Bioassay-guided development and in vitro characterization of lectin-drug conjugates for the targeted intravesical therapy of bladder cancer

Im Rahmen der vorliegenden Arbeit wurde ein Delivery-System entwickelt, das, basierend auf dem Konzept des aktiven Drug-Targetings, zur Verbesserung der adjuvanten Therapie des nicht-muskelinvasiven Harnblasenkarzinoms oder anderer intravesikal zugänglicher Erkrankungen beitragen soll. Das entwickelte Prodrug-System besteht aus fluoreszenzmarkiertem BSA (Bovines Serum Albumin), das über Divinylsulfon kovalent mit WGA (Wheat Germ Agglutinin) verknüpft wurde, welches als Targeting-Protein fungieren soll. Das Hauptaugenmerkt zunächst auf einer Optimierung der Kopplungs- und Reinigungsprozedur, wobei das Endprodukt zellkulturgestützt evaluiert wurde. Dabei erwies sich die Verwendung eines 20- fachen molaren Überschusses an WGA bezogen auf BSA als am geeignetsten. Um ein reines Produkt zu garantieren, wurden für die weitere Charakterisierung nach der Aufreinigung nur die ersten 10-12 Fraktionen des erhaltenen Peaks gepoolt. Unter den gewählten Bedingungen wurde so ein Konjugatgemisch mit durchschnittlichem Modifikationsgrad von 3-5 Molekülen WGA pro Molekül fBSA erhalten. Diese Parameter konnten dabei auch mit Hilfe alternativer Analyseverfahren (GEMMA, Acrylamid-Gelelektrophorese) bestätigt werden. Anhand von Zellstudien wurde nachgewiesen, dass das Konjugat zur spezifischen Bindung an die Zelloberfläche befähigt ist und in weiterer Folge relativ rasch von den Zellen internalisiert wird. Das konnte auch durch Fluoreszenzemissionsmessung vor und nach Behandlung mit Monensin untermauert werden. Um die Vermutung, dass WGA und somit auch das Kopplungsprodukt mit höherer Präferenz an maligne Zellen bindet, zu überprüfen, wurden konzentrationsabhängige Bindungsstudien neben SV-HUC-1 (Zellen ohne maligen Hintergrund) zusätzlich auch an 5637 (Tumorgrad 2) und HT1376 (Tumorgrad 3) durchgeführt. Hierbei konnte gezeigt werden, dass das Konjugat im höheren Konzentrationsbereich tatsächlich eine verstärkte Tendenz zur Bindung an Zellen mit malignem Hintergrund aufweist Außerdem wurden Konjugate mit PNA (Peanut Agglutinin) als Lektin entwickelt und deren Bindungsspezifität überprüft. Die RFI-Werte der Zelloberflächenbindung von fBSA/PNA-Konjugaten sind verglichen mit jenen der fBSA/WGA-Konjugate insgesamt sehr gering, was eventuell auf eine mangelnde Kopplungseffizienz im Fall von PNA hindeuten könnte. Zusätzlich wude fHSA (fluoreszenzmarkiertes humanes Serum Albumin) an WGA (Wheat Germ Agglutinin) gekoppelt. Allerdeings erwieß sich HSA als weniger homogen wie BSA. Die Feinjustierung der Konjugataufreinigung erschien deshalb mit dem homogeneren fBSA einfacher, überdies bietet der Einsatz von kostengünstigerem fBSA einen ökonomischen Vorteil Die Ergebnisse der vorliegenden Diplomarbeit belegen, dass fBSA/WGA-Konjugate einen ersten Schritt in Richtung Verbesserung der adjuvanten Therapie des nicht-muskelinvasiven Harnblasenkarzinoms beruhend auf dem Konzept des aktiven Drug-Targetings darstellen könnten. Hinsichtlich der Spezifität der Bindung und deren Aufnahme in maligne Zellen sind weitere Untersuchungen in ArbeitThe structural and compositional characteristics of the urothelial tissue account for a problematically low bioavailability in intravesical therapy, and call for the implementation of penetration-promoting delivery modalities to improve treatment impact. This study presents the first biorecognitive approach for intravesical drug delivery, based on the specific interaction between lectins and the corresponding carbohydrates at the cell membrane. Fluorescence-labeled bovine serum albumin (fBSA) or human serum albumin (fHSA) was covalently coupled to wheat germ agglutinin (WGA) to yield a model prodrug (fBSA/WGA conjugate) for the characterization of cytoadhesive and cytoinvasive potential in cell culture models. Coupling protocol and purification procedure were optimized and validated by a comparative screening in bioassays and physicochemical analysis of molecular weight. Preliminary studies demonstrated the general applicability of the coupling procedure to lectins of different carbohydrate specificity. Binding and uptake by human urothelial cells of non-malignant origin (SV-HUC-1) were compared to that by cells deriving from grade 2 (5637) or grade 3 (HT-1376) urothelial carcinoma. In contrast to unmodified fBSA, WGA-conjugates were effectively internalized (>50%) and processed to acidic compartments. Specificity of the conjugate-cell interaction was validated via competitive inhibition with the complementary carbohydrate. Intracellular accumulation was followed microscopically and verified by monensin-induced de-quenching. Utilizing receptor-mediated endocytotic pathways, glycotargeted delivery platforms may thus provide a potent tool for the establishing of novel therapy concepts for intravesical application

Teaching an old pET new tricks: tuning of inclusion body formation and properties by a mixed feed system in E. coli

