Identification of lamprey variable lymphocyte receptors that target the brain vasculature

Abstract

The blood–brain barrier (BBB) represents a significant bottleneck for the delivery of therapeutics to the central nervous system. In recent years, the promise of coopting BBB receptor-mediated transport systems for brain drug delivery has increased in large part due to the discovery and engineering of BBB-targeting antibodies. Here we describe an innovative screening platform for identification of new BBB targeting molecules from a class of lamprey antigen recognition proteins known as variable lymphocyte receptors (VLRs). Lamprey were immunized with murine brain microvessel plasma membranes, and the resultant repertoire cloned into the yeast surface display system. The library was screened via a unique workflow that identified 16 VLR clones that target extracellular epitopes of in vivo-relevant BBB membrane proteins. Of these, three lead VLR candidates, VLR-Fc-11, VLR-Fc-30, and VLR-Fc-46 selectively target the brain vasculature and traffic within brain microvascular endothelial cells after intravenous administration in mice, with VLR-Fc-30 being confirmed as trafficking into the brain parenchyma. Epitope characterization indicates that the VLRs, in part, recognize sialylated glycostructures. These promising new targeting molecules have the potential for brain targeting and drug delivery with improved brain vascular specificity.

Protein Engineering and High‐Throughput Screening by Yeast Surface Display: Survey of Current Methods

“…Recent work demonstrated the utility of YDIP screening using an in vivo‐sourced cell lysate antigen pool which holds the promise of increased in vivo relevance of the output binding pool. [ 25 ] A YSD library of variable lymphocyte receptors (VLRs) was generated from lamprey that were immunized with brain endothelial cell plasma membranes directly fractioned from mouse brain microvessels. [ 25 ] To enrich the library for brain endothelial cell membrane targeted VLRs, YDIP was performed using an antigen pool comprising detergent‐solubilized, biotinylated lysates of the same mouse brain microvessel plasma membranes that were used for lamprey immunization.…”

“…[ 25 ] A YSD library of variable lymphocyte receptors (VLRs) was generated from lamprey that were immunized with brain endothelial cell plasma membranes directly fractioned from mouse brain microvessels. [ 25 ] To enrich the library for brain endothelial cell membrane targeted VLRs, YDIP was performed using an antigen pool comprising detergent‐solubilized, biotinylated lysates of the same mouse brain microvessel plasma membranes that were used for lamprey immunization. [ 25 ] YDIP with one round of magnetic activated cell sorting (MACS) followed by one round of FACS led to a tenfold enrichment in yeast‐displayed VLRs capable of binding brain microvessel plasma membrane compared to the starting library.…”

“…[ 25 ] To enrich the library for brain endothelial cell membrane targeted VLRs, YDIP was performed using an antigen pool comprising detergent‐solubilized, biotinylated lysates of the same mouse brain microvessel plasma membranes that were used for lamprey immunization. [ 25 ] YDIP with one round of magnetic activated cell sorting (MACS) followed by one round of FACS led to a tenfold enrichment in yeast‐displayed VLRs capable of binding brain microvessel plasma membrane compared to the starting library. [ 25 ] Subsequent selection of the library for VLRs that could bind extracellular epitopes was performed by biopanning against mouse brain endothelial cell cultures.…”

“…[ 25 ] YDIP with one round of magnetic activated cell sorting (MACS) followed by one round of FACS led to a tenfold enrichment in yeast‐displayed VLRs capable of binding brain microvessel plasma membrane compared to the starting library. [ 25 ] Subsequent selection of the library for VLRs that could bind extracellular epitopes was performed by biopanning against mouse brain endothelial cell cultures. [ 25 ] While biopanning for enrichment of VLRs binding to extracellular targets was performed using cell cultured substrates rather than in vivo sourced cell lysates, the initial enrichment of the library using the brain endothelial plasma membranes ensured that the resulting pool of VLRs had many variants (16 of 26 clones tested) that could bind relevant antigens in brain microvessels.…”

An innovative strategy to identify new targets for delivering antibodies to the brain has led to the exploration of the integrin family

“…At the time we submitted our article, a lamprey immunization with murine brain microvessels plasma membranes with the aim of discovering new brain targeting receptors was published by J.M. Lajoie et al [ 32 ]. The first goal of the present report is to describe such an approach of generating an immune library from immunized mice and screening the resulting antibodies based on binding, internalization and finally transcytosis.…”