NO316917B1 - Process for the preparation of a pharmaceutical composition containing an interleukin-1 inhibitor - Google Patents

Description

The present invention comprises a method for producing a pharmaceutical preparation containing an interleukin-1 inhibitor (IL-linhihbitor) or an IL-1 inhibitor with an N-terminal leader sequence.

This application is a continuation-in-part application of US Patent Application No. 238,713

by the same inventors, filed Aug. 31, 1988, which is a continuation-in-

party to US Patent Application No. 199,915 filed May 27, 1988.

A. IL-1

Interleukins-1 are a class of proteins produced by a variety of cell types, including monocytes and some macrophages. This class includes at least two proteins of 17-18 kilodaltons known as interleukin-1-a and interleukin-1-fl. These proteins have important physiological effects on a number of different target cells that are involved in inflammation and immune reactions. The proteins are co-mitogens (with phytohemagglutinin) for T cells. They cause both fibroblasts and chondrocytes to secrete latent collagenase and increase the surface adhesion ability of endothelial cells with respect to neutrophils. Moreover, they act on the hypothalamus as pyrogens, they stimulate the catabolism of muscle protein, and they cause hepatocytes to synthesize a class of proteins known as "acute phase reactants". Thus, interleukin-1 (IL-1) is obviously an important part of an organism's reaction to

infection and injury.

B. Pathological roles of IL-1

Despite their normally beneficial effects, however, it has emerged

cases where the effects of IL-1 are harmful. For example, IL-1 can increase the level of collagenase in an arthritic joint and has been indicated as a mediator of both the acute and chronic stages of immunopathology in rheumatoid arthritis. IL-1 may be responsible for the alteration of endothelial cell function, for directing chemotaxis and migration of leukocytes and lymphocytes into the joint tissue, inducing capillary proliferation and stimulating macrophage accumulation in the synovium during the acute phase of this disease. In the tissue-destructive phase, IL-1 has been indicated as a mediator for the induction of tissue damage by stimulating the release of enzymes from fibroblasts and chondrocytes.

Furthermore, excessive IL-1 production has been demonstrated in the skin of patients with

psoriasis, and high levels of IL-1 can be found in the synovial fluid of patients with psoriatic arthritis. IL-1 released by cells in the inflamed synovium in psoriatic arthritis can cause tissue destruction through stimulation of enzyme release from other cells. The joint pathology of Reiter's syndrome is similar to that observed in psoriatic arthritis and in rheumatoid arthritis. IL-1 has been implicated as a mediator of tissue destruction in these three different forms of inflammatory arthritis. Furthermore, IL-1 can be found in the synovial fluid of patients with osteoarthritis. Release of IL-1 by chondrocytes has been indicated in connection with the destruction of articular cartilage in this disease. IL-1 can also increase the severity of autoimmune diseases. For example, reduced IL-1 production from peripheral blood cells has been described in people suffering from systemic lupus erythematosus. Moreover, some of the changes in B-lymphocyte function may be related to abnormalities in IL-1 production or IL-1 availability.

Excessive IL-1 production has been demonstrated in the peripheral monocytes of patients with scleroderma, and IL-1 has been indicated as a possible agent of fibrosis through the stimulation of collagen production by fibroblasts. The mechanism of tissue damage in dermatomyositis may also include cell-mediated immunity, and IL-1 may therefore be involved as a mediator in this pathophysiological process.

Acute and chronic interstitial lung disease is characterized by excessive collagen production by lung fibroblasts which can be stimulated by IL-1. Later investigations on an animal model of pulmonary hypertension indicate that IL-1 may be responsible for the induction of endothelial cell changes that lead to narrowing of the pulmonary arteries. It is this narrowing that leads to pulmonary hypertension and further secondary damage. Thus, IL-1 inhibitors may be useful in the treatment of these lung diseases.

In later research, it has been reported that IL-1 can directly damage the beta cells in the islets of Langerhans that are responsible for the production of insulin. IL-1 damage to the cells is now, according to a hypothesis, a primary occurrence in the acute phase of juvenile diabetes mellitus.

Monocyte and macrophage infiltration in the kidney predominates in many forms of acute and chronic glomerulonephritis. IL-1 release by these cells can lead to local accumulation of other inflammatory cells and ultimately lead to inflammatory damage and fibrotic reaction in the kidney.

It has been shown that the crystals found in tissues or fluids in gout or pseudogout can directly stimulate macrophages to release IL-1. Thus, IL-1 may be an important mediator in the inflammatory cycle in these diseases. IL-1 can induce the loss of calcium from bone and may be responsible for the osteoporosis seen in inflammatory joint diseases.

Keratinocytes from patients with psoriasis release large amounts of IL-1. This mediator may be responsible for the secondary cell proliferation and accumulation that occurs in the skin of patients with this disease. IL-1 is one of the most important endogenous pyrogens and may be responsible for inducing the marked degree of fever observed in some infectious diseases such as acute febrile illnesses caused by bacteria or viruses.

Sarcoidosis is characterized by granulomatous lesions in many different organs of the body. IL-1 has been shown to induce granuloma formation in vitro and may be involved in this process in patients with sarcoidosis.

Excessive IL-1 production has been demonstrated in peripheral monocytes from both Crohn's disease and ulcerative colitis. Local IL-1 release in the gut may be an important mediator in stimulating the inflammatory cycle in these diseases.

Certain lymphomas are characterized by fever, osteoporosis and even secondary arthritis. Excessive IL-1 release has been demonstrated by some lymphoma cells in vitro and may be responsible for some of the clinical manifestations of these malignancies. Furthermore, IL-1 production by some malignant lymphocytes may be responsible for some of the fever, acute phase reaction and cachexia observed with leukemia. IL-1 release by astrocytes in the brain is thought to be responsible for inducing the fibrosis that can result after injury to the brain from vascular occlusion.

C. Areas of application for an IL-1 inhibitor

In these and other circumstances where IL-1 has a deleterious effect, there is a clear clinical application for an inhibitor of IL-1 action. As IL-1 is a co-mitogen for T cells, it is central to the development of autoimmune diseases and other immune diseases. Thus, when given systemically, the IL-1 inhibitors may be useful immunosuppressive agents. Used locally, such IL-1 inhibitors can serve to prevent tissue destruction in an inflamed joint and other places with inflammation. In order to prevent tissue destruction, some IL-1 inhibitors can actually be even more effective when given in conjunction with collagenase inhibitors.

Therapeutic intervention against the action of IL-1 may be possible at the level of synthesis, secretion or the target cell's binding or reaction to the protein. IL-1 is synthesized by monocytes/macrophages and other cells in response to lipopolysaccharides, complement fragments and viruses. Any molecule that blocks the binding of these inducers to producer cells or that interferes with their effects on the physiology of these cells will serve as a regulator of IL-1 action. IL-1 is not secreted by a traditional secretion system, as mRNA has been isolated that encodes at least two 30 kd precursors of the proteins, but which do not contain a hydrophobic signal sequence. Liberation of

the active protein from the inactive precursor probably requires proteolysis of this precursor. An inhibitor of the release of one or more types of IL-1 from their precursors could theoretically regulate IL-1 action. IL-1 probably acts on target cells through a classical receptor-mediated pathway, although this receptor has not yet been isolated. Thus, it may be that a molecule that interferes with the binding of IL-1 to its receptors or that down-regulates these receptors, can also regulate the effect of IL-1. Furthermore, although the intracellular events that

since receptor binding of IL-1 is not yet fully understood, it is possible that there exist agents that can interfere with cell reactions to other receptor-mediated events and therefore block the action of IL-1. For the reasons stated above, proteins and small molecules capable of inhibiting IL-1 in one or more of these ways have been sought.

It is therefore an aim to provide a method for the production of pharmaceutical preparations containing IL-1 inhibitor without the above-mentioned disadvantages. This purpose has been achieved by the present invention, characterized by what appears in the attached claims.

Surprisingly, at least two IL-1 inhibitor proteins with IL-1 inhibitory properties have now been found. These molecules have been obtained in a purified form which will allow one skilled in the art to determine their amino acid sequence. Furthermore, a preparation of cells has been characterized that produces these proteins, and an mRNA that leads to its synthesis has been characterized. Finally, an antiserum has been developed that will facilitate screening of cDNA expression libraries for the genes that code for these inhibitors. Together, these reagents will allow the cDNA encoding the IL-1 inhibitors to be cloned. These genes will in turn enable the large-scale production of IL-1 inhibitors suitable for use in pharmaceutical formulations useful in the treatment of pathophysiological conditions mediated by IL-1.

The invention relates to the production of a pharmaceutical preparation containing IL-1 inhibitors ("IL-li") in general and more specifically a monocyte-derived (monocyte-derived) IL-1 inhibitor, or biologically active analogues of these inhibitors, as well as purified forms of IL-1 inhibitors that are active against IL-1-α or IL-1-B or a combination thereof.

Further purposes and advantages of the invention will be indicated in the description that follows and will in part also be obvious from the description or can be learned by

execution of the invention. The purposes and benefits can be fulfilled and achieved by using the instrumentalities and combinations that are specifically pointed out in the associated requirements.

In order to achieve the purposes and in accordance with the purposes of the present invention, IL-1 inhibitors are indicated which exhibit inhibitory activity against IL-1. The preferred inhibitors have been isolated in a purified form from monocyte-conditioned medium with monocytes cultured on IgG-coated plates.

