This study utilizes an iPSC-derived BBB model combined with phage display and histidine mutation strategies to screen and evaluate the brain-targeting antibody 46.1's capacity for BBB crossing, offering new optimization pathways for antibody engineering.
Literature Overview
This article, titled 'Strategies to Screen and Evaluate Brain Targeting Antibodies Using an iPSC-Derived Blood–Brain Barrier Model', published in the journal Antibodies, describes the identification of 46.1 antibody variants with enhanced BBB-crossing capability through phage display library and site-directed histidine mutagenesis strategies, and evaluates their transcytosis efficiency in vitro. The study highlights how engineering approaches can enhance antibody performance in receptor-mediated BBB transport, providing new insights for CNS drug delivery.Background Knowledge
The blood-brain barrier (BBB) is a major obstacle in central nervous system (CNS) drug delivery. In recent years, receptor-mediated transcytosis (RMT) strategies have emerged as a promising approach for delivering therapeutics across the BBB by targeting surface receptors such as transferrin receptor (TfR) or insulin receptor (IR). Although several TfR-targeting antibodies have entered clinical trials, there remains a demand for novel RMT targets and efficient antibody screening methods. The 46.1 antibody, a new BBB-crossing tool not targeting TfR or IR, has not yet been optimized for BBB transcytosis efficiency. Therefore, this study employs two complementary approaches—random mutagenesis phage display screening and CDR site-directed histidine mutagenesis—to enhance its transcytosis capability. The study also utilizes iPSC-derived BMEC cells to construct a BBB model with high TEER values, ensuring the rigor of screening and reliability of functional validation.
Research Methods and Experiments
The research team first constructed a random mutagenesis phage display library of the 46.1 single-chain antibody (scFv), followed by multiple rounds of screening using an iPSC-derived BMEC cell model to evaluate BBB transcytosis efficiency at different time points (60, 90, and 120 minutes). Subsequently, the I-TASSER homology modeling tool was used to identify the most solvent-exposed residues within the CDRs, guiding the construction of histidine point mutants. These mutants were then evaluated for binding, endocytosis, and transcytosis capacity. All variants were quantitatively assessed for transcytosis using scFv-Fc-nLuc fusion proteins in the BBB model and further validated through flow cytometry, SDS-PAGE, fluorescence labeling, and luciferase activity assays.Key Conclusions and Perspectives
Research Significance and Prospects
This study demonstrates the application potential of iPSC-derived BBB models in antibody screening and evaluation, while validating the effectiveness of histidine mutagenesis in improving transcytosis efficiency. Future studies could combine multiple CDR histidine mutations or develop more complex mutation libraries to further enhance the BBB-crossing efficiency of the 46.1 antibody. Additionally, this model can be extended to engineer and optimizing other RMT-targeting antibodies, advancing the development of CNS-targeted therapeutics.
Conclusion
This study systematically evaluated the application of phage display screening and CDR site-directed mutagenesis in enhancing the BBB-crossing capability of the 46.1 antibody. Through construction and screening of a random mutagenesis phage library, multiple enriched variants were identified, yet none demonstrated improved transcytosis efficiency in soluble scFv form. In contrast, the histidine mutagenesis approach successfully identified the R162H variant, which exhibited a 1.4-fold increase in transcytosis efficiency compared to the WT antibody, suggesting that pH-sensitive engineering within the CDR regions can influence BBB-crossing performance. These findings provide an operational screening platform and evaluation system for the development of next-generation brain-targeting antibodies and establish a methodological foundation for the optimization of non-TfR-targeting RMT antibodies. The study further emphasizes that in vitro model optimization should be coupled with in vivo pharmacokinetic and pharmacodynamic assessments to validate their true utility in CNS applications.
