IL103052A - Soluble interferon-alpha receptors,their preparation and pharmaceutical compositions containing them - Google Patents

Description

Soluble interferon-a receptors, their preparation and pharmaceutical compositions containing them on:on ,o>o o» a-) aio N 3θΐ7ΐ οτηκ o ? !»3n ηιηρΐΊ 'TWDJII Yeda Research and Development Co. 103052/2 FIELD OF THE INVENTION The present invention relates to soluble interferon-a receptors, their muteins and fused proteins and the salts, functional derivatives and active fractions thereof, as well as to their production and pharmaceutical compositions containing them.

BACKGROUND OF THE INVENTION Interferon (IFN)-a constitute a family of structurally related cytokines, defined by their ability to confer resistance to viral infections. Many other biological activities of IFN-have been reported, including inhibition of cell proliferation, induction of class I MHC antigens and several other immunoregulatory activities (1). IFN-a is useful for the treatment of several viral diseases including hepatitis-C (2) and viral warts (3) as well as certain malignancies such as hairy cell leukemia (4)' chronic myelogeous leukemia (5) and Kaposi's sarcoma (6).

As in the case of other cytokines, IFN-a exerts its biological activities by binding to a cell surface receptor, which is specific for all IFN-a subtypes as well as IFN-β. Human IFN-a receptor was identified and closed from Daudi cells (7). When expressed in murine cells this receptor confers them responsive to human IFN-aB and to a lesser extent to other IFN-a species, indicating that additional receptors or accessory proteins may be involved in the response to various IFN-a subtypes. The cloned receptor had a single transmembrane domain, an extracellular and an intracellular domain. Lutfalla et al. the same research group as (7) report in J. Biol. Chem. on the same receptor protein, i.e. the cellbound receptor.

EP369,877 relates to a protein L7 which is on the surface of cells, i.e. not soluble. 103052/2 Soluble cytokine receptors which correspond to the extracellular ligand binding domains of the respective cell associated receptors have been identified in the past. These include the soluble receptors of IL-6, IFN- gamma ( 8, 9, 10 ), TNF(1 1), IL-1(12), IL-4(13) and IL-2(14).

SUMMARY OF THE INVENTION The present invention provides human soluble IFN-a receptors obtainable by affinity chromatography of a body fluid with IFN-a2, their muteins and fused proteins and the salts, functional derivatives and active fractions thereof, herein reffered to as sIFN-a.

The body fluids may be e.g. sera obtained from healthy or sick people, or urine.

The receptors may also be produced by recombinant DNA methods.

The present invention thus also provides methods for the preparation of the soluble IFN-a receptors.

One such method comprises isolation of the soluble receptor from human fluids by passing the fluid through a column to which IFN-a is coupled, and elution of the bound soluble receptor. 103052/2 Another method comprises isolation of the soluble receptor from human fluids by passing the fluids through a column to which anti-IFN-a receptor antibody is coupled and elution of the bound soluble receptor.

Preferably the human fluids are urine or human serum.

The invention also relates to pharmaceutical compositions comprising a soluble IFN-a receptor.

DESCRIPTION OF THE FIGURES Figure 1 shows (a) Western blotting of human sera with anti-IFN-aR Mab. Lanes:-A: molecular weight markers (K); B: normal human serum (NHS, 5μ1); C and D; serum from a hairy cell leukemia (HCL) patient (5 and 1 μΐ respectively). (b) Autoradiogram of cross-linked complexes consisting of 12SI-labeled-IFN-a and IFN-a receptor in body fluids. Lanes:- E: molecular weight markers (K) ; F: serum (5 μΐ) from HCL patient cross-linked to 125I-IFN-a; G: NHS (5 μΐ) cross-linked to 125I-IFN-a. (c) Autoradiogram of cross-linked complexes following Iptn with anti-IFN-α MAb No. 74-3<15>. Lanes: H-K: serum (50 μΐ) from HCL patient cross-linked to 125I-IFN-a in the absence (lanes H and J) or in the presence of an excess of cold IFN-a (Lanes I and K) . Lanes L and M: normal human serum (50 μΐ) cross-linked to 125I-IFN-a in the absence (lane L) or in the presence (lane M) of cold IFN-a; Lane N: molecular weight markers (K) .

