Heterogenous Gene Expression of Bicellular and Tricellular Tight Junction-Sealing Components in the Human Intestinal Tract.

A major site for the absorption of orally administered drugs is the intestinal tract, where the mucosal epithelium functions as a barrier separating the inside body from the outer environment. The intercellular spaces between adjacent epithelial cells are sealed by bicellular and tricellular tight junctions (TJs). Although one strategy for enhancing intestinal drug absorption is to modulate these TJs, comprehensive gene (mRNA) expression analysis of the TJs components has never been fully carried out in humans. In this study, we used human biopsy samples of normal-appearing mucosa showing no endoscopically visible inflammation collected from the duodenum, jejunum, ileum, colon, and rectum to examine the mRNA expression profiles of TJ components, including occludin and tricellulin and members of the claudin family, zonula occludens family, junctional adhesion molecule (JAM) family, and angulin family. Levels of claudin-3, -4, -7, -8, and -23 expression became more elevated in each segment along the intestinal tract from the upper segments to the lower segments, as did levels of angulin-1 and -2 expression. In contrast, expression of claudin-2 and -15 was decreased in the large intestine compared to the small intestine. Levels of occludin, tricellulin, and JAM-B and -C expression were unchanged throughout the intestine. Considering their segment specificity, claudin-8, claudin-15, and angulin-2 appear to be targets for the development of permeation enhancers in the rectum, small intestine, and large intestine, respectively. These data on heterogenous expression profiles of intestinal TJ components will be useful for the development of safe and efficient intestinal permeation enhancers.

Association of Plasma Claudin-5 with Age and Alzheimer Disease.

The blood-brain barrier (BBB) plays pivotal roles in synaptic and neuronal functioning by sealing the space between adjacent microvascular endothelial cells. BBB breakdown is present in patients with mild cognitive impairment (MCI) or Alzheimer disease (AD). Claudin-5 (CLDN-5) is a tetra-spanning protein essential for sealing the intercellular space between adjacent endothelial cells in the BBB. In this study, we developed a blood-based assay for CLDN-5 and investigated its diagnostic utility using 100 cognitively normal (control) subjects, 100 patients with MCI, and 100 patients with AD. Plasma CLDN-5 levels were increased in patients with AD (3.08 ng/mL) compared with controls (2.77 ng/mL). Plasma levels of phosphorylated tau (pTau181), a biomarker of pathological tau, were elevated in patients with MCI or AD (2.86 and 4.20 pg/mL, respectively) compared with control subjects (1.81 pg/mL). In patients with MCI or AD, plasma levels of CLDN-5-but not pTau181-decreased with age, suggesting some age-dependent BBB changes in MCI and AD. These findings suggest that plasma CLDN-5 may a potential biochemical marker for the diagnosis of AD.

Brain endothelial tricellular junctions as novel sites for T cell diapedesis across the blood-brain barrier.

The migration of activated T cells across the blood-brain barrier (BBB) is a critical step in central nervous system (CNS) immune surveillance and inflammation. Whereas T cell diapedesis across the intact BBB seems to occur preferentially through the BBB cellular junctions, impaired BBB integrity during neuroinflammation is accompanied by increased transcellular T cell diapedesis. The underlying mechanisms directing T cells to paracellular versus transcellular sites of diapedesis across the BBB remain to be explored. By combining in vitro live-cell imaging of T cell migration across primary mouse brain microvascular endothelial cells (pMBMECs) under physiological flow with serial block-face scanning electron microscopy (SBF-SEM), we have identified BBB tricellular junctions as novel sites for T cell diapedesis across the BBB. Downregulated expression of tricellular junctional proteins or protein-based targeting of their interactions in pMBMEC monolayers correlated with enhanced transcellular T cell diapedesis, and abluminal presence of chemokines increased T cell diapedesis through tricellular junctions. Our observations assign an entirely novel role to BBB tricellular junctions in regulating T cell entry into the CNS. This article has an associated First Person interview with the first author of the paper.

Claudin-4: A New Molecular Target for Epithelial Cancer Therapy.

