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

Abstract

Background 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. Objectives 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. Methods 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. Results 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. Conclusions 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.

Development of a human phage display-derived anti-PD-1 scFv antibody: an attractive tool for immune checkpoint therapy

“…Antibody isolation using the hybridoma technology or transgenic mice needs long-term immunization procedures and screening [ 32 ]. Phage display is a technology to select recombinant antibodies like scFvs that bind specifically to various target molecules such as proteins, peptides, cell-surface glycans, and receptors with high affinity [ 23 ]. Until now, 14 phage display-derived therapeutic antibodies have been approved by FDA and/or EMA that the first one was against anti-tumor necrosis factor α (TNFα), named adalimumab (Humira) [ 35 ].…”

“…Phage display technology has broadly been used for selection of recombinant antibody fragments. This technique makes it easy to select specific binders against various target molecules [ 23 , 24 ].…”

Pyruvate dehydrogenase complex—enzyme 2, a new target for Listeria spp. detection identified using combined phage display technologies

“…In fact, most of the studies on antibody generation via phage display describe the use of purified antigens, suggesting that this approach may be more successful in providing high-quality antibodies [52][53][54][55][56] . Accordingly, in the present study, 16 novel antibodies were generated with this strategy, of which four showed better diagnostic performance than the best of the initial four antibodies generated against cell fractions (see Table 3).…”

Molecular Docking and Molecular Dynamics (MD) Simulation of Human Anti-Complement Factor H (CFH) Antibody Ab42 and CFH Polypeptide

“…pCFH was the ligand and Ab42 was the receptor whose CDRs was defined as the binding region in the docking calculation. The RMSD Cutoff and the Interface Cutoff were set to six and nine angstroms, respectively [27,28]. A total of 2000 docking poses and 100 cluster were generated.…”