Claudin-Targeted Suicide Gene Therapy for Claudin-Overexpressing Tumor Cells by Using Modified Clostridium perfringens Enterotoxin (CPE).

Bacterial toxins gain growing attention as potential cancer treatment due to their potent cytotoxic effects. Among the very different toxins with diverse modes of action, the Clostridium perfringens enterotoxin (CPE) is in focus to treat solid cancers. This toxin targets the tight junction proteins claudin-3 and -4 (Cldn-3/4), which are frequently overexpressed in solid cancers. Binding to these claudins induces pore formation in the host cell plasma membrane leading to rapid oncoleaking cell death of tumor cells. Based on this, extending the targeting of CPE beyond Cldn-3/4 is of interest, since other claudins, such as claudin-1 or -5 are often overexpressed in various cancer entities such as non-small-cell lung cancer (NSCLC) or papillary thyroid carcinoma. In this chapter we describe the modification of a CPE-encoding vector by structure-directed mutagenesis to either preferentially target Cldn-1 and -5 or to expand targeting to Cldn1-9 for improved broadened cytotoxic targeting of claudin-overexpressing tumors such as but not limited to lung cancer via CPE gene transfer.

Targeting claudin-overexpressing thyroid and lung cancer by modified Clostridium perfringens enterotoxin.

Clostridium perfringens enterotoxin (CPE) can be used to eliminate carcinoma cells that overexpress on their cell surface CPE receptors - a subset of claudins (e.g., Cldn3 and Cldn4). However, CPE cannot target tumors expressing solely CPE-insensitive claudins (such as Cldn1 and Cldn5). To overcome this limitation, structure-guided modifications were used to generate CPE variants that can strongly bind to Cldn1, Cldn2 and/or Cldn5, while maintaining the ability to bind Cldn3 and Cldn4. This enabled (a) targeting of the most frequent endocrine malignancy, namely, Cldn1-overexpressing thyroid cancer, and (b) improved targeting of the most common cancer type worldwide, non-small-cell lung cancer (NSCLC), which is characterized by high expression of several claudins, including Cldn1 and Cldn5. Different CPE variants, including the novel mutant CPE-Mut3 (S231R/S313H), were applied on thyroid cancer (K1 cells) and NSCLC (PC-9 cells) models. In vitro, CPE-Mut3, but not CPEwt, showed Cldn1-dependent binding and cytotoxicity toward K1 cells. For PC-9 cells, CPE-Mut3 improved claudin-dependent cytotoxic targeting, when compared to CPEwt. In vivo, intratumoral injection of CPE-Mut3 in xenograft models bearing K1 or PC-9 tumors induced necrosis and reduced the growth of both tumor types. Thus, directed modification of CPE enables eradication of tumor entities that cannot be targeted by CPEwt, for instance, Cldn1-overexpressing thyroid cancer by using the novel CPE-Mut3.

Use of Modified Clostridium perfringens Enterotoxin Fragments for Claudin Targeting in Liver and Skin Cells.

Claudins regulate paracellular permeability in different tissues. The claudin-binding domain of Clostridium perfringens enterotoxin (cCPE) is a known modulator of a claudin subset. However, it does not efficiently bind to claudin-1 (Cldn1). Cldn1 is a pharmacological target since it is (i) an essential co-receptor for hepatitis C virus (HCV) infections and (ii) a key element of the epidermal barrier limiting drug delivery. In this study, we investigated the potential of a Cldn1-binding cCPE mutant (i) to inhibit HCV entry into hepatocytes and (ii) to open the epidermal barrier. Inhibition of HCV infection by blocking of Cldn1 with cCPE variants was analyzed in the Huh7.5 hepatoma cell line. A model of reconstructed human epidermis was used to investigate modulation of the epidermal barrier by cCPE variants. In contrast to cCPEwt, the Cldn1-binding cCPE-S305P/S307R/S313H inhibited infection of Huh7.5 cells with HCV in a dose-dependent manner. In addition, TJ modulation by cCPE variant-mediated targeting of Cldn1 and Cldn4 opened the epidermal barrier in reconstructed human epidermis. cCPE variants are potent claudin modulators. They can be applied for mechanistic in vitro studies and might also be used as biologics for therapeutic claudin targeting including HCV treatment (host-targeting antivirals) and improvement of drug delivery.

