C-terminal Fragment Of Clostridium Perfringens Enterotoxin Research Articles

Application of C-Terminal Clostridium Perfringens Enterotoxin in Treatment of Brain Metastasis from Breast Cancer.

Simple SummaryBrain metastasis occurs in primary cancers, such as breast cancer, and is correlated with mortality. There are limited options available for treatment, but Clostridium perfringens Enterotoxin (CPE) and its interaction with Claudin-4, a possible diagnostic biomarker for breast cancer, can provide a molecular pathway basis for the development of treatment options for metastatic brain cancer. Analysis of the literature reveals that Claudin-4 plays an important role as a receptor for CPE, allowing for the disruption of cell membrane permeability, an influx of calcium ions, and subsequent cell death. The negligible presence of Claudin-4 in normal brain cancer cells and the high abundance of Claudin-4 in breast cancer cells metastasized to the brain, allow for the targeted binding of CPE to tumor cells in the brain. We show that the C-terminal of CPE conjugated to nanoparticles that cross the blood–brain barrier could serve as a drug delivery tool to treat metastatic cells in the brain.Claudin-4 is part of the Claudin family of transmembrane tight junction (TJ) proteins found in almost all tissues and, together with adherens junctions and desmosomes, forms epithelial and endothelial junctional complexes. Although the distribution of Claudin-4 occurs in many cell types, the level of expression is cell-specific. Claudin proteins regulate cell proliferation and differentiation by binding cell-signaling ligands, and its expression is upregulated in several cancers. As a result, alterations in Claudin expression patterns or distribution are vital in the pathology of cancer. Profiling the genetic expression of Claudin-4 showed that Claudin-4 is also a receptor for the clostridium perfringens enterotoxin (CPE) and that Claudin-4 has a high sequence similarity with CPE’s high-affinity receptor. CPE is cytolytic due to its ability to form pores in cellular membranes, and CPE treatment in breast cancer cells have shown promising results due to the high expression of Claudin-4. The C-terminal fragment of CPE (c-CPE) provides a less toxic alternative for drug delivery into breast cancer cells, particularly metastatic tumors in the brain, especially as Claudin-4 expression in the central nervous system (CNS) is low. Therefore, c-CPE provides a unique avenue for the treatment of breast–brain metastatic tumors.

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).

Development of Adjuvant-Free Bivalent Food Poisoning Vaccine by Augmenting the Antigenicity of Clostridium perfringens Enterotoxin.

Clostridium perfringens enterotoxin (CPE) is a common cause of food poisoning and hyperkalemia-associated death. Previously, we reported that fusion of pneumococcal surface protein A (PspA) to C-terminal fragment of CPE (C-CPE) efficiently bound mucosal epithelium so that PspA-specific immune responses could be provoked. In this study, we found that fusion of C-CPE with PspA augmented the antigenicity of C-CPE itself. These findings allowed us to hypothesize that fusion of C-CPE and another food poisoning vaccine act as a bivalent food poisoning vaccine. Therefore, we constructed an adjuvant-free bivalent vaccine against CPE and cholera toxin (CT), which is a major food poisoning in developing country, by genetically fusing CT B subunit to C-CPE. Because of the low antigenicity of C-CPE, immunization of mice with C-CPE alone did not induce C-CPE-specific immune responses. However, immunization with our vaccine induced both C-CPE- and CT-specific neutralizing antibody. The underlying mechanism of the augmented antigenicity of C-CPE included the activation of T cells by CTB. Moreover, neutralizing antibodies lasted for at least 48 weeks and the quality of the antibody was dependent on the binding activity of CTB–C-CPE to its receptors. These findings suggest that our fusion protein is a potential platform for the development of an adjuvant-free bivalent vaccine against CPE and CT.

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.

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.

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.

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.

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.

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.

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.

Creation and biochemical analysis of a broad-specific claudin binder

Claudins (CL) are a family of tetra-transmembrane proteins that are the structural and functional components of tight junctions (TJ). CLs are promising targets for drug development because of their role in mucosal drug absorption and cancer. However, CL-targeted drug development has been delayed because CLs have low antigenicity and preparing CL proteins is difficult. We developed a CL binder by using the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) and a baculoviral display system. After screening CL binders from a C-CPE mutant-displaying library by using CL-displaying budded baculovirus (BV) we isolated a C-CPE mutant called m19, which bound to CL1, CL2, CL4 and CL5. A 3-dimensional analysis showed that m19 has a structural backbone similar to C-CPE. The charge density of the CL-binding domains of m19 and C-CPE differed, suggesting that electrostatic interactions may occur between m19 and CLs. Treatment of epithelial cells with m19 decreased the paracellular but not transcellular integrity, and m19 enhanced jejunal absorption. Thus, we successfully created a CL binder with broad specificity. These findings will contribute to future preparation of CL binders for CL-targeted drug development.

