HUMAN INTERFERONS

Human-specific glycosylation of human interferons

Interferons (IFNs), initially discovered fifty years ago as virus inhibiting agents¹, are now known to be a family of pleiotropic cytokines with additional antiproliferative, antitumour, and immunomodulatory properties. Current clinical uses of IFNs include treatment of viral infections, cancers and autoimmune diseases². The clinically relevant IFNs include IFN-alpha 2a and 2b, IFN-beta and IFN-gamma. IFN-alpha 2 isoforms are used to treat malignant melanoma, hairy cell leukemia, chronic hepatitis B, chronic hepatitis C, follicular (non-Hodgkin's) lymphoma, and AIDS-related Kaposi's sarcoma. IFN-beta has been approved for treatment of multiple sclerosis whereas IFN-gamma is used to treat chronic granulomatous disease and osteopetrosis.

At present, all commercial sources of therapeutic IFNs are produced from E.coli, except IFN-beta 1a, which is produced from CHO cells³. IFNs occur naturally as glycoproteins, although the effect of glycosylation on the in vivo activity of IFNs has not been extensively characterised. Nevertheless, it is known that glycosylation has major effects on pharmacokinetics, solubility, stability, and receptor binding of therapeutic proteins.⁴

In particular, glycosylation of IFN-beta has been associated with 10-fold higher antiviral, antiproliferative and immunomodulatory activity. Reduced antiviral activity has been attributed to the tendency of IFN-beta 1b to form large, insoluble aggregates due to the lack of glycosylation⁵ as the glycans have a stabilising effect on the structure. Glycosylation is also critical for human IFN-gamma protease resistance⁶. Thus glycosylation is of critical importance for IFN function and therefore using human cell expressed proteins could potentially be beneficial for experimental outcomes.

Apollo's recombinant hcx™ proteins are unique in being produced from human cells and containing human-specific post-translational modifications including glycosylation, making them more similar to naturally occurring proteins compared to E.coli or CHO expressed recombinant human proteins. Apollo produces human cell expressed (hcx™) IFN-alpha 2b and IFN-gamma as well as the IFN-gamma receptor 1 extracellular domain fused to the Fc region of human IgG1. IFN-gamma^hcx, for example, has greater antiproliferative activity than E. coli expressed IFN-gamma (Figure 1), demonstrating the advantage of using human cell expressed IFNs.

Figure 1

IFN-gamma

IFN-gamma is a secreted protein with 2 potential N-linked glycoslation sites produced by lymphocytes upon activation by specific antigens or mitogens. It is a potent activator of macrophages, it has antiproliferative effects on transformed cells and it can potentiate the antiviral and antitumor effects of the type I interferons. In the clinic it is used as an anticancer drug for its antiproliferative activity, and for reducing the frequency and severity of serious infections associated with chronic granulomatous disease (CGD).

Apollo's IFN-gamma (hcx) consists of 10-30% carbohydrate by weight and has been shown experimentally to have N-linked oligosaccharides.

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IFN-gamma R1 - Fc Chimera

IFN-gamma R1 is a type I membrane protein that contains two fibronectin type III domains, 2 Ig-like C2-type (immunoglobulin-like) domains and five potential N-linked glycosylation sites. IFN-gamma R1 binds to IFN-gamma resulting in dimerization of the heterodimer receptor into a heterotetramer complex.

Apollo's hcx IFN-gamma R1-Fc Chimera has been shown experimentally to be N-glycosylated and contains 10-35% carbohydrate by weight.

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IFN-alpha 2b

IFN-alpha 2b is a secreted protein produced by macrophages shown to have an O-linked glycan at 129. IFN-alpha have antiviral activities and stimulate the production of two enzymes: a protein kinase and an oligoadenylate synthetase. Clinically IFN-alpha 2b is used as an anticancer drug for its antiproliferative activity.

Apollo's IFN-alpha 2b (hcx) consists of up to 5% carbohydrate by weight and has been shown experimentally to be O-glycosyalted.

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IFNAR2 - Fc Chimera

IFNAR2 is a receptor for the alpha and beta type interferons. Apollo's hcx IFNAR2 - Fc Chimera consists of 15-40% carbohydrate by weight and has been shown experimentally to have N-linked and probably O-linked oligosaccharides.

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References

1. Isaacs and Lindermann (1957) Proc. R. Soc. Lond. B Biol.Sci. 147:258-267
2. Maher et. al. (2007) Curr Med Chem. 14:1279-1289
3. Walsh G. (2006) Nature Biotechnology 24:769 – 776
4. Werner et. al. (2007) Acta Paediatr Suppl. 96:17-22
5. Runkel et. al. (1998) Pharm Res 15:641-649
6. Sareneva et. al. (1995) 308:9-14