Mack M, Riethmüller G, Kufer P
Institut für Immunologie, Munich, Germany.
Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):7021-5. doi: 10.1073/pnas.92.15.7021.
Construction of a bispecific single-chain antibody derivative is described that consists of two different single-chain Fv fragments joined through a Gly-Ser linker. One specificity of the two Fv fragments is directed against the CD3 antigen of human T cells and the other is directed against the epithelial 17-1A antigen; the latter had been found in a clinical trial to be a suitable target for antibody therapy of minimal residual colorectal cancer. The construct could be expressed in CHO cells as a fully functional protein, while its periplasmic expression in Escherichia coli resulted in a nonfunctional protein only. The antigen-binding properties of the bispecific single-chain antibody are indistinguishable from those of the corresponding univalent single-chain Fv fragments. By redirecting human peripheral T lymphocytes against 17-1A-positive tumor cells, the bispecific antibody proved to be highly cytotoxic at nanomolar concentrations as demonstrated by 51Cr release assay on various cell lines. The described bispecific construct has a molecular mass of 60 kDa and can be easily purified by its C-terminal histidine tail on a Ni-NTA chromatography column. As bispecific antibodies have already been shown to be effective in vivo in experimental tumor systems as well as in phase-one clinical trials, the small CD3/17-1A-bispecific antibody may be more efficacious than intact antibodies against minimal residual cancer cells.
本文描述了一种双特异性单链抗体衍生物的构建方法，该衍生物由通过甘氨酸 - 丝氨酸连接子连接的两个不同单链Fv片段组成。两个Fv片段中的一个特异性针对人T细胞的CD3抗原，另一个针对上皮17 - 1A抗原；后者在一项临床试验中被发现是微小残留结直肠癌抗体治疗的合适靶点。该构建体可在CHO细胞中表达为具有完全功能的蛋白质，而其在大肠杆菌周质中的表达仅产生无功能的蛋白质。双特异性单链抗体的抗原结合特性与相应的单价单链Fv片段无法区分。通过将人外周血T淋巴细胞重定向至17 - 1A阳性肿瘤细胞，双特异性抗体在纳摩尔浓度下被证明具有高度细胞毒性，这在对各种细胞系的51Cr释放试验中得到了证实。所描述的双特异性构建体分子量为60 kDa，可通过其C末端组氨酸尾在Ni - NTA色谱柱上轻松纯化。由于双特异性抗体已在实验性肿瘤系统以及一期临床试验中显示出体内有效性，这种小型CD3/17 - 1A双特异性抗体可能比完整抗体对微小残留癌细胞更有效。
