Download Development and Preclinical Evaluation of a

[CANCER RESEARCH 63, 1280 –1287, March 15, 2003]
Development and Preclinical Evaluation of a Bacillus Calmette-Guérin-MUC1-based
Novel Breast Cancer Vaccine1
Maureen A. Chung, Yi Luo, Michael O’Donnell, Claire Rodriguez, Walter Heber, Surendra Sharma,2 and
Helena R. Chang
Departments of Surgery [M. A. C.], Pediatrics [S. S.], and Obstetrics and Gynecology [W. H.], Women and Infants Hospital, Brown University, Providence, Rhode Island 02905;
Department of Urology, University of Iowa, Iowa City, IA 52242 [Y. L., M. O.]; and Department of Surgery, University of California Los Angeles School of Medicine, Los
Angeles, California 90095 [H. R. C.]
ABSTRACT
Due to the high incidence of breast cancer and associated mortality
rate, the development of an effective vaccine may be beneficial for the
prevention or adjuvant treatment of this malignancy. We have constructed a novel breast cancer vaccine, Bacillus Calmette-Guérin (BCG)hIL2MUC1, that consists of BCG and expresses a truncated form of
MUC1 and human interleukin (IL)-2. In vitro analysis of the BCGhIL2MUC1 construct confirmed coexpression of MUC1 and human IL-2.
The ability of BCG-hIL2MUC1 to inhibit breast cancer growth was
evaluated in hu-PBL-SCID mice (severe combined immunodeficient mice
reconstituted with 50 ⴛ 106 human peripheral blood lymphocytes) that
received three biweekly injections of BCG-hIL2MUC1 (0.5 colony-forming unit). Control animals received PBS, MUC1 peptide (100 ␮g), or
empty vector BCG-261 (0.5 colony-forming unit) vaccination. After immunization, hu-PBL-SCID mice (n ⴝ 8 in each group) were xenografted
with 4 ⴛ 106 ZR75-1 human breast cancer cells. Whereas mice receiving
the control vaccines developed a tumor, only 87% of BCG-hIL2MUC1immunized animals developed a palpable tumor with a slower rate of
tumor growth (P < 0.001). Histological analysis of the primary tumors in
BCG-hIL2MUC1-immunized animals revealed areas of reduced MUC1
expression. CD8-positive human lymphocytes were detected only in tumors grown in BCG-hIL2MUC1-immunized animals. These results imply
a critical role of coexpressed IL-2 and MUC1 in eliciting tumor-specific
immune response. To our knowledge, this is the first report of BCG
engineered to express a tumor-associated antigen. Our results suggest that
BCG-hIL2MUC1 immunization inhibited breast cancer growth in huPBL-SCID mice. Therefore, BCG-hIL2MUC1 may be a promising candidate as a breast cancer vaccine.
INTRODUCTION
Breast cancer is a prevalent and often fatal disease (1). Despite
advances in detection and chemotherapy, approximately 25% of
women with breast cancer continue to die of this malignancy. Breast
cancer immunotherapy is currently being explored as a novel therapy.
One of the difficulties in designing a breast cancer vaccine or a
general cancer vaccine is ensuring that the immune response elicited
is targeted specifically for the tumor cells, with little or no adverse
effect on the host. To aid in vaccine specificity, a tumor antigen is
often included in the construction of cancer vaccines. One of
the candidate tumor-associated antigens for breast cancer is MUC1
(MUCIN-1) protein. MUC1 protein is known by a variety of names
including DF3 and episialin (2, 3). The human MUC1 gene encodes
for a large transmembrane polypeptide (⬎400 kDa) consisting of a
variable number of tandem repeats of 20 amino acids (4). MUC1 is
Received 4/22/02; accepted 1/15/03.
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.
1
Supported in part by grants from Association of Academic Surgery (to M. A. C.),
Expeditions Inspirations Outstanding Young Investigator Award (to M. A. C.), and
HD41701-01 (to S. S.).
2
To whom requests for reprints should be addressed, at Department of Pediatrics,
Women and Infants’ Hospital-Brown University, 101 Dudley Street, Providence, RI
02905. Phone: (401) 274-1122, ext. 1289; Fax: (401) 453-7571; E-mail: ssharma@
wihri.org.
expressed on a variety of epithelial-derived cells including breast
ductal cells. In the benign state, MUC1 is heavily glycosylated, and its
distribution is limited to the apical surface of the ductal cell (5). In the
malignant state, there is increased expression of underglycosylated
MUC1, which is distributed along the entire cell surface (6). Underglycosylation of MUC1 in breast malignancies unmasks novel
epitopes on the protein that are unique to the malignant state (7).
MUC1, in its malignant form, has been shown to be immunogenic,
with each tandem repeat containing an epitope (8). MUC1 antibodies
have been detected in breast cancer patients, albeit at a low rate (9).
It has been postulated that underglycosylated MUC1 may be capable
of stimulating a potent immune response and in this manner can serve
as a target for vaccine immunotherapy (10).
There have been several attempts at using MUC1 as a cancer
vaccine. Most of the work has focused on the use of a synthetic
peptide containing five or seven of the tandem repeats, either by itself
or conjugated to a carrier protein (11–13). These MUC1 vaccines have
been able to stimulate a modest humoral MUC1 response. Importantly, reactivation of patient-derived memory T cells has also proved
to be efficacious against breast cancer cells in a non-obese diabetic/
SCID3 mouse model (14). As a matter of fact, MUC1-specific cytotoxic T cells have been isolated from MUC1 transgenic mice that,
when adoptively transferred in vivo, eradicate tumors (15). However,
despite the encouraging results obtained from these approaches, it
would be preferable to use a live MUC1-cytokine-coexpressing molecular construct as a vaccine. Attempts at using viral vectors have
met with limited success (16). The MUC1 protein expressed by host
cells infected with MUC1-viral vectors is predominantly glycosylated,
mimicking the benign form of MUC1 protein. Additionally, the
MUC1 proteins expressed in other settings are heterogenous, suggesting a varying pattern of glycosylation or some instability in expression
of the recombinant protein (17).
