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In this thesis, the overall goal was to establish linear polyglycerol as PEG-alternative polymer platform for the half-life extension of therapeutic proteins and peptides. The aim was to design polymer-protein conjugates with a 1:1 ratio of polymer to biomolecule by employing mono-functional LPG and site-selective coupling chemistries. Furthermore, this work aimed to reveal differences between LPG- and PEG-bioconjugates in regard to their hydrodynamic size, bioactivity, and stability in vitro and their extended half-life and therapeutic efficacy in vivo. In the first project, a site-selective N-terminal conjugation strategy was developed which enabled modification of the therapeutic protein anakinra by employing LPG-aldehyde of 5, 10, 20 and 40 kDa. PEG-conjugates of the same protein were synthesized as reference. The molecular weights of the isolated mono-conjugates were systematically analyzed by multi-angle light scattering and mass spectrometry and confirmed close values for LPG- and PEG-conjugates of same nominal weight, with dispersities below 1.1. The hydrodynamic size of the bioconjugates increased with ascending polymer length, where the LPG-conjugates displayed slightly smaller values than their PEG-analogs. This finding confirms the general picture of LPG as a more compact polymer than PEG, even though differences were not as large as found for the free polymers. Studies on enzymatic and thermal degradation revealed a somewhat better performance for the more hydrophilic LPG-conjugates, which aids in designing more stable protein-conjugates in the future. Additionally, binding affinity to IL-1 receptor 1 decreased about factor 3–7 upon polymer conjugation, with no significant differences between LPG- and PEG-conjugates observed. Finally, a pharmacokinetic study of a selected Anakinra-40-LPG conjugate revealed a similar performance and terminal half-life than its PEGylated analog of same molecular weight. The second project focused on the site-specific C-terminal conjugation of the propargyl-modified diabetes therapeutic exenatide. LPG-N3 of 10, 20 and 40 kDa was employed to synthesize conjugates by Cu-catalyzed click-chemistry. PEG-conjugates of similar molecular weights again served as reference. The secondary structure of exenatide was not altered by polymer conjugation, whereas its hydrodynamic size increased with ascending polymer length. The values were higher than expected, as exenatide is known to exhibit oligomerization in solution, which was not prevented by conjugation of LPG or PEG. Exenatide-LPG-conjugates displayed smaller sizes and a more compact structure than their PEG-analogs of same molecular weight. Furthermore, thermal denaturation studies showed a slightly larger retainment of exenatide’s -helical structure upon PEG-conjugation, which was independent of the length of the polymer. Determination of the in vitro bioactivity of the conjugates by GLP-1 receptor-mediated cAMP-response revealed no significant differences between LPG- and PEG-exenatide of same molecular weight. Despite the bioactivity diminished after conjugation, maximum receptor response was still enabled at slightly higher concentrations. The main goal of this project was the evaluation of LPG-conjugates in a therapeutic disease model. A single injection of a selected exenatide-40-LPG conjugate in diabetic mice led to a significant blood glucose reduction for up to 72 h. This extended therapeutic activity was comparable to its exenatide-40-PEG analog, but 9-fold longer than native exenatide (8 h). The third project aimed to focus on the plasma characterization of LPG-and PEG-conjugates. The same coupling strategy as in project 1 was employed to generate N-terminal bioconjugates of interleukin-4. Comparable masses and hydrodynamic sizes between LPG- and PEG-IL-4 of same nominal weights were confirmed by SEC-MALS, MALDI-TOF