Preferred inhibitors for use in the invention are 1, 2 and 3. Inhibitors 1 and 2 are proteins that run at positions characteristic of 22-23 kDa proteins on SDS-PAGE and elute at 52 mM and 60 mM NaCl, respectively, from a

Mono Q FPLC column under specified conditions. Inhibitor 3 is a protein that runs at a position characteristic of a 20 kD protein on SDS-PAGE and elutes at 48 mM NaCl from a Mono Q FPLC column under the specified conditions. Moreover, in order to achieve the purposes and in accordance with the purposes of the present invention, pharmaceutical compositions containing at least one of the active ingredients, an IL-1 inhibitor according to the invention or its biologically active analogue as indicated herein, are described .

Furthermore, in order to achieve the purposes and in accordance with the purposes of the present invention, a recombinant DNA system for the production of these IL-1 inhibitors and their analogs is also provided. A preferred embodiment of this system includes at least one cDNA clone or its synthetic equivalent that encodes at least one IL-1 inhibitor together with vectors and cells constituting an expression system capable of expressing the IL-1 inhibitors indicated herein. Antisera for use in identifying these cDNA clones have also been obtained. Expression systems for the production of these IL-1 inhibitors using these cDNA clones, their analogs or other DNA sequences encoding these inhibitors have also been provided.

Brief description of the figures

Fig. 1a and 1b show the protein profile by Mono Q chromatography of two metabolically labeled monocyte supernatants. The cells were grown on plates coated with IgG (1a) or fetal calf serum (1b). Fig. 2a shows silver-stained gels of fractions from the areas indicated in fig. let and lb.

Fig. 2b is an autoradiogram of the gels shown in Fig. 2a.

Fig. 3a, b and c show data for the purified IL-1 i according to example 1. Fig. 3a shows chromatography data with the radioactivity pattern superimposed. Fig. 3b shows gels stained with silver run on samples of the fractions indicated in fig. 3a. Fig. 3c shows autoradiograms of the gels from fig. 3b. Fig. 4a and b show the results of gel filtration chromatograms of Mono Q-purified IL-li. Fig. 5a and b show Western analysis of mouse antisera.

Fig. 6 shows the construction of plasmid pSVXVPL2IL-li.

Fig. 7 shows the construction of plasmid pMK-SGE:IL-li.

Fig. 8a-d shows data on IL-1 i-a. Fig. 8a and b show chromatography data. Fig. 8c shows a gel stained with silver run on samples of fractions as indicated in fig. 8b. Fig. 8d shows an autoradiogram. Fig. 9a and 9b show data for IL-li-13. Fig. 9a shows chromatography data. Fig. 9b shows SDS-PAGE data.

Fig. 10 shows data for IL-H-α peptide separation.

Fig. 11 shows data for IL-11-B peptide separation.

Fig. 12a is a photograph of the gel with GT10-ILH-2A digested with EcoRI after electrophoresis according to Example 6. Fig. 12b shows data for an autoradiogram of a Southern blot of the gel shown in Fig. 12a. Fig. 13 shows part of the DNA sequence for the protein coding region of lambda GT10-ILH-2A and the predicted amino acid sequence according to example 6.

Fig. 14 shows the nucleotide sequence of GT-10-illI-2A.

Fig. 15 shows a peptide which comprises, among other things, an IL-li sequence and a secretory leader sequence.

Description of different designs

Reference will now be made in detail to the individual embodiments which together with the following examples serve to explain the principles of the invention.

A. Inhibitor from human monocvtter

As stated above, the invention relates to the production of pharmaceutical preparations containing IL-1 inhibitors which have been isolated in a purified form. Preferably, the IL-1 inhibitors used in the invention are derived from human monocyte conditioned medium where monocytes are cultured on IgG-coated containers. Moreover, the invention covers largely purified IL-1 inhibitors of any origin

which are biologically equivalent to the inhibitors obtained from human monocyte

rich medium.

In the case of "biological equivalent" as used in the description and in the requirements, shall

are understood to be mixtures according to the invention which can prevent IL-1 action in a similar way, but not necessarily to the same extent as the native IL-1 inhibitor which is isolated from monocytes. By "substantially homologous" as used in the following description and claims, is meant a degree of homology with the native IL-1 inhibitor isolated from monocyte-conditioned medium that exceeds that exhibited by any previously reported IL-1 -inhibitors. Preferably, the degree of homology is greater than 70%, preferably greater than 80% and most preferably greater than 90%. In a particularly preferred group of inhibitors, more than 95% are homologous to the native inhibitor. The percentage of homology as described is calculated as the percentage of amino acid residues found in the smaller of the two sequences that align with identical amino acid residues in the sequence being compared, when four openings 100 amino acids in length can be introduced to assist in this alignment , as set forth by Dayhoff, M. in Atlas of Protein Sequence and Structure Vol. 5, p. 124 (1972), National Biochemical Research Foundation, Washington, D.C..

The preferred IL-1 inhibitors according to the invention have been derived from monocyte-conditioned medium and have been isolated for the first time in a purified form. For the purposes of the present invention, the terms "pure form" or "purified form", when applied to the IL-1 inhibitors set forth herein, shall mean a preparation that is substantially free of other proteins other than IL-1 -proteins. Preferably, the IL-1 inhibitors according to the invention are at least 90% pure and preferably 95% pure.

At least three purified IL-1 inhibitors have been isolated by the methods of the example. These include inhibitor 1, inhibitor 2 and inhibitor 3. Inhibitor 1

behaves as a 22-23 kDa molecule on SDS-PAGE with an approximate isoelectric point of 4.8 and elutes from a Mono Q FPLC column at ca. 52 mM NaCl in Tris buffer, pH 7.6. Inhibitor 2 is also a protein of 22-23 kDa, pl=4.8, but elutes from a Mono Q column at 60 mM NaCl. Inhibitor 3 is a protein on

20 kDa and elutes from a Mono Q column at 48 mM NaCl. Inhibitors 1, 2 and 3

are related immunologically and functionally. Obtaining these inhibitors in purified forms has made it possible to obtain their amino acid sequences. Using purified inhibitors as described here for the first time and methods such as those described in and by the ABI Protein Sequencer technical manuals supplied with the ABI Protein Sequencer, a significant proportion of the amino acid sequences of these inhibitors can be deduced.

Example 3 shows amino acid sequence data obtained for three IL-1 inhibitors, namely IL-lj-X, IL-lj-ct and IL-lj-B.

At least one antibody has been found that is formed against an IL-1 inhibitor. Other polyclonal and monoclonal antibodies against this and other IL-1 inhibitors can be prepared by methods known to those skilled in the art. A particular polyclonal antibody is described in Example 4.

B. Recombinant inhibitor

1. General

A recombinant DNA method for the production of an IL-1 inhibitor is now disclosed. In one embodiment of the invention, the active seat works in a way that is

. biologically equivalent to that of native IL-1 inhibitor isolated from humans. A natural or synthetic DNA sequence can be used to direct the production of the IL-1 inhibitors. This method comprises: (a) producing a DNA sequence capable of directing a host cell to produce a protein having IL-1 inhibitory activity, (b) cloning the DNA sequence into a vector which can be transferred into and replicated in a host cell, which vector contains operational elements necessary to express the DNA sequence, (c) transferring the vector containing the synthetic DNA sequence and operational elements into a host cell capable of expressing the DNA encoding the IL the -1 inhibitor, (d) culturing the host cell under conditions suitable for amplification of the vector and expression of the inhibitor;

(e) harvesting the inhibitor, and

(f) allowing the inhibitor to adopt an active tertiary structure so that it has IL-1 inhibitory activity.

2. DNA sequencing

DNA sequences which are intended for use in this method are partly discussed in Example 5 and partly in Example 6. It is intended that these sequences include synthetic and natural DNA sequences. The natural sequences also contain cDNA segments or genomic DNA segments.

Example 6 obtains a molecular clone of DNA encoding a protein identical to that isolated according to Examples 1-3. In Example 6, a plaque, GT10-ILH-2A, was isolated from a GT10 library. The phage inside this plaque was propagated and the DNA was isolated and digested with EcoRI. An EcoRI fragment of 1850 base pairs carries the coding sequence for ILI inhibitor. Fig. 13 shows the partial DNA sequence for the EcoRI fragment.

In light of what is shown here and the known methods, other synthetic polynucleotide sequences will be available to those skilled in the art. As an example of the state of the art regarding polynucleotide synthesis, reference is made to Matteucci, M.D.

and Caruthers, M.H. in J. Am. Chem. Soc. 103:3185 (1981) and Beaucage, S. L. and Caruthers, M. H. in Tetrahedron Lett. 22:1859 (1981) and to the instructions provided with an ABI oligonucleotide synthesizer.

These synthetic sequences may be identical to the natural sequences described in more detail below or they may contain different nucleotides. In one embodiment, it is thought that, if the synthetic sequences contain nucleotides that are different from those found in the natural DNA sequences according to the invention, that these different sequences will still code for a polypeptide that has the same primary structure as IL-li isolated from monocytes. IN

in an alternative embodiment, the synthetic sequence containing different nucleotides will encode a polypeptide having the same biological activity as the IL-1 described herein.

Moreover, the DNA sequence may be a fragment of a natural sequence, i.e. a fragment of a polynucleotide which occurs in nature and which has been isolated and purified for the first time by the inventors. In one embodiment, the DNA sequence is a restriction fragment isolated from a cDNA library.

In an alternative embodiment, the DNA sequence is isolated from a human genomic library. An example of such a library useful for this embodiment,

is stated by Lawn et al. in Cell 15:1157-1174 (1978).

