Research On Tumor-therapeutic Peptide Vaccine Modified With Cell-penetrating Peptide Or Endoplasmic Reticulum Retention Signal

The antigen specific CD8+ cytotoxic T lymphocytes (CTLs) are thought to play a key role in tumor immunotherapy. The stimulation of specific CTL therefore represents one major goal in the design of vaccines for tumor immunotherapy. CTLs recognize short peptides of 8~10 amino acids (derived from tumor-associated antigen (TAA), known as CTL epitopes) in the context of Major Histocompatibility Complex (MHC) class I molecules on the surface of antigen-presenting cells (APCs) such as malignant cells. As the result of the specific interaction between the T cell receptor (TCR) and MHC class I /peptide complexes, CTLs are able to kill target cells expressing TAAs. In consequence, manufacturing these TAAs derived-epitopes by conventional organic peptide synthesis methods is relatively simple, expedient, and cost-effective way to produce safe, therapeutic tumor vaccines. Identification of TAAs and CTL epitopes provides more opportunities for the development of synthetic peptide vaccines. Unfortunately, peptide vaccines are always unsuccessful at stimulating CTL response, either because of their inherent lack of immunogenicity, or because of their rapid biodegradation, or because of poor uptaken by APCs.Generally, the majority of MHC class I-associated peptides are derived from the cytosolic degradation of endogenous protein. Exogenous proteins, however, enter the MHC class II-presentation pathway through endocytosis and are degraded in lysosomes, thus the resulting peptides generally do not contribute to MHC class I-associated presentation. A few of exogenous soluble antigens can find their way to associate with MHC class Imolecules (i.e., "cross-presentation"), but this is not an effective way to activate CTL. This basic dichotomy explains why antigen-specific CD8+ CTLs are generated most effectively against intracellular antigens or when immunogens are delivered into the cytosol, e.g., using viral vectors, and why immunization with soluble proteins or peptides rarely induces CTL.In the case of peptide-based vaccines, the way of delivering exogenous peptides into the MHC class I presentation pathway is critical to elicite CTL responses. On the one hand, an appropriate delivery system may represent a method to transport exogenous antigenic peptides into the cytosol of APCs. Over the past few years, there has been an increased interest in using cell-penetrating peptides (CPP) to transport exogenous molecules into living cells. Several CPPs have been identified from proteins, including antennapedia protein of Drosophila, VP22 proteins of herpes simplex virus and Tat protein of HIV-1. Among them, the short carriers derived from Tat protein have been well studied for the transduction of biologically active proteins into cells both in vitro and in vivo. The region of Tat necessary for translocation has been identified as a short basic sequence corresponding to residues 49~57 (RKKRRQRRR). We hypothesized that exogenous synthetic peptides fused with HIV-Tat49-57 sequence could efficiently cross the cell membrane and be transported rapidly from the extracellular milieu into the cytosol of most cells, and then directly enter into classic MHC class I presentation pathway.On the other hand, the association of antigenic peptides with MHC class I molecules in the ER is, in some respects, a curious evolutionary choice, because nascent peptide-receptive MHC class I molecules and molecules chaperones such as immunoglobulin heavy chain-binding protein (BiP), calnexin, calreticulin and Tapasin, which are beneficial for peptide assembling with MHC molecules, are present in the ER at very high concentration. Thus an appropriate antigen delivery system may also represent a method to increase the number of defined epitopes in the ER lumen. It's well known that an important step in MHC class I presentation pathway is the translocation of processedproteins from the cytosol across the endoplasmic reticulum (ER) membrane mediated by transporter associated with antigen processing (TAP). Functional TAP is required for the optimal assembly of...