AU633831B2 - Interleukin-1 inhibitors - Google Patents

Description

OPI DATE 12/12/89 AOJP DATE 25/01/90 APPLN. ID 37469 89

PC

PCT NUMBER PCT/US89/02275 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 C12P 21/.0, C07K 13/00 C07H 15,112

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(11) International Publicatioa Number: WO 89/11540 Al (43) International Publication Date: 30 November 1989 (30.11.89) (21) International Application Number: (22) International 7Fling Date: Priority data: 199,915 27 May 1 238,713 31 Augus 266,531 3 Noveml PCT/US89/02275 25 May 1989 (25.05.89) 988 (27.05.88) t 1988 (31.08.88) ber 1988 (03.11.88) (71) Applicant: SYNERGEN, INC. [US/US]; 1888 33rd Street, Boulder, CO 80301 (US).

(72) Inventors: HANNON, Charles, H. 6150 Willow Lane, Boulder, CO 80301 EISENBERG, Stephen, P. 2325 Panorama Avenue, Boulder, CO 80302 (US).

THOMPSON, Robert, C. 1820 Lehigh Street, Boulder, CO 80303 AREND, William, P. 4157 Montview Blvd., Denver, CO 80207 JOSLIN, Fenneke, G. 1900 Maghoha Street, Denver, CO 80220 SOM- MER, Andreas 117 Oak Grove Road, Concord, CA 94518 (US).

(74) Agents: PATTERSON, Herbert, W. et al.; Finnegan, Henderson, Farabow, Garrett Dunner, 1775 K Street, Washington, DC 20006 (US).

(81) Designated States: AT, AU, BB, BF (OAPI patent), BG, BJ 'OAPI patent), BR, CF (OAPI patent), CG (OAPI patent), CH, CM (OAPI patent), DE, DK, FI, GA (OA- PI patent), GB, HU, JP, KP, KR, LK, LU, MC, MG, ML (OAPI patent), MR (OAPI patent), MW, NL, NO, RO, SD, SE, SN (OAPI patent), SU, TD (OAPI patent), TG (OAPI patent).

Published With international search report.

633831 1'

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(54) Title: INTERLEUKIN-1 INHIBITORS (57) Abstract A substantially purified interleukin-1 inhibitor (IL-li) which is active against interleukin-1 alpha or interleukin-1 beta or both of these substances. A recombinant-DNA method for the production of an interleukin-1 inhibitor (IL-li), and an isolated DNA sequence encoding a physiologically functional interleukin-1 inhibitor (IL-li).

i WO 89/11540 PCT/US89/02275 INTERLEUKIN-1

INHIBITORS

Backoround of the Invention This application is a continuation-in-part application of United States Patent Application Serial No. 238,713, of the same inventors filed August 31, 1988, which is a Scontinuation-in-part of United States Patent Application Serial No. 199,915, filed May 27, 1988.

A. IL-1 Interleukins-1 are a class of proteins produced by numerous cell-types, including monocytes and some macrophages. This class includes at least two 17-18 kilodalton proteins known as interleukin-1 alpha and interleukin-1 beta.

These proteins have important physiological effects on a number of differen: target cells involved in the inflammatory and immune responses. The proteins are co-m.togens (with phytohemaglutinin) for T-cells, cause both fibroblasts and chondrocytes to secrete latent collagenase, and increase the surface adhesive powers of endothelial cells for neutrophils.

In addition, 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, interleukins-1 (IL-1) are obviously an important part of an organism's response to infection and injury.

B. Pathological Roles of IL-1 However, despite their normally beneficial effects, circumstances have come to light in which the actions of IL-1 are harmful. For example, IL-1 may increase the level of collagenase in an arthritic joint and has been implicated as Ia mediator of both the acute and chronic stages of immunopathology in rheumatoid arthritis. IL-1 may be responsible for altering endothelial cell function, directing the chemotaxis and migration of leukocytes and lymphocytes into the synovial tissue, inducing capillary proliferation, and stimulating macrophage accumulation in the synovial lining during the acute phase of this disease. In the phase of tissue destruction, IL-1 has been implicated as a mediator in induction of tissue damage through stimulating release of enzymes from fibrobias-s and chondrocytes.

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i i- -n WO 89/11540 PCT/US89/02275 -2- In addition, 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 Swith psoriatic arthritis. IL-1 released by cells in the inflamed synovium in psoriatic arthritis may mediate tissue destruction through stimulation of enzyme release from other cells. The joint pathology of Reiter's syndrome is similar to that seen 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. Moreover, IL-1 may be found in the synovial fluid of patients with osteoarthritis. The release of IL-1 by chondrocytes has been implicated in the destruction of j articular cartilage in this disease.

IL-

1 may also increase the severity of autoimmune diseases. For example, decreased IL-1 production has been described from peripheral blood cells in persons suffering from systemic lupus erythematosus. Moreover, some of the alterations 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 implicated a. a possible agent of fibrosis through stimulation of collagen production by fibroblasts.

The mechanism of tissue damage in dermatomyositis might also involve 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 may be stimulated by IL-1. Recent studies on an animal model of pulmonar.y hypertension indicate that IL-1 may be responsible for induction of endothelial cell changes that result in narrowing of pulmonary arteries. It is this narrowing that leads to pulmonary hypertension and further secondary damage. Thus, IL-1 inhibitors could be useful in treating these lung diseases.

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1^ i "WO 89/11540 W89/11540 PCT/US89/02 27 -3- Recent studies have described that IL-I is capable Sof directly damaging the beta cells in the Islets of Langer- I hans that are responsible for the production of insulin.

IL- damage to the cells is now hypothesized to be a primary Sevent in the acute phase of juvenile diabetes mellitus.

I d Monocyte and macrophage infiltration in the kidneys predominates in many forms of acute and :hronic glomerulonephritiS.

IL-

1 release by these cells may result in local accumulation of other inflammatory cells, eventually leading to inflannatory damage and fibrotic reaction in the kidneys.

SIt has been demonstrated that the crystals found in tissues or fluids in gout or pseudogout can directly stimulate macrophages to release IL-l. Thus, IL- may be an important mediator in the inflammatory cycle in these diseases.

IL-I is capable of inducing loss of calcium from bones and may be responsible for the osteoporosis that is seen in inflammatory joint diseases.

Keratinocytes from patients with psoriasis release large amounts of IL-I. This mediator may be responsible for the secondary cell proliferation and accumulation which occurs in the skin in patients with this disease.

IL-I is one of the important endogenous pyrogens and may be responsible for inducing the marked degree of fever seen in some infectious diseases such as acute febrile illnesses due to bacteria or viruses.

Sarcoidosis is characterized by granulomatous le- Ssions in many different organs in the body.

IL-

1 has been Sshown to be capable of inducing granuloma formation in vitr and may be involved in this process in patients with sarcoidosis.

Excessive IL-I production has been demonstrated in peripheral monocytes from both Crohn's disease and ulcerative colitis. Local IL-3 releas6 in the intestine may be an important mediator in stimulating the inflammatory cycle in these diseases.

ISUBSTITUTE SHEET WO 89/11540 PCT/US89/02275 -4- 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. Also, IL-1 production by some malignant lymphocytes may be responsible for some of the fever, acute phase response and cachexia seen with leukemias.

IL-1 release by astrocytes in the brain is thought to be responsible for inducing the fibrosis that may result after damage to the brain from vascular occlusion.

C. Uses for an IL-1 Inhibitor In these and other circumstances in which IL-1 has a harmful effect, there is clearly a clinical use 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 and other immune diseases. Thus, systemically administered, IL-1 inhibitors could be useful immunosuppressive agents. Locally applied, such IL-1 inhibitors could serve to prevent tissue destruction in an inflamed joint and other sites of inflammation. Inceed, to prevent tissue destruction some IL-1 inhibitors could be even more effective when administered in conjunction with collagenase inhibitors.

Therapeutic intervention against the action of IL-1 might be possible at the level of synthesis, secretion, or the target cell's binding or response to the protein. IL-1 is synthesized by monocyte/macrophages and other cells in response to lipopolysaccharides, complement fragments and viruses. Any molecule that blocks binding of these irucing U agents to producer cells or which interferes with their effects on the physiology of these cells would serve as a -regulator of IL-1 action. IL-1 is not secreted by a traditional secretion system since mRNAs have been isolated that code for at least two 30 kd precursors of the proteins but which do not contain a hydrophobic signal sequence. Release of the active protein from the inactive precursor probably requires proteolysis of that precursor. An inhibitor of the 'SUBSTITUTE SHEET release of IL- 1 or IL-Is from their precursors could theoretically regulate IL-I action.

IL-

1 probably acts on target cells through a classical receptor-mediated pathway, although that receptor has not yet been isolated. Thus, it could be that a molecule that interferes with IL-1 binding to its receptors, or down-regulates these receptors, could also regulate IL-1 action. Moreover, although the intracellular events following receptor binding of IL-1 are not yet fully understood, it is possible that agents exist that can interfere with the cellular responses to other recEptor-mediated events and therefore block

IL-

1 action. For the reasons stated above, proteins and small molecules capable of inhibiting IL-l in one or more of these manners have been sought.

Surprisingly, the present inventors have found at Least two IL-I inhibitor proteins with IL-1 inhibiting prop- erties. These molecules have been obtained in a purified form which will enable one of ordinary skill in the art to determine their amino acid sequence. Furthermore, a preparation of cells has been characterized which produce these proteins, and an mRNA that leads to its synthesis has been characterized. Finally, an a-Se has been developed that will facilitate screening of CDNA expression libraries for the genes coding for these inhibitors. Together these reagents will allow cDNAs encoding the IL-1 inhibitors to be cloned.

These genes will, in turn, make possible the large scale production of IL-1 inhibitors suitable for use in pharmaceutical formulations useful in treating pathophysicological conditions mediated by IL-1.

Summary of the Invention This invention relates to IL-I inhibitors ("IL-li") generally and, more specifically, to a monocyte-derived IL-1 inhibitor. Additionally, the present invention relates to biologically-active analogs of these inhibitors.

iAn object of the present invention is to provide purified forms of IL-I inhibitors which are active against SIL-la or IL- 1 8 or a combination thereof. An additional SI ~CiU-TiTUTE

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WO 89/11540 PCT/US89/02275 object of the present invention is to provide these inhibitors in purified forms to enable the determination of their amino acid sequence. A further object is to provide the amino acid sequences of certain IL-1 inhibitors. Furthermore, the identification of biologi-ally-active analogs of such IL-1 inhibitors with enhanced or equivalent properties is also one of the objects of the invention.

Additionally, it is an object of this invention to provide a recombinant-DNA system for the production of the IL-1 inhibitors described herein. A further object of the present invention includes providing purified forms of IL-1 inhibitors which would be valuable as pharmaceutical preparations exhibiting activity against IL-1.

Additional objects and advantages of the invention will be set 'orth in part in the description which follows, and in part will be obvious from the description or may be learned from the practice of the invention. The objects and advantages may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.

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

Preferred inhibitors of the present invention are 1, 2 and 3. Inhibitors 1 and 2 are proteins running at positions characteristic of 22-23 kDa proteins on SDS-PAGE and eluting at 52 mM and 60 mM NaCl, respectively, from a Mono Q FPLC column under specified conditions. Inhibitor 3 is a protein running at a position characte:istic of a 20kD protein on SDS-PAGE and eluting at 48 mM NaCl from a Mono Q FPLC column under the specified conditions. Additionally, to achieve the objects and in accordance with the purposes of the present invention, pharmaceutical compositions

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1 SWO89/11540 PCT/US89/02275 -7containing, at least one of the active ingredients, an IL-1 inhibitor in accordance with the present invention or its biologically-active analog as set forth herein are disclosed.

Moreover, to achieve the objects and in accordance with the purposes of the present invention, a recombinant-DNA system for the creation of these IL-1 inhibitors and their analogs is also disclosed. A preferred embodiment of this system includes at least one cDNA clone or its synthetic equivalent encoding at least one IL-1 inhibitor along with vectors and cells constituting an expression system capable of expressing the IL-1 inhibitors disclosed herein. Antiser- for use in identifying these cDNA clones is also provided. Expression systems for producing these IL-1 inhibitors using these cDNA clones, their analogs, or other DNA sequences encoding these inhibitors are also provided.

Brief Description of the Figures Figures la and lb depict the protein profile of the Mono Q chromatography of two metabolically-labelled monocyte supernatants. The cells were cultured on IgG (la) or fetal calf serum (Ib) coated plates.

Figure 2a shows silver stained gels of fractions from the regions indicated in Figures la and lb.

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

Figures 3a, b and c present data on the purified IL-li of Example 1. Figure 3a presents chromatography data with the radioactivity pattern superimposed. Figure 3b presents silver stained gels run on iamples of the fractions indicated in Figure 3a. Figure 3c presents autoradiograms of the gels in Figure 3b.

Figures 4a and b present the results of gel filtration chromatograms of Mono Q-purified IL-li.

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

Figure 6 depicts the construction of plasmid I pSVXVPL2IL-li.

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~-LII ~-~PI11 0~.~1 WO 89/11540 PCUS9/02275 -8-

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

Figures 8a-d present data on IL-li-. Figures 8a and 8b present chromotography data. Figure 8c presents a silver stained gel run on samples of fractions indiated in figure 8b. Figure 8d presents an autoradiogram.

Figures 9a and 9b present data on IL-li-. Figure 9a presents chromotography data. Figure 9b presents

SDS-PAGE

data.

Figure 10 presents data of IL-li- peptide separation.

Figure 11 presents data of IL-li-B peptide separation.

F-igure l 2a is a photograph of the gel with the GT0-ILli-2A digested with EcoRI after electrophoresis according to Example 6.

Figure 12b presents data of an autoradiogram of a Southern blot of the gel shown in Figure 12a.

Figure 13 depicts a part of the DNA sequence of the protein coding region of lambda GT0-ILli-2A and the pred i c t e d am i nio acid 2Adi and the p r e dicted amino acid sequence according to Example 6.

Figure 14 depicts the nucleotide sequence of GT-10-iII-2A.

Figure 15 depicts a peptide including, inter alia, an IL-li sequence and a secre.ory leader sequence.

eP- tiotn of the Preferred mbodimen Reference will now be made in detail to the presently preferred embodiments of the invention, which, together with the following examples, serve to explain the principles of the invention.

A. Inhibitor from Human Monocytes As noted above, the present nvention relates to IL-1 inhibitors which have been isolated in a purified form.

Preferably, the IL-1 inhibitors of the present invention are derived from human monocyte conditioned medium where the monocytes are grown on IgG coated vessels. :n addition, the OUSTS3TUTE S E T WO 89/11540 PCT/US89/02275 -9invention encompasses substantially purified IL-1 inhibitors of any origin which are biologically equivalent to t.e inhibitor derived from human monocyte-contained medium.

By "biologically equivalent," as used throughout the specification and claims, we mean compositions of the present invention that are capable of preventing IL-1 action in a similar fashion, but not necessarily to the same degree, as the native IL-1 inhibitor isolated from monocytes. By "substantially homologous" as used throughout the ensuing specification and claims, is meant a degree of homology to the native IL-1 inhibitor isolated from monocyte-conditioned medium in excess of that displayed by any previously reported IL-1 inhibitors. Preferably, the degree of homology in excess of 70 percent, more preferably in excess of 80 percent and even more preferably in excess of 90 per cent. A particularly preferred group of inhibitors are in excess of 95 percent homologous with 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 ali' with identical amino acid residues in the sequence being compared when four gap; in a length of 100 amino acids may be introduced to assist in that alignment as set forth by Dayhoff, M. in Atlas of Protein Sequence and Structure p. 124 (1972), National Biochemical Research Foundation, Washington, specifically incorporated herein by reference.

The preferred IL-1 inhibitors of the present invention have been derived from monocyte-conditioned medium and, for the first time, have been isolated in a purified form.

S For the purposes of the present application, "pure form" or "purified form" when used to refer to the IL-1 inhibitors disclosed herein, shall mean a preparation which is substantially free of other proteins which are not IL-1 inhibitor proteins. Preferrably, the"IL-1 inhibitors of the present invention are at least 90% pure and preferably 95% pure.

.ITE! SHE-ET A .Li i V LaCl7 this 26th lay of December 19 q -i- Positi-on xecutive i ce resident GRIFFITH HACK CO PATENT AN D TRADE MARK ATTORNEYS ME L B OUR N E SYD N E Y PERTH WO 89/11540 PCT/US89/02275 At least three purified IL-1 inhibitors have been isolated by the methods of the Example. These include inhibitor iitor inhitor 2 and inhibitor 3. Inhibitor 1 is behaving as a 22-23 kDa molecule on SDS-PAGE with an approximate isoelectric point of 4.8 and eluting from a Mono Q FPLC column at around 52 mM NaCI in Tris buffer, pH 7.6. Inhibitor 2 is also a 22-23 kDa protein, pl=4.8, but eluting from a Mono Q column at 60 mM NaC1. Inhibitor 3 is a 20kDa protein and elutes from a Mono Q column at 48 mM NaC1. Inhibitors 1, 2 and 3 are related immumologically and functionally. Having obtained these inhibitors in purified forms has enabled the present inventors to obtain their amino acid sequences.