Figure 2 shows (a) Western blotting of sIFN-aR from urine. Lanes:- A: molecular weight markers (K); B: crude urine (1 μΐ). (b) Autoradiogram of a cross-linked complex of urinary sIFN-aR and 125I-IFN-a following Iptn. Lanes:- C: urinary proteins, 100-fold concentrated (100 μΐ) cross-linked to a-25I-IFN-a and immunoprecipitated with anti-IFN-a MAb No. 74-3; D: same as C except that excess unlabeled IFN-a was added prior to cross-linking; E: molecular weight markers (K) .

DETAILED DESCRIPTION OF THE INVENTION Soluble IFN-a receptors were identified in human serum and urine with the aid of anti-IFN-a receptor monoclonal antibodies. High levels of soluble IFN-a receptors were found in sera of hairy cell leukemia patients that were undergoing IFN-a treatment, while the level of soluble IFN-a receptors in normal human serum was low. This indicates that IFN treatment may increase shedding or release of the soluble IFN-a receptors.

The calculated molecular weight of the entire extracellular domain of the IFN-aR prior to post-translational modifications is 47,000. Since there are 12 potential glycosylation sites in this extracellular domain, it's actual molecular weight may be as high as 70,000. The soluble IFN-a receptor found in serum is a protein of molecular weight 55K as was determined by Western blotting. Using the same techniques, we obtained indications that a soluble IFN-a receptor of similar size is also present in culture medium of human Daudi cells. The results obtained with normal urine indicate that it contains a soluble IFN-a receptor having a molecular weight which is lower by 10K than the molecular weight of the soluble receptor from serum and cell culture medium. This urinary receptor (45K) was probably generated by a further truncation of the soluble receptor. Thus it appears that the naturally ocς ring soluble receptors are truncated forms of the extracellular portion of the IFN-a receptor. The truncation could occur at either terminal of the extracellular portion of the receptor and may consist of both a polypeptide and a polysaccharide. Interestingly, the two soluble receptors for TNF were found to be both C- and N-terminally truncated <1β>.

The soluble IFN-a receptors regained the ability to bind their ligand and following covalent cross-linking complexes of molecular weight 65K (urine) and 75K (serum) ere formed. In both cases the molecular weight corresponded to a 1:1 complex of the respective soluble receptor and the 20K IFN-a. An excess of IFN-a added to the cross-linking reactions reduced significantly the signal, thereby proving the specificity of the interaction between these soluble receptors and IFN-a.

Based on the sensitivity of the Western blotting it is estimated that the concentration of the soluble receptor in normal human urine is in the range of 0.1-1 ng/ml, while the level in serum from HCL patient is in the range of 10-100 ng/ml. Similar levels were found in the case of soluble IL-6 receptor (1 ng/ml for urine, 20-40 ng/ml for normal human serum and elevated levels, 150-250 ng/ml, in HIV seropositive patients) .

The soluble IFN-aR is a newly discovered member of the family of soluble cytokine receptors. So far the physiological role of the soluble cytokine receptors was not established. Two mechanisms of formation of these receptors have so far been proposed: proteolytic cleavage of the membrane anchored receptor, eg. the soluble IL-2B <21' and alternative splicing of mRNA as in the case of IL-4<22> and IL-7<23>. The soluble receptors bind their specific ligands and modulate their activity either by inhibiting the biological activity, as was shown in the TNF system 24 ' 25 > , or by enhancing their activity as demonstrated with theIL-6 system 26 >. The recombinant soluble TNF receptor was found to prevent septic shock in animal models <27> and soluble forms of IL-1 receptor were found to have profound inhibitory effects on the development of in vivo alloreactivity in mouse allograft recipients <2e>.

