Fragment Of Clostridium Perfringens Enterotoxin Research Articles

Autophagic degradation of claudin‐5 mediated by its binding to a Clostridium perfringens enterotoxin fragment modulates endothelial barrier permeability

The blood-brain barrier (BBB) protects the central nervous system (CNS) from harmful elements, while it also restricts efficient drug delivery into the CNS. Previously, we generated a mutated fragment of Clostridium perfringens enterotoxin (cCPEYWSH ) which specifically binds to the endothelial tight junction protein claudin-5. Here, we explore the mechanisms regulating the dynamics of membranous claudin-5 and BBB permeability. Following cCPEYWSH binding to claudin-5, caveolin-1 mediates the redistribution of claudin-5 to the cytosol. This abnormal cytosolic aggregation triggers the autophagic degradation of claudin-5, leading to an increase in BBB permeability. Enhancement or inhibition of autophagy accelerates or inhibits the degradation of cytosolic claudin-5, respectively. Our findings may pave the way for improving BBB permeability for drug delivery.

A time‐resolved fluorescence resonance energy transfer‐based screen for identification of a novel claudin‐4 binder that modulates the epithelial tight junction barrier

Claudins, a 27‐member family of proteins with four transmembrane domains each, play a pivotal role in maintaining tight junction (TJ) seals in epithelial and endothelial tissue. The C‐terminal fragment of Clostridium perfringens enterotoxin (C‐CPE) binds to claudin‐4, which is a key functional and structural component of the TJ in mucosal epithelial cells. Upon binding, C‐CPE reversibly modulates TJ seals, yielding an enhancement of the paracellular transport of solutes. However, the use of C‐CPE as an absorption enhancer is limited by the molecule’s immunogenicity and manufacturing cost. In the present study, we developed a high‐throughput screening system using Time‐Resolved Fluorescence Resonance Energy Transfer (TR‐FRET) to identify small‐molecule compounds that bind to claudin‐4. Using this system, we performed a claudin‐4 binding screen of a library containing 2642 biologically active small molecules, and identified thiostrepton as a novel claudin‐4 binder. Thiostrepton decreased the transepithelial electrical resistance and increased flux of 4‐kDa FITC‐dextran in Caco‐2 cell monolayers without cell toxicity. Immunoblotting analysis revealed that thiostrepton changed the level of claudin‐4 in the Triton‐X soluble fraction, which contains TJ components, without altering the localization of claudin‐4. Furthermore, treatment of isolated rat jejunum with thiostrepton increased absorption of 4‐kDa FITC‐dextran. The TR‐FRET‐based screening system described here is expected to be of use in identifying compounds that modulate the permeability of the intestinal epithelial cell barrier; these compounds might serve as novel absorption enhancers in the intestine.Support or Funding InformationThis study was supported in part by Grants‐in‐Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (19H04468, 18K19400); a grant for Research on Development of New Drugs from the Japan Agency for Medical Research and Development (AMED); and grants from the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) of AMED (JP19am0101077, JP19am0101084, JP19am0101090).

Parameters for Optoperforation-Induced Killing of Cancer Cells Using Gold Nanoparticles Functionalized With the C-terminal Fragment of Clostridium Perfringens Enterotoxin.

Recently, we used a recombinant produced C-terminus (D194-F319) of the Clostridium perfringens enterotoxin (C-CPE) to functionalize gold nanoparticles (AuNPs) for a subsequent specific killing of claudin expressing tumor cells using the gold nanoparticle-mediated laser perforation (GNOME-LP) technique. For a future in vivo application, it will be crucial to know the physical parameters and the biological mechanisms inducing cell death for a rational adaptation of the system to real time situation. Regarding the AuNP functionalization, we observed that a relationship of 2.5 × 10−11 AuNP/mL to 20 µg/mL C-CPE maximized the killing efficiency. Regardingphysical parameters, a laser fluence up to 30 mJ/cm2 increased the killing efficiency. Independent from the applied laser fluence, the maximal killing efficiency was achieved at a scanning velocity of 5 mm/s. In 3D matrigel culture system, the GNOME-LP/C-CPE-AuNP completely destroyed spheroids composed of Caco-2 cells and reduced OE-33 cell spheroid formation. At the biology level, GNOME-LP/C-CPE-AuNP-treated cells bound annexin V and showed reduced mitochondria activity. However, an increased caspase-3/7 activity in the cells was not found. Similarly, DNA analysis revealed no apoptosis-related DNA ladder. The results suggest that the GNOME-LP/C-CPE-AuNP treatment induced necrotic than apoptotic reaction in tumor cells.

