Objective: To systematically review and summarize the available literature regarding the effectiveness and safety of biologics in the treatment of juvenile-onset systemic lupus erythematosus. Methods: PubMed was systematically searched for relevant literature (2012–2017 inclusive) using the following criteria: (1) patients diagnosed with juvenile-onset systemic lupus erythematosus (≤18 years at diagnosis); (2) treatment with any biological agent; and (3) outcome measures assessing effectiveness and safety. Systematic literature reviews, meta-analyses, randomized controlled trials, cohort studies, case control studies, cross sectional surveys and case-series with ≥3 patients were included. Independent extraction of articles by two authors using predefined criteria was performed. The quality of each study was assessed using CASP tools and Oxford CEBM Levels of Evidence. Results: Nine articles met inclusion criteria: six cohort studies, two case series and one pilot study, totalling 230 patients. All but one article reported the effects of rituximab, the other those of belimumab. Overall, patients had active disease refractory to standard of care regimens using corticosteroids and immunosuppressants. Available evidence for rituximab demonstrated improvements in disease activity, complement levels and anti-dsDNA titres accompanying a steroid-sparing effect. Conclusion: Rituximab can be considered an effective treatment in juvenile-onset systemic lupus erythematosus patients with severe disease manifestations and/or refractory disease. Based on current evidence, use of belimumab in juvenile-onset systemic lupus erythematosus patients cannot be recommended. The long-term safety of these biological agents remains uncertain. Further prospective studies, ideally robust randomized controlled trials, are urgently needed to obtain more accurate data on the effectiveness and long-term safety of rituximab, belimumab and other biologics in juvenile-onset systemic lupus erythematosus.

During the last decades the number of biologics increased dramatically and several biopharmaceutical drugs such as peptides, therapeutic proteins, hormones, enzymes, vaccines, monoclonal antibodies and antibody-drug conjugates conquered the market. Moreover, administration and local delivery of growth factors has gained substantial importance in the field of tissue engineering. Despite progress that has been made over the last decades formulation and delivery of therapeutic proteins is still a challenge. Thus, we worked on formulation and delivery strategies of therapeutic proteins to improve their biological performance. Phase I of this work deals with protein stability with the main focus on a liquid protein formulation of the dimeric fusion protein PR-15, a lesion specific platelet adhesion inhibitor. In order to develop an adequate formulation ensuring the stability and bioactivity of PR-15 during storage at 4 °C, a pH screening, a forced degradation and a Design of Experiments (DoE) was performed. First the stability and bioactivity of PR-15 in 50 mM histidine buffer in relation to pH was evaluated in a short-term storage stability study at 25 °C and 40 °C for 4 and 8 weeks using different analytical methods. Additionally, potential degradation pathways of PR-15 were investigated under stressed conditions such as heat treatment, acidic or basic pH, freeze-thaw cycles, light exposure, induced oxidation and induced deamidation during the forced degradation study. Moreover, we were able to identify the main degradation product of PR-15 by performing LC/ESI-MS analysis. Further optimization of the injectable PR 15 formulation concerning pH, the choice of buffer and the addition of excipients was studied in the following DoE and finally an optimal PR-15 formulation was found. The growth factors BMP-2, IGF-I and TGF-β3 were selected for the differentiation of stem cells for tissue engineering of cartilage and bone in order to prepare multifunctionalized osteochondral implants for the regeneration of cartilage defects. Silk fibroin (SF) was chosen as biomaterial because of its biocompatibility, mechanical properties and its opportunity for biofunctionalization. Ideal geometry of SF scaffolds with optimal porosity was found in order to generate both tissues on one scaffold. The growth factors BMP-2 and IGF-I were modified to allow spatially restricted covalent immobilization on the generated porous SF scaffolds. In order to perform site-directed covalent coupling by the usage of click chemistry on two opposite sides of the scaffold, we genetically engineered BMP-2 (not shown in this work; performed by Barbara Tabisz) and IGF-I for the introduction of alkyne or azide bearing artificial amino acids. TGF β3 was immobilized to beads through common EDC/NHS chemistry requiring no