Title of Invention	"AN IFNα-FC HYBRID RECOMBINANT PROTEIN CONTAINING AN INTERFERON MOLECULE JOINED AT ITS C-TERMINAL END THROUGH A PEPTIDE LINKER"
Abstract	A hybrid recombinant protein comprising an interferon molecule joined at its C-terminal end through a peptide linker to the N-terminal end of the immunoglobulin Fc fragment, the peptide linker comprising the sequence Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser (SEQ ID NO: 1).

Full Text

Background of the invention Interferon-α ("IFNσ") was among the first of the cytokines to be produced by recombinant DNA technology and has been shown to have therapeutic value in conditions such as inflammatory, viral, and malignant diseases. Several IFNσ preparations, including those purified from the natural sources and those generated by recombinant DNA technology, have been used or are being tested in a variety of malignant and viral diseases. IFNσ can cause regression of some established tumors and induce positive responses in some viral infections. So far, IFNα has been approved or tested in many countries for indications such as: Kaposi's sarcoma; hairy ceil leukemia; malignant melanoma; basal cell carcinoma; multiple myeloma; renal cell carcinoma, hepatitis B; hepatitis C; venereal warts, Herpes Ifll, varicella/herpes zoster; and mycosis fungoides. Most cytokines, including IFNα, have relatively short circulation half-lives since they are produced in vivo to act locally and transiently. The serum half-life of IFNα is only about two to eight hours (Roche Labs. Referon A, Schering Intron A, Physicians' Desk Reference, 47th edition, 1993, pp. 2006-2008, 2194-2201). To use IFNσ as an effective systemic therapeutic, one needs relatively large doses and frequent administrations. For example, one of the recommended therapeutic strategies for the AIDS-related Kaposi's sarcoma starts with an induction dose of 36 million III daily for 10 to 12 weeks, administered as an intramuscular or subcutaneous injection, followed by a maintenance dose of 36 million III, three times a week. (Roche labs. Referon A, Physicians' Desk Reference, 47th edition, 1993, pp. 2006-2008). Such frequent parenteral administrations are inconvenient and painful. Further, toxic effects, which are probably caused by the high dosage, are a problem for certain patients. Skin, neurologic, endocrine, and immune toxicity have been reported. To overcome these disadvantages, one can modify the molecule to increase its circulation half-life or change the drug's formulation to extend its release time. The dosage and administration frequency can then be reduced while increasing the efficacy. It was reported that doses of less than nine million units had been well tolerated, while doses more than 36 million units can induce severe toxicity and significantly alter patient status. (Quesada, J.R. et al., J. Clin. Oncol., 4:23443, 1986). It is possible to decrease substantially the toxic effects by producing a new form IFNα which is more stable in the circulation and requires smaller doses. Efforts have been made to create a recombinant IFNα-gelatin conjugate with an extended retention time (Tabata, Y. et al., Cancer Res. 51:5532-8, 1991). A iipid-based encapsulated IFNα formulation has also been tested in animals and achieved an extended release of the protein in the peritoneum (Bonetti, A. and Kim, S. Cancer Chemother Pharmacol. 33:258-261, 1993). Immunoglobulins of IgG and IgM class are among the most abundant proteins in the human blood. They circulate with half-lives ranging from several days to 21 days. IgG has been found to increase the half -lives of several iigand binding proteins (receptors) when used to form recombinant hybrids, including the soluble CD4 molecule, LHR, and IFN receptor (Mordenti J. et al., Nature, 337:525-31, 1989; Capon, D.J. and Lasky, LA., U.S. Patent number 5,116,964; Kurschner, C. et al., J. Immunol. 