Studies on the generation and application of fusion protein of single chain antibody for P-glycoprotein and chemokine

• Project/Area Number: 13557053
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 展開研究
• Research Field: Respiratory organ internal medicine
• Research Institution: The University of Tokushima
• Principal Investigator: SONE Saburo The University of Tokushima, School of Medicine, Professor, 医学部, 教授 (40145024)
• Co-Investigator(Kenkyū-buntansha): YANO Seiji The University of Tokushima, University Hospital, Assistant Professor, 医学部附属病院, 講師 (30294672) ; NISHIOKA Yasuhiko The University of Tokushima, School of Medicine, Assistant Professor, 医学部, 講師 (70274199) ; TANI Kenji The University of Tokushima, School of Medicine, Associate Professor, 医学部, 助教授 (70207166)
• Project Period (FY): 2001 – 2002
• Project Status: Completed (Fiscal Year 2002)
• Budget Amount: ¥9,800,000 (Direct Cost: ¥9,800,000); Fiscal Year 2002: ¥4,800,000 (Direct Cost: ¥4,800,000); Fiscal Year 2001: ¥5,000,000 (Direct Cost: ¥5,000,000)
• Keywords: Multi-drug resistance / P-glycoprotein / MRK16 / single chain antibody / P糖蛋白 / single chan抗体 / ケモカイン / 薬剤耐性 / 抗体治療 / 免疫療法 / MRK-16 / MCP-1

Research Abstract

The MDR1 gene product P-glycoprotein plays a key role in multidrug resistance of cancer cells. P-glycoprotein is the energy-dependent pump that extrudes a variety of chemotherapeutic drugs. Overexpression of P-glycoprotein results in multidrug resistance of tumor cell lines in vitro as well as in cancer patients. Therefore, much attention has been paid to P-glycoprotein as a molecular target for cancer treatment. MRK16 is the monoclonal antibody against P-glycoprotein and binds to an external domain of P-glycoprotein. It partially inhibits the efflux of certain chemotherapeutic drugs in multidrug-resistant cells, and leads the tumor regression in vivo. Due to the limitations of using intact murine antibodies in human clinical settings, we engineered a single-chain antibody fragment of MRK16 (scFvMRK16). After cloning the variable region of MRK16, both heavy and light chain fragments were fused with linker, and recombinant protein were produced from Escherichia coli. Although the binding activity of scFvMRK16 to P-glycoprotein was 200-300 fold less compared to MRK16, we confirmed by flow cytometric analysis that it does recognize the P-glycoprotein. Furthermore, scFvMRK16 could inhibit the activity of MRK16 to bind P-glycoprotein. These results indicated the therapeutic potential of scFvMRK16.