The present study reports on the use of the attenuated form of
Mycobacterium bovis, BCG, as the vehicle for delivering MUC1
protein. BCG, in its attenuated form, has been shown to be safe in
humans. We have engineered BCG to express a truncated form of
MUC1 protein containing 22 tandem repeats and to simultaneously
secrete hIL-2. The ability of BCG-hIL2MUC1 to inhibit the growth of
MUC1-positive tumor cells in a xenograft model of human breast
cancer was evaluated in hu-PBL-SCID mice (SCID mice reconstituted
with human PBLs).
MATERIALS AND METHODS
Vectors and Primers. For experiments involving BCG, it is important to
use a shuttle plasmid capable of functioning in Escherichia coli and BCG.
BCG replicates slowly, at 24 –36-h intervals, and testing of the plasmid in E.
coli permits more rapid experimentation. PMOD12, derived from pMV261
3
The abbreviations used are: SCID, severe combined immunodeficient; BCG, Bacillus
Calmette-Guérin; hIL, human interleukin; IL, interleukin; cfu, colony-forming unit(s);
HSP, heat shock protein; MCS, multiple cloning site; HA, hemagglutinin; ATCC, American Type Culture Collection; PBL, peripheral blood lymphocyte; ORF, open reading
frame.
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DEVELOPMENT AND PRECLINICAL EVALUATION OF BCG-hIL2MUC1
(18), is a shuttle plasmid capable of replicating and expressing recombinant
proteins in E. coli and BCG. PMOD12, developed in Michael O’Donnell’s
laboratory, differs from its parent plasmid in that it contains two independent
and constitutively active promoters from BCG HSP60 and HSP70 and two
MCSs. A schematic diagram of PMOD12 is presented in Fig. 1A. The first
MCS is located after the first promoter, a start codon, the BCG ␣ secretion
signal (Fig. 1A, SS), and a marker epitope, the influenza virus HA epitope tag
(Fig. 1A, TAG). The second MCS is located after the HSP70 promoter. A
selectable antibiotic marker for kanamycin (Fig. 1A, KAN) is also encoded by
the plasmid. The plasmid PMOD12 was genetically modified to create
pIL2MUC1, a plasmid capable of secreting IL-2 and expressing a truncated
form of MUC1 protein. A schematic diagram of pIL2MUC1 is illustrated in
Fig. 1B. Plasmid pIL2MUC1 was constructed in a stepwise manner by inserting the cDNA for IL-2 into PMOD12 to create P12IL2 (intermediate plasmid),
followed by the cDNA for the truncated form of MUC1 containing 22 tandem
repeats to create pIL2MUC1.
Several primers were used for the experiments described here. The sequences of the oligonucleotide primers used for amplification of the hIL2 DNA fragment were 5⬘-CAAGGGATCCGCACCTACTTCAAGTTCTACAAAG-3⬘ (IL-2 sense) and 5⬘-GCCGGAATTCTTATCAAGTTAGTGTTGAGATGAT-3⬘ (IL-2 antisense). The sequence of the primer used for
sequencing pIL2MUC1 was 5⬘-ATTTGACAGCACACCGCCGT-3⬘. All
oligonucleotides were obtained from Integrated DNA Technologies, Inc.
(Coralville, IA).
Construction of pIL2MUC1 Plasmid. The plasmid containing the human
IL-2 cDNA was obtained from ATCC (Manassas, VA), and the cDNA was
amplified by PCR using IL-2 sense and antisense primers encompassing the
restriction enzyme sequences for BamHI and EcoRI, respectively. The purified
IL-2 cDNA fragment was ligated into PMOD12 to create the intermediate
plasmid, P12IL2. Using P12IL2, pIL2MUC1 was constructed by inserting a
cDNA containing a truncated form of MUC1 with 22 tandem repeats through
standard subcloning techniques. Briefly, a 1.7-kb fragment containing 22
tandem repeats of MUC1 was derived from pDKOF (kindly provided by Dr.
O. J. Finn; Ref. 19). The MUC1 1.7-kb fragment was subcloned into P12IL2
using cohesive and blunt-end ligations. P12IL2 was digested with NcoI and
treated with calf intestinal phosphatase to prevent self-ligation. The two DNA
fragments were then ligated at the NcoI site, and the free ends were bluntended by filling in the missing nucleotides with Klenow polymerase. The
newly created blunt ends were religated with T4 ligase to create the new
plasmid, phIL2MUC1. The new plasmid phIL2MUC1 was used to transform
competent bacteria and colonies grown on selectable media. All restriction
endonucleases and enzymes were obtained from New England Biolabs (Beverly, MA), and the reactions were performed according to the manufacturer’s
guidelines.
The presence of the correct DNA inserts in phIL2MUC1 was confirmed
using restriction enzyme mapping. The plasmid DNA was digested with
BamHI, EcoRI, HindIII, and NcoI. The hIL-2 insert was released from the
plasmid by codigestion with BamHI and EcoRI, and the truncated version of
MUC1 was released by codigestion with HindIII and NcoI. The site at which
the majority of the DNA manipulations for inserting the truncated MUC1
fragment had occurred involved the first series of ATG after the TATAA box.
To ensure that the correct ORF had been maintained after the HSP70 promoter
(the second promoter), phIL2MUC1 was sequenced from the second promoter
using primer 3 and fluorescent end-labeled nucleotides (Brown Sequencing
Facility; Ref. 20). Of 14 clones sequenced, only 4 maintained the correct
reading frame. After confirmation of the correct DNA sequence of
phIL2MUC1 by restriction enzyme analysis and DNA sequencing,
phIL2MUC1 was used to transform competent BCG (Pasteur strain; Laval,
Canada) by electroporation. BCG-conditioned media were tested for the presence of the recombinant proteins, hIL-2 and truncated MUC1.
