DK175969B1 - Interleukin-1 inhibitor - obtd. from urine of febrile patients or by recombinant DNA techniques, used in immunosuppressive and anti-inflammatory compsns. - Google Patents

Abstract

A pure interleukin-1 inhibitor (IL-1 INH) is claimed characterised by an inhibitory activity to the LAF activity of IL-1, to the MCF activity of IL-1, to IL-1 mediated fibroblast proliferation and non-inhibitory activity to the INF alpha mediated prodn. of PGE2 and collagenase. Also claimed is a recombient DNa molecule comprising a DNA sequence which codes on expression for the IL-1 INH.

Description

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The present invention relates to interleukin 1 inhibitors (IL-1 INHs) which selectively inhibit interleukin 1 activity. The invention also relates to a method for purifying such IL-1 INHs from urine. These methods and agents are useful for immunosuppressive and anti-inflammatory applications and therapies.

5 Interleukin 1 (IL-1) is a protein cytokine produced primarily by macrophage / monocyte lineage cells. There are two distinct IL-1 genes capable of encoding IL-1 polypeptides - IL-1 and IL-1 (P. Auron et al., "Nucleotide Sequence of Human Monocyte Interleukin-1 Precursor cDNA" , Proc. Natl. Acad. Sci., USA, 81, p.

7907 (1984), C. March et al., "Cloning Sequences and Expression of Two Distinct 10 Human Interleukin 1 Complementary DNA's Eye β", Nature. 315, p. 641 (1985)).

Studies of their respective biological activities using recombinant IL-1 α and β have thus far shown that both forms of IL-1 exhibit multiple biological activities.

IL-1 exhibits several specific biological activities. One of these activities — lymphocyte activating factor (LAF) activity — results in IL-1 as an immunologic response mediator stimulating IL-1 maturation, differentiation, and growth of many cell types such as immature T and B lymphocytes (P. Auron et al., Proc. Natl. Acad. Sci.

USA. 81, supra). Another IL-1 activity - mononuclear cell factor (MCF) activity - results in IL-1 playing a key role in the regulation of various inflammatory responses (C. Dinarello, "An Update of Human Interleukin 1", J, Clin Immun. 5, p. 287 (1985), and as such, IL-1 stimulates several cells, e.g., fibroblasts and chondrocytes, to produce prostaglandin E2 (PGE2) and collagenase, respectively. These responses are involved in the pathogenesis of joint disease. such as rheumatoid arthritis or diseases associated with tissue destruction (J. Dayer, "Cytokines and Other Mediators in Rheumatoid Arthritis", Springer Semin. Immunopath. 7, p. 387 (1984)). -1 induces the production of IL-2 (JW Lowenthal et al., "IL-1 Dependent Induction of Both IL-2 Secretion and IL-2 Receptor Expression by Thymoma Cells", J. Imm. 137, pp. 1226-1231 ( 1986)), which is involved in T cell proliferation. Finally, it is also known that IL-1 stimulates molecules on endothelial cells. r to capture white blood cells (J. Oppenheim et al., "There is More Than One Interleukin 1". Immune. Todav. 7. P. 45 (1986) 1.

Therefore, it would be interesting to identify and isolate an IL-1 inhibitor that suppresses antigen-specific T cell and B cell proliferation and suppresses prostaglandin and collagenase synthesis in fibroblasts. Such a compound would be useful in the treatment of disorders involving immune and inflammatory responses. In addition, it would also be desirable to isolate an IL-1 inhibitor capable of suppressing IL-1-mediated IL-2 production. In addition, it is also interesting to identify a compound that selectively inhibits the activities of IL-1 without simultaneously

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In inhibiting other proteins, e.g. tumor necrosis factor such as TNF-σ having multiple biological I

Characteristics coincide with IL-1, i.e. PGE2 and collagenase production in human I

In dermal fibroblasts (J. Dayer et al., "Cacchectin / Tumor Necrosis Factor Stimulates I

In Collogenase and Prostaglandin E2 Production by Human Synovial Cells and Dermal I

In 5 Fibroblasts ", J. Exp. Med. 162, p. 2163 (1985)) or induction of fibroblast proliferation I.

(P. Seckinger et al., "A Urine Inhibitor of Interleukin 1 Activity Affects Both Interleukin 1 and I

I And B But Not Tumor Necrosis Factor 0, J. Immun. 139, p. 1541 (1987). I

Currently, compounds that are reported to exhibit inhibitory effects I work

In contrast to IL-1, such as prostaglandins, primarily as non-selective inhibitors. So are you

In 10 it has been reported that urine from fever patients contains a 20-30 kD selective inhibitor for I

IL-1 (Z. Liao et al., "Identification of a Specific Interleukin 1 Inhibitor in the Urine of I

In Febrile Patients, "J. Exp. Med. 159, p. 126 (1984)). Liao does not suggest that this

compound occurs in other than very crude form and that it inhibits MCF activity I

I of IL-1, inhibits the binding of IL-1 to target cell receptors or inhibits I

In 15 fibroblast proliferation in the presence of IL-1. Another compound which is reported to have

IL-1 Inhibitory Effects (W. Arend et al., "Effects of Immune Complexes On I

I Production By Human Monocytes of Interleukin or an Interleukin 1 Inhibitor ", J. Immun. I

I 134, p. 3868 (1985)) is produced by human monocytes grown on adherent I

In immune complexes. However, the Arend report is unclear as to whether compound I

In both, LAF and MCF inhibit the activity of IL-1 (see p. 3874). Under all I

In the circumstances, it is not reported in the Arend article. that the compound is essentially I

pure or that it blocks IL-1 binding to target cell receptors or inhibits fibro-I

blast proliferation in the presence of IL-1. A third compound is reported to have IL-1 I

In Inhibitory Effects (J-F. Balavoine et al., "Prostaglandin E2 And Collagenase I

I 25 Production By Fibroblasts And Synovial Cells Is Regulated By Urine-derived Human I

