Isolation and Characterization of Human ScFv Antibody Fragments to Non-metastatic Gastric Cancer Cells (AGS) from a Semisynthetic Phage Display Library

Abstract

Introduction: Generation of antibodies which potentially discriminate between malignant and healthy cells is an important prerequisite aimed at early diagnosis and therapy of gastric cancer (GC). Comparative analysis of cell surface protein landscape will provide an experimental basis for biomarker discovery which is essential for molecular targeted therapies. The objective of cur-rent study was to isolate phage-antibodies recognizing cell surface proteins which were differen-tially expressed between two closely related GC cell lines namely AGS and MKN-45. Methods: For this purpose, we selected and screened a semisynthetic phage-scFv library on AGS, MKN-45, and NIH-3T3 cell lines utilizing a tailored selection scheme that was designed to isolate phage-scFvs that not only recognize the differentiated AGS cells but also distinguish them from NIH-3T3 fibroblasts and the poorly differentiated MKN-45 cells. Results: After four rounds of subtractive whole cell panning, 14 unique clones were identified by ELISA screening and nucleotide sequencing. For further characterization, we focused on 4 phage-scFvs with strong signals in screening and their specificity was confirmed by cell-based ELISA. Furthermore, the selected phage-scFvs were able to stain AGS cells with 38.74 (H1), 11.04 (D11), 76.93 (G11), and 69.03% (D1) in flow cytometry analysis, supporting the ability of distinguishing AGS from MKN-45 and NIH-3T3 cells. Conclusion: Combined with other techniques, such phage-scFvs can be applied for proteome analysis and subsequently identifying novel tumor-related antigens mediating proliferation and differentiation of cells. Furthermore, the antibody fragments can be exploited for diagnostic pur-poses, as well as targeted drug delivery of GC.