Western Blotting. Production of IL-2 and MUC1 by phIL-2MUC1 plasmid was confirmed by immunoblotting as described below. Protein extracts
from equivalent optical densities of cultured BCG were prepared in the
following manner for one-dimensional protein electrophoresis. One ml of
cultured media of BCG-hIL2MUC1 was centrifuged, and the pellet was
resuspended in SDS sample buffer and boiled for 10 min. The supernatant was
loaded on a 15% bis-acrylamide resolving gel, and the protein was separated
by electrophoresis. Molecular mass determinations were made by calibration
of the gels with protein standards. At the completion of electrophoresis, the
proteins were transferred to a nitrocellulose membrane (Bio-Rad Laboratories,
Hercules, CA), and nonspecific sites were blocked with 10% nonfat powdered
milk in PBS. The presence of hIL-2 was determined by immunoblotting with
an anti-hIL-2 antibody (Amersham Pharmacia Biotech, Piscataway, NJ). After
completion of the primary incubation, the membranes were incubated with
goat antimouse peroxidase-labeled secondary antibody. The immunoblot was
developed by the enhanced chemiluminescence method (Amersham Pharmacia
Biotech) as directed by the manufacturer. To confirm that the IL-2 detected
arose from BCG-hIL2MUC1, a parallel Western blot was performed with a
primary antibody against HA, the epitope present on our recombinant IL-2
(Amersham Pharmacia Biotech). Cultured media of BCG-261 (BCG containing vector plasmid pMV261) were used as a negative control for these
experiments. Commercially available IL-2 (Boehringer Mannheim, Indianapolis, IN) was used as a positive control. MUC1 was detected essentially as
described above, except that the protein mass was separated on a 10% gel.
Anti-MUC1 antibody was obtained from Sigma (St. Louis, MO).
Human Breast Cancer Cells. Three human breast cancer cell lines,
ZR75-1, MCF-7, and MDA-MB-175, were obtained from ATCC and evaluated for MUC1 expression. ZR75-1 (ATCC CRL 1500) is a human breast
carcinoma cell line derived from the malignant ascites of a postmenopausal
Caucasian female with infiltrating ductal carcinoma (21). MCF-7 (ATCC HTB
22) is a breast carcinoma cell line established from the pleural effusion of a
postmenopausal Caucasian female (22). MDA-MB-175-VII (ATCC HTB 25)
is also a breast carcinoma cell line derived from a pleural effusion, but in this
instance, the source was a postmenopausal African-American female (23). The
cell lines were maintained in RPMI 1640 supplemented with 10% fetal bovine
serum using standard cell culture technique. Total MUC1 expression was
determined by immunocytochemistry. Trypsinized cells were incubated with
BC3 (anti-MUC1 antibody; a gift from Dr. O. J. Finn) in a 1:20 dilution. The
antigen-antibody complex was then detected by a goat antimouse antibody and
Fig. 1. A, schematic diagram for PMOD12, the starting plasmid for pIL2MUC1.
HSP60 and HSP70 are two constitutively expressed promoters. SS, BCG ␣ signal
visualized using the streptavidin peroxidase method. Cell surface expression of
sequence. TAG, epitope tag of influenza virus hemagglutinin. KAN, kanamycin antibiotic
MUC1 expression was determined by flow cytometry. Equivalent aliquots of
selection. MCS1 and MCS2 are the MCSs. The DNA sequence for each MCS is included.
exponentially growing breast cancer cells were stained with BC3 followed by
A potential triplet for a stop codon is illustrated in italics. B, schematic diagram of
incubation with antimouse fluorescence-labeled antibodies. Presence of the
pIL2MUC1. This is an extrachromosomal plasmid with two constitutively expressed and
independent promoters (P). After the first promoter are the sequences for the BCG ␣
antigen-antibody complex was quantified in a Becton Dickinson FASCalibur
secretion signal (SS), the influenza virus hemagglutinin antigen tag sequence (TAG), and
machine. MUC1 staining was localized to the cell surface, and ⬎95% of the
IL-2. After the second promoter lies the sequence for 22 tandem repeats of MUC1. There
cells stained strongly for this protein in the three cell lines tested. ZR75-1 cells
is also a selectable antibiotic marker (KAN) on the plasmid. Both IL-2 and MUC1 DNA
sequences are flanked by unique restriction enzyme sites as illustrated.
were chosen as the source of human breast cancer cells for the animal model
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DEVELOPMENT AND PRECLINICAL EVALUATION OF BCG-hIL2MUC1
because of abundant MUC1 expression on the cell surface and because of their
easy and rapid growth in culture. PANC1 (ATCC CRL-1469), a pancreatic
carcinoma cell line known to express MUC1, was used as a positive control for
these experiments. The negative control included incubation of the ZR75-1
breast cells without any primary antibody.
Reconstitution of SCID Mice (hu-PBL-SCID Mice). Female SCID mice
(CB17 scid/scid; 3– 4 weeks old; Taconic Farms, Inc., Germantown, NY) were
reconstituted with 50 ⫻ 106 human PBLs to create a xenograft of human
lymphocytes in SCID mice (hu-PBL-SCID mice). PBLs were procured as
described from human buffy coats obtained from the Rhode Island Blood
Center (24). The buffy coats were resuspended in HBSS (pH 7.4) and layered
under a high-density solution of Ficoll-Paque plus (Amersham Pharmacia
Biotech, AB, Sweden) and centrifuged for 30 min at 1500 rpm. The interface
containing the PBLs was then harvested and washed twice in HBSS. The
washed cell pellet was resuspended in PBS, and 50 ⫻ 106 PBLs were injected
i.p. into the SCID mice.
