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    Research Grants Awarded

    Enhanced Immunotherapy Of Breast Cancer Using Modified Tlr And Rlr Pathway Activating Adenoviral Vaccines Targeting Her2/Neu.

    Grant Mechanism:
    Postdoctoral Fellowships

    Scientific Abstract:
    Targeted anti-cancer therapies are providing new opportunities to improve the outcome for people with high risk or advanced breast cancers. Recent success in immunotherapeutics has raised hopes for this type of approach, as monoclonal antibodies to key cancer signaling genes (e.g. the targeting of HER2/neu with Herceptin/Trastuzumab) has offered proof that an immunotherapeutic approach is clinically efficacious in breast cancer. But despite this promise, current cancer vaccines against well-known Tumor Associated Antigens (TAAs) such as HER2/neu have achieved only limited success in clinical trials. In part, this is because the magnitude and duration of the T cell and antibody response to the vaccine is limited, a phenomenon attributable to strong tolerance to tumor associated self-antigens (TAAs) (Curigliano, Spitaleri et al., 2007;Salazar & Disis, 2005). Clinical data supports this claim and further demonstrates a broad correlation between the size of tumors and the strength of tolerance (Curigliano, Spitaleri et al., 2006;Tabi & Man, 2006;Salazar & Disis, 2005;Overwijk, Theoret et al., 2003;Curigliano, Spitaleri et al., 2007). Thus, in order to achieve efficacy, a vaccine must overcome a strong tolerance generated in vivo to tumor associated self-antigens. Recent investigation into the mechanisms of tolerance and the initiation of adaptive immunity has revealed an important role for Toll-Like Receptor and Rig-I-Like Receptors. In particular, deficiency in TLR and RLR genes resulted in significantly diminished innate and adaptive responses to Ad vectors (Hartman, Black et al. 2007; Hartman, Kiang et al. 2007). While TLR and RLR pathways have been shown to play critical roles in mediating immunity to many pathogens, in particular adenovirus, it is unclear how important these pathways are in mediating tumor immunity. We propose that activation of these pathways will enhance the innate and adaptive immune responses to adenoviral vector expressed HER2/neu and reduce tolerance to the HER2/neu tumor-associated antigen, thus leading to more effective immunotherapy of breast cancer. Furthermore, we expect that manipulation of these pathways will have the greatest effect in HER2/neu tolerant models and in those animals with larger HER2/neu-positive tumor burdens. To explore this hypothesis, we have designed a series of Adenoviral vectors that express human tumor antigens in combination with TLR or RLH signaling adaptors, such as MyD88, TRIF, and MAVS. Our hypothesis is that dendritic cells infected in situ or animals infected in vivo with these vectors will have enhanced immune responses to the HER2/neu TAA transgene because of the co-expression of large amounts of signaling adaptor proteins. We further propose that this augmented immunity will help break tolerance animal models and may help overcome tolerance in animals with a high HER2/neu+ tumor burdens. To specifically test the hypothesis of adaptor signaling augmenting anti-HER2/neu tumor immunity and elucidate its mechanism in these settings, we propose to investigate: 1) Effect of adaptor genes on cellular signaling and adaptive stimulation in mouse and human dendritic cells in vitro, 2) Effect of adaptor genes on cellular (CTL, Th, NK, Treg) and humoral responses (antibody titer, CDC, ADCC, anti-proliferative effects) in human HER2/neu na‹ve and tolerant transgenic mice, 3) effects of adaptor overexpression on anti-tumor responses in tolerant mouse tumor models with pre-existing HER2/neu tumor burdens. Our preliminary data strongly suggests that overexpression of TLR and RLR signaling adaptors leads to profound immune stimulation in Dendritic Cells and elicits activation of multiple gene networks and pathways. Our preliminary data also suggests that stimulation of these pathways elicits enhanced cell-mediated responses in mice and may yield high-titer humoral immunity and inhibit the toleragenic capacity of Treg cells. Other data work also suggests that stimulation of humoral immunity is likely to mediate Complement Dependant Cytotoxicity (CDC) and Antibody Dependent Cell Cytotoxicity (ADCC) responses as well as anti-proliferative effects in HER2/neu+ tumor cells. Anti-HER2/neu antibodies are also expected to hinder cell signaling via the HER2/neu pathway by antibody-receptor mediated endocytosis in tumor cells. Finally, preliminary data from animals with other types of pre-existing tumor burdens suggests that TLR/RLR adaptor over-expression strongly enhances anti-tumor responses and we predict that we will observe similar outcomes with the HER2/neu breast cancer model. While the clinical impact of immunotherapy in breast cancer is currently limited, the success of the approach through the use of monoclonal antibodies demonstrates its great potential in breast cancer targeting of HER2/neu. The widely acknowledged impediment to achieving the full potential of immunotherapy remains the initiation of robust immunity in the face of tolerance. To address this problem we propose to generate adenoviral vectors expressing signaling adaptors to activate key innate immune pathways that have recently been demonstrated to play critical role in the initiation of adenoviral derived immune responses. These strategies offer the potential to augment the already promising anti-tumor immunity we have obtained with Ad-HER2 to date to create a uniquely potent vaccine strategy for breast cancer. Significantly, these vaccine strategies may also help overcome tolerance in the midst of pre-existing tumor burdens, perhaps the largest impediment to realizing the effective immunotherapy of breast cancer.

