Screening for antibodies against intact cancer cells with a naïve large phage antibody library.
Screening for antibodies against intact cancer cells with a naïve large phage antibody library.
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DOI：
10.3892/ijmm.2011.800
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发表时间：
2011-09
影响因子：
5.4
通讯作者：
Y. Qiao;Lijun Zhou;Yuxiao Wang;Fang Liu;Peng Xie;Yuping Chen;Dajin Zhang;Xiaohang Zhao
中科院分区：
文献类型：
--
作者：
Y. Qiao;Lijun Zhou;Yuxiao Wang;Fang Liu;Peng Xie;Yuping Chen;Dajin Zhang;Xiaohang Zhao
大型噬菌体抗体库有望成为获得人源化抗肿瘤抗体的有效技术。然而，很少有报道关注完整癌细胞作为靶点的选择策略。本研究利用LoxP-cre系统构建了2 × 10 ~（11）个来自正常成人血液和新生儿脐带血B淋巴细胞的天然单链抗体库。将三种人食管癌细胞系均匀混合用作靶标。根据细胞的处理方法（分别用2%PFA固定或不固定），将这些完整细胞分为PF和NF两组。在4个粘附、洗脱和扩增的淘选循环后鉴定阳性噬菌体抗体。用细胞ELISA法检测，发现用酸性溶液洗涤后的细胞沉淀直接感染XL 1-Blue细菌的附加步骤可有效地减少阳性噬菌体的损失，阳性筛选率为11.6%（61/525）。大多数噬菌体抗体显示与所有三种食管癌细胞系的结合活性。此外，其他的转录因子（如NFc 53 a和NFc 70 a）似乎对某些细胞系具有特异性。关于用于处理靶细胞的方法，NF组的阳性筛选率和抗体可变区的遗传多样性（分别为12.9%和71.4%）均显著高于PF组（分别为10.5%和14.2%）。免疫组化结果显示，该单链抗体对食管癌具有良好的特异性。该技术有助于开发小分子示踪剂和恶性肿瘤的靶向治疗。
Large phage antibody libraries are expected to be an efficient technology for obtaining humanized anti-tumor antibodies. However, few reports have been concerned about the selection strategies for intact cancer cells as targets. In this study, a 2x1011 large naïve scFv library from blood B lymphocytes of normal adult donors and neonatal cord blood was constructed using the LoxP-cre system. Three human esophageal cancer cell lines were equally mixed for use as the target. These intact cells were divided into two groups, PF and NF, according to the treatment method of the cells (fixed with 2% PFA or not, respectively). Positive phage antibodies were identified following 4 panning cycles of adhesion, elution and amplification. Using a cell ELISA assay, it was found that the additional procedure of directly infecting XL1-Blue bacteria with the cell pellet following washing with an acidic solution can effectively decrease the loss of positive phage, yielding a positive screening rate of 11.6% (61/525). Most of the phage antibodies displayed binding activity with all three esophageal cancer cell lines. Moreover, other phages (such as NFc53a and NFc70a) appeared to be specific for certain cell lines. Regarding the method used to treat the target cells, both the positive screening rate and the genetic diversity of the antibody variable region were significantly higher in the NF group (12.9 and 71.4%, respectively) than in the PF group (10.5 and 14.2%, respectively). Immunohistochemical analysis demonstrated that the scFv phages have good specificity for esophageal cancer. This technology is helpful for developing small molecular tracers and targeted therapies for malignant tumors.