Similarly the soluble forms of IFN-aR may find use as modulators of IFN-a activity in autoimmune disease in which abberant expression of IFN-a was reported <2s>, e.g. systemic lupus erythematosus .

As used herein the term "muteins" refers to analogs of the soluble IFN-a receptor in which one or more of the amino acid residues of the natural soluble IFN-a receptor are replaced by different amino acid residues or are deleted, or one or more amino acid residues are added to the natural sequence of the soluble IFN-a receptor, without changing considerably the antiviral activity of the resulting product. These muteins are prepared by known synthesis and/or by site-directed mutagenesis techniques, or any other known technique suitable therefor.

The term "fused protein" refers to a polypeptide comprising the soluble IFN-a receptor or a mutein thereof fused with another protein which has an extended residence time in body fluids. The soluble IFN-a receptor may thus be fused to another protein, polypeptide or the like, e.g. an immunoglobulin or a fragment thereof .

The term "salts" herein refers to both salts of carboxyl groups and to acid addition salts of amino groups of the soluble IFN-a receptor, muteins and fused proteins thereof. Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like. Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids such as, for example, acetic acid or oxalic acid.

"Functional derivatives" as used herein cover derivatives of the soluble IFN-a receptor and its fused proteins and muteins, which may be prepared from the functional groups which occur as side chains on the residues or the N- or C- terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e. they do not destroy the activity of the protein and do not confer toxic properties on compositions containing it. These derivatives may, for example, include polyethylene glycol side-chains which may mask antigenic sites and extend the residence of the soluble IFN-a receptor in body fluids. Other derivatives include aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues formed with acyl moieties (e.g. alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl groups ( for example that of seryl or threonyl residues ) formed with acyl moieties .

As "active fractions" of the soluble IFN-a receptor, its fused proteins and its muteins , the present invention covers any fragment or precursors of the polypeptide chain of the protein molecule alone or together with associated molecules or residues linked thereto, e.g. sugar or phosphate residues, or aggregates of the protein molecule or the sugar residues by themselves, provided said fraction has the same biological and/or pharmaceutical activity.

The soluble IFN-a receptor and its muteins, fused proteins and their salts, functional derivatives, and active fractions thereof are indicated for the treatment of autoimmune diseases and other inflammations in mammals.

The present invention further relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and the soluble IFN-a receptor of the invention or its active muteins, fused proteins and their saltsA functional derivatives or active fractions thereof.

The method of administration can be via any of the accepted modes of administration for similar agents and will depend on the condition to be treated, e.g. intravenously or intramuscularly or subcutaneously, in case of systemic viremia, or local injection or topical application in case of a localized infection, or continuously by infusion, etc.

The pharmaceutical compositions of the invention are prepared for administration by mixing the soluble IFN-a receptor its derivatives, with physiologically acceptable carriers, stabilizers and excipients, and prepared in dosage form, e.g. by lyophilization in dosage vials. The amount of active compound to be administered will depend on the route of administration, the disease to be treated and the condition of the patient. Local injection, for instance, will require a lower amount of the protein on a body weight basis than will intravenous infusion in case of systemic viremia.

The invention will now be illustrated by the following non-limiting examples: Materials Sera of patients were obtained from Dr. Dan Aderka (Ichilov Hospital, Tel-Aviv, Israel). Crude urinary proteins of normal individuals, concentrated 1000-fold ; di-N-hydroxy- succinimidyl suberate (DSS) was from Pierce.

Interferons Recombinant IFN- 2 (2X10e units/mg) was kindly provided by Dr. C. Weismann, University of Zurich. IFN-a was labeled by a modification of the Chloramine T method c3-6). Briefly, 7 g of IFN-a was labeled with 1 mCi of Na125I in the presence of 1 mg/ml of chloramine T (20 sec. on ice), to a specific activity of 4X107 cpm/μg.

