Antennapedia transduction sequence promotes anti tumour immunity to epicutaneously administered CTL epitopes.

The identification of tumor antigens has spurred the development of efficient adjuvants and novel delivery systems for cancer immunotherapy. To this end, a peptide-based vaccine consisting of the Antennapedia transduction sequence (ANTP) attached to an antigenic peptide was designed to enhance per-cutaneous delivery into cells of the epidermis and dermis. Here we show that the topical application of OVA(257-264) linked to ANTP in mice onto tape-stripped skin resulted in enhanced delivery of the antigen through the skin whereas OVA(257-264) alone remained distributed uniformly on the skin surface. This delivery correlated with an increase in the CTL response against OVA. When mixed with CpG oligodinucleotides (ODN), the recombinant antigen protected mice from tumor challenge. These data provide the first indication that in vivo use of a translocation sequence can enhance delivery of therapeutic peptides and increase anti-tumor immunity through a simple and safe mechanism involving enhanced penetration of the skin barrier.

Effective induction of CD8+ T-cell response using CpG oligodeoxynucleotides and HER-2/neu-derived peptide co-encapsulated in liposomes.

CpG oligodeoxynucleotides (CpG ODN) have been shown to have potent adjuvant activity for a wide range of antigens. Of particular interest is their improved activity when closely associated with the antigen. The purpose of this study is to determine the potential benefit of liposomes as a co-delivery vehicle to enhance the adjuvant activity of CpG ODN for a HER-2/neu-derived peptide to induce CD8+ T-cell response. Immunization studies were performed to evaluate the effectiveness of the liposomal vaccine in BALB/c mice. Mice were immunized with p63-71 encapsulated in liposomes alone or in combination with CpG ODN, as well as p63-71 alone in saline or with peptide-pulsed dendritic cells (DC) as controls. Enzyme-linked immunospot assay (ELISPOT) assay was performed to measure the frequency of splenocytes secreting IFN-gamma as a means to determine the antigen-specific response. It was found that immunization using p63-71 co-encapsulated with CpG ODN within the same liposomes enhanced the antigen-specific IFN-gamma response by more than 100-fold when compared with mice immunized with p63-71 alone. Immunization using free CpG ODN plus p63-71 encapsulated in liposomes or p63-71 and CpG ODN encapsulated in separate liposomes could not achieve the same effect. Using CD8 as a second marker and intracellular flow cytometric analysis, it was found that the IFN-gamma response was contributed by CD8+ T-cells, confirming the induction of cytotoxic T-lymphocytes (CTL) by this vaccination method. This indicates that a close association of HER-2/neu peptide and CpG ODN inside liposomes enhances the CTL epitope delivery and induces CD8+ mediated immune response. These results suggest that a vaccinal approach using liposome delivery system carrying in self-tumoral epitope and CpG ODN as adjuvant may have important implications for cancer therapy.

Attaching histidine-tagged peptides and proteins to lipid-based carriers through use of metal-ion-chelating lipids.

The therapeutic potential of selected peptides and proteins is enormous, with applications ranging from use as therapeutic vaccines, as modulators of intracellular signaling pathways and as highly selective agents capable of recognizing unique extracellular targets. We have been pursuing development of hybrid lipid-based carrier formulations designed to take advantage of the therapeutic benefits of peptides selected for their ability to act in a complementary fashion with the carrier system. In this regard, it is critical to have simple and versatile methods to promote and control the binding of diverse peptides to a broad range of carrier formulations. As demonstrated here, recombinant proteins and synthetic peptides containing poly-histidine residues (4 to 10) can be specifically bound to liposomes containing a metal-ion-chelating lipid, DOGS-NTA-Ni. The potential of this approach is demonstrated using two functional peptides, AntpHD-Cw3 (applications for vaccine production) and AHNP (specificity for Her-2 expressing cells).

Liposomal delivery of CTL epitopes to dendritic cells.

The induction of strong and long lasting T-cell response, CD4+ or CD8+, is a major requirement in the development of efficient vaccines. An important aspect involves delivery of antigens to dendritic cells (DCs) as antigen presenting cells (APCs) for the induction of potent antigen-specific CD8+ T lymphocyte (CTLs) responses. Protein or peptide-based vaccines become an attractive alternative to the use of live cell vaccines to stimulate CTL responses for the treatment of viral diseases or malignancies. However, vaccination with proteins or synthetic peptides representing discrete CTL epitopes have failed in most instances due to the inability for exogenous antigens to be properly presented to T cells via major histocompatibility complex (MHC) class I molecules. Modern vaccines, based on either synthetic or natural molecules, will be designed in order to target appropriately professional APCs and to co-deliver signals able to facilitate activation of DCs. In this review, we describe the recent findings in the development of lipid-based formulations containing a combination of these attributes able to deliver tumor- or viral-associated antigens to the cytosol of DCs. We present in vitro and pre-clinical studies reporting specific immunity to viral, parasitic infection and tumor growth.