149:4096-4100, 1992). The US patent 5,349,053 describes chimeric molecules which are characterized to generally increase the specificity and improve the binding affinity of immunoglobulins beyond the immunoglobulin gene superfamily, while retaining their other useful characteristics. The described chimeric molecules comprise a Iigand molecule linked to a constant region of an immunoglobulin molecule. The patent describes, overall, 30 specific or generic examples for corresponding ligands. However, the document does not comprise any pointer to any member of the interferon family. Furthermore, US patent 5,349,053 describes only chimeric molecules in which the Iigand molecule is directly linked to the constant region without the use of a peptide linker. WO 91/16353 describes inter alia antibody derivatives consisting of two variable regions of an antibody (e.g. a VH- and a VL-region). Said two regions are linked to each other via a linker polypeptide. The document provides examples for peptide linker which are described to be suitable in this specific context. However, such hybrids can present problems in that the peptide at the C-termrrral of the active moeity and the peptide at the -terminal of the Fc portion at the fusion point creates a new peptide sequence, which is a neoantigen, and which can be immunogenic. The invention relates to a IFNσ-Fc hybrid which is designed to overcome this problem and extend the half-life of the IFNα. Summary of the invention The present invention relates to a hybrid recombinant protein which consists of two subunits. Each subunit includes a human interferon, preferably IFNσ, joined by a peptide linker which is primarily composed of a T cell inert sequence, linked to a human immunoglobulin Fc fragment, preferably the 4 chain. The γ4 chain isr preferred over the y} chain because the former has little or no complement activating ability. The C-terminal end of the IFNa is linked to the N-terminal end of the Fc fragment. An additional IFNa (or other cytokine) can attach to the N-terminal end of any other unbound Fc chains in the Fc fragment, resulting in a homodimer for the y4 chain. If the Fc fragment selected is another chain, such as the fj chain, then, because the Fc fragments form pentamers with ten possible binding sites, this results in a molecule with interferon or other cytokine linked at each of ten binding sites. The two moieties of the hybrid are linked through a T cell immunologically inert peptide, e.g., Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser. This peptide itself is immunologically inactive. The insertion of this peptide at the fusion point eliminates the neoantigenicity created by the joining of the two peptide moeities. The linker peptide also increases the flexibility of these moieties and allows retention of the biological activity. This relatively long linker peptide helps overcome the possible steric hindrance from the Fc portion of the hybrid, which could interfere with the activity of the hybrid. The hybrid has a much longer half-life than the native IFNa. Due to the linker, it is also designed to reduce the possibility of generating a new immunogenic epitope (a neoantigen) at what would otherwise be the fusion point of the IFNa and the immunoglobulin Fc segment. Cytokines are generally small proteins with relatively short half-lives which dissipate rapidly among various tissues, including at undesired sites. It is believed that small quantities of some cytokines can cross the blood-brain barrier and enter the central nervous system, thereby causing severe neurological toxicity. The IFNa linked to fey of the present invention would be especially suitable for treating hepatitis B or C, because these products will have a long retention time in the vasculature (upon intravenous administration) and will not penetrate undesired sites. The specific hybrid described can also serve as a model for the design and construction of other cytokine-Fc