In a preferred version of this embodiment, it is envisaged that the natural DNA sequence will be obtained by a method comprising: (a) Preparation of a human cDNA library from cells, preferably monocytes, which can generate an IL-1 inhibitor in a vector and cell that can amplify and express all or part of this cDNA, (b) the human DNA library is probed with at least one probe that can bind to the IL-1 inhibitor gene or its protein product, (c) at least one clone containing the gene encoding the inhibitor is identified by the ability of the clone to bind to at least one probe for the gene or its protein product, (d) isolation of the gene or part of the gene encoding the inhibitor from the clone or clones which is selected, (e) linking the gene or suitable fragments thereof to operational elements necessary to maintain and express the gene in a host cell.

The natural DNA sequences useful in the above process can also be identified and isolated by a method comprising: (a) Preparation of a human genomic DNA library, preferably propagated in a recArecBC E. coli host, (b ) the human genomic DNA library is probed with at least one probe capable of binding to an IL-1 inhibitor gene or its protein product, (c) at least one clone containing the gene encoding the inhibitor is identified by the clone's ability to bind to i at least one probe for the gene or its protein product, (d) isolating the gene encoding the inhibitor from the identified clone(s), and (e) linking the gene or suitable fragments thereof to operational elements necessary to maintain and express the gene in a host cell.

When isolating a natural DNA sequence suitable for use in the above-mentioned method, it is preferred to identify the two restriction sites which are arranged within and closest to the end portions of the appropriate gene or sections of this gene. The DNA segment containing the correct gene is then removed from the rest of the genomic material using appropriate restriction endonucleases. After being excised, the 3' and 5' ends of the DNA sequence and any exon junctions are reconstructed to obtain appropriate DNA sequences that can encode the N- and C-termini of the IL-1 inhibitor protein and can add the DNA sequence to its operational elements.

3. Vectors

(a) Microorganisms, especially E. coli

The vectors contemplated for use in the present invention include any vectors into which the DNA sequence set forth above can be inserted, together with any preferred or necessary operative elements, and which vector can then be transferred into a host cell and is replicated in such a cell. Preferred vectors are those whose restriction sites have been well documented and which may contain the operational elements preferred or required for transcription of the DNA sequence. However, one can also imagine certain embodiments of the invention that use hitherto undiscovered vectors that will contain one or more of the cDNA sequences described here. More specifically, it is preferred that all of these vectors have some or all of the following characteristics: (1) have a minimal number of host organism sequences; (2) can be stably maintained and propagated in the desired host; (3) can be present in high copy number in the desired host; (4) has a regulatable promoter arranged to promote transcription of the gene in question; (5) has at least one marker DNA sequence that codes for a selectable property that exists on a part of the plasmid separately from where the DNA sequence is to be inserted, and (6) has a DNA sequence that can terminate transcription.

In various preferred embodiments, these cloning vectors which contain and can express the DNA sequences according to the invention have different operational elements. These "operational elements" as described herein include at least one promoter, at least one Shine-Dalgarno sequence and initiator codon and at least one stop codon. Preferably, these "operational elements" also include at least one operator and at least one leader sequence for proteins to be exported from intracellular compartments, at least one gene for a regulatory protein and any other DNA sequences necessary or preferred for proper transcription and subsequent translation of the vector - DNA.

Certain of these operational elements may be present in each of the preferred vectors according to the invention. It is believed that any additional operational elements that may be necessary can be added to these vectors using methods known to those skilled in the art, particularly in light of what is set forth herein.

In practice, it is possible to construct each of these vectors in a way that allows them to be easily isolated, assembled and exchanged. This facilitates the assembly of a number of functional genes from combinations of these elements and the coding region of the DNA sequences. Furthermore, many of these elements will be applicable in more than one host. It is also intended that these vectors in certain preferred embodiments will contain DNA sequences that can function as regulators ("operators") and other DNA sequences that can code for regulator proteins.

(i) Regulators

These regulators will in one embodiment serve to prevent expression of the DNA sequence in the presence of certain environmental conditions and will in the presence of other environmental conditions allow transcription and subsequent expression of the protein coded for by the DNA sequence. More specifically, it is preferred that regulatory segments are inserted into the vector so that the expression of the DNA sequence will not take place or will take place to a greatly reduced extent in the absence of e.g. isopropylthio-beta-D-galactoside. In this situation, the transformed microorganisms containing the DNA sequence can be grown to a desired density before initiating the expression of IL-11. In this embodiment, the expression of the desired protein is induced by adding a substance to the microbial environment which can cause expression of the DNA sequence after the desired density has been achieved.

(ii) Promoters

The expression vectors must contain promoters that can be used by the host organism for expression of its own proteins. Although the lactose promoter system is commonly used, other microbial promoters have been isolated and characterized, enabling one skilled in the art to use them for expression of the recombinant IL-1i.

(iii) Transcription terminator

The transcription terminators are intended here to serve to stabilize the vector. Specifically, the sequences described by Rosenberg, M. and Court, D.,

in Ann. Fox. The gene. 13:319-353 (1979) intended for use in the present invention.

(iv) Untranslated sequence

It should be noted that in the preferred embodiment it may also be desirable to reconstruct the 3' or 5' end of the coding region to allow incorporation of 3'-

or 5-untranslated sequences into the gene transcript. Included among these untranslated sequences are those that stabilize mRNA as identified by Schmeissner, U., McKenney, K., Rosenberg, M. and Court, D. in J. Mol. Biol. 176:39-53 (1984).

(v) Ribosome binding sites

The microbial expression of foreign proteins requires certain operational elements including but not limited to ribosome binding sites. A ribosome binding site is a sequence to which a ribosome recognizes and binds at the initiation of protein synthesis as set forth in Gold, L. et al., Ann. Fox. Microbio. 35:557-580 or Marquis, D.M. et al., Gene 42:175-183 (1986). A preferred ribosome binding site is GAGGCGCAAAA(ATG).

(vi) Leader sequence and translational coupling element

It is further preferred that DNA encoding a suitable secretory leader sequence (signal sequence) is present at the 5' end of the DNA sequence as indicated by

Watson, M.E. in Nucleic Acids Res. 12:5145-5163, if the protein is to be secreted from the cytoplasm. The DNA for the leader sequence must be in a position that allows the production of a fusion protein where the leader sequence is in close proximity to and covalently joined to the inhibitor, i.e. there must be no transcription or translation termination signals between the two DNA coding sequences. The presence of the leader sequence is desirable, in part for one or more of the following reasons. First, the presence of the leader sequence may facilitate host processing of IL-li. More specifically, the leader sequence can direct cleavage of the original translation product by a leader peptidase to remove the leader sequence and leave a polypeptide with the amino acid sequence that has potential protein activity. Second, the presence of the leader sequence may facilitate the purification of IL-1i by directing the protein out of the cell cytoplasm. In some species of host microorganism, the presence of an appropriate leader sequence will allow transport of the finished protein into the periplasmic space, as is the case for some E. coli. In the case of certain E. coli, Saccharomyces and strains of Bacillus and Pseudomonas, the correct leader sequence will allow transport of the protein through the cell membrane and into the extracellular medium. In this situation, the protein can be purified from extracellular protein. Third, in the case of some of the proteins produced according to the invention, the presence of the leader sequence may be necessary to place the finished protein in an environment where it can fold to assume its active structure, which structure has the correct protein activity .

In a preferred embodiment of the invention, a further DNA sequence is arranged just before the DNA sequence which codes for the IL-1 inhibitor. The additional DNA sequence can function as a translational coupling element (coupler), i.e. it is a DNA sequence that codes for an RNA that serves to position the ribosomes close to the ribosome binding site of the inhibitor RNA that it encounters . In one embodiment of the invention, the translational linker can be obtained using the DNA sequence

and methods currently known in the field of translational link elements.

(vii) Translation Terminator

The translation terminators contemplated according to the invention serve to stop the translation of mRNA. They can be either natural as described by Kohli, J., Mol. Gen. The gene. 182:430-439, or synthesized as described by Petterson, R.F. Gene 24:15-27 (1983).

(viii) Selectable cursor

It is further preferred that the cloning vector contains a selectable marker such as a drug-resistant marker or other marker which causes expression of a selectable feature of the host microorganism. In one embodiment of the invention, the gene for ampicillin resistance is included in the vector, while in other plasmids the gene for tetracycline resistance or the gene for chloramphenicol resistance is included.

Such a drug resistance or other selectable marker is partly intended to facilitate the selection of transformants. Moreover, the presence of such a selectable marker in the cloning vector may be useful in keeping contaminating microorganisms from multiplying in the culture medium. In this embodiment, a pure culture of the transformed host microorganisms will be obtained by culturing the microorganisms under conditions that require the induced phenotype for survival.

The operational elements described here are chosen according to routines by professionals in the light of previous literature and the guidelines stated here. General examples of such operational elements are given in B. Lewin, Genes. Wiley & Sons, New York (1983). Various examples of suitable operational elements can be found among the vectors indicated above and can be elucidated by a review of the publications that discuss the basic properties of the vectors indicated above.

Upon synthesis and isolation of all necessary and desired component parts of the above-mentioned vector, the vector is assembled by methods generally known to those skilled in the art. Composition of such vectors is believed to be within the duties and tasks performed by those with ordinary knowledge in the field, and as such can be performed without unnecessary experimentation. For example, similar DNA sequences have been ligated into suitable cloning vectors as indicated by Maniatis et al. in Molecular Cloning, Cold Spring Harbor Laboratories (1984).

