Selection platform for biparatopic fusion molecules using designed ankyrin repeat proteins (DARPins) and antibody fragments

Abstract

Harnessing the intrinsic properties of antibodies for the generation of therapeutic drugs has been major driving force for the development of robust molecular biotechnology and protein engineering methods. Consequently, antibodies and antibody fragments (such as fragment antibody binding, Fab and single-chain fragment variable, scFv) can be efficiently selected from combinatorial libraries and are routinely used as affinity reagents and therapeutic drugs. Alternative scaffolds are a diverse class of proteins that were developed as alternatives for antibodies. Small size, high stability and easy production in prokaryotic hosts are major benefits of alternative scaffolds. During the last decade designed ankyrin repeat proteins (DARPins) have been the most researched class of alternative scaffolds for therapeutics as well as other applications. DARPins are highly stable 14-21 kDa proteins that consist of usually two to six ankyrin repeat motifs. Synthetic DARPin libraries have been used to select and isolate DARPins with nano- and picomolar affinities against various targets.

Affinity maturation, where the complementary determining regions (CDRs) are mutated, does not necessarily lead to increased affinity of antibody. The aim in this study was to study if joining another protein domain to an antibody fragment could be a viable way to boost the affinity. Therefore, a selection platform for biparatopic fusion molecules that consisted of DARPins and antibody fragments Fab and scFv was established. Using a stage-wise randomised DARPin library, binders were first enriched against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein (N). The selected binder pools and N specific antibody RS16D01 were then amplified with linker primers using PCR. Then, the pieces were joined together with type IIS restriction enzymes. These fusions were designed to include a flexible (G4S)n-linker (n = 1, 2, 4 or 6) in between the two molecules to ensure that there was enough space to fold and bind two different epitopes. DARPins were attached to both N- and C-terminus of scFv, and N-terminus of Fab. After one round of selection, the fusion clones were screened as phages followed by characterisation with phage immunoassay and sequencing.

The scFv fusions performed better had higher signal towards N than parental RS16D01 whereas with DARPin-Fab fusions the signal level was similar to RS16D01-Fab. It was hypothesised that the display of Fab fusions might be limited due to the big size of fusion molecules, leading to e.g., lower signals in phage immunoassays. The best scFv-DARPin fusion JT007H02 had 8.2-fold higher signal to background ratio compared to the parental based on phage immunoassay. The randomised positions in DARPin diversity repeats contained negative, aromatic, and hydrophobic residues. The (G4S)2 linker was found in 11/24 sequenced clones. Even though the characterisation of the fusion clones was limited, this proof-of-concept study demonstrates that functional biparatopic DARPin-RS16D01 fusion molecules can successfully be created using flexible linkers. The results indicate that especially the scFv fusions had enhanced binding properties towards N. Therefore, attaching enriched DARPins to existing parental antibodies via simple, flexible linkers, could be an alternative and effective approach to boosting antibody affinities.