Using the purified inhibitors disclosed for the first time herein and methods such as those described in and by ABI Protein Sequencer technical manuals supplied with the ABI Protein Sequencer, a substantial proportion of the amino acid p -1 sequences of these inhibitors can be deduced.

Example 3 shows amino acid sequence data obtained of three species of IL-1 inhibitors, namely IL-li-X, IL-li-a and IL-li-B.

The present invf:'tors have discovered at least one antibody raised against an IL-1 inhibitor. Other polyclonal and monoclonal antibodies against this and other IL-1 inhibitors may be prepared by methods known to those of ordinary skill in the art. One particular polyclonal antibody is described in Example 4.

B. Recombinant Inhibitor 1. General A recombinant DNA method for the manufacture of an IL-1 inhibitor is now disclosed. In one embodiment of the invention, the active site functions in a manner biologically equivalent to that of the native IL-1 inhibitor isolated from human. A natural or synthetic DNA sequence may be used to direct production of the IL-1 inhibitors. This method comprises: 7 EI i I i i-i rc- rr- WO89/11540 PCT/US89/02275 1 1 Preparation of a DNA sequence capable of directing a host cell to produce a protein having IL-1 inhibitor activity; Cloning the DNA sequence into a vector capable of being transferred into and replicated in a host cell, such vector containing operational elements needed to express the DNA sequence; Transferring the vector containing the synthetic DNA sequence and operational elements into a host cell capable of expressing the DNA encoding IL-1 inhibitor; Culturing the host cells under conditions appropriate for amplification of the vector and expression of the inhibitor; Harvesting the inhibitor; and Permitting the inhibitor to assume an active tertiary structure whereby it possesses IL-1 inhibitory activity.

2. DNA Sequences DNA sequences contemplated for use in this method are discussed in part in Example 5 and in part in Example 6. It is contemplated that these sequences include synthetic and natural DNA sequences. The natural sequences further include cDNA or genomic DNA segments.

Example 6 provides a molecular clone of DNA encoding a protein identical to that isolated in Examples 1- 3. In Example 6, a plaque, GT10-ILli-2A, was isolated from a Library. The phage within 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 ILl inhibitor. Figure 13 shows the partial DNA sequence of the EcoRI fragment.

In light of the teachings contained herein and procedures known, other synthetic polynucleotide sequences will be available to one of ordinary skill in the art. As an example of the current state of the art relating to F UT E.S' 6 i1u~. uerieuKn-1 inhibitor (IL-li) comprising a DNA sequence encoding a polypeptide having an amino acid sequence sufficiently duplicative of IL-li to U allow possession of at least one of IL-li's biological properties.

/2 i WO 89/11540 PCT/US89/02275 -12polynucleotide synthesis, one is directed to Matteucci, M.D.

and Caruthers, in J. Am. Chem. Soc. 103:3185 (1981) and Beaucage, S.L. and Caruthers, M.H. in Tetrahedron Lett.

S22:1859 (1981), and to the instructions supplied with an ABI oligonucleotide synthesizer, each of which is specifically incorporated herein by reference.

These synthetic sequences may be identical to the natural sequences described in more detail below or they may contain different nucleotides. In one embodiment, if the synthetic sequences contain nucleotides different from those found in the natural DNA sequences of this invention, it is contemplated that these different sequences will still encode a polypeptide which has the same primary structure as IL-li isolated from monocytes. In an alternate embodiment, the synthetic sequence containing different nucleotides will encode a polypeptide which has the same biological activity as the IL-li des:ribed herein.

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

SIn an alternative embodiment, the DNA sequence is isolated from a human genomic library. An example of such a library useful in this embodiment is set forth by Lawn et al.

in Cell 15:1157-1174 (1978), specifically incorporated herein by reference.

In a preferred version of this embodiment, it is contemplated that the natural DNA sequence will be obtained by a method comprising: Preparation of a human cDNA library from cells, preferably monocytes, capable of generating an IL-1 inhibitor in a vector and cell capable of l amplifying and expressing all or part of that cDNA; l TTUESHEET WO 89/11540 PCT/US89/02275 -13- Probing the human DNA library with at least one probe capable of binding to the IL-1 inhibitor gene or its protein product; Identifying at least one clone containing the gene coding for the inhibitor by virtue of the ability of the clone to bind at least one probe for the gene or its protein product; Isolation of the gene or portion of the gene coding for the inhibitor from the clone or clones chosen; Linking the gene, or suitable fragments thereof, to operational elements necessar-y to maintain and express the gene in a host cell.

The natural DNA sequences useful in the foregoing process may also be identified and isolated through a method comprising: Preparation of a human genomic DNA library, preferably propagated in a recArecBC E. coli host; Probing the human- genomic DNA library with at least one probe capaole of binding to an IL-1 inhibitor gene or its protein product; Identification of at least one clone containing the gene coding for the inhibitor by virtue of the ability of the clone to bind at least one probe for the gene or its protein product; Isolation of the gene coding for the inhibitor from the clone(s) identified; and V Linking the gene, or suitable fragments thereof, to operational elements necessary to maintain and express the gene in a host cell.

In isolating a natural DNA sequence suitable for use in the above-method, it is preferred to identify the two restriction sites located within and closest to the end portions of the appropriate gene or sections of the ge:e. The DNA segment containing the appropriate gene is then removed UstTIM TE SHEET WO 89/11540 PCr/US89/02275 roam toe remainder of the genomic material using appropriate restriction endonucleases. After excision, the 3' and ends of the DNA sequence and any exon junctions are reconstructed to provide appropriate DNA sequences capable of coding for the N- and C- termini of the IL-1 inhibitor protein and capable of fusing the DNA saquence to its operational elements.

3. Vectors Microorganismns, especially E. coli The vectors contemplated for use in the present invention include any vectors into which a DNA sequence as discussed above can be inserted, along with any preferred or required operational elements, and which vector can then be subsequently transferred into a host cell and replicated in such cell. Preferred vectors are those whose restriction sites have been well documented and which contain the operational elements preferred or required for transcription of the DNA sequence. However, certain embodiments of the present invention are also envisioned which employ currently undiscovered vectors which would contain one or more of the cDNA sequences described herein. in particular, it is preferred that all of these vectors have some or all of the following characteristic-: possess a minimal number of host-organism sequences; be stably maintained and propagated in the desired host; be capable of being present in a high copy number in the desired host; possess a regulatable promoter positioned so as to promote tr~inscription of the gene of interest; have at least one marker DNA sequence coding~ for a selectable trait present on a portion of the plasmid separate from that where the DNA sequence will be inserted; and a DNA sequence capable of terminating transcription.

in various preferred embodiments, these cloning vectors containi g and capable of expressing the DNA sequences of the present invention contain various operational elements. These "operational elements," as discussed herein, ~i~T1UTE SHEET 'd Lu^' .CIa I j I j Uj.IL iLQL-IUn, CLu stimulating macrophage accumulation in the synovial lining during the acute phase of this disease. In the phase of tissue destruction, IL-1 has been implicated as a mediator in induction of tissue damage through stimulating release of enzymes from fibroblas:s and chondrocytes.

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WO 89/11540 PCT/US89/02275 include at least one promoter, at least one Shine-Dalgarno sequence and initiator codon, and at least one terminator codon. Preferably, these "operational elements" also include at least one operator, at least one leader sequence for proteins to be exported from intracellular space, at least one gene for a regulator protein, and any other DNA sequences necessary or preferred for appropriate transcription and subsequence translation of the vector DNA.

Certain of these operational elements may be present in each of the preferred vectors of the present invenii 4'~ tion. It is contemplated tnat any additional operational elements which may be required may be added to these vectors using methods known to those of ordinary skill in the art, particularly in light of the teachings herein.

In practice, it is possible to construct each of these vectors in a way that allows them to be easily isolated, assembled and interchanged. This facilitates assembly of numerous functional genes from combinations of these elements and the coding region of the DNA sequences. Further, many of these elements will be applicable in more than one host. It is additionally contemplated that the vectors, in certain preferred embodiments, will contain DNA sequences zapable of functioning as regulators ("operators"), and other DNA sequenes capable of coding for regulator proteins.

Requlators These regulators, in one embodiment, will serve to prevent expression of the DNA sequence in the presence of certain environmental conditions and, in the presence of other environmental conditions, will allow transcription and subsequent expression of the protein coded for by the DNA sequence. In particular, it is preferred that regulatory segments be inserted into the vector such that expression of the DNA sequence will not occur, or will occur to a greatly reduced extent, in the absence of, for example, isopropylthio-beta-D-galactoside. In this situation, the transformed microorganisms containing the DNA sequence may be SUBSTITUTE SHEET Ia=pulusiUe zor inauction of endothelial cell changes that result in narrowing of pulmonary arteries. It is this narrowing that leads to pulmonary hypertension and further secondary damage. Thus, IL-1 inhibitors could be useful in treating these lung diseases.

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WO089/11540 PCT/US89/02275 -16grown toa at a desired density prior to initiation of the expression of IL-li. In this embodiment, expression of the desired protein is induced by addition of a substance to the microbial environment capable of causing expression of the DNA sequence after the desired density has been achieved.

(ii) Promoters The expression vectors must contain promoters which can be used by the host organism for expression of its own proteins. While 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-li.

(iii) Transcription Terminator The transcription terminators contemplated herein serve to stabilize the vector. In particular, those sequences as described by Rosenberg, M. and Court, in Ann.

Rev. Genet. 13:319-353 (1979), specifically incorporated herein by reference, are contemplated for use in the present invention.

(iv) Non-Translated Sequence It is 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' non-translated sequences into the gene transcript. Included among these non-translated sequences are those which stabilize the mRNA as they are identified by Schmeissner, McKenney, K., Rosenberg, M and Court, D. in J. Mol. Biol. 176:39-53 (1984), Sspecifically incorporated herein by reference.

Ribosome Binding Sites The microbial expression of foreign proteins requires certain operational elements which include, but are not limited to, ribosome binding sites. A ribosome binding site is a sequence which a ribosome recognizes and binds to in the initiation of protein synthesis as set forth in Gold, et al., Ann. Rev. Microbio. 35:557-580; or Marquis, D.M., et al., Gene 42:175-183 (1986), both of which are SUBSTITUTE SHEET Excessive IL-1 production has been demonstrated in peripheral monocytes from both Crohn's disease and ulcerative colitis. Local IL-1 release in the intestine may be an important mediator in stimulating the inflammatory cycle in these diseases.

j SUBSTITUTE SHEET WO 89/11540 PCT/US89/02275 specifically incorporated herein by reference. A preferred ribosome binding site is GAGGCGCAAAAA(ATG).

(vi) Leader Sequence and Translational Couoler K Additionally, it is preferred that DNA coding for L an appropriate secretory leader (signal) sequence be present at the 5' end of the DNA sequence as set forth by Watson, M.E. in Nucleic Acids Res. 12:5145-5163, specifically incorporated herein by reference, if the protein is to be excreted from the cytoplasm. The DNA for the leader sequence must be in a position which allows the production of a fusion protein in which the leader sequence is immediately adjacent to and covalently joined to the inhibitor, there must be no transcription or translation termination signals between the two DNA coding sequences. The presence of the leader sequence is desired in part for one or more of the following reasons. First, the presence of the leader sequence may facilitate host processing of the IL-li. In particular, the leader sequence may direct cleavage of the initial translation product by a leader peptidase to remove the leader sequence and leave a polypeptide with the amino acid sequence which has potential protein activity. Second, the presence of the leader sequence may facilitate purification of the IL-li, through directing the protein out of the cell cytoplasm. In some species of host microorganisms, the presence of an appropriate leader sequence will allow transport of the completed protein into the periplasmic space, as in the case of some E. coli. In the case of certain E. coli, Saccharomyces and strains of Bacillus and Pseudomonas, the appropriate leader sequence will allow transport of the protein through the cell membrane and into the extracellular medium. In this situation, the protein may be purified from extracellular protein. Thirdly, in the case of some of the proteins prepared by the present invention, the presence of the leader sequence may be necessary to locate the completed protein in an environment where it may fold to assume its S STTUTE SHEET east two 30 kd precursors of the proteins but which do not contain a hydrophobic signal sequence. Release of the active protein from the inactive precursor probably Srequires proteolysis of that precursor. An inhibitor of the SSUBSTITUTE SHEET

:I

WO 89/11540 PCT/US89/02275 i -18active structure, which structure possesses the appropriate protein activity.

In one preferred embodiment of the present invention, an additional DNA sequence is located immediately preceding the DNA sequence which codes for the IL-1 inhibitor.

The additional DNA sequence is capable of functioning as a translational coupler, -it is a DNA sequence that encodes an RNA which serves to position ribosomes immediately adjacent to the ribosome binding site of the inhibitor RNA with which it is contiguous. In one embodiment of the presxi ent invention, the translational coupler may be derived using the DNA sequence

TAACGAGGCGCAAAAAATGAAAAAGACAGCTATCGCGATCTTGGAGGATGATTAAATG

and method,, currently known to those of ordinary skill in the art related to translational couplers.

(vii) Translation Terminator The translation terminators contemplated herein serve to stop the translation of mRNA. They may be either natural, as described by Kohli, Mol. Gen. Genet.

182:430-439; or synthesized, as described by Pettersson, R.F.

Gene 24:15-27 (1983), both of which references are specifically incorporated herein by reference.

(viii) Selectable Marker Additionally, it is preferred that the cloning vector contain a selectable marker, such as a drug resistance marker or other marker which causes expression of a selectable trait by the host microorganism. In one embodiment of the present 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 intended in part to facilitate in'the selection of transformants. Additionally, the presence of such a selectable marker in the cloning vector may be of use in keeping contaminating microorganisms from multiplying in the cul.ure C*.d SGT1TUTE SHEET Y, L~e present invention relates to biologically-active analogs of these inhibitors.

An object of the present invention is to provide I purified forms of IL-1 inhibitors which are active against SIL-la or IL-18 or a combination thereof. An additional -,4o C WO 89/11540 PCT/US89/02275 19medium. In this embodiment, a pure culture of the transformed host microorganisms would be obtained by culturing the microorganisms under conditions which require the induced phenotype for survival.

The operational elements as discussed herein are routinely selected by those of ordinary skill in the art in light of prior literature and the teachings contained herein.

General examples of these operational elements are set forth in B. Lewin, Genes, Wiley Sons, New York (1983), which is specifically incorporated herein by reference. Various examples of suitable operational elements may be found on the vectors discussed above and may be elucidated through review of the publications discussing the basic characteristics of the aforementioned vectors.

Upon synthesis and isolation of all necessary and desired component parts of the above-discussed vector, the vector is assembled by methods generally known to those of ordinary skill in the art. Assembly of such vectors is believed to be within the duties and tasks performed by those with ordinary skill in the art and, as such, is capable of being performed without undue experimentation. For example, similar DNA sequences have been ligated into appropriate cloning vectors, as set forth by Maniatis et al. in Molecular Cloning, Cold Spring Harbor Laboratories (1984), which is specifically incorporated herein by reference.

In construction of the cloning vectors of the present invention, it should additionally be noted that multiple copies of the DNA sequence and its attendant operational elements may be inserted into each vector. In such an embodiment, the host organism would produce greater amounts per vector of the desired IL-1 inhibitor. The number of multiple copies of the DNA sequence which may be inserted into the vector is limited only by the ability of the resultant vector, due to its size, to be transferred into and replicated and transcribed in an appropriate host cell.

SUBSTITUTE

SHEET

tein on SDS-PAGE and eluting at 48 mM NaC! from a Mono Q FPLC column under the specified conditions. Additionally, to achieve the objects and in accordance with the purposes of the present invention, pharmaceutical compositions QC S ET WO 89/11540 PCTIUS89/02275 Other Microorganisms Vectors suitable for use in microorganisms other than E. coli are also contemplated for this invention. Such vectors are described in Table 1. In addition, certain preferred vectors are discussed below.

