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L-BLP25 Vaccine plus Letrozole Induces a TH1 Immune Response and has Additive
Antitumor Activity in MUC-1 Expressing Mammary Tumors in Mice
Neelima R. Mehta, 1 Gregory T. Wurz, 1 Rebekah A. Burich, 1
Brittany E. Greenberg, 1 Stephen Griffey, 2 Audrey Gutierrez 1
Katie E. Bell, 3 Jamie L. McCall1, Michael Wolf, 4 and Michael DeGregorio,1†
1Department of Internal Medicine, Division of Hematology and Oncology, University of
California, Davis. 2Comparative Pathology Laboratory, UC Davis School of Veterinary
Medicine, 3Center of Comparative Medicine, University of California, Davis, and 4Merck
Serono Research, Merck KGaA, Darmstadt, Germany.
Running title: L-BLP25/letrozole combination for breast cancer Key Words: L-BLP25, breast cancer, aromatase inhibitor, immunotherapy Grant support: This work was supported by a grant from Merck-KGaA, Darmstadt, Germany. †Corresponding Author: Michael DeGregorio, Professor of Medicine, Department of Internal
Medicine, Division of Hematology and Oncology, Cancer Center, University of California,
Davis, 4501 X Street Suite 3016, Sacramento, CA 95817, Tel: 916-734-2360, Fax: 916-734-
2070, e-mail: [email protected]
Conflict of Interest: The authors declare no competing interest
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Translation Relevance
At present tamoxifen and raloxifene are the only hormonal agents approved for reducing the risk
of breast cancer. Unfortunately, these agents are only effective in women at risk of developing
hormone dependent cancer. Patients that develop hormone-independent and/or triple negative
(ER-, Her2/neu-, PR-) breast cancer have limited therapeutic options. A new therapeutic
direction is the development of an immunotherapy that targets a tumor associated antigen
overexpressed in breast cancer, such as mucin1 (MUC1). Several vaccines such as L-BLP25 that
target MUC1 are under development for a variety of epithelial cancers, such as lung, breast and
pancreatic. In the present study, we examine the immunomodulatory effects and antitumor
activity of L-BLP25 when combined with tamoxifen or letrozole in the hMUC1-expressing
mammary tumor (MMT) mouse model. The results of the present study support clinical
development of L-BLP25 immunotherapy combined with hormonal therapy as a new treatment
and prevention strategy for breast cancer.
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Abstract
Purpose: In the present study, we examine the immunomodulatory effects and antitumor
activity of tamoxifen and letrozole when combined with the human epithelial mucin (hMUC1)
specific vaccine, L-BLP25, in the hMUC1-expressing mammary tumor (MMT) mouse model.
Experimental Design: Dose-finding studies were conducted for both tamoxifen and letrozole.
Letrozole and L-BLP25 combination studies used 69 MMT female mice assigned to five groups:
untreated, cyclophosphamide + placebo, cyclophosphamide + L-BLP25, letrozole 0.8 mg/kg and
cyclophosphamide + L-BLP25 + letrozole. Tamoxifen and L-BLP25 combination studies used
48 MMT female mice assigned to five treatment groups: untreated, cyclophosphamide + placebo,
cyclophosphamide + L-BLP25, tamoxifen 50 mg/kg and cyclophosphamide + L-BLP25 +
tamoxifen 50 mg/kg group. Mice were injected subcutaneously with L-BLP25 (10 µg) weekly
for eight weeks. Serum cytokines were serially measured using a Luminex assay while
splenocytes at termination were analyzed by ELISpot to determine Th1/Th2 polarization of
immune response.
Results: Daily oral doses of 50 and 0.8 mg/kg of tamoxifen and letrozole, respectively, resulted
in a significant survival advantage over controls (p<0.05). A predominant Th1 polarized immune
response in vaccinated mice was seen with or without tamoxifen or letrozole treatments. In the
L-BLP25 plus letrozole treatment group, statistically significant (p<0.05) additive antitumor
activity was observed, whereas tamoxifen plus L-BLP25 was not significantly different (p>0.05).
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Conclusion: The results of the present study demonstrate that hormonal therapy does not
interfere with L-BLP25 induced predominant Th1 response, and the combination of L-BLP25
with letrozole has additive antitumor activity in the MMT mouse model.
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Breast cancer is one of the leading causes of cancer death in women and accounts for one-third
of all cancer diagnoses worldwide (1). The majority of breast cancer patients will develop
estrogen receptor positive tumors and will receive a hormonal agent during the course of their
treatment. Unfortunately, a subset of patients will develop hormone-independent and/or triple
negative (ER-, Her2/neu-, PR-) breast cancer (2). For these patients with limited treatment
options it is important to consider a new immunotherapy that targets a tumor associated antigen
(TAA) that is expressed on tumors irrespective of hormone status.
