HYBRID WITH INTERFERON- alpha AND AN IMMUNOGLOBULIN Fc LINKED THROUGH A NON-IMMUNOGENIC PEPTIDE

Abstract: Disclosed is a hybrid recombinant protein consisting of human interferon, preferably interferon- alpha (IFN alpha ), and human immunoglobulin Fc fragment, preferably gamma 4 chain, joined by a 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).

Description: Hybrid with Interferon σ and an I munoglobuiin Fc Linked through a Non immuπogeπic Peptide Background of the invention lπterferon-σ ("IFNσ") was among the first of the cγtokines 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 cγtokines, 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). However, such hybrids can present problems in that the peptide at the C-termiπal 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 is preferred over the γ\ chain because the former has little or no complement activating ability. The C-terminal end of the IFNα is linked to the N-termiπal end of the Fc fragment. An additional IFNα (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 γ4 chain. If the Fc fragment selected is another chain, such as the μ 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 (SEQ ID NO:!)). 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 IFNα. Due to the linker, it is also designed to reduce the possibility of generating a new immunogenic epitope (a πeoantigen) at what would otherwise be the fusion point of the IFNσ 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 IFNα 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 adminstration) 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 interleukiπ-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 γ4 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 (SEQ ID N0:1), was created to link the two moieties. The complete nucleotide sequence of the preferred | hybrid (including the linker and the Fc moiety) appears in SEQ ID NO: 7. 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 yA 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 SEQ ID N0:7, 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 IFNα 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. Mol. Bio/. 33:251-300, 1986; Zoon, K.C., Interferon, 9:1-12, 1987). IFNσ2 (also called IFNσA) is one of the most intensively studied interferon species. The recombinant version of IFNσ2 has been used as a therapeutic for several years. Two IFNα2 recombinant products, IFNα2a and IFNσ2b, 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. Bur. J. Biochem. 190:257-61, 1990). IFNσ2a was selected as the fusion partner for the interferon hybrid of the invention, although the IFNα2b 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 γ4 chain as the Fc moiety in the hybrid is that it is stable in the human circulation. The γ4 chain (unlike the yl chain) also avoids the wide spectrum of secondary biological properties, such as complement fixation and antibody-dependant cell-mediated cytotoxicitγ (ADCC), which may be undesirable properties. The cDNA of the IFNσ2a can be obtained by reverse transcription and PCR, using RNA extracted from leukocytes which express IFNα. 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 at., Methods in Enzymology, 78A:29-38, Adacemic Press, 1981). As mentioned above, IFNα is a collection of IFN species and each cell expresses several different IFNα subspecies at the same time. The DNA sequence ho ology among these species is so high that RT-PCR would probably amplify a group of them instead a specific one. To obtain specifically the IFNα2a 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 IFNσ2a. These are position S22 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. et al., 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 IFNσ-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 SEQ ID N0:1. 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 ceils or other expression systems. The prokaryotic system, £ coli, is not able to do post-translationai modification, such as glycosylation. But this is probably not a serious problem for the IFNσ-Fc hybrid since the native IFNσ and immunoglobulin γ4 molecule are not heavily glycosylated. Further, it has been reported that recombinant IFNα without any glycosylation retained its biological activity (Baron, E. and Narula, S., Bio/technology, 10:179-190, 1990). HouveuEr, the purification of recombinant protein from the £ coli lysate can be difficult. The foreign proteins expressed by £ coli often aggregate and form insoluble inclusion bodies. Thus, solubilization and subsequent refolding of the inclusion bodies is usually required (Schein, CH. 