HK1081124B - Intracellular isoform of the interleukin-1 receptor antagonist - Google Patents

Description

intracellular isoforms of interleukin-1 receptor antagonists

The present application is a divisional application of the invention patent application of Chinese No. 95195664.7.

Technical Field

The invention belongs to the field of biotechnology. It describes a new interleukin-1 (IL-1) antagonist active on both IL-1a and IL-1B, a new DNA sequence coding for the IL-1 antagonist and a process for obtaining the IL-1 antagonist by DNA recombination techniques. It also describes the prophylactic, therapeutic and diagnostic use of the novel IL-1 antagonists in pathologies resulting from the production of IL-1.

Background

There are two distinct interleukin-1 encoding genes, designated IL-1a and IL-1B, which encode the IL-1a protein and the IL-1B protein, respectively.

The interleukins IL-1a and IL-1B are pleiotropic cytokines that, although showing little similarity in sequence, have a myriad of similar effects on different tissues and on many pathologies in humans, in particular in the course of the immune response and inflammation of tissues.

Both proteins have a molecular weight of about 17.5kDa and have been previously synthesized as larger precursor molecules having a molecular weight of about 31 kDa.

IL-1 is a potent inflammatory and pyrogenic cytokine, which generally has a beneficial effect on tissues but can also have very adverse effects.

For example, they can be involved in the pathogenesis of autoimmune pathologies, such as lupus erythematosus and in particular, for example, in rheumatoid arthritis, as mediators which cause damage to tissues.

Many of the physiological effects of IL-1 are similar to those observed during sepsis. Recent studies have shown that intravenous administration of IL-1 at doses of 1 to 10ng/kg can cause fever, drowsiness, anorexia, generalized myalgia, arthralgia and migraine.

Because IL-1 has pleiotropic physiological activities, many of which negatively affect tissues, the potent potency of IL-1 must be under strict physiological control.

IL-1 synthesis is inhibited by anti-inflammatory cellular mediators, prostaglandins and glucocorticoids, while the presence of multiple levels of inhibition of IL-1 is required for tight control of this mediator.

IL-1 is the only cytokine among the receptor polypeptide antagonists described so far: the third known component of the IL-1 family to date is an antagonist of the IL-1 receptor (IL-1 ra).

All three components (IL-1a, IL-1B, IL-1ra) are able to recognize and bind to the same receptor (IL-1R) on the cell surface: IL-1a and IL-1B bind to IL-1R and transmit signals, whereas IL-1ra does not.

There are two types of IL-1 receptors, known as IL-1RI and IL-1 RII. IL-1ra is a polypeptide which binds to IL-1RI with poor affinity to IL-1RII and no agonistic activity.

IL-1ra is induced by IgG, cytokines and bacterial products in different cell types, including mononuclear phagocytes, polymorphonuclear cells (PMNs) and fibroblasts.

To date, two molecular forms of IL-1ra have been identified and cloned: 1) secreted IL-1ra (sIL-1ra) contains a 25 amino acid typical leader sequence, giving a 152 amino acid mature protein; 2) intracellular IL-lra (iciL-1ra) lacks a leader sequence, thus indicating that the protein is retained intracellularly.

sIL-1ra and iciL-1ra from the same gene. The iciL-lra transcript was generated from the start of either and from the first alternative exon spliced into an internal splice acceptor site located in the first exon of sIL-1 ra. Thus, the predicted proteins remove their NH₂The terminal is identical, in the end sIL-1ra of the first 21 amino acids in the iciL-lra by four amino acid substitution.

The expression of the transcriptional codes for sIL-1ra and iciL-lra are differentially regulated. The biological significance of iciL-1ra is still unclear.

Given the involvement of IL-1 in the pathology of many diseases, the need for commercially available drugs to limit the adverse effects of IL-1 is evident.

Summary of the invention

It is an object of the present invention to provide an IL-1 antagonist that is active on IL-1a and IL-1B and combinations thereof.

It is a further object of the present invention to provide a DNA sequence encoding an IL-1 antagonist and a method for obtaining this novel antagonist by recombinant DNA techniques.

It is another object of the invention to provide the antagonist in a substantially purified form suitable for use in a pharmaceutical composition active when inhibition of IL-1 pathology is desired.

Other objects and advantages of the present invention will become apparent from the following description.

Brief description of the figures and sequence listing

FIG. 1 depicts the DNA sequence and protein sequence of iciL-1ra II (SEQ ID NO: 8 and SEQ ID NO: 9) as compared to the non-common portions of typical sIL-lra (iciL-1ra I: SEQ ID NO: 6) and sIL-lra (SEQ ID NO: 4 and SEQ ID NO: 5), which also depicts the DNA sequence and encoded protein of the IL-1ra common portion (SEQ ID NO: 13 and SEQ ID NO: 14).

FIG. 2 depicts RT-PCR analysis of expression of iciL-lraII in different cell types.

FIG. 3 depicts Western blot analysis of recombinant iciL-1 raiI.

FIG. 4 depicts the effect of iciL-1ra II on IL-1-induced E-selectin expression in endothelial cells.

SEQ ID NO: 1 reports the sequence called IRA5 oligonucleotide used in RT-PCR.

SEQ ID NO: 2 reports the oligonucleotide sequence corresponding to nucleotides 69-70 of B-actin cDNA used in RT-PCR.

SEQ ID NO: 3 reports the oligonucleotide sequence reverse complementary to nucleotide 430-449 for RT-PCR.

SEQ ID NO: 4 reports the DNA sequence encoding the non-common part of sIL-1 ra.