SUBSTITUTE

SHEET

REGULATED

PROMOTERS INDUCER TRANS

CRIPTION

TERMINATOR

TABLE 1

MRNA

STABSIL-

IZATION

TRANSCRI P- TI ON AL START SITE LEADER PEPTIDE

RS

BINDING

SITE,

HOST MARKER E. coli Lac TaG 2 Lampda pL Trp I PTG i ncreased temperature IAA addition or tryptophan depletion rrnB 6 rrnC 7 ompA 8 lambda trp 1 bla 11 OMPAI 2 phoS ampicillinl 4 tetra-14 cycline 1 ,1 chloram-1 Bacillus *alpha.~ E. coli rrn B.amy nlral Kanr 24 B.amy neural amylasel rrn BT.T2 protease Camnr 25 protease *sub- 18 B.amy a~gha- B.amy a, hatii 9amylase amylase- *-3B.subt. spac-T 26 IPTG subtilisin 3 Pseudo- Trp 27 IAA addition, phos- 28 sulfo p Trp coli) nonas coli) or tryptophan pholipase 59 amide Lac depletion exotoxin A 9 strep- 3 coil) IPTG tomyc in 0 Tac coli) Yeast Ga 3 2 3 Glucose Cyc 1 Invertase 6 U ra3 3 8 depletion Una Acid ph- Leu 23 A 13, and phatase 4galactose Alpha Alpha His 3 IIGlucose Factor Factor Tap 1 Pho 5 depletion Sac 2 Phosphate depletion *nnregula ted L11n1101tors Which have been isolated in a purified form.

Preferably, the IL-1 in'hibitors of the present invention are derived from human monocyte conditioned medium where the mronlocytes are grown on Ig,G coated vessels. :n addition, the WO 89/11540 PCT/US89/02275 1. Backmnan, Ptashne, M. and Gilbert, W. Proc. Nat!.

'cad. Sci. USA 73, 417 4-Al178 (1976) 2. de Boer, Comstock, and Vasser, M. Proc.

Natl. Acad. Sci. USA 80, 21-25 (1983).

3. Shimatake, H. and Rosenberg, M. Nature 292, 128-132 (1981).

4. Derom. Gheysen, D. and Fiers, W. Gene 17, 15-51 (1982).

Hallewell R.A. and Entage, S. Gene 9, 27-47 (1980).

6. Grosius, Dull, T. Sleeter, D. and Noller, H.

J. Mol. Biol. 148 107-127 (1981).

7. Normanly, J. Ogden, Hiorvath, S.J. and Abelson, J.

Nature 321, 213-219 (1986).

8. Belasco, Nilsson, von Gabain, A. and Conen, S.N. Cell 46, 245-251 (1986).

9. Schrnelssner, McKenney, Rosenberg M. and Court, D. J. Mol. Biol. 176, 39-53 (1984).

Mott. Galloway, J.LJ. and Platt, T. EMBO J. 4, 1887-1891 (1985).

11. Koshland, D. and Botstein, D. Cell 20, 749-760 (1980).

12. Movva. Kakamura, K. and Inouye, M. J. Mol. Biol.

143, 317-328,(1980).

13. Surin, Jans, Fimmel, Shaw, Cox, G.B. and Rosenberg, M.J. Bacteriol. 157, 772-778 (1984).

14. Su .liffe, J.G. Proc. Natl. Acad. Sci. USA 75, 3737-3741 Peden, K.W.C. Gene 22, 277-280 (1983).

16. Alton, N.K. and Vapnek, D. Nature 282, 864-869 (1979).

17. Yang, Galizzi, and Henner, D. Nuc. Acids Res.

11(2), 237-248 (1983).

18. Wong. Price, Goldfarb, and Doi, R.M.

Proc. Natl.. Acad. Sci. USA 81, 1184-1188 (1984).

19. Wang. and DoiL, R.M. J. Biol. Chem. 259, 8619-8625, (1984).

C-J7, 3T -17U TE SH E ET proteins. Preferrably, the-IL-1 inhibitors of the present invention are at least 90% pure and preferably 95-) pure.

SY~T1TUTESH~ET WO 89/1154' 21.

22.

23.

24.

0 PCT/US89/022l7 -23- LsIn. Quinn, L.A. Rodriquez, R.L. J. Cell Biochem.

Suppl. 19B), p. 198 (1985)1.

Vasantha, Thompson, Rhodes, Banner, C., Nagle, and Filpula, D. J. Bact. 159(3), 811-819 (1984).

Palva, Sarvas, Lehtovaara, Sibazkov, and Kaariainen, L. Proc. Natl. Acad. Sci. USA 79, 5582-5586 (1982).

Wong. Pricee, Goldfarb, and Doi, R.H.

Proc. Natl. Acad. Sci. USA 81, 1184-1188 (1984).

Sullivan, Yasbin, and Young, F.E. Gene 29, 21-46 (1984).

Vasantha, Thompson, Rhodes, Banner, C.

Nagle, and Filpula, D. J. Bact. 811-819 (1984).

26. Yansura, D.G. and H-enner, D.J. PNAS 81, 439-443 (1984).

27. Gray, McKeown, Jones, Seeburg, P.H.

and Iieyrieker, H.L. Biotechnology, 161-165 (1984).

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

29. Liu, P.V. J. Infect. Dis, 130 (suppl), 594-599 (1974).

Wood, Hollinger, and Tinrqo1, M.B. J. Bact.

145, 1448-1451 (1981).

31. St. John, T.P. and Davis, R.W. J. Mol. Biol. 152, 285-315 (1981).

32. Hopper, and Rowe, L.B. J. Biol. Chem. 253, 7566-7569 (1978).

33. Denis, Ferguson., J. and Young, E.T. J. Biol. Chem.

258, 1165-1171 (1983).

34. Lutsdorf. L. and Megnet, R. Archs. Biochen. Biophys.

126, 933-944 (1968).

Meyhack, B. Bajwa, N. Rudolph, M. and IHinnen, A. EMBO.

J. S, 675-680 (1982).

36. Watson, M.E. Nucleic Acid Research 12, 5145-5164 (1984).

37. Gerband, C. and Guerineala, M. Curr. Genet. 1, 219-228 EUSTFTUTE SHEE 11141 1idauz-a. or synitfetic DNA sequence may be used to direct production o 'the IL-1 inhibitors. This method comprises: CJ22TrrUTES:::Jr WO 89/11540 PCT/UIS89/02275 -24- 33.

39.

Hiinnen, A. Hiicks J.B. and Fink, G.R. Proc. Nat!. Acad.

Sci. USA 75, 1929-1933 (19,78).

~jabbar, Sivasubramamian, N. and Nayak, D.P. Proc.

Natil. Acad. Sci. USA 82, 2019-2023 (1985).

i;3 T 1T UT E S H EET -uu es nown, other synthetic polynucleotide sequences will be available to one of ordinary skill in the art. As an example of the current state of the art relating to q i *;i.l -i u.

'wo,5WO540 PCI!US89/02275 Pseudomonas Vectors Several vector plasmids which autonomously replicate in a broad range of Gram negative bacteria are preferred for use as cloning vehicles in hosts of the genus Pseudomonas. Certain of these are described by Tait, Close, T.J., Lundquist, Hagiya, Rodriguez, 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 Topic in Microbiology and Immunology 96:31-45 (1982), each of which is specifically incorporated herein by reference.

One particularly preferred construction would employ the plasmid RSF1010 and derivatives thereof as described by Bagdasarian, Bagdasarian, Coleman, and Timmis, K.N. in Plasmids of Medical, Environmental'and Commercial Imoortance, Timmis, K.N. and Puhler, A. eds., Elsevier/North Holland Biomedical Press (1979), specifically incorporated herein by reference. The advantages of RSF1010 are that it is relatively a small, high copy number plasmid which is readily transformed into and stably maintained in both E_ coli and Pseudomonas species. In this system, it would be preferred to use the Tac expression system as described for Escherichia, since it appears that the E. coli trp promoter is readily recognized by Pseudomonas RNA polymerase as set forth by Sakagucki, K. in Current Topics in Microbiology and Immunology 96:31-45 (1982) and Gray, McKeown, K.A., Jones, Seeburg, and Heyneker, H.L. in Biotechnology, Feb. 1984, pp. 161-165, both of which are specifically incorporated herein by reference. Transcriptional activity may be further maximized by requiring the exchange of the promoter with, an E. coli or P. aeruqinosa trp promoter. Additionally, the lacd gene of E. coli would also be included in the plasmid to effect regulation.

Translation may be coupled to translation initiation for any of the Pseudo*,onas proteins, as well as to initiation CU~iTUTE SHEET an IL-1 inhibitor in a vector and cell capable of amplifying and expressing all or part of that CDNA; suTITLTE SHEET 1 WO 89/11540 PCT/US89/02275 sites for any of the highly expressed proteins of the type chosen to cause intracellular expression of the inhibitor.

In those cases where restriction minus strains of a host Pseudomonas species are not available, transformation efficiency with plasmid constructs isolated from E. coli are poor. Therefore, passage of the Pseudomonas cloning vector through an r- m+ strain of another species prior to transformation of the desired host, as set forth in Bagdasarian, M., et al., Plasmids of Medical, Environmental and Commercial Imoortance, pp. 411-422, Timmis and Puhler eds., Elsevier/North Holland Biomedical Press (1979), specifically incorporated herein by reference, is desired.

iii) Bacillus Vectors Furthermore, a preferred expression system in hosts of the genus 3acillus involves using plasmid pUB10 as the cloning vehicle. As in other host vectors system, it is possible in Bacillus to express the IL-li of the present invention as either 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 constructing and testing various genes as described by Dubnau, Gryczan, Contente, and Shivakumar, A.G.

in Genetic Engineering, Vol. 2, Setlow and Hollander eds., Plenum Press, New York, New York, pp. 115-131 (1980), specifically incorporated herein by reference. For the expression and secretion of the IL-li from B. subtilis, the signal sequence of alpha-amylase is preferably coupled to the coding region for the protein. For synthesis of intracellular inhibitor, the portable DNA sequence will be translationally coupled to the ribosome binding site of the alpha-amylase leader sequence.

Transcription of either of these constructs is preferably directed by the alpha-amylase promoter or a derivative thereof. This derivative contains the RNA polymerase recog- Snition sequence of the native alpha-amylase promoter but incorporates the lac operator region as well. Similar hybrid SUBSTITUTE SHEET I i 1 I 1 S uL y cne two restriction sites located within and closest to the end portions of the appropriate gene or sections of the ge:e. The DNA segment containing the appropriate gene is then removed Si UBSTITUTE SHEET WO 89/11540 PCT/US89/02275 -27promoters constructed from the penicillinase gene promoter and the lac ooerator have been shown to function in Bacillus hosts in a regulatable fashion as set forth by Yansura, D.G.

and Henner in Genetics and Biotechnology of Bacilli, Ganesan, A.T. and Hoch, eds., Academic Press, pp. 249-263 (1984), specifically incorporated by reference. The lacI gene of E. coli would also be included in the plasmid to effect regulation.

(iii) Clostridium Vectors One preferred construction for expression in Clostridium is in plasmid pJUl2, described by Squires, C.H. et al., in J.

Bacteriol. 159:465-471 (1984) and specifically incorporated herein by reference, transformed into C. perfrinaens by the method of Heefner, D.L. et al., as described in J. Bacteriol.

159:460-464 (1984), specifically incorporated herein by reference. Transcription is directed by the promoter of the tetracycline resistance gene. Translation is coupled to the Shine-Dalgarno sequences of this same tetr gene in a manner strictly analogous to the procedures outlined above for vectors suitable for use in other hosts.

(iv) Yeast Vectors Maintenance of foreign DNA introduced into yeast can be effected in several ways as described by Botstein, D.

and Davis, in The Molecular Biology of the Yeast Saccharomyces, Cold Spring Harbor Laboratory, Strathern, Jones and Broach, eds., pp. 607-636 (1982), specifically incorporated hereby by reference. One preferred expression system for use with host organisms of the genus Saccharomvces harbors the IL-li gene on the 2 micron plasmid. The advantages of the 2 micron circle include relatively high copy number and stablility when introduced into cir° strains.

These vectors preferably incorporate the replication origin and at least one antibiotic resistance marker from DBR322 to allow replication and selection in E. coli. In addition, the plasmid will preferably have the two micron sequence and the yeast LEU2 gene to serve the same purposes in LEU2 defective mutants of yeast.

S' TiTu TE SHEET L nvention contain various operational elements. These "operational elements," as discussed herein, S9STiTUTE

SHEET

I

WO 89/11540 PCT/US89/02275 If it is contemplated that the recombinant IL-1 inhib- Sitors will ultimately be expressed in yeast, it is preferred that the cloning vector first be transferred into Escherichia coli, where the vector would be allowed to replicate and from which the vector would be obtained and purified after amplification. The vector would then be transferred into the yeast for ultimate expression of the IL-1 inhibitor.

Mammalian Cells The cDNA for the IL-1 inhibitor will serve as the gene for expression of the inhibitor in mammalian cells. It should have a secuence that will be efficient at binding ribsomes such as that described by [Kozak, in Nucleic Acids Research 15:8125-8132 (1987), specifically incorporated herein by reference,] and should have coding capacity for a leader sequence (see sectien to direct the mature protein out of the cell in a processed form. The DNA restriction fragment carrying the complete cDNA sequence can U be inserted into an expression vector which has a transcriptional promoter and a transcriptional enhancer as described by Guarente, L. in Cell 52:303-305 (1988) and Kadonaga, J.T.

et al., in Cell 51:1079-1090 (1987), both of which are specifically incorporated herein by reference. The promoter may be regulatable as in the plasmid pMSG (Pharmacia Cat. No.

27450601) if 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), specifically incorporated herein by reference, so that the mRNA transcribed from this vector is processed properly. Finally, the vector will have the replication origin and at least one antibiotic resistance marker from pBR322 to allow replication and selection in E. coli.

In order to select a stable cell line that produces the IL-1 inhibitor, the expression vector can carry the gene for a selectable marker such as a drug resistance marker or carry a complementary gene for a deficient cell line, such as a SUBSTITUTE SHEET duced extent, in the absence of, for example, i isopropylthio-beta-D-galactoside. In this situation, the transformed microorganisms containing the DNA sequence may be SUBSTITUTE SHEET WO89/11540 PCT/US89/02275 dihydrofolate reductase (dhfr) gene for transforming a dhfrcell line as described by Ausubel et al., supra. Alternatively, a separate plasmid carrying the selectable marker can be cotransformed along with the expression vector.

4. Host Cells/Transformation i The vector thus obtained is transferred into an appropriate host cell. These host cells may be microorganisms or mammalian cells.

Microorganisms It is believed that any microorganism having the ability to take up exogenous DNA and express those genes and attendant operational elements may be chosen. After a host organism has been chosen, the vector is transferred into the host organism using methods generally known to those of ordinary skill in the art. Examples of such methods may be found in Advanced Bacterial Genetics by R. W. Davis et al., Cold Spring Harbor Press, Cold Spring Harbor, New York, (1980), which is specifically incorporated herein by reference. It is preferred, in one embodiment, that the transformation occur at low temperatures, as temperature regulation is contemplated as a means of regulating gene expression through the use of operational elements as set forth above. In another embodiment, if osmolar regulators have been inserted into the vector, regulation of the salt concentrations during the transformation would be required to insure appropriate control of the foreign genes.

It is preferred that the host microorganism be a facultative anaerobe or an aerobe. Particular hosts which may be preferable for use in this method include yeasts and bacteria. Specific yeasts include those of the genus Saccharomvces, and especially Saccharomvces cerevisiae. Specific bacteria include those of the genera Bacillus, Escherichia, and Pseudomonas, especially Bacillus subtilis and Escherichia coli. Additional host cells are listed in Table I, supra.

SUBSTITUTE SHEET LAo squecr WI I a rDOosome recognizes dnu amu. Lu in the initiation of protein synthesis as set forth in Gold, L.,et al., Ann. Rev. Microbio. 35:557-580; or Marquis,

D.M.,

et al., Gene 42:175-183 (1986), both of which are SUBSTITUTE

SHEET

WO 89/11540 PCT/US89/02275 Mammalian Cells The vector can be introduced into mammalian cells in culture by several techniques such as calcium phosphate:DNA coprecipitation, electroporation, or protoplast fusion. The preferred method is coprecipitation with calcium phosphate as described by Ausubel et al., supra.

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

Culturina Enoineered Cells rhe host cells are cultured under conditions appropriate for the expression of the IL-1 inhibitor. These conditions are generally specific for the host cell, and are readily determined by one of ordinary skill in the art in 4 light of the published literature regarding the growth conditions for such cells and the teachings contained herein. For example, Bergey's Manual of Determinative Bacteriology, 8th Ed., Williams Wilkins Company, Baltimore, Maryland, which is specifically incorporated herein by reference, contains information on conditions for culturing bacteria. Similar information on culturing yeast and mammalian cells may be obtained from Pollack, R. Mammalian Cell Culture, Cold Spring Habor Laboratories (1975), specifically incorporated herein dl by reference.

