Utilistions d'anticorps monoclonaux est une partie prometteuse de la thérapie du cancer. À ce jour, il existe plus de 30 anticorps monoclonaux approuvés pour la thérapie contre le cancer. Plus de 350 anticorps se situent également dans différentes phases du développement clinique. La mésothéline est l'une des cibles les plus prometteuses pour l'immunothérapie. La mésothéline est présente à des niveaux relativement faibles dans les cellules mésothéliales de la plèvre, du péritonéum et du péricarde normaux, mais est fortement exprimée dans un certain nombre de cancers différents, y compris les mésothéliomes, le cancer de l'estomac, les carcinomes à cellules squameuses, le cancer de la prostate, le cancer du pancréas, le cancer du poumon et le cancer de l'ovaire. La mésothéline est une glycoprotéine liée au glycosylphosphatidylinositol (GPI) synthétisée sous la forme d'un précurseur de 69 kDa et transformée de façon protéolytique en une forme sécrétée à 30 kDa (anciennement appelée Facteur de potentialisation des mégacaryocytes (MPF)) et une forme liée à la membrane de 40 kDa. Par ailleurs, il peut être clivé par une protéase et peut produire une forme de mésothéline soluble. Il a été déjà montré que cette forme soluble de mésothéline agit comme un ligand et neutralise les anticorps thérapeutiques ciblant la mésothéline. Par conséquent, les anticorps ne pouvaient pas atteindre les cellules cancéreuses et reste inefficaces. Dans notre travail, nous avons décidé de développer un anticorps discriminant spécifique à la forme associée à la membrane pour surmonter l'antagonisme produit par les formes solubles de mésothéline. Pour ce but, nous avons utilisé une nouvelle méthode d'immunisation de souris, que nous avons d'abord toléré la souris avec une mésothéline soluble et ensuite ré-immunisée avec des cellules exprimant la mésothéline. En utilisant la technologie de phage display, nous avons obtenu près de 150 clones de ciblant mésothéline dans 34 familles de VH-CDR3 parmi lesquelles nous avons identifié seulement 2 familles qui se lient à la mésothéline membranaire avec une affinité élevée et ne reconnaissent aucune autre forme soluble de mésothéline. Ici, nous proposons qu'ils puissent être des bons candidats pour être utilisés pour la thérapie contre le cancer de qui permet de passer à travers la barrière de mésothéline soluble. Pour démontrer leur efficacité pour une utilisation thérapeutique, nous avons construit une CAR avec le sc-Fv d'un anticorps discriminant de la forme membranaire. / Antibody based immune treatment is a promising component of cancer therapy. To date there are more than 30 approved monoclonal antibodies for cancer therapy. More than 350 antibodies are also in different phases of clinical development. Mesothelin is one of the most promising targets for immunotherapy. It is present at relatively low levels in mesothelial cells of the pleura, peritoneum and pericardium of healthy individuals, but is highly expressed in a number of different cancers, including mesotheliomas, stomach cancers, squamous cell carcinomas, as well as prostate, pancreatic, lung, and ovarian cancers. Mesothelin is a glycosylphosphatidylinositol (GPI)-linked glycoprotein synthesized as a 69 kDa precursor and proteolytically processed into a 30 kDa NH2-terminal secreted form (formerly referred to as Megakaryocyte Potentiating Factor (MPF)) and a 40 kDa membrane-bound form. Besides that it can be cleaved by a protease leading to the production of a soluble, shedded, form of mesothelin. It has already been shown that this soluble form of mesothelin acts as a ligand and neutralizes the mesothelin targeting therapeutic antibodies. Therefore antibodies could not reach cancer cells and remained inefficient. In our work we decided to develop discriminating antibodies specific to a membrane associated form so as to overcome the antagonism produced by soluble forms of mesothelin. To this aim we used a novel method of mouse immunization, in which we first tolerized the mouse with soluble mesothelin before immunization with mesothelin expressing cells. By using phage display technology we obtained nearly 150 mesothelin recognizing clones in 34 VH-CDR3 families, among which we identified only 2 families that bind membrane mesothelin with high affinity and do not recognize any other soluble form of mesothelin. Here we suggest that this Fab can be effective candidates to be used for mesothelin expressing cancer therapy being allowed to pass through the soluble mesothelin barrier. To show their efficacy for therapeutic use we constructed a CAR with the sc-Fv of a membrane-form discriminating antibody