EXAMPLE 1; Immunization of mice, cell fusion and screening Balb/c mice were injected 6 times with a partially purified preparation of E. coli IFN- R fused to protein A (10 μ9/πιου8β/ injection) . The mouse showing the highest titer by inverted sRIA (see below) was chosen for fusion. Its splenic lymphocytes were fused with an NSO-1 myeloma variant (NSO) cells, kindly provided by C. Milstein, MRC, Cambridge, U.K.). Hybridoma supernatants were tested for the present of anti-IFN-aR antibodies by an inverted sRIA. 96-well plates were coated with affinity purified goat anti-mouse antibodies followed by the addition of hybridoma supernatants and 125I-IFN-aR. Hybridomas that were found to secrete anti-IFN-aR antibodies were cloned and recloned by the limiting dilution technique. Supernatant of hybridoma No. 21.4 was used.

EXAMPLE 2; Western blotting Samples were subjected to SDS-PAGE under non-reducing conditions and electroblotted (in 25 mM Tris, 1.92 mM glycine, 20% methanol) onto nitrocellulose sheets (Schleicher and Schuell, 0.45 μπι) . Following electroblotting the sheet was incubated with a Blocking Buffer (10% non-fat milk in PBS containing 0.05% Tween-20 and 0.02% sodium azide) and then for 2 hrs at room temperature with the anti-IFN-aR antibody. The nitrocellulose sheet was washed with 0.05% Tween-20 in PBS and incubated overnight at 4°C with 125I-goat anti-mouse antibodies (0.7X106 cpm/ml, in the Blocking Buffer). The blot was then washed, dried and autoradiographed .

EXAMPLE 3: Cross-linking and Immunoprecipitation Samples of serum or urine were incubated (1 hr at 4°C) with 12SI-IFN-a (300,000 cpm) in the absence or in the presence of a 100-fold excess of unlabeled IFN-a. DSS dissolved in dimethyl sulfoxide (Me2S0) was then added to a final concentration of 1 mM and the mixture was left for 20 min. at 4°C. The reaction was stopped by the addition of 1M Tris-HCl pH 7.5, and 1M NaCl to a final concentration of 100 mM. The samples were immuno-precipitated by the addition of anti-IFN-a MAb immobilized on agarose hydrazide (25 μΐ, 7 mg/ml) <15'. Following incubation (overnight at 4°C) , the beads were washed 3 times with PBS, suspended in a sample buffer containing 2% mercaptoethanol and the supernatants were analysed by SDS-PAGE followed by autoradiography .

SDS Polvacrylamide Gel Electrophoresis SDS-PAGE (7.5% or 10% acrylamide gels) was performed by the method of Laemmli <17>.

As is illustrated in the figures, sera from two hairy cell leukemia (HCL) patients and normal human sera (NHS) were subjected to SDS-PAGE and Western blotting with anti-IFN-aR MAb. The HCL serum exhibited a band of molecular weight 55K (Figure 1, lanes C and D), while no such band was observed in NHS (Figure 1, lane B) . The high molecular weight protein seen in lanes B-D (ca. 150K) was most probably immunoglobulin. In order to characterize the 55K protein and to check its ligand binding capacity, aliquots of the various sera were covalently cross-linked to 125I-IFN-a. A specific but weak band of molecular weight 75K could be observed (lane F). The cross-linked product was enriched by Iptn with immobilized anti-IFN-a MAb. Indeed a broad band centering around 75K, probably consisting of the 55K protein cross-linked to 125I-IFN-a, was clearly observed in the HCL sera (lane H and J) but not in the NHS (lane L) . The specificity of this binding was verified by cross-linking experiments in the presence of an excess of cold IFN-a. Indeed the 75K band was significantly reduced (lanes I and K). In addition to the specific (displacable) 75K band, some non-displacable bands (50, 80, 97 and >100K) were observed. The 8OK band (clearly seen in lanes H-M) could not be completely resolved from the 75K band. It should be noted that in some experiments traces of sIFN-aR could be detected in NHS by cross-linking and Iptn (data not shown).

