Production of soluble ScFvs with C-terminal-free thiol for site-specific conjugation or stable dimeric ScFvs on demand
Production of soluble ScFvs with C-terminal-free thiol for site-specific conjugation or stable dimeric ScFvs on demand
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DOI：
10.1021/bc030018
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发表时间：
2004-01-01
影响因子：
4.7
通讯作者：
DeNardo, SJ
中科院分区：
文献类型：
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作者：
Albrecht, H;Burke, PA;DeNardo, SJ
ScFv重组抗体片段可以为靶向药物提供特异性的肿瘤结合模块。在构建多聚体肿瘤靶向药物的过程中，先决条件是功能性scFv抗原结合结构域的保守，从而排除scFv与载体分子或另一种scFv的随机缀合。对pCANTAB 5E噬菌体展示/表达载体进行遗传工程改造，以将任何scFv基因表达为具有额外C-末端半胱氨酸的scFv（scFv-Cys），使得从结合结构域去除特异性缀合位点。将来自抗MUC-1 scFv噬菌体文库的所选scFv在pCANTAB 5E及其修饰版本pCANTAB 5E Cys载体中表达，并比较关键特征。比较了scFv和scFv-Cys在摇瓶和发酵罐中的产量。在不存在还原剂的情况下，稳定的二聚体（共价scFv同二聚体（scFv-Cys）（2））是scFv-Cys的主要形式。这些双抗体为组织的免疫组织化学染色提供了显著的信号增强。在还原剂存在下，scFv-Cys分子保持单体，游离SH可用于缀合至PEG（马来酰亚胺）（2）支架以形成免疫反应性PEG（scFv）（2）生物缀合物。来自pCANTAB 5E Cys的ScFv表达允许从大肠杆菌产生可溶性scFv-Cys蛋白，作为稳定的scFv-Cys或（scFv-Cys）（2）。ScFv-Cys可用于与PEG缀合以形成二价PEG（scFv-Cys）（2）分子或用作（scFv-Cys）（2）以提高IHC中的灵敏度。
ScFv recombinant antibody fragments can provide specific tumor binding modules for targeting drugs. In the process of building multimeric tumor targeting pharmaceuticals, a prerequisite is the conservation of functional scFv antigen binding domains, thereby excluding scFv random conjugation to a carrier molecule or to another scFv. The pCANTAB 5E phage display/expression vector was genetically engineered to express any scFv gene as scFv with an additional C-terminal cysteine (scFv-Cys) such that the specific conjugation site is removed from the binding domain. Selected scFvs derived from an anti-MUC-1 scFv phage library were expressed in pCANTAB 5E and its modified version pCANTAB 5E Cys vectors, and compared for key characteristics. Production yields of scFv and scFv-Cys in shaker flask and biofermentor were compared. In the absence of a reducing agent, stable dimers (covalent scFv homodimers (scFv-Cys)(2)) were the major form of scFv-Cys. These diabodies provided substantial signal enhancement for immunohistochemical staining of tissues. In the presence of a reducing agent, scFv-Cys molecules remained monomeric, with the free SH available for conjugation to a PEG(maleimide)(2) scaffold to form immunoreactive PEG(scFv)(2) bioconjugates. ScFv expression from pCANTAB 5E Cys allowed for the production of soluble scFv-Cys protein from E.coli, either as stable scFv-Cys or (scFv-Cys)(2). ScFv-Cys can be used for conjugation to PEG to form bivalent PEG (scFv-Cys)(2) molecules or used as (scFv-Cys)(2) for increased sensitivity in IHC.