Schultz J, Lin Y, Sanderson J, Zuo Y, Stone D, Mallett R, Wilbert S, Axworthy D
NeoRx Corporation, Molecular Biology Research, Seattle, Washington 98119-4007, USA.
Cancer Res. 2000 Dec 1;60(23):6663-9.
Single-chain Fv antibody fragments from the CD20-specific murine monoclonal antibody B9E9 were genetically engineered as streptavidin fusions [single-chain Fv-streptavidin (scFvSA) fusion protein] for use in pretargeted radioimmunotherapy. The scFvSA constructs were expressed as soluble, tetrameric species in the periplasm of Escherichia coli. Expression levels were affected by the order of the variable regions and the length and composition of the single-chain Fv linker. The best expressor was obtained with the variable regions in the heavy chain-light chain configuration separated by a 25-mer Gly4Ser linker. This construct produced 250-300 mg of soluble, tetrameric fusion protein per liter of fermentor culture. The fusion protein (Mr 173,600) was purified from crude lysates by iminobiotin affinity chromatography with an overall yield of about 50% and was analyzed for functionality both in vitro and in vivo. Immunoreactivity of the scFvSA fusion protein and its nanomolar affinity to CD20-positive Ramos cells were comparable with the B9E9 monoclonal antibody. The fusion protein had a biotin dissociation rate identical to recombinant streptavidin and bound an average of 3.6 biotins/molecule of a possible 4 biotins/molecule. Labeled fusion protein cleared from the blood of BALB/c mice with a P half-life of about 16 h. In nude mice bearing Ramos xenografts, the fusion protein demonstrated sufficient tumor localization of functional streptavidin to enable efficient, tumor-specific targeting of a subsequently administered radionuclide-chelate/biotin molecule. These results suggest that large quantities of functional scFvSA can be produced for clinical testing as a therapy for non-Hodgkin's lymphoma.
将来自CD20特异性鼠单克隆抗体B9E9的单链Fv抗体片段进行基因工程改造，制成链霉亲和素融合体[单链Fv-链霉亲和素（scFvSA）融合蛋白]，用于预靶向放射免疫治疗。scFvSA构建体在大肠杆菌周质中表达为可溶性四聚体。表达水平受可变区顺序以及单链Fv接头的长度和组成影响。以重链-轻链构型的可变区由25聚体Gly4Ser接头隔开时获得最佳表达体。该构建体每升发酵罐培养物产生250 - 300毫克可溶性四聚体融合蛋白。融合蛋白（Mr 173,600）通过亚氨基生物素亲和层析从粗裂解物中纯化，总产率约为50%，并在体外和体内分析其功能。scFvSA融合蛋白的免疫反应性及其对CD20阳性Ramos细胞的纳摩尔亲和力与B9E9单克隆抗体相当。融合蛋白的生物素解离速率与重组链霉亲和素相同，每个分子平均结合3.6个生物素（每个分子可能结合4个生物素）。标记的融合蛋白从BALB/c小鼠血液中清除，P半衰期约为16小时。在携带Ramos异种移植物的裸鼠中，融合蛋白显示出功能性链霉亲和素在肿瘤中有足够的定位，能够有效、肿瘤特异性地靶向随后给予的放射性核素螯合物/生物素分子。这些结果表明，可以生产大量功能性scFvSA用于临床试验，作为非霍奇金淋巴瘤的一种治疗方法。