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.

Impaired airway mucociliary function reduces antigen-specific IgA immune response to immunization with a claudin-4-targeting nasal vaccine in mice

Vaccine delivery is an essential element for the development of mucosal vaccine, but it remains to be investigated how physical barriers such as mucus and cilia affect vaccine delivery efficacy. Previously, we reported that C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) targeted claudin-4, which is expressed by the epithelium associated with nasopharynx-associated lymphoid tissue (NALT), and could be effective as a nasal vaccine delivery. Mice lacking tubulin tyrosine ligase-like family, member 1 (Ttll1-KO mice) showed mucus accumulation in nasal cavity due to the impaired motility of respiratory cilia. Ttll1-KO mice nasally immunized with C-CPE fused to pneumococcal surface protein A (PspA-C-CPE) showed reduced PspA-specific nasal IgA responses, impaired germinal center formation, and decreased germinal center B-cells and follicular helper T cells in the NALT. Although there was no change in the expression of claudin-4 in the NALT epithelium in Ttll1-KO mice, the epithelium was covered by a dense mucus that prevented the binding of PspA-C-CPE to NALT. However, administration of expectorant N-acetylcysteine removed the mucus and rescued the PspA-specific nasal IgA response. These results show that the accumulation of mucus caused by impaired respiratory cilia function is an interfering factor in the C-CPE-based claudin-4-targeting nasal vaccine.

Identification of claudin-4 binder that attenuates tight junction barrier function by TR-FRET-based screening assay

Claudins are key functional and structural components of tight junctions (TJs) in epithelial cell sheets. The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) binds to claudin-4 and reversibly modulates intestinal TJ seals, thereby enhancing paracellular transport of solutes. However, the use of C-CPE as an absorption enhancer is limited by the molecule’s immunogenicity and manufacturing cost. Here, we developed a high-throughput screening system based on the Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) method to identify claudin-4 binders in a library collection of 32,560 compounds. Thiostrepton, identified from the screen, decreased transepithelial electrical resistance and increased flux of 4-kDa fluorescein isothiocyanate–labelled dextran (FD-4) in Caco-2 cell monolayers, a model of intestinal epithelium. Thiostrepton changed the expression, but not the localisation, of TJ components. Treatment of rat jejunum with thiostrepton increased the absorption of FD-4 without tissue toxicity, indicating that thiostrepton is a novel claudin-4 binder that enhances intestinal permeability. The screening system may therefore be a useful tool for identifying claudin-4 binders to enhance drug absorption in mucosa.

Specific binding of a mutated fragment of Clostridium perfringens enterotoxin to endothelial claudin-5 and its modulation of cerebral vascular permeability

The vertebrate blood–brain barrier (BBB) creates an obstacle for central nervous system-related drug delivery. Claudin-5 (Cldn5), expressed in large quantities in BBB, plays a vital role in restricting BBB permeability. The C-terminal domain of Clostridium perfringens enterotoxin (cCPE) has been verified as binding to a subset of claudins (Cldns). The Cldn5-binding cCPE194–319 variant cCPEY306W/S313H was applied in this study to investigate its ability to modulate the permeability of zebrafish larval BBB. In vitro results showed that cCPEY306W/S313H is able to bind specifically to Cldn5 in murine brain vascular endothelial (bEnd.3) cells, and is transported along with Cldn5 from the cell membrane to the cytoplasm, which in turn results in a reduction in transendothelial electrical resistance (TEER). Conversely, this effect can be reversed by removal of cCPEY306W/S313H. In an in vivo experiment, this study estimates the capability of cCPEY306W/S313H to modulate Cldn5 using a rhodamine B-Dextran dye diffusion assay in zebrafish larval BBB. The results show that cCPEY306W/S313H co-localized with Cldn5 in zebrafish cerebral vascular cells and modulated BBB permeability, resulting in dye leakage. Taken together, this study suggests that cCPEY306W/S313H has the capability – both in vitro and in vivo – to modulate BBB permeability temporarily by specific binding to Cldn5.

