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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 2999-3007
Inhibition of Human Breast Carcinoma Growth by a Soluble Recombinant
Human CD40 Ligand
By
Akio Hirano,
Dan L. Longo,
Dennis D. Taub,
Douglas K. Ferris,
Lawrence S. Young,
Arisitides G. Eliopoulos,
Angelo Agathanggelou,
Nicky Cullen,
James Macartney,
William C. Fanslow, and
William J. Murphy
From the Laboratory of Leukocyte Biology, National Cancer
Institute-Frederick Cancer Research and Development Center, Frederick;
Intramural Research Support Program, Science Applications
International Corporation (SAIC)-Frederick, Frederick, MD; National
Institute on Aging, Baltimore, MD; Cancer Research
Campaign (CRC) Institute for Cancer Studies, University of Birmingham
Medical School, Birmingham, UK; Histopathology Department, Walsgrave
Hospitals, Coventry, UK; and Immunex Inc, Seattle, WA.
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ABSTRACT |
CD40 is present on B cells, monocytes, dendritic cells, and
endothelial cells, as well as a variety of neoplastic cell types, including carcinomas. CD40 stimulation by an antibody has previously been demonstrated to induce activation-induced cell death in aggressive histology human B-cell lymphoma cell lines. Therefore, we wanted to
assess the effects of a recombinant soluble human CD40 ligand (srhCD40L) on human breast carcinoma cell lines. Human breast carcinoma
cell lines were examined for CD40 expression by flow cytometry. CD40
expression could be detected on several human breast cancer cell lines
and this could be augmented with interferon- . The cell lines were
then incubated with a srhCD40L to assess effects on in vitro growth.
srhCD40L significantly inhibited the proliferation of the
CD40+ human breast cancer cell lines. This inhibition
could also be augmented with interferon-. Viability was also
affected and this was shown to be due to increased apoptosis of the
cell lines in response to the ligand. Treatment of tumor-bearing mice
was then performed to assess the in vivo efficacy of the ligand.
Treatment of tumor-bearing SCID mice with the ligand resulted in
significant increases in survival. Thus, CD40 stimulation by its ligand
directly inhibits human breast carcinoma cells in vitro and in vivo.
These results suggest that srhCD40L may be of clinical use to inhibit human breast carcinoma growth.
This is a US government work. There are no restrictions on its use.
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INTRODUCTION |
CD40 IS A MEMBER of the tumor necrosis
factor (TNF)/nerve growth factor (/NGF) receptor superfamily of
molecules, which includes fas and CD30.1,2 CD40 has
been demonstrated to be present on a variety of cell types ranging from
B cells to dendritic and endothelial cells.3,4 CD40 has
been shown to play a critical role in normal B-cell development and
function.2,5 The ligand for CD40 (CD40L) is expressed
predominantly on activated T cells.2,5 Interestingly, CD40
has also been demonstrated to be expressed at high levels on a variety
of human carcinomas, including bladder, breast, and ovarian cancers, as
well as melanomas.6,7 Little is known concerning the
function of CD40 on these neoplastic cells.
We and others have previously shown that stimuli inducing activation of
normal lymphocytes (eg, anti-CD3, IgM, etc) often can lead to death of
transformed lymphocytes.8,9 This is postulated to occur by
activation induced cell death, which may or may not involve
apoptosis.8,9 We have shown that CD40 stimulation can
promote normal B-cell growth and development yet inhibit neoplastic
B-cell growth both in vitro and in vivo.10,11 Even though
the role of CD40 on carcinoma cells remains largely speculative, its
high-level expression on neoplastic epithelial cells may make it an
attractive target for therapy. A recombinant soluble human CD40 ligand
(srhCD40L) has been developed and we therefore wanted to evaluate its
biologic effects on human breast carcinoma cells in vitro and, if
effective, in vivo.
Breast cancer remains a significant cause of mortality among women.