Claudin-4 (CLDN4) is a key component of tight junctions (TJs) in epithelial cells. CLDN4 is overexpressed in many epithelial malignancies and correlates with cancer progression. Changes in CLDN4 expression have been associated with epigenetic factors (such as hypomethylation of promoter DNA), inflammation associated with infection and cytokines, and growth factor signaling. CLDN4 helps to maintain the tumor microenvironment by forming TJs and acts as a barrier to the entry of anticancer drugs into tumors. Decreased expression of CLDN4 is a potential marker of epithelial-mesenchymal transition (EMT), and decreased epithelial differentiation due to reduced CLDN4 activity is involved in EMT induction. Non-TJ CLDN4 also activates integrin beta 1 and YAP to promote proliferation, EMT, and stemness. These roles in cancer have led to investigations of molecular therapies targeting CLDN4 using anti-CLDN4 extracellular domain antibodies, gene knockdown, clostridium perfringens enterotoxin (CPE), and C-terminus domain of CPE (C-CPE), which have demonstrated the experimental efficacy of this approach. CLDN4 is strongly involved in promoting malignant phenotypes in many epithelial cancers and is regarded as a promising molecular therapeutic target.

Homoharringtonine is a transdermal granular permeation enhancer.

Although modulation of claudin-1-based tight junction (TJ) in stratum granulosum is an option for transdermal absorption of drugs, granular permeation enhancers have never been developed. We previously found that homoharringtonine (HHT), a natural alkanoid, weakened intestinal epithelial barrier with changing expression and cellular localization of TJ components such as claudin-1 and claudin-4. In the present study, we investigated whether HHT is an epidermal granular permeation enhancer. Treatment of normal human epidermal keratinocytes (NHEK) cells with HHT decreased claudin-1 and claudin-4 but not zonula occludens-1 and E-cadherin. HHT lowered TJ-integrity in NHEK cells, accompanied by permeation-enhancement of dextran (4 kDa) in a dose-dependent manner. Transdermal treatment of mice with HHT weakened epidermal barrier. HHT treatment enhanced transdermal absorption of dextran with a molecular mass of up to 10 kDa. Together, HHT may be a transdermal absorption enhancer.

Translocation of LSR from tricellular corners causes macropinocytosis at cell-cell interface as a trigger for breaking out of contact inhibition.

Withdrawal from contact inhibition is necessary for epithelial cancer precursor cells to initiate cell growth and motility. Nevertheless, little is understood about the mechanism for the sudden initiation of cell growth under static conditions. We focused on cellular junctions as one region where breaking out of contact inhibition occurs. In well-differentiated endometrial cancer cells, Sawano, the ligand administration for tricellular tight junction protein LSR, which transiently decreased the robust junction property, caused an abrupt increase in cell motility and consequent excessive multilayered cell growth despite being under contact inhibition conditions. We observed that macropinocytosis essentially and temporarily occurred as an antecedent event for the above process at intercellular junctions without disruption of the junction apparatus but not at the apical plasma membrane. Collectively, we concluded that the formation of macropinocytosis, which is derived from tight junction-mediated signaling, was triggered for the initiation of cell growth in static precancerous epithelium.

Hypomethylation of CLDN4 Gene Promoter Is Associated with Malignant Phenotype in Urinary Bladder Cancer.

The tight junction (TJ) protein claudin-4 (CLDN4) is overexpressed in bladder urothelial carcinoma (BUC) and correlates with cancer progression. However, the mechanism of CLDN4 upregulation and promotion of malignant phenotype is not clear. Here, we analyzed 157 cases of BUC and investigated the hypomethylation of CpG island in the CLDN4 promoter DNA and its correlation with cancer progression. In hypomethylated cases, CLDN4 expression, cell proliferation, stemness, and epithelial-mesenchymal transition were increased. Treatment of three human BUC cell lines with the demethylating agent aza-2'-deoxycytidine (AZA) led to excessive CLDN4 expression, and, specifically, to an increase in CLDN4 monomer that is not integrated into the TJ. The TJ-unintegrated CLDN4 was found to bind integrin ß1 and increase stemness, drug resistance, and metastatic ability of the cells as well as show an anti-apoptosis effect likely via FAK phosphorylation, which reduces upon knockdown of CLDN4. Thus, CLDN4 is overexpressed in BUC by an epigenetic mechanism and the high expression enhances the malignant phenotype of BUC via increased levels of TJ-unintegrated CLDN4. CLDN4 promoter DNA methylation is expected to be a novel indicator of BUC malignant phenotype and a new therapeutic target.

