Background: While the benefit of passive immunotherapy is commonly accepted, active immunization may have advantages for the patient’s quality of life. We identified a new epitope of Mab CH401 against Her-2/neu extracellular domain (N: 167-175), and evaluated the effect of active immunization of the 20mer peptide containing the epitope (CH401 peptide). Materials and Methods: Epitopemapping was performed using ELISA with Her-2/neu-related multiple antigen peptides (MAP). BALB/c mice were transplanted with Her-2/neu-expressing lymphoma cell line and immunized with the peptides. For monitoring the condition, ELISA and flow cytometry was performed. Results: CH401 peptide induced Her-2/neu-specific IgG antibody. Tumor growth in immunized mice was suppressed and tumorinfiltrating lymphocytes comprised more CD8+ T-cells, which secreted larger amounts of interleukin-2 after the peptide restimulation. Conclusion: The new Her-2/neu peptide contained epitopes for CD4+ and CD8+ T-cells, which contributes to the suppressive effect on Her-2/neu-expressing tumor cell growth. Molecular targeting reagents have been extensively developed for cancer therapy. Among them, antibodies against tumor-associated antigen (TAA) are considered to be powerful tools through their multiple functions, such as antibody-dependent cell cytotoxicity and blocking of signals for cell proliferation (1-3). The antitumor effect of anti-TAA antibody has been investigated in two ways: via passive immunization with monoclonal antibody (Mab) and active immunization with TAA vaccination (4, 5). However, the processes including the clinical application of Mab therapy require huge amounts of antibodies, and consequently may impose considerable physiological, psychological and financial burdens upon the patients. As for the strategy for overcoming the disadvantage of passive immunization with Mab, active immunization with tumor-specific antigen may avoid adverse effects induced by transfusing large amounts of antibodies repeatedly. However, most of the trials with immunization of whole TAA molecules have not been successful (4), partially because the antibodies sometimes function positively for cancer growth (6-9). Thus, peptides composed of limited amino acid sequences, which do not produce tumor-promotory antibodies, may be useful for effective antitumor antibody production. In past studies, however, the B-cell epitope was tentatively estimated simply from the biochemical/structural nature of the antigen, which is still problematic because epitopes may include the region agonistic to tumor growth (10, 11). Otherwise, B-cell epitopes or mimotopes were artificially found by screening with phage display (12-14). Thus, identifying an epitope of TAA-specific monoclonal antibody, which is already characterized to induce an antitumor effect, and designing a peptide that includes the epitope sequence might be an effective protocol for the peptide design of anti-TAA vaccination. In contrast, T-cell epitope, particularly of CD8+ cytotoxic Tcells (CTL), has been well investigated since the characterization/identification of TAA (15). The techniques for purifying peptide presented on major histocompatibility complex (MHC) class I and II molecules have identified numerous anchoring peptides and categorized (15, 16). 3361 Correspondence to: Yoshie Kametani, Ph.D., Department of Immunology, Tokai University School of Medicine, 143, Shimokasuya, Isehara-shi, Kanagawa, 259-1193, Japan. Tel: +81 463931121 ext. 2589, Fax:+81 463942976, e-mail: y-kametn@is.icc.u-tokai.ac.jp

BACKGROUND:While the benefit of passive immunotherapy is commonly accepted, active immunization may have advantages for the patient's quality of life. We identified a new epitope of Mab CH401 against Her-2/neu extracellular domain (N: 167-175), and evaluated the effect of active immunization of the 20mer peptide containing the epitope (CH401 peptide).MATERIALS AND METHODS:Epitope-mapping was performed using ELISA with Her-2/neu-related multiple antigen peptides (MAP). BALB/c mice were transplanted with Her-2/neu-expressing lymphoma cell line and immunized with the peptides. For monitoring the condition, ELISA and flow cytometry was performed.RESULTS:CH401 peptide induced Her-2/neu-specific IgG antibody. Tumor growth in immunized mice was suppressed and tumor-infiltrating lymphocytes comprised more CD8(+) T-cells, which secreted larger amounts of interleukin-2 after the peptide re-stimulation.CONCLUSION:The new Her-2/neu peptide contained epitopes for CD4(+) and CD8(+) T-cells, which contributes to the suppressive effect on Her-2/neu-expressing tumor cell growth.

