Piggybacking on the Cholera Toxin: Identification of a CTB-Binding Protein as an Approach for Targeted Delivery of Proteins to Motor Neurons

Abstract

A significant unmet need exists for the delivery of biologic drugs such as polypeptides or nucleic acids to the central nervous system for the treatment and understanding of neurodegenerative diseases. Naturally occurring bacterial toxins have been considered as tools to meet this need. However, due to the complexity of tethering macromolecular drugs to toxins and the inherent dangers of working with large quantities of recombinant toxins, no such route has been successfully exploited. Developing a method where a bacterial toxin's nontoxic targeting subunit can be assembled with a drug immediately prior to in vivo administration has the potential to circumvent some of these issues. Using a phage-display screen, we identified two antibody mimetics, anticholera toxin Affimer (ACTA)-A2 and ACTA-C6 that noncovalently associate with the nonbinding face of the cholera toxin B-subunit. In a first step toward the development of a nonviral motor neuron drug-delivery vehicle, we show that Affimers can be selectively delivered to motor neurons in vivo.

In-Depth Characterization of a Re-Engineered Cholera Toxin Manufacturing Process Using Growth-Decoupled Production in Escherichia coli

“…The binding of rCTC increased with concentration as expected, showing that each variant produced soluble and active rCTC, but the slow dissociation of rCTC from the GM1 receptor prevented K d determination. Therefore, Isothermal Titration Calorimetry (ITC) was used to compare the K d for each variant binding to GM1 oligosaccharide (Figures 8 and S10) [5,50]. The values of ∆H (enthalpy change) and log 10 [K AB ] (log-binding affinity), for all rCTC samples, agreed with those for commercial CTxB (Sigma), within a 68.3% confidence interval (Table S1).…”

“…Injection of CTxB into a muscle can, thus, allow uptake of the protein into motor neurons for retrograde trafficking to the cell body, which is located in the spinal cord or brainstem. Wild-type CTxB, re-engineered CTxB proteins, and their complexes have, therefore, been widely exploited as a neuronal tracer [5][6][7][8]. Non-toxic AB 5 complexes of analogous bacterial toxins have, also, been reported for the targeted delivery of proteins to the cytosol of neurons [9].…”

Synthesis of cholera toxin B subunit glycoconjugates using site-specific orthogonal oxime and sortase ligation reactions

“…Oxime ligation at the N-terminus of CTB was first described for the construction of well-defined CTB-viral peptide immunogens ( Chen et al, 2003 ). We have previously applied oxime ligation of CTB for the preparation of multivalent neoglycoprotein bacterial toxin inhibitors and for N-terminal biotinylation of CTB for phage display ( Branson et al, 2014 ; Balmforth et al, 2021 ). To explore if oxime and C-terminal sortase-mediated ligation could be used orthogonally, the N-terminus of CTB–LPETGA was first biotinylated before the peptides and glycopeptides ( 10 , 13 – 18 ) were conjugation to the C-terminus using CBD-Sortase 7M.…”

“…This reaction typically reaches completion within 5 min in sodium phosphate ( Figures 7A,B ), whereas the reaction does not reach completion in phosphate-buffered saline containing potassium ions, which are known to hinder periodate reactivity ( Brabham et al, 2020 ). Previously oxime ligation of alkoxyamine–PEG 4 –biotin was used for the N-terminal biotinylation of wt CTB; this was sufficient to allow effective binding to streptavidin for phage display screening ( Balmforth et al, 2021 ). Using the same alkoxyamine–PEG 4 –biotin (10 eq) in the presence of aniline (1% v / v ) at 37°C, oxime biotinylation of the oxidized CTB reached completion within 16 h ( Figure 7C ).…”

“…However, the non-toxic CTB subunit is also widely used as a neuronal cell tracer ( Vercelli et al, 2000 ; Saleeba et al, 2019 ), and it has also been used as a scaffold for the preparation of multivalent neoglycoprotein inhibitors of bacterial toxin adhesion and as a mucosal adjuvant ( Holmgren et al, 1993 , 2005 ; Pizza et al, 2001 ; Branson et al, 2014 ). It is our interest in these applications of the CTB protein that has led us to develop a range of ligation methods for the site-specific modification of this protein at its N-terminus and also through site-specific incorporation of azidohomoalanine for bioorthogonal ligation reactions ( Branson et al, 2014 ; Machin et al, 2019 ; Haigh et al, 2020 ; Balmforth et al, 2021 ). The work reported here extends our toolbox of methods to C-terminal modification of CTB by exploring the feasibility of using sortase to conjugate naturally occurring glycopeptide epitopes to produce a series of well-defined glycoconjugates.…”