title: Screening for antibodies against intact cancer cells with a naïve large             phage antibody library.
abstract: 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.
authors.affiliation: Graduate School of Southern Medical University, Guangzhou, PR China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Qiao
authors.forename: Lijun
authors.initials: L
authors.lastname: Zhou
authors.forename: Yuxiao
authors.initials: Y
authors.lastname: Wang
authors.forename: Fang
authors.initials: F
authors.lastname: Liu
authors.forename: Peng
authors.initials: P
authors.lastname: Xie
authors.forename: Yuping
authors.initials: Y
authors.lastname: Chen
authors.forename: Dajin
authors.initials: D
authors.lastname: Zhang
authors.forename: Xiaohang
authors.initials: X
authors.lastname: Zhao
mesh_terms: D000918:Antibody Specificity; D001402:B-Lymphocytes / Q000276:immunology; D045744:Cell Line, Tumor; D003001:Cloning, Molecular / Q000379:methods*; D016503:Drug Delivery Systems; D004797:Enzyme-Linked Immunosorbent Assay; D004938:Esophageal Neoplasms / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D019151:Peptide Library; D011485:Protein Binding; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology*
chemical_list: D019151:Peptide Library; D057127:Single-Chain Antibodies