hybrids. The same or a similar linker could be used in order to reduce the possibility of generating a new immunogenic epitope while allowing retention of the biological activity. Cytokine-Fc hybrids in which interleukin-2 is the cytokine, or hybrids including other cytokines, could be made using the same techniques. Detailed Description of Making and Using the Invention The hybrid molecule of the invention includes an interferon moiety linked through a unique linker to an immunoglobulin Fc moiety. Preferably, the C-terminal ends of two interferon moieties are separately attached to each of the two N-terminal ends of a heavy chain >4 Fc fragment, resulting in a homodimer structure. A unique linker peptide, Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser, having the nucleotide sequence shown in Appendix "A", was created to link the two moieties. The complete nucleotide sequence of the preferred y4 hybrid (including the linker and the Fc moiety) is set forth in Appendix "B", and the linker is located at amino acid residue numbers 189 to 204. The advantage of the hybrid over the native cytokine is that the half-life in vivo is much longer. The hybrid including interferon and the y4 chain Fc homodimer is larger than the native interferon. Because the pores in the blood vessels of the liver are large, this larger molecule is more suitable for use in treating hepatitis, where the virus responsible primarily affects the liver. The linker peptide is designed to increase the flexibility of the two moieties and thus maintain their biological activity. Although the interferon and the immunoglobulin are both of human origin, there is always a possibility of generating a new immunogenic epitope at the fusion point of the two molecules. Therefore, the other advantage of the linker of the invention, which consists mainly of a T cell inert sequence, is to reduce immunogenicity at the fusion point. Referring to Appendix "B" it can be seen that if the linker (residue numbers 189-204) was not present, a new sequence consisting of the residues immediately before number 189 and immediately after 204 would be created. This new sequence would be a neoantigen for the human body. Human IFNor is derived from a family of several different genes. More than 24 species have been identified so far, from gene and protein sequence data. They differ from each other by anywhere from a few to a maximum of 35 amino acids. Most of the species have a signal peptide sequence of 23 amino acid residues and a mature amino acid sequence of 166 amino acid residues (Goeddel, D.V. et al., Nature, 290:20-26, 1981; Weissmann, C. and Weber, H., Prog. Nuc. Acid Res. Mo/. Biol. 33:251-300,1986; Zoon, K.C., Interfemn, 9:1-12, 1987). IFNorZ (also called IFNcrA) is one of the most intensively studied interferon species. The recombinant version of IFNcr2 has been used as a therapeutic for several years. Two IFNa2 recombinant products, IFNcrZa and IFNa2b, are now commercially available. They differ only in one amino acid at position 23, and there is no significant difference in biological activity between them (von Gabain, A., et al., Eur. J. Biochem. 190:257-61, 1990). IFNa2a was selected as the fusion partner for the interferon hybrid of the invention, although the IFNorZb or any other interferon species (including IFN/?) can be used as well. It is also possible to make similar constructs with other cytokines, such as interleukin-1 or interleukin-2. The same linker could be used, or another one which is not immunogenic and which maintains the biological activity of the contruct could be substituted. The advantages of the )4 chain as the Fc moiety in the hybrid is that it is stable in the human circulation. The )4 chain (unlike the y\ chain) also avoids the wide spectrum of secondary biological properties, such as complement fixation and antibody-dependant cell-mediated cytotoxicity (AOCC), which may be undesirable