Identification of antigenic peptides expressed on cancer cells enables us to treat cancer patients with peptide-based immunotherapy. Although optimal protocols for peptide-based vaccines have not yet been elucidated, boosting the immune system could be a better approach than priming the immune system to elicit prompt and potent peptide-specific T-cell responses in cancer patients. With this possibility in mind, the authors undertook a clinical trial in which cancer patients were vaccinated with peptides (maximum 4) after confirmation of pre-existing peptide-specific cytotoxic T-lymphocyte (CTL) precursors in the periphery. Fourteen patients (seven with melanoma and seven with other types of cancer) positive for either HLA-A24 or HLA-A2 were enrolled in this study. Fourteen and 16 peptides were used to screen for HLA-A24+ and HLA-A2+ patients, respectively. The vaccination was well tolerated, and the only adverse effects were local pain and fever. Kinetic analysis revealed that peptide-reactive CTLs increased after peptide vaccination in 7 of 14 patients. Immunoglobulin G (IgG) reactive to the administered peptides was detected in 2 patients before vaccination, although it became detectable in 8 of the other 12 patients after the peptide vaccination. Stable disease for more than 6 months was observed in five patients (one with melanoma and four with other types of cancer); all of these patients showed increased levels of peptide-specific IgG. These results indicate that peptide vaccination of patients showing evidence of pre-existing peptide-specific CTL precursors can be applied in further clinical trials aimed at the treatment of melanoma and other types of cancer.

Cyclophilin B (CypB) possesses two antigenic epitopes (CypB(84-92) and CypB(91-99) ) recognized by HLA-A24-restricted and tumor-specific cytotoxic T lymphocytes (CTLs). To determine the safety of CypB-derived peptides and its ability to generate antitumor immune responses, patients with advanced lung cancer received subcutaneous vaccinations of these peptides or their modified peptides. All 16 patients were vaccinated with CypB(91-99) or its modified peptide, whereas only two patients were vaccinated with the modified CypB(84-92), as immediate-type hypersensitivity to CypB(84-92) or its modified peptide was observed in the remaining patients. No severe adverse events were associated with the vaccination. No significant increase in cellular responses to either peptides or tumor cells was observed in the postvaccination PBMCs by the conventional CTL assays in any patients tested. These results suggest that the vaccination of CypB(91-99) peptide was safe, but failed to induce objective immune responses at this regimen.

Nuclear lamins are a type of intermediate filament (IF) proteins. They have a characteristic tripartite domain structure with a alpha-helical rod domain flanked by non-alpha-helical N-terminal head and C-terminal tail domains. While the head domain has been shown to be important for the formation of head-to-tail polymers that are critical assembly intermediates for lamin IFs, essential structural elements in this domain have remained obscure. As a first step to remedy this, a series of mouse lamin A mutants in which the head domain (30 amino acid residues) was deleted stepwise from the N-terminus at intervals of 10 residues were bacterially expressed. The assembly properties in vitro of the purified recombinant proteins were explored by electron microscopy. We observed that while a lamin A mutant lacking N-terminal 10 residues formed head-to-tail polymers, a mutant lacking N-terminal 20 residues or the whole head domain (30 residues) showed significantly decreased potency to form head-to-tail polymers. These results suggest that the last 20 residues (from Arg-11 to Gln-30) of the head domain of mouse lamin A contain essential structures for the formation of head-to-tail polymers. The last 20 residues of the head domain include several conserved residues between A- and B-type lamins and also the phosphorylation site for cdc2 kinase, which affects lamin IF organization in vivo and in vitro. Our results provide clues to the molecular mechanism by which the head domain plays a crucial role in lamin polymerization.

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The amino-terminal head domain of vimentin is the target site for several protein kinases and phosphorylation induces disassembly of the vimentin intermediate filaments in vivo and in vitro. To better understand molecular mechanisms involved in phosphorylation-dependent disassembly, we examined domain interactions involving the head domain and the effect of phosphorylation on the interaction, using surface plasmon resonance. We observed that the head domain binds to the carboxyl-terminal helix 2B in the rod domain, under physiological ionic strength. This interaction was interfered with by Akinase phosphorylation of the head domain. Deletion of the carboxyl-terminal 20 amino acids of helix 2B resulted in loss of the interaction. Furthermore, peptide representing the carboxylterminal 20 residues of helix 2B had a substantial affinity with the head domain but not with the phosphorylated one. These findings support the idea that the interaction between the head domain and the last 20 residues of helix 2B is essential for association of vimentin tetramers into the intermediate filaments and that the phosphorylation-dependent disassembly is the result of loss of the interaction. ß