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Selection of Potential Therapeutic Human Single-Chain Fv Antibodies against Cholecystokinin-B/Gastrin Receptor by Phage Display Technology

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Abstract

Background and Objective

Gastric/gastrointestinal cancers are associated with high mortality worldwide. G-protein coupled receptor (GPCR) superfamily members such as gastrin/cholecystokinin-B receptor (CCK-BR) are involved in progression of gastric tumors, thus CCK-BR is considered as a potential target for immunotherapy. However, production of functional monoclonal antibodies (mAbs) against GPCR seems to be very challenging, in part due to its integration in cell membranes and inaccessibility for selection. To tackle this problem, we implemented phage display technology and a solution-phase biopanning (SPB) scheme for production of mAbs specific to the native conformation of CCK-BR.

Methods

To perform the SPB process, we utilized a synthetic biotinylated peptide corresponding to the second extracellular loop (ECL2) of CCK-BR and a semi-synthetic phage antibody library. After enzyme-linked immunosorbent assay (ELISA) screening, the CCK-BR specificity of the selected single-chain variable fragments (scFvs) were further examined using immunoblotting, whole-cell ELISA, and flow cytometry assays.

Results

After performing four rounds of selection, we identified nine antibody clones which showed positive reactivity with the CCK-BR peptide in an ELISA assay. Of these, eight clones were unique scFv antibodies and one was a VL single domain antibody. Specificity analysis of the selected scFvs revealed that five of the selected scFvs recognized a denatured form of CCK-BR, while the majority of the selected scFvs were able to recognize the native conformation of CCK-BR on the surface of human gastric adenocarcinoma cells and cervical carcinoma HeLa cells.

Conclusion

For the first time, we report on the establishment of a diverse panel of scFv antibody fragments that are specific to the native conformation of CCK-BR. Based on these results, we suggest the selected scFv antibody fragments as potential agents for diagnosis, imaging, targeting, and/or immunotherapy of cancers that overexpress CCK-BR.

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Acknowledgments

This work was supported by the Digestive Disease Research Center (DDRC) at Tehran University of Medical Sciences and the Research Center for Pharmaceutical Nanotechnology (RCPN) at Tabriz University of Medical Sciences. The authors are grateful to Mr. Abolfazl Barzegari, Dr. Hossein Zareh and Dr. Safar Farajnia (Tabriz University of Medical Sciences) and Dr. Masoumeh Rajabi Bazli (Shaid Behesti University of Medical Sciences) for their useful comments.

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Authors and Affiliations

  1. Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran

    Mohammad R. Tohidkia, Jaleh Barar & Yadollah Omidi

  2. Digestive Disease Research Center, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran

    Mohammad R. Tohidkia & Farzad Asadi

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

    Mohammad R. Tohidkia & Farzad Asadi

  4. Ovarian Cancer Research Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Jaleh Barar & Yadollah Omidi

  5. Ovarian Cancer Research Center, Translational Research Center, 8th Floor, Office #8-104, 3400 Civic Center Blvd, Building 421, Philadelphia, PA, 19104-5156, USA

    Yadollah Omidi

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  1. Mohammad R. Tohidkia
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  2. Farzad Asadi
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Correspondence to Farzad Asadi or Yadollah Omidi.

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Tohidkia, M.R., Asadi, F., Barar, J. et al. Selection of Potential Therapeutic Human Single-Chain Fv Antibodies against Cholecystokinin-B/Gastrin Receptor by Phage Display Technology. BioDrugs 27, 55–67 (2013). https://doi.org/10.1007/s40259-012-0007-0

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