properties. The cONA of the IFNor2a can be obtained by reverse transcription and PCR, using RNA extracted from leukocytes which express IFN a. One such cell line, KG-1, can be obtained from the American Type Culture Collection (ATCC) in Rockville, Maryland, where it is held under number CCL 246. In the procedure used in making the hybrid of the invention, before the RNA extraction, the cells were challenged by Sendai virus to increase their transcription of interferons (Cantell, K. et al., Methods in Enzymology, 78A:29-38, Adacemic Press, 1981). As mentioned above, IFNor is a collection of IFN species and each cell expresses several different IFNa subspecies at the same time. The DNA sequence homology among these species is so high that RT-PCR would probably amplify a group of them instead a specific one. To obtain specifically the IFNa2a cDNA, the PCR primers were designed so that the last nucleotides of the two primers ended at positions where the amino acids coded are unique for IFNa2a. These are position 822 and 161, respectively (See Zoon, K.C. Interferon, 9:1-12, 1987). By using an overlapping PCR technique (Daugherty, B.L et al., Nucleic Acids Res. 19:2471-6, 1991), one can easily ligate two gene segments at any site as desired. However, one drawback of PCR amplification is the relatively high mutation rate (Saiki, R.K. eta/., Science, 239:487,1988). Thus, DNA sequencing was also done to check every DNA segment obtained through PCR for lack of mutation. Sequencing can be tedious and time consuming when the size of the segment is over 1kb, as is the full length IFNa-Fc cDNA. However, a restriction endonuclease site, BamH I, can be incorporated into the linker nucleotide sequence without changing its amino acid sequence. This site is located between the nucleotide numbers 15 and 16 in Appendix "A". The two gene segments from PCR can be separately cloned into cloning vectors. This makes the DNA sequencing easier and quicker since both segments are only a few hundred base pairs in length. Once the clones with the correct DNA sequences are identified, the two gene segments can be linked together through the BamH I site. No second round overlapping PCR and subsequent DNA sequencing of the full length segment are required. There are several ways to express the recombinant protein in vitro, including in £ coli, baculovirus, yeast, mammalian cells or other expression systems. The prokaryotic system, £ coli, is not able to do post- translational modification, such as glycosylation. But this is probably not a serious problem for the IFNor-Fc hybrid since the native IFNcr and immunoglobulin y4 molecule are not heavily glycosylated. Further, it has been reported that recombinant IFNcr without any glycosylation retained its biological activity (Baron, E. and Narula, S., Bio/technology, 10:179-190, 1990). However, the purification of recombinant protein from the£ co//lysate can be difficult. The foreign proteins expressed by £ calf often aggregate and form insoluble inclusion bodies. Thus, solubilization and subsequent refolding of the inclusion bodies is usually required (Schein, C.H. and Noteborn, H.M., Bio/technology, 6:291-294, 1988; Wilkinson, D.L and Harrison, R.G., Bio/technology, 9:443-448, 1991). The yeast expression system Pichia Pastoris (Invitrogen, San Diego, CA) overcomes some of the problems encountered when using the bacterial system. It usually gives a high yield and has the ability to do various post-translational modifications. The expressed foreign protein can be secreted into the culture supernatant where not many other proteins reside, making protein purification and process scale-up much easier. This system was tried first to express either the IFNor-Fc hybrid or the wild type IFNaZa. Unfortunately the IFNcr-Fc secreted was found to be partially degraded on SOS-PAGE, whereas the IFNor2a alone was not. The degradation was believed to be caused by the