HK1017261B - HYBRID WITH INTERFERON-α AND AN IMMUNOGLOBULIN FC LINKED THROUGH A NON-IMMUNOGENIC PEPTIDE - Google Patents

Description

Hybrid with interferon alpha and immunoglobulin Fc linked together via a non-immunogenic peptide

Background

Interferon-alpha ("IFN α") is the first cytokine produced by recombinant DNA technology and has been shown to be of therapeutic value in conditions such as inflammation, viral and malignant diseases. A variety of IFN alpha preparations, including purified from natural sources and produced by recombinant DNA techniques, have been used and tested in a variety of malignant and viral diseases. IFN α can cause regression of certain developed tumors and induce negative responses in certain viral infections. To date, IFN α has been licensed or tested in many countries for the following indications: such as kaposi's sarcoma; hairy cell leukemia; malignant melanoma; basal cell carcinoma; multiple myeloma; renal cell carcinoma; hepatitis B; hepatitis C; willow warts; I/II herpes, varicella/herpes zoster and mycosis fungoides.

Most cytokines, including IFN α, have a relatively short circulating half-life due to their local and transient effects produced in vivo. Serum half-life of IFN alphaThe period is only about 2 to 8 hours (Roche labs. Referenon A, Schering Intron A, Physicians' desk reference, 47)^thedition, 1993, pp.2006-2008, 2194-. To use IFN α as an effective systemic therapeutic, it is administered in extremely large doses and often. For example, one of the recommended courses of treatment for AIDS-related kaposi's sarcoma is to start with an induction dose of 3 thousand 6 million IU per day for 10 to 12 weeks, given by intramuscular or subcutaneous injection, followed by a maintenance dose of 3 thousand 6 million IU 3 times a week. (Roche labs. Reference a, Physicians' Desk Reference, 47th edition, 1993, pp.2006-2008). Such frequent parenteral administration is inconvenient and painful. Furthermore, the toxic effects that may be caused by high doses are a difficult problem for some patients. Cutaneous, neurological, endocrine and immunotoxicity have been reported. To overcome these disadvantages, the molecules may be modified to increase their circulatory half-life or the drug formulation may be modified to prolong their release time. The dosage and frequency of administration can be reduced while still increasing its efficacy. Doses below 9 million units are reported to be tolerable, while doses above 3 thousand 6 million units often induce severe toxicity and significantly alter patient condition. (Quesada, J.R.et al, J.Clin, Oncol., 4: 234-43, 1986). It is also possible to substantially reduce toxic effects by generating new IFN α, which may be more stable in circulation and require lower doses. Efforts have been made to produce recombinant IFN α -gelatin conjugates with extended residence times (Tabata, Y.et al., Cancer Res.51: 5532-8, 1991). Lipid-based encapsulated IFN α formulations have also been tested in animals and achieve prolonged release of protein in the peritoneum (Bonetti, A.and Kim, S.cancer chemotherPharmacol.33: 258-261, 1993).

IgG and IgM immunoglobulins are the most abundant in human blood. The circulation half-life is from several days to 21 days. IgG, when used to form recombinant hybrids, was found to increase the half-life of a number of ligand-binding proteins (receptors), including the soluble CD4 molecule, LHR and IFN-gamma receptor (Mordenti J.et al, Nature, 337: 525-31, 1989; Capon, D.J.and Lasky, L.A., U.S. Pat. No. 5,116,964; Kurschner, C.et al, J.Immunol.149: 4096-. However, such hybrids present the problem that the peptide at the C-terminus of the active moiety, and the peptide at the N-terminus of the Fc moiety, at the fusion, generate a new peptide sequence, which is a new antigen and is immunogenic. The present invention relates to an IFN α -Fc hybrid designed to overcome this problem and to extend the half-life of IFN α.

