JPH03505279A - interleukin-1 inhibitor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] interleukin-1 inhibitor Background of the invention This application is filed in U.S. Patent Application No. 238.713, August 1988, by the same inventor. This is a continuation in part of the application filed on the 31st, and furthermore, this U.S. Patent Application No. 238.713 No. 199.915, a continuation in part of U.S. Patent Application No. 199.915, filed May 27, 1988. The application Interleukin-1 is found in a large number of cells, including monocytes and some macrophages. A class of proteins produced by different cell types. This class has at least There are also two types, interleukin-1 alpha and interleukin-1 beta. 17-18 kilodalton proteins known as These tampers Proteins are important physiological targets for many different target cells involved in inflammation and immune responses. exerts a scientific effect. This protein is a comitodiene (phytomolecule) for T-cells. (acting together with tohemagglutinin) and both fibroblasts and chondrocytes This induces the secretion of inactive collagenase and induces the secretion of endothelial cells against neutrophils. Increases surface adhesion. Furthermore, interleukin-1 is a pyrogen. acts on the hypothalamus, stimulates muscle protein catabolism, and induces “acute triggers the synthesis of certain proteins known as "phase reactants". therefore , interleukin-1 (rL-1) is important in biological responses to infection and wounding. It is clear that it is a part of

B. Pathological role of IL-1 However, although IL-1 usually has beneficial effects, the effects of IL-1 A situation has become clear that would be harmful. For example, IL-1 is involved in arthritis. can increase collagenase levels in joints and reduce chronic rheumatoid arthritis. It has been implicated as a mediator of both acute and chronic immunopathology in the community. ing.

IL-1 alters endothelial cell function and increases the chemical contribution of leukocytes and lymphocytes to the sulcus membrane tissue. directs chemotaxis and migration, induces capillary proliferation, and inhibits synovial lining during the acute phase of the disease. can stimulate the accumulation of macrophages in the macrophages.

At the stage of tissue destruction, IL-1 stimulates enzyme release from fibroblasts and chondrocytes. Balls have been implicated as mediators in tissue damage by stimulation.

Furthermore, overproduction of IL-1 in the skin of shoe polish patients has been revealed, and High levels of 11. -1 was found. Displaced arthritis Inflammation can mediate tissue destruction by stimulating enzyme release from cells. La The joint pathology of Iter syndrome is similar to that seen in displaceable arthritis and rheumatoid arthritis. It is similar to ! L-1 induces tissue destruction in these three different forms of inflammatory arthritis. He is involved as a mediator of destruction.

Additionally, IL-1 has been found in the synovial fluid of osteoarthritis patients.

IL-1 release by chondrocytes is involved in the destruction of articular cartilage in this disease. Ru.

Additionally, IL-1 can exacerbate autoimmune diseases. For example, systemic lupus erythematosus A decrease in IL-1 producers from peripheral blood cells of affected individuals has been described. difference Additionally, some changes in B lymphocyte function are associated with IL-1 production or IL-1 availability. It may be related to sexual abnormalities.

Overproduction of IL-1 has been shown in peripheral monocytes of scleroderma patients, and fibroblasts I as a substance that can cause fibrosis by stimulating collagen production by L-1 is involved. The mechanism of tissue damage in dermatomyositis is also cell-mediated immunity. Therefore, IL-1 may be involved in such pathophysiological He is probably involved in the process as a mediator.

Acute and chronic interstitial lung diseases are caused by overproduction of IL-4 or stimulated lung fibroblasts. It is characterized by collagen production. In a recent study on an animal model of pulmonary hypertension, It is possible that TL-1 induces changes in endothelial cells that lead to pulmonary artery stenosis. It is shown that there is sex. causing pulmonary hypertension and further secondary damage. Which stenosis is this? Therefore, IL-1 inhibitors may be useful in the treatment of these pulmonary diseases. It would be useful.

As shown in recent studies, IL-1 is a Langerhan protein responsible for the production of insulin. can directly harm the beta cells of the islets. Currently IL- It is hypothesized that damage caused by 1 is the initial event in the acute phase of juvenile diabetes. It is.

In many forms of acute and chronic glomerulonephritis, monocytes and macrophages in the kidney Lophage infiltration is mainly seen. The release of IL-1 from these cells is associated with other flames. causing a local accumulation of diseased cells, eventually leading to inflammatory damage and results in a fibrogenic reaction.

Crystals found in gout and pseudogout tissues or joint fluid are macrophages It has been shown that it is possible to directly stimulate IL-1 release by Therefore, IL- 1 may be an important mediator in the inflammatory cycle of these diseases.

IL-1 can induce calcium loss from bones and cause inflammatory joints. This may be the cause of osteoporosis seen in the disease.

Keratinocytes from shoe shine patients release large amounts of IL-1. This mediator is This 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 important endogenous pyrogienes and is found in bacteria or viruses. cause a marked fever, as seen in some infectious diseases, such as acute febrile illness caused by It will probably wake you up.

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

Peripheral monocytes derived from Crohn's disease and ulcerative colitis both show hyperregulation of IL-1. Surplus production is shown. Localized IL-1 release within the intestine may contribute to the inflammation of these diseases. It may be an important mediator that stimulates the symptomatic cycle.

Some types of lymphoma can cause fever, osteoborosis, and even secondary arthritis. It also features Excessive IL-1 release by some lymphoma cells in vitro has been shown to be the cause of some of the clinical signs of these malignant tumors. I can do it. In addition, due to IL-1 production by some malignant lymphocytes, the fever, acute phase response, and cachexia.

IL-1 release by brain astrocytes may result from vascular occlusion after brain injury This is thought to be the cause of the disease.

C. Uses of IL-1 inhibitors In these or other situations where IL-1 has a deleterious effect, IL-1 There is clearly clinical use for single-acting inhibitors. IL-1 is a T cell The development of autoimmune and other immune diseases due to the comitogenes against This is the main cause. Therefore, IL-1 inhibitors are useful if administered systemically. It can be a powerful immunosuppressant. When applied topically, such IL-1 inhibitors reduce inflammation. This may prevent tissue destruction within the joints and other sites of inflammation. case Certain IL-1 inhibitors are administered with collagenase inhibitors. would be more effective in preventing tissue destruction if

at the level of synthesis, secretion, or target cell binding or response to IL-1; Therapeutic intervention against rL-1 effects may be possible. IL-1 is lipopolysaccharide, complement Monocytes/macrophages and other cells in response to fragments and viruses more synthesized. Any amount that prevents these inducers from binding to producing cells. or any molecules that interfere with their effects on the physiology of these cells. It may serve as a modulator of L-1 action. at least of the IL-1 protein Two 30 kd precursor mRNAs were isolated, which are hydrophobic and Because it does not contain a signal sequence, IL-1 is not secreted by the normal secretion system. . The release of active proteins from inactive precursors probably involves proteolysis of the precursors. or is necessary. for the release of IL-1 from the precursor. Inhibitors should theoretically control the effects of IL-1. IL-1 is Probably a classical receptor (although no such receptor has yet been isolated) It acts on target cells through a pathway mediated by Therefore, IL- to the receptor Molecules that prevent binding of IL-1 or down-regulate this receptor are The effect of -1 could also be adjusted. Furthermore, following receptor binding of IL-1, cells Although the events that occur within the receptor are still not completely understood, it is possible that other events mediated by the receptor may occur. Substances that interfere with the cellular response to ectopia and therefore block the action of IL-1 may exist. For the reasons stated above, the above format - or more Proteins and small molecules that can inhibit IL-1 are being sought.

Surprisingly, the inventors have identified at least two IL-1 inhibitory properties. -1 inhibitor protein was discovered. These molecules are obtained in purified form. , one skilled in the art would be able to determine its amino acid sequence. In addition, these proteins can be produced The cell preparations that produce it have been characterized, and the mRNAs that lead to its synthesis have been characterized. sex has been determined. Finally, c regarding the genes encoding these inhibitors. Antisera have been developed that will facilitate the screening of DNA expression libraries. It is. By using these substances together, IL-1 inhibitors can be inhibited. It will be possible to clone the cDNA encoding the

These genes may then be useful in treating pathophysiological conditions mediated by IL-1. enables large-scale production of IL-1 inhibitors suitable for use in pharmaceutical formulations. Good morning.

Summary of the invention The present invention relates generally to IL-1 inhibitors (“IL-1i”) and More specifically, the present invention relates to a monocyte-derived IL-1 inhibitor. Furthermore, the present invention It also relates to biologically active analogs of inhibitors.

The object of the present invention is to provide active drugs against IL-1α or IL-1β, or a combination thereof. The purpose of the present invention is to provide a highly purified IL-1 inhibitor. Another eye of the invention The goal is to provide these inhibitors in purified form and to determine their amino acid sequences. The goal is to make it possible. Yet another objective is to The purpose is to provide a amino acid sequence. Furthermore, such IL-1 inhibitors It is also essential to identify biologically active analogues with stronger or equivalent properties. This is one of the purposes of the invention.

Additionally, we provide a recombinant DNA system for the production of the IL-1 inhibitors described herein. It is also an object of the present invention to.

Still another object of the invention is that the present invention has a value as a pharmaceutical preparation showing activity against IL-1. It also includes the use of purified IL-1 in vitro.

Other objects and advantages of the invention are set forth in part in the description that follows, and in part. are obvious from the description of the invention, or may be learned from practice of the invention. Especially attached These objects and advantages can be achieved by the means and combinations indicated in the appended claims. May it be realized and achieved.

In order to achieve these objectives and in accordance with the objectives of the present invention, Disclosed are IL-1 inhibitors that exhibit inhibitory activity. The preferred inhibitor is Monocytes using monocytes grown on IgG-coated plates - monocytes in purified form from conditioned medium separated.

Preferred inhibitors of the invention include 1,2. and 3. inhibitor 1 and 2 in a position characteristic of a 22-23 kDa protein on 5DS-PAGE. 52mM and 52mM respectively from a MonoQ FPLC column under specific conditions. and 60mM NaC]. Inhibitor 3 is 5DS -Moves to a position characteristic of the 20kD protein on PAGE and under certain conditions Mo This is a protein eluted from the no Q FPLC column at 48mM NaCl.

Furthermore, in order to achieve the object and for the purpose of the present invention, the active ingredient at least one IL-1 inhibitor according to the invention or an agent mentioned in the text; Pharmaceutical compositions containing biologically active analogs thereof are disclosed.