A sample of crude urinary proteins (1 μΐ 1000-fold concentrated) was subjected to SDS-PAGE followed by Western blotting with anti-IFN-aR MAb. A protein band of molecular weight 45K was observed (Fig. 2a). The high molecular weight protein seen in the same lane (150K) was most identified by protein sequence analysis as human immunoglobulin that could be detected in 1000-fold concentrated urine due to cross-reaction of the second antibody (125I-goat anti-mouse antibodies) with the human immunoglobulins. The ligand binding capacity of the 45K protein was checked by incubating a crude urine sample with 125I-IFN-a in the presence or absence of excess of unlabeled IFN-a, and then cross-linking with DSS. Following Iptn with anti-IFN-a MAb and SDS-PAGE, a specific cross-linked product of molecular weight 65K was detected (Figure 2b). This complex was smaller by 10K than that obtained from serum.

EXAMPLE 4; Isolation of soluble IFN- receptor from urine IFN-a2 (3 mg) is coupled to Affigel (1ml, BioRad) according to the manufacturer's instructions and packed into a column. Crude urinary proteins (1000-fold concentrated, 250ml) are loaded onto the column at a flow rate of 0.2 ml/min. The column is washed with 250 ml 1M NaCl in phosphate buffered saline (PBS) followed by PBS (10ml). Bound proteins are then eluted with 0.25mM citric acid, pH 2.2, immediately neutralized by 1M Hepes, pH 9, and 1ml fractions are collected. The fractions are analyzed by SDS-PAGE and silver staining and the protein content is determined with fluorescamine . The fractions exhibiting a major protein band of molecular weight 45K are subjected to protein microsequence analysis which identifies the protein as part of the extracellular domain of the IFN-a receptor.

Instead of employing IFN-a coupled to the Agarose column, a monoclonal antibody to the IFN-a receptor can be coupled to the Agarose column and similarly employed.

EXAMPLE 5; Recombinant DNA production of IFN-a receptors The production of a recombinant soluble IFN-a receptor may be carried out by different techniques. According to one approach, the known cDNA of the soluble IFN-a receptor is taken from plasmid pAB1023 (Uze et al . , Cell 60, 225-234, 1990). The DNA is subjected to site directed mutagenesis with appropriate oligonucleotides so that a termination codon and a polyadenylation site are inserted after codon 436 (or an earlier codon) of the IFN-a receptor precursor. This construct is then inserted into appropriately constructed expression vectors by techniques well known in the art (see Maniatis, T. et al . , Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor (1982)). Double-stranded cDNA is linked to plasmid vectors by homopolymeric tailing or by restriction linking involving the use of synthetic DNA linkers or blunt-ended ligation techniques. DNA ligases are used to ligate the DNA molecules and undesirable joining is avoided by treatment with alkaline phosphatase.

The production of a fused protein comprising the extracellular domain of the IFN-a receptor and eg. the constant region of IgG2 heavy chain may be carried out as follows: the DNA of pAB1023 is subjected to site-directed mutagenesis with appropriate oligonucleotides so that a unique restriction site is introduced immediately after codon 436 (or an earlier codon) of the IFN-a receptor precursor. A plasmid bearing the constant region of IgG2 heavy chain, e.g. pRKC042Fci (Byrn R.A. et al . , 1990, Nature (London) 344 , 667-670) is subjected to similar site-directed mutagenesis to introduce the same unique restriction site as close as possible to Asp 216 of IgGx heavy chain in a way that allows translation in phase of the fused protein. A dsDNA fragment consisting of 5' untranslated sequences and encoding the first 436 amino acids (or less) of the IFN-a receptor precursor is prepared by digestion of the mutated pAB1023 at the Smal and the unique restriction sites. The mutated pRKCD42Fci is similarly digested to generate a large fragment containing the plasmid and the IgGi sequences. The two fragments are then ligated to generate a new plasmid encoding a polypeptide precursor consisting of the N-terminal 436 amino acids (or less) of the IFN-a receptor precursor and about 227 C-terminal amino acids of IgGi heavy chain ( hinge region and CH2 and CH3 domains ) . The DNA encoding the fused protein may be isolated from the plasmid by digestion with appropriate restriction enzymes and then inserted into an efficient expression vector.

