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.


Introduction
The boundaries between the inside of the body and the outside environment in the airway and gastrointestinal tract, and between the systemic circulation and tissues in the brain, eye, testis, and placenta, are separated by epithelial and endothelial cell sheets, respectively. The paracellular spaces between the adjacent cells in these sheets are sealed by a structural and functional component called the tight junction (TJ) [1]. TJs control the diffusion of ions, solutes, and water across the paracellular space to maintain homeostasis, and the loss of TJ integrity appears to be associated with the development of intestinal diseases [2,3], atopic dermatitis [4], and psychiatric disorders [5,6]. TJs also There is currently no structural information available for angulins. The secondary structural elements are shown as cylinders (α-helices) and arrows (β-strands). aa, amino acid.

First-Generation Binders: Fragments of Bacterial Toxins
Clostridium perfringens enterotoxin (CPE) has two domains: The N-terminal cytotoxic domain, which is involved in oligomerization and pore formation, and the C-terminal receptor binding domain (C-CPE) [35] (Figure 2a). The CPE receptor (CPE-R) was identified, and CPE-R has significant similarity to the rat androgen withdrawal apoptosis protein (RVP1) in 1997 [36]. Two years after the identification of CPE-R and RVP1, claudin-3 and -4 have been identified to be RVP1 and CPE-R, respectively [37]. C-CPE binds to claudin-3 and -4 [38]. However, C-CPE also binds to claudin-6, -7, -8, -9, -14, and -19 [22,39,40]. The affinity of C-CPE to claudin-4 is approximately 0.5 nM [41]. The treatment of MDCK cells with C-CPE decreases TJ integrity [38]. The research into the generation of claudin binders using C-CPE as a template for site-directed mutagenesis has produced several C-CPE mutants: One broad-spectrum binder to claudin-1 to -5 and four relatively specific binders for claudin-3, -4, and -5 (Table 1). , respectively. TM domain 3 is much longer than the other three TM domains. Claudins also contain an extracellular helix (ECH) and two variable regions (V1 and V2). (b) Angulins are 60-70 kDa type I TM proteins containing an extracellular immunoglobulin-like domain and an intracellular tail. There is currently no structural information available for angulins. The secondary structural elements are shown as cylinders (α-helices) and arrows (β-strands). aa, amino acid.

Second-Generation Binders: Monoclonal Antibodies
The TJ components are promising targets for the development of drugs to treat cancers and inflammatory bowel disease, for preventing infection by hepatitis C virus, and for the development of regenerative medicines. Antibodies are promising therapeutics for TJ-directed drug development because they bind to target proteins with high affinity and high specificity [51,52]. Thus, mAbs

Second-Generation Binders: Monoclonal Antibodies
The TJ components are promising targets for the development of drugs to treat cancers and inflammatory bowel disease, for preventing infection by hepatitis C virus, and for the development of regenerative medicines. Antibodies are promising therapeutics for TJ-directed drug development because they bind to target proteins with high affinity and high specificity [51,52]. Thus, mAbs against the extracellular domains of the TJ components have been generated and their pharmaceutical activities are being investigated (Table 2).

Drug Delivery Using Claudin and Angulin Binders
The authors have recently completed a series of studies examining TJ binders that has provided new insights for TJ-directed drug development. Here, the proofs-of-concept for TJ-directed drug development using first-and second-generation claudin and angulin binders are introduced.

Mucosal Absorption
Drug absorption across epithelia is either transcellular or paracellular [70]. One strategy for paracellular drug absorption is to loosen the TJs between adjacent epithelial cells. C-CPE increased jejunal absorption of dextran (4 kDa) 400-fold compared with sodium caprate, an absorption enhancer in current clinical use [17]. C-CPE also enhanced jejunal, nasal, and pulmonary absorption of a biologically active peptide [41]. The treatment of cells with angubindin-1 enhances the permeability of tricellular TJs to solutes up to 10 kDa (Figure 3). Angubindin-1 also enhanced jejunal absorption of dextran (4 kDa) [50]. These results demonstrate that modulation of bicellular and tricellular TJs could be useful strategy for the development of non-invasive drug-delivery systems. enhancer in current clinical use [17]. C-CPE also enhanced jejunal, nasal, and pulmonary absorption of a biologically active peptide [41]. The treatment of cells with angubindin-1 enhances the permeability of tricellular TJs to solutes up to 10 kDa (Figure 3). Angubindin-1 also enhanced jejunal absorption of dextran (4 kDa) [50]. These results demonstrate that modulation of bicellular and tricellular TJs could be useful strategy for the development of non-invasive drug-delivery systems.