Reversible opening of the blood-brain barrier by claudin-5-binding variants of Clostridium perfringens enterotoxin's claudin-binding domain.

The blood-brain barrier (BBB) prevents entry of neurotoxic substances but also that of drugs into the brain. Here, the paracellular barrier is formed by tight junctions (TJs) with claudin-5 (Cldn5) being the main sealing constituent. Transient BBB opening by targeting Cldn5 could improve paracellular drug delivery. The non-toxic C-terminal domain of Clostridium perfringens enterotoxin (cCPE) binds to a subset of claudins, e.g., Cldn3, -4. Structure-based mutagenesis was used to generate Cldn5-binding variants (cCPE-Y306W/S313H and cCPE-N218Q/Y306W/S313H). These cCPE-variants were tested for transient TJ opening using multiple in vitro BBB models: Primary porcine brain endothelial cells, coculture of primary rat brain endothelial cells with astrocytes and mouse cerebEND cells. cCPE-Y306W/S313H and cCPE-N218Q/Y306W/S313H but neither cCPE-wt nor cCPE-Y306A/L315A (not binding to claudins) decreased transendothelial electrical resistance in a concentration-dependent and reversible manner. Furthermore, permeability of carboxyfluorescein (with size of CNS drugs) was increased. cCPE-Y306W/S313H but neither cCPE-wt nor cCPE-Y306A/L315A bound to Cldn5-expressing brain endothelial cells. However, freeze-fracture EM showed that cCPE-Y306W/S313H did not cause drastic TJ breakdown. In sum, Cldn5-binding cCPE-variants enabled mild and transient opening of brain endothelial TJs. Using reliable in vitro BBB models, the results demonstrate that cCPE-based biologics designed to bind Cldn5 improve paracellular drug delivery across the BBB.

In Colon Epithelia, Clostridium perfringens Enterotoxin Causes Focal Leaks by Targeting Claudins Which are Apically Accessible Due to Tight Junction Derangement.

Clostridium perfringens enterotoxin (CPE) causes food poisoning and antibiotic-associated diarrhea. It uses some claudin tight junction proteins (eg, claudin-4) as receptors to form Ca2+-permeable pores in the membrane, damaging epithelial cells in small intestine and colon. We demonstrate that only a subpopulation of colonic enterocytes which are characterized by apical dislocation of claudins are CPE-susceptible. CPE-mediated damage was enhanced if paracellular barrier was impaired by Ca2+ depletion, proinflammatory cytokine tumor necrosis factor α, or dedifferentiation. Microscopy, Ca2+ monitoring, and electrophysiological data showed that CPE-mediated cytotoxicity and barrier disruption was limited by extent of CPE-binding. The latter was restricted by accessibility of non-junctional claudin molecules such as claudin-4 at apical membranes. Focal-leaks detected in HT-29/B6 colonic monolayers were verified for native tissue using colon biopsies. These mechanistic findings indicate how CPE-mediated effects may turn from self-limiting diarrhea into severe clinical manifestation such as colonic necrosis-if intestinal barrier dysfunction, eg, during inflammation facilitates claudin accessibility.

A cCPE-based xenon biosensor for magnetic resonance imaging of claudin-expressing cells.

The majority of malignant tumors originate from epithelial cells, and many of them are characterized by an overexpression of claudins (Cldns) and their mislocalization out of tight junctions. We utilized the C-terminal claudin-binding domain of Clostridium perfringens enterotoxin (cCPE), with its high affinity to specific members of the claudin family, as the targeting unit for a claudin-sensitive cancer biosensor. To overcome the poor sensitivity of conventional relaxivity-based magnetic resonance imaging (MRI) contrast agents, we utilized the superior sensitivity of xenon Hyper-CEST biosensors. We labeled cCPE for both xenon MRI and fluorescence detection. As one readout module, we employed a cryptophane (CrA) monoacid and, as the second, a fluorescein molecule. Both were conjugated separately to a biotin molecule via a polyethyleneglycol chemical spacer and later via avidin linked to GST-cCPE. Nontransfected HEK293 cells and HEK293 cells stably expressing Cldn4-FLAG were incubated with the cCPE-based biosensor. Fluorescence-based flow cytometry and xenon MRI demonstrated binding of the biosensor specifically to Cldn4-expressing cells. This study provides proof of concept for the use of cCPE as a carrier for diagnostic contrast agents, a novel approach for potential detection of Cldn3/-4-overexpressing tumors for noninvasive early cancer detection.