SEQ ID NO: 5 report sIL-1ra non common part of the amino acid sequence.

SEQ ID NO: 6 reports the DNA sequence encoding three amino acids of the non-common part of iciL-lra I.

SEQ ID NO: 7 reports three amino acids of the non-common part of iciL-1-ra I.

SEQ ID NO: 8 report the coding of iciL-1ra II non common part of the DNA sequence.

SEQ ID NO: 9 report the ICIL-1ra II non common part of the amino acid sequence.

SEQ ID NO: 10 reports the DNA sequence encoding the common part of IL-1 ra. Regarding the problems associated with the "Patentin EPO" program of sequence preparation, a G nucleotide is added at the first position of the sequence in order to allow the first amino acid to be encoded as Glu and more so to avoid the formation of a stop codon within the sequence.

SEQ ID NO: 11 report IL-1ra common part of the amino acid sequence.

SEQ ID NO: 12 reports a 21 amino acid sequence representing a non-common fragment of iciL-1ra II with other IL-1 ra.

SEQ ID NO: 13 reports the DNA sequence encoding the complete iciL-1ra II.

SEQ ID NO: 14 report the complete amino acid sequence of iciL-1ra II.

Disclosure of Invention

To accomplish the object of the present invention, the present invention relates to a purified protein having antagonistic activity against at least one protein selected from the group consisting of interleukin 1a and interleukin 1B, said protein having the amino acid sequence as shown in SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof. It is obtained by recombinant DNA technology.

The invention also relates to a method for encoding a polypeptide having the sequence shown in SEQ ID NO: 14, and an isolated DNA sequence of an IL-1 antagonist having the amino acid sequence set forth in SEQ ID NO: 13, or a pharmaceutically acceptable salt thereof.

In addition, the present invention relates to a polypeptide comprising a nucleotide sequence encoding a polypeptide comprising SEQ ID NO: 14, or a DNA sequence of a protein having the amino acid sequence described in (1). It also relates to cells transfected with DNA encoding said purified protein. Such cells are mammalian cells.

The invention also relates to a method for obtaining the IL-1 antagonist by recombinant DNA technology, which comprises the following steps:

(1) culturing a host cell containing a DNA sequence encoding said purified protein;

(2) collecting and isolating the encoded protein.

In the above method, the DNA sequence is SEQ ID NO: 13.

this novel IL-1 antagonist is generated by inserting a novel 63 base pair (bp) sequence between the internal receptor of the first specific exon of iciL-lra and the first exon of sIL-lra in the DNA framework encoding iciL-1 ra.

Through RT-PCR experiments, the present inventors found that this novel transcript can be expressed in activated monocytes and fibroblasts and in polymorphonuclear cells (PMNs).

Expression in COS cells revealed that the novel antagonist was generally intracellular and had a Molecular Weight (MW) of approximately 25kDa in SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

The novel recombinant antagonists show inhibitory activity of IL-1.

For reasons of clarity and ease, the presently known iciL-1ra is referred to herein as iciL-lra type I (iciL-lra I), while the novel antagonists described herein and the objects of the present invention are defined as iciL-1ra type II (iciL-1ra II).

Examples of pathologies in which the antagonists according to the invention may be advantageously used for prophylactic, therapeutic or diagnostic purposes are rheumatoid arthritis, septic shock, acute myelomonocytic leukemia, organ transplant immune responses against a subject, Acquired Immune Deficiency Syndrome (AIDS), ulcerative colitis and all autoimmune diseases in general.

An example of the present invention is the administration of a pharmacologically active amount of icIL-lra II to a person at high risk of sensing a pathology requiring IL-1 inhibition or to a person already exhibiting a pathology such as sepsis.

An example of the above type is a patient awaiting surgery.

Any route of administration that is not exclusive of the active ingredient may be used, but a route of parenteral administration is particularly preferred because it can exert systemic effects in a short time.

For this reason, large intravenous doses are the preferred route, just before, during or after surgery. The dose of iciL-1ra II administered depends on the drug prescription based on the age, weight and individual response of the patient.

The dosage may be between 0.05 and 30mg/kg body weight, and the preferred dosage is between 0.1 and 10mg/kg body weight.

Pharmaceutical compositions for parenteral use can be prepared in injectable dosage forms containing the active ingredient and a suitable carrier. Carriers for parenteral administration are well known in the art and include, for example, water, saline solution, ringer's solution and dextrose.

The vehicle may contain a small amount of excipients to maintain stability and isotonicity of the solution.

The preparation of the above solution can be carried out in the usual manner, preferably the content of iciL-lra II will be between 1mg/ml and 10 mg/ml.

Further pathological examples, in which the novel antagonists according to the invention can advantageously be used for prophylactic, therapeutic, diagnostic purposes, are rheumatoid arthritis, septic shock, acute myelomonocytic leukemia, the immune response of organ transplants to subjects, Acquired Immune Deficiency Syndrome (AIDS), ulcerative colitis and all autoimmune diseases in general.

The present invention has been described with reference to specific examples, but modifications and substitutions which may be made by those skilled in the art are not to be considered as beyond the purpose and meaning of the claims.

In the following sections, some methods of achieving the invention will be described, although equivalent materials and methods may be used. The following examples are, therefore, purely illustrative and are not intended to limit the invention.

Detailed Description

Example 1

Cloning and characterization of iciL-1ra II

Materials and methods

Reagent

The following commercially available reagents were used for cell culture and isolation: pyrogen-free saline and distilled water for clinical use: RPMI 1640 medium; DMEM medium; m199 medium; l-glutamine; percoll (Percoll); Ficoll-Hipaque (Ficoll-Hipaque); aseptically collected fetal bovine serum; endothelial Cell Growth Supplements (ECGS) prepared from bovine brain; heparin.