In order to be capable of expressing a soluble IFN-a receptor, its muteins or the fused proteins, an expression vector should comprise also specific nucleotide sequences containing transcriptional and translational regulatory information linked to the DNA coding for the desired protein in such a way as to permit gene expression and production of the protein. First, in order for the gene to be transcribed, it must be preceded by a promoter recognizable by RNA polymerase, to which the polymerase binds and thus initiates the transcription process. There are a variety of such promoters in use, which work with different efficiencies (strong and weak promoters). They are different for prokaryotic and eukaryotic cells.

The promoters that can be used in the present invention may be either constitutive, for example, the int promoter of bacteriophage lambda, the bla promoter of the β-lactamase gene of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene of pPR325, etc., or inducible, such as the prokaryotic promoters including the major right and left promoters of bacteriophage lambda (PL and PR), the trp, recA, lacZ, lacl , ompF and gal promoters of E. coli, or the trp-lac hybrid promoter, etc. (Glick, B.R. (1987) J. Ind. Microbiol.1:277-282).

Besides the use of strong promoters to generate large quantities of mRNA, in order to achieve high levels of gene expression in prokaryotic cells, it is necessary to use also ribosome-binding sites to ensure that the mRNA is efficiently translated. One example is the Shine-Dalgarno sequence (SD sequence) appropriately positioned from the initiation codon and complementary to the 3 '-terminal sequence of 16S RNA.

For eukaryotic hosts, different transcriptional and translational regulatory sequences may be employed, depending on the nature of the host. They may be derived from viral sources, such as adenovirus, bovine papilloma virus, Simian virus, or the like, where the regulatory signals are associated with a particular gene which has a high level of expression. Examples are the TK promoter of Herpes virus, the SV40 early promoter, the yeast gal4 gene promoter, etc. Transcriptional initiation regulatory signals may be selected which allow for repression and activation, so that expression of the genes can be modulated.

The DNA molecule comprising the nucleotide sequence coding for the soluble IFN-a receptor of the invention or its fragments or muteins or fused proteins thereof, and the operably linked transcriptional and translational regulatory signals is inserted into a vector which is capable of integrating the desired gene sequences into the host cell chromosome. In order to be able to select the cells which have stably integrated the introduced DNA into their chromosomes, one or more markers which allow for selection of host cells which contain the expression vector is used. The marker may provide for prototrophy to an auxotropic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like. The selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by cotransfection. Additional elements may also be needed for optimal synthesis of single chain binding protein mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals. cDNA expression vectors incorporating such elements include those described by Okayama, H. , (1983) Mol . Cel. Biol. 3..280.

In a preferred embodiment, the introduced DNA molecule will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species .

Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coli, for example, pBR322, ColEl, pSClOl, pACYC 184, etc. (see Maniatis et al . , op.cit.); Bacillus plasmids such as pC194, pC221, pT127, etc. (Gryczan, . , "The Molecular Biology of the Bacilli", Academic Press, NY (1982), pp. 307-329); Streptomyces plasmids including pIJlOl (Kendall, K.J. et al., (1987) J. Bacteriol . 169 : 4177-4183 ^ : Streptomyces bacteriophages such as §C31 (Chater, KF. et al . , in "Sixth International Symposium on Actinomycetales Biology" , Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54), and Pseudomonas plasmids (John, J.F., et al . (1986) Rev. Infect. Dis. 8.:693-704), and Izaki, K. (1978) Jpn. J. Bacteriol . 33 ; 729-742 ) .

Preferred eukaryotic plasmids include BPV, vaccinia, SV40, 2-micron circle, etc., or their derivatives. Such plasmids are well known in the art (Botstein, D., et al . (1982) Miami Wint.Symp. 19.-265-274; Broach, JR., in "The Molecular Biology of the Yeast Saccharomyces : Life Cycle and Inheritance", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 445-470 (1981); Broach, J.R. , (1982) Cell .28.: 203-204 ; Bollon, D.P., et al . (1980) J. Clin. Hematol. Oncol. 10:39-48; Maniatis, T. , in "Cell Biology: A Comprehensive Treatise, Vol. 3: Gene Expression", Academic Press, NY, pp. 563-608 (1980)).