protease activities present in the yeast expression system, as reported by Scorer, C.A. et al., Gene, 136:111-9, 1993. The relatively weak spot in the hinge region is the possible target for the proteases. A mammalian cell expression system for the IFNor-Fc hybrid was also tried. The mammalian expression vector, pCDNA3 (Invitrogen, San Diego, CA) which contains a CMV promoter and a NEO resistance gene, was employed. The host cells, NSO cells, were transfected by the pCDNA3/IFNor-Fc expression vector using the electroporation method. The cells were selected by G418 at a concentration of 0.8 mg/ml. The IFNa-Fc expressing clones were identified by ELISA. The hybrid was successfully expressed in this system and there was no degradation. There are several advantages to this mammalian expression system. First, the recombinant protein is secreted into the culture supernatant and there is no aggregation, thereby simplifying purification. One chromatography step using a protein A column yields a purified IFNa-Fc protein. Also, the protein produced in this system has a glycosylation pattern very similar to the natural molecules since it is expressed by mammalian cells. Further, a native IFNa2a signal peptide sequence is included in the expression vector. Therefore the protein secreted from the cells has an authentic N-terminal, whereas in the £ coli or yeast expression systems there either is no signal peptide or a non-IFNa signal peptide is used. Either way, it will bring in additional artificial amino acid residue(s) at the N-terminal end of the recombinant IFNa-Fc. As mentioned above, the purification of the IFNa-Fc recombinant protein from the culture supernatant is relatively straightforward. The protein with a purity of more than 90%, as judged by SDS-PAGE, can be easily obtained by one step of affinity chromatography with a protein A column. There are several assay methods available for the measuring of the IFNa bioactivity. Using an antiviral assay, it was demonstrated that the hybrid of Appendix "B" had a specific activity about 5 to 10 fold higher than a related IFNa-Fc hybrid, in which the linker molecule had the sequence Gly Gly Ser Gly Gly Ser, and the Fc portion of the hybrid was derived from human lgG1 rather than lgG4. Nevertheless, although the biologicial activity of the hybrid shown in Appendix "B" was improved substantially, it was still lower than that of the native IFNa. However, it is expected that this hybrid will have a longer half-life in vivo. This expectation is based on results demonstrating that the related IFNa hybrid with the linker sequence Gly Gly Ser Gly Gly Ser and an lgG1 Fc portion showed a much longer half-life, in a mouse model, than did the native IFNa. Because the hybrid of Appendix "B" is expected to have a longer half-life in vivo than native IFNa, even though its specific activity is lower, this novel hybrid is expected to be preferred to the native IFNa for clinical use. This is because, as a result of the longer half-life, the Cxt (the area under the concentration vs. time curve) fvould be up to several hundred times greater than for the native IFNa. This means that at the equivalent molar dosage of the native IFNa and the hybrid, the latterwould be up to several hundred times greater than for the native IFNα. This means that at the equivalent molar dosage of the native IFNα and the hybrid, the latter would' pro-vide a several hundred fold increased exposure to IFNα, resulting in vastly increased efficacy at the same dosage, and less frequent administration. In measuring specific activity, molar dosage is preferred instead of expressing activity as units per mass of protein. This is because interferons function through the binding to their specific receptors, which is directly related to the number of molecules present. Also, the molecular weight of the IFNα-FCΓ4 110 Kd, is more than five-fold larger than that of the wild type