When constructing cloning vectors according to the invention, it should also be noted that multiple copies of the DNA sequence and its accompanying operational elements can be inserted into each vector. In such an embodiment, the host organism will produce larger quantities per vector of the desired IL-1 inhibitor. The number of multiple copies of the DNA sequence that can be inserted into the vector is limited only by the resulting vector's ability, due to its size, to be transferred into and replicated and transcribed in a suitable host cell.

(b) Other micro-organisms

Vectors suitable for use in microorganisms other than E. coli are also contemplated for the invention. Such vectors are described in Table 1. Furthermore, certain preferred vectors are discussed below.

(i) Pseudomonas vectors

A variety of vector plasmids that autonomously replicate in a wide range of Gram-

negative bacteria are preferred for use as cloning vehicles in hosts of the genus Pseudomonas. Some of these are described by Tait, R. C, Close, T. J.,

Lundquist, R.C, Hagiya, M., Rodriquez, R.L. and Kado, C.I. in Biotechnology, May 1983, pp. 269-275; Panopoulos, N.J. in Genetic Engineering in the Plant Sciences, Praeger Publishers, New York, New York, pp. 163-185 (1981), and Sakagucki, K. in Current Topics in Microbiology and Immunology 96:31-45 (1982).

A particularly preferred construct uses the plasmid RSF1010 and derivatives

thereof as described by Bagdasarian, M., Bagdasarian, M. M., Coleman, S. and Timmis, K. N. in Plasmids of Medical, Environmental and Commercial Importance, Timmis, K. N. and Puhler, A. eds., Elsevier/North Holland Biomedical Press (1979 ). The advantages of RSF1010 are that it is a relatively small plasmid with a high copy number

which is readily transformed into and stably maintained in both E. coli and Pseudomonas species. In this system, it is preferred to use the Tac expression system as described for Escherichia, as it appears that the E. coli trp promoter is readily recognized by Pseudomonas RNA polymerase, as indicated by Sakagucki, K. in Current Topics in Microbiology and Immunology 96:31-45 (1982) and Gray, G.L., McKeown, K.A., Jones, A.J.S., Seeburg, P.H. and Heyneker, H.L. in

Biotechnology, Feb. 1984, pp. 161-165. Transcription activity can be further maximized by arranging for exchange of the promoter with e.g. an E. coli

or P. aeruginosa trp promoter. In addition, the lacI gene of E. coli will also be included in the plasmid to effect regulation.

Translation can be coupled to translation initiation for any of the Pseudomonas proteins, as well as to initiation sites for any of the

highly expressed proteins of the type selected to effect intracellular expression of the inhibitor.

In those cases where restriction-minus strains of a host Pseudomonas species are not available, the transformation efficiency with engineered plasmid fragments

isolated from E. coli, poor. Consequently, passage of the Pseudomonas cloning vector through an r-m+ strain of another species is desirable prior to transformation of

the desired host, as set forth in Bagdasarian, M. et al., Plasmids of Medical,

Environmental and Commercial Importance, pp. 411-422, Timmis and Puhler eds., Elsevier/North Holland Biomedical Press (1979).

(ii) Bacillus vectors

A preferred expression system in hosts of the genus Bacillus also includes using plasmid pUB1 10 as the cloning vehicle. As in other host vector systems, it is possible in Bacillus to express (express) IL-li according to the invention, either as an intracellular or a secreted protein. The present embodiments include both systems. Shuttle vectors that replicate in both Bacillus and E. coli are available for the construction and testing of various genes as described by Dubnau, D., Gryczan, T., Contente, S. and Shivakumar, A. G. in Genetic Engineering, Vol. 2, Setlow and Hollander eds., Plenum Press, New York, New York, pp. 115-131 (1980). For expression and secretion of IL-1i from B. subtilis, the signal sequence of alpha-amylase is preferably linked to the coding region of the protein. For intracellular inhibitor synthesis, the mobile DNA sequence will be translationally linked to the ribosome binding site of the alpha-amylase leader sequence.

Transcription of any of these engineered fragments is preferably controlled by the alpha-amylase promoter or a derivative thereof. This derivative contains the RNA polymerase recognition sequence of the native alpha-amylase promoter, but incorporates the lac operator region as well. Similar hybrid promoters constructed from the penicillinase gene promoter and the lac operator have been shown to function in Bacillus hosts in a regulatable manner as reported by Yansura, D. G. and Henner in Genetics and Biotechnology of Bacilli, Ganesan, A. T. and Hoch, J. A., eds., Academic Press, pp. 249-263 (1984). The lacI gene of E. coli will also be included in the plasmid to effect regulation.

(iii) Clostridium vectors

A preferred construct for expression in Clostridium is in plasmid pJU12, described by Squires, C. H. et al. in J. Bacteriol. 159:465-471 (1984), transformed into C. perfringens by the method of Heefner, D. L. et al. as described in J. Bacteriol. 159:460-464 (1984). Transcription is controlled by the promoter for the tetracycline resistance gene. Translation is associated with the Shine-Dalgarno sequences of this same tet gene in a manner entirely analogous to the procedures set forth above for vectors suitable for use in other hosts.

(iv) Give respect vectors

Maintenance of foreign DNA introduced into yeast can be accomplished in a number of different ways as described by Botstein, D. and Davis, R.W. in The Molecular Biology of the Yeast Saccharomyces, Cold Spring Harbor Laboratory, Strathern, Jones and Broach, eds., pp , 607-636 (1982). A preferred expression system for use with host organisms of the genus Saccharomyces contains the IL-lig gene on the 2 micrometer plasmid. The advantages of the 2-micrometer circle include a relatively high copy number and stability when introduced into eir strains. These vectors preferably incorporate the origin of replication and at least one antibiotic resistance marker from pBR322 to allow replication and selection in E. coli. Also, the plasmid will preferably have the 2-μm sequence and the yeast LEU2 gene to serve the same purpose in LEU2-defective mutants of yeast.

If it is intended that the recombinant IL-1 inhibitors will eventually be expressed in yeast, it is preferred that the cloning vector is first transferred into Escherichia coli, where the vector will be allowed to replicate and from which the vector can be obtained and purified after amplification. The vector will then be transferred to the yeast for final expression of the IL-1 inhibitor.

(c) Patted<y>rcells

The cDNA for the IL-1 inhibitor will serve as the gene for expression of the inhibitor in mammalian cells. It should have a sequence that will be effective in binding ribosomes such as that described by Kozak in Nucleic Acids Research 15:8125-8132

(1987), and should have the coding capacity for a leader sequence (see Section 3(a)(vi)) to

directing the mature protein out of the cell in a processed form. The DNA restriction fragment carrying the complete cDNA sequence can be inserted into an expression vector having a transcription promoter and a transcription enhancer as described by Guarente, L. in Cell 52:303-305 (1988) and Kadonaga, J. T. et al. ., in Cell 51:1079-1090 (1987). The promoter can be regulated as in the plasmid pMSG (Pharmacia Cat. no. 27450601) if the constitutive expression of the inhibitor is harmful to cell growth. The vector should have a complete polyadenylation signal as described by Ausubel, F. M. et al. in Current Protocols in Molecular Biology, Wiley (1987), so that the mRNA transcribed from this vector is processed correctly. Finally, the vector will have the origin of replication and at least one antibiotic resistance marker from pBR322 to allow replication and selection in E. coli.

To select a stable cell line that produces the IL-1 inhibitor, the expression vector may carry the gene for a selectable marker such as a drug resistance marker or carry a complementary gene for a defective cell line such as a dihydrofolate reductase gene (dhfr) for transformation of a dhfr" cell line as described by Ausubel et al. above. Alternatively, a separate plasmid carrying the selectable marker can be co-transformed with the expression vector.

4. V ert scel 1 er/ transformas i ons

The vector thus obtained is transferred into a suitable host cell. These host cells can be microorganisms or mammalian cells.

(a) Microorganisms

It is believed that any microorganism that has the ability to take up

exogenous DNA and express these genes and accompanying operational elements, can be selected. After a host organism has been selected, the vector is transferred into the host organism using methods generally known to those skilled in the art. Examples of such methods can be found in Advanced Bacterial Genetics by R.W. Davis et al., Cold Spring Harbor Press, Cold Spring Harbor, New York

(1980). It is preferred in one embodiment that the transformation takes place at low temperatures, as temperature regulation is contemplated as a way to regulate gene expression using operational elements as indicated above. In another embodiment, if osmolar regulators have been inserted into the vector, the regu-

lering the salt concentration during the transformation be necessary to

ensure proper control over the foreign genes.

It is preferred that the host microorganism is a facultative anaerobe or an aerobe. Special hosts that may be preferred for use in this procedure

includes yeast and bacteria. Special types of yeast include yeasts of the genus Saccharomyces and especially Saccharomyces cerevisiae. Specific bacteria include bacteria of the genera Bacillus, Escherichia and Pseudomonas, especially Bacillus subtilis and Escherichia coli. Additional host cells are listed in Table I above.

(b) Patted<y>rcells

The vector can be introduced into mammalian cells in culture by various techniques such as calcium phosphate:DNA co-precipitation, electroporation or protoplast fusion. The preferred method is coprecipitation with calcium phosphate as described by Ausubel et al. above.

Many stable cell types exist that are transformable and capable of transcribing and translating the cDNA sequence, processing precursor IL-11 and secreting the mature protein. Cell types may, however, be variable with regard to glycosylation of secreted proteins and post-translational modification of amino acid residues, if present. Thus, the ideal cell types are those that produce a recombinant IL-1 inhibitor that is identical to the natural one

molecule.

