Development of an Anti–Claudin-3 and -4 Bispecific Monoclonal Antibody for Cancer Diagnosis and Therapy

Abstract

Most malignant tumors are derived from epithelium, and claudin (CLDN)-3 and CLDN-4 are frequently overexpressed in such tumors. Although antibodies have potential in cancer diagnostics and therapy, development of antibodies against CLDNs has been difficult because the extracellular domains of CLDNs are too small and there is high homology among human, rat, and mouse sequences. Here, we created a monoclonal antibody that recognizes human CLDN-3 and CLDN-4 by immunizing rats with a plasmid vector encoding human CLDN-4. A hybridoma clone that produced a rat monoclonal antibody recognizing both CLDN-3 and -4 (clone 5A5) was obtained from a hybridoma screen by using CLDN-3-and -4-expressing cells; 5A5 did not bind to CLDN-1-, -2-, -5-, -6-, -7-, or -9-expressing cells. Fluorescenceconjugated 5A5 injected into xenograft mice bearing human cancer MKN74 or LoVo cells could visualize the tumor cells. The human-rat chimeric IgG1 monoclonal antibody (xi5A5) activated FcgRIIIa in the presence of CLDN-3-or -4-expressing cells, indicating that xi5A5 may exert antibody-dependent cellular cytotoxicity. Administration of xi5A5 attenuated tumor growth in xenograft mice bearing MKN74 or LoVo cells. These results suggest that 5A5 shows promise in the development of a diagnostic and therapeutic antibody for cancers.

Monoclonal Antibodies against Extracellular Domains of Claudin-1 Block Hepatitis C Virus Infection in a Mouse Model

“…These FLAG-tagged CLDN1 vectors were transiently introduced into 293T cells by use of X-tremeGENE HP DNA transfection reagent (Roche Diagnostics). Mouse CLDN1 and human CLDN1, -2, -4, -5, -6, -7, and -9 cDNAs were generated via PCR, using primer pairs specific to each CLDN (23). The resultant cDNAs were cloned into pcDNA3.1(Ϫ) (Invitrogen, CA).…”

“…Six-week-old male BXSB mice were immunized with a eukaryotic expression vector that encoded hCLDN1 by use of proprietary Genovac technology (Genovac GmbH) (18,23). The lymphocytes were removed 7 days after the final immunization and fused with P3-UI cells in the presence of polyethylene glycol 1000.…”

“…The resultant cDNAs were cloned into pcDNA3.1(Ϫ) (Invitrogen, CA). The CLDN expression vectors were then introduced into HT1080 cells, and G418-resistant clones were selected, resulting in the isolation of cells that stably expressed each CLDN (23).…”

“…Immunoglobulin classes/subclasses of clones were determined using a mouse immunoglobulin isotyping enzyme-linked immunosorbent assay (ELISA) kit (BD Biosciences). A rat MAb recognizing extracellular domains of CLDN4 was prepared as described previously (23).…”

Claudin-1 Binder Enhances Epidermal Permeability in a Human Keratinocyte Model

“…Human intestinal epithelial cells (Caco-2; American Type Culture Collection, Manassas, VA) were grown under a 5% CO 2 atmosphere at 37°C in modified Eagle's medium containing 10% fetal bovine serum. Human embryonic kidney 293T cells (American Type Culture Collection) and HT1080 cells stably transfected with mock treatment, human CLDN-1, CLDN-2, CLDN-4, or CLDN-5 (mock/HT1080 cells, CLDN-1/HT1080 cells, CLDN-2/HT1080 cells, CLDN-4/HT1080 cells, or CLDN-5/ HT1080 cells) were grown under a 5% CO 2 atmosphere at 37°C in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Li et al, 2014).…”