Anti‐mouse CD98hc nanobodies for brain delivery of a therapeutic moiety

Abstract

AbstractBackgroundThe blood‐brain barrier (BBB) limits the therapeutic perspective for central nervous system (CNS) disorders such as Alzheimer’s disease. Interestingly, monoclonal antibodies generated against receptor‐mediated transcytosis receptors are able to transport therapeutic proteins across the BBB. Nanobodies, which are the variable domain isolated from camelid heavy‐chain only antibodies have also shown their potential to deliver therapeutics into the CNS. Here we aimed to expand the panel of nanobodies targeting other brain endothelial cell receptors.MethodCamelids were immunized with a pool of DNA encoding for several brain endothelial cell receptors. One target was chosen to validate the immunization and discovery approach, CD98hc, previously suggested to allow drug shuttling across the BBB. Nanobody libraries were selected on Chinese Hamster Ovarian (CHO) cells overexpressing the target of interest. Selected nanobodies were screened in vivo based on their potential to deliver into the CNS either a biologically active neuropeptide neurotensin to reduce body temperature, or a BACE1 inhibiting antibody that reduces the accumulation of Aβ1‐40 species.ResultDNA immunization followed by selections on CHO cells overexpressing the target of interest resulted in specific anti‐mouse CD98hc binders with affinities in the low nanomolar range. In vivo screening of mouse CD98hc nanobody binders revealed some discrepancy between the two methods used. Whereas none of the tested nanobodies fused to neurotensin was able to reduce body temperature, nanobodies fused to the anti‐BACE1 antibody 1A11 effectively reduced Aβ1‐40 levels in the brain. We suggest that the neurotensin approach, although having shown robustness with anti‐TfR nanobodies, might lack sensitivity when screening non‐TfR binders. We suggest BACE1 inhibition as a more robust model to validate CD98hc‐mediated transport across the BBB.ConclusionThis is a proof of concept study in which we have optimized our nanobody discovery approach to find nanobodies against BBB targets, and validated CD98hc as a receptor to target in order to shuttle drugs into the brain. In the future we will explore whether nanobodies raised against novel brain endothelial cell receptors could also improve brain penetration of biologics.