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    Research Grants Awarded

    Combination Chemo-Immunotherapy For Established Breast Cancer

    Grant Mechanism:
    Investigator Initiated Research

    Scientific Abstract:
    Rationale: A combination of chemotherapy and trastuzumab (a monoclonal antibody (mAb) against ErbB2) has greatly reduced the risk of recurrence in patients with ErbB2-positive breast cancer. However, there remains an important unmet medical need for new therapies for both ErbB2-positive breast cancer patients and for patients with ErbB2-negative breast cancer refractory to current treatment. This proposal builds on a previously funded Komen grant where we demonstrated that targeting of the TNF-related apoptosis-inducing ligand (TRAIL) pathway with an agonistic anti-DR5 mAb represents a potential new form of treatment against breast cancer. We now aim to tackle the major problems of TRAIL-resistance and/or trastuzumab-resistance in breast cancer. Our preliminary data suggest that anti-ErbB2 mAb or histone deacetylase inhibitors (HDCAi) can significantly enhance anti-DR5 mAb therapy. Moreover, we have shown that inducing such potent tumor cell apoptosis with these novel combinations also triggers robust and long-lived tumor-specific immunity. Hypotheses: Aim 1: We hypothesize that anti-ErbB2 mAb will sensitize ErbB2 positive breast cancer cells to anti-DR5 mAb-mediated apoptosis. We propose that anti-ErbB2 mAb may regulate the expression of apoptotic proteins and pathways involved in DR5-mediated apoptosis and will synergize with anti-DR5 mAb in the activation of innate and/or adaptive anti-tumor immune responses. Aim 2: We hypothesize that HDACi will sensitize breast cancer cells to anti-DR5 mAb therapy or anti-ErbB2 mAb therapy. We propose that HDACi may regulate the expression of oncogenic and/or apoptotic proteins that control sensitivity to biological agents that target the TRAIL and ErbB2 pathways and stimulate anti-tumor immune responses. Aim 3: We hypothesize that the therapeutic modalities that we are establishing in mouse models can be translated to a human setting. Experimental design: Aim 1: Our preliminary data demonstrated that anti-ErbB2 mAb sensitizes ErbB2-positive mouse mammary tumors to anti-DR5 mAb therapy. The planned experiments can be divided into two categories. Firstly, experiments will be undertaken to determine what underpins the synergistic apoptotic activity. We will assess the modulation of proteins involved in DR5-mediated apoptosis such as Bcl-2 family members, inhibitors of apoptosis (IAPs), death-inducing signalling complex (DISC) proteins, proteins that regulate DR5 glycosylation, and proteins involved in ErbB2 signalling. We will engineer cancer cells derived from ErbB2/Neu-transgenic mice to over-express or under-express candidate proteins and assess treatment efficacy in vitro and in vivo. The second set of experiments will determine whether the combined treatment acts in part via immune stimulation and by what mechanisms that immunity is exerted. Combination therapy will be assessed in mice depleted of specific immune cell subsets, adaptive immunity will be assessed by lymphocyte transfer and tumor challenge experiments and antigen-specific CD8+ T cell responses will be monitored with labeled class I tetramers. Aim 2: Our preliminary data suggest that the HDACi LBH589 sensitizes 4T1.2 mouse mammary tumors to anti-DR5 mAb therapy. An approach similar as detailed above will be undertaken in order to determine how HDACi synergizes with anti-DR5 mAb and whether tumor immunity is generated. We will further assess whether LBH589 synergizes with anti-ErbB2 mAb therapy. The therapeutic activities of various combinations of the three agents will then be assessed in two mouse models of breast cancer, i.e. 4T1.2 transplanted tumors and spontaneous tumors arising in ErbB2/Neu-transgenic mice. Aim 3: We will test human-specific agents currently approved (trastuzumab) or in clinical development (the anti-DR5 mAb LBY135 and the HDACi LBH589) for their ability to mediate synergistic anti-breast cancer activities. We will test, in vitro, various human breast carcinoma lines for sensitivity to increasing doses of these agents in various combinations. Finally, human breast cancer cell lines identified as being sensitive to specific combinations will be tested for sensitivity to the combinations in vivo in immunodeficient mice. Clinical impact: Monoclonal antibodies are proving to be powerful therapeutic agents in the treatment of cancer owing to the selective targeting of antigens differentially expressed on cancer cells. Our previous work demonstrated that anti-DR5 mAb therapy can induce selective tumor cell apoptosis and stimulate adaptive anti-tumor immunity in a mouse model of breast cancer. We here propose that targeting breast cancer cells with either an anti-ErbB2 mAb or HDACi in addition to anti-DR5 mAb will significantly improve the treatment of breast cancer patients including those refractory to current treatment regimens. We aim to demonstrate the principle that three separate pathways to tumor reduction can be synergistic with one another and in particular can be effective against refractory disease. Ultimately, we hope that patients will be stratified, not only based on Her2/ErbB2 status, but also responsiveness to TRAIL receptor agonists and HDACi. We will determine the most effective and safe combination using the most relevant pre-clinical models to take these approaches into clinical trial. By determining the mechanism of action of these synergistic combinations and the role of anti-tumor immune responses, our research should open up avenues to even more effective treatment approaches.