Once the vector or DNA sequence containing the construct(s) has been prepared for expression, the expression vector may be introduced into an appropriate host cell by any of a variety of suitable means, such as transformation, transfection, lipofection, conjugation, protoplast fusion, electroporation, calcium phosphate precipitation, direct microinjection, etc.

Host cells to be used in this invention may be either prokaryotic or eukaryotic. Preferred prokaryotic hosts include bacteria such as E. coli. Bacillus, Streptomyces , Pseudomonas , Salmonella, Serratia, etc. The most preferred prokaryotic host is E. coli. Bacterial hosts of particular interest include E. coli K12 strain 294 (ATCC 31446), E. coli X1776 (ATCC 31537), E. coli W3110 (F~, lambda-, prototropic (ATCC 27325)), and other enterobacterium such as Salmonella typhimurium or Serratia narcescens and various Pseudomonas species. Under such conditions, the protein will not be glycosylated. The prokaryotic host must be compatible with the replicon and control sequences in the expression plasmid.

However, since the soluble IFN- receptor is a glycosylated protein, eukaryotic hosts are preferred over prokaryotic hosts. Preferred eukaryotic hosts are mammalian cells, e.g., human, monkey, mouse and Chinese hamster ovary (CHO) cells, because they provide post-translational modifications to protein molecules including correct folding, correct disulfide bond formation as well as glycosylation at correct sites. Also yeast cells and insect cells can carry out post-translational peptide modifications including high mannose glycosylation. A number of recombinant DNA strategies exist which utilize strong promoter sequences and high copy number of plasmids which can be utilized for production of the desired proteins in yeast and in insect cells. Yeast cells recognize leader sequences on cloned mammalian gene products and secrete peptides bearing leader sequences .

After the introduction of the vector, the host cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of the soluble IFN-a receptor, a fusion protein, or a mutein or a fragment thereof. The expressed protein is then isolated and purified by any conventional procedure involving extraction, precipitation, chromatography, electrophoresis, or the like, or by affinity chromatography, using anti-soluble IFN-a receptor monoclonal antibodies immobilized on a gel matrix contained within a column. Crude preparations containing said recombinant soluble IFN-a receptor are passed through the column whereby the soluble IFN-a receptor will be bound to the column by the specific antibody while the impurities will pass through. After washing, the protein is eluted from the gel at a high or a low pH, eg. pH 11 or pH 2.

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Claims (11)

103052/4

1. A soluble IFN-a receptor protein obtainable by affinity chromatography of a body fluid with IFNa2, its muteins and fused proteins, their salts, functional derivatives and active fractions (as herein defined) having the same activity as the soluble IFN-a receptor protein.

2. The soluble IFN-a receptor protein according to claim 1, having a molecular weight of about 45kD.

3. The soluble IFN-a receptor protein according to claim 1, having a molecular weight of about 55kD.

4. A process for the preparation of a soluble IFN-a receptor protein according to claim 1 , comprising isolation of the soluble receptor protein from human fluids by passing the fluid through an affinity chromatographic column, followed by purification.

5. A process according to claim 4, in which a column to which IFN-a2 is coupled is employed.

6. A process according to claim 4, in which a column to which anti- IFN-a receptor antibodies are coupled is employed.

7. A process according to any one of claims 4 to 6, wherein the fluid is urine.

8. A process according to any one of claims 4 to 6, wherein the fluid is serum.

9. A process according to claim 8, wherein the serum is obtained from healthy individuals. 103052/2

10. A process according to claim 8, wherein the serum is obtained from hairy cell leukemia patients undergoing IFN-a treatment.

11. A pharmaceutical composition comprising a soluble IFN-a receptor protein according to any one of claims 1-3.