) containing 1% bovine serum albumin.
B3/25 MAb binds to human transferrin receptor, which internalizes rapidly at 37°C. 16 After washing three times with cold
PBS, the cells were incubated at 4°C or 37°C for 15 minutes
to 1 hour and then reacted for 1 hour at 4°C with 200 ILtLof
fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin G (Cappel, Cochranville, PA) diluted 1:100 in PBS
containing 1% bovine serum albumin. After washing threetimes with cold PBS and fixed with 2% paraformaldehyde,
fluorescence was detected in a Becton Dickinson FACScan(Becton Dickinson, Mountain View, CA). Results are expressed
as mean percentage of positive staining cells.

174 TAKAHASHIETAL.

GASTROENTEROLOGYVol. 108, No. 1

Complement-Mediated Cytotoxicity Assay
LS 180 cells were used as target cells. Confluent LS
180 cells were harvested with 0.04% EDTA solution and radiolabeled by incubating 1 × 106 cells with 100 IlCi of 5~Cr
(New England Nuclear, Boston, MA) for 30 minutes at 37°C.
The radiolabeled cells were washed with PBS and adjusted to1 × 105 mL in RPMI 1640. Guinea pig, mouse, and human
complement serum (Sigma Chemical Co., St. Louis, MO) wereabsorbed before use by incubating 1 mL of each serum with
10 v LS 180 cells for 30 minutes at 4°C and centrifuged. One
hundred microliters of target cells and 50 IlL of chimeric SF25 MAb ( 1 - 1 0 0 0 Ilg/mL in RPMI 1640) were pipetted into
96-well U-bottom plates, and then 100 IlL of 1:5 dilution ofcomplement serum was added to each well. Plates were incubated in a CO2 incubator at 37°C for 30 minutes. After centrifugation of the U-bottom plates at 1500 rpm for 15 minutes,
200 IlL of culture supernatant was collected and radioactivity
was determined by a gamma well counter. As a positive control, we used a n t i - L S 180 polyclonal antibody (a gift fromDr. Sabro Sone, Toray Industries, Tokyo, Japan) that was prepared by immunization of rabbit with LS 180 cells andFreund's complete adjuvant. Spontaneous release of 5~Cr wasmeasured after incubation of target ceils alone with RPMI
1640, and total count was determined after incubation of cells
in 1.0 N HCI. Spontaneous release was < 10% of total releasein all experiments. The percent specific lysis was determinedby the following formula:
Observed cpm - Spontaneous cpm
Total cpm - Spontaneous cpm

X 100= % Specific Lysis.

Antibody-Dependent Cell-MediatedCytotoxicity Assay
An antibody-dependent cell-mediated cytotoxicity(ADCC) assay was performed using 51Cr-labeled LS 180 cells
as the target cells. One hundred microliters of target cells
adjusted to 2 × 105/mL of RPMI 1640 and 50 IlL of MAb(100 Ilg/mL in RPMI 1640) were pipetted into 96-well Ubottom plates, and then 100 IlL of various concentrations ofmurine effector cells prepared according to the methods described below were added to each well. Final concentrationof MAb in ADCC was adjusted to 20 Ilg/mL because thisconcentration was optimal as shown by preliminary experiments. After plates were incubated in a CO2 incubator at 37°C
for 8 hours, radioactivity of supernatant was determined by agamma well counter and the percent specific lysis was determined as described above.

Animals
Four to five-week-old female athymic Balb/c nude mice
were obtained from the Harlan Sprague-Dawley Inc. (Indianapolis, IN). Throughout the experiments, these animals weremaintained under specific pathogen-free conditions. All animal
experiments were approved by the Committee on Research

Animal Care protocol review group and performed according
to Massachusetts General Hospital guidelines.

Preparation of Thioglycollate-Elicited
Macrophages
Nude mice were inoculated intraperitoneally with 1
mL of thioglycollate medium (Difco) (VWR Scientific Corp.,
Philadelphia, PA). Four days later, thioglycollate-elicited macrophages were collected from the peritoneal cavity. The purityof macrophages was > 9 0 % when examined by flow cytometry
using F4/80 MAb, a rat MAb specific to murine macrophages, l 7