IFNα2a, which is 20kd. Talcing this into consideration, measuring activity in units/µmol instead of the units/mg provides a better comparison of activity specifitv. The present invention relates to an IFNα-Fc hybrid recombinant protein containing an interferon molecule joined at its C-terminal end through a peptide linker to the N-terminal end of the immunoglobulin γ4 Fc fragment, the peptide linker having the sequence Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser (SEQ ID NO: 1). Example I: Cloning human IFNcr cDNA and constructing the IFNa-Fc expression vector 6x106 KG-1 cells (ATCC 246) were incubated with 200 units of Sendai virus at 37°C overnight. The cells were harvested and washed with PBS throughly. The total RNA was extracted by using the RNA-ZOL RNA isolation kit (BIOTEX, Houston, TX) following the procedure provided by the manufacturer. The first-strand cDNA was synthesized by reverse transcription using AMV reverse transcriptase with oligo(dT) as 3' primer in 50mM Tris-HCI (pH 8.3), 60mMKCI, and 6mM MgCI2, incubated at 42°C for 1 hour. The reaction mixture was used directly as the template for PCR to amplify IFNα cDNA. The 5' primer for PCR contained a Hind III site and the coding sequence for the first 21 amino acids from the IFNα2a leader peptide: CATAAGCTTC ATCTACAATG GCCTTGACCT TTGCTTTACT GGTGGCCCTC CTGGTGCTCA GCTGCAAGTC AAGCTGCTCT G. The 3' primer contained the sequence coding for part of the linker Appendix "A" and the last five amino acids of the IFNα2a, and a BamH I site integrated in the linker sequence CTCTGCGGAT CCACCTGAGC CACCTTCCTT ACTTCTTAAA. The PCR buffer contained 50mM KCI, 10mMTris-Hcl (pH8.3), 1.5mM MgCI2, 0.01% gelatin,0.1 mmol each of dNTP, 0.5 µmol of each primers, 5 µ\ RT reaction mixture, and 1 unit of Taq DNA polymerase in a total of 50 µl volume. The PCR condition was 94°C (1 min), 55°C (2 min), and 72°C (2 min) for 40 cycles on a GeneAmp PCR System 9600 (Perkin Elmer, Norwalk, CT). The cDNA of the human immunoglobulin y4 Fc was obtained by reverse transcription and PCR performed the same way as described above. The RNA was extracted from the human tonsil B cells. The 5' primer had the sequencez; AATGGATCCG GTGGAGGCGG AAGCGGCGGT GGAGGATCAG AGTCCAAATA TGGTCCCC. The 3' primer had the sequence: ATCGAATTCT ATTTACCCAG AGACAGGGAG AGGCTCTTCT GT. The two PCR amplified DNA segments were cloned into pUC18 vectors at sites Hind lll/BamH I or sites BamH 1/EcoR I respectively. After their DNA sequences were confirmed by DNA sequencing using the kit from USB (Cleveland, Ohio), the two segments were ligated together through the BamH I site by a second round cloning. The full length IFNa-Fc cDNA was then inserted into a mammalian expression vector pCDNAS (Invitrogen, San Diego, CA) through the Hind III and EcoR I sites. Example 2: Expressing IFNa-Fc in mammalian cells 107 NSO cells were mixed with 10/vg linearized pCDNA3/IFNa-Fc plasmid in 0.8 ml PBS and kept on ice for 5 min. Electroporation was performed at 200v, 960//F using Gene Pulser (BioRad, Hircules, CA). The cells were then put back on ice for 20 minutes and transferred to a 100mm tissue culture plate in 10ml DMEM supplied with 2% FCS. After incubation at 37°C for two days, the cells were washed and resuspended in the same medium. 0.6 mg/ml G418 was added to start the selection. The cells were plated out in eight 96-well micro plates and incubated at 37°C. Colonies appeared in one week and they were ready for screening in two weeks. The supernatants from each well with a single colony growing were collected. The IFNa-Fc in the supernatant was quantitatively determined by an ELISA assay employing goat anti-human IgG and anti-human Fc conjugated with horseradish peroxidase. The clones with higher ELISA readings and smaller colony size were selected for subcloning. These colonies were transferred to a 24-well plate and supplied with a medium containing G418. The clone with the highest