Breast SPORE Developmental Awards

Measles Virotherapy in the Treatment of Breast Cancer
Investigator: Evanthia Galanis, M.D.

The Gene and Virus Therapy Program research group has introduced measles virus vaccine strains as in the treatment of solid tumors and hematologic malignancies. Infected tumor cells fuse with neighboring tumor cells to form giant cell aggregates (syncytia), which subsequently undergo programmed cell death. As a result, for each tumor cell infected by the virus, 80 to 100 neighboring cells die. In contrast, measles virus infection has minimal effect in normal cells. This is due to very low or undetectable expression of the measles virus receptor in normal cells. Mayo's laboratory was first to demonstrate activity of measles virus derivatives against breast cancer lines and animal models. The investigators showed that the virus has potent antitumor activity against breast cancer lines and animal models, independently of hormone receptor status and HER-2 status. Measles virotherapy can therefore be developed as an attractive option for patient populations such as hormone receptor negative and HER-2 positive breast cancer patients for whom only toxic treatment alternatives exist.

Breast cancer is a systemic disease and therefore systemic, intravenous delivery of the virus could increase the applicability of this approach to include the majority of breast cancer patients with disease refractory to other treatment options. A possible obstacle that the virus may have to overcome in this setting is the patient's own immunity against the virus. Measles virus is a cell bound virus. As an example, the virus uses peripheral blood mononuclear cells to disseminate in the case of a natural infection. Dr. Galanis and her team therefore hypothesized that cell delivery can be used to effectively deliver measles virus to treat metastatic disease in breast cancer patients. The aims of this developmental project are to:

1. Optimize cells as delivery vehicles for the measles virus, both in vitro and in vivo in a therapeutic setting; and
2. Rapidly translate this approach to a clinical trial for breast cancer patients.

The investigators tested a variety of human derived carrier cells regarding their ability to deliver measles infection to breast cancer tumor cells. They chose cell carriers for this purpose that can be reliably isolated from patients in adequate numbers and/or expanded in vitro to allow effective delivery of the virus in a therapeutic setting. Human cells tested for their potential as cell carriers include peripheral blood mononuclear cells, immature dendritic cells, mature dendritic cells, and CIK cells (subpopulation of NK(natural killer)/T cells).

Among these cell lines, dendritic cells and epithelial cells showed the best infectivity by the virus (greater than or equal to 50 percent). Using cell carriers, the team has been able to successfully transfer measles virus infection to breast cancer cells in vitro. The ability of these cell carriers to deliver the virus in the presence of neutralizing antibodies was 40 to 160 times higher than that of free viral particles, indicating that cell-mediated delivery can effectively bypass immune response to the virus.

Optimization of a novel therapeutic approach prior to testing in humans clearly depends on the use of an appropriate animal model that simulates the therapeutic target as close as is possible. Developing such a metastatic model, simulating the burden of disease in metastatic lung cancer patients could more accurately predict the activity of the proposed therapeutic approach in patients and would allow the researchers to optimize the therapeutic treatment protocol.

The team performed extensive in vivo work in mice with three breast cancer cell lines (MCF7 cells, MDA-MB-435 and MDA-MB-231 cells) in order to develop such a model, and they have been able to successfully develop a reproducible metastatic breast cancer model to the lung using MDA-MB-231 cells. Following intravenous administration of a lung passaged subpopulation of MDA-MB-231 breast cancer cells, all animals developed lung metastases with symptomatic disease as early as four to five weeks and a median survival of only seven to eight weeks. In addition, with Institutional Review Board (IRB) approval, the investigators collected measles immune serum from healthy individuals.

Mayo is now able to use this reagent in its animals in order to simulate the immune status of human patients. Using this animal model, Dr. Galanis' team demonstrated that repeat administration of carrier cells infected with the virus results in massive infection of metastatic breast cancer deposits in the lungs, which is significantly higher as compared to using free virions.

The researchers are currently generating comparative efficacy data that can be used to support a new IND (Investigational New Drug) application to the U.S. Food and Drug Administration (FDA) in order to initiate a clinical trial in breast cancer patients. Parameters that need to be optimized include comparative efficacy of endothelial cells versus dendritic cells in delivering the virus in vivo, dose of the virus to infect the cells, number of cells per administration, and number of doses. The optimal cell delivery approach will be compared to administration of free virus at the presence of neutralizing antibodies. Furthermore, the team plans to compare the measles virus strain MV-CEA with the MV-NIS virus, a viral strain encoding for the human sodium iodine symporter, that is currently tested clinically in the treatment of multiple myeloma, the latter giving the advantage of being able to employ radioactive iodine in order to further increase the efficacy of the virus in a therapeutic setting.

