Investigating receptor-mediated antibody transcytosis using blood–brain barrier organoid arrays

In recent years, development of technology platforms to deliver biologics to the brain parenchyma from the circulation has accelerated. Robust evidence from independent groups shows that targeting receptors present on endothelial cells at the blood–brain barrier (BBB) can lead to enhanced brain uptake of therapeutic proteins in preclinical species [1‐5]. The diversity of shuttle formats (e.g. bispecific antibodies [1], single domain antibodies [6‐9], Fc-engineered binders [3]) together with different receptor targets in brain endothelial cells (e.g. transferrin receptor [2, 10‐12], insulin receptor [13], CD98 [14], TMEM30 [15]) expand the design space for successful brain delivery in a combinatorial manner. Despite these rapid advances in protein delivery technologies to the central nervous system (CNS), the understanding of the precise molecular mechanisms of receptor-mediated antibody transport across the BBB is lagging.

Recent studies have shed light on candidate pathways for antibody transcytosis across the BBB. We previously found that intracellular endosome tubules regulate transferrin receptor-based Brain Shuttle sorting for transcytosis [16]. Sorting tubules are also involved during transcytosis of the low-density lipoprotein receptor–related protein 1 [17] and a specific serotype of adeno-associated virus (AAV9) [18]. In contrast, FC5 transcytosis depends on multivesicular body transport [15]. These results highlight that receptor-mediated transcytosis may occur via a variety of independent pathways. Identifying the molecular regulators of these transport pathways requires functional genomic tools to evaluate the effect of individual genes on transcytosis, as was recently shown for polarized epithelial cells [19]. However, most work on the regulation of transcytosis at the BBB is still performed in rodents [20‐22]. While these landmark efforts identified novel regulators for specific pathways, such as Mfsd2a [20] and ALPL [23], in vivo studies do not enable sufficiently high throughput and are not optimal to perform unbiased system-level analysis of transport mechanisms.

Multiple groups have recently established a novel 3D model of the BBB formed by the self-organization of human brain endothelial cells, pericytes and astrocytes [24‐27]. Self-assembled 3D BBB models allow direct cell–cell interactions in the absence of artificial membranes or substrates and, importantly, recapitulate key cellular and molecular properties of the BBB, including: (1) tight junction formation, (2) efflux pump expression and activity and (3) receptor-mediated transport of peptides [26]. Since these 3D cellular assemblies undergo self-organization to display functional and morphological features of the BBB in vivo, they fulfill the criteria to be defined as organoids [28, 29]. Although BBB organoids are a major advance in the field of in vitro BBB modelling, organoid production, processing and analysis still require extensive manual work [30]. Thus, the complexity of the protocol and its relative low-throughput hinder its widespread adoption for screening purposes.

Here, we used a recently developed technology based on hydrogel micro-scaffolds [31] to generate patterned BBB organoid arrays, which allowed the formation of more than 3000 viable organoids with reproducible sizes in a 96-well plate format. In agreement with previously described human in vitro models, BBB organoid arrays restricted the passage of large molecules to the organoid core but recapitulated receptor-mediated transcytosis of a transferrin receptor-targeting antibody shuttle. Finally, we showed that BBB organoids can be combined with CRISPR/Cas9 gene editing to knock out specific genes in brain endothelial cells to evaluate their role in receptor-mediated transcytosis. Together, our data show that BBB organoid arrays are a high-throughput platform to discover new mechanisms of antibody transport across the BBB.

