Helfrich W, Kroesen B J, Roovers R C, Westers L, Molema G, Hoogenboom H R, de Leij L
GUIDE, University Hospital, Department of Clinical Immunology, Groningen, The Netherlands.
Int J Cancer. 1998 Apr 13;76(2):232-9. doi: 10.1002/(sici)1097-0215(19980413)76:2<232::aid-ijc11>3.0.co;2-e.
We describe the construction of a recombinant bispecific antibody fragment in the diabody format with specificity for both the well-established human pancarcinoma associated target antigen EGP2 (epithelial glycoprotein 2, also known as the CO17-1A antigen or KSA) and the CD3epsilon chain of human TCR/CD3 complex. The murine anti-EGP2 (MOC31) single chain variable fragment (scFv) and the humanized anti-CD3 (Ucht1v9) scFv were cast into a diabody format (designated Dia5v9) using a short 5 amino acid Gly-Ser linker between immunoglobulin heavy-chain and light-chain variable domains. Purification of the poly-histidine tagged Dia5v9 was achieved from extracts of protease deficient Escherichia coli by IMAC chromatography. The Dia5v9 diabody showed strong binding to both EGP2 and CD3 in transfected cells. The in vitro efficacy of Dia5v9 in mediating tumor cell lysis by interleukin-2 activated human T cells appeared to be similar to that of the hybrid-hybridoma-derived BsF(ab')2 Bis1 (anti-EGP2/anti-CD3) in a standard 4-hr 51Cr-release assay. This small and partially humanized recombinant bispecific antibody fragment may be valuable for T-cell-based immunotherapeutical treatment protocols, retargeting activated peripheral blood T lymphocytes to lyse various human carcinomas in vivo.
我们描述了一种双特异性抗体片段的构建，其为双体形式，对已明确的人泛癌相关靶抗原EGP2（上皮糖蛋白2，也称为CO17-1A抗原或KSA）和人TCR/CD3复合物的CD3ε链均具有特异性。使用免疫球蛋白重链和轻链可变域之间短的5个氨基酸的甘氨酸-丝氨酸接头，将鼠抗EGP2（MOC31）单链可变片段（scFv）和人源化抗CD3（Ucht1v9）scFv构建成双体形式（命名为Dia5v9）。通过IMAC色谱法从蛋白酶缺陷型大肠杆菌提取物中纯化多组氨酸标记的Dia5v9。Dia5v9双体在转染细胞中显示出与EGP2和CD3均有强烈结合。在标准的4小时51Cr释放试验中，Dia5v9在介导白细胞介素-2激活的人T细胞裂解肿瘤细胞方面的体外效力似乎与杂交杂交瘤衍生的BsF(ab')2 Bis1（抗EGP2/抗CD3）相似。这种小的、部分人源化的重组双特异性抗体片段对于基于T细胞的免疫治疗方案可能具有重要价值，可将活化的外周血T淋巴细胞重新靶向以在体内裂解各种人类癌症。