A Novel Drug Delivery System for the Human Nasal Epithelium.

The epithelium of upper respiratory tissues such as the human nasal mucosa forms a continuous barrier via tight junctions (TJs). The development of a drug delivery system for use across the nasal mucosa is being reconsidered. In intranasal administration across the nasal mucosa, the paracellular pathway regulated by TJs is extremely important. It is known that the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) binds the TJ protein claudin and disrupts the tight junctional barrier without inducing a cytotoxic effect. We investigated the effects of C-CPE mutants on the function of TJs of human nasal epithelial cells (HNECs) and on the permeability of human recombinant insulin across HNECs treated with C-CPE 194 and C-CPE m19. We recently reported that C-CPE mutants 194 and m19 can regulate the permeability of insulin across HNECs via the MAPK pathway and may play a crucial role in therapy for various diseases via direct intranasal insulin administration. On the other hand, microRNAs (miRNAs) are known to regulate the expression of TJs as direct or indirect targets in genes to maintain barrier function. We investigated the effects of miRNAs on the epithelial barrier of HNECs and found that miRNA-146a plays crucial roles in the maintenance of the TJ barrier and innate immune response against invading pathogens. This chapter reviews a novel drug delivery system across the nasal mucosa from the point of view of the epithelial barrier function.

Claudin-4 binder C-CPE 194 enhances effects of anticancer agents on pancreatic cancer cell lines via a MAPK pathway.

The C‐terminal fragment of Clostridium perfringens enterotoxin (C‐CPE) modulates the tight junction protein claudin and disrupts the tight junctional barrier. It also can enhance the effectiveness of anticancer agents. However, the detailed mechanisms of the effects of C‐CPE remain unclear in both normal and cancerous cells. The C‐CPE mutant called C‐CPE 194 binds only to claudin‐4, but the C‐CPE 194 mutant called C‐CPE m19 binds not only to claudin‐4 but also to claudin‐1. In the present study, to investigate the mechanisms of the effects of C‐CPE on claudin expression, the tight junctional functions and the cytotoxicity of anticancer agents, human pancreatic cancer cells, and normal human pancreatic duct epithelial cells (HPDEs) were treated with C‐CPE 194 and C‐CPE m19. In well‐differentiated cells of the pancreatic cancer cell line HPAC, C‐CPE 194 and C‐CPE m19 disrupted both the barrier and fence functions without changes in expression of claudin‐1 and ‐4, together with an increase of MAPK phosphorylation. C‐CPE 194, but not C‐CPE m19, enhanced the cytotoxicity of the anticancer agents gemcitabine and S‐1. In poorly differentiated pancreatic cancer cell line PANC‐1, C‐CPE 194, but not C‐CPE m19, decreased claudin‐4 expression and enhanced MAPK activity and the cytotoxicity of the anticancer agents. In normal HPDEs, C‐CPE 194 and C‐CPE m19 decreased claudin‐4 expression and enhanced the MAPK activity, whereas they did not affect the cytotoxicity of the anticancer agents. Our findings suggest that the claudin‐4 binder C‐CPE 194 enhances effects of anticancer agents on pancreatic cancer cell lines via a MAPK pathway.

