2. CLINICAL SCIENCE

RESEARCH REPORT FOR JOINT PROJECT

  1. Title of Project:
    Evaluation of immunosurveillance mechanisms and its application to develop 2nd generation of tumor vaccine therapy protocol
  2. Duration: April 1, 2004 — March 31, 2006
  3. Project Organization
    1. Japanese Principal Investigator (JPI)
      Takashi Nishimura (Professor, Institute for Genetic Medicine, Hokkaido University)
    2. U.S. Principal Investigator (USPI)
      Lloyd J. Old (Director, Ludwig Institute for Cancer Research)
    3. Research Associate (Japanese / US)
      Takemasa Tsuji (Postdoctoral fellow, Institute for Genetic Medicine, Hokkaido University)
    4. List of Other Project Participants (except for PIs and RA)

      Japanese-side:
      Hiroaki Ikeda (Associate Professor, Inst. for Genetic Med. Hokkaido Univ., Div. of Immunoregulation )
      Hirotoshi Akita (Professor, Sch. of Med. Hokkaido Univ., Dept of Medical Oncology)
      Tsukasa Seya (Professor, Sch. of Med. Hokkaido Univ. Dept of Pathophysiological Science)
      Jun Yoshida (Professor, Nagoya Univ. Graduate Sch. of Med., Department of Neurosurgery)
      Tadatoshi Takahashi (Director, Aichi Cancer Center, Res. Inst., Div. of Immunology)
      Hiroshi Siku (Professor, Mie Univ. Sch. of Med., Second Dept. Internal Med.)
      Eiichi Nakayama (Professor, Okayama Univ. Graduate Sch. of Med., Dept. Immunology)
      Yutaka Kawakami (Professor, Inst. For Advanced Medical Res. Keio Univ. Sch. of Med. Div. of Cellular Signaling)
      Hideaki Tahara (Professor, Inst. of Medical Science, The University of Tokyo, Dept. Surgery and Bioengineering)
      Norimitsu Kadowaki (Associate Professor, Graduate Sch. of Med, Kyoto Univ., Dept. of Hematology and Oncology)
      Masaki Yasukawa (Professor, Ehime Univ. Sch. of Med., First Dept. of Internal Med.)
      Setsuya Aiba (Professor, Tohoku Univ. Graduate Sch. of Med., Dept. of Dermatology)
      Mari Kannagi (Professor, Graduate School, Tokyo Medical and Dental Univ., Dept. Of Immunotherapeutics)
      Kenji Chamoto (Ph.D. candidate, Inst. for Genetic Medicine Hokkaido Univ., Div. of Immunoregulation)
      Eiichi Sato (Assistant Professor, Tokyo Medical Univ., Diagnostic Pathology)
      Satoshi Kondo (Professor, Sch. of Med. Hokkaido Univ., Dept of Surgical Oncology)
      Masaki Miyamoto (Assistant Professor, Sch. of Med. Hokkaido Univ., Dept of Surgical Oncology)
      Koichi Yamazaki (Assistant Professor, Sch. of Med. Hokkaido Univ., Dept of Surgical Oncology)
      Kozo Imai (President, Sapporo Medical Univ.)
      Noriyuki Sato (Professor, Sapporo Medical Univ., Department of Basic Medical Science Dept.of Pathology)
      Yasuhihro Nagata (Assistant Professor, Nagasaki Univ., Sch. of Medicine, Dept.of Surgical Oncology)
      Kiyotaka Kuzushima (Chief, Aichi Cancer Center, Res. Inst., Div. of Immunology)
      Yoshiki Akatsuka (Project Leader, Aichi Cancer Center, Res. Inst., Div. of Immunology)
      Yuitchiro Sato (Associate Professor, Teikyo Univ. of Science and Technology, Dept. of Bioscience)

      US-side:
      Eric W. Hoffman (Director, Office of Clinical Trials Management, Ludwig Inst. for Cancer Res.,)
      Gerd Ritter (Associate Director, Ludwig Inst. for Cancer Res.)
      Sacha Gnjatic (Associate Member, Ludwig Inst. for Cancer Res.)
      Hiroyoshi Nishikawa (Research Fellow, Ludwig Inst. for Cancer Res.)
      Yao-Tseng Chen (Associate Professor, Weill Medical College of Cornell University Center for Immunology)
      Robert D. Schreiber (Professor, Washington Univ. Sch. of Med., Dept. of Pathology and Immunology)
      Esteban Celis (Professor, Mayo Clinic, Dept of Immunology)
      Rongfu Wang (Professor, Center for Cell and gene Therapy and Dept of Immunology, Baylor College of Med.)
      Steven J. Burakoff (Director, Skirball Inst. Biomolecular Med.)
      Andrew Sinpson (Ludwig, Inst. for Cancer Res.)
  4. Activities of Research Associate (RA)
    1. Duration in US: March 29, 2005- March 31, 2006
    2. Institution Visited in US: Ludwig Inst. for Cancer Res.
      Research Purposes: Immunological monitoring in Cancer vaccine trials Development of efficient cancer vaccine therapy
    3. Scientific Results/Achievements
      1. Analysis of CD4+ T cell response against tumor antigen peptide vaccination.
        Recently, it was demonstrated that spontaneous NY-ESO-1-specific CD4+ and CD8+ T cell response is detectable from blood lymphocytes of cancer patients who have antibody against NY-ESO-1 (S. Gnjatic, et al. Survey of naturally occurring CD4+ T cell responses against NY-ESO-1 in cancer patients: correlation with antibody responses. Proc Natl Acad Sci U S A. 2003; 100: 8862), (E. Jager, et al. Monitoring CD8 T cell responses to NY-ESO-1: correlation of humoral and cellular immune responses. Proc Natl Acad Sci U S A. 2000; 97: 4760). This suggests that tumor-specific helper T cells play a critical role in inducing tumor-specific antibody and tumor-specific cytotoxic T cells. On the other hand, Nishikawa et al. showed naive CD4+ T cell precursors specific for NY-ESO-1 exist in sero-negative cancer patient or healthy individuals, while their activation is strongly inhibited by CD4+CD25+ regulatory T cells. (H. Nishikawa, et al. CD4+CD25+ regulatory T cells control the induction of antigen-specific CD4+ helper T cell responses in cancer patients. Blood. 2005; 106: 1008) Therefore, the method to activate the precursor of tumor-specific helper T cells in vivo would become a powerful tool to enhance an anti-tumor immunity in the body. In the present study, we examined the activation of NY-ESO-1-specific helper T cells in the blood of cancer patients who received HLA class II binding peptide vaccination. It was found that peptide-specific helper T cells were induced in all vaccinated patients and these T cells were less-sensitive against the suppressive action of regulatory T cells. However, peptide vaccine-induced helper T cells were found to be low-avidity against antigen peptide and were not able to recognize naturally-processed antigen peptide. These results suggest that novel vaccination strategy, such as vaccination with tumor antigen protein, use of strong adjuvant, or efficient delivery systems, is need to generate high-avidity helper T cells that are able to recognize processed tumor antigen peptide presented on tumor cells or antigen presenting cells
      2. Analysis of immuno-stimulating activity of synthetic or natural compounds using monocyte-derived dendritic cells.
        Some microbes such as Bacillus Calmette-Guerin (BCG) or tuberculosis have been known to stimulate immune system and they were utilized as anti-cancer reagent. Recently, toll-like receptors (TLR) were found to be responsible for the activation of innate immune cells by microbes and many synthetic and natural compounds that activate immune cells through TLR were identified. Dendritic cells (DC) are the most potent antigen presenting cells and the activation of DC is a critical factor in inducing antigen-specific T cell response. Because DC were known to express various TLR family molecules, we compared the stimulating activity of known TLR ligands against dendritic cells. Monocyte-derived DC were reported to express TLR 1, 2, 3, 4, 5, 6, and 8. (Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nat Immunol. 2004; 5: 987) As expected, activation of monocyte-derived DC by these TLR ligands induced up-regulation of various cell surface molecules such as HLA-class II, CD80, CD83, CD86. However, in response to peptidoglycan (TLR2 ligand), LPS (TLR4 ligand) or poly(I:C) (TLR3 ligand), DC produce both IL-12 and IL-10, which stimulate Th1 and Th2 immune responses, respectively. On the other hand, zymosan, which is a ligand for TLR2/6 heterodimer, induced only IL-10 production from DC. Similarly, DC showed distinct cytokine production activity against various microbes. Induction of Th1-type immune response is important for tumor immunotherapy. These results suggest that it is important to compare cytokine responses against synthetic or natural compounds, or microbes for the development of strong adjuvant which induce more Th1-cytokine and less Th2-cytokine.