MUC1 or Mucin1 glycoprotein is a TAA overexpressed in greater than 90% of human breast
cancers (3). Although MUC1 is a molecule physiologically expressed on a variety of mammalian
cells, its expression is significantly altered in tumorigenesis. Overall expression of MUC1
increases on cancer cells and it is underglycosylated revealing variable number of tandem repeat
regions (VNTR) on a peptide backbone. T cells specific for antigenic epitopes of MUC1 that
bind to HLA class I molecules have been identified and isolated from the blood and bone
marrow of breast cancer and myeloma patients, making MUC1 a potential target for a cellular
immune response (4-10). In addition, the immunodominant peptides from the VNTR region are
recognized by cytotoxic T-lymphocytes (CTL) that are thought to destroy tumor cells expressing
the underglycosylated form of MUC1 (11-14).
L-BLP25, a liposomal vaccine, consists of immunodominant peptides from the VNTR region of
MUC1 (13, 15). This vaccine generates CTLs capable of destroying MUC1-expressing tumor
cells and produces Th1 polarized cytokine responses (16, 17). L-BLP25 liposomal vaccine (L-
BLP25, Merck KGaA, Darmstadt, Germany) is in Phase III clinical development for non-small
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cell lung cancer (NSCLC). L-BLP25 in NSCLC demonstrated increased survival rates among
vaccinated patients (18). In addition to NSCLC, clinical studies evaluating the benefits of L-
BLP25 immunotherapy combined with breast cancer hormonal therapies such as tamoxifen or
aromatase inhibitors (e.g. letrozole, anastrozole) are being considered.
Babina et.al. reported that tamoxifen is capable of inducing a shift from a cellular (Th1) to a
humoral (Th2) immunity (19, 20). Tamoxifen also induces latent TGFβ in breast cancer cell
lines (21), and prevents maturation of antigen presenting cells (22). TGFβ can inhibit CTLs and
induce regulatory T-cells (T-regs), creating an immunosuppressive tumor microenvironment
(23). The aromatase inhibitor anastrozole increased levels of proinflammatory cytokines IFN-γ
and IL-12 (Th1), and decreased levels of IL-4 and IL-10 (Th2) cytokines in an experimental
polyarthritis study. Anastrozole also suppressed the differentiation of naïve T-cells into T-regs,
which are known to produce immunosuppressive cytokines in the tumor microenvironment (24,
25). These observations make it necessary to investigate the immunomodulatory properties of the
commonly used hormonal therapies on the L-BLP25-induced immune response.
Materials and Methods
Chemicals. Tamoxifen citrate was a gift from the Orion Corporation, Orion-Pharma (Espoo,
Finland). Letrozole was purchased from Toronto Research Chemicals, Inc. (North York, Ont.,
Canada). Carvedilol, used as the internal standard for letrozole bioanalysis, was purchased from
Tocris Bioscience (Ellisville, MO, USA). Human MUC1 peptide (BP25)
STAPPAHGVTSAPDTRPAPGSTAPP, scrambled human MUC1 peptide (BP l-424), L-BLP25
lyophilisate vaccine and L-BLP25 placebo were provided by Merck KGaA (Darmstadt,
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Germany). 17β-estradiol and cyclophosphamide (CTX) was obtained through Sigma Chemical
Company (St. Louis, MO, USA).
Animals. The hMUC1-expressing mammary tumor (MMT) mouse model was developed by
Gendler et al (26). Breeding pairs (MTag.Tg; males and MUC1.Tg; females) were purchase from
Mayo Clinic (26). Following breeding protocols, 267 female MMT mice were supplied for these
studies by the UC Davis Mouse Biology Program housed at the UC Davis Center for Laboratory
Animal Science vivarium. All animal studies were conducted under a protocol approved by the
University of California Davis Institutional Animal Care and Use Administrative Advisory
Committee. UC Davis is an Association for Assessment and Accreditation of Laboratory Animal
Care accredited institution.
Genotype Screening. MTag forward and reverse primers were 5'-TTGGAGAATGTTTTT-
GTCTTGAATG and 3'-CAGCACATCTCGG-GTTGGT. The MTag TaqMan probe (ACATG-
CAATGGTTTGGAA) carrying a 6´ FAM reporter label and a 3´ MGBNFQ quencher group was
used. For MUC1, forward and reverse primers were 5´-CACTCTTCCCCCAACCTTAAGTG
and 3´-GGGTGGGTGGTGGTCATG. The MUC1 TaqMan probe (ACCAGTCCCTCCC-
TACG) carrying a 5´ VIC reporter label and a 3´ MGBNFQ was used. The PCR amplification
program consisted of one cycle of 2 minutes at 95ºC and 40 cycles of 15 seconds each at 60ºC
and 95ºC.