In Interleukin 1 And Inhibitors), J. Clin. Invest. 78, p. 1120 (1986))

In very crude form and its mode of action is not described. IN

The present invention solves the above problems by:

To provide substantially pure IL-1 inhibitors ("IL-1 INHs") which selectively inhibit I

In 30 IL-1 LAF and IL-1 MCF activities, IL-1 mediated production of IL-2, inhibited IL-I

I-1-mediated fibroblast proliferation and binds to the IL-1 receptors on target cells, to I

For use in immunosuppressive or anti-inflammatory preparations, I

procedures and therapies. The IL-1 INHs of the invention do not inhibit TNFα1

In mediated production of PGE2 and collagenase in target cells. The IL-1 INHs of the invention I

Thus, 35 are useful in many different compositions and in methods of I

In immunosuppressive and anti-inflammatory applications. The IL-1 INHs of I

The invention is characterized by being polypeptides having a molecular weight of approx. 25 I

3 DK 175969 B1 kD according to SDS / PAGE and an isoelectric point of 4.7 as determined by chromatofocusing.

Preparation of these IL-1 INHs includes concentration of crude urine from fever patients, precipitation of crude IL-1 INH from the urine and fractionation of IL-1 INH from the other 5 proteins of this precipitate by either ion exchange chromatography, hydrophobic chromatography, gel filtration and / or immune absorption.

The IL-1 INHs of the invention can be used to determine the I molecular structures and localize the active IL-1 INH sites, and this information can then be used to design fragments and peptides for use as IL-110 INHs in immunosuppressants. or anti-inflammatory preparations and methods.

FIG. 1 depicts the activity profile of urinary IL-1 INH obtained from the QAE-Sepharose step.

FIG. 2 depicts the activity profile of urinary IL-1 INH obtained from the DEAE-Sepharose step.

FIG. 3 depicts the activity profile of urine IL-1 INH from the AcA54 filtration fraction.

FIG. Figure 4 depicts the dose response for urinary IL-1 INH pools after each purification step as measured by IL-1 / LAF and IL-1 / receptor binding assay.

FIG. 5 depicts IL-1 INH activity measured by IL-1 / LAF assay.

FIG. 6 depicts IL-1 INH activity as measured by IL-1 / MCF assay.

FIG. Figure 7 shows that IL-1 iNH does not affect hrTNFo-induced PGEg production.

FIG. 8 depicts IL-1 INH activity measured by IL-1 / fibroblast proliferation assay.

FIG. 9 depicts specific IL-1 INH activity measured after each purification step by IL-1 receptor binding assay, LAF assay, EL-4 / CTLL assay and MCF assay.

The invention is illustrated in more detail in the following detailed description.

In the specification, the following terms are used: MCF - "mononuclear cell factor." The "MCF activity" of IL-1 indicates its ability to stimulate prostaglandin E and collagenase production in numerous target cells, e.g. fibroblasts, synovial cells.

LAF - "lymphocyte activating factor". The "LAF activity" of IL-1 indicates its ability to stimulate the proliferation and differentiation of T and B lymphocytes.

CTLL - "" cytotoxic T lymphocyte cell line ". CTLL cells are incubated with EL-4 cells and used to assay the stimulatory effect of IL-1 on the production of IL-2 from EL-4 cells. IL-2 then stimulates proliferation. of the CTLL cells, which can be measured.

The present invention relates to substantially pure IL-1 INHs. "Essentially pure" IL-1 INHs, as defined in the present invention, are essentially free of essential contaminants, especially apolipoprotein A1 and

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retinol binding protein, and moves as a single band according to SDS / PAGE. IN

The IL-1 INHs of the invention selectively inhibit IL-1 LAF and IL-1 MCF I

activities, IL-1-mediated IL-2 inhibits production by EL-4 cells, inhibits IL-1- I

mediated fibroblast proliferation and binds to the IL-1 receptors on target cells. The definition I

I 5 on such selective inhibition is that it has the ability to block IL-1 mediated activity,

However, it lacks the ability to block other compounds with similar activities

I as IL-1, such as human recombinant TNF10 (hrTNFff), which is a mediator for PGE2-1

I and collagenase production. This specificity is an important factor for selectively blocking I

IL-1 without interfering with the necessary activity of other mediators in the immune system. IN

This specificity of the IL-1 INHs of the invention was demonstrated by comparing I

In the effect of the IL-1 INHs of the invention on the activity of IL-1 against hrTNFo I

using IL-1 / MCF and fibroblast proliferation assay. IN

In most preferred, the IL-1 INHs of the invention have a molecular weight of approx. 25 I

In kD according to SDS-PAGE and their isoelectric point is 4.7 as determined by I

In chromatofocusing. IN

The IL-1 INHs of the invention are capable of inhibiting an IL-1 mediated response I

I in an LAF assay, an MCF assay, an EL-4 / cytotoxic T lymphocyte assay ("EL-4 / CTLL") and an I

In fibroblast proliferation assay. In IL-1 / LAF assay, the inhibition of IL-1 is or is measured

In Li-induced T cell proliferation by detecting a reduction in the level of incorporation I

In 20 O of [H1-1-thymidine in the presence of various dilutions of IL-1 INH of the invention. Do you know

In IL-1 / MCF assay, the inhibition of IL-1 mediated PGE2 production is measured by a double I antibody radioimmunoassay using antiserum against PGE ~ in the presence of I I in various dilutions of IL-1 INH of the invention. The decrease of PGE2 in medium I as detected by a radioimmunoassay indicates IL-1 INH activity. By EL-4 / CTLL-I assay, the inhibition of IL-1-mediated IL-2 production is measured in the presence of various dilutions of IL-1 INH according to the invention. The inhibition is observed as a reduction in the uptake level of [H ^ J-thymidine ([H ^ j-TdR) used as a measure of CTLL cell proliferation. Proliferation occurs only in the presence of IL-1 that induces IL-I2 in a dose-dependent manner. In fibroblast proliferation assay, the inhibition 30 of [H + -TdR uptake of IL-1-induced fibroblasts is measured in the presence of different I dilutions of IL-1 INH of the invention.