Claudins are essential for cell shape changes and convergent extension movements during neural tube closure.

During neural tube closure, regulated changes at the level of individual cells are translated into large-scale morphogenetic movements to facilitate conversion of the flat neural plate into a closed tube. Throughout this process, the integrity of the neural epithelium is maintained via cell interactions through intercellular junctions, including apical tight junctions. Members of the claudin family of tight junction proteins regulate paracellular permeability, apical-basal cell polarity and link the tight junction to the actin cytoskeleton. Here, we show that claudins are essential for neural tube closure: the simultaneous removal of Cldn3, -4 and -8 from tight junctions caused folate-resistant open neural tube defects. Their removal did not affect cell type differentiation, neural ectoderm patterning nor overall apical-basal polarity. However, apical accumulation of Vangl2, RhoA, and pMLC were reduced, and Par3 and Cdc42 were mislocalized at the apical cell surface. Our data showed that claudins act upstream of planar cell polarity and RhoA/ROCK signaling to regulate cell intercalation and actin-myosin contraction, which are required for convergent extension and apical constriction during neural tube closure, respectively.

Polar and charged extracellular residues conserved among barrier-forming claudins contribute to tight junction strand formation.

Claudins (Cldn) form the backbone of tight junction (TJ) strands and thereby regulate paracellular permeability for solutes and water. Polymeric strands are formed by homo- and heterophilic cis- and trans-interactions between claudin protomers. Crystal structures of some claudins have been resolved; however, the mechanism by which claudins assemble into TJ strands remains unclear. To elucidate strand architecture, TJ-like strands were reconstituted in HEK293 cells by claudin transfection. Determinants of prototypic, classic barrier-forming claudins (Cldn1, -3, and -5) involved in strand formation were analyzed by mutagenesis. The capability of claudin constructs to interact in trans and to form strands was investigated by cell contact-enrichment assays and freeze-fracture electron microscopy. Residues in extracellular loops 1 and 2 of the claudins affecting strand formation were identified. Using homology modeling and molecular docking, we tested working concepts for the arrangement of claudin protomers within TJ strands. We show that the charge of Lys65 in Cldn1 and Glu158 in Cldn3, but not of Arg30 or Asp145 in Cldn3, and the polarity of Gln56 and Gln62 in Cldn3 and of Gln57 in Cldn5 are necessary for TJ strand formation. These residues are all conserved among barrier-forming classic claudins. The results contribute to mechanistic understanding of claudin-based regulation of paracellular permeability.

Targeting and alteration of tight junctions by bacteria and their virulence factors such as Clostridium perfringens enterotoxin.

The integrity of tight junctions, which regulate paracellular permeability, is challenged by many bacterial pathogens. This is caused by inflammatory responses triggered by pathogens and direct interaction of bacteria or their toxins with host epithelial cells. In some cases, tight junction proteins represent receptors for cell surface proteins or toxins of the pathogen, such as Clostridium perfringens enterotoxin (CPE). CPE causes diarrhea and cramps-the symptoms of a common foodborne illness, caused by C. perfringens type A. It uses a subgroup of the claudin family of tight junction proteins as receptors and forms pores in the membrane of intestinal epithelial cells. Ca2+ influx through these pores finally triggers cell damage. In this review, we summarize tight junction targeting and alteration by a multitude of different microorganisms such as C. perfringens, Escherichia coli, Helicobacter pylori, Salmonella typhimurium, Shigella flexneri, Vibrio cholerae, Yersinia enterocolitica, protozoan parasites, and their proteins. A focus is drawn towards CPE, the interaction with its receptors, cellular, and pathophysiological consequences for the intestinal epithelium. In addition, we portend to the use of CPE-based claudin modulators for drug delivery as well as diagnosis and therapy of cancer.

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.

Specific binding of Clostridium perfringens enterotoxin fragment to Claudin-b and modulation of zebrafish epidermal barrier.