Current treatments include chemotherapy, hormone therapy, and bone
marrow transplantation, but mortality rates remain high for women with
advanced disease.12 The demonstration that CD40 is
expressed at high levels on human carcinomas and not on corresponding normal epithelial tissue lends promise for the idea of using CD40 to
target therapy to the tumor. Since CD40 is expressed on a variety of
hematopoietically derived cells, the use of monoclonal antibodies (MoAbs) (either nonconjugated or conjugated with a toxin or
radioisotope) directed towards CD40 could result in their depletion. If
stimulation of CD40 by its ligand could be demonstrated to adversely
affect neoplastic cell growth, the use of a recombinant soluble CD40 ligand would be an attractive alternative in that the normal
CD40+ cells would be spared and in fact may be functionally
stimulated. Additionally, use of soluble recombinant human ligand is
least likely to induce potentially neutralizing antibodies that often occur when murine proteins are used clinically.
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MATERIALS AND METHODS |
Mice.
C.B-17 scid/scid mice with severe combined immune deficiency
(SCID) were obtained from the Animal Production Area (National Cancer
Institute-Frederick Cancer Research and Development Center [NCI-FCRDC], Frederick, MD) and were not used until 6 to 8 weeks of
age. SCID mice were kept under specific-pathogen-free conditions at all
times. The mice were housed in microisolator cages and all food, water,
and bedding were autoclaved before use. SCID mice received
trimethoprim/sulfamethoxazole (40 mg trimethoprim and 200 mg
sulfamethoxazole per 320 mL drinking water) in suspension in their
drinking water.
Antibodies, srhCD40L, and cytokines.
Anti-human CD40 (M3 hybridoma, a mouse IgG1 antibody) was purified and
characterized as described.13 Normal mouse sera and mouse
IgG1 myeloma proteins (msIgG1) used as control antibodies were
purchased from Cappel (West Chester, PA). srhCD40L derived from Chinese
hamster ovary (CHO) cells was manufactured by Immunex (Seattle, WA),
and contains the extracellular domain of the CD40L fused to an
amino-proximal modified leucine zipper motif.13 Goat-anti-mouse IgG1 antibody (GamIgG1) used for cross-linking anti-CD40 was purchased from Southern Biotechnology Associates (Birmingham, AL). Anti-fas MoAb was obtained from Pharmingen
(San Diego, CA). Recombinant human interferon- was obtained by the Repository at the NCI-FCRDC. For studies involving interferon-, 500 U/mL was used, either as a pretreatment in the surface expression studies or concurrently as in the proliferation/viability studies.
Breast cancer and normal mammary epithelial cell lines.
The human breast carcinoma line, MDA-231, was kindly provided by Dr
Jack Pearson (NCI-FCRDC). MCF-7, BT-20, and T-47D were obtained from
American Type Culture Collection (Rockville, MD). Normal mammary
epithelial cells were obtained by Clonetics (BioWhittaker, Walkersville, MD) and were cultured in Mammary Epithelial Cell Growth
Medium Bullet kit (MEGM-Bullet kit) also obtained by Clonetics. MDA-231
cells were isolated from a pleura effusion of a 51-year-old woman with
poorly differentiated adenocarcinoma in 1973.14 BT-20 cells
were isolated from original tumor of a 74-year-old woman with
infiltrating ductal carcinoma in 1958.14 T-47D cells were isolated from a pleural effusion of a 54-year-old woman with
infiltrating ductal carcinoma.14 The cell lines were
maintained in monolayers cultures using RPMI-1640 medium (BioWhittaker,
Walkersville, MD) supplemented with 10% fetal bovine serum (FBS;
Atlanta Biologicals, Norcross, GA), L-glutamine (BioWhittaker,
Walkersville, MD), penicillin, and streptomycin (BioWhittaker,
Walkersville, MD). The cells were incubated at 37°C in a
humidified, 5% CO2 atmosphere and subcultured every 3 to 7 days. Only cultures in the log phase of growth were used.
Flow cytometric analysis and immunofluorescence studies.
Expression of CD40 on various human breast cancer cell lines was
determined by flow cytometric analysis. The protocol for flow
cytometric analysis has been described previously.11
Briefly, the cells were washed and counted using Coulter counter
(Coulter Electronic, Hialeah, FL). The cells were then adjusted to the appropriate cell concentration and were blocked with 2% human AB serum
to prevent nonspecific binding of immunoglobulin. The cells were then
incubated with the appropriate primary antibody of either anti-CD40 or
an isotype-matched mouse IgG1 myeloma protein. The cells were then
washed and incubated with a fluoresceinated (fluorescein isothiocyanate
[FITC]) secondary antibody; a goat-anti-mouse IgG (kindly provided
by Dr Kristin Komschilies, Science Applications International Corporation [SAIC] Frederick, Frederick, MD) or HLA-ABC
from Olympus (Lake Success, NY) as control. After washing, the cells
were fixed in 1% paraformaldehyde (Fluka Chemical, Ronkonkoma, NY) and
analyzed on an EPICS flow cytometer (Coulter Electronic).
