DARPin

DARPin stands for designed ankyrin repeat protein, a class of engineered binding proteins derived from ankyrin repeat motifs. DARPins are designed to recognize target proteins with high affinity and specificity, offering an antibody-like binding surface without using immunoglobulin frameworks. The technology was developed in the late 1990s and early 2000s, with key contributions from Albert Plückthun and colleagues, and has since been pursued by research groups and biotechnology companies.

Structure and properties: A DARPin consists of a series of ankyrin repeat units arranged in a modular, single-domain scaffold. Each repeat contributes a contact surface, allowing a customizable binding interface. DARPins are small, typically about 14–18 kDa, and are highly stable, soluble, and straightforward to produce in simple expression systems such as Escherichia coli. They lack disulfide crosslinks, which simplifies production and enhances stability. Their modularity enables straightforward affinity maturation and the creation of multispecific formats.

Generation and selection: Diversity is introduced in the binding loops of the ankyrin repeats, and target-binding variants are isolated through display technologies such as phage, ribosome, or yeast display. Affinity can reach picomolar to low-nanomolar ranges, and clones can be engineered for increased stability, specificity, or altered pharmacokinetics.

Applications and formats: DARPins are explored for therapeutics, diagnostics, and research tools. They can be used as monovalent binders, multispecifics that engage two targets, or fused to Fc or other half-life extension modules. They are also used as high-quality reagents for imaging and target validation. While several DARPins have entered clinical trials for cancer and other diseases, as of the mid-2020s none had achieved regulatory approval in major markets.