The extracellular matrix (ECM) provides mechanical and biochemical support to tissues and cells. It is crucial for cell attachment, differentiation, and migration, as well as for ailment-associated processes such as angiogenesis, metastases and cancer development. An approach to study these phenomena is through emulation of the ECM by synthetic gels constructed of natural polymers, such as collagen and fibronectin, or simple but tunable materials such as poly(ethylene glycol) (PEG) crosslinked with short peptide sequences susceptible to digestion by metalloproteases and cell-binding domains. Our lab uses PEG gels to study cell behavior in three dimensions (3D). Although this system fosters cell attachment and crosslinking peptides mentioned, the regenerative process of the ECM has not been mimicked yet in 3D synthetic gels. In an attempt to build in this functionality to PEG-based gels, I performed phage display to identify short oligopeptides that bind either collagen or fibronectin to assess them as potential nucleation points for crosslinking elements in order to emulate the in vivo reconstitution process. A phage display is a library of random oligopeptides expressed on a M13 bacteriophage that allows identification of a phenotype and a genotype with a single screening step. This inexpensive strategy could yield a short oligopeptide with high specificity. I identified the conditions under which phage display is compatible with our targets, and I isolated and identified five peptide candidates for fibronectin binding and two for collagen. Future work includes assessing whether these candidates could facilitate the formation of cell-created crosslinking in 3D synthetic hydrogels.

Development of nanochemical tools targeting receptors in the tumor microenvironment Targeting the receptors in the tumor microenvironment is crucial for the future development of targeted therapies, precision medicine and immunotherapy of cancer. The options available now are, however, limited by the availability of specific ligands. The advances in the field strongly rely on the use of antibodies and genetic modifications of immune cells. Availability of small molecules targeting the receptors of interest would allow further development of alternative strategies as well as deepen our understanding of the underlying mechanisms of cancer development, progression and clearance. In the search for new small-molecule ligands and their use for receptor targeting, the prostate-specific membrane antigen (PSMA) and the immune receptors CD3 and CD64 were selected as model targets. The selected method - the phage display of bicyclic peptides - utilizes chemical modification of the displayed three-cysteine peptides to achieve their cyclization and formation of bicycles. The panning of a peptide library displayed on the phages and probed with PSMA revealed a reproducibly-selected amino acid sequence. Interestingly, the phage clone carrying this sequence was a specific binder of PSMA, but the synthesized peptide alone...

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Diese Arbeit beschreibt die Kombination von Phagen-Display-Biopanning und Illumina Next-Generation DNA-Sequencing (NGS) zur Identifizierung peptidbasierter Adhäsionsdomänen für Polypropylen (PP). Eine Biopanning-Runde gefolgt von NGS liefert PP-bindende Peptide, die durch Sanger-Sequenzierung nicht erkennbar sind. NGS bietet den Vorteil eines enorm umfangreichen Datensatzes, welcher tiefgreifende Sequenzanalysen erlaubt. Die selektierten Sequenzen werden als wasserbasierte Primer für PP–Metallhaftung zur Vorbehandlung von PP-Oberflächen eingesetzt und erhöhen die Haftfestigkeit um 100 % gegenüber nicht vorbehandeltem PP. / This thesis describes the combination of phage display biopanning and Illumina Next-Generation DNA-Sequencing (NGS) to identify peptide-based adhesion domains for polypropylene (PP). One round of biopanning followed by NGS yields PP-binding peptides that are undetectable by Sanger sequencing. NGS has the advantage of an extensive data set, which allows in-depth sequence analysis. The selected peptide sequences are then used as water-based primers for PP metal adhesion for the pretreatment of PP surfaces and increase the adhesion by 100% compared to non-pretreated PP.

