Selection of potential therapeutic human single-chain Fv antibodies against cholecystokinin-B/gastrin receptor by phage display technology.
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 V(L) 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.


CONCLUSIONS
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.
Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
Mohammad R
MR
Tohidkia
Farzad
F
Asadi
Jaleh
J
Barar
Yadollah
Y
Omidi
D000818:Animals; D001666:Binding Sites, Antibody; D061505:Cell Surface Display Techniques; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005770:Gastrointestinal Neoplasms / Q000188:drug therapy* / Q000276:immunology; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D019151:Peptide Library; D043887:Receptor, Cholecystokinin B / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000627:therapeutic use
D019151:Peptide Library; D043887:Receptor, Cholecystokinin B; D057127:Single-Chain Antibodies