Hayashi Hiroki, Asano Ryutaro, Tsumoto Kouhei, Katayose Yu, Suzuki Masanori, Unno Michiaki, Kodama Hideaki, Takemura Shin-ichi, Yoshida Hiroshi, Makabe Koki, Imai Kohzoh, Matsuno Seiki, Kumagai Izumi, Kudo Toshio
Division of Gastroenterological Surgery, Department of Surgery, Graduate School of Medicine, Tohoku University, Seiryomachi 1-1, Aoba-ku, 980-8574 Sendai, Japan.
Cancer Immunol Immunother. 2004 Jun;53(6):497-509. doi: 10.1007/s00262-003-0465-9. Epub 2003 Nov 25.
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.
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.
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.
The Ex3 diabody can be considered a highly promising reagent for study of specific targeting immunotherapy against bile duct and other EGFR-positive carcinomas.
在癌症免疫治疗研究领域，用特异性抗体靶向效应细胞是一种非常有前景的方法。基因工程的最新进展使得利用细菌表达系统制备由可变域组成的免疫球蛋白片段成为可能。
我们在大肠杆菌（E. coli）表达系统中采用重折叠方法制备了一种抗表皮生长因子受体（EGFR）x抗CD3双特异性双抗体（Ex3双抗体）。Ex3双抗体将具有T细胞表型的淋巴因子激活的杀伤细胞（T-LAK细胞）靶向至EGFR阳性胆管癌细胞，在体外显著增强了细胞毒性。针对EGFR和CD3抗原的亲本单克隆抗体IgG完全抑制了对EGFR阳性细胞的这种特异性杀伤。
当T-LAK细胞与EGFR阳性肿瘤细胞在Ex3双抗体存在的情况下共培养时，它们产生的干扰素-γ、粒细胞-巨噬细胞集落刺激因子和肿瘤坏死因子-α水平比不存在时高得多，这可能是特异性抗肿瘤活性的潜在机制。Ex3双抗体在37℃下测试48小时时显示出良好的稳定性，并且还显著抑制了严重联合免疫缺陷（SCID）小鼠胆管癌异种移植瘤的生长。当静脉注射Ex3双抗体（20微克/小鼠），同时注射T-LAK细胞和白细胞介素-2（IL-2）时，六只小鼠中有三只肿瘤完全治愈，另外三只显示肿瘤生长明显迟缓。
Ex3双抗体可被认为是用于研究针对胆管癌和其他EGFR阳性癌的特异性靶向免疫治疗的一种非常有前景的试剂。