Preparation of Murine Splenocytes andIsolation of Natural Killer Cells
Mice were anesthetized with ether and killed by axillary bleeding. The peritoneal cavity was exposed under sterile
conditions, and the spleen was excised into a plastic dish containing 5 mL of RPMI 1640 medium. Next, a small incision
was made in the spleen, and splenocytes were isolated bysqueezing the spleen repeatedly with forceps. Splenocytes werethen transferred to a plastic tube and washed twice with RPMI
1640 medium. Adherent cells were removed from murine
splenocytes after incubation in a plastic dish for 2 hours at
37°C, and murine natural killer (NK) cells were isolated bydiscontinuous density gradient centrifugation. 18'19 Nonadherent splenocytes were adjusted to 5 × 107 cells/gradient andseparated into fractions by centrifugation at 300g for 1 hour
at 20°C on a discontinuous density gradient of Percoll at concentrations of 20%, 40%, 50%, 60%, 70%, and 100% (osmotic pressure adjusted at 320 mOsm/kg using 10 × PBS).
Large granular lymphocytes were collected from the interphases between 20% and 40% and between 40% and 50%
concentrations of Percoll. The purity of murine N K cells was
examined by flow cytometric analysis using rabbit anti-serum
to ganglio-N-tetraosylceramide (anti-asialo GM1) (Wako
Chemicals, Dallas, TX) 2° and was more than 90%.

Hepatic Metastasis of Human Colon
Cancer Established in Athymic Nude Mice
Hepatic metastasis of human colon cancer was established by the methods described by Kozlowski et al. with somemodification. 21 A human colonic carcinoma cell line, LS 180,
was harvested from tissue culture by overlaying subconfluent
monolayer cultures with 0.05% trypsin and 0.02% EDTA
solution in Hank's balanced salt solution (Gibco Laboratories,
Grand Island, NY) for 5 minutes and washed three times with
PBS. In some experiments, when treatment started immediately after the tumor cell injection, cells were harvested with
0.04% EDTA solution as described above. Nude mice wereanesthetized with 0.4 mL of 2 % chloral hydrate (Sigma Chemical Co.) by intraperitoneal injection and placed in the decubitus
position. A transverse incision was made in the left flankthrough the skin and peritoneum, exposing the spleen. Mice
were injected with 1.0 × 106 LS 180 cells in 0.25 mL PBS
into the portal vein through splenic hilus using a 27-gauge

January 1995

INHIBITION OF COLON CANCER METASTASES 175

needle. After waiting a few minutes, when hematostasis ofspleen was ascertained, the abdominal cavity was closed. Visible tumor loci are generally formed in the liver within 5 days
after tumor cell injection. They grew to a few millimeters indiameter by day 7.

lo4~

L

A

103

In Vivo Effect of Chimeric SF-25 MAb
102
The in vivo antitumor effect of chimeric SF-25 MAb
was studied using the above described hepatic metastatic
model. Mice were intravenously injected into the tail veinwith 100 ~L of PBS (untreated control group), chimeric SF25 MAb, or F(ab')2 fragment of chimeric SF-25 MAb at thedose as indicated in Results. Levels of endotoxin concentrationin MAbs used in our experiments were tested by Limulus
Amebocyte Lysate test (QCL-1000 Chromogenic LAL; BioWhittaker, Inc., Walkersville, MD) and were always < 0 . 2 EU
per mg of MAb. If necessary, we removed endotoxin fromMAbs using E N D - X B 15 endotoxin removal kit (Associationof Cape Cod, Inc., Woods Hole, MA). W e killed mice 5 weeks
after the tumor cell injection when untreated control animals
begin to die because of the large tumor mass. The liver was
fixed in formaldehyde, and the weight of all hepatic tumors
was determined after careful separation from the normal surrounding liver. The survival rate of mice treated with chimeric
SF-25 MAb was also examined and compared with the life
span of untreated animals.

In Vivo Depletion of Murine Macrophageand NK Cells
Antitumor effect of chimeric SF-25 MAb after in vivo
depletion of murine macrophages and N K cells was examinedusing animals bearing 5-day hepatic micrometastases. Because
t-carrageenan (CGN) (Sigma Chemical Co.) is toxic to macrophages, 22'23 this reagent was chosen as a macrophage-depleting
agent. However, native t - C G N is not water soluble at low
temperature, and the sulfate groups contained in the t - C G N
have been shown to produce mitogenic effects. 23 To prepare awater-soluble reagent and to avoid nonspecific stimulation of
the immune system in vivo, t - C G N was desulfated according
to the method of Ishizaka et al. 2z After tumor cell injection,
mice were injected intraperitoneally with 3 mg of this desulfated C G N dissolved in PBS every other day for 5 days (totalof 9 mg/mouse) to deplete macrophages and then treated withchimeric SF-25 MAb. To confirm that macrophages were depleted, flow cytometric analysis was performed using antimacrophage MAb (F4/80). It was found that macrophages inperipheral blood decreased from 18.0% to 1.9% by intraperitoheal injection of desulfated CGN.
For N K cell d e p l e t i o n in vivo, nude mice were injected
intravenously w i t h 20 ~ g o f anti-asialo G M 1 r a b b i t ser u m 5, 7, 9, and 12 days after t u m o r cell injection, andthe a n t i t u m o r effect of c h i m e r i c SF-25 M A b was tested.
T h e percentage of N K cells in p e r i p h e r a l blood reduced
from 7 % to an u n d e t e c t a b l e level after N K cell d e p l e t i o n