Furthermore, to achieve the objectives and in accordance with the objectives of the present invention, these rules are - Recombinant DNA systems for producing inhibitors and analogs thereof are also disclosed. It will be done. Preferred embodiments of this system include the IL-1 inhibitors disclosed herein. Together with the vector and cells constituting the expression system capable of expressing at least one I. at least one CDNA clone encoding an L-1 inhibitor or Synthetic equivalents are included. used to identify these cDNA clones Antisera are also provided.

encode these CDNA clones, their analogs, or their inhibitors. Expression systems for the production of these lu1 inhibitors using other DNA sequences are also provided. It will be done.

Brief description of the drawing Figures 1a and 1b show MonoQ chroma of two metabolically labeled supramonocyte grooves. Figure 2 shows the protein profile of the tograph. Cells are IgG(la) or bovine Cultured on plates coated with fetal serum (lb).

Figure 2a shows the fraction of silver from the area shown in Figures 1a and 1b. A stained gel is shown.

Figure 2b is an autoradiogram of the gel shown in Figure 2a.

Figures 3a, b and 0 show data regarding purification roux 11 of Example 1. 3rd a The figure shows chromatography data overlaid with radioactivity patterns. Figure 3b is the third Figure a shows a silver-stained gel performed on the fraction sample shown in figure. Figure 3c is Figure 3b The autoradiogram of the gel is shown.

Figures 4a and 4b are gel filtration chromatograms of MonoQ purified IL-1i. The results are shown below.

Figures 5a and 5b show Western analysis of mouse antisera.

Figure 6 shows the construction of plasmid psVXVPL21L-1i.

Figure 7 shows the construction of plasmid pMK-SGE:IL-1i.

Figures 8a-d show data for IL-1i-α. Figures 8a and 8b are Showing romarographie data.

Figure 8c shows a silver stained gel run on the fraction sample shown in Figure 8b. vinegar. Figure 8d shows the autoradiogram.

Figures 9a and 9b show data for IL-1i-β. Figure 9a is chroma The tography data is shown. Figure 9b shows 5DS-PAGE data.

FIG. 10 shows peptide separation data for IL-1i-α.

FIG. 11 shows peptide separation data for IL-1i-β.

FIG. 12a shows GTIO-ILI digested with ECORI after electrophoresis according to Example 6. It is a photograph of the gel of 1-2A.

Figure 12b is an autoradiogram of the Southern plot of the gel shown in Figure 12a. Show data.

FIG. 13 shows the protein code of lambda GTIO-IL1i-2A according to Example 6. A portion of the DNA sequence and predicted amino acid sequence of the domain region is shown.

Figure 14 shows the nucleotide sequence of GTIO-ILI 1-2A.

Figure 15 specifically depicts a peptide containing an IL-1i sequence and a secretory leader sequence.

Description of preferred embodiments A detailed description of the presently preferred embodiments of the invention will now be provided. , this description, together with the following examples, provides a detailed explanation of the invention.

A, human monocyte-derived inhibitor As mentioned above, the present invention relates to IL-1 inhibitors that have been isolated in purified form. Ru. From human monocyte conditioned medium in which monocytes were grown in containers coated with IgG, the Preferably, an IL-1 inhibitor is obtained. Furthermore, the present invention provides human monocyte-containing culture media. Substantially purified Rue 1 of any source that is biologically equivalent to the naturally derived inhibitor Including inhibitors.

The term “bioequivalent” used throughout this specification and claims Currently, it works in the same manner as the natural IL-1 inhibitor isolated from monocytes! L-1 of the invention that can inhibit (but not necessarily to the same extent) the action of means a composition. As used throughout this specification and claims, "substantially" "homologous to" refers to the native +L-1 inhibitor isolated from monocyte conditioned media. , with greater homology than previously reported and shown by any IL-1 inhibitor. It means degree. A degree of homology of 70% or more is preferred, preferably 80% or more. Preferred, and even more preferred is 90% or more. A particularly preferred group of insulators The inhibitor is more than 95% homologous to the natural inhibitor. The homology mentioned here The percentage is calculated based on the number of identical amino acid residues in the sequences being compared. Calculated as the percentage of amino acid residues found on the smaller side of the sequence. It will be done. In this case, Dayhoff, M, and D are At1as of Prote. inSequence and 5structure Volume 5, page 124 (1 972), National Biochemical Re5earch F foundation, Washington, D, C, (details for reference) (incorporated herein) helps arrays for such comparisons, as shown in For this reason, four gaps can be introduced in the length of 100 amino acids.

Preferred IL-1 inhibitors of the invention are obtained from monocyte conditioned media and purified for the first time. isolated in type. For the purposes of this application, the IL-1 inhibitors disclosed in the text "Pure" or "purified" when used to refer to IL-1 protein It means a standard material that does not substantially contain proteins other than inhibitor proteins. Main departure Preferably, Ming's IL-1 inhibitor is at least 90% pure; % pure is even more preferred.

At least three purified IL-1 inhibitors were isolated by the method of the Examples. There is. These include inhibitor 1, inhibitor 2 and inhibitor 3. be done. Inhibitor 1 showed similar behavior as a 22-23 kDa molecule on 5DS-PAGE. 52 in Tris buffer, pH 7.6, and has an isoelectric point of about 4.8. Elutes from the Mono Q FPLC column at around 1 mM NaCl. inhibitor -2 is also a 22-23 kDa protein and I) I = 4.8, but Mono Q Elution from ram is 60mM NaCl. Inhibitor 3 is 20kDa tan The protein is eluted from the Mono Q column with 48mM NaCl. inhibitor -1.2 and 3 are immunologically and functionally related. These inhibitors By obtaining them in purified form, we were able to obtain their amino acid sequences. came. Purified inhibitors and AB+ proteins clearly demonstrated for the first time in the text. In-sequencer technical manual (ABI Protein 5equ amino acids of these inhibitors using methods such as those described in The substantial proportion of amino acid sequences can be estimated.

Example 3 shows three types of IL-1 inhibitors, namely IL-1i-X, IL-1 Amino acid sequence data obtained for 1i-α and IL-1doβ are shown.

The present inventors have identified at least one antibody generated against an IL-1 inhibitor. It was expressed. This IL-1 inhibitor and its Additional polyclonal and monoclonal antibodies against conventional IL-1 inhibitors body can be prepared. One particular polyclonal antibody is described in Example 4. do.

B1 recombinant inhibitor 1. Overview A recombinant DNA method for producing IL-1 inhibitors is presented here. A fruit of the present invention In embodiments, the active site is a naturally occurring rL-1 inhibitor isolated from humans. functions biologically equivalent to To direct the production of IL-1 inhibitors , natural or synthetic NA sequences can be used. This method does not include: (a) instructing host cells to produce proteins with IL-1 inhibitor activity; Preparation of DNA sequences that can be produced; (b) a vector containing, for example, the necessary operational elements to express the DNA sequence; DNA sequences into vectors that are imported into and replicated in host cells, such as vectors. Cloning of: (C) IL-1 infusion of vectors containing synthetic NA sequences and operational elements. Transfer into a host cell capable of expressing DNA encoding the inhibitor: (d) host cells under conditions suitable for amplification of the vector and expression of the inhibitor. culture; (e) Harvesting the inhibitor; and (f) causing the obtained inhibitor to adopt the active tertiary structure, thereby rL- 1 inhibitory activity.

2. DNA sequence The DNA sequences intended for use in this method are shown in part in Example 5 and in part in Example 5. This will be discussed in Example 6. These sequences may include synthetic and natural DNA sequences. is intended. The native sequence may also include cDNA or genomic DNA segments. is included.

Example 6 is a DNA encoding the same protein as that isolated in Examples 1-3. provides a molecular clone of A. In Example 6, plaque, GTIO-IL- 1i-2A.

was isolated from the GTIO library. The phages within this plaque are multiplied, The DNA was isolated and digested with EcoR1. 1850 base pair EcoRI The fragment carries the coding sequence for an IL-1 inhibitor. Figure 13 is A partial NA sequence of this EcoRI fragment is shown.

In view of the teachings contained herein and known methods, those skilled in the art will know that other synthetic polynucleotides It would be possible to use a sequence. Industry updates regarding polynucleotide synthesis As an example, Matteucci, M. D., and Caruthers.

M, H,, J, Am, Chem, Sac, 103:3185 (198 5) and Beaucage, S.L., and Caruthers, M.H. , Tetrahedron Lett, 22:1859 (1981), and and AB■ Indicates the instructions for use supplied with the oligonucleotide synthesizer. vinegar. Each of these is incorporated herein in detail by reference.

These synthetic sequences may be identical to the native sequences detailed below, or they may be different. It can also contain nucleotides. In some embodiments, if the synthetic sequence contains nucleotides different from those found in the naturally occurring DNA sequences of the invention. If so, these different sequences share the same primary structure as IL-1i isolated from monocytes. It is intended that it still encode a polypeptide having the following. Or something else In embodiments of the invention, synthetic sequences containing different nucleotides are It would encode a polypeptide with the same biological activity as IL-1i.

Furthermore, the DNA sequence is a fragment of the native sequence, i.e. Polynucleotide fragment isolated and purified for the first time by the present inventors It may be In some embodiments, the DNA sequence is a cDNA live Restriction fragments isolated from the library.

In another embodiment, the DNA sequence is isolated from a human genomic library. Ru. Examples of such libraries useful in this embodiment include Lawn et al., Ce1l. 15:1157-1174 (1978), which is hereby incorporated by reference. This will be incorporated in detail.

In a preferred form of this embodiment, the native DNA sequence is subjected to a method comprising: Therefore, it is intended to be obtained.

(a) Vectors or vectors capable of amplifying and expressing all or part of cDNA. and human cells that produce IL-1 inhibitors intracellularly, preferably monocytes. Preparation of DNA library: (b) Binding to the TL-1 inhibitor gene or its protein product Searching a human DNA library using at least one probe capable of C) The clone contains at least one gene related to the inhibitor gene or its protein product. Genes encoding inhibitors based on their ability to bind to one type of probe identification of at least one clone containing; (d) the selected one or more clones; the gene encoding the inhibitor or a portion of the gene from a clone of Isolation: (e) inserting the gene or a suitable fragment of the gene into a host cell. It is coupled to the operating elements necessary to maintain it within and express it.