modification and distributed in the pores of the entire scaffold. For this reason protein modification, protein engineering, protein immobilization and bioconjugation are investigated in phase II. Beside the synthesis the focus was on the characterization of such modified proteins and its conjugates. The field of protein engineering offers a wide range of possibilities to modify existing proteins or to design new proteins with prolonged serum half-life, increased conformational stability or improved release rates according to their clinical use. Site-directed click chemistry and non-site-directed EDC/NHS chemistry were used for bioconjugation and protein immobilization with the aim to underline the preferences of site-directed coupling. We chose three strategies for the incorporation of alkyne or azide functionality for the performance of click reaction into the protein of interest: diazonium coupling reaction, PEGylation and genetic engineering. Azido groups were successfully introduced into SF by implementation of diazonium coupling and alkyne, amino or acid functionality was incorporated into FGF-2 as model protein by means of thiol PEGylation. The proper folding of FGF-2 after PEGylation was assessed by fluorescence spectroscopy, WST-1 proliferation assay ensured moderate bioactivity and the purity of PEGylated FGF-2 samples was monitored with RP-HPLC. Moreover, the modification of native FGF-2 with 10 kDa PEG chains resulted in enhanced thermal stability. Additionally, we genetically engineered one IGF-I mutant by incorporating the unnatural amino acid propargyl-L-lysine (plk) at position 65 into the IGF-I amino acid sequence and were able to express hardly verifiable amounts of plk-IGF-I. Consequently, plk-IGF-I expression has to be further optimized in future studies in order to generate plk-IGF-I with higher yields. Bioconjugation of PEGylated FGF-2 with functionalized silk was performed in solution and was successful for click as well as EDC/NHS chemistry. However, substantial amounts of unreacted PEG-FGF-2 were adsorbed to SF and could not be removed from the reaction mixture making it impossible to expose the advantages of click chemistry in relation to EDC/NHS chemistry. The immobilization of PEG-FGF-2 to microspheres was a trial to increase product yield and to remove unreacted PEG-FGF-2 from reaction mixture. Bound PEG-FGF-2 was visualized by fluorescence imaging or flow cytometry and bioactivity was assessed by analysis of the proliferation of NIH 3T3 cells. However, immobilization on beads raised the same issue as in solution: adsorption caused by electrostatic interactions of positively charged FGF-2 and negatively charged SF or beads. Finally, we were not able to prove superiority of site-directed click chemistry over non-site-directed EDC/NHS. The skills and knowledge in protein immobilization as well as protein characterization acquired during phase II helped us in phase III to engineer cartilage tissue in biofunctionalized SF scaffolds. The approach of covalent immobilization of the required growth factors is relevant because of their short in vivo half-lives and aimed at controlling their bioavailability. So TGF-β3 was covalently coupled by means of EDC/NHS chemistry to biocompatible and biostable PMMA beads. Herein, we directly compared bioactivity of covalently coupled and adsorbed TGF-β3. During the so-called luciferase assay bioactivity of covalent coupled as well as adsorbed TGF-β3 on PMMA beads was ensured. In order to investigate the real influence of EDC/NHS chemistry on TGF-β3’s bioactivity, the amount of immobilized TGF-β3 on PMMA beads was determined. Therefore, an ELISA method was established. The assessment of total amount of TGF-β3 immobilized on the PMMA beads allowed as to calculate coupling efficiency. A significantly higher coupling efficiency was determined for the coupling of TGF-β3 via EDC/NHS chemistry compared to the reaction without coupling reagents indicating a small amount of adsorbed TGF-β3. These results provide opportunity to determine the consequence of coupling by means of EDC/NHS chemistry for TGF β3 bioactivity. At first sight, no statistically significant difference between covalent immobilized and adsorbed TGF-β3 was observed regarding relative luciferase activities. But during comparison of total and active amount of TGF-β3 on PMMA beads detected by ELISA or luciferase assay, respectively, a decrease of TGF-β3’s bioactivity became apparent. Nevertheless, immobilized TGF β3 was further investigated in combination with SF scaffolds in order to drive BMSCs to the chondrogenic lineage. According to the results obtained through histological and immunohistochemical studies, biochemical assays as well as qRT-PCR of gene expression from BMSCs after 21 days in culture immobilized TGF-β3 was