6
101

10~0

t t Illl~

I t Iltl~

10-4

I I tltt~

10-3

I I Illiid

10-2

I I Jlltld

10-1

100

t I lllitdi I Jllfld

101

I i fltit|

102

103

COLD mAb ADDED (/~g/m/)
Figure 1. Competitive inhibition of 12Sl-labeled chimeric SF-25 MAb
binding to LS 180 cells by unlabeled chimeric SF-25 and murine SF25 MAbs. The binding of 1251-labeledchimeric SF-25 MAb was inhibitedby both chimeric SF-25 and murine SF-25 MAbs. In contrast, thisbinding was not inhibited by nonrelevant MAb (B2Tr: antitetanus toxoid MAb). These results show that the chimeric SF-25 MAb recognizes
the same epitope as the murine MAb. O, chimeric SF-25 MAb; e ,murine SF-25 MAb; A, nonspecific MAb (B2TI-).

when e x a m i n e d by flow c y t o m e t r i c analysis using antiasialo G M 1 serum.

Expression of 8 F - 2 5 Antigen in LS 1 8 0
Tumor Cells After T r e a t m e n t With
Chimeric SF-25 MAb
To examine the expression of SF-25 antigen in LS 180
tumor cells after chimeric SF-25 MAb treatment, a single cellsuspension of LS 180 cells was prepared from hepatic tumors
derived from chimeric SF-25 MAb-treated and -untreatedmice, and the expression of SF-25 antigen was examined byflow cytometry using fluorescein isothiocyanate-tabeled SF25 MAb.

Statistical Analysis
The differences in tumor weights were tested by
K r u s k a l - W a l l i s test followed by M a n n - W h i t n e y rank sum
test with Bonferroni correction to conduct multiple comparison. 24 The survival rate was compared with the untreated control group using the Mantel-Haenszel test. 25

Results
Properties of Chimeric SF-25 MAb
T h e chimeric SF-25 and m u r i n e SF-25 M A b s
showed identical physical properties w i t h respect to its
i m m u n o r e a c t i v i t y and association constant. F i g u r e 1
shows a c o m p e t i t i v e i n h i b i t i o n assay u s i n g 125I-labeled
chimeric SF-25 M A b . T h e b i n d i n g o f 125I-labeled c h i m eric SF-25 M A b to the LS 180 cell line was c o m p l e t e l y176 TAKAHASHIET AL.

GASTROENTEROLOGYVol. 108, No. 1

Table 1. Flow Cytometric Analysis of SF-25 MAb Binding to
LS 1 8 0 Cells
Percentage of positive staining cells (min) (%)

SF-25 MAb
4°C
37°C
B3/25 MAb
4°C
370C

0

15

30

60

98.4
98.4

97.2
97.0

97.7
89.7

96.5
85.6

49.8
49.8

42.9
28.0

43.8
20.8

49.0
18.7

NOTE. Because SF-25 antigen did not internalize or shed, the bindingof SF-25 MAb to the tumor cell surface was stable at both 4°C and
37°C. By contrast, percentage of B3/25 MAb-positive cells decreased
at 37°C because of the internalization of transferrin receptor.

inhibited by both cold murine SF-25 and chimeric SF25 but not by B2TT, a nonspecific MAb. Thus, the chimeric SF-25 and murine SF-25 MAbs recognize the sameor structurally very close epitopes. The association constant of chimeric SF-25 and murine SF-25 MAbs were
calculated to be 2.4 × 109 L/mol and 1.9 × 109 L/tool,
respectively. These binding constants indicate that the
antigen-antibody interaction by both chimeric SF-25 androutine SF-25 MAbs is of high avidity. The number ofantibody binding sites per cell (LS 180) was approximately 2.5 × 105 for both the chimeric and original
murine MAbs. The binding of MAbs to the tumor cellsurface after incubation at 4°C or 37°C was examinedusing flow cytometry (Table 1). It was found that the
number of B3/25 MAb-positive cells rapidly decreased
from 49.8% to 18.7% after 60 minutes of incubation at
37°C because bound B3/25 MAb was internalized alongwith the transferrin receptor at this temperature. In contrast, SF-25 MAb-positive cells did not change significantly at both 4°C and 37°C. This data indicates that
SF-25 MAb is not internalized or shed from the tumor
cell surface on binding to the antigen. Similar results
were obtained when 125I-labeled SF-25 MAbs were used
to examine the amount of cell-bound, free, and internalized antibodies after various incubation times at 37°C
(unpublished data, October 1990).