A major obstacle for accelerating the development of new brain targeting biologics is the lack of high-throughput in vitro BBB models that are translatable to humans. Here, we bridged this gap by using a novel bioengineering approach relying on hydrogel-based patterned microcavity arrays to grow homogeneous organoids [31, 40, 41]. Previous work established that BBB organoids recapitulate key properties of the BBB observed in vivo [25, 26, 30] and constituted a major advance in the field. Specifically, organoid assembly increased expression of receptors involved in transcytosis, transporters, and tight junction proteins compared to a monoculture of endothelial cells [24, 26, 27]. Furthermore, it was shown that BBB organoids assembled with hCMEC/D3 cells reproduced the expected permeability of angiopep-2 whereas the same cells grown in a transwell model failed to do so [26]. Altogether, these data strongly suggested that BBB organoids are a superior alternative to BECs grown in a transwell configuration. Nevertheless, we need to stress that the fact that non-conjugated FITC accumulated within BBB organoids (Figure S1) suggests that further optimization is needed to fully recapitulate the low permeability of the BBB observed in vivo for small molecules [42]. The model and workflow we developed confirms the key properties and functionality of BBB organoids and builds upon previous work by making substantial improvements to facilitate its widespread adoption for drug discovery. First, microwell arrays increased the BBB organoid yield more than 30 fold per experiment compared to previous protocols using agarose [26, 30] or hanging-drop culture [24, 25]. This yield increase is accompanied by a substantial reduction in experimental handling time, as hydrogel production can be automated [31] and replaces manual preparation of individual plates [30]. Second, BBB organoid formation in arrays was highly reproducible between independent experiments, whereas assembly in agarose resulted in inter-experimental variation of up to 40% in organoid size. Third, we used an automated image analysis script on confocal microscopy images to measure the accumulation of antibodies within organoids. This streamlined workflow enabled the measurement of nearly 10 times more organoids compared to previous studies [25, 30]. This larger data set allowed us to robustly estimate the kinetics of human Brain Shuttle transcytosis. Interestingly, accumulation of human Brain Shuttle in BBB organoids reached a steady-state around 60 min (Fig. 3d), which could be the result of receptor saturation or the balance between transcytosis and efflux/recycling [43, 44]. Via this workflow, various scenarios can be evaluated in more detailed mechanistic studies with the same in vitro platform. Fourth, the transport assay in BBB organoid arrays allows the characterization of native, non-labelled antibodies, whereas previous work measured BBB-transport of biologics using molecules that were covalently coupled with organic fluorophores [18, 26]. While detection of fluorescent conjugates with microscopy requires no additional sample preparation, it does require the prior chemical labelling of each one of the molecules in the test set, which may not be feasible during lead identification and optimization phases. On the other hand, a limitation of detecting non-labelled antibodies by immunofluorescent labeling as done in this study is that it prevents absolute quantification of the amount of transcytosed molecules. Therefore, quantifying permeability coefficients for direct comparison with in vivo observations as done in studies with microfluidic platforms [35, 45] is currently not possible with BBB organoids arrays. This could be overcome by using radiolabelled molecules or by performing antibody measurements from organoid lysates with analytical methods such as ELISA. However, unlike our approach of quantifying the 3D distribution of fluorescence intensity in organoids, these analytical methods cannot distinguish between antibodies bound at the surface and those that underwent transcytosis. Ultimately, this would overestimate the extent of transport across the BBB and yield false-positive hits during a screening campaign. Instead, we consider that combining single cell sorting with analytical methods as recently done in vivo [46] could be a more promising strategy for absolute quantification in organoid models in the future. Altogether, we consider that these improvements make BBB organoid arrays an optimal platform for drug discovery to identify biologics that can cross the BBB in a scalable manner. The use of high-throughput human in vitro models of the BBB will advance translatability of early discovery work into the clinic.

The reproducibility and high sensitivity of the transport assay in BBB organoid arrays make them a useful tool to dissect the molecular mechanisms of transcytosis. We showed that CRISPR-based gene editing can be combined with BBB organoid arrays to evaluate the role of individual genes in receptor-mediated transcytosis. Therefore, this platform will enable the execution of functional large scale screens to identify novel regulators of transcytosis. As a first proof-of-concept we focused on well-characterized genes and investigated caveolin-1 depletion as it has been proposed to be a key regulator of transcytosis across the BBB [20, 21, 47]. However, our data shows that clathrin, but not caveolin-1, is required for transferrin-receptor mediated transcytosis of a human Brain Shuttle. This finding supports the presence of multiple transcytosis pathways with different molecular machineries in brain endothelial cells [48]. Overall, we consider this approach will be useful to evaluate whether different formats (e.g. bispecific antibodies, single domain antibodies, multivalent coated particles) or receptors utilize the same transport pathways across the BBB.

Human BBB organoid arrays are a high-throughput platform to generate homogeneous and reproducible BBB organoids that allow the identification and characterization of antibodies undergoing receptor-mediated transcytosis. We show here that combining high-throughput organoid analysis and gene editing technologies can lead to novel insights into the molecular regulation of transcytosis across the BBB. Implementation of transport assays with BBB organoid arrays will help accelerate the discovery and development of new brain targeting modalities.