Fluids and Barriers of the CNS (Mar 2022)
Exploring ITM2A as a new potential target for brain delivery
Abstract
Abstract Background Integral membrane protein 2A (ITM2A) is a transmembrane protein expressed in a variety of tissues; little is known about its function, particularly in the brain. ITM2A was found to be highly enriched in human brain versus peripheral endothelial cells by transcriptomic and proteomic studies conducted within the European Collaboration on the Optimization of Macromolecular Pharmaceutical (COMPACT) Innovative Medicines Initiative (IMI) consortium. Here, we report the work that was undertaken to determine whether ITM2A could represent a potential target for delivering drugs to the brain. Methods A series of ITM2A constructs, cell lines and specific anti-human and mouse ITM2A antibodies were generated. Binding and internalization studies in Human Embryonic Kidney 293 (HEK293) cells overexpressing ITM2A and in brain microvascular endothelial cells from mouse and non-human primate (NHP) were performed with these tools. The best ITM2A antibody was evaluated in an in vitro human blood brain barrier (BBB) model and in an in vivo mouse pharmacokinetic study to investigate its ability to cross the BBB. Results Antibodies specifically recognizing extracellular parts of ITM2A or tags inserted in its extracellular domain showed selective binding and uptake in ITM2A-overexpressing cells. However, despite high RNA expression in mouse and human microvessels, the ITM2A protein was rapidly downregulated when endothelial cells were grown in culture, probably explaining why transcytosis could not be observed in vitro. An attempt to directly demonstrate in vivo transcytosis in mice was inconclusive, using either a cross-reactive anti-ITM2A antibody or in vivo phage panning of an anti-ITM2A phage library. Conclusions The present work describes our efforts to explore the potential of ITM2A as a target mediating transcytosis through the BBB, and highlights the multiple challenges linked to the identification of new brain delivery targets. Our data provide evidence that antibodies against ITM2A are internalized in ITM2A-overexpressing HEK293 cells, and that ITM2A is expressed in brain microvessels, but further investigations will be needed to demonstrate that ITM2A is a potential target for brain delivery.
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