chimeric SF-25 MAb using mouse or human complement(data not shown).
ADCC Produced by Murine NK Cells andMacrophages
ADCC activities produced by murine NK cells
and macrophages in the presence of chimeric SF-25 MAb
were examined by a 51Cr release assay. Figure 3 shows
the results of an 8-hour cytotoxicity assay because a 4hour assay did not produce substantial cytotoxicity. Figure 3A shows the ADCC by murine NK cells isolated
from splenocytes by discontinuous density gradient centrifugation. The purified murine NK cells showed ADCC
in the presence of chimeric SF-25 MAb. Figure 3B shows
the results obtained with thioglycollate-elicited murine
macrophages and shows that these cells mediated ADCC
at various effector to target ratios.
Animal Model of Hepatic Metastatic
DiseaseHepatic metastases of human colon adenocarcinoma were established by injecting LS 180 cells into theportal vein of nude mice. Figure 4 shows a representative
example of hepatic metastases established in nude mice
5 weeks after injection of LS 180 ceils.
Inhibition of Tumor Cells Grafted Into
Portal Vein
It was first determined if chimeric SF-25 MAb
would inhibit the growth of tumor cells that have been
seeded into the portal vein. Chimeric SF-25 MAb (1.0,
10, and 100 ~g/mouse) was intravenously injected intomice for 4 consecutive days (immediately after and 1, 2,

,,,,I

10

Complement-Mediated Cytotoxicity
Against LS 1 8 0 Cells
Figure 2 shows the percent specific lysis of LS 180
cells by complement-mediated cytotoxicity using guinea
pig serum as a complement source. Rabbit anti-LS 180
polyclonal antibody produced substantial cytotoxicity
against LS 180 in the presence of guinea pig complement,
but cytolysis was not elicited by chimeric SF-25 MAb
under the same experimental conditions. Similarly, complement-mediated cytotoxicity was not produced by

0 ~Complement

c-SFo25 (pg/ml)
Anti-LS 180

÷

÷

÷

~

÷

0

0.2

2

20

200

÷

200

0

0

+

+

Figure 2. Cytolysis of LS 180 cells by complement-mediated cytotoxicity using guinea pig complement. Cytolysis was elicited by rabbit
anti-LS 180 polyclonal antibody but not by chimeric SF-25 MAb in
the presence of guinea pig complement.

January 1995

INHIBITION OF COLON CANCER METASTASES 177

and 3 days after tumor cell grafting into the portal vein).
Five weeks later, the weight of hepatic metastatic tumors
from each mouse was measured. Results are shown in
Figure 5A. T u m o r weight of mice injected with PBS
was 1.11 + 1.51 g (mean + SD; n = 10). The chimeric
SF-25 MAb treatment significantly inhibited the tumor
growth ( K r u s k a l - W a l l i s test; P = 0.0005). In particular, mice injected with 10 or 100 btg (n = 10, respectively) were free of detectable tumor in the liver at thetime of killing (1.0 ~g/mouse; 0.37 + 0.83 g; n = 10).
I n h i b i t i o n s of 5- a n d g - D a y H e p a t i c
Metastases

The treatment was initiated 5 days after tumor
cell injection to test if chimeric SF-25 MAb inhibits the

2O

A

.a

lO

outgrowth of tumors derived from hepatic micrometasrases already present at the time of treatment. A single
intravenous injection of chimeric SF-25 MAbs (100 pg/
mouse) produced a substantial antitumor effect on 5-day
hepatic tumors (Figure 5B). Nine of 10 mice were foundto be free of detectable hepatic tumors at the time ofkilling, and only 1 mouse developed a hepatic tumorof < 1 rag. The antitumor effect of chimeric SF-25 MAb
was statistically significant by M a n n - W h i t n e y rank sum
test with Bonferroni correction (P = 0.0001 vs. untreated; 1.14 + 1.30 g; n = 18). In contrast, the F(ab')2
fragment of chimeric SF-25 MAb was less effective, andstatistically significant antitumor effect was not observed(1.20 + 1.77 g; n = 10; P = 0.3228 vs. untreatedgroup). Mice were also treated with various doses ofchimeric SF-25 MAb (0.1, 1.0, or 10 ~lg per mouse) 7
days after tumor cell injection when there were larger
tumor burdens in the liver. Antitumor effect was observed in a dose-dependent manner (untreated, 1.87 _+1.80 g, n = 15; 0.1 ~tg of chimeric SF-25 MAb/mouse,
1.37 + 1.67 g, n = 9; 1.0 ~g of chimeric SF-25 MAb/
mouse, 0.53 + 0.70 g, n = 8; 10 btg of chimeric SF-25
MAb/mouse, 0.07 + 0.09 g, n = 7) (P = 0.025 by
K r u s k a l - W a l l i s test), and statistically significant antitumor effect was observed at 10 [.tg per mouse (P = 0.025
by M a n n - W h i t n e y rank sum test) (Figure 5B).