and DLS, respectively. HPLC analysis revealed the large impact of LPG on the overall hydrophilicity of the conjugate, which increased with ascending polymer length. In contrast, PEG-conjugation resulted in a more hydrophobic character of IL-4. Polymer modification diminished the bioactivity of IL-4 in a proportional manner, where each 10 kDa polymer unit attached resulted approximately in a three-fold reduction of potency compared to the unmodified protein. The hydrophilicity had no substantial impact on the bioactivity in vitro, as LPG- and PEG-modified IL-4 displayed similar activation of the respective receptor. All conjugates remained stable in human plasma for up to 24 h and displayed diminished binding to human serum albumin (HSA), with no significant impact of polymer type or length observed. To conclude, this thesis has demonstrated comparable in vitro and in vivo performances of LPG- and PEG-conjugates of various therapeutic proteins and thereby verified polyglycerol’s great potential as an alternative to PEG for half-life extension. The impact of LPG on the bioactivity, terminal half-life and in vivo efficacy of selected biopharmaceuticals was similar to PEG. Differences were observed in thermal and proteolytic stability, overall hydrophilicity, and hydrodynamic size of the conjugates. In the future, questions regarding the immunogenicity and antigenicity of LPG need to be answered. Several setups employing ELISA or SPR already exist that enable the detection of anti-polymer specific antibodies in serum from healthy animals that received weekly doses of a specific PEGylated protein. Other studies evaluated polymers of different chemical structures on their cross-reactivity towards anti-PEG antibodies. This could be useful to determine the antigenicity of LPG outruling concerns about its reactivity towards pre-existing anti-PEG antibodies in the healthy population, which might impede the therapeutic efficacy of PGylated proteins. Another aspect covers the lyophilization of biopharmaceuticals. Due to its high content of hydroxy groups, LPG (free or conjugated) could serve as a stabilization tool during freeze-drying of therapeutic proteins. So far, high concentrations of sugars (trehalose, sucrose) are used to replace the water hydration shell upon drying. Conjugation of LPG might serve as a similar tool and could potentially maintain protein structure, stability and bioactivity after drying without the need of complex formulation studies., Ziel dieser Arbeit war es, Polyglycerol als PEG-alternative Polymer-Plattform zur Halbwertszeitverlängerung von therapeutischen Proteinen und Peptiden zu etablieren. Der Fokus lag dabei auf Polymer-Protein Konjugaten mit definiertem Verhältnis von einer Polymerkette pro Protein, was durch die Anwendung von monofunktionalem LPG und ortsspezifischer Konjugationschemie erreicht werden sollte. Weiterhin sollten Unterschiede zwischen LPG- und PEG-Biokonjugaten bezüglich deren hydrodynamischer Größe, Bioaktivität und Stabilität in vitro sowie ihrer verlängerten Halbwertszeit und therapeutischen Effektivität in vivo untersucht werden. Im ersten Projekt dieser Arbeit wurde eine ortsspezifische, N-terminale Konjugationsstrategie entwickelt, bei der LPG-Aldehyd mit Molekulargewichten von 5, 10, 20 und 40 kDa an das therapeutische Protein Anakinra konjugiert wurde. PEG-Konjugate des gleichen Proteins wurden als Vergleichsmaterial hergestellt. Die Molekulargewichte der gereinigten Mono-Konjugate wurden systematisch mittels multi-angle light scattering und Massenspektrometrie untersucht, wobei ähnliche Werte für LPG- und PEG-Konjugate gleichen, nominellen Molekulargewichts erhalten wurden. Die Dispersität lag hierbei bei unter 1,1. Die hydrodynamische Größe der Biokonjugate nahm mit steigender Polymerlänge zu, wobei die LPG-Konjugate ein etwas kleineres Volumen als ihre jeweiligen PEG-analoga zeigten. Die Ergebnisse