Any conditions necessary for the regulation of the expression of the DNA sequence, dependent upon any operational elements inserted into or present in the vector, would be in effect at the transformation and culturing stages. In one embodiment, cells are grown to a high density in the presence of appropriate regulatory conditions which inhibit the expression of the DNA sequence. When optimal cell density is SUc TTUTE

SHEET

S' i r- j _l -u L xI. eu r rom extracellular protein. Thirdly, in the case of some of the proteins prepared by the present invention, the presence, of the leader sequence may be necessary to locate the completed protein in an environment where it may fold to assume its SUBSTITUTE SHEET I WO 89/11540 PCT/US89/02275 approached, the environmental conditions are altered to those appropriate for expression of the DNA sequence. It is thus contemplated that the production of the IL-1 inhibitor will occur in a time span subsequent to the growth of the host cells to near optimal density, and that the resultant IL-1 inhibitor will be harvested at some time after the regulatory conditions necessary for its expression were induced.

6. Purification IL-li Produced From Microorganisms In a preferred embodiment of the present invention, the recombinant IL-1 inhibitor is purified subsequent to harvesting and prior to assumption of its active structure.

This embodiment is preferred as the inventors believe that recovery of a high yield of re-folded protein is facilitated if the protein is first purified. However, in one preferred, alternate embodiment, the IL-1 inhibitor may be allowed re-fold to assume its active structure prior to purification.

In yet another preferred, alternate embodiment, the IL-1 inhibitor is present in its re-folded, active state upon recovery from the culturing medium.

In certain circumstances, the IL-1 inhibitor will assume its proper, 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 occur if DNA coding for an appropriate leader se- Squence has been linked to the DNA coding for the recombinant protein. If the IL-1 inhibitor does not assume its proper, \I active structure, any disulfide bonds which have formed Sand/or any noncovalent interactions which have occurred will first be disrupted by denaturing and reducing agents, for example, guanidinium chloride and beta-mercaptoethanol, before the IL-1 inhibitor is allowed to assume its active structure following dilution and oxidation of these agents under controlled conditions.

For purification prior to and after refolding, some combination of the following steps is preferably used: anion SSUBTIT UTE SHEE

IT

ys. i.Lade in tne selection oz transformants. Additionally, the presence of such a selectable marker in the cloning vector may be of use in keeping contaminating microorganisms from multiplying in the culcure SUBSTITUTE SHEET WO89/11540 PCT/US89/02275 axcnange ciiromatography (MonoQ or DEAE-Sepharose) gel filtration chromatography (superose), chromatofocusing (MonoP), and hydrophobic interaction chromatography (octyl or phenyl sepharose). Of particular value will be antibody affinity chromatography using the IL-li-specific monoclonal antibodies S(described in Example 3).

IL-li Produced from Mammalian Cells IL-li produced 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 will be apparent to those skilled in the art that various modifications and variations can be made in the processes and products of the present invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.

It is to be understood that application of the teachings of the present invention to a specific problem or environment will be within the capabilities of one having ordinary skill in the art in light of the teachings contained herein. Examples of the products of the present invention and representative processes for their isolation and manufacture appear in the following.

The following examples illustrate various presently preferred embodiments of the present invention. The publications provided in this examples are specifically incorporated Sby reference herein.

i <EXAMPLES Example 1 Protein Preparation 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 SUBSTITUTE SHEET cated and transcribed in an appropriate host cell.

SUBSTITUTE

SHEET

WO 89/11540 PCT/US89/02275 incomiplete Freund's adjuvants, hypoxanrhine, 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 Drs. John Kappler and Philippa 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 the NJC/IRM, Denver, Colorado. Monoclonal mouse anti-rabbitt IgG antibodies were purchased from AIA reagents, Aurora, Colorado. Low methionine RPMI was made using a Select-Amine kit from GIBCO Labcratories, Grand Island, N.Y.

35 S]-methionine, diphenyloxazole, and 14 C]-iodoacetic acid Swere 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, SInc., 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. Trifluroacetic acid and guanidine hydrochloride were obtained from Pierce Chemicals, Rockford, Illinois. Endoproteinase Lys C was obtained from Boehringer Mannheim Biochemicals, Indianapolis, Indiana. The microtitering plates used for PGE2 ELISA were Nunc-Immuno Plate I obtained from Intermountain Scientific Corporation, 1 Bountiful, Utah. The plates used for hybridoma production were from Costar, Cambridge, Massachusetts.

B. Generation of Monocvte 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, underlayed with Co3aSTTUTE SHEETl CUBSTITUTE SHEET WO 89/11540 PCTIJS89/0'22575

A

V

Lymphoprep and spun at 400 xg for 30' at room temperature.

The mononuclear fraction was taken (typically 4-5 X 109 cells were obtained per donor), washed 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 papx G200 (6 X 107 cells in 10ml per 100 -m dish).

All reagents contained less than 10 pg/ml LPS. The cells were cultured 24-48 hr, and the resulting conditioned medium constituted the crude IL-1 inhibitor (IL-li) supernatant.

Typically, the cells from one donor yielded 700-900 ml crude IL-li supernatant.

C. Assays for the IL-1 Inhibitor Two IL-1 assays have been used routinely to detect the IL-li. Thymocytes (1 x 106 cells from 4 to 6 week old C3H/HeJ mice) respond to 1.0 unit/ml of recombinant human IL-1 plus 1 ug/ml phytohaemaglutinin by proliferating half-maximally, as measured by 3 H-thymidine incorporation or uptake of the tetrazolium salt MTT (Mosmann, J. Immunol.

Method, 65:55-61 (1983)) after three days of stimulation.

Crude IL-li supernatant fully inhibits this proliferative response at a 1/10 dilution. Human dermal fibroolasts (1 x 10 cells per well in a 96 well plate) typically respond to units/ml recombinant human IL-1 by secreting, at 6 hours of stimulation, approximately 50,000 pg/ml PGE 2 that can be measured by ELISA. This assay is as sensitive to IL-li as is the thymocyte assay.

D. Metabolic Labelina of the IL-1 Inhibitor The IL-li was metabolically labeled by culturing mononuclear leukocytes for 48 hours on IgG-coated plates (as described in B) in serum-free RPMI containing only 0.75 ug/ml cold methionine (15ug/ml is normal) and to which was added mCi 35 S-methionine (1151 Ci/mmol) per 107 cells. Control labelings were performed identically except that the plates were coated with fetal calf serum rather than IgG. Assays on such control supernatants showed that very little IL-li was secreted when the cells were cultured on fetal calf serum-coated plates.

SIS uiSTiTUT£ SHEET 0 =oI I P1 Ui aU hr U0 C 0 SSUBSTTUTE

SHEET

WO89/11540 PCT/US89/02275 E. Purification of the IL-1 Inhibitor Protein Crude IL-li suoernatants were made 1.0 M in sodium chloride, incubated on ice for 1 hour and centrifuged at 10,000 rpm for 15 minutes. The supernatants, which contained all of the inhibitor activity but only 20% of the initial protein, were then dialyzed extensively at 4 0 C versus 0.025M Tris, pH 7.6 containing 0.1% sucrose (the A buffer) for gradient fractionation of proteins on a Mono Q anion exchange column.

Following dialysis the inhibitor-containing solutions were recentrifuged at 10,000 rpm for 15 minutes and then passed through 0.22u nylon filters. The supernatants were typically combined with 10 ml of similarly prepared supernatant from a metabolic labeling and loaded onto Mono Q-Superose (Pharmacia SPLC) columns with bed volumes of either 1.0 ml or 8,0 ml, Swashed with A buffer until the OD 280 of the effluent returned to baseline, and carefully chromatographed using a linear sodium chloride gradient (.025M to .10M) in buffer A. Column fractions were collected and analyzed for radioactivity and bioactivity. Samples of each fraction were also run on reduced 12.5% SDS-PAGE, silver stained, permeated with diphenyloxazole, dried and put onto film to obtain autoradiographic data. Figure la shows the protein profile of the Mono Q chromatography of 40 ml crude Il-li supernatant mixed with 3 ml of metabolically labeled IL-li supernatant.

Superimposed are the amount of radioactivity found in 50 ul of each fraction as well as the IL-li bioactivity as measured in the PGE 2 -production assay. Two major and one minor radio- Sactive species are shown that perfectly correlate with three Speaks of bioactivity. Figure lb shows the similar chromatography of 15 ml of crude Il-li supernatant mixed with 3 ml of supernatant from monocytes metabolically labeled on plates coated with fetal calf serum (FCS) rather than IgG.

The levels of the three radioactive species discussed above are markedly diminished. Figure 2a shows silver stained gels run on the fractions from the regions of interest in the chromatographies shown in Figures la and lb. Note that the ;.C'STsTUTE SHEET J Jr 1 4-J 0 J.0 4).

19. Wang. and Doi, R.M. J. Biol. Chem. 259, 8619-8625, (1984).

S.J=STiTUTESHEET WO 89/11540 PCT!US89/02275 -36- fractions of peak radioactivity and bioactivity in Figure la (fractions 52 and 59) both show a major band at 22 Kd (marked with arrows) on SDS-PAGE. The third species (fraction 48 in Figure la) shows a band at 20kD on SDS-PAGE. Gel filtration experiments on crude IL-li have shown that the active imolecule has a molecular weight of 18-25 Kd. Figure 2b is an autoradiogram of the gels shown in Figure 2a. It can be readily seen that the protein bands at 20 and 22 Kd are the major radioactive species in those fractions.

Summarizing these results, we have shown that the metabolic labeling of monocytes plated on petri dishes coated with IgG results in radioactive species that are only poorly produced if the cells are plated on dishes coated with FCS.

These induced radioactive species perfectly co-chromatograph with several species of IL-li bioactivity on Mono Q, and gels and resulting autoradiograms show that the three major induced molecules are proteins of the predicted molecular weight for IL-li.

The IL-li molecules were further purified for sequencing in two ways. First, Mono Q fractions with peak bioactivity Sand radioactivity were loaded onto a C4-reversed phase column and eluted with an H 2 0/0.1%TFA: acetonitrile/0.1%TFA gradient. Since the IL-li molecule was trace labeled, samples from each fraction were directly counted for radioactivity and were also analyzed by SDS-PAGE followed by autoradiography. Figure 3a shows such a chromatograph with the radioacti ity pattern superimposed. The silver stained gels run on samples from each fraction (Figure 3b) and subsequent autoradiograms of the gels (Figure 3c) shows that the IL-li molecule is found in fractions 32-36. These fractions were dried down and sequenced. Alternatively, the peak Mono Q fractions were dried by Speed Vac, resuspended in 0.4 ml 0.05 M NH 4

HCO

3 and directly chromatographed two times on a X 300mm Superose 12 gel filtration column (Pharmacia FPLC) equilibrated in the same buffer, as shown in Figs. 4a and 4b.

Fractions were collected and samples of each were tested for S3uSTiTUTE SHEET 37. Gerband, C. and Guerineau, M. Curr. Genet. 219-228 (1-30).

S-SUBSTiTUTE SHEET WO 89/11540 PCT/US89/02275 ad ioactiviLy and bioact ivty and were analyzed by silver stained and autoradiographed SDS-PAGE. Appropriate fractions were then dried on a speed vac and sequenced.

Example 2 Proposed Secuencing of the IL-1 Inhibitor Prior to sequencing, samples were dissolved in 6 M guanidine-HC, pH 8.6, reduced for 4 hours at 37 0 C under N 2 with 100-fold molar excess dithiothreitol over protein, and alkylated for 1 hour with 400-fold excess 14 C-iodoacetic acid. In that case, the reactions would be 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 which may have been reduced and alkylated would be digested with cyanogen bromide or proteolytic enzymes using methods known to those of ordinary skill in the art. Reactions will be dried, dissolved in 0.1% TFA/H 2 0, and peptides will be separated using a C8-reverse phase column.

Example 3 Purification and Secuencing of the Soecies of IL-1 Inhibitors A. IL-li-X, IL-li-a and IL-li-b Soecies The Mono Q purification of IL-li resolves the biological activity into three major species, as shown in Figure la 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 Figure 2a, reveal pertinent species at kD, 22 kD, and 22kD, respectively. Western analysis of such gels, using the mouse antisera discussed in Example 4 below, stains all three of these species. When IL-li is prepared from cells metabolically labeled with 35 S-methionine, during growth on plates coated with IgG, each of these bands is radioactive (as shown in Figure 2b, the autoradiogram o the above-mentioned gel). Based on the logic discussed in Example 1, name that parallel cells incubated in a norn- i inducing condition do not produce the IL-li bioactivity and do not produce these radioactive bands, we can conclude that i UTE SHEET WO89'11540 PCT/US89/02275 rchlese three species account for the biological activity. We have tentatively named these species IL-li-X, IL-li-a, and IL-li-b, respectively.

B. Purification and Seauencino of IL-li-X Mono Q fractions containing IL-li-X and/or IL-li-a were further purified by reversed-phase HPLC chromatography on a Synchropak RP-4 (C4) column, and radioactive species were submitted for sequence analysis. Numerous attempts at directly sequencing RP-HPLC-purified IL-li-a and IL-li-b have failed, suggesting that they are chemically blocked at their N-termini. However, one preparation of IL-li-a (IL-li-aB2p42) yielded the following sequence: 1 5 10 15 RP S G RK SS K M Q A F I S D V N Q and subsequent preparations of IL-li-X, similarly purified by I C4 RP-HPLC, have produced the same sequence: PrepKxF24 1 5 10 15 and M A F I D V N K F PrepKxF23 RP RK L KM Q A F I These are obviously part of the sequence found in the A initial attempt at sequencing IL-li-a. It is the inventors' conclusion that the sequence data shown is the N-terminus of the 20 kD species called IL-li-X.

In these and all subsequent sequences an underlined position indicates either an inability to identify a residue or that ambiguity 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 that Ssequencing step, and the more likely correct residue is on top.

C. Generation, Purification, and Sequencina of PeDtides of IL-li-a and IL-li-b Since 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 CI /U-ST7TUTE H£ETi u a yene or coil woul also be incluued in the plasmid to effect regulation.

Translation may be coupled to translation initiation for any Of the Pseudoh :nas proteins, as well as to initiation USSTJTUTE

SHEET

WO089/11540 PCT/US89/02275 -29- 4.6x25 am C3-RPHPLC column (Zorbax Protein Plus), an acceptable alternative to the C-4 columns used in all previous experiments. Very gradual gradients acetonitrile per minute at 0.5 ml/min) resolved the IL-li-a (Figure 8a,b) or IL-li-b (Figure 9a) away from the major contaminating radioactive species, human lysozyme. The identities of the purified species were confirmed by the presence of a single, radioactive, 22 kD protein on SDS-PAGE and subsequent autoradiograms (Figures 8c,d and 9b). The proteins were hand-collected into siliconized glass tubes and to each was added 25 ml of a 0.2% Tween-20 solution. The IL-li-containing fractions were then reduced in volume on a Speed-Vac to 50 ul, brought up to 300 pl by the addition of [1 1% NH4HCO3, 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 carried out at 37°C for 16 hr, and then the volume of the reaction mix was reduced to 50 ml on a Speed Vac.

In the case of IL-li-a, the sample was directly chromatographed, whereas the IL-li-b sample was first reduced by the addition of 5ml of 50 mM dithiothreitol in 2 M Tris, pH reacted for 30 min at 37 0 C, and then carboxymethylated by addition of 1.1 umole 3 H-iodoacetic acid in 10 ml ethanol (reacted 30 min at 37 0 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 pl/min 1 using a Beckman HPLC outfitted with microbore hardware and microbore-compatible pumps. A 200 min 0-100% linear gradient was used (H20/0.1% TFA to acetonitrile/0.1% TFA). The peptide separations are shown in Figures 10 and 11. The sequence information obtained is as follows: SO "zGTITUTE SNE L~eeQ ii:~ UtLE1 Vdt IVL Lull Ld.±u ,L LI±= Ju~ L OZ nition sequence of the native aipha-amylase promoter butin corporates the lac operator region as well.

Similar hybrid SUBSTITUTE SHEE T 11 WO 89/11540 PCTIIJS89/02275, -a4na v z *Z.

mQAF RaLysc-53 D V N 1 5 10 15 20 K F YL N N Q LVA Y LQ GP N VN L E E01D N N RaLysC-61 F AT T y R HV RaLysC-31 1 F Y F Q E RaLysC-37 G E 0 OD F T

V

s L QL E AN R LQ L E:V 15 N I T s RO0SO0L GEO0 15 T DL LE N E T RBAspN-51 1 Q K T F D V NP I E PY RNNQ LV A Y L QG PN VN L RBAspN- 43 1 5 D E G V M TKF YFO0 RBAsnN-3 9 1 5 P PSG R K 101 K S SF MQ AF RT0 4. D K RF AF I?'R S U 1jT ITv'-;E S H2E T The method of claimn 9 wherein said t sequence allow replication and selection in E. ccli. in addition, the plasmid will preferably have the two micron sequence and the yeast LEU2 gene to serve the same purposes in LEU2 defectilve mutants of yeast.