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-transiational 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 IFNσ-Fc hybrid or the wild type IFNσ2a. Unfortunately the IFNσ-Fc secreted was found to be partially degraded on SDS- PAGE. whereas the IFNα2a alone was not. The degradation was believed to be caused by the protease activities present in the yeast expression system, as reported by Scorer, CA. 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 IFNσ-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 ceils, NSO cells, were transfected by the pCDNA3|IFNσ-Fc expression vector using the electroporation method. The cells were selected by G418 at a concentration of 0.8 mg/ml. The IFNσ-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 IFNσ-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 IFNσ2a 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-IFNσ signal peptide is used. Either way, it will bring in additional artificial amino acid residue(s) at the N-terminal end of the recombinant IFNσ-Fc. As mentioned above, the purification of the IFNσ-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 IFNα bioactivity. Using an antiviral assay, it was demonstrated that the hybrid of SEQ ID NQ:7 had a specific activity about 5 to 10 fold higher than a related IFNσ-Fc hybrid, in which the linker molecule had the sequence Gly Gly Ser Gly Gly Ser (SEQ ID N0:2), and the Fc portion of the hybrid was derived from human IgGI rather than lgG4. Nevertheless, although the bioiogicial activity of the hybrid shown in SEQ ID N0:7 was improved substantially over the similar hybrid, it was still lower than that of the native IFNα. However, it is expected that this hybrid will have a longer half-life in vivo, than the native IFNσ. This expectation is based on results demonstrating that the related IFNα hybrid with the linker sequence shown in SEQ ID N0:2 and an IgGI Fc portion showed a much longer half-life, in a pharmacokiπetic study in a mouse model, than did the native IFNα. Because the hybrid of SEQ ID N0:7 is expected to have a longer half-life in vivo than native IFNσ, even though its specific activity is lower, this novel hybrid is expected to be preferred to the native IFNσ for clinical use. This is because, as a result of the longer half -life, the Cxt (the area under the concentration vs. time curve) would 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 provide a several hundred fold increased exposure to IFNσ, 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 iπterferons function through the binding to their specific receptors, which is directly related to the number of molecules present. Also, the molecular weight of the IFNσ-Fc 4, 110 Kd, is more than five-fold larger than that of the wild type IFNσ2a, which is 20kd. Taking this into consideration, measuring activity in units/μmol instead of the units/mg provides a better comparison of activity specifity. Example I: Cloning human IFNσ cDNA and constructing the IFNσ-Fc expression vector 6x10 β KG-1 cells (ATCC 246) were incubated with 200 units of Seπdai 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 oiigo(dT) as 3' primer in 50m Tris-HCI (pH 8.3), SOmMKCI, and 6mM MgCI,, incubated at 42° C for 1 hour. The reaction mixture was used directly as the template for PCR to amplify IFNσ cDNA. The 5' primer for PCR contained a Hind III site and the coding sequence for the first 21 amino acids from the IFNσ2a leader peptide (SEQ ID NQ:3). The 3' primer contained the sequence coding for part of the linker (SEQ ID NQ:1) and the last five amino acids of the IFNσ2a, and a BamH I site integrated in the linker sequence (SEQ ID N0:4). The PCR buffer contained 50mM KCI, lOmMTris-Hcl (pH8.3), 1.5mM MgCI 2 , 0.01% gelatin, 0.1 mmol each of dNTP, 0.5 μmol of each primers, 5 μl RT reaction mixture, and 1 unit of Taq DNA polγmerase 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 sequence shown in SEQ ID NQ:5. The 3' primer had the sequence shown in SEQ ID N0:6. The two PCR amplified DNA segments were cloned into pUC18 vectors at sites