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    Research Grants Awarded

    Preclinical Development Of An Innovative Mammaglobin-A Single Chain Trimer Dna Vaccine

    Grant Mechanism:
    Postdoctoral Fellowships

    Scientific Abstract:
    RATIONALE: The mammaglobin-A gene was first identified by Timothy Fleming, one of the mentors/co-investigators of this application. Mammaglobin-A has several unique properties that make it an exceptional target for breast cancer vaccine therapy. (1) Mammaglobin-A is expressed almost exclusively in normal breast epithelium and in breast cancer. This exquisite tissue specificity decreases the potential for autoimmunity. (2) Mammaglobin-A is dramatically overexpressed in over 80% of primary breast cancers, suggesting that almost all breast cancer patients are likely to be candidates for vaccine therapy. (3) There is considerable evidence that mammaglobin-A is capable of eliciting an immune response in breast cancer patients. One of the limitations of many conventional vaccine strategies is the requirement for intracellular processing and presentation of encoded antigens by MHC class I molecules, a very inefficient process. Project mentor/co-investigator Ted Hansen has engineered MHC class I peptide complexes as single-chain trimers (SCT) to bypass this process. First generation SCT have proven to be potent stimulators of T cells, and represent an innovative and attractive vaccine strategy. Second and third generation SCT were designed to enhance peptide binding. The fellowship candidate has further engineered SCT constructs to improve interaction with the CD8 coreceptor (fourth generation SCT), and to elicit CD4 help, dramatically improving the efficacy of the SCT DNA vaccine. Specific accomplishments to date include: (1) demonstration that third generation SCT can elicit tumor immunity; (2) design and creation of fourth generation SCT, and validation that these constructs have higher affinity interactions with CD8 T cells; (3) design of a vector that expresses both the SCT and a generic helper epitope and demonstration that this construct dramatically improves the efficacy of SCT DNA vaccines; and (4) successful creation of third and fourth generation mammaglobin-A SCT constructs. HYPOTHESIS: The hypothesis of this application is that an optimized mammaglobin-A SCT DNA vaccine is capable of eliciting an enhanced mammaglobin-A-specific immune response in preclinical animal models. SPECIFIC AIMS: 1. Validate novel mammaglobin-A SCT for enhanced peptide binding and CD8 interaction. 2. Test the ability of mammaglobin-A SCT DNA vaccines to generate breast cancer immunity in an established preclinical model. 3. Test the ability of helper epitopes to enhance the efficacy of mammaglobin-A SCT DNA vaccine. EXPERIMENTAL DESIGN: Specific Aim 1: We have created third and fourth generation mammaglobin-A SCT. These constructs will require in vitro validation prior to preclinical assessment. In third generation SCT, a disulfide bond has been engineered between the F pocket and the first flexible linker to "trap" peptides into the binding groove of MHC class I heavy chain. Resistance to exogenous peptide exchange will be assessed to confirm that the third generation mammaglobin-A SCT has enhanced peptide binding. In fourth generation SCT, a single residue has been substituted (Q115E) in the ?2 domain of the heavy chain to enhance interaction with CD8. Enhanced interaction with CD8 will be confirmed by tetramer ?off-rate? experiments. T2 cells transfected with SCT will be used in cytotoxicity assays to confirm that the mammaglobin-A SCT constructs can be recognized by mammaglobin-A peptide-specific T cells. Specific Aim 2: To test the ability of mammaglobin-A SCT to generate breast cancer immunity, HLA-A2*0201/hCD8 double transgenic mice will be vaccinated with mammaglobin-A SCT. Cytotoxicity assays will be performed using HLA-A2+/mammaglobin-A+ and control breast cancer cell lines as targets. To measure antitumor immunity, cells from vaccinated double transgenic mice will be adoptively transferred into nude mice bearing breast cancer xenografts. Tumor growth and tumor-free survival of the control and vaccinated mice will be closely monitored. Specific Aim 3: Preliminary data suggests that co-expression of a CD4 helper epitope dramatically enhances the efficacy of mammaglobin-A SCT DNA vaccine. A defined CD4 helper epitope derived from mammaglobin-A will be given as peptide, stand-alone DNA, or co-expressed with the SCT construct. An optimal strategy for CD4 help will be identified in the context of preclinical model described above. RELEVANCE: It has been predicted that 1 in 8 American women will develop breast cancer during her lifetime. Progress in basic and translational immunology has confirmed the importance of the immune system in cancer prevention and prognosis, and has renewed interest in vaccine therapy for cancer. Cancer vaccines are safe, well tolerated, and can typically be given in an ambulatory facility. One of the most compelling advantages of a mammaglobin-A DNA vaccine is the potential application of this strategy in the majority of patients with breast cancer, and the strong possibility that a successful vaccine could be used in breast cancer prevention strategies. Dr. Gillanders and colleagues have successfully manufactured a cDNA vaccine targeting mammaglobin-A and are currently preparing an IND application for a phase I clinical trial. The preliminary studies performed by the fellowship candidate suggest that a mammaglobin-A SCT vaccine has the potential to be significantly more effective than a conventional cDNA vaccine. This application represents a unique opportunity to test a novel therapeutic in an established preclinical model with the potential for clinical translation through a defined pathway.

