Technology
CytoDel's Technology - Recombinant BoNT
CytoDel’s patented and proprietary technology uses the tools of 21st century molecular biology to produce recombinant derivatives of botulinum neurotoxin (BoNT). The technology allows the Company to manipulate the BoNT molecule into a “Trojan Horse” drug delivery vehicle that can deliver therapeutic molecules to the inside of neurons, with important implications for both Biodefense and advancing the treatment of nervous system disorders, chronic pain and neurodegenerative diseases. The Company’s recombinant BoNT derivatives retain the biological properties of wild type (wt) BoNT, preserving the toxin’s native structure and trafficking properties. CytoDel is the first company to produce an engineered BoNT/A derivative for pharmaceutical applications, which reduces toxicity while maintaining pharmacologic activity. CytoDel has also created novel first-in-class BoNT-based Antibody Fusion Proteins (AFPs), which specifically deliver functional single chain antibodies to the cytoplasm of neurons. These AFPs provide access to previously inaccessible intraneuronal targets, without the need for delivery via a viral vector.
BoNT Structure
The active form of BoNT is a disulfide-bonded heterodimer consisting of a Heavy Chain (HC) and a Light Chain (LC), which are structurally held together in the active protein by a disulfide bond (S-S). This structural composition makes it difficult to produce active recombinant derivatives in bacterial systems at high yield.
Bioengineering
Production of recombinant BoNTs 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 modern molecular biology to engineer novel BoNT derivatives, creating de-novo molecules
for the BoNT pharmaceutical category. The Company’s recombinant BoNT derivatives can be customized for improved performance in established indications
and enable indications currently not approved for treatments using Botulinum neurotoxins (e.g. Botox®, Xeomin®, Dysport®).
Current botulinum 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.
The current Clostridia-derived BoNT products are difficult to engineer to specifically improve their pharmaceutical properties.
The products on the market differentiate themselves based only on purity, therapeutic potency, formulation, serotype and market familiarity.
Expression and Purification
CytoDel’s expression and purification platform produces de-novo engineered BoNT 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.
Antibody Fusion Proteins
CytoDel bioengineers Antibody Fusion Proteins (AFPs) to provide a first-in-class mechanism that specifically delivers antibodies to therapeutic targets inside the neuronal cytoplasm without a viral vector. The enzymatic activity of CytoDel’s BoNT-based AFPs has been eliminated, to create a molecular vehicle that can specifically carry therapeutic drugs into the neuronal cytoplasm.
Our modular technology allows us to design recombinant pharmaceutical BoNTs for tailored indications
Intra-Neuronal Delivery Platform
The CytoDel Delivery Platform is based on the natural trafficking pathway of the BoNT molecule. BoNT passes across the epithelia in the gut or lung and
can circulate stably in the blood until it specifically binds to the surface of peripheral neurons. The BoNT molecule is then internalized into an acidified
endocytic compartment, where its “translocation domain” creates a channel through which the toxic protease portion of the molecule translocates into the presynaptic
neuronal cytoplasm. This toxic protease disables the molecular machinery for neurotransmitter release and does so at lower doses than any other known protein toxin
or drug. Interestingly, while BoNT can completely shut down neurotransmitter release, it has no known cytotoxic effects on the neuron itself.
CytoDel has coopted the BoNT trafficking pathway to create an entirely new class of drugs directed to intra-neuronal targets. Recombinant
derivatives of BoNT are engineered to render them atoxic by disabling the toxin protease, while retaining the trafficking properties of the native
molecule. These atoxic BoNT-based molecular vehicles can deliver drugs to the cytoplasm of neurons via a Trojan Horse type mechanism, enabling delivery
to previously inaccessible targets inside neurons. The high specificity of neuronal targeting is a consequence of BoNT having a “dual receptor” on the
neuronal surface: it requires both a protein receptor and a specific lipid receptor for binding and internalization. CytoDel has developed and patented
two ways to use atoxic BoNT molecular vehicles for intra-neuronal drug delivery: 1) via a genetically inserted peptide that acts as an acceptor for
enzymatically attaching a drug with an intra-neuronal target; 2) by genetic fusion of a peptide drug, such as a single domain antibody, which is directly
carried to an intra-neuronal site of action.
This diagram shows the structure of Cyto-111 composed of an atoxic derivative of BoNT and a single-domain antibody. B8 is a single domain antibody (VHH) that specifically inactivates the cytoplasmic form of the LC protease of BoT/A. B8 is genetically fused to CytoDel’s BoNT-based molecular vehicle (BoNT/C1ad) for delivery to the neuronal cytoplasm.
Biobetter Cyto-BoNTs
CytoDel’s technology platform is the first to produce recombinant biobetter BoNTs. The enzymatic activity of BoNTs can be bioengineered to modulate its enzymatic activity to achieve specific medical improvements aimed at BoNTs current and new pharmaceutical indications.
Technological Advantage
CytoDel has made significant progress for applications in the pharmaceutical BoNT and the biodefense (neuronal drug delivery) markets. The foundation has also been laid to develop products that address important neurological diseases, by providing access to therapeutic targets in the neuronal cytosol that have been previously difficult to access.