The application of an alanine-substituted mutant of the C-terminal fragment of Clostridium perfringens enterotoxin as a mucosal vaccine in mice

Efficient delivery of antigen to mucosal immune tissues is an essential part of mucosal vaccination. Claudin-4 is expressed on the epithelial cells that cover the mucosal immune tissues. We previously found that claudin-4-targeting is a promising strategy for mucosal vaccination by using a claudin-4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE). Substitution of Asn and Ser at positions 309 and 313, respectively, with alanine increased the affinity of C-CPE for claudin-4. However, application of the C-CPE mutant as a mucosal vaccine has never been tried. Here, we investigated whether the C-CPE mutant could serve as a mucosal vaccine. We used ovalbumin (OVA) as a model antigen and fused the C-CPE mutant to it. The resultant fusion protein was bound to claudin-4. When mice were immunized with the C-CPE mutant-fused OVA, OVA-specific serum IgG and nasal IgA increased relative to levels in mice immunized with a C-CPE-fused antigen. Immunization with the C-CPE mutant-fused OVA activated Th1- and Th2-type responses and led to increased anti-tumor activity against OVA-expressing thymoma cells relative to that of mice immunized with the C-CPE-fused antigen. These findings suggest that the alanine-substituted C-CPE mutant shows promise as a claudin-targeted mucosal vaccine.

Development of novel claudin‐4 binder and its application in mucosal vaccine

Mucosal vaccines activate both mucosal and systemic immune responses, but mucosal administration is needle‐free and less painful. Thus, mucosal vaccination appears to be an ideal vaccination strategy. The effective delivery of antigens to mucosa‐associated lymphoid tissues (MALT) is critical to the development of potent mucosal vaccines. We previously found that claudin‐4‐targeting is a promising strategy for mucosal vaccination using a claudin‐4 ligand, the C‐terminal fragment of Clostridium perfringens enterotoxin (C‐CPE). In the present study, we identified the functional domain of C‐CPE, and we attempted to prepare a novel claudin binder with higher affinity for claudin using the functional mapping information of C‐CPE. Substitution of some amino acids with alanine increased affinity of C‐CPE to claudin‐4, and Kd values decreased from 455 pM to 46 pM. To investigate mucosal vaccination using the mutant C‐CPE, ovalbumin (OVA) was used as a model antigen. When mice were immunized with the C‐CPE mutant‐fused antigen, antigen‐specific serum IgG and nasal IgA increased 10‐fold when compared with mice immunized using C‐CPE‐fused antigen. Immunization with the C‐CPE mutant‐fused antigen OVA activated Th1‐ and Th2‐type responses more strongly than in mice immunized with C‐CPE‐fused antigen. These results indicate that the alanine‐substituted C‐CPE mutant is a promising lead for claudin‐targeted mucosal vaccines.

A Novel Screening System for Claudin Binder Using Baculoviral Display

Recent progress in cell biology has provided new insight into the claudin (CL) family of integral membrane proteins, which contains more than 20 members, as a target for pharmaceutical therapy. Few ligands for CL have been identified because it is difficult to prepare CL in an intact form. In the present study, we developed a method to screen for CL binders by using the budded baculovirus (BV) display system. CL4-displaying BV interacted with a CL4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), but it did not interact with C-CPE that was mutated in its CL4-binding region. C-CPE did not interact with BV and CL1-displaying BV. We used CL4-displaying BV to select CL4-binding phage in a mixture of a scFv-phage and C-CPE-phage. The percentage of C-CPE-phage in the phage mixture increased from 16.7% before selection to 92% after selection, indicating that CL-displaying BV may be useful for the selection of CL binders. We prepared a C-CPE phage library by mutating the functional amino acids. We screened the library for CL4 binders by affinity to CL4-displaying BV, and we found that the novel CL4 binders modulated the tight-junction barrier. These findings indicate that the CL-displaying BV system may be a promising method to produce a novel CL binder and modulator.

A Claudin-Targeting Molecule as an Inhibitor of Tumor Metastasis

Tumor metastasis of epithelium-derived tumors is the major cause of death from malignant tumors. Overexpression of claudin is observed frequently in malignant tumors. However, claudin-targeting antimetastasis therapy has never been investigated. We previously prepared a claudin-4-targeting antitumor molecule that consisted of the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) fused to protein synthesis inhibitory factor (PSIF) derived from Pseudomonas exotoxin. In the present study, we investigated whether claudin CPE receptors can be a target for tumor metastasis by using the C-CPE-fused PSIF as a claudin-targeting agent. One of the most popular murine metastasis models is the lung metastasis of intravenously injected B16 cells. Therefore, we first investigated the effects of the C-CPE-fused PSIF on lung metastasis of claudin-4-expressing B16 (CL4-B16) cells. Intravenous administration of the C-CPE-fused PSIF suppressed lung metastasis of CL4-B16 cells but not B16 cells. Injection of C-CPE-fused PSIF also inhibited tumor growth and spontaneous lung metastasis of murine breast cancer 4T1 cells inoculated into the subcutis. Treatment with C-CPE-fused PSIF did not show apparent side effects in mice. These findings indicate that claudin targeting may be a novel strategy for inhibiting some tumor metastases.