Introduction

Due to the global increase in age-related central nervous system (CNS) diseases, there is substantial demand for new CNS medicines. Biologics has become the new trend for CNS drug discovery. Despite the large number of candidates in the pipeline and clinical trials, the success rate of CNS biologics drug is extremely low [1]. Low blood-brain barrier (BBB) permeability has been one of the major causes of failure for new CNS drug candidates. The BBB is formed by specialized brain microvascular endothelial cells (BMECs) and other supporting cells of the neurovascular unit including pericytes, astrocytes, and neurons [2]. The low permeability is caused by decreased transcytotic activity and by tight junctions and adherents’ junctions that limit paracellular passage [3]. BBB plays a key role as a critical protecting barrier for the CNS against toxic and infectious agents while maintaining the ionic and volumetric environments. The barrier properties also create an obstacle for effective systemic drug delivery to the CNS from blood in which 0.01-0.4% percent of all large molecule drugs gets access [4]. Therefore, there has been a great interest in cell models which mimic BBB permeation properties for the purposes of drug screening and engineering [5, 6].

Receptor-mediated transcytosis (RMT) has been proven as a feasible approach for enhancing protein drug delivery to brain [7-10]. Specific BBB receptors such as transferrin receptor (TfR), insulin receptor, and insulin-like growth factor 1 receptor (IGF1R) are triggered by ligand binding to internalize and traffic through brain endothelial cells (BEC), and in some cases to release ligand cargo on the abluminal surface of the BBB [11-13]. Targeting these endogenous mechanisms of transcytosis is considered one of the most promising approaches to develop a non-invasive, safe, and specific cross-BBB delivery of biologics [14, 15]. The RMT delivery strategy is based on using the coupling of a non-transportable therapeutic protein to a transportable peptide or protein, which undergoes receptor-mediated transcytosis through the BBB. TfR is the best-known BBB target. Currently, there are approved drug [16] and clinical stage biologics [10] utilizing TfR targeted fusion protein approach to enhance brain uptake of therapeutic biologics through the RMT mechanism.

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Affinity and species reactivity characteristic of antibody require the development of species-specific in vitro transcytosis model. Models with human origin are more relevant for screening of human BBB targeted biologics. Models from primary human brain capillary endothelial cells (BCEC) [17-20] and immortalized cells, such as human cerebral microvascular endothelial cell line hCMEC/D3 [21], have been used in some studies despite their high paracellular permeabilities. More recently, human induced pluripotent stem cell (iPSC)-derived BBB models with high trans-epithelial electrical resistance (TEER) value have been developed [22, 23]. Also, more sophisticated models with sheer stress and multi-cellular support are reported [24, 25]. These models are validated by endothelial phenotype, expression of tight junction proteins and small molecule transport [26]. Given the lack of human brain exposure data of biologics, the applications of these models to biologics screening have not been validated. Therefore, even though many in vitro human BBB models exist, the prediction power of these models for brain penetration to biologics remains unknown.

At early stages of drug development, in vivo studies are mostly performed in rodents. Rodent brain exposure data of biologics are available [8, 9, 14, 27] and validation data can be readily generated. Rat primary or immortalized BEC-based model, such as rat RBE4 [27-30] have been used to some extent for BBB-targeted antibody selection. The lack of sufficient barrier properties of the immortalized mouse brain endothelial cell line bEND3 cells [31], has hindered its potential as an accurate model. The utility of rodent in vitro BBB models in biologics screening and in vitro in vivo correlation has not been fully evaluated.

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To better understand the utility of these in vitro BBB models for screening of biologics, we have performed systematic transcytosis of anti-TfR antibodies and / or bispecifics in mouse cells, monkey cells and human cells models. Our data indicates that high TEER value is required for a successful transcytosis model. Epithelial cell models, such as the murine epithelial cell line mIEC or the human colorectal adenocarcinoma cell line Caco-2, can be useful for biologics screening. We showed a good correlation between in vitro transcytosis of anti-mouse TfR molecules and their in vivo brain uptake in mice. Good differentiation of anti-human TfR antibodies was also observed in Caco-2-based transcytosis assay.

Anti-hTfR.B1 has comparable binding potency to human and cynomolgus monkey TfR. In vitro transcytosis of anti-hTfR.B1 antibody showed better transcytosis in both Caco-2 and monkey brain transcytosis (MBT) models compared to control antibody. Enhanced brain radioactivity concentration of radiolabeled anti-hTfR.B1 was observed in a non-human primate (NHP) positron emission tomography (PET) study relative to control antibody. The validation using the mouse model strengthened our confidence on the predictive value of the human in vitro BBB model despite there is no human in vivo brain uptake data and limited NHP brain uptake data for biologics. Both mouse and human in vitro models will serve as important screening assays for brain targeted biologics selection in CNS drug development.

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