Development and characterization of a human single-chain antibody fragment against claudin-3: a novel therapeutic target in ovarian and uterine carcinomas.

Romani, Chiara; Comper, Fabrizio; Bandiera, Elisabetta; Ravaggi, Antonella; Bignotti, Eliana; Tassi, Renata A; Pecorelli, Sergio; Santin, Alessandro D · Am J Obstet Gynecol · 2009

basic_science · Level V

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Abstract

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). 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. 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. scFv H6 may represent a novel antitumor agent against chemotherapy-resistant ovarian and serous papillary carcinomas and other human malignancies that overexpress CLDN3.

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