5. Cultivation of engineered cells

The host cells are cultured under conditions that are appropriate for the expression of the IL-1 inhibitor. These conditions are generally specific to the host cell and can be readily determined by those skilled in the art in light of the published literature regarding culture conditions for such cells and what is set forth in the present specification. For example, Bergey's Manual of Determinative Bacteriology, 8th ed., Williams & Wilkins Company, Baltimore, Maryland, contains information on the conditions for culturing bacteria. Similar information regarding the cultivation of yeast and mammalian cells can be obtained from Pollack, R. Mammalian Cell Culture, Cold Spring Harbor Laboratories (1975).

Any conditions necessary to regulate the expression of the DNA sequence, depending on any operational elements inserted into or present in the vector, will act at the transformation and cultivation step. In one embodiment, the cells are grown to a high density in the presence of appropriate regulatory conditions, which inhibit the expression of the DNA sequence. As the optimal cell density is approached, the environmental conditions are changed to those suitable for expression of the DNA sequence. It is thus contemplated that the production of the IL-1 inhibitor will take place in a period of time following the cultivation of the host cells to near optimal density, and that the resulting IL-1 inhibitor will be harvested some time after the regulatory conditions that were necessary for its expression, were induced.

6. Purification

(a) IL-li produced from microorganisms

In a preferred embodiment of the invention, the recombinant IL-1 inhibitor is purified after harvesting and before it assumes its active structure. This embodiment is preferred, as it is believed that high-yield recovery of refolded protein is facilitated if the protein is first purified. In a preferred alternative. however, in one embodiment, the IL-1 inhibitor may be allowed to refold to assume its active structure prior to purification. In yet another preferred embodiment, the 1L-1 inhibitor is present in its refolded active state upon extraction from the culture medium.

Under certain circumstances, the IL-1 inhibitor will assume its correct, active structure upon expression in the host microorganism and transport of the protein through the cell wall or membrane or into the periplasmic space. This will generally take place if the DNA encoding a suitable leader sequence has been linked to the DNA encoding the recombinant protein. If the IL-1 inhibitor does not assume its correct, active structure, any disulfide bonds that have formed and/or any non-covalent interactions that have taken place will first be broken up by denaturing and reducing agents, e.g. guanidinium chloride and beta-mercaptoethanol, before the IL-1 inhibitor is allowed to assume its active structure after dilution and oxidation of these agents under controlled conditions.

For purification before and after refolding, some combination of the following steps is preferably used: anion exchange chromatography (MonoQ or DEAE Sepharose), gel filtration chromatography (Superose), chromatofocusing (MonoP) and hydrophobic interaction chromatography (octyl or phenyl Sepharose). Antibody affinity chromatography using IL-1 specific monoclonal antibodies (described in Example 3) is of particular value.

(b) IL-1 in prepared from patted<y>r cells

IL-1 in prepared from mammalian cells will be purified from conditioned medium by steps that will include ion exchange chromatography and immunoaffinity chromatography using monoclonal antibodies described in Example 3.

It should be understood that applying what is shown by the present invention to a specific problem or environment will be within the abilities of a person skilled in the art in light of the indications given here. Examples of the products according to the invention and representative methods for their isolation and preparation are given below.

The following examples show various currently preferred embodiments of the invention. The publications indicated in these examples are incorporated as part of the description.

EXAMPLES

Example 1 - Protein production line

A. Materials

Hank's balanced salt solution (HBSS) and RPMI were purchased from Mediatech, Washington, D.C. Lymphoprep was obtained from Accurate Chemical and Scientific Corp., Westbury, N.Y. Human IgG, MTT, rabbit anti-prostaglandin E2 antiserum, ammonium bicarbonate, dithiothreitol, complete and incomplete Freund's adjuvants, hypoxanthine, aminopterin, and thymidine were purchased from Sigma Chemical Co., St. Louis, Missouri. C3H/HeJ mice were purchased from Jackson Labs, Bar Harbor, Maine. BALB/c mice and P3 myeloma cells were obtained from Dr. John Kappler and Dr. Phi Hippa Marrack at the National Jewish Center for Immunology and Respiratory Medicine (NJC/IRM), Denver, Colorado. Recombinant human IL-1 was obtained from Cistron Biotechnology, Pine Brook, N.J. Purified phytohemagglutinin was purchased from Wellcome Diagnostics, Research Triangle Park, N.C. Human foreskin fibroblasts from primary cultures were obtained from Dr. Richard Clark at NJC/IRM, Denver, Colorado. Monoclonal mouse anti-rabbit IgG antibodies were purchased from AIA reagents, Aurora, Colorado. Low methionine RPMI was prepared using a Select-Amine kit from GIBCO Laboratories, Grand Island, N.Y. [<35>S]-methionine, diphenyloxazole, and [<14>C]-iodoacetic acid were obtained from DuPont-NEN, Chicago, Illinois. Fetal calf serum was purchased from HyClone Laboratories, Logan, Utah. Mono Q and Superose-12 columns were purchased from Pharmacia, Inc., Piscataway, N.J.' C4 reversed-phase columns were obtained from Synchrom, Inc., Lafayette, Indiana. C8 reversed-phase columns were obtained from Applied Biosystems, Inc., Foster City, California. Acetonitrile and polyethylene glycol 8000 were purchased from J.T. Baker Chemical Co., Phillipsburg, N.J. Trifluoroacetic acid and guanidine hydrochloride were obtained from Pierce Chemicals, Rockford, Illinois. Endoproteinase Lys C was obtained from Boehringer Mannheim Biochemicals, Indianapolis, Indiana. The microtiter plates used for the PGE2 ELISA were Nunc-Immuno Plate I obtained from Intermountain Scientific Corporation, Bountiful, Utah. The plates used for hybridoma production were from Coster, Cambridge, Massachusetts.

B. Generation of monocyte IL-1 inhibitor

Human leukocytes were obtained from normal donors by leukophoresis, resuspended in Hank's balanced salt solution (HBSS) at 1 part packed cells to 1 part HBSS, coated with Lymphoprep and spun at 400 x g for 30 min at room temperature. The mononuclear fraction was harvested (typically 4-5 x 10<9> cells were obtained per donor), grown in HBSS without Ca<++> or Mg"", suspended in serum-free RPMI and plated on Petri dishes coated with normal human -IgG made LPS-free by chromatography over Sephapex G200 (6 x 107 cells in 10 ml per 100 mm dish). All reagents contained less than 10 pg/ml LPS.. The cells were cultured for 24-48 h, and the resulting conditioned medium constituted the untreated IL-1 inhibitor supernatant (IL-11).Typically, the cells from one donor yielded 700-900 ml of crude IL-11 supernatant.

C. Assay for IL-1 inhibitor

Two IL-1 assays have been used routinely to detect IL-11. Thymocytes (1 x 10<6> cells from 4-6 week old C3H/HeJ mice) react to 1.0 unit per ml recombinant human IL-1 plus 1 µg/ml phytohemagglutinin at half-maximal proliferation, as measured by H-thymidine incorporation or uptake of the tetrazolium salt MTT (Mosmann, T., J. Immunol. Method, 65:55- 61 (1983)) after three days of stimulation. Crude IL-11 supernatant completely prevents this proliferative response at a dilution of 1/10. Human dermal fibroblasts (1 x 10<5> cells per well in a 96-well plate) typically respond to 0.5 units per well. ml of recombinant human IL-1 by secretion after 6 h stimulation of approximately 50,000 pg/ml PGE2, which can be measured by ELISA. This assay is as sensitive to IL-1 as the thymocyte assay.

D. Metabolic labeling of the IL-1 inhibitor

IL-11 was metabolically labeled by culturing mononuclear leukocytes for 48 h on IgG-coated plates (as described in B) in serum-free RPMI containing only

0.75 µg/ml cold methionine (15 µg/ml is normal) and to which was added 0.5 mCi 3SS-methionine (1151 Ci/mmol) per 10<7> cells. The control labeling was carried out in the same way except that the plates were coated with fetal calf serum instead of IgG. Analyzes of such comparison supernatants showed that very little IL-11 was secreted when the cells were grown on plates coated with fetal calf serum.

E. Rensin<g> of the IL-1 inhibitor protein

Crude IL-1β supernatants were made 1.0 M in sodium chloride, incubated on ice for 1 h and centrifuged at 10,000 rpm for 15 min. The supernatants, which contained all the inhibitor activity but only 20% of the original protein, were subjected to extensive dialysis at 4°C against 0.025 M Tris, pH 7.6, containing 0.1%

sucrose (buffer A) for gradient fractionation of proteins on a Mono Q anion exchange column. After dialysis, the inhibitor-containing solutions were centrifuged again at 10,000 rpm for 15 min and then passed through 0.22 µm nylon filters. The supernatants were typically combined with 10 ml of similarly prepared supernatant from a metabolic labeling and loaded onto Mono Q-Superose columns (Pharmacia FPLC) with layer volumes of either 1.0 ml or 8.0 ml, washed with A buffer until the OD280 of the effluent was returned to baseline and carefully chromatographed using a linear sodium chloride gradient (0.025M-0.1 OM) in buffer A. Column fractions were collected and analyzed for radioactivity and bioactivity. Samples of each fraction were also run at reduced 12.5%

SDS-PAGE, stained with silver, permeated with diphenyloxazole, dried and placed on film to obtain autoradiographic data. Fig. 1a shows the protein profile of the Mono Q chromatography of 40 ml of crude IL-1i supernatant mixed with 3 ml of metabolically labeled IL-1i supernatant. On top of this is added the amount of radioactivity found in 50 av

each fraction as well as the IL-1 bioactivity measured in the PGE2 production assay. Two major and one minor radioactive component (species) are shown which correlate perfectly with three peaks (peaks) of bioactivity. Fig. 1b shows the similar chromatography of 15 ml of crude IL-1 in supernatant mixed with 3 ml of supernatant from metabolically labeled monocytes on plates coated with fetal calf serum (FCS) rather than IgG. The levels of the three radioactive components listed above have been significantly reduced. Fig. 2a shows gels stained with silver and run on the fractions from the regions of interest in the chromatographies shown in fig. let and lb. It should be noted that the fractions of highest radioactivity and bioactivity in fig. 1a (fractions 52 and 59) both show a major band at 22 Kd (marked with arrows) on SDS-PAGE. The third component (fraction 48 in Fig. 1a) shows a band at 20 kD on SDS-PAGE. Gel filtration experiments on crude IL-li have shown that the active molecule has a molecular weight of 18-25 Kd. Fig. 2b is an autoradiogram of the gels shown in fig. 2a. It can easily be seen that the protein bands at 20 and 22 Kd are the most important radioactive components in these fractions.