Against the outdated belief that inclusion bodies (IBs) in Escherichia coli are only inactive aggregates of misfolded protein, and thus should be avoided during recombinant protein production, numerous biopharmaceutically important proteins are currently produced as IBs. To obtain correctly folded, soluble product, IBs have to be processed, namely, harvested, solubilized, and refolded. Several years ago, it was discovered that, depending on cultivation conditions and protein properties, IBs contain partially correctly folded protein structures, which makes IB processing more efficient. Here, we present a method of tailored induction of recombinant protein production in E. coli by a mixed feed system using glucose and lactose and its impact on IB formation. Our method allows tuning of IB amount, IB size, size distribution, and purity, which does not only facilitate IB processing, but is also crucial for potential direct applications of IBs as nanomaterials and biomaterials in regenerative medicine.COMET6676761

Production strategies for active heme-containing peroxidases from E. coli inclusion bodies – a review

Heme-containing peroxidases are frequently used in medical applications. However, these enzymes are still extracted from their native source, which leads to inadequate yields and a mixture of isoenzymes differing in glycosylation which limits subsequent enzyme applications. Thus, recombinant production of these enzymes in Escherichia coli is a reasonable alternative. Even though production yields are high, the product is frequently found as protein aggregates called inclusion bodies (IBs). These IBs have to be solubilized and laboriously refolded to obtain active enzyme. Unfortunately, refolding yields are still very low making the recombinant production of these enzymes in E. coli not competitive. Motivated by the high importance of that enzyme class, this review aims at providing a comprehensive summary of state-of-the-art strategies to obtain active peroxidases from IBs. Additionally, various refolding techniques, which have not yet been used for this enzyme class, are discussed to show alternative and potentially more efficient ways to obtain active peroxidases from E. coli

The production of a recombinant tandem single chain fragment variable capable of binding prolamins triggering celiac disease

Abstract Background Celiac disease (CD) is one of the most common food-related chronic disorders. It is mediated by the dietary consumption of prolamins, which are storage proteins of different grains. So far, no therapy exists and patients are bound to maintain a lifelong diet to avoid symptoms and long-term complications. To support those patients we developed a tandem single chain Fragment variable (tscFv) acting as a neutralizing agent against prolamins. We recombinantly produced this molecule in E. coli, but mainly obtained misfolded product aggregates, so-called inclusion bodies, independent of the cultivation strategy we applied. Results In this study, we introduce this novel tscFv against CD and present our strategy of obtaining active product from inclusion bodies. The refolded tscFv shows binding capabilities towards all tested CD-triggering grains. Compared to a standard polyclonal anti-PT-gliadin-IgY, the tscFv displays a slightly reduced affinity towards digested gliadin, but an additional affinity towards prolamins of barley. Conclusion The high binding specificity of tscFv towards prolamin-containing grains makes this novel molecule a valuable candidate to support patients suffering from CD in the future

A combination of HPLC and automated data analysis for monitoring the efficiency of high-pressure homogenization

Abstract Background Cell disruption is a key unit operation to make valuable, intracellular target products accessible for further downstream unit operations. Independent of the applied cell disruption method, each cell disruption process must be evaluated with respect to disruption efficiency and potential product loss. Current state-of-the-art methods, like measuring the total amount of released protein and plating-out assays, are usually time-delayed and involve manual intervention making them error-prone. An automated method to monitor cell disruption efficiency at-line is not available to date. Results In the current study we implemented a methodology, which we had originally developed to monitor E. coli cell integrity during bioreactor cultivations, to automatically monitor and evaluate cell disruption of a recombinant E. coli strain by high-pressure homogenization. We compared our tool with a library of state-of-the-art methods, analyzed the effect of freezing the biomass before high-pressure homogenization and finally investigated this unit operation in more detail by a multivariate approach. Conclusion A combination of HPLC and automated data analysis describes a valuable, novel tool to monitor and evaluate cell disruption processes. Our methodology, which can be used both in upstream (USP) and downstream processing (DSP), describes a valuable tool to evaluate cell disruption processes as it can be implemented at-line, gives results within minutes after sampling and does not need manual intervention

The E. coli pET expression system revisited—mechanistic correlation between glucose and lactose uptake

The final publication is available at Springer via https://doi.org/10.1007/s00253-016-7620-7.Therapeutic monoclonal antibodies are mainly produced in mammalian cells to date. However, unglycosylated antibody fragments can also be produced in the bacterium Escherichia coli which brings several advantages, like growth on cheap media and high productivity. One of the most popular E. coli strains for recombinant protein production is E. coli BL21(DE3) which is usually used in combination with the pET expression system. However, it is well known that induction by isopropyl β-d-1-thiogalactopyranoside (IPTG) stresses the cells and can lead to the formation of insoluble inclusion bodies. In this study, we revisited the pET expression system for the production of a novel antibody single-chain variable fragment (scFv) with the goal of maximizing the amount of soluble product. Thus, we (1) investigated whether lactose favors the recombinant production of soluble scFv compared to IPTG, (2) investigated whether the formation of soluble product can be influenced by the specific glucose uptake rate (qs,glu) during lactose induction, and (3) determined the mechanistic correlation between the specific lactose uptake rate (qs,lac) and qs,glu. We found that lactose induction gave a much greater amount of soluble scFv compared to IPTG, even when the growth rate was increased. Furthermore, we showed that the production of soluble protein could be tuned by varying qs,glu during lactose induction. Finally, we established a simple model describing the mechanistic correlation between qs,lac and qs,glu allowing tailored feeding and prevention of sugar accumulation. We believe that this mechanistic model might serve as platform knowledge for E. coli