Naturally occurring DNA sequences useful in the methods described above can also be prepared by a method comprising: can be determined and isolated: (a) Preferably recArecBc E, human amplified in a coli host Preparation of genomic DNA library; (b) IL-1 inhibitor gene or human genome using at least one probe capable of binding to the protein product. (C) If the clone is an inhibitor gene or its based on the ability to bind at least one probe for the protein product. Identification of at least one clone containing the gene encoding the inhibitor: ( d) inhibitor-encoding genes from the identified clone or clones; Isolation of the gene; (e) the gene or a suitable fragment of the gene is isolated from the gene; maintain the offspring within the host cell and attach it to the necessary operating elements to express it .

Isolation of native DNA sequences suitable for use in the methods described above involves the isolation of a suitable gene or two restrictions within and closest to the end of the section of the gene Preferably, the site is identified. Next, using an appropriate restriction endonuclease, A DNA segment containing the appropriate gene is excised from the remaining genomic material. Cut After extraction, reconstruct the 3° and 5′ ends of the DNA sequence and any exon joins. can encode the N- and C-terminus of the IL-1 inhibitor protein, and Fuse that DNA sequence to the operating element to provide the appropriate DNA sequence. do.

3. Vector (a) Microorganisms, especially E. coli Vectors intended for use in the present invention may include any preferred or necessary manipulations. Any vector into which the above DNA sequences can be inserted together with the elements, The vector can then be transferred into a host cell and replicated within that cell. Vectors that can be used are included.

Preferred vectors are those whose restriction sites are well-documented and whose restriction sites are well suited for transcription of the DNA sequence. A vector containing operational elements preferred or necessary for transcription. but However, certain embodiments of the invention provide that one of the cDNA sequences described herein It is also assumed that currently undiscovered vectors may be used that may contain ing.

In particular, all of these vectors may have some or all of the following characteristics: preferable. (1) possessing the minimum number of host organism sequences; (2) the desired host; (3) be present in a large number of copies in the desired host; (4) a regulatable protein positioned to promote transcription of the gene of interest; (a part of the plasmid where the 51 DNA sequence is inserted and another part of the plasmid) at least one marker DNA encoding a selectable feature present on the portion and (6) a DNA sequence capable of terminating transcription.

In various preferred embodiments, a DNA sequence of the present invention is contained and expressed. These cloning vectors contain various operational elements. child These "operating elements" include at least one pr romator, at least one Shine-Dalgarno sequence and a start codon, and and at least one stop codon. Preferably, these “operating elements” "ment" also includes at least one operator, a tank exported from the intracellular space. at least one leader sequence for proteins; at least one leader sequence for regulatory proteins; for proper transcription and subsequent translation of one gene and the vector DNA. Any other DNA sequences necessary or preferred are also included.

Certain of these operational elements are present in each of the preferred vectors of the invention. can. using methods known to those skilled in the art, especially in light of the teachings herein. , adding any additional operational elements that may be needed to these vectors. It is also intended.

In fact, these vectors can be easily isolated, assembled and interchanged in a way that It is possible to construct each of the following. These elements and It has become easy to assemble numerous functional genes from combinations of coding regions of DNA sequences. Ru. Furthermore, many of these elements will apply to more than one host. be In a preferred embodiment of the species, the vector has a regulator (“operator”) ”), and codes for regulator proteins. It is further intended to include other DNA sequences that can be used.

(i) Regulator In one embodiment, these regulators respond to the presence of certain environmental conditions. in the presence of DNA sequences and in the presence of other environmental conditions. enable transcription and subsequent expression of the protein encoded by the A sequence. cormorant. In particular, the regulatory segment is e.g. of isopropylthio-β-D-galactoside. In the absence, expression of the DNA sequence does not occur or occurs only to a very reduced extent. It is preferable to insert the vector into the vector in such a manner that it does not cause any damage. In this situation, D The transformed microorganism containing the NA sequence is grown to the desired density before the start of IL-1i expression. Can be cultivated. In this embodiment, the expression of the desired protein is expressed at the desired density. The microorganisms achieved are induced by adding to the environment.

(ii) Promoter Expression vectors can be used by host organisms for their own protein expression Must contain a promoter. The lactose promoter system is commonly used. Although other microbial promoters have been isolated and characterized, allows those skilled in the art to use them for the expression of recombinant IL-1i. It became.

(i) Transcription terminator Transcription terminators contemplated herein serve to stabilize the vector. Special Rosenberg, M., and Court, D., Ann. Rev, Genet, 13:319-353 (1979X reference here (incorporated as a reference) is intended for use in the present invention. It will be done.

(iv) Untranslated sequences In a preferred embodiment, 3" or 5° untranslated sequences are incorporated into the gene transcript. It is also possible to reconstruct the 3' or 5' end of the coding region to allow It is noteworthy that this is desirable. Included in these untranslated sequences are Schmeissner, tJ, McKen, incorporated by reference. ney, K., Rosenberg, M. and Court, D., J. Mo1. Biol, 176:39-53 (1984). This is a sequence that stabilizes mRNA.

(v) Liposome binding site Microbial expression of foreign proteins involves liposome binding sites, but Requires certain operating elements without limitation. The liposome binding site is G old, L, et al, Ann, I? ev, Microbio, 35:55 7-580 or Marquis, D. M. et al. Gene 42:175-1 83 (1986), liposomes are used in the initiation of protein synthesis. It is an array that recognizes and binds to the system. These documents are incorporated herein by reference. Ru. A preferred liposome binding site is GAGGCGCAAAAAA (ATG). Ru.

(vi) Leader sequence and translation coupler Additionally, if the protein or from the cytoplasm When excreted, the DNA encoding the appropriate secretory leader (signal) sequence is Nucleic Ac1ds Res by Atson, M.E.

12:5145-5163 (this article is incorporated herein by reference). ’) is preferably present at the 5° end of the DNA sequence. Lee The leader sequence DNA is such that the leader sequence is directly adjacent to and covalently bonded to the inhibitor. must be in a position that allows the production of a fusion protein that is There must be no transcription or translation termination signal between the two DNA coding sequences. do not have. The presence of a leader sequence is due in part to one or more of the following reasons: desired. First, the presence of a leader sequence inhibits host processing of IL-1i. It hangs easily. In particular, the leader sequence is the first translation product produced by the leader peptidase. It directs the cleavage of proteins, removes the leader sequence, and has beneficial protein activity. A polypeptide having an amino acid sequence that

Second, the presence of the leader sequence guides IL-1i out of the cell's cytoplasm. , which can facilitate purification of the protein. Several species of host microorganisms In some Escherichia coli, due to the presence of appropriate leader sequences, (E. coli), transporting the completed protein into the periplasmic space. It will make it possible to transport. Some species of E. coli, Satucharomyces (S. accharomyces) and Bacillus and In the case of Pseudomonas strains, appropriate leader sequences Transport of proteins across the cell membrane and into the extracellular medium would be possible. in this situation In this case, the protein can be purified from extracellular proteins. Thirdly, according to the present invention In the case of some proteins prepared by Folds the protein to assume its active structure where it has the appropriate protein activity. It may be necessary to place it in a possible environment.

In one preferred embodiment of the invention, additional NA sequences 11. It is located immediately before the DNA sequence encoding the -1 inhibitor. This additional The NA sequence can function as a translational coupler. That is, its DNA sequence is RN A, and its RNA is a contiguous liposome of inhibitor RNA. serves to position the liposome directly adjacent to the membrane binding site. The details of the present invention In one embodiment, the DNA sequence ACGAGGCGCAAAAAAT GAAAAAAGA CAGCTATCGCGAT CTTGGAG GATGA using methods currently known to those skilled in the art relating to TTAAATG and translation couplers. can induce translational couplers.

(vII) Translation terminator Translation terminators contemplated here serve to stop translation of mRNA . They are Kohli, J., Mol.

Gen, Genet, 182:430-439. or Pettersson, R.F., Gene 24:1 5-27 (1983); Both documents are incorporated herein by reference.

(vii) Selectable marker In addition, cloning vectors are selected by drug resistance markers or host microorganisms. Contains selectable markers such as other markers that result in expression of possible characteristics is preferable. In one embodiment of the invention, the ampicillin resistance gene is included in the vector, while other plasmids contain tetracycline resistance or Includes genes for chloramphenicol resistance.

Such drug resistance or other selectable markers may, in part, permit selection of transformants. intended to be easy. Additionally, such selection in cloning vectors The presence of selectable markers may be useful in preventing contaminating microorganisms from growing in the culture medium. It's possible. In this embodiment, a pure culture of the transformed host microorganism microorganisms under conditions such that the induced phenotype is necessary for survival. It can be obtained by culturing.

The operational elements as discussed here are based on prior literature and the teachings contained herein. is routinely selected by one skilled in the art in view of the indications. One of these operating elements A general example is B. Lewin, which is incorporated herein by reference.

Genes, Wiley & 5ons, New York (1983) It is described in. Various examples of suitable operational elements can be found in the vectors discussed above. Reference is made to publications that can be found and discuss the basic characteristics of said vectors. It will become clearer.

Once all necessary and desired components of the vector described above have been synthesized and isolated, Vectors are constructed by methods commonly known to those skilled in the art. vector The assembly of the This can be done without undue experimentation. For example, Maniati s, T.

et al., Mo1ecular Cloning: Co1d Spring Ha rbor Laboratories, N. Y. (1984). If similar DNA sequences are ligated into an appropriate lon- ing vector, This document is incorporated herein by reference.

In constructing the cloning vector of the present invention, the DNA sequence and its associated It is also worth noting that multiple copies of an operational element can be inserted into each vector. It is. In such embodiments, the host organism contains the desired IL-1 per vector. Let's produce more inhibitors. Multiple DNA sequences inserted into a vector The number of copies depends on whether the resulting vector is transferred into a suitable host cell due to its size. limited only by its ability to be copied, copied and transcribed.

(b) Other microorganisms B. Vectors suitable for use in microorganisms other than coli are also contemplated by the present invention. be done. Such vectors are listed in Table 1. Additionally, certain preferred vectors - will be discussed below.

(margin below essence) 5, Hallewell R, A, anci Entage, S, G ene 9.27-47 (1980).

