APPLICATION OF EXCHABODY TECHNOLOGY FOR PAIRING VHHS TO ACHIEVE SYNERGISTIC EFFECTS

Abstract Objective Single-domain antibodies, such as VHH and nanobody, have shown potential for use in therapy and diagnostics. One application of VHHs is the tethering of two fragments to different epitopes on the same target, which is difficult to achieve with conventional antibodies. Synergistic heterologous VHH dimers have higher affinity, better specificity, and broad applications in developing high-affinity monoclonal antibodies, bispecific antibodies, ADCs, and CAR-Ts. However, finding the best pair of VHHs for these applications requires combinational screening, which is traditionally a time-consuming and costly process. The objective of this study is to develop a technology that can quickly screen and pair two synergistic VHHs without the need to express tandem VHH dimers. Methods The researchers developed proprietary tags and specific dockers that, when stabilized on a solid station, can capture any VHHs that the dockers recognize and pull them together to form a non-covalent dimer. This platform is called ExchaBody technology, and the VHH dimers formed this way are ExchaBodies. The researchers used this technology to conduct a bi-epitope screening campaign, where VHHs were first expressed as monomers with tags and then binned and grouped into different categories. VHHs were then paired with all reasonable combinations using ExchaBody technology, and these ExchaBodies were evaluated for their combined activities. Results ExchaBody technology was able to link any two VHHs together within one hour, and the resulting ExchaBodies had bivalent or bifunctional VHH activities. The bi-epitope VHH screening campaign, which would have taken months to complete using traditional methods, was finished within two weeks using ExchaBody technology, saving time and cost. The researchers were able to construct two lead molecules, a bi-specific VHH-Fc fusion protein and a tri-valent VHH molecule, using ExchaBody technology. These lead molecules were found to be superior to their counterparts on the market based on affinity and functional assays. Conclusion ExchaBody technology is a bispecific VHH screening and pairing platform that can quickly and cost-effectively create non-covalent, bispecific VHHs (ExchaBodies) without the need to express them. ExchaBodies possess the binding and cellular activities of a covalently linked, bispecific, tandem VHH dimer. This technology has broad applications in developing high-affinity monoclonal antibodies, bispecific antibodies, ADCs, and CAR-Ts.