Vaccination of hu-PBL-SCID Mice. The vaccines used in this experiment
included BCG-hIL2MUC1 (0.5 cfu; experimental vaccine), MUC1 peptide
(100 ␮g; MUC1 control), BCG-261 (0.5 cfu; BCG control vaccine), and PBS
(sham vaccine) diluted in a total volume of 200 ␮L of PBS. BCG-hIL2MUC1
is our recombinant vaccine consisting of BCG that expresses a truncated form
of MUC1 protein with 22 tandem repeats while simultaneously secreting
human IL-2. MUC1 synthetic peptide was a custom peptide consisting of five
tandem repeats of MUC1 (GVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAH; Boston Biomolecules, Warhem, MA).
BCG-261 vaccine consisted of the starting plasmid used to construct
pIL2MUC1 and served as the control for BCG-stimulated antineoplastic activity. PBS was a sham vaccine and served as an internal control. hu-PBLSCID mice were reconstituted as described above with 50 ⫻ 106 PBLs on day
0. One day after lymphocyte reconstitution, hu-PBL-SCID mice received three
i.p. vaccine injections at biweekly intervals. Each group of animals had eight
mice. Two weeks after the third vaccination, 4 ⫻ 106 ZR75-1 breast cancer
cells were injected s.c. into the right flank. The mice were observed until
impending death or 150 days after tumor inoculation and then sacrificed.
Outcomes of interest were time to tumor detection, size of primary tumor, and
rate of tumor growth. Median time to tumor onset was determined for each
experimental group. Mean tumor size was plotted over time after tumor
engraftment for each group, and polynomial regression analysis was performed
to determine whether the rate of tumor growth differed between the groups of
mice.
To further verify that the tumor growth inhibition observed in animals
immunized with BCG-hIL2MUC1 was solely due to a MUC1-specific response and was not due to the enhanced T-cell function from BCG-mediated
IL-2 expression, hu-PBL-SCID mice were immunized with BCG-IL2 with and
without exogenous MUC1 peptide and xenografted with 4 ⫻ 106 ZR75-1
human breast cancer cells. The primary tumors obtained from these animals
were evaluated for MUC1 expression and infiltration of CD8-positive human
lymphocytes.
When the animals became gravely ill, as manifested by inability to move or
groom, they were sacrificed. All surviving mice were sacrificed 24 weeks (168
days) after tumor engraftment. Three mice receiving BCG-hIL2MUC1 and one
mouse receiving MUC1 peptide were sacrificed at 10 weeks after receiving the
tumor xenograft to allow for completion of the experiments in a timely fashion.
The mice were anesthetized by 100% CO2 insufflation, and necropsy was
performed on all mice. All tumor deposits were carefully measured and fixed
in a 10% formalin solution. All enlarged masses or abnormalities within the
abdominal cavity or detected on the liver and spleen were harvested, measured,
and fixed in formalin. Liver, spleen, and lung samples were also obtained. The
harvested tissue was fixed in a 10% formalin solution and embedded in
paraffin wax. The tissue blocks were then sectioned for H&E staining and
immunohistochemistry.
Histological Analysis of Tissue Samples. Immunohistochemistry of tissue
sections was performed using the LSAB⫹ peroxidase kit (DAKO Corp.,
Carpinteria, CA). Briefly, slides containing the tissue sections were deparaffinized and rehydrated with alcohol and xylene. Endogenous peroxidase activity was suppressed with a 3% hydrogen peroxide solution, and nonspecific
antigen binding was blocked by incubation with swine serum. The tissue was
then incubated with the appropriate primary antibodies. The following anti-
bodies were used: anti-episialin antibody (Sigma) was used for detection of
MUC1; anti-cytokeratin clone AE1/AE2 antibody was used for detection of
cytokeratins, antihuman CD8 was used for detection of CD8-positive human
lymphocytes; anti-mycobacterial antibody was used to detect the presence of
BCG; and anti-CD45 antibody was used to detect human lymphocytes (DAKO
Corp.). Incubation of tissue sections with murine-derived control IgG served as
the negative control for these experiments (DAKO Corp.). After incubation
with the primary antibody, the secondary antibody consisting of a biotinylated
antirabbit, antimouse, and antigoat immunoglobulin antibody was used.
Streptavidin peroxidase was then added. Color development followed incubation with a substrate chromogen solution (3⬘,3⬘-diaminobenzidine chromogen
solution). Slides were then counterstained with hematoxylin (Mayer’s hematoxylin; Lillie’s modification; DAKO Corp.). Slides were finally dehydrated
with alcohol and xylene and mounted for histological evaluation.
Statistical Analysis. The Kaplan-Meier method was used to estimate survival curves. Polynomial regression analysis of tumor size over time and
within groups showed a P ⬍ 0.01.
RESULTS
Characterization and Propagation of pIL2MUC1. The presence
of the DNA inserts for hIL-2 and the truncated form of MUC1 were
confirmed by restriction enzyme mapping (data not shown). Although
restriction enzyme mapping is a quick method to identify the presence
of DNA inserts, it does not permit the determination of the DNA
sequence. The site at which the majority of the DNA manipulations
for inserting the truncated MUC1 fragment had occurred involved the
first series of ATG after the TATAA box, and it was possible that the
ORF for MUC1 had been altered. To ensure that the correct ORF had
been maintained after HSP70 promoter (the second promoter),
pIL2MUC1 was sequenced from the second promoter. Of 14 clones
sequenced, only 4 maintained the predicted sequence and the correct
ORF for the truncated MUC1 protein. The four clones found to have
the correct sequence were then used to transform BCG. After electroporation of pIL2MUC1 into BCG, colonies were grown on selectable agar media.
Production of IL-2 and MUC-1 in BCG-hIL2MUC1. Cultured
BCG-hIL2MUC1 mycobacteria samples were evaluated for the expression of hIL-2 and MUC1 (Fig. 2) by immunoblotting as described
in “Materials and Methods.” Fig. 2A shows data for detection of IL-2.
