(649c) Engineered Protein-Polymer Conjugates for Drug Delivery to the Central Nervous System

The blood-brain barrier (BBB) protects the central nervous system (CNS) from potentially harmful compounds that circulate in the blood, presenting a major obstacle to delivering drugs intended to treat disorders of the CNS like Alzheimer’s disease, Parkinson's disease, or brain tumors. In order to transport specific molecules that are needed for CNS function into the brain, there are dedicated receptors on the BBB to facilitate active transport from the blood into the CNS. One strategy to address the challenge of drug delivery to the CNS is the use of receptor mediated transport, utilizing native transport systems to carry non-native cargo, such as a therapeutic, into the brain.

In our work, we have engineered proteins that bind to the human transferrin receptor (TfR) and are developing methods to conjugate these proteins to novel polymer systems to create drug delivery complexes. TfR is a BBB receptor whose native function is in iron regulation, and the receptor has previously been shown to effectively transport therapeutic cargo across the BBB into the CNS in animal studies. Despite much progress in pursuing receptor mediated transport into the brain, there are still no FDA approved therapeutics that use receptor mediated drug transport. In previous studies, several essential qualities needed for a successful use of TfR mediated drug transport have been elucidated. The binding affinity of an engineered molecule that targets TfR should optimally be ~100s nanomolar dissociation constant, so that the drug complex can bind to the TfR in brain blood vessels and be released after crossing into CNS tissue. To avoid serious toxic side effects, TfR binding molecules should not interfere with native transferrin ligand binding. Furthermore, if a targeting molecule is to be used as part of a drug delivery complex, the targeting molecule should be suitable for appropriate chemical modifications, such as conjugation to a polymer system.

Using yeast surface display and directed evolution, we have engineered TfR binding proteins based on the 10^th domain of human fibronectin type III (Fn3), beginning with a naïve Fn3 library. Our engineered Fn3 proteins bind TfR in the range of the optimal binding affinity to specifically bind TfR and then be released into the CNS. Our results indicate that the candidate drug delivery proteins do not compete with the transferrin ligand binding epitope. The highly stable, single domain structure of the approximately 10 kDa Fn3 proteins are amenable to further chemical modification in a variety of solvent systems. We are currently conjugating our engineered targeting proteins to multifunctional linear polymer and micelle systems towards the ultimate goal of developing a modular drug delivery complex for the central nervous system.