unterstützen hierbei das Bild von LPG als etwas kompakterem Polymer, verglichen mit PEG, obwohl die Unterschiede bei den Konjugaten nicht so deutlich waren wie bei den freien Polymeren in Lösung. Studien zum enzymatischen und thermischen Abbau zeigten eine höhere Stabilität der hydrophilen LPG-Konjugate, was hilfreich beim zukünftigen Design von stabileren Proteinkonjugaten sein könnte. Zusätzlich wurde die Affinität der Konjugate an den IL-1 Rezeptor 1 untersucht, wobei die Bindung um Faktor 3–7 abnahm und die LPG- und PEG-Konjugate ähnliches Verhalten zeigten. Schließlich bestätigte eine pharmakokinetische Studie eines ausgewählten Anakinra-40-LPG Konjugats eine vergleichbare Zirkulationsdauer zum jeweiligen PEG-Analogon. Im zweiten Projekt lag der Fokus auf der ortsspezifschen, C-terminalen Konjugation des Diabetestherapeutikums Exenatid. LPG-Azid (10, 20 und 40 kDa) wurde mittels Kupfer-katalysierter „Klick“-Chemie an das Propargyl-modifizierte Peptid angebracht, wobei PEG-Konjugate gleichen Molekulargewichts erneut als Vergleichsmaterial dienten. Die Sekundärstruktur von Exenatid wurde durch die Polymerkonjugation nicht beeinflusst, wohingegen die hydrodynamische Größe mit aufsteigender Polymerkettenlänge zunahm. Die erhaltenen Werte waren hierbei größer als erwartet, da Exenatid zur Bildung von Oligomeren in Lösung neigt, was durch Konjugation von LPG oder PEG nicht unterbunden werden konnte. Die hydrodynamischen Durchmesser der LPG-Exenatid-Konjugate waren kleiner als die der PEG-Konjugate gleichen Molekulargewichts. Die -helikale Sekundärstruktur von Exenatid konnte bei Erhitzen besser durch PEG stabilisiert werden als durch LPG, wobei die Länge der Polymerkette keine große Rolle spielte. Eine Aktivierung des GLP-1 Rezeptors wurde für LPG-Exenatid in ähnlichem Maße erreicht wie für PEG-Exenatid gleichen Molekulargewichts. Hierbei wurden jedoch höhere Konzentrationen der größeren Konjugate benötigt, um die gleiche, maximale Rezeptorantwort von unmodifiziertem Exenatid zu generieren. Schließlich konnte gezeigt werden, dass ein ausgewähltes LPG-Exenatid-Konjugat die Blutglukose in diabetischen Mäusen ähnlich stark und lange reduzieren kann wie ein PEG-Konjugat gleichen Molekulargewichts (bis zu 72 Stunden). Im dritten Projekt wurde der Fokus auf die Charakterisierung von LPG- und PEG-Konjugaten gegenüber Plasmabestandteilen gelegt. Hierfür wurde die etablierte Kupplungsstrategie aus dem ersten Projekt angewandt, um N-terminale Konjugate des Proteins Interleukin-4 (IL-4) zu erhalten. LPG- und PEG-IL-4 gleichen, nominellen Molekulargewichts zeigten vergleichbare Massen und hydrodynamische Größen. HPLC-Analysen bestätigten den großen Einfluss von LPG auf die generelle Hydrophilie des Konjugats, die mit steigender LPG-Kettenlänge zunahm. Im Gegensatz dazu führte die Konjugation von PEG zu einem eher hydrophoben IL-4-Konstrukt. Die Bioaktivität wurde durch Polymerkonjugation reduziert, wobei das Anbringen einer 10 kDa Polymereinheit zu einer ungefähr 3-fachen Abnahme der in vitro Wirksamkeit führte. Große Unterschiede zwischen LPG- und PEG-IL-4 konnten hierbei nicht festgestellt werden. Alle Konjugate waren stabil in Humanplasma (bis 24 Stunden) und zeigten verminderte Affinität zum Plasmaprotein Human Serum Albumin (HSA). Zusammenfassend konnte in dieser Arbeit dargestellt werden, dass LPG- und PEG-Konjugate verschiedener therapeutischer Proteine ein ähnliches in vitro und in vivo Verhalten zeigen, was das große Potential von Polyglycerol als PEG-Alternative zur Halbwertszeitverlängerung unterstreicht. Der Einfluss von LPG auf Bioaktivität, terminale Halbwertszeit und in vivo Wirksamkeit von ausgewählten Biopharmazeutika war ähnlich wie der von PEG. Unterschiede konnten in den Bereichen thermischer und proteolytischer Stabilität, genereller Hydrophilie und hydrodynamischer Größe festgestellt werden. Um die Anwendbarkeit von Polyglycerol als PEG-Alternative weiter