As described above, the IL-1 INHs of the invention specifically bind to IL-1- I receptors on target cells. This binding was demonstrated using a series of IL-1-I binding assays. First, the effect of increased concentrations of IL-1 INH on the binding of labeled IL-1 to receptors on target cells was measured. It was observed that higher I concentrations of IL-1 INH reduced the amount of bound labeled IL-1. Next, an excess of unlabeled IL-1 INH was added, and the effect of this excess on the tagged IL-1 bound to a target cell bound was hunted. This analysis shows that excess IL-1 INH successfully competed with and replaced the labeled IL-1 bound to the surface of the target cells. An additional excess of retinol binding protein was added and it was observed that this did not prevent the binding of IL-1 INH to the target cells. This assay 5 showed that the IL-1 INHs of the invention compete specifically with IL-1 for binding to IL-1 receptors on target cells.

The present invention relates to a purification method for isolating IL-1 INH according to the invention from natural sources, e.g. patients with high fever or AIDS patients suffering from opportunistic infections. This process comprises several ί 10 steps. Broadly, these steps include (1) concentration of urine, (2) precipitation of crude IL-1 INH from the concentrated urine, and fractionation of IL-1 INH from the other proteins in this precipitate by ion exchange chromatography, hydrophobic chromatography, gel filtration and / or immune absorption.

In a preferred embodiment of the method of the invention, first, crude urine from fever patients was first concentrated using standard procedures, e.g. ultrafiltration. Next, a crude fraction containing the IL-1 INH was precipitated from the concentrated urine pool using ammonium sulfate. After replacing the ammonium sulfate by dialysis, the fractions containing IL-1 INH activity were separated from other proteins using ion exchange chromatography. Especially in the most preferred embodiment, two anion exchangers - one diethyl (2-hydroxypropyl) - were used in the aminoethyl Sepharose column ((QAE) -Sepharose column) and a diethylaminoethyl Sepharose column (DEAE) -Sepharose column). separately or in combination, the DEAE-Sepharose column being preferably used after the QAE-Sepharose column. To monitor the activity of the various fractions, LAF and receptor binding assay was used. In this embodiment of the invention, the IL-1 INH active fractions are then fractionated on a molecular weight basis using gel filtration, most preferably on an AcA54 gel, again selecting the active fractions as above. The selected ions fractions are characterized by a molecular weight of approx. 25 kD and at least 90% of the protein content. The major contaminants are apolipoprotein A1 and re-30 tinol binding protein. Although these contaminants can be removed in different ways | the immune absorption method is preferred using monoclonal or polyclonal antibodies induced against apolipoprotein and retinol binding protein. j

The above-mentioned active IL-1 INH fractions are further purified by hydrophobic chromatography on a phenyl-Sepharose column which fractionates proteins according to their hydrophobicity. Apolipoprotein A1 and retinol binding protein are e.g. more hydrophobic than proteins of the present invention and will therefore be retained on the cavity.

i i i

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Using the preferred method described above, I was increased

In the specific activity of the IL-1 INH of the invention, i. the amount of IL-1 INH that I

You were required to produce half of the maximum inhibition after each I

purification steps. See FIG. 9. Per an IL-1 receptor binding assay, an LAF assay, and I were used

In 5 EL-4 / CTLL assays and an MCF assay to measure the specific activity. Several others I

However, analyzes could also have been used. IN

In the IL-1 INH which has been purified by the method I described above

I or a preferred method can be used directly in immunosuppressants and I

anti-inflammatory preparations. Such purified proteins can be used as a source I

I 10 for amino acid sequence data to enable the design of DNA probes for use I

I by isolating and selecting a DNA sequence encoding an IL-1 INH of I

In the invention. Such DNA sequences, recombinant DNA molecules containing I

these sequences, as well as single-cell hosts that have been transformed with these I

In sequences, can then be used to prepare large amounts of IL-1 INHs contained in it

Essentially, it is free of other human proteins, for use in the compositions and for I

therapies. IN

In the above application, the amino acid sequences are determined by different I

In parts and fragments of the purified IL-1 INH. Next, these sequences and the I

In derived DNA sequences encoding therefore, to produce a series of DNA probes which I

Are potentially useful for screening various DNA libraries for DNA sequences, I

In coding for the IL-1 INHs. Such libraries include chromosomal gene banks and I

DNA or cDNA libraries prepared from tissues or cell lines for which it has I

have been shown to produce the IL-1 INHs. Such cell lines include monocytic I

In cell lines well known in the art. j I

As a means of producing cDNA and finally cloning and expression of IL-1 I

In INH polypeptides, poly A + mRNA is isolated from an IL-1 INH producing cell source such as I

In stimulated macrophages, using conventional methods, e.g. as in

As described in Land et al., "5-Terminal Sequences Of Eukaryotic mRNA Can Be Cloned I

I With High Efficiency. "Nucleic Acids Research. 9. pp. 2251-66 (1981). Okavoma and, I

Berg, "High Efficiency Cloning of Full-Length cDNA, Mol, and Cell. Biol. 2, pp. 161-70 in I

I I

(19821. et al. Maniatis et al. In "Molecular Cloning1" bold. Cold Spring Harbor Laboratory. Cold I

In Spring Harbor, New York), pp. 229-46 (1982). Next, a cDNA library I was built

I isolated from the above poly A + mRNA using conventional I

In methods, e.g. as described in Wickens et al. "Synthesis Of Double-Stranded I

In 35 DNA Complementary To Lysosyme, Ovomucoid and Ovalhuman mRNAs ", J. Biol. I

In Chem., 253, pp. 2483-95 (1978), Maniatis et al. in "Molecular Cloning" (ed. Cold Spring I

In Harbor Laboratory, Cold Spring Harbor, New York), pp. 229-46 (1982), and V. Gubler et al. "A Simple and Very Efficient Method for Generating cDNA Libraries", Gene. 25, pp.