In vitro proliferation assay.
Proliferation was measured by using a microculture tetrazolium (MTT)
assay as described by Carmichael et al15 or by
3H-thymidine incorporation.10 Briefly, 3 × 103 to 1 × 104 cells were
incubated in each well of 96-well flat-bottom microtiter plates
(Corning, Corning, NY) in a volume of 200 µL containing various doses
of reagents for up to 72 hours. After incubation, either 1 µCi of
3H-thymidine (specific activity, 6.7 Ci/mmol; New England
Nuclear Research Products, Boston, MA) or 100 µg of MTT (3-[4,5
dimethylthiozol-2-yl]-2,5-diphenyl tetrazolium bromide; Boehringer
Mannheim, Mannheim, Germany) was added to each well at 37°C in a
humidified, 5% CO2 atmosphere. After 4 hours of culture,
100 µL of 10% sodium dodecyl sulfate (SDS) in 0.01 mol/L HCl
(Boehringer Mannheim) was added to solubilize the MTT formazan
crystals. The spectrophotometric absorbance at 570 nm was determined
using a scanning multiwell spectrophotometer (EI 900; Bio-Tec
Instruments, Winooski, VT) or counts per minutes (cpm) was determined
by liquid scintillation, and the surviving fraction of cells and the
percentage growth inhibition were calculated. When no significant
departure from linearity was detected, the regression of slopes for the
control and treated samples were compared by t-test. Cell
viability was assessed using the trypan blue exclusion method (Life
Technology, Grand Island, NY). After culturing breast cancer cells for
24 to 72 hours in the presence of various amounts of anti-CD40,
srhCD40L, or control MsIgG1, the cells were stained with the vital dye
trypan blue and the number of dead cells, which do not exclude the
trypan blue dye, was counted under a phase contrast microscope (CD2,
Olympus, Tokyo, Japan). Each experiment was performed 3-4 times with a
representative experiment being shown.
In vivo experiments.
All SCID mice received 20 µL of anti-asialo GM1 (anti-ASGM1; Wako
Chemicals, Richmond, VA) in 0.2 mL phosphate-buffered saline (PBS) by
intravenous (IV) injection 1 day before tumor transfer to remove host
natural killer (NK) cells.11 One million MDA-231 breast
cancer cells were then administrated by IV injection. SCID recipients
then received either 10 µg of anti-CD40, 100 µg of srhCD40L, or
isotype-matched control antibody in 0.2 mL Hanks' balanced saline
solution (HBSS) intraperitoneally (IP) every day for a total of five
injections starting 2 days after tumor injection. Tumor-bearing mice
were then monitored for tumor development and progression. Moribund
mice were euthanized and all mice underwent necropsy for evidence of
tumor. There were eight to 10 mice per group. Survival data were
plotted by the Kaplan-Meier method and analyzed by the log-rank test. A
P value  .05 was considered significant.
Immunohistology of primary tumors.
Sections cut from formalin-fixed and paraffin-embedded tissues were
dewaxed by incubating in xylene for 10 minutes. Xylene was then removed
with three changes of acetone and the tissue sections were rehydrated
by incubating in 80%, 50%, and 20% acetone solutions for 1 minute
each. Endogenous peroxidase activity was blocked by incubating the
tissue sections in 3% hydrogen peroxide/methanol for 10 minutes. After
rinsing under a running tap for 2 minutes, the tissues were immersed in
300 mL of citric acid buffer (pH 6.0) at room temperature and subjected
to microwave irradiation at full power until boiling. The tissue
sections were then subjected to a further 5 minutes of microwave
irradiation. The citric acid buffer was then replenished and the tissue
sections were subjected to another 5 minutes of microwave irradiation.