zu verfestigen, sollten in Zukunft Fragen zu dessen Immunogenität und Antigenizität beantwortet werden. Mehrere Möglichkeiten zur Detektion von anti-Polymer-spezifischen Antikörpern im Serum mittels ELISA oder SPR wurden bereits beschrieben. Zeitlich begrenzte, wöchentliche Injektionen eines bestimmten PG-Konjugats im gesunden Versuchstier würden hierbei eingesetzt, um die entsprechenden Polymer-Antikörper zu erzeugen. Andere Studien befassen sich mit der Kreuz-Reaktivität verschiedener Polymertypen gegenüber anti-PEG Antikörpern.113 Dies könnte hilfreich sein, um Bedenken gegenüber der Reaktivität von LPG auf bereits bestehende anti-PEG Antikörper in der gesunden Bevölkerung auszuräumen, die eine Anwendbarkeit von PGylierten Proteinen limitieren würde. Ein anderer Aspekt betrifft die Gefriertrocknung (Lyophilisation) von Biopharmazeutika. Durch den hohen Gehalt an Hydroxygruppen könnte LPG (frei oder konjugiert) die Struktur von therapeutischen Proteinen während des Gefriertrocknungsprozesses stabilisieren. Bisher werden hierfür hohe Konzentrationen von Zuckern (Trehalose, Sucrose) eingesetzt, die die Hydrathülle des Wassers nach Trocknung ersetzen. Konjugation von LPG könnte einen ähnlichen Effekt bewirken und somit die Durchführung komplexer Formulierungsstudien zur Proteinstabilität bei Lyophilisation vereinfachen.

Life-threatening infections with Staphylococcus aureus are often difficult to treat due to the increasing prevalence of antibiotic-resistant bacteria and their ability to persist in protected niches in the body. Bacteriolytic enzymes are promising new antimicrobials because they rapidly kill bacteria, including drug-resistant and persisting cells, by destroying their cell wall. However, when injected into the bloodstream, these enzymes are not retained long enough to clear an infection. Here, we describe a modification to increase blood circulation time of the enzymes and enhance treatment efficacy against S. aureus-induced bloodstream infections. This was achieved by preselecting enzyme candidates for high activity in human blood and coupling them to serum albumin, thereby preventing their elimination by kidney filtration and blood vessel cells., Staphylococcus aureus is a human pathogen causing life-threatening diseases. The increasing prevalence of multidrug-resistant S. aureus infections is a global health concern, requiring development of novel therapeutic options. Peptidoglycan-degrading enzymes (peptidoglycan hydrolases, PGHs) have emerged as a highly effective class of antimicrobial proteins against S. aureus and other pathogens. When applied to Gram-positive bacteria, PGHs hydrolyze bonds within the peptidoglycan layer, leading to rapid bacterial death by lysis. This activity is highly specific and independent of the metabolic activity of the cell or its antibiotic resistance patterns. However, systemic application of PGHs is limited by their often low activity in vivo and by an insufficient serum circulation half-life. To address this problem, we aimed to extend the half-life of PGHs selected for high activity against S. aureus in human serum. Half-life extension and increased serum circulation were achieved through fusion of PGHs to an albumin-binding domain (ABD), resulting in high-affinity recruitment of human serum albumin and formation of large protein complexes. Importantly, the ABD-fused PGHs maintained high killing activity against multiple drug-resistant S. aureus strains, as determined by ex vivo testing in human blood. The top candidate, termed ABD_M23, was tested in vivo to treat S. aureus-induced murine bacteremia. Our findings demonstrate a significantly higher efficacy of ABD_M23 than of the parental M23 enzyme. We conclude that fusion with ABD represents a powerful approach for half-life extension of PGHs, expanding the therapeutic potential of these enzybiotics for treatment of multidrug-resistant bacterial infections.

