WO1994000491A1

WO1994000491A1 - INTERLEUKIN-1β DELETION MUTANT

Info

Publication number: WO1994000491A1
Application number: PCT/SE1993/000562
Authority: WO
Inventors: Tamas Bartfai; Jesper Bristulf; Miklos CSERZÖ; Sandor Pongor; Andras Simoncsits; Marie-Louise TJÖRNHAMMAR
Original assignee: Trion Forskning- Och Utvecklings Ab
Priority date: 1992-06-25
Filing date: 1993-06-23
Publication date: 1994-01-06
Also published as: SE9201966D0; EP0647238A1; JPH07508408A; AU4519193A

Abstract

An Interleukin-1β deletion mutant, which has a quasi conserved sequence of three amino acids deleted from the amino acid sequence of an endogenous mammalian Interleukin-1β is disclosed. The deletion is Ser Asn Asp (numbers 52-54 of mature human IL-1β) in the case of human, mouse, rat and rabbit, and Arg Asp Asn in the case of bovine and sheep Interleukin-1β. The Interleukin-1β deletion mutant is shown to be an Interleukin-1 receptor ligand which binds to both Interleukin-1 type I receptors and Interleukin-1 type II receptors with substantially different binding-affinities. The ligand may have additional amino acid deletion(s) and/or point mutation(s). The Interleukin-1β deletion mutants may be used as carriers and adjuvants for biologically active molecules as well as in therapeutic and/or prophylactic agent.

Description

INTERLEUKIN-1β DELETION MUTANT

The present invention relates to an Interleukin-1β deletion mutant which has a quasi conserved sequence of three amino acids deleted from the amino-acid sequence of an endogenous mammalian Interleukin-1β. In the case of a human Interleukin-1β deletion mutant, said three deleted amino acids are Ser Asn Asp (numbers 52-54 of mature human IL-1β), which mutant is also called ΔSND.

Background

The cytokines Interleukin-1α and β (IL-1α and IL-1β), are mediators of a multitude of processes involving the immune endocrine and nervous system (cf for review [1]). The role of interleukin-1α and β as inflammatory mediators, as lymphocyte activating factors, endogenous pyrogens and radiation-protective agents render these two small proteins of approximately 153 amino acids extremely interesting in both physiological and pharmacological studies. However, widespread use of IL-1α and IL-1β in therapy has been hampered by their high pyrogenicity.

Since the molecular identification and cloning of the cDNA coding for the murine IL-1α by Lomedico et al in 1984, [2] the interleukin-1α and β have been in the focus of interest by molecular biologists, protein chemists and crystallographers. The cDNA sequences for IL-1α and IL-1β are known from several species. Two IL-1 receptors have been cloned. Type I from T-lymphocytes [3] and type II from B-lymphocytes [4]. Discovery and cloning of an endogenously occuring IL-1 receptor antagonist (IL-1 ra), which belongs to the same protein family as IL-1α and β [e.g. 5], and which antagonizes the actions of these agonists by binding to the same receptor, has accelerated studies on the IL-1 receptor-ligand interactions. A large number of modifications, such as point-mutations, of the human IL-1β have been examined with respect to their effect on biological and receptor binding properties of the IL-1β molecule [e.g.6, 7, 8]. Important residues in both the N-terminal portion of IL-1β [9, 10, 11] and in its C-terminal region [12] have been identified. However, to our knowledge no interesting internal deletion mutants of IL-1β have been reported. Description of the invention

The amino acid sequences of IL-1β from a variety of species, have now been compared with the sequence of the human IL-1 ra, which is the only known human IL-1 antagonist. A structural motif, which is quasi conserved in interleukin-β from different species but is missing from the human Interleukin-1 receptor antagonist, was identified. In this context it can be mentioned that there also exists receptor antagonist sequences from rat and mouse, and both these also lack said motif [5]. Deletion of Ser Asn Asp from said motif from the human

IL-1β (numbers 52-54 of the mature protein), yields a mutant, here called ΔSND, which binds with the same high affinity to Interleukin-1 receptors (type II) on human Raji cells as does interleukin-1β. ΔSND binds with a tenfold less affinity to Interleukin-1 receptors (type I) on mouse El-4 thyoma cells.

