Original Article

Cancer Immunology, Immunotherapy

, Volume 53, Issue 6, pp 497-509

First online:

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

  • Hiroki HayashiAffiliated withDivision of Gastroenterological Surgery, Department of Surgery, Graduate School of Medicine, Tohoku University
  • , Ryutaro AsanoAffiliated withCell Resource Center for Biomedical Research, Institute of Development Aging and Cancer, Tohoku University
  • , Kouhei TsumotoAffiliated withDepartment of Biochemistry and Engineering, Graduate School of Engineering, Tohoku University
  • , Yu KatayoseAffiliated withDivision of Gastroenterological Surgery, Department of Surgery, Graduate School of Medicine, Tohoku University
  • , Masanori SuzukiAffiliated withDivision of Gastroenterological Surgery, Department of Surgery, Graduate School of Medicine, Tohoku University
  • , Michiaki UnnoAffiliated withDivision of Gastroenterological Surgery, Department of Surgery, Graduate School of Medicine, Tohoku University
  • , Hideaki KodamaAffiliated withDivision of Gastroenterological Surgery, Department of Surgery, Graduate School of Medicine, Tohoku University
  • , Shin-ichi TakemuraAffiliated withDivision of Gastroenterological Surgery, Department of Surgery, Graduate School of Medicine, Tohoku University
  • , Hiroshi YoshidaAffiliated withDivision of Gastroenterological Surgery, Department of Surgery, Graduate School of Medicine, Tohoku University
    • , Koki MakabeAffiliated withDepartment of Biochemistry and Engineering, Graduate School of Engineering, Tohoku University
    • , Kohzoh ImaiAffiliated withDepartment of Internal Medicine, Sapporo Medical University School of Medicine
    • , Seiki MatsunoAffiliated withDivision of Gastroenterological Surgery, Department of Surgery, Graduate School of Medicine, Tohoku University
    • , Izumi KumagaiAffiliated withDepartment of Biochemistry and Engineering, Graduate School of Engineering, Tohoku University
    • , Toshio KudoAffiliated withCell Resource Center for Biomedical Research, Institute of Development Aging and Cancer, Tohoku University Email author 

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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.

Keywords

Bispecific diabody Refolding system Bile duct carcinoma EGF receptor Targeting immunotherapy