        Publication
        Nishikawa H, Qian F, Tsuji T, Ritter G, Old LJ, Gnjatic S, Odunsi K. Influence of CD4+CD25+ regulatory T cells on low/high-avidity CD4+ T cells following peptide vaccination. J. Immunol., in press.
  5. Number of Exchanges during Project Period
    1. from Japan to US
      Takemasa Tsuji:
      2004.7.19-2004.7.24: Canada, Montreal, 12th International Congress of Immunology
      2004.10.3-2004.10.6: USA, N.Y., Ludwig, Inst. for Cancer Res.
      Kenji Chamoto:
      2004.7.19-2004.7.24: Canada, Montreal, 12th International Congress of Immunology
      Hiroaki Ikeda:
      2004.10.3-2004.10.8: USA, N.Y., Ludwig, Inst. for Cancer Res.
      Takashi Nishimura:
      2004.12.8-2004.12.12: USA, N.Y., Ludwig, Inst. for Cancer Res
    2. from US to Japan
      Robert D. Schreiber:
      2004.3.9-2004.3.14: Hokkaido University
      Hiroyoshi Nishikawa:
      2004.8.3-2004.9.3: Mie University
      Hiroyoshi Nishikawa:
      2005.8.1-2005.8.24: Mie University
  6. Purpose and Content of Project
    Purpose
    The purpose of this project is to establish 2nd generation of tumor vaccine therapy protocol that facilitates the generation of Th1-dependent tumor-specific CTL in tumor patients.
    Tumor vaccine therapy using MHC class I-binding peptide has been tried in the world. Although tumor-peptide therapy provided a great impact that immunotherapy is one of the effective ways to treat cancer, it is not still sufficient protocol to conquer tumors. Namely, while clinical studies have indicated that class I-binding TRA peptides can elicit antigen-specific tetramer-positive CTL in vivo, the increased frequency of CTL did not lead to tumor rejection in these patients. Therefore, it is necessary to develop 2nd generation of tumor vaccine therapy that can overcome a strong immunosuppression in tumor-bearing hosts. The close communication among many cancer immunologists engaged in immunosurveillance work and clinical trial is essential to develop a novel 2nd generation of tumor vaccine protocol.

    Content of project
    1. Basic study on cancer immunosurveillance
      1. Evaluation of immunosurveillance mechanisms in tumor-bearing hosts.
        Cellular and molecular mechanisms in tumor-bearing hosts is evaluated in both animal and human system. Especially, the significance of immunoediting concept in tumor immunology and a precise mechanisms of immunosuppression in tumor-bearing hosts should be evaluated.
        [(Immunoediting, Schriber and Ikeda (Washington Univ.), Nishimura (Hokkaido Univ.; Immunosuppression, Shiku (Mie Univ.), Wang (Baylor College of Med.), Nishikawa, Gnjatic, Ritter and Old (Ludwig Inst.))
      2. Development of an efficient tumor vaccine protocol in animal models
        An efficient vaccine protocol for induction of tumor-specific CTL should be established. [(Nishimura (Hokkaido Univ.), Shiku (Mie Univ.), Nishikawa, Gnjatic, Ritter and Old (Ludwig Inst.))
    2. Application of 2nd generation of tumor vaccine therapy into clinical trial
      Dr. Lloyd J Old is a pioneer of tumor-immunology and found a novel tumor antigen, NY-ESO-1, which is distributed on a variety of tumor cells including colon, breast, esophagus, stomach, melanoma, B cell lymphoma. We initially evaluated the precise role for Th1/Th2 cells in tumor immunity and have proposed that Th1-dominant immunity is essential to overcome immunosuppression of tumor-bearing animals. Based on these basic evidences, we will aim to develop a novel tumor vaccine therapy that facilitates the activation of Th1-dependent immunity in tumor-patients using NY-ESO-1 protein and some Th1-activating adjuvant. As a Th1-activating adjuvant, CpG and Th1 cells will be applied to clinical trial. Tumor vaccine therapy using NY-ESO-1 protein and CpG encapsulated in liposome will be applied to melanoma, colon and B cell lymphoma patients. Tumor vaccine cell therapy using NY-ESO-1 protein and Th1 cells will be applied to melanoma patients. The project mainly will be carried out by the collaborative works between Hokkaido University, Nagoya University and Ludwig Inst. for Cancer Research.
      (i) Tumor vaccine therapy using NY-ESO-1 and CpG encapsulated with liposome will be applied to clinical trial. [(Nishimura and Shimizu (Hokkaido Univ.), Yoshida (Nagoya Univ.), Yasukawa (Ehime Univ.), Nishikawa, Gnjatic, Ritter and Old (Ludwig Inst.))
      (ii) Tumor vaccine cell therapy using NY-ESO-1 and Th1 cells will be applied to clinical trial. [(Nishimura and Shimizu (Hokkaido Univ.), Yoshida (Nagoya Univ.), Nishikawa, Gnjatic, Ritter and Old (Ludwig Inst.))
    3. Seminar
      Hold a seminar entitled "Japan-US collaborative meeting for cancer immunosurveillance and cancer vaccine clinical trials" in Sapporo, Japan. Promote the basis of basic research of cancer immunology and its clinical application in Japan, and establish the collaboration of US and Japan on its issue.
    4. Exchange of a research associate
      A research associate, Takemasa Tsuji visits Ludwig Institute for Cancer Research LICR) in US. He contributes to the collaboration of institutes in Japan and LICR that is essential for this project. He performs the immunological monitoring of cancer vaccine clinical trials, and develops an new and efficient cancer vaccine therapy.
  7. Status Report of Project Implementation
    Status in 2004
    1. Evaluation of immunosurveillance mechanisms in tumor-bearing hosts.
      Schreiber and Ikeda identified candidate genes that are the targets for the cancer immunoediting process by comparing the gene expression profiles of tumors derived from immunocompetent versus immunodeficient mice. Started to evaluate the molecules and cells involved in cancer immunoediting process other than IFN-γ. Shiku and Nishikawa fond that immunization with SEREX defined self antigen resulted into the activation of regulatory T cells by itself, but helper T cells when immunized with a CTL epitope.
    2. Development of an efficient tumor vaccine protocol in animal models
      Nishimura evaluated the importance of helper T cells in anti-tumor immunity in mouse model, and established the model system of immunotherapy utilizing CpG, liposome, and OVA as an model antigen. By this model, it is suggested that this protocol can show much better efficacy compared to the existing peptide vaccine protocols of cancer immunotherapy.
    3. Application of 2nd generation of tumor vaccine therapy into clinical trial
      Old and the members in LICR established the system to supply GMP level recombinant NY-ESO-1 protein. Yoshida in Nagoya Univ. established the system to supply the GMP level liposome. Nishimura and Ikeda developed a cancer vaccine clinical trial protocol with NY-ESO-1 protein /CpG/liposome in Hokkaido University Hospital. This protocol entered the process of evaluation in clinical trial committee in LICR. In Hokkaido University, a project team for cancer vaccine and cell therapy was established by Institute for Genetic Medicine and 10 clinical departments in Hokkaido University Hospital. The screening of the expression of tumor antigens in patients was performed in Hokkaido University Hospital that includes 157 lung cancers, 17 esophageal cancer, 20 breast cancers, 31 stomach cancers, 10 colon cancers, and 18 bill-duct cancers. NY-ESO-1 was expressed in 29% of esophageal cancer, 30% of breast cancer, and 8.3% of lung cancer. MAGE-A4 was expressed in 29% of lung cancer, 59% of esophageal cancer, 33% of colon cancer, and 17% of bill-duct cancer. In lung cancer patients, the expression of MAGE-A4 tumor antigen and the infiltration of T cells showed prognostic significance (Int J Oncology, 2006).
    4. Exchanges
      Takemasa Tsuji and Kenji Chamoto visited 12th International congrsss of Immunology, Montreal, Canada from July 19 to 24, 2004, and presented the results and achievements of this project in Hokkaido University. Hiroaki Ikeda and Takemasa Tsuji attended Cancer Vaccine 2004 annual meeting of Cancer research Institute in NY, USA from Oct. 3 to 8 2004, and visited LICR. They discussed about this project with members of LICR. Takashi Nishimura visited LICR in NY, USA from Dec. 8 to 12 2004, and discussed about this project with Old and the members of LICR.

