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    Awarded Grants
    Delivery of Recombinant Mucin 1(MUC1) Protein and MUC1 Cytotoxic T Cell Epitopes into the Cytoplasm of Antigen Presenting Cells Using a Membrane Permeable Peptide for the Immunotherapy of Breast Cancer

    Scientific Abstract:
    Background. The incidence of many types of cancer continues to rise in developed and underdeveloped countries, in particular, the incidence of breast cancer in women is currently 1 in 11. With the exception of a few treatable cancers, the emergence of new drugs, conventional therapies such as cytotoxic therapy, radiotherapy or combination therapies have not improved the prognosis. Immunotherapy is the best alternative to be considered for further investigation. A large number of different strategies are being investigated in preclinical and clinical settings to develop new vaccine candidates. Several tumor associated antigens and their cytotoxic T cell epitopes have been identified and used as targets for immunotherapy. MUC1 is a high molecular weight glycoprotein with a number (40-80) of 20 amino acid repeats (PDTRPAPGSTAPPAHGVTSA) denoted variable number of tandem repeats (VNTR). The MUC1 molecule is expressed in breast, colon cancer and other adenocarcinomas in levels 10-100 times greater than on normal tissues and is an ideal target for immunotherapy. MHC Class I molecules can present peptides derived from endogenous, nuclear, cytosolic and exogenous protein antigens. For MHC Class I presentation, exogenous antigens are taken up by APC into endosomes, escape into the cytosol (either directly or after degradation) where they enter the proteosome, which is responsible for their further degradation. The presence of peptide in the cytosol is required for processing to the Class I pathway. We have shown that the OVA SIINFEKL Class I epitope linked in tandem to penetratin peptide (RQIKIWFQNRRMKWKK) can be taken up into the cytoplasm of antigen presenting cells and presented by Class I molecules. Mice immunized with these peptides were protected from a challenge of ovalbumin expressing tumor cells. We have also shown in preliminary studies that the penetratin peptides can be linked to a MUC1 Class I epitope present in the VNTR region of MUC1 and splenocytes of mice immunised with these peptides generate IFN-ƒ× detected in ELISPOT assays. We now would like to extend these studies to incorporate penetratin conjugates of intact MUC1 proteins and peptides specifically from the non-VNTR region of MUC1. This should overcome cross-reactivity of MUC1 VNTR peptides with human natural anti-galƒÑ (1,3)gal antibodies in future clinical studies. We will also examine the immune responses of complexes of penetratin peptides incorporating a cytotoxic T cell epitope together with universal T helper epitopes prepared using multiple antigen peptides (MAP) or using a novel synthetic trifunctional crosslinker . Objective/Hypothesis. Recombinant MUC1 antigens and MUC1 cytotoxic T cell epitopes linked to the membrane translocating peptide, penetratin will generate superior immune responses when used to immunize mice alone or as pulsed dendritic cells. Specific aims: 1. Link penetratin peptide to MUC1 protein and MUC1 CTL epitopes. 2. Immunize mice using different routes (ip, id and pulsed DC) with penetratin-peptide and penetratin-protein conjugates and measure cellular and humoral responses in HLA-A2xMUC1 transgenic mice. 3. Compare immune responses to various complexes and perform tumour protection studies. Study design. Penetratin will be linked to recombinant MUC1 using a commercially available heterobifunctional crosslinker. Penetratin-MUC1 peptide complexes with and without T helper cell epitopes will also be made using MAP or a novel heterotrifunctional crosslinker. These conjugates or in vitro-pulsed mouse dendritic cells will be used to immunize mice and study cellular and humoral responses using ELISPOT , CTL, proliferation and ELISA assays. Mice will be immunized ip, id, sc and pulsed DC, with the various immunogens and challenged with MUC1+ve tumors to ascertain antitumor efficacy. Potential outcomes and benefits of the research. This project will identify optimal procedures for design of penetratin complexes and their processing, cellular uptake and immunisation of mice to obtain tumor protection. Novel procedures to develop new candidate cancer vaccines, eg by peptide design, appropriate conjugation and targeting to APC will be defined. There is a great urgency to develop a cure or vaccine for breast cancer. Any breakthroughs for a potential vaccine for breast cancer that emerges from this project will greatly benefit vaccine approaches to other cancers such as cervical cancer and prostate cancer because the methodology will be similar except for the antigen.

    Lay Abstract:
    Delivery of Recombinant Mucin 1(MUC1) Protein and MUC1 Cytotoxic T Cell Epitopes Into The Cytoplasm Of Antigen Presenting Cells Using A Membrane Permeable Peptide For The Immunotherapy Of Breast Cancer. The incidence of many types of cancer continues to rise in developed and underdeveloped countries, in particular, the incidence of breast cancer in women is currently 1 in 11. With the exception of a few treatable cancers, the emergence of new drugs, conventional therapies such as cytotoxic therapy, radiotherapy or combination therapies have not improved the prognosis. Immunotherapy is the best alternative to be considered for further investigation. A large number of different strategies are being investigated in preclinical and clinical settings to develop new vaccine candidates. Mucin 1 (MUC1) is a protein highly expressed in breast and other solid cancers such as lung and colon cancer. Proteins present in the cytoplasm of cells is cleaved by specialized machinery and fragments of the protein are displayed on the surface of the cell by specialized proteins called Class I molecules. These displayed fragments stimulate special immune cells called T cells to kill. We propose to introduce tumor associated proteins or peptides into cells so that killer T cells that are induced as a consequence of this will seek out and destroy tumor cells presenting the same fragments. We propose that by incorporating a membrane translocating peptide onto proteins and synthetic peptides we can introduce tumor associated antigens and peptides into antigen presenting cells. To do this we will chemically synthesize suitable membrane translocating peptides that can be linked to MUC1 protein or linked in tandem to MUC1 peptides. We will also use novel synthetic chemistry techniques to incorporate a number of other relevant peptides in addition to the membrane translocating peptide to optimise the immunogenicity of the complexes. The membrane translocating protein/peptide complexes will be used to immunize mice and the immune responses will be studied using immunological assays. If enhanced immune responses are observed the ability of the complexes to protect mice from a tumor challenge will be studied. The research in this proposal if successful would greatly benefit breast cancer patients by producing a vaccine. At the end of two years we will be ready to take this study further using human cells and eventually a clinical trial. The strategies developed in this proposal will be equally applicable to other cancers that a tumor associated protein is known.