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A highly effective and stable bispecific diabody for cancer immunotherapy: cure of xenografted tumors by bispecific diabody and T-LAK cells

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Abstract

Purpose

In the field of cancer immunotherapy research, the targeting of effector cells with specific antibodies is a very promising approach. Recent advances in genetic engineering have made it possible to prepare immunoglobulin fragments consisting of variable domains using bacterial expression systems.

Methods

We have produced an anti-epidermal growth-factor receptor (EGFR) × anti-CD3 bispecific diabody (Ex3 diabody) in an Escherichia coli (E. coli) expression system with refolding method. The Ex3 diabody targets lymphokine-activated killer cells with a T-cell phenotype (T-LAK cells) to EGFR positive bile duct carcinoma cells with dramatic enhancement of cytotoxicity in vitro. This specific killing of EGFR-positive cells was completely inhibited by parental mAb IgGs directed to EGFR and the CD3 antigen.

Results

When T-LAK cells were cultured with EGFR-positive tumor cells in the presence of Ex3 diabody, they produced much higher levels of IFN-γ, GM-CSF, and TNF-α than in its absence, this being a possible mechanism underlying specific antitumor activity. The Ex3 diabody showed good stability when tested at 37°C for 48 h, and also markedly inhibited tumor growth of bile duct carcinoma xenografts in severe combined immunodeficient (SCID) mice. When Ex3 diabody (20 μg/mouse) was administrated intravenously, together with T-LAK cells and interleukin-2 (IL-2), complete cure of tumors were observed in three of six mice, and the other three showed marked retardation of tumor growth.

Conclusion

The Ex3 diabody can be considered a highly promising reagent for study of specific targeting immunotherapy against bile duct and other EGFR-positive carcinomas.

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Acknowledgements

We thank F. Koizumi for her help in preparation of the manuscript and H. Saeki for valuable advice for this study.

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Authors and Affiliations

  1. Division of Gastroenterological Surgery, Department of Surgery, Graduate School of Medicine, Tohoku University, Seiryomachi 1-1, Aoba-ku, 980-8574, Sendai, Japan

    Hiroki Hayashi, Yu Katayose, Masanori Suzuki, Michiaki Unno, Hideaki Kodama, Shin-ichi Takemura, Hiroshi Yoshida & Seiki Matsuno

  2. Cell Resource Center for Biomedical Research, Institute of Development Aging and Cancer, Tohoku University, Seiryomachi 4-1, Aoba-ku, 980-8575, Sendai, Japan

    Ryutaro Asano & Toshio Kudo

  3. Department of Biochemistry and Engineering, Graduate School of Engineering, Tohoku University, Aoba, Aoba-ku, 980-8579, Sendai, Japan

    Kouhei Tsumoto, Koki Makabe & Izumi Kumagai

  4. Department of Internal Medicine, Sapporo Medical University School of Medicine, S-1, W-16, Chuo-ku, 060-0061, Sapporo, Japan

    Kohzoh Imai

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  1. Hiroki Hayashi
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Correspondence to Toshio Kudo.

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Hayashi, H., Asano, R., Tsumoto, K. et al. A highly effective and stable bispecific diabody for cancer immunotherapy: cure of xenografted tumors by bispecific diabody and T-LAK cells. Cancer Immunol Immunother 53, 497–509 (2004). https://doi.org/10.1007/s00262-003-0465-9

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