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Dose-Finding Studies
Tamoxifen treatment: A total of 22 MMT female mice (4-5 weeks old) were weighed and
randomly assigned to cages such that the average age and weight in each treatment arm was
approximately equal. There were a total of four treatment groups: control (N=7) and tamoxifen
1.0, 25, and 50 mg/kg (N=5 at each dose).
Letrozole treatment: A total of 52 MMT female mice were weighed and randomly assigned to
cages in the four treatment arms as described above. The treatment groups consisted of: control
and letrozole 0.08, 0.8, and 8.0 mg/kg (N=13, all groups).
Study Design
The drugs were dissolved in DMSO and then diluted in peanut oil such that 100 µl delivered the
appropriate doses. The final concentration of DMSO in all solutions was 2.0%. Control mice
received a solution of peanut oil with 2% DMSO. All mice were dosed daily by oral gavage
using a stainless steel, 20-gauge gavage needle (Popper & Sons, Inc., NY, USA). All mice were
weighed and palpated for the presence of new mammary tumors once a week. When possible,
tumors were measured in three dimensions using calipers, and an approximate volume was
calculated using the formula v=l×w×h, where v= volume, w= width, l= length, and h= height of
the tumor. Once the tumor(s) reached the cumulative volume of 1.5 cm3, or was abscessed or
visibly impairing the mobility of the mouse, the mice were euthanized by CO2 asphyxiation.
Whole blood was collected by cardiac puncture and placed in heparinized tubes. The blood was
centrifuged to collect plasma for HPLC analysis. Following blood collection, the following
tissues were harvested for HPLC analysis: liver, tumor, and spleen. Mice were gavaged daily
with tamoxifen, letrozole or control. Tumor incidence and histology were then compared among
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treatment groups. The estrogen receptor (ER) status of the tumors was determined, as SERMs
and aromatase inhibitors have only been shown to be effective in ER-positive tumors. Tissue and
plasma concentrations of tamoxifen and letrozole were assessed using high performance liquid
chromatography (HPLC)(27-29). The lowest dose of each agent that provided a statistically
significant survival advantage compared to control was used in later studies.
Immune Phenotype in Immunized Female MMT Mice. This study was designed to examine
the impact of immunization on Th1 or Th2 cytokine polarization in female MMT mice. A total
of 16 female MMT mice were divided into four treatment groups: untreated, cyclophosphamide
only, placebo and vaccine (N=4, all groups). To reduce T-suppressor lymphocyte activity, all
mice except those in the untreated group received a single intraperitoneal (i.p.) injection of
cyclophosphamide (100 mg/kg) three days before beginning the L-BLP25 vaccine regimen (30-
33). Mice in the vaccine group were then subcutaneously (s.c.) injected with 10 µg of L-BLP25
(17) in 100 µl using a 25-gauge needle once each week for eight weeks. Placebo mice were
injected with 100 µl of the empty liposomes. Following the 4th, 6th, and 8th dose of vaccine or
placebo, blood was collected via submandibular bleeds. Blood was pooled within a treatment
group and serum was isolated for cytokine analysis.
Effects of Hormonal Therapy on L-BLP25 Immune Response.
A total of 60 MMT female mice (approximately 4 weeks old) were weighed and assigned to
cages. On study day 119, mice were assigned to four treatment arms with approximately equal
average weights and tumor volumes: control, tamoxifen 50 mg/kg, letrozole 0.8 mg/kg and
estradiol 0.5 mg/kg (N=15, all groups). Control mice received peanut oil containing 2% DMSO.
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All mice were dosed daily and monitored weekly for cumulative tumor volume. On the 7th day
of drug treatment, seven mice from each treatment group with the greatest tumor burdens were
euthanized.
The remaining 32 mice in this study were: control, tamoxifen 50 mg/kg, letrozole 0.8 mg/kg,
and estradiol 0.5 mg/kg (N=8, all groups). All mice were given a single i.p. injection of
cyclophosphamide (100 mg/kg). Daily oral dosing was continued until the conclusion of the
study. Three days after cyclophosphamide treatment, one mouse from each treatment group was
removed and placed in a separate cage. These mice were not part of the vaccine treatment. The
other 28 mice were injected s.c. with 100 µl of L-BLP25 (10 µg) weekly until the mice reached
maximum tumor burden. Approximately 24 hours after the 3rd and 4th dose of vaccine, whole
blood was collected via submandibular bleeds. Whole blood was pooled within a treatment group
and serum was isolated for cytokine analysis. Mice achieved maximum tumor burden after the
6th dose of vaccine and were euthanized. Serum and splenocytes were collected for cytokine
analysis.
