# Translational applicability of human blood-brain barrier spheroid models for the development of brain-penetrant therapeutic antibodies

## TL;DR

This study shows that human blood-brain barrier spheroid models can effectively predict how antibodies cross the brain barrier and explain their permeability differences.

## Contribution

The study demonstrates the translational applicability of hMCS-BBB models for evaluating and understanding brain-penetrant antibody permeability.

## Key findings

- Three out of four hTfRMAbs showed BBB permeability with distinct Km values.
- hMCS-BBB model results correlated with in vivo monkey brain permeability profiles.
- Lysosome co-localization revealed intracellular localization differences between permeable and non-permeable antibodies.

## Abstract

Drug delivery systems play a key role in the development of new therapeutic strategies for brain diseases. Antibodies capable of crossing the blood–brain barrier (BBB) have attracted considerable attention as promising drug carriers. To facilitate their development, we previously established human multi-cellular spheroidal (hMCS)-BBB models as microphysiological system-based platforms. In this study, we aimed to enhance the applicability of these hMCS-BBB models for the development of brain-permeable antibodies using four anti-human transferrin receptor monoclonal antibodies (hTfRMAbs). Permeability assays using hMCS-BBB models revealed BBB permeability in three out of four hTfRMAbs, with two antibodies exhibiting Km values of 6.5 and 12.0 µg/mL, respectively. Importantly, differences in the BBB permeability of tested hTfRMAbs were consistent with the brain permeability profiles reported in monkeys in vivo. Moreover, these differences were not detected via simple uptake assays using monoculture brain microvascular endothelial cells, highlighting the superior evaluation potential of hMCS-BBB models. Lysosome co-localization assays using hMCS-BBB models revealed the distinct intracellular localization patterns of BBB-permeable and non-permeable hTfRMAbs with lysosomes, presumably explaining their different permeability properties. Overall, hMCS-BBB models effectively characterized the BBB permeability of hTfRMAbs, demonstrated strong potential for in vivo extrapolation, and proved suitable for mechanistic studies of the cellular pathways regulating their BBB permeability. Therefore, hMCS-BBB models are considered promising tools for advancing the development of brain-permeable therapeutic antibodies.

The online version contains supplementary material available at 10.1186/s12987-025-00752-8.