Surface-engineered bacteria in drug development

Abstract

Bacterial surface display in combination with fluorescence-activated cell sorting is a versatile and robust system and an interesting alternative approach to phage display for the generation of therapeutic affinity proteins. The system enables real-time monitoring and sorting of cell populations, which presents unique possibilities for drug development. It has been used to develop several affibody molecules currently being evaluated preclinically for the treatment and diagnosis of, for example, cancer and neurodegenerative diseases. Additionally, it can be implemented in other areas of drug design, such as for mapping epitopes and evolving enzyme specificities.

FIGURE 1

(A) Schematic overview of bacterial cell surface display methodology. I. A DNA library coding for millions or billions of different protein variants is used to transform bacterial cells, typically by heat shock or electroporation methods. II. Cells express the protein library, often fused to a normalization tag such as an albumin‐binding domain (ABD), and display it on the surface through different cellular mechanisms. III. The cells are incubated with fluorescently labelled target antigen and reporter (for example, human serum albumin). Here, the stringency of the selection round can be adjusted by changing the number of washing steps or dissociation times. IV. The cells are sorted through a flow cytometer to be discarded or kept depending on the desired level of fluorescent signal determined through gating. V. The entire binding cell population can be amplified for additional selection rounds or single clones can be analysed by sequencing and by direct binding analysis using flow cytometry. (B) Mechanisms by which the protein library (POI) can be displayed on the cell surface. For display on the Gram‐negative E. coli, a method using the autotransporter secretion pathway of AIDA‐I can be used to express, secrete and anchor the protein of interest on the cell surface. (C) For display on Gram‐positive S. carnosus, a cell‐wall anchoring sequence (XM) is used to anchor the protein library on the cell wall following secretion, directed by the signal peptide (S), through the cell membrane.

FIGURE 2

Illustration of drug strategies that have been explored using affibody molecules generated from bacterial cell display. (A) A bivalent HER3‐targeting affibody molecule comprised of two identical HER3‐binding domains that bind an epitope overlapping with that of the natural ligand heregulin and an albumin‐binding domain that prolongs the circulatory half‐life by association with human serum albumin (HSA). (B) An EGFR‐binding affibody molecule is fused by a protease substrate linker sequence to an anti‐idiotypic affibody masking domain with affinity for its binding surface, allowing the drug to be conditionally activated in protease‐rich tumour environments while remaining inert in circulation and healthy tissues. (C) VEGFR2‐targeting affibody molecules that either act as an inhibitor for the ligand VEGF when formatted as a biparatopic molecule but promote proliferation and angiogenesis when formatted as a tetrameric molecule by acting as an artificial ligand. (D) Affibody molecules can be functionalized site‐specifically using unique cysteine residues with, for example, cytotoxic small molecular drugs for increasing drug potency or radioactive compounds for diagnostic radioimaging applications. (E) Affibody‐based sequestrin molecules with a unique conformation that binds to monomeric amyloid ß peptides by enveloping them with high affinity, thus preventing the formation of large aggregates.