HM10660A, a long-acting hIFN-α-2b, is a potent candidate for the treatment of hepatitis C through an enhanced biological half-life
Graphical abstract
Introduction
Human interferon α-2b (IFN-α-2b) has antiviral effects and inhibits tumor cell proliferation by enhancing innate immune functions (Friedman, 2008). Recombinant human interferon α-2b (rhIFN-α-2b) is widely used to treat chronic hepatitis B and C virus (HBV and HCV) as well as certain types of cancer such as hairy cell leukemia, malignant melanoma, follicular lymphoma, and Kaposi’s sarcoma caused by AIDS (Davis et al., 1998, Flanigan et al., 2001, McHutchison et al., 1998, Reichard et al., 1998).
Although IFN-α has shown potent therapeutic effects against HCV, it has a short half-life in serum (4–5 h) (Glue et al., 2000). To overcome the short half-life, IFN-α must be frequently administered at a high dose. The limited duration of the therapeutic effect and relapse prevention consequently interferes with patient compliance. To improve the serum half-life and reduce renal excretion, various alternatives have been developed such as PEGylated IFNs (Glue et al., 2000, Perry and Jarvis, 2001, Kaminskas et al., 2013), albumin-linked IFN-α (Osborn et al., 2002), and Fc-fusion IFN-α (Jones et al., 2004). PEGylated IFNs increased the serum half-life by 10–20 fold compared to native IFN, which has the potential to reduce the dosing frequency to once weekly (Glue et al., 2000, Perry and Jarvis, 2001). Human serum albumin-linked IFN-α showed an 18-fold increase in serum half-life in cynomolgus monkeys compared to native IFN-α (Osborn et al., 2002). Despite the prolonged serum half-life of PEGylated or HSA-fusion IFN-α, their in vitro specific activity is very low (1–20% of native IFN-α activity) (Bailon et al., 2001, Grace et al., 2001, Grace and Cutler, 2004, Osborn et al., 2002). In addition, the published report on the Fc-fusion IFN-α did not describe pre-clinical data.
To improve the therapeutic effects, as well as the serum half-life, the feasibility of modified IFN-α-2b was studied using an Fc-linked-cytokine expression strategy. This expression strategy has been widely used to improve serum half-life through the reduced renal clearance of cytokines due to the dramatically increased molecular weight and resulting larger size (Lo et al., 1998, Zheng et al., 1995). Among the various types of globulin fragments, the human IgG4 fragment has been utilized as a stabilizing agent because it is the most prevalent blood protein and it has a half-life of several weeks without unwanted effector functions such as CDC (complement-dependent cytotoxicity) or ADCC (antibody-dependent cellular cytotoxicity) (Jiang et al., 2011). Furthermore, the half-life extension of IgG- and Fc-fusion proteins is caused by a neonatal Fc receptor (FcRn)-mediated recycling mechanism (Suzuki et al., 2010). FcRn protects IgG from degradation through an interaction with the Fc portion of IgG in the acidic endosome, resulting in IgG’s release to plasma (Lobo et al., 2004). Because FcRn is primarily expressed in vascular endothelial cells in adults (Roopenian and Akilesh, 2007), Fc-fusion proteins remain for a long time in the plasma due to recycling through binding FcRn. Therefore, enhanced FcRn binding properties of Fc-fusion proteins might increase their circulation time in plasma. (Datta-Mannan et al., 2007, Deng et al., 2010, Petkova et al., 2006, Yeung et al., 2009)
In this study, IFN-α-2b was site-specifically conjugated with the Fc region of human IgG4 via a non-peptidyl PEG linker to create HM10660A, which was compared with native IFN-α-2b and peginterferon α-2a (PEGASYS®). The feasibility of HM10660A as an antiviral treatment was confirmed using both in vitro studies, including IFN alpha receptor (IFNAR2)-binding measured with surface plasmon resonance and antiviral effects against encephalomyocarditis virus (EMCV), and in vivo studies including its efficacy in human hepatocyte-bearing mice and its pharmacokinetics (PK) and pharmacodynamics (PD) in monkeys.