    Status in 2005
    1. Evaluation of immunosurveillance mechanisms in tumor-bearing hosts.
      Schreiber and Ikeda evaluated the physiological significance of the candidate genes identified in 2004, and found that CD1d was involved in the process of cancer immunoediting (manuscript in preparation). They also showed the involvement of type 1 IFN in cancer immunoediting process (Nature Immunol, 2005), and found the downregulation of the expression of H60, a ligand of NKG2D on tumors (J Immunol, 2006. Shiku and Nishikawa fond that immunization with SEREX defined
      self-antigen resulted into the increased development of tumors by the activation of regulatory T cells (J Exp Med, 2005, Proc Natl Acad Sci USA, 2005, J Immunol, 2005). Wang found that toll-like receptor 8-mediated stimulation could reverse the regulatory T cell function in cancer patients (science, 2005). Nishimura developed the mouse model of methylcholanthrene-induced squamous cell carcinoma, and found that activation of type 1 immunity by CpG could prevent the growth of primary squamous cell carcinoma (manuscript in preparation).
    2. Development of an efficient tumor vaccine protocol in animal models
      Nishimura developed a mouse model of tumor vaccine immunotherapy with CpG and OVA to investigate the application of the regulation of immunological balance. By immunizing tumor-bearing mice with OVA and CpG encapsulated in liposome, 8mm tumor of EG7 was regressed and therefore showed that it is a cancer vaccine therapy superior to the existing vaccines with antigenic peptides. This vaccination effect depended on the type 1 IFNs and was mediated with the activation of asialo GM1+CD8+ CTL (Int Immunol, 2006). Regarding Th1 therapy, It was shown that Th1 cells interact with APC in draining Lymph nodes, and tumor specific CTL was induced in lymph node by the cytokines from activated Th1 cells. These CTL migrated to tumor site and eradicated tumor (Cancer Res, 2006). Moreover, Nishimura showed that Th1 cells could act as an "cell adjuvant" as CpG when injected into tumor site with tumor antigen. These Th1 cells inhibited the induction of regulatory T cells in tumor site or draining lymph nodes, activated tumor specific CTL, and induced complete tumor regression (manuscript in preparation). On the basis of this fundamental research, clinical application of Th1 cell therapy is intended, and MHC class II restricted peptide derived from MAGE-A4 tumor antigen was identified (manuscript in preparation). Nishimura also succeeded in the development of the induction of peptide-reactive tumor-specific Th1 cells or gene-modified tumor-specific Th1 cells (Cancer Res, 2005, Blood, 2005).
    3. Application of 2nd generation of tumor vaccine therapy into clinical trial.
      The protocol of the clinical trial entitled "Immunization with NY-ESO-1 protein and cytosine-phosphorothionate-guanine (CpG) 7909 alone or encapsulated in liposome in patients with tumors expressing NY-ESO-1 antigen or LAGE-1 antigen" developed by Nishimura and Ikeda was approved by the ethics committee of Hokkaido University School of Medicine on June 3, 2005. The clinical trial will evaluate immunogenicity in patients with NY-ESO-1 or LAGE-1 expressing tumors who receive NY-ESO-1 protein given with CpG, or with CpG and liposome. Patients must have progressive or metastatic disease after standard treatment or have refused standard treatment. At first, 3 patients will receive 100µg of NY-ESO-1 protein and 2.5 mg of CpG subcutaneously 4 times every 2 weeks. After confirming safety of this stage 1 treatment, 9 patients will receive 100µg of NY-ESO-1 protein and 2.5 mg of CpG encapsulated in 1mg liposome subcutaneously 4 times every 2 weeks as stage 2 treatment. The Primary objective is to assess the safety, and secondary objectives are to evaluate immunological reactions and clinical impacts (see appendix 1). MTA for GMP level recombinant NY-ESO-1 protein was exchanged between Hokkaido University and LICR and NY-EsO-1 protein was sent to Hokkaido University on Sep. 20, 2005. MTA for GMP level CpG was exchanged between Hokkaido University and Coley Pharmaceutical Group on Oct 18, 2005. However, Coley Pharmaceutical Group unexpectedly informed us that they can not send the CpG (see appendix 2), and sent us a termination notice of this MTA on Nov. 18, 2005 (see appendix 2 and 3). They explain that it happened because of the agreement with Pfizer about the rights on CpG. Because of this unexpected trouble, the start of this clinical trial delayed. To overcome this trouble, Hokkaido University started to discuss with Pfizer US, and succeeded to exchange MTA about CpG supply with Pfizer US on Feb 28, 2006, and received CpG drug. Now the patient enrollment is open in Hokkaido University Hospital. By the basic research of Nishimura, Th1 cell therapy of Cancer patient is also approved by ethical committee in Hokkaido University School of Medicine. Th1 cell therapy is planned to start as a clinical trial in the near future.
    4. Seminar
      The seminar entitled "Japan-US collaborative meeting for cancer immunosurveillance and cancer vaccine clinical trials" was hold in Sapporo, Japan, on August 19 and 20, 2005. In this seminar, the status and the problems in the cancer vaccine clinical trials are discussed. This seminar promoted the collaboration of US and Japan to accelerate the fundamental research of cancer immunology and application of these findings into clinical trials.
    5. Research Associate
      Takemasa Tsuji visited LICR in NY, USA as research associate from March 29, 2005 to March 31, 2006. He contributed to the collaboration of institutes in Japan and LICR that was essential for this project. He performed the immunological monitoring of cancer vaccine clinical trials, and developed an new and efficient cancer vaccine therapy.
  8. Seminar
    1. Title:
      Japan-US collaborative meeting for cancer immunosurveillance and cancer vaccine clinical trials
    2. Period: August 19th, 20th, 2005
    3. Site: Sapporo, Japan
    4. Participants: Total 30 (Japan side 22 , US side 8 , Other country 0 )
    5. Agenda, Topics and Scientific Achievements