Research Products

• [Publications] Nishimura et al.: "Enhanced efficiency by centrifugal manipulation of adenovirus-mediated interleukin 12 gene transduction into human monocyte-derived dendritic cells"Hum Gene Ther. 12. 333-346 (2001)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Goto H et al.: "Activity of a new vascular targeting agent, ZD6126, in pulmonary metastases by human lung adenocarcinoma in nude mice"Cancer Res. 62. 3711-3715 (2002)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Shiraga M et al.: "Organ heterogeneity of host-derived metalloproteinase expression and its involvement in multiple-organ metastasis by lung cancer cell lines"Cancer Res.. 62. 5967-5973 (2002)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Nishioka M et al.: "MYO18B, a candidate tumor suppressor gene at chromosome 22q12.1, deleted, mutated, and methylated in human lung cancer"P Natl Acad Sci USA. 99・19. 12269-12274 (2002)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Yamamoto A et al.: "A Third-Generation Matrix Metalloproteinase (MMP) Inhibitor (ONO-4817) Combined with Docetaxel Suppresses Progression of Lung Micrometastasis of MMP-Expressing Tumor Cells in Nude Mice"Int J Cancer. 103. 822-828 (2002)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Nishioka Y et al.: "Differential effects of IL-12 on the generation of alloreactive CTL mediated by murine and human dendritic cells : A critical role for nitric oxide"J Leukoc Biol. (In press). (2003)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Nishimura, N., Nishioka, Y., Shinohara, T. and Sone, S.: "Enhanced efficiency by centrifugal manipulation of adenovirus-mediated interleukin 12 gene transduction into human monocyte-derived dendritic cells."Hum. Gene Ther.. 12. 333-346 (2001)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Goto, H., Yano, S., Zhang, H., Matsumori, Y., Ogawa, H., David C. Blakey., and Sone, S.: "Activity of a New Vascular Targeting Agent, ZD6126, in Pulmonary Metastases by Human Lung Adenocarcinoma in Nude Mice"Cancer Res. 62. 3711-3715 (2002)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Shiraga, M., Yano, S., Yamamoto, A., Ogawa, H., Goto, H., Miki, T., Miki, K., Zhang, H. and Sone, S.: "Organ Heterogeneity of Host-derived Matrix Metalloproteinase Expression and Its Involvement in Multiple-Organ Metastasis by Lung Cancer Cell Lines"Cancer Res. 62. 5967-5973 (2002)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Nishioka, M., Kohno, T., Tani, M., Yanaihara, N., Tomizawa, Y., Otsuka, A., Sasaki, S., Kobayashi, K., Niki, T., Maeshima, A., Sekido, Y., J.D.Minna., Sone, S. and Yokota, J.: "MYO18B, a candidate tumor suppressor gene at chromosome 22q12.1, deleted, mutated, and methylated in human lung cancer."PNAS. 99(19). 12269-12274 (2002)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Yamamoto, A., Yano, S., Shiraga, M., Ogawa, H., Goto, H., Miki, T., Zhang, H. and Sone, S.: "A Third-Generation Matrix Metalloproteinase (MMP) Inhibitor (ONO-4817) Combined with Docetaxel Suppresses Progression of Lung Micrometastasis of MMP-Expressing Tumor Cells in Nude Mice"Int J Cancer. 103. 822-828 (2003)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Nishioka Y, Wen H, Mitani K, Robbins PD, Lotze MT, Sone S and Tahara H.: "Differential effects of IL-12 on the generation of alloreactive CTL mediated by murine and human dendritic cells : A critical role for nitric oxide."J Leukoc Biol. (In press). (2003)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Goto H et al.: "Activity of a new vascular targeting agent, ZD6126, in pulmonary metastases by human lung adenocarcinoma in nude mice"Cancer Res. 62. 3711-3715 (2002)
• [Publications] Shiraga M et al.: "Organ heterogeneity of host-derived metalloproteinase expression and its involvement in multiple-organ metastasis by lung cancer cell lines"Cancer Res.. 62. 5967-5973 (2002)
• [Publications] Nishioka M et al.: "MYO18B, a candidate tumor suppressor gene at chromosome 22q12.1, deleted, mutated, and methylated in human lung cancer"P Natl Acad Sci USA. 99・19. 12269-12274 (2002)
• [Publications] Yamamoto A et al.: "A Third-Generation Matrix Metalloproteinase (MMP) Inhibitor (ONO-4817) Combined with Docetaxel Suppresses Progression of Lung Micrometastasis of MMP-Expressing Tumor Cells in Nude Mice"Int J Cancer. 103. 822-828 (2002)