In comparison with interleukin-1β, ΔSND exhibits a significantly reduced biological activity in a mouse thymocyte proliferation assay. ΔSND is shown to have a lower pyrogenic activity than IL-1β in a rabbit model. It is now contemplated that the SND motif is an important structural feature of a receptor ligand for its binding-affinity to the two types of interleukin-1 receptors. The Il-1β deletion mutants of the invention are therefore useful in the development of new therapeutic and/or prophylactic agents, and as carriers and adjuvants for biologically active molecules. One aspect of the invention is directed to an Interleukin-1β deletion mutant, which has a quasi conserved sequence of three amino acids deleted from the amino-acid sequence of an endogenous mammalian Interleukin-1β, said sequence of amino acids being Ser Asn Asp (numbers 52-54 of mature human IL-1β) in the case of human, mouse, rat and rabbit, and Arg Asp Asn in the case of bovine and sheep Interleukin-1β.

In one embodiment of this aspect of the invention the endogenous mammalian Interleukin-1β is an endogenous human Interleukin-1β.

In another embodiment of this aspect of the invention the Interleukin-1β deletion mutant is an Interleukin-1 receptor ligand which binds to both Interleukin-1 type I receptors (found i.a. on T-lymphocytes) and Interleukin-1 type II receptors (found i.a. on B-lymphocytes) with substantially different binding-affinities. The Interleukin-1β deletion mutant according to the last mentioned embodiment may have additional amino-acid deletion(s) and/or point mutation(s), as long as the mutant is a ligand to the Interleukin-1 receptor and binds to both Interleukin-1 type I receptors and Interleukin-1 type II receptors with substantially different binding-affinities.

Another aspect of the invention is directed to a carrier for biologically active molecules which is an Interleukin-1β deletion mutant according to the invention.

Yet another aspect of the invention is directed to an adjuvant for biologically active molecules which is an Interleukin-1β deletion mutant according to the invention. The carrier and adjuvant according to the invention are to be used in association with biologically active molecules, especially synthetic vaccines. Still another aspect of the invention is directed to a therapeutic and/or prophylactic agent which comprises an Interleukin-1β deletion mutant according to the invention. It is believed that such a therapeutic and/or prophylactic agent will be used for radiation-protection, as lymphocyte activating factor, anti-inflammatory agent and as IL-1 antagonist in the treatment of sepsis and rheumatoid

arthritis. It can be mentioned that in a preliminary test the compound of the invention was useful in radiation-protection.

The Interleukin-1β deletion mutants of the invention can be produced in per se known manner, e.g. by constructing a gene which codes for the desired mutant, followed by insertion of said gene into a suitable vector, which in turn is inserted into a host capable of expressing said gene, propagating said host and finally isolating said desired mutant.

Guidance on how to proceed can be found in the description of the preparation of the ΔSND of the invention.

Sequence alignment

The sequences of Il-1β from rabbit, sheep, bovine, mouse and man as well as the human IL-lra were taken from the EMBL Data Bank, Heidelberg. The sequence of IL-1β from rat was taken from the literature [13]. Multiple alignments of the sequence of human IL-lra with IL-1β from the above mentioned species was performed using the Pile up program from the University of Wisconsin Genetic Computer Group [14]. The result is given in Table 1.

The result of the alignments of the mature proteins using the one-letter code for the amino acids given in Table 1 shows that at numbers 52-54 of the IL-1β mature proteins from the different species the interesting motif (a quasi conserved sequence) is found, which is missing from the IL-lra. Preparation of the ΔSND

Enzymes, bacterial strains and vectors Restriction enzymes and T4 ligase were obtained from New England Biolabs, T4 polynucleotide kinase from Boehringer Mannheim, Klenow fragment of DNA polymerase I from Pharmacia. The M13 in vitro mutagenesis kit was obtained from Bio-Rad, including the E. coli strains CJ236 and MV1190 [15]. E. coli strains JM101 [16] and JM109 [17] as well as the M13K07 helper phage [18] and the Ml3mpl8 vector [17] were from

Pharmacia. pZ152 phagemid [19] was purchased from Anglian Biotec (Colchester, England) and α-[³⁵S]-dATP was obtained from Amersham. The oligonucleotides used for mutagenesis, vector construction and for sequencing were synthesized by the phosphoramidite method using PAC-amidites [20] and a Pharmacia Gene Assembler. The deprotected oligonucleotides were purified by polyacrylamide gel electrophoresis in the presence of 8 M urea. Recombinant vectors were purified on Qiagen-tips (Diagen, GmbH, Germany) as recommended by the supplier.