263-69 (1983).

There are several methods for screening a clone library for a clone containing specially recombinant DNA molecule, ie. a clone containing an IL-1 INH insert. For example, on the basis of a partial amino acid sequence of the purified IL-1 INH, DNA probes can be constructed which include a variety of synthetic DNA fragments encoding selected portions of the IL-1 INHs. Techniques for determining amino acid sequences are well known in the art. Following the determination of amino acid

the sequences of different IL-1 INH regions can be pooled by degenerate IL-1 INH

10 probes are chemically synthesized using conventional phosphoramide DNA synthesis techniques for use in the screening of various DNA libraries to select related DNA sequences by hybridization. The DNA probes are then labeled 32 at the 5-end with P using P-ATP and T4 polynucleotide kinase, essentially as described in A.M. Maxam and W. Gilbert, "A New Method for Sequencing 15 DNA," Proc. Natl. Acad. Sci. USA. 74, pp. 560-64 (1977). These DNA probes are then used to screen cDNA or genome libraries, e.g. cDNA libraries derived from monocytic leukemia cell lines U937, THP-I and HL60 for DNA sequences encoding IL-1 INHs using conventional methods. These selected sequences can then be manipulated for expression of IL-1 INHs in prokaryotic and 20 eukaryotic hosts, which have been transformed with these sequences by methods well known in the art. The sequences are also useful as screening probes for selecting other related DNA sequences encoding mammalian IL-1 INHs.

The DNA sequences and DNA molecules can be expressed using many different host / vector combinations. For example, useful vectors may consist of 25 segments of chromosomal, non-chromosomal and synthetic DNA sequences such as various known derivatives of SV40 and known bacterial plasmids, e.g. plasmids from E. coli, including co1E1, pCR1, pBR322, pMB9 and RP4, phage DNAs, e.g. the various derivatives of phage, e.g. NM 989, and other DNA phages, e.g. M13 and other single-stranded Filamentous DNA phages, vectors that can be used in yeast species such as the 2β plasmid, j. 30 vectors which can be used in eukaryotic cells, such as vectors useful in

animal cells, such as e.g. vectors containing DNA sequences derived from SV-40 adenovirus and retrovirus, and vectors derived from combinations of plasmids and phage I

DNAs such as plasmids that have been modified to use phage DNA or other derivatives thereof.

Such expression vectors are also characterized in that they contain at least one expression control sequence operatively linked to the IL-1 INH DNA sequence inserted into the vector to control and regulate the expression of the DK 175969 B1.

I I

cloned DNA sequence. Examples of useful expression control sequences are lac-I

the system, the three system, the tac system, the three system, the main operator— and I

the promoter regions in phage λ, the control region for the former coating protein, the glycolytic promoters in yeast, e.g. the promoter of 3-phosphoglycerate kinase, the promoters of yeast acid phosphatase, e.g. Pho5, the promoters of the yeast o-adaptation factors, and promoters derived from polyoma, adenovirus, retrovirus and simian virus, e.g. the early and late promoters of SV40, as well as other sequences known to control the expression of genes of prokaryotic or eukaryotic cells and their viruses or combinations thereof.

Such useful expression vectors include vectors that enable the expression of the cloned IL-1 INH DNA sequences in eukaryotic hosts such as in animal and human cells (e.g., P. J. Southern and P. Berg, J. Mol. AppI. .

327-41 (1982), S. Subramani et al., Mol. Cell. Biol. 1, pp. 854-64 (1981), R.J. Kaufmann

I and P.A. Sharp, “Amplification And Expression Of Sequences Cotransfected With A

Modular Dihydrofolate Reductase Complementary DNA Gene ", J. Mol. Biol. 159, p.

I, 601-21 (1982). R.J. Kaufmann oo P.A. Sharp. Moth. Cell. Biol., 159. pp. 601-64 (19821.

S. I. Scahill et at., "Expression And Characterization Of The Product Of A Human Immune In Interferon DNA Gene In Chinese Hamster Ovary Cells", Proc. Natl. Acad. Sci. U.S.A ..

In 80, pp. 4654-59 (1983). G. Uriaub and L.A. Chasin. Proc. Natl. Acad. Sci. USA. 77. pp.

In 4216-20 (1980)).

In each specific expression vector, different sites may also be selected for insertion of the IL-1 INH DNA sequence. These sites are usually denoted by the restriction endonuclease that cuts them. They are well known to those skilled in the art. It is apparent that a useful expression vector in accordance with the invention does not necessarily have to have a restriction endonuclease site for insertion of the selected DNA fragment. Instead, the vector can be bound to the fragment alternatively.

In the Expression Vector, and in particular the space selected therein for inserting a selected DNA I fragment and the operative binding of the fragment therein to an expression control sequence I is determined by various factors, e.g. the number of sites affected by a particular I restriction enzyme, the size of the protein to be expressed, the sensitivity of the desired protein to proteolytic degradation by host cell enzymes, pollution I, or binding of the protein to be expressed to host cell proteins which are difficult to remove during purification, expression characteristics such as the location of start and stop codons relative to the vector sequences, and other factors known to those skilled in the art.

In Vector selection and insertion site for a DNA sequence, by balancing I 35 these factors are determined, as not all choices are equally effective in a given case.

Useful expression hosts include well-known eukaryotic and I prokaryotic hosts such as strains of E. coli, e.g. E. coli SG-936, E. coli HB 101, E. coli 9 E. 175969 B1 W3110, E. coli X1776, E. coli X2282, E. coli DHI and E. coli MRC1, Pseudomonas.

Bacillus, e.g. Bacillus subtilis, Steptomyces, herbs and other fungi, animal cells, e.g. COS cells and CHO cells, and human cells and plant cells in tissue culture.