Hind lll/BamH I or sites BamH l/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 IFNσ-Fc cDNA was then inserted into a mammalian expression vector pCDNA3 (Invitrogen, San Diego, CA) through the Hind III and EcoR I sites. Example 2: Expressing IFNσ-Fc in mammalian cells 10 7 NSO cells were mixed with lOμg linearized pCDNA3/IFNσ-Fc plas id in 0.8 ml PBS and kept on ice for 5 min. Electroporation was performed at 200v, 960μF 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 ceils 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 IFNσ-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 eiuted by 50 mM citric acid (pH 3.0) and concentrated by lyophilization. Example 3: Characterization of the IFNσ-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 IFNσ-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-IFNσ antibodies, confirming that it consists of two moieties, the IFNα and the Fc fragment. The bioactivity assay for the IFNσ-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 σ, β, and < . Current Protocols in Immunology, 1994, pp. 6.9.1- 6.9.8). Briefly, human lung carcinoma cells (A549, ATCC#CCL 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 serially diluted IFNσ-Fc hybrid or native IFNσ (NIH# GxaOI-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 encephalomγocarditis virus (ATCC #VR 129B) at a concentration of about 0.1 MOIfcell 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 IFNα- Fc hybrid was calculated by comparing it with the IFNα standard, and was found to be about 30 to 60% of the activity of the IFNσ 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. SEQUENCE LISTING 1) General Information: i) Applicant Yu, Liming; Chang, Tse Wen ii) Title of Invention: Hybrid with Interferon-αr and an Immunoglobulin Fc Linked through a Non-Immunogenic Peptide in) Number of Sequences: 7 IV) Correspondence Address: A) Addressee: Tanox Biosysteπis, Inc. B) Street: 10301 Stella Link Rd. C) City: Houston D) State: Texas E) Country: USA F) Zip: 77025 v) Computer Readable Form: A) Medium Type: Diskette, 3.5 inch B) Computer: Addonics C142 SVGA C) Operating System: DOS 3.30 D) Software: WordPerfect S.l vi) Current application data A) Application Numbe - B) Filing Date: C) Classification: vn) Prior Application Data. A) Application Number: 08/579,211 B) Filing Date: 12/28/95 vm) Attorney/Agent Information: A) Name: Mirabel, Eric P B) Registration Number- 31,211 C) Reference/Docket Number- 95-2-PCT x) Telecommunication Information: A) Telephone- (713) 664-2288 B) Telefax: (713) 664-8914 2) Information for SEQ ID NO:l. i) Sequence Characteristics: A) Length: 48 nucleic acids B) Type: nucleotide C) Strandedness: double stranded D) Topology linear xi) Sequence Description SEQ ID NO-1 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 1 5 10 GGT GGA GGA TCA 48 Gly Gly Gly Ser 15 (2) Information for SEQ ID NO.2 (i) Sequence Characteristics (A) Length: 6 amino acids (B) Type: ammo acid (D) Topology: unknown (xi) Sequence Description SEQ ID NO:2 Gly Gly Ser Gly Gly Ser 1 5 (2) Information for SEQ ID NO 3 (i) Sequence Characteristics (A) Length: 81 nucleic acids (B) Type: nucleic acid (C) Strandedness : single stranded (D) Topology: linear (xi) Sequence Description: SEQ ID NO:3: CATAAGCTTC ATCTACAATG GCCTTGACCT TTGCTTTACT 40 GGTGGCCCTC CTGGTGCTCA GCTGCAAGTC AAGCTGCTCT G 81 (2) Information for SEQ ID NO:4: (i) Sequence Characteristics: (A) Length: 40 nucleic acids (B) Type: nucleic acid (C) Strandedness: single stranded (D) Topology: linear (xi) Sequence Description: SEQ ID NO:4: CTCTGCGGAT CCACCTGAGC CACCTTCCTT ACTTCTTAAA 40 (2) Information for SEQ ID NO: 5: d) Sequence Characteristics: (A) Length: 58 nucleic acids (B) Type: nucleic acid (C) Strandedness: single stranded (D) Topology: linear (xi) Sequence Description: SEQ ID NO:5: AATGGATCCG GTGGAGGCGG AAGCGGCGGT GGAGGATCAG 40 AGTCCAAATA TGGTCCCC 58 (2) Information for SEQ ID NO:6: (i) Sequence Characteristics: (A) Length: 42 nucleic acids (B) Type: nucleic acid (C) Strandedness: double stranded (D) Topology: linear (xi) Sequence Description: SEQ ID NO:6: ATCGAATTCT ATTTACCCAG AGACAGGGAG AGGCTCTTCT GT 42 (2) Information for SEQ ID NO: 7- (i) Sequence Characteristics: (A) Length: 1302 nucleic acids (B) Type: nucleic acid (C) Strandedness: double stranded (D) Topology: linear (xi) Sequence Description: SEQ ID NO:7: 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 1 5 10 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 15 20 25 CCT CAA ACC CAC AGC CTG GGT AGC AGG AGG ACC TTG ATG 117 Pro Gin Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met 30 35 CTC CTG GCA CAG ATG AGG AAA ATC TCT CTT TTC TCC TGC 156 Leu Leu Ala Gin Met Arg Lys lie Ser Leu Phe Ser Cys 40 45 50 TTG AAG GAC AGA CAT GAC TTT GGA TTT CCC CAG GAG GAG 195 Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gin Glu Glu 55 60 65 TTT GGC AAC CAG TTC CAA AAG GCT GAA ACC ATC CCT GTC 234 Phe Gly Asn Gin Phe Gin Lys Ala Glu Thr lie Phe Val 70 75 CTC CAT GAG ATG ATC CAG CAG ATC TTC AAT CTC TTC AGC 273 Leu His Glu Met lie Glu Glu lie Phe Asn Leu Phe Ser 80 85 90 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 95 100 GAC AAA TTC TAC ACT GAA CTC TAC CAG CAG CTG AAT GAC 351 Asp Lys Phe Tyr Thr Glu Leu Tyr Gin Gin Leu Asn Asp 105 110 115 CTG GAA GCC TGT GTG ATA CAG GGG GTG GGG GTG ACA GAG 390 Leu Glu Ala Cys Val He Gin Gly Val Gly Val Thr Glu 120 125 130 ACT CCC CTG ATG AAG GAG GAC TCC ATT CTG GCT GTG AGG 429 Thr Pro Leu Met Lys Glu Asp Ser He Leu Ala Val Arg 135 140 AAA TAC TTC CAA AGA ATC ACT CTC TAT CTG AAA GAG AAG 468 Lys Tyr Phe Gin Arg He Thr Leu Tyr Leu Lys Glu Lys 145 150 155 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 160 165 ATC ATG AGA TCT TTT TCT TTG TCA ACA AAC TTG CAA GAA 546 He Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gin Glu 170 175 180 AGT TTA 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 185 190 195 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 200 205 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 210 215 220 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 225 230 GAC ACT CTC ATG ATC TCC CGG ACC CCT GAG GTC ACG TGC 741 Asp Thr Leu Met He Ser Arg Thr Pro Glu Val Thr Cys 235 240 245 GTG GTG GTG GAC GTG AGC CAG GAA GAC CCC GAG GTC CAG 780 Val Val Val Asp Val Ser Gin Glu Asp Pro Glu Val Gin 250 255 260 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 265 270 AAG ACA AAG CCG CGG GAG GAG CAG TTC AAC AGC ACG TAC 858 Lys Thr Lys Pro Arg Glu Glu Gin Phe Asn Ser Thr Tyr 275 280 285 CGT GTG GTC AGC GTC CTC ACC GTC CTG CAC CAG GAC TGG 897 Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp 290 295 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 300 305 310 GGC CTC CCG TCC TCC ATC GAG AAA ACC ATC TCC AAA GCC 975 Gly Leu Pro Ser Ser He Glu Lys Thr He Ser Lys Ala 315 320 325 AAA GGG CAG CCC CGA GAG CCA CAG GTG TAC ACC CTG CCC 1014 Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro 330 335 CCA TCC CAG GAG GAG ATG ACC AAG AAC CAG GTC AGC CTG 1053 Pro Ser Gin Glu Glu Met Thr Lys Asn Gin Val Ser Leu 340 345 350 ACC TGC CTG GTC AAA GGC TTC TAC CCC AGC GAC ATC GCC 1092 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala 355 360 GTG GAG TGG GAG AGC AAT GGG CAG CCG GAG AAC AAC TAC 1131 Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr 365 370 375 AAG ACC ACG CCT CCC GTG CTG GAC TCC GAC GGC TCC TTC 1170 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 380 385 390 TTC CTC TAC AGC AGG CTA ACC GTG GAC AAG AGC AGG TGG 1209 Phe Lys Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 395 400 CAG GAG GGG AAT GTC TTC TCA TGC TCC GTG ATG CAT GAG 1248 Gin Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 405 410 415 GCT CTG CAC AAC CAC TAC ACA CAG AAG AGC CTC TCC CTG 1287 Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu 420 425 TCT CTG GGT AAA TAG 1302 Ser Leu Gly Lys 430

Claims: What Is Claimed Is: 1. A hybrid molecule 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). 2. The hybrid molecule of claim 1 in which another interferon molecule is joined 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 molecule of claim 2 in which the interferon molecule is IFNα2a or IFNα2b. 4. The hybrid molecule of claim 2 in which the Fc fragment is a γ4 chain Fc fragment. 5. A method of treating hepatitis, hairy cell leukemia, multiple myeloma, or other cancers or viral diseases, comprising administering the hybrid molecule of any of claims 1 to 4.

Inventors: CHANG TSE WEN, YU LIMING, CHANG, TSE, WEN, YU, LIMING
Applicants: TANOX BIOSYSTEMS INC, TANOX BIOSYSTEMS, INC.
Application year: 1996
Main classification: A61K38
IPC codes: A61K38, A61K39, A61P1, A61P31, A61P35, A61P37, C07K14, C07K16, C07K19, C12N5, C12N15, C12P21, C12R1