Construction of the mutant IL-lβ σenes The human IL-1β gene was purchased from British Bio-technology (BBG25). [The nucleotide and amino acid sequences are shown in Table 2.] It was subcloned as an EcoRI-HindIII fragment into M13mpl8 and was then subjected to oligonucleotide- directed in vitro mutagenesis [15] using an M13 in vitro mutagenesis kit (Bio-Rad) . Deletion mutagenesis was performed with the following oligonucleotide primer:

TGTACAAGGAGAAGAA|AAAATACCTGTGGCC (Δ52-54 mutant)

where the vertical bar separating the mutagenic oligonucleotides into two arms, shows the fusion point obtained after deletion. The template-oligonucleotide annealing temperature for the arms was calculated as described [21], otherwise the suppliers instructions were followed. Recombinants were randomly picked (2-4 plaques per mutagenesis), sequenced [22] and replicative forms were prepared from the correct clones as described [23]. [The nucleotide and amino acid sequences of ΔSND are shown in Table 3.]

Construction of the expression vector pRIZ'

The pPEX E. coli expression vector was constructed from pM23, a pBR322 based expression vector which utilizes the lac repressor controlled rrnB P2 promoter [24], as previously described [25]. It was further modified to obtain the pRIZ' vector as follows. 1; The ribosomal binding site of the β-galactosidase gene was replaced with the AGGAGGAAATAACCATGG sequence, which contains the consensus Shine-Dalgarno sequence GGAGG and includes the ATG initiation codon as part of the CCATGG Ncol restriction site. 2; The M13 phage intergenic region was introduced by using the Ndel-PstI fragment of pZ152 [19] to obtain a phagemid. 3; The 1.7 kbp EcoRI fragment of pMC9 [26] containing the lac I^q gene, the lac Z control region and a part of the lac Z gene was inserted at the unique Ndel site after rendering the cohesive ends blunt with Klenow polymerase and dNTP. The resulting pRIZ' vector was then cleaved with Ncol and EcoRI and the genes coding for human recombinant IL-1β (hrlL-1β) or its analogs were cloned between these sites, using JM109 as E. coli host for transformation and for recombinant phagemid isolation. The recombinants were sequenced using the oligonucleotide primer

TGTAGCGGGAAGGCGTATTAT which corresponds to a part of the promoter region (from -28 to -8) and allows checking of the amino terminal half of the IL-1β genes.

Expression and purification of ASND

JM101 E. coli cells transformed with the expression constructions were grown in LB medium containing 100 mg/l ampicillin at 37°C and the exponentially growing cultures (A₆₀₀=0.3-0.4) were induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.7 mM and further grown for 4-6 h. Pellets of the induced cells were extracted by using a repeated freeze-thaw procedure followed by hydrophobic interaction chromatography over phenyl Sepharose Cl-4B column [27]. Further purification on Mono S column (Pharmacia) FPLC chromatography with a linear gradient of NaCl

(0-0.2 M) in a 50 mM Na-acetate, pH 5.5, buffer. The elution was followed by SDS-polyacrylamide gel electrophoresis [28] and the homogeneous fractions were pooled and dialysed against 5 mM Na-phosphate, pH 7.5.

Receptor binding studies and biological assay of ASND

Cell cultures;

The EL-4 mouse thyoma cells, possessing IL-lr type I were grown in RPMI-1640 medium, supplemented with 5% fetal calf serum, 25 μM β-mercaptoethanol and 50 μg/ml gentamycin. Human β-lymphoma cell line Raji (possessing IL-lr type II) was maintained in RPMI-1640, containing 10% fetal calf serum and 50 μg/ml gentamycin.

Human recombinant IL