Of course, not all host / expression vector combinations are equally effective in expressing the DNA sequences or producing IL-1 INH-like polypeptides.

However, the choice of a particular host / expression vector combination can be made by one of ordinary skill in the art with due regard to the principles set forth herein without departing from the scope of the invention. The selection should e.g. is based on the trade-off of a number of factors. These include e.g. host-vector compatibility, toxicity of the proteins encoded by the DNA sequence to the host, the ease with which the desired protein can be recovered, the expression characteristics of the DNA sequences and the expression control sequences operatively linked thereto, biosecurity, cost, and folding, shape or other necessary modifications of the desired protein after expression.

The IL-1 INHs, which have been prepared by fermentation of the prokaryotic and eukaryotic hosts transformed with the DNA sequences, can then be used in the immunosuppressive and anti-inflammatory preparations.

The IL-1 INHs can also be assayed to determine the active sites therein for the production of fragments or peptides, including synthetic peptides with activity as IL-1 INHs. Among the known techniques for determining such active sites are X-ray crystallography, nuclear magnetic resonance, circular dichroism, UV spectroscopy, and site-specific mutagenesis. These fragments or peptides can be used for immunolysis. suppressive or anti-inflammatory purposes and methods.

Administration of the IL-1 INH polypeptides, or peptides derived or synthesized therefrom or obtained using their amino acid sequences, or their salts or pharmaceutically acceptable derivatives thereof, can be accomplished via any of the conventionally accepted modes of administration which exhibit agents. immunosuppressive or anti-inflammatory activity. These include oral, parenteral, subcutaneous, intravenous, intralessential or topical administration. Local, intravenous or intravenous injection is preferred.

The compositions used in these therapies may also have many different forms. These include e.g. solid, semi-solid or liquid dosage forms such as tablets, pills, powders, liquid solutions or suspensions, suppositories, in injection solutions and infusion solutions. The preferred form is dependent on the intended mode of administration and therapeutic use. The compositions will preferably also contain conventional pharmaceutically acceptable carriers and may comprise other drugs, carriers, adjuvants, excipients, etc., e.g. human serum albumin or in plasma preparations. Preferably, the compositions take the form of one in unit dose and are usually administered one or more times a day.

The invention is illustrated in more detail in the following examples. Obviously, these examples are intended to serve as an illustration only and not to limit the invention.

Example 1

Purification of IL-1 INH

a) Concentration of human urine protein I 10 1001 combined urine from high fever patients (> 38.5 ° C), which patients not suffering from urinary tract infections, were concentrated to 21 at 4 ° C in an Amicon ultrafiltration hollow fiber apparatus. The resulting solution exhibited a specific activity of 12 U / mg I protein as determined by the IL-1 receptor assay described in Example 2, 166 15 U / mg protein as determined by the LAF assay described in Example 3, 32 U / mg I protein as determined by the EL-4 / CTLL assay described in Example 4 and 125 U / mg protein as determined by the MCF assay described in Example 5. "U" or I unit is defined as the amount of IL-1 INH (pg) that produces half the maximum inhibition in each bioassay. See FIG. 9th

I b) Precipitation of Human Urine Protein I First, the combined concentrated urine with solid ammonium sulfate I was saturated by slow addition of the ammonium sulfate with constant stirring at 4 µC to I25 to obtain an ammonium sulfate saturation of 40%. Next, precipitate was removed from the solution by centrifugation in a Sorvall RC-5B (E.L. Du Pont, New Town, Conn.) Using a fixed angle GSA rotor at 10,000 rpm for 1 hour. Next, the pellet obtained was discarded and the supernatant adjusted to 80% saturation with 1 ammonium sulfate and centrifuged at 10,000 rpm for 1 hour. The resulting pellet I was resuspended in 20 mM sodium phosphate (pH 7.2) with 150 mM NaCl (650 mL).

The solution was then dialyzed for 24 hours against 451 (twice) containing 10 mM Tris I HCl (pH8), 2 mM EDTA and 5 mM benzamidine HCl to remove the ammonium sulfate.

In c) lonebinotine chromatography I

Next, IL-1 INH activity in the combined fraction was separated from other proteins using the strong binding affinity of IL-1 IHNs for two different anion exchangers. Each anion exchanger was used alone or in combination, the DEAE-Sepharose column being preferably used after the QAE-Sepharose column.

1) Diethyl (2-hydroxypropyraminoethyl) (QAE) Sepharose column 5

Although many chromatographic anion exchange systems are well known to those skilled in the art, a 5 cm diameter x 45 cm (Pharmacia Fine Chemicals, Piscataway, New Jersey) first was chosen to use a QAE-Sepharose column. After introducing the above dialyzed solution, the column was washed until unbound proteins were eluted (optical density at 280 nm). Oe bound proteins were eluted with four column volumes of a salt gradient from 0 to 0.8 M NaCl dissolved in the equilibration buffer.

The flow rate of the column was 120 ml / h. The activity of the various fractions was monitored by LAF and receptor binding assay (below). See FIG. 1. The fractions exhibiting the biological activity of IL-1 INH were eluted at ca. 150 mM NaCl. The 15 combined active fractions had a specific activity of 33 U / mg protein as determined by the IL-1 receptor binding assay described in Example 2, 63 U / mg protein as determined by the LAF assay described in Example 3, 27 U / mg protein as determined by the EL-4 / CTLL assay described in Example 4 and 200 U / mg protein as determined by the MCF assay described in Example 5. See FIG. 9th

2) Diethylaminoethyl (DEAEI Sepharose column

The combined active fractions were dialyzed against 10 mM Tris HCl (pH 7) loaded onto a rapid-flow DEAE-Sepharose column, 2.5 cm x 30 cm (Pharmacia Fine Chemicals, Piscataway, New Jersey). The column containing the active combined fractions was washed with equilibration buffer (10 mM Tris HCl, pH 7) until the optical density (at 280 nM) was approx. 0. Next, the bound protein was eluted with a gradient of 0 to 0.2 M NaCl dissolved in the equilibration buffer. The gradient was 10 times the volume of the column. The flow rate of the column was 78 ml / hr. Again, the activity of the various fractions was monitored as above. This elution gave elution of the fractions containing IL-1 INH activity at the end of the washing step with 90 mM NaCl. FIG. Figure 2 shows the activity profile of IL-1 inhibitor in urine on DEAE-Sepharose, where the inhibitory activity was followed by (A) IL-1 / LAF assay and (B) IL-1 / receptor binding assay (below).