11. Koshland, D+and Botstein, D, Ca1 l　20.749-760　(19801゜14, 5utcliffe, J. G, Proc, Natl, Acad, Sci, USA 75.373 7-3741 (197J3), -15, Pecian, LW, C, Gene 22.277 -280 (1983).

16, Alton, N-ni, and Vapnek, D, Natur e 2B2.864-869 (1979).

26- Yansura, D, G-and Henner, D, J, P NAS 81.439-443 (1984).

28, Lory, S, and Tai, P, C, Gene 22. 95-101 (1983).

29, Liu, P, V, J, Engineering, Dis, 130 (s upply, 594-599 L1974)-36, Watson, M, E , Nucleic Ac1d Re5earch 12, 5145-5164 (1984).

(margin below essence) (i) Pseudomonas vector Several vector plasmids that replicate autonomously in a wide range of Durham-negative bacteria are Preferred for use as a cloning vehicle in a Pseudomonas host.

Some of these are Talt, R, C, C1ose, T, J,.

Lundquist, R.C., Haqiya, M., Rodrigue z, R,L, and Kado, C,1,, In Biotechnolog y, May, 1983. pp.

269-275; Panopoulos, N. J., Genetic E. ngineeringin the Plant 5 sciences, Pra eger Publishers, New York, New York, pp, 163-185 (1981); and SakaguCki.

K., Current Topic in Microbiology an d Rmmunology 96:31-45 (1982), Each document is incorporated herein by reference.

One particularly preferred construction method is Bagdasarian.

M., Bagdasarian, M.M., Coleman, S. and and Timm1s.

K, N, Plasmids of Medical, Environme ntal andComn+ercial Importance, Timm 1s, K, N, and Puhler.

A, (ii>, Elsevier/North Ho1land Biomed ical Press (1979) (incorporated herein by reference). By using plasmid R3FIOIO and its derivatives, as shown in Probably. The advantage of R3FIOIO is that it is A relatively small copy that is easily transformed and stably maintained in both species. - It is a large number of plasmids. In this system, Escherichia It would be preferable to use the Tac expression system described in because For example, the B. coli trp promoter is Sakagucki, K., C. currentTopics in Microbiology and I+n o+unology 96:3l-45 (1982) and Gray, G.L. ,, McKeown, K.A., Jones.

A. J. S., Seeburg, P. H., and Heyneker, H. L.

Biotechnology, Feb. 1984. pp, 161-165 Pseudomonas as described in (both documents are incorporated herein by reference) This is because it is thought to be easily recognized by RNA polymerase. Transcriptional activity is The promoter can be used, for example, in ε, coli or Pseudomonas aeruginosa. (P. aeruginosa) requires replacement with the trp promoter. This can be further maximized by Furthermore, l:, 1a of coli The cl gene may also be included in the plasmid for regulation.

Translation initiation of any Pseudomonas protein and intracellular expression of inhibitors Initiation of any abundantly expressed protein of the type selected to elicit Translation can be attached to the site.

If a strain lacking restriction of the host Pseudomonas species is not available, a simple strain from E. coli can be obtained. Transformation efficiency with isolated plasmid constructs is low. Therefore, Bagdas arian, M. et al., Plasmids of Medical, Envi ronmental and commercial importance, pp, 411-422. Timms and Puhler (eds.), El sevier/North Ho1land Biomedical Pre ss (1979) (incorporated herein by reference). , transform the Pseudomonas cloning vector into another one before transformation of the desired host. It is desirable to pass it to two species r-m''.

(ii) Bacillus vector Additionally, preferred expression systems in Bacillus hosts include cloning vehicles. This includes the use of plasmid pUB110 as a. Other host-vector systems The TL-1i of the present invention can be administered intracellularly or as a secreted protein in Bacillus. It is possible to express it as either. Embodiments of the invention include both systems. be done.

A shuttle vector that replicates in both Bacillus and E. coli is Dub nau, D., Gryczan, T., Contente, S.

and Shivakumar, A.G., Genetic Engine ering.

Vol.2. Setlow and Ho1lander, Plenum Press.

New York, New York, IMp, 115-131 (19 Construction of various genes as described in 80X (incorporated herein by reference) and available for inspection. B, Expression and secretion of IL-1i from C. subtilis. In order to Preferably, they are combined. Mobile DNA sequences are required for intracellular inhibitor synthesis. Translationally linked to the liposome binding site of the alpha amylase leader sequence. cormorant.

Preferably, the promoter is directed by a enzyme promoter or a derivative thereof. This derivative contains the RNA polymerase recognition sequence of the native alpha-amylase promoter. However, it also incorporates a lac operator field. penicillinase gene pro Similar hybrid promoters built from motor and lac operators Yansura, D.G., which is hereby incorporated by reference in detail.

and Henner's Genetics and Biotechnology of Bacilli, Ganesan, A. T., and Hoch, J. A, (print), Academic Press, pp, 249-263 (19 It has been shown to function in the Bacillus host in a regulatable manner as described in 84). has been done. The E. coli 1acl gene is also included in the plasmid and is regulated. will do.

(i) Clostridium vector One of the preferred constructs for expression in Clostridium is incorporated herein by reference. J, Bacteriol.

159:460-464 (1984). transformed into C. perfringens by the method incorporated herein by reference. 5quires, C.H., et al., J.

Bacteriol, 159: 465-471 (1984). It is lasmid pJU12. Transcription occurs at the promoter of the tetracycline resistance gene Directed by. Translation is as described above for vectors suitable for use in other hosts. of this same tet gene in a manner closely analogous to the method outlined in Combined with Shine-Dalgarno sequence.

(iv) Yeast vector Maintenance of foreign DNA introduced into yeast is described by Botst, which is incorporated herein by reference. ein, D., and Davis, R.W.

The Mo1ecular Biology of the Yeast S accharomyces.

Co1d Spring Harbor Laboratory, 5trath Ern, Jonesand Broach (ed.), pp, 607-636. (1982). Satsukalomi One preferred expression system for use in host organisms of the genus Ses is the 2 micron plasmid. Contains the IL-1i gene on the card. The advantages of 2 micron circles include cir’ stocks This includes relatively high copy number and high stability when introduced into the system.

These vectors are pBR3 to enable replication and selection in E. coli. 2 microorganisms of origin of replication and at least one antibiotic resistance marker. lon sequence, and an enzyme that serves the same purpose in LEL12-deficient mutants of yeast. It would be preferable to have the mother LEL12 gene.

If the recombinant IL-1 inhibitor is intended to be ultimately expressed in yeast , the cloning vector is first introduced into E. coli, where the vector or clone is Preferably, the vector is then obtained and purified after propagation. Then the vector - will be transferred into yeast for final expression of the IL-1 inhibitor.

(c) Mammalian cells The cDNA for an IL-1 inhibitor can be used to detect the inhibitor in mammalian cells. It will serve as a gene for expression. It is incorporated here for reference. Kozak, Nucleic Ac1ds Re5earch 15:8 125-8132 (1987). It should have good sequence and the mature protein in its processed form. Ability to encode a leader sequence (see section 3(aXvi)) for leading out of the cell should have. DNA restriction fragments carrying the complete cDNA sequence are Copy Promoter and Guarente, L., incorporated herein by reference. Ce1l 52:303-305 (1988) and Kadonaga, J. , T. et al., Ce1l 51:1079-1090 (1987). It can be inserted into an expression vector having a hanger. If constitutive expression of the inhibitor If it is detrimental to cell growth, the promoter may be added to the plasmid pMSG. As in (Pharmacia Cat, No. 27450601) It can be adjusted. Vector is A, which is incorporated herein by reference. u5ubel, F, M, et al. Current Protocols in M o1ular Biology, Wiley (1987). This vector contains a complete polyadenylation signal, thereby allowing the Transcribed mRNA is properly processed. Finally, the vector is E, Cori The origin of replication from pBR322 to allow for replication and selection in and have at least one antibiotic resistance.

To select stable cell lines that produce IL-1 inhibitors, the expression vector - carry genes for selectable markers, such as drug resistance markers, or To transform the bhfr- cell line described by Ausubel et al., supra. Complementary inheritance of deletion cell lines such as the dihydrofolate reductase (dhfr) gene in Can carry a child. Another plasmid also carrying a selectable marker can also be transformed together with an expression vector.

4. Host cells/transformation The vector thus obtained is introduced into a suitable host cell. These host details The cells can be microbial or mammalian cells.

(a) Microorganisms It takes in foreign DNA and expresses these genes and associated operating elements. It is conceivable that any microorganism can be selected that has the ability to host organism selected The vector is then introduced into the host organism using methods commonly known to those skilled in the art.

Examples of such methods can be found in Advanced Bacte, which is incorporated herein by reference. real Genetics.

RoW, Davis et al., Cold Spring Harbor Pres. s, Cold Spring Harbor, New York (i98 0) can be found.

In one embodiment, temperature regulation is achieved through the use of the operating elements described above. Transformation can occur at low temperatures, as it is considered a means of regulating gene expression. is preferred. In another embodiment, if the osmotic agent is present in the vector. If inserted, regulating salt concentration during transformation will ensure proper control of the foreign gene. It will be necessary to make it a reality.

Preferably, the host microorganism is facultatively anaerobic or aerobic. In this method, Special hosts of particular use include yeasts and bacteria. For detailed yeast Included are yeasts of the genus Satucharomyces, especially Satucharomyces ceravisiae. detail Bacteria include bacteria of the genera Bacillus, Escherichia and Pseudomonas, especially Bacillus. Includes T. subtilis and E. coli. Other host cells are listed in Table 1 above. Are listed.

(b) Mammalian cells Vector is calcium phosphate: DNA co-precipitation method, electroporation , or mammals in culture by some techniques such as protoplast fusion Can be introduced into cells. A preferred method is described by Ausubel et al., supra. can be transformed by co-precipitation with calcium phosphate, allowing for transcription and transcription of cDNA sequences. processing of the precursor IL-1i and secretion of the mature protein. Many stable cell types exist. However, the glycosylation of secreted proteins Regarding zylation and post-translational modifications of amino acid residues, the details, if any, are Varies depending on cell type. Therefore, the ideal cell type is a recombinant I that is identical to the native molecule. This is the cell type that produces L-1 inhibitor.

5. Culture host cells of genetically engineered cells to produce IL-1 inhibitors. Cultivate under appropriate conditions. These conditions are generally specific for the host cell and Having regard to the published literature regarding growth conditions for such organisms and the teachings contained herein, can be easily determined by one skilled in the art. For example, Berg, which is incorporated here for reference. ey’s Manual of Determinative Bacteri ology.