      Agenda

      Day 1

      10:00
      Opening remark and overview
      Takashi Nishimura, Hokkaido University, Institute for Genetic Medicine

      Session I: Experimental basic research in cancer immunosurveillance
      10:20 - 11:40.
      Tsukasa Seya, Hokkaido University, Graduate School of Medicine, Department of Microbiology and Immunology
      Molecular mechanism whereby Toll-activating adjuvants induce tumor regression.

      Hiroaki Ikeda, Hokkaido University, Institute for Genetic Medicine
      Cancer immunoediting: Molecular mechanisms and therapeutic implications

      Hiroyoshi Nishikawa, Ludwig Institute for Cancer Research, New York Branch at MSKCC Role of CD4+ CD25+ regulatory T cells in tumor immunity - from mouse models to cancer patients-

      Takashi Nishimura, Hokkaido University, Institute for Genetic Medicine Effective strategies for selective activation of type1 immunity essential for inducing tumor-specific CTL in tumor-bearing host

      Session II: Basic Research for Tumor Immunity in Human
      13:40 - 15:20

      Mari Kannagi, Tokyo Medical Dental University, Graduate School
      A crossing of infection and tumor immunity against adult T-cell leukemia.

      Esteban Celis, Louisiana State University Health Sciences Center, Cancer Immunology/Immunotherapy Program
      Development of a Peptide-Based Vaccine for the Treatment of Prostate Cancer.

      Rong-fu Wang, Baylor College of Medicine, The Center for Cell and Gene Therapy
      Suppression mechanisms and functional control of regulatory T cells in cancer therapy

      Yoshiki Akatsuka, Kiyotaka Kuzushima, Toshitada Takahashi, Aichi Cancer Center
      Identification of minor histocompatibility antigens involved in graft-versus-leukemia effect and GVHD following allogeneic hematopoietic cell transplantation

      Eiichi Sato, Tokyo Medical University
      Clinico-pathological significance of tumor infiltrating lymphocytes.

      Session III: Cancer vaccine clinical trials
      15:40 - 17:00

      Eric W. Hoffman, Ludwig Institute for Cancer Research, New York
      Cancer vaccine clinical trials

      Yutaka Kawakami, Keio University School of Medicine, Institute for Advancer Medical Research
      Individualized immunotherapy based on the analysis of human tumor antigens -Intratumoral administration of dendritic cells-

      Gerd Ritter, Ludwig Institute for Cancer Research, New York
      Development of cancer vaccines: Bridging laboratory and clinical discovery

      Sacha Gnjatic, Ludwig Institute for Cancer Research, New York Branch at MSKCC
      Clinical trials with NY-ESO-1 protein or DNA: Strategy for immunization and monitoring of antibody and T cell response

      Day 2

      Session III (continues from August 19): Cancer vaccine clinical trials
      9:00 - 11:20

      Hiroshi Shiku, Mie University School of Medicine
      HER2 protein-based cancer vaccine with a novel antigen delivery system of cholesteryl hydrophobized polysaccharides (CHP).

      Eiichi Nakayama, Okayama University Graduate School of Medicine and Dentistry, Department of Immunology
      Immunization of patients with tumors expressing NY-ESO-1 antigen with a complex of cholesterol-bearing hydrophobized pullulan and NY-ESO-1 protein.

      Masataka Yasukawa, Ehime University School of Medicine
      Cellular immunotherapy for leukemia

      Jyun Yoshida, Yuichiro Sato, Nagoya University Graduate School of Medicine
      New therapeutic strategy against malignant brain tumor utilizing liposome

      Hideaki Tahara, University of Tokyo, Institution of Medical Science, Advanced Clinical Research Center
      Anti-angiogenic cancer therapy with vaccination using epitope peptides derived from human vascular endothelial growth factor receptor 2 (VEGFR2)

      Norimitsu Kadowaki, Kyoto University Graduate School of Medicine
      Basic Immunology of human dendritic cells and its application for Cancer Immunotherapy.

      11:20
      Closing remark
      Eric W. Hoffman, Ludwig Institute for Cancer Research, New York

      Topics and Scientific Achievements
      Discovery of tumor antigen peptides and the demonstration of the existence of cancer immnunosurveillance clearly showed that the immune system can recognize and destroy the arising tumor in host. However, immunotherapy of cancer patients is still difficult because of the immunosuppressive mechanisms in the tumor-bearing host, and we have not achieved the development of efficient cancer vaccine therapies. To overcome this problem, researchers discussed the issues in three sessions. Experimental basic research in cancer immunosurveillance was discussed in session 1. Basic research for tumor immunity in human was discussed in session 2. Cancer vaccine clinical trials were discussed in session3.

      In session 1, Ikeda demonstrated and the concept of cancer immunosurveillance. He showed a new finding that type 1 interferon was involved in the immunosurveillance system. The implication of this finding for clinical application was discussed. Seya presented the importance of toll-like receptors and their ligands in innate immunity. The possible role of them in the rejection of tumor was discussed. Nishikawa presented the activation of CD4+ CD25+ regulatory T cells by immunization with SEREX defined self antigens. He also presented the effect of the depletion of regulatory T cells in the PBMC from cancer patients. The role of regulatory T cells in anti-tumor immunity was discussed. Lastly Nishimura demonstrated the importance of the selective activation of type 1 immunity to induce tumor specific CTL in tumor-bearing host. The strategy of clinical trials with tumor antigen protein and CpG was discussed. As a summary of this session, the necessity of the precise understanding of the integrated anti-immune response including the innate and adoptive immunity as well as the mechanisms of immunosuppression in tumor bearing host. It is also pointed out that we need to design the clinical trials on these newest findings and feedback study from these clinical trials.