Claudin-binder C-CPE mutants enhance permeability of insulin across human nasal epithelial cells

Objective: Intranasal insulin administration has therapeutic potential for Alzheimer's disease and in intranasal administration across the nasal mucosa, the paracellular pathway regulated by tight junctions is important. The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) binds the tight junction protein claudin and disrupts the tight junctional barrier without a cytotoxic effect. The C-CPE mutant called C-CPE 194 binds only to claudin-4, whereas the C-CPE 194 mutant called C-CPE m19 binds not only to claudin-4 but also to claudin-1.Methods: In the present study, to investigate the effects of C-CPE mutants on the tight junctional functions of human nasal epithelial cells (HNECs) and on the permeability of human recombinant insulin across the cells, HNECs were treated with C-CPE 194 and C-CPE m19.Results: C-CPE 194 and C-CPE m19 disrupted the barrier and fence functions without changes in expression of claudin-1, -4, -7, and occludin or cytotoxicity, whereas they transiently increased the activity of ERK1/2 phosphorylation. The disruption of the barrier function caused by C-CPE 194 and C-CPE m19 was prevented by pretreatment with the MAPKK inhibitor U0126. Furthermore, C-CPE 194 and C-CPE m19 significantly enhanced the permeability of human recombinant insulin across HNECs and the permeability was also inhibited by U0126.Conclusion: These findings suggest that C-CPE mutants 194 and m19 can regulate the permeability of insulin across HNECs via the MAPK pathway and may play a crucial role in therapy for the diseases such as Alzheimer's disease via the direct intranasal insulin administration.

C-Terminal Clostridium perfringens Enterotoxin-Mediated Antigen Delivery for Nasal Pneumococcal Vaccine.

Efficient vaccine delivery to mucosal tissues including mucosa-associated lymphoid tissues is essential for the development of mucosal vaccine. We previously reported that claudin-4 was highly expressed on the epithelium of nasopharynx-associated lymphoid tissue (NALT) and thus claudin-4-targeting using C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) effectively delivered fused antigen to NALT and consequently induced antigen-specific immune responses. In this study, we applied the C-CPE-based vaccine delivery system to develop a nasal pneumococcal vaccine. We fused C-CPE with pneumococcal surface protein A (PspA), an important antigen for the induction of protective immunity against Streptococcus pneumoniae infection, (PspA-C-CPE). PspA-C-CPE binds to claudin-4 and thus efficiently attaches to NALT epithelium, including antigen-sampling M cells. Nasal immunization with PspA-C-CPE induced PspA-specific IgG in the serum and bronchoalveolar lavage fluid (BALF) as well as IgA in the nasal wash and BALF. These immune responses were sufficient to protect against pneumococcal infection. These results suggest that C-CPE is an efficient vaccine delivery system for the development of nasal vaccines against pneumococcal infection.

Claudin-4 SPECT Imaging Allows Detection of Aplastic Lesions in a Mouse Model of Breast Cancer.

The expression of claudin-4, a protein involved in tight junction complexes, is widely dysregulated in epithelial malignancies. Claudin-4 is overexpressed in several premalignant precursor lesions, including those of cancers of the breast, pancreas, and prostate, and is associated with poor survival. A noncytotoxic C-terminal fragment of Clostridium perfringens enterotoxin (cCPE) is a natural ligand for claudin-4. Here, we demonstrate whole-body quantitative SPECT imaging of preneoplastic breast cancer tissue using (111)In-labeled cCPE. cCPE.GST or GST (GST is glutathione S-transferase) was conjugated to the metal ion chelator benzyl-diethylenetriaminepentaacetic acid to allow (111)In radiolabeling. The affinity of radiolabeled cCPE.GST for claudin-4 was confirmed using claudin-4-expressing MDA-MB-468 and SQ20b cells, compared with claudin-4-negative HT1080 cells. In vivo SPECT imaging was performed using athymic mice bearing MDA-MB-468 or HT1080 xenografts and using genetically modified BALB/neuT mice, which spontaneously develop claudin-4-expressing breast cancer lesions. The uptake of (111)In-cCPE.GST in claudin-4-positive MDA-MB-468 xenograft tumors in athymic mice was significantly higher than in (111)In-GST or claudin-4-negative HT1080 tumors (6.72 ± 0.18 vs. 3.88 ± 1.00 vs. 2.36 ± 1.25 percentage injected dose per gram [%ID/g]; P < 0.0001). No other significant differences were observed in any of the examined organs. BALB/neuT mice, expressing rat neuT under mmtv promotor control, spontaneously developed tumorous lesions within their mammary fat pads over the course of 130 d. Overt mammary tumors were claudin-4-positive, and (111)In-cCPE.GST uptake was 3.2 ± 0.70 %ID/g, significantly higher than (111)In-GST (1.00 ± 0.60 %ID/g; P < 0.05). Mammary fat pads in mice aged 80 d bore claudin-4-positive aplastic lesions and accumulated (111)In-cCPE.GST (3.17 ± 0.51 %ID/g) but not (111)In-GST (0.99 ± 0.39 %ID/g; P < 0.001). Taken together, (111)In-cCPE.GST targets claudin-4 expression in frank tumors and preneoplastic tissue, and cCPE imaging may be used as an early detection tool for breast, prostate, and pancreatic cancer.