Next, the combined active fractions were concentrated to 6 ml with an Amicon ultrafiltration apparatus using a YM-10 membrane. The resulting solution had a specific activity of 50 U / mg protein as determined by IL-1 in DK 175969 B1 receptor binding assay (below), 125 U / mg protein as determined by LAF assay (below), 40 U / mg protein as determined by EL-4 / CTLL assay (below) and 500 U / mg protein as determined by MCF assay (below). See 5 Of course, it is obvious that other anion exchange columns will also be selectable without departing from the scope of the invention.

d) Ultroael AcA54 10 Next, the preparation prepared was fractionated twice as described above according to molecular weight using gel filtration. Although a number of suitable gel filtration systems are well known to those skilled in the art, it was chosen to use an AcA54 gel (LKB, Sweden) with a fractionation range of 6,000 * 70,000 daltons. The activity of the fractions was monitored again as above. See FIG. 3. The resulting combined 15 active fractions showed a specific activity of 1666 U / mg protein as determined by an IL-1 receptor binding assay (below), 526 U / mg protein as determined by LAF assay (below), 333 U / mg protein as determined by EL-4 / CTLL assay (below), 1110 U / mg protein as determined by MCF assay (below) (see Figure 9), and a molecular weight of approx. 25 kD. Again, it is obvious that other filtration systems may also be used.

FIG. 4 illustrates the dose response of the various combined urinary IL-1 INHs in the (A) IL-1 / LAF assay described below and (B) IL-1 / receptor binding assay.

After each purification step, i.e. Concentrated urine, QAE-Sepharose, DEAE-Sepharose, and AcA54 (twice), reduced the concentration (pg / ml) of IL-1 INH required to achieve inhibition, indicating the presence of a purer protein.

e) Negative Immune Absorption 30

The combined active fractions from the gel filtration were analyzed by amino acid sequencing using conventional Edman automated degradation, and the two major contaminants constituting at least 90% of the protein content were apolipoprotein A1 and retinol binding protein. Although there are 35 different methods for removing these proteins, immune absorption and hydrophobic chromatography were chosen.

Thus, the main contaminants were then separated from the combined active IL-1 INH AcA54 by immune absorption. Monoclonal and polyclonal antibodies against retinol binding protein and apolipoprotein A1 were generated using standard immunization methods. Next, immunoglobulin G (IgG) was partially purified by precipitation with 40% ammonium sulfate saturation. The resulting IgG pellet was resuspended in 0.2 M sodium phosphate and dialyzed against the same buffer. The combined IgG was then coupled to vinyl sulfone-activated agarose as described by the manufacturer (KEM-EN-TEC, Biotechnology Corp., Denmark). After equilibrating the immunosorbent with phosphate buffer-adjusted saline, the contaminants from the combined IL-1 INHs were absorbed by passing them several times over the immunosorbent until any retinol-binding protein and apolipoprotein Al1 (SD) -sulfur-polyacrylate-acrylate-acetylsalcyl acetic acid-acetylsecyl acetate ). The resulting solution exhibited a specific activity of 3334 U / mg protein as determined by IL-1 receptor binding assay (below), 2500 U / mg protein as determined by LAF assay (below), 1250 U / mg protein as determined by EL-assay. 4 / CTLL assay (below), 2160 U / mg protein as determined by MCF assay (below) (see FIG.

9), and showed a single peak on SDS / PAGE.

'20 0 Phenyl Sepharose

The combined immune-absorbed IL-1 inhibitors were adjusted to 1 M NaCl by the addition of 1 volume of 2 M NaCl dissolved in 10 mM Tris HCl, pH 7, and introduced onto a phenyl-Sepharose column (0.5 x 5 cm, this resin was obtained from Pharmacia Fine in 25 Chemicals, Sweden). The resin was pre-equilibrated with 10 mM Tris HCl, pH 7 containing 0.2 M NaCl (equilibration buffer). After introduction to the column, the equilibration solution was washed with 3 column volumes to elute all unbound proteins, and the bound proteins were eluted with a gradient from 0.2 M NaCl to 0 M dissolved in 10 mM Tris HCl, pH 7. The overall gradient was 50 times the column volume.

The 30 column flow rate was 30 ml / h. IL-1 inhibitor activity was eluted at ca. 0.160 M NaCl yielding a protein with a molecular weight of 25 KDa and a PI of 4.7. The resulting solution had a specific activity of 38461 U / mg protein as determined by the IL-1 receptor binding assay described in Example 2, 2,500 U / mg protein as determined by the LAF assay described in Example 3, 35,020 U / mg 35 protein according to determination by the EL-4 / CTLL assay described in Example 4, 30,203 U / mg protein as determined by the MCF assay described in Example 5 (see FIG.

9), and showed a single peak on SDS / PAGE.

In DK 175969 B1 14 H Example 2

H Receptor Binding Capability of IL-1 INH

To determine the binding properties of IL-1 INH according to the invention above

5 for the IL-1 receptor on EL-4-6.1 target cells, it was first investigated whether IL-1 INH

disrupted the binding of [IJ-IL-1 to the target cells. IL-1 was labeled with I by

Chloramine T method (Lowenthal et al., "Binding and Internalization of Interleukin-1 by T

Cells, J. Exp. Med .. 164, p. 1060) and incubated with an excess of unlabeled IL-1 INH followed by washing on an oil gradient. Using a gamma counter, the material retained with the cells was then measured and it was found, H 125, that an increase in IL-1 INH concentration decreased the amount of [Q-IL-1 bound to the surface of the target cells.