    Lay Abstract:
    It has been predicted that 1 in 8 American women will develop breast cancer during her lifetime. Clearly, innovative strategies for breast cancer prevention and therapy must be considered a public health priority. There is increasing evidence that the immune system helps to protect us against cancer. As we learn more about the immune system, it is clear that cancer vaccines may develop into a very effective way to prevent and treat cancer. Cancer vaccines are safe, well tolerated, and can typically be given in an ambulatory facility. The Principal Investigator of this application and collaborators at Washington University School of Medicine have been studying the molecular biology and immunology of breast cancer. As a group, we were the first to identify and characterize a molecule named mammaglobin-A. The function of mammaglobin-A is still not known. What is known is that mammaglobin-A seems to be expressed only in normal breast tissue and breast cancer. Expression in breast cancer is much higher than in normal breast tissue, and this is one reason why we have worked very hard to study this molecule. In our previous studies, we have determined that mammaglobin-A has a number of unique characteristics that make it an ideal target for a breast cancer vaccine. Mammaglobin-A is overexpressed in almost all breast cancers, and so if we successfully develop a vaccine we can use it in almost all breast cancer patients. Other molecules that are considered to be good targets for breast cancer vaccines are overexpressed in only about 30% of breast cancers. We have also been able to detect immune cells in blood from breast cancer patients that are capable of recognizing and killing breast cancer. The molecule that is recognized by the immune system in these experiments is mammaglobin-A. This provides additional evidence that a vaccine strategy targeting mammaglobin-A deserves exploration. In this application, we will target mammaglobin-A with a novel molecular therapeutic, known as a MHC class I single-chain trimer (SCT). In previous studies we created a first generation mammaglobin-A SCT. This molecule appears to be able to generate breast cancer immunity in an established preclinical model. In proof-of-principle experiments we have demonstrated that third and fourth generation SCT, engineered to enhance peptide binding and interaction with CD8, are significantly more effective than first generation SCT. We have now created third and fourth generation SCT specifically targeting mammaglobin-A, and we are now poised to test these novel therapeutics in a preclinical model as a first step towards clinical translation. The hypothesis of this grant application is that these optimized mammaglobin-A SCT DNA vaccines are capable of generating an enhanced mammaglobin-A-specific breast cancer immune response. To test this hypothesis, we will perform some preliminary validation studies in the laboratory to confirm that the third and fourth generation mammaglobin-A SCT have all of the biochemical properties of the SCT that we created for the proof-of-principle studie . Next, we will test the efficacy of the third and fourth generation SCT for breast cancer therapy in our established preclinical model. We will vaccinate mice, harvest immune cells from the vaccinated mice, and test their ability to kill breast cancer cells. Finally, we will investigate strategies to activate helper T cells, which have the potential to further enhance the efficacy of the mammaglobin-A SCT. Although DNA vaccines are new, they have a number of advantages over conventional vaccine strategies and there is considerable enthusiasm in the immunology community for these vaccines. DNA vaccines appear to be very safe, they are inexpensive to manufacture, and may represent an ideal platform for modular vaccine development. The Principal Investigator and colleagues have successfully manufactured a cDNA vaccine targeting mammaglobin-A and are currently preparing an IND application for a phase I clinical trial. The preliminary studies performed by the fellowship candidate suggest that a mammaglobin-A SCT vaccine has the potential to be significantly more effective than a conventional cDNA vaccine. This application represents a unique opportunity to test a novel therapeutic in an established preclinical model with the potential for clinical translation through a defined pathway.