Fagmider är ett kraftfullt verktyg som använts för att uttrycka heterologa proteiner på fagvirus i metoder som exempelvis riktad evolution via fagdisplay av proteinbinliotek. Monovalent uttryck genom fagemider lider av ett överflöd av kala fager som är svåra att rensa bort. Effektiviteten hos detta system kan förbättras genom att använda tekniker för att selektivt fånga uttryckande fag. Biotinylering och infångning med streptavidin kan vara ett användbart verktyg för sådana ändamål och enzymatisk biotinylering med AviTag™-BirA-systemet är ett lovande alternativ. En fagemidvektor skapades genom amplifiering och kloning av sekvensen av AviTag™ in i en linjäriserad pAffi1#336-vektor. Sekvenser av två affikroppar, Zher2 och Zwt, som binder till två olika mål, Her2 och trastuzumab, klonades in i denna Avi-märkta vektor. Denna sammansatta fagemidvektor transformerades därefter till tre Escherichia coli-stammar: XL1Blue, ER2738, TG1. En plasmid innehållande BirA-enzym avsett för platsspecifik biotinylering av proteinerna som bär AviTag™ samtransformerades tillsammans med fagemiden i vissa transformationer för att undersöka biotinylering in vivo. Produktionsnivåerna för var och en av dessa stammar tillsammans med deras tillväxtkurvor beräknades för att analysera om plasmidbelastningen påverkar någon av stammarnas tillväxt. Det observerades att XL1Blue kunde växa i en takt som var jämförbar med de andra stammarna och samtidigt lyckades producera tillräckligt högre titrar. Superinfektion av hjälparfager för dessa produktioner var också mycket lovande. ER2738 och TG1 var avsevärt dåliga, den förra i termer av alla aspekter och den senare saknade bra superinfektionskarraktär men uppvisade anmärkningsvärt högre tillväxt och avsevärt högre titrar. Fagen biotinylerades in vitro genom att använda BirA som producerades och renades internt och dess biotinyleringsgrad jämfördes med den biotinylerade in vivo fagen som producerades i dessa tre stammar. Dessa fager utvärderades för deras förmåga att binda till sina mål och deras grad av biotinylering. Ett märkbart mönster som observerades fag visade minskad bindning till humant serumalbumin, vilket gjorde deras målbindning svår att tolka. / Phagemids have been a convenient and powerful tool used to display heterologous proteins on phage in methods such as directed evolution by phage display of protein libraries. Monovalent display through phagemids suffers from an overabundance of bald phage which are difficult to deplete. The efficiency of this system can be improved by using techniques to selectively capture expressing phage. Biotinylation and capture using streptavidin can be a useful tool for such purposes and enzymatic biotinylation using the AviTag™-BirA system is a promising option. A phagemid vector was created by the amplification and cloning of the sequence of AviTag™ into a linearized pAffi1#336 vector. Sequences of two affibodies, Zher2 and Zwt, binding to two different targets, Her2 and human IgG, were cloned into this Avi-tagged vector. This assembled phagemid vector was subsequently transformed into three Escherichia coli strains: XL1Blue, ER2738, TG1. A plasmid containing BirA enzyme intended for site-specific biotinylation of the proteins that carry the AviTag™ was co-transformed along with the phagemid in some transformations to investigate in vivo biotinylation. The production levels of each of these strains along with their growth curves were calculated to analyse if the plasmid burden impacts either of the strains’ growth. It was observed that XL1Blue was able to grow at a pace comparable to the other strains and managed to produce sufficiently high titers. The superinfection rate of these productions was also very promising. ER2738 and TG1 were considerably poor, the former in terms of all aspects and the latter only lacking in the superinfection rate but notable displaying higher growth and considerably higher titers. The phage was biotinylated in vitro by using BirA that was produced and purified in-house and its biotinylation degree was compared against the in vivo biotinylated phage that were produced in these three strains. The phage were then evaluated for their ability to bind to their targets and their degree of biotinylation. One noticeable pattern that was observed was that the Avi- tagged phage displayed reduced binding to the Human Serum Albumin which made their target binding hard to interpret.

