Functional construction of the anti-mucin core protein (MUC1) antibody MUSE11 variable regions in a bacterial expression system.
Functional construction of the anti-mucin core protein (MUC1) antibody MUSE11 variable regions in a bacterial expression system.
复制标题
细菌表达系统中抗粘蛋白核心蛋白 (MUC1) 抗体 MUSE11 可变区的功能构建。
DOI：
10.1093/oxfordjournals.jbchem.a022656
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发表时间：
2000
影响因子：
2.7
通讯作者：
I. Kumagai
中科院分区：
文献类型：
--
作者：
R. Asano;S. Takemura;K. Tsumoto;N. Sakurai;A. Teramae;S. Ebara;Y. Katayose;M. Shinoda;M. Suzuki;K. Imai;S. Matsuno;T. Kudo;I. Kumagai
构建了抗MUC1肿瘤抗原抗体MUSE11可变区片段的细菌表达系统。该Fv片段与TFK-1细胞具有结合特异性，但亲和力略低于其亲本抗体。单链Fv片段按两个顺序排列：VH-接头-VL和VL-接头-VH。然而，用柔性多肽接头(GGGGS)(3)或用较短的接头(GGGGS)连接这些区域会导致在这两种排列中针对靶抗原的生物活性急剧下降，这表明当这些区域与多肽接头连接时，MUSE11抗体失去了活性。这些结果表明，抗MUC1抗体MUSE11的可变区仅在Fv形式下具有特异性，连接这些区域会强烈地降低与其靶抗原的联系。凝胶过滤分析表明，单链抗体具有二聚体结构，表明MUSE11单链抗体的失活是由于单链抗体链的分子间结合不良所致。据我们所知，这是第一次报道由于连接两种排列中的可变域，即VH-VL和VL-VH导致亲和力显著降低。
A bacterial expression system for the variable region fragments (Fvs) of the anti-MUC1 tumor antigen antibody MUSE11 has been constructed. The Fv fragment showed binding specificity toward TFK-1 cells, with slightly reduced affinity compared to its parent IgG. The single-chain Fv fragment was arranged in two orders, VH-linker-VL and VL-linker-VH. However, linking the regions with a flexible peptide linker (GGGGS)(3) or with a shorter linker (GGGGS) led to a dramatic decrease in the biological activity toward the target antigen in both arrangements, suggesting that the MUSE11 antibody loses its activity when the domains are linked with polypeptide linkers. These results indicate that the variable region domains of the anti-MUC1 antibody MUSE11 have specificity only in the Fv form, and that linking the domains strongly reduces the association with its target antigen. Gel filtration analysis indicates that the scFv has a dimeric structure, suggesting that the inactivation of MUSE11 scFv is due to unfavorable intermolecular associations of the scFv chains. To our knowledge, this is the first report of a significant reduction in affinity caused by linking the variable domains in both arrangements, i.e., VH-VL and VL-VH.