BioDrugs

, Volume 28, Issue 4, pp 393–402 | Cite as

Purification, Characterization and Plasma Half-Life of PEGylated Soluble Recombinant Non-HA-Binding CD44

  • Anne Pink
  • Aili Kallastu
  • Marina Turkina
  • Marianna Skolnaja
  • Priit Kogerman
  • Taavi Päll
  • Andres Valkna
Original Research Article

Abstract

Background and Objectives

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 CD44HABDR41AY78SY79S (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.

Abbreviations

AUC

Area under curve

C0

Initial plasma protein concentration

CD44HABD

CD44 hyaluronan binding domain

CD44-3MUT

Non-hyaluronan binding mutant of CD44HABD–CD44HABDR41AR78SY79S

CL

Total body clearance

CV

Column volumes

FT

Flow through fraction

GF

Gel filtration chromatography

GST-CD44-3MUT

CD44-3MUT GST-fusion protein

HA

Hyaluronan

IB

Inclusion bodies

ID

Initial dose of injected protein

IEC

Ion exchange chromatography

MLEC

Mouse lung endothelial cells

MS

Mass-spectrometric analysis

PEG

Polyethylene glycol

T1/2

Plasma half-life

%TBW

Percent of total body weight

Vd

Volume of distribution

Supplementary material

40259_2014_89_MOESM1_ESM.pdf (1.2 mb)
Supplementary material 1 (PDF 1257 kb)