H 125

In addition, it was investigated whether IL-1 INH disrupted the binding of [IJ-IL-1H 125 INH to the target cells, whether binding of [1] -IL-1 INH was able to compete with 125 unlabeled IL-1 or whether binding of [J-IL-1 INH to the target cells was able to compete with retinol binding protein or apolipoprotein A1. IL-1 INH was labeled with H 125 [I] according to the method described by Bolton and Hunter (Bolton and Hunter The 125

Labeling of Proteins to High Specific Radioactivities by Conjugation to a Containing I Acylating Agent ", Biochem. J. 133, p. 529 (1975)). Incubation of the material with EL-4- I 2 0 6.2 target cells followed by washing on an oil gradient as well as analysis on SDS PAGE showed that a material with a molecular weight of about 25 kD binds to the EL-4 cells, it was hunted, I 125, to incubate [IJ-IL-1 INH together with an excess of unlabeled IL-1 INH inhibited 125 binding of the labeled 25 kD material. [IJ-IL-1-INH was also incubated together with 50 ng of unlabeled IL-1 IX, and this was found to inhibit the binding of the labeled 125 [IJ-IL-1 INH was also incubated with 1 µg of immune purified I retinol binding protein and with 1 µg of recombinant apolipoprotein A1, and it was found that this did not inhibit the binding of the labeled 25 kD material. In labeled IL-1 INH preparation present 25 kD material to the surface of intact I EL-4-6.1 cells, and this binding is subject to competition from unlabeled inhibitor and IL-1, I30 but not from retinol binding protein or apolipoprotein A1. Example 3 In IL-1 / LAF Assay I The inhibitory activity of IL-1 INH in an IL-1 / LAF assay was demonstrated by the determination of thymocyte (T cell) proliferation in C mice (JM Dayer et al., "Human Recombinant IL-1 Stimulates Collagenase and Prostaglandin E2 Production by I Human Synovial Cells", J. Clin. Invest. 77, p. 645 (1986)). Thymocyte cells were co-stimulated for 72 hours with PH A (1 pg / ml) in the presence of either hr1L-10 (P. Wingfield et al., "Purification and Characterization of Human Interleukin-1 Expressed in E. coli" , Eur. J.

Biochem .. 165, p. 537 (1987)) or IL-β (P. Wingfield et al., "Purification and Characterization of Human Interleukin-1ft Expressed in Recombinant E. coli", Eur. J.

5 Biochem. 160, p. 491 (1986)) at various final concentrations ranging from 20 pg / ml to 2,000 pg / ml hrlL-1 as shown in FIG. 5. Complete inhibition of the co-stimulated hlL-1α or hlL-1 ft thymocyte proliferation was achieved when the IL-1 INH fractions of Example 1 (e) were added to the cells. The inhibitory activity was monitored in three dilutions of IL-1 INH: 1 / 20.1 / 40 and 1/80.

Since addition of IL-1 INH to PHA-stimulated cells in the absence of hrlL-1 did not affect [H] -TdR incorporation, it was shown that inhibition was not due to a cytotoxic or nonspecific effect but inhibition of the biological activity of IL-1.

Example 4 EL-4 / CTLL Assay

The inhibitory activity of IL-1 INHs was demonstrated by observing whether the ability of IL-10 or IL-1 ft to induce production of IL-2 in EL-4 cells could be inhibited (see, e.g., A.J.H.

3

Geanng et al., "A Simple Sensitive Bioassay for IL-1 which is Unresponsive to 10 U / ml of IL-2", J. Immun. Met .. 99, p. 7 (1987)). This was measured by co-culturing a subclone of 20 EL-4 cells that were unable to take up thymidine from surrounding medium with CTLL-2 cells and to observe whether the CTLL-2 cells proliferated.

EL-4.6.1 c10 cells and CTLL-2 cells were cultured together at a concentration of 4 10 cells of each type per cell. microtiter well (96 well plates) in the presence of IL-1a or IL-1ft at ca. 1 µg / ml together with IL-1 INH of Example 1 (e). 1 microCurie [H] -TdR 2 was added after 18 hours of co-culture and incubated for an additional 6 hours at 37 ° C in a humid atmosphere. Cells were harvested with a MASH cell harvesting device on fiberglass strips, dried and prepared with scintillation cocktail for counting in a beta counter. Complete inhibition of [H] -TdR incorporation with IL-1 INH dilutions of 1 / 20.1 / 40 and 1/80 was achieved.

30

Example 5 IL-1 / MCF Assay

The inhibitory activity of IL-1 INH in an IL-1 / MCF assay was further demonstrated by determination of fibroblasts obtained from human infantile foreskin (J. Dayer et al., 35 "Participation of Monocyte-Macrophage and Lymphocyte in the Production of a Factor that Stimulates Collagenase and Prostaglandin Release by Rheumatoid Synovial Cells ", J. Clin. Invest. 64, p. 1386 (1979), J. Dayer et al.," Induction of Human lterleukin-1 DK 175969 B1 16 mRNA Measured by Collagenase and Prostaglandin E2-Stimulating Activity in

Rheumatoid Synovial Cells ", Eur. J. Immunol. 14, p. 898 (1984)). The fibroblasts were stimulated with hrlL-1a or hrlL-1b at the same concentrations as IL-1 / LAF assay, and the inhibitory activity was monitored by the same final dilutions of 5 IL-1 INH as by IL-1 / LAF assay. After culturing the cells for 72 hours, prostaglandin E2 production was measured in fibroblast supernatants by a double antibody radioimmunoassay (J. Dayer (1979 ), above) using an antiserum against prostaglandin Ε2 · As shown in Fig. 6, a dose response for prostaglandin E2 production up to concentrations of 100 pg / ml with either hrlL-1o or hrlL-1B was chased. inhibiting this biological activity by adding the IL-1 INH fractions from Example 1 (e) It was shown that IL-1 INH was again effective against hrlL-1a and hrlL-1l.