Summary of The Invention

The present invention relates to hybrid recombinant proteins composed of two subunits. Each subunit comprises a human interferon, preferably IFN α, linked by a peptide linker consisting essentially of a T cell inert sequence, to a human immunoglobulin Fc fragment, preferably the γ 4 chain. The γ 4 chain is preferred over γ 1 because the former has little or no complement activation capacity. The invention also relates to the use of the hybrid molecule in the manufacture of a medicament for the treatment of hepatitis, hairy cell leukemia, multiple myeloma or other cancer or viral disease.

The C-terminus of IFN α is linked to the N-terminus of the Fc fragment. Additional IFN α (or other cytokine) can adhere to the N-terminus of any other unbound Fc chain of the Fc fragment, producing a homodimer of γ 4 chains. If the Fc fragment selected is another chain, such as a μ chain, then since the Fc fragment can form a pentamer with 10 possible binding sites, this can result in a molecule with interferon or other cytokine attached at each of the 10 binding sites.

The two parts of the hybrid are linked via a peptide that is immunologically inert to T cells, such as GlyGly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser (SEQ ID NO: 1). The peptide itself is immunologically inactive. Insertion of this peptide at the fusion site eliminates the new antigenicity that results from the joining of the two peptide moieties. Linker peptides may also increase the flexibility of these moieties and may retain biological activity. This relatively long linker peptide helps to overcome possible steric hindrance from the Fc portion of the hybrid, which can interfere with the activity of the hybrid.

The hybrid has a longer half-life than native IFN α. Due to the linker, it is also possible to design so that the probability of generating new immunogenic epitopes (neoantigens) is reduced, which is the point of fusion of IFN α and the Fc fragment of an immunoglobulin.

Cytokines are generally small proteins with a relatively short half-life that rapidly disappear in a variety of tissues, including unwanted locations. It is believed that small amounts of certain cytokines can cross the blood brain barrier and enter the central nervous system, thus causing severe neurotoxicity. IFN α, linked to the Fc γ of the present invention, would be particularly useful for the treatment of hepatitis B or C, since these products have a long residence time in the blood vessel (once administered intravenously) and do not penetrate into undesired locations.

The specific hybrids described herein can also serve as models for the design and construction of other cytokine-Fc hybrids. The same or similar linkers can be used to reduce the likelihood of generating new immunogenic epitopes while retaining biological activity. cytokine-Fc hybrids, in which interleukin-2 is a cytokine, or hybrids including other cytokines, can be made using the same techniques.

Detailed Description

The hybrid molecules of the present invention include an interferon moiety linked to an immunoglobulin Fc moiety via a unique linker. Preferably, the C-termini of the two interferon moieties are attached to the two N-termini of the heavy chain γ 4Fc fragment, respectively, to create a homodimeric structure. A unique linker peptide, Gly Gly Ser Gly Gly Ser Gly Gly Gly GlySer Gly Gly Gly Gly Ser (SEQ ID NO: 1), can be generated to link the two moieties. The complete nucleotide sequence (including linker and Fc portion) of the preferred γ 4 hybrid is shown in SEQ id no: 7 and the linker is located at amino acid residues 189 to 204.

The advantage of hybrids, even native cytokines, is a longer half-life in vivo. Hybrids comprising interferon and gamma 4 chain Fc homodimers are larger than native interferons. Due to the larger pore size of the blood vessels in the liver, this macromolecule is more suitable for the treatment of hepatitis, where the virus is primarily responsible for eroding the liver.

Linker peptides are designed to increase the flexibility of the two moieties and thus maintain their biological activity. Although both interferons and immunoglobulins are derived from the human body, there is always the possibility of generating new immunogenic epitopes at the point of two-molecule fusion. Thus, another advantage of the linker of the invention, which consists essentially of a T cell inert sequence, is that immunogenicity can be reduced at the point of fusion. In SEQ ID NO: 7, it can be seen that if the linker (residue 189-204) is absent, a new sequence consisting of residue 189 and residue 204 can be generated. The new sequence will be a new antigen for the human body.