The Robo4-TRAF7 complex suppresses endothelial hyperpermeability in inflammation.

Roundabout guidance receptor 4 (Robo4) is an endothelial cell-specific receptor that stabilizes the vasculature in pathological angiogenesis. Although Robo4 has been shown to suppress vascular hyperpermeability induced by vascular endothelial growth factor (VEGF) in angiogenesis, the role of Robo4 in inflammation is poorly understood. In this study, we investigated the role of Robo4 in vascular hyperpermeability during inflammation. Endotoxemia models using Robo4-/- mice showed increased mortality and vascular leakage. In endothelial cells, Robo4 suppressed tumor necrosis factor α (TNFα)-induced hyperpermeability by stabilizing VE-cadherin at cell junctions, and deletion assays revealed that the C-terminus of Robo4 was involved in this suppression. Through binding and localization assays, we demonstrated that in endothelial cells, Robo4 binds to TNF receptor-associated factor 7 (TRAF7) through interaction with the C-terminus of Robo4. Gain- and loss-of-function studies of TRAF7 with or without Robo4 expression showed that TRAF7 is required for Robo4-mediated suppression of hyperpermeability. Taken together, our results demonstrate that the Robo4-TRAF7 complex is a novel negative regulator of inflammatory hyperpermeability. We propose this complex as a potential future target for protection against inflammatory diseases.

Claudin-5: A Pharmacological Target to Modify the Permeability of the Blood-Brain Barrier.

Claudin-5 is the dominant tight junction protein in brain endothelial cells and exclusively limits the paracellular permeability of molecules larger than 400 Da across the blood-brain barrier (BBB). Its pathological impairment or sustained down-regulation has been shown to lead to the progression of psychiatric and neurological disorders, whereas its expression under physiological conditions prevents the passage of drugs across the BBB. While claudin-5 enhancers could potentially act as vascular stabilizers to treat neurological diseases, claudin-5 inhibitors could function as delivery systems to enhance the brain uptake of hydrophilic small-molecular-weight drugs. Therefore, the effects of claudin-5 manipulation on modulating the BBB in different neurological diseases requires further examination. To manipulate claudin-5 expression levels and function, several claudin-5 modulating molecules have been developed. In this review, we first describe the molecular, cellular and pathological aspects of claudin-5 to highlight the mechanisms of claudin-5 enhancers/inhibitors. We then discuss recently developed claudin-5 enhancers/inhibitors and new methods to discover these molecules.

Effects of HMGB1 on Tricellular Tight Junctions via TGF-ß Signaling in Human Nasal Epithelial Cells.

The airway epithelium of the human nasal mucosa acts as a physical barrier that protects against inhaled substances and pathogens via bicellular and tricellular tight junctions (bTJs and tTJs) including claudins, angulin-1/LSR and tricellulin. High mobility group box-1 (HMGB1) increased by TGF-ß1 is involved in the induction of nasal inflammation and injury in patients with allergic rhinitis, chronic rhinosinusitis, and eosinophilic chronic rhinosinusitis. However, the detailed mechanisms by which this occurs remain unknown. In the present study, to investigate how HMGB1 affects the barrier of normal human nasal epithelial cells, 2D and 2.5D Matrigel culture of primary cultured human nasal epithelial cells were pretreated with TGF-ß type I receptor kinase inhibitor EW-7197 before treatment with HMGB1. Knockdown of angulin-1/LSR downregulated the epithelial barrier. Treatment with EW-7197 decreased angulin-1/LSR and concentrated the expression at tTJs from bTJs and increased the epithelial barrier. Treatment with a binder to angulin-1/LSR angubindin-1 decreased angulin-1/LSR and the epithelial barrier. Treatment with HMGB1 decreased angulin-1/LSR and the epithelial barrier. In 2.5D Matrigel culture, treatment with HMGB1 induced permeability of FITC-dextran (FD-4) into the lumen. Pretreatment with EW-7197 prevented the effects of HMGB1. HMGB1 disrupted the angulin-1/LSR-dependent epithelial permeability barriers of HNECs via TGF-ß signaling in HNECs.

