Production and efficacy test of a recombinant fluorescence-labelled single chain variable fragment against trophoblast cell surface antigen 2 protein in vitro

Abstract

Trophoblast cell surface antigen 2 (Trop2) is a transmembrane glycoprotein encoded by the single-exon gene tumor-associated calcium signal transducer 2 (TACSTD2) gene and was first identified in human placental trophoblasts. Trop2 expression was detected in widely distributed healthy epithelial cells of many organs. A recent study showed that overexpression of Trop2 was presented and played a critical role in tumor progression and was involved in numerous signaling pathways associated with the development of many types of cancer. A single chain variable fragment (scFv) is a recombinant antibody that can be developed and modified for several applications in medicine including cancer diagnosis and therapy. The purpose of this study is to develop scFv against Trop2 and investigate the efficiency of scFv against Trop2 with mCherry labeling (Trop2mCherry scFv) for the detection of Trop2 molecules expressing on the surface of cancer cell lines. In this project, a gene of Trop2mCherry scFv has been constructed in pET-23a(+) plasmid and expressed by a bacterial system (Escherichia coli). Following the production process, Trop2mCherry36 scFv was tested for the specificity binding of Trop2mCherry scFv to Trop2 protein on human cholangiocarcinoma (CCA) cell lines including KKU-213A (high Trop2 expression) and KKU-055 (low Trop2 expression) by immunocytochemistry (ICC) analysis using inverted fluorescence microscopy and confocal microscopy analysis, in comparison to the staining by commercial anti-Trop2 antibody and Trop2mCherry31 scFv, a known Trop2-specific scFv. The results from the ICC analysis staining by either commercial anti-Trop2 antibody or Trop2mCherry31 scFv showed the stainings of KKU-213A and KKU-055 were correlated with their properties. However, Trop2mCherry36 scFv staining showed a positive signal in KKU-055. This may be a non-specific binding. The staining of Trop2mCherry 31 scFv was significantly reduced in Trop2-knockdown KKU-213A cells when compared with un-transfected cells. Overexpression of Trop2 was performed in KKU-055 cells, the results showed the binding of antibody had no difference between commercial antibody and Trop2mCherry scFv. Confocal microscopy analysis was correlated with ICC analysis and Trop2mCherry scFv staining showed membranous patterns on Trop2-expressing cell lines. In summary, the non-specific binding of Trop2mCherry36 scFv may be from the effect of denaturation during the protein extraction process, or the property of this scFv itself. Trop2mCherry31 scFv can be used for the detection of specific Trop2-expressing cell in vivo models for further study. Implication of Thesis: Trop2 overexpression has been identified in various human epithelial cancers. One of the most popular formats for developing recombinant antibody derivatives is scFv, which can be utilized for numerous applications. Producing a Trop2-mCherry labeled scFv may help in the detection of Trop2 molecules. The Trop2mCherry 31 scFv can be used as a specific detector for Trop2-expressing CCA cells. However, modifications are required for further studies of the Trop2mCherry 36 scFv.