Search and Characterization of ITM2A as a New Potential Target for Brain Delivery

Background Integral membrane protein 2A (ITM2A) is a transmembrane protein whose function is not well described. This target was identified as highly enriched in human brain vs peripheral endothelial cells by transcriptomic and proteomic studies during the European Collaboration on the Optimization of Macromolecular Pharmaceutical Innovative Medicines Initiative (COMPACT IMI) consortium. The object of the present paper is to report the work we have undertaken to characterize this protein as a potential brain delivery target. Methods A series of ITM2A constructs, cell lines and specific anti-human and mouse ITM2A antibodies were generated. Binding and internalization 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 antibody was evaluated in an in vitro human blood brain barrier (BBB) model and in a mouse in vivo pharmacokinetic study to validate blood brain barrier crossing. Results Antibodies specifically recognizing extracellular parts of ITM2A or tags inserted in its extracellular domain showed selective binding and uptake in ITM2A-expressing cells. However, despite high RNA expression in mouse and human microvessels, this protein, is rapidly downregulated upon endothelial cells culture, probably explaining why transcytosis could not be evidenced in vitro. An attempt to directly demonstrate in vivo transcytosis in mice turned out unconvincing, using a cross reactive anti ITM2A antibody on one hand and in vivo phage panning of an anti ITM2A phage library on the other hand. Conclusions The present article describes the work we have undertaken to explore the potential of ITM2A as target to mediate transcytosis through BBB. This work highlights the multiple challenges linked to the identification of new brain delivery targets.