u~
Effect of C h i m e r i c S F - 2 5 M A b on A n i m a lSurvival
50:1

25:1

12.5 : 1

The length of survival of nude mice injected withchimeric SF-25 MAb (100 ~g per mouse) 5 days afterE/ l" Ratio

1

sO

2O

10

50:1

25:1

12.5:11¸

E l F Ratio

Figure 3. ADCC by murine effector cells in an 8-hour S*Cr releaseassay. (A) Murine NK cells purified from splenocytes showed ADCC
activity against LS 180 cells in the presence of chimeric SF-25 MAb.
(B) Thioglycollate-elicited murine macrophages also elicited ADCC
against LS 180 cells in the presence of chimeric SF-25 MAb. IB,
chimeric SF-25 MAb; m, MAb.

Figure 4. Hepatic metastases of human colon adenocarcinoma established in nude mice. The majority of mice injected with LS 180
cells developed large "cannon ballfike" metastasis in the liver as
indicated by a r r o w s .

178

TAKAHASHI ET AL.

GASTROENTEROLOGY Vol. 108, No. 1

'
~:~>

•

A

.o

Treated
Untreated

t~
LU
•

..j

2

i

,.

0

c-SF25 c-SF25 c-SF25

PBS

1/~g

10/~g

Day 5
IBm

100/~g3;0Figure 6. Kaplan-Meyer curves of mice treated with chimeric SF-25
MAb. All mice treated with chimeric SF-25 MAb survived more than 6
weeks, whereas all untreated animals died within 6 weeks after LS
180 cell injection. The survival rate of treated animals was significantly longer than untreated controls (P < 0.000001 vs. untreatedby ManteI-Haenszel test),

Day 7
•

B

"
$

~

2;0

DAYS AFTER TUMOR CELL INJECTION

LU
:1=

"~ >3

i

2

•
PBS c-8F25 F(ab')2£

PBS c-SF25 c-SF25 c-SF25
0.1pg
l#g
t0pg

Figure 5. Effect of chimeric SF-25 MAb on hepatic metastases. Tumors were carefully separated from surrounding normal liver after
formaldehyde fixation and weighed in a balance. Vertical axis denotes
the weight of hepatic metastases. The differences in tumor weights
were tested by Kruskal-Wallis test followed by Mann-Whitney rank
sum test with Bonferroni correction. (A) Inhibition of tumor cells
grafted into porLal vein. The effect of different treatments (KruskalWallis test; P = 0.0005) is given as follows: untreated mice vs. micetreated with 1 pg chimeric SF-25 MAb, 10 pg chimeric SF-25 MAb, or
100 Pg chimeric SF-25 MAb (P = 0.0344, P = 0.0003, and P =0.0003, respectively). Statistically significant (~ level is 0.05/3 =0.0167. (B) Inhibition of 5-day and 7-day hepatic micrometastases.
The effect of different treatments on 5-day hepatic micrometastases(Kruskal-Wallis test; P = 0.005) is given as follows: untreated micevs. mice treated with 100 Ixg chimeric SF-25 MAb or 100 pg F(ab')2
fragment of chimeric SF-25 MAb (P = 0.0001 and P = 0.3228, respectively); mice treated with 100 pg chimeric SF-25 MAb vs. mice treatedwith 100 pg F(ab')2 fragment of chimeric SF-25 MAb (P = 0.0021).
Statistically Significant c( level is 0 . 0 5 / 3 = 0.0167. The effect ofdifferent treatments on 7-day hepatic micrometastases (Kruskal-Wallis test; P = 0.025) is given as follows: untreated mice vs. micetreated with 0.1 pg chimeric SF-25 MAb, 1 ixg chimeric SF-25 MAb,or 10 I~g chimeric SF-25 MAb (P = 0.1949, P = 0.0192, and P =0.0009, respectively). Statistically significant (~ level is 0.05/3 =0.0167.

the tumor cell injection was determined. Figure 6 shows
the survival curve of mice treated with chimeric SF-25
compared with untreated animals. It is noteworthy thatall untreated mice died within 6 weeks after tumor cell
injection due to massive hepatic metastasis (survival, 3 2 48 days; median survival, 38 days; n = 19). In contrast,
all animals treated with chimeric SF-25 MAb survived
longer than untreated mice and 3 of 10 animals were
free of hepatic tumor when removed from the study 1
year after tumor cell injection (survival, 4 9 - > 3 6 5 days;
median survival, 80 days; n = 10). The improved survival
rate of treated animals was highly significant (P <0.000001 vs. untreated by Mantel-Haenszel test).

