Design and Construction of Antibody Fusion Proteins Incorporating Variable New Antigen Receptor (VNAR) Domains

Antibody therapeutics have become a cornerstone of the pharmaceutical market due to their precise molecular targeting, favorable pharmacokinetic properties, and multitiered mechanisms of action. Since the first monoclonal antibody was clinically approved 35 years ago, there have been considerable advances in antibody technology. A major breakthrough has been the design of multispecific antibodies and antibody fusion proteins, which introduce the possibility of recognizing two or more targets with a single molecule. However, despite tremendous progress in the antibody engineering field, challenges in formulation, stability, and tissue penetration necessitate the design of novel antibody formats with improved pharmaceutical properties. There is a growing interest in development of single-domain antibodies, which harbor advantages such as high solubility, robust thermostability, and unique geometries that allow for access to cryptic epitopes. Chondrichthyes such as sharks and rays provide a source of single-domain antibody fragments known as variable new antigen receptors (VNARs), which have been exploited as molecular targeting agents. Here, we describe methods to engineer antibody fusion proteins that incorporate VNARs. We present multiple fusion topologies, and detail the design, expression, and purification for each format. These novel antibody fusion proteins hold great promise for a range of applications in biomedical research and therapeutic design.