secretion level was expanded and adapted to grow in a spinner. For large scale preparation, the culture supernatant was collected and passed through a protein A agarose column equilibrized by PBS. The protein bound to the protein A was eluted by 50 mM citric acid (pH 3.0) and concentrated by lyophilization. Example 3: Characterization of the IFNa-Fc hybrid. The purity of the recombinant protein isolated from NSO culture medium was examined by SDS-PAGE and Western blot. Only one protein band was visible on the blotted membrane stained by ponceau s for total proteins, showing a homogeneity of the protein preparation. The apparent molecular weight of this protein is about 55kd under reducing conditions and 110kd under non-reducing conditions, which is exactly the predicted size for the IFNa-Fc hybrid. The doubling of its apparent molecular weight under non-reducing conditions suggests that the hybrid is in a dimeric form. The recombinant protein can be recognized by both anti-Fc and anti-IFNcr antibodies, confirming that it consists of two moieties, the IFNa and the Fc fragment. The bioactivity assay for the IFNa-Fc was an antiviral assay. Specifically, the assay method used was a modification of the protocol described by Robert M. Friedman et al (Measurement of antiviral activity induced by interferons a, /?, and y, Current Protocols in Immunology, 1994, pp. 6.9.1-6.9.8). Briefly, human lung carcinoma cells (A549, ATCCICCL 185) were seeded in 96-well plates at a density of 40,000 cells/well and incubated at 37°C for 24 hours. 1:2 serial diluted IFNa-Fc hybrid or native IFNa (NIH# Gxa01-901-535) were added and incubated at 37°C for 24 hours. Every sample was done in triplicate. The culture medium was replaced with a fresh one containing encephalomyocarditis virus (ATCC #VR 129B) at a concentration of about 0.1 MOI/cell and incubated at 37°C for a further 48 hours. The dead cells were washed away by pipetting up and down vigorously with PBS. The attached cells were fixed by 2% formaldehyde and stained by giemsa stain. The plates were rinsed with tap water and allowed to dry. The stained cells were dissolved by methanol and the samples were read spectrophotometrically at 595nm. The antiviral activity of IFNa-Fc hybrid was calculated oy comparing it with the IFNa standard, and was found to be about 30 to 60% of the activity of the IFNa standard. It should be understood that the terms and expressions used herein are exemplary only and not limiting, and that the scope of the invention is defined only in the claims which follow, and includes all equivalents of the subject matter of those claims. APPENDIX "A" GGT GGC TCA GGT GGA TCC GGT GGA GGC GGA AGC GGC 36 GLY GLY SER GLY GLY SER GLY GLY GLY GLY SER GLY 12 GGT GGA GGA TCA 48 GLY GLY GLY SER 16 APPENDIX "B" ATG GCC TTG ACC TTT GCT TTA CTG GTG GCC CTC CTG GTG 39 MET ALA LEU THR PHE ALA LEU LEU VAL ALA LEU LEU VAL 13 CTC AGC TGC AAG TCA AGC TGC TCT CTG GGC TGT GAT CTG 78 LEU SER CYS LYS SER SER CYS SER LEU GLY CYS ASP LEU 26 CCT CAA ACC CAC AGC CTG GGT AGC AGG AGG ACC TTG ATG 117 PRO GLN THR HIS SER LEU GLY SER ARG ARG THR LEU MET 39 CTC CTG GCA CAG ATG AGG AAA ATC TCT CTT TTC TCC TGC 156 LEU LEU ALA GLN MET ARG LYS ILE SER LEU PHE SER CYS 52 TTG AAG GAC AGA CAT GAC TTT GGA TTT CCC CAG GAG GAG 195 LEU LYS ASP ARG HIS ASP PHE GLY PHE PRO GLN GLU GLU 65 TTT GGC AAC CAG TTC CAA AAG GCT GAA ACC ATC CCT GTC 234 PHE GLY ASN GLN PHE GLN LYS ALA GLU THR ILE PHE VAL 78 CTC CAT GAG ATG ATC CAG CAG ATC TTC AAT CTC TTC AGC 273 LEU HIS GLU MET ILE GLU GLU ILE PHE ASN LEU PHE SER 91 ACA AAG GAC TCA TCT GCT GCT TGG GAT GAG ACC CTC CTA 312 THR LYS ASP SER SER ALA ALA TRP ASP GLU THR LEU LEU 104 GAC AAA TTC TAC ACT GAA CTC TAG CAG CAG CTG AAT GAC 351 ASP LYS PHE TYR THR GLU LEU TYR GLN GLN LEU ASN ASP 117 CTG GAA GCC TGT GTG ATA CAG GGG GTG GGG GTG ACA GAG 390 LEU GLU ALA CYS VAL ILE GLN GLY VAL GLY VAL THR GLU 130 ACT CCC CTG ATG AAG GAG GAC TCC