      In session 2, Kannagi reported that the stem-cell transfusion into ATL patients resulted in the induction of Tax specific CTL. The possibility of the immunological therapy targeting tax epitope was discussed. Celis reported the identification of epitope peptides derived from PAMA, TARP, and STEAP for CD8 and CD4 positive T cells. He demonstrated the enhanced anti-tumor immune response in the protocol utilizing a toll-like receptor ligand. Efficacy of the clinical protocol with toll-like receptor ligand was discussed. Wang reported an amazing finding that poly-G, a stimulator of toll-like receptor, can reverse immunosuppression of tumor- patients. The possible effective immunotherapy of cancer patients to overcome the immunosuppressive state of cancer patients by the use of this finding was discussed. Akatsuka reported the induction of CTLs recognizing Minor histocompatibility antigens (mHAgs), BCL1A1 and TMSB4Y. The possible application of mHAgs as targets for the immunotherapy of hematological malignancies was discussed. Sato reported that the number of CD8 positive TIL, CD8/CD4 in TIL, and CD8/Treg in TIL showed prognostic significance in ovarian malignancy. The necessity of the investigation of cellular populations that show prognostic significance in a variety of malignancies was discussed. As a summary of this session, the requirement of the strategy to reverse immunosuppression not only in animal models but also in cancer patients for the efficient eradication of tumor was clearly recognized, and future design of the cancer vaccine therapy was discussed.

      In session 3, Hoffman overviewed the cancer vaccine clinical trials in USA especially in LICR. Kawakami reported the basic data of dendritic cells activated by toll-like receptor ligands, and reported the clinical trial with these dendritic cells in Keio University. Ritter pointed out that the existing academic and commercial structures were not well suited to support an efficient and timely transfer of laboratory discoveries into early phase clinical trials. Better structures for translational research such as Cancer Vaccine Collaborative (CVC) was discussed. Gnjatic reported the phase 1 clinical trials with NY-ESO-1 protein or DNA aiming the activation of both CD4 positive and CD8 positive T cells. Both protocol were safe, but sporadic activation of CD8 T cells was observed so far. Shiku reported a phase 1 clinical trial utilizing HER2 protein and a novel antigen delivery system, CHP. The vaccine was safe. Specific CD4 positive and CD8 positive T cell activation with antibody production were observed. Nakayama reported a phase 1 clinical trial with NY-ESO-1 protein with CHP. Vaccine was safe, and antibody production was increased in all 8 patients. Some patients showed specific CD4 positive and CD8 positive T cell activations. Yasukawa demonstrated the induction of WT-1 specific MHC class I restricted or class II restricted T cells, and their cancer therapy experiments in nude mice with these T cells. He reported that they started a phase 1 clinical trial with the identified WT-1 derived peptide. Yoshida reported the development of novel liposome which not only work as a delivery system but also as a adjuvant. The effect of siRNA encapsulated in their liposome against malignant glioma cells was shown. Tahara reported the identification of epitope peptide derived from human vascular endothelial growth factor receptor 2 (VEGFR2). He informed that they started a phase 1 clinical trial in University of Tokyo based on their findings. Kadowaki reported that plasmacytoid DC (pDC) express TLR7 and TLR9, and therefore can react to CpG suggesting a positive rile in cancer immunotherapy. On the other hand, pDC can produce IFNα which sometimes induce regulatory T cells, suggesting a paradoxical role for pDC in anti-tumor immunity. As a summary of this session, it was clear that many researchers have realized the importance to induce a variety sets of immune cells including cells in innate and acquired immunity, and to overcome the immunosuppressive state of cancer patients. For this purpose, several strategies including (1) use of proteins and DNAs as antigen, (2) use of adjuvant such as CpG to activate innate immunity especially type 1 sifted immunity, (3) use of delivery systems such as liposome or CHP, (4) use of cell therapy such as dendritic cells or Th1 cells, have been developed. Many of them were already in phase 1 clinical trial. It was too early to evaluate the clinical effect of these trials, but it was suggested that they were promising. It was realized that the system for the translational research in Japan is not efficient enough, and that it was an urgent requirement to develop an efficient translational research by the collaboration with LICR, CRI, and CVC in USA.
      During this seminar, it was shown that the development of the 2nd generation of cancer vaccine therapy is on going in many institutions. It will require approximately 5 years to test the clinical effect of these 2nd generation of cancer vaccine therapy. The collaboration of US researchers and Japan researchers was suggested to facilitate strongly the development of efficient 2nd generation of cancer vaccine therapy to benefit many cancer patients.

  9. Research Results of Project
    1. Evaluation of immunosurveillance mechanisms in tumor-bearing hosts.
      This project contributed in the many new findings made in the mechanisms of cancer immunoediting. Schreiber and Ikeda evaluated the physiological significance of the candidate genes identified in 2004, and found that CD1d is involved in the process of cancer immunoediting (manuscript in preparation). They also showed the involvement of type 1 IFN in cancer immunoediting process (Nature Immunol, 2005), and found the downregulation of the expression of H60, a ligand of NKG2D on tumors (J Immunol, 2006. Shiku and Nishikawa fond that immunization with SEREX defined self-antigen resulted into the increased development of tumors by the activation of regulatory T cells (J Exp Med, 2005, Proc Natl Acad Sci USA, 2005, J Immunol, 2005). Wang found that toll-like receptor 8-mediated stimulation could reverse the regulatory T cell function in cancer patients (science, 2005). Nishimura developed the mouse model of methylcholanthrene-induced squamous cell carcinoma, and found that activation of type 1 immunity by CpG could prevent the growth of primary squamous cell carcinoma (manuscript in preparation), and this system to make squamous cell carcinoma in mouse skin as a first time in the world will contribute to the future research of cancer immunology.
    2. Development of an efficient tumor vaccine protocol in animal models
      Nishimura developed a mouse model of tumor vaccine immunotherapy with CpG and OVA to investigate the application of the regulation of immunological balance. By immunizing tumor-bearing mice with OVA and CpG encapsulated in liposome, 8mm tumor of EG7 was regressed and therefore showed that it is a cancer vaccine therapy superior to the existing vaccines with antigenic peptides. This vaccination effect depended on the type 1 IFNs and was mediated with the activation of asialo GM1+CD8+ CTL (Figure 1, Figure 2, Int Immunol, 18: 425-434, 2006). Regarding Th1 therapy, It was shown that Th1 cells interact with APC in draining Lymph nodes, and tumor specific CTL was induced in lymph node by the cytokines from activated Th1 cells. These CTL migrated to tumor site and eradicated tumor (Cancer Res, 2006). Moreover, Nishimura showed that Th1 cells could act as an “cell adjuvant” as CpG when injected into tumor site with tumor antigen. These Th1 cells inhibited the induction of regulatory T cells in tumor site or draining lymph nodes, activated tumor specific CTL, and induced complete tumor regression (manuscript in preparation). On the basis of this fundamental research, clinical application of Th1 cell therapy is intended, and MHC class II restricted peptide derived from MAGE-A4 tumor antigen was identified (manuscript in preparation). Nishimura also succeeded in the development of the induction of peptide-reactive tumor-specific Th1 cells or gene-modified tumor-specific Th1 cells (Figure 3, Cancer Res, 2005, Blood, 2005). As a achievement of this project, Nishimura collaborated with many researchers in US and Japan on the issue of induction of tumor specific human Th1 cells.