able to engineer cartilage tissue. These findings support the thesis that local presentation of TGF β3 is superior towards exogenous TGF β3 for the development of hyaline cartilage. Furthermore, we conclude that covalent immobilized TGF β3 is not only superior towards exogenously supplemented TGF-β3 but also superior towards adsorbed TGF-β3 for articular hyaline cartilage tissue engineering. Diffusion processes were inhibited through covalent immobilization of TGF-β3 to PMMA beads and thereby a stable and consistent TGF-β3 concentration was maintained in the target area. With the knowledge acquired during phase II and III as well as during the studies of Barbara Tabisz concerning the expression and purification of plk-BMP-2 we made considerable progress towards the formation of multifunctionalized osteochondral implants for the regeneration of cartilage defects. However, further studies are required for the translation of these insights into the development of multifunctionalized osteochondral SF scaffolds. / In den letzten Jahrzehnten stieg die Zahl der Biologika dramatisch an und mehrere biopharmazeutische Arzneimittel wie Peptide, therapeutische Proteine, Hormone, Enzyme, Impfstoffe, monoklonale Antikörper und Antikörper-Wirkstoff-Konjugate eroberten den Markt. Darüber hinaus hat die Applikation und lokale Verabreichung von Wachstumsfaktoren im Bereich des Tissue Engineerings eine wesentliche Bedeutung erlangt. Trotz der in den letzten Jahrzehnten erzielten Fortschritte ist die Formulierung und Verabreichung therapeutischer Proteine noch immer eine Herausforderung. Daher haben wir uns in dieser Arbeit mit der Formulierung und Verabreichung therapeutischer Proteine beschäftigt und Strategien entwickelt, um deren biologische Wirkung zu verbessern. In Phase I dieser Arbeit konzentrieren wir uns auf die Stabilität des dimeren Fusionsproteins PR 15, einem Inhibitor der Adhäsion von Plättchen an arterielle Gefäßläsionen. Um eine geeignete flüssige Formulierung zu entwickeln, welche die Stabilität und Bioaktivität von PR-15 während der Lagerung bei 4 °C sicherstellt, wurde ein pH Screening, eine Forced Degradation Studie und ein Design of Experiments (DoE) durchgeführt. Zuerst wurde die Stabilität und Bioaktivität von PR-15 bei verschiedenen pH Werten in 50 mM Histidinpuffer in einer Kurzzeitstabilitätsstudie bei 25 °C und 40 °C nach 4 und 8 Wochen mit Hilfe verschiedener analytischer Methoden beobachtet. Des Weiteren wurden mögliche Abbauwege von PR-15 unter Stressbedingungen wie erhöhter Temperatur, saurem oder basischem pH-Wert, Einfrier-Auftau-Zyklen, Lichteinwirkung, induzierter Oxidation sowie induzierter Deamidierung während der Forced Degradation Studie untersucht. Darüber hinaus konnten wir das Hauptabbauprodukt von PR-15 durch LC/ESI-MS Analysen identifizieren. Im folgenden DoE wurde die injizierbare PR-15 Formulierung weiter optimiert und bezüglich pH, der Wahl des Puffers sowie der Zugabe von Hilfsstoffen analysiert, bis letztendlich eine optimale PR 15-Formulierung gefunden wurde. Die Wachstumsfaktoren BMP-2, IGF-I und TGF-β3 wurden zur Differenzierung von Stammzellen für das Tissue Engineering von Knochen und Knorpel ausgewählt, um multifunktionalisierte osteochondrale Implantate zur Regeneration von Knorpeldefekten herzustellen. Seidenfibroin (SF) wurde aufgrund seiner Biokompatibilität, seiner mechanischen Eigenschaften und seiner Möglichkeiten zur Biofunktionalisierung als Biomaterial gewählt. Zuerst wurden SF-Scaffolds mit idealer Geometrie und optimaler Porosität erzeugt, um sowohl Knochen also auch Knorpel auf einem Scaffold herzustellen. Um eine räumlich begrenzte kovalente Immobilisierung der Wachstumsfaktoren BMP-2 und IGF-I auf den porösen SF-Scaffolds zu ermöglichen, wurden diese mit unnatürlichen Aminosäuren genetisch modifiziert. Das Einführen von Alkin- bzw. Azidresten in die Aminosäuresequenz von BMP-2 (in dieser Arbeit nicht gezeigt; von Barbara Tabisz durchgeführt) und IGF-I erlaubt unter Verwendung der Click-Chemie eine ortsgerichtete kovalente Kopplung der Wachstumsfaktoren an zwei gegenüberliegenden Seiten der Scaffolds. TGF-β3 wurde durch gewöhnliche EDC/NHS-Chemie, welche keine Modifikation erforderte, kovalent an Mikrosphären immobilisiert und in den Poren des gesamten SF-Scaffolds verteilt. Daher beschäftigen wir uns in Phase II mit der Modifikation von Proteinen, dem Protein Engineering, der Immobilisation von Proteinen und mit Biokonjugation. Neben der Synthese lag der Fokus auf der Charakterisierung modifizierter Proteine und deren Konjugaten. Das Gebiet des Protein Engineerings bietet eine Vielzahl von Möglichkeiten, bestehende Proteine zu modifizieren oder neue Proteine mit verlängerter Serumhalbwertszeit, erhöhter konformativer Stabilität oder verbesserten Freisetzungsraten entsprechend der klinischen