Phage antibody library screening for the selection of novel high-affinity human single-chain variable fragment against gastrin receptor: an in silico and in vitro study
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As a membrane G protein coupled receptors (GPCRs) family, gastrin/cholecystokinin-2 receptor (CCK2R) plays a key role in the initiation and development of gastric cancer.
Targeting CCK2R by immunotherapeutics such as single-chain variable fragments (scFvs) may provide an effective treatment modality against gastric cancer. Thus, the main objective of this study was to isolate scFvs specific to CCK2R.
To isolate scFvs specific to the CCK2R, we capitalized on a semi-synthetic diverse phage antibody library (PAL) and a solution-phase biopanning process. The library was panned against a biotinylated peptide of the second extracellular loop (ECL2) of CCK2R. After four rounds of biopanning, the selected soluble scFv clones were screened by enzyme-linked immunosorbent assay (ELISA) and examined for specific binding to the peptide. The selected scFvs were purified using immobilized metal affinity chromatography (IMAC). The binding affinity and specificity of the scFvs were examined by the surface plasmon resonance (SPR), immunoblotting and flow cytometry assays and molecular docking using ZDOCK v3.0.2.
Ten different scFvs were isolated, which displayed binding affinity ranging from 0.68 to 8.0 (nM). Immunoblotting and molecular docking analysis revealed that eight scFvs were able to detect the denatured form of CCK2R protein. Of the isolated scFvs, two scFvs showed high-binding affinity to the human gastric adenocarcinoma AGS cells.
Based on our findings, a couple of the selected scFvs showed markedly high-binding affinity to immobilized CCK2R peptide and CCK2R-overexpressing AGS cells. Therefore, these scFvs are proposed to serve as targeting and/or treatment agents in the diagnosis and immunotherapy of CCK2R-positive tumors.
KeywordsPhage display technology Biopanning Single-chain variable fragment Gastric cancer Gastrin receptor Molecular docking
G protein coupled receptors
Single-chain variable fragments
Phage antibody library
Enzyme-linked immunosorbent assay
Immobilized metal affinity chromatography
Surface plasmon resonance
Mucosa-associated lymphoid tissue lymphomas
Phage antibody display
Gene 3 minor coat protein
Phage display technology
The authors are grateful to the technical support provided by the Research Center for Pharmaceutical Nanotechnology (RCPN) at Tabriz University of Medical Sciences.
SJR and MM contributed to the data analysis and interpretation, and MMP was involved in the bioinformatics analysis, YP checked the grammar, MRT, and YO designed and supervised the study, conducted data analysis and interpretation, SJR, JB, MMP, MRT and YO wrote the manuscript.
The authors are grateful for the financial support provided by the Tabriz University of Medical Sciences (grant# 93003 and 93010).
Compliance with ethical standards
Conflict of interest
The authors of this study declare that they have no conflict of interest.
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