Smal binding protein molecules with high specificity and affinity as an alternative to complex molecules of human antibodies.
Laboratory of Ligand Engineering
Group leader: RNDr. Petr Malý, CSc.
Artificial binding proteins derived from small protein scaffolds represent a valuable alternative to commonly used antibodies. Novel binders with engineered affinity, high specificity or designed inhibitory function attract attention as key components for the development of novel biosensing devices, in vivo diagnostics and next-generation therapeutics. Small, robust, and soluble proteins with high thermal and hydrodynamic stability and without disulphide bonds are amenable to rational improvement and can be easily modified by gene-fusion approaches.
In our laboratory, which operates since 2008, we focus on engineering novel binders raised against human cytokines, their receptors or tumour markers. Recently we have established a model of three-helix bundle of albumin-binding domain (ABD) of streptococcal protein G as a master scaffold for the generation of high-complex combinatorial libraries. Using randomization of 11 pre-determined amino acid residues we engineered a library of a theoretical complexity of 1014 protein variants which was successfully used, in combination with ribosome display selection, for the generation of unique binders of human interferon gamma (hIFNg) with KD in the nanomolar range (Ahmad et al 2012). High-affinity binders of hIFNg were then used as key components for the construction of a novel SPR biosensor with the improved sensitivity for detection of hIFNg in diluted blood plasma (Šípová et al. 2012).
The assembled combinatorial library has been used in another project leading to the generation of inhibitory ABD variants targeting human IL-23 and its receptor (IL-23R), key elements of the Th17-mediated pro-inflammatory pathway. In this running project we have already identified a collection of ABD variants (REX binders) with sub- to nanomolar KD value for the binding of recombinant hIL-23R or IL-23R-Fc chimera. These so-called REX binders inhibit binding of p19 protein (a subunit of IL-23) to the IL-23 receptor in several arrangements of direct competition ELISA. We further demonstrate that REX variants bind to human cell lines K-562 and THP-1 and this binding correlates with IL-23R cell-surface expression. As binding of REX proteins to THP-1 cells can be substantially diminished by a high dose of the p19 protein, we conclude that we identified novel IL-23R-binding inhibitors that might be useful in designing novel anti-inflammatory tools. This is of special value as the molecular structure of the IL-23/IL-23R complex has not been described yet and, therefore, designing efficient inhibitors of IL-23 function with a promising therapeutic potential remains cumbersome. Currently, we have been working on the characterization of IL-23 binding ABD variants (ILP binders) and investigation of a possible immunomodulatory function of the most promising REX and ILP candidates.
In another part of our research activities, we closely collaborate with several Czech biotech companies in the project AFFIBINDER supported by the Ministry of Industry and Trade of the Czech Republic. Within this project, we develop a novel fluorescence-based screening method for large-scale identification of high-affinity binders raised against selected human oncomarkers. As a part of this project we develop novel high-affinity binders of human KLK2 and PSP94 prostate cancer biomarkers.