ZT TUTE SHIEET

J

m 'WO 89/11540 PCT/YUS89/02275 -al ,1V.

Z,

Q

iN Lir. K K 1 S Two Of the oeptide sequences are obviously related to that which was obtained earlier from IL-li--X. One of these, RaLysC-4l, is an IL-li-a seq uence, arid the other, is an IL-li-b sequence, arguing that the three species of IL-li are at least closely related proteins if not chemically and/or physically modified forms of a single origir.il IL-li molecule. If the listed sequences are combined, the following composite sequences result: RQLYSC-41 HaLysC-53 I I 1L-1mB2p42 IGKP R PSG R KS S K W0A FR ISD VN Q KT F Y L R NQL VAY LQ GP N VN L E E 0D N I -RaLyzC-31- I

I

RaLyzC-35,37

S

G I G 0 N D E T R &IQ L E A V R T D L h RUY&C-61----l RBAS5P-2 D KR FA F I R~~ These composite sequences appear to be present in no other known polypeptides listed in the most recently updated Protein Identification Resource Database (PIR 16.0). The inventors believe that these sequences, or minor variants thereof, represent a class of molecules that are capable of acting as IL-l inhibitors.

Example 4 Prenaration of Antibodies Soecific For the IL-l inhibitor Ten week old BALB/c mic e were injected subcutaneously with IL-li that was partially purified (400-fold) from crude suriernatants by Mono Q-chromar-ography, dialyzed versus PBS, 6, 61TUTirwSHEET 4 A WO089/11540 I I u I C.iA i P CT/US 89/0 2275- 3 0 350 360 a selectable marker such as a drug resistance marker or carry a complementary gene for a deficient cell line, such as a S USSTITUTE

SHEET

WO89/11540 PCT/US89/0227 and emulsified with Complete Freund's Adjuvant. Each mouse received the IL-li purified from 5 ml of crude supernatant.

The mice were boosted every two weeks with an equivalent amount of IL-li emulsifed with Incomplete Freund's Adjuvant, and serum samples were taken from the tails seven days after each boost. Antisera were tested for anti-IL-li activity by Western analysis of transblots of the immunogen run on SDS-PAGE, as shown in Fig. 5a. Fig. 5b shows that all of the mice were making anti-IL-li antibodies after three injections of IL-li.

Since monoclonal antibodies will be of great value in cloning the IL-li gene from an expression library, purifying the recombinant IL-li protein, and studying the biology of the molecule, we have begun the process of making a battery of monoclonal antibodies specific for Il-li. To produce B cell hybridomas, the above mice were injected intravenously with the same amount of IL-li in saline 24 hours prior to removal of the spleens. Splenocytes were teased out of the spleens into cold balanced salt solution (BSS), washed two times with BSS, mixed with P3 myeloma cells at a ratio of 2 x 7 08 107 P3 cells per 10 splenic B cells and spun down. The cells were fused by the dropwise addition of 1 ml of warm, gassed C0 2 PEG 6000 (40% polyethylene glycol 6000 minimal essential medium) to the dry pellet. Fused cells were washed with BSS and resuspended in 10 ml of rich media FBS) containing 2 x 105 peritoneal cells per ml and the pellet was gently broken up using a 10 ml pipet. The volume was adjusted to 20 ml with the addition of more peritoneal cells in media, and the cells were plated out in 96 well plates at 0.1 ml/well. Plates were placed in a gas incubater and treated in the following manner thereafter: Day 1 Add 3x HAT (hypoxanthine, aminopterin, thymidine) in rich medium to a final concentration of lx Day 5 Change medium, replacing with 200 ul lx HAT in rich medium SSUBSTiTUTE SHEET SpCT./' S89/02275 WO 89/11540 24. The i so tred D4A sequence of claim 17, wherein said and Escherichia coli. Additional host cells are listed in Table I, suDra.

SUBSTITUTE

SHEET

'WO 89/11540 PCT/US89/02275 Day 10 Begin c.ec ing for hybrid growth. Change medium replacing with 200 ul Ix HAT in rich medium containing 1.5 x 106 peritoneal cells per ml.

When hybrid cells are nearly confluent in a well the supernatants are transfered for testing, and the cells are gently scraped with a pipet tip and transfered to 1 ml culture wells containing lx HAT in rich medium plus 3 x 106 peritoneal cells per ml.

The supernatants from the confluent wells are tested for anti-IL-li activity using an ELISA in which partially purified IL-li (Mono Q-purified material identical to that injected into the mice) is bound to microtitering wells.

Normal mouse sera and hyperimmune antisera are used as the if negative and positive controls, respectively. Positive Ssupernatants will be retested by ELISA on plates coated with homogeneously purified IL-li and by immunoprecipitation of purified metabolically labeled IL-li. Positive cells will then be cloned by limiting dilution and injected into pristane-treated mice for the generation of ascites. Large quantities or IL-li-specific antibodies can be produced by tissue culture or by massive generation and collection of ascitic fluid in mice. Purification of these antibodies and attachment thereof to insoluble beads will produce affinity adsorbents for the purification of the recombinant IL-li protein.

Example SClonina the Il-li cDNA It was shown that monocytes plated on IgG-coated petri dishes and cultured for 24 hours in the presence of 35 S]-methionine produced 35 S]-IL-li which could be identified by its chromotographic properties on Mono Q.

In order to determine when (during the 24 hour period) IL-li was being produced at a maximal rate, plated monocytes were exposed to 35 S)-methionine (pulsed) for a short, twohour period, at which time a large excess of unlabelled S o 3.u ou tIne DNA sequence. When optimal cell density is T 1 rITUTE SHEET Ii F l ~11: i i E ift

'WO

89/11540 PCT/TS89/02275 -dadmethionine was added and incubated for an additional two hours. The medium was then collected and analyzed for ratiolabelled IL-li. This procedure was applied to monocytes at various times after'plating of IgG-coated plates and it was found that exposing monocytes to 35 S]-methionine at hours after plating produced the maximal amount of 35 S]-IL-li, indicating that IL-li mRNA in monocytes was at its maximal level 15 hours after plating on IgG.

Fresh monocytes were then plated on LPS free IgG obtained as in Example IB. After incubating in RPMI media for 15 hours at 37 0 C, the cells are washed with phosphate buffered saline then lysed with 4M guanidinium thiocyanate; mM sodium citrate, pH 7, 0.5% sarcosyl, 0.1M 2-mercaptoethanol. Total RNA was then isolated from this lysate by the AGPC method of 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) Proc.

Natl. Acad. Sci. (USA) 69:1408-1412 precipitated with ethanol and dissolved to a concentration of 0.36 ug/ul. One microgram to poly A RNA was used to prepare cDNA according to Gubler, U. and Hoffman, B. J. (1983) Gene 25:263-169.

The cDNA was incorporated into a lambda gtll expression library using Eco R1 linkers from Boehringer Mannheim catalog No. 988448 or New England Bio Lab No. 1070 and instructions provided by these manufacturers.

The resulting library, which contains 106 independent clones, was screened on E. col. Y1090 rk- (Promega Biotec) with an appropriate polyclonal antibody to 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 strepavidin-alkaline phosphatase conjugate (Bethesda Research Labs), as described by Bayer, E. A. and TT TUTE SHEET structure following dilution and oxidation of tnese agen1i under controlled conditions.

For purification prior to and after refolding, some combination of the following steps is preferably used: anion 1T3TUTE

SHEE'

i -;1 r; WO 89/11540 V PCT/US89/0227

L

I

4 WVLchek, (1979) in Methods in Biochemical Analysis, and Guesdon, J. L. Ternynch, T. and Avrameas, S. (1979) J. Histochem. Cvtochem. 27:1131-1138 and according to manufacturer's instructions.

Example 6 Preparation and Secuencing of Gene Encodino IL-li 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 attached 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 ug of linkered, sizeselected cDNA and 1 ug of EcoRI-cut and phosphatase-treated lambda GT10 was performed, and the products of this ligation reaction were packaged using GIGAPACK GOLD packaging extracts (Stratagene). This yielded a library of lx10 7 members.

In order to screen this GT10 library, oligonucleotide (antisense) probes were synthesized based on protein and peptide sequence presented in Example 3. The sequences of the probes and of their corresponding peptide sequence are as follows.

Probe #ILlil-3 TT

T

TAC GT C G NA A C C G Lys Met Gin Ala Phe Probe #ILlil-4 T TT A A A A A G T .C T T C T C G G G C C Lys Phe Phe Gin Glu Asp Probe #ILlil-5 Probe #ILlil-6 Probe #ILlil-7 T A C CA N T G N TT T A A

A

A T

A

AA C G G Met Val Thr Lys Phe Tyr Phe C TA CANTTAGT TT T T G G G C C Aso Val Asn Gin Lys Thr T TA G T T T T G N A A A T

I

'7SZIT UTE SHEET cnasea trom 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, dizhiothreitol, complete and BSTiTUT SHEET WO 89/11540 PCT/''IS89/02275 -46- G r C ,r Asn Gin Lys Thr Phe Thr Note: N A, G, C, and T Probe #ILlil-3 was 3 2P-phphophorylated at its 5' end and used to screen 3x10 5 plaques of the library. The probe hybridized reproducibly to three plaques, and out of these, one plaque was shown to also hybridize to probe #ILlil-4. This plaque, GT10-ILli-2A, was cultivated and the DNA was isolated using Lambdasorb (Promega) according to the manufacturer's instructions. GT10-ILli-2A has been deposited at American Type Culture Collection (ATCC) in Rockville, Maryland under Accession No. 40488. The DNA was digested with EcoRI, divided into five equal aliquots, and electrophoresed on a 1% agarose gel.

After electrophoresis, this gel was stained with ethidium bromide, A photograph of this gel is shown in Figure 12 a. Lanes 6, 8, 10, 12, and 14 contain the five aliquots from the EcoRI digestion. Lane 5 contains a mixture of wild-type lambda DNA cut with HindIII and OX174 RF DNA cut with HaeIII (New England Biolabs) which are useful as molecular weight markers. Figure 12a shows that GTl0-ILli-2A contains an EcoRI fragment that is 1850 base pairs in lIsigth.

In order to demonstrate more conclusively that this 1850 bp fragment carries coding sequence for the IL1 inhibitor, a Southern blot was performed as follows. The DNA fragments in the gel shown in Figure 12a were blotted onto nitrocellulose using standard methods. The nitrocellulose was then cut lengthwise into five strips such that each strip contained the DNA from lanes 6, 8, 10, 12, and 14, The strips were then individually hybridized to each of the five oligonucleotide probes (above) which were labeled at the end with 3 2 P phosphate. The oligonucleotide concentration was 1 pmole/ml and the hybridization temperatures were as follows.

LANE PROBE TEMPERATURE :-ZWUTE SHEET Human leukocvtes were obtained from normal donors by leukophoresis, resuspended in Hank's balanced salt solution (HBSS) at 1 part packed cells to 1 part HBSS, underlayed with STiTUTE SHE WO 89/11540 PCT/US89/02275 6 6 #!Llil-3 35°C 8 #ILlil-4 42°C #ILlil-5 42°C 12 #ILlil-6 14 #ILlil-7 After washing, the strips were lined up and taped together to reform the original nitrocellulose sheet. This was autoradiographed in the presence of an intensifying screen at 0 C for 24 hours. Figure 12b is a photograph of this autoradiograph. It provides evidence that all of the probes hybridize specifically to the 1850 bp fragment, proving that this fragment carries substantial coding sequences for the IL1 inhibitor.

In order to determine its DNA sequence, GT10-IL1I-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 M13 mpl9 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 protein coding sequence. In Figure 13, the DNA sequence is shown that was obtained for the protein coding region of the cDNA.

Figure 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 hypothesized amino acid sequence. The predicted amino acid sequence from the amino acid Proline to the 78th amino acid Proline agrees with the peptide sequences described in Example 3.

GUBSTTUTE SHEET latelings were performed identically except that the plates were coated with fetal calf serum rather than IgG. Assays on isuch control supernatants showed that very little IL-li was secreted when the cells were cultured on fetal calf ,.serum-coated plates.

ITUTE SHEET %TLT 8 WO 89/11540 PC T!US89/02275 Example 7 Seuuencina GT10-IL-11-2A and IL-li A portion of GT10-ILlI-2A has been sequenced and is set forth in Figure 14. The DNA encodes a protein containing amino acid sequences that are characteristic of IL-li (nucleotides 99-557). However, it is believed that several modifications may be made to this protein before it is secreted into the extracellular milieu. These modifications may or may not be essential for the protein to have activity as an IL-li.

GT10-ILli-2A encodes at least 32 amino acids N-terminal (nucleotides 3-98) to the amino terminus of the form of IL-li known as X. It is believed that included in these 32 amino acids is a secretory leader sequence that starts at the M encoded by nucleotides 24-26, directs the nascent IL-li to the extracellular milieu, and is then removed by a leader peptidase, and possible other peptidases. The extent to which this sequence is removed in forms alpha and beta of IL-li is presently unknown, 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 required for the protein to have effective IL-li activity.

Nucleotides 349-351 of GT10-ILlI-2A encode an N residue that is a part of a concensus N-glycosylation site. On the basis of their susceptibility to digestion wih N-glycanase it is believed that forms alpha and beta of IL-li are glycosylated. Since form X is not believed to be susceotible to Sdigestion with this enzyme it is believed that it is not Ss glycosylated, although this remains a possibility that could easily be demonstrated by one of ordinary skill in the art of protein sequencing using the information provided here. It is believed that glycosylation at this N residue is not required for the protein to show effective IL-li activity.

Nucleotides 99-101 of GTl0-ILli-2A encode a P (see Figure 15), but no P has been detected at this position (the N-terminus) of form X of IL-li. It is possible that this SUBSTITUTE SHEET me levels or the three radioactive species discussed above are markedly diminished. Figure 2a shows silver stained gels run on the fractions from the regions of interest in the chromatographies shown in Figures la and lb. Note that the c iTUTE SHEET S WO 89/11540 PCT/US89/02275 -a9- Sresidue has been modified in rne mature protein. It is obelieved tha modification of this residue is not essential for effective IL-li activity.

The presently unknown N-terminus residues of forms alpha and beta are not wholly detectable by Edman degradation and are likely to be modified following removal of some of the N-terminal residues of the protein encoded by GT0l-ILli-2A.

It is believed that this modification is not essential for effective IL-li activity.

Example 8 Exoression of Genes Encoding IL-li in Animal Cells Animal-cell expression of IL-li requires the following steps: a. Construction of an expression vector b. Choice of a host cell line c. Introduction of the expression vector into host cells d. Manipulation of recombinant host cells to increase expression levels of IL-li 1. IL-li expression vectors designed for use in animal cells can be of several types including strong consitutitve expression constructs, inducible gene constructs, as well as those designed for expression in particular cell types. In all cases promoters and other gene regulatory regions such as enhancers (inducible or not) and polyadenylation signals are placed in the appropriate location in relation to the cDNA sequences in plasmid-based vectors. Two examples of such constructs follow: A construct using a strong constitutive promoter region should be made using the simian virus 40 (SV40) gene control signals in an arrangement such as that found in the plasmid pSV2CAT as described by Gorman et al. in Mol. Cel. Biol. 2:1044-1051, 1982, specifically incorporated herein by reference. This plasmid should be manipulated in such a way as to substitute the IL-li cDNA for the chloramphenicol acetyltransferase (CAT) coding sequences SUBSTITUTE

SHEET

v.U L. nn4nuu3 and directly chromatographed two times on a X 300mm Superose 12 gel filtration column (Pharmacia

FPLC)

equilibrated in the same buffer, as shown in Figs. 4a and 4b.

Fractions were collected and samples of each were tested for SWO 89/11_540 PCT/US89/02275 using s'andcard olecular biological techniques (Maniatis et dl., suora), as shown in Fig. 6. An inducible gene construct should be made utilizing the plasmid PMK which contains 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 yield a metal-inducible gene construct.

2. A number of animal cell lines should be used to Sexpress IL-li using the vectors described above to produce Sactive protein. Two potential cell lines that have been well-characterized for their ability to promote foreign gene expression are mouse Ltk and Chinese hamster ovary (CHO) dhfr- cells, although expression of Il-li is not limited 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 employed here involves the calcium phosphate-DNA precipitation technique described by S.L. Graham A.S. van der Eb (Virology 52:456-467, 1973) in which the expression vector for IL-li is co-precipitated with a second 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. 1:155-175, 1981.