The tissue sections were then cooled under a running tap, immersed in
PBS, ringed with a Dako pen (Dako, Carpenteria, CA), and incubated with
the rabbit polyclonal antibody N-16 (Santa Cruz Biotech, Santa Cruz, CA) at 1/160 PBS overnight in a humidified atmosphere. The tissue sections were then washed in PBS for 10 minutes at room temperature (RT); incubated with biotinylated goat-anti-rabbit (Dako) in 10% heat
inactivated goat serum in PBS for 30 minutes at RT; washed in PBS for
10 minutes at RT incubated with biotinylated horse radish peroxidase
(HRP)/streptavidin complex (Dako) 30 minutes at RT, and washed in PBS
for 10 minutes at RT. Bound antibody was then visualized by incubation
with a substrate solution containing 1 mg/mL diaminobenzidine (Sigma,
St Louis, MO), 3% hydrogen peroxide/PBS for 10 minutes. Tissue
sections were then lightly counterstained with hematoxylin and mounted
with Immunomount (Shandon, Inc, Pittsburgh, PA).
Assays for assessment of apoptosis.
MDA-231 cells were grown in RPMI 1640 with 5% FBS as an adherent
monolayer. The tumor cells were harvested in log-phase growth (705 to
80% confluent) by brief trypsinization and replated at 50,000 cells
per well in 96-well flat-bottom tissue culture-treated microplate wells
(Corning, Acton, MA) in the presence or absence of media alone or media
containing srhCD40L at 3 µg/mL and cultured for an additional 18 hours. The cells were harvested by brief trypsinization and then were
assessed for levels of apoptosis/necrosis by flow cytometry on a
FACScan (Becton Dickinson, Sunnyvale, CA) using the Annexin V-FITC
apoptosis kit according to the manufacturer's instructions (R&D
Systems, Minneapolis, MN).
Apoptosis was also assessed by examining nuclear matrix protein (NMP)
levels in the culture supernatants of treated cells using an
enzyme-linked immunosorbent assay (ELISA) kit (Oncogene Research
Products, Cambridge, MA) according to the manufacturer's instructions.
The results are expressed as NMP units per milliliter of culture supernatant.
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RESULTS |
CD40 expression on human breast carcinoma cell lines and upregulation
by interferon-.
Several human breast carcinoma cell lines were examined for CD40
expression by flow cytometry. The MDA-231 and T-47D human breast
carcinoma cell lines demonstrated surface expression of CD40 (Fig
1A and B). Another common breast
carcinoma line MCF-7 was negative for CD40 staining (data not shown).
Normal mammary epithelial cells were only slightly positive for CD40
expression (Fig 1C). It has been reported that CD40 expression can be
augmented with interferon-.6,16 We therefore cultured
the breast cancer cell lines with interferon- for 12 hours before
staining for CD40 expression. The results demonstrate that
interferon- pretreatment of the cell lines resulted in an increase
in CD40 expression (Fig 1A and B). Interestingly, normal mammary
epithelial cells still expressed low levels of CD40 despite
interferon- pretreatment (Fig 1C). These results indicate that human
breast carcinoma cell lines can express CD40 and that CD40 expression
can be increased by interferon-. We then examined primary human
breast carcinomas for CD40 expression (Fig
2). CD40 expression was detected in the majority of breast carcinoma cases examined (25 of 27 cases). CD40
staining of this ductal carcinoma was predominantly on the membrane of
carcinoma cells with a weaker cytoplasmic component (Fig 2). Smaller
CD40+ infiltrating lymphocytes, probably B cells, were also
frequently observed. These results demonstrate that CD40 is expressed
on breast carcinomas, both cell lines and primary tumors.
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| Fig 1.
Expression of CD40 on various human breast carcinoma cell
lines. White shade with solid line is the msIgG1 control staining. Dark
gray with dotted lines is CD40 staining. Light gray shade with solid
lines is CD40 staining after overnight incubation of the cells with 500 U/mL interferon-. (A) T-47D, (B) MDA-231, and (C) normal mammary
epithelial cell lines.
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| Fig 2.
Expression of CD40 on vitually all tumor cells in a case
of ductal carcinoma. Lower magnification (bottom) demonstrated
that CD40 can also be observed in smaller infiltrating
lymphocytes. Original magnification: top, ×400; bottom, ×200.