Targeting claudin-4 enhances chemosensitivity in breast cancer.

Triple negative breast cancer (TNBC) is characterized by highly aggressive phenotype, limited treatment options and a poor prognosis. In the present study, we examined the therapeutic effect of anti-claudin (CLDN)-4 extracellular domain antibody, 4D3, on TNBC. When the expression of CLDN4 and CLDN1 in invasive ductal carcinoma (IDC) was examined in 114 IDC (78 cases from 2004 to 2009 in a single center and 36 cases of tissues array), CLDN1 had lower expression than CLDN4 and was correlated with histological grade. In contrast, expression of CLDN4 was correlated with histological grade, receptor subtype, and stage. CLDN4 expression in human IDC cell lines MCF-7 (luminal subtype) and MDA-468 (TNBC) was at the same level. In both cells, paclitaxel (PTX)-induced growth suppression was enhanced by 4D3. Furthermore, 4D3 increased both intracellular PTX concentration (in both cells) and apoptosis. In the mouse model, 4D3 promoted the antitumor effect of PTX on subcutaneous tumors and reduced lung metastasis. The combination of PTX and 4D3 reduced M2 macrophages and mesenchymal stem cells in the tumor. 4D3 also reduced stemness of the tumors and increased the intratumoral pH. Moreover, concurrent treatment with 4D3, PTX and tamoxifen, or with PTX and tamoxifen in MDA-468 also showed the same level of antitumor activity and survival as MCF-7. Furthermore, in a bone metastasis model, combination of PTX and bisphosphonate with 4D3 promoted tumor growth in both cells. Thus, CLDN4 targeting of the antibody facilitated existing therapeutic effects.

Tricellular tight junction protein LSR/angulin-1 contributes to the epithelial barrier and malignancy in human pancreatic cancer cell line.

Lipolysis-stimulated lipoprotein receptor (LSR)/angulin-1 is a crucial molecule of tricellular contacts in the epithelial barrier of normal cells and the malignancy of cancer cells. To investigate whether LSR/angulin-1 affects the epithelial barrier and malignancy in human pancreatic cancer, human pancreatic cancer cell line HPAC was used. Treatment with EGF or TGF-ß increased the expression of LSR, but not tricellulin (TRIC), and induced the localization of LSR and TRIC to bicellular tight junctions from tricellular tight junctions. TGF-ß receptor type-1 inhibitor EW-7197 prevented changes of the distribution and the barrier function of LSR by TGF-ß. Knockdown of LSR increased cell migration, invasion, proliferation and EGF ligand amphiregulin expression and decreased the epithelial barrier. Treatment with amphiregulin induced cell migration and invasion and knockdown of amphiregulin prevented the increases of cell migration, invasion and proliferation caused by knockdown of LSR. Treatment with LSR ligand peptide angubindin-1 decreased the epithelial barrier and the expression of LSR, but not TRIC, and increased cell invasion. Knockdown of TRIC decreased cell migration and the epithelial barrier. In immunohistochemical analysis of human pancreatic cancer tissues, LSR and TRIC were found to be localized at the cell membranes of normal pancreatic ducts and well-differentiated pancreatic ductal adenocarcinomas (PDAC), whereas in poorly differentiated PDAC, LSR was weakly detected in the cytoplasm. Amphiregulin was highly expressed in the cytoplasm of well- and poorly differentiated PDAC. In pancreatic cancer, LSR contributes to the epithelial barrier and malignancy via growth factors and may be a potential targeting molecule in the therapy.

Development of a bivalent food poisoning vaccine: augmented antigenicity of the C-terminus of Clostridium perfringens enterotoxin by fusion with the B subunit of Escherichia coli Shiga toxin 2.

