Pathomechanism of Clostridium perfringens enterotoxin-mediated damage of colonic epithelia and generation of novel toxin variants as therapeutics for claudin overexpressing tumors

Lebensmittelvergiftungen durch Clostridium perfringens Typ A sind weit verbreitet. C. perfringens Enterotoxin (CPE) gilt hier als wichtigster Verursacher gastrointestinaler (GI) Symptome und spielt zudem eine Rolle bei Antibiotika-assoziierter Diarrhö. Das β-Poren-formende Toxin schädigt Zellen in Dünndarm und Kolon. Es bindet über die C-terminale Domäne (cCPE) an seine Rezeptoren – einige (z.B. Claudin-3, -4, -7) aber nicht alle (z.B. Claudin-1, -5) Mitglieder der Tight-Junction(TJ)-Proteinfamilie der Claudine (Cldn1-27). Durch CPE-Poren einströmendes Ca2+ führt dann zum Zelltod. Ziel dieser Arbeit war die Untersuchung des Pathomechanismus CPE-vermittelter Schädigungen des Kolon-Epithels sowie die Generierung neuartiger Toxin-Varianten für die Claudin-gerichtete Therapie von Tumoren. Es zeigte sich, dass nur eine Subpopulation von Enterozyten des Kolon-Epithels CPE-sensitiv ist. Entscheidend ist hierfür die Lokalisation des CPE-Rezeptors Cldn4: Während Cldn4 in CPE-insensitiven Zellen für eine CPE-Bindung unzugänglich in TJs vorlag, ist es bei CPE-sensitiven Zellen im apikalen, dem Darmlumen zugewandten Plasmamembrankompartiment lokalisiert, wo es CPE als Angriffspunkt dient. Störung der epithelialen Barriere durch den inflammatorischen Faktor TNFα, EGTA oder Dedifferenzierung erhöhte die Zugänglichkeit von CPE- Rezeptoren und so die CPE-Sensitivität. Diese mechanistischen Daten erklären, wie CPE in Einzelfällen zu schwerwiegenden Manifestationen wie intestinalen Nekrosen führen kann, wenn die intestinale Barriere – z.B. bei Entzündung – gestört und so der Zugang zu Claudinen erleichtert ist. Neben seiner Rolle in GI-Pathologien besitzt CPE großes Potential in der Krebsmedizin, denn in vielen Tumoren werden Claudine überexprimiert. Bei Therapie solcher Tumoren wirken allerdings zwei Faktoren limitierend: (i) Da auch in gesundem Gewebe – wie Leber und Niere – CPE-Rezeptoren zugänglich sind, kann es zu Nebenwirkungen kommen. (ii) Nur Tumoren, die CPE-Rezeptoren, nicht aber solche die andere Claudine (Nicht-CPE-Rezeptoren wie Cldn1 und -5) überexprimieren, können angegriffen werden. CPE-Varianten, deren claudinspezifische Toxizität auf das Claudinexpressionsprofil des jeweiligen Tumors zugeschnitten ist, könnten diese Limitierungen überwinden helfen. Dass sich die claudinspezifische Toxizität gezielt über strukturgeführte Mutationen in CPE verändern lässt, wurde hier gezeigt. Mit CPE-L254A/S256A/I258A/D284A konnte eine CPE-Variante generiert werden, die toxische Effekte bevorzugt über Bindung an Cldn4 und kaum über andere CPE-Rezeptoren vermittelt. Die Mutationen S305P/S307R/S313H und Y306W/S313H erlauben eine Bindung an die Nicht-CPE-Rezeptoren Cldn1 und -5. Mit entsprechenden CPE-Varianten konnten toxische Effekte, über Nicht-CPE-Rezeptoren wie Cldn1 und -5 vermittelt werden. Schließlich konnte CPE-S305P/S307R/S313H erfolgreich in vitro eingesetzt werden, um Cldn1-exprimierende Thyroidkarzinomzellen (K1) anzugreifen, die aufgrund fehlender CPE-Rezeptorclaudine insensitiv gegenüber CPE-wt sind. Dies demonstriert das Potential optimierter CPE-basierter Biologika. Hierauf aufbauend können künftig weitere CPE-basierte Tumortherapeutika und -diagnostika generiert werden.Food poisoning caused by Clostridium perfringens type A is common. C. perfringens enterotoxin (CPE) is responsible for its gastrointestinal (GI) symptoms and also plays a role in antibiotic-associated diarrhea. The β-pore- forming toxin damages epithelial cells in the small intestine and the colon. As cellular receptors serve some (e.g. claudin-3, -4, -7) but not all (e.g. claudin-1, -5) claudins – a family of tetraspan membrane proteins (Cldn1-27). Ca2+-influx through CPE-pores leads to cell death. This study aimed to investigate the mechanism of CPE-induced damage to the colonic epithelium and to generate novel CPE-variants for claudin-targeted cancer therapy. This work demonstrates that only a subpopulation of colonic enterocytes is sensitive towards CPE. CPE sensitivity depends on localization of CPE receptor Cldn4: In insensitive cells Cldn4 was found inaccessible for CPE binding at the TJ. In CPE-sensitive cells Cldn4 was localized in the apical membrane compartment. Facing the lumen of the gut it here is easily accessible for CPE. If the epithelial barrier was disturbed by inflammatory factor TNFα, EGTA or dedifferentiation, enhanced accessibility of CPE receptors led to enhanced CPE sensitivity. These mechanistic details provide an explanation of how CPE sometimes may lead to severe clinical manifestations like intestinal necrosis if claudin accessibility is facilitated by a disturbed intestinal barrier – e.g. during inflammation. Besides its role in GI pathologies CPE bears great potential in cancer medicine, as claudins are frequently overexpressed in tumors. However, tumor therapy using CPE has its limitations: (i) as claudins are also expressed in healthy tissue (e.g. liver and kidney) there are side effects. (ii) Only tumors expressing CPE receptors but not those overexpressing other claudins (such as Cldn1 or -5) can be targeted. Novel CPE variants with claudin subtype specific affinities, which are tailored to the claudin expression profiles of distinct tumors could help to overcome these limitations. Here it is demonstrated that claudin subtype specific toxicity can be altered by structure-based site directed mutagenesis. CPE variant CPE- L254A/S256A/I258A/D284A preferentially mediates toxicity via Cldn4 but barely via other CPE receptors. Mutations S305P/S307R/S313H and Y306W/S313H allow binding to non-CPE receptors Cldn1 und -5. Accordingly, the respective CPE variants allowed to mediate a toxic effect via Clnd1 and -5. Finally, CPE- S305P/S307R/S313H was successfully used in vitro to target Cldn1-expressing thyroid carcinoma cells (K1), which were insensitive towards CPE-wt due to lack of high levels of CPE receptor claudins. These results demonstrate the potential of optimized CPE-based biologicals and may serve as foundation for further developments in the field of CPE-based therapeutics and diagnostics