We discuss scaling the equivalent oxide thickness (EOT) of Hf-based high-k gate dielectrics by post-deposition annealing (PDA). Thin HfON/SiON gate stacks with EOT=0.57 nm were successfully formed by repeating ultra thin (0.6 nm) HfO2 deposition and high-temperature (950 °C) PDA on a previously formed SiON interfacial layer. Physical and electrical analyses revealed that the reduction in EOT was due to crystallization of HfON to the tetragonal phase which has a higher dielectric constant than the amorphous and other crystalline phases. It was also found that Hf diffusion in the SiON interfacial layer was induced by the high-temperature PDA treatment. This also improved the k-value of the interfacial layer and enabled aggressive scaling even when using a SiO2-based interfacial layer. The electron mobility of the gate stack is higher than those in the previous reports, indicating that a high quality interface is realized using this approach. The reduction in EOT together with the excellent interfacial quality demonstrated in the present study shows that this technique is a promising solution for the 22-nm-node and beyond.

Peptides which mimic functional activities of glycosphingolipids were prepared by a technology of phage-displayed peptide library using monoclonal antibodies against glycosphingolipids. These peptides were named glyco-replica peptides. Peptides prepared with anti-GD1alpha antibody by this technology were found to contain WHW as common motif, and they showed suppressive activity not only on adhesion between hepatic sinusoidal endothelial cells and lymphosarcoma RAW117-H10 cells, but also on metastasis of the tumor cell to the liver and lung. The WHW motif seems to be important to mimic the functional activity of the ganglioside GD1alpha. Next, we prepared GD3-replica peptides using a monoclonal antibody against GD3 (4F6). A peptide, GD3-P4 with highest affinity to 4F6 was used to immunize mice to examine if the mice show their immune response to raise antibodies against GD3. We confirmed the immune response and succeeded in the production of a monoclonal antibody (3D2) against GD3. The monoclonal antibody 3D2 showed specific binding to GD3 on a thin-layer chromatography plate and also melanoma tissues. Interestingly, the amino acid sequence of the CDR regions of light and heavy chains showed high similarity with those of the original GD3 monoclonal antibody (4F6) used for the preparation of GD3-replica peptide. The technology of the phage-displayed peptide library was applied to in vivo bio-panning study using an angiogenesis experimental model. The obtained peptides were found to show strong binding property to the neo-vasculature system and to be quite useful to carry an anti-tumor drug to the tumor tissue. Based on these experimental results, we discuss about some applications of this method to drug discovery.

Dai Ishikawa, Satoshi Kamiyama, Etsuo Kurosawa, Takayuki Aoyama, and Yasuo Nara Research Dept. 1, Semiconductor Leading Edge Technologies (Selete), Inc 16-1 Onogawa, Tsukuba-city, Ibaraki, 305-8569, Japan E-mail: ishikawa.dai@selete.co.jp Tel: +81-29-849-1261

Fabrication and application of nano-sized noble metal particles are introduced for newly, ecology and economy system. In this method, noble metal oxides and alcohol as starting materials are low emission (LE) materials, and we use ultrasonic irradiation as reaction energy. We have reduced noble metal oxides by ultrasound and fabricated noble nano metal nanoparticles at room temperature, and investigated various applications. By choosing suitable process and conditions, it is reasonable to be expected that these ultrasonic fabrications can be extended to obtain simply various noble metal nanoparticles related materials at total eco-design.

For the successful integration of high-k gate dielectrics into advanced complementary metal-oxide-semiconductor (CMOS) processes, it is important to determine the stability of high-k materials during exposure to an ambient atmosphere. In this work, we investigated the effect of exposure to air on the nitrogen concentration in HfSiON films formed by sequentially combining HfSiO chemical vapor deposition (CVD), plasma nitridation, and postnitridation annealing (PNA). We observed that exposure to air after the nitridation step reduces the nitrogen concentration due to a reaction between the HfSiON surface and the constituents of atmospheric air. We also found that exposure to air for even a short time between nitridation and PNA leads to a significant loss of nitrogen concentration, indicating that in situ PNA is critical for achieving precise control of the nitridation. These results confirmed the importance of using clustered multichamber platforms for successful high-k fabrication.

