Albrecht Huguette, Burke Patricia A, Natarajan Arutselvan, Xiong Cheng-Yi, Kalicinsky Mark, DeNardo Gerald L, DeNardo Sally J
Radiodiagnosis and Therapy, Molecular Cancer Institute, University of California Davis Medical Center, Sacramento, California 95816, USA.
Bioconjug Chem. 2004 Jan-Feb;15(1):16-26. doi: 10.1021/bc030018+.
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.
单链抗体片段（ScFv）重组抗体片段可为靶向药物提供特异性肿瘤结合模块。在构建多聚体肿瘤靶向药物的过程中，一个先决条件是保留功能性ScFv抗原结合域，从而排除ScFv与载体分子或另一个ScFv的随机缀合。对pCANTAB 5E噬菌体展示/表达载体进行基因工程改造，以表达任何ScFv基因，使其作为带有额外C末端半胱氨酸（ScFv-Cys）的ScFv，从而将特异性缀合位点从结合域中去除。从抗MUC-1 ScFv噬菌体文库中筛选出的ScFv在pCANTAB 5E及其修饰版本pCANTAB 5E Cys载体中表达，并对关键特性进行比较。比较了摇瓶和生物发酵罐中ScFv和ScFv-Cys的产量。在没有还原剂的情况下，稳定的二聚体（共价ScFv同型二聚体（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。在有还原剂的情况下，ScFv-Cys分子保持单体状态，游离的SH可用于与聚乙二醇（马来酰亚胺）2支架缀合，以形成免疫反应性聚乙二醇（ScFv）2生物缀合物。pCANTAB 5E Cys表达的ScFv可使大肠杆菌产生可溶性ScFv-Cys蛋白，既可以是稳定的ScFv-Cys，也可以是（ScFv-Cys）2。ScFv-Cys可用于与聚乙二醇缀合，形成二价聚乙二醇（ScFv-Cys）2分子，或用作（ScFv-Cys）2以提高免疫组织化学的灵敏度。