As a summary, it should be said that these results show that the metabolic

labeling of monocytes plated on petri dishes coated with IgG leads to radioactive

components that are only poorly produced if the cells are plated on dishes coated with FCS. These induced radioactive components co-chromatograph perfectly with several different components with IL-1 bioactivity on Mono Q, and gels and resulting autoradiograms show that the three major induced molecules are proteins of the molecular weight predicted for IL-1. The IL-11 molecules were further purified for sequencing in two ways. First, Mono Q fractions with the highest bioactivity and radioactivity were loaded onto a C4 reversed-phase column and eluted with a HiO/0.1% TFA: acetonitrile/0.1% TFA gradient. As the IL-11 molecule was trace-labeled, samples from each fraction were directly counted for radioactivity and were also analyzed by SDS-PAGE followed by autoradiography. Fig. 3a shows such a chromatography with the radioactivity pattern superimposed. The silver-stained gels run on samples from each fraction (Fig. 3b) and subsequent autoradiograms of the gels (Fig. 3c) show that the IL-1 molecule is present in fractions 32-36. These fractions were dried down and sequenced. Alternatively, the highest Mono Q fractions were dried by a speed evaporator (Speed Vac), resuspended in 0.4 ml of 0.05 M NH4HCO3 and directly chromatographed twice on a 10 x 300 mm Superose-12 gel filtration column (Pharmacia FPLC) equilibrated in the same buffer as shown in fig. 4a and 4b. Fractions were collected and samples of each were tested for radioactivity and bioactivity and were analyzed by silver-stained and autoradiographed SDS-PAGE. Appropriate fractions were then dried on a speed vac (Speed Vac) and sequenced.

Example 2

Proposed sequencing of the IL-1 inhibitor

Before sequencing, samples were dissolved in 6 M guanidine-HCl, pH 8.6, reduced for 4 h at 37°C under N2 with a 100-fold molar excess of dithiothreitol over protein, and alkylated for 1 h with a 400-fold excess of MC iodoacetic acid. In this case, the reactions are desalted on a C8 reversed phase column, eluted and partially dried. N-terminal sequences will be determined using an Applied Biosystems protein sequencer. To obtain internal sequences, samples that may have been reduced and alkylated will be digested with cyanogen bromide or proteolytic enzymes using methods known to those skilled in the art. The reacted mixtures will be dried, dissolved in 0.1% TFA/H 2 O, and peptides will be separated using a C8 reverse phase column.

Example 3

Purification and sequencing of the components of IL-1 inhibitors

A. Components IL- li- X. IL- 1 i- a and IL- li- b

The Mono Q purification of IL-li dissolves the biological activity into three main components as shown in fig. 1 a and described in example 1, where the peak fractions for this activity are 48, 52 and 59. SDS-PAGE on samples of these fractions, as shown in fig. 2a, shows relevant components at 20 kD, 22 kD and 22 kD respectively. Western analysis of such gels, using the mouse antisera discussed in Example 4 below, stains all three of these components. When IL-11 is prepared from cells metabolically labeled with <35->methionine during growth on plates coated with IgG, each of these bands is radioactive (as shown in Fig. 2b, the autoradiogram of the above gel). Based on the logic discussed in Example 1, namely that parallel cells incubated in a non-inducing condition do not produce the IL-li bioactivity and do not produce these radioactive bands, it can be concluded that these three components account for the biological activity . These components have tentatively been called IL-li-X, IL-1 i-a and IL-li-b, respectively.

B. Purification and sequencing of IL-li-X

Mono Q fractions containing IL-11-X and/or IL-11-a were further purified by reversed phase HPLC chromatography on a Synchropak RP-4 (C4) column and radioactive components were submitted for sequence analysis. A number of attempts at direct sequencing of RP-HPLC-purified IL-li-a and IL-li-b have been unsuccessful, suggesting that they are chemically blocked at their N-termini. However, a preparation of IL-11-a (IL-11-aB2p42) gave the following sequence: and subsequent preparations of IL-11-X in the same way purified by C4 RP-HPLC have given the same sequence:

These are obviously part of the sequence found in the original attempt to sequence IL-li-a. It is believed that the sequence data shown is the N-terminus of the 20 kD component called IL-11-X.

In these and all subsequent sequences, an underlined position indicates either the inability to identify a residue or that doubt exists with respect to the residue identified. When two or more residues are put in one position, it indicates that more than one amino acid was detected at this sequencing step, and that the residue most likely to be correct is at the top.

C. Generation, purification and sequencing of peptides of

IL-li-a and IL-li-b

As IL-li-a and IL-li-b are apparently chemically blocked at their N termini, peptides of each were generated by endoproteinase digestion. Specifically, Mono Q fractions containing either IL-li-a or IL-li-b were passed through a 4.6x250 mm C3-RPHPLC column (Zorbax Protein Plus), an acceptable alternative to the C-4 columns that were used in all previous experiments. Very gradual gradients (0.2% acetonitrile per min at 0.5 ml/min) dissolved IL-li-a (Fig. 8a,b) or IL-li-b (Fig. 9a) away from the main contaminating radioactive component, human lysozyme. The identities of the purified components were confirmed by the presence of a single, radioactive, 22 kD protein on SDS-PAGE and subsequent autoradiograms (Figs. 8c,d and 9b). The proteins were collected by hand in siliconized glass tubes, and 25 ml of a 0.2% Tween-20 solution was added to each tube. The IL-1 containing fractions were then reduced in volume on a Speed-Vac to 50 µl, brought up to 300 µl by the addition of 1% NH 4 HCO 3 , followed by the addition of 1 mg of endoproteinase. In the case of IL-li-a, the enzyme used was Endoproteinase Lys C (Boehringer-Mannheim), while IL-li-b was cleaved with Endoproteinase Asp N (Boehringer-Mannheim). Cleavage was performed at 37°C for 16 h, and then the volume of the reaction mixture was reduced to 50 ml on a Speed Vac.

In the case of IL-li-a, the sample was directly chromatographed, while the IL-1 i-b sample was first reduced by adding 5 ml of 50 mM dithiothreitol in 2 M Tris, pH 8.0, reacted for 30 min at 37°C and then carboxymethylated by addition of 1.1 umol <3>H-iodoacetic acid in 10 ml of ethanol (reacted for 30 min at 37°C in the dark). Separation of the peptides was performed on a 2.1x250 mm Brownlee Aquapore RP-300 (C8) narrow-bore column at a flow rate of 100 µl/min using a Beckman HPLC equipped with microbore hardware and microbore-compatible pumps. A 200 min 0-100% linear gradient was used (H 2 O/0.1% TFA to acetonitrile/0.1% TF A). The peptide separations are shown in fig. 10 and 11. The sequence information obtained is as follows:

Two of the peptide sequences are obviously related to that obtained previously from IL-11-X. One of these, RaLysC-41, is an IL-li-a sequence, and the other, RbAspN-51, is an IL-li-b sequence, suggesting that the three components of IL-li are in the least closely related proteins, if not chemically and/or physically modified forms of a single original IL-li molecule. If the specified sequences are combined, the following composite sequences are obtained:

These composite sequences do not appear to be present in any other known polypeptides listed in the most recently updated Protein Identification Resource Database (PIR 16.0). It is believed that these sequences or smaller variants thereof represent a class of molecules that can act as IL-1 inhibitors.

Example 4

Production of antibodies specific for the IL-1 inhibitor

Ten-week-old BALB/c mice were injected subcutaneously with IL-1 that was partially purified (400-fold) from crude supernatants by Mono Q chromatography, dialyzed against PBS, and emulsified with complete Freund's adjuvant. Each mouse received IL-li purified from 5 ml of crude supernatant. The mice were given a booster dose every two weeks with an equivalent amount of IL-1 in emulsified with incomplete Freund's adjuvant, and serum samples were taken from the tails seven days after each booster injection. Antisera were tested for anti-lL-li activity by Western transblot of the immunogen run on SDS-PAGE, as shown in Fig. 5a. Fig. 5b shows that all mice produced anti-IL-li antibodies after three injections of IL-li.