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srhCD40L inhibits the proliferation of human breast carcinoma cell
lines in vitro.
The effect of srhCD40L or an anti-CD40 MoAb on the growth of breast
carcinoma cell lines was examined using an MTT assay or 3H-thymidine incorporation. The results demonstrate that
srhCD40L significantly inhibited the proliferation of all of the
CD40+ cell lines tested (Fig 3A
through C; MDA-231, BT-20, and T-47D respectively; and Table
1), with an optimal inhibition occurring at
human CD40L concentrations of 1 to 10 µg/mL. No effect of the ligand
was observed with the CD40 line MCF-7 (Fig 3D and
Table 1). Interestingly, in direct contrast to results
previously reported with B-cell lymphomas, the anti-CD40 MoAb (M3
clone) did not appear capable of exerting significant inhibitory
effects on the breast carcinoma cell lines (Fig 3).
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| Fig 3.
Effect of srhCD40L on proliferation of breast carcinoma
cell lines in vitro. After 72 hours, proliferation was assessed by MTT.
( ) srhCD40L, ( ) anti-CD40 MoAb, and ( ) msIgG1 control. (A)
MDA-231, (B) T-47D, (C) BT20, and (D) MCF-7 (CD40) cell
line. Incubation with either 1 or 10 µg/mL srhCD40L resulted in
significant (P < .001) inhibition of proliferation of the 3 CD40+ lines (A, B, and C).
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We have previously demonstrated that prior crosslinking of the antibody
was necessary to produce optimal inhibition of lymphomas.10 Crosslinking of the antibody had no effect on breast cancer cell line
proliferation and, consistent with this, prior crosslinking of the
ligand had no effect on the inhibition by the srhCD40L (data not
shown). These results indicate that binding of CD40 by its ligand can
result in direct antiproliferative effects on human breast carcinoma
cell lines. Because interferon- can increase CD40 expression on the
cell lines, we then examined whether interferon- incubation would
result in greater growth inhibition of the cells. The results
demonstrate that coincubation of the T-47D and MDA-231 cell lines with
interferon- and srhCD40L resulted in greater inhibition of cell
proliferation (Fig 4). Interferon- alone
had no effect on viability of this dose (data not shown). Thus,
interferon- can both increase CD40 expression and augment the growth
inhibitory effects of srhCD40L with human breast carcinoma cell lines
in vitro.
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| Fig 4.
Effect of interferon- and srhCD40L on proliferation.
In some wells, interferon- (500 U/mL) was added with srhCD40L (10 µg/mL) during the assay; 72 hours later, an MTT assay was performed.
Values presented as percent control(msIgG1). (A) MDA-231 and (B) T-47D.
Significant (P < .05) differences in proliferation were
detected.
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srhCD40L induces apoptosis in human breast carcinoma cell lines.
It has been shown that signals which cause the activation of normal
cells can result in growth inhibition of transformed cells. This
"activation-induced cell death" (AICD) has been extensively characterized on lymphocytes and has been shown to involve apoptosis, cell-cycle arrest, and necrosis.8,9 To ascertain the
mechanism underlying the antiproliferative effects of srhCD40L, we
examined the effects of the ligand on cell viability and apoptosis on
human breast carcinoma cell lines. The results demonstrate that
incubation of the T-47D and MDA-231 cell lines with srhCD40L resulted
in a significant reduction of viable cells after 72 hours (Fig
5). Coincubation with interferon-
augmented this ligand-mediated decrease in cell viability. When the
cells were assessed by flow cytometry for effects on apoptosis using
Annexin-V dye binding, it was found that srhCD40L induced apoptosis and
necrosis of the MDA-231 to a significant extent. The percentage of
ligand-treated cells in the Annexin-V single-positive (indicative of
apoptosis) quadrant increased to 5% and the amount in the
Annexin-V/propidium iodide (PI) double-positive quadrant (indicative of
apoptosis and necrosis) increased to 31%
(Fig 6). NMP release, another indicator of
apoptosis, was also assessed. It was found that srhCD40L induced NMP
release comparable with an anti-fas MoAb (Table 1). Thus, the
antiproliferative effects of the srhCD40L on human breast carcinoma
cell lines are due, at least in part, to the induction of apoptosis and
necrosis in the growth-inhibited cells.