8th Edition, Williams & Wilkins Company, Bal timore.

Maryland contains information regarding bacterial culture. yeast and mammalian cells Similar information regarding the cultivation of , Mammalian Ce1lCulture, Co1d Spring Habor Laboratories (1975).

Depending on whether any operational elements are inserted or present in the vector Any conditions necessary to regulate the expression of the DNA sequence are included in the transformation and culture steps. would be effective. In one embodiment, the agent inhibits expression of the DNA sequence. In the presence of appropriate regulatory conditions, cells grow to high densities. Close to optimal cell density If found, the environmental conditions are changed to conditions appropriate for expression of the DNA sequence. Therefore Therefore, production of IL-1 inhibitor occurs after host cell proliferation reaches near optimal density. IL-1 inhibitors that occur and arise during the time period are sometimes necessary for its expression. It is contemplated that the samples will be taken after the necessary regulatory conditions have been induced.

6. Precision made (a) IL-1i produced from microorganisms In a preferred embodiment of the present invention, Recombinant IL-1 inhibitor is purified after harvest and before it assumes the active conformation. . The inventors believe that if the protein is first purified, it will be refolded and the protein This embodiment is preferred because we believe it facilitates the recovery of high yields of . However, in one preferred alternative embodiment, the IL-1 inhibitor The tar can be refolded into its active conformation prior to purification. Still others In a preferred embodiment, when the IL-1 inhibitor is recovered from the culture medium, the IL-1 inhibitor is It exists in its folded active state again.

In certain situations, IL-1 inhibitors are expressed in the host microorganism and when the protein is transported through the cell wall or membrane or into the periplasmic space , will assume its proper active conformation. If D encodes a suitable leader sequence When NA is attached to DNA encoding a recombinant protein, this is commonly will happen. If an IL-1 inhibitor does not adopt its proper active conformation, If not, any disulfide bonds formed and/or any non-covalent bonds generated The interaction is first destroyed with suitable denaturing and reducing agents, then diluted and controlled by these agents. The rL-1 inhibitor assumes its active conformation following oxidation under controlled conditions. You can do it.

Use some combination of the following steps for purification before and after refolding: It is preferable. That is, anion exchange chromatography (Mono Q or DEAE-Sepharose), gel filtration chromatography (Superose) ), isoelectric focusing chromatography (Mono P), and hydrophobic interaction chromatography Matography (octyl or phenyl Sepharose). especially valuable Among these, IL-1i-specific monoclonal antibodies (described in Example 3) Antibody affinity chromatography using

(b) IL-1 produced from mammalian cells I produced from mammalian cells L-1i was obtained by ion exchange chromatography and monoclonal analysis as described in Example 3. by methods involving immunoaffinity chromatography using native antibodies. It will be purified from culture medium.

It will be apparent to those skilled in the art that various modifications and changes can be made to the methods and products of the invention. It will be clear. Accordingly, the invention resides in the claims appended hereto and their equivalents. It is intended to cover modifications and variations of the present invention insofar as they come within the scope of the invention.

Applying the teachings of the present invention to a particular problem or environment may be difficult to apply without reference to the teachings contained herein. It is to be understood that this would be within the ability of those skilled in the art. present invention Examples of products and representative methods for their isolation and production are shown below. .

The following examples illustrate various presently preferred embodiments of the invention. In this example The publications provided in are incorporated by reference.

Example Example 1 Protein preparation A. Material Hanks Balanced Salt Solution (HBSS) and RPMI from Mediatech.

Purchased from D.C., Washington. Lymphoprep is Ac curateChemical and 5cientific Corp. Obtained from Westbury, N.Y. Human IgG, MTT, rabbit anti Prostaglandin E2 antiserum, ammonium bicarbonate, dithiothreitol, complete Complete and incomplete Freund's adjuvant, hyboxanthin, aminopterin and and thymidine from Sigma Chemical Co., Ltd., St. Louis, Purchased from Missouri.

C3H/) IeJ vus is Jackson Labs, Bar Harbo r, purchased from Maine. BALB/c mice and P3 myeloma cells is the National Jewish Center for Irnmunol. ogy and Respiratory Medicine (NJC/IRM ), Denver, Colorado.

John Kappler and Ph1lippa Marrack Obtained from. Recombinant human IL-1 is produced by C15tron Biotechnology , PineBrook, N.J. Purified phytohemagglutini Wellcome Diagnostics, Re5earch Tri Purchased from anglePark, N.C. Primary cultured human foreskin fibroblasts Dr. Richa from NJC/IRM, Denver, Colorado Obtained from rdClark. Monoclonal mouse anti-rabbit IgG antibody is AIA Purchased from reagents, Aurora, Colorado.

Low concentration methionine RPMI is available from GIBCO Laboratories, Gran Use the Select-Amine kit from dIsland, N, Y. It was. 353-methionine, diphenyloxazole and [”C]-iodo vinegar Acid was obtained from DuPont-NEN, Chicago, llinois . Fetal bovine serum is from HyClone Laboratories.

Purchased from Logan, Lltah. Mono Q and 5uperose Column 12 is from Pharmacja Inc., Piscataway, N. Purchased from J. The C4 reverse phase column was manufactured by Synchrom, Inc., L afayette.

Obtained from Indiana. C8 reverse phase column is Applied Biosystem ms.

Inc., Faster C1ty, California. Aceto Nitrile and polyethylene glycol 8000 from J, T, BakerChe purchased from Mical Co., Phillipsburg, N.J.

Triple O-acetic acid and guanidine hydrochloride from Pierce Chemicals; Obtained from Rockford, 11linois. endobrotinase L ysC is Boehringer Mannheim Biochemicals .

Obtained from Indianapolis, Indiana. PGE2 ELI The microtiter plate used for SA was Intermountain Sci. entific Corporation, Bountiful, Utah It was Nunc-Immuno Plate I obtained from Nunc-Immuno Plate I. Hybridoma product The plates used for raw materials were Co5tar, Cambridge, and Massac. Obtained from hu-setts.

B, Production of monocyte IL-1 inhibitor. Human leukocytes were obtained from normal blood donors by leukophoresis, and were collected in Hanks' balanced salt solution. Suspend at a ratio of 1 part of HBSS to 1 part of cells packed in (HBSS), and Mphoprep was placed underneath and centrifuged at 400×g for 30 minutes at room temperature.

The mononuclear cell fraction is typically collected from a blood donation of 4-5 x 10” cells. (obtained per person), washed in Ca" and Mg-free HBSS, and bloodless. Suspended in clear RPMI and chromatographed on 5ephapex G200. Normal human IgG from which LPS had been removed was applied to a Petri dish. (1 (6X1O' cells in 7) per 100 mm dish. All reagents contain 10 LPS 1] g/mj or less. Cells were cultured for 24-48 hours and the resulting habituation was The culture medium constituted the crude IL-1 inhibitor (IL-1i) supernatant. Typically, , cells from one blood donor yielded 700-900 yd of crude IL-1i supernatant. .

C, IL-1 inhibitor assay Two IL-1 assays are routinely used to detect IL-1i. 3 days 3H-thymidine incorporation or tetrazolium salt MTT incorporation after stimulation for (Mosmann, T., J. rmmunol, Me. thod, 65:55-61 (1983)) thymocytes (C3H/ lXl0' cells from HeJv mice) were injected with 1.0 units of recombinant human IL-1. /−+1 μg/− of phytohemagglutinin with half-maximal proliferation. Crude I L-1j supernatant completely inhibits this proliferative response at a 1/10 dilution. human skin fibers Blast cells (txio' cells per well in a 96-well plate) are typically For 6 hours of stimulation with 0.5 units/rILl of recombinant human IL-1, E Approximately 50. secretes OOOpg/rd PGE2 respond. This assay is as sensitive to IL-1i as the thymocyte assay. Good.

D. Metabolic labeling of the IL-1 inhibitor IL-1i in an IgG-coated preform 0.75 u g/mj of cold methionine (usually 15 μg /rnI and 0.5 mC1”S-methionine (115 Mononuclear leukocytes were cultured for 48 hours in serum-free RPMI supplemented with 1 Ci/mmol). Metabolic labeling was performed by The plates are coated with fetal bovine serum rather than IgG. Control labeling was performed otherwise identically. cells coated with fetal bovine serum In assays for such control supernatants, most rL-1 It was shown that it does not secrete i.

(margin below essence) E. Purification of IL-1 inhibitor protein. Crude IL-1i superior groove was purified with sodium chloride. Make 1.0 M in microorganisms, incubate on ice for 1 hour, and at 10.00 rpm. Centrifuged for 15 minutes. Next, the first protein that contains all the inhibitor activity The upper groove, which is only 20% of the protein, was analyzed using a Mono Q anion exchange column. 0,025M solution containing 0.1% sucrose to perform the gradient fractionation. Long dialysis was performed at 4° C. against Lis, pH 7.6 (Buffer A). Inhibition following dialysis 10. Centrifuge again for 15 minutes at OOOrpm, then 0.2 It was passed through a 2μ nylon filter. The superior groove was metabolically labeled and prepared in the same way. Usually with 10 rnl of supernatant in a bed volume of 1.0 rnl or 8.0 rnl. A Mono Q-3uperose (Pharmacia FPLC) column and ODt* of the effluent. Wash with buffer A until the A linear sodium chloride gradient (0,025-0,10M) in buffer A was used. Chromatography was performed carefully using Collect column fractions and collect radioactivity and analyzed for biological activity. In addition, the sample of each fraction is reduced type 12゜5. %5DS-PAGE, silver staining, diphenyloxazole infiltration, Autoradiogram data was obtained by drying and placing on film. Figure 1a 40-ml of crude TL-1i supernatant mixed with 3 ml each of metabolically labeled IL-1i supernatant. The protein profile of MonoQ chromatography is shown. Overlapping Each fraction is 50μ! Most of the radioactivity and PGE2 production found in Figure 3: Biological activity of IL-1i measured in the assay. Three peaks of biological activity and Two large and one small radioactive species are shown to be perfectly correlated. . Figure 1b shows a plate coated with fetal bovine serum (Fe2) instead of IgG. A similar chromatogram of 15 mN' of crude IL-1i mixed with 3 ml of purified monocytes was prepared. Figure 2 shows the topography. The radioactivity levels of the three species mentioned above have been significantly reduced. Second Figure a is the region of interest in the chromatography shown in Figures 1a and lb. A silver-stained gel was performed on the fractions. Figure 1a (fraction 5 The peak radioactivity and bioactive fractions in 2 and 59) are both 5DS - It should be noted that PAGE shows a large band (indicated by an arrow) at 22Kd. be. The third species (fraction 48 in Figure 1a) was analyzed at 20K on 5O3-PAGE. D indicates one band. Gel filtration experiments of crude IL-1i revealed that the active molecule was 18- It is shown to have a molecular weight of 25 Kd. Figure 2b shows the game shown in Figure 2a. This is an autoradiogram of Le. The protein bands at 20 and 22 Kd are It is easily seen that this is the major radioactive species in these fractions.

