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    Research Grants Awarded

    Characterization Of Trps-1 As Novel Active Vaccine Target For Breast Cancer Immunotherapy

    Grant Mechanism:
    Postdoctoral Fellowships

    Scientific Abstract:
    Immunotherapy is a promising new method to treat breast cancer (BC). For example, monoclonal antibodies, such as anti-HER2, are now standard of care for a about 25-30% of BC patients with HER2+ BC. Another form of immunotherapy showing potential therapeutic benefit is active vaccination against mutated or over-expressed tumor-associated antigens (TAAs) using peptide, whole cell, and viral-based approaches. Vaccines can be used to help prolong life in Stage IV patients or as a long-term regimen in earlier stage patients to prevent disease recurrence in the adjuvant treatment setting. Vaccination of Stage I/II BC patients, or patients with defined early stage lesions (e.g., DCIS) in the adjuvant setting represents a more rationale approach not only due to the better ?immunocompetence? of these patients, but also due to the opportunities to combine vaccine with synergistic targeted therapies, such as anti-hormone drugs. Recent clinical trials by George Peoples testing HER2 peptide vaccination has clearly shown the great promise for this approach to treat HER-2 over-expressing cancer. However, in order to make this form of vaccination a reality in the clinic, we need vaccines targeting additional antigens applicable to the majority of BC patients, especially those having ER+ BC, representing up to 70% of cases. One of the current limitations of anti-BC vaccination, especially in the adjuvant/preventative setting, is the lack of new clearly-defined immunogenic antigens highly over-expressed at early BC stages. Apart from the ?usual suspects?, such as MUC1, HER2, p53, telomerase, CEA, Cyclin B1, that have had limited efficacy so far, very few new players have emerged in the field of BC vaccination. We have recently found 15 new potential BC-specific antigens after a comprehensive DNA microarray and IHC screen of 54 ER+ and ER- (HER2 +) invasive BC samples in comparison to over 289 normal tissue samples (Radvanyi et al. Proc. Natl. Acad. Sci. 102: 11005-11010). One of these new targets, called TRPS-1, is over-expressed in >90% of invasive BC with expression also found in early stage disease, such as DCIS and ADH. Initial immunological characterization found a number of immunogenic HLA-A*0201 (A2.1)-restricted TRPS-1 peptides inducing the expansion of CD8+ CTL, some of which showed killing activity against BC cell lines. TRPS-1 has been cloned into plasmid DNA and viral vectors and used to immunize C57BL/6 and HLA-A2/Kb transgenic mice where they generated antigen-specific CD8+ T-cell responses. More recently, we also found that TRPS-1 is over-expressed in 17 beta-estradiol (E2)-induced mammary gland (MG) cancers in ACI rats in collaboration with Dr. Sara Li (University of Kansas). TRPS-1 is highly conserved over evolution with the human and rat protein sequence being 93% similar. Moreover, the ACI rat breast ductal cancers are similar in morphology and phenotype as human BC, and show similar stages of progression from ADH-DCIS-invasive cancer as human ER+ BC. Thus, the ACI rat E2-induced BC model would be a valuable tool to test whether vaccination against TRPS-1 (and potentially other of our newly found BC antigens) can curtail the development of invasive ER+ BC from DCIS and/or other earlier stage of ductal hyperplasia. Based on these findings, we hypothesize that TRPS-1 is a potent new TAA in BC patients that can be targeted in an active vaccine approach to treat early stage BC in the adjuvant setting or prevent the development of invasive cancer from in situ ductal carcinomas and hyperplasias. These hypotheses will be tested experimentally under the following specific aims: Aim #1: To identify HLA-A2.1-restricted epitopes from the TRPS-1 BC antigen capable of activating CD8+ T-cells to kill HLA-matched BC cells. Aim #2: To characterize CD8+ T-cell responses against TRPS-1 epitopes in PBMC and TIL from primary BC patients. Aim #3: To develop a therapeutic vaccine approach targeting TRPS-1 to prevent development of ER-induced ductal carcinoma in the ACI rat model. We will use PBMC from HLA-A2.1+ normal donors to generate peptide-specific T-cell lines to screen for immunogenic TRPS-1 peptides inducing CTL activity against A2.1+ BC cell lines (Aim #1). We have identified and synthesized a library of 9-mer peptide for these experiments, including overlapping 15-mer peptides covering the entire sequence of TRPS-1. In parallel, using designated reactive 9-mer and overlapping 15-mer peptides we will screen PBMC from Stage I/II BC patients for de novo reactivity against TRPS-1 (Aim #2). We predict that a high proportion of patients will have CD8+ T-cell responses that can be boosted by vaccination. Lastly, in Aim #3 we will use the ACI rat BC tumor model to determine whether vaccination against full-length TRPS-1 using DNA plasmid or pox virus vectors can prevent the progression of E2-induced BC. This project will not only characterize a novel TAA highly over-expressed in BC, but will also utilize a versatile animal model of ER+, estrogen-responsive BC closely resembling human BC to develop a novel therapeutic vaccine approach to eradicate and/or prevent invasive BC. The results can help guide the development of new active vaccine in BC patients in the adjuvant or DCIS setting. TRPS-1 can also be combined with other BC TAAs in multi-antigenic vaccines to further enhance immune responses against BC progression. Thus, our work offers a valuable new approach to treat BC, especially in early stage disease or in the adjuvant setting to prevent disease recurrence.