Lane A represents commercial IL-2 protein that served as our positive
control. The protein mass extracted from BCG-261 served as a negative control (Lane B). Proteins extracted from BCG-hIL2MUC1 were
found to contain IL-2 as shown in Lanes C and D. Immunoblotting
with an anti-IL-2 antibody resulted in the detection of a unique protein
in Lanes A and C. The protein detected in Lane C represented IL-2 and
was approximately 20 kDa in size. The size of the BCG-expressed
IL-2 was slightly larger than that obtained for its commercially
available counterpart (Lane A) and is due to the additional sequences
for the BCG ␣ secretion signal and the HA marker epitope tag. To
confirm that the IL-2 detected by immunoelectrophoresis was from
BCG-IL2MUC1 and of the same size, a parallel immunoblot was
performed, but the blot probed with an antibody directed against HA,
the epitope tag (Lane D).
Because we were interested in the simultaneous expression of hIL-2
and MUC1, extracts from BCG-hIL2MUC1 clones known to express
hIL-2 were used for MUC1 detection. Our results are summarized in
Fig. 2B. Recombinant MUC1 protein with a molecular mass of 87
kDa was expressed by BCG-hIL2MUC1 (Lane F). Lanes A and C
represent our positive controls (ZR-75-1 and E. coli-phIL2MUC1,
respectively). Lanes B, D, and E are the negative controls consisting
of extracts from E. coli-PMOD12, BCG-261, and BCG-hIL2, respectively. MUC1 expressed by BCG-hIL2MUC1 was a single band,
indicating that no heterogeneity occurred due to glycosylation as has
been described with recombinant viral vectors expressing MUC1 (25).
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DEVELOPMENT AND PRECLINICAL EVALUATION OF BCG-hIL2MUC1
Fig. 2. A, Western blotting of BCG-hIL2MUCI with anti-IL-2 antibody. Protein was
extracted from BCG-hIL2MUC1 and immunoblotted for the presence of hIL-2 with an
anti-IL2 antibody. Lanes B and C represent proteins extracted from BCG-261 (negative
control) and BCG-hIL2MUC1, respectively. The positive control was commercially
available hIL-2, which is shown in Lane A. A unique band can be seen in Lane C,
corresponding to the recombinant IL-2 expressed by BCG-hIL2MUC1. The molecular
mass of the hIL-2 is slightly larger than that in Lane A because of the secretion signal and
HA epitope tag. Lane D represents protein extracted from BCG-hIL2MUC1 probed with
anti-HA antibody (TAG). B, Western blotting of BCG-hIL2MUC1. Immunoblotting with
anti-MUC1 antibody of proteins extracted from BCG-hIL2MUC1 revealed a unique
protein of approximately 87 kDa corresponding to the truncated form of MUC1 protein
(Lane F). Controls for this experiment included proteins extracted from ZR75-1 and E.
coli-phIL2MUC1 (Lanes A and C, respectively) and E. coli-phIL2MUC1, BCGPMOD12, and BCG-hIL2 (Lanes B, D, and E), which served as the positive and negative
controls, respectively.
Tumor Kinetics of ZR75-1 Cells in hu-PBL-SCID Mice. Before
embarking on our vaccine experiments, it was important to determine
the growth kinetics of ZR75-1 cancer cells in our animal model. As
described earlier, SCID mice were reconstituted with 50 ⫻ 106 human
PBLs on day 0 to create a xenograft of a human immune system
(hu-PBL-SCID). One day after lymphocyte reconstitution of the huPBL-SCID mice, varying concentrations of exponentially growing
ZR-75-1 cells were injected s.c. into the right flanks of these mice.
Sham inoculation of the contralateral flank with PBS was performed.
There were three groups with 4 mice/group. Group A received
1 ⫻ 106 ZR75-1 cells, group B received 2 ⫻ 106 ZR75-1 cells, and
group C received 4 ⫻ 106 ZR75-1 cells. The mice were observed until
death or 150 days, whichever was later. Outcomes of interest were
time to tumor detection, size of primary tumor, and the rate and
pattern of metastatic disease. At the time of necropsy, primary tissue,
liver, lung, and abnormal masses were harvested for histological
analysis. All animals developed a gross primary tumor with “the mean
time to gross tumor detection” inversely proportional to tumor inoculum. Fifty percent of mice developed metastatic breast cancer irrespective of tumor inoculum. One animal (in group C) developed a
lymphoma and died on day 133. The results indicated that 4 ⫻ 106
ZR75-1 cells was the suitable concentration for the aggressive tumor
model described in this study.
Overall Survival of Vaccinated Animals. The overall survival of
the animals was evaluated to determine the toxicity of the various
vaccines as well as death from breast cancer. All of the vaccines (see
details in “Materials and Methods”) were well tolerated by the animals with the exception of BCG-261. Nine weeks after receiving their
first immunization, and 3 weeks after tumor engraftment, 75% of the
hu-PBL-SCID mice that had been vaccinated with BCG-261 had died.
The cause of death was not breast cancer because it was observed in
animals who had received the vaccine but were never xenografted
with breast cancer. The two remaining mice who had received BCG261 vaccine subsequently died at days 52 and 57 after tumor engraftment. These results indicated that BCG-261 alone, at the concentration used, was lethal to the hu-PBL-SCID mice. Necropsy of these
animals did not reveal an obvious cause of death. However, it is
important to point out that coexpression of hIL-2 or the truncated form
of MUC1 had protected the hu-PBL-SCID mice from the toxic effects
of the same dose of BCG.
All of the animals who received BCG-hIL2MUC1 vaccine survived
until termination of the experiment, with the exception of three mice
in the group sacrificed at the 10-week interval. All of the animals in
the MUC1 peptide vaccine group had died by 16 weeks after tumor
engraftment; one was sacrificed at the 10-week interval. Animals with
sham inoculation (PBS) had died by week 9 after tumor engraftment.
The cause of death in the MUC1 peptide vaccine- and sham-inoculated animals was similar. These animals developed large intraabdominal masses and were lethargic and preterminal within a week
of detection of these masses. The etiology of these masses will be
discussed later.