Next, the above analysis was performed, replacing hrlL-1 with | hrTNFα (A. Marmenout et al., "Molecular Cloning and Expression of TNF and Comparison with Mouse TNF", Eur. J. Biochem. 152, pp. 515-22 (1985)) to determine the specificity of IL-1. 1 INH, as TNFor also mediates prostaglandin E2 and H collagenase production. As shown in FIG. 7, the hrTNF-induced prostaglandin I E2 production was not significantly affected by the addition of IL-1 INH of the invention, illustrating the specificity of IL-1 INH of the invention.

In Example 6

Fibroblastproliferationsassav

The biological activity of IL-1 in the presence of IL-1 INH of Example 1 (e) I was subjected to further study using a fibroblast proliferation assay to determine [H] -TdR incorporation. After culturing 25 human foreskin fibroblasts in Eagle's MEM supplemented with 10 mM HEPES, 100 U / ml I penicillin, 100 µg / ml streptomycin, 1% glutamine, 1% non-essential amino acids and 2% I FCS, cells were placed in 96-well plates. wells (2000 cells / well) and cultured for 24 l hours in a 5% CO 2 incubator at 37 ° C. After removal of the medium, the fibroblast cells were stimulated by the addition of either hr11L-10 or hr11L-1b and IL-1 INH was added in the same dilutions as under the assays listed above. After standing for 48 hours in 3, cells were treated with [H] -TdR for an additional 16 hours. The medium was removed from the cells, I and the cells washed with PBS and treated with trypsin at 37 ° C for an additional 15 I minutes. The cells were collected with a cell harvesting device (Skatron, Lier, Norway) I on glass filters (Skatron, Inc., Sterling, Virginia, USA), washed with water and air-dried, I5, and the cpm incorporation determined using a scintillation counter. As shown in FIG. 8, fibroblast proliferation was dose dependent with respect to the hrlL-1 o and hrlL-1 S dose.

I 3

A maximum [H] -TdR incorporation was obtained with 250 µg / ml hr11L-1a or B.

17 DK 175969 B1

Addition of the AcA54 inhibitory fraction also resulted in a complete decrease in hr1L-1-induced proliferation. Complete reversibility of the inhibition was also achieved by increasing IL-1 concentration or by diluting IL-1 INH.

The specificity of this inhibition was also determined by stimulation of the fibroblasts with hrTNFo, which induces fibroblast proliferation in a dose-dependent manner up to concentrations of 250 µg / ml. The addition of the AcA54 inhibitory fraction did not result in the inhibition of hrTNFo bioactivity. This confirms the specificity of IL-1 INH.

10

Example 7

Determination of the isoelectric point

The combined eluted proteins of Example 1 (e) were used for loading onto a PBE 94 chromatofocusing column (Pharmacia Fine Chemicals, Sweden) (2.5 x 10 15 cm) previously equilibrated with 25 mM imidazole (pH 7.5) . A 74 HCl, pH 4 polyp buffer was added which resulted in elution of bound proteins depending on their isoelectric points. IL-1 INH was shown to have a pI of 4.7.

Although a number of embodiments of the invention have been described above, it is obvious that the basic measures can be changed to provide other embodiments employing the methods and compositions of the invention.

It is therefore apparent that the scope of the present invention is defined by the claims and not by the specific embodiments set forth in the examples.

25

Claims (12)

A method of purifying substantially pure IL-1 INH, wherein the purified IL-1 INH II moves as a single band on an SDS / PAGE gel and is essentially free of apolipoprotein II A1 and retinol binding protein, and exhibits II (a) inhibitory activity against the LAF activity of IL-1; In I (b) inhibitory activity against the MCF activity of IL-1; In I (c) inhibitory activity against IL-1 mediated fibroblast proliferation; In I (d) inhibitory activity against IL-1 binding to IL-1 receptors; I (e) non-inhibitory activity against TNFα-mediated production of PGE2 and I I collagenase; and I (f) specific activity of at least 1.2 x 10 3 U / mg in an IL-1 mediated IL-2 production assay; The method of purification is characterized by fractionation of IL-1 INH from other I contaminants by either ion exchange chromatography, hydrophobic chromatography, gel filtration or I I immunoabsorption. IN Method according to claim 1, characterized in that before said fractionation I I a) concentrates urine from fever patients and I I b) precipitates crude IL-1 INH from the concentrated urine I Process according to claim 2, characterized in that the crude IL-1 INH is precipitated from the concentrated urine using ammonium sulphate. IN A method according to any one of claims 1-3, characterized in that the I I ion exchange chromatography is performed on QAE or DEAE Sepharose columns, alone or in I I combination. IN Process according to any one of claims 1-3, characterized in that the gel filtration 11 is carried out on an Aca54-gei. IN A method according to any one of claims 1-3, characterized in that the immunoabsorption I is performed using monoclonal or polyclonal antibodies against retinol binding protein I and apolipoprotein A1. In DK 175969 B1 Process according to any one of claims 1-6, characterized in that the purified IL-1 INH has a molecular weight of approx. 25,000 daltons on SDS / PAGE. Process according to claim 7, characterized in that the purified IL-1 INH has an isoelectric point of 4.7 by chromatographic focusing. The method according to claim 1, characterized in that the purified IL-1 INH has a specific activity of at least 3.8 x 10 4 U / mg in an IL-1 receptor binding assay. The method according to claim 1, characterized in that the purified IL-1 INH has a specific activity of at least 6.2 x 10 4 U / mg in an IL-1 / LAF assay. The method according to claim 1, characterized in that the purified IL-1 INH has a specific activity of at least 3.5 x 10 4 U / mg in an EL-4 / CTLL assay. The method of claim 1, wherein the purified IL-1 INH has a specific activity of at least 3.0 x 10 4 U / mg in an IL-1 / MCF assay.