All reagents contained less than 0.125EU/ml endotoxin as checked by Limulus amebocyte lysate assay.

Cells

Human circulating PMNs and monocytes are isolated from peripheral blood of healthy donors by centrifugation with an isotonic (285mOsm) peltier discontinuous (46% PMN for monocytes is 62%) gradient, such as Colotta f, Peri g, vila sa, Mantovani a, Rapid bagging of actinomycin D great watt cells by human mononarbler cells j.immunol.132: 936, 1984. Cells were recovered at the interface, washed twice in saline and resuspended in culture medium.

Recovery of PMNs and monocytes was higher than 90% and purity higher than 98%, evaluated by morphological examination of stained cytocentrifuge cells. The cell culture medium conventionally used for PMNs and monocytes was RPMI 1640 with 2 mML-glutamine and 10% FCS.

Human Endothelial Cells (ECs) are obtained from umbilical veins and cultured as described in detail in the literature (allivena p., pagannin c., Martin-Padura i., Peri g., Gaboli m., Dejana e., marchicio p.c., manovani a., Molecules and Structures infusion of natural kiler cells to vascular endothelial, j.exp.med., 173: 439, 1991).

The fused cells from the second to fifth passages were routinely maintained using M199 medium containing 10% FCS and fed with ECGS (50 μ/ml) and heparin (100 μ/ml).

COS cells were cultured in DMEM medium containing 10% FCS, while 8387 fibroblasts were cultured in RPMI 1640 medium containing 10% FCS.

After appropriate treatment, cells were observed for IL-1ra mRNA or IL-1ra protein in the following manner.

RT-PCR

Whole RNA was extracted using a slightly modified guanidinium isothiocyanate protocol.

RT-PCR was performed as described in Colotta f., polentrutti n., siroi m., mantovani a., j.biol.chem., 267: 18278, 1992.

Briefly, 1 μ bulk RNA was reverse transcribed in reverse transcriptase buffer (5mM MgCl. sub.5 mM), random hexamer, 1mM deoxynucleotide triphosphates, 1 unit/ml RNase inhibitor and 2.5 units/ml moloney murine leukemia virus transcriptase (Perkin Elmer Cetus. Norwalk, CT)₂50mM KCl, 10mM Tris-HCl; pH 8.3).

The samples were incubated at 25 ℃ for 10 minutes followed by 42 ℃ for 45 minutes. Then, to the cDNA reaction, specific primer pairs designed to amplify cDNAs encoding iciL-1ra I or iciL-1ra II, and human B-actin as an internal control were added.

Amplification was at 2mM MgCl₂50mM KCl, 0.2M deoxynucleotide triphosphate each, 2.5 units/100 ml Taq polymerase (Perkin Elmer Cetus) and 4mg/ml of each specific primer (below). Amplification (30 cycles) was performed in an automatic heating cycler (Perkinelmer Cetus) at 95 deg.C, 55 deg.C and 72 deg.C for 1.5 minutes each.

The amplification products were passed through a 1% ethidium bromide stained agarose gel along with molecular weight standards (Boehringer Mannheim, Mannheim, Germany).

The oligonucleotides are bound by the phosphoramidite method. Oligonucleotide sequences for selective amplification of icIL-1ra are described in conjunction with Haskill s, et al, natl.acad., USA, 88: 3681, 1991.

In particular, the authors used the oligonucleotides GM397 (denoted here as IRA 1) and GM368(IRA 4).

For amplification of iciL-lra II, the authors used IRA4 and IRA 5(SEQ ID NO: 1), which specifically recognized the superexons included in the iciL-lra II sequence described herein.

For amplification of B-actin, the forward oligonucleotide is set forth in SEQ ID NO: 2, corresponding to nucleotides 60-79 of the B-actin cDNA.

The reverse oligonucleotide is represented in SEQ ID NO: 3, is complementary to nucleotide sequence 430-449. The amplified products were subcloned (TA Cloning System, Invitrogen, san Diego, Calif.) and sequenced using the dideoxy-bond termination method.

Expression of iciL-lra product in COS cells

cDNAs containing 32bp of the 5 '-untranslated region, the entire open reading frame and 6bp of the 3' -untranslated region (including the stop codon) of iciL-1ra I and iciL-1ra II were obtained by RT-PCR with oligonucleotides IRA4 and IRA5 as detailed above and then ligated back into the pSF5 expression vector, and the fidelity and amplification of reverse transcription was confirmed by sequencing.

Plasmids containing the cDNA in the correct orientation were purified by CsCl gradient and transfected into COS cells using the calcium precipitation method as described in Sambrook J, et al, Cold Spring Harbor Laboratory Press, 1989.

After two days, the culture supernatants and sonicated cell lysates were examined by ELISA or immunoblotting as described in detail below, with empty plasmid (untransfected) as control.

Identification of immunologically active IL-1ra

A commercial ELISA assay (Amersham, Buckinamshire, UK) was used for the identification of sIL-1ra and iciL-1 ra. Polyclonal antisera from two rabbits and one goat were used for Western blot analysis.

COS cell lysate samples and supernatants were electrophoresed on 12.5% SDS-PAGE and blotted onto nitrocellulose membranes (Stratagene, La Jolla, Calif., USA).

The primary and secondary antibody incubations were performed according to standard protocols, with the primary antibody being an anti-IL-lra rabbit polyclonal antibody.