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

Therapeutic drug monitoring-based dose optimisation of piperacillin/tazobactam to improve outcome in patients with sepsis (TARGET): a prospective, multi-centre, randomised controlled trial

Background: Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection with a hospital mortality in excess of 40%. Along with insufficient and delayed empirical antimicrobial therapy, inappropriate antimicrobial exposure has been identified to negatively affect patient outcomes. Receipt of prolonged infusion (i.e. extended or continuous infusion) of piperacillin/tazobactam (TZP) improves antimicrobial exposure and is associated with reduced mortality in patients with sepsis. Using therapeutic drug monitoring (TDM) with dosing tailored to the altered pharmacokinetics of the individual patient to avoid under-and overdosing may be a further strategy to improve patient outcomes. This current trial will address the question whether a TDM-guided therapy with TZP administered by continuous infusion will result in a greater resolution of organ dysfunction and hence better clinical outcome compared to continuous infusion of the total daily dose of TZP without TDM. Methods: The study is an investigator-initiated, multi-centre, parallel-group, single-blinded, randomised controlled trial. The trial will be conducted in several centres across Germany. Adult patients (aged >= 18 years) with severe sepsis or septic shock will be eligible for study participation. Participants will be randomly assigned to receive either TZP by continuous infusion guided by daily TDM of piperacillin (experimental group) or by continuous infusion without TDM guidance (total daily dose in normal renal function 13.5 g TZP) (control group). The pharmacokinetic (PK)/ pharmacodynamic (PD) target will be 100% f T->4MIC (percentage of time during a dosing interval that the free [f] drug concentration exceeds 4 times the minimum inhibitory concentration). The primary efficacy endpoint is the change in mean total Sequential Organ Failure Assessment score from day 1 after randomisation until day 10 or discharge from the intensive care unit or death, whichever comes first. Secondary outcomes include mortality, clinical cure, microbiological cure, overall antibiotic use, individual components of the primary outcome, adverse events and analysis of PK and (PD) indices. Discussion: This trial will assess for the first time whether continuous infusion of TZP guided by daily TDM in patients with sepsis will result in a greater resolution of organ dysfunction and hence better clinical outcome compared to continuous infusion without TDM