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| Fig 5.
Effect of srhCD40L on cell viability. (A) MDA-231 or (B)
T-47D cells were incubated with 10 µg/mL srhCD40L and/or 500 U/mL
interferon . After 72 hours, viability was assessed by trypan blue
exclusion. Significant differences (P < .05) in viability
were noted.
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| Fig 6.
Effect of srhCD40L on apoptosis. MDA-231 cells were
cultured with 6 µg srhCD40L as described in Materials and Methods.
Annexin /PI staining was then performed to assess apoptosis/necrosis 24 hours after treatment. No interferon- was present in this assay.
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srhCD40L exerts antitumor effects in SCID mice bearing human breast
carcinoma cells.
We then wanted to assess the efficacy of the ligand against human
breast carcinoma cells in vivo. Immunodeficient SCID mice were injected
with MDA-231 cells IV. After 2 days, the mice were treated with either
srhCD40L (100 µg IP given every day for five injections) or an
anti-CD40 MoAb (10 µg IP using the same regimen). The results
demonstrate that treatment with either the anti-CD40 antibody or the
srhCD40L resulted in significant increases in survival of the
tumor-bearing recipients (Fig 7). The
antitumor effects of the ligand were dose-dependent as lowering the
amount of the ligand resulted in diminished antitumor effects (data not shown). No adverse effects of the ligand were detected at this dose and
schedule. These results indicate that srhCD40L exerts antitumor effects
against human breast carcinoma cells in vitro and in vivo.
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| Fig 7.
Effect of srhCD40L on survival in MDA-231 tumor-bearing
SCID mice. Mice were treated as described in Materials and Methods.
Mice were treated 2 days after tumor cell injection. Treatment with
srhCD40L or anti-CD40 resulted in significant (P < .01) increases in survival.
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DISCUSSION |
Breast cancer remains a significant cause of mortality among women.
Current treatments include surgery, chemotherapy, radiation therapy,
and hormone therapy. There has been increasing interest in the use of
immunotherapy for breast cancer, particularly in the treatment of the
clinically undetectable residual disease that remains after
conventional cytoreductive therapy. The results presented here suggest
that stimulation of CD40 by its ligand may offer promise in the
treatment of breast cancer. More work needs to be performed assessing
the expression of CD40 on primary breast carcinomas and correlating it
with disease progression. The results presented here also suggest that
CD40 may be a target receptor for attacking the neoplastic cell itself.
CD40 stimulation appears to induce apoptosis and inhibit growth in
transformed epithelial cells. More work also needs to be performed to
understand the mechanism underlying the growth-inhibitory effects of
the CD40-CD40L interaction in carcinomas. It is possible that srhCD40L may be capable of predisposing the breast carcinoma cells for apoptosis
by inducing fas expression.17 In that regard, it is also of interest that the inhibition and cell death can be enhanced with interferon-. Interferon- may simply induce more CD40
expression on the surface of the tumor making it more accessible for
the ligand. Alternatively, interferon- can also induce fas
expression or may even be playing a more direct role in the
increased inhibition seen. We are currently performing experiments to
assess the effects of interferon- and srhCD40L in vivo. The
observations that interferon- can augment CD40 expression and growth
inhibition by the ligand in vitro suggests that combination
immunotherapy involving srhCD40L and cytokines, such as interferon-,
may offer additional benefits clinically.
It is of interest that while in vitro culture of the breast carcinoma
cell lines with the anti-CD40 MoAb did not result in growth inhibition,
there were significant antitumor effects noted in the tumor-bearing
SCID recipients. The anti-CD40 MoAb is a murine IgG1 and therefore
capable of inducing antibody-dependent cell-mediated cytotoxicity
(ADCC) in the antibody-deficient SCID recipients. Indeed, we have
previously demonstrated that the antitumor effects of this anti-CD40
MoAb in SCID recipients bearing human lymphomas could be partially
attributed by its ability to induce ADCC in vivo.18 Thus,
the ability of the srhCD40L to inhibit breast carcinoma growth in vivo
appears to correlate with its direct inhibitory effects as seen in
vitro, whereas the anti-CD40 MoAb we assessed appears to exert
antitumor effects in vivo not seen in vitro. The MoAb used in our
studies is a partial antagonist and may therefore not bind regions
capable of triggering apoptotic signals. However, other anti-CD40 MoAbs
that recognize other epitopes may be found to be able to exert
growth-inhibitory effects on carcinoma cells. It may also be of
interest to combine the ligand with anti-CD40 MoAbs to assess potential
synergistic effects.