Time to Tumor Detection and Tumor Growth After Immunization. hu-PBL-SCID xenografted with human breast cancer cells
were examined for the development of primary tumors. Mice with the
MUC1 synthetic peptide-based or sham immunization developed a
primary tumor with a median time to tumor detection of 34 and 32
days, respectively. Most of the animals receiving BCG-hIL2MUC1
vaccine developed a gross tumor at the site of injection of the breast
cancer cells. The median time for gross tumor detection in this group
of mice was 43 days. As mentioned earlier, only 2 mice in the
BCG-261 group survived beyond week 6 after tumor engraftment. In
all of the surviving BCG-261-immunized mice, a primary tumor was
detected with a median time to tumor detection of 33 days. The delay
in median time to tumor detection in the group of mice immunized
with BCG-hIL2MUC1 was different from that of the other groups and
suggested that the experimental vaccine, BCG-hIL2MUC1, was capable of suppressing the growth of breast cancer cells.
The primary tumors detected in the animals were measured and
plotted over time (Fig. 3). The size of the primary tumors in the
control animals (PBS, MUC1 synthetic peptide, and BCG-261)
showed a continual increase in size with time. The curves for these
three groups of animals were essentially identical. However, the rate
of tumor growth in the animals immunized with BCG-hIL2MUC1
was much slower than that observed in the control groups. In these
animals, the mean tumor size, stratified by treatment group and time
after tumor inoculation, was significantly smaller (polynomial regression analysis, P ⬍ 0.001). The rate of tumor growth in BCGhIL2MUC1-immunized animals was also significantly slower than
that observed in the control animals (P ⬍ 0.01). These results suggest
that the control vaccines, i.e., MUC1 peptide or BCG-261, exhibited
no inhibitory effect on the growth of the primary tumor. However,
BCG-hIL2MUC1 vaccination inhibited the rate of tumor growth.
Histological Evaluation of Primary Breast Cancer Grown in
hu-PBL-SCID Mice. The primary breast cancers detected in our
mice were examined with standard histological techniques and immunohistology. In the groups of mice vaccinated with MUC1 synthetic peptide, BCG-261, or PBS, the primary breast cancers detected
in the hu-PBL-SCID mice had the same histological characteristics as
human breast cancers (Fig. 4). The breast cancer cells formed ductallike structures and had a Bloom-Richardson appearance with a nuclear
grade of 2. The breast cancer cells were moderately differentiated.
The primary breast tumors from mice that had received BCGhIL2MUC1 were of a similar morphology as that detected in the
control groups (Fig. 4E).
All of the primary tumors were stained for MUC1 protein. The
primary tumors in MUC1 synthetic peptide-, BCG-261-, and shaminoculated animals were strongly positive for MUC1 protein. A representative case is shown in Fig. 4. The MUC1 protein was present on
the cell surface of virtually all breast cancer cells. There was no
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DEVELOPMENT AND PRECLINICAL EVALUATION OF BCG-hIL2MUC1
Fig. 3. Mean tumor size after tumor engraftment in animals immunized with sham (PBS), BCG-261, MUC1 peptide, or BCG-hIL2MUC1.
Induction of Cell-mediated Immunity by BCG-hIL2MUC1. It
was not possible to obtain viable human lymphocytes for evaluation
of anti-MUC1 cell-mediated cytotoxicity in hu-PBL-SCID mice. Alternatively, immunohistochemistry of primary tumors for CD8-positive human lymphocytes was performed. As illustrated in Fig. 5D,
only tumors obtained from animals immunized with BCG-hIL2MUC1
had any detectable infiltration of CD8-positive cells. There were no
detectable CD8-positive cells in tumors obtained from mice immunized with BCG-hIL2, with or without MUC1 peptide, or shamimmunized animals (Fig. 5).
In Vivo BCG Retention of phIL2MUC1. One of the unexpected
results from this study was the observed lethality of BCG-261 in
hu-PBL-SCID mice, questioning the potential safety of BCGhIL2MUC1 in humans and the length of time that the plasmid
phIL2MUC1 was retained by BCG. Spleens obtained from mice
immunized with BCG-hIL2MUC1 were stained for the presence of
BCG and MUC1 protein. The antibody used in our study does not
react with mouse MUC1, and therefore any MUC1 detected must be
from BCG-hIL2MUC1. As illustrated in Fig. 6, A and B, spleens from
BCG-hIL2- and BCG-hIL2MUC1-treated animals were strongly positive for mycobacteria. However, only spleens from BCGhIL2MUC1-immunized animals expressed MUC1 protein 168 days
after inoculation (Fig. 6D). This result suggested that BCG retained
phIL2MUC1 for a significant time in vivo without any selectable
pressure.
Fig. 4. Histology and MUC1 expression in primary breast tumors. Histology and
MUC1 expression of primary breast tumors derived from mice immunized with PBS (A),
MUC1 peptide (B), BCG-hIL2 (C), BCG-hIL2 and MUC1 peptide (D), and BCGhIL2MUC1 (E). The histological pattern was consistent with a moderately differentiated
breast carcinoma. Immunostaining with MUC1 antibody showed abundant cell surface
expression in primary tumors derived from control animals (A–D). There was markedly
decreased MUC1 expression in tumors grown in mice immunized with BCG-hIL2MUC1
(E). F consists of tumors grown in BCG-hIL2MUC1-immunized animals stained with
control mouse IgG1 antibody (negative control).
