Cloutier S M, Couty S, Terskikh A, Marguerat L, Crivelli V, Pugnières M, Mani J C, Leisinger H J, Mach J P, Deperthes D
Institute of Biochemistry, University of Lausanne, CH-1066, Epalinges, Switzerland.
Mol Immunol. 2000 Dec;37(17):1067-77. doi: 10.1016/s0161-5890(01)00023-2.
Phage display is a powerful method of isolating of antibody fragments from highly diverse naive human antibody repertoires. However, the affinity of the selected antibodies is usually low and current methods of affinity maturation are complex and time-consuming. In this paper, we describe an easy way to increase the functional affinity (avidity) of single chain variable fragments (scFvs) by tetramerization on streptavidin, following their site-specific biotinylation by the enzyme BirA. Expression vectors have been constructed that enable addition of the 15 amino acid biotin acceptor domain (BAD) on selected scFvs. Different domains were cloned at the C-terminus of scFv in the following order: a semi-rigid hinge region (of 16 residues), the BAD, and a histidine tail. Two such recombinant scFvs directed against the carcinoembryonic antigen (CEA) were previously selected from human non-immune and murine immune phage display libraries. The scFvs were first synthesized in Escherichia coli carrying the plasmid encoding the BirA enzyme, and then purified from the cytoplasmic extracts by Ni-NTA affinity chromatography. Purified biotinylated scFvs were tetramerized on the streptavidin molecule to create a streptabody (StAb). The avidity of various forms of anti-CEA StAbs, tested on purified CEA by competitive assays and surface plasmon resonance showed an increase of more than one log, as compared with the scFv monomer counterparts. Furthermore, the percentage of direct binding of 125I-labeled StAb or monomeric scFv on CEA-Sepharose beads and on CEA-expressing cells showed a dramatic increase for the tetramerized scFv (>80%), as compared with the monomeric scFv (<20%). Interestingly, the percentage binding of 125I-labeled anti-CEA StAbs to CEA-expressing colon carcinoma cells was definitely higher (>80%) than that obtained with a reference high affinity murine anti-CEA mAb (30%). Another advantage of using scFvs in a StAb format was demonstrated by Western blot analysis, where tetramerized anti-CEA scFv could detect a small quantity of CEA at a concentration 100-fold lower than the monomeric scFv.
噬菌体展示是一种从高度多样化的天然人抗体库中分离抗体片段的强大方法。然而，所选抗体的亲和力通常较低，并且当前的亲和力成熟方法复杂且耗时。在本文中，我们描述了一种简便的方法，通过在链霉亲和素上进行四聚化来提高单链可变片段（scFv）的功能亲和力（亲合力），这是在其经BirA酶进行位点特异性生物素化之后。构建了表达载体，能够在所选scFv上添加15个氨基酸的生物素受体结构域（BAD）。不同结构域按以下顺序克隆到scFv的C末端：一个半刚性铰链区（16个残基）、BAD和一个组氨酸尾。先前已从人非免疫和鼠免疫噬菌体展示文库中筛选出两种针对癌胚抗原（CEA）的重组scFv。scFv首先在携带编码BirA酶质粒的大肠杆菌中合成，然后通过Ni-NTA亲和色谱从细胞质提取物中纯化。纯化的生物素化scFv在链霉亲和素分子上进行四聚化，以产生链霉抗体（StAb）。通过竞争分析和表面等离子体共振在纯化的CEA上测试各种形式的抗CEA StAb的亲合力，结果显示与scFv单体相比增加了一个对数以上。此外，125I标记的StAb或单体scFv在CEA-琼脂糖珠和表达CEA的细胞上的直接结合百分比显示，四聚化的scFv（>80%）与单体scFv（<20%）相比有显著增加。有趣的是，125I标记的抗CEA StAb与表达CEA的结肠癌细胞的结合百分比肯定高于参考高亲和力鼠抗CEA单克隆抗体（30%）。使用StAb形式的scFv的另一个优点通过蛋白质印迹分析得到证明，其中四聚化的抗CEA scFv能够检测到浓度比单体scFv低100倍的少量CEA。
