Syntaxin has established an extensive ‘toolbox’ of genes encoding the components of its recombinant fusion proteins. These genes are encoded and cloned in a proprietary manner developed within Syntaxin to maximise the flexibility with which domains can be exchanged to create genes encoding new proteins. The proteins are expressed as single chain polypeptides with a specified enzyme cleavage site between the endopeptidase and the remainder of the polypeptide.
This enables selective activation of the expressed protein using a defined exogenous protease to produce the active di-chain protein. The linker region containing this activation site can be varied to optimise activation of the particular protein. Spacer regions are incorporated to optimise the spacing of the various components within the engineered protein, and the precise organisation of the component domains within the overall protein can be varied to suit the specific requirements of the final active protein. All of these elements of the design and construction of the active di-chain recombinant protein are components of Syntaxin’s proprietary technology and know-how in regard to producing functional TSI.
To date, Syntaxin has cloned over 1200 novel genes using this approach. The genes encode proteins targeted at a wide diversity of receptor targets across a range of ligand classes. Syntaxin has demonstrated the tolerability of TSI to the incorporation of peptide and protein ligands of between 8 amino acids and 50 kDa in size to facilitate targeted delivery into cells. In addition to selecting suitable targeting ligands to achieve selective and potent delivery of the endopeptidase domain, selection of the serotype of endopeptidase from the botulinum toxin family of proteins enables a specified SNARE pathway to be modulated and determines the duration of inhibition of secretion.
The resulting recombinant proteins are purified and are assessed for solubility, purity and integrity, before being put through a series of relevant pharmacological screens to confirm functionality and target tractability. Functionality of the TSI is measured via an assay cascade to ensure that the proteins have all of the required characteristics. The targeting component of the protein is assessed for both receptor binding affinity and activation. The endopeptidase domain is assessed for SNARE cleavage activity. The ability of the protein to deliver the endopeptidase into the cell cytosol and enable intracellular SNARE protein cleavage is assessed in cell models possessing both the relevant receptor/ binding site and also the appropriate SNARE protein, thus confirming receptor dependent delivery of the endopeptidase. Finally, cell assays involving all of the components of the target: receptor, SNARE protein and secretory function are used to demonstrate that the SNARE protein cleavage results in the inhibition of the intended secretion process within the target cell. Having characterised and established functionality of the TSI, lead candidates are assessed in vivo for their pharmacological properties and therapeutic potential.
Syntaxin continues to develop its technology in respect of creating novel TSI. This builds on its existing dominant position and enhances the opportunity for applying the technology to the creation of novel therapeutic proteins.
To date the ‘work-horse’ expression system that Syntaxin has employed to great success in expression of its proteins has been E. coli. This has enabled the successful development and transfer to a CMO of a GMP-compatible manufacturing process for the drug candidate in pain. Production of Syntaxin’s proprietary proteins in standard prokaryotic expression systems means that manufacture is highly scaleable and economic. Syntaxin has explored alternative expression systems for production of its proteins. To date, expression of our proteins has been demonstrated in both yeast and insect cell line systems.
Nature has provided a large number of natural neurotoxin endopeptidase variants that cleave different SNARE proteins, and selection of the appropriate serotype has enabled Syntaxin to develop a large variety of TSI that address specific secretion pathways. Not all SNARE proteins involved in vesicular secretion are, however, substrates for the natural neurotoxin endopeptidases and the company is exploring opportunities to overcome this constraint. Based upon understanding of the enzyme – substrate interaction at the active site, Syntaxin is developing rationally designed mutants of native neurotoxin endopeptidase able to cleave previously non-substrate SNARE proteins. This further enhances the breadth of options available to the company for creation of TSI.
Syntaxin has so far focused on using peptide or protein ligands to cell surface receptors as the binding domains for cell secretion inhibitors, and this has proved very successful at producing potent and selective proteins for targeting cells of choice. Alternative binding approaches, in particular use of antibodies, antibody domains and fragments and other binding scaffolds, would complement this approach and open up categories of cell surface binding sites not suited to traditional receptor ligand approaches and also receptors to non-peptide ligands. Syntaxin has demonstrated an ability to express proteins using both scFv and dAb fragments as the binding domain component. There is a particular opportunity to combine Syntaxin’s expertise and capability in regard to TSI with partners possessing novel cell targeting capabilities to create proteins able to impact secretory pathways in defined cell targets of therapeutic value.