Anwendung zu entwickeln. Die ortsspezifische Click-Chemie und die nicht-ortsspezifische EDC/NHS-Chemie wurden für die Biokonjugation und die Immobilisierung von Proteinen verwendet mit dem Ziel, die Vorzüge der ortsgerichteten Kopplung hervorzuheben. Für den Einbau der für die Durchführung der Click-Reaktion erforderlichen Alkin- bzw. Azidfunktionalität in das betreffende Protein wurden drei Strategien ausgewählt: die Azokupplung, die PEGylierung und die gentechnische Modifizierung. Azidgruppen wurden mittels Azokupplung erfolgreich in SF eingebaut und die Alkin-, Amino- oder Säurefunktionalität wurde mittels PEGylierung der Cysteine in das Modellprotein FGF-2 integriert. Die korrekte Faltung von FGF-2 nach erfolgreicher PEGylierung wurde durch Fluoreszenzspektroskopie bestätigt, im WST-1 Proliferationsassay wurde eine angemessene Bioaktivität festgestellt und die Reinheit von PEGylierten FGF-2 wurde mittels RP-HPLC analysiert. Darüber hinaus führte die Modifikation von nativem FGF-2 mit 10 kDa PEG-Ketten zu einer erhöhten thermischen Stabilität. Des Weiteren wurde ein IGF-I-Mutant gentechnisch hergestellt, indem die unnatürliche Aminosäure Propargyl-L-Lysin (Plk) an Position 65 in die IGF-I-Sequenz eingebaut wurde. Da letztendlich lediglich kaum nachweisbare Mengen an Plk-IGF-I exprimiert werden konnten, muss die Plk-IGF-I-Expression in anschließenden Studien weiter optimiert werden, um Plk-IGF-I mit höheren Ausbeuten erzeugen zu können. Die Biokonjugation von PEGyliertem FGF-2 und funktionalisierter Seide wurde sowohl mittels Click- als auch mittels EDC/NHS-Chemie erfolgreich durchgeführt. Allerdings wurden erhebliche Mengen PEG-FGF-2 lediglich an SF adsorbiert und nicht kovalent gekoppelt und konnten schlussendlich nicht aus dem Reaktionsgemisch entfernt werden. Die anschließende Immobilisierung von PEG-FGF-2 an Mikrosphären, war ein Versuch die Ausbeute der Reaktion zu erhöhen und adsorbiertes PEG-FGF-2 leichter zu entfernen. Immobilisiertes PEG-FGF-2 wurde mittels Fluoreszenzmikroskopie und/oder Durchflusszytometrie nachgewiesen und die Bioaktivität wurde durch die Analyse der Proliferation von NIH-3T3-Zellen ermittelt. Die Immobilisierung auf Mikrosphären führte jedoch zu demselben Problem wie in Lösung: Adsorption von positiv geladenem FGF-2 an negativ geladenes SF bzw. negativ geladenen Mikrosphären durch elektrostatische Wechselwirkungen. Schließlich waren wir nicht in der Lage, die Überlegenheit der ortsgerichteten Click-Chemie gegenüber der nicht-ortsgerichteten EDC/ NHS-Chemie zu beweisen. Die während Phase II erworbenen Fähigkeiten und Kenntnisse im Bereich der Immobilisierung und Charakterisierung von Proteinen halfen uns in Phase III Knorpelgewebe in biofunktionalisierten SF-Scaffolds zu erzeugen. Der Ansatz der kovalenten Immobilisierung, der für das Tissue Engineering von Knorpel erforderlichen Wachstumsfaktoren, ist aufgrund ihrer kurzen in vivo Halbwertszeiten von Bedeutung und zielt darauf ab, ihre Bioverfügbarkeit zu kontrollieren. So wurde TGF-β3 mittels EDC/NHS-Chemie kovalent an biokompatible und biostabile PMMA-Mikrosphären gekoppelt. Mit Hilfe des sogenannten Luciferase-Assays wurden die Bioaktivitäten von kovalent gekoppeltem sowie von adsorbiertem TGF-β3 auf PMMA-Mikrosphären ermittelt. Um die Kopplungseffizienz zu berechnen und den tatsächlichen Einfluss der EDC/NHS-Chemie auf die Bioaktivität von TGF-β3 zu untersuchen, wurde die Menge an immobilisiertem TGF-β3 auf PMMA-Mikrosphären mittels ELISA bestimmt. Für die Kopplung von TGF-β3 mittels EDC/NHS-Chemie wurde eine signifikant höhere Kopplungseffizienz im Vergleich zu der Reaktion ohne Kopplungsreagenzien, welche eine geringe Menge an adsorbiertem TGF-β3 zeigte, bestimmt. Bei alleiniger Betrachtung der Ergebnisse des Luciferase-Assays, bei welchem kein statistisch signifikanter Unterschied zwischen kovalent immobilisiertem und adsorbiertem TGF-β3 bezüglich der relativen Luciferase-Aktivität beobachtet wurde, scheint es als hätte die EDC/NHS-Kopplung keinen Einfluss auf die Bioaktivität von TGF β3. Beim Vergleich der mittels ELISA bestimmten TGF β3 Gesamtmenge und der mittels Luciferase-Assay bestimmten Menge an aktivem TGF-β3 auf den PMMA-Mikrosphären, wurde jedoch ein Verlust der Bioaktivität von TGF-β3 durch die EDC/NHS-Kopplung deutlich. Ungeachtet dessen, wurde immobilisiertes TGF-β3 genutzt, um Knorpelgewebe in SF-Scaffolds aus BMSCs zu generieren. Nach den Ergebnissen der histologischen und immunhistochemischen Untersuchungen, der biochemischen Assays sowie der qRT-PCR der Genexpression von BMSCs nach 21 Tagen in Kultur, gelang es uns unter Verwendung von immobilisiertem TGF-β3 Knorpelgewebe aufzubauen. Diese Ergebnisse unterstützen die These, dass die lokale Präsentation von TGF-β3 gegenüber exogen zugegebenem TGF-β3 für die Entwicklung von hyalinem