Isolation and characterization of antibodies against three consecutive Tn-antigen clusters from a phage library displaying human single-chain variable fragments
Isolation and characterization of antibodies against three consecutive Tn-antigen clusters from a phage library displaying human single-chain variable fragments
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DOI：
10.1093/jb/mvq014
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发表时间：
2010-06-01
影响因子：
2.7
通讯作者：
Fujita-Yamaguchi, Yoko
中科院分区：
文献类型：
--
作者：
Sakai, Keiko;Yuasa, Noriyuki;Fujita-Yamaguchi, Yoko
Tn 抗原 GalNAc α-Ser/Thr 是一种肿瘤相关碳水化合物抗原，可为癌症的临床前检测提供敏感且特异的标记物，并成为癌症治疗的靶点。我们最近报道 MLS128 单克隆抗体治疗显着抑制结肠癌细胞和乳腺癌细胞的生长。根据我们的观察，本研究旨在生产具有与 MLS128 单克隆抗体相似的特异性的人抗 Tn 抗原抗体，该抗体可识别三个连续 Tn 抗原（Tn3）的结构。通过用合成 Tn3 肽进行淘选和筛选，从新构建的噬菌体文库中分离出 6 个展示人单链可变片段 (scFv) 的噬菌体克隆。推导的 6 个抗 Tn3 scFv 的氨基酸序列表现出高度的同源性。其中，抗 Tn3 4E10 和 4G2 scFv 蛋白已成功从噬菌体感染的大肠杆菌中纯化至接近同质。表面等离子共振分析显示，纯化的scFv蛋白对Tn3的K(D)约为10(-7) M，这对于碳水化合物特异性单价抗体来说很高。进一步的分析表明，这两种 scFv 蛋白还可以与 Tn2 以及培养的结肠癌细胞和乳腺癌细胞结合。这些结果证明了使用这些 scFv 开发针对结肠癌和乳腺癌的抗体疗法的潜力。
The Tn-antigen, GalNAc alpha-Ser/Thr, is a tumour-associated carbohydrate antigen that may provide a sensitive and specific marker for pre-clinical detection of carcinoma and a target for cancer therapies. We recently reported that MLS128 monoclonal antibody treatment significantly inhibited colon and breast cancer cell growth. On the basis of our observations, the present study aimed to produce human anti-Tn-antigen antibodies with specificity similar to that of MLS128 monoclonal antibody, which recognizes a structure of three consecutive Tn-antigens (Tn3). Six phage clones displaying human single-chain variable fragments (scFvs) were isolated from a newly constructed phage library by panning and screening with a synthetic Tn3-peptide. Deduced amino-acid sequences of six anti-Tn3 scFvs exhibited a high degree of homology. Of those, anti-Tn3 4E10 and 4G2 scFv proteins were successfully purified from phage-infected Escherichia coli to near homogeneity. Surface plasmon resonance analyses revealed a K(D) of purified scFv proteins for Tn3 on an order of 10(-7) M, which is high for carbohydrate-specific monovalent antibodies. Further analyses suggested that both scFv proteins also bind to Tn2 and cultured colon and breast cancer cells. These results demonstrated the potential for use of these scFvs in developing antibody therapeutics targeting colon and breast cancer.