Non-Antibody Scaffolds as Alternative Therapeutic Agents

In the field of protein engineering and design, the term "scaffold" describes a multifaceted group of compact and stably folded proteins - differing in size, structure, and origin - that serve as a starting point for the generation of novel binding reagents. Inspired by the structure-function relationships of antibodies (immunoglobulins), such an alternative protein scaffold provides a robust, conserved structural framework that supports an interaction site which can be reshaped for the tight and specific recognition of a given (bio)molecular target. Compared with antibodies, engineered protein scaffolds offer a much smaller size and simpler architecture, which facilitates recombinant gene expression or the construction of bifunctional fusion proteins as well as better tissue penetration in vivo, among other beneficial features. As this approach exclusively relies on in vitro techniques, including rational design and molecular evolution procedures, it allows the specific tailoring of various molecular properties in addition to target affinity and specificity. In particular, this includes stability, solubility, and pharmacokinetic behavior, thus matching the requirements for a desired application, for example, with regard to biopharmaceutical development. Starting from conceptual studies in an academic environment about two decades ago, several protein scaffolds have been promoted to a commercial setting, mainly within biotech companies. Meanwhile, the most advanced concepts, including engineered Kunitz domains, Adnectins, Affibodies, Anticalins, and DARPins, have entered the clinical trial stage, promising a new class of biological drugs, which also has prompted increasing attention by the pharmaceutical industry. This review provides detailed insight into the main strategies for the generation of protein scaffold-based binding reagents and covers several approaches since their beginning. Finally, an overview of the development activities for the first scaffold-derived drug candidates is given, illustrating the increasing level of clinical validation in this area.