4. Cells that express the IL-li gene constructs should then be grown under conditions that will increase the levels of production of IL-li. Cells carrying the metallothionein promoter constructs can now be grown in the presence of heavy metals such as cadmium which will lead to a 5-fold increased utilization of the MT-1 promoter (Mayo et al., Cell 29:99-108) subsequently leading to a comparable increase in IL-li protein levels. Cells containing IL-li expression vectors (either SV40- or MT-1-based; along with a DHFR u S3TITUTE SHEET i 1 Example 1, name that parallel cells incubated in a noninducing condition do not produce the IL-li bioactivity and do not produce these radioactive bands, we can conclude that 1

'T

i i:: WO 89/11540 PCT/US89/02275 expression vector: an e taken thr ough the gene amplification protocol described by Ringold et al, Mol. Appl. Genet, 1:165-175, 1981) using methotrexate, a competitive antagonist of DHFR. This leads to more coies of the DHFR genes present in the cells and, concomitantly, increased copies of the IL-li genes which, in turn, can lead to more IL-li protein being produced by the cells.

Example 9 Purification of Il-li From Recombinant Animal Cells Since the IL-li are expected to be secreted from cells like the natural material, it is anticipated that the methods described above for purification of the natural protein will allow similar purification and characterization of the recombinant protein.

I"

I

Example Seauence of IL-li The amino terminal residue of IL-li has been identified several times by direct protein sequencing as an arginine The result of such sequencing is shown in Example 3.

In contrast, the amino terminal residue of IL-li predicted by the sequence of the cDNA is a proline This amino terminal residue corresponds to nucleotides 85-87 in Fig. 13, and is circled in Figs. 14 and 15. This apparent disagreement between the cDNA sequence and the direct protein sequence can be resolved by assuming that an error in the cDNA sequence was incorporated during the reverse transcriptase-catalyzed synthesis from its mRNA. That is, a CGA (arginine) condon, located on the mRNA where it would code for that amino terminal residue, could have been -changed during the reversetranscriptase reaction to a CCA (proline) codon in the cDNA.

This type of reverse transscriptase problem has been reported in the literature before, by B.D. Clark et al. in Nucleic Acids Research 14:7897 (1986).

The present inventors believe that the correct amino acid sequence of the protein is as predicted by the cDNA except that the amino terminal amino acid is an arginine SUBSTITUTE SHEET it!e L)d eI Ip~ eiL IVL Iiy i ILy blocked at their N-terMIni, peptides of each were generated by endoproteinase dig-estion. Specifically, Mono Q fractions containing either IL-li-a or IL-li-bD were oasse6 through a WO 89/115Z40 ~8/27 ±nsea -h ze pro±iine residue indicated in Figs. 13-15. The nve ntors cont-emolate that both DNA se-uences and "eir corresponiding peptide sequences fall wit-hin the scope of their inventlion although the amino terminal arginine sequence is preferred.

Example 11 A protein having the sequence: M E I X R G L R S H L I S ET I X Z P S G R K S W D V N Q K T F YZ L R N Q G P N V N L EE K I D L F L G I H G G K M X L 100 T R L Q L EA V N IT D 130 K R F A F I RS D S G P 150 X P G W F L X T A ME A 170 M P D E G V M V T K F Y TT L L F L F H4 S KM Q A FR I N Q L V A G Y I V V P I P H 100 S X V K S G D E 120 L S EN R. K Q D 140 T T S F E S A A 160 D Q P V S L T N F Q E D E wherein X is cysteine, serine or alaNine; and Z is arginine or proline is also included in the invention.

OUSTTUTE SHEET

Claims (30)

1. An interleukin-1 inhibitor (IL-li) capable of in.ibiting IL-1 induced PGE 2 production and sufficiently pure such that the amino acid sequence of such IL-li can be determined.

2. The IL-li of claim 1 wherein said IL-li is obtained from mammalian cells.

3. The IL-li of claim 1 wherein said IL-li is obtained from monocytes.

4. The IL-li of claim 3 wherein said IL-li is obtained from human monocytes.

5. The IL-li of claim 1 wherein said IL-li is produced by recombinant-DNA methods.

6. A recombinant-DNA method for the production of an interleukin-1 inhibitor (IL-li) comprising: Preparation of a DNA sequence encoding a polypeptide having IL-1 inhibitor activity; Cloning the DNA sequence into a vector capable of being transferred into and replicated in a host cell, such vector containing operational elements needed to express the DNA sequence; Transferring the vector containing the DNA sequence and operational elements into a host cell capable of expressing the DNA sequence; Culturing the host cells under conditions appropriate for expression of the inhibitor; and Harvesting the inhibitor.

7. The method of claim 6 wherein said DNA sequence is a cDNA.

8. The method of claim 6 wherein said DNA sequence is a genomic sequence.

9. The method of claim 6 wherein said DNA sequence is derived from mammalian cells. vt ~it 11 I Ir 1 D KRFAF I R 11 1 1 1 1 S UBSQTITUTE 'SHE te_^| 1 k g i. l 3V 1 L o ET. r 1~ 'Ax The method of clair 9 wherein said DNA sequence is derived from human monocyte cells.

11. The method of claim 6 wherein said host cell is a microorganism.

12. The method of claim 11 wherein said microorganism is E. coli.

13. The method of claim 6 wherein said host cell is a mammalian cell.

14. The method of claim 13 wherein said mammalian cell is a CHO cell. The method of claim 6 wherein said DNA sequence is a cDNA polynucleotide.

16. The method of claim 6 wherein said DNA sequence is a genomic polynucleotide sequence.

17. An isolated DrA sequence encoding a physiologically functional interleukin-1 inhibitor (IL-li) comprising a DNA sequence encoding a polypeptide having an amino acid sequence sufficiently duplicative of IL-li to allow possession of at least one of IL-li's biological properties.

18. The isolated DNA sequence of claim 17, wherein said DNA sequence includes the nucleic acids between position 99 and 554 from the sequence which follows: 70 EO 90 100 110 120 CTCTCCTCCTCTTCCTGTTCCATTCAGAGACGATCTGCCCACCCTCTGGGAGAAAATCCA T L L L F L F H S E T I C P P S G R F S 170 140 150 160 170 180 GCAAGATGCAAECCTTCAGAATCTGGGATETTAACCAGAAGACCTTCTATCTGAGGAACA S K M Q A F R I W D V N Q K T F Y L R N 190 200 210 220 230 240 ACCAACTAGTTGCTGGATACTTGCAAGGACCAAATGTCAATTTAGAAGAAAAGATAGATS N O L V A G Y L Q G -P N V N E E K: D 250 260 270 280 290 300 TGGTACCCATTGAGCCTCATGCTCTGTTCTTGGGAATCCATGGAGGGAAGATGTGCCTGT V V P I E P H A L LF L G I H G G K M C L I. r I I r: cZ II r r r I I WVO 89/111540 PCT!LUS89/02275 350 360 CCT GT TCAA-TCTGGGa TGAG AC CAGACT CCAGcCTGGACGCAG TAA%.%CAT CACTGAC C rC 17 W S GD E T R L Q E A V N I T D 370 380 30400 410 420 T ,GAGCG AGAACAGAA'A GCAGGACAAGCGCTTCGCCTTCATCCGCTCAGACAGTGGCCCC A L S EN R K Q D K R F A F IR S D S G P 4-30 440 450 460 470 480 CCACCAGTmTTTGAGTCTGCCGCCTGCCCCGGTTGGTTCCTCTGCACAGCGATGGAAGCTG T T S F E S A A C P G W F L C T A M E A 490 500 510 520 530 540 ACCAGCCCGTCAGCCTCACCAATATGCCTGACGAAGGCGTCATGGTCACCAAATTGTACT D Q P V S L T N M P D E G V M V T K F Y H.550 560 570 580 509 510 TCCAGGAGGACGAGTAGTACTGCCCAGGCCTGCTGTTCCATTCTTGCATGGCAAGGACTG F Q E D E* 4Z4

19. The recombinant DNA molecule GT10-lLli-2A 4 A substantially purified interleukin-l inhibitor (IL-li) comprising at least one of IL--li-X, IL-li-a and IL-li-B.

21. Interleukin-l inhibitor of claim 20, wherein said IL-li is IL-li-X.

22. Interleukin-l inhibitor of claim 2D, wherein said IL-li is IL-li-a.

23. Interieukin-l inhibitor of claim 20, wherein said jIL-li is IL-li-S. C3iTUTE SHEET j WO 89/11540 PD1 1 I /T275 '414 DNA sequence of c'aim 17, wherein said DNA sequence includes the nucleic cr bet-ween posit~l 99 and 55%- from the sequence which follows: 80 90 100 110 120 CTCTCCTCCTICrjTr-CTGTTCCATTCAGAGACGATCTGCCGACCCTCTGGGAGAAIAATCCA Tp L L L F L F H S E T I C R P S G R M S 130 140 150 160 170 180 GCAAGATGCAAGCCTTCAGAATCTGGGATGTTAACCAGAAGACCTTCTATCTGAGGAACA S K M Q A FR I W D V N Q K T F Y L R N 190 200 210 220 230 240 ACCAACTAGTTGCTGGATACTTGCAAGGACCAAATGTCAATTTAGAAGAAAAGATAGATG N Q L V A G Y L Q G P N V N L EE K I D 250 260 270 280 290 300 TGGTACCCATTGAGCCTCATGCTCTGTTCTTGGGAATCCATGGAGGGAAGATGTGCCTGT V V P1I E P H A L F L G I H G G K M C L 310 320 330 340 350 360 CCTGTGTCAAGTCTGGTGATGAGACCAGACTCCAGCTGGAGGCAGTTA.ACATCACTGACC ~C V K S G D E T R L Q L E A V N I T D 370 380 390 400 410 420 TGAGCGAGAACAGAAAGCAGGACAAGCGCTTCGCCTTCATCCGCTCAGACAGTGGCCCCA L SE N R K D K R F A F IR S D S G P 430 440 450 460 470 480 CCACCAGTTTTGAGTCTGCCGCCTGCCCCGGTTGGTTCCTCTGCACAGCGATGG%-AAGCTG T T S F E S A A 2 P G W F L C T A ME A 490 500 510 520 530 540 ACCAGCCCGTCAGCCTCACCAATATGCCTGACGAAGGCGTCATGGTCACCAAATTCTACT D Q P V S L T N M P D E G V M V T K F Y 550 560 570 580 590 600 TCCAGGAGGACGAGTAGTACTGCCCAGGCCTGCTGTTCCATTCTTGCATGGCAAGGACTG F Q E D E SUBTITTESHEET

