Home | Clinical Cancer Research - L-BLP25 Vaccine plus Letrozole … · 2012. 3. 20. · Patients that develop hormone-independent and/or triple negative (ER-, Her2/neu-, PR-) breast

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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

Research. on January 24, 2021. © 2012 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 20, 2012; DOI: 10.1158/1078-0432.CCR-12-0168

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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




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.




22




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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




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).




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.






















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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