Even though our understanding of mechanisms such as protein folding and molecular recognition is relatively poor, antibodies and alternative affinity proteins with entirely novel functions are today generated in a routine manner. The reason for this success is an engineering approach generally known as directed evolution. Directed evolution has provided researchers with a tool for circumventing our limited knowledge and hence the possibility to create novel molecules that by no means could have been designed today. The approach is based on construction of high-complexity combinatorial libraries from which protein variants with desired properties can be selected. Engineered proteins are already indispensable tools in nearly all areas of life science and the recent advent of mainly monoclonal antibodies as therapeutic agents has directed even more attention to the field of combinatorial protein engineering. In this thesis, I present the underlying research efforts of six original papers. The overall objective of the studies has been to develop and investigate a new staphylococcal surface display method for protein engineering and protein characterization. The technology is based on display of recombinant proteins on surface of the Gram-positive bacteria Staphylococcus carnosus. In two initial studies, two key issues were addressed in order to improve the protein engineering method in regard to affinity discrimination ability and transformation efficiency. The successful results enabled investigation of the staphylococcal display system for de novo generation of affibody molecules from large combinatorial libraries. In this study, a high-complexity protein library was for the first time displayed on surface of Gram-positive bacteria and by means of fluorescence-activated cell sorting, specific affinity proteins for tumor necrosis factor-alpha were isolated. Moreover, in following papers, the staphylococcal display method was further improved and investigated for affinity determination, soluble protein production and epitope mapping purposes in order to facilitate downstream characterizations of generated affinity proteins. Taken together, in these studies we have demonstrated that the staphylococcal display system is a powerful alternative to existing technologies for protein engineering and protein characterization. / QC 20100809

<p>QC 20150922</p>

This thesis, based on five research papers, presents results from development and evaluation ofmethods for identifying the interaction site of antibodies on their antigens and the functional investigation of these in different assays. As antibodies have proven to be invaluable tools in diagnostics, therapy and basic research, the demand of characterizing these binding molecules has increased. Techniques for epitope mapping in a streamlined manner are therefore needed, particularly in high throughput projects as the Human Protein Atlas that aims to systematically generate two antibodies with separate epitopes towards all human proteins.  In paper I we describe an approach to map the epitopes of polyclonal and monoclonal antibodies for the first time using staphylococcal display. This method was combined with peptide scanning and alanine scanning using suspension bead arrays, to create a streamlined approach of highresolution characterization of epitopes recognized by antibodies as demonstrated in paper II. Single epitopes were identified for the monoclonal antibodies and several (one to five) separate epitopes scattered throughout the antigen sequence were determined for each polyclonal antibody. Further, antibodies of different species origin showed overlapping binding epitopes. In paper III we studied the epitope patterns of polyclonal antibodies generated with the same antigen in different animals. Although common epitope regions could be identified the exact epitope pattern was not repeated, as some epitopes did not reoccur in the repeated immunizations. In paper IV, a potential biomarker for colon cancer, RBM3, was investigated using validated antibodies by epitope mapping and siRNA analysis. Finally, in paper V, a method for generating epitope-specific antibodies based on affinity purification of a polyclonal antibody is described. The generated antibodies were used in several immunoassays and showed a great difference in functionality. Paired antibodies with separate epitopes were successfully generated and could be used in a sandwich assay or to validate each other in immunohistochemistry. Taken together, in these studies we have demonstrated valuable concepts for the characterization of antibody epitopes. / QC 20120111

Antibodies have become indispensable tools in diagnostics, research and as therapeutics. There are several strategies to generate monoclonal antibodies (mAbs) in order to avoid the drawbacks of polyclonal antibodies (pAbs) for therapeutic use. Moreover, the growing interest in precision medicine requires a well-characterized target and antibody to predict the responsiveness of a treatment. This thesis describes the use of epitope information and display technologies to generate and characterize antibodies. In Paper I, we evaluated if the epitope information of a well-characterized pAb could be used to generate mAbs with retained binding characteristics. In Paper II, the epitope on the complement protein C5 towards Eculizumab was mapped with surface display, the results of which explained the non-responsiveness of Eculizumab treatment among a patient group due to a mutated C5 gene. With this in mind, we showed efficacy in treatment of the mutated C5 variants using a drug binding to another site on C5, suggesting that our approach can be used to guide treatment in precision medicine. In Paper III, a Gram-positive bacterial display platform was evaluated to complement existing platforms for selection of human scFv libraries. When combined with phage display, a thorough library screening and isolation of nano-molar binders was possible. In Paper IV, a solid phase method for directed mutagenesis was developed to generate functional affinity maturation libraries by simultaneous targeting of all six CDRs. The method was also used to create numerous individual mutants to map the paratope of the parent scFv. The paratope information was used to create directed libraries and deep sequencing of the affinity maturation libraries confirmed the viability of the combination approach. Taken together, precise epitope/paratope information together with display technologies have the potential to generate attractive therapeutic antibodies and direct treatment in precision medicine. / <p>QC 20170418</p>

Aggregation of microtubule-associated protein tau is involved in the pathology of several neurodegenerative diseases, including Alzheimer’s disease. The affibody TP4 has been shown to inhibit this aggregation process, and its target-binding positions were previously grafted onto a dimeric affibody scaffold, creating the sequestrin seqTP4. This project constitutes a part of the affinity maturation process of seqTP4, using two different bacterial display methods. An error-prone PCR library was first expressed on Staphylococcus carnosus cells for selection of variants with improved tau-binding properties, resulting in a library of 1.4×107 transformants. Flow cytometric tests indicated difficulties in the setup due to nonspecific interactions, and whereas several different approaches to alleviate the problems were investigated, two cell sorting attempts were ultimately unsuccessful. Subcloning of seqTP4 and the library to an Escherichia coli surface display vector resulted in functional surface expression of seqTP4 on E. coli JK321 and BL21 cells, and a BL21 library size of 1.6×109 transformants. An initial flow cytometric test of this library indicates the presence of improved tau-binding variants, paving the way for future cell sorting. / Aggregering av mikrotubuli-associerat protein tau är involverad i patologin av flera neurodegenerativa sjukdomar, däribland Alzheimers sjukdom. Affibodymolekylen TP4 har visat sig inhibera denna aggregeringsprocess, och överföring av dess målbindande positioner till ett dimeriskt affibodyprotein har tidigare gett upphov till seqTP4, en så kallad sequestrin. Detta projekt utgör ett led i processen att affinitetsmaturera seqTP4, med hjälp av två olika metoder för presentation av proteiner på ytan av bakterieceller. Ett error-prone PCR-bibliotek uttrycktes först på ytan av Staphylococcus carnosus-celler för selektion av varianter med ökad affinitet för tau, vilket resulterade i ett bibliotek av 1.4×107 transformanter. Flödescytometriska tester tydde på svårigheter i detta upplägg på grund av ospecifika interaktioner, och emedan flera olika angreppssätt för att förmildra dessa problem undersöktes, misslyckades slutligen två cellsorteringsförsök. Omkloning av seqTP4 och biblioteket till en vektor för ytpresentation på Escherichia coli resulterade i funktionellt ytuttryck av seqTP4 på E. coli JK321- och BL21-celler, och ett BL21-bibliotek bestående av 1.6×109 transformanter. Ett första flödescytometriskt test av detta bibliotek tyder på närvaron av varianter med förbättrad förmåga att binda tau, och vägen ligger nu relativt öppen för cellsortering.