The reason that CD40 is expressed at higher levels on certain carcinoma
cells as compared with normal epithelium is unclear. CD40 has been
reported on bladder, ovarian, and breast carcinomas, as well as on
melanomas.6,7 In another study, CD40 was found to be
expressed on 31% of breast, 40% of lung, and 24% of ovarian primary
tumors.19 Thus, CD40 stimulation may also be of benefit in
the treatment of other solid tumors as well. Preliminary data indicate
that ovarian carcinomas and bladder carcinomas are also inhibited by
the ligand for CD40 in vitro (data not shown). It will be important to
determine the role of CD40 in the progression of the tumors and to
assess whether tumors heterogeneous for CD40 expression can be selected
for outgrowth of CD40 cells under the influence of
exposure to CD40L.
The use of a recombinant human soluble ligand offers significant
advantages over the current use of MoAbs to target the cancer cell.
MoAbs (either conjugated with a toxin or radioisotope or nonconjugated)
often have the potential to deplete normal cells positive for the
marker towards which they are directed. Thus, antibodies to CD40 might
deplete normal B cells,1-3 monocytes,20 endothelial cells,4 dendritic cells,3 and other
normal cell types and, thus, could be deleterious. Another advantage of
using the recombinant human ligand would be the reduced risk of
inducing potentially neutralizing human anti-mouse antibodies that
often develop when murine MoAbs are used clinically. Other additional benefits of the ligand would be that not only are immune cell types not
depleted, but certain functions of these cells may be augmented after
ligand administration. A potential disadvantage of a ligand or agonist
MoAb may be the triggering of nonspecific and polyclonal host B cells
or induction of cytokine release by monocytes. As the human ligand does
not bind murine CD40 with the same affinity of human CD40
(unpublished observations, January 1998) it is difficult
to assess toxicity to murine cells in the xenograft model. Another
potential problem with the xenograft model is the lack of binding of
the srhCD40L to nontransformed cells (ie, B cells, etc), which would
occur clinically and thus potentially allow for an overestimation of
its efficacy. It would be useful therefore to assess the antitumor
effects of a soluble murine CD40L in a mouse model. With regard to
toxicity we have recently demonstrated that treatment of mice with a
recombinant murine CD40L after syngeneic bone marrow transplantation
resulted in accelerated immune and hematopoietic recovery with no overt toxicity.21 Thus, the srhCD40L may augment immune function, as well as provide direct antitumor effects clinically. Recent evidence
also appears to suggest that CD40L is capable of augmenting antitumor
immunity in tumor-bearing mice.22 Taken together, these
results suggest that srhCD40L may offer potential clinical benefits in
the treatment of CD40+ carcinomas.
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ACKNOWLEDGMENT |
We thank Dr Frank Ruscetti for critically reviewing the manuscript. We
are also grateful for the excellent technical assistance by Steve Stull
and the superb secretarial assistance by Laura Knott and Karen Hughes.
Animal care was provided in accordance with the procedures outlined in
the "Guide for the Care and Use of Laboratory Animals" (NIH
Publication No. 86-23. 1985)
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FOOTNOTES |
Submitted August 27, 1998; accepted December 17, 1998.
Supported in part with Federal funds from the National Cancer
Institute, National Institutes of Health under Contract No. N01-CO-56000.
The content of this publication does not necessarily reflect the views
or policies of the Department of Health and Human Services, nor does
mention of trade names, commercial products, or organizations imply
endorsement by the US government.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to William J. Murphy, PhD,
SAIC-Frederick, Bldg 567, Room 210, Frederick, MD; e-mail:
murphyw{at}ncifcrf.gov.
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The expansive role of CD40 and its ligand, gp39, in immunity.
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Van Kooten C, Banchereau J:
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Curr Opin Immunol
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ABSTRACT
INTRODUCTION