      Figure1
      Figure 1 Regression of OVA+ tumor by OVA/CpG/liposome cancer immunotherapy
      Figure2
      Figure 2 IFNα/β dependent induction of OVA specific CTL by OVA/CpG/liposome
      Figure3
      Figure 3 Strategy to prepare tumor-specific Th1 cells


    3. Application of 2nd generation of tumor vaccine therapy into clinical trial
      In Hokkaido University, a project team for cancer vaccine and cell therapy was established by Institute for Genetic Medicine and 10 clinical departments in Hokkaido University Hospital. The screening of the expression of tumor antigens in patients was performed in Hokkaido University Hospital that includes 157 lung cancers, 17 esophageal cancer, 20 breast cancers, 31 stomach cancers, 10 colon cancers, and 18 bill-duct cancers. NY-ESO-1 was expressed in 29% of esophageal cancer, 30% of breast cancer, and 8.3% of lung cancer. MAGE-A4 was expressed in 29% of lung cancer, 59% of esophageal cancer, 33% of colon cancer, and 17% of bill-duct cancer. In lung cancer patients, the expression of MAGE-A4 tumor antigen and the infiltration of T cells showed prognostic significance (Int J Oncology, 2006). The protocol of the clinical trial entitled “Immunization with NY-ESO-1 protein and cytosine-phosphorothionate-guanine (CpG) 7909 alone or encapsulated in liposome in patients with tumors expressing NY-ESO-1 antigen or LAGE-1 antigen” developed by Nishimura and Ikeda was approved by the ethics committee of Hokkaido University School of Medicine on June 3, 2005. The clinical trial will evaluate immunogenicity in patients with NY-ESO-1 or LAGE-1 expressing tumors who receive NY-ESO-1 protein given with CpG, or with CpG and liposome. Patients must have progressive or metastatic disease after standard treatment or have refused standard treatment. At first, 3 patients will receive 100µg of NY-ESO-1 protein and 2.5 mg of CpG subcutaneously 4 times every 2 weeks. After confirming safety of this stage 1 treatment, 9 patients will receive 100µg of NY-ESO-1 protein and 2.5 mg of CpG encapsulated in 1mg liposome subcutaneously 4 times every 2 weeks as stage 2 treatment. The Primary objective is to assess the safety, and secondary objectives are to evaluate immunological reactions and clinical impacts (see appendix1). MTA for GMP level recombinant NY-ESO-1 protein was exchanged between Hokkaido University and LICR and NY-EsO-1 protein was sent to Hokkaido University on Sep. 20, 2005. MTA for GMP level CpG was exchanged between Hokkaido University and Coley Pharmaceutical Group on Oct 18, 2005. However, Coley Pharmaceutical Group unexpectedly sent a termination notice of this MTA on Nov. 9, 2005 (see appendix2 and 3). They explain that it happened because of the agreement with Pfizer about the rights on CpG. Because of this unexpected trouble, the start of this clinical trial delayed. To overcome this trouble, Hokkaido University started to discuss with Pfizer US, and succeeded to exchange MTA about CpG supply with Pfizer US on Feb 28, 2006, and received CpG drug. Now the patient enrollment is open. By the basic research of Nishimura, Th1 cell therapy of Cancer patient is also approved by ethical committee in Hokkaido University School of Medicine. Th1 cell therapy is planned to start as a clinical trial in the near future.
    4. Seminar
      The seminar entitled "Japan-US collaborative meeting for cancer immunosurveillance and cancer vaccine clinical trials" was hold in Sapporo, Japan, on August 19 and 20, 2005. In this seminar, the status and the problems in the cancer vaccine clinical trials are discussed. This seminar promoted the collaboration of US and Japan to accelerate the fundamental research of cancer immunology and application of these findings into clinical trials. During this seminar, it was shown that the development of the 2nd generation of cancer vaccine therapy is on going in many institutions. It will require approximately 5 years to test the clinical effect of these 2nd generations of cancer vaccine therapy. The collaboration of US researchers and Japan researchers was suggested to facilitate strongly the development of efficient 2nd generation of cancer vaccine therapy to benefit many cancer patients.
    5. Research Associate
      Takemasa Tsuji visited LICR in NY, USA as research associate from March 29, 2005 to March 31, 2006. In this project, he examined the activation of NY-ESO-1-specific helper T cells in the blood of cancer patients who received HLA class II binding peptide vaccination. By this analysis, it was suggest that novel vaccination strategy, such as vaccination with tumor antigen protein, use of strong adjuvant, or efficient delivery systems, is need to generate high-avidity helper T cells that are able to recognize processed tumor antigen peptide presented on tumor cells or antigen presenting cells (J Immunol in press). By analyzing the response to microbial component-derived adjuvant on DC, responses against synthetic or natural compounds, or microbes were found to be important for the development of strong adjuvant which induce more Th1-cytokine and less Th2-cytokine.
  10. Papers and Publications (Project-related papers that have or will be published)
    1. Chamoto, K., Wakita, D., Narita, Y., Zhang, Y., Noguchi, D., Ohnishi, H., Iguchi, T., Sakai, T., Ikeda, H.,Nishimura, T.: An essential role of antigen-presenting cell/T-helper type 1 cell-cell interactions in draining lymph node during complete eradication of class II-negative tumor tissue by T-helper type 1 cell therapy. Cancer Res, 66: 1809-1817, 2006.
    2. Wakita, D., Chamoto, K., Zhang, Y., Narita, Y., Noguchi, D., Ohnishi, H., Iguchi, T., Sakai, T., Ikeda, H.,Nishimura, T.: An indispensable role of type-1 IFNs for inducing CTL-mediated complete eradication of established tumor tissue by CpG-liposome co-encapsulated with model tumor antigen. Int Immunol, 18: 425-434, 2006.
    3. Yoshida, N., Abe, H., Ohkuri, T., Wakita, D., Sato, M., Noguchi, D., Miyamoto, M., Morikawa, T., Kondo, S., Ikeda, H., Nishimura, T. Expression of the MAGE-A4 and NY-ESO-1 cancer-testis antigens and T cell infiltration in non-small cell lung carcinoma and their prognostic significance. Int J Oncology, 28(5): 1089-1098, 2006.