The secondary antibody was a goat anti-rabbit immunoglobulin moiety linked to horseradish peroxidase (Amersham). Bands of immunologically active protein moieties are shown using chemiluminescence-based procedures (ECL Detection, Amersham) according to the manufacturer's instructions.

IL-1-induced expression of E-selectin on EC

The fused EC cultured in 96-well plates (Falcon) were incubated for 30 minutes with an amount of transfected COS cell lysate (see above) corresponding to 25 to 100ng of recombinant IL-1ra (either iciL-1ra I or iciL-1ra II) as assessed by a specific ELISA assay (Amersham).

An equal amount of COS lysates from mock transfected cells were used in parallel as controls. Next, EC was exposed to 0.1-1ng/ml human recombinant IL-1B for 6 hours. Expression of E-selectin was determined by ELISA analysis using adherent EC with the anti-E-selectin monoclonal antibody BBIG-E2 as the primary antibody and rabbit anti-mouse Ig antiserum conjugated to horseradish peroxidase as the secondary antibody. O.d. of the samples was determined using a spectrophotometer (Flow) at 405 wavelength detection well plate.

Results

identification of iciL-1raiI

To obtain the complete coding sequence of iciL-1ra (FIG. 1) by RT-PCR, specific oligonucleotide primers (IRA 1 and IRA4 as indicated in FIG. 1) were designed. The product amplified from the human PMN was subcloned and sequenced.

In addition to the previously known iciL-1ra sequence, the present inventors isolated clones whose sequence was identical to the published iciL-1ra coding sequence, with the notable exception of an additional 63bp sequence between nucleotides 132 and 133 of the iciL-1ra sequence. Given the depicted iciL-1ra exon-intron boundaries, the additional sequence is inserted between the first leader-free exon of iciL-1ra and the internal acceptor site of the first exon of sIL-1ra (FIG. 1).

The predicted amino acid sequence is shown in FIG. 1. The novel protein (hereinafter referred to as iciL-1ra type II) is bound to NH₂The first three terminal amino acids are common to typical iciL-1ra (iciL-1ra type I), followed by a new sequence of 21 amino acids. The remainder of the two proteins are identical.

Surprisingly, the junction with the internal accepting site of the first exon of sIL-1ra often produces the same amino acid residues, i.e., glutamic acid (FIG. 1), as do both sIL-1ra and iciL-1raI and iciL-1ra II.

The most prominent feature of the inserted amino acid extra sequence is the occurrence of seven glycine residues, six of which are consecutive. The glycine residue is flanked on both sides by glutamic acid residues. iciL-1ra II consists of 180 amino acids.

The overall hydrophilicity pattern of iciL-1ra II is similar to iciL-1ra I, still in NH₂The termini lack a hydrophobic leader peptide.

Expression of iciL-ra II

An RT-PCR analysis was performed using a pair of specially designed oligonucleotides (IRA 5 and IRA4, FIG. 1) with the desired 33bp amplification product to identify the iciL-1ra II transcript.

As shown in FIG. 2, transcripts encoding iciL-1ra II were detectable in PMA-, IL-1-and TNF-activated fibroblasts. There was clearly a weak but measurable band in LPS-treated monocytes.

The PMNs, whether untreated or activated (fig. 2), also showed a very weak band of the desired size.

The specificity of the amplification products indicated in FIG. 2 was determined by subcloning and sequencing.

Expression of recombinant iciL-1ra II

COS cells were transfected with the DNA sequence encoding iciL-1ra II and, for comparison, with the DNA sequence encoding iciL-1 raI. Next, cell lysates and supernatants were examined by Western blotting.

The polyclonal antisera used in these experiments recognized both iciL-1ra II and iciL-1ra I equally well (FIG. 3). Most, if not all of iciL-1ra II and iciL-1ra I in cell lysate found.

Recombinant iciL-1ra I mainly with 22kDa band movement, and iciL-1ra II shows approximately 25kDa quality.

Inhibitory Activity of recombinant iciL-1ra II on IL-1B

The inhibitory activity of recombinant iciL-lra II on IL-1 was examined. For this purpose the authors chose the expression of IL-1-induced E-selectin in endothelial cells, since this assay was sensitive (measurable induction at 100pg/ml IL-1, or less) and rapid (6 h incubation with IL-1).

Lysates that mimic transfected COS cells did not significantly attenuate IL-1 activity.

There is no agonist activity of iciL-1ra II.

As shown in FIG. 4, recombinant iciL-1ra II in a dose-dependent form inhibits IL-1 activity.

These data provide evidence that iciL-1ra II is indeed an IL-1 inhibitor.

Discussion:

the present invention describes a novel molecular form of iciL-1 ra. This new molecule was generated by inserting 63bp between the first leader-free exon of iciL-1ra and the internal acceptor site of the first exon of sIL-1 ra.

Because the protein formed is not located in NH₂The terminal 21 amino acids are partially equivalent to typical iciL-1ra, the inventor suggests that this new form is called IL-1ra type II, referred to the typical iciL-1ra sequence as iciL-1ra form I.

RT-PCR experiments showed that the iciL-1ra II transcript is inducible in monocytes and fibroblasts. Recombinant iciL-1ra II expressed in COS cells has a molecular weight of approximately 25kDa and IL-1 inhibitor activity comparable to the effect of iciL-1ra I production expressed under the same experimental conditions.

The iciL-1ra and sIL-1ra encoding transcripts were generated from the same gene using a specific (differential) splicing approach. Production of iciL-1ra is initiated by transcription of another exon embedded within the internal acceptor site of the first exon containing the sIL-1ra leader sequence.

