title: Extension of in vivo half-life of biologically active peptides via chemical conjugation to XTEN protein polymer.
abstract: XTEN, unstructured biodegradable proteins, have been used to extend the in vivo half-life of genetically fused therapeutic proteins and peptides. To expand the applications of XTEN technology to half-life extension of other classes of molecules, XTEN protein polymers and methods for chemical XTENylation were developed. Two XTEN precursors were engineered to contain enzymatically removable purification tags. The proteins were readily expressed in bacteria and purified to homogeneity by chromatography techniques. As proof-of-principle, GLP2-2G peptide was chemically conjugated to each of the two XTEN protein polymers using maleimide-thiol chemistry. The monodisperse nature of XTEN protein polymer enabled reaction monitoring as well as the detection of peptide modifications in the conjugated state using reverse phase-high performance liquid chromatography (RP-HPLC) and electrospray ionization mass spectrometry. The resulting GLP2-2G-XTEN conjugates were purified by preparative RP-HPLC to homogeneity. In comparison with recombinantly fused GLP2-2G-XTEN, chemically conjugated GLP2-2G-XTEN molecules exhibited comparable in vitro activity, in vitro plasma stability and pharmacokinetics in rats. These data suggest that chemical XTENylation could effectively extend the half-life of a wide spectrum of biologically active molecules, therefore broadening its applicability.
authors.affiliation: Amunix Operating Inc., 500 Ellis Street, Mountain View, CA 94043, USA.
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Podust
authors.forename: Bee-Cheng
authors.initials: BC
authors.lastname: Sim
authors.forename: Dharti
authors.initials: D
authors.lastname: Kothari
authors.forename: Lana
authors.initials: L
authors.lastname: Henthorn
authors.forename: Chen
authors.initials: C
authors.lastname: Gu
authors.forename: Chia-wei
authors.initials: CW
authors.lastname: Wang
authors.forename: Bryant
authors.initials: B
authors.lastname: McLaughlin
authors.forename: Volker
authors.initials: V
authors.lastname: Schellenberger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002626:Chemistry, Pharmaceutical / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D005260:Female; D053767:Glucagon-Like Peptide 2 / Q000097:blood / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics; D006207:Half-Life; D008969:Molecular Sequence Data; D010455:Peptides / Q000097:blood / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics*; D011108:Polymers / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D053767:Glucagon-Like Peptide 2; D010455:Peptides; D011108:Polymers
keywords: Chemical conjugation; GLP2; XTEN; half-life extension; protein polymer