2007 International Conference on Solid State Devices and Materials,Production-Worthy HfSiON Gate Dielectric Fabrication Enabling EOT Scalability Down to 0.86 nm and Excellent Reliability by Polyatomic Layer Chemical Vapor Deposition Technique

To clarify the mechanism of platelet production from megakaryocytes, expression of target proteins by gene transfection was examined using various gene delivery techniques. Transfection into hematopoietic cells, including megakaryocytes, by conventional gene delivery techniques such as electroporation and lipofection are known to be difficult. In this study, in addition to electroporation and lipofection, we tested other gene-transfer methods (nucleofection, transfection using inactivated virus envelope, and transferrin-linked cationic polymer) with the green fluorescent protein (GFP) gene into the human megakaryocytic cell line MEG-01. We found that nucleofection, which uses a combination of special electrical parameters and specific solutions, was the best, judging from the expression ratio of GFP-positive cells (approximately 70% of cells) and low toxicity. The efficiency of GFP expression was not related to the amount of pDNA delivered into the MEG-01 cells. To verify the utility of nucleofection, the thrombopoietin (TPO) receptor c-mpl was transfected into MEG-01 cells. Transfected cells showed a higher responsiveness to TPO than mock-transfected MEG-01 cells. We propose that nucleofection is a useful method for transfecting target genes to megakaryocytic cells when addressing the mechanism of platelet production.

GD3-replica peptides were obtained from a phage peptide library and an anti-GD3 monoclonal antibody (Mab) (4F6), and anti-GD3 Mabs were generated by immunizing a peptide GD3P4. A Mab, 3D2 was found to recognize GD3 by immunohistochemical approaches. Amino acid analysis of heavy and light chain variable regions of 4F6 and 3D2 showed that the respective chains had the same length, and only a few different amino acid substitutions were found. The present data indicate that the immunogenic GD3P4 is processed in a certain size and exposed on the antigen-presenting cells with a molecular shape quite similar to that of the GD3 epitope in 4F6.

Objective. Numerous monoclonal antibodies have been developed for the purpose of medical treatments, including cancer treatment. For clinical application, the most useful are humanderived antibodies. In this study, we tried to prepare designed antigen-specific antibodies of completely human origin using immunodeficient mouse.Methods. Nonobese diabetic/severe combined immunodeficient/IL-2 receptor gamma null mouse (NOG) mouse was used to reconstitute the human immune system with umbilical cord blood hematopoietic stem cells (CB-NOG mouse) and to prepare human-derived Her-2-epitope-specific antibodies. Hybridoma lines were prepared by fusing the human myeloma cell line Karpas707H.Results. Serum of immunized NOG mouse contained human-derived immunoglobulin M (IgM) antibodies specific for a short peptide sequence of 20 amino acids, including the epitope peptide of apoptotic Her-2 antibody CH401. Hybridoma lines were successfully prepared with spleen B cells obtained from the immunized CB-NOG mouse. One of these cell lines produced human IgM against the epitope peptide that can recognize surface Her-2 molecule.Conclusion. We could produce human-derived IgM antibody against Her-2 epitope peptide in CBN-OG mouse, succeeding in generation of human hybridoma-secreting IgM against a given peptide. (c) 2006 International Society for Experimental Hematology. Published by Elsevier Inc.

Cancer chemotherapy targeted to angiogenic vessels is expected to cause indirect tumor regression through the damage of the neovasculature without the induction of drug resistance. To develop a tool for neovasculature-specific drug delivery, we isolated novel peptides homing to angiogenic vessels formed by a dorsal air sac method from a phage-displayed peptide library. Three distinct phage clones that markedly accumulated in murine tumor xenografts presented PRPGAPLAGSWPGTS-, DRWRPALPVVLFPLH- or ASSSYPLIHWRPWAR-peptide respectively. After the determination of the epitope sequences of these peptides, we modified liposomes with epitope penta-peptides. Liposome modified with APRPG-peptide showed high accumulation in murine tumor xenografts, and APRPG-modified liposome encapsulating adriamycin effectively suppressed experimental tumor growth. Finally, specific binding of APRPG-modified liposome to human umbilical endothelial cells, and that of PRP-containing peptide to angiogenic vessels in human tumors, i.e., islet cell tumor and glioblastoma, were demonstrated. The present study indicates the usefulness of APRPG-peptide as a tool for anti-neovascular therapy, a novel modality of cancer treatment.