If Jesus was preaching to his disciples today, he might think about altering his ideas about who can be saved, by proclaiming, “A macromolecule has more chance of crossing the blood-brain barrier than for a rich man to enter the kingdom of God.” That is because blood-brain barrier (BBB) is one of the most tightly regulated physiological interfaces, regulated by physical, transport and metabolic barrier mechanisms to maintain the proper influx and efflux of metabolites to and from the brain (Abbott et al., 2010). The impermeable nature of the BBB importantly regulates the neuronal signalling microenvironment, but at the same time hinders the delivery of therapeutic agents. Aside from invasively delivering therapeutics to the neuronal microenvironment, current systemic delivery approaches typically rely on therapeutic interventions that can readily diffuse across the BBB and are no larger than 400 Da in size (Pardridge, 2012) or the blood-cerebrospinal fluid barrier (BCSFB), whose potential for drug delivery to the brain is currently the focus of multiple studies (Strazielle & Ghersi-Egea, 2016). This of course hampers and limits treatment strategies aimed at tackling neuronal disorders.
Many researchers worldwide are trying to find effective, safe therapeutic avenues to circumvent the molecular pathology evident in neurodegenerative diseases and other brain diseases. Protein based biological drugs are the fastest growing field in drug development, with a quarter of the newly approved drugs being proteins. Protein based biologics are uniquely adept in binding specifically to a disease target which enables them to treat diseases that small molecules cannot treat (Stanimirovic et al., 2018). The most recognized strategy to shuttle large biologics across the selectively permeable endothelial cell layer is to use the receptor-mediated endocytosis/transcytosis (RMT) pathways of the BBB (Jones & Shusta, 2007). The methodology relies on discovering receptors found on the apical side of the endothelial cell unit of the BBB that normally regulate the transport of essential nutrients and growth factors from the blood into the brain. Artificial, protein-based transporters that bind to these receptors can then be designed and these can in the ideal situation cross the endothelium via endocytosis/transcytosis into the extracellular environment of the brain. Since the endosome is large, one can recombinantly link therapeutic payloads to these transporters. We have successfully used such “Trojan horse” strategies to deliver intravenously injected antibodies against the pathological Ab protofibrils to the Alzheimer diseased brain in mice (Hultqvist et al., 2017). Our transporter bound the transferrin receptor that expressed promiscuously on the apical membrane of the endothelial cells, which is responsible for the transport of transferrin and iron to the brain parenchyma. The uptake compared to the antibody without the transporter was increased approximately 80 times. The same transporter has also been employed to similar effect to efficiently transport both antibody fragments and peptides into the brain (Fang et al., 2019; Rofo, Sandbaumhüter, et al., 2021; Rofo, Yilmaz, et al., 2021).
Alongside the advancement of novel methods to non-invasively deliver therapeutics to the brain, new pre-clinical in vitro analytical methodologies also need to be developed, to not only complement the necessary in vivo brain shuttling efficacy studies of the therapeutic in question, but also to abide by the directives of the EU concerning the reduction, replacement and refinement of animals used in research (European Parliament, 2010). The development of an in vitro cell culture model that mimics the in vivo blood brain barrier could be one such technique. It allows the user to pulse or load a therapeutic in the apical chamber of a cell-coated permeable support membrane (transwell), followed by the collection and analysis of media in the basolateral chamber at various time-points (referred to as the chase). In this set up, the apical chamber mimics the abluminal blood flow seen in the arterial and venous capillaries of the BBB, whereas the basolateral compartment resembles the luminal brain milieu. Ideally, if the therapeutic does have brain shuttling properties, its presence should be detectable within the basolateral chamber during the chase phase of the in vitro assay. There are several in vitro BBB model systems of varying complexity that are under development, which can be employed to mimic the in vivo BBB in certain physiological settings (Linville & Searson, 2021). However, the majority of the published in vitro BBB cell culture systems are focused on creating a model that closely resembles the in vivo BBB, rather than creating a cell culture system that can be used to test the brain shuttling efficacy of therapeutics. An in vitro model of the human BBB, based upon an adapted in vitro protocol developed using primary cultures of bovine brain microvessel endothelial cells (Raub & Newton, 1991), has been used to assess the role of pH on transcytosis rates of antibodies directed against potential brain shuttle receptors (Sade et al., 2014). However, there is a lack of simple, descriptive mouse in vitro BBB systems that can be used pre-emptively to assess the efficacy of protein-based BBB penetrating therapeutics.
To this end we have developed the In-Cell BBB-Trans assay, a standardised mouse monolayer transwell culture system that can be used to assess the brain shuttling properties of antibodies conjugated to the transferrin receptor binder 8D3, which has been shown on numerous occasions to be an excellent BBB transporter in mice (Boado et al., 2009; Hultqvist et al., 2017; Zuchero et al., 2016). Utilising a “pulse-chase” strategy, the cell culture assay is streamlined so the entire assay can be completed within four-days, from initial plating of the cells to analysis. In addition, a highly sensitive enzyme-linked immunosorbent assay (ELISA) has been developed to work alongside the assay, detecting antibodies in cell medium down to a concentration as low as 0.5 pM. Furthermore, using transverse cryosections of the transwell membranes and subsequent immunohistochemical staining techniques, affords the user the ability to detect the molecular orchestrators of antibody transcytosis in an apical/basolateral orientation. Initial findings obtained using the In-Cell BBB-Trans assay shows a significant increase in transcytosis of scFv8D3 conjugated antibodies compared to unconjugated antibodies, as well as verifying the requirement of endocytosis pathways in transferrin receptor mediated transcytosis of scFv8D3 conjugated antibodies (Fig. 1). Together, the described In-Cell BBB-Trans assay provides a robust methodology for quickly and efficiently validating possible transferrin receptor related brain shuttling antibodies, as well as providing a platform for delineating the molecular mechanisms behind these processes.