Knorpel überlegen ist. Außerdem schließen wir daraus, dass kovalent immobilisiertes TGF-β3 nicht nur gegenüber exogen zugegebenem TGF-β3 für die Entwicklung von hyalinem Knorpelgewebe überlegen ist, sondern auch gegenüber adsorbiertem TGF-β3. Diffusionsprozesse konnten durch kovalente Immobilisierung von TGF-β3 an PMMA-Mikrosphären verhindert werden und damit eine stabile und gleichmäßige TGF β3-Konzentration am Wirkort aufrechterhalten werden. Mit den in Phase II und III gewonnenen Erkenntnissen und den Untersuchungen von Barbara Tabisz zur Expression und Aufreinigung von plk-BMP-2 haben wir erhebliche Fortschritte bei der Entwicklung multifunktionaler osteochondraler Implantate zur Regeneration von Knorpeldefekten gemacht. Für die Umsetzung dieser Erkenntnisse zur Herstellung multifunktionaler osteochondraler SF-Scaffolds sind jedoch weitere Studien erforderlich.

Dans la prise en charge de la polyarthrite rhumatoïde (PR), l’identification de biomarqueurs associés à un diagnostic, un pronostic mais surtout à une bonne réponse thérapeutique est un des enjeux de la prochaine décennie, d’autant que de nombreux outils sont maintenant disponibles grâce à l’étude du génome, du transcriptome et du protéome. Au cours de mon travail de thèse, je me suis intéressée à l’identification de différentes classes de biomarqueurs prédictifs de la réponse au traitement par biothérapies.Tout d’abord par une approche protéomique, j’ai identifié une combinaison de biomarqueurs protéiques sériques, MCP-1 et EGF, permettant à l’initiation du traitement par étanercept (anti-TNFα), puis par rituximab (anti-CD20), de prédire la réponse clinique à 3 mois. En parallèle, toujours chez des patients PR traités par rituximab, j’ai recherché un biomarqueur cellulaire par cytométrie de flux. J’ai ainsi pu montrer que la densité de CCR5 à la surface des lymphocytes T CD4+ circulants est faible chez les patients PR . Celle-ci est corrélée positivement à l’activité de la maladie et négativement au taux intracellulaire d’ARNm de CCR5. Trois mois après l’initiation du traitement, la densité de CCR5 à la surface des lymphocytes T CD4+ circulants augmente proportionnellement à la diminution de l’activité de la maladie. J’ai également étudié l’intérêt de biomarqueurs génétiques. Tout d’abord, en analysant le profil d’expression de 723 micro(mi)ARNs dans le sang de patients PR, j’ai montré que l’expression de 37 miARNs était dérégulée par rapport à des donneurs sains. Par ailleurs, j’ai montré que le niveau d’expression de miR-125b dans le sang des patients PR à l’initiation du traitement par rituximab permet de prédire la réponse à 3 mois. J’ai enfin exploré les polymorphismes de gènes de cytokines et de récepteurs aux cytokines afin de prédire la réponse au traitement par rituximab à 6 mois chez 63 patients atteints de PR active. Douze polymorphismes de nucléotides uniques (SNPs) ont été génotypés et ont permis d’identifier 2 SNPs du facteur de croissance transformant beta 1 (TGFb1) codon 25 et codon 10 prédictifs d’une bonne réponse au rituximab.Je me suis aussi intéressée à la thérapie génique qui est un outil thérapeutique pour délivrer des agents anti-TNF dans la PR. En effet le transfert de gène permet à l’organisme de synthétiser in situ la protéine médicament et d’éviter des administrations répétées comme c’est le cas avec les biothérapies. Le défi de demain réside plus dans le développement de vecteurs fiables et efficients. Dans un premier travail réalisé dans l’équipe de recherche du Pr Hirsch à Pittsburgh (E.U), j’ai évalué in vitro l’intérêt d’un virus adéno-associé recombinant de sérotype 2 (rAAV2) contenant un ARN double brin pour le transfert de gène dans les synoviocytes humains. Dans un contexte inflammatoire, celui-ci s’est révélé efficace lorsqu’associé à un inhibiteur du protéasome, le zLLL. Lors d’un deuxième stage effectué au sein du laboratoire du Pr Jorgensen (Montpellier), j’ai évalué le potentiel thérapeutique d’un rAAV de sérotype 5 (rAAV5) exprimant un petit ARN interférant ciblant le TNF-α dans un modèle expérimental d’arthrite. L’inhibition du TNF-α a été validée in vitro sur des macrophages murins, puis in vivo dans le modèle murin d’arthrite au collagène, après injection dans les articulations arthritiques. Depuis la fin des années 90, l’avènement des biothérapies a permis de bouleverser l’évolution de la PR. Les biomarqueurs, en particulier ceux permettant le suivi des traitements, sont des outils de choix à développer pour notre pratique clinique courante afin de choisir d’emblée le traitement le plus efficace et le mieux toléré pour chaque patient. D’autres thérapies innovantes, telles que la thérapie génique, s’appuient sur les succès des biothérapies pour permettre d’autres avancées en choisissant d’emblée les cibles les plus