CYP2D6 Pharmacogenetics and Tamoxifen: Implications for Patient Care
Award 2005
Investigator: Matthew Goetz, M.D.

Approximately half a million women in the United States currently take the drug tamoxifen for the prevention of breast cancer recurrence following surgery, or as a chemopreventive agent for those at high risk of the disease.

Research has shown that tamoxifen needs to be changed (activated) within the body to a much stronger form of the medication in order to be fully effective in the treatment of breast cancer. This process is called metabolism. An enzyme in the body, cytochrome (CYP) 2D6, is an important part of tamoxifen metabolism. This ability of the CYP2D6 enzyme to fully metabolize tamoxifen in patients is highly variable, and previous research has demonstrated that this variability is influenced by two main factors: the genetic variation in the CYP2D6 gene, which influences the enzyme activity (up to 15 percent of Caucasian women are born with little or no functional CYP2D6 activity); and the co-administration of drugs which inhibit the CYP2D6 enzyme (such as medicines used to treat hot flashes).

Dr. Goetz's research was conducted in the context of a prospective randomized trial conducted by the North Central Cancer Treatment Group, in which postmenopausal women were administered five years of tamoxifen therapy for estrogen-receptor-positive breast cancer. Both CYP2D6 genotype and medications that interfere with CYP2D6 were known for 180 women randomly assigned to the tamoxifen-only control arm of the trial.

The researchers classified 65 women as having decreased metabolism of tamoxifen based on expected genetic or drug-induced inhibition of CYP2D6 and 115 patients expected to metabolize tamoxifen normally.

Tamoxifen's clinical benefit was greatly decreased for women with factors that negatively affected CYP2D6 metabolism. These women had significantly shorter time to disease recurrence and significantly worse disease-free survival compared with women able to metabolize the drug normally. Women with the largest decrease in CYP2D6 metabolism (CYP2D6 poor metabolizers or those taking a potent CYP2D6 inhibitor like paroxetine) had a threefold higher risk of breast cancer recurrence.

On October 18, 2006, a presentation of this study and related historical data by Dr. Goetz to the U.S. Food and Drug Administration led to an advisory committee unanimously recommending a label change for tamoxifen. This change will include information about the increased risk both from genetic factors and drug interactions affecting CYP2D6.

Dr. Goetz is continuing to study the effect of CYP2D6 genetic changes in the context of a completed and ongoing international clinical trials comparing tamoxifen to aromatase inhibitors in both pre and post menopausal breast cancer.

Determinants of sensitivity to insulin-like growth factor 1 receptor inhibition in breast cancer
Award 2007
Investigator: Paul Haluska, M.D.

Dr. Haluska's team has been focusing on investigating new therapy that blocks an important new target in breast cancer. This target is called the insulin-like growth factor 1 receptor (IGF-1R). This protein receives signals that promote growth in breast (and other) cancers.

These signals also contribute to resistance to breast cancer therapies. Because of this, agents that target the IGF-1R have great promise for treating breast cancer. However, it may not work in all patients. In order to identify markers that may predict patients may or may not respond to such a therapy, Dr. Haluska's lab has developed a model breast cancer cell line that is resistant to the IGF-1R inhibitor BMS-536924.

Using this model, Dr. Haluska has identified several markers that may be important in predicting sensitivity to IGF-1R-direct therapy. For instance, HER2, which is an important target in breast cancer, may predict resistance to IGF-1R inhibition. As there are approved therapies to target HER2, this support the combination of IGF-1R and HER2- targeted agents. Additionally, IGFBP5, which can regulate the IGF system, is very high in breast cancer cells resistant to IGF-1R inhibition. As this may be measured in patient serum, this could be tested prior to consideration of an IGF-1R targeting agent. These and other information support the rationale for clinical trials in breast cancer patients.

A multi-antigen DNA vaccine for the prevention of breast cancer recurrence
Investigator: Keith L. Knutson, Ph.D.

The goal of this project is to design, produce and test a multi-antigen DNA vaccine for use in the prevention of recurrence of breast cancer. There are several reasons for the development of this vaccine. Recurrence is the major cause of mortality and morbidity among breast cancer patients today. Prevention of recurrence is an important goal. A DNA vaccine is attractive because it is fairly easy to produce and can be used regardless of immunologic backgrounds (e.g. major histocompatibility complex and other transplantation antigens). The multi-antigen strategy also makes broad application possible and minimizes the chances of immune escape.

