Exploring ITM2A as a new potential target for brain delivery

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.

Keywords: Antibodies; Blood brain barrier; ITM2A; Transcytosis.

Fig. 1

Validation of ITM2A as a potential brain delivery target and stable cell line generation. A Flowchart, go steps are highlighted with blue arrow, no-go steps are highlighted with red arrow. B Human or mouse tagged ITM2A plasmid design. Each construct has intracellular domain (ICD) in blue, transmembrane domain (TM) in yellow and extracellular domain (ECD) in green with Brichos domain; HA tag localization in red, GFP tag in fluo green. C Human or mouse GFP tagged ITM2A expression in HEK293 cells; HEK293 cells are stably recombined with human or mouse ITM2A with a GFP tag at different positions; GFP-tagged ITM2A is visualized in green, and nuclei are stained in blue by Hoechst. D Highlighting the ITM2A colocalization in HEK293 human ITM2A N-Ter GFP cell compartments. Confocal images of N-Ter GFP ITM2A in green and anti-Giantin antibody (ab37266) in red or anti-LAMP1 antibody (ab24170) in red. Yellow color indicates colocalization in the same focal plan, red and green colors indicate no colocalization in the same focal plan

Fig. 2

ITM2A uptake and internalization of several antibodies (anti-ITM2A, anti-GFP or anti-HA) on HEK293 human and mouse. A, B internalization of anti ITM2A revealed with anti-species Alexa 647 in red on HEK293 hITM2A wt, hITM2A GFP N-Ter and hITM2A GFP C-Ter. C internalization of anti-tag antibodies: anti-GFP A31852 Alexa 647 in red, anti-GFP A11122 revealed with anti-Rabbit Alexa 647 in red and anti-HA 901509 Alexa 488 in green on HEK293 hITM2A wt, hITM2A tag N-Ter and hITM2A tag C-Ter. D internalization of anti GFP A31852 revealed with anti-Rabbit Alexa 647 in red on HEK293 mouse ITM2A

Fig. 3

ITM2A mRNA and protein detection in endothelial cells from different species. A ITM2A mRNA detection in monkey endothelial cells from cortex analyzed with RNA-seq at different times of culture. P0D0 (blue) = fresh microvessels, P0D7 (green) = endothelial cells after 1 week in culture, P1D7 (magenta) = endothelial cells after 1 thawing then 1 week in culture. B Differences in the mRNA levels of Itm2a (orange) and CD31 (blue) in mouse endothelial bEnd.3 cells. Microvessels named P0D0, primary cells selected by puromycin named P0D7, primary cells selected by CD31 + named P0D0 CD31 + selected and P0D7 CD31 + selected. Actin was used as a housekeeping gene. C Immunofluorescence staining of mouse ITM2A protein in rodent endothelial cells with three ITM2A antibodies. D Immunofluorescence staining of hCMEC/D3 with TFRC and ITM2A antibodies. Labelling is visualized with secondary anti-species Alexa 647 antibodies in red. Nuclei are labelled with Hoechst in blue

Fig. 4

ITM2A RNA and protein levels. A Relative quantification of ITM2A protein expression in different cells by Western Blot. Green: protein at 60 kDa (ITM2A + GFP), pink: protein at 30 kDa (ITM2A). Western Blot membranes are in Additional file 2. B Quantification of ITM2A in samples of human brain microvessels by LC/MS. ITM2A in green and TFRC in red are expressed as mean ± S.D. (4 quantified peptides for ITM2A and 5 quantified peptides for hTFRC). C Relative quantification of ITM2A protein expression in newborn mice by Western Blot. HEK293 mouse ITM2A GFP was used as control. Green: protein at 60 kDa (ITM2A + GFP), pink: protein at 30 kDa (ITM2A). Western Blot membranes in Additional file 3. D itm2a mRNA relative quantity (RQ) in new-born and adult mouse brain homogenates. Housekeeping gene was gapdh. Results are expressed as mean ± S.D. (n = 1 experiment, performed in triplicates). p values were obtained by Ordinary One-way ANOVA with Dunnett’s multiple comparisons test vs P1. E Quantification of ITM2A in samples of mouse cells and tissues by LC/MS. Cortex in purple, muscles in blue and cells in green. Results are expressed as mean ± SD. (3 quantified peptides)

Fig. 5

ITM2A transport assays. A In vitro human brain transcytosis model from BrainPlotting. P_app antibody anti- ITM2A in blue and P_app antibody anti-TFRC in yellow normalized to P_app Control mouse IgG. Results are expressed in P_app ratio (target/control) means ± Standard Deviation p values were obtained by Mann–Whitney test *p = 0.0444. B In vitro human brain transcytosis controls were Papp control antibody, Papp Fluoresceine and TEER measurements in each Transwell. Results are expressed in means ± Standard Deviation p values were obtained by Mann–Whitney test NS: p > 0.9999. C In vivo mouse brain exposure of an ITM2A monoclonal antibody. 5 mg/kg iv injection of Yu093-G04 and anti-TNP to C57Bl6 mice; samples were collected 5 h after injection. Results are expressed as mean ± S.D. (1 experiment, n = 3 animals). p values were obtained by Two-way ANOVA with Sidak’s multiple comparisons test ns: no statistical significance = p > 0.05, ****p < 0.0001