The overall goal of this revised application is to test immunogens and strategies of new conception for vaccination against MUC-1. MUC-1, a highly glycosylated mucin expressed on the luminal side of glandular epithelial, consists of 30-90 tandem repeats of 20 amino acids within which there exists an immunodominant epitope. During malignant transformation mucin is over-expressed, and mucin-producing cells lose luminal polarity with mucin becoming expressed all around the cell surface rendering tumor cells less susceptible to immune effector mechanisms and favoring apoptosis of T cells. Vaccination against MUC-1 is intended at generating two effects: (1) antibodies of sufficient specificity and avidity could cause redistribution (via cross-linking and capping) of the MUC-1 antigen at the surface of tumor cells hence reinstating the full immunological potential of the host against tumor cells expressing MUC-1; (2) T cells with cytotoxic activity that kill mucin-expressing tumor cells, particularly micro-metastasis. The vaccination experiments proposed herein are based on methods and principled developed in the laboratory and designed at maximizing the immunogenicity of MUC-1 oligopeptides by expressing them in the hyper-variable loops of antibodies, i.e., structures with limited flexibility anchored into the conserved immunoglobulin fold. This approach allows for their expression as conformationally-constrained units, a necessary prerequisite for the induction of biologically active B cell responses. Heterologous epitopes expressed in hyper-variable loops of antibodies are also site of preferential intracellular cleavage so that peptides for the activation of T cells are easily generated. Combined with the advantages of this pro protein engineering approach will be the use of a newly-developed method of DNA-based vaccination (Somatic Transgene Immunization), a method through which the genes of antibodies coding for MUC-1 epitopes are themselves utilized in vivo as vaccines to induce both antibodies and T cells. Studies will be done in normal C57B16 mice, in human MUC-1 transgenic mice to evaluate the limitations (if any) proposed by self tolerance, and in mice knock-out for the alpha (1->3) galactosyl transferase to determine if naturally-occurring antibodies (which are present in these KO mice) limit the extent to which specific cellular responses can be induced by vaccination. The efficacy of this type of vaccination will be evaluated in experiments of tumor protection (prevention or down-regulation of growth) in normal as well as transgenic mice.
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