Food poisonings caused by Clostridium perfringens and Shiga toxin (Stx)-producing Escherichia coli (STEC) occur frequently worldwide; however, no vaccine is currently available. Therefore, we aimed to develop a bivalent vaccine against C. perfringens and STEC infections. Although it has been considered that the C-terminal region of C. perfringens enterotoxin (C-CPE) could be a good vaccine antigen to block the binding to its receptor, it was insufficient for induction of a protective immune response because of the low antigenicity. However, the fusion of C-CPE with Stx2 B subunit (Stx2B) augmented the antigenicity of C-CPE without affecting the antigenicity of Stx2B. Indeed, high levels of C-CPE-specific neutralizing IgG were found in the serum of mice immunized with the fusion protein Stx2B-C-CPE. Additionally, comparable and substantial levels of Stx2B-specific neutralizing IgG were induced in mice receiving Stx2B-C-CPE or Stx2B alone. These antibody responses against C-CPE and Stx2B lasted for at least 48 weeks, which were sufficient for protective immunity in vitro and in vivo, indicating that Stx2B-C-CPE could induce long-term protective immunity. As an underlying mechanism, ex vivo stimulation with Stx2B, but not with C-CPE, induced cytokine production from splenic T cells collected from mice immunized with Stx2B-C-CPE, suggesting that Stx2B-specific, but not C-CPE-specific, T cells were induced by the immunization with Stx2B-C-CPE and plausibly promoted immunoglobulin class switching of both Stx2B- and C-CPE-specific B cells from IgM to IgG. These findings collectively indicate that Stx2B-C-CPE is a T-cell-antigen-supplement-type bivalent vaccine, which could be an efficient against C. perfringens and STEC infections.

New Japanese Regulatory Frameworks for Post-Marketing Management of Pharmaceutical Products.

Dossiers on approved pharmaceutical products must be kept updated and current during the products' life cycles. The coalition, merger and acquisition along with corporate strategy that pursues efficiency and profitability of pharmaceutical companies have led to the globalization of supply chains for pharmaceutical ingredients and instruments in the post-marketing phase, and progress in manufacturing technologies can improve manufacturing processes during this phase. Regulatory requirements for post-marketing management of pharmaceutical products sometimes differ among countries around the world depending on national/regional policies or situations, even though the basic concepts of each regulation are the same. Therefore, an understanding of up-to-date region specific regulatory management frameworks is important for the optimal provision of pharmaceutical products by pharmaceutical industries. The amendment of the Japanese Pharmaceutical and Medical Device Act (Act No. 63 of 2019) was promulgated in December 2019, and will be enforced from September 2020 onwards. The amended Act sets out regulatory frameworks for post-marketing management systems, including inspection for good manufacturing practice of drugs, quasi-drugs, and gene-, cell-, and tissue-based products; and post-approval change-management protocols. Here, we review these new Japanese post-marketing management frameworks.

Role of Clostridium perfringens Enterotoxin on YAP Activation in Colonic Sessile Serrated Adenoma/ Polyps with Dysplasia.

Sessile serrated adenoma/polyp with dysplasia (SSA/P-D) is an SSA/P with cellular dysplasia and has a higher risk of progressing to colon carcinogenesis. Previously, we reported that tight junction impairment by Clostridium perfringens enterotoxin (CPE) leads to activation of the transcriptional co-activator yes-associated protein (YAP) in oral squamous cell carcinoma. Here, we investigated whether CPE activates YAP to promote the malignant progression of SSA/P. E-cadherin expression was lower in the 12 cases with SSA/P-D examined than that in normal mucosa, SSA/P, or tubular adenoma (TA). Furthermore, intracellular translocation of claudin-4 (CLDN4) and nuclear translocation of YAP were observed. The CPE gene was detected in DNA extracted from SSA/P-D lesions, but not in SSA/P or TA. Treatment of the rat intestinal epithelial cell line IEC6 with low-dose CPE resulted in intracellular translocation of CLDN4 to the cytoplasmic membrane. Cytoplasmic CLDN4 showed co-precipitation with transcriptional co-activator with PDZ-binding motif, zonula occludens (ZO)-1, large tumor suppressor, and mammalian Ste20-like. Additionally, YAP co-precipitated with ZO-2 under CPE treatment led to decreased YAP phosphorylation and nuclear translocation. YAP activation promoted increase in nuclear TEA domain family member level, expression of cyclin D1, snail, vimentin, CD44, NS and decrease in E-cadherin levels, thereby inducing stemness and epithelial-mesenchymal-transition (EMT). The Hippo complex with the incorporation of CLDN4 increased stability. Upon low-dose CPE treatment, HT29 cells with BRAFV600E gene mutation showed increased growth, enhanced invasive potential, stemness, and induced EMT phenotype, whereas HCT116 cells, which carry KRASG13D gene mutation, did not show such changes. In an examination of 10 colorectal cancers, an increase in EMT and stemness was observed in CPE (+) and BRAF mutation (+) cases. These findings suggest that C. perfringens might enhance the malignant transformation of SSA/P-D via YAP activation. Our findings further highlight the importance of controlling intestinal flora using probiotics or antibiotics.

