Strategies to identify, engineer, and validate antibodies targeting blood-brain barrier receptor-mediated transcytosis systems for CNS drug delivery

Abstract

Introduction: Numerous therapeutics for neurological diseases have been developed, but many have failed in clinical trials in part due to limited brain bioavailability, mainly stemming from inefficient transport through the blood-brain barrier (BBB). One potential approach to noninvasive, BBB-targeted drug delivery to the brain is the use of engineered antibodies as delivery vehicles that can transport conjugated drug cargo across the BBB and into the brain via receptor-mediated transcytosis (RMT). Effective development of these RMT targeting systems includes novel target discovery, along with antibody engineering and subsequent validation.

Areas covered: This review focuses on both known and emerging RMT systems, targeting antibody properties in relation to BBB trafficking, and antibody validation strategies.

Expert opinion: Clinical development of known RMT targeting systems and identification of novel BBB RMT targets will be complementary strategies for overcoming the BBB in central nervous system (CNS) disease treatment. The search for new RMT targets with higher brain specificity and enriched expression in the brain has given rise to some new targets which may offer unique benefits. It is our opinion that the expansion of BBB RMT system identification, along with targeting molecule engineering and validation strategies, will substantially contribute to the treatment of a wide range of neurological diseases.

Keywords: Receptor-mediated transcytosis; antibody; blood–brain barrier; drug delivery; engineering; screening.

Figure 1:

Intracellular pathways that RMT targeting systems can take at the BBB. After 1. binding and 2. endocytosis, the cargos are either 3a. recycled, 3b. trafficked across the BBB or 3c. be subject to lysosomal degradation before 4. Exocytosis to the brain parenchyma. Created with BioRender.com.

Figure 2:

New target identification methods. a. Omics typically involves transcriptomic or proteomic profiling. Brain endothelial cells are purified/isolated from brain tissue. For transcriptomics, RNAseq or scRNAseq are typically performed; for proteomics, mass spectrometry is performed. From the data analyses, potential RMT targets are identified and targeting antibodies developed b. Screening involves antibody libraries displayed on phage or yeast can be screened in vivo and in vitro. For an in vivo screen, the library is intravenously injected, left to circulate for a time period; then, the brain is harvested and analyzed for protein variants with the highest brain uptake. For an in vitro screen, the library is dosed on the top (apical, blood side) chamber of a BBB model; the cells are incubated with the library for a period of time; then, the bottom (basolateral, brain side) chamber is harvested analyzed for protein variants with the highest BBB crossing. Subsequently, these antibodies are then used as reagents to identify the targeted BBB RMT receptor. Created with BioRender.com.

Figure 3:

Summary of affinity, valency, and pH effects of TfR-targeting agents on their BBB transcytosis ability. Affinity: Enhanced transcytosis is achieved with lower affinity variants of Genentech’s anti-TfR antibody, intermediate affinity variant of OX26, higher affinity anti-TfRscFv for Regeneron Pharmaceutical’s anti-TfRscFv:GAA gene therapy. Valency: Enhanced transcytosis is achieved with monovalent targeting compared to bivalent targeting for both Roche’s anti-TfR shuttle and anti-BACE1 Fab-anti-TfR VHH fusion protein. pH: Enhanced transcytosis is achieved with lower affinity at endosomal pH (pH 5.5) for anti-TfR antibody MEM-189 and anti-TfR VHH-NT fusion proteins. Created with BioRender.com.

Figure 4:

After engineering the delivery system, proper validation is required to a. discern parenchymal antibody uptake from vascular binding/uptake, and demonstrate that the antibody can deliver pharmacologically relevant amounts of therapeutic by using PD analyses in wild-type animals (b. and c). and c. disease models. Created with BioRender.com.