    Lay Abstract:
    This proposal seeks to test several new approaches to augment anti-cancer vaccines for HER2+ metastatic breast cancer patients. While vaccines currently undergoing phase I trials have demonstrated potential to stimulate anti-tumor immune responses in animal models, this response has noted limitations, particularly in regard to achieving efficacy with larger tumors. To overcome these limitations, we are using known innate pathways proven to be involved the initiation of the adaptive immune response and will assess the enhanced activity against breast cancer in patient cells and stringent mouse models of human breast cancer. The immune system is the bodies? ?army? that is used to fight off invaders such as disease causing bacteria and viruses. A great deal of work over the last 30 years has shown that a cancer patients? own immune system respond to tumors and has the ability to actually kill tumors. This is due to the tumors? unchecked growth and unusual characteristics that can make them appear as ?invaders? to the immune system. We are targeting one such unusual characteristic of breast cancer, a tumor antigen (HER2 protein) that is over-produced by approximately a third of breast tumors. The immune system fights cancer in a 2 pronged assault, using killer T cells (soldiers/infantry) and antibodies (artillery) to destroy tumor cells. The current clinical therapeutic for combating HER2/neu expressing breast cancer is Herceptin, an antibody that attacks the HER2 protein on cancer cells. In contrast, vaccines elicit responses from both arms of the immune system (killer T-cells and antibodies). Our adenovirus-based HER2/neu vaccine stimulates the immune system, producing killer T-cells to destroy HER2/neu expressing cancer cells and antibodies that attack many different targets on the HER2/neu protein. However, most large tumors can effectively reduce the size of the immune response elicited by typical viral vaccines, resulting in a ?recruitment? problem. We propose that activation of innate immune pathways known to help ?recruitment? will allow a much larger recruitment and thus a larger army to effectively combat the tumor (both large and small). We believe that our approach will be more effective against larger tumors for which Herceptin and other viral vaccines are minimally effective. Additionally, our approach can be applied to other targets, thus could have drastic effects on other types of cancer. This proposal has several major goals. We will develop new adenoviral Her2/neu vaccines that express proteins known to stimulate innate immunity (stimulatory vaccines), critical for increasing recruitment. The first goal is to investigate the effect of these stimulatory vaccines on the immune system. Since there have been very few studies investigating immunity by these newly discovered pathways , our investigation of these pathways may yield important new insights into mechanisms behind immune responses. Second, this is the first adenoviral vaccine approach to take advantage of new innate immunity findings in a cancer vaccine. We anticipate that the combination of the immune stimulation and anti-HER2/neu targeting will prove a potent ?one-two punch? that will trigger strong anti-tumor immune responses in patient cells as well as mouse preclinical models of breast cancer. Finally, we expect that this strongly amplified HER2/neu targeting approach will generate greatly enhanced immune responses in animals that have large pre-existing breast tumor burdens, a limitation of current clinical immunotherapies. There is potential for these studies to lead to a paradigm shift in breast cancer therapy. Recent studies have indicated that vaccines can make tumors more easily killed by conventional drugs. Thus, the immune stimulatory vaccines being tested in this proposal could be used in combination with existing vaccines or small molecule drugs, to improve the effectiveness of breast cancer therapy. Finally, it should be emphasized that therapies that use the immune system to fight cancer have generally proven to be safe and to have low toxicity and we expect the same to be true for these vaccines. We believe a significant strength of this proposal is the outstanding team of investigators assembled from multiple departments all part of the Comprehensive Cancer Center at Duke. This proposal is the result of longstanding collaborations by these investigators and leverages the extensive experience of the PI and his collaborators in translating ?cutting-edge? research into clinical trials. If the approach proves successful in patient cells and preclinical mouse models of breast cancer, this strong translational team is well positioned to rapidly move this therapy into their existing breast cancer immunotherapy clinical trials.