Human IFN α is derived from a family of many different genes. From gene and protein sequence data, more than 24 species have been identified. It varies from a few to up to 35 amino acids throughout. Most 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. Nature, 290: 20-261981: Weissmann, C.and Weber, H., prog.Nuc. acid Res.mol. biol.33; 251. Bufonic 300, 1986; Zoon, K.C., Interferon, 9: 1-12, 1987).

IFN alpha 2 (also known as IFN alpha A) is the most fully studied interferon. Recombinant forms of IFN α 2 have been used as therapeutics for several years. Two recombinant products of IFN α that are currently available are IFN α 2a and IFN α 2b, which differ by only one amino acid at position 23 and do not differ significantly in their biological activity (von Gabain, a., et al., eur.j. biochem.190: 257-61, 1990).

IFN alpha 2a is selected as the fusion partner of the interferon hybrids of the present invention, although IFN alpha 2b or other interferons (including IFN beta) may be used. Similar constructs, such as interleukin-1 or interleukin-2, may also be made with other cytokines. The same linkage can be used and can be substituted for those that are not immunogenic and retain the biological activity of the construct.

The advantage of having the γ 4 chain as the Fc portion in the hybrid is that it is stable in the human circulation. γ 4 (as opposed to γ 1 chains) avoids a broad spectrum of secondary biological properties such as complement fixation and antibody-dependent cell-mediated cytotoxicity (ADCC), which may be undesirable properties.

The cDNA of IFN alpha 2a can be obtained by reverse transcription and PCR, using IFN alpha expression of leukocyte extraction of RNA and. One such cell line, KG-1, is available from American Type Culture Collection (ATCC), Rockville, Maryland, under the number CCL 246. In the step of preparing the hybrids of the present invention, cells are challenged with Sendai virus to increase transcription of interferons prior to RNA extraction (Cantell, K.et al, methods in Enzymology, 78A: 29-38, Adacemic Press, 1981).

As mentioned above, IFN alpha IFN is a collection of IFN, and each cell can be at the same time expression of many different IFN alpha subtypes. The homology among these types of DNA sequences is so high that RT-PCR may amplify a population rather than a specific one. To obtain IFN alpha 2a cDNA specifically, PCR primers are designed such that the last nucleotide of the two primers ends at the amino acid code unique to IFN alpha 2 a. Which are positions S22 and 161, respectively (see Zoon, k.c. interferon, 9: 1-12, 1987).

Two gene fragments can be readily ligated at any desired position using overlapping PCR techniques (Daugherty, B.L.et. al., Nucleic Acids Res, 19: 2471-6, 1991). However, one disadvantage of PCR amplification is the rather high mutation ratio (Saiki, R.K.et al., Science, 239: 487, 1988). Therefore, DNA sequencing was also performed to examine whether each DNA fragment obtained by PCR had a mutation. When the fragment size exceeds 1kb, sequencing is tedious and time consuming, as is the full-length IFN α -Fc cDNA. However, the restriction enzyme BamHI site can be introduced into the linker nucleotide sequence without changing its amino acid sequence. This site is located in SEQ ID NO: 1 between nucleotides 15 and 16.

Two gene fragments from PCR were cloned into cloning vectors separately. This makes DNA easier and faster to sequence because two fragments are only hundreds of base pairs long. Once a clone with the correct DNA sequence was identified, the two gene fragments were ligated together via the BamHI site. No second round of overlapping PCR and subsequent DNA sequencing of the full-length fragment was required.

There are many ways to express recombinant proteins in vitro, including in E.coli, baculovirus, yeast, mammalian cells or other expression systems. Coli cannot perform post-translational modifications such as glycosylation. However, this is not a serious problem for IFN alpha-Fc hybrids, since native IFN alpha and immunoglobulin gamma 4 molecules are not heavily glycosylated. Furthermore, it has been reported that recombinant IFN α without any glycosylation retains its biological activity (Baron, E.and Narula, S., Bio/technology, 10: 179-190, 1990). However, purification of recombinant proteins from E.coli lysates is very difficult. Foreign proteins expressed by E.coli often aggregate and form insoluble inclusion bodies. Thus, solubilization and subsequent refolding of inclusion bodies is often 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, Calif.), overcomes some of the problems encountered with bacterial systems, typically results in high yields and the ability to perform various post-translational processing modifications. The expressed foreign protein can be secreted into the culture supernatant without many other proteins remaining, making it easier to expand the purification and processing of the protein. The system can express IFN alpha-Fc hybrid or wild-type IFN alpha 2 a. Unfortunately, IFN alpha Fc secretion after found in SDS-PAGE partially degraded, but IFN alpha 2a alone was not. Degradation is believed to be caused by the protease activity present in the yeast expression system, as described by Scorer, c.a.et.al, Gene, 136: 111-9, 1993. A point that is rather weak in the pivot region may be a target for the protease.