Abstract 4929: Radiolabeled cCPE for molecular imaging of tight junction changes during breast oncogenesis

Abstract Expression of Claudin-4, a member of the Claudin protein family that is integral in adherens and tight junction complexes, is widely dysregulated in epithelial malignancies and is overexpressed in a number of premalignant precursor lesions, including those of cancers of the breast, pancreas and prostate. Increased Claudin-4 expression in breast cancer is also associated with poor prognosis. Optical imaging of Claudin-4 has been shown previously to be an effective way to detect precancerous lesions of the pancreas, using a fluorescently labelled form of a non-cytotoxic c-terminal fragment of Clostridium perfringens enterotoxin (cCPE, aa184-319), the natural ligand for Claudin-4. Here, we demonstrate whole-body quantitative SPECT imaging of Claudin-4 in preneoplastic breast cancer tissue using 111In-labelled cCPE. Our aim is to improve overall outcome by detection of lesions at an earlier stage. cCPE was produced as a GST-fusion protein from a pGEX plasmid. cCPE.GST or GST was conjugated to the metal ion chelator, benzylDTPA, to allow radiolabelling with 111In. Radiolabeling yield was &amp;gt;95%. Affinity of radiolabelled cCPE-GST for Claudin-4 was confirmed by binding to Claudin-4 expressing MDA-MB-468 and SQ20b cells, compared to Claudin-4 negative HT1080 cells. Radioactivity associated with MDA-MB-468 cells was 24.1±0.9 times higher for 111In-cCPE.GST vs. 111In-GST; P&amp;lt;0.001. Association of 111In-cCPE.GST was 4.7±0.2 times higher with MDA-MB-468 vs. HT1080 cells; P&amp;lt;0.001. 111In-cCPE.GST, but not 111In-GST, was internalised in MDA-MB-468 and SQ20b cells, but not in HT1080 cells. Athymic balb/c mice carrying subcutaneous xenograft tumors of MDA-MB-468 or HT1080 cells were injected intravenously with 5 µg 111In-labelled cCPE.GST or GST (5 MBq). After 24 h, SPECT/CT images were acquired, and the amount of radioactivity in selected organs was determined after dissection. Uptake of 111In-cCPE.GST in Claudin-4 positive MDA-MD-468 tumors was significantly higher compared to 111In-GST or HT1080 tumors (26.14±12.31 vs. 4.84±1.29 vs. 2.36±1.25 percent of the injected dose per gram of tumor tissue (%ID/g); p=0.021). No other significant differences were observed in any of the examined organs. Balb/neuT mice, expressing rat neuT under mmtv promotor control, spontaneously obtain tumorous lesions within their mammary fat pads over the course of 130 days. Balb/neuT mice were imaged monthly using 111In-cCPE.GST or 111In-GST. Mammary fat pads in mice aged around 90 days, bore aplastic lesions which were positive for Claudin-4, as determined by immunohistochemistry, and attracted 111In-cCPE.GST (3-5 %ID/g), but not 111In-GST (&amp;lt;1 %ID/g). In summary, 111In-cCPE.GST targets Claudin-4 expression in frank tumors and preneoplastic tissue, and cCPE imaging may be used as an early detection tool for breast, prostate, pancreas. Citation Format: Michael Mosley, James Knight, Albrecht Neesse, Patrick Michl, Veerle Kersemans, Bart Cornelissen. Radiolabeled cCPE for molecular imaging of tight junction changes during breast oncogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4929. doi:10.1158/1538-7445.AM2014-4929