The results obtained by the present inventors suggest a novel structure of the IL-1ra gene in which an additional exon is located between the first exons of each of the classical iciL-1ra and sIL-1 ra. This new exon was used to generate a polypeptide molecule which still lacks the signal peptide, differs from iciL-1ra I by the insertion of 21 amino acids at the N-terminus, and still retains the ability to inhibit IL-1.

The use of additional splicing to produce different IL-1ra molecules appears to be highly regulated. The iciL-1ra II transcript was induced in fibroblasts with IL-1, TNF and phorbol esters and in monocytes with LPS. Phorbol esters were found in fibroblasts to selectively induce iciL-1ra transcripts, while IL-1 and TNF induced both sIL-1ra and iciL-1ra mRNA. In monocytes, IL-13 (which expands transcripts of sIL-1ra and iciL-1ra I) cannot induce iciL-1ra II.

Finally, PMNs (where sIL-1ra and iciL-1ra can be expressed and induced substantially) express only a few transcripts as indicated by RT-PCR. Taken together, these data indicate that the mechanism of inducing specific splicing that produces the three forms of IL-1ra is specific regulation of the response to external signals.

The amino acid sequence of the additional sequence described herein is unexpectedly such that it comprises seven glycine residues, six of which are contiguous.

Glycine-rich sequences are present in different biologically active molecules including cardiac sodium clearance receptors, HOX11 homeobox genes, intermediate fibrokeratin and nucleoproteins involved in centromere binding or RNA splicing.

However, there was no apparent homology between these proteins and the amino acid sequence flanking the glycine-rich region of iciL-1ra II, except for the glycine residues.

The IL-1 system shows a very high degree of complexity and comprises two agonists, two receptors, one of which is an inhibitor of IL-1, and one receptor antagonist, for which at least three different forms of the molecule are possible, taking into account the results obtained.

Although the biological significance of intracellular forms of IL-1ra has not been clearly established, the data reported herein indicate that in response to an external stimulus of choice, with additional splicing, two different forms of iciL-1ra with different N-termini can be produced.

The existence of multiple and complex levels of IL-1 control points to an absolute need for tight physiological control of the inflammatory potential of this cytokine.

Description of the drawings

FIG. 1 shows a schematic view of a

The DNA sequence and predicted protein sequence of iciL-1ra II compared to typical iciL-1ra (ieIL-1ra I) and sIL-1 ra.

The upper part of FIG. 1 shows DNA and protein sequences specifically representing sIL-1ra, iciL-1ra I and iciL-1ra II. The sequence common to the three forms of IL-1ra is shown in the lower part of FIG. 1.

The complete sequence of each molecule is generated by joining the respective specific part to the common part. For clarity, the DNA sequence of iciL-1ra begins with nucleotide 91 of the published 5 'untranslated sequence, and only 6bp of the 3' untranslated sequence is reported.

The common IL-1ra sequence starts with an internal acceptor site located in the first exon of sIL-1ra, corresponding to nucleotide 133 of the complete iciL-1ra I sequence and nucleotide 88 of the complete sequence of sIL-1 ra.

Arrows indicate the forward (IRA 1 and IRA 5) and reverse (IRA4) oligonucleotides used for RT-PCR analysis, as described in the content. Oligonucleotide IRA5 recognizes only iciL-1ra II DNA.

FIG. 2

RT-PCR analysis of iciL-1ra II expression in different cell types

RNAs starting from 8387 fibroblasts (plate A), monocytes (B) and PMN (C) were reverse transcribed. Each DNA synthesis reaction was divided into two samples, one amplified with the oligonucleotides IRA5 (forward) and IRA4 (reverse) in order to determine transcripts of iciL-1ra II; another sample was amplified with B-actin specific oligonucleotides (see materials and methods).

The amplification products were visualized on an ethidium bromide stained agarose gel. Amplification products corresponding to B-actin are reported on the left of the standard, and amplification products corresponding to iciL-1ra II (on the right) are indicated by arrows. The specificity of these bands was determined by subcloning and sequencing.

FIG. 3

Western blot analysis of recombinant iciL-1ra II

Cell lysates from COS cells transfected with DNA encoding either iciL-1ra I (2) or iciL-1ra II (3) or an empty vector without DNA (1) were examined by immunoblotting with anti-IL-lra rabbit polyclonal antibody. Molecular weight standards have been described.

FIG. 4

Effect of iciL-1ra II on IL-1-induced E-selectin expression on endothelial cells

Using the methods as explained in detail in the materials and methods section, endothelial cells were treated with 0.1 or 1ng/ml human IL-1B with or without the addition of 25-100ng/ml iciL-1ra II or equivalent amounts of COS cell lysates obtained by mock transfection with empty vector.

After 6 hours of incubation, expression of E-selectin was investigated using ELISA assays performed on adherent cells.

Data reported are percent of IL-1 induced E-selectin expression control.