Folate receptor (FR) has proven to be a valuable target for chemotherapy using folic acid (FA) conjugates. However, FA-conjugated chemotherapeutics still have low therapeutic efficacy accompanied with side effects, resulting from complications such as short circulation half-life, limited tumor delivery, as well as high kidney accumulation. Herein, we present a novel FA-conjugated paclitaxel (PTX) prodrug which was additionally conjugated with an Evans blue (EB) derivative for albumin binding. The resulting bifunctional prodrug prolonged blood circulation, enhanced tumor accumulation, and consequently improved tumor therapeutic efficacy. Methods: Fmoc-Cys(Trt)-OH was coupled onto PTX at the 7'-OH position for further synthesis of ester prodrug FA-PTX-EB. The targeting ability was investigated using confocal microscopy and flow cytometry. The pharmacokinetics of this bifunctional compound was also studied. Meanwhile, cell viability was evaluated in normal cells and three cancer cell lines by MTT assay. In vivo therapeutic effect was tested on FR-α overexpressing MDA-MB-231 tumor model. Results: Compared with free PTX, the FA-PTX, PTX-EB and FA-PTX-EB prodrugs increased circulation half-life in mice from 2.19 to 3.82, 4.41, and 7.51 h, respectively. Pharmacokinetics studies showed that the FA-PTX-EB delivered more PTX to tumors than FA-PTX and free PTX. In vitro and in vivo studies demonstrated that FA-EB-conjugated PTX induced potent antitumor activity. Conclusion: FA-PTX-EB showed prolonged blood circulation, enhanced drug accumulation in tumors, higher therapeutic index, and lower side effects than either free PTX or monofunctional FA-PTX and EB-PTX. The results support the potential of using EB for the development of long-acting therapeutics.

Background: Gardnerella vaginalis is a bacterial vaginosis-associated vaginal bacterium, which produces toxin vaginolysin (VLY). VLY is a pore-forming toxin suggested to be the main virulence factor of G. vaginalis. The high recurrence rate of BV and emergence of antibiotic-resistant bacterial species demonstrate the need for development of recombinant antibodies as novel therapeutic agents for disease treatment. Single-chain variable fragments (scFvs) generated against VLY exhibited reduced efficacy to neutralize VLY activity compared to the respective full-length antibodies. To improve properties of scFvs the monospecific dimeric scFvs were generated by a genetic fusion of two anti-VLY scFv molecules connected with the alpha-helix-forming peptide linker. Results: N-terminally hexahistidine-tagged d imeric scFvs were constructed, produced in Escherichia coli and purified using metal-chelate affinity chromatography. Inhibition of VLY-mediated human erythrocyte lysis by dimeric and monomeric scFvs was detected by the in vitro hemolytic assay. Circulating half-life of purified scFvs in blood plasma of mice was accessed using ELISA. Dimeric anti-VLY scFvs showed higher neutralizing potency and extended circulating half-life in comparison with parental monomeric scFv. Conclusions: The protein obtained by a genetic fusion of two anti-VLY scFvs into dimeric molecule exhibited increased properties in comparison with monomeric scFv. This new recombinant antibody might implement new possibilities for the prophylaxis and treatment of the diseases caused by bacteria G. vaginalis . Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tabla normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Calibri",sans-serif; mso-bidi-font-family:"Times New Roman";}

To improve the pharmacological properties of maize ribosome-inactivating protein (maize RIP) for targeting HIV-infected cells, the previously engineered TAT-fused active form of maize RIP (MOD) was further engineered for cysteine-directed PEGylation. In this work, two potential antigenic sites, namely Lys-78 and Lys-264, were identified. They were mutated to cysteine residue and conjugated with PEG5k or PEG20k. The resultant PEG derivatives of MOD variants were examined for ribosome-inactivating activity, circulating half-life and immunogenicity. Our results showed that MOD-PEG conjugates had two- to five-fold lower biological activity compared to the wild-type. Mutation of the two sites respectively did not decrease the anti-MOD IgG and IgE level in mice, but the conjugation of PEG did dramatically reduce the antigenicity. Furthermore, pharmacokinetics studies demonstrated that attachment of PEG20k prolonged the plasma half-life by five-fold for MOD-K78C and 17-fold for MOD-K264C, respectively. The site-specific mutation together with PEGylation therefore generated MOD derivatives with improved pharmacological properties.