pertinentes. / Rheumatoid arthritis (RA) is a chronic inflammatory disease. The effective use of biologics, which block key molecules involved in the pathogenesis of RA, has dramatically improved the treatment of this chronic disease over recent years. However, for at least one-third of patients with RA biologic treatments will produce an inadequate response. Therefore, the use of predictive biomarkers of response to identify individuals who are likely to respond to specific treatments will provide benefit. An individualised approach will allow patients to receive effective treatment without unnecessary exposure to potentially toxic side effects.During my thesis, I identified predictive biomarkers in RA patients treated with biologics by using proteomics, genetics and cellular biomarkers.Genen therapy could be used to optimise drug administration by avoiding repetitive administration. I validated the interest of a recombinant adeno-associated virus in RA synovial fibroblasts.

Introduction The fundamental concept for modern inflammatory bowel disease (IBD) treatment algorithm is an early induction of mucosal healing and its maintenance. Biological therapies are becoming mainstays of IBD therapy. It is however still unclear if there is a difference between Crohn’s disease (CD) and Ulcerative Colitis (UC) in time from diagnosis to stable maintenance treatment when biological treatment is introduced. Aim To investigate the comparative efficacy of biological agents in CD and UC by studying the time course when biological treatment is introduced. Methods Retrospective study of patients suffering from IBD at Falu Hospital, receiving a new start with biologics between 2015-01-01 and 2016-12-31. Remission rate after 3 months of induction therapy was analyzed. Subsequently, when 6 months passed without active disease, it was considered a stable remission. Results Through database extraction 58 patients were identified. A total of 52 % patients fulfilled the criteria for remission. Of patients with CD 49 % got in remission, whereof 33 % had treatment with infliximab, 48 % adalimumab and 19 % vedolizumab. Of patients with UC 60 % got in remission, whereof 33 % had treatment with infliximab and 67 % with adalimumab. Log rank test showed no significant difference in efficacy of biologics in patients suffering from CD or UC in time from diagnosis respective after initiation with biologics to stable maintenance treatment. ConclusionsIn this study patients with CD and UC responded equally to biological treatment.

Les bio-médicaments sont aujourd’hui confrontés à un manque d’adhésion thérapeutique due principalement à leur voie d’administration parentérale. Pour pallier à cela, un système de double encapsulation composé d’une gélule gastro-résistante renfermant un système de nanoparticules (NPs) polymériques à base de PLGA a été développé apportant protection et absorption pour in fine une administration par voie orale. L’objectif de ce travail a été de mettre au point le système de NPs renfermant de l’insuline, en jouant sur la charge de surface afin d’augmenter la biodisponibilité. Ainsi, nous avons montré un meilleur passage intestinal des nanoparticules grâce à l’apport d’une charge de surface négative confirmé par une diminution de la glycémie in vivo. Ensuite les NPs ont été recouvertes d’acide hyaluroniqueafin de réduire la perte intestinale d’insuline. Une fois validé avec l’insuline, le système nanoparticulaire a été transposé à un second bio médicament d’intérêt, l’héparine. Ainsi, nous avons obtenu des paramètres physico-chimiques semblables à ceux obtenus avec l’insuline prouvant le transfert de notre technologie. / Biologics are administrated by parenteral route due to their degradation in the gastrointestinal tract. This administration mode leads to lowadherence, showed by the low adherence for chronic diseases treatment. In order to increase adherence by reducing pain and adverse effects associated with treatment we developed a double encapsulation system to protect and increase biologic absorption for a future oral administration. Our system consists in encapsulated biologics inside PLGA nanoparticles (NPs), themselves encapsulated inside a gastroresistant vehicle. The aim of this work was to improve the system efficiency with insulin by NPs charges modification and intestinal release reduction and to transfer the technology to another molecule: the heparin. Negative charges improved the intestinal nanoparticle crossing as shown by the insulinopenic rat model with a decrease of glycaemia. To stabilize the NPs system in intestinal medium, a hyaluronic acid coating was tested and validated in vitro. When transferred to another biologics, we showed similar NPs chacacteristics with heparin than with insulin, revealing the transposition of our technic.