As monoclonal antibodies will be of great value for the cloning of the IL-li gene from an expression library, purification of the recombinant IL-li protein and investigation of the molecule's biology, the process of producing a set of monoclonal antibodies specific for IL-li. To produce B-cell hybridomas, the above mice were injected intravenously with the same amount of IL-1 in saline 24 h before removal of the spleens. Splenocytes were carefully removed from the spleens into cold balanced salt solution (BSS), washed twice with BSS, mixed with P3 myeloma cells at a ratio of 2 x 10<7> P3 cells per 10<*> splenic B cells and spun down. The cells were joined by the dropwise addition of 1 ml of warm, gassed (5% CO 2 ) PEG 6000 (40% polyethylene glycol 6000 : 60% minimal essential medium) to the dry pellet. Adherent cells were washed with BSS and resuspended in 10 ml rich medium (10% FBS) containing 2 x 10 s peritoneal cells per ml and the pellet was carefully broken up using a 10 ml pipette. The volume was adjusted to 20 ml by adding several peritoneal cells in medium, and the cells were plated out in 96 well plates at 0.1 ml per well. well. The plates were placed in a gas incubator and then treated as follows: Day 1 - Add 3 x HAT (hypoxanthine, aminopterin, thymidine) in rich medium to a final concentration of lx

Day 5 - Change medium, replace with 200 ul 1 x HAT in rich medium

Day 10 - Begin survey for hybrid growth. Change medium, replace with 200 ul 1 x HAT in rich medium containing 1.5 x 10<6> peritoneal cells per ml.

When hybrid cells are nearly confluent in a well, the supernatants are transferred for testing, and the cells are gently scraped with the tip of a pipette and transferred to 1 ml culture wells containing lx HAT in rich medium plus 3 x 10<6 >peritoneal cells per ml.

The supernatants from the confluent wells are tested for anti-IL-li activity using an ELISA, where partially purified IL-li (Mono Q-purified material identical to that injected into the mice) is bound to microtiter wells. Normal mouse sera and hyperimmune antisera are used as the negative and positive comparison samples, respectively. Positive supernatants will be retested by ELISA on plates coated with homogeneously purified IL-1 and by immunoprecipitation of purified metabolically labeled IL-1. Positive cells will then be cloned at limiting dilution and injected into pristane-treated mice to generate ascites. Large amounts of IL-1-specific antibodies can be produced by tissue culture or by massive generation and collection of ascitic fluid in mice. Purification of these antibodies and attaching them to insoluble beads will produce affinity adsorbents for the purification of the recombinant IL-1 protein.

Example 5

Cloning of IL-1i cDNA

It was shown that monocytes plated on IgG-coated Petri dishes and cultured for 24 h i

presence of [<35>S]-methionine produced [<35>S]-IL-li which could be identified by its chromatographic properties on Mono Q.

To determine when (during the 24-hour period) IL-11 was produced at a maximal rate, plated monocytes were exposed to [<3S>S]-methionine (pulsed) for a short two-hour period, at which time a large excess of unlabeled methionine was added and incubated for an additional 2 h. The medium was then collected and analyzed for radiolabeled IL-1i. This procedure was applied to monocytes at various time points after plating IgG plates, and it was found that exposing monocytes to [<35>S]-methionine at 15 h after plating produced the maximal amount of [<35>S]-IL-1, suggesting that IL-1 mRNA in monocytes was at its highest level 15 h after plating on IgG.

Fresh monocytes were then plated on LPS-free IgG obtained as in Example IB.

After incubation in RPMI medium for 15 h at 37°C, the cells were washed with

phosphate-buffered saline and then lysed with 4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7, 0.5% sarcosyl, 0.1 M 2-mercaptoethanol. The total RNA was then isolated from this lysate by the AGPC method according to P. Chomczynski and N. Sacchi described in Analytical Biochemistry, vol. 162, pp. 156-159 (1987).

Poly A<+> RNA was isolated by oligo dT cellulose chromatography by the method of Aviv, H. and Leder, P. (1972) Proe. Nati. Acad. Pollock. (US) 69:1408-

1412 precipitated with ethanol and dissolved to a concentration of 0.36 ng/uL One microgram of poly A<+> RNA was used to prepare cDNA according to Gubler, U.

and Hoffman, B.J. (1983) Gene 25: 263-169.

cDNA was incorporated into a lambda gtl 1 expression library using Eco RI linkers from Boehringer Mannheim catalog #988448 or New England Bio Lab #1070 and instructions obtained from these manufacturers.

The resulting library, containing 10<6> independent clones, was screened

E. coli Yl 090 rk" (Promeca Biotec) with an appropriate polyclonal antibody against

IL-li as described previously using screening conditions described by R. A. Young and R. W. Davis [(1983) PNAS 80:1194-1198]. Positive signals will be detected using a biotinylated second antibody (such as goat anti-mouse IgG, Bethesda Research Labs) followed by a streptavidin/alkaline phosphatase conjugate

(Bethesda Research Labs.) as described by Bayer, E. A. and Wilchek, M. (1979) in Methods in Biochemical Analysis, and Guesdon, J. L., Ternynch T. and Avrarhas, S.

(1979) J. Histochem. Cytochem. 27:1131-1138 and according to the manufacturer's instructions.

Example 6

Production and sequencing of the gene that codes for IL-1 i

cDNA prepared as described in Example 5 was incorporated into the cloning vector lambda GT10. This cDNA was first methylated using EcoRI methylase with S-adenosyl-methionine as the substrate, EcoRI linkers were added in a ligation reaction, and excess linkers were removed by digestion with EcoRI endonuclease and chromatography on a CL6B spin column. A ligation reaction containing 0.124 µg of ligated size-selected cDNA and 1 µg of EcoRI-capped and phosphatase-treated lambda GT10 was performed, and the products of this ligation reaction were packaged using GIGAPACK GOLD packaging extracts (Stratagene). This gave a library of lxlO<7> components.

To screen this GT10 library, oligonucleotide probes (antisense) were synthesized based on the protein and peptide sequence presented in Example 3. The sequence of the probes and their corresponding peptide sequence is as follows:

Note. N = A, G, C and T.

Probe No. ILlil-3 was <32>P-phosphorylated at its 5'-end and is used to screen 3xl0<5 >plaques of the library. The probe reproducibly hybridized to three plaques, and out of these, one plaque was shown to also hybridize to probe no. ILlil-4. This plaque, GT10-ILH-2A, was cultured and DNA was isolated using Lambdasorb (Promeca) according to the manufacturer's instructions. GT10-ILH-2A has been deposited at the American Type Culture Collection (ATCC) in Rockville, Maryland under accession no. 40488. DNA was digested with EcoRI, divided into five equal portions and subjected to electrophoresis on a 1% agarose gel.

After electrophoresis, the gel was stained with ethidium bromide. A photograph of this gel is shown in fig. 12a. Lanes 6, 8, 10, 12 and 14 contain the five portions from the EcoRI digestion. Lane 5 contains a mixture of wild type lambda DNA cut with HindlH and 0X174 RF DNA cut with Haelll (New England Biolabs) useful as molecular weight markers. Fig. 12a shows that GT10-ILH-2A contains an EcoRI fragment that is 1850 base pairs in length.

To show more conclusively that this 1850 bp fragment carries the coding sequence for the ILI inhibitor, a Southern blot was performed as follows. The DNA fragments in the gel shown in fig. 12a was blotted onto nitrocellulose using standard methods. The nitrocellulose was then cut lengthwise into five strips, so that each strip contained DNA from lanes 6, 8, 10, 12, and 14. The strips were then individually hybridized to each of the five oligonucleotide probes (listed above) labeled at the 5'- end with P-phosphate. The oligonucleotide concentration was 1 pmol per ml, and the hybridization temperatures were as follows:

After washing, the strips were placed side by side and taped together to re-form the original nitrocellulose sheet. This was autoradiographed in the presence of an intensifying screen at -70°C for 24 h. Fig. 12b is a photograph of this autoradiography. It provides evidence that all probes hybridize specifically to the 1850 bp fragment, proving that this fragment carries significant coding sequences for the ILI inhibitor.

To determine its DNA sequence, GT10.-IL11-2A DNA was digested with EcoRI. electrophoresed on a 1% agarose gel, and the 1850 bp fragment was isolated. This fragment was ligated with EcoRI-digested Ml3 mp 19 and transformed into

E. coli strain JM109. Transformants were screened by looking for those lacking beta-galactosidase activity. Five such transformants were isolated, single-stranded DNA was prepared and sequencing was performed according to Sanger et al. The DNA sequence of three of the transformants corresponded to the 3' end of the mRNA, while two transformants provided the protein coding sequence. In fig. 13 shows the DNA sequence that was obtained for the protein-coding region of the cDNA.

Fig. 13 also shows the predicted amino acid sequence. The amino acid sequence from the first amino acid alanine to the 29th amino acid proline and from the 79th amino acid isoleucine to the end is the amino acid sequence according to the hypothesis. The predicted amino acid sequence from the 30th amino acid proline to the 78th amino acid proline agrees with the peptide sequences described in Example 3.

Example 7

Sequencing of GT10- IL- 1I- 2A oe IL- li

A portion of GT10-IL1I-2A has been sequenced and is indicated in Fig. 14. DNA encodes a protein containing amino acid sequences characteristic of IL-1 (nucleotides 99-557). However, it is believed that a number of modifications can be carried out on this protein before it is secreted into the extracellular environment. These modifications may or may not be necessary for the protein to have activity as an IL-1.

GT10-ILH-2A encodes at least 32 amino acids N-terminal (nucleotides 3-98) relative to the amino terminus of the form IL-li known as X. It is believed that included in these 32 amino acids is a secretory leader sequence starting at the M which is encoded by nucleotides 24-26, directs nascent IL-1 to the extracellular environment and is then removed by a leader peptidase and possibly other peptidases. The extent to which this sequence is removed in the alpha and beta forms of IL-1 is currently not known, but the N-terminus of these forms is thought to be close to that of form X. Removal of the secretory leader sequence is probably necessary for the protein must have effective IL-li activity.

