title: Purification, characterization and plasma half-life of PEGylated soluble recombinant non-HA-binding CD44.
abstract: OBJECTIVE
  The aim of this study was to increase the serum half-life of recombinant CD44 hyaluronan (HA) binding domain by PEGylation. We have previously found that recombinant soluble CD44 HA binding domain (CD44HABD) and its non-HA-binding triple mutant CD44HABD(R41AY78SY79S) (CD44-3MUT) inhibits angiogenesis and subcutaneous tumor growth. However, this ~12 kDa recombinant protein displays a high serum clearance rate.
  
  
  METHODS
  Here, we report the purification of monomeric CD44-3MUT from urea solubilized inclusion bodies using weak anion exchange chromatography and gel filtration. To increase the serum residence time of CD44-3MUT we PEGylated the resulting protein using 20 kDa methoxy-PEG-propionaldehyde.
  
  
  RESULTS
  PEGylation of CD44-3MUT prolonged its in vivo serum half-life about 70-fold from 0.03 to 1.8 hours. Along with extended plasma residence time, PEGylation also increased the systemic exposure. By cell impedance assay we confirmed that PEGylated CD44-3MUT maintained its in vitro function. The results from the impedance assay additionally demonstrate that the CD44-3MUT effect on endothelial cells is mediated by vimentin.
  
  
  CONCLUSIONS
  In summary, we have developed a purification protocol for large-scale production of CD44-3MUT and generated a PEGylated form of CD44-3MUT. HA binding domain of CD44(CD44HABD) and its modified non-HA binding form (CD44-3MUT) inhibit angiogenesis and tumor growth in vivo without disturbing HA-binding functions. CD44-3MUT has been PEGylated for use as a new type of anti-angiogenic human drug. PEGylation of CD44-3MUT improved pharmacokinetic properties but retains its functional activity.
authors.affiliation: Department of Gene Technology, Tallinn University of Technology, Akadeemia tee 15, 12618, Tallinn, Estonia.
authors.forename: Anne
authors.initials: A
authors.lastname: Pink
authors.forename: Aili
authors.initials: A
authors.lastname: Kallastu
authors.forename: Marina
authors.initials: M
authors.lastname: Turkina
authors.forename: Marianna
authors.initials: M
authors.lastname: Skolnaja
authors.forename: Priit
authors.initials: P
authors.lastname: Kogerman
authors.forename: Taavi
authors.initials: T
authors.lastname: Päll
authors.forename: Andres
authors.initials: A
authors.lastname: Valkna
mesh_terms: D020533:Angiogenesis Inhibitors / Q000008:administration & dosage* / Q000493:pharmacokinetics / Q000494:pharmacology; D000818:Animals; D005260:Female; D006207:Half-Life; D018960:Hyaluronan Receptors / Q000008:administration & dosage / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000378:metabolism*; D051379:Mice; D011092:Polyethylene Glycols / Q000737:chemistry*; D011485:Protein Binding; D051381:Rats; D011916:Rats, Inbred F344; D011994:Recombinant Proteins / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism
chemical_list: D020533:Angiogenesis Inhibitors; D018960:Hyaluronan Receptors; D011994:Recombinant Proteins; D014746:Vimentin; D011092:Polyethylene Glycols; D006820:Hyaluronic Acid