Design, biochemical characterisation and analysis of the therapeutic relevance

Titelblatt, Inhaltsverzeichnis, Abkürzungen, Zusammenfassung 1\. Einleitung 1 2\. Material und Methoden 18 3\. Ergebnisse 42 4\. Diskussion 77 5\. Literatur 94In den letzten Jahren wurden vielfach neue Medikamente entwickelt, die konventionelle Behandlungsmethoden von Krebserkrankungen verbessern. Diese verwenden oft Antikörper für eine spezifische Erkennung von Oberflächenstrukturen, die auf Tumorzellen überexprimiert werden. Während durch die Anwendung von Antikörpern bereits beachtliche Verbesserungen in den Therapiemöglichkeiten erreicht wurden, werden weitere vielversprechende Untersuchungen zu kombinierten Wirkstoffen vorangetrieben. Durch Kombination von Antikörpern oder Wachstumsfaktoren mit toxischen Substanzen entstehen Immunotoxine oder chimäre Toxine, die nach spezifischer Bindung eine Aufnahme der Toxine in die Tumorzelle vermitteln, wodurch die Zelle stirbt. Damit kann die Behandlung von Krebs durch spezifische Wirkstoffe erheblich verbessert werden, während gleichzeitig Nebenwirkungen minimiert werden. In der vorliegenden Arbeit wurden chimäre Toxine entwickelt und charakterisiert, bei denen das Toxin mit dem Liganden über einen spaltbaren Adapter verbunden ist. Der Adapter dient der Senkung von Nebenwirkungen und der Verbesserung der Toxinaufnahme. Zur Analyse von chimären Toxinen mit Diphtheriatoxin wurde ein colorimetrischer Festphasenassay entwickelt, der eine quantitative Analyse der Enzymaktivität in vitro erlaubt und durch seine Flexibilität und hohe Sensitivität auch zur Diagnose von Patientenproben mit Diphtherieverdacht hervorragend geeignet ist. Die Analyse von drei chimären Toxinen, die jeweils Liganden für den Interleukin-2 Rezeptor tragen, zeigte zwar Enzymaktivität der Toxine, jedoch keine Cytotoxizität und Bindung an Interleukin-2 Rezeptor exprimierende Zellen. Wahrscheinlich ist die für diese chimären Toxine gewählte Kombination von Toxin und Ligand zur Ausbildung aktiver chimärer Toxine durch sterische Effekte nicht geeignet. Um die Verbesserung der Toxinaufnahme durch den Adapter in den chimären Toxinen weiter zu charakterisieren, wurden innerhalb des Adapters sechs unterschiedliche Membrantransferpeptide verwendet, für die eine Vermittlung der Wirkstoffaufnahme beschrieben wurde. Für diese Analysen wurden chimäre Toxine mit epidermal growth factor als Ligand verwendet. Die Einführung der unterschiedlichen Membrantransferpeptide in die chimären Toxine ergab in Cytotoxizitätsassays und Bindungsstudien keine erhöhte Aufnahme, jedoch konnte für einen Teil der Sequenzen eine gesteigerte unspezifische Bindung an Zellen beschrieben werden, weshalb sie sich für die Verwendung in tumorspezifischen Wirkstoffen nicht eignen. Mit dem Ziel, die Wirksamkeit der chimären Toxine zu verbessern, wurde die Steigerung der Cytotoxizität durch Kombination Saponinen charakterisiert. Alle sieben verwendeten Saponine konnten in einem synergistischen Prozess die Cytotoxiztität erhöhen, jedoch konnten nur Quillajasaponin und Saponinum album die Wirkung mehr als 10fach steigern. Saponinum album erhöhte die Cytotoxizität sogar 13600fach. Saponinum album erhöhte dabei gezielt die Ligand abhängige und damit spezifische Cytotoxizität, während die Ligand unabhängige, unspezifische Cytotoxizität nur wenig beeinflusst wurde. Damit bietet sich Saponinum album als Zusatz für spezifische Tumorwirkstoffe in Maus-Tumormodellen an, da durch die Kombination mit Saponinen das therapeutische Fenster erweitert wird und tumorspezifische Wirkungen gesteigert und Nebenwirkungen gesenkt werden.Mutation-based, uncontrolled growth of cells leads to cancer. Several innovative new treatments have been developed in the recent years to improve conventional cancer treatments. These biological treatments are based on the use of antibodies or growth factors for specific binding to over-expressed tumor-specific structures on the cell surface. Treatment with antibodies in cancer therapies is promising, but the development of new drugs is ongoing. By conjugating antibodies or growth factors to toxic substances, immunotoxins or chimeric toxins have been developed. These conjugates are able to bind specifically to tumor cells and the resulting uptake of the toxin causes cell death. Thus, immunotoxins or chimeric toxins can be designed to treat specific cancer types as well as reducing side effects on healthy cells. In the present study chimeric toxins with a molecular adapter linking the toxin and the ligand were generated and characterized. The adapter has been developed to reduce side effects and to improve the uptake of the toxins into cells. A new colorimetric solid phase assay has been established to determine the enzymatic activity of chimeric toxins composed of diphtheria toxin or pseudomonas exotoxin. This colorimetric solid phase assay permits quantitative examinations in vitro and its enhanced sensitivity enables it for use in diagnostics. The analyses of three chimeric toxins composed of diphtheria toxin A-chain and ligands for interleukin-2 receptor showed the enzymatic activity of diphtheria toxin but failed to elicit cytotoxicity. The combination of diphtheria toxin A-chain with the ligands probably produced a sterical effect that prevents the ligand binding to interleukin-2 receptor expressing cells. Six different membrane transfer peptides within the adapter of chimeric toxins targeting the epidermal growth factor receptor were used to characterize the adapter-mediated improvement of toxin uptake into cells. The investigations of the chimeric toxins in cytotoxicity assays and binding studies revealed no increased uptake of the toxins into cells regardless of the membrane transfer peptide used. However, some of the sequences showed higher nonspecific binding to cells. This result demonstrates that these sequences are not suitable for use in tumor-specific drugs. To further enhance the efficacy of chimeric toxins the combination with saponins, plant steroidal or triterpenoidal glycosides, was characterized. All seven analysed saponins caused higher cytotoxicity in a synergistic process, but only Quillajasaponin and Saponinum album elicited a more than 10-fold enhancing effect on cytotoxicity. Saponinum album even caused a 13600-fold higher cytotoxicity of the chimeric toxin. Furthermore, its enhancement of cytotoxicity is dependent on receptor-ligand interactions. Saponinum album is thus a suitable additive for tumor therapies with specific drugs in mouse tumor models. The combination of saponins and tumor-specific compounds increases the prospect for therapeutic treatments, as tumor-specific compounds will have increased cytotoxicity and fewer side effects