MUC1 staining observed on the surrounding stromal cells or in the
negative control (Fig. 4F). In addition, a satellite deposit detected in
a MUC1 peptide-immunized animal was also strongly positive for
MUC1 protein (data not shown). The MUC1 staining pattern detected
in mice receiving BCG-hIL2MUC1 vaccine was different from that
detected in the control animals. Although many of the breast cancer
cells maintained their cell surface expression of MUC1 protein, a
significant proportion of the breast cancer cells were negative for
specific MUC1 staining (Fig. 4E). These cells maintained the histological characteristics of breast cancer cells. This observation suggested that our vaccine, BCG-hIL2MUC1, inhibited the growth of
MUC1-positive breast cancer cells, resulting in the maintenance of
probably less aggressive MUC1-negative breast cancer cells.
Fig. 5. Infiltration of primary tumors with CD8-positive lymphocytes. Primary tumors
obtained from mice immunized with PBS (A), BCG-hIL2 (B), BCG-hIL2 plus MUC1
peptide (C), and BCG-hIL2MUC1 (D) were stained for the presence of CD8-positive
tumor-infiltrating lymphocytes. Tumor-infiltrating C8-positive lymphocytes could be
detected only in tumors obtained from animals vaccinated with BCG-hIL2MUC1.
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DEVELOPMENT AND PRECLINICAL EVALUATION OF BCG-hIL2MUC1
Etiology of Intra-abdominal Masses. In addition to the primary
breast tumors, mice in the PBS (62.5%) and MUC1 synthetic peptide
(50%) and BCG-hIL2MUC1 (12.5%) vaccine groups developed large
intra-abdominal masses. In our preliminary work with the tumor
kinetics of ZR75-1 growth in hu-PBL-SCID mice, similar intraabdominal masses were not detected. The intra-abdominal masses
arose from the upper abdomen and measured greater than 1 cm (Fig.
7A). The exact primary source of the masses was not clear because of
their large size but involved the mesenteric lymph nodes, peripancreatic region, and liver. In one mouse (in the PBS group), the mass arose
in the retroperitoneum and impinged on the nerves of the left hind leg.
The masses contained large areas of necrosis on gross inspection.
Histological examination of these masses showed a diffuse infiltration
of small cells that lacked an organoid pattern (Fig. 7B). This lack of
histological arrangement was different from the primary breast tumors. The histological pattern observed in these masses was that of a
lymphoma or poorly differentiated carcinoma. In addition to the cells
noted, large areas of necrosis were documented by histology. Immunohistochemistry with anti-CD45 antibody (a marker of human lymphocytes), a pan-cytokeratin antibody (a marker of epithelial cells),
and MUC1 antibody was performed. As shown in Fig. 7C, the
malignant cells were strongly positive for CD45. Only the cells within
the masses were CD45 positive (Fig. 7C, solid arrow). There was no
appreciable CD45 staining identified in the adjacent organs such as
the pancreas or liver (Fig. 7C, open arrow). The cells within these
abdominal masses were also negative for both cytokeratin (Fig. 7D)
and MUC1 staining (Fig. 7E). Specificity of the staining was confirmed by a negative control consisting of mouse-derived sera (Fig.
7F). Based on these results, the masses were compatible with a
lymphoma.
DISCUSSION
In this study, we report a series of observations that, taken together,
provide evidence for the immunopotency of a novel MUC1 vaccine
against breast cancer cells. The vaccine, BCG-hIL2MUC1, was designed with the following features in mind: (a) MUC1 and the
stimulatory cytokine IL-2 should be coexpressed; (b) MUC1 should
Fig. 6. BCG retention of phIL2MUC1 in vivo. Spleens from mice immunized with
BCG-hIL2 and BCG-hIL2MUC1 were stained for the presence of mycobacteria and
MUC1 expression. Abundant mycobacteria were detected in the spleens of BCG-hIL2
(A)- and BCG-hIL2MUC1 (B)-vaccinated animals. There was expression of MUC1 by
spleens from BCG-hIL2MUC1-immunized animals 168 days after immunization (D).
There was no detectable MUC1 expression in the spleens of BCG-hIL2-immunized
animals (C).
Fig. 7. Histology of intra-abdominal masses. A illustrates the large intra-abdominal
masses that were found in animals immunized with MUC1 peptide and PBS. These
masses appeared to arise within the mesentery and retroperitoneum. Histological examination of these masses revealed a lesion with small cells that lacked an organizational
pattern (B). Immunostaining with CD45 (C), MUC1 (D), and AE1/AE3 (E) confirmed that
the masses were compatible with a human lymphoma. Specificity of CD45 staining was
confirmed by IgG1 negative control antibody (F) and the lack of CD45 staining of
adjacent organs such as the liver (C, open arrow). In all of the figures, the solid arrow
points to the lymphoma, and the open arrow points to adjacent liver.
be expressed as a nonglycosylated protein; and (c) BCG should be
used in vivo as an adaptive immune stimulator. It is demonstrated here
that a BCG-derived vehicle vector simultaneously expressed two
recombinant proteins. The MUC1 protein that was expressed by the
vaccine vector contained 22 tandem repeats that gave rise to a single
protein of 87 kDa. The expression of MUC1 as a single native peptide
is likely to be advantageous for its use as a breast cancer vaccine.
Because proteins are not N-glycosylated in BCG, the recombinant
MUC1 protein expressed by BCG-hIL2MUC1 was expressed in the
malignant form. This is different from the MUC1 protein expressed by
viral vectors; in these circumstances, MUC1 is usually heterogeneously glycosylated (17, 25). IL-2 detected in our studies was clearly
derived from the BCG construct because it was also detected using an
antibody against HA, the epitope placed to tag the recombinant
protein.
Analysis of the ability of BCG-hIL2MUC1 to prevent and/or inhibit
the growth of xenografted human breast cancer cells in hu-PBL-SCID
mouse model reveals that human breast cancer cells were capable of
growing a primary tumor. There was a high rate of tumor engraftment
observed in all animals, as observed by others (26). The tumors in
BCG-hIL2MUC1-immunized mice grew significantly more slowly
than the tumors observed in animals immunized with PBS, BCG-261,
or MUC1 peptide. The size of the tumor detected in the current
protocol was approximately 1.0 cm, which was much smaller than that
reported from other studies involving SCID mice (27, 28). This
difference in maximal tumor size may have been due to the presence
of the reconstituted human immune system in hu-PBL-SCID mice
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DEVELOPMENT AND PRECLINICAL EVALUATION OF BCG-hIL2MUC1
because a smaller tumor size has been reported in SCID mice reconstituted with human lymphocytes and grafted with human tumors (29).