In the field of biologics production, productivity and stability of the transfected gene of interest are two very important attributes that dictate if a production process is viable. To further understand and improve these two traits, we would need to further our understanding of the factors affecting them. These would include integration site of the gene, gene copy number, cell phenotypic variation and cell environment. As these factors play different parts in the development process, they lead to variable productivity and stability of the transfected gene between clones, the well-known phenomenon of “clonal variation”. A study of this phenomenon and how the various factors contribute to it will thus shed light on strategies to improve productivity and stability in the production cell line. Of the four factors, the site of gene integration appears to be one of the most important. Hence, it is proposed that work is done on studying how different integration sites affect the productivity and stability of transfected genes in the development process. For the study to be more industrially relevant, it is proposed that the Chinese Hamster Ovary dhfr-deficient cell line, CHO-DG44, is used as the model system. / Singapore-MIT Alliance (SMA)

Current anti-VEGF biologics, such as bevacizumab and VEGF trap, have been successfully used as therapeutic agents for cancer and age-related macular degeneration (AMD). Since these strategies target VEGF systemically, their toxicity profile, including proteinuria and thromboembolic events, and need for frequent eye injections in AMD treatment, prevail. Therefore, the aim of this PhD thesis was to generate novel anti-VEGF biologics that inhibit VEGF activity specifically at the desired target site. Two classes of biologics were engineered that simultaneously bind VEGF and either: 1) the extracellular matrix (ECM) or 2) target-site specific antigens. The first subgroup, “sticky-traps”, is composed of VEGF trap linked to a sequence of hydrophobic amino acids, with affinity for heparin sulfate proteoglycans of the ECM. The second subgroup, “lassos”, is composed of a C-terminus positioned form of VEGF trap linked to single-chain variable domain antibodies specific for either HER2 (HER2/V lasso) or fibronectin extra domain B (EDB; EDB/V lasso), expressed on breast cancer cell surfaces or in the vascular bed of solid tumours, respectively. ii Using a novel transgenic method, piggyBac transposons, biologics were expressed in transgenic cancer cell lines in a doxycycline inducible manner. They were shown to inhibit VEGF activity and also retain the native function of their constituent domains. Specifically, the sticky-traps adhered to the ECM and the HER2/V lasso inhibited the proliferation of HER2 positive cancer cell lines. Sticky-traps as well as lassos were able to inhibit or delay tumour growth of A-673, Pc-3, SKOV-3 and HT-29 xenografts. In contrast to soluble VEGF trap, sticky-traps were retained at the tumour site and were undetectable in the circulation. Moreover, sticky-traps, in contrast to VEGF trap, did not delay wound healing and regression of trachea blood vessels. Furthermore, transgenic studies indicated that HER2/V lasso is more effective compared to anti-HER2 Ab and VEGF trap used alone or in combination. These novel classes of antiangiogenic molecules could be advantageous in a clinical setting. Using the principles established in my PhD thesis work, similar dual function biologics can be designed for inhibition of other molecules with disease relevance.

