The blood brain barrier (BBB) presents a challenge for the delivery of brain therapeutics. Trans-BBB delivery methods that use targeting vectors to coopt the vesicle trafficking machinery of BBB endothelial cells have been developed, but these are often hampered by limited flux through the BBB. A solution to this problem lies in the semi-rational engineering of BBB targeting vectors. Leveraging knowledge of intracellular trafficking, researchers have begun to tune selected binding properties of the vector-receptor interaction. Engineered binding affinity, avidity and pH-sensitivity have been shown to affect binding, intracellular sorting and release, ultimately leading to increased brain uptake of the targeting vector and its associated cargo. However, each targeted receptor may exhibit differential responses to engineered binding properties, illustrating the need to better understand vector-receptor interactions and trafficking dynamics.

Drug delivery across the blood‐brain barrier (BBB) remains a significant obstacle for the development of neurological disease therapies. The low penetration of blood‐borne therapeutics into the brain can oftentimes be attributed to the restrictive nature of the brain microvascular endothelial cells (BMECs) that comprise the BBB. One strategy beginning to be successfully leveraged is the use of endogenous receptor‐mediated transcytosis (RMT) systems as a means to shuttle a targeted therapeutic into the brain. Limitations of known RMT targets and their cognate targeting reagents include brain specificity, brain uptake levels, and off‐target effects, driving the search for new and potentially improved brain targeting reagent‐RMT pairs. To this end, we deployed human‐induced pluripotent stem cell (iPSC)‐derived BMEC‐like cells as a model BBB substrate on which to mine for new RMT‐targeting antibody pairs. A nonimmune, human single‐chain variable fragment (scFv) phage display library was screened for binding, internalization, and transcytosis across iPSC‐derived BMECs. Lead candidates exhibited binding and internalization into BMECs as well as binding to both human and mouse BBB in brain tissue sections. Antibodies targeted the murine BBB after intravenous administration with one particular clone, 46.1‐scFv, exhibiting a 26‐fold increase in brain accumulation (8.1 nM). Moreover, clone 46.1‐scFv was found to associate with postvascular, parenchymal cells, indicating its successful receptor‐mediated transport across the BBB. Such a new BBB targeting ligand could enhance the transport of therapeutic molecules into the brain.

The equilibrium binding affinity of receptor-ligand or antibody-antigen pairs may be modulated by protonation of histidine side-chains, and such pH-dependent mechanisms play important roles in biological systems, affecting molecular uptake and trafficking. Here, we aimed to manipulate cellular transport of single-chain antibodies (scFvs) against the transferrin receptor (TfR) by engineering pH-dependent antigen binding. An anti-TfR scFv was subjected to histidine saturation mutagenesis of a single CDR. By employing yeast surface display with a pH-dependent screening pressure, scFvs having markedly increased dissociation from TfR at pH 5.5 were identified. The pH-sensitivity generally resulted from a central cluster of histidine residues in CDRH1. When soluble, pH-sensitive, scFv clone M16 was dosed onto live cells, the internalized fraction was 2.6-fold greater than scFvs that lacked pH-sensitive binding and the increase was dependent on endosomal acidification. Differences in the intracellular distribution of M16 were also observed consistent with an intracellular decoupling of the scFv M16-TfR complex. Engineered pH-sensitive TfR binding could prove important for increasing the effectiveness of TfR-targeted antibodies seeking to exploit endocytosis or transcytosis for drug delivery purposes.

Expressing antibodies as fusions to the non‐self‐cleaving Mxe GyrA intein allows for site‐specific chemical functionalization via expressed protein ligation. It is highly desirable to maximize the yield of functionalizable protein; and previously an evolved intein, 202‐08, was identified that could increase protein fusion production in yeast. Given that the −1 amino acid residue upstream of inteins can affect cleavage efficiency, we examined the effects of amino acid variability at this position on 202‐08 intein cleavage efficiency and secretion yield. Varying the −1 residue resulted in a wide range of cleavage behaviors with some amino acids yielding substantial autocleaved product that could not be functionalized. Autocleavage was noticeably higher with the 202‐08 intein compared with the wild‐type Mxe GyrA intein and resulted directly from the catalytic activity of the intein. Refeeding of production cultures with nitrogen base and casamino acids reduced, but did not eliminate autocleavage, while increasing protein‐intein production up to seven‐fold. Importantly, two amino acids, Gly and Ala, at the −1 position resulted in good cleavage efficiency with no undesirable autocleavage, and can be used in concert with refeeding strategies to increase total functionalizable protein yield for multiple protein fusion partners. Taken together, we describe an optimized yeast expression platform for protein‐intein fusions. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2736, 2019