It has been observed that human cancers, after transplantation into
SCID mice, maintain the histological features of the donor (30). Thus,
the primary tumors were stained for MUC1 expression to determine
whether our vaccine had selected for MUC1 negative cells. Although
some heterogeneity in MUC1 expression has been reported in SCID
mice (31, 32), virtually all of the breast cancer cells in the primary
neoplasm stained positive for MUC1 protein in the hu-PBL-SCID
mice receiving MUC1 peptide, BCG-261, or sham vaccine. The
staining pattern in the hu-PBL-SCID mice vaccinated with BCGhIL2MUC1 was different from that of the control animals. A substantial proportion of the cancer cells in these primary neoplasms did not
stain for MUC1 protein. Furthermore, the presence of CD8-positive
cells in the primary tumor was detected only in animals immunized
with BCG-hIL2MUC1. These observations suggested that BCGhIL2MUC1 had stimulated a cell-mediated immune response resulting in the selective deletion of MUC1-positive cells and are supported
by recent findings. It has been shown that a single transfer of restimulated bone marrow cells from breast cancer patients caused regression of xenografted autologous tumor in NOD/SCID mice (14). Such
a phenomenon has also been observed in MUC1 transgenic mice,
in which MUC1-specific cytotoxic T cells, when adoptively transferred, were effective in controlling mammary gland and melanoma
tumors (15).
When the survival trends were compared in the vaccine groups, it
appeared that control BCG-261 was lethal to SCID mice. These mice
died within 9 weeks after lymphocyte reconstitution, with some dying
before engraftment of the human cancer cells. This fatal outcome with
BCG-261 was not expected from our prior studies; however, those
experiments were completed within 6 weeks. The cause of death
remains unclear but was not due to disseminated breast cancer. Even
though our vaccine was BCG-based, a similar fatal outcome was not
observed in animals that had received BCG-hIL2MUC1. A more rapid
intrinsic growth of BCG-261 in animals is unlikely because both
BCG-261 and BCG-hIL2MUC1 have similar doubling rates in vitro
(data not shown). It is feasible that the presence of either IL-2 or
MUC1 protein protected the animals from the toxic effects of BCG
(33). If BCG were lethal, then the length of time that BCG retained
phIL2MUC1 is important. Spleens obtained from BCG-hIL2MUC1immunized animals and stained for MUC1 protein were positive for
MUC1 protein. Therefore, despite no in vivo selective pressure, our
data suggest that the plasmid hIL2MUC1 is retained by the BCG for
at least 168 days. Similar results reported by Luo et al. (34) indicate
that the recombinant BCG is stable in vivo for up to 16 weeks. An
explanation for the lethality of BCG-261 observed in our model is the
use of SCID mice reconstituted with a limited supply of human
peripheral lymphocytes. In the absence of IL-2, this small compartment of T cells cannot control the BCG infection. It would be
expected that in a competent animal, the lethality of BCG-261 would
not be observed. Competent mice have been vaccinated with BCG261 with no morbidity. Duda et al. (35) reported no morbidity or
lethality in C57BL/6 mice immunized with BCG-261 after a total dose
of 3 ⫻ 106 cfu administered intratumorally. Taken together, these
results suggest that BCG-hIL2MUC1 is a safe vaccine.
The majority of hu-PBL-SCID mice in the sham-inoculated and
MUC1 peptide-vaccinated groups developed large intra-abdominal or
retroperitoneal masses. Only one mouse in the BCG-hIL2MUC1immunized group developed a similar mass. Few mice immunized
with BCG-hIL2 or BCG-hIL2MUC1 developed these masses. Histological examination of these masses indicated that they had different
histological characteristics than those observed in the primary tumors,
appeared to arise from nodal tissue, and were consistent with a
lymphoma by immunostaining. The development of lymphoma in
hu-PBL-SCID mice has been reported previously and is not a unique
phenomena (36). Lymphomas that arise in hu-PBL-SCID mice are
derived from B cells, and virtually all are EBV positive (37). Most
adult humans are EBV seropositive, and therefore the source of the
lymphomas observed in our animal model was most likely secondary
to the use of EBV-positive lymphocytes for reconstitution. However,
the incidence of lymphoma development and the rate of growth were
delayed in the BCG-hIL2MUC1-immunized animals. It has been
reported that in hu-PBL-SCID mice, T cells are critical for control of
EBV infection (38). In hu-PBL-SCID mice, the administration of low
doses of human IL-2 may abolish the development of lymphoproliferative disease in these animals (33). Therefore, it is feasible that
animals vaccinated with BCG-hIL2MUC1 were protected from lymphoma development by IL-2 secreted by the recombinant vaccine. Our
data thus warrant the use of EBV-negative human PBLs in SCID mice
reconstitution experiments.
In summary, we have constructed a novel BCG-based MUC1
vaccine for the treatment and prevention of breast cancer. This is the
first report of a BCG-based vaccine engineered to express a tumor
antigen (in this case, MUC1). Our vaccine, BCG-hIL2MUC1, inhibited the growth of breast cancer in hu-PBL-SCID mice and, in a
minority of animals, even prevented primary tumor development.
Future experiments are planned to evaluate the efficacy of this vaccine
in the adjuvant treatment of breast cancer and the immune mechanisms involved in tumor immunity.
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Development and Preclinical Evaluation of a Bacillus
Calmette-Guérin-MUC1-based Novel Breast Cancer Vaccine
Maureen A. Chung, Yi Luo, Michael O'Donnell, et al.
Cancer Res 2003;63:1280-1287.
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