Extension of in vivo half-life of biologically active molecules by XTEN protein polymers
Journal of Controlled Release
XTEN™ is a class of unstructured hydrophilic, biodegradable protein polymers designed to increase the half-lives of therapeutic peptides and proteins. XTEN polymers and XTEN fusion proteins are typically expressed in Escherichia coli and purified by conventional protein chromatography as monodisperse polypeptides of exact length and sequence. Unstructured XTEN polypeptides have hydrodynamic volumes significantly larger than typical globular proteins of similar mass, thus imparting a bulking
... rting a bulking effect to the therapeutic payloads attached to them. Since their invention, XTEN polypeptides have been utilized to extend the half-lives of a variety of peptide-and protein-based therapeutics. Multiple clinical and preclinical studies and related drug discovery and development efforts are in progress. This review details the most current understanding of physicochemical properties and biological behavior of XTEN and XTENylated molecules. Additionally, the development path and status of several advanced drug discovery and development efforts are highlighted. The second half-life extension approach exploits recycling through the FcRn receptor (reviewed in [14, 17, 18] ). Two plasma proteins, serum albumin and IgG, have exceptionally long half-lives in circulation. When taken up by cells through nonspecific endocytosis, both proteins bind to the FcRn receptor in acidic endosomes and become protected from intracellular degradation. The protein-receptor complexes are recycled to the cell surface, where albumin and immunoglobulin molecules are released back into circulation at the neutral pH of the plasma. Several methods have been used to exploit the increased halflife of IgG or albumin for therapeutic payloads: (1) genetic fusion of the payload to the Fc domains of IgG or to the albumin polypeptide; (2) chemical conjugation of the payload to the albumin, IgG or Fc domain; (3) fusion or conjugation to mediator molecules which physically bind to IgG or albumin. Examples of such mediators are IgG-binding peptides, fatty acids, albumin-binding peptides, and albumin-specific antibody fragments [14, 16] . This review focuses on XTEN, a class of unstructured biodegradable protein polymers developed by Amunix to increase the half-lives of therapeutic peptides and proteins genetically fused or chemically conjugated to them. XTEN polymers were conceived as non-immunogenic polypeptides consisting of the six hydrophilic, chemically stable amino acids A, E, G, P, S and T. In order to create genetically and chemically stable polypeptides that lack secondary structure and that are highly expressed in Escherichia coli, a library of non-repetitive 36 amino acidlong segments was generated, and nearly 1500 unique segments were screened for expression and biophysical properties. Selected segments were iteratively ligated, and the resulting molecules were rescreened for maximal expression, yielding a series of 864 residue-long polypeptides. The five most highly expressed of these polypeptides were tested for genetic stability, solubility in aqueous media, heat stability, and propensity to aggregate. Emerging from the selection process was the polypeptide designated as XTEN  . The prototype molecule was followed by development and characterization of multiple derivatives based on the parental XTEN molecule, and today, the name XTEN applies to the entire group of polypeptides. The physicochemical and biological properties of XTEN protein polymers, their manufacturability and specific applications are discussed below.