Effects of Combining L-BLP25 with Hormonal Therapy on Overall Survival.
Letrozole and L-BLP25 combination studies used 69 MMT female mice assigned to five groups
(N= 13-14): untreated, cyclophosphamide + placebo, cyclophosphamide + L-BLP25, letrozole
0.8 mg/kg and cyclophosphamide + L-BLP25 + letrozole. Tamoxifen and L-BLP25
combination studies used 48 MMT female mice assigned to five treatment groups (N= 9-10):
untreated, cyclophosphamide + placebo, cyclophosphamide + L-BLP25, tamoxifen 50 mg/kg
and cyclophosphamide + L-BLP25 + tamoxifen 50 mg/kg group. All mice were dosed by oral
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gavage on a daily basis with tamoxifen 50 mg/kg, letrozole 0.8 mg/kg, or control according to
treatment group assignment starting from the age of four weeks. All mice except for those in
untreated groups were given a single i.p. injection of CTX (100 mg/kg). Three days after CTX
treatment, mice were injected s.c. with 100 µl (delivering 10 µg) of L-BLP25 or placebo
according to treatment group once each week for eight weeks. Daily oral dosing was continued
until the conclusion of the study. The tamoxifen combination study was terminated on day 135
after approximately 70% of untreated mice had reached maximum tumor burden. The letrozole
combination study was first analyzed on study day 116, when 85% of the untreated mice had
reached maximum tumor burden. The letrozole combination study was terminated on study day
137, when 85% of mice in the CTX+vaccine and CTX+placebo groups had reached maximum
tumor burden. Serum and splenocytes were collected for cytokine analysis.
Immunohistochemistry (IHC). The breast tumors, left kidney and spleen were harvested from
each mouse at the time of sacrifice. Tissues were then paraffin embedded and step-sectioned at 4
µm for immunohistochemical analysis. IHC was performed using a MUC1 antibody (CD227,
550486; 1:400; BD Pharmingen). ER-α staining was performed using a 1:1200 diluted rabbit
polyclonal primary antibody (MC-20, Santa Cruz).
Western Blotting Analysis. The following antibodies were used to assess protein expression:
ER-α (E115; Novus Biologicals NB110-56961 lot#YF112101C, 1:1500), ER-β (Abcam, ab3576,
lot#GR14900-1, 1:1500) and β-actin (AC-15; Sigma A5441, 1:50,000). All images were
obtained on a FluorChem E System with AlphaView (SA) software, version 3.2.2 (Cell
Biosciences, Santa Clara, CA.).
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Luminex. We used the mouse cytokine 10-plex panel (Invitrogen, Carlsbad, CA) to evaluate the
cytokine levels in collected mouse serum. The cytokines assessed consisted of interleukin (IL)-
1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IFN-γ, GM-CSF, and TNF-α. The assay was performed
according to manufacturer’s protocol. Cytokine concentrations were acquired on a BioPlex
System using BioPlex software version 5.0 (BioRad, Hercules, CA, USA).
ELISpot Assay. Splenocytes from vaccinated and unvaccinated mice were examined for
Th1/Th2 polarization by analyzing two key cytokines: IFN-γ for Th1 and IL-4 for Th2
polarization. BD™ ELISPOT mouse IFN-γ (BD, San Jose, CA, USA) and dual color ELISpot
mouse IFN-γ/IL-4 (R&D systems, MN., USA) kits were used for this assay. The BP25 peptide
and scrambled peptide (BP 1-424) were prepared at a final concentration of 500 ng/ml in culture
medium. The lymphocytes were incubated with either no peptide (medium only), BP25, or
scrambled peptide at 37°C overnight.
Cytotoxic T Lymphocyte (CTL) Assay. From an ongoing study we took female MMT mice
belonging to three treatment groups: untreated, vaccine and placebo. All mice in the vaccine and
placebo groups had received a single i.p. injection of CTX (100 mg/kg) three days before
beginning the L-BLP25 vaccine regimen. One day after the 5th weekly dose of vaccine/placebo
was administered the mice were euthanized and the spleens were collected for cytotoxic T
lymphocyte assay. For effector cells: Spleens were harvested aseptically from mice given L-
BLP25 or placebo and ground through 100-mesh sterile sieves into 5 mL of 1x PBS. For
separation of CD8-positive T-Cells, Isolation Kit II (Miltenyi Biotec, cat # 130-095-236,
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Auburn, CA, USA) was used. Cell concentration was adjusted to 1 x 107 cells/ml. For target
cells: hMUC1-expressing murine breast cancer cells were derived from MMT tumors. Cells were
resuspended in PanToxiLux wash buffer (OncoImmunin Inc., cat# PTL 802-8, Gaithersburg,
MD, USA), at a final concentration of 2x106 cells/ml. Flow cytometry was performed on an
LSRFortessa™ (BD Biosciences, San Jose, CA, USA) and data was collected using BD
FACSDIVA software. Data was analyzed using FCS Express 4 (De Novo Software, Los
Angeles, CA, USA) software.
