Isolation and Characterization of Metastatic Gastric Cancer Cell line (MKN-45)-Specific scFv Fragments by Whole Cell Panning Using Phage Display Technology

Abstract

Among various solid tumors, gastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide. Expansion into peritoneal cavity which results from dissemination of diffuse cancer cells is the main cause of mortality in gastric adenocarcinoma patients. The aim of present study is identification of putative biomarkers involved in metastasis of diffuse gastric adenocarcinoma cell line (MKN-45) using phage display derived scFv fragments.

Methods: A semi-synthetic human scFv library (Tomlinson I) was used to isolate novel antibody fragments recognizing MKN-45, a poorly differentiated diffuse gastric adenocarcinoma cell line. Four rounds of subtractive selection each consisting of extensive pre-absorption of phage library with NIH-3T3 (murine embryonic fibroblasts) and AGS (a well-differentiated intestinal gastric adenocarcinoma) cell lines were carried out prior to positive selection on MKN-45 target cells. Specificity analysis of the selected scFv fragments were determined through whole cell ELISA and flow cytometry. LC-MS/MS analysis was carried out to identify target antigens. Immunohistochemistry was performed on paraffin-embedded tissue sections of gastric adenocarcinoma biopsies.

Result: ELISA-based screening of 192 phage-displayed scFv clones indicated 21 high affinity binders with specific staining of MKN-45 compared to NIH-3T3 and AGS cells. Diversity analysis of the selected phage-scFvs resulted in 5 distinct sequences with multiple frequency. Further analysis by ELISA and flow cytometry verified three clones specifically recognized MKN-45 cells. LC-MS/MS analysis of immunoperecipitated proteins has led to identification of c-MET, HSP90 α and HSP90 β as candidate proteins associated with diffuse GC. Immunohistochemistry revealed capability of purified scFvs to differentiate diffuse and intestinal gastric adenocarcinoma. Conclusion: In summary, we successfully isolated a panel of scFv fragments which can discriminate diffuse gastric adenocarcinoma cell line, MKN-45, from intestinal ones, AGS. The isolated scFvs and their cognate antigens would be beneficial in screening, management, targeting and therapy of the diffuse gastric adenocarcinoma.