ATT CTG GCT GTG AGG 429 THR PRO LEU MET LYS GLU ASP SER ILE LEU ALA VAL ARG 143 AAA TAC TTC CAA AGA ATC ACT CTC TAT CTG AAA GAG AAG 468 LYS TYR PHE GLN ARG ILE THR LEU TYR LEU LYS GLU LYS 156 AAA TAC AGC CCT TGT GCC TGG GAG GTT GTC AGA GCA GAA 507 LYS TYR SER PHE CYS ALA TRP GLU VAL VAL ARG ALA GLU 169 ATC ATG AGA TCT TTT TCT TTG TCA ACA AAC TTG CAA GAA 546 ILE MET ARG SER PHE SER LEU SER THR ASN LEU GLN GLU 182 AGT HA AGA AGT AAG GAA GGT GGC TCA GGT GGA TCC GGT 585 SER LEU ARG SER LYS GLU GLY GLY SER GLY GLY SER GLY 195 GGA GGC GGA AGC GGC GGT GGA GGA TCA GAG TCC AAA TAT 624 GLY GLY GLY SER GLY GLY GLY GLY SER GLU SER LYS TYR 208 GGT CCC CCG TGC CCA TCA TGC CCA GCA CCT GAG TTC CTG 663 GLY PRO PRO CYS PRO SER CYS PRO ALA PRO GLU PHE LEU 221 GGG GGA CCA TCA GTC TTC CTG TTC CCC CCA AAA CCC AAG 702 GLY GLY PRO SER VAL PHE LEU PHE PRO PRO LYS PRO LYS 234 GAC ACT CTC ATG ATC TCC CGG ACC CCT GAG GTC ACG TGC 741 ASP THR LEU MET ILE SER ARG THR PRO GLU VAL THR CYS 247 GTG GTG GTG GAC GTG AGC CAG GAA GAC CCC GAG GTC CAG 780 VAL VAL VAL ASP VAL SER GLN GLU ASP PRO GLU VAL GLN 260 TTC AAC TGG TAC GTG GAT GGC GTG GAG GTG CAT AAT GCC 819 PHE ASN TRP TYR VAL ASP GLY VAL GLU VAL HIS ASN ALA 273 AAG ACA AAG CCG CGG GAG GAG CAG TTC AAC AGC ACG TAC 858 LYS THR LYS PRO ARG GLU GLU GLN PHE ASN SER THR TYR 286 CGT GTG GTC AGC GTC CTC ACC GTC CTG CAC CAG GAC TGG 897 ARG VAL VAL SER VAL LEU THR VAL LEU HIS GLN ASP TRP 299 CTG AAC GGC AAG GAG TAC AAG TGC AAG GTC TCC AAC AAA 936 LEU ASN GLY LYS GLU TYR LYS CYS LYS VAL SER ASN LYS 312 GGC CTC CCG TCC TCC ATC GAG AAA ACC ATC TCC AAA GCC 975 GLY LEU PRO SER SER ILE GLU LYS THR ILE SER LYS ALA 325 AAA GGG CAG CCC CGA GAG CCA CAG GTG TAC ACC CTG CCC 1014 LYS GLY GLN PRO ARG GLU PRO GLN VAL TYR THR LEU PRO 338 CCA TCC CAG GAG GAG ATG ACC AAG AAC CAG GTC AGC CTG 1053 PRO SER GLN GLU GLU MET THR LYS ASN GLN VAL SER LEU 351 '/fCC TGC CTG GTC AAA GGC TTC TAG CCC AGC GAG ATC GCC 1092 THR CYS LEU VAL LYS GLY PHE TYR PRO SER ASP ILE ALA 364 GTG GAG TGG GAG AGC AAT GGG CAG CCG GAG AAC AAC TAG 1131 VAL GLU TRP GLU SER ASN GLY GLN PRO GLU ASN ASN TYR 377 AAG ACC ACG CCT CCC GTG CTG GAG TCC GAC GGC TCC TTC 1170 LYS THR THR PRO PRO VAL LEU ASP SER ASP GLY SER PHE 390 TTC CTC TAG AGC AGG CTA ACC GTG GAC AAG AGC AGG TGG 1209 PHE LYS TYR SER ARG LEU THR VAL ASP LYS SER ARG TRP 403 CAG GAG GGG AAT GTC TTC TCA TGC TCC GTG ATG CAT GAG 1248 GLN GLU GLY ASN VAL PHE SER CYS SER VAL MET HIS GLU 416 GCT CTG CAC AAC CAC TAG ACA CAG AAG AGC CTC TCC CTG 1287 ALA LEU HIS ASN HIS TYR THR GLN LYS SER LEU SER LEU 429 TCT CTG GGT AAA TAG 1302 SER LEU GLY LYS 433 WE CLAIM: 1. An IFNα-Fc hybrid recombinant protein containing an interferon molecule joined at its C-terminal end through a peptide linker to the N-terminal end of the immunoglobulin γ4 Fc fragment, the peptide linker having the sequence Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser (SEQ ID NO: 1). 2. The hybrid recombinant protein as claimed in claim 1 wherein said hybrid optionally joined to another interferon molecule at its C-terminal end through the peptide linker to the N-terminal end of a chain of the immunoglobulin Fc fragment, thereby forming a homodimer. 3. The hybrid recombinant protein as claimed in claim 1 and 2 wherein the interferon molecule is IFNα2a, IFNα2b or IFNß.

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2931-DEL-1996-Claims-(02-09-2008).pdf

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2931-del-1996-description (complete).pdf

2931-DEL-1996-Form-1-(02-09-2008).pdf

2931-DEL-1996-Form-1-(29-08-2008).pdf

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2931-del-1996-form-29.pdf

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2931-DEL-1996-PCT-210-(29-08-2008).pdf

2931-del-1996-pct-210.pdf

2931-DEL-1996-Petition-137-(29-08-2008).pdf

2931-DEL-1996-Petition-138-(29-08-2008).pdf

2931-del-1996-petition-138.pdf

2931-delnp-2005-complete specification (granted).pdf