Current anti-VEGF biologics, such as bevacizumab and VEGF trap, have been successfully used as therapeutic agents for cancer and age-related macular degeneration (AMD). Since these strategies target VEGF systemically, their toxicity profile, including proteinuria and thromboembolic events, and need for frequent eye injections in AMD treatment, prevail. Therefore, the aim of this PhD thesis was to generate novel anti-VEGF biologics that inhibit VEGF activity specifically at the desired target site. Two classes of biologics were engineered that simultaneously bind VEGF and either: 1) the extracellular matrix (ECM) or 2) target-site specific antigens. The first subgroup, “sticky-traps”, is composed of VEGF trap linked to a sequence of hydrophobic amino acids, with affinity for heparin sulfate proteoglycans of the ECM. The second subgroup, “lassos”, is composed of a C-terminus positioned form of VEGF trap linked to single-chain variable domain antibodies specific for either HER2 (HER2/V lasso) or fibronectin extra domain B (EDB; EDB/V lasso), expressed on breast cancer cell surfaces or in the vascular bed of solid tumours, respectively. ii Using a novel transgenic method, piggyBac transposons, biologics were expressed in transgenic cancer cell lines in a doxycycline inducible manner. They were shown to inhibit VEGF activity and also retain the native function of their constituent domains. Specifically, the sticky-traps adhered to the ECM and the HER2/V lasso inhibited the proliferation of HER2 positive cancer cell lines. Sticky-traps as well as lassos were able to inhibit or delay tumour growth of A-673, Pc-3, SKOV-3 and HT-29 xenografts. In contrast to soluble VEGF trap, sticky-traps were retained at the tumour site and were undetectable in the circulation. Moreover, sticky-traps, in contrast to VEGF trap, did not delay wound healing and regression of trachea blood vessels. Furthermore, transgenic studies indicated that HER2/V lasso is more effective compared to anti-HER2 Ab and VEGF trap used alone or in combination. These novel classes of antiangiogenic molecules could be advantageous in a clinical setting. Using the principles established in my PhD thesis work, similar dual function biologics can be designed for inhibition of other molecules with disease relevance.

The emerging class of biologic drugs, including proteins, peptides, and gene therapies, are widely administered by injection, despite potential systemic side effects. Rational design of targeted carriers that can be delivered non-invasively, with reduced side effects, is essential for the success of these therapies, as well as for the improvement of patient compliance and quality of life. One potential approach is to take advantage of specific physiological cues, such as enzymes, which would trigger drug release from a drug carrier. Enzymatic cleavage is highly specific and could be tailored for certain diseased tissues where specific enzymes are up regulated. Enzymatically-degradable hydrogels, which incorporate an enzyme- cleavable peptide into the network structure, have been extensively reported for releasing drugs for tissue engineering applications. These studies showed that a rapid response and corresponding drug release occurs upon enzyme exposure, whereas minimal degradation occurs without enzyme. Recently, Michael addition reactions have been developed for the synthesis of such enzymatically-degradable hydrogels. Michael addition reactions occur under mild physiological conditions, making them ideally suited for polymerizing hydrogels with encapsulated biologic drugs without affecting its bioactivity, as in traditional polymerization and particle synthesis. The focus of my research was to create enzymatically-degradable hydrogel microparticles, using Michael addition chemistry, to evaluate for use as an inhalable, disease-responsive delivery system for biologic drugs and nanoparticles. In this dissertation, I utilize bioconjugation and Michael addition chemistries in the design and development of enzymatically-degradable hydrogels, which may be tailored to a multitude of disease applications. I then introduce a new method of hydrogel microparticle, or microgel, synthesis known as the Michael Addition During Emulsion (MADE) method. These microgel carriers were evaluated in vitro, and found to exhibit triggered release of encapsulated biologic drugs in response to enzyme, no significant cytotoxic effects, and the ability the avoid rapid clearance by macrophages. Lastly, in vivo studies in mice were conducted, and microgels were found to exhibit successful delivery to the deep lung, as well as prolonged pulmonary retention after intratracheal aerosol delivery. In conclusion, a new class of enzymatically-degradable microgels were successfully developed and characterized as a versatile and promising new system for pulmonary, disease-responsive delivery of biologic drugs. / text

Après les médicaments génériques, copies de médicaments d’origine chimique, c’est désormais le marché des médicaments biologiques qui s’ouvre à la concurrence de « copies » avec les bio-similaires. De nombreuses incertitudes demeurent concernant l’essor de ce marché aujourd’hui émergent et la capacité des bio-similaires à entrer en compétition avec les autres bio-médicaments innovants.Comme dans le cas des génériques, l’objectif avec les bio-similaires est d’assurer un accès optimal et équitable des patients à des traitements aussi efficaces et sûrs d’utilisation que leurs médicaments de référence, mais à des coûts sensiblement moindres. Suite à l’analyse des premières expériences avec ces produits dans l’Union européenne et au Japon, des limites des politiques mises en place pour encourager leur utilisation et des écueils au développement de ce marché, nous proposons des recommandations pour la construction d’une politique des bio-similaires ambitieuse et cohérente en France. / After generics, which are copies of chemical medicines, "copies" of already patented biologics (biosimilars) are entering the biologics market and competing with their originators. Many uncertainties remain regarding the development of this newly emerging market, and the ability of biosimilars to compete with other innovative biologics. As in the case of generics, the objective with biosimilars is to ensure optimal and equitable patient access to treatment as effective and safe to use as their originators, but at significantly lower costs. Following the analysis of the first experiences with these products in the European Union and Japan, we have noted the limits of existing policies to encourage their use as well as noting pitfalls in the development of this market. To remedy these problems, we make recommendations for the construction of an ambitious and coherent policy for biosimilars in France.