Tumor Targeting and Drug Delivery by Anthrax Toxin

Anthrax toxin is a potent tripartite protein toxin from Bacillus anthracis. It is one of the two virulence factors and causes the disease anthrax. The receptor-binding component of the toxin, protective antigen, needs to be cleaved by furin-like proteases to be activated and to deliver the enzymatic moieties lethal factor and edema factor to the cytosol of cells. Alteration of the protease cleavage site allows the activation of the toxin selectively in response to the presence of tumor-associated proteases. This initial idea of re-targeting anthrax toxin to tumor cells was further elaborated in recent years and resulted in the design of many modifications of anthrax toxin, which resulted in successful tumor therapy in animal models. These modifications include the combination of different toxin variants that require activation by two different tumor-associated proteases for increased specificity of toxin activation. The anthrax toxin system has proved to be a versatile system for drug delivery of several enzymatic moieties into cells. This highly efficient delivery system has recently been further modified by introducing ubiquitin as a cytosolic cleavage site into lethal factor fusion proteins. This review article describes the latest developments in this field of tumor targeting and drug delivery

Diving through Membranes: Molecular Cunning to Enforce the Endosomal Escape of Antibody-Targeted Anti-Tumor Toxins

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The activity of myeloid cell-specific VHH immunotoxins is target-, epitope-, subset- and organ dependent

The central role of myeloid cells in driving autoimmune diseases and cancer has raised interest in manipulating their function or depleting them for therapeutic benefits. To achieve this, antibodies are used to antagonize differentiation, survival and polarization signals or to kill target cells, for example in the form of antibody-drug conjugates (ADC). The action of ADC in vivo can be hard to predict based on target expression pattern alone. The biology of the targeted receptor as well as its interplay with the ADC can have drastic effects on cell apoptosis versus survival. Here we investigated the efficacy of CD11b or Ly-6C/Ly-6G-specific variable fragments of camelid heavy chain-only antibodies (VHH) conjugated to Pseudomonas exotoxin A to deplete myeloid cells in vitro and in vivo. Our data highlight striking differences in cell killing in vivo, depending on the cell subset and organs targeted, but not antigen expression level or VHH affinity. We observed striking differences in depletion efficiency of monocytes versus granulocytes in mice. Despite similar binding of Ly-6C/Ly-6G-specific VHH immunotoxin to granulocytes and monocytes, granulocytes were significantly more sensitive than monocytes to immunotoxins treatment. Our results illustrate the need of early, thorough in vivo characterization of ADC candidates

Label-Free Immunodetection in High Ionic Strength Solutions Using Carbon Nanotube Transistors with Nanobody Receptors

Nanomaterial-based field-effect transistors (FETs) have been proposed for real-time, label-free detection of various biological species. However, screening of the analyte charge by electrolyte ions (Debye screening) has so far limited their use in physiological samples. Here, this challenge is overcome by combining FETs based on single-walled semiconducting carbon nanotube networks (SWCNTs) with a novel surface functionalization comprising: (1) short nanobody receptors, and (2) a polyethylene glycol layer (PEG). Nanobodies are stable, easy-to-produce, short biological receptors (~2–4 nm) that enable analyte binding closer to the sensor surface. The addition of PEG enhances the signal in high ionic strength environment. Using green fluorescent protein (GFP) as a model antigen, high selectivity and sub-picomolar detection limit with a dynamic range exceeding 4 orders of magnitude is demonstrated in physiological solutions. The presented immunoassay is fast, label-free, does not require any sample pre-treatment or washing steps