Prostate cancer is one of the most common cancer types worldwide. However, current diagnostic approaches and treatments are invasive and unspecific. Prostate-specific membrane antigen (PSMA) is an ideal biomarker for prostate cancer and can act as a target for therapeutic or diagnostic agents. Previous attempts to develop an affibody with affinity towards PSMA have been unsuccessful, therefore this thesis aimed at making the affibody selections against PSMA more efficient. In this thesis HEK293 cells expressing a modified version of PSMA containing a 3C protease cleavage site were generated, to enable extraction of the extracellular domain of PSMA during the selections. However, further analyses must be performed to determine if the extracellular domain can be successfully cleaved off. To develop an affibody that can be used both in vitro and in vivo, selections will be carried out against recombinant PSMA as well. The recombinant PSMA was previously produced incorporating an Avi tag for site-specific biotinylation and immobilization for the selections. To biotinylate the recombinant PSMA, the enzyme BirA that catalyzes the biotinylation of the Avi tag, was produced. A protein yield of 8.95 mg/liter culture was obtained and the site-specific biotinylation was highly efficient. To evaluate the proposed affibody selection strategy the next step is to determine if cleavage of the PSMA expressed on the HEK293 cells is possible, optimize the cleavage conditions and to start initial selections using the generated HEK293 cells and the produced BirA enzyme. / Prostatacancer är en av de mest förekommande cancertyperna över hela världen. Nuvarande diagnostiska metoder och terapeutiska behandlingar är dock invasiva och ospecifika. Prostataspecifikt membranantigen (PSMA) är en idealisk biomarkör för prostatacancer och kan agera som en målmolekyl för terapeutiska eller diagnostiska ändamål. Tidigare försök att utveckla en affibody med affinitet mot PSMA har inte lyckats, därför var målet med detta examensarbete att effektivisera selekteringen av affibodies mot PSMA. I detta projekt har HEK293 celler som uttrycker en modifierad version av PSMA, innehållande ett 3C-proteas- klyvningsställe, genererats för att möjliggöra extraktion av den extracellulära domänen av PSMA under selekteringen. Ytterligare analyser måste dock utföras för att avgöra om den extracellulära domänen kan klyvas av. För att utveckla en affibody som kan användas både in vitro och in vivo kommer selekteringen att utföras även mot rekombinant PSMA. Rekombinant PSMA har producerats tidigare med en Avi tag för specifik biotinylering och immobilisering under selekteringen. För att biotinylera det rekombinanta PSMA producerades enzymet BirA, som katalyserar biotinyleringen av en Avi tag. Ett proteinutbyte av 8,95 mg/liter kultur erhölls och den specifika biotinyleringen var effektiv. För att utvärdera den föreslagna strategin för selektering av affibodies är nästa steg att avgöra om klyvning av PSMA uttryckt av HEK293 cellerna är möjlig, optimera klyvningsförhållandena och starta initiala selektioner med de genererade HEK293-cellerna och det producerade BirA-enzymet.

Standard purification processes in large scale antibody production are largely dependent on Protein A chromatography. Protein A binds specifically to many subclasses of IgG with high affinity. However, in order to elute the proteins, the pH needs to be lowered. Since lowering of the pH can be detrimental to some antibodies, a milder purification process is of great interest. A variant of Protein A, called ZCa, has previously been engineered to bind to IgG in a calcium-dependent manner. The antibody binds to ZCa when calcium is present and releases when calcium is removed. For the IgG1 subclass, the elution still requires a slight lowering of the pH, which is why there is room for improvement of the molecule. A phage display selection has been performed with the aim to obtain calcium-dependent IgG1 binders that are able to release the antibodies upon calcium depletion at neutral pH. In addition, an attempt on increasing the alkaline stability of the binders was made. Sequence analysis of the selection output showed almost no indications of increased alkaline stability. Instead, M13K07 helper phages were exposed to new selections for increased alkaline tolerance which might be useful in future phage display selections. Even though the binders selected for in this project did show some promising characteristics, none of them were able to elute upon calcium depletion at neutral pH as aimed for. However, one of the variants did show promising results during most of the performed characterizations. Most interestingly, the elution properties of this variant could indicate a higher calcium-dependence in the interaction with the target than that of ZCa, although further characterizations need to be performed in order to draw any conclusions about possible improvements of this protein domain.

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.