Antitumor Effect of Chimeric SF-25 MAb
After Macrophage and NK Cell Depletion
Because F(ab')2 of chimeric SF-25 MAb was lesseffective than the whole chimeric SF-25 MAb, the Fc
fragment of the antibody seemed to be essential for its
antitumor effect, and such observed effects may be cell
mediated. Macrophages and N K cells are major effector
cells that express immunoglobulin G Fc receptors innude mice. Therefore, we examined if antitumor activityof chimeric SF-25 MAb is inhibited by macrophage andN K cell depletion. Mice were injected intraperitoneallywith desulfated C G N to deplete macrophages after the
injection of LS 180 cells, and then the antitumor effects
of chimeric SF-25 MAb (100 [lg per mouse 5 days after
tumor cell injection) were retested under these conditions. The results are shown in Figure 7A. The injectionof desulfated C G N did not affect the growth of hepaticJanuary1995

A

INHIBITION OF COLON CANCER METASTASES 179

DS-CGN (-)

DS-CGN (+)

eo

T

.....

PBS

S

J=, .....

C-sF25--PBS

Anti-NK ( - )c-SF25Anti-NKm (+)

410

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o

e

",

"

metastasis (untreated mice, 2.61 + 1.69 g, n = 13; micetreated with desulfated C G N , 1.50 --- 1.61 g, n = 10;
P = 0.1561). A single intravenous injection of chimeric
SF-25 MAbs (100 I.tg per mouse) produced a substantial
antitumor effect, and 12 of 13 mice were found to be
free of detectable hepatic tumors at the time of killingin this experiment (0.00 + 0.00 g; n = 13; P = 0.00003
vs. untreated). This antitumor effect of chimeric SF-25
MAb was substantially inhibited by desulfated C G N(mice treated with chimeric SF-25 MAb and desulfatedC G N , 0.36 + 0.96 g, n = 11, P = 0.00005 vs. micetreated with chimeric SF-25 MAb alone).
Nude mice were injected with 20 btg of anti-asialo
GM1 rabbit serum 5, 7, 9, and 12 days after tumor cell
injection to deplete N K cells and treated with a single
intravenous injection of chimeric SF-23 MAb (100 }.tg
per mouse 7 days after tumor cell injection). Results areshown in Figure 7B. The treatment of mice with antiN K serum did not affect the growth of hepatic metastasis(untreated mice, 1.31 + 1.31 g, n = 15; mice treatedwith anti-asialo GM1 rabbit serum, 1.33 -+- 1.65 g, n =18; P = 0.3745) but significantly inhibited the antitumot effect of chimeric SF-25 MAb (mice treated withchimeric SF-25 MAb, 0.14 + 0.25 g, n = 14; micetreated with chimeric SF-25 MAb and anti-asialo GM1
serum, 1.09 + 1.33 g, n = 8; P = 0.0023).

Expression of SF-25 Antigen in Liver
Metastases After Chimeric SF-25 MAb
Treatment

I

•

_

i_

_÷_

~

"

PBS

c-8F25PBS

c-SF25Figure 7. Antitumor effect of chimeric 8F-25 MAb after in vivo depletion of murine macrophages and NK cells. (A) Macrophage depletionby desulfated CGN. The effect of different treatments (Kruskal-Wallis
test; P = 0.0001) is given as follows: untreated mice vs. mice treatedwith 100 #g chimeric SF-25 MAb alone, desulfated CGN alone, or
desulfated CGN and 100 pg chimeric SF-25 MAb (P = 0.00003, P =0.1561, and P = 0.00034, respectively); mice treated with 100 pg
chimeric SF-25 MAb alone vs. mice treated with desulfated CGN alone
or desulfated CGN and 100 I~g chimeric SF-25 MAb (P = 0.00003
and P = 0.00005, respectively); mice treated with desulfated CGN
alone vs. mice treated with desulfated CGN and 100 ~g chimeric SF25 MAb (P = 0.0244). Statistically significant c~ level is 0 . 0 5 / 6 =0.0083. (B) NK cell depletion by anti-asialo GM1 antibody. The effectof different treatments (Kruskal-Wallis test; P = 0.005) is given as
follows: untreated mice vs. mice treated with 100 l~g chimeric SF-25
MAb alone, anti-NK alone, or anti-NK and 100 pg chimeric SF-25 MAb(P = 0.0006, P = 0.3745, and P = 0.3485, respectively); micetreated with 100 i~g chimeric SF-25 MAb alone vs. mice treated with
anti-NK alone or anti-NK and 100 lag chimeric SF-25 MAb (P = 0.0023
and P = 0.0023, respectively); mice treated with anti-NK alone vs.
mice treated with anti-NK and 100 pg chimeric SF-25 MAb (P =0.4286). Statistically significant c~ level is 0 . 0 5 / 6 = 0.0083.

