Expression of single-chain Fv-Fc fusions in Pichia pastoris
Expression of single-chain Fv-Fc fusions in Pichia pastoris
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DOI：
10.1016/s0022-1759(00)00290-8
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发表时间：
2001-05-01
影响因子：
2.2
通讯作者：
Marks, JD
中科院分区：
文献类型：
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作者：
Powers, DB;Amersdorfer, P;Marks, JD
噬菌体展示技术使得直接分离单价单链抗体片段成为可能。然而，对于许多应用，恢复Fc介导的抗体功能如亲合力、效应子功能和延长的血清半衰期是有用的。我们已经构建了载体，方便，快速表达的单链抗体Fv结构域（scFv）融合到Fc部分的人IgG 1的甲醇营养型酵母巴斯德毕赤酵母。scFv-Fc融合蛋白作为二硫键连接的糖基化同二聚体从培养基中分泌和回收。二聚体的增加的尺寸（类似于106 kDa对类似于25 kDa的scFv）导致体内血清半衰期延长，小鼠中β清除期的t（1/2）从典型scFv的3.5小时增加到scFv-Fc融合体的93小时。scFv-Fc融合体能够使用人外周血单核细胞作为效应器介导针对肿瘤靶细胞的抗体依赖性细胞毒性。最后，Fc结构域是用于纯化和免疫化学应用的方便、稳健的亲和手柄，消除了对scFv上的蛋白水解敏感表位和/或亲和标签的需要。(C)2001 Elsevier Science B. V.保留所有权利。
Phage display technology makes possible the direct isolation of monovalent single-chain Fv antibody fragments. For many applications, however, it is useful to restore Fc mediated antibody functions such as avidity, effector functions and a prolonged serum half-life. We have constructed vectors for the convenient, rapid expression of a single-chain antibody Fv domain (scFv) fused to the Fc portion of human IgG1 in the methylotrophic yeast Pichia pastoris. The scFv-Fc fusion protein is secreted and recovered from the culture medium as a disulfide-linked, glycosylated homodimer. The increased size of the dimer (similar to 106 kDa vs. similar to 25 kDa for a scFv) results in a prolonged serum half-life in vivo, with t(1/2) of the beta phase of clearance increasing from 3.5 h for a typical scFv to 93 h for a scFv-Fc fusion in mice. The scFv-Fc fusion is capable of mediating antibody-dependent cellular cytotoxicity against tumor target cells using human peripheral blood mononuclear cells as effecters. Finally, the Fc domain is a convenient, robust affinity handle for purification and immunochemical applications, eliminating the need for proteolytically sensitive epitope and/or affinity tags on the scFv. (C) 2001 Elsevier Science B.V. All rights reserved.