Epidermal Absorption
The epidermis covers the outer body, preventing the passage of solutes and the absorption of drugs. However, epidermal administration is a potentially useful route of administration because it is noninvasive, can easily be stopped, and avoids first-pass metabolism [71]. The epidermal barrier comprises the stratum corneum and TJs in the stratum granulosum [72]. The analyses using knockout mice have revealed that claudin-1 is critical for TJ integrity in the stratum granulosum [73]. The treatment with an anti-claudin-1 mAb (7A5) weakened TJ integrity and enhanced the permeation of dextran (4 kDa) in an in vitro human epidermal model [53]. Thus, claudin-1-mediated modulation of the permeability of TJs in the stratum granulosum is a promising means of increasing epidermal drug absorption.
The various claudin-targeting molecules, including toxins, toxin fragments, and antibodies, have been generated for claudin-targeted cancer therapy [32,33,85]. For example, CPE has been used as an anti-cancer agent against pancreatic cancer overexpressing claudin-4 [86]. One issue with claudin-targeted therapies is that claudins are expressed not only in malignant tissues, but also in non-malignant tissues. However, most claudins in non-malignant tissues are localized within TJ complexes, whereas their localization is often dysregulated from TJ complexes to the cell surface in malignant tissues [87,88]. This study found that C-CPE fused with protein synthesis inhibitory factor (C-CPE-PSIF) may recognize claudins with aberrant localization, resulting in less binding and therefore, less toxicity to the normal cells [89]. Caco-2 is a human colon carcinoma cell line. Caco-2 cells form a polarized cell monolayer with well-developed TJs when confluent, and they are frequently used as a model of polarized normal epithelial cells. The claudin-4 protein level in the confluent culture (normal epithelial-like cells) was higher than in the subconfluent culture (carcinoma cells), and C-CPE-PSIF was cytotoxic to preconfluent (immature TJs) but not to postconfluent Caco-2 cells (mature TJs) (Figure 4) [89,90]. Similarly, anti-claudin-4 mAbs systemically administered to mice

Targeting Tissues Involved in Immunological Processes
Mucosal vaccination may be a useful immunization strategy because it is non-invasive and it activates both the mucosal and systemic immune responses. Epithelial cells associated with the mucosa-associated lymphoid tissues (MALT) include Peyer's patches and nasopharynx and play pivotal roles in preventing the invasion of pathological microorganisms into the body by inducing the secretion of IgA [92]. MALT comprises various immune cells, including T cells, B cells, and dendritic cells, and is covered by follicle-associated epithelium. M cells are specialized epithelial cells in the follicle-associated epithelium that transport luminal antigens to immune cells in MALT by transcytosis [93]. In general, when antigen alone is orally or nasally administered, it fails to reach the MALT and so immune responses are not induced. Thus, the efficient delivery of antigen to MALT may provide effective mucosal vaccines.
Follicle-associated epithelium contains claudin-4-expressing cells, some of which are highly capable of capturing luminal antigen [94,95]. Claudin-4 is also expressed on the luminal surface of M cells [96]. Thus, claudin-4 targeting may be a promising strategy for delivering antigens to MALT. A nasally administered ovalbumin fused with C-CPE induced mucosal IgA production, systemic IgG production, and antigen-specific immune responses for preventing tumor growth ( Figure 5) [97]. Of note, a simple mixture of C-CPE and antigen did not induce IgA production, indicating that the vaccination efficacy may be depending on the binding affinity of the C-CPE to claudins [97]. Nasal immunization with chimeric C-CPE-antigen did not induce mucosal injury [98]. The augmentation of the antigenicity of the first-generation binder C-CPE has been used to develop an adjuvant-free bivalent food poisoning vaccine [99]

