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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

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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.

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Abbreviations

CCK2R:

Gastrin/cholecystokinin-2 receptor

GPCRs:

G protein coupled receptors

scFvs:

Single-chain variable fragments

PAL:

Phage antibody library

ECL2:

Extracellular loop

ELISA:

Enzyme-linked immunosorbent assay

IMAC:

Immobilized metal affinity chromatography

SPR:

Surface plasmon resonance

TM:

Transmembrane

ICL:

Intracellular loops

ECL:

Extracellular loops

MALT:

Mucosa-associated lymphoid tissue lymphomas

mAbs:

Monoclonal antibodies

PAD:

Phage antibody display

Ag:

Antigen

g3p:

Gene 3 minor coat protein

PDT:

Phage display technology

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Acknowledgments

The authors are grateful to the technical support provided by the Research Center for Pharmaceutical Nanotechnology (RCPN) at Tabriz University of Medical Sciences.

Funding

The authors are grateful for the financial support provided by the Tabriz University of Medical Sciences (grant# 93003 and 93010).

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Author notes

  1. Sepideh Jalilzadeh-Razin and Malihe Mantegi contributed equally to this work.

Authors and Affiliations

  1. Research Center for Pharmaceutical Nanotechnology, Biomedicine Institute, Tabriz University of Medical Sciences, Tabriz, Iran

    Sepideh Jalilzadeh-Razin, Malihe Mantegi, Mohammad R. Tohidkia, Mohammad M. Pourseif, Jaleh Barar & Yadollah Omidi

  2. Islamic Azad University of Urmia, Urmia, Iran

    Malihe Mantegi

  3. Department of Biochemistry, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

    Yaghub Pazhang

  4. Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran

    Jaleh Barar & Yadollah Omidi

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  1. Sepideh Jalilzadeh-Razin
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Contributions

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.

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Correspondence to Mohammad R. Tohidkia or Yadollah Omidi.

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Jalilzadeh-Razin, S., Mantegi, M., Tohidkia, M.R. et al. 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. DARU J Pharm Sci 27, 21–34 (2019). https://doi.org/10.1007/s40199-018-0233-1

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