title: XTEN-annexin A5: XTEN allows complete expression of long-circulating protein-based imaging probes as recombinant alternative to PEGylation.
abstract: The coupling of polyethylene glycol (PEG) to proteins (PEGylation) has become a standard method to prolong blood circulation of imaging probes and other proteins, liposomes, and nanoparticles. However, concerns have arisen about the safety of PEG, especially with respect to its poor biodegradability and antibody formation, including new evidence about preformed anti-PEG antibodies in a quarter of healthy blood donors. Here, we apply a new hydrophilic polypeptide XTEN to extend the blood half-life of an imaging probe. As an example, we chose annexin A5 (AnxA5), a recombinant 35-kD protein extensively used for the in vitro and in vivo detection of apoptosis, that has a blood half-life of less than 7 min in mice, limiting its accumulation in target tissues and therefore limiting its utility as an imaging reagent.
  
  
  METHODS
  The sequence of XTEN was developed by Volker Schellenberger and colleagues by evolutionary in vitro optimization to yield PEG-like properties but provides several key advantages in comparison to PEG. The DNA of a 288-amino-acid version of XTEN with an additional N-terminal cysteine for site-directed coupling was fused to AnxA5 (XTEN-AnxA5). The fusion protein could be highly expressed in Escherichia coli and efficiently purified using XTEN conveniently as a purification tag. It was labeled with a thiol-reactive fluorescent dye and via a chelator with a radionuclide.
  
  
  RESULTS
  SPECT/CT imaging revealed a blood half-life of about 1 h in mice, markedly longer than the 7-min blood half-life for unmodified AnxA5, which should allow improved imaging of target tissues with low perfusion. In comparison to AnxA5, XTEN-AnxA5 demonstrated a substantially higher accumulation in tumors under chemotherapy in near-infrared fluorescence imaging.
  
  
  CONCLUSIONS
  The presented method allows the expression and production of high amounts of long-circulating XTEN-AnxA5 without the necessity of PEGylation, thereby simplifying the synthesis while avoiding labeling-induced inactivation of AnxA5 and potential adverse effects of PEG. It is readily applicable to other recombinant protein or peptide-based imaging probes and allows fine-tuning of the desired blood half-life, because longer XTEN variants yield longer blood half-lives.
authors.affiliation: Institute of Radiology-Molecular Imaging Group, Charité, Berlin, Germany.
authors.forename: Akvile
authors.initials: A
authors.lastname: Haeckel
authors.forename: Franziska
authors.initials: F
authors.lastname: Appler
authors.forename: Lena
authors.initials: L
authors.lastname: Figge
authors.forename: Harald
authors.initials: H
authors.lastname: Kratz
authors.forename: Mathias
authors.initials: M
authors.lastname: Lukas
authors.forename: Roger
authors.initials: R
authors.lastname: Michel
authors.forename: Jörg
authors.initials: J
authors.lastname: Schnorr
authors.forename: Marietta
authors.initials: M
authors.lastname: Zille
authors.forename: Bernd
authors.initials: B
authors.lastname: Hamm
authors.forename: Eyk
authors.initials: E
authors.lastname: Schellenberger
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000097:blood* / Q000737:chemistry / Q000378:metabolism; D000970:Antineoplastic Agents / Q000627:therapeutic use; D015870:Gene Expression; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D008958:Models, Molecular; D057054:Molecular Imaging / Q000379:methods*; D010455:Peptides / Q000235:genetics*; D010718:Phosphatidylserines / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry*; D011487:Protein Conformation; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000097:blood* / Q000737:chemistry / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry; D015899:Tomography, Emission-Computed, Single-Photon; D014057:Tomography, X-Ray Computed
chemical_list: D017304:Annexin A5; D000970:Antineoplastic Agents; D010455:Peptides; D010718:Phosphatidylserines; D011993:Recombinant Fusion Proteins; D013438:Sulfhydryl Compounds; D011092:Polyethylene Glycols
keywords: XTEN; annexin A5; long-circulating; polyethylene glycol; recombinant protein