Statistical Analysis. Overall survival was compared graphically for different treatment groups
using Kaplan–Meier estimates. Kaplan-Meier survival curves and log-rank tests for significance
were generated using GraphPad Prism® 5 software. Bonferroni's adjustment for multiple tests
was used to lessen the likelihood of a false positive result. For the ELISPOT assay, spot forming
cells (SFC) are presented as the mean value of triplicate wells. The differences in SFC levels
were analyzed by two-way ANOVA.
Results
MMT Tumors Respond to SERM/AI Treatment
To determine the most effective doses of tamoxifen and letrozole in the MMT mouse model, we
conducted a series of dose-finding studies. In the tamoxifen dose-finding study, animals in the
highest dose group (50 mg/kg) showed a significant survival advantage over controls (p=0.0210)
(Fig 1A). For the letrozole dose-finding study, both the 0.8-mg/kg and 8.0-mg/kg treatment
groups were associated with significantly improved survival versus the control group,
(p=0.0085) and (p=0.0052), respectively (Fig 1B). No survival advantage was seen with 8 mg/kg
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versus 0.8 mg/kg. The bioavailabilty of tamoxifen and letrozole was verified following oral
dosing using HPLC bioanalytical methods (data not shown).
Estrogen Receptor Status
In order to examine the ER status and potential antiestrogen sensitivity of the MMT tumors, mice
were allowed to develop tumors, which were then isolated for Western blotting. ER-α and
hMUC1 expression in an MMT tumor was confirmed by IHC (Supplementary Fig. 1). Using
selected samples from various studies, we confirmed that ERα and hMUC1 expression were
retained. A cell line established from an MMT tumor (MMT-494) shows preservation of the
same protein expression patterns of ER-α and ER-β in vitro (Fig 1C).
L-BLP25 Induces Predominant Th1 Cytokine Response
Noticeable differences were observed in cytokine levels of vaccinated versus untreated,
cyclophosphamide, and placebo-treated mice. The levels of IFN-γ were greater in vaccinated
mice in comparison to placebo treated mice at 24 hours following the 4th, 6th and 8th doses of
vaccine (Fig. 2). Levels of IL-4 in vaccinated mice were not different at any time point. The
levels of IL-6 were elevated in vaccinated mice in comparison to placebo-treated mice. While
levels of TNF-α were not elevated in all vaccinated mice, a trend was evident. The observed
elevated levels of IL-6 and TNF-α in vaccinated mice may be related to the adjuvant
(monophosphoryl lipid A) used in the vaccine and support immunogenicity of the vaccine. In
summary, the results indicate a predominant Th1 polarization of cytokines following
vaccination.
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Hormonal Therapy does not Interfere with L-BLP25-induced Immune Response
This study was conducted to examine the impact of pretreatment with hormonal therapy on the
overall immune response induced by L-BLP25 vaccine. Serial cytokine analysis of serum
specimens following the 3rd and 4th doses of vaccine showed a clear Th1 response with
noticeably higher levels of IFN-γ in vaccinated mice from all treatment groups (Fig. 3). The
levels of IL-4 in vaccinated mice were unchanged compared to unvaccinated mice.
Terminal serum cytokines levels in each treatment group were analyzed (Fig. 4). These results
confirmed the serial cytokine response observed showing that the vaccinated mice have elevated
IFN-γ levels compared to unvaccinated mice. Serum IL-5 levels were noticeably elevated in all
vaccinated mice. There was a trend of elevated IL-6 and TNF-α levels in vaccinated mice, which
is associated with the immunogenicity of the vaccine. The IL-10 levels were unchanged in
vaccinated mice compared to non-vaccinated mice, while IL-12 levels were higher in only
vaccinated mice. Although the IL-4 and IL-5 levels were elevated in some vaccinated mice, on
balance the predominant immune response was Th1 polarized as evidenced by the IFN-γ and IL-
12 cytokine response.
This data was supported by the ELISpot analysis of splenocytes from the aseptically collected
spleens. Control vaccinated mice showed a highly significant increase in IFN-γ SFC levels
(p<0.001) in comparison to non-vaccinated control mice (Fig. 5A). The IL-4 SFC levels were not
statistically different from splenocytes stimulated with scrambled peptide (Fig. 5B).
