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Research Grants Awarded
Penetratin-Based Vaccines for the Treatment of Breast Cancer
Tumor Cell Biology III
Background: We have utilized a membrane translocating peptide (Int) to deliver cytotoxic T cell epitopes (CTL) into antigen presenting cells (APC). We have showed that a tandem peptide consisting of the Int with an ovalbumin (OVA) or mucin 1(MUC1) CTL epitope was rapidly taken up by (APC) and entered the class I Ag presentation pathway. Mice immunized with a single dose of these peptides were protected from OVA +ve or MUC1 +ve tumors. Objective/Hypothesis: To investigate the utility in human in vitro cell culture systems of the delivery system based on Int that has already been demonstrated to be a potent method to generate both CD4 and CD8 T cell responses in in vitro and in vivo mouse models. Specific aims: Produce rec (rec) human MUC1 and hTERT proteins. Prepare novel Ag constructs of Int to rec protein, peptides incorporating Int with the various cytotoxic/helper T cell epitopes and rec fusion proteins incorporating Int. Investigate the presentation of the Int linked proteins and peptides to Ag-specific T cell clones by human APC. Study the mechanism of Int linked peptide and protein presentation by human APC. Compare the Int mediated delivery of Ag to human APC with oxidized mannan mediated delivery. Study Design: Aim 1 and 2. We intend to produce various forms of rec MUC1 and hTERT for conjugation to Int. We will also produce rec proteins that have an Int fused to the N- or C-terminal of the tumor Ags and also make synthetic tandem peptides of the known CTL epitopes of MUC1 and hTERT. Aim 3. To ascertain if human APC can process and present CTL epitopes delivered using Int, we will utilize Ag-specific T cell clones. hTERT T cell clones will be used to measure Ag presentation using cellular and cytokine assays. We will also utilize HLA-A2, HLA-DR 1, 3 and 4 transgenic mice to investigate processing of tandem peptides incorporating CD8 epitopes as well at CD4 epitopes of tumor Ags. Aim 4. Various biochemical inhibitors will be used to decipher the Ag presentation pathway. Aim 5. We will compare Int mediated delivery with a mannan-based Ag delivery method using similar assays to those used in aim 3. Potential outcomes and benefits of research: At the end of this project novel methodology will be available to deliver any tumor Ag or CTL peptide for breast cancer vaccine development. The major benefit of these studies is a vaccine for MUC1 expressing cancers, such as breast that can be administered by simple injection.
The incidence of cancers continues to rise, wherein 1 in 8 women will develop breast cancer. A large number of different strategies are being investigated in preclinical and clinical settings to develop new vaccines with none yet showing exceptional therapeutic effects in clinical trials. Several tumor associated antigens and their peptides that are recognized by our immune cells (T cells) have been identified and used as targets for immunotherapy. MUC1 is a transmembrane glycoprotein with a protein core consisting of a 20 amino acid variable number of repeat (VNTR) region and is frequently expressed by breast and other cancer cells in levels 10-100 times greater than on normal tissues. In this overexpressed form it is incompletely glycosylated and thus express novel antibody (Ab) and T cell epitopes not detected on normal cells. MUC1 is an important target for vaccines for breast cancer. Telomerase is a similar tumor associated antigen expressed on all types of cancers. To stimulate the immune system to recognize tumor antigens as foreign and destroy the cancers the tumor antigen must be delivered to the the cells known as dendritic cells (APC) that stimulate the T cells. We have previously demonstrated that when tumor antigens are delivered to APC in mice using a membrane translocating peptide they stimulate the mouse T cells to produce various hormones. When mice are immunized with these constructs the mice are protected from a challenge of breast cancer cells In this project we will investigate if the constructs that we generated can be processed in human APC in the same way they were processed in mouse APC to stimulate human T cell lines . We will make synthetic peptides that have the membrane translocating sequence and various peptides that can be recognized by T cells. These peptides will be chosen from the two tumor associated antigens MUC1 and hTERT. We will include peptides that can stimulate the T cells that kill tumors as well as peptides that can stimulate T cells that can help the killer T cells. By choosing different peptides from MUC1 and hTERT as well as peptides that will work in a broader population of breast cancer patients it will be possible to generate constructs that could be used as a vaccine. The end product is a vaccine for breast cancer that can be tested in future clinical trials.