A highly effective and stable bispecific diabody for cancer immunotherapy: cure of xenografted tumors by bispecific diabody and T-LAK cells.
OBJECTIVE
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) x 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-gamma, GM-CSF, and TNF-alpha than in its absence, this being a possible mechanism underlying specific antitumor activity. The Ex3 diabody showed good stability when tested at 37 degrees 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 microg/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.


CONCLUSIONS
The Ex3 diabody can be considered a highly promising reagent for study of specific targeting immunotherapy against bile duct and other EGFR-positive carcinomas.
Hayashi
Hiroki
H
Division of Gastroenterological Surgery, Department of Surgery, Graduate School of Medicine, Tohoku University, Seiryomachi 1-1, Aoba-ku, 980-8574 Sendai, Japan.
Asano
Ryutaro
R
Tsumoto
Kouhei
K
Katayose
Yu
Y
Suzuki
Masanori
M
Unno
Michiaki
M
Kodama
Hideaki
H
Takemura
Shin-ichi
S
Yoshida
Hiroshi
H
Makabe
Koki
K
Imai
Kohzoh
K
Matsuno
Seiki
S
Kumagai
Izumi
I
Kudo
Toshio
T
D000818:Animals; D018033:Antibodies, Bispecific / Q000276:immunology* / Q000627:therapeutic use; D001650:Bile Duct Neoplasms / Q000276:immunology / Q000628:therapy*; D017252:CD3 Complex / Q000276:immunology*; D002478:Cells, Cultured; D066246:ErbB Receptors / Q000276:immunology*; D004926:Escherichia coli / Q000378:metabolism; D005260:Female; D005822:Genetic Vectors / Q000235:genetics; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D007167:Immunotherapy; D007371:Interferon-gamma / Q000378:metabolism; D007376:Interleukin-2 / Q000378:metabolism; D015979:Killer Cells, Lymphokine-Activated / Q000276:immunology*; D051379:Mice; D008813:Mice, Inbred ICR; D016513:Mice, SCID; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000627:therapeutic use; D013601:T-Lymphocytes / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
D018033:Antibodies, Bispecific; D017252:CD3 Complex; D007074:Immunoglobulin G; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D066246:ErbB Receptors