SBIR-STTR Award

Protein drugs have been approved for many therapeutic indications and represent a rapidly growing segment of the pharmaceutical industry. However, many approved protein drugs and candidates in development fail to reach their potential efficacy due to suboptimal pharmacokinetic properties and immunogenicity concerns. These properties include short circulating half-lives, short shelf lives, low solubility, rapid kidney clearance and susceptibility to proteolytic degradation. The modification of proteins with hydrophilic chemical polymers like polyethylene glycol (PEG) is a clinically validated approach to addressing these limitations. However, the challenges associated with chemical modification procedures required for the attachment of these polymers present significant challenges. Our ultimate goal is to generate amino acid sequences that mimic the physiochemical properties of hydrophilic chemical polymers like PEG. Our proposal is based on the observation that glycine rich sequences (GRS) which contain few hydrophobic amino acids will not fold into compact 3-dimensional structures but will adopt random conformations with large hydrodynamic radii similar to PEG. We hypothesize that they will confer similar pharmacokinetic improvements when attached to therapeutic proteins. These sequences can be attached to proteins using conventional recombinant technology and thus completely obviates the need for chemical modifications steps. We have synthesized a 198 amino acid glycine rich sequence based on sequences that occur in human proteins. We aim to express this protein and systematically test its physiochemical and biological properties relevant to pharmacokinetic enhancement. Our specific aims are: 1) Produce 5 mg of a purified GRS protein in E. coli expression system for downstream characterization and studies. 2) Characterize serum stability, protease resistance and biophysical properties of the GRS protein. 3) Characterize plasma pharmacokinetics and immunogenic potential of the GRS protein in animal models. In Phase II, we will perform detailed optimization of GRS for high-level expression, plasma half-life extension and reduced immunogenicity. We aim to advance optimized GRS which are fused to pharmaceutically active proteins like interferon-alpha or G-CSF into animal and clinical studies. Our ultimate goal is to validate and make this method readily applicable to therapeutic proteins. Our project aims to generate glycine rich sequences that can be attached recombinantly to therapeutic proteins to improve their pharmacokinetic properties. This approach would circumvent the difficulties associated with the modifying proteins with hydrophilic polymers like PEG.

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The market for protein-based biopharmaceuticals is rapidly expanding. However, the clinical utility of many proteins is limited by their short serum half-life, requiring frequent injections. The most common approach to improve serum half-life is PEGylation. The chemical conjugation of Polyethylene glycol (PEG) to therapeutic proteins typically results in product mixtures that include inactive isomers and reduce the overall potency of the product. The chemical PEGylation of proteins significantly increases manufacturing costs, requiring precise process control and analytical assays to ensure reproducibility of product composition. The current project describes polypeptide chains (called rPEGs) that mimic the physicochemical properties of chemical PEG. rPEGs are hydrophilic and have very large hydrodynamic radii. Most importantly, rPEGs can be recombinantly fused to therapeutic proteins resulting in homogeneous products. The fusion of rPEGs to biopharmaceutical is expected to provide benefits that are similar to chemical PEGylation (long serum half-life, reduced immunogenicity) while offering improved product potency, homogeneity, and significantly reduced manufacturing costs. Phase I of this project was extremely successful and demonstrated the feasibility of the rPEG technology. rPEG sequences that closely mimic the properties of chemical PEG were developed. We demonstrated that a 20 kDa rPEG sequence has an apparent molecular weight of 180 kDa. Fusion of rPEG to the model protein GFP increased its serum half-life in rats from 1-3 h to approximately 10 h, similar to the effect of chemical PEGylation. Furthermore, this rPEG-GFP fusion elicited only a very weak immune response in rats. Our phase II goal is to apply rPEG technology to human growth hormone (hGH). hGH is currently used for the treatment of dwarfism in children. 2006 sales exceeded $3B. Due to its rapid plasma elimination, hGH treatment requires daily injections. No long-acting form of hGH has been approved and chemical PEGylation of hGH had limited success due to the formation of mixtures containing inactive isomers. We aim to develop methods for the production, purification, and formulation of rPEG-hGH. The resulting product will be thoroughly characterized in vitro and in vivo. The resulting data package will allow rPEG- hGH to enter clinical development. The methods and data developed during the project will validate rPEG technology and enable its broad application to other protein pharmaceuticals.

Public Health Relevance:
The utility of many biopharmaceuticals is limited by their short serum half-life, which requires frequent injections. The goal of this project is to develop recombinant peptide chains (called rPEG) that mimic the properties of polyethylene glycol. These rPEGs can be directly fused to protein pharmaceuticals to increase their serum half-life. We will validate rPEG technology by developing a long-acting version of human growth hormone for the treatment of dwarfism in children.

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