Combinatorial protein engineering for gene therapy and affinity protein applications

QC 2026-10-15

Abstract

Today, affinity proteins are used as important tools for studying biological functions and play major roles in therapeutic and bioprocessing applications. Many of these applications require protein binders with specific functional properties, such as recognition of different epitopes and protein conformations, modulation of protein activity, or selective targeting of cells and tissues. The work presented in this thesis concentrates on the generation and evaluation of diverse affinity proteins with such distinct functional properties, aiming to support the development of more selective gene therapies and improved bioprocessing technologies. 

Introducing deep-sequencing to phage display workflows, as presented in Paper I and IV, resulted in the identification of Affibody and nanobody binders with distinct kinetic profiles and preferential binding to different target conformations and epitopes. Paper III describes the thorough characterization of two isolated nanobodies for their properties and antagonistic role in protease inhibition, while Paper IV further expands the characterization of nanobody families from the same selection process. 

Aiming to achieve selective gene delivery to specific cells or organs, Paper II describes a modular platform for integration of Affibody targeting ligands into the Adeno-associated virus (AAV) viral surface. These gene delivery vehicles were shown to selectively transduce target-expressing cells in vitro and reduced liver uptake while redirecting the transduction to other organs in vivo. Binders generated in Paper I and IV were also evaluated as targeting ligands on the AAV surface, showing selective transduction capabilities in vitro. Transduction capabilities do not solely depend on target binding, but can be impacted by factors such as binding epitope, kinetics and capsid integration site. This highlights the value of maintaining binder diversity throughout affinity protein development, as the best functional properties cannot always be predicted or screened for. 

A new purification ligand for AAV9 affinity chromatography is presented in Paper V. This Affimer-based ligand showed comparable recovery and product quality to commercial resins, while eluting under milder conditions. 

Lastly, Paper VI evaluates the impact of plasmid purification methods on rAAV production at developmental scale, showing that the choice of buffer and production system can have a major effect on production titers. 

Taken together, the work presented in this thesis contributes to the discovery and application of affinity proteins and highlights their potential to support the development of new therapies and bioprocessing technologies.

Link to DiVA