Under senare år har målinriktad terapi varit ett växande område inom cancerterapi som en mer målinriktad behandling än kemoterapi. Dessa behandlingar baseras främst på antikroppsbaserade läkemedel som är ganska stora och komplexa, vilket ökar den totala kostnaden för behandlingen. Därför måste man hitta en alternativ metod för både upptäckt och behandling för att hjälpa patienterna. Små affinitetsdomäner har skapats med målet att förbättra vävnadspenetrationen och samtidigt upprätthålla en hög grad av målspecificitet, vilket leder till färre biverkningar. Ett av exemplen på detta är Albumin Binding Domain-Derived Affinity Protein (ADAPT). Det har baserats på en av de albuminbindande domänerna (ABD) i streptokockproteinet G, med en storlek på 6,5 kDa. Nyligen modifierades ADAPT ytterligare för att samtidigt binda albumin och ett annat relevant målprotein av intresse, vilket tyder på en längre halveringstid i patientserum och möjliggör utveckling av nyare och terapeutiska läkemedel. I detta projekt presenteras den fjärde generationen av ADAPT-biblioteket som utformats för att ha förbättrad stabilitet. Selektioner med fagdisplay utfördes mot tre målproteiner: carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), en biomarkör för kolorektalcancer, epithelial cell adhesion molecule (EpCAM), en markör för flera gastrointestinala karcinom och trophoblast cell-surface antigen 2 (Trop2) som är överuttryckt i trippel-negativ bröstcancer. Resultaten visar bindning till CEACAM5, EpCAM och Trop2, vilket har visats med monoklonal fag-ELISA. Bindningsaffiniteten, sekundärstrukturen hos de utvalda bindarna och den bispecifika bindningen till serumalbumin återstår att utvärdera ytterligare. Projektet visar således att de ADAPTs som valts ut mot målen CEACAM5, EpCAM och Trop2 har en enorm potential för framtida kliniska tillämpningar som syftar till utveckling av diagnostik och terapi för dessa cancerbiomarkörer. / In recent years, targeted therapy has been a growing field of cancer therapy as a more targeted treatment than chemotherapy. These treatments are primarily based on antibody-based pharmaceuticals which are quite large and complex, increasing the overall cost of the treatment. Thus, an alternative method of both detection and treatment needs to be found to aid patients. Small affinity domains have been created with the goal of enhancing tissue penetration while maintaining a high level of target specificity, leading to fewer side effects. One of the examples for these is the Albumin Binding Domain-Derived Affinity Protein (ADAPT). It has been based on one of the albumin binding domains (ABD) of the streptococcal protein G, with a size of 6.5 kDa. Recently, the ADAPTs were further modified to simultaneously bind albumin and another pertinent target protein of interest, suggesting a longer half-life in patient serum, and enabling the development of newer therapeutics. This project presents the 4th generation of the ADAPT library designed to have improved stability. Phage display selections were performed against three target proteins: carcinoembryonic antigen- related cell adhesion molecule 5 (CEACAM5), a biomarker for colorectal cancer, epithelial cell adhesion molecule (EpCAM), a marker for several gastrointestinal carcinomas and trophoblast cell-surface antigen 2 (Trop2) which is overexpressed in triple-negative breast cancer. The results demonstrate binding towards CEACAM5, EpCAM and Trop2, which has been shown by monoclonal phage ELISA. The binding affinity, secondary structure of the selected binders and bispecific binding towards serum albumin remain to be further assessed. The project thus reveals that the ADAPTs selected against the targets CEACAM5, EpCAM and Trop2 present a massive potential for future clinical applications aimed towards development of diagnostics and therapeutics for these cancer biomarkers.