To summarize these results, the metabolism of table tennis applied to a Petri dish coated with IgG The labeling generates a radioactive species, which if the cells are plated on an FC3-coated vetri dish. We have shown that this occurs only slightly when These inductions The radioactive species obtained were completed with MonoQ along with some species of IL-1i bioactivity. All chromatography runs simultaneously and the gel and the resulting autoradiolog Lamb is a protein whose three main molecules have the predicted molecular weight of rL-1i. It shows that there is.

The IL-1i molecule was further purified for sequencing in two ways. First of all Transfer the biologically active and radioactive Mono Q fraction to a C4 reverse phase column. Load, H, Olo, 1% TFA Niacetonitrile 10.1% TFA gradient It was eluted at the eluting agent. Since the IL-1i molecule was only trace-labeled, each flux Samples from the section were directly counted for radioactivity and analyzed by 5DS-PAGE before autoclaving. I did traradiography. Figure 3a shows such a graph with radioactivity patterns superimposed on it. Showing matography. A silver-stained gel ( Figure 3b) and the subsequent gel autoradiogram (Figure 3c) show that IL- 1i molecules are found in fractions 32-36. These frames The tract was dried and sequenced. Alternatively, the peak MonoQ 0.4-0.05 of 0.4-0.05 Resuspend in M NH4HCO3 and as shown in Figures 4a and 4b. IOX 300mm 5uperose 12 gel filtration column equilibrated with the same buffer The sample was directly chromatographed twice on a Pharmacia FPLC. Hula Each sample was tested for radioactivity and biological activity and analyzed by silver staining. Then, 5DS-PAGE was autoradiographed. Next, select an appropriate fraction. The samples were dried in a speed vac and sequenced.

Example 2 Proposed Sequencing of IL-1 Inhibitors Samples were treated with 6M guar prior to sequencing. Nigin-MCI, dissolved in pH 8.6, 10% against protein under N2 Reduction with a 0-fold molar excess of dithiothreitol at 37°C for 4 hours resulted in a 400-fold molar excess of dithiothreitol. Alkylated with +4c iodoacetic acid for 1 hour.

In that case, the reaction may be desalted on a C8 reverse phase column, eluted and partially dried. cormorant. N-terminal sequence is Applied Biosystems Protein 5e quencer.

To obtain the internal sequence, the reduced and alkylated sample is prepared using methods known in the art. may be digested with cyanogen bromide or proteolytic enzymes. The reactants are dried; Dissolved in 0.1% TPA/H20, and the peptides were separated using a C8 reverse phase column. Let's be separated.

Example 3 Purification and sequencing of IL-1 inhibitors A, IL-1i-X, I ManoQ purification of L-1i-a and IL-1i-b species IL-1i The biological activity or the three main It is broken down into different species. As shown in Figure 2a, samples of these fractions 20KD', 22KD, and 22KD by 5DS-PAGE, respectively. Related species are indicated. using the mouse antiserum described in Example 4 below. Western analysis of such gels stains all three species. coated with IgG Cells that were metabolically labeled with 3fiS-methionine while growing on plates If IL-1i was prepared from (shown in Figure 2b in the autoradiogram). Based on the logic discussed in Example 1 i.e., parallel cells incubated under non-inducing conditions exhibit IL-1i biological activity. We believe that these three species do not produce these radioactive bands. It can be concluded that this is the cause of bioactivity. we have these seeds Tentatively named IL-1i-X, IL-1i-a, and IL-1i-b, respectively. Let's go.

B. Purification and sequencing of IL-1i-X and/or IL-1i-X RP the MonoQ fraction containing -1i-a. Further purification by reverse phase HPLC chromatography on a -4 (C4) column, The radioactive species were then used for sequence analysis. RP-HPLC purified IL-1i-a and Numerous attempts to directly sequence IL-1i-b have failed; indicates that their N-termini are chemically blocked. However, I The following sequence from one preparation of L-11-a (IL-11-aB2p42): was obtained and then similarly purified by C4RP-HPLC. Preparations of X also produced the same sequence.

PrepKxF23 RP --, RM --! , K M Q A F -I These clearly correspond to the sequences found in the first attempts to sequence IL-1i-a. is part of a column. The sequence data shown is for 0 out of 20 species called IL-1i-X. The inventors' conclusion is that it is the N-terminus.

In these and all subsequent sequences, the underlined positions are where residues can be identified. Indicates that there is no or that there is ambiguity regarding the groups identified. 2 toes or more residues are placed in one position, this means that one or more indicates that the amino acid was detected at its sequencing step, and is more likely to be correct. Possible residues are at the top.

C. Production, purification, and sequence of IL-1i-a and IL-1i-b peptides. decision IL-1i-a and IL-1i-b are apparently chemically blocked at their N-termini. Each peptide was generated by endobrotinase digestion. In detail is a MonoQ fraction containing IL-1i-a or IL-1i-b. The 04 column used in all previous experiments is an acceptable substitute for the 4,6 column. X250mm C3-RP HPLC column (Zorbax Protein P lus). Very gradual gradient (0.5 yd/min) 0.2% acetonitrile) to remove the major radioactive species, human lysozyme. Separate IL-11-a (Figures 8a and b) or IL-1-b (Figure 9a). Ta. The identity of the purified species was determined by 5DS-PAGE and subsequent autoradiography. The presence of a single, radioactive, 22KD protein in Confirmed by. Collect proteins by hand into siliconized glass tubes and 25 g of 0.2% Tween-20 solution was added to each. Next, IL-1 The volume of the i-containing fraction was reduced to 50 yd by speedback, and the volume was reduced to 1%. 300 i by addition of NH, HCO, then 1 µ of endoproteinase. was added. In the case of IL-1i-a, the enzyme used was Endoproteina se LysC (Boehringer-Mannheim), while IL -1i-b is Endoproteinase AspN (Boehringer -Mannhe im). Cutting was carried out at 37°C for 16 hours, then The volume of the reaction mixture was reduced by 50 mJ i by speedback.

In the case of IL-1i-a, the sample was directly chromatographed, whereas in the case of IL-1i -b sample was initially 5ml of 50mM dithiothreitol in 2M Tris, pH 8.0. Reduction by addition of 1.0% in ethanol and reaction at 37°C for 30 minutes, followed by addition of 1.0% in ethanol. Carboxymethylated by addition of 1 μmol 3H-iodoacetic acid (37 React for 30 min at °C). Peptide separation is performed using micropore equipment and micropores. Using a Beckman HPLC with an arch-compatible pump, 2.1 x 250+nm Brownlee Aquapore R at a flow rate of It was performed using a P-300 (C8) narrow bore column. 200 minutes o -1oo% direct A linear gradient was used (H, Olo, 1% TFA to acetonitrile 10 ．． up to 1% TFA). The separation of the peptides is shown in Figures 10 and 11. The obtained distribution The column information is as follows.

(margin below essence) RaLysC-1'LM Q A F -: -D V N Q KRaLys C-Col' FYFQEDI S S 10 i s　　　　　　20RaLysC-35---Eτ1LQLEAV-ITDLIE NL S 10 L! S 20 25R JAipN-41D E G V M V T K F Y r QRJJup M-250K IL r A F I R (margin below essence) Two of the peptide sequences were clearly related to those previously obtained from IL-1i-X. There is. One of these, RaLysC-41, is likely an IL-1i-a sequence. The other RbAspN-51 has an IL-1i-b sequence, which indicates that If the three types of IL-1i are chemically and/or chemically linked to a single original IL-1i molecule, If it is not a physically modified form, at least it indicates that it is a closely related protein. are doing. Combining the listed arrays yields the following composite array: IF SC IKi S INQA rat S 0VNQ E? FYLIIIQLV &1lLQG y*vsLtcgt tI-’GJ (margin below essence) These composite sequences are from the most recently updated Protein Identification Resource Database (P rotein Identification Resource Databa There are no other known polypeptides listed in seXPIR16,0). It seems unlikely.

We believe that these sequences, or minor variants thereof, may be used as IL-1 inhibitors. This is considered to be a type of molecule that can act as a molecule.

Example 4 Preparation of TL-1 inhibitor monospecific antibodies The supernatant was partially purified (400 times) by MonoQ-chromatography, and PB Subcutaneous injection of IL-1i dialyzed with S and emulsified in complete Freund's adjuvant. did. Each mouse has 5th crude supernatant! were given IL-1i purified from. child These mice received 2 doses of the same amount of IL-1i emulsified in incomplete Freund's adjuvant. Weekly boosts were given, and serum samples were taken from the tail 7 days after each boost.

The antiserum was analyzed on immunogen wafers by 5DS-PAGE as shown in Figure 5a. Anti-IL-1i activity was tested by Stan Trans plot analysis. Fifth b. All mice produced anti-IL-1i antibodies after three injections of IL-1i. It shows what you did.

Monoclonal antibodies were created by cloning the IL-1i gene from an expression library. , highly useful for the purification of recombinant IL-1i protein and the study of its molecular biology. Because of its value, we will produce a panel of monoclonal antibodies specific for IL-1i. started. To generate B-cell hybridomas, the mice are placed in saline. The same amount of IL-1i was injected intravenously, and the spleen was removed 24 hours later. spleen cells Loosen the spleen into cold balanced salt solution (BSS), wash twice with BSS, and remove the spleen. P3 myeloma cells at a ratio of 2x10' P3 myeloma cells per 10' visceral B cells and centrifuged. Warm, gas addition to dry pellets (5 %C02) PEG 6000 (40% polyethylene glycol 6000: Cells were fused by dropping IrILl (60% minimum essential medium) . The fused cells were washed with BSS and rich medium containing 2X10'/mj' of peritoneal cells. (10% FBS) and using a ]0rnl pipette. The pellet was gently crushed. Increase the volume by adding more peritoneal cells in the medium. The volume was adjusted to 20 rnl, and the cells were plated in 96-well plates at 0.0 rnl. Irn1/u It was applied with Elle. Place the plate in a gas incubator and then proceed as follows. Processed by formula.