    Lay Abstract:
    Breast cancer (BC) is one of the most prevalent forms of cancer among women in the US and in Europe, both in incidence and cause of mortality. More than 1.2 million women will be diagnosed with BC this year worldwide, according to the World health Organization. The chance of developing invasive breast cancer during a woman?s lifetime is approximately 1 in 7 (13.4%). New treatments are critically needed to prevent and halt the clinical course of this disease. Recent clinical evidence suggests that activating the immune system against BC and other cancers using vaccines or using antibodies to block factors stimulating BC growth are showing great promise in slowing progression of cancer in patients who have spread of their disease (metastases), and may also play a role in secondary prevention. This new approach is collectively called ?immunotherapy?. For example, an antibody called trastuzumab (Herceptin?) used to treat BCs over-expressing the HER2/neu+ growth factor receptor has been a great success story with clinical responses >80% of patients, especially in combination with chemotherapy. In addition, several active vaccines activating cytotoxic or killer T cells targeting HER2/neu+ BCs are also currently being tested in the ?adjuvant? setting, i.e., as a means of preventing disease recurrence after removal of the primary tumor(s). A recent Phase II clinical trial (now expanded to a nationwide Phase III trial) has found a distinct benefit of using a HER2/neu peptide vaccine in preventing disease recurrence. However, HER2/neu-over-expressing BCs account for only 20-30% of human BC incidence and, thus, the identification of vaccine targets that can be used to help treat the majority of patients with Estrogen responsive (ER+) BC in the adjuvant setting are still needed. At present, there are relatively only a few good antigen targets highly specific for ER+ BC that would focus a vaccine immune response only against the cancer. To help solve this problem, our group has searched for new BC-specific vaccine targets using a number of different molecular biology and immunology approaches. Our efforts have led to the discovery of a number of new, highly over-expressed proteins that show highly specific expression in BC with little or no expression in normal tissues. One of these new breast cancer-specific proteins we found is called TRPS-1. TRPS-1 is especially interesting because it is expressed in both early and later stage breast cancer (pre-neoplastic hyperplasias, ductal carcinoma in situ, and invasive cancers) in over 90% of patients. We also found that the human immune system can be programmed to generate killer T cells (CD8+ T cells) against TRPS-1 that may kill BC cells. Based on these findings, we hypothesize that TRPS-1 can be the target of a new and potent vaccine in BC patients to prevent tumor recurrence after primary surgery or prevent further spread of metastatic disease. In this Susan G. Komen Foundation project we aim to characterize how TRPS-1 can be recognized by CD8+ T cells in BC patients and by normal, disease-free individuals. In these experiments, we will isolate blood lymphocytes containing T cells and determine how these T cells react against TRPS-1. We will also identify fragments of the TRPS-1 protein (called peptides) that trigger CD8+ T-cell responses able to kill BC cells. These peptides can be manufactured in large quantities in order to immunize many patients. It is our hope that these immunogenic peptides from TRPS-1 can be later tested in a clinical trial in ER+ BC patients (and also HER2/neu+ BC patients also expressing TRPS-1 in their tumors). In the other major part of our project, we propose to use test our hypothesis that TRPS-1 vaccination can control BC by using an animal model closely mimicking ER+ human BC. This model uses the so-called ACI rat that progressively developed estrogen-dependent ductal BC. We will immunize these rats at different stages of disease (early pre-invasive cancer and later invasive stages) to determine whether anti-TRPS-1 vaccines can prevent BC development and/or eradicate existing disease. If we succeed in doing this, we will be the first to demonstrate that ER+ BC can be prevented through antigen-specific vaccination. This would open the door to a whole new paradigm for BC prevention and management. Another, important aspect of our work is that our TRPS-1 target can be combined in so-called ?multi-antigen vaccines? with other BC-specific proteins to prevent any BC cells that lose expression of TRPS-1 from ?escaping? the immune response. The success of this project will directly drive our efforts in the clinic to begin testing vaccines against TRPS-1 and other antigens in BC patients.