Tissue distribution and safety evaluation of a claudin-targeting molecule, the C-terminal fragment of Clostridium perfringens enterotoxin

We previously found that claudin (CL) is a potent target for cancer therapy using a CL-3 and -4-targeting molecule, namely the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE). Although CL-3 and -4 are expressed in various normal tissues, the safety of this CL-targeting strategy has never been investigated. Here, we evaluated the tissue distribution of C-CPE in mice. Ten minutes after intravenous injection into mice, C-CPE was distributed to the liver and kidney (24.0% and 9.5% of the injected dose, respectively). The hepatic level gradually fell to 3.2% of the injected dose by 3h post-injection, whereas the renal C-CPE level gradually rose to 46.5% of the injected dose by 6h post-injection and then decreased. A C-CPE mutant protein lacking the ability to bind CL accumulated in the liver to a much lesser extent (2.0% of the dose at 10min post-injection) than did C-CPE, but its renal profile was similar to that of C-CPE. To investigate the acute toxicity of CL-targeted toxin, we intravenously administered C-CPE-fused protein synthesis inhibitory factor to mice. The CL-targeted toxin dose-dependently increased the levels of serum biomarkers of liver injury, but not of kidney injury. Histological examination confirmed that injection of CL-targeted toxin injured the liver but not the kidney. These results indicate that potential adverse hepatic effects should be considered in C-CPE-based cancer therapy.

A toxicological evaluation of the C‐terminal fragment of Clostridium perfringens enterotoxin as a claudin‐3/‐4 binder

Claudins (CLs) are tetratransmembrane proteins that function as intercellular sealing components of tight junctions. Recent progress in epithelial pharmaceutical sciences provides us new insight into the use of CLs as potent targets for drug development. The C‐terminal fragment of Clostridium perfringens enterotoxin (C‐CPE) was the first identified CL‐3/‐4 binder. We previously showed that CL‐3/‐4 could play a role in the mucosal absorption of drugs, in cancer therapy, and in mucosal vaccination using C‐CPE as a CL binder. Since CL‐3/‐4, are highly expressed in the liver and kidney, evaluations of the safety and antigenicity of C‐CPE are needed for future clinical applications. Here, we evaluated whether C‐CPE in mice leads to tissue injury or production of antibodies. Intravenous administration of 5 mg/kg C‐CPE, which is a dose over 25‐fold higher than that used in a murine mucosal absorption model, did not increase biochemical markers of liver and kidney injury, even after 11 weekly injections. Similarly, nasal administration of 2 mg/kg C‐CPE weekly for 11 weeks did not increase these biochemical markers, but 6 administrations of C‐CPE resulted in elevation of C‐CPE‐specific serum IgG. These results indicate that development of a CL modulator with lower antigenicity will be essential for future clinical application of a C‐CPE‐based mucosal absorption enhancer.

Tissue‐distribution of claudin‐3/‐4 binder, the C‐terminal fragment of Clostridium perfringens enterotoxin, in mice

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 mutant C‐CPE 10 min after administration. The distribution of C‐CPE gradually decreased in the liver, thyroid gland and intestine, but the distribution of C‐CPE increased in the kidney until 48 h after administration. These findings suggest that the influence of CL‐3/‐4 binders on the liver, thyroid gland, intestine and kidney should be considered for future therapeutic applications.

