Generation of a stable anti-human CD44v6 scFv and analysis of its cancer-targeting ability in vitro
Generation of a stable anti-human CD44v6 scFv and analysis of its cancer-targeting ability in vitro
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DOI：
10.1007/s00262-010-0819-z
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发表时间：
2010-06-01
影响因子：
5.8
通讯作者：
Wu, Ying
中科院分区：
文献类型：
--
作者：
Chen, Yinting;Huang, Kaihong;Wu, Ying
CD 44 v6是一种肿瘤相关抗原，主要在腺癌的一个亚群中表达。因此，在本研究中，抗人CD 44 v6单链可变片段（scFv）已被选择和表征，因为它是构建用于癌症诊断和治疗的新型癌症靶向药物的首要步骤。在我们的研究中，抗人CD 44 v6单链抗体是从人噬菌体展示的单链抗体库中选择的，基于其体外结合CD 44 v6抗原的能力。随后，进行免疫荧光染色和Western印迹分析以测量该scFv的结合特性。此外，通过流式细胞术分析验证其体外肿瘤靶向能力。用流式细胞仪测定其平衡解离常数（K（D））。最后，选择并表征了一种功能性抗CD 44 v6 scFv。核苷酸测序证实它是一个不完整的scFv基因，但只有一个可变重链（V-H）。免疫荧光染色和Western blot分析表明，抗CD 44 v6单链抗体具有细胞结合和抗原结合活性。此外，流式细胞术分析证明该scFv在体外特异性靶向表达CD 44 v6的癌细胞，而不是不表达CD 44 v6的正常细胞或肿瘤细胞。该scFv的K（D）计算为7.85 +/- A 0.93 × 10（-8）M。总之，所选择的抗CD 44 v6的人scFv具有特异性结合活性和有利的结合亲和力，尽管缺乏可变轻链（V-L）。此外，它可以有效和特异性地靶向表达CD 44 v6的癌细胞。所有这些特性使得抗CD 44 v6单链抗体成为一种有希望的癌症检测和抗癌治疗试剂。
CD44v6 is a cancer-associated antigen that mainly expresses in a subset of adenocarcinomas. Therefore, in this study, anti-human CD44v6 single-chain variable fragment (scFv) has been selected and characterized because it is the first step of primary importance towards the construction of a novel cancer-targeted agent for cancer diagnosis and therapy. In our study, anti-human CD44v6 scFv was selected from a human phage-displayed scFv library based on its ability to bind in vitro to CD44v6 antigen. Subsequently, immunofluorescent staining and Western blot analyses were performed to measure the binding characteristics of this scFv. In addition, flow cytometric analysis was done to verify its cancer-targeting ability in vitro. And a flow cytometry-based assay was used to determine its equilibrium dissociation constant (K (D)). Finally, one functional anti-CD44v6 scFv was selected and characterized. Nucleotide sequencing verified that it was an incomplete scFv gene but had a variable heavy chain (V-H) alone. However, anti-CD44v6 scFv demonstrated cell-binding and antigen-binding activities by immunofluorescent staining and Western blot analyses. Furthermore, flow cytometric analysis proved that this scFv specifically targeted CD44v6-expressing cancer cells other than CD44v6 non-expressing normal cells or tumor cells in vitro. The K (D) of this scFv was calculated to be 7.85 +/- A 0.93 x 10(-8) M. In summary, the selected human scFv against CD44v6 has specific binding activity and favorable binding affinity despite lacking a variable light chain (V-L). Moreover, it can effectively and specifically target CD44v6-expressing cancer cells. All these characteristics make anti-CD44v6 scFv a promising agent for cancer detection and anti-cancer therapy.