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We then compared serum cytokine profiles following 24 hrs versus 120 hrs after vaccination for
the purpose of monitoring the polarization of cytokine response to Th1 or Th2. Only 24 hr time
points showed a Th1 response suggesting that serum cytokine profiles are not sufficient for
monitoring the immune response 120 hrs post-vaccination. However, the ELISpot assay on
splenocytes 120 hrs post-vaccination showed highly significant IFN-γ SFC levels for all
vaccinated mice (p<0.001). In comparison, the IL-4 SFC levels were not significantly different
from those of media/scrambled peptide stimulated splenocytes (p>0.05) (Fig. 5C, 5D and 5E).
These results confirm the Th1 polarization is retained 120 hrs post-vaccination. The difference
between Luminex and ELISpot is probably due to the delayed nature of splenocytes vs. transient
plasma cytokine changes.
In a subset of 32 tumor bearing mice, tamoxifen 50 mg/kg, letrozole 0.8 mg/kg, and estradiol 0.5
mg/kg (N=8, all groups) were examined for antitumor activity. Survival was compared between
vaccine treatment alone with antiestrogen/AI treated and vaccinated treatment groups. The tumor
progression rates for vaccinated mice treated with tamoxifen or letrozole were not statistically
different from the vaccine alone (log rank test: p=0.5117 and 0.886) (Supplementary Fig. 2A and
2B). Mice which were vaccinated and treated with estradiol had significantly shorter survival
compared to vaccine treatment alone (log rank test: p=0.0429) (Supplementary Fig. 2C).
Letrozole and Tamoxifen in Combination with L-BLP25
The main aim of these L-BLP25 combination studies was to determine whether combining L-
BLP25 with hormonal therapy provides any overall survival advantage over L-BLP25 or
hormonal therapy alone. These studies were designed to use untreated and CTX+placebo groups
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as controls. As in the dose-finding studies, survival data was compiled for both combination
studies to generate Kaplan-Meier survival curves.
Letrozole
Interim analysis on study day 113, as measured by mean tumor volumes for all surviving mice,
showed that treatment with L-BLP25 in combination with letrozole reduced mean tumor burden
(Fig. 6A). In the letrozole and L-BLP25 combination study on day 116, when 85% of the
untreated mice had reached maximum tumor burden, mice in the CTX+L-BLP25 and
letrozole+CTX+L-BLP25 treatment groups showed a clear and significant survival advantage
over untreated mice (log rank test: p<0.001), whereas the CTX+placebo group was not
significantly different from the untreated group (log rank test: p=0.3457). Letrozole in
combination with L-BLP25 showed significantly improved survival compared to letrozole alone
(log rank test: p=0.0405) (data not shown).
As per our design, we continued the study until 85% of mice in the CTX+placebo group were
euthanized due to excessive tumor burden, at which time (day 137) survival data was calculated
again for this study (Fig. 6B). Confirming our previous findings, animals in the CTX+L-BLP25
and letrozole+CTX+L-BLP25 treatment groups showed a clear and significant survival
advantage over the untreated group, (log rank test: p=0.0086 and p<0.0001), respectively.
Letrozole in combination with L-BLP25 showed significantly improved survival compared to the
CTX+L-BLP25 alone treatment group (log rank test: p=0.0024). Letrozole in combination with
L-BLP25 was, however, not significantly different when compared to the letrozole alone
treatment group (log rank test: p=0.0895).
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Tamoxifen
Interim analysis on study day 113, as measured by mean tumor volumes for all surviving mice,
showed that treatment with L-BLP25 in combination with tamoxifen reduced mean tumor
burden, although not to the same extent as tamoxifen monotherapy (Fig. 6C). On study day 135,
only the mice in the tamoxifen 50 mg/kg group showed a significant survival advantage over the
untreated group (log rank test: p<0.0083) (Fig. 6D). It was also noted that neither CTX+L-
BLP25 nor tamoxifen alone were significantly different from the CTX+L-BLP25 plus tamoxifen
treatment group, p=0.1229 and p=0.1451, respectively (Fig.6D). Although not significantly
different, both interim tumor analysis and overall survival data showed that combining L-BLP25
with tamoxifen does not provide any added advantage over tamoxifen monotherapy.
L-BLP25 Elicited CTL’s Specific for hMUC1
To determine whether cytotoxic T lymphocyte activity was induced, we exposed splenocytes
(effector cells) from vaccinated mice to hMUC1 expressing breast cancer cells established from
an MMT tumor (target cells). With a single-cell cytotoxicity assay kit that detects both granzyme
B and upstream caspase activation, a higher cytotoxicity was observed with effector cells (CD8+
T cells) from vaccinated mice in comparison to placebo and untreated mice (Supplementary Fig.