    4. Matsuzaki, J., Tsuji, T., Zhang, Y., Wakita, D., Imazeki, I., Sakai, T., Ikeda, H.,Nishimura, T.: 1alpha,25-Dihydroxyvitamin D3 downmodulates the functional differentiation of Th1 cytokine-conditioned bone marrow-derived dendritic cells beneficial for cytotoxic T lymphocyte generation. Cancer Sci, 97: 139-147, 2006.
    5. Imazeki, I., Matsuzaki, J., Tsuji K., Nishimura, T.: Immunomodulating effect of vitamin D3 derivatives on type-1 cellular immunity. Biomedical Research, 27:1-9, 2006.
    6. Nishikawa, H., Qian, F., Tsuji, T., Ritter, G., Old, LJ., Gnjatic, S., Odunsi, K.: Influence of CD4+CD25+ regulatory T cells on low/high-avidity CD4+ T cells following peptide vaccination. J. Immunol., In press.
    7. Kobayashi, H., Nagato, T., Aoki N., Sato, K., Kimura, S., Tateno, M., and Celis, E. Defining MHC class II T helper epitopes for WT1 tumor antigen. Cancer Immunol. Immunother. 55:850-860, 2006.
    8. Rodeberg, D.A., Nuss, R.A., Elsawa, S.F., Erskine, C.L., and Celis, E. Generation of tumoricidal PAX3 peptide antigen specific cytotoxic T lymphocytes. Int . J. Cancer. [Epub ahead of print], 2006.
    9. Wang, R. F.: Immune suppression by tumor-specific CD4+ regulatory T-cells in cancer. Semin Cancer Biol, 16: 73-79, 2006.
    10. Wang, R. F.: Functional control of regulatory T cells and cancer immunotherapy. Semin Cancer Biol, 16: 106-114, 2006.
    11. Wang, R. F., Peng, G.,Wang, H. Y.: Regulatory T cells and Toll-like receptors in tumor immunity. Semin Immunol, 18: 136-142, 2006.
    12. Bui, J. D., Carayannopoulos, L. N., Lanier, L. L., Yokoyama, W. M.,Schreiber, R. D.: IFN-dependent down-regulation of the NKG2D ligand H60 on tumors. J Immunol, 176: 905-913, 2006.
    13. Yamaguchi, A., Koda, T., Abe, H., Sato, M., Li, J., Sakai, T., Togashi, Y., Shinohara, Y., Ikeda, H.,Nishimura, T.: Development of a functional cDNA array for evaluation of the Th1/Th2 balance. Immunol Lett, 101: 95-103, 2005.
    14. Tsuji, T., Yasukawa, M., Matsuzaki, J., Ohkuri, T., Chamoto, K., Wakita, D., Azuma, T., Niiya, H., Miyoshi, H., Kuzushima, K., Oka, Y., Sugiyama, H., Ikeda, H.,Nishimura, T.: Generation of tumor-specific, HLA class I-restricted human Th1 and Tc1 cells by cell engineering with tumor peptide-specific T-cell receptor genes. Blood, 106: 470-476, 2005.
    15. Kijima, M., Saio, M., Oyang, G. F., Suwa, T., Miyauchi, R., Kojima, Y., Imai, H., Nakagawa, J., Nonaka, K., Umemura, N., Nishimura, T.,Takami, T.: Natural killer cells play a role in MHC class I in vivo induction in tumor cells that are MHC negative in vitro. Int J Oncol, 26: 679-684, 2005.
    16. Matsuzaki, J., Tsuji, T., Imazeki, I., Ikeda, H.,Nishimura, T.: Immunosteroid as a regulator for Th1/Th2 balance: its possible role in autoimmune diseases. Autoimmunity, 38: 369-375, 2005.
    17. Itoh, T. and Celis, E. Transcutaneous immunization with cytotoxic T-cell peptide epitopes provides effective antitumor immunity in mice. J Immunother, 28: 430-437, 2005.
    18. Rodeberg, D. A., Nuss, R. A., Heppelmann, C. J., and Celis, E. Lack of effective T-lymphocyte response to the PAX3/FKHR translocation area in alveolar rhabdomyosarcoma. Cancer Immunol Immunother, 54: 526-534, 2005.
    19. Rodeberg, D. A., Nuss, R. A., Elsawa, S. F., and Celis, E. Recognition of six-transmembrane epithelial antigen of the prostate-expressing tumor cells by peptide antigen-induced cytotoxic T lymphocytes. Clin Cancer Res, 11: 4545-4552, 2005.
    20. Radhakrishnan, S., Celis, E., and Pease, L. R. B7-DC cross-linking restores antigen uptake and augments antigen-presenting cell function by matured dendritic cells. Proc Natl Acad Sci U S A, 102: 11438-11443, 2005.
    21. Kobayashi, H., Nagato, T., Oikawa, K., Sato, K., Kimura, S., Aoki, N., Omiya, R., Tateno, M., and Celis, E. Recognition of prostate and breast tumor cells by helper T lymphocytes specific for a prostate and breast tumor-associated antigen, TARP. Clin Cancer Res, 11: 3869-3878, 2005.
    22. Kennedy, R., Undale, A. H., Kieper, W. C., Block, M. S., Pease, L. R., and Celis, E. Direct cross-priming by Th lymphocytes generates memory cytotoxic T cell responses. J Immunol, 174: 3967-3977, 2005.
    23. Smith CL, Dunbar PR, Mirza F, Palmowski MJ, Shepherd D, Gilbert SC, Coulie P, Schneider J, Hoffman E, Hawkins R, Harris AL, Cerundolo V.Recombinant modified vaccinia Ankara primes functionally activated CTL specific for a melanoma tumor antigen epitope in melanoma patients with a high risk of disease recurrence. Int J Cancer. 10;113(2):259-66. 2005.
    24. Wang, H. Y.,Wang, R. F.: Antigen-specific CD4+ regulatory T cells in cancer: implications for immunotherapy. Microbes Infect, 7: 1056-1062, 2005.
    25. Schreiber, R. D.: Cancer vaccines 2004 opening address: the molecular and cellular basis of cancer immunosurveillance and immunoediting. Cancer Immun, 5 Suppl 1: 1, 2005.
    26. Peng, G., Guo, Z., Kiniwa, Y., Voo, K. S., Peng, W., Fu, T., Wang, D. Y., Li, Y., Wang, H. Y.,Wang, R. F.: Toll-like receptor 8-mediated reversal of CD4+ regulatory T cell function. Science, 309: 1380-1384, 2005.
    27. Nishikawa, H., Kato, T., Tawara, I., Takemitsu, T., Saito, K., Wang, L., Ikarashi, Y., Wakasugi, H., Nakayama, T., Taniguchi, M., Kuribayashi, K., Old, L. J.,Shiku, H.: Accelerated chemically induced tumor development mediated by CD4+CD25+ regulatory T cells in wild-type hosts. Proc Natl Acad Sci U S A, 102: 9253-9257, 2005.
    28. Nishikawa, H., Kato, T., Tawara, I., Saito, K., Ikeda, H., Kuribayashi, K., Allen, P. M., Schreiber, R. D., Sakaguchi, S., Old, L. J.,Shiku, H.: Definition of target antigens for naturally occurring CD4(+) CD25(+) regulatory T cells. J Exp Med, 201: 681-686, 2005.
    29. Nishikawa, H., Kato, T., Tawara, I., Ikeda, H., Kuribayashi, K., Allen, P. M., Schreiber, R. D., Old, L. J.,Shiku, H.: IFN-gamma controls the generation/activation of CD4+ CD25+ regulatory T cells in antitumor immune response. J Immunol, 175: 4433-4440, 2005.
    30. Miyahara, Y., Naota, H., Wang, L., Hiasa, A., Goto, M., Watanabe, M., Kitano, S., Okumura, S., Takemitsu, T., Yuta, A., Majima, Y., Lemonnier, F. A., Boon, T.,Shiku, H.: Determination of cellularly processed HLA-A2402-restricted novel CTL epitopes derived from two cancer germ line genes, MAGE-A4 and SAGE. Clin Cancer Res, 11: 5581-5589, 2005.