• [Publications] Nishioka Y et al.: "Differential effects of IL-12 on the generation of alloreactive CTL mediated by murine and human dendritic cells : A critical role for nitric oxide"J Leukoc Biol. (in press). (2003)
• [Publications] Takeuchi, E. et al.: "IL-12-induced production of IL-10 and interferon-gamma by mononuclear cells in lung cancer-associated malignant pleural effusions"Lung Cancer. 35. 171-177 (2002)
• [Publications] Shinohara, T. et al.: "NF-kB-dependent expression of metastasis suppressor KAI1/CD82 gene in lung cancer cell lines expressing mutant p53"Cancer Res.. 61. 673-678 (2001)
• [Publications] Shinohara, T. et al.: "Transduction of KAI1/CD82 cDNA promotes hematogenous spread of human lung-cancer cells in natural killer cell-depleted SCID mice"Int. J. Cancer. 94. 16-23 (2002)
• [Publications] Goto, H. et al.: "Gamma interferon-dependent induction of thymidine phosphorylase/platelet-derived endothelial growth factor through gamma-activated sequence-like element in human macrophages"Cancer Res.. 61. 469-473 (2001)
• [Publications] Nishimura, N. et al.: "Enhanced efficiency by centrifugal manupulation of adenovirus-mediated interleukin 12 gene transduction into human monocyte-derived dendritic cells"Hum. Gene Ther.. 12. 333-346 (2001)
• [Publications] Nishimura, N. et al.: "Novel centrifugal method for simple and highly efficient adenovirus-mediated green fluorescence protein gene transduction into human monocyte-deried dendritic cells"J. Immunological. Methods.. 253. 113-124 (2001)
• [Publications] Takeuchi, E. et al.: "Interleukin (IL-) 15 has less activity than IL-2 to promote type 2 cytokine predominance in tumour-associated mononuclear cells form lung cancer patients"Cytokine. 13. 119-123 (2001)
• [Publications] Parajuli, P. et al.: "Humanized anti-ganglioside GM2 antibody is effective to induce antibody-dependent cell-mediated cytotoxicity in mononuclear cells from lung cancer patients"Cancer Lett.. 163. 179-184 (2001)
• [Publications] Hanibuchi, M. et al.: "Immunological circumvention of multiple organ metastases of multidrug resistant human small cell lung cancer cells by mouse-human chimeric anti-ganglioside GM2 antibody KM966"Clin. Exp. Metastasis. 18. 353-360 (2001)
• [Publications] Cui, P.et al.: "A novel bioactive 31-amino acid endothelin-1 is a potent chemotactic peptide for human neutrophils and monocytes"J. Leukoc. Biol.. 70. 306-312 (2001)
• [Publications] Miki, T. et al.: "Bone metastasis model with multi-organ dissemination of human small cell lung cancer (SBC-5) cells in natural killer-cell depleted SCID mice"Oncol. Res.. 12. 209-217 (2001)
• [Publications] Tanaka, H. et al.: "CD40 and IFN-gamma dependent T cell activation by human bronchial epithelial cells"J. Med. Invest.. 48. 109-117 (2001)
• [Publications] Suzuki, Y. et al.: "Efficient generation of dendritic cells from alveolar and pleural macrophages as well as blood monocytes in patients with lung cancer"Lung Cancer. 34. 195-205 (2001)
• [Publications] Ge, N.et al.: "Interferon-gamma activates outwardly rectifying chloride channels in the human bronchial epithelial cell line BEAS-2B"J. Med. Invest.. 48. 97-101 (2001)
• [Publications] Sone, S. et al.: "Molecular pathogenesis of lung cancer and its molecular targeted therapy"Internal Med.. 40(2). 167-170 (2001)
• [Publications] Huang, L. et al.: "Thrombin promotes fibroblast proliferation during the early stages of experimental radiation pneumonitis"Radiation Res.. 156. 45-52 (2001)
• [Publications] Nokihara, H. et al.: "A new quinoline derivative MS-209 reverses multidrug resistance and inhibits multiorgan metastases by P-glycoprotein-expressing human small cell lung cancer cells"Jpn. J. Cancer Res.. 92. 785-792 (2001)
• [Publications] Ogushi, F. et al.: "Autoantibodies to IL-1α in sera from rapidly progressive idiopathic pulmonary fibrosis"J. Med. Invest.. 48. 181-189 (2001)
• [Publications] Tani, K. et al.: "Protease-induced leukocyte chemotaxis and activation : Roles in host defense and inflammation"J. Med. Invest.. 48. 133-141 (2001)
• [Publications] Hase, K. et al.: "Increased CCR4 expression in active systemic lupus erythematosus"J. Leukoc. Biol.. 70. 749-755 (2001)
• [Publications] Nishioka, Y. et al.: "Human monocyte-derived and CD83+ blood dendritic cells enhance NK cell-mediated cytotoxicity"Eur. J. Immunol.. 31. 2633-2641 (2001)