3). We also saw 35% cytotoxicity in CTX plus placebo treatment group. One explanation for
such a high cytotoxicity in this treatment group may be that the CTX treatment eliminates T
suppressor cells, therefore allowing tumor-sensitized T cells to attack the tumor cells to a greater
extent (34). These mice already had existing tumor burden, and the presence of MUC1 specific
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CTL’s was confirmed by the 18% cytotoxicity we saw in the untreated group. CTX followed by
the vaccine regimen augmented this existing cytotoxicity to approximately 45%.
Discussion
In the present study we investigated the immunomodulatory effect of tamoxifen and letrozole on
the L-BLP25 induced immune response in an MUC1-expressing breast cancer mouse model.
Our results showed both tamoxifen and letrozole do not interfere with the Th1 polarized cytokine
response induced by L-BLP25, although only letrozole in combination with L-BLP25 showed a
significant survival advantage. To our knowledge this is the first demonstration of a hormonal
therapy combined with a vaccine that achieved additive antitumor activity and survival benefit in
a preclinical model.
Gendler et al. first utilized the MMT model to test a vaccine formulation comprised of L-BLP25
and human recombinant IL-2 (26). In this study, MMT mice were vaccinated with liposomal-
MUC1 formulation every two weeks starting at 7 weeks of age for a total of six doses. The
immunized mice were positive for T cells that express intercellular INF-γ, were reactive with
MHC class I H-2Db/MUC1 tetramer, and were cytotoxic to MUC1-expressing tumor cells in
vitro. Immunized MMT mice had significantly lower tumor burden at 18 weeks of age compared
to controls. However, by 20-24 weeks of age, no significant difference in tumor burden was
observed between immunized and control mice (26). In a recent study by this group, MUC1.Tg
mice were immunized with a glycosylated MUC1-derived glycopeptide covalently linked to a
Toll-like Receptor (TLR) agonist (6). After 35 days, mice were transplanted with 1× 106 MMT
mammary tumor cells, followed by additional vaccination 7 days after implantation. On day 14
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post-implantation mice were euthanized and the efficacy of the vaccine was determined by tumor
wet weight. The results of this study suggest that the vaccine elicited potent antitumor response
(6).
In agreement with previous findings of Gendler et al.(26), our study demonstrated that
vaccination with L-BLP25 does not produce a durable antitumor response when administered to
mice with large tumor burdens. An increase in tumor burden is associated with increasing T-reg
populations and an overall immunosuppressive tumor microenvironment which can affect a
vaccine-induced immune response. It is also well known that the elicitation of cancer-specific
immunity is best when tumor burden is at its lowest (35-37), and perhaps the timing of our
vaccinations influenced the results of the current study. Furthermore, it is becoming more
common to integrate strategies that enhance T cell responsiveness and down regulate existing
immune suppression for the purpose of augmenting cancer immunotherapies (38). In our studies,
mice were administered cyclophosphamide before beginning the L-BLP25 vaccine regimen to
reduce T- suppressor lymphocyte activity.
Of interest is the relationship between pregnancy and reduced incidence of breast cancer (39,
40). Many multiparous women have circulating T-cells which function in a MUC1-specific
manner as well as a nonspecific immune response (41). It is thought that hormone levels, such
as estrogens, during pregnancy contribute to the reduced risk, but other factors such as age at
first completed pregnancy also contribute (42). In fact, it is estimated that a woman must have a
full-term pregnancy before the age of 25 to reduce the lifetime risk of developing breast cancer
(43). Recent work has shown that high estrogen levels are associated with an increased risk of
breast cancers diagnosed before age 40 and a decreased risk after the same age (44). The authors
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21
conclude that this increased risk is associated with hormone-negative tumors and therefore their
findings mainly apply to premenopausal women. Their data also support the theory that
pregnancy protects against hormone receptor-positive cancers (44). Other work has shown that
multiparous women have an even lower risk of developing breast cancer than uniparous women
(43). The reduction of breast cancer risk after full-term pregnancy may be related to a MUC1
response. Vaccines that elicit a MUC-1 immune response may be useful in mimicking a
multiparous woman as a preventive strategy.
In conclusion, we demonstrated that: 1) Th1 polarization of L-BLP25-induced immune response
is unaffected by concurrent administration of tamoxifen or letrozole, and 2) L-BLP25 vaccine
when combined with letrozole has additive antitumor activity. Both observations support the
clinical testing of a combination therapy of L-BLP25 with letrozole for the treatment and
prevention of breast cancer.
