Development of a Bispecific Shuttle for Efficient and Long-Lived Brain Delivery of Antibodies

Abstract

Monoclonal antibodies are an emerging class of therapeutics with over 100 antibody-based therapeutics approved for treatment of a plethora of diseases, ranging from cancer to autoimmune and infectious diseases. While antibodies have been more successful in certain disease areas, their use for treatment of neurodegenerative diseases is limited. The greatest hurdle hindering the therapeutic success of antibodies for neurological disorders is arguably the blood-brain barrier (BBB). Some small and lipophilic molecules can easily cross into the brain, but larger molecules such as antibodies are largely excluded from the brain. Relevant to this work, there are certain BBB pathways that facilitate transport of proteins. Once such process is receptor-mediated transcytosis (RMT), which enables specific proteins such as transferrin to be transported into the brain via the transferrin receptor (TfR-1). In addition to this receptor, previous work has shown that CD98 heavy chain (CD98hc), a widely expressed extracellular protein on the BBB, can also transport proteins (antibodies) into the brain. In this project, we aimed to develop a bispecific molecular shuttle for improving brain delivery of validated ‘off-the-shelf’ antibodies. We showed that these antibodies, when reformatted into a bispecific shuttle, retain similar affinities and avidities as the parental antibodies, while being able to engage specific receptors (TfR-1 and CD98hc) on the BBB and facilitate transport into the brain. We further characterized the pharmacokinetics of these bispecific shuttles using radiotracing techniques to determine antibody concentrations in the brain and blood at different time points. Interestingly, we found that bispecific shuttles targeting TfR-1 led to higher brain concentrations, while those targeting CD98hc led to much longer brain retention. We also used fluorescence imaging and immunostaining techniques to characterize antibody distribution and localization in the brain. We found that TfR-1 shuttles led to internalization into brain cells even without delivery of a targeted IgG, while CD98hc shuttles did not. We also sought to deliver target-specific IgGs that can reach certain cell types in the brain (i.e., neurons and astrocytes) by utilizing this bispecific shuttle platform. We identified two highly characterized IgGs specific for cell-surface proteins on neurons and astrocytes and reformatted them into bispecific CD98hc shuttles. We showed strong in vivo colocalization of these bispecific antibodies to either neurons or astrocytes after intravenous administration, as confirmed by co-staining with cell-specific markers (NeuN and GFAP) over a period of five days. To demonstrate a functional pharmacodynamic effect, we delivered a validated TrkB agonist antibody (clone 29D7) as a bispecific antibody. We showed that this TrkB antibody can be delivered into the brain and induce a cascade of signaling pathways, as evidenced by co-staining with phospho-AKT and phospho-ERK 1/2 markers. In conclusion, our CD98hc bispecific shuttle technology has been extensively characterized to demonstrate feasibility as a platform technology for brain delivery of any sequence-defined IgG. We anticipate that this bispecific antibody technology will accelerate the development of novel therapeutics for treating a wide range of challenging neurodegenerative and brain diseases.