Bifunctional ADAPTs: Opportunity for serological Half-life extension and Targeted therapy

Sammanfattning: Small engineered scaffold proteins (ESPs) gain more and more popularity as biological drugs, due to their specificity and applicability in diagnostics and therapy. Thanks to their high stability, low immunogenicity and low production cost, they present themselves as a promising alternative to the market-leading antibodies. However, their relatively small size poses the risk of fast blood clearance, a circumstance advantageous for imaging purposes but a disadvantage in a therapeutic setting.This thesis has focused on introducing bifunctionality, the ability of the same engineered scaffold protein to exert more than one function, by applying different engineering approaches. Therefore, a new combinatorial protein library based on ABD-derived affinity proteins (ADAPTs) was generated, originating from a bacterial albumin-binding domain. From this library, it was possible to achieve protein modules with the ability to simultaneously bind to its intended target as well as to human serum albumin (HSA), a feature that has been shown to increase the binder’s half-life in the body. Specific binding modules were achieved by performing phage display selections towards the targets Tumor necrosis factor alpha (TNF⍺) and Interleukin-17c (IL-17c), both proinflammatory cytokines involved in many different inflammatory diseases and therefore interesting targets for therapeutic applications. The selection output was analyzed through sequencing and promising candidates were cloned and produced in Escherichia coli (E. coli), followed by a detailed characterization of each candidate including target binding, stability and their oligomeric state using methods like Surface Plasmon Resonance (SPR), Circular Dichroism (CD) and Size Exclusion Chromatography (SEC). It was possible to generate binders that passed all characterization criteria, most importantly showing simultaneous bispecificity to either TNF⍺ or IL-17c in combination with albumin. Each binder was then examined for their usefulness as a real therapeutic by successfully evaluating its ability to block the interaction of the cytokine and its specific receptor in vitro. These newly developed protein binders, showing high affinity towards their targets as well as keeping their initial binding to HSA, present another possibility to combine the advantages of small engineered scaffold proteins with those of typical larger proteins, allowing for more convenient production in bacteria leading to lower production costs and making them ideal candidates for future therapeutics.Furthermore, a previously developed ADAPT targeting the human epidermal growth factor receptor 2 (HER2) was genetically fused to an improved Horseradish Peroxidase (HRP) variant, thereby combining the idea of tumor-targeted therapy through the ADAPT with the utilization of HRP to enzymatically catalyze the prodrug IAA into its active form. After proving these new fusion proteins have similar binding kinetics to the target, as well as comparable enzymatic activities, as their free counterpart, the cytotoxic effects were put to the test in vitro. Hereby, the variants showed to benefit immensely through the addition of an ADAPT by being selectively effective only on HER2-positive cells. The evident advantage of these fusion proteins and their competency to be functionally produced in E. coli as well as the possibility to avoid an additional step of conjugation or coupling of affinity proteins to cytotoxic payloads, makes this approach a promising alternative for current procedures and another reason why ESPs are on the rise.