Role of Nuclear Claudin-4 in Renal Cell Carcinoma.

Claudin-4 (CLDN4) is a tight junction protein to maintain the cancer microenvironment. We recently reported the role of the CLDN4 not forming tight junction in the induction of epithelial-mesenchymal transition (EMT). Herein, we investigated the role of CLDN4 in renal cell carcinoma (RCC), focusing on CLDN4. CLDN4 expression in 202 RCCs was examined by immunostaining. CLDN4 phosphorylation and subcellular localization were examined using high metastatic human RCC SN12L1 and low metastatic SN12C cell lines. In 202 RCC cases, the CLDN4 expression decreased in the cell membrane and had no correlation with clinicopathological factors. However, CLDN4 was localized in the nucleus in 5 cases (2%), all of which were pT3. Contrastingly, only 6 of 198 nuclear CLDN4-negative cases were pT3. CLDN4 was found in the nuclear fraction of a highly metastatic human RCC cell line, SN12L1, but not in the low metastatic SN12C cells. In SN12L1 cells, phosphorylation of tyrosine and serine residues was observed in cytoplasmic CLDN4, but not in membranous CLDN4. In contrast, phosphorylation of serine residues was observed in nuclear CLDN4. In SN12L1 cells, CLDN4 tyrosine phosphorylation by EphA2/Ephrin A1 resulted in the release of CLDN4 from tight junction and cytoplasmic translocation. Furthermore, protein kinase C (PKC)-ε phosphorylated the CLDN4 serine residue, resulting in nuclear import. Contrarily, in SN12C cells that showed decreased expression of EphA2/Ephrin A1 and PKCε, the activation of EphA2/EphrinA1 and PKCε induced cytoplasmic and nuclear translocation of CLDN4, respectively. Furthermore, the nuclear translocation of CLDN4 promoted the nuclear translocation of Yes-associated protein (YAP) bound to CLDN4, which induced the EMT phenotype. These findings suggest that the release of CLDN4 by impaired tight junction might be a mechanism underlying the malignant properties of RCC. These findings suggest that the release of CLDN4 by impaired tight junction might be one of the mechanisms of malignant properties of RCC.

Human-rat chimeric anti-occludin monoclonal antibodies inhibit hepatitis C virus infection.

Occludin (OCLN), an integral tetra-spanning plasma membrane protein, is a host entry factor essential for hepatitis C virus (HCV) infection, making it a promising host-targeting molecule for HCV therapeutic intervention. We previously generated rat anti-OCLN monoclonal antibodies (mAbs) that strongly prevented HCV infection in vitro and in vivo. In the present study, we attempted to improve the druggability of the extracellular loop domain-recognizing anti-OCLN mAbs, namely clones 1-3 and 37-5, using genetic engineering. To avoid adverse reactions induced by antibody-dependent cellular cytotoxicity and enhance the antibody stability, we developed human-rat chimeric immunoglobulin G4 S228P mutant (IgG4m) forms of clones 1-3 and 37-5 (named Xi 1-3 and Xi 37-5, respectively) by grafting the variable regions of the light and heavy chains of each rat anti-OCLN mAb into those of human IgG4m. The constructed Xi 1-3 and Xi 37-5 chimeras demonstrated levels of affinity and specificity similar to each parental rat anti-OCLN mAb, and the Fcγ receptor Ⅲa was not activated by the antigen-bound chimeric mAbs, as expected. Both chimeric mAbs inhibited in vitro infection with various HCV genotypes. These results indicate that the IgG4m forms of human-rat chimeric anti-OCLN mAbs may be potential candidate molecules of host-targeting antivirals with pan-genotypic anti-HCV activity.

Anti-Claudin Antibodies as a Concept for Development of Claudin-Directed Drugs.