Section snippets
Materials
Bi-functional aldehyde PEG (MW 3400 Da) was purchased from IDB Inc. (Ulsan, Korea). All other chemicals used were of analytical grade. HBS-P buffer (10 mM HEPES, 150 mM NaCl, 0.005% polysorbate 20, pH 7.4) was obtained from GE Healthcare Life Sciences (Piscataway, NJ, USA). The pET-22b-derived and pET-14b-derived vectors were purchased from Merck (Darmstadt, Germany). The IFNAR2 ECD domain was obtained from R&D systems (Minneapolis, MN, USA). IFN-α-2b and peginterferon α-2a (PEGASYS®) were
Results and discussion
The purification processes of Fc, IFN-a-2b, and HM10660A were presented in supplement data as a flow diagram (Fig. S2). Recombinant human IFN-α-2b was conjugated to the N-terminus of a recombinant human immunoglobulin G4 (IgG4) Fc fragment (HMC001) through a non-peptidyl linker to increase its half-life. HMC001 was chosen as the carrier protein because IgG4 has an enhanced in vivo half-life of several weeks without exhibiting effector functions such as complement-dependent cytotoxicity (CDC) or
Conclusion
HM10660A is feasible as an alternative treatment for HCV due to its longer circulation time. It displays improved in vitro activity compared to both native IFN-α-2b and peginterferon α-2a (PEGASYS®). Furthermore, HM10660A had superior PK and PD properties, specifically stronger efficacy and prolonged duration of action, compared to peginterferon α-2a in a normal cynomolgus monkey model. Based on these results, HM10660A could be an attractive therapeutic candidate for clinical use, providing
Declaration of interest
The authors declare that there is no conflict of interest associated with this manuscript.
Acknowledgement
This study was supported by a grant of the Korea Drug Development Fund R&D Project (KDDF-201204-03).
References (31)
- et al.
Polyethylene glycol-conjugated pharmaceutical proteins
Pharma. Sci. Technol. Today
(1998) - et al.
Monoclonal antibody clearance impact of modulating the interaction of IgG with the neonatal Fc receptor
J. Biol. Chem.
(2007) - et al.
Alternative glycosylation modulates function of IgG and other proteins—implications on evolution and disease
Biochimica et Biophy. Acta (BBA)-Gen. Sub.
(2012) Recombinant antibody therapeutics: the impact of glycosylation on mechanisms of action
Trends Pharmacol. Sci.
(2009)- et al.
PEGylation of interferon α2 improves lymphatic exposure after subcutaneous and intravenous administration and improves antitumour efficacy against lymphatic breast cancer metastases
J. Controlled Release
(2013) - et al.
Antibody pharmacokinetics and pharmacodynamics
J. Pharm. Sci.
(2004) - et al.
Randomised, double-blind, placebo-controlled trial of interferon é-2b with and without ribavirin for chronic hepatitis C
Lancet
(1998) - et al.
Rational design of a potent, long-lasting form of interferon: a 40 kDa branched polyethylene glycol-conjugated interferon α-2a for the treatment of hepatitis C
Bioconjugate Chem.
(2001) - et al.
The binding affinity of human IgG for its high affinity Fc receptor is determined by multiple amino acids in the CH2 domain and is modulated by the hinge region
J. Exp. Med.
(1991) - et al.
Effects of interferon-alpha (IFN-α) administration on leucocytes in healthy humans
Clin. Exp. Immunol.
(1997)
Interferon alfa-2b alone or in combination with ribavirin for the treatment of relapse of chronic hepatitis C
New Engl. J. Med.
Pharmacokinetics of humanized monoclonal anti-tumor necrosis factor-α antibody and its neonatal Fc receptor variants in mice and cynomolgus monkeys
Drug Metab. Dispos.
Nephrectomy followed by interferon alfa-2b compared with interferon alfa-2b alone for metastatic renal-cell cancer
N. Engl. J. Med.
Clinical uses of interferons
Br. J. Clin. Pharmacol.
Pegylated interferon-α2b: pharmacokinetics, pharmacodynamics, safety, and preliminary efficacy data
Clin. Pharmacol. Therapeu.
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2021, BiologicalsCitation Excerpt :In this study, to improve in vivo half-life of FGF21, the conjugated FGF21 using PEG linker and Fc fragment of IgG4, named as LAPS-FGF21, was devised and evaluated. We have reported this type of therapeutic candidate protein structure in previous studies that showed prolong of half-life in various species [23–25]. In this novel structure, the Fc works as a long-acting core region using a neonatal Fc receptor (FcRn)-mediated recycling mechanism, and the PEG linker works as a connector for the site-specificity as well as protection of proteolytic cleavage region of proteins.