Also tried are mammalian cell expression systems for IFN α -Fc hybrids. A mammalian expression vector, pCDNA3(Invitrogen, San Diego, Calif.), containing a CMV promoter and a NEO resistance gene was used. Host cells, NSO cells, were transfected with pCDNA3/IFN α -Fc expression vector using electroporation. Cells were selected at a concentration of 0.8 mg/ml of G418. Clones expressing IFN α -Fc were identified by ELISA. The hybrid in this system can be successfully expressed without degradation.

There are many advantages in this mammalian expression system. First, the recombinant protein is secreted into the culture supernatant without aggregation, so that the purification can be simplified. A chromatography step using a protein A column can yield a purified IFN α -Fc protein. Meanwhile, the glycosylation pattern of the protein produced by the system is very similar to that of the natural molecule, and the protein is expressed by mammalian cells. Furthermore, natural IFN alpha 2a signal peptide sequence included in the expression vector. Thus, the protein secreted by the cell has a true N-terminus, however, in E.coli or yeast expression systems, either no signal peptide or a non-IFN α signal peptide is used. In any case, the recombinant IFN alpha Fc N-terminal brought additional artificial amino acid residues.

As mentioned above, the purification of IFN-. alpha.Fc recombinant proteins from culture supernatants is quite straightforward. Proteins with a purity of 90% or more (judged by SDS-PAGE) can be easily obtained by single-step affinity chromatography on a protein A column.

A number of assays are available for detecting IFN α biological activity. Using antiviral assays, it has been demonstrated that SEQ ID NO: 7, having a specific activity about 5 to 10 fold higher than that of the related IFN α -Fc hybrid, wherein the linker molecule has the sequence Gly Gly Ser Gly Gly Ser (SEQ ID NO: 2) and the Fc portion of the hybrid is derived from human IgG1 rather than IgG 4. However, although shown in SEQ ID NO: 7 has a substantial improvement in biological activity, which is still lower than natural IFN alpha. However, it is expected that this hybrid will have a longer half-life in vivo. This expectation is based on the demonstration that related IFN α hybrids with Gly Gly Ser Gly Gly Ser linker sequences and IgGl Fc portions have longer half-lives than native IFN α in a mouse model.

Because SEQ ID NO: 7 is expected to have a longer in vivo half-life than native IFN α, so even though its specific activity is lower, the new hybrid should be superior to native IFN α for clinical use. This is because the longer half-life results, Cxt (concentration versus time curve area) can be hundreds of times greater than natural IFN alpha. This means that at the same molarity dose of native IFN α and hybrids, the latter can provide hundreds of times increased IFN α exposure, resulting in greatly increased efficacy at the same dose, and the dosing frequency can be lower.

In detecting specific activity, the molar concentration is preferably substituted for the expression activity in units of mass per protein. This is because interferons act by binding to their specific receptors, which is directly related to the number of molecules present. Meanwhile, the molecular weight (110Kd) of IFN α -Fc γ 4 is about 5 times greater than that of the wild-type IFN α 2a (20 Kd). In this regard, detection of activity in units/micromolar instead of units/mg provides a more specific comparison of activity.