Day 1 - 3X HAT (hypoxanthine, aminopterin, thymidine) in rich medium ) to the final concentration x 1, Day 5 - Medium conversion by replacing 200 μl of IXHAT in rich medium; Day 10 - Start of testing for hybrid growth. Abdominal lung cells 1.5X10@/ By replacing 200 μm of IXHAT in a rich medium containing yd, exchange.

Once the hybrid cells are almost confluent in the wells, examine the supernatant. IX in rich medium by gently scraping the cells with a pipette tip. HAT plus peritoneal cells 3X10'/mA! Transfer to a lit’ culture well containing did.

Supernatants from confluent wells were partially purified IL-1i (same as injected into mice, ELISA in which Mono Q-purified substance) was bound to microtiter wells. to test for anti-IL-1i activity. Normal mouse serum and hyperimmune anti- Sera are used as negative and positive controls, respectively.

Positive supernatants were analyzed by ELISA and on plates coated with homogeneously purified IL-1i. This will be retested by immunoprecipitation of purified and metabolically labeled IL-1i. Next Positive cells were cloned by limiting dilution and injected into Bristane-treated mice. and produce ascites. For tissue culture or bulk production and collection of mouse ascites fluid A larger amount of IL-1i specific bodies can be produced. Purification of these antibodies and their Attachment to insoluble beads provides an aperture for recombinant IL-1i protein purification. A affinity adsorbent will be produced.

Example 5 Cloning of IL-1i cDNA Apply to one IgG-coated plate and incubate for 24 hours in the presence of [”S]-methionine. The cultured monocytes can be identified by MonoQ chromatography [”S]- It was shown to produce IL-1i.

To determine when L-1i is generated at the maximum rate during a 24-hour period, The coated monocytes were exposed to [35Sl-methionine (pulse) for a brief, 2 hour period. Once cooled, add a large excess of unlabeled methionine and incubate for an additional 2 hours. Ta. The medium was then collected and analyzed for radiolabeled IL-1i. This operation Applications were applied to monocytes at various times after application to the IgG-coated plates and applied at application 15. Exposure of monocytes to ["S]-methionine after hours produces the maximum amount of ["5l-IL- It was found that IL-1i is produced in monocytes, which means that IL-1i mRNA in monocytes is It is shown that the maximum level was reached 15 hours after application to G.

Fresh monocytes were then infused with LPS-free 1gG obtained as in Example IB. I clothed it. After incubation for 15 hours at 37°C in RPM l medium, cells were Wash with saline, then 4M guanicinium thiocyanate); 25mM citric acid. sodium acid pH 7, 0.5% sarcosyl, 0.1M 2-mercaptoethanol It was dissolved in Next, total RNA was divided into P, Chomc-zynski and N, 5 acchi's Analytical Biochemistry.

162:156-159 (1987) from this lysate by the AGPC method described in isolated.

PolyA'' RNA was converted to Aviv, H., and Leder, P. (1972) P. roc.

Natl. Acad. Sci. (LISA) 69:1408-1412. Oligo dT cellulose isolated by chromatography and ethanol It was precipitated and dissolved to a concentration of 0.36 μg/μl. Gubler, Ll , and Hoffman, B. J. (1983) Gene 25:263-16 cDNA using 1 microgram for polyA'' RNA according to the method described in 9. A was prepared.

This cDNA was obtained from Boehringer Mannheim Catalog No. 988. From 448 or New England Bio Lab No. 1070 lambda gtl1 expression using the EcoRI linker and according to the manufacturer's instructions. included in the library.

The resulting library containing 10 independent clones was divided into R, A, Y ung and R.W., Davis [(1983) PNAS 80:] 194 -1198] using the screening conditions described by IL-1198]. Using an appropriate polyclonal antibody against E. coli Y1090r k- (Promega Biotec). Positive signals are Bayer, Methods by E., A., and Wilchek, M. (1979) in Biochemical Analysis, and Guesdon, J.L., Ternynch, T. and Avrameas, S. (19 79) by J. Histochem, Cytochem.

27:1131-1138 and according to the manufacturer's instructions. biotinylated second antibody (e.g. goat anti-mouse IgG, Bethesda Re5search Labs) followed by streptavidin-alkaline phosph Detected using a phatase conjugate (Bethrsda Research Labs). Let's be served.

Example 6 Preparation and Sequencing of the Gene Encoding IL-1i As described in Example 5. The prepared cDNA was incorporated into the cloning vector lambda GTIO. this The cDNA was purified using EcoRI methionine using S-adenosyl-methionine as a substrate. The enzyme is first methylated, an EcoRI linker is attached by a ligation reaction, and Excess linker was removed by digestion with εcoRI endonuclease and CL6B spin. It was removed by column chromatography. linked by a linker and selected by molecular weight. 0.124 μg of the selected cDNA and cleaved with EcoRI and treated with phosphatase. The ligation reaction was performed with a reaction containing 1 μg of purified lambda GTIO, and this The product of the ligation reaction was converted into GIGAPACK GOLD packaging extract (Str Atagene). As a result, the lX10' member's la library was obtained.

20 Proteins presented in Example 3 for selection of GTIO library and synthesize oligonucleotide (antisense) probes based on the peptide sequence. accomplished. The sequences of the probes and their corresponding peptide sequences are as follows: Ru.

h-San-E-11-4 Ding stone ＾ in λτ included ＾ in ACT τC ττC ding 5 ILye Pha Tyr Pha Gin Glu ~ A f’reba [ uLl-5τ included CCλNTGNτ Teite included, A included λ 5° Mat Van k LF Pha Tp PhaProlxlXLlil-6Cτ 1C NττAG, 7-cho: TG5’$ Van Asn GLn L ys Thrp-r-oba reference Iuil-7τT’GTτTτTding GW entry included Entry 5'CC Input an　　　Gin　　　Lys　　Thr　　n＊　　　Ty rNote:　　N　-A, G, C, axv! r T probe #IL1i1-3 is 32P-phosphorylated at its 5′ end and 3X 10′ of the library. used to select plaques. This probe can replicate in 3 plaques and one of those plaques was probe #4L1i. 1-4 was also shown to hybridize. This plaque GTIO-ILI 1-2A and Lambdasorb (Pro DNA was isolated using GTIO-ILli-2A is Amer icanType Culture Co11ection (ATCC), Ro ckville, Maryland under accession number 40488. Ru. This DNA was digested with EcoRI, divided into 5 equal parts, and run on a 1% agarose gel. electrophoresed.

After electrophoresis, the gel was stained with ethidium bromide. Photo of this gel Shown in Figure 12a. Lanes 6, 8.10.12 and 14 are from EcoRI digestion. Contains five equal parts of. Lane 5 is HindIII, which is useful as a molecular weight marker. Cut wild-type lambda DNA and HaeI [[cut φX174RF DNA (New England Biolabs)' mixture. Figure 12a shows G Tlo-ILli-2A contains an EcoRI fragment with a length of 1850 base pairs. It shows that it has.

This 1850 bp fragment carries the coding sequence for the ILI inhibitor. In order to demonstrate this more conclusively, a Southern bloat test was carried out as follows.

The DNA fragments in the gel shown in Figure 12a are nitrotransferred using standard methods. Plotted on cellulose. This nitrocellulose is then coated with each strip. 6.8° 5 strips containing DNA from 10, 12 and 14. It was cut lengthwise into strips. These strips were then attached with ``''PP-phosphorus at the 5°-end. hybridize to each of the five identified oligonucleotide probes (described above). I made it zoom. The oligonucleotide concentration was 1 pmol/ml and the hybridization The oxidation temperatures were as follows.

6 #IL1i1-3 35℃8 #IL1il-4 42℃ 10 #IL1i1-5 42℃12 #IL1i1-6 40℃14 #IL1i1-7 After washing at 35℃, arrange the strips The original nitrocellulose sheet was regenerated by pricking it with tape. Autoradiography was performed for 24 hours at -70°C in the presence of clean. 12b The figure is a photograph of this autograph. This will cause all probes to be 1850b Evidence is provided that hybridizes specifically to the p fragment, indicating that this Demonstration that the fragment carries the substantial coding sequence of the rL1 inhibitor are doing.

To determine its DNA sequence, GTIO-ILli-2A DNA was Digested with RI, electrophoresed on a 1% agarose gel and generated a 1850 bp fragment. isolated. This fragment was ligated with EcoRI-digested M13 mp19, The cells were transformed into E. coli strain JM 109. Transformants were transformed into β-galactin Selection was made by searching for those lacking tosidase activity. It takes 5 kinds Transformants were isolated, -heavy chain DNA was prepared, and according to the method of Sanger et al. The sequence was determined. The DNA sequences of the three transformants correspond to the 3° end of mRNA. However, two transformants provided protein coding sequences. Figure 13 shows the CD The DNA sequence obtained for the protein coding region of NA is shown.

Figure 13 also shows the predicted amino acid sequence. From the first amino acid alanine to the up to the 29th amino acid proline and from the 79th amino acid isoleucine The amino acid sequence up to the end is the hypothetical amino acid sequence. 30th amino acid pro The predicted amino acid sequence from phosphorus to the 78th amino acid proline is as described in Example 3. Matches the peptide sequence.

Example 7 Sequencing of GTIO-IL-I l-2A and IL-1i GTIO-IL1i The -2A portion was sequenced and shown in FIG. This DNA is specific to IL-1i. encodes a protein containing a characteristic amino acid sequence (nucleotides 99-5 57).

However, some modifications occur before this protein is secreted into the extracellular environment. It is thought that this will be done.

These modifications are essential for the protein to have TL-1i activity. It may or may not.