128. The process of claim 124 wherein the IL-i IL-li was being produced at a maximal rate, plated monocytes were exposed to 35 S]-methionine (pulsed) for a short, two- hour period, at which time a large excess of unlabelled 57 A substantially purified interleukin-i inhibitor having the sequence: ME: X R G L R S H LIT L L L FL F H S T X Z P S G R K S S K M Q A F R 7 W D V N Q K T F Y L R N N Q L V A G Y L Q G P N V N L E E K I D V V P I E P H A 100 L F L G I H G G K M X L S X V K S G D E 100 120 T R L Q L E A V N I T D L S E N R K Q D 130 140 K R F A F I R S D S G P T T S F E S A A 150 160 X P G W F L X T A M E A D Q P V S L T N 170 M P D E G V M V T K F Y F Q E D E wherein X is cysteine, serine or alanine; and Z is arginine or proline. 26. A recombinant DNA molecule comprising a DNA sequence according to any one of claims 17, 18 or 24. 27. A method for producing an interleukin-I inhibitor (IL-li) according to any one of claims 1 to 5, 20, 21 and comprising the steps of culturing a host cell transformed with a recombinant DNA molecule according to claim 26 and harvesting the inhibitor. 28. A recombinant DNA vector comprising the DNA sequence of claim 17. 29. The vector of claim 28 wherein said vector is an expression vector and further comprises at least one I IV. C Iai yudc. anil-mouse IgG, Bethesda Research Labs) fol- lowed by a strepavidin-alkaline phosphatase conjugate (Bethesda Research Labs), as described by Bayer, E. A. and C TiT UTE SHEET' -q 58 regulatory element needed for the expression vector and further comprises at least one regulatory element needed for the expression of the DNA sequence in a host. The vector of claim 29 wherein said DNA sequence is capable of being expressed in bacteria. 31. The vector of claim 29 wherein said DNA sequence is capable of being expressed in mammalian cells. 32. The vector of claim 28 wherein said DNA sequence comprises a DNA sequence that encodes IL-li X, Il-li alpha or IL-li beta. 33. A cell host including the vector of claim 28 inserted therein. 34. The host cell of claim 33 wherein said host cell is capable of expressing said DNA sequence. The host cell of claim 34 wherein said host cell is a microorganism. 36. The host cell of claim 35 wherein said host cell is a bacterial cell. 37. The host cell of claim '36 wherein said host cell is Escherichia coli. 38. The host cell of claim 35, wherein said host cell is a mammalian cell. 39. An isolated DNA sequence encoding a physiologically functional interleukin-1 inhibitor (IL-li) comprising a DNA sequence that is selected from the group consisti,,ng of a DNA sequence that encodes IL-li X, IL-li alpha, or IL-li beta and a DNA sequence that cross- hybridizes to a DNA sequence that encodes IL-li X, IL-li alpha, or IL-li beta or (ii) that cross-hybridizes to a DNA sequence that is complementary to a DNA sequence that encodes IL-li X, IL-li alpha, or IL-li beta, wherein said DNA sequence of or (ii) encodes a protein having IL-1 inhibitor activity. The recombinant DNA molecule GT10-1Lli-2A. WO 89/11540 u: C C AsD Val Asn Gin Lys Thr Pobe "IL -7 T TA G T TTT G NA A A A T TUTE S H EET 59 41. The isolated DNA sequence of claim 39 wherein said DNA sequence comprises a DNA sequence that encodes IL- Ii X, IL-li alpha or IL-li beta. 42. The isolated DNA sequence of claim 39 wherein said DNA base sequence includes the nucleic acids from position 99 to 554 from the sequence which follows: so CTCTCCT CCTCTrCCTG TTCC ATTC AG AG ACG A TCT G T L L L F L F H S E T I C 100 110 120 130 CCCACCCTCTGGG AG AAAATCCACA AG A TG CA AGCC p P S G R K S S K M Q A 140 150 160 170 T'TCAGXATCTGGG ATGTTAACCAGAAGACCTTCTATCT F R I 0 V N Q K T F Y L 180 190 200 210 GAGGAACkACCA.ACTAGTTGCTGCATAC GCAAGGA R N N Q L VA G Y L Q C 220 230 2410 CCAAATGTCAATTrAG AAGAAAAGATAGATGTGGTAC P N V N L E E K I D V V 250 260 270 280 CCATTGAAGCCTCATGCTCTGTTCTTGG A ATCCATGGA P I E P H A L F L G 1 H G 290 302 3!0 320 GGGAAGATGTGCCTGTCCTGTGTCAAGTCTGGTGATG G K M C L S C V 'K S GD 330 340 350 AGACCAGACTCCAGCTGGAGGCAGTTAACATCACTG E T R L Q L E A V N 1 T 360 38070 38039 ACCTGAGCGAGAACAG&AAGCAGGACAAGCGCTTCG D L S E N R K Q D K R F 400 410 420 430 so CCTTCATCCGCTCAGACAGTGGCCCCACCACCAGTTTG A F I R S D S O P T T S F 440 450 AGTCTGCCGCCTGCCCCGGTTrGG'TTCCTCTGCACA E S A A C P G F L C T **470 480 490 500 GCGATGGA AGCTGACCAGCCCCTCAGCCTCACCAATA A Mi E A D Q P V S L T N 510 520 530 540 TGCCTGACGAAGGCGTCATGGTCACCAAATCTACTTC M P D E G V NI V T K F Y F 550 560 570 CAGGAGGACGAGTAGTACTGCCCAGGCCTGCTGT7 Q E D E 580 590 600 CCATTCTTGCATGGCAACACTG dflQ, tne hYbridiz at iontme-au were-as" follows. temperatures LANE PROBE TEMPERATURE [1 b II 43. The isolated DNA sequence of claim 39 wherein said DNA base sequence includes the nucleic acids from position 99 to 554 from the sequence which follows: 80 CTCTCCTCCT=cTCCTGTTCCATTCAGAGACGATCTG T L L L F L F H S E T I C 100 110 120 130 CCrGACCCTCTG AG.kAA-ATCCAGCAAGATGCAACCC R P S G R K S S K M Q A 140 150 160 170 TTCAGA-ATCTGGC ATGTTAACCAG AAGACCTTCTATCT F R I NV D V N Q K T F Y L 180 190 200 GAGGAACAACCAACTAGTTGCTGC ATACTfGCA kAG R N N Q L V A G Y L Q 210 220 230 240 OACCA-AATGTCAATTTAGAAGAAAAGATAG ATGTGGT G P N V N L E E K I D V V 250 260 270 280 ACCCATTG AGCCTCATGCTCTGTTTTGGG AATCCATG PI E PH AL FL G I H 290 300 310 GAG-C AAGATGTGCCTGTCCTGTGTCAAGTCTGT GOG K M C L S C V KS G 320 330 340 350 GATGAGACCAGACTCCAGCTGGATGCAGTAACATCA D E T R L Q L E A V NI 360 370 80 390 CTGACCTGAGCGAGAACAGAAACAGGACAAGCGC-r T D L S E N R K Q D K R 40 410 420 TCGCCTTCATCCGCTCAGACAGTGGCCCCACCACCAG F A F 1 R S D S G PT T S 430 4"40 450 460 TTTTGAGTCTGCCGCCTGCCCCGGTTGGTTCCTCTGCA F E S A A C P G W F L C 470 480 490 500 CAGCGATOGAAGCTGACCAGCCCGTCAGCCTCACCA'A T A M E A D Q P V S L T N 510 520 530 TATGCCTGACGAAGGCGTCATGGTCACCAAATTCT M P D E G V NM V T K F W4 550 560 570 ACCAGGAGACGAGTAGTACTGCCCAGGCCTGCT Y F Q E D E 580 590 600 GTTCCATTCrrGCATGCAAGGACTG 4 r ,r r i I rr r rir~ r rr r. c I i t I (QL-11 amino acid proj. me agrees with the peptide sequences descriBed in Example 3. J T iT U TE S HET 61 44. A recombinant DNA vector comprising the DNA sequence of any one of claims 39 to 43. The vector of claim 44 wherein said vector is an expression vector and further comprises at least one regulatory element needed for the expression vector and further comprises at least one regulatory element needed for the expression of the DNA seq-uence in a host. 46. The vector of claim 45 wherein said DNA sequence is capable of being expressed in bacteria. 47. The vector of claim 45 wherein said DNA sequence is capable of being expressed in mammalian cells. 48. The vector of any one of claims 44 to 47 wherein said DNA sequence comprises a DNA sequence that encodes IL- ii X, IL-li alpha or IL-li beta. 49. A cell host including the vector of any one of claims 44 to 48 inserted therein. The host cell of claim 49 wherein said host cell is capable of expressing said DNA sequence. 51. The host cell of claim 50 wherein said host cell is a microorganism. 52. The host cell of claim 51 wherein said host cell is a bacterial cell. 53. The host a-all of claim 52 wherein said host cell is Esclherichia coZ-4. 54. The host cell of claim 51, wherein said host cell is a mammalian cell. A recombinant-DNA method for the production of an interleukin-1 inhibitor (IL-li) comprising the steps of: preparing a DNA sequence encoding a protein having IL-i inhibitor activity wherein said DNA sequence is selected from the group consisting of a DNA sequence that encodes IL-li X, IL-li alpha, or IL- l i beta and a DNA sequence that cross- A hybridizes to a DNA sequence that encodes IL-li X, IL- LI ,ue purotein to show effective IL-li activity. Nucleotides 99-101 of GT10-ILli-2A encode a P (see Fig- ure 15), but no P has been detected at this position (the N-terminus) of form X of IL-li. It is oossible that this It susSTITUTE SHELE-7 i 62 li alpha or IL-li beta or (ii) that cross-hybridizes to a DNA sequence that is complementary to a DNA sequence that encodes IL-li X, IL-li alpha, or IL-li beta, wherein said DNA sequence of or (ii) encodes a protein having IL-I inhibitor activity; subcloning the DNA sequence into a vector capable of being inserted into and replicated in a host cell, such vector containing at least one regulatory element needed to express the DNA sequence; inserting the vector containing the DNA sequence and at least one regulatory element into a host cell capable of expressing the DNA encoding the IL-1 inhibitor; culturing the host cell under conditions appropriate for replication of the vcator and expression of the IL-li inhibitor; and harvesting the IL-1 inhibitor. 56. The method of claim 55 wherein said DNA sequence comprises a DNA sequence that encodes IL-li X, IL-li alpha or IL-li beta. 57. The method of claim 55 wherein said DNA sequence is a cDNA. 58. The method of claim 55 wherein said DNA sequence is a genomic sequence. 59. The method of claim 55 wherein said DNA sequence is derived from mammalian cells. The method of claim 59 wherein said DNA sequence is derived from human monocytes. 61. The method of claim 55 wherein said host cell is a microorganism. 62. The method of claim 61 wherein said microorganism is E. coll. 63. The method of claim 55 wherein said host cells are mammalian cells. SI* 1 i* II i e 1e q o* J t:\ 7 in- corporated herein by reference. This plasmid should be ma- nipulated in such a way as to substitute the IL-li cDNA for the chloramphenicol acetyltransferase (CAT) coding seauences CU2STITUTE SHEET 63 64. The method of claim 63 wherein said mammalian cells are CHO cells. The method of claim 55 wherein said DNA sequence is a sy-.hetic polynucleotide. 66. A recombinant DNA method for the production of an interleukin-1 inhibitor (IL-li) comprising the steps of: culturing a host cell that includes inserted therein a vector comprising the DNA sequence of claim 39 operatively linked to at least one regulatory element needed for the expression of the DNA sequence in the host cell; harvesting the protein having IL-1 inhibitor activity. 67. A recombinant-DNA method for the construction of an interleukin-1 inhibitor (IL-li) expressing vector comprising: preparing the DNA sequence of claim 39; and subcloning the DNA sequence into a vector capable of being inserted into and replicated in a host cell, such vector containing at least one regulatory element needed for the expression of the DNA sequence. 68. The method of claim 66 wherein said DNA sequence comprises a DNA sequence that encodes IL-li X, IL-li alpha or IL-li beta. S69. The method of claim 67 wherein said DNA sequence comprises a DNA sequence that encodes IL-li X, IL-li alpha or J IL-li beta. An IL-1 inhibitor (IL-li) in substantially pure form, capable of inhibiting IL-1 induced thymocyte proliferation and having an N-terminal amino acid sequence as il' follows: P S G R K S S K M Q A FR I W D V N Q Swherein is R or P. 8. 97 i- utilization of the MT-1 promoter (Mayo et al., Cell 29:99-108) subsequently leading to a comparable increase in IL-li protein levels. Cells containina IL-li expression vectors (either SV40- or MT-1-based) along with a DHFR TITUTE SHEET 64 71. The IL-1 inhibitor of claim 70 which is at least about 90% pure. 72. The IL-1 inhibitor of claim 70 which is at least about 95% pure. 73. The IL-1 inhibitor of claim 70 which is derived from monocytes stimulated with IgG. 74. The IL-I inhibitor of claim 73 wherein the monocytes are human monocytes. The IL-1 inhibitor of claim 70 which is derived froQ a host cell transformed by recombinant DNA. 76. The IL-1 inhibitor of claim 70, 73 or 75 which is a glycosylated polypeptide. 77. The IL-1 inhibitor of claim 70, 73 or 75 which is a substantially unglycosylated polypeptide. 78. The IL-1 inhibitor of claim 70 which is selected from the group consisting of IL-1 inhibitor alpha, IL-1 inhibitor beta, IL-1 inhibitor X and mixtures thereof. 79. An IL-1 inhibitor in substantially pure form, capable of inhibiting IL-1 induced thymocyte proliferation and having at least a part of the following amino acid Ssequence or at least a part of a substantially homologous sequence: S(X) P S G R K S S K M Q A FR I W D V N Q K T F Y L R N N Q L VAG Y L Q G P NVNL E E K ID V VP I E P HA L FLGI H G G KM C L S C V KS G D E TR L Q LE AV N I T D L S E NR K Q D KR F A F I R S D S G P T T S F E SAAC P GWF L C TAMEAD QPVS L TNMP D E G V M V T K F Y F Q E D E wherein is R or P. The IL-1 inhibitor of claim 79, further including a N-terminal secretion leader sequence which is capable of dq q' irecting the inhibitor out of a cell in a processed form. 8 SCU STITUTE SHEET 81. The IL-1 inhibitor of claim 80 wherein the secretion leader sequence has all or part of the following amino acid sequence: M E I C R G L R S H L I T L L L F L F H S E T I C. 82. The IL-1 inhibitor of claim 79, which is at least about 70% homologous to the sequence shown in claim 79. 83. The IL-1 inhibitor of claim 79, which is at least about 80% homologous to the sequence shown in claim 79. 84. The IL-1 inhibitor of claim 79, which is at least about 90% homologous to the sequence shown in claim 79. I 85. A process for producing the IL-1 inhibitor (IL- li) of claim 70 comprising the steps of: cultur±ng monocytes in a culture medium on an IgG coated surface; and i isolating the IL-li from the culture medium. 86. The process of claim 85 wherein the cells are cultured on a human IgG coated surface. 87. The process of claim 85 wherein the cells are cultured in a culture medium which is substantially serum- free. 88. The process of claim 85 wherein the monocytes are Sisolated from normal human donors. 2 89. A process for preparing the IL-1 inhibitor (IL- li) of claim 70 comprising the steps of: fractionating a source of impure IL-li by anion exchange chromatography and collecting the fractions containing the IL-li; subjecting the Il-li containing fractions Sobtained in step to gel filtration chromatography and collecting the fractions containing the IL-li; and e B J4 IR o a S...TITUTE SHEET 66 subjecting the IL-li containing fractions obtained in step to reversed phase chromatography and collecting the fractions containing the IL-li. The process of claim 89 wherein the reversed phase chromatography is performed on a hydrophobic chromatography resin in which the absorbing group is selected from the group consisting of C4, octyl and phenyl. 91. The process of claim 89 wherein in step the reversed phase chromatography is substituted by a second cycle of gel filtration chromatography. 92. A process for preparing the IL-1 inhibitor (IL- li) of claim 70 comprising the steps of: contacting a source of impure IL-li with an antibody which specifically binds the IL-li; and separating the IL-li from the antibody. 93. The process of claim 92 wherein the source of impure IL-li is contacted with a polyclonal antibody. 94. The process of claim 92 wherein the source of impure IL-li is contacted with a monoclonal antibody. The process of claim 92, 93 or 94 wherein the source of impure IL-li is contacted with an antibody which is immobilized on a solid support. 1; 96. An isolated DNA sequence coding for the IL-1 I inhibitor of claim S 97. An. isolated DNA sequence coding for an IL-1 Sinhibitor (IL-li) capable of inhibiting IL-1 induced "'thymocyte proliferation comprising a cross-hybridizing DNA sequence that is detectable by cross-hybridization to the following DNA sequence: mTne method of claim 6 wherein said DNA sequence is derived from mammalian cells. CYA ATC CAA GAA TTC AAG ATC GCC TCT GCT GTC CCC TCT GGG AGA AAA TCC AGC AAG ATG CAA GCC TTC AGA TGG GAT GTT AAC CAG AAG ACC TTC TAT CTG AGG AAC AAC CTA GTT GCT GGA TAC TTG CAA GGA CCA AAT GTC AAT TTA GAA AAG ATA GAT GTG GTA CCC ATT GAG CCT CAT GCT CTG TTG GGA ATC CAT GGA GGG AAG ATG TGC CTG TCC TGT GTC TCT GGT GAT GAG ACC AGA CTC CAG CTG GAG GCA GTT AAC ACT GAC CTG AGC GAG AAC AGA AAG CAG GAC AAG CGC TTC TTC ATC CGC TCA GAC AGT GGC CCC ACC ACC AGT TTT GAG GCC GCC TGC CCC GGT TGG TTC CTC TGC ACA GCG ATG GAA GAC CAG CCC GTC AGC CTC ACC AAT ATG CCT GAC GAA GGC ATG GTC ACC AAA TTC TAC TTC CAG GAG GAC GAG 3' i r I wherein Y is C or G. 98. An isolated DNa sequence coding for an IL-1 inhibitor (IL-li) capable of inhibiting IL-1 induced thymocyte proliferation comprising a DNA sequence that encodes an amino acid sequence substantially homologous to an amino acid sequence encoded by the cross-hybridizing DNA sequence of claim 97. 99. The isolated DNA sequence of claim 97 or 98 comprising all or part of the DNA sequence listed in claim 97. 100. An isolated DNA sequence which is the complement of the DNA sequence of any one of claims 97, 98 or 99. 101. The isolated DNA sequence of any one of claims 97, 98 or 99 further including at the 5' end a DNA segment coding for a secretory leader sequence which is capable of directing IL-li out of a cell in a processed form. 102. The isolated DNA sequence of claim 101 wherein the segment coding for the secrtory leader sequence comprises all or part of the following nucleotide sequence: ATG GAA ATC TGC AGA GGC CTC CGC AGT CAC CTA ATC ACT CTC v CTC CTC TTC CTG TTC CAT TCA GAG ACG ATC TGC 3'. S' 250 260 0 2 280 290 300 TGGTACCCATTGAGCCTCATGCTCTGTTCTTGGGAATCCATGGAGGGAAGAGTGCC-TGT V V V P I E P H A L F L G I H G K M C L 68 103. The isolated DNA of any one of claims 97, 98, 99 or 101 which is selected from cDNA, genomic or synthetic DNA. 104. An isolated DNA which is substantially equivalent to the nucleotide sequence of claim 97, 98, 99 or 101 by i virtue of codon degeneracy. 105. The DNA sequence of claim 96 or 97 which is contained in lambda GT10-IL-li-2A, having ATCC Accession No. l 40488. 106. An isolated DNA sequence which codes for a second IL-1 inhibitor which is biologically equivalent and substantially homologous to the IL-li of claim 107. A recombinant DNA vector comprising the DNA sequence of claim 96, 97, 98 or 106. 108. The vector of claim 107 which is an expression vector for IL-1 inhibitor. 109. The vector of claim 107 or 108 further comprising at the 5' end of the DNA sequence coding for the IL-li, a second DNA sequence coding for a secretory leader sequence which is capable of directing the IL-li out of a cell in Sprocessed form. 110. The vector of claim 109 wherein the second DNA sequence codes for a naturally-occurring IL-li secretory leader sequence. Sill. The vector of claim 110 further comprising a translational coupler immediately preceding the DNA sequence coding for the IL-li. S 112. The vector of claim 111 wherein the translational Scoupler includes the following nucleotide sequence: TAACGAGGCGCAAAAAATGAAAAAGACAGCTATCGCGATCTTGGAGGATGATTAAATG. I 113. The vector of claim 108 for use in expressing IL- li in mammalian cells. j I TUTE SHEET 1 69 114. The vector of claim 113 for use in expressing IL- li in CHO cells. 115. The vector of claim 113 or 114 which further comprises a strong constitutive promoter region upstream of the IL-li coding region. 116. The vector of claim 115 wherein the promoter is an SV40 promoter. 117. The vector of claim 108 for use in expressing IL- li in microbial cells. 118. The vector of claim 117 for use in expressing IL- ii in E. coli cells. 119. A transformed host cell carrying the vector of at least one of the claims 107 to 118. 120. The host cell of claim 119 which is a microbial cell. 121. The host cell of claim 120 which is an E. coli cell. 122. The host cell of claim 119 which is a mammalian cell. 123. The host cell of claim 122 which is a CHO cell. 124. A process of using the host cell of claim 119 to 123 to produce an IL-li inhibitor comprising: culturing the transformed host cell under conditions which allow expression of the IL-I inhibitor Sand isolating the IL-I inhibitor. step 125. The process of claim 124 further comprising the steps of denaturing the IL-I inhibitor and allowing the Il-i inhibitor to refold to assume an active structure. 126. The process of claim 125 wherein the IL-I inhibitor is allowed to refold prior to isolation. 127. The process of claim 125 wherein the IL-1 iA inhibitor is allowed to refold subsequent to isolation. SUSSTiTUTE SHEET 128. The process of claim 124 wherein the IL-1 inhibitor is isolated from a culture medium.

129. The process of claim 124 wherein the IL-1 inhibitor is isolated according to the method of claim 92, 93 or 94.

130. An antibody which binds to the IL- inhibitor of claim

131. An antibody which binds to an IL-1 inhibitor substantially homologous to the IL-1 inhibitor of claim

132. The antibody of claim 130 or 131 which is a polyclonal antibody.

133. The antibody of claim 130 or 131 which is a monoclonal antibody.

134. An immunoaffinity adsorbent comprising the antibody of at least one of the claims 130 to 133 attached to an insoluble support.

135. A pharmaceutical composition comprising, in a pharmaceutically acceptable preparation, the IL-1 inhibitor of at least one of the claims 70 to 84.

