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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. Graphical abstract ?.

SUBMITTER: Jalilzadeh-Razin S

PROVIDER: S-EPMC6593017 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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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.

Jalilzadeh-Razin Sepideh S Mantegi Malihe M Tohidkia Mohammad R MR Pazhang Yaghub Y Pourseif Mohammad M MM Barar Jaleh J Omidi Yadollah Y

Daru : journal of Faculty of Pharmacy, Tehran University of Medical Sciences 20190103 1

<h4>Background</h4>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.<h4>Objectives</h4>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.<h4>Methods</h4>To isolate scFvs specific to the CCK2R, we c ...[more]

PMID: 30607886

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Project description:Phage-display selection of immunoglobulin (IG) or antibody single chain Fragment variable (scFv) from combinatorial libraries is widely used for identifying new antibodies for novel targets. Next-generation sequencing (NGS) has recently emerged as a new method for the high throughput characterization of IG and T cell receptor (TR) immune repertoires both in vivo and in vitro. However, challenges remain for the NGS sequencing of scFv from combinatorial libraries owing to the scFv length (>800?bp) and the presence of two variable domains [variable heavy (VH) and variable light (VL) for IG] associated by a peptide linker in a single chain. Here, we show that single-molecule real-time (SMRT) sequencing with the Pacific Biosciences RS II platform allows for the generation of full-length scFv reads obtained from an in vivo selection of scFv-phages in an animal model of atherosclerosis. We first amplified the DNA of the phagemid inserts from scFv-phages eluted from an aortic section at the third round of the in vivo selection. From this amplified DNA, 450,558 reads were obtained from 15 SMRT cells. Highly accurate circular consensus sequences from these reads were generated, filtered by quality and then analyzed by IMGT/HighV-QUEST with the functionality for scFv. Full-length scFv were identified and characterized in 348,659 reads. Full-length scFv sequencing is an absolute requirement for analyzing the associated VH and VL domains enriched during the in vivo panning rounds. In order to further validate the ability of SMRT sequencing to provide high quality, full-length scFv sequences, we tracked the reads of an scFv-phage clone P3 previously identified by biological assays and Sanger sequencing. Sixty P3 reads showed 100% identity with the full-length scFv of 767?bp, 53 of them covering the whole insert of 977?bp, which encompassed the primer sequences. The remaining seven reads were identical over a shortened length of 939?bp that excludes the vicinity of primers at both ends. Interestingly these reads were obtained from each of the 15 SMRT cells. Thus, the SMRT sequencing method and the IMGT/HighV-QUEST functionality for scFv provides a straightforward protocol for characterization of full-length scFv from combinatorial phage libraries.

Dataset Information

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.

Publications

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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