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Claudin‐4 (CLDN‐4), a tight‐junction protein, is overexpressed in various malignant tumors, including gastric, colorectal, pancreatic, and breast cancers. However, CLDN‐4 is also expressed in normal tissues, including the liver, pancreas, kidney, and small intestine. Whether CLDN‐4 is an effective and safe target for cancer therapy has been unclear owing to the lack of a binder with both CLDN‐4 specificity and cross‐reactivity to human and murine cells. In this study, we successfully generated a rat anti‐CLDN‐4 monoclonal antibody (5D12) that was specific to, and cross‐reactive with, human and mouse CLDN‐4. 5D12 recognized the second extracellular domain of human CLDN‐4 in a conformation‐dependent manner. A human–rat chimeric IgG1 of 5D12 (xi‐5D12) activated the Fcγ IIIa receptor, indicating the activation of antibody‐dependent cellular cytotoxicity in CLDN‐4‐expressing cells. Moreover, xi‐5D12 significantly suppressed tumor growth in mice bearing human colorectal and gastric tumors without apparent adverse effects, such as weight loss or liver and kidney damage. These results suggest that CLDN‐4 is a potent target for cancer therapy and that an anti‐CLDN‐4 antibody is a promising candidate anticancer agent.

Adult T-cell leukemia/lymphoma (ATLL) is an aggressive form of human leukemia/lymphoma. Although this disease is initiated by infection with human T-lymphotropic virus type 1 (HTLV-1), many HTLV-1 carriers survive for a long period without aggressive illness, suggesting that other factors may play roles in the progression of ATLL to an aggressive state. However, the mechanism involved in this progression still remains unclear. Previously, we have reported that ASY/Nogo mRNA was markedly down-regulated in human small-cell lung carcinomas, whereas it was expressed in normal tissues and other lung carcinomas, such as adenocarcinoma and squamous cell carcinoma. To understand whether or not ASY/Nogo gene is involved in the progression of ATLL, we examined the expression of ASY/Nogo mRNA in smoldering, chronic and aggressive ATLL, and found that the expression level of ASY/Nogo mRNA was markedly reduced in clinically aggressive ATLL. HTLV-1 Tax expression was not affected by the down-regulation of ASY/Nogo mRNA. These results indicate that the ASY/Nogo gene may act as a suppressor against ATLL progression, independent of Tax expression.

Epithelium plays pivotal roles in barriers that separate the inside of the body from the outside environment. Passage across epithelial barriers is the first step in drug absorption. Epidermal absorption is an ideal route for drug administration because it is non-invasive and easily controlled. Claudin-1 (CLDN1) seals tight junctions in the stratum granulosum, but whether modulation of CLDN1 enhances epidermal permeability is unclear. We investigated the effect of an anti-CLDN1 antibody on the epidermal epithelial barrier by using human epidermal keratinocytes (NHEK), a popular in vitro model of human epithelial cell sheets. Immunoblot and FACS analyses showed that CLDN1 was expressed on NHEK cell membranes. Treatment of NHEK monolayers with anti-CLDN1 antibody decreased the integrity of tight junctions between adjacent cells and enhanced the permeation of solutes across the cell sheets. Furthermore, treatment of Caco-2 cell monolayers, a popular model of the human intestinal barrier that expresses CLDN1 ...

Epithelial cell sheets have two types of intercellular tight junctions (TJs): bicellular tight junctions (bTJs) and tricellular tight junctions (tTJs). Modulation of bTJs has been investigated in n...

Claudin (CLDN) is a key component of the epithelial tight junction. Expression of the various CLDNs differs among tissues and is deregulated in malignant tumors. For example, CLDN4 is frequently overexpressed in pancreatic, breast, and ovarian cancers and therefore may be an attractive target for anticancer drugs. However, the low antigenicity of CLDN4 has hampered the development of antibody-based CLDN4-targeted cancer therapy. We successfully generated monoclonal antibodies (mAbs) to CLDN4 and evaluated their anti-tumor activity. Briefly, we immunized rats with plasmids containing the CLDN4 coding sequence. We fused the resulting B cells with myeloma cells and screened 8 hybridoma clones that produced anti-CLDN4 mAbs. All 8 mAbs bound to CLDN4-expressing cells but not to those expressing CLDN1, 2, 5, 6, 7or 9. All 8 mAbs showed CLDN4 binding-dependent activation of the Fcγ receptor. A clone with high affinity to CLDN4 suppressed tumor growth in vitro and in vivo. Interestingly, treatment of cancer cells...

Claudins (CLDNs) comprise a tetra-transmembrane protein family with ~20 members, which are located in the lateral membrane between adjacent epithelial cells. Because of loss of cellular polarity during malignant transformation, CLDN often migrates from the lateral membrane and becomes exposed on epithelial cell surfaces. CLDN3 and CLDN4 are targets for tumor therapy because they are overexpressed in various malignant tumors. We previously optimized a protocol for immunization with CLDN and developed 8 rat anti-CLDN4 monoclonal antibodies, one of which recognized both CLDN3 and CLDN4. In this study, we focused on the CLDN3/4 antibody. The CLDN3/4 antibody showed high affinity for CLDN4 (KD value: 4.3 nM). We fluorescently labeled the CLDN3/4 antibody and intravenously injected it into mice bearing tumors (human colon cancer Lovo cells, or human gastric cancer MKN74 cells). The CLDN3/4 antibody accumulated in tumor tissues and attenuated tumor growth without apparent adverse effects in the Lovo- and MKN74-b...

The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) is the most well-characterized claudin-3 and -4 (CL-3 and -4) binder. The mucosal absorption-enhancing activity of C-CPE is over 400-fold more potent than a clinically used enhancer. C-CPE has potential in cancer therapy and for mucosal vaccination. Since CL-3 and -4 are ubiquitously expressed in heart, lung, liver, kidney, thyroid gland, stomach, and intestine, safety evaluations are important for their future therapeutic use. Here, we investigated the tissue distribution of C-CPE in mice after intravenous administration. We prepared CF750-labeled wild-type C-CPE and C-CPE harboring mutant CL-3/-4 binding regions. FACS analysis showed that labeled wild-type C-CPE bound to CL-4 but that labeled mutant C-CPE did not, indicating that labeling did not alter the CL-binding properties of C-CPE. Most wild-type and mutant C-CPE promptly accumulated in the kidney, and C-CPE were more distributed in the liver, thyroid gland and intestine than m...