136. A pharmaceutical composition comprising, in a pharmaceutically acceptable preparation, an IL-1 inhibitor 'that is biologically equivalent and substantially homologous to the IL-1 inhibitor of claim *o DATED this 30th day of June 1992 By Their Patent Attorneys: S..GRIFFITH HACK CO Fellows Institute of Patent SAttorneys of Australiaf a :-i sequence of claim 17. 29. The vector of claim 28 wherein said vector is an expression vector and further comprises at least one Iwo 89/11540 PCT/UJS89/02275- */22 0 4s 1 rMET LABEL WITH M6 j~YTES PLATED ON PETRI DISHES COATED 0WITH IgG. OD280 -~-o-RADIOACTIVITY TIVI TY -NcCU GRADIENT 35 S- MET LABEL WITH MONOCYTES PLATED ON PETRI DISHES COATED WITH FOS. 0D280 Q*RADIOACTIVITY vS:TITULTESH EET sequence of or (ii) encodes a protein having IL-i inhibitor activity. The recombinant DNA molecule GT10-lLli-2A. WO089/11540 PCTIUS89/022754 0 0 C= h$9*Me z C= -1M C\J C' 4 IgG PLATE FCS PLATE SUBSTITUTE SHEET sQVHoinv 0 ou a U U U 0 t 0 ->0 <LU 0 U <0 <LU <0' U q4? 91i 5059060 GTTCCATCTGCATGCAGGAC-r WO 89/11540 p M U S8 91 n2 2 4w 4w'h~ FIG. 3a 04-REVERSE PHASE HPLC ON MONO Q-PURI-FIED IL-1i (TRACE 3 0D 2 1 0 D 2 8 0 RDOCTVT S~ST1UTE SHET DNA sequence that encodes IL-li X, IL-li alpha, or IL- 'l i beta and a DNA sequence that cross- hybridizes to a'DNA sequence that encodes IL-li X, IL- mE 4 B j T P IA IWO089/11540 PCT/US89/022-75 __-06-43 KD SILVER-STAINED GELS P/.1G c N ft' Ln r- (VI en n Cn -0-43 KD KD AU TO RAD SUBSTITUTE SHEET V c3.laim bL wferein said microorganism is E. coli. 63. The method of claim 55 wherein said host cells are mammalian cells. I 1 11 1 I 11 1- I I WO 89/11540 PCT/US89/02275 6y/ 19 FIG. 4a CONSECUTIVE SUPEROSE 12 (SIZING) CHROMATO- GRAPHIC SEPARATIONS ON MONOQ PURIFIED IL-Ij E E E C "T CoT C 1 I I I I I 5 10 15 20 I E E E 30 35 40 RERUN I I 45 50 55 FRACTIONS 37+38 OD 28 0 RADIOACTIVITY BIOACTIVITY FIG. 4b I 5 10 15 25I I 25 I I i SUBSTITUTE SHEET low,- FIG (D 09 rJJ C 3: m 4 0H ril WESTERN ANALYSIS OF IMMUNOGEN (MONO 0- 43 KD PURE IL-li) USING NORMAL MOUSE SERUM 25KD (NMS) AND A MIX OF ANTISERA FROM ALL IL-li 5 MICE LMWM LMWM I MWMM NJS* ANTISERUM WESTERN SILVER STAIN C' FIG C!) U) Co H C H PJ ~cn El' B C D E Koo, ~-43KD WESTERN ANALYSIS OF IMMUNOGEN USING THE SEPARATE BLEEDS OF ALL MICE (A-E) 13 coi ItU IL-ti WO 89/11540 PCT/US89/02275, FIG. 6 Hind M I H pal DIGEST WITH HpaI- Hind~ll AND _-HpaI 2. ISOLATE LARGE FRAGMENT. P0 LY LIN KER L. INCLUDES FOLLOWING RESTRICTION SITES: Hind III? Sma 1, Bg ll NarI, EcoRI, Hp a 1?T EcoRI EcoRI %G Ti 1-IL-li I. CUT WITH EcoRI 1. CUT WITH EcoRI pSVXV P 2. PURIFY THE SMALL 2. ISOLATE LINEA (IL-li cDNA) FRAGM\ENT FRAGMENT. vvP FRAGMENTS. LIGATE EcoRI 3. SELECT E.cali TRANSFORMANTS CONTAINING THE PLASMID PmnPTSHOWN BELOW. %c .,f.;D7.,STITUTE SHEET INTERNATIONAL SEARCH REPORT International Aiclic.,o' No. PCT/US889 /02275 1. CLASSIFICATION OF SUBJECT MATTER s..;eral CIassiticat-on syn;1oiS _0o~y, -oicate all) Ac coc in o1e rnallonal P3e.11 Clidsfiction (IPC) or to otm, National Classirication and IPC 1IPC(4: C12P 21/00; C07K 13/000; C07H 15/12 1u.. C, d-I z/c0 r WO 89/1 1540 PCT/US89/02275 fl I FIG. 7 DIGEST WITH EcoRI AND EcoRl FILL IN EcoRI STICKY END TO FORM BLUNT END. EcoRI MT-I PROMOTER Bg 0l pMKHSV- tk DIGEST WITH EcoRI FILL IN ENDS TO FORM BLUNT ENDS. PURIFY 4kb FRAGMENT CONTAINING MT-i PROMOTERBq2 SITE, AND HSV -ltk BLUNT END MT-i PROMOTER BgtlI H lSV-ltk BLUNT END -MIX FRAGMENTS 1 AND 2 LIGATE TRANSFORM E. coli, ISOLA1YE PLASMID DNA, CHECK STRUCTURE BY' RESTRICTION DIGESTS. THE PLASMID SHOULD BE 8kb IN LENGTH WITH NO EcoRI SITES AND ONE Bg9 fl SITE. MT-I 'ROMOTER Bg IT pMK-SGE 1 Eco RI CU'S7ITUTE S H E .WO 89/1!540PfIS8O27 PCTl/US89/022775 11/22 FIG. 7 (cont) MT-i PROMOTER BgIII pMK-SG~lDIGEST pMK-GE 12.GCOMB I SYNTHE GATCGGI cc. 3. LIGATE WITH BgtII NE WITH TIC LINKER AATTCC TTAAGGCTAG MT-i P ROMOTER EcoRI -S G E2 CUT WITH EcoRI EcoR I EcoRI LAMBDA GTI-IL-i I -F101 CUT WITH Eco l, PURIFY THE SMALL (IL- i cONA) FRAG- MENT BY AGAROSE GEL ELECT RO)PH ORESI S. COMBINE EcoRI CUT pMK-SGE2 AND IL-l 1 DNA AND LIGATE. TRANSFORM E. coll AND ISOLATE PLASM ID. CHECK FOR THE PRESENCE AND ORIENTATION OF IL-l cONA FRAGMENT. MT-I PROMOTER fEco RI E:IL-1 IL-li cDNA SUT ITUTE SHEET I- FIG. 8a U 10 PROTEIN PLUS (C3) RUN ON FRACTION 48 OF THE ABOVE RUN 30 40 (RERUN OF F48) TIME (MIN) FIG. 8b -i i i r f. p 1~ L WO 89/11540 PCT/US89/02275 13122 FROM FIG. 8b 0 FIG. 8c FIG. 8d SUBSTITUTE SHEETi V W089/11540 1~ L C I ft I I Li PCT/US89/02275 1 4/ 22 I -START- PREP R 8(F31 -1-)32 FILE 3 Ca) 0.5 mi/ min PROTEIN PLUS (4.6 X 250 mm) 7 ITUTE SHEMT .WO 89/11540 PCTF/US89/02217 15/22 TV f SUBSTITUTE SHEET IWO 89/11540 PCTIUS89/02275 I1c/ 22 73 44 V42 Al RVOW 41 37- 31 AUTO Z 0 AUTO 12- 26 C; TITUTE SHEET IL-11 R BEIAENDO ASP N- ~I 3H 35S PEPIDE

314.00 21.00 13 88.00 25.00 16 122.00 16.00 20 ORDER FOR SEQUENCING 84.00 16.00 22 51,43,39,25,30,50,28,41,48,54,27,46 106.00 21.00 23 15.00 21.00 83.00 32.00 21 11.00 31.00 28 50.00 23.00 16.00 33.00 34 11.00 16.00 31 58.00 26.00 38 69.00 38.00 39 w 141.00 21.00 41 C 89.00 38.00 43 -I 111.00 15.00 46 m1 76-00 21.00 48 co) 83.00 16.00 3: 216.00 56.00 51!ac Iii 13.00 18.00 53 P n p 61.00 23.00 54 C 11.00 16-00 51 10.00 23.00 58 C 80.00 22.00 138.00 23.00 62 c-"D- jA V I. 17 I;' p II WO 89/11540 PCr/US89/0227 ,18/22 /2?A LANE 5 6 8 10 12 14 SUBSTITUTE SHEET IWO 89/11540 19/22 PCT/US89,'022 7 .4 II ii VW. F117 /29 LANE 5 6 8 10 12 14 SUBSTITUTE SHEET WO 89/11540 PCT/US89/02275 20/22 FI1G. 3 GCG TCA CAG AAT GGA AAT CTG CAG Ala Ser Gin Asn Giv Asn Leu Gin CCT CTT CTG ATC ATT CAG AGA CCG Pro Leu Leu Ile Ile Gui Arg Pro AAG ATG CAA GCC TTC AGA ATC TGG Lys MET Gin Ala Phe Arg Ile Trp A.AC AAC CAA CTA GTT GCT GGA TAC asn Asn Gin Leu Val Ala Gly Tyr AAG ATA GAT GTG GTA CCC ATT GAG .Lys Ile Asp Vai Val Pro Ile Giu 27 AGG Arg 81 ATC Ile 135 GAT Asp 189 TTG Leu 243 CCT Pro CCT CCG CAG TCA CCT AAT CAC TCT Pro Pro Gin Ser Pro Asn His Ser TGC CCA CCC TCT GGG AGA AAA TCC Cys Pro Pro Ser Gly Arg Lys Ser GTT AAC CAG AAG ACC TTC TAT CTG Val Asn Gin Lys Thr Phe Tyr Leu CAA GGA CCA AAT GTC AAT TTA GAA Gin Gly Pro Asn Val Asn Leu Giu CAT GCT CTG TCT TGG GAA TCC ATG His Ala Leu Ser Trp Giu Ser MET 54 CCT Pro 108 AGC Ser 162 AGG Arg 216 GAA Glu 270 GAG Glu 8-USSTIT UTE SHEET ,WO 89/ ~11540 PCT/1JS89/02275 21/22 FIG. 14 20 30 40 50 GAATTCCGGGCTGCAGTCACAGAkATGGAAATCTGCAGAGGCCTCCGCAGTCACCTAATCA M E IC R G L R S H L I 80 90 160 110 120 CTCTCCTCCTCTTCCTGTTCCATTCAGAGACGATCTGCCACCCTCTGGGAGAAAkATCCA T L L L FL F'H SET IC UP PS G RK S 130 140 150 160 170 180 GCAAGATGCAAGCCTTCAGAATCTGGGATGTTAACCAGAAGACCTTCTATCTGAGGAACA S K M Q A F RI W D V N Q K T F Y L R N 190 200 210 220 230 240 ACCAACTAGTTGCTLGGATACTTGCAAGGACCAAATGTCAATTTAGAAGAAAkAGATAGATG N QLVAG YLQ G PNVN LEEK I D 250 260 270 280 290 300 TGGTACCCATTGAGCCTCATGCTCTGTTCTTGGGAATCCATGGAGGGAAGATGTGCCTGT V VP I E P H AL F L G I H G GK MC L 310 320 330 340 350 360 CCTGTGTCAAGTCTGGTGATGAGACCAGACTCCAGCTGGAGGCAGTTAACATCACTGACC S C VK S G D ET R L Q L E A VN I T D 370 380 390 400 410 420 TGAGCGAGAACAGAAAGCAGGACAAGCGCTTCGCCTTCATCCGCTCAGACAGTGGCCCCA L S E N R K Q DK R F A F I RS D S G P 430 440 450 460 470 480 CCACCAGTTTTGAGTCTGCCGCCTGCCCCGGTTGGrjTCCTCTGCACAGCGATGGAAGCTG TT S F E S AA C P G W F L C TA M E A 490 500 510 520 530 540 ACCAGCCCGTCAGCCTCACCAATATGCCTGACGAAGGCGTCATGGTCACCAAATTCTACT D Q P V S L T NM P D E G V M VT K F Y 550 +560 570 580 590 600 TCCAGGAGGACGAGTAGTACTGCCCAGGCCTGCTGTTCCATTCTTGCATGGCAAGGACTG F Q ED E* sUBSTITUTE S-HET, *SUBSTITUTE SHEET WO089/11540 PCT/US89/02275 22/22 FIG. M E IC R G L R S H LI T L L L F L S ET I C®P S G R K S SK M Q A F RI W D V N QK T F Y L R N N QL V A G Y L s0 QG P N V N L E E K I D V V P I E P H A 100 L F L G I H G G K M C L S C V K S G D E 110 120 T R L QL E A V N I T D L S E. N R K QD 130 140 K R F A F I R S D S G P T T S F E S A A 150 160 C P G W F L C T A M E A D Q P V S L T N 170 M P D E G VM V T K F Y F Q E D E SUIBSTITUTE SHEF2T INTERNATIONAL SEARCH REPORT International Aochc.ion No. PCT/US89 /0227 I. CLASSIFICATION OF SUBJECT MATTER ,lf sitral ciassriti.r, Fvmr~ns nnv "rii:aie all)t f r Accicmg a Internaianal Patent Ciassificaon (IPC) or to oon National Classication and IPC IPC(4): C12P 21/00; C07K 13/00; C07H 15/12 U.S. Cl.: 435/68; 530/350; 536/27 II. FIELDS SEARCHED Minimum Documenttion Searcneo 7 Classfication System Classification Symools U.S. 435/68, 70, 91, 172.1, 172.3, 252.33, 320 1536/27 530/350 935/4, 18, 29, 32, 34, 38, 56, 57 Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included in the Fields Searched a Chemical Abstracts DataBase (CAS) 1967-1989 Key works: interleukin, inhibitor ill. DOCUMENTS CONSIDERED TO BE RELEVANT 9 Category Citation ol Document, 11 with indication, where aOpproiate, of the relevant passages 12 Relevant to CL. m No. '1 X Journal of Experimental Medicine, volume 1-4 Y 163, issued March 1986, (ROBERTS et al) 5-25 "Interleukin 1 and interleuKin 1 inhibitor production by human macrophages exposed to influenza virus or respiratory syncytial virus", See pages 511-519, see especially the abstract and figure X Journal of Immunology, volume 134, 1-4 Y issued June 1985, (LIAO et al) 5-25 "Characterization of a human interleukin 1 inhibitor", see pages 3882-3886, see especially the abstract and figure 8. SSpecial categories ot cited documents: o later document published after the international filing date document dofining the general state of the art which is not or plorty date and not in conflic with the aolication but ont t h e gneratlstate of t h e a rt w h i c hiscited to understand the principle or theory underlying the considered to be of particular relevance invention earlier document but published on or after the international document of particular relevance; the claimed invention filing date cannot be considered novel or cannot be considered to document which may throw doubts on priority claim(s) or involve an inventive step which is cited to establish the publication date of another document of particular relevance: the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when thwJ document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document oublished prior to the international filing date but in the art. later than the prjority date claimed document member of the same patent family IV. CERTIFICATION Oate of the Actual Completion of the International Search Date of Mailing of this International Search Report 7 Auoust 19890 6SEP 99 International Searching Authority Snalure of Authorized Officer ISA/US Jan Ellis Fomh PCTASAi0 (Msaond (Re.ltt4) 07rS;'i UTE SHEET International AoPlcation No. PCT/US89/02275 Ill. DOCUMENTS CONSIDERED TO 13E RELEVANT (CONTINUED FROM THE SECOND SHEET) Category Citation at Document. aitt indication, where appropriate, ot the relevant oassages Relevant to Claim No x Journal of Immunology, volume 139,14 Yissued Sep ;tember 1987, (SECKINGER et al) 52 "A urine inhibitor of interleukin 1 activity affects both interleukin 1&4-and interleukin lp but not tumor necrosis Ii factor see pages 1541-1545, see especially the abstract x Chemical Abstracts, volume 105, no. 17, 1-4 Y issued 1986, L.T. Hall, "Isolation and 5-25 partial purification of an inhibitor to interleukin see page 539, column 2, the abstr-act no. 151238 w Diss. Abstr. Int. B, 1986, 46(12), pt. 1, 4191. A Chemical Abstracts, volume 107, no. 25, 1-25 issued 1987, K. Williamson "Bioassay for interleuKin-l inhibitors", see page column 1, the abstract no. 234307K. J. Immunol. Methods, 1987, 102(2), 283-4 (Eng). Chemical Abstracts, volume 108, no. 17, 1-25 Y, P issued 1988, D. L. Rosenstreich, "Human interleuKin-±L inhibitors", see :1 page 559, column 1. the abstract no. 148372s. lymphokines, 1987, 14, 63-89 (Eng). FamPCTj't~ijex. (maea) (R~v.ila7)