Although the outgrowth of hepatic micrometastases was inhibited by chimeric SF-25 MAb and thesurvival rate of mice was significantly improved, the invivo purging of tumor cells was not perfect under our
experimental conditions. Therefore, the expression of SF25 antigen in the tumor cells from chimeric SF-25 M A b treated mice was examined to investigate whether treatment selected for the growth of tumor cells that no longer
bore the SF-25 antigen. However, as shown by flow cytometric analysis (Figure 8), the chimeric SF-25 MAbtreated tumor cells expressed SF-25 antigen at a similarlevel as those obtained from untreated mice. These experiments show that the outgrowth of tumor in treated mice
was not the result of selection of SF-25-negative tumor
cells.

Discussion
The properties of chimeric SF-25 MAb were determined in the present investigation, and it was foundthat (1) the chimeric SF-25 MAb construct retains binding specificity of the murine SF-25 MAb, (2) the antibody
is not internalized or shed from the tumor cell surface

180

TAKAHASHI El" AL.

GASTROENTEROLOGY Vol. 108, No. 1~A

iveControl

Treotedwith

J
I I II1#111

I IIIIIII]~eotedI I IIIIIJ

I

I IllUl

100 101 102 103 104
FLUORESCENCEINTENSITY
Figure 8. Expression of SF-25 antigen in LS 180 tumor after chimeric
SF25 MAb treatment. SF25 antigen was examined by flow cytometricanalysis using fluorescein isothiocyanate-labeled SF25 MAb. The
antigen was uniformly expressed at high density in the majority of thetumor cell populations (>99%) derived from both untreated andtreated mice. Fluorescein isothiocyanate-labeled irrelevant MAb(B2TT) served as a negative control.

on binding to the antigen, (3) chimeric SF-25 MAb elicits an ADCC by N K cells and macrophages but does not
induce complement-mediated cytotoxicity, and (4) the
observed high number of antibody binding sites(2.5 × 105 colon adenocarcinoma cell) and high association constant (2.4 × 109 tool/L) suggest that this MAb
is delivered to the tumor cells in high density in vivo.
All of these properties of chimeric SF-25 MAb are ofinterest and suggest that it may be effective as a potential
immunotherapeutic reagent. 26'27
An experimental animal model of hepatic metastasisof human colon cancer using athymic nude mice wasused to study the in vivo antitumor effect of chimeric
SF-25 MAb. Although this animal model is not a spontaneous metastatic model and does not represent all of thesteps of the metastatic cascade as defined by Fidler, 28 it
simulates metastatic growth of tumor cells in the liver
after vascular spread. The subcutaneous xenograft models
have often been used to test the antitumor effects ofMAbs directed against tumor antigens. 29'3° However, thesubcutaneous model of colon cancer rarely produces spontaneous hepatic metastasis. In addition, all mice with
hepatic metastasis died from massive hepatic tumor
involvement and hepatic dysfunction 5 - 7 weeks after
tumor cell injection. Therefore, it is possible to objectively test the antitumor effects of a reagent such as

chimeric SF-25 MAb by examining the survival curvesof the mice.
W e have shown that the chimeric SF-25 MAb inhibited the outgrowth of the experimental metastatic tumorsof human colon cancer established in the liver of nude
mice. Especially noteworthy was the inhibition of tumor
growth when treatment was started immediately after
tumor cell injection. A complete inhibition of tumor
growth was achieved and no tumor was detectable in theliver at the time of killing when control untreated animals are about to die from the large hepatic tumor burden. A substantial antitumor response was also produced
against 5- and 7-day hepatic metastases by a single intravenous injection of chimeric SF-25 MAb. In these experiments, antitumor effects were achieved in a dose-dependent manner and inhibition of tumor growth was greaterat higher doses than at lower doses of chimeric SF-25
MAb. Furthermore, the survival of the tumor-bearing
animals was improved by chimeric SF-25 MAb administration as well. These results strongly support the conceptthat chimeric SF-25 MAb has in vivo antitumor properties. In this regard, possible antitumor mechanisms mediated by MAbs have been shown to include (1) inductionof tumor cytotoxicity by effector cells such as N K cells
and macrophages, 31-33 (2) activation of complement andinduction of complement-mediated cytotoxicity,34 (3) interference with cell growth or differentiation by binding
growth factors or receptors on the surface of tumor
cells, 35'36 and (4) induction of anti-idiotypic antibodies
that subsequently act as novel vaccines against the tumor. 37,38
It was found that F(ab' )2 fragment had little effect on
the inhibition of hepatic tumor growth. This observation
indicates that the Fc portion of the antibody was essential
for a possible effector function of chimeric SF-25 MAb.
Because chimeric SF-25 MAb was not capable of mediating complement-mediated cytotoxicity against LS 180
cells or the direct anti-tumor effect in vitro (unpublisheddata, January 1992), we further examined if cell-mediated mechanisms were involved as an explanation for the
antitumor effects of chimeric SF-25 MAb. Because theimmunologic defect of nude mouse is limited to T lymphocytes, N K cell and macrophage functions are intact. 29'39'4° Therefore, such effector cell populations may
mediate tumor cell cytotoxicity in nude mice under our
experimental conditions. It was found that the administration of anti-asialo GM1 antibody significantly inhibited the in vivo antitumor effects mediated throughchimeric SF-25 MAb. Previous studies have shown thatsystemic injection of this antiserum decreases the numberof hepatic asialo GMl-positive cells and N K cell functional activities in the liver) 9'41'42 Hepatic N K cell activ-