Example 1 cloning of human IFN alpha cDNA and construction of IFN alpha-Fc expression vector

6×10⁶One KG-1 cell (ATCC 246) was incubated with 200 units of Sendai virus overnight at 37 ℃. The cells were recovered and washed well with PBS. Total RNA was extracted using the RNA-ZOL RNA isolation kit (BIOTEX, Houston, TX) following the manufacturer's instructions. Reverse transcription was performed using AMV reverse transcriptase with oligo (dT) as 3' primer in 50mM Tris-HCl (pH 8.3), 60mM KCl and 6mM MgCl₂In (3), first strand cDNA was synthesized by incubating at 42 ℃ for 1 hour. The reaction mixture was directly used as a template for PCR to amplify IFN α cDNA. The 5' primer used for PCR contained a Hind III site and the coding sequence from the first 21 amino acids of the IFN α 2a leader (SEQ ID NO: 3). The 3' primer contains a sequence (SEQ ID NO: 4) encoding part of the linker (SEQ ID NO: 1) and 5 amino acids after IFN α 2a, and integrated in one BamHI site in the linker sequence. The PCR buffer solution contained 50mM KCl，10mMTris-Hcl(pH8.3)，1.5mMMgCl₂0.01% gelatin, 0.1 mmoles of each dNTP, 0.5. mu. moles of each primer, 5. mu.l of RT reaction mix, and 1 unit of Taq DNA polymerase in a total volume of 50. mu.l. PCR conditions were 94 deg.C (1 min), 55 deg.C (2 min) and 72 deg.C (2 min), 40 cycles in GeneAmp PCR System 9600(Perkin Elmer, Norwalk, CT).

The cDNA of human immunoglobulin gamma 4Fc was obtained by reverse transcription and PCR as described above. RNA was extracted from human tonsillar B cells. The 5' primer has the sequence shown in SEQ ID NO: 5, and (c) a sequence shown in the specification. The 3' primer has the sequence of SEQ ID NO: 6.

Two PCR-amplified DNA fragments were cloned into pUC18 at HindIII/BamHI sites or BamHI/EcoRI sites, respectively. After confirmation of the DNA sequence with the kit by DNA sequencing from USB (Cleveland, Ohio), the two fragments were ligated together in a second round of cloning via the BamH I site. The full-length IFN α -Fc cDNA was re-embedded into the mammalian expression vector pCDNA3(Invitrogen, San Diego, Calif.) via HindIII and EcoRI sites.

Example 2: expression of IFN alpha-Fc in mammalian cells

Will 10⁷Individual NSO cells were mixed with 10 μ g of linearized pcDNA3/IFN α -Fc plasmid in 0.8 ml PBS and placed on ice for 5 minutes. Electroporation was performed at 200v, 960. mu.F using GenePulser (BioRad, Hircules, Calif.). The cells were placed back on ice for 20 minutes and transferred to 100 mm tissue culture dishes supplemented with 10 ml DMEM (and 2% FCS). After 2 days of incubation at 37 ℃, the cells were washed and resuspended in the same medium. The screening was started by adding 0.6 mg/ml G418. Cells were plated on 8 96-well microplates and incubated at 37 ℃. Colonies appeared after one week, which could be easily screened within 2 weeks. The supernatant from each well, if a single colony grows, is collected. IFN α -Fc in the supernatant was determined quantitatively by ELISA assay using goat anti-human IgG and anti-human Fc conjugated with horseradish peroxidase. Clones with higher ELISA readings and smaller colony sizes were selected for further subcloning. These colonies were transferred toIn a 24-well plate, and supplied with a medium containing G418. Clones with the highest secretion level were expanded and grown in a spinner. In large scale preparations, culture supernatants were collected and passed through a protein a sepharose column equilibrated with PBS. The protein bound to protein A was eluted with 50mM citrate (pH3.0) and concentrated by freeze-drying.

