Forschung Protein Engineering

CD Laboratory for Next Generation CAR T Cells

In 2019 the "CD Laboratory for Next Generation CAR T Cells" was started in collaboration with Manfred Lehner at the Children´s Cancer Research Institute (CCRI, Vienna), as well as with Miltenyi Biotec as our industrial partner. In this collaborative project, we work on novel strategies to engineer CAR T cells, i.e. T cells genetically manipulated to express chimeric antigen receptors (CARs). One of the crucial stengths of this CD Laboratory is the combination of the CAR T cell expertise of the group of Manfred Lehner with our expertise in protein engineering, with the aim of constructing CAR molecules with improved tumor specificity.

In addition, we are also working on novel concepts for controlling the function of CAR molecules in patients in vivo by administration of safe drugs to prevent over-activation. For that purpose, we have constructed molecular ON-switches, i.e. protein-protein interaction modules which can be turned on by administration of a clinically applicable small molecule. The figure on the right presents the crystal structure of the assembled ON-switch, in which the human lipocalin RBP4 (cyan) is recognized by an engineered rcSso7d-based binder (red) only in the presence of the small molecule A1120 (green).

More detailed information on this "CD Laboratory for Next Generation CAR T Cells" can be found here.

Engineering artificial mini-binders

Due to the availability of in vitro display technologies such as yeast display, also non-antibody-based proteins can be engineered for binding to virtually any given target antigen. Thus, those engineered binders can be considered as alternatives to antibodies for molecular recognition. We use the small (7 kDa), hyperthermostable protein rcSso7d (reduced charge Sso7d) for the construction of artificial mini-binders, which have been shown to interact specifically and with high affinity (typically low nM) with their respective antigen. Moreover, due to the high stability of the engineered protein scaffold, the obtained binders are usually stable, monomeric proteins that can easily be produced in E. coli at high expression titers. The picture on the right shows the NMR structure of the parental protein Sso7d (PDB-ID 1SSO) with the engineered binding site being highlighted in green.

Engineering IgG1-Fc for antigen recognition and improved stability

In collaboration with the group of Prof. Florian Rüker (BOKU, Vienna) and with the company F-star, novel artificial binding sites were engineered in the CH3 domains of human IgG1-Fc, resulting in the generation of so-called Fcabs (Fc antigen binding).

We obtained the first crystal structures of Fcabs in complex with their antigens. This figure shows the crystal structure of an Fcab-Her2 complex. Since an Fcab is usually a homodimeric protein with two identical antigen binding sites, one Fcab can potentially bind two target molecules, provided that there are no steric constraints. In this example, two extracellular domains of Her2 simultaneously interact with one Fcab molecule.

Moreover, we have developed a novel yeast display-based method for stability engineering of proteins. This experimental strategy was applied to improve the thermostability of human IgG1-Fc and of Fcabs, as well as for the construction of a “stability landscape” of the CH3 domain of human IgG1-Fc. That is, at each amino acid position in the CH3 domain the mutational tolerance was measured in a yeast display-based high throughput screen.

Directed evolution of EGFR to study drug resistance

We have developed a novel flow-cytometry-based high-throughput assay for the identification of drug resistance mutations in the receptor tyrosine kinase EGFR. By using this novel selection method, EGFR-mutations conferring resistance to the tyrosine kinase inhibitor erlotinib have successfully been identified. Importantly, drug resistance mutations reported in the clinic were also enriched in this assay, thus validating this novel experimental approach.