The specific aims of this development project are to:

1. Determine the efficacy and safety of multi-antigen DNA-based vaccine strategies
2. Synthesize a multi-antigen DNA vaccine for prevention of relapse in breast cancer patients

Specific Aim 1 is a murine modeling aim with the major goals to obtain all of the necessary preclinical data required to justify moving this into a clinical trial which includes immunologic, safety and efficacy data. The aim involves the synthesis and testing of about 11 different DNA constructs. The HER-2/neu transgenic model of human breast cancer was chosen because it is a well characterized model and breast tumor cell lines have been developed that show overexpression of all the antigens.

Specific Aim 2 is to prepare the backbone of a future clinical trial vaccine DNA construct to be used in humans. This consists of a panel of constructs for which eventual use will be guided by the murine modeling experiments above. Part of this aim also includes the identification of epitopes that can be used to monitor the progress of vaccination in humans.

The DNA vaccine proposed will encode cyclin D1, folate receptor alpha, and IGFBP2. These antigens are highly restricted to malignant tissue and are overexpressed in 50 percent or greater of patients. Cyclin D1 is overexpressed in 80 percent of patients, folate receptor alpha in 50 percent to 80 percent, and IGFBP2 in about 100 percent. Dr. Knutson and his team have substantial evidence that patients are responding to these antigens naturally which is one reason why the investigators chose this group to research. Another reason is that prior work indicates that these antigens are biologically important for breast cancer (i.e. they are needed to help the tumors growth and spread). Along with the DNA vaccines, three immune boosting co-stimulatory molecules (CD80, CD86, and CD137L) for both mouse and human will also be produced as adjuvant DNA constructs. The researchers' is to produce the antigens as a single protein that will be rapidly recognized by the immune system as foreign.

Significant progress has been made. All of the planned constructs have been synthesized and testing shows that they can be expressed. The researchers are developing the most appropriate immunization strategies in the mouse model of breast cancer. Once developed, the safety and efficacy data will be collected.

The panel of human constructs has also been prepared for the eventual translation of this approach into human. All of the single antigen constructs (e.g. the co-stimulatory molecules) have been prepared and the 2 multi-antigen fusion constructs are nearly completed. The Mayo Breast SPORE development funds have been used to extend related studies (funded by another National Cancer Institute grant) determining the epitopes for immune monitoring of the forthcoming clinical trial. In these studies, 23 novel peptides within each of the antigens have been identified and most have been verified as being capable of binding to MHC class II binding (i.e. T cell epitopes).

The ultimate goal is to test multi-antigen DNA vaccines into phase I clinical trials vaccinating disease-free breast cancer patients at high risk for disease relapse. The Mayo Breast SPORE has been instrumental as source of funding for these early preclinical studies to develop the concepts and help ensure the clinical translation.

Development of a MUC1-driven conditionally replicating adenovirus containing the sodium iodide symporter (NIS) gene for imaging and therapy of MUC1-positive breast cancer approved
Award 2006
Investigator: John Morris III, M.D.

The sodium iodide symporter (NIS) is the protein that allows uptake and concentration of iodide by thyroid cells. Normal NIS expression in the thyroid allows diagnosis and effective therapy of metastatic thyroid cancer with radioactive iodine, the most effective of systemic radiotherapy available to the clinician today. However this therapy is only available today for thyroid cancer because other cancers, including breast cancer, do not express the NIS gene sufficiently to allow high level uptake of iodine.

Dr. Morris and his team are looking for a way to increase uptake of iodine in breast cancer cells and thus allow treatment of breast tumors using radioactive iodide. The specific aims of their project, funded through a Breast SPORE Developmental Grant, include:

1. Develop a replicating adenovirus containing the MUC1 promoter driving replication of the viral backbone, and expression of NIS
2. Image and treat breast tumor xenografts in nude mice using radioactive iodide. ¹²³I imaging will confirm targeted expression of the gene and permit optimization of viral, and radioiodide dose for ¹³¹I therapy

The investigators have developed a method to transfer and express the NIS gene in breast cancer cells and induce them to concentrate iodide similar to that of thyroid cells. This has been accomplished by producing an adenovirus capable of replicating in breast cells by use of a promoter that is highly active in the majority of breast cancers, MUC1. This virus also contains the NIS gene so that after infection and replication in breast cancer cells, it induces high level uptake or radioactive iodine. The work is similar to that Dr. Morris has pursued previously for prostate cancer, which has led to the development of a Phase I clinical trial of NIS mediated gene therapy in men with recurrent prostate cancer.

To date the researchers have demonstrated that the virus effectively targets and replicates in breast cancer cells and stimulates uptake of iodine in cell culture. They have just started experiments to examine the activity of the virus in animal models of breast cancer and expect to complete those studies in the next few months. The goal of these studies is evaluate the effectiveness of this cancer gene therapy approach in breast cancer models so that clinical studies in women with breast cancer can be pursued.