Claudin (CLDN) proteins, a tetra-transmembrane family containing over 20 members, have been identified as key structural and functional components of intercellular seals, tight junctions (TJs). CLDNs are involved in the barrier and fence functions of TJs. Loosening the TJ barrier is one strategy for increasing drug absorption and delivery to the brain. Due to aberrant CLDN expression, the TJ fence function is frequently dysregulated in carcinogenesis. In addition, CLDN-1 is a co-receptor for the hepatitis C virus. Together these characteristics indicate CLDNs as promising targets for drug development, and CLDN binders are potential candidates for delivering drugs, treating cancer, and preventing viral infection. Before 2008, a receptor-binding fragment of Clostridium perfringens enterotoxin was the only CLDN binder available. Since then, several challenges regarding the generation of monoclonal antibodies against CLDNs have been surmounted, leading to breakthroughs in CLDN-targeted drug development. Here, we provide an overview of the recent progress in technology using created CLDN binders-anti-CLDN monoclonal antibodies.

Monoclonal Antibodies against Occludin Completely Prevented Hepatitis C Virus Infection in a Mouse Model.

Hepatitis C virus (HCV) entry into host cells is a multistep process requiring various host factors, including the tight junction protein occludin (OCLN), which has been shown to be essential for HCV infection in in vitro cell culture systems. However, it remains unclear whether OCLN is an effective and safe target for HCV therapy, owing to the lack of binders that can recognize the intact extracellular loop domains of OCLN and prevent HCV infection. In this study, we successfully generated four rat anti-OCLN monoclonal antibodies (MAbs) by the genetic immunization method and unique cell differential screening. These four MAbs bound to human OCLN with a very high affinity (antibody dissociation constant of <1 nM). One MAb recognized the second loop of human and mouse OCLN, whereas the three other MAbs recognized the first loop of human OCLN. All MAbs inhibited HCV infection in Huh7.5.1-8 cells in a dose-dependent manner without apparent cytotoxicity. Additionally, the anti-OCLN MAbs prevented both cell-free HCV infection and cell-to-cell HCV transmission. Kinetic studies with anti-OCLN and anti-claudin-1 (CLDN1) MAbs demonstrated that OCLN interacts with HCV after CLDN1 in the internalization step. Two selected MAbs completely inhibited HCV infection in human liver chimeric mice without apparent adverse effects. Therefore, OCLN would be an appropriate host target for anti-HCV entry inhibitors, and anti-OCLN MAbs may be promising candidates for novel anti-HCV agents, particularly in combination with direct-acting HCV antiviral agents.IMPORTANCE HCV entry into host cells is thought to be a very complex process involving various host entry factors, such as the tight junction proteins claudin-1 and OCLN. In this study, we developed novel functional MAbs that recognize intact extracellular domains of OCLN, which is essential for HCV entry into host cells. The established MAbs against OCLN, which had very high affinity and selectivity for intact OCLN, strongly inhibited HCV infection both in vitro and in vivo Using these anti-OCLN MAbs, we found that OCLN is necessary for the later stages of HCV entry. These anti-OCLN MAbs are likely to be very useful for understanding the OCLN-mediated HCV entry mechanism and might be promising candidates for novel HCV entry inhibitors.

Potential for Tight Junction Protein-Directed Drug Development Using Claudin Binders and Angubindin-1.

The tight junction (TJ) is an intercellular sealing component found in epithelial and endothelial tissues that regulates the passage of solutes across the paracellular space. Research examining the biology of TJs has revealed that they are complex biochemical structures constructed from a range of proteins including claudins, occludin, tricellulin, angulins and junctional adhesion molecules. The transient disruption of the barrier function of TJs to open the paracellular space is one means of enhancing mucosal and transdermal drug absorption and to deliver drugs across the blood-brain barrier. However, the disruption of TJs can also open the paracellular space to harmful xenobiotics and pathogens. To address this issue, the strategies targeting TJ proteins have been developed to loosen TJs in a size- or tissue-dependent manner rather than to disrupt them. As several TJ proteins are overexpressed in malignant tumors and in the inflamed intestinal tract, and are present in cells and epithelia conjoined with the mucosa-associated lymphoid immune tissue, these TJ-protein-targeted strategies may also provide platforms for the development of novel therapies and vaccines. Here, this paper reviews two TJ-protein-targeted technologies, claudin binders and an angulin binder, and their applications in drug development.