January 1995

ities have been found to be important in inhibiting the
growth of tumor metastasis. 18'19'41-43 Our data is consistent with the hypothesis that chimeric SF-25 MAb may
have augmented this N K cell activity in an antibodymediated fashion. Other experiments examined the roleof the macrophages as effector cells in this experimental
animal model system. Desulfated C G N was used to deplete macrophages in vivo because desulfated C G N has
been previously shown to be effective for the depletionof resident macrophages in systemic organs as well as
macrophages in peripheral blood. 22 The suppression ofmacrophages by desulfated C G N significantly inhibitedthe antitumor effect of chimeric SF-25 MAb. It is possible that the circulating macrophage populations and theliver resident macrophages (Kupffer's cells)44 play an important role as effector cells in the inhibition of tumor
growth mediated by chimeric SF-25 MAb.
Although chimeric SF-25 MAb elicits ADCC by murine effector cells in vitro like other chimeric MAbs, 45
this finding does not imply that ADCC is the only important mechanism for the inhibition of metastatic tumor
growth in vivo. It was possible that chimeric SF-25 MAb
has augmented the tumoricidal properties of effector cells
by unknown mechanisms other than ADCC, and theseeffects may not have been identified and tested in our invitro assays. Furthermore, the data obtained in the nude
mice model may as well underestimate as overestimate
what may be achieved with this MAb in humans. Indeed,human effector cells are more cytotoxic to LS 180 humancolon adenocarcinoma cells than these of nude mice in
the ADCC assay (unpublished data, July 1990). Furthermore, antibodies injected into human subjects might
induce a T cell-mediated immune response against colon
tumors, and this effector mechanism is not testable inathymic nude mice. Thus, there are several limitations
inherent in the nude mice xenograft model; nevertheless,this experimental animal model provides a functional
system to investigate some of the in vivo antitumor properties of the chimeric SF-25 MAb.
Because chimeric SF-25 MAb was not able to completely irradicate hepatic micrometastasis, we determined
if chimeric SF-25 MAb treatment may have selected SF25 antigen-negative tumor cells that were resistant tochimeric SF-25 MAb treatment. The expression of SF25 antigen was studied in the tumors derived from micetreated with chimeric SF-25 MAb. It was found that SF25 antigen was indeed expressed in chimeric SF-25 MAbtreated tumor cells as shown by flow cytometric analysis.
Therefore, the outgrowth of tumor in treated mice was
not the result of selection of SF-25 antigen-negative tumors but may have resulted from insufficient dose or
efficacy of chimeric SF-25 MAb for the tumor burden

INHIBITION OF COLON CANCER METASTASES

181

and growth rate of the LS 180 ceils. In addition, chimeric
SF-25 MAb strikingly inhibited the outgrowth of tumor
cells seeded into the portal vein and micrometastases
established in the liver of nude mice and thus was apotent reagent for the prevention of the hepatic metastasis. The requirement of Fc fragment of the antibody aswell as the depletion studies of N K ceils and macrophages indicates that the antitumor effects of this antibody may be through cell-mediated mechanisms.

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Received January 25, 1994. Accepted September 22, 1994.
Address requests for reprints to: Hiroshi Takahashi, M.D., Ph.D.,
Gastrointestinal Unit, Jackson 7, Massachusetts General Hospital,
Fruit Street, Boston, Massachusetts 02114. Fax: (617) 726-3673.
Supported by grants CA 57584, DK 43351, and CA 35711 fromthe National Institutes of Health.
The authors thank Rolf Carlson and Issabelle Puisieux for excellent
technical assistance, Dr. Ken-ichi Kodera for help with statisticalanalysis, and Drs. Jiro Fujimoto and Shuichi Hanada for discussion.