Developing a Fusion Protein Platform to Improve Blood-Brain Barrier Transcytosis of Immunoglobulin G Therapeutics

摘要

Introduction: The clinical success of antibody-based therapies is evident by the efficacious treatment of a wide variety of diseases including autoimmunity, inflammation, cancer, and more. However, the application of this class of therapeutics to diseases of the central nervous system (CNS) is limited by their inability to cross the blood-brain-barrier (BBB). Recently, it has been shown that bispecific antibodies can overcome this hindrance by hijacking receptor-mediated transcytosis (RTM). Despite the improved transcytosis observed with RTM-hijacking bispecifics, their challenging production prevents broad application as well as side-to-side comparison of multiple constructs. To facilitate this process, we have developed a platform for the rapid transformation of any off-the-shelf antibody into a bispecific antibody that is capable of traversing the BBB. Materials and Methods: Our bispecific production technology relies on fusing BBB-penetrating antibody fragments with a photoreactive antibody-binding domain (pAbBD). This domain contains the unnatural amino acid benzoylphenylalanine, which is able to covalently link to immunoglobulin G heavy chains under ultraviolet light. These BBB-penetrating fusion proteins are cloned into a bacterial expression vector that co-expresses two key folding chaperones. This plasmid is co-transformed with a vector containing the unnatural amino acid integration machinery into an E. Coli expression cell line that stabilizes disulfide bond formation.To demonstrate the utility of our platform, Cetuximab, an FDA-approved monoclonal antibody cancer therapeutic, was converted to a BBB-penetrating bispecific. Binding affinity and specificity of the modified Cetuximab to both targets is being characterized using a fluorescent cell-based titration assay. Next, the ability of the bispecific to traverse across the BBB versus that of unmodified Cetuximab will be compared in a Boyden chamber transmigration model and in mice. Results and Discussion: We have designed two types of BBB-penetrating pAbBD fusion proteins: one contains an anti-transferrin single-chain variable fragment and the other two utilize llama single-domain antibodies. Bacterial expression has been optimized to repeatable yields exceeding 80 mg protein per liter of culture with high purity. Using our pAbBD technology, nearly 100% crosslinking of the three BBB-penetrating fusions to Cetuximab was obtained in two hours. Bispecific antibody binding following the photo-crosslinking procedure has been confirmed by fluorescent imaging. Binding affinity and in vitro BBB transcytosis quantifications are currently under evaluation.Conclusion: Our fusion protein platform provides a modular, rapid method to convert any off-the-shelf IgG therapeutic into a BBB-traversing bispecific. This technology harnesses the benefits of existing antibody-based therapies in the context of CNS diseases and it speeds up the evaluation of novel BBB-traversing antibody fragments.