GTlo-ILli-2A is present at the amino terminus of the form of IL-1i known as encodes at least 32 amino acids (nucleotides 3-98) that are N-terminal to do. Among these 32 amino acids are those encoded by nucleotides 24-26. begins with M, which directs the nascent IL-1i toward the extracellular environment, and then the leader – a secretory leader that is removed by peptidases and possibly other peptidases - Arrays are considered to be included. This arrangement can be used for IL-1i alpha and base. The N-terminus of these forms is It is thought that it is close to that of feeling. Removal of the secretory leader sequence probably This may be necessary for the protein to have effective IL-1i activity.

Nucleotides 349-351 of GTIO-ILI 1-2A are consensus N- Encodes an N-residue that is part of the glycosylation site. They are N-Glycana The alpha and beta forms of IL-1i are grouped based on their ease of digestion by It is thought to be lycodylated. It is unlikely that the X form would be easily digested by this enzyme. Since the information provided here has not yet been established, the information provided here can be used to There remains a possibility that can be easily demonstrated by those with the skills, but it is It is thought that it will not be standardized. Glycosylation at 20N residues is present in the protein. It is thought that it is not necessary to exhibit effective IL-1i activity.

Nucleotides 99-101 of GTlo-ILli-2A are P (see Figure 15) However, P is not detected at this position ('N-terminus) in the X form of IL-1i. Not yet. It is possible that this residue is modified in the mature protein. Second Modification of residues is not believed to be a prerequisite for effective IL-1i activity.

The currently unknown N-terminal residues of the alpha and beta forms can be determined by Edman degradation. is not fully detected and is coded by GTIO-ILI 1-2A. The modification is likely to occur following removal of some of the N-terminal residues of the protein. child It is believed that modification of is not a necessary requirement for effective IL-1i activity.

Example 8 Expression of the gene encoding IL-1i in animal cells Expression of IL-1i in animal cells Currently, the following steps are required.

Construction of a2 expression vector; b. Selection of host cell line; Introduction of the expression vector into C0 host cells; d. Increase the expression level of IL-1i for the manipulation of recombinant host cells.

1. IL-1l expression vector designed for use in animal cells is a powerful construct expression constructs, inducible gene constructs, and expression in specific cell types. They can be of several types, including those designed to in all cases Promoters and other gene regulatory regions such as enhancers (inducible or not) and polyadenylation signals in the plasmid vector. placed in the appropriate position with respect to the cDNA sequence. Two examples of such preparations are: It is.

(1) Constructions using strong constitutive promoter regions are included here as a reference. Gorman et al.'s Mol incorporated.

eel, Biol, 2+1044-1051. Plas listed in 1982 Sumido pSV2 Simian virus in an arrangement like that found in CAT SV40 (SV40) gene control signals. This program The lasmid was prepared using standard molecular biology techniques (Mamiati Chloramphenicol acetyltransferase (CA T) Must be engineered to include IL-1i cDNA in place of the coding sequence. stomach. (2) The inducible gene construct is mouse metallothionein (MT-1) protein. Motor region (Brinster et al. Ce1l 27:228-231.19 81) must be made using the plasmid pMK containing the plasmid pMK.

This plasmid can be used as a starting material and contains genes inducible by metals. In order to produce the article, it must be operated as shown in FIG.

2. In order to produce an active protein, the above-mentioned vector is used to produce a large number of animals. A cell line must be used to express IL-1i. Promotes foreign gene expression Two effective cell lines that have been well characterized for their ability to induce IL- Expression of 1i is not limited to these cell lines, but is present in murine Ltk- and Chinese cells. These are hamster ovary (CHO) dhfr- cells.

3. Vector DNA can be transferred to these cell lines using any of a number of gene transfer techniques. must be introduced. The method used here includes S.L., Graham & A, S, van der Eb (Virology 52:456-46 7, 1973), in which IL-1 i at the same time as a second expression vector encoding a selectable marker. It is precipitated. For Ltk-cell transfections, the selectable marker is thymidine kinase gene and its selection was carried out by Wigler, et al. (Cell 16:777-785.1979) and CHOdhfr-Details. In the case of cysts, the selectable marker is dihydrofolate reductase (DHPR). The selection was made by Ringold et al.

Mo1. Appl, Genet, 1:165-175.1981. It is.

4. Cells expressing the IL-1l gene construct are then treated to increase IL-1i production levels. It must be grown under conditions that will allow it to grow. Metallothionein promoter structure The cells carrying the construct now have the MT-1 promoter (Mayo et al., Ce1l 29:99-108) and thereby increase IL-1i protein levels. can be grown in the presence of heavy metals like cadmium, which also increase Ru. IL-1i expression vector (SV40 or M T-1), which is a competitive antagonist of DHPR. Using totrexate, Ringold et al. (J.

Mo1. Appl, Genet, 1:165-175.1981) It can be collected using gene amplification protocols. This allows DHP present in cells to The number of copies of the R gene increases, and at the same time the number of copies of the IL-1i gene increases, which More IL-1i protein can be produced by the cell.

Example 9 Purification of IL-1i from recombinant animals IL-1i is expected to be secreted from cells in the same way as natural substances, so it is a natural protein. Recombinant proteins can be similarly purified and purified using the methods described above for protein purification. It is expected that it will be possible to read and characterize it.

Example 10 The amino-terminal residues of IL-1i were identified several times by direct protein sequencing. R). The results of such sequencing are shown in Example 3. Contrary to this In contrast, the amino terminal residue of IL-1i predicted by the cDNA sequence is proline. (P). This amino-terminal residue corresponds to nucleotides 85-87 in Figure 13. and is surrounded by O in FIGS. 14 and 15. cDNA sequence and direct protein This apparent discrepancy between the cDNA sequences may be due to an error in the cDNA sequence. Assume that it is incorporated during the synthesis catalyzed by reverse transcriptase from A. This can be solved by that is, present on the mRNA, where its amino-terminal residue The CGA (arginine) codon that encodes the CDNA during the reverse transcriptase reaction could have been changed to a CCA (proline) codon in This kind of reverse The transcription enzyme problem has previously been addressed, for example, in Nucleic Ac by B, D, C1ark et al. Reported in the literature by 1dsResearch 14 Near 897 (1986) ing.

The present inventors have determined that the amino terminal amino acid is the proline residue shown in Figures 13-15. The exact amino acid sequence of this protein, except that it is instead arginine, is We believe that this is as predicted by the cDNA. The present inventors have determined that the amino-terminal The luginine sequence is preferred, but the DNA sequences and their corresponding peptide sequences Both are intended to fall within the scope of this invention.

Example 11 Proteins having the following sequences are also included in the present invention: 302° SET! XZPSGRKSSKMQAFR1K RF A F I RS D 5 G P T m, S F E S A AXPGWFLXTAMEADQPV5 L 7 N (in the formula, X is cysteine, serine or alanine and Z is arginine or proline ).

Figure 2a silver stain gel Figure 2b auto radio durham Figure 3b silver stain gel Figure 3C -□43 KD Saiichi Trazi Said Durham Figure 5b Figure 6 Figure 7 Figure 7 (continued) From figure 8b From Figure 9a Figure 12A Lane 568101214 Figure 12B Lane 56 8 10 12 14 Figure 13 Figure 14 Figure 15 international search report

Claims (25)

[Claims] 1. Consists of interleukin-1 alpha and interleukin-1 beta a substantially purified substance active against at least one substance selected from the group Interleukin-1 inhibitor (IL-1i). 2. 2. The IL-1i of claim 1, wherein said IL-1i is derived from a mammalian cell. 3. 2. The IL-1i of claim 1, wherein said IL-1i is isolated from monocytes. 4. 4. The IL-1i of claim 3, wherein said IL-1i is isolated from human monocytes. 5. The IL according to claim 1, wherein the IL-1i is produced by a recombinant DNA method. -1i. 6. (a) instructs the host cell to produce a protein with IL-1 inhibitor activity; (b) preparation of a DNA sequence necessary to express the DNA sequence; into a vector containing the agent and capable of transferring into and replicating in a host cell. cloning the DNA sequence; (c) cloning the synthetic DNA sequence and operational elements; A vector containing a host capable of expressing DNA encoding an IL-1 inhibitor. Import into principal cells; (d) host cells under conditions suitable for amplification of the vector and expression of the inhibitor. culture; (e) Harvesting the inhibitor; and (f) causing the inhibitor to adopt an active three-dimensional structure, such that the inhibitor will have L-1 inhibitor activity, A set for interleukin-1 inhibitor (IL-1i) production consisting of Replacement DNA method. 7. 7. The method according to claim 6, wherein said DNA sequence is cDNA. 8. The method according to claim 6, wherein the DNA sequence is a genomic polynucleotide sequence. Law. 9. 7. The method of claim 6, wherein said DNA sequence is derived from a mammalian cell. 10. 10. The method of claim 9, wherein said DNA sequence is derived from human monocytic cells. 11. 7. The method of claim 6, wherein the host cell is a microorganism. 12. The microorganism is E. 12. The method according to claim 11, wherein coli is used. 13. 7. The method of claim 6, wherein the host cell is a mammalian cell. 14. 14. The method of claim 13, wherein the mammalian cell is a CHO cell. 15. The method according to claim 6, wherein the DNA sequence is a cDNA polynucleotide. Law. 16. 7. The method according to claim 6, wherein the DNA sequence is a genomic polynucleotide sequence. Method. 17. IL-1i is sufficient to have at least one of the biological properties of IL-1i. DNA sequences encoding polypeptides having amino acid sequences that are 1i redundant. interleukin-1 inhibitors (IL-1 i) An isolated DNA sequence encoding. 18. The above DNA base sequence is the following sequence: [There is a sequence] The isolated D according to claim 17, comprising the nucleic acid from position 99 to position 557 of NA sequence. 19. Recombinant DNA molecule GT10-IL1i-2A. 20. At least one of IL-1i-X, IL-1i-α and IL-1i-β A substantially purified interleukin-1 inhibitor (IL-1i) consisting of . 21. The interlocutor according to claim 20, wherein the IL-1i is IL-1i-X. Ikin-1 inhibitor. 22. The interlocutor according to claim 20, wherein the IL-1i is IL-1i-α. Ikin-1 inhibitor. 23. The interlocutor according to claim 20, wherein the IL-1i is IL-1i-β. Ikin-1 inhibitor. 24. The above DNA base sequence is the following sequence: [There is a sequence] The isolated D according to claim 17, comprising the nucleic acid from position 99 to position 557 of NA sequence. 25. The following array: (wherein X is cysteine, serine or alanine and Z is arginine or is proline). Tar.