TECHNOLOGY
Wild Type BoT
The active form of BoT is a disulfide-bonded heterodimer, consisting of the toxic Light Chain (LC, 50 kD) protease and a Heavy Chain (HC, 100kD) which mediates specific binding to neurons and translocation of the LC protease into the neuronal cytoplasm.
The HC consists of two domains: the receptor binding domain (RBD) mediates highly specific binding to neuronal membranes.
The translocation domain (TD) mediates the translocation of the toxic LC protease to the neuronal cytoplasm.
CytoDel's Technology Recombinant BoT (rBoT)
CytoDel’s rBoT platform enables the creation of a new class of antibody fusion proteins, that specifically deliver their payload to previously inaccessible targets in the neuronal cytoplasm.
CytoDel’s technology platform is based on the unique ability of botulinum toxin (BoT) to specifically target neurons, and deliver its toxic protease to the neuronal cytoplasm at sub-nanogram doses. The BoT protease cleaves SNARE proteins in the neuronal cytoplasm that are essential for the fusion of synaptic vesicles with the neuronal membrane, thereby disabling neurotransmitter release and muscle activation. The approved clinical indications for BoT are all based on weakening overactive muscles by direct intramuscular injection, to alleviate movement disorders and associated pain.
Bioengineering
Production of recombinant BoTs is difficult because of the protein’s large size and complex structure. CytoDel’s proprietary genetic constructs, expression systems and purification methods use the tools of molecular biology to engineer novel rBoT derivatives, creating de-novo molecules for the BoT pharmaceutical category. Current BoT products cannot be engineered because they are all produced from the same native bacterial source, cultures of Clostridium botulinum, and thus cannot be readily manipulated using available molecular biology techniques.
Expression and Purification
CytoDel’s expression and purification platform produces de-novo engineered BoT derivatives, synthesized as precursors. These precursors are then purified using a gentle two-step affinity purification process and activated as required during a final production step before packaging.
Intraneuronal delivery via rBoT Antibody Fusion Proteins
CytoDel is developing a new class of Antibody Fusion Proteins (AFPs) based on rBoTs designed to deliver therapeutic antibodies to the neuronal cytoplasm. The LC protease is disabled by genetic substitution, but the BoT’s native trafficking to the neuronal cytoplasm is retained. This enables the construction of rBoT AFPs, designed to deliver therapeutic antibodies to the neuronal cytoplasm and access targets not accessible to conventional antibodies.
This diagram shows the structure of CytoDel’s lead rBoT AFP, Cyto-111, an atoxic rBoT derivative genetically fused to the B8 camelid antibody. B8 is a single chain antibody (VHH) that specifically inactivates the cytoplasmic form of the LC protease of BoT/A. CytoDel genetically fuses B8 to CytoDel’s atoxic rBoT molecular vehicle (C1ad, atoxic derivative of BoT serotype C1) to create the B8-C1ad antibody fusion protein (AFP), now proven to deliver its B8 therapeutic cargo to the neuronal cytoplasm in three species.
“ The modular structure of BoT enables CytoDel to engineer rBoT pharmaceuticals tailored to diverse indications”
- Philip Band, CEO
Biobetter BoTs
The global market for BoT pharmaceuticals is approximately $5B, and still growing. CytoDel’s technology platform enables the design of “biobetter” rBoTs that have clinical advantages customized to specific BoT indications. The enzymatic activity of rBoTs can be modulated to achieve specific medical improvements aimed at current and new pharmaceutical indications.