Investigations of chemical and enzymatic functionalization of affinity proteins

Abstract Affinity proteins are important reagents in research, diagnostics and therapeutic settings. The focus of this thesis has been on investigating different chemical and enzymatic strategies for engineering of affinity proteins to generate affinity reagents with improved or changed functionality. The modifications introduced in affibodies, representing a class of small, three-helix engineered scaffold proteins, and antibodies were selected and implemented through rational design, using a combination of solid phase peptide synthesis, genetic engineering and enzymatic conjugation, depending on the case. In a first study, thioether crosslinks were introduced between internally positioned lysines and cysteines of the human epidermal growth factor receptor (hEGFR)-targeting affibody Z EGFR:1907 , to test the possibility to increase the proteolytic stability of the affibody scaffold. Three different variants of crosslinked affibodies were produced, containing one or two crosslinks. All three variants showed similar affinities to EFGR, and secondary structure contents, as the unmodified control protein. The crosslinked affibodies were challenged with the endopeptidases pepsin, found in the stomach, and trypsin and chymotrypsin, found in the gut. All affibodies showed improved stability towards at least one of the proteases, but the largest improvement was seen for the affibody harboring two crosslinks, which displayed the greatest stability in both assays. Improvement in proteolytic stability of affibodies was further explored. In another study a sortase A-catalyzed intramolecular head-to-tail conjugation of the dimeric human epidermal growth factor 2 (HER2)-targeting affibody (Z HER2:342 ) 2 was performed. Analysis showed no change in α-helicity for the cyclic dimer compared to the linear control, and a slight increase in melting temperature. Interestingly, in contrast to the linear variant, the cyclic dimer showed no signs of proteolytic degradation after 60 min exposure to the exopeptidase carboxypeptidase A. The ability to change protein functionality by chemical modification was explored in two studies. The immunoglobulin-binding Z domain, from which the affibody scaffold is derived, was used as a model protein in one study, where light-induced affinity modulation was investigated. An azobenzene switch that isomerizes from a trans to a cis state was introduced end-to-end to one of the helices in three different designs of the Z domain. The conformationa