    Lay Abstract:
    Despite current "state-of-the-art" treatment, it is predicted that in 2007 there will be over 40,000 deaths due to breast cancer in the United States. Drugs such as HerceptinTM, which belong to a class of drugs called antibodies, have recently proven to be powerful yet safe agents in the treatment of breast cancer owing to their selective killing of cancer cells with minimal effect on normal cells. As such, HerceptinTM currently benefits approximately one-third of patients with breast cancer. Nevertheless, a great proportion of patients will develop resistance to HerceptinTM and to other forms of therapies. Our main objective is to improve the treatment of patients refractory to current therapies. Recently, using mouse models of human breast cancer our group has shown that another antibody (named MD5.1) that also specifically kills cancer cells although by a different mechanism than HerceptinTM, can induce profound anti-breast cancer effects. Remarkably, we showed that treatment of breast tumors with this new antibody can further stimulate immune cells to recognize and kill breast cancer cells. Thus, treatment of breast cancer with this new antibody (MD5.1) not only directly kills breast tumors, but exploits the body's own immune system against established breast cancer. Our initial experiments revealed that when combined together, MD5.1 greatly enhances the treatment efficacy of an Herceptin-like antibody. Moreover, our experiments showed that a new class of drugs know as HDAC inhibitors can further sensitize breast cancer cells to MD5.1 therapy. Based on these results, we now wish to test various combination therapies incorporating HerceptinTM, MD5.1 and HDAC inhibitors in even more rigorous mouse models of breast cancer, examine how the therapy works and why some mice are not cured. The specific aims of this proposal are: (1) to test the therapeutic activity and investigate the mechanism of action of HerceptinTM therapy in combination with MD5.1 therapy, (2) to test the therapeutic activity and investigate the mechanism of action of HDAC inhibitors in combination with MD5.1 or Herceptin-like antibody; (3) to test whether the therapeutic modalities that we are establishing in mouse models can be translated to a human settings. In order to test our study, the therapeutic activities of various combinations of the three agents will firstly be assessed in two mouse models of breast cancer, including a model where mice spontaneously develop breast tumors. Secondly, various types of human breast tumors will be tested for sensitivity to humanised versions of these mouse-specific agents in various combinations. Using these original and innovative approaches, our team is at the forefront of efforts to study the effect of new therapies that combine standard approaches to new approaches aimed at boosting the immune system against cancer. We anticipate that the proposed studies will lead to several possible new combination for treating more established breast cancer. These studies will bring us closer to helping all those afflicted by breast cancer.