More research needed, nanoparticles might be better if you need something delivered right now.
Exploring ITM2A as a new potential target for brain delivery
Fluids and Barriers of the CNS volume 19, Article number: 25 (2022)
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.
Background
Under normal physiological conditions, endothelial cells form biological barriers that regulate exchanges and maintain a low and selective permeability to fluid and solutes. The endothelial cells lining blood vessels in the brain, unlike those found in peripheral blood vessels, are extremely tightly packed, non-fenestrated and equipped with many efflux systems. These brain endothelial cells are part of the blood brain barrier, which, with its network of tight junctions, efflux pumps and specific metabolic systems, is mostly permeable to very small lipophilic compounds but actively prevents most molecules, in particular large or polar molecules such as biotherapeutics and antibodies, from entering the brain [1, 2]. As a consequence, there are few biologics in drug development in therapeutic areas such as neurology, oncology (e.g., central nervous system (CNS) lymphoma or glioblastoma) or rare brain diseases. For instance, the use of therapeutic antibodies for CNS disorders such as Alzheimer’s, Parkinson’s, and Huntington’s diseases or brain cancers has been very limited so far [3], largely owing to the presence of the BBB. The biologics that are on the market in neurology act peripherally (or else are given intrathecally) [4]. Therefore, strategies to increase brain exposure of biotherapeutics will be key to their success in this field.
So far, the most successful strategy to deliver biotherapeutics to the brain following systemic administration has been to use a ligand or antibody against receptor-mediated transcytosis (sometimes referred to as the ‘Trojan horse’ approach). Several receptors such as insulin [5], transferrin [6], lipoprotein-related proteins [7, 8], low density lipoprotein [9] or Insulin-like Growth Factor 1 [10] receptors have been used in preclinical models and in the clinic for the three first ones. The most advanced results have been reported with the transferrin receptor, with a few clinical candidates in development; one of these, IZCARGO®, a fusion of an anti-TfR antibody and iduronate sulfatase has been approved in Japan as an enzyme replacement therapy for the treatment of mucopolysaccharidosis [11, 12]. Several challenges however remain in the field. One of them is linked to the fact that all these receptors are ubiquitously expressed, leading to exposure in other tissues than the brain, which could potentially lead to pleiotropic and adverse effects. Discovering brain-specific mechanisms remains the ultimate unreached goal and is the active focus of current research in this area.
Identifying endothelial cell-specific or enriched membrane proteins is an essential first step towards facilitating drug delivery to specific organs. Two main workflows have been described for the search of new mechanisms of brain delivery. The first strategy relies on transcriptomic and proteomic approaches from either brain microvessels or endothelial cells of human [40], cynomolgus monkey [41], bovine [42], rat [33, 43] or mouse [14, 44,45,46,47], including human [34, 48,49,50] diseased brains. A second approach is based on phenotypic in vitro or in vivo screening of antibodies and peptide libraries displayed in various formats including phage and yeast [51,52,53]. Only a few attempts have uncovered new brain delivery targets. Proteomics of rodent BECs have led to identifying CD98 heavy chain (a solute carrier) and Basigin (a matrix metalloprotease) along with the previously known LDL Receptor Related Protein 1 (Lrp1) and Insulin receptor (InsR). Phenotypic panning of naive lama single-domain antibody phage display for binding and internalization in primary human BEC versus primary human lung endothelial cells led to the discovery of FC5 and FC44 [52]. It was later shown that FC5 binds to Cdc50A (energy-dependent clathrin endocytosis) [54]. Our approach combines both strategies.
Within the COMPACT IMI consortium (https://www.imi.europa.eu/projects-results/project-factsheets/compact) a collaborative effort aimed at understanding biological differences among tissue barriers and developing drugs to target specific organs, a variety of proteomic, MicroArray and RNA sequencing studies were conducted using human primary endothelial cells from brain, liver and lung [13]. The resulting over 60,000 RNAs were then processed through several filters, downsizing to mRNAs not detected at all in liver and/or lung or with high differential levels in the brain, mRNAs with mostly transmembrane expression, association to the BBB vasculature, expression and selectivity comparable between rodents and humans and a high degree of conservation between orthologs. Finally annotation of human tissue, cell type and membrane localization using several public databases led to a few mRNAs such as those corresponding to basigin [14] and Low-density lipoprotein receptor-related protein 8 (LRP8) [15], which had already been identified as brain transport mechanisms, thus validating the approach. ITM2A was among these mRNAs. ITM2A had been identified in previous omics studies in rat [33, 34] and pig [35] BECs and reported to be specifically expressed in brain endothelial cells. However, the function of the ITM2A protein remains largely unknown, and it has never been characterized as a transporter in the brain.
ITM2A (alias E25A or BRICD2A) is a 263-amino acid protein with a single transmembrane domain [16]. Its ubiquitous expression is high in thymus, where it was shown to be an activation marker of thymocyte development [17]. ITM2A is mainly believed to be associated with cell differentiation during myogenesis [18, 19], chondrogenesis [20,21,22,23,24] and odontogenesis [25]. The overall homology between mouse and human ITM2A is more than 95% in the extracellular domain [26]. The ITM2A protein has a motopsin-binding Brichos domain within the C-terminal extracellular domain, but the significance of this domain is poorly understood [27]. The Brichos domains appear to bear a chaperone function in different biological situations and has been shown to bind amyloid fibrils [28]. The expression of ITM2A has recently been shown to negatively regulate autophagic flux by inhibiting lysosomal function, through a physical interaction with vacuolar Adenosyl Tri Phosphatase [29].
Transcriptional expression of Itm2a in mouse and human brain has been reported to be homogenous in all brain regions (Protein Atlas) and quite specific of endothelial cells versus neurons, microglia, oligodendrocytes or astrocytes as shown in brain RNA-seq databases: ITM2A mRNA quantification by RNA-seq from http://www.brainrnaseq.org/ shows high levels in mouse and human brain endothelial cells [30]. In fact, ITM2A is commonly referred to as an endothelial cell-specific gene [30]. An analysis of five human and murine cell type-specific transcriptome-wide RNA expression datasets generated within the past several years also identified ITM2A as one of the top expressed genes in endothelial cells [31]. A recent single cell RNA-seq analysis of 20 organs in mice also established the Itm2a gene as specific for brain endothelial cells [32].
Two precedents point to an association of Itm2a with the blood brain barrier. Itm2a was identified as microvasculature-specific through the screening of subtractive cDNA libraries from rat brain capillaries versus kidney/liver on one hand [33, 34] and from porcine brain and aortic endothelial cells on the other hand [35]. In addition, the expression of Itm2a found in freshly isolated porcine Brain Microvascular Endothelial Cells (BMECs) was shown to be lost when these cells were grown in culture, similarly to some other known BBB markers [35].
ITM2A as an endothelial brain specific transmembrane protein has not been associated in the literature with brain transcytosis or transport. The present report describes the work we undertook to characterize ITM2A and investigate the potential of targeting ITM2A to enhance drug delivery to the CNS.
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