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.

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.