Targeting receptor-mediated transport for delivery of biologics across the blood-brain barrier.

Biologics are an emerging class of medicines with substantial promise to treat neurological disorders such as Alzheimer's disease, stroke, and multiple sclerosis. However, the blood-brain barrier (BBB) presents a formidable obstacle that appreciably limits brain uptake and hence the therapeutic potential of biologics following intravenous administration. One promising strategy for overcoming the BBB to deliver biologics is the targeting of endogenous receptor-mediated transport (RMT) systems that employ vesicular trafficking to transport ligands across the BBB endothelium. If a biologic is modified with an appropriate targeting ligand, it can gain improved access to the brain via RMT. Various RMT-targeting strategies have been developed over the past 20 years, and this review explores exciting recent advances, emphasizing studies that show brain targeting in vivo.

Identification of lamprey variable lymphocyte receptors that target the brain vasculature.

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.

A yeast display immunoprecipitation screen for targeted discovery of antibodies against membrane protein complexes.

Yeast display immunoprecipitation is a combinatorial library screening platform for the discovery and engineering of antibodies against membrane proteins using detergent-solubilized membrane fractions or cell lysates as antigen sources. Here, we present the extension of this method for the screening of antibodies that bind to membrane protein complexes, enabling discovery of antibodies that target antigens involved in a functional protein-protein interaction of interest. For this proof-of-concept study, we focused on the receptor-mediated endocytosis machinery at the blood-brain barrier, and adaptin 2 (AP-2) was chosen as the functional interaction hub. The goal of this study was to identify antibodies that bound to blood-brain barrier (BBB) membrane protein complexes containing AP-2. Screening of a nonimmune yeast display antibody library was carried out using detergent-solubilized BBB plasma membranes as an antigen pool, and antibodies that could interact with protein complexes containing AP-2 were identified. Downstream characterization of isolated antibodies confirmed targeting of proteins known to play important roles in membrane trafficking. This functional yeast display immunoprecipitation screen may be applied to other systems where antibodies against other functional classes of protein complexes are sought.

Identification of variable lymphocyte receptors that can target therapeutics to pathologically exposed brain extracellular matrix.

Diseases that lead to blood-brain barrier (BBB) disruption will pathologically expose normally inaccessible brain extracellular matrix (ECM) to circulating blood components. Therefore, we hypothesized that brain ECM-targeting moieties could specifically target the disrupted BBB and potentially deliver therapies. Variable lymphocyte receptors (VLRs) that preferentially associate with brain ECM were identified from an immune VLR library via yeast surface display biopanning coupled with a moderate throughput ECM screen. Brain ECM binding of VLR clones to murine and human brain tissue sections was confirmed. After systemic administration, P1C10, the lead brain ECM-targeting VLR candidate, specifically accumulated in brains with mannitol-disrupted BBB and at disrupted BBB regions in two different intracranial glioblastoma models. We also demonstrate P1C10's ability to deliver doxorubicin-loaded liposomes, leading to significantly improved survival in glioblastoma-bearing mice. Thus, VLRs can be used to selectively target pathologically exposed brain ECM and deliver drug payloads.

Introducing glycophage arrays: facile production, purification and patterning of glycophages.

Glycosylation is a widespread post-translational modification that plays important roles in health and disease. As glycan sequence and structure are not directly coded into the genome, our understanding of glycans and their functions in biological systems is much more primitive than that of DNA and proteins.Recently, printed glycan microarrays (glycoarrays) have emerged as powerful, high-throughput tools for screening glycan-protein interactions[1,2], and have been applied in disease detection [3], drug discovery [4], the study of immunity [5], and host-pathogen interactions [1, 2], among others.Unfortunately, glycoarray applications are currently limited by the expensive and complex methods available to synthesize glycans or alternatively, by the challenges in identifying and tagging glycans from natural sources [6, 7]. In this issue of Biotechnology Journal, Çelik et al. [8] introduce a potentially powerful new method for facile, scalable production, and purification of glycans compatible with microarray patterning. Çelik et al.'s [8] approach is based on innovative deployment of filamentous phage display so that the displayed proteins can be tagged with specific glycans of interest (glycophages) and subsequently patterned in array format.

Yeast Display-Based Antibody Affinity Maturation Using Detergent-Solubilized Cell Lysates.

It is often desired to identify or engineer antibodies that target membrane proteins (MPs). However, due to their inherent insolubility in aqueous solutions, MPs are often incompatible with in vitro antibody discovery and optimization platforms. Recently, we adapted yeast display technology to accommodate detergent-solubilized cell lysates as sources of MP antigens. The following protocol details the incorporation of cell lysates into a kinetic screen designed to obtain antibodies with improved affinity via slowed dissociation from an MP antigen.