ACKNOWLEDGEMENTS: We would like thank Kate Wasson (UC Davis, Mouse Biology
Program), and Beverly Packard (Oncoimmunin, Inc.) for their valuable technical assistance. We
would like to extend a special thanks to Dr. Oscar Kashala (EMD Serono, Inc, Rockland, USA)
for his expert opinions and feedback.
FINANCIAL SUPPORT: This work was supported by a grant from Merck-KGaA, Darmstadt,
Germany.
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Figure Legends
Figure 1. Tamoxifen and letrozole dose-finding studies
Treatment with tamoxifen (50 mg/kg) (A) and letrozole (0.8 and 8.0 mg/kg) (B) showed a
statistically significant survival benefit compared to controls (p<0.05). For tamoxifen, N=7 for
control and N=5 for each dose level, and for letrozole, N=13, all groups. MMT tumors and a
derived cell line expressed ER-α and –β, as determined by Western blot analysis (C). MCF-7
cell lysates serve as a positive control while β-actin served as loading control.
Figure 2. Serum cytokine levels in vaccinated MMT females versus placebo/untreated and CTX-
treated mice.
Whole blood was collected via submandibular bleeds 24 hours after the 4th (A), 6th (B) and 8th
(C) dose of vaccine or placebo. Whole blood was pooled within each treatment group (at least 3
mice per group) and serum was analyzed using Luminex technology. The levels of IFN-γ were
noticeably higher in vaccinated vs. placebo-treated mice at all time-points. IL-4 levels in
vaccinated vs. placebo-treated mice were not noticeably different at any time point. Serum levels
of IL-5 appeared to be higher in all vaccinated mice vs. placebo-treated mice. Concentration
values are means of duplicate samples and error bars represent the range. Missing bars represent
values below detection limits.
Figure 3. Representative serum cytokine responses following hormonal therapy in vaccinated
and unvaccinated mice. Whole blood was collected via submandibular bleeds 24 hours after the
3rd (A) and 4th (B) dose of vaccine and pooled for each treatment group (at least 3 mice per
group). The levels of IFN-γ were higher in all vaccinated mice regardless of treatment. No
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30
obvious changes in levels of IL-4 were noted in vaccinated mice compared to unvaccinated mice.
Concentration values are means of duplicate samples and error bars represent the range. Missing
bars represent values below detection limits.
Figure 4. Representative cytokine patterns in hormonally treated vaccinated and unvaccinated
mice.
Mice were euthanized 24 hours after the 5th dose of vaccine, and serum was analyzed for 10
cytokines. Vaccinated mice, regardless of treatment, had elevated IFN-γ and IL-12 levels, which
are indicative of a Th1 immune response. Although levels of the Th2-associated cytokines IL-4
and IL-5 were elevated in some vaccinated mice, on balance the predominant immune response
was Th1 polarized as evidenced by the strong IFN-γ and IL-12 responses. Concentration values
are means of duplicate samples and error bars represent the range. Missing bars represent values
below detection limits.
Figure 5. Detection of MUC1-specific splenocytes by ELISpot assay in hormonally treated
vaccinated and unvaccinated mice.
Media, a scrambled peptide, and a 25-amino acid peptide (BP25) spanning the VNTR region of
MUC1 were used to stimulate splenocytes at 1×106 cells/well before counting IFN-γ or IL-4
positive SFC (mean ± S.D. of triplicate wells). Splenocytes from a vaccinated mouse stimulated
with BP25 showed elevated IFN-γ SFC levels (A), while IL-4 SFC levels were unchanged (B).
The average IFN-γ SFC levels in letrozole vaccinated (N=4) (C) and tamoxifen vaccinated (N=3)
(D) mice in response to BP25 stimulation were significantly higher (p<0.001) independent of
hormonal therapy. IL-4 SFC levels were not significantly different (p>0.05).
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31
Figure 6. Effect of L-BLP25 combined with hormonal therapy on antitumor activity and
survival.
Mean tumor volumes (± SEM) for all surviving mice and Kaplan-Meier survival curves are
shown for letrozole (A, B) and tamoxifen (C, D). Tumor volumes were evaluated on study day
113, and overall survival was evaluated on study days 137 and 135 for letrozole and tamoxifen,
respectively.
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Published OnlineFirst March 20, 2012.Clin Cancer Res Neelima R. Mehta, Gregory T. Wurz, Rebekah A. Burich, et al. Expressing Mammary Tumors in MiceResponse and has Additive Antitumor Activity in MUC-1 L-BLP25 Vaccine plus Letrozole Induces a TH1 Immune
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