Development and characterization of a human single-chain antibody fragment against claudin-3: a novel therapeutic target in ovarian and uterine carcinomas.
OBJECTIVE
The purpose of this study was to develop and characterize a human antibody in a single-chain antibody fragment format (scFv) that is directed specifically against claudin-3 (CLDN3).


METHODS
The synthetic ETH-2 Gold human antibody phage display library was used to select scFv specific against CLDN3. scFv binding properties were analyzed by surface plasmon resonance; specificity was confirmed with enzyme-linked immunosorbent assay, immunofluorescence, and flow cytometry on a panel of ovarian and uterine serous carcinoma cell lines.


RESULTS
Surface plasmon resonance studies indicated scFv H6 to be the clone with the highest affinity against CLDN3 (K(D) of 23.60 nmol/L). scFv H6 efficiently stained CLDN3-expressing cells and recognized its epitope in enzyme-linked immunosorbent assay that was performed with uterine serous papillary carcinoma native protein extract, which suggested that a conformational epitope is recognized by this antibody. Cell surface immunofluorescence with laser scanning confocal microscopy confirmed the specific binding to the native membrane CLDN3.


CONCLUSIONS
scFv H6 may represent a novel antitumor agent against chemotherapy-resistant ovarian and serous papillary carcinomas and other human malignancies that overexpress CLDN3.
Romani
Chiara
C
Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, University of Brescia, Brescia, Italy.
Comper
Fabrizio
F
Bandiera
Elisabetta
E
Ravaggi
Antonella
A
Bignotti
Eliana
E
Tassi
Renata A
RA
Pecorelli
Sergio
S
Santin
Alessandro D
AD
D002291:Carcinoma, Papillary / Q000276:immunology* / Q000378:metabolism; D045744:Cell Line, Tumor; D002469:Cell Separation; D062465:Claudin-3; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D015723:Gene Library; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology* / Q000378:metabolism; D008565:Membrane Proteins / Q000276:immunology* / Q000378:metabolism; D018613:Microscopy, Confocal; D010051:Ovarian Neoplasms / Q000276:immunology* / Q000378:metabolism; D021161:Self-Sustained Sequence Replication; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance; D002583:Uterine Cervical Neoplasms / Q000276:immunology* / Q000378:metabolism
C568122:CLDN3 protein, human; D062465:Claudin-3; D007128:Immunoglobulin Fragments; D008565:Membrane Proteins