Development of claudin‐1‐specific ligand

Claudins (CLs) are a family of tetratransmembrane proteins consisting of 27 members and are potential targets for enhancing mucosal drug absorption, treating cancer, and mucosal vaccination using the C‐terminal fragment of Clostridium perfringens enterotoxin (C‐CPE), a CL‐3/‐4 binder. The development of efficient CL binders is important for their future clinical application. We previously developed a broadly specific CL binder by using C‐CPE as a prototype; however, we did not successfully generate a CL subtype‐specific binder. Here, we focused on developing a CL subtype‐specific antibody. Because of the low antigenicity of CLs, we performed three different immunization procedures. BXSB mice with an autoimmune disorder were immunized with CL‐1‐displayed budded baculovirus, CL‐1‐expressing cells, or plasmid DNA encoding CL‐1. The DNA‐based immunization showed the highest production of serum anti‐CL‐1 antibodies. Therefore, B cells were isolated from the DNA‐immunized mice, and the B cells were fused with myeloma cells to form hybridomas. Hybridomas were screened for the production of the anti‐CL‐1 antibody. The antibodies bound to CL‐1‐expressing cells, but not to CL‐2, ‐4 or ‐5‐expressing cells. These results indicate that immunization of BXSB mice with CL DNA is a potentially useful method for the development of specific CL ligands.

Recent Advances in Claudin-Targeting Technology

Most malignant tumors are derived from epithelium, and pathologic microorganisms often invade the body through the mucosal epithelium. Thus epithelial tissues are potent targets for drug delivery. The tight junction (TJ) is the intercellular seal in epithelial cell sheets. Claudins (CLs) are a family of tetratransmembrane proteins with a molecular mass of approximately 23 kDa. CLs are key structural and sealing components of TJs. CLs are often overexpressed in malignant tumors. CL-4 is highly expressed in the epithelial cells covering mucosal immune tissues. Therefore CLs may be potent targets for drug delivery, cancer therapy, and mucosal vaccination. Herein, we overview a series of our studies using the C-terminal fragment of Clostridium perfringens enterotoxin to target and bind CLs; we also discuss the efficacy of CL-targeted drug delivery.

Comparison of mucosal absorption-enhancing activity between a claudin-3/-4 binder and a broadly specific claudin binder

Intercellular spaces between adjacent mucosal epithelial cells are sealed by tight junctions (TJs) that prevent the free movement of solutes across the epithelium. Claudins (CLs), a family of 27 integral membrane proteins, are essential components for TJ seals. We previously used a CL-3/-4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), to show that CL modulation is a promising method to enhance mucosal absorption. Recently, by using a C-CPE mutant library, we developed a CL binder (m19) with broad specificity to CL-1, -2, -4, and -5. Here, we compared the mucosal absorption-enhancing activity of C-CPE and m19. Both CL binders enhanced jejunal absorption of dextran with a molecular mass of 4000 and 150,000Da and nasal absorption of dextran with a mass of 4000Da but not 150,000Da in rats. Although both binders showed similar nasal absorption-enhancing activity of dextran (4000Da), m19 exhibited a more potent jejunal absorption-enhancing effect than that of C-CPE. These findings suggest that mucosal absorption-enhancing activity may be modified by modulating CL specificity.

Preparation of a broad‐specific claudin binder by using Clostridium perfringens enterotoxin

Claudins (CLs), tetra‐transmembrane proteins, are potent targets for drug development. However, development of CL binders has been delayed because of their low antigenicities. A C‐terminal fragment of Clostridium perfringens enterotoxin (C‐CPE) is the most characterized CL binder. We previously developed a screening system for CL binders by using a C‐CPE mutant library and CL‐displaying baculovirus (CL‐BV), and used this system to identify a novel CL‐1 binder (m19). In the present study, we characterized the mode of binding of m19. Functional domain mapping of m19 revealed that substitution of three amino acids in C‐CPE was critical for the CL‐1 binding function of m19. We showed that m19 bound not only to CL‐1 but also to CL‐2 and ‐5. These findings suggest that m19 is a broad‐specific CL binder, the first reported to date. The X‐ray structural analysis of m19 showed that the structure of the putative CL‐binding region of m19 is similar to that of C‐CPE. In contrast, the electrostatic maps differed between C‐CPE and m19. The putative CL‐binding region of m19 is more positively charged than that of C‐CPE, and the putative C‐CPE binding regions of CL‐1, ‐2 and ‐5 are more negatively charged than that of CL‐4, suggesting that electrostatic forces are involved in the interaction between m19 and CLs. These findings will contribute to the further development of CL binders for CL‐targeted drug development.