Signaling through ephrin-A ligand leads to activation of Src-family kinases, Akt phosphorylation, and inhibition of antigen receptor-induced apoptosis.

Eph receptor tyrosine kinases and ephrins play important roles in diverse biological processes such as migration, adhesion, and angiogenesis. Forward and reverse signaling has been reported in receptor- and ligand-bearing cells. The ligands can be divided into the transmembrane ephrin-B family and the GPI-anchored ephrin-A family. Here, we show expression of ephrin-A ligands on CD4+ T cells cultured in medium with human serum and the T cell line Jurkat TAg and on cells isolated from patients with T cell lymphomas and T cell leukemias. Functional role and identification of proteins involved in ephrin-A signaling were investigated here in the T cell line Jurkat TAg. Signaling through ephrin-A induces phosphorylation of several proteins, including the Src kinases Lck and Fyn. In addition, PI-3K is activated, shown by induced phosphorylation of the Akt kinase. An ephrin-A signaling complex could be isolated, containing several phosphorylated proteins including Lck and Fyn. Interestingly, we show that signaling through ephrin-A in Jurkat TAg cells, initiated by interaction with the EphA2 receptor, leads to inhibition of activation-induced cell death. To conclude, ephrin-A signaling in Jurkat TAg cells leads to induced phosphorylation of several proteins including Lck, Fyn, and Akt. A consequence of ephrin-A signaling is inhibition of antigen receptor-induced apoptosis.

Mucosal vaccination with phage-displayed tumour antigens identified through proteomics-based strategy inhibits the growth and metastasis of 4T1 breast adenocarcinoma.

Cancer immunotherapy relies on the identification and characterization of tumour antigens that can be recognized by effector T cells. Here, we used a proteomics-based approach to identify tumour antigens recognized by serum antibodies from patients with breast cancer. Specific reactivity against a set of spots was identified and their identity was revealed by MALDI-TOF peptide mass fingerprinting. They include disintegrin and metalloprotease 10, aldolase A, beta-ATPase F1, heat shock protein 27, deaminase, pyruvate dehydrogenase protein X component, and Vimentin. Western blot analysis using recombinant proteins expressed in E. coli confirmed the specific reactivity with patient sera. Several tumour antigens were expressed on the surface of the T7 phage and shown to trigger specific immune responses in BALB/c mice following oral immunisation. Furthermore, these immune responses inhibited tumour growth and metastasis of the 4T1 mammary adenocarcinoma cell line. Collectively, the present data indicate that proteomics-based strategy can identify tumour antigens whose surface display on phages or bacteria can provide an effective strategy for mucosal cancer vaccines. In addition, arrayed phage-displayed tumour antigens could be useful as a serum-based screening test for the detection of several tumour antigens.

Selection of peptides for specific delivery of oligonucleotides into cancer cells.

In this chapter, phage-display peptide technology has been used to select peptides that internalize into breast-cancer cells. The used biopanning procedure provides information on the best peptide to be used. When one of the selected peptides was conjugated to an antisense oligonucleotide against the ErbB2 receptor, specific delivery to breast-cancer cells was demonstrated. The established biopanning procedure should help in the rational selection of cancer-targeting peptides for specific delivery of DNA and RNA oligonucleotides into cancer cells.

Identification of novel carrier peptides for the specific delivery of therapeutics into cancer cells.

Cancer therapy is currently limited by the difficulty of achieving efficient delivery into target cells. To investigate whether therapeutics can be delivered specifically to cancer cells, we have explored the possibility of selecting small peptides that bind specifically, or preferentially, to breast cancer cell lines. By using random peptide phage libraries and an experimental approach that allows the selection of internalized peptides, cell-specific binding peptides have been identified. The peptides define a major core motif (LTVXPWY) that was not found in negative phages. Phage displaying LTVSPWY peptide sequence exhibited a specific binding to breast cancer cells. None of the selected peptides bound to human primary cells from different tissue origin (e.g., epithelial, endothelial, hematopoetic). The potential of the selected peptides to mediate cellular internalization in the context of phages and recombinant GFP-peptide fusions was demonstrated. By linking the LTVSPWY peptide to an antisense phosphorothioate oligonucleotide against the ErbB2 receptor, specific delivery to cancer cells was achieved. In contrast to free antisense, the peptide-antisense conjugates inhibited ErbB2 gene expression. Thus, efficient delivery of antisense oligonucleotides can be achieved by coupling them to cancer cell-specific peptides, identified by a method that did not require any knowledge about their corresponding receptors.

Selective targeting of cancer cells using synthetic peptides.

To establish efficient and reliable therapeutic delivery into cancer cells, a number of delivery agents and concepts have been investigated in the recent years. Among many improvements in targeted and controlled delivery of therapeutics, cell-targeting peptides have emerged as the most valuable non-immunogenic approach to target cancer cells. Peptides can be incorporated into multicomponent gene-delivery complexes for cell-specific targeting. In contrast to larger molecules such as monoclonal antibodies, peptides have an excellent tumor penetration, which make them ideal carriers of therapeutics to the site of primary tumor and the distant metastatic sites. Here we give an update on the progress made during the last two years on the identification and potential of specific synthetic tumor targeting peptides.