References

  1. 1.
    Cichy J, Bals R, Potempa J, Mani A, Puré E. Proteinase-mediated release of epithelial cell-associated CD44. Extracellular CD44 complexes with components of cellular matrices. J Biol Chem. 2002;277:44440–7.PubMedCrossRefGoogle Scholar
  2. 2.
    Guo YJ, Liu G, Wang X, Jin D, Wu M, Ma J, Sy MS. Potential use of soluble CD44 in serum as indicator of tumor burden and metastasis in patients with gastric or colon cancer. Cancer Res. 1994;54:422–6.PubMedGoogle Scholar
  3. 3.
    Katoh S, McCarthy JB, Kincade PW. Characterization of soluble CD44 in the circulation of mice. Levels are affected by immune activity and tumor growth. J Immunol. 1994;153:3440–9.PubMedGoogle Scholar
  4. 4.
    Nakamura H, Suenaga N, Taniwaki K, Matsuki H, Yonezawa K, Fujii M, Okada Y, Seiki M. Constitutive and induced CD44 shedding by ADAM-like proteases and membrane-type 1 matrix metalloproteinase. Cancer Res. 2004;64:876–82.PubMedCrossRefGoogle Scholar
  5. 5.
    Ristämaki R, Joensuu H, Grön-Virta K, Salmi M, Jalkanen S. Origin and function of circulating CD44 in non-Hodgkin’s lymphoma. J Immunol. 1997;158:3000–8.PubMedGoogle Scholar
  6. 6.
    Okamoto I, Kawano Y, Tsuiki H, Sasaki J, Nakao M, Matsumoto M, Suga M, Ando M, Nakajima M, Saya H. CD44 cleavage induced by a membrane-associated metalloprotease plays a critical role in tumor cell migration. Oncogene. 1999;18:1435–46.PubMedCrossRefGoogle Scholar
  7. 7.
    Ahrens T, Sleeman JP, Schempp CM, Howells N, Hofmann M, Ponta H, Herrlich P, Simon JC. Soluble CD44 inhibits melanoma tumor growth by blocking cell surface CD44 binding to hyaluronic acid. Oncogene. 2001;20:3399–408.PubMedCrossRefGoogle Scholar
  8. 8.
    Päll T, Gad A, Kasak L, Drews M, Strömblad S, Kogerman P. Recombinant CD44-HABD is a novel and potent direct angiogenesis inhibitor enforcing endothelial cell-specific growth inhibition independently of hyaluronic acid binding. Oncogene. 2004;23:7874–81.PubMedCrossRefGoogle Scholar
  9. 9.
    Banerji S, Day AJ, Kahmann JD, Jackson DG. Characterization of a functional hyaluronan-binding domain from the human CD44 molecule expressed in Escherichia coli. Protein Expr Purif. 1998;14:371–81.PubMedCrossRefGoogle Scholar
  10. 10.
    Takeda M, Terasawa H, Sakakura M, Yamaguchi Y, Kajiwara M, Kawashima H, Miyasaka M, Shimada I. Hyaluronan recognition mode of CD44 revealed by cross-saturation and chemical shift perturbation experiments. J Biol Chem. 2003;278:43550–5.PubMedCrossRefGoogle Scholar
  11. 11.
    Pasut G, Veronese FM. State of the art in PEGylation: the great versatility achieved after forty years of research. J Control Release. 2012;161:461–72.PubMedCrossRefGoogle Scholar
  12. 12.
    Kontermann RE. Strategies for extended serum half-life of protein therapeutics. Curr Opin Biotechnol. 2011;22:868–76.PubMedCrossRefGoogle Scholar
  13. 13.
    Päll T, Pink A, Kasak L, Turkina M, Anderson W, Valkna A, Kogerman P. Soluble CD44 interacts with intermediate filament protein vimentin on endothelial cell surface. PLoS ONE. 2011;6:e29305.PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Studier FW. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif. 2005;41:207–34.PubMedCrossRefGoogle Scholar
  15. 15.
    Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, Friman O, Guertin DA, Chang JH, Lindquist RA, Moffat J, Golland P, Sabatini DM. Cell Profiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 2006;7:R100.PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    R Development Core Team. R: a language and environment for statistical computing. Vienna, Austria; 2012.Google Scholar
  17. 17.
    Bajorath J, Greenfield B, Munro SB, Day AJ, Aruffo A. Identification of CD44 residues important for hyaluronan binding and delineation of the binding site. J Biol Chem. 1998;273:338–43.PubMedCrossRefGoogle Scholar
  18. 18.
    Cattelino A, Liebner S, Gallini R, Zanetti A, Balconi G, Corsi A, Bianco P, Wolburg H, Moore R, Oreda B, Kemler R, Dejana E. The conditional inactivation of the beta-catenin gene in endothelial cells causes a defective vascular pattern and increased vascular fragility. J Cell Biol. 2003;162:1111–22.PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Teriete P, Banerji S, Noble M, Blundell CD, Wright AJ, Pickford AR, Lowe E, Mahoney DJ, Tammi MI, Kahmann JD, Campbell ID, Day AJ, Jackson DG. Structure of the regulatory hyaluronan binding domain in the inflammatory leukocyte homing receptor CD44. Mol Cell. 2004;13:483–96.PubMedCrossRefGoogle Scholar
  20. 20.
    Banerji S, Wright AJ, Noble M, Mahoney DJ, Campbell ID, Day AJ, Jackson DG. Structures of the Cd44-hyaluronan complex provide insight into a fundamental carbohydrate-protein interaction. Nat Struct Mol Biol. 2007;14:234–9.PubMedCrossRefGoogle Scholar
  21. 21.
    Ouellette T, Destrau S, Ouellette T, Zhu J, Roach JM, Coffman JD, Hecht T, Lynch JE, Giardina SL. Production and purification of refolded recombinant human IL-7 from inclusion bodies. Protein Expr Purif. 2003;30:156–66.PubMedCrossRefGoogle Scholar
  22. 22.
    Pasut G, Veronese FM. PEGylation, successful approach to drug delivery. Drug Discov Today. 2005;10:1451–8.PubMedCrossRefGoogle Scholar
  23. 23.
    Bailon P, Palleroni A, Schaffer CA, Spence CL, Fung WJ, Porter JE, Ehrlich GK, Pan W, Xu ZX, Modi MW, Farid A, Berthold W, Graves M. Rational design of a potent, long-lasting form of interferon: a 40 kDa branched polyethylene glycol-conjugated interferon alpha-2a for the treatment of hepatitis C. Bioconjug Chem. 2001;12:195–202.PubMedCrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2014

Authors and Affiliations

  • Anne Pink
    • 1
    • 2
  • Aili Kallastu
    • 2
  • Marina Turkina
    • 2
  • Marianna Skolnaja
    • 1
    • 2
  • Priit Kogerman
    • 1
    • 2
  • Taavi Päll
    • 1
    • 2
  • Andres Valkna
    • 1
    • 2
  1. 1.Department of Gene TechnologyTallinn University of TechnologyTallinnEstonia
  2. 2.Competence Centre for Cancer ResearchTallinnEstonia

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