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.

Glucagon-like peptide 2 and its beneficial effects on gut function and health in production animals

“…Because of GLP-2's short (approximately 7-min) half-life in humans [44], administration of DPP-IV inhibitors has been used to enhance the therapeutic effects of GLP-2 [45][46][47], and synthetic GLP-2 analogs that are resistant to DPP-IV degradation have been used to prolong and increase its biological activity. Modifications include replacing the Ala (2) residue with Gly [40,42], which increases the half-life of GLP-2 to approximately 3 h in humans [48], or conjugating the peptide to various polymers, which extends the half-life in humans to as great as 10 d, based on theoretical projections from animal models [49][50][51]. Further advances in our understanding of factors that increase GLP-2 synthesis and means to enhance its biological activity will support its potential use as an enhancer of animal productivity, health, and well-being.…”

Unraveling the bioactivity of anticancer peptides as deduced from machine learning

“…However, it should be noted that it is this particular characteristic of therapeutic peptides that allows it to escape resistance unlike other oncogenic therapies. However, research on improving the half-life of peptides without compromising their potency is currently an active area of research (Hao et al, 2015[25]; Podust et al, 2013;[68] Schellenberger et al, 2009[74]; Garay et al, 2012[20]; Penchala et al, 2015[67]). Despite some limitations, no other class of peptides have been able to surpass the multi-functionality of bioactive/therapeutic peptides and thus, these peptides possess high potential for use in many avenues of clinical applications.…”

Multivalent Antiviral XTEN–Peptide Conjugates with Long in Vivo Half-Life and Enhanced Solubility

“…To characterize the stability of the succinimide thioethers, we previously evaluated the in vitro plasma stability of the thioether bond in XTENylated proteins produced using the same chemistry, and the half-life of the bond was determined to be greater than 10 days. 11 …”