Example 3: identification of IFN alpha-Fc hybrids

The purity of the recombinant protein isolated from NSO medium was checked by SDS-PAGE and Western blotting. Only one protein band was visible on blotted membranes stained with ponceau S for all proteins, indicating homogeneity of the protein preparation. The apparent molecular weight of this protein was about 55kd under reducing conditions and 110kd under non-reducing conditions, which is indeed the expected size of the IFN α -Fc hybrid. Under non-reducing conditions, a doubling of its apparent molecular weight suggests that this hybrid is in the dimeric form. The recombinant protein was identified by anti-Fc and anti-IFN alpha antibodies, confirming that it consists of two parts, IFN alpha and Fc fragment.

The analysis of the biological activity of IFN alpha-Fc is an antiviral assay. In particular, the analytical method used is a modification of the strategy described by Rodert M.Friedman et al (Measurement of antigenic activity induced by interferons. alpha., beta. and. gamma., Current Protocols in Immunology, 1994, pp.6.9.1-6.9.8). Briefly, human lung cancer cells (a549, ATCC # CCL185) were seeded into 96-well plates at a density of 40,000 cells/well and incubated at 37 ℃ for 24 hours. IFN α -Fc hybrids or native IFN α (NIH # GXA01-901-535) were added in a 1: 2 dilution series and incubated at 37 ℃ for 24 hours. Each sample was run in triplicate. The medium was replaced with fresh medium containing encephalomyocarditis virus (ATCC # VR 129B) at a concentration of about 0.1 MOI/cell and cultured at 37 ℃ for another 48 hours. Dead cells were washed off by aspiration and washed extensively with PBS. Adherent cells were fixed with 2% formaldehyde and stained with giemsa dye. The plates were rinsed with tap water and allowed to dry. The stained cells were dissolved in methanol and the sample was read spectrophotometrically at 595 nm. The antiviral activity of the IFN α -Fc hybrids was evaluated in comparison to IFN α standards and was found to be about 30-60% of the activity of the IFN α standards.

It is to be understood that the terminology and expressions used herein are used for purposes of illustration and not of limitation, and that the scope of the invention is defined only by the claims which follow, and includes all equivalents of the subject matter of the claims.

Sequence listing

(1) General information:

(i) the applicant: yu Liming; chang, Tse Wen

(ii) The invention name is as follows: hybrid with interferon alpha and immunoglobulin Fc linked together via a non-immunogenic peptide

(iii) Sequence number: 7

(iv) Communication address:

(A) the addressee: teno Kers biosystems Ltd

(B) Street: 10301 Stella Link Rd.

(C) City: houston

(D) State: state of Texas

(E) The state is as follows: united states of America

(F) And E, postcode: 71025

(v) Computer readable form

(A) Type of medium: 3.5 inch floppy disk

(B) A computer: addonics C142 SVGA

(C) Operating the system: DOS 3.30

(D) Software: wordpeffect

(vi) Information of the present application

(A) Application No.:

(B) application date:

(C) and (4) classification:

(vii) application of prior art

(A) Application No.: 08/579,211

(B) Application date: 1995.12.28

(viii) Lawyer/attorney information:

(A) name: mirabel, Eric D.

(B) Registration number: 31,211

(C) Volume/document number: 95-2-PCT

(ix) Electric communication information:

