Extension of in vivo half-life of biologically active peptides via chemical conjugation to XTEN protein polymer†

Vladimir N.Podust 0 Bee-Cheng Sim 0 Dharti Kothari 0 Lana Henthorn 0 Chen Gu 0 Chia-wei Wang 0 Bryant McLaughlin 0 Volker Schellenberger 0 0 Amunix Operating Inc. , 500 Ellis Street, Mountain View, CA 94043 , USA 1To whom correspondence should be addressed. E-mail: 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 GLP2-2GXTEN, 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. Introduction One of the major challenges associated with the development of therapeutic drugs is ensuring that the intact pharmaceutical molecule persists long enough in the blood to carry out its intended function. While some drugs are more susceptible to in vivo degradation than others, the problem of half-life extension is relevant to all major classes of therapeutics, and extensive efforts are made in designing and testing them for This publication is dedicated to the memory of Willem Pim Stemmer (19572013), a co-founder of Amunix Operating Inc. pharmacokinetic stability while retaining physiological efficacy (Kontermann, 2012). Several methods have been described to prolong the plasma half-life of biologically active molecules, and perhaps the most widely employed is their conjugation to polyethylene glycol (PEG) (Veronese and Pasut, 2005; Ryan et al., 2008; Bailon and Won, 2009; Kang et al., 2009). This water-soluble, polar polymer acts as a bulking agent, providing extra mass and therefore slowing glomerular filtration of the drug. PEG can also act as a physical barrier to prevent the drug breakdown induced by other plasma factors and to decrease its immunogenicity (Caliceti and Veronese, 2003; Fishburn, 2008). However, PEG itself may be more immunogenic than initially believed. Population exposure to PEG and PEG-containing compounds in cosmetics, pharmaceuticals and processed food products led to the significant increase of anti-PEG antibodies occurrence in healthy blood donors (Garay et al., 2012). Since PEG is a chemical polymer that cannot be metabolized, cellular vacuolation has been observed following treatment with PEGylated compounds in animal studies (Bendele et al., 1998; European Medicinal Agency, 2012; Rudmann et al., 2013). We have developed protein polymers called XTEN, specifically engineered to mimic the beneficial properties of PEG while avoiding some of its disadvantages, e.g. immunogenicity and lack of biodegradability. Highly hydrophilic, unstructured polypeptides, XTEN are soluble, stable and non-immunogenic (Schellenberger et al., 2009). Their fusion to protein and peptide drugs resulted in half-life extension by 60- to 130-fold over native molecules, in addition to conferring increased heat stability and solubility (Schellenberger et al., 2009; Geething et al., 2010; Alters et al., 2012; Cleland et al., 2012; Yuen et al., 2013). Two recombinant-based XTENylated products are currently being evaluated in the clinic, namely, VRS-859 (ExenatideXTEN) and VRS-317 (human growth hormone-XTEN). Under Phase 1a studies, VRS-859 was found to be well-tolerated and efficacious in patients with type 2 diabetes (http://www. diartispharma.com/content/newsandevents/releases/100212 .htm), and VRS-317 reported superior pharmacokinetic and pharmacodynamic properties compared with previously studied recombinant human growth hormone products and has the potential for once-monthly dosing (Yuen et al., 2013). The genetic fusion of therapeutic peptides and proteins to XTEN offers the convenience of expression and purification of a single, homogenous drug molecule. However, recombinant XTEN fusion does come with some limitations. Both XTEN and its partner payload molecule must be composed of natural L-amino acids and fusion proteins are produced as linear, unidirectional polypeptides. Also, either the N- or C-terminus of the therapeutic payload must be dispensable for biological activity to allow for attachment of the XTEN. To increase the applicability of XTEN for a wider range of drug classes, XTEN protein polymers containing reactive groups for chemical conjugation were created, and GLP2-2G peptide was chosen as an example payload. Glucagon-like peptide 2 (GLP2), a specific gastrointestinal growth factor, has been shown to possess potent intestinotrophic properties as well as strong reparative activity for the mucosal epithelium of the small and large intestine (Drucker et al., 1996; Brubaker et al., 1997; Tsai et al., 1997). Teduglutide, a human GLP2-2G variant resistant to dipeptidyl peptidase IV, has been approved by the US Food and Drug Administration (FDA) for treatment of short bowel syndrome in 2012 and is in clinical evaluation for Crohns disease (Buchman et al., 2010). A major limitation of teduglutide, however, is its short half-life of 13 h in humans, resulting in a cumbersome daily dosing regimen. We have previously evaluated recombinant GLP2-2G-XTEN protein in a rat model of Crohns disease and have demonstrated that in addition to significant half-life extension of .75-fold, fusion to XTEN also resulted in improved in vivo efficacy plus dose-sparing potential relative to GLP2-2G peptide (Alters et al., 2012). This genetically fused GLP2-2G-XTEN was used as a reference for the chemically conjugated versions. Here, the design, production, analysis and use of reactive XTEN to create conjugates linked to GLP2-2G are described, and the potential of this novel half-life extension tool to be applied to a broad range of therapeutics is discussed. Materials and methods Peptides GLP2-2G peptide was purchased from American Peptide Company (cat. #304076). Synthetic peptides GLP2-2G-Cys (HGDGSFSDEMNTILDNLAARDFINWLIQTKITDC) and GLP2-2G-Mal (HGDGSFSDEMNTILDNLAARDFINWLIQ TKITDK-Ahx-Mpa, where Ahx is 6-aminohexanoic acid conjugated to t (...truncated)