Targeting Tissues Involved in Immunological Processes
Mucosal vaccination may be a useful immunization strategy because it is non-invasive and it activates both the mucosal and systemic immune responses. Epithelial cells associated with the mucosa-associated lymphoid tissues (MALT) include Peyer's patches and nasopharynx and play pivotal roles in preventing the invasion of pathological microorganisms into the body by inducing the secretion of IgA [92]. MALT comprises various immune cells, including T cells, B cells, and dendritic cells, and is covered by follicle-associated epithelium. M cells are specialized epithelial cells in the follicle-associated epithelium that transport luminal antigens to immune cells in MALT by transcytosis [93]. In general, when antigen alone is orally or nasally administered, it fails to reach the MALT and so immune responses are not induced. Thus, the efficient delivery of antigen to MALT may provide effective mucosal vaccines.
Follicle-associated epithelium contains claudin-4-expressing cells, some of which are highly capable of capturing luminal antigen [94,95]. Claudin-4 is also expressed on the luminal surface of M cells [96]. Thus, claudin-4 targeting may be a promising strategy for delivering antigens to MALT. A nasally administered ovalbumin fused with C-CPE induced mucosal IgA production, systemic IgG production, and antigen-specific immune responses for preventing tumor growth ( Figure 5) [97]. Of note, a simple mixture of C-CPE and antigen did not induce IgA production, indicating that the vaccination efficacy may be depending on the binding affinity of the C-CPE to claudins [97]. Nasal immunization with chimeric C-CPE-antigen did not induce mucosal injury [98]. The augmentation of the antigenicity of the first-generation binder C-CPE has been used to develop an adjuvant-free bivalent food poisoning vaccine [99] Int

Targeting Inflamed Tissues
Ulcerative colitis is a chronic, relapsing inflammatory bowel disease characterized by severe diarrhea and mucosal inflammation in the colon. The disruption of the colonic mucosal barrier leads to the activation of immune responses against bacterial and food fragments in the colon, followed by the development of ulcerative colitis [2,3]. Although claudin-2 is rarely expressed in normal colonic epithelial cells, its expression in the colon is upregulated in ulcerative colitis patients [80]. Inflammatory cytokines, including tumor necrosis factor-α (TNF-α), decrease the epithelial barrier integrity and upregulate the expression of claudin-2 [100]. Claudin-2 decreases the integrity of TJs by facilitating the formation of discontinuous TJ strands [101]. This suggests that the inhibition of claudin-2 may restore the disrupted mucosal barrier. Indeed, an anti-claudin-2 mAb (1A2) ameliorated TNF-α-induced reduction of TJ integrity in Caco-2 cells. Moreover, the co-treatment of the cells with anti-claudin-2 mAb and an anti-TNF-α mAb showed an additive effect on the restoration of the barrier [56]. (c) Mice were nasally immunized with vehicle, OVA, a mixture of OVA and C-CPE, OVA-C-CPE, or OVA-C-CPE303 (5 µg OVA in each vaccine) once a week for 3 weeks. Seven days after the final immunization, the mice were injected subcutaneously with 1 × 10 6 OVA-expressing EL4 (H-2b) cells. The tumor volumes were measured over time. The data are shown as the mean ± S.D. (n = 4). * p < 0.05. The data are reproduced from reference [97] with slight modifications and permission from the copyright holder.

Targeting Inflamed Tissues
Ulcerative colitis is a chronic, relapsing inflammatory bowel disease characterized by severe diarrhea and mucosal inflammation in the colon. The disruption of the colonic mucosal barrier leads to the activation of immune responses against bacterial and food fragments in the colon, followed by the development of ulcerative colitis [2,3]. Although claudin-2 is rarely expressed in normal colonic epithelial cells, its expression in the colon is upregulated in ulcerative colitis patients [80]. Inflammatory cytokines, including tumor necrosis factor-α (TNF-α), decrease the epithelial barrier integrity and upregulate the expression of claudin-2 [100]. Claudin-2 decreases the integrity of TJs by facilitating the formation of discontinuous TJ strands [101]. This suggests that the inhibition of claudin-2 may restore the disrupted mucosal barrier. Indeed, an anti-claudin-2 mAb (1A2) ameliorated TNF-α-induced reduction of TJ integrity in Caco-2 cells. Moreover, the co-treatment of the cells with anti-claudin-2 mAb and an anti-TNF-α mAb showed an additive effect on the restoration of the barrier [56].