Claudins (Cldn) are the major components of tight junctions (TJs) sealing the paracellular cleft in tissue barriers of various organs. Zebrafish Cldnb, the homolog of mammalian Cldn4, is expressed at epithelial cell-cell contacts and is important for regulating epidermal permeability. The bacterial toxin Clostridium perfringens enterotoxin (CPE) has been shown to bind to a subset of mammalian Cldns. In this study, we used the Cldn-binding C-terminal domain of CPE (194-319 amino acids, cCPE 194-319 ) to investigate its functional role in modulating zebrafish larval epidermal barriers. In vitro analyses show that cCPE 194-319 removed Cldn4 from epithelial cells and disrupted the monolayer tightness, which could be rescued by the removal of cCPE 194-319. Incubation of zebrafish larvae with cCPE 194-319 removed Cldnb specifically from the epidermal cell membrane. Dye diffusion analysis with 4-kDa fluorescent dextran indicated that the permeability of the epidermal barrier increased due to cCPE 194-319 incubation. Electron microscopic investigation revealed reversible loss of TJ integrity by Cldnb removal. Collectively, these results suggest that cCPE 194-319 could be used as a Cldnb modulator to transiently open the epidermal barrier in zebrafish. In addition, zebrafish might be used as an in vivo system to investigate the capability of cCPE to enhance drug delivery across tissue barriers.

Directed structural modification of Clostridium perfringens enterotoxin to enhance binding to claudin-5.

Clostridium perfringens enterotoxin (CPE) binds to distinct claudins (Clds), which regulate paracellular barrier functions in endo- and epithelia. The C-terminal domain (cCPE) has the potential for selective claudin modulation, since it only binds to a subset of claudins, e.g., Cld3 and Cld4 (cCPE receptors). Cld5 (non-CPE receptor) is a main constituent in tight junctions (TJ) of the blood-brain barrier. We aimed to reveal claudin recognition mechanisms of cCPE and to create a basis for a Cld5-binder. By utilizing structure-based interaction models, mutagenesis and assays of cCPE-binding to the TJ-free cell line HEK293, transfected with human Cld1 and murine Cld5, we showed how cCPE-binding to Cld1 and Cld5 is prevented by two residues in extracellular loop 2 of Cld1 (Asn(150) and Thr(153)) and Cld5 (Asp(149) and Thr(151)). Binding to Cld5 is especially attenuated by the lack of a bulky hydrophobic residue like leucine at position 151. By downsizing the binding pocket and compensating for the lack of this leucine residue, we created a novel cCPE-variant; cCPEY306W/S313H binds Cld5 with nanomolar affinity (K d 33 ± 10 nM). Finally, the effective binding to endogenously Cld5-expressing blood-brain barrier model cells (murine microvascular endothelial cEND cell line) suggests cCPEY306W/S313H as basis for Cld5-specific modulation to improve paracellular drug delivery, or to target claudin overexpressing tumors.

Molecular and structural transmembrane determinants critical for embedding claudin-5 into tight junctions reveal a distinct four-helix bundle arrangement.

The mechanism of TJ (tight junction) assembly and the structure of TJ strand-forming Cldns (claudins) are unclear. To identify determinants of assembly of blood-brain barrier-related Cldn3 and Cldn5, chimaeric mutants were analysed by cellular reconstitution of TJ strands and live-cell imaging. On the basis of the rescue of mutants deficient for strand formation, we identified Cldn5 residues (Cys128, Ala132, Ile142, Ala163, Ile166 and Leu174) involved in Cldn folding and assembly. Experimental results were combined with structural bioinformatics approaches. Initially the experimentally validated previous model of the ECL2 (extracellular loop 2) of Cldn5 was extended to the flanking transmembrane segments (TM3/TM4). A coiled-coil interface probably caused by alternating small and large residues is supported by concomitant knob-into-hole interactions including Cldn5-specific residues identified in the present paper. To address arrangement of the TMs in a four-helix bundle, data from evolutionary sequence couplings and comparative modelling of intramolecular interfaces in the transmembrane region of Cldns led to a complete Cldn5 model. Our suggested Cldn subtype-specific intramolecular interfaces that are formed by conserved coiled-coil motifs and non-conserved residues in distinct TM positions were confirmed by the recently released crystal structure of Cldn15. The identified molecular and structural determinants essentially contribute to assembly of Cldns into TJ strands.