Whole protein and defined CD8 + and CD4 + peptides linked to penetratin targets both MHC class i and II antigen presentation pathways

Cytoplasmic delivery and cross-presentation of proteins and peptides is necessary for processing and presentation of antigens for the generation of cytotoxic T cells. We previously described the use of the 16 amino acid peptide penetratin from the Drosophila Antennapedia homeodomain (penetratin, Antp) to transport cytotoxic T lymphocyte epitopes derived from ovalbumin (OVA) or the Mucin-1 tumor-associated antigen into cells. We have now shown that penetratin covalently conjugated to OVA protein and linked in tandem to CD4+ and/or CD8+ T-cell epitopes from OVA-stimulated T cells in vitro (B3Z T-cell hybridoma and OT-I and OT-II T cells). The induction of these responses was directly mediated by the penetratin peptide as linking a nonspecific 16-mer peptide to OVA or mixing did not induce CD8+ or CD4+ T-cell responses in vitro. Furthermore, interferon (IFN)-γ-secreting CD4+ and CD8+ T cells were induced which suppressed B16.OVA tumor growth in C57BL/6 mice. Tumor protection was mediated by a CD8+ T-cell-dependent mechanism and did not require CD4+ help to protect mice 7 days after a boost immunization. Alternatively, 40 days after a boost immunization, the presence of CD4+ help enhanced antigen-specific IFN-γ-secreting CD8+ T cells and tumor protection in mice challenged with B16.OVA. Long-term CD8 responses were equally enhanced by antigen-specific and universal CD4 help. In addition, immunization with AntpOVA significantly delayed growth of B16.OVA tumors in mice in a tumor therapy model.