Nucleotides 349-351 of GT10-ILH-2A encode an N-residue that is part of a consensus N-glycolysis site. On the basis of their susceptibility to digestion with N-glycanase, the alpha and beta forms of IL-1 are believed to be glycosylated. As form X is not believed to be amenable to digestion by this enzyme, it is assumed that it is not glycosylated, although this is a possibility that can be readily demonstrated by one skilled in the art of protein sequencing using the information provided herein. It is assumed that glycosylation at this N-residue is not necessary for the protein to have effective IL-1 activity.

Nucleotides 99-101 of GT10-ILH-2A encode a P (see Fig. 15), but no P has been detected at this position (N-terminus) in form X of IL-11. It is possible that this residue has been modified in the mature protein. It is believed that modification of this residue is not absolutely required for efficient iL-li activity.

The currently unknown N-terminal residues of the alpha and beta forms are not fully detectable by Edman digestion and it is likely that they have been modified after removal of some of the N-terminal residues of the protein encoded by GT10-IL-H- 2A. It is believed that this modification is not required for efficient IL-1 activity.

Example 8

Expression of genes encoding IL-11 in dwarf cells

Animal cell expression of IL-1 requires the following steps:

a. Construction of an expression vector

b. Selection of a host cell line

c. Introduction of the expression vector into the host cells.

d. Manipulation of recombinant host cells to increase expression levels of

IL-li.

1. IL-1α expression vectors engineered for use in animal cells can be of various types including strong constitutive expression fragments, inducible gene fragments as well as those engineered for expression in particular cell types. In all cases, promoters and other gene regulatory regions such as enhancers (inducible or non-inducible) and polyadenylation signals are placed in the correct location relative to the cDNA sequences in plasmid-based vectors. Two examples of such engineered pieces follow: (1) An engineered piece using a strong constitutive promoter region should be made using simian virus 40 gene control signals (SV40) in an arrangement such as that found in plasmid pSV2CAT as described by Gorman et al. in Mol. Cel. Biol. 2:1044-1051, 1982. This plasmid should be manipulated in such a way as to substitute the IL-1 cDNA for chloramphenicol acetyltransferase (CAT) coding sequences using standard molecular biology techniques (Maniatis et al, supra) as shown in fig. 6. (2) An inducible gene fragment should be prepared using the plasmid PMK containing the mouse metallothionein (MT-1) promoter region (Brinster et al., Cell 27:228-231, 1981). This plasmid can be used as a starting material and should be manipulated as shown in fig. 7 to provide a metal-inducible gene fragment. 2. A number of animal cell lines should be used to express IL-1 using the vectors described above for production of active protein. Two potential cell lines that have been well characterized for their ability to promote foreign gene expression are mouse Ltk" cells and Chinese hamster ovary (CHO) dhff cells, although expression of IL-11 is not restricted to these cell lines 3. Vector DNA should be introduced into these cell lines using any of a number of gene transfer techniques. The method used here includes the calcium phosphate DNA precipitation technique described by S. L. Graham & A. S. van der Eb (Virology 52:456-467 , 1973), where the expression vector for IL-11 is coexpressed with another expression vector encoding a selectable marker. In the case of Ltk" cell transfection, the selectable marker is a thymidine kinase gene and the selection is as described by Wigler et al. (Cell 16:777-785, 1979) and in the case of CHO dhfr' cells the selectable marker is dihydrofolate reductase (DHFR) whose selection is as described by Ringold et al. in J. Mol. Appl. Genet, 1:165-175, 1981. 4. Cells expressing the IL-1 i gene fragments should then be cultured under conditions that will increase the production amount of IL-11. Cells carrying metallothionein promoter fragments can now be cultured in the presence of heavy metals such as cadmium, which will lead to a fivefold increased utilization of the MT-1 promoter (Mayo et al., Cell 29:99-108), which then leads to a comparable increase in IL-1 protein levels. Cells containing IL-11 expression vectors (either SV40 or MT-1 based) together with a DHFR expression vector can be taken through the gene amplification protocol as described by Ringold et al. (J. Mol. Appl. Genet, 1:165-175, 1981) using methotrexate, a competitive antagonist for DHFR. This leads to more copies of the DHFR genes present in the cells and at the same time increased copies of the IL-li genes, which in turn can lead to more IL-li protein being produced by the cells.

Example 9

Rensina of IL-1 from recombinant dwarf cells

Since IL-1 is expected to be secreted from cells like the native material, it is expected that the methods described above for purification of the native protein will allow similar purification and characterization of the recombinant protein.

Example 10

IL-li sequence

The amino-terminal residue of IL-1i has been identified several times by direct protein sequencing as an arginine (R). The result of such sequencing is shown in example 3.1 in contrast to this, the amino-terminal residue of IL-1 as predicted by the sequence of the cDNA, is a proline (P). This amino-end corresponds to nucleotides 85-87 in fig. 13 and is surrounded by a circle in fig. 14 and 15. This apparent discrepancy between the cDNA sequence and the direct protein sequence can be explained if one assumes that an error in the cDNA sequence was introduced during the reverse transcriptase-catalyzed synthesis from its mRNA. That is, a CGA codon (arginine) located on the mRNA where it would code for this amino terminus could have been changed during the reverse transcriptase reaction to a CCA codon (proline) in the cDNA. This type of reverse transcriptase problem has been reported in the literature previously, e.g. by B.D. Clark et al. in Nucleic Acids Research 14:7897 (1986).

It is assumed that the correct amino acid sequence for the protein is as predicted by cDNA, except that the amino terminal amino acid is an arginine instead of the proline residue indicated in fig. 13-15. It is considered that both the DNA sequences and their corresponding peptide sequences fall within the scope of the invention, although the amino-terminal arginine sequence is preferred.

Example 11

A protein that has the sequence:

where X is cysteine, serine or alanine, and

Z is arginine or proline,

is also used in the invention.

Claims (13)

1. Process for producing a pharmaceutical preparation containing an interleukin-1 inhibitor (IL-1) or an IL-1 with an N-terminal leader sequence, in which IL-1 is able to inhibit interleukin-1 (IL-1) and wherein IL-li or IL-li with an N-terminal leader sequence is encoded by a DNA sequence selected from; (A) the following nucleotide sequence from position 99 to 554, wherein X is C or G, and Z is R or P; (B) the coding region of the sequence of (A) or a portion of the sequence of (A); (C) a sequence degenerate in the coding region of the sequence in (A) or part thereof; (D) a sequence encoding all or part of the following amino acid sequence: wherein (U) is M or none or an N-terminal secretion leader sequence and (X) is R or P; or (E) a sequence encoding a polypeptide that is at least about 70% homologous to the amino acid sequence set forth in (D). characterized in that it comprises cultivating a recombinant host cell comprising a DNA sequence encoding an interleukin-1 inhibitor (IL-li) operably linked to at least one regulatory element necessary to express the DNA sequence in the host cell under appropriate conditions to express the DNA sequence to produce IL-1i in the polypeptide or IL-1i with an N-terminal secretion leader sequence; and harvesting the IL-li polypeptide or IL-li with an N-terminal secretion leader sequence, where the harvested polypeptide is combined with pharmaceutically acceptable carriers, fillers and any excipients to form pharmaceutical compositions. 2. Procedure as stated in claim 1, wherein the DNA sequence comprises a sequence encoding a polypeptide that is at least 80% homologous to the amino acid sequence: wherein (U) is M or nil and (X) is R or P, characterized by the fact that a corresponding expression method is used. 3. Method as stated in claim 1, in which the DNA sequence comprises a sequence that codes for a polypeptide that is at least 90% homologous to the amino acid sequence: wherein (U) is M or nil and (X) is R or P, characterized by the fact that a corresponding expression method is used. 4. Method as stated in claim 1, in which the DNA sequence comprises a sequence that codes for a polypeptide that is at least 95% homologous to the amino acid sequence: wherein (U) is M or nil and (X) is R or P, characterized by the fact that a corresponding expression method is used. 5. Method as stated in claim 1, in which said IL-inhibitor comprises a sequence consisting of the amino acid sequence: where (U) is M or nothing, characterized by the fact that a corresponding expression method is used. 6. Method as stated in claim 1, in which the DNA sequence comprises a DNA sequence encoding IL-li X, IL-li-a or IL-li-fi, characterized by the fact that a corresponding expression method is used. 7. Method as stated in claim 1, wherein said DNA sequence is a cDNA, characterized in that a corresponding expression method is used. 8. Method as stated in claim 1, in which said DNA sequence is derived from mammalian cells, characterized by the fact that a corresponding expression method is used. 9. Method as stated in claim 1, in which said DNA sequence is derived from human monocytes, characterized by the fact that a corresponding expression method is used. 10. Method as stated in claim 1, in which the DNA base sequence comprises the nucleic acid sequences from position 99 to 554 as follows; characterized by the fact that a corresponding expression method is used. 11. Method as set forth in claim 1, wherein the DNA base sequence comprises the nucleic acid sequences from position 99 to 554 of the sequence that follows; characterized by the fact that a corresponding expression method is used. 12. Method as set forth in claim 1, in which the DNA sequence comprises a sequence that encodes the following amino acid sequence; characterized by the fact that a corresponding expression method is used. 13. Method as set forth in claim I, in which the DNA sequence comprises a sequence encoding the following amino acid sequence; characterized by the fact that a corresponding expression method is used.