    31. Dunn, G. P., Sheehan, K. C., Old, L. J.,Schreiber, R. D.: IFN unresponsiveness in LNCaP cells due to the lack of JAK1 gene expression. Cancer Res, 65: 3447-3453, 2005.
    32. Dunn, G. P., Bruce, A. T., Sheehan, K. C., Shankaran, V., Uppaluri, R., Bui, J. D., Diamond, M. S., Koebel, C. M., Arthur, C., White, J. M.,Schreiber, R. D.: A critical function for type I interferons in cancer immunoediting. Nat Immunol, 6: 722-729, 2005.
    33. Dunn, G. P., Ikeda, H., Bruce, A. T., Koebel, C., Uppaluri, R., Bui, J., Chan, R., Diamond, M., White, J. M., Sheehan, K. C.,Schreiber, R. D.: Interferon-gamma and Cancer Immunoediting. Immunol Res, 32: 231-246, 2005.
    34. Tsuji, T., Chamoto, K., Gyobu, H., Ikeda. H., Nishimura, T.: Application of genetically engineered tumor-specific Th1 cells to adoptive tumor immunotherapy. Annals of Cancer Research and Therapy, 12: 71-86, 2004.
    35. Suzuki, Y., Wakita, D., Chamoto, K., Narita, Y., Tsuji, T., Takeshima, T., Gyobu, H., Kawarada, Y., Kondo, S., Akira, S., Katoh, H., Ikeda, H.,Nishimura, T.: Liposome-encapsulated CpG oligodeoxynucleotides as a potent adjuvant for inducing type 1 innate immunity. Cancer Res, 64: 8754-8760, 2004.
    36. Ikeda, H., Chamoto, K., Tsuji, T., Suzuki, Y., Wakita, D., Takeshima, T.,Nishimura, T.: The critical role of type-1 innate and acquired immunity in tumor immunotherapy. Cancer Sci, 95: 697-703, 2004.
    37. Yamamoto, S., Tsuji, T., Matsuzaki, J., Zhange, Y., Chamoto, K., Kosaka, A., Togashi, Y., Sekikawa, K., Sawada, K. I., Takeshima, T., Koike, T.,Nishimura, T.: Unexpected role of TNF-a in graft versus host reaction (GVHR): donor-derived TNF-a suppresses GVHR via inhibition of IFN-γ -dependent donor type-1 immunity. Int Immunol, 16: 811-817, 2004.
    38. Sato, T., Saito, R., Jinushi, T., Tsuji, T., Matsuzaki, J., Koda, T., Nishimura, S., Takeshima, H.,Nishimura, T.: IFN-Γ-induced SOCS-1 regulates STAT6-dependent eotaxin production triggered by IL-4 and TNF-a. Biochem Biophys Res Commun, 314: 468-475, 2004.
    39. Hoshino, A., Tsuji, T., Matsuzaki, J., Jinushi, T., Ashino, S., Teramura, T., Chamoto, K., Tanaka, Y., Asakura, Y., Sakurai, T., Mita, Y., Takaoka, A., Nakaike, S., Takeshima, T., Ikeda, H.,Nishimura, T.: STAT6-mediated signaling in Th2-dependent allergic asthma: critical role for the development of eosinophilia, airway hyper-responsiveness and mucus hypersecretion, distinct from its role in Th2 differentiation. Int Immunol, 16: 1497-1505, 2004.
    40. Gyobu, H., Tsuji, T., Suzuki, Y., Ohkuri, T., Chamoto, K., Kuroki, M., Miyoshi, H., Kawarada, Y., Katoh, H., Takeshima, T.,Nishimura, T.: Generation and targeting of human tumor-specific Tc1 and Th1 cells transduced with a lentivirus containing a chimeric immunoglobulin T-cell receptor. Cancer Res, 64: 1490-1495, 2004.
    41. Fujimura, T., Chamoto, K., Tsuji, T., Sato, T., Yokouchi, H., Aiba, S., Tagami, H., Tanaka, J., Imamura, M., Togashi, Y., Koda, T.,Nishimura, T.: Generation of leukemia-specific T-helper type 1 cells applicable to human leukemia cell-therapy. Immunol Lett, 93: 17-25, 2004.
    42. Chamoto, K., Tsuji, T., Funamoto, H., Kosaka, A., Matsuzaki, J., Sato, T., Abe, H., Fujio, K., Yamamoto, K., Kitamura, T., Takeshima, T., Togashi, Y.,Nishimura, T.: Potentiation of tumor eradication by adoptive immunotherapy with T-cell receptor gene-transduced T-helper type 1 cells. Cancer Res, 64: 386-390, 2004.
    43. Lu, J., Higashimoto, Y., Appella, E., and Celis, E. Trojan antigen peptide vaccines for the induction of antitumor CTL and Th immune responses. J Immunol, 172:4575-4582, 2004.
    44. Ruiz, M, Kobayashi H., Lasarte, J.J., Prieto, J., Borras-Cuesta, F., Celis, E. and Sarobe, P. Identification and characterization of a T-helper peptide from carcinoembryonic antigen.. Clin Cancer Res, 10:2860-2867, 2004.
    45. Undale, A.H., Van Den Elsen, P. J., and Celis, E. Antigen-independent acquisition of MHC class II molecules by human T lymphocytes. Intl Immunol, 16:1523-1533, 2004
    46. Roden, A.C., Moser, M.T., Tri, S.D.., Mercader, M., Kuntz, S.M., Dong, H., Hurwitz, A.A., McKean, D.J., Celis, E., Leibovich ,B.C., Allison, J.P., and Kwon, E.D. Augmentation of T cell levels and responses induced by androgen deprivation. J Immunol, 173:6098-6108, 2004
    47. Kobayashi, H., Nagato, T., Yanai ,M., Oikawa, K., Sato, K., Kimura, S., Tateno, M., Omiya, R., and Celis, E. Recognition of adult T-cell leukemia/lymphoma cells by CD4+ helper T lymphocytes specific for human T-cell leukemia virus type I envelope protein. Clin Cancer Res, 10:7053-7062, 2004.
    48. Elsawa, S.F., Rodeberg , D.A., and Celis, E. T-cell epitope peptide vaccines. Expert Rev Vaccines, 3:563-575, 2004.
    49. Skipper J, Hoffman EW, O'Donnell-Tormey J, Old LJ.Translation of cancer immunotherapies. Nat Med.;10(11):1154-5; 2004.
    50. Davis ID, Chen W, Jackson H, Parente P, Shackleton M, Hopkins W, Chen Q, Dimopoulos N, Luke T, Murphy R, Scott AM, Maraskovsky E, McArthur G, MacGregor D, Sturrock S, Tai TY, Green S, Cuthbertson A, Maher D, Miloradovic L, Mitchell SV, Ritter G, Jungbluth AA, Chen YT, Gnjatic S, Hoffman EW, Old LJ, Cebon JS.Recombinant NY-ESO-1 protein with ISCOMATRIX adjuvant induces broad integrated antibody and CD4(+) and CD8(+) T cell responses in humans. Proc Natl Acad Sci U S A. 20;101(29):10697-702. 2004.
    51. Speiser DE, Pittet MJ, Guillaume P, Lubenow N, Hoffman E, Cerottini JC, Romero P.Ex vivo analysis of human antigen-specific CD8+ T-cell responses: quality assessment of fluorescent HLA-A2 multimer and interferon-gamma ELISPOT assays for patient immune monitoring. J Immunother.;27(4):298-308. 2004.
    52. Dunn, G. P., Old, L. J.,Schreiber, R. D.: The three Es of cancer immunoediting. Annu Rev Immunol, 22: 329-360, 2004.
    53. Dunn, G. P., Old, L. J.,Schreiber, R. D.: The immunobiology of cancer immunosurveillance and immunoediting. Immunity, 21: 137-148, 2004.
  11. Any Comments
    We are grateful to the Japan Society for Promotion of Science (JSPS), NCI, and their staffs for the wonderful support of this project that resulted into a fruitful foundation of the collaboration of Japanese and US researchers on the development of novel and efficient cancer vaccine therapies. We believe that this project will benefit the many people in both countries in the near future.