A Hyperstable Minimalist Protein for Molecular Recognition

Antibodies represent the most rapidly growing class of therapeutic proteins. They are used for the treatment of a variety of diseases, including various types of cancer and immunological disorders. The most important property of antibodies is their ability to bind to virtually any antigen with high affinity and specificity. Nowadays, display technologies such as ribosome, phage or yeast display enable the development of alternative binding scaffolds, which, like antibodies, have the ability to strongly and specifically bind to any given target. Those binding scaffolds may be used for a variety of applications, such as the construction of multispecific antibodies by attaching them to full-size IgG, intracellular targeting, affinity chromatography or detection assays. Desired properties of those scaffolds include high stability, high expression yield, small size, high solubility, low aggregation and negligible stickiness. Therefore, in the present study we aim to develop a novel binder scaffold based on a small, highly stable protein from a hyperthermophilic organism. In addition to being highly stable, extremely pH resistant and highly soluble, this protein also lacks cysteines. This property allows low-cost expression in E. coli, as well as usage for intracellular targeting applications. First, we will further improve the biophysical properties of this protein by computational design. This will be done in collaboration with Bruce Tidor, whose lab is located at MIT and who has already successfully collaborated with the Wittrup-lab (the host lab for this study, also located at MIT) in various previous studies. Next, the optimized protein will be expressed as a fusion to IgG in order to prove that this novel scaffold is suitable for constructing multispecific antibodies. Subsequently, based on the optimized scaffold, a library containing mutated surface residues will be constructed and displayed on yeast. By using a novel strategy for library design it will be possible to sample all possible binding domains in one yeast surface display library. This library will be used for selection of binders against a variety of model antigens. Finally, selected clones will be tested for usability in affinity chromatography, as well as for intracellular targeting. Importantly, our developed binders will not only target the intracellular antigen by simply binding to it, but we will develop a new system for targeted intracellular protein degradation. We hypothesize, that the library based on the optimized scaffold will enable the rapid development of highly stable binders. Those binders will be highly useful for the construction of multispecific antibodies, as well as for intracellular targeting applications. Moreover, due to the possibility of low-cost expression in E. coli, binders selected from this library will represent a low-cost alternative to antibodies in various biotechnological applications, such as detection assays (e.g. ELISA) and affinity chromatography.

Antibodies are molecules in our body, which specifically bind to foreign molecules and mark them for destruction by the immune system. Their ability to bind to virtually any given target molecule with high specificity and affinity has resulted in their broad usage for a variety of applications, including diagnostic assays, various molecular biology-based methods in research, as well as therapeutic applications, e.g. for the treatment of cancer and autoimmune disorders. However, the usage of antibodies is also associated with various disadvantages, such as the necessity to produce them in mammalian cells, the fact that antibodies are assembled from several units, as well as their tendency to be unstable and to aggregate. Therefore, in this project we have developed an alternative to antibodies. We have engineered a small and highly stable protein (here referred to as mini-protein) for binding to any given target molecule (antigen), like antibodies do. However, in contrast to antibodies, this mini-protein is highly stable, only composed of a single unit and it can be produced in bacteria at low cost. First, we further developed this mini-protein to avoid non-specific stickiness to human cells. Subsequently, one side of this optimized mini-protein was modified in such a way as to enable specific binding to the given target molecule. Importantly, the obtained binding strengths (affinities) and specificities were comparable to those of antibodies. For example, mini-proteins were engineered for binding to molecules that are present on the surface of cancer cells. Moreover, we also developed a mini-protein that binds to a protein that is frequently mutated (modified) in cancer cells. Remarkably, this mini-protein shows increased affinity to the mutated protein found in cancer cells compared to the non-mutated protein in healthy cells. To sum up, in this project we have developed a system for engineering mini-proteins for binding to virtually any given target molecule of interest. During the return phase this system was successfully implemented at the University of Natural Resources and Life Sciences in Vienna. Moreover, binders based on these engineered mini-proteins have already been used in a collaboration project with the Childrens Cancer Research Institute (CCRI, also based in Vienna).