Sequence listing

(1) General data:

(i) the applicant:

(A) name: ARS shares of applied research systems

(B) Street: john B Gauss Wejie No. 14

(C) City: storehouse guy cable

(E) The state is as follows: leusi island of Hessian

(F) And (3) post code: is free of

(G) Telephone: 599-9639300

(H) Faxing: 599-9614129

(ii) The invention name is as follows: intracellular isoforms of interleukin-1 receptor antagonists

(iii) Sequence number: 14

(iv) A computer-readable form:

(A) type of medium: optical disk

(B) A computer: IBM PC compatible

(C) Operating the system: PC-DOS/MS-DOS

(D) Software: issued patent #1.0, version #1.30(EPO)

(2) SEQ ID NO: 1, data:

(i) sequence characteristics:

(A) length: 25 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) The anatomy: linearity

(ii) Molecular type: cDNA

(iii) Suppose that: is free of

(iv) Antisense: is free of

(ix) Is characterized in that:

(A) the name/main point is as follows: various features

(B) Position: 1..25

(C) Other data: the RT-PCR oligonucleotide acid is named IRA5 ″)

(xi) Description of the sequence: SEQ ID NO: 1:

CTGACTTGTA TGAAGAAGGA GGTGG 25

(2) SEQ ID NO: 2, data:

(i) sequence characteristics:

(A) length: 20 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) The anatomy: linearity

(ii) Molecular type: DNA

(iii) Suppose that: is free of

(iv) Antisense: is free of

(ix) Is characterized in that:

(A) the name/main point is as follows: various features

(B) Position: 1..20

(D) Other data: the RT-PCR oligonucleotide corresponds to 60-79 of B-actin

(xi) Description of the sequence: SEQ ID NO: 2:

GCGCTCGTCG TCGACAACGG 20

(2) SEQ ID NO: 3, data:

(i) sequence characteristics:

(A) length: 21 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) The anatomy: linearity

(ii) Molecular type: cDNA

(iii) Suppose that: is free of

(iv) Antisense: is free of

(ix) Is characterized in that:

(A) the name/main point is as follows: various features

(B) Position: 1..21

(D) Other data: the RT-PCR reverse oligonucleotide is complementary to 430-

(xi) Description of the sequence: SEQ ID NO: 3:

GATAGACAAC GTACATGGCT G 21

(2) SEQ ID NO: 4, data:

(i) sequence characteristics:

(A) length: 87 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) The anatomy: linearity

(ii) Molecular type: cDNA

(iii) Suppose that: is free of

(iv) Antisense: is free of

(ix) Is characterized in that:

(A) the name/main point is as follows: CDS

(B) Position: 24..86

(ix) Is characterized in that:

(A) name/requirement: various features

(B) Position: 1..87

(D) Other data: (ii) note ═ sIL-1ra non-consensus sequence

(xi) Description of the sequence: SEQ ID NO: 4:

GAATTCCGGG CTGCAGTCAC AGA ATG GAA ATC TGC AGA GGC CTC CGC AGT 50

Met Glu Ile Cys Arg Gly Leu Arg Ser

1 5

CAC CTA ATC ACT CTC CTC CTC TTC CTG TTC CAT TCA G 87

His Leu Ile Thr Leu Leu Leu Phe Leu Phe His Ser

10 15 20

(2) SEQ ID NO: 5, data:

(i) sequence characteristics:

(A) length: 21 amino acids

(B) Type (2): amino acids

(D) The anatomy: linearity

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 5:

Met Glu Ile Cys Arg Gly Leu Arg Ser His Leu Ile Thr Leu Leu Leu

1 5 10 15

Phe Leu Phe His Ser

20

(2) SEQ ID NO: data of 6:

(i) sequence characteristics:

(A) length: 42 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) The anatomy: linearity

(ii) Molecular type: cDNA

(iii) Suppose that: is free of

(iv) Antisense: is free of

(ix) Is characterized in that:

(A) the name/main point is as follows: CDS

(B) Position: 33..41

(ix) Is characterized in that:

(A) the name/main point is as follows: various features

(B) Position: 1..42

(D) Other data: "non-common sequences of intracellular IL-1ra type I

(xi) Description of the sequence: SEQ ID NO: 6:

CAGAAGACCT CCTGTCCTAT GAGGCCCTCC CC ATG GCT TTA G 42

Met Ala Leu

1

(2) SEQ ID NO: 7, data:

(i) sequence characteristics:

(A) length: 3 amino acids

(B) Type (2): amino acids

(D) The anatomy: linearity

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 7:

Met Ala Leu

1

(2) SEQ ID NO: data of 8:

(i) sequence characteristics:

(A) length: 105 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) The anatomy: linearity

(ii) Molecular type: cDNA

(iii) Suppose that: is free of

(iv) Antisense: is free of

(ix) Is characterized in that:

(A) the name/main point is as follows: CDS

(B) Position: 33..104

(ix) Is characterized in that:

(A) the name/main point is as follows: various features

(B) Position: 1..1O5

(C) Other data: "intracellular IL-1ra type II non-common sequence

(xi) Description of the sequence: SEQ ID NO: 8:

CAGAAGACCT CCTGTCCTAT GAGGCCCTCC CC ATG GCT TTA GCT GAC TTG TAT 53

Met Ala Leu Ala Asp Leu Tyr

1 5

GAA GAA GGA GGT GGA GGA GGA GGA GAA GGT GAA GAC AAT GCT GAC TCA 101

Glu Glu Gly Gly Gly Gly Gly Gly Glu Gly Glu Asp Asn Ala Asp Ser

10 15 20

AAG G 105

Lys

(2) SEQ ID NO: 9, data:

(i) sequence characteristics:

(A) length: 24 amino acids

(B) Type (2): amino acids

(D) The anatomy: linearity

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 9:

Met Ala Leu Ala Asp Leu Tyr Glu Glu Gly Gly Gly Gly Gly Gly Glu

1 5 10 15

Gly Glu Asp Asn Ala Asp Ser Lys

20

(2) SEQ ID NO: data of 10:

(i) sequence characteristics:

(A) length: 474 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) The anatomy: linearity

(ii) Molecular type: cDNA

(iii) Suppose that: is free of

(iv) Antisense: is free of

(ix) Is characterized in that:

(A) the name/main point is as follows: CDS

(B) Position: 1..468

(ix) Is characterized in that:

(A) the name/main point is as follows: various features

(B) Position: 1..474

(D) Other data: -note ═ IL-1ra common sequence; for software reasons, G is added in the first position, so that the first codon encodes Glu and thus the generation of a stop codon "within the sequence is avoided

(xi) Description of the sequence: SEQ ID NO: 10:

GAG ACG ATC TGC CGA CCC TCT GGG AGA AAA TCC AGC AAG ATG CAA GCC 48

Glu Thr Ile Cys Arg Pro Ser Gly Arg Lys Ser Ser Lys Met Gln Ala

1 5 10 15

TTC AGA ATC TGG GAT GTT AAC CAG AAG ACC TTC TAT CTG AGG AAC AAC 96

Phe Arg Ile Trp Asp Val Asn Gln Lys Thr Phe Tyr Leu Arg Asn Asn

20 25 30

CAA CTA GTT GCT GGA TAC TTG CAA GGA CCA AAT GTC AAT TTA GAA GAA 144

Gln Leu Val Ala Gly Tyr Leu Gln Gly Pro Asn Val Asn Leu Glu Glu

35 40 45

AAG ATA GAT GTG GTA CCC ATT GAG CCT CAT GCT CTG TTC TTG GGA ATC 192

Lys Ile Asp Val Val Pro Ile Glu Pro His Ala Leu Phe Leu Gly Ile

50 55 60

CAT GGA GGG AAG ATG TGC CTG TCC TGT GTC AAG TCT GGT GAT GAG ACC 240

His Gly Gly Lys Met Cys Leu Ser Cys Val Lys Ser Gly Asp Glu Thr

65 70 75 80

AGA CTC CAG CTG GAG GCA GTT AAC ATC ACT GAC CTG AGC GAG AAC AGA 288

Arg Leu Gln Leu Glu Ala Val Asn Ile Thr Asp Leu Ser Glu Asn Arg

85 90 95

AAG CAG GAC AAG CGC TTC GCC TTC ATC CGC TCA GAC AGT GGC CCC ACC 336

Lys Gln Asp Lys Arg Phe Ala Phe Ile Arg Ser Asp Ser Gly Pro Thr

100 105 110

ACC AGT TTT GAG TCT GCC GCC TGC CCC GGT TGG TTC CTC TGC ACA GCG 384

Thr Ser Phe Glu Ser Ala Ala Cys Pro Gly Trp Phe Leu Cys Thr Ala

115 120 125

ATG GAA GCT GAC CAG CCC GTC AGC CTC ACC AAT ATG CCT GAC GAA GGC 432

Met Glu Ala Asp Gln Pro Val Ser Leu Thr Asn Met Pro Asp Glu Gly

130 135 140

GTC ATG GTC ACC AAA TTC TAC TTC CAG GAG GAC GAG TAGTAC 474

Val Met Val Thr Lys Phe Tyr Phe Gln Glu Asp Glu

145

(2) SEQ ID NO: data of 11:

(i) sequence characteristics:

(A) length: 156 amino acids

(B) Type (2): amino acids

(D) The anatomy: linearity

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 11:

Glu Thr Ile Cys Arg Pro Ser Gly Arg Lys Ser Ser Lys Met Gln Ala

1 5 10 15

Phe Arg Ile Trp Asp Val Asn Gln Lys Thr Phe Tyr Leu Arg Asn Asn

20 25 30

Gln Leu Val Ala Gly Tyr Leu Gln Gly Pro Asn Val Asn Leu Glu Glu

35 40 45

Lys Ile Asp Val Val Pro Ilu Glu Pro His Ala Leu Phe Leu Gly Ile

50 55 60

His Gly Gly Lys Met Cys Leu Ser Cys Val Lys Ser Gly Asp Glu Thr

65 70 75 80

Arg Leu Gln Leu Glu Ala Val Asn Ile Thr Asp Leu Ser Glu Asn Arg

85 90 95

Lys Gln Asp Lys Arg Phe Ala Phe Ile Arg Ser Asp Ser Gly Pro Thr

100 105 110

Thr Ser Phe Glu Ser Ala Ala Cys Pro Gly Trp Phe Leu Cys Thr Ala

115 120 125

Met Glu Ala Asp Gln Pro Val Ser Leu Thr Asn Met Pro Asp Glu Gly

130 135 140

Val Met Val Thr Lys Phe Tyr Phe Gln Glu Asp Glu

145 150 155

(2) SEQ ID NO: data of 12:

(i) sequence characteristics:

(A) length: 21 amino acids

(B) Type (2): amino acids

(C) Chain:

(D) the anatomy: linearity

(ii) Molecular type: peptides

(iii) Suppose that: is free of

(v) Fragment type: n-terminal end

(ix) Is characterized in that:

(A) the name/main point is as follows: peptides

(B) Position: 1..21

(D) Other data: (iv) non-common part of intracellular IL-1ra type II

(xi) Description of the sequence: SEQ ID NO: 12:

Ala Asp Leu Tyr Glu Glu Gly Gly Gly Gly Gly Gly Gly Gly Glu Asp

1 5 10 15

Asn Ala Asp Ser Lys

20

(2) SEQ ID NO: 13, data:

(i) sequence characteristics:

(A) length: 579 base pairs

(B) Type (2): nucleic acids

(C) Chain: single strand

(D) The anatomy: linearity

(ii) Molecular type: cDNA

(iii) Suppose that: is free of

(iv) Antisense: is free of

(ix) Is characterized in that:

(A) the name/main point is as follows: CDS

(B) Position: 34..573

(ix) Is characterized in that:

(A) the name/main point is as follows: various features

(B) Position: 1..579

(D) Other data: "intracellular IL-1ra type II

(xi) Description of the sequence: SEQ ID NO: 13:

CAGAAGGACC TCCTGTCCTA TGAGGCCCTC CCC ATG GCT TTA GCT GAC TTG TAT 54

Met Ala Leu Ala Asp Leu Tyr

1 5

GAA GAA GGA GGT GGA GGA GGA GGA GAA GGT GAA GAC AAT GCT GAC TCA 102

Glu Glu Gly Gly Gly Gly Gly Gly Glu Gly Glu Asp Asn Ala Asp Ser

10 15 20

AAG GAG ACG ATC TGC CGA CCC TCT GGG AGA AAA TCC AGC AAG ATG CAA 150

Lys Glu Thr Ile Cys Arg Pro Ser Gly Arg Lys Ser Ser Lys Met Gln

25 30 35

GCC TTC AGA ATC TGG GAT GTT AAC CAG AAG ACC TTC TAT CTG AGG AAC 198

Ala Phe Arg Ile Trp Asp Val Asn Gln Lys Thr Phe Tyr Leu Arg Asn

40 45 50 55

AAC CAA CTA GTT GCT GGA TAC TTG CAA GGA CCA AAT GTC AAT TTA GAA 246

Asn Gln Leu Val Ala Gly Tyr Leu Gln Gly Pro Asn Val Asr Leu Glu

60 65 70

GAA AAG ATA GAT GTG GTA CCC ATT GAG CCT CAT GCT CTG TTC TTG GGA 294

Glu Lys Ile Asp Val Val Pro Ile Glu Pro His Ala Leu Phe Leu Gly

75 80 85

ATC CAT GGA GGG AAG ATG TGC CTG TCC TGT GTC AAG TCT GGT GAT GAG 342

Ile His Gly Gly Lys Met Cys Leu Ser Cys Val Lys Ser Gly Asp Glu

90 95 100

ACC AGA CTC CAG CTG GAG GCA GTT AAC ATC ACT GAC CTG AGC GAG AAC 390

Thr Arg Leu Gln Leu Glu Ala Val Asn Ile Thr Asp Leu Ser Glu Asn

105 110 115

AGA AAG CAG GAC AAG CGC TTC GCC TTC ATC CGC TCA GAC AGT GGC CCC 438

Arg Lys Gln Asp Lys Arg Phe Ala Phe Ile Arg Ser Asp Ser Gly Pro

120 125 130 135

ACC ACC AGT TTT GAG TCT GCC GCC TGC CCC GGT TGG TTC CTC TGC ACA 486

Thr Thr Ser Phe Glu Ser Ala Ala Cys Pro Gly Trp Phe Leu Cys Thr

140 145 150

GCG ATG GAA GCT GAC CAG CCC GTC AGC CTC ACC AAT ATG CCT GAC GAA 534

Ala Met Glu Ala Asp Gln Pro Val Ser Leu Thr Asn Met Pro Asp Glu

155 160 165

GGC GTC ATG GTC ACC AAA TTC TAC TTC CAG GAG GAC GAG TAGTAC 579

Gly Val Met Val Thr Lys Phe Tyr Phe Gln Glu Asp Glu

170 175 180

(2) SEQ ID NO: 14, data of:

(i) sequence characteristics:

(A) length: 180 amino acids

(B) Type (2): amino acids

(D) The anatomy: linearity

(ii) Molecular type: protein

(xi) Description of the sequence: SEQ ID NO: 14:

Met Ala Leu Ala Asp Leu Tyr Glu Glu Gly Gly Gly Gly Gly Gly Glu

1 5 10 15

Gly Glu Asp Asn Ala Asp Ser Lys Glu Thr Ile Cys Arg Pro Ser Gly

20 25 30

Arg Lys Ser Ser Lys Met Gln Ala Phe Arg Ile Trp Asp Val Asn Gln

35 40 45

Lys Thr Phe Tyr Leu Arg Asn Asn Gln Leu Val Ala Gly Tyr Leu Gln

50 55 60

Gly Pro Asn Val Asn Leu Glu Glu Lys Ile Asp Val Val Pro Ile Glu

65 70 75 80

Pr0 His Ala Leu Phe Leu Gly Ile His Gly Gly Lys Met Cys Leu Ser

85 90 95

Cys Val Lys Ser Gly Asp Glu Thr Arg Leu Gln Leu Glu Ala Val Asn

100 105 110

Ile Thr Asp Leu Ser Glu Asn Arg Lys Gln Asp Lys Arg Phe Ala Phe

115 120 125

Ile Arg Ser Asp Ser Gly Pro Thr Thr Ser Phe Glu Ser Ala Ala Cys

130 135 140

Pro Gly Trp Phe Leu Cys Thr Ala Met Glu Ala Asp Gln Pro Val Ser

145 150 155 160

Leu Thr Asn Met Pro Asp Glu Gly Val Met Val Thr Lys Phe Tyr Phe

165 170 175

Gln Glu Asp Glu

180

Claims (2)

1. Use of a purified protein having antagonistic activity against at least one substance selected from interleukin 1a and interleukin 1B for the preparation of a pharmaceutical composition active against autoimmune pathology, septic shock, acute myelomonocytic leukemia, an immune response of an organ transplant to a subject or acquired immune deficiency syndrome AIDS, characterized in that the purified protein has the amino acid sequence as shown in SEQ id no: 14, or a pharmaceutically acceptable salt thereof.

2. Use according to claim 1, wherein the autoimmune pathology is selected from rheumatoid arthritis and ulcerative colitis.