ArmaGen Technologies-Enabling Biotechnology for the Brain

ArmaGen Technologies' molecular Trojan horses deliver protein neurotherapeutics to the brain

POTENTIAL OF PROTEIN-BASED THERAPEUTICS IN THE TREATMENT OF BRAIN DISEASES

Many disorders of the brain have proven refractory to small molecule therapeutics, and could be treated with protein therapeutics. Indeed, many protein lead drug candidates have been identified for brain diseases, but these proteins do not enter CNS drug development, because the proteins do not cross the blood-brain barrier (BBB). Instead, attempts are made to isolate small molecule peptidomimetics. However, it is very difficult to produce small molecule peptidomimetics. Moreover, in the rare case that a small molecule agonist is identified, this lead drug candidate most likely will not cross the BBB. Those small molecules that have a molecular weight > 400 Daltons, or form even a few hydrogen bonds, will not cross the BBB in pharmacologically significant amounts. (See Targeting Small Molecules). An alternative strategy is to re-formulate the protein drug candidate to enable transport across the BBB in vivo. This is done by genetically engineering a novel fusion protein, wherein the protein drug is fused to ArmaGen Technologies' molecular Trojan horse (MTH). The MTH part of the fusion protein triggers transport across the BBB via an endogenous receptor-mediated transport system. The following Table is a partial list of protein drug candidates for brain disease:

Brain Disorder	Protein Drug
Stroke	multiple neurotrophins
Alzheimer's disease	anti-Abeta monoclonal antibody
Acute brain injury; spinal cord injury	multiple neurotrophins
chronic brain injury (neurogenesis)	BDNF, FGF-2
Parkinson's disease	GDNF
Amyotrophic lateral sclerosis	BDNF, CNTF
Brain cancer	anti-EGF receptor monoclonal antibody
Lysosomal storage disorders of the brain	lysosomal enzymes
Schizophrenia	neuregulin-1
Depression	BDNF

None of these drugs can enter CNS drug development, because the drugs do not cross the BBB. However, if the drug was re-formulated to enable BBB transport, then the protein drug could enter into clinical CNS drug development with routes of administration no more invasive than an intravenous or subcutaneous injection. For many of the above CNS disorders, there is no effective treatment for the millions of individuals suffering from these disorders of the brain.

RECEPTOR-MEDIATED TRANSPORT (RMT) OF ENDOGENOUS PEPTIDES ACROSS THE BBB

Certain endogenous peptides cross the BBB via one of several receptor-mediated transport (RMT) systems. The RMT systems are endogenous transporters for circulating peptides such as insulin, transferrin, or the insulin-like growth factors (IGFs). The luminal and abluminal plasma membranes of the brain capillary endothelium express high amounts of the insulin receptor (IR), the transferrin receptor (TfR), the IGF receptor (IGFR), as well as other receptors, as illustrated in the adjacent figure. These endothelial receptor transport systems are depicted in the adjacent figure as the blue portals on the luminal membrane of the capillary endothelium in brain. The endogenous RMT systems are portals of entry to the brain from blood of the molecular Trojan horses (MTH) developed by ArmaGen Technologies.

The transcytosis of peptides across the BBB has been verified by electron microscopy, and occurs in 3 sequential steps: (1) receptor-mediated endocytosis of the blood-borne peptide at the luminal endothelial membrane, (2) movement through the thin endothelial compartment, which is only about 200 nm wide, and (3) exocytosis of the peptide at the abluminal endothelial membrane with release into brain interstitial space. The reverse transcytosis of peptides such as transferrin in the brain to blood direction also occurs, owing to the bi-directionality of the BBB transferrin receptor. In addition to the endogenous peptides, certain peptidomimetic monoclonal antibodies (MAb) also traffic across the BBB via these receptor transport systems. Either the endogenous peptide or the receptor-specific MAb can cross the BBB via the endothelial RMT systems. In order for the MAb to be an effective drug delivery vector, this MAb must be an 'endocytosing antibody', and bind the receptor at an exofacial epitope that enables endocytosis, and the MAb must bind a site on the receptor that does not interfere with the transport of the endogenous peptide. If an MAb is chosen for development as a BBB MTH, then the MAb must retain its affinity for the BBB receptor following the genetic engineering of the MAb to enable chronic use in humans.

RE-FORMULATION OF PEPTIDES, RECOMBINANT PROTEINS, ENZYMES, OR MONOCLONAL ANTIBODIES AS FUSION PROTEINS TO ENABLE TRANSPORT ACROSS THE BBB

Scientists at ArmaGen Technologies have developed proprietary MTHs that cross the primate and human BBB faster than neuroactive small molecules such as morphine. The lead MTHs, AGT-1 and AGT-2, have been genetically engineered to enable chronic use in humans without immune reactions. With the genes encoding AGT-1 or AGT-2, ArmaGen scientists can create novel fusion genes that encode for fusion proteins that have dual functions: (a) cross the BBB on one of the endogenous BBB RMT systems, and (b) bind the neuronal or glial receptor in brain to trigger the desired pharmacological effect. This bi-functionality is illustrated in the adjacent figure: the "head" of the fusion protein binds a BBB receptor to trigger uptake of the fusion protein into the brain, and the "tail" of the fusion protein then activates an endogenous receptor on brain cell membranes. In the case of enzyme drugs, where the enzyme must also distribute into the intra-cellular space of brain cells, the MTH also causes the receptor-mediated endocytosis of the enzyme into brain cells.

Drug developers may own the intellectual property of novel recombinant proteins, enzymes or monoclonal antibodies, that by themselves do not cross the BBB, and have little chance of being successful as a brain drug in clinical trials. ArmaGen can partner with CNS drug developers to re-formulate their protein-based neurotherapeutic and create a novel fusion protein that both crosses the human BBB and acts at specific drug receptors once inside the brain. ArmaGen Technologies has genetically engineered and expressed several fusion proteins, and these recombinant proteins retain the bi-functionality of both the BBB delivery part and the protein drug part. See Products.