Drug Deliavery to the Brain
Unlike peripheral capillaries, those in the brain lack fenestrations and have well-developed TJs that form the blood-brain barrier (BBB). More than 98% of small-molecular-weight drugs cannot pass the BBB [102]. Claudin-5 and angulin-1 are abundantly expressed by brain endothelial cells in mice [103]. Claudin-5-or angulin-1-knockout mice have a size-selectively loosened BBB [104,105]. These data suggest that claudin-5 and angulin-1 are candidate targets for opening the BBB. Indeed, a C-CPE mutant that can bind to claudin-5, angubindin-1, and anti-claudin-5 mAb was able to reduce the transepithelial/transendothelial electrical resistance (TER) in an in vitro model of the BBB [54,106]. Furthermore, in the mice, an angubindin-1-, but not claudin-5-binding C-CPE mutant increased the permeability of the BBB to allow passage of a 16-mer gapmer antisense oligonucleotide (5.3 kDa) [106]. No obvious adverse effects were observed in the mice in these experiments.

Safety of Claudin-and Angulin-Targeted Therapies
A series of proof-of-concept studies examining claudin and angulin targeting has provided insights into enhancing drug absorption, treating cancer and inflammatory diseases, improving vaccines, and obtaining drug delivery to the brain. No apparent adverse effects were observed in these studies. However, claudins and angulins play roles in the formation of the intercellular seal between and among epithelial cells and endothelial cells in many tissues. Therefore, ensuring the safety of claudinand angulin-targeted drugs is critical for future drug development.
The knockout and knockdown analyses of the genes encoding claudins and angulins have shown that there are risks associated with claudin-and angulin-targeted therapeutics (Table 3). For instance, the inhibitors of claudin-1 and -5 may induce atopic dermatitis and schizophrenia-like symptoms via the inhibition of the epidermal barrier and the BBB, respectively [4,6]. Claudin-2, -4, and angulin-2-targeted drugs may induce renal impairment with respect to the reabsorption of ions and water [107][108][109]. A deletion in exon 1 of claudin-1 results in neonatal ichthyosis and sclerosing cholangitis syndrome in humans [110]. A deletion of 1.5 to 3.0 Mb of human chromosome 22q11.2 that includes the claudin-5 gene is associated with the development of schizophrenia [111]. A single nucleotide polymorphism in claudin-5 is also associated with the development of schizophrenia [112,113]. Table 3. Phenotypes of representative claudin-or angulin-knockout or -knockdown mice.

Phenotype of Knockout (KO) or Knockdown (KD) Mice
Ref. Impaired renal water reabsorption and colonic water absorption (KO) [109] The expression profiles of claudins and angulins differ among tissues ( Table 4). The specific claudin ratio is critical for the functions of TJs [115]. Thus, the toxicity of claudin-directed drugs should be carefully investigated, especially if the target claudins are also expressed in non-target tissues. Claudins and angulins in TJs are embedded in the lateral cell membranes and extend into the intercellular space. The TJ cavity is estimated to be 0.5 nm under physiological conditions [116,117]. Large binders, such as antibodies, which are unable to access proteins embedded in TJs, are a promising modality for treating cancers and for improving the effectiveness of vaccines because in these conditions the target claudins are exposed on the cell surfaces [34,87,88,96]. TJ components are potent targets for the development of many novel therapies, but targets and drug modalities must be optimized to afford an acceptable risk-benefit balance.

Conclusions
TJ binders are classified as first-generation binders (toxins and their fragments), and secondgeneration binders (antibodies) [32,33,85]. The augmentation of the antigenicity of the first-generation binder, C-CPE, has been used to develop an adjuvant-free vaccine [97,99]. Second-generation binders are being used to develop cancer therapies ( Table 2). For example, an anti-claudin-18.2 mAb is undergoing clinical study for the use in the treatment of gastric (phase III study) and pancreatic cancer (phase II study) [NCT03504397; NCT03816163].
The other application of TJ binders is to enhance the mucosal and epidermal absorption of drugs and to deliver drugs to the brain by modulating the permeability of TJs. The currently available TJ binders are toxin fragments and antibodies. However, the generation of novel TJ binders, such as peptides and chemicals, is now needed because of the potential antigenicity of toxins and the costs associated with antibody preparation. A high-throughput screening system for claudin-4 binders based on the time resolved fluorescence resonance energy transfer in a chemical library was developed [126]. In the future, the generations of peptide-and chemical-type of binders are expected to accelerate.