Nanoparticle (NP) drug delivery systems (5−250 nm) have the potential to improve current disease therapies because of their ability to overcome multiple biological barriers and releasing a therapeutic load in the optimal dosage range. Rapid clearance of circulating nanoparticles during systemic delivery is a critical issue for these systems and has made it necessary to understand the factors affecting particle biodistribution and blood circulation half-life. In this review, we discuss the factors which can influence nanoparticle blood residence time and organ specific accumulation. These factors include interactions with biological barriers and tunable nanoparticle parameters, such as composition, size, core properties, surface modifications (pegylation and surface charge), and finally, targeting ligand functionalization. All these factors have been shown to substantially affect the biodistribution and blood circulation half-life of circulating nanoparticles by reducing the level of nonspecific uptake, delaying opsonization, and increasing the extent of tissue specific accumulation.

The clearance from blood circulation of fluorinated liposomes made with perfluoroalkylated phosphatidylcholines was investigated in mice using liposome-entrapped 5(6)-carboxyfluorescein. The presence of a fluorinated core inside the membrane strongly retards their blood clearance. The fluorinated vesicles showed circulation half-lives of up to 8.6 h, which are 6–13 and 3–6 times larger than those of similarly sized conventional distearoylphosphatidylcholine and distearoylphosphatidylcholine/cholesterol liposomes, respectively. Their blood clearance was similar to that of some polyethylene glycol (PEG)-labelled ‘stealth’ liposomes and was dose-independent in a 3.3–330 μmol kg body weight dose range.

Staphylococcus aureus is a human pathogen causing life-threatening diseases. The increasing prevalence of multidrug-resistant S. aureus infections is a global health concern, requiring development of novel therapeutic options. Peptidoglycan-degrading enzymes (peptidoglycan hydrolases, PGHs) have emerged as a highly effective class of antimicrobial proteins against S. aureus and other pathogens. When applied to Gram-positive bacteria, PGHs hydrolyze bonds within the peptidoglycan layer, leading to rapid bacterial death by lysis. This activity is highly specific and independent of the metabolic activity of the cell or its antibiotic resistance patterns. However, systemic application of PGHs is limited by their often low activity in vivo and by an insufficient serum circulation half-life. To address this problem, we aimed to extend the half-life of PGHs selected for high activity against S. aureus in human serum. Half-life extension and increased serum circulation were achieved through fusion of PGHs to an albumin-binding domain (ABD), resulting in high-affinity recruitment of human serum albumin and formation of large protein complexes. Importantly, the ABD-fused PGHs maintained high killing activity against multiple drug-resistant S. aureus strains, as determined by ex vivo testing in human blood. The top candidate, termed ABD_M23, was tested in vivo to treat S. aureus-induced murine bacteremia. Our findings demonstrate a significantly higher efficacy of ABD_M23 than of the parental M23 enzyme. We conclude that fusion with ABD represents a powerful approach for half-life extension of PGHs, expanding the therapeutic potential of these enzybiotics for treatment of multidrug-resistant bacterial infections., mBio, 11 (5), ISSN:2150-7511, ISSN:2161-2129