(A) telephone: (713)664-2288

(B) Faxing: (713)664-8914

(2) SEQ ID NO: 1 information

(i) Sequence characterization

(A) Length: 48 nucleotides

(B) Type (2): nucleotide, its preparation and use

(C) Chain property: double chain

(D) Topology: linearity

(xi) Description of the sequence: SEQ ID NO: 1

GGT GGC TCA GGT GGA TCC GGT GGA GGC GGA AGC GGC 35

Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10

GGT GGA GGA TCG 48

Gly Gly Gly Ser

15

(2) SEQ ID NO: 2 information

(i) Sequence characterization

(A) Length: 6 amino acids

(B) Type (2): amino acids

(D) Topology: is unknown

(xi) Description of the sequence: SEQ ID NO: 2

Gly Gly Ser Gly Gly Ser

1 5

(2) SEQ ID NO: 3 information

(i) Sequence characterization

(A) Length: 81 nucleotides

(B) Type (2): nucleotide, its preparation and use

(C) Chain property: single strand

(D) Topology: linearity

(xi) Description of the sequence: SEQ ID NO: 3

CATAAGCTTC ATCTACAAT GGCCTCACCT TTGCTTTACT 40

GGTGGCCCTC CTGGTGCTCA GCTGCAGTC AAGCTGCTCT G 81

(2) SEQ ID NO: 4 information

(i) Sequence characterization

(A) Length: 40 nucleotides

(B) Type (2): nucleotide, its preparation and use

(C) Chain property: single strand

(D) Topology: linearity

(xi) Description of the sequence: SEQ ID NO: 4

CTCTGCGGAT CCACCTGAGC CACCTTCCTT ACTTCTAAA 40

(2) SEQ ID NO: 5 information

(i) Sequence characterization

(A) Length: 58 nucleotides

(B) Type (2): nucleotide, its preparation and use

(C) Chain property: single strand

(D) Topology: linearity

(xi) Description of the sequence: SEQ ID NO: 5

AATGGATCCG GTGGAGGCG AAGCGGCGGT GGAGGATCAG 40

AGTCCAAATA TGTCCCC 58

(2) SEQ ID NO: 6 information

(i) Sequence characterization

(A) Length: 42 nucleotides

(B) Type (2): nucleotide, its preparation and use

(C) Chain property: double chain

(D) Topology: linearity

(xi) Description of the sequence: SEQ ID NO: 6

ATCGAATTCT ATTTACCCAG AGACAGGGAG AGGCTCTTCT GT 42

(2) SEQ ID NO: 7 information

(i) Sequence characterization

(A) Length: 1302 nucleotides

(B) Type (2): nucleotide, its preparation and use

(C) Chain property: double chain

(D) Topology: linearity

(xi) Description of the sequence: 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 Cya 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 Gln 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 Gln Met Arg Lys Ile Ser Leu Phe Ser Cya

40 45 50

TTG AAG GAC AGA CAT GAC TTT GGA TTT CCC CVG GAG GAG 195

Leu Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu

55 60 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

70 75

CTG 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

80 85 90

ACA AAG GAC TCA TCT GCT GCT TGG GAT GAG ACC CTC CTA 312

Thr Lya Asp Ser Ser Ala Ala Trp Asp Gln 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 Gln Gln 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 Ile Gln 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 Ile Leu Ala Val Arg

135 140

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

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

Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln 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 GTG 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 Ile Sar 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 Gln Glu Asp Pro Glu Val Gln

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 Gln Phe Asn Ser Thr Tyr

275 280 265

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 Typ

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 Ile Glu Lys Thr Ile Ser Lys Ala

315 320 325

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

330 335

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

340 345 350

ACC TGC CTG GTC AAA GGC TTC TAC CCC AGC GAC ATC GCC 1092

Thr Cya Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

355 360

GTG GAG TGG GAG AGC AAT GGG CAG CCG GAG AAC AAC TAC 1131

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asp 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 Aep 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

Gln Gln Gly Asn Val Phe Ser Cye 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 Gln Lys Ser Leu Ser Leu

420 425

TCT CTG GGT AAA TAG 1302

Ser Leu Gly Lys

430

Claims (5)

1. A hybrid molecule in monomeric form consisting of an interferon molecule linked at the C-terminus to the N-terminus of an immunoglobulin Fc fragment via a peptide linker consisting of the sequence Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly GlyGly Gly Ser.

2. A hybrid molecule in homodimeric form consisting of two subunits, each comprising one interferon molecule linked at the C-terminus to the N-terminus of an immunoglobulin Fc fragment via a peptide linker consisting of the sequence Gly Ser GlyGly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser.

3. The hybrid molecule according to claim 2 wherein the interferon molecule is IFN α 2a or IFN α 2 b.

4. The hybrid molecule according to claim 2 wherein the Fc fragment is a γ 4 chain Fc fragment.

5. Use of the hybrid molecule of any of claims 1-4 in the manufacture of a medicament for the treatment of hepatitis, hairy cell leukemia, multiple myeloma or other cancer or viral disease.