This preparation retained complete inhibitory activity toward the MMP-3 catalytic domain and partial inhibitory activity toward full length MMP-9. Pharmacokinetic evaluation showed that PEGylation extended the plasma half-life of rhTIMP-1 in mice from 1.1 h to 28 h. In biological assays, PEG20K-TIMP-1 inhibited both MMP-dependent cancer cell invasion and tumor cell associated gelatinase activity. Overall these results suggest that PEGylated TIMP-1 exhibits improved potential for development as an anti-cancer recombinant protein therapeutic, and additionally may offer potential for clinical applications in the treatment of other diseases.
Citation: Batra J, Robinson J, Mehner C, Hockla A, Miller E, et al. (2012) PEGylation Extends Circulation Half-Life While Preserving In Vitro and In Vivo Activity of Tissue Inhibitor of Metalloproteinases-1 (TIMP-1). PLoS ONE 7(11): e50028. doi:10.1371/journal.pone.0050028 Editor: Nikos K. Karamanos, University of Patras, Greece Received September 11, 2012; Accepted October 15, 2012; Published November 20, 2012 Copyright: ?2012 Batra et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by Florida Department of Health grants 08KN12 and 09BB17 (to ESR) and 1BD01 (to JB), National Cancer Institute grant R01 CA122086 (to DCR), and by the Mayo Clinic Breast Cancer Specialized Program of Research Excellence (SPORE) grant P50 CA116201 (PI James Ingle). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
Introduction
The matrix metalloproteinases (MMPs) are a family of 23 zincdependent endopeptidases with important functions in tissue morphogenesis, wound healing, and other physiological processes that require remodeling of the extracellular matrix [1,2,3,4]. MMP activity is regulated in vivo by a family of four endogenous protein protease inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), that bind to MMPs in 1:1 stoichiometry and block the protease active site [5,6]. Disruption of the balance between MMPs and TIMPs is evidenced under many pathological conditions, and excess MMP activity has long been recognized for important contributions to the development and progression of many diseases including cardiovascular, vascular, and pulmonary diseases, arthritis, multiple sclerosis, and cancer [2,7,8,9,10]. Diverse roles in disease development and progression have led MMPs to be regarded as promising therapeutic targets, resulting in development of many small-molecule MMP inhibitors, but clinical trials of early-generation MMP inhibitors in cancer and arthritis proved disappointing [11,12,13,14]. Broad-spectrum MMP inhibitors produced serious dose-limiting musculoskeletal
toxicity, failed to reach therapeutic plasma levels, and failed to extend progression-free survival in cancer trials [11,12,14]; these disappointing outcomes have been attributed both to the toxicity and off-target effects of the drugs and to inadequate specificity for target MMPs. A less toxic alternative to synthetic MMP inhibitors might be offered by TIMPs. Studies using many preclinical cancer models have shown that overexpression of natural TIMPs in tumors often leads to reduced tumor growth and metastasis [15]. Systemic gene transfer of TIMPs in animal models of cancer has likewise produced antitumor effects, with minimal toxicity [15]. In a handful of studies investigating the suppressive effect of TIMP-1 on tumor cell proliferation and metastasis, mice have been treated with recombinant human TIMP-1 (rhTIMP-1) protein at doses of 2?0 mg/kg with no reported toxicity [16,17,18,19]. Recombinant human TIMPs -1 and -2 have also been investigated as inhibitors of airway inflammation in a murine model of asthma, via intranasal instillation, with promising results [20]. For many applications, one barrier that will likely need to be addressed for TIMPs to enter the clinic as recombinant therapeutics is the short half-life in circulation of these small proteins. Persistence in the circulation is desirable because protein therapeutics generally cannot be administered orally and typically are administered by subcutaneous, intramuscular, or intravenous injection or infusion. Animal studies using recombinant TIMPs have thus far been limited in part by rapid clearance of the protein; the plasma clearance of murine TIMP-1 in rats was reported to occur within minutes [21], and the blood elimination half-life of human TIMP-1 in mice was reported to be ,4 hours [22]. Chemical modification has been used to improve the pharmacokinetic profiles of several protein therapeutics now in the clinic [23,24,25,26]; one successful strategy is PEGylation, the covalent conjugation of polyethylene glycol chains to a protein. In general, PEGylation reduces renal clearance, increases circulatory half-life by a factor of 5?00-fold, and improves biological activity; it may also confer resistance to proteolysis and reduce immunogenicity [23,24]. While some PEGylated molecules demonstrate decreased binding in vitro to their natural ligands or receptors, these effects tend to be offset in vivo, with striking improvements in functional pharmacodynamic properties [23,24]. Furthermore, losses in target affinity can sometimes be minimized by site-directed PEGylation [23]; for example, by chemical conjugation of activated PEG to an unpaired cysteine residue introduced through genetic engineering [27,28]. TIMP-1 is a potent biological inhibitor of MMPs including MMP-9 (gelatinase B), a metalloproteinase that has been implicated as a potential therapeutic target in a wide variety of inflammatory and vascular diseases and in cancer [29]. Here, we tested several approaches to the covalent PEGylation of rhTIMP1, and evaluated PEGylated rhTIMP-1 for retention of MMP inhibitory activity in biochemical and biological assays, as well as the impact of PEGylation on circulation half-life in mice.were assessed by silver stained SDS-PAGE. Recombinant human MMP-3 catalytic domain (MMP-3cd) was expressed in E. coli, purified, refolded, and activated as described previously [31,32]. Full-length recombinant human MMP-9 was purchased from Calbiochem, San Diego, CA, USA.