Purpose
Two recombinant Interferon-beta (IFN-ß) products have been approved for the treatment of Multiple Sclerosis.

A GlycoSylated form with the predicted natural Amino Acid sequence (IFN-ß-1a) and a Non-GlycoSylated form that has a Met-1 deletion and a Cys-17 to Ser mutation (IFN-ß-1b).

The structural basis for activity differences between IFN-ß-1a and IFN-ß-1b, is determined.

Methods
In Vitro AntiViral, AntiProliferative and ImmunoModulatory assays were used to directly compare the two IFN-ß products.

Size Exclusion Chromatography (SEC), SDS-PAGE, thermal denaturation, and X-Ray Crystallography were used to examine structural differences.

Results
IFN-ß-1a was 10 times more active than IFN-ß-1b with specific activities in a standard AntiViral assay of 20 x 10(7) IU/mg for IFN-ß-1a and 2 x 10(7) IU/mg for IFN-ß-1b.

Of the known structural differences between IFN-ß-1a and IFN-ß-1b, only GlycoSylation affected In Vitro activity.

DeGlycoSylation of IFN-ß-1a produced a decrease in total activity that was primarily caused by the formation of an insoluble disulfide-linked IFN precipitate.

DeGlycoSylation also resulted in an increased sensitivity to thermal denaturation. SEC data for IFN-ß-1b revealed large, soluble aggregates that had reduced AntiViral activity (approximated at 0.7 x 10(7) IU/mg).

Crystallographic data for IFN-ß-1a revealed that the Glycan formed H-bonds with the Peptide backbone and shielded an uncharged surface from solvent exposure.

Conclusions
Together these results suggest that the greater biological activity of IFN-ß-1a is due to a stabilizing effect of the Carbohydrate on structure.

Interferons (IFNs) are potent ExtraCellular protein mediators of host defence and Homoeostasis. This article reviews the structure of human IFN-ß (HuIFN-ß), in particular in relation to its activity.

The recently determined crystal structure of Hu IFN-ß provides a framework for understanding of the mechanism of differentiation of Type I IFNs by their common receptor.

Insights are generated by comparison with the structures of other Type I IFNs and from the interpretation of existing mutagenesis data.

The details of the observed Carbohydrate structure, together with biochemical data, implicate the GlycoSylation of Hu IFN-ß, which is uncommon among Type I IFNs, as an important factor in the solubility, stability and, consequently, activity of the protein.

Finally, these structural implications are discussed in the context of the clinical use of Hu IFN-ß.

Today Interferon-beta (IFN-ß) generally is accepted as a therapeutic approach in Multiple Sclerosis (MS). All human IFN-ß preparations contain Interferon in a monomeric, biologically active and inactive polymeric form.

Therefore, both the specification of the mass of IFN-ß (microgram) and the biological activity (IU) are used for the characterization of any particular drug.

Nevertheless, not all publications present data on both measurements, apparently resulting in inconsistent statements in some cases.

To directly compare pharmacological data from studies distinguishing in methodology or time; only the specification of mass is valid.

In Europe, basic PharmaCoKinetic and PharmacoDynamic studies are available from three different IFN-ß-1a preparations. The standard drug, with the brand name Rebif, is clinically tested and approved for treatment in MS.

This overview compares results from the three preparations: Rebif, the so called reference drug, which is the very first IFN-ß-1a clinically tested in MS, but unfortunately not approved up to now.

And another preparation, which is approved by means of BioEquivalence studies only, and therefore called comparison drug here.

According to the data available now, it can be stated that the IFN-ß-1a drugs probably show identical results in bioavailability, when given subcutaneously (s.c.) or intramuscularly (i.m.).

The data further indicate that all drugs reach the same level of activity in vivo, and will cause identical efficacy, if the same masses are given either s.c. or i.m.

Type I Interferons (IFNs) are helical Cytokines that have diverse biological activities despite the fact that they appear to interact with the same receptor system.

To achieve a better understanding of the structural basis for the different activities of and ß-IFNs, we have determined the crystal structure of GlycoSylated human IFN-ß at 2.2 - a resolution by molecular replacement.

The molecule adopts a fold similar to that of the previously determined structures of murine IFN-ß and human IFN-2b but displays several distinct structural features.

Like human IFN-2b, human IFN-ß contains a Zinc-binding site at the interface of the two molecules in the asymmetric unit, raising the question of functional relevance for IFN-ß dimers.

However, unlike the human IFN-2b dimer, in which homologous surfaces form the interface, human IFN-ß dimerizes with contact surfaces from opposite sides of the molecule.

The relevance of the structure to the effects of point mutations in IFN-ß at specific exposed residues is discussed.

A potential role of Ligand-Ligand interactions in the conformational assembly of IFN receptor components is discussed.

Interferon-alpha (IFN-) subtypes were separated by HPLC from the IFN mixtures produced by Virus-stimulated human LymphoBlastoid Cells and Leukocytes.

Together with preparations of LymphoBlastoid IFN and recombinant IFN-ß, these were tested in three human Tumor Cell lines derived from Liver, Lung, and NeuroBlasts.

Their relative AntiViral activities differed markedly: subType IFN- 8 was the most potent and IFN- 1 the least. The results were broadly similar in all three cells, with some minor differences.

When the same preparations were tested for inhibition of Thymidine incorporation, the relative activities were quite different:

1. Subtypes IFN- 10, IFN- 17, IFN- 21, and IFN- 5 were now the most active

2. IFN- 2 was the least active

3. IFN- 1 and IFN- 8 had comparable intermediate activity

Thus, the differences in activity were not caused by degradation of some subtypes during their separation.

IFN- 8 not only had the greatest AntiViral activity but also, like IFN-ß, induced an AntiViral state in U1 mutant cell lines.

Which lack the Tyrosine kinase, Tyk2, required for signal transduction by other IFN- subtypes.

The present study focused on the investigation of the chemical stability of recombinant human Interferon-beta (rhIFN-ß) tested In Vitro by chemical treatments.

That simulate stress-induced conditions that may occur during handling, storage or ageing of protein samples.

Mild Oxidation and/or Alkylation of the recombinant protein showed that the four methionines occurring in the Interferon displayed different chemical susceptibility.

In that Met36 and Met117 were fully modified, whereas Met1 showed only little modification and Met62 was completely resistant.

Moreover, incubation of rhIFN-ß under Alkaline conditions resulted in the formation of a covalent dimeric species stabilized by an intermolecular DiSulphide bridge involving the free SH group of Cys17 from each polypeptide chain.

Analysis of biological activity of the different IFN-ß derivatives showed that rhIFN-ß fully retains its specific activity following mild Oxidation treatments.

Whereas reaction with a high concentration of Alkylating agents or incubation under Alkaline conditions strongly reduce its specific AntiViral activity.

The ExtraCellular portions of the chains that comprise the human Type I Interferon receptor, IFN-AR1 and IFN-AR2, have been expressed and purified as recombinant soluble His-tagged proteins.

And their interactions with each other and with human Interferon-beta-1a (IFN-ß-1a) were studied by gel filtration and by cross-linking.

By gel filtration, no stable binary complexes between IFN-ß-1a and IFNAR1, or between IFN-AR1 and IFN-AR2 were detected. However, a stable binary complex formed between IFN-ß-1a and IFNAR2.

Analysis of binary complex formation using various molar excesses of IFN-ß-1a and IFN-AR2 indicated that the complex had a 1:1 stoichiometry, and reducing SDS-PAGE of the binary complex treated with the cross-linking reagent DisSucinimidyl Glutarate (DSG).

Indicated that the major cross-linked species had an apparent Mr consistent with the sum of its two individual components.

Gel filtration of a mixture of IFN-AR1 and the IFN-ß-1a/IFN-AR2 complex indicated that the three proteins formed a stable ternary complex.

Analysis of ternary complex formation using various molar excesses of IFN-AR1 and the IFN-ß-1a/IFN-AR2 complex indicated that the ternary complex had a 1:1:1 stoichiometry.

And reducing SDS-PAGE of the ternary complex treated with DSG indicated that the major cross-linked species had an apparent Mr consistent with the sum of its three individual components.

We conclude that the ternary complex forms by the sequential association of IFN-ß-1a with IFN-AR2, followed by the association of IFN-AR1 with the preformed binary complex.

The ability to produce the IFN-ß-1a/IFN-AR2 and IFN-ß-1a/IFN-AR1/IFN-AR2 complexes make them attractive candidates for X-ray crystallography studies aimed at determining the molecular interactions between IFN-ß-1a and its receptor.

Recombinant human Interferon-gamma (Hu-IFN-γ) produced by Chinese-hamster ovary (CHO) cells was analyzed by ImmunoPrecipitation and SDS/PAGE.

Up to twelve molecular-mass variants were secreted by this cell line.

Three variants were recovered after enzymic removal of all N-linked OligoSaccharides or when GlycoSylation was inhibited by Tunicamycin.

The presence of three polypeptide forms rather than a single form suggested that ProteoLytic cleavage had occurred at two sites in both the GlycoSylated and Non-GlycoSylated forms.

ProteoLytically cleaved IFN-γ was more prevalent in cell lysates than in the secreted GlycoProtein.

In common with naturally produced IFN-γ, both fully GlycoSylated IFN-γ (Asparagine residues 28 and 100 occupied) and partially GlycoSylated product (thought to be substituted at position Asn28) were secreted.

This was deduced from the Mr of the GlycoSylated products and the relative amounts of Sialic Acid expressed by each variant.

In contrast with naturally produced IFN-γ, Non-GlycoSylated IFN-γ was also secreted by the transfected CHO cells.

When the cells were grown in batch culture in Serum-free medium under pH and dissolved-Oxygen control, the proportion of Non-GlycoSylated IFN-γ increased from 3 to 5% after 3 h, to 30% of the total IFN-γ present after 195 h.

This change in the proportion of GlycoSylated protein produced was not seen when metabolically labelled IFN-γ was incubated for 96 h with cell-free supernatant from actively growing CHO cells.

This implied that an alteration in IntraCellular GlycoSylation was occurring rather than a degradation of OligoSaccharide side chains after secretion.

The decrease in IFN-γ GlycoSylation was independent of the Glucose concentration in the culture medium.

But could be related to specific growth and IFN-γ production rates, as these declined steadily after 50 h of culture.

In line with the increased production of Non-GlycoSylated IFN-γ.

Human Interferon-gamma (IFN-γ) has two N-linked GlycoSylation sites at positions 25 and 97 of the 143-Amino-Acid-long secretory form.

To study the role of Glycan residues in the PharmacoKinetics of IFN-γ, we produced recombinant IFN-γ molecules lacking either one or both of the GlycoSylation sites (Asn mutated to Gln) by Baculovirus expression in insect cells.

In addition, we produced the fully GlycoSylated forms both in insect cells and in human Leukocyte cultures.

Two million units of each IFN were injected intravenously or intramuscularly into rabbits. The GlycoSylated IFN-γ molecules from the insect cells were rapidly eliminated from the blood.

This is probably due to the fact that their OligoSaccharides are of a high mannose type that are rapidly taken up by the Liver.

The UnGlycoSylated IFN-γ persisted longer in the blood than the GlycoSylated recombinant forms.

However, the natural IFN-γ exhibited the longest survival in the blood.

The results emphasize the importance of the Carbohydrate groups in human IFN-γ to its PharmacoKinetic properties.

Human FibroBlast-derived Interferon-beta (IFN-ß) labeled in vivo with 35S-Methionine and purified to greater than 90% RadioChemical purity has been used to study the effect of GlycoSidases on its biological activity and its molecular weight.

Incubation of the radioactive Interferon with the GlycoSidases causes a reduction in the estimated molecular weight from 23,000 to 18,000 as determined by ElectroPhoresis on SDS-gels.

There are intermediate transient sizes that are generated prior to 18,000, but not Proteins are observed that are smaller than 18,000 even after prolonged incubation.

The DeGlycoSylated IFN-ß (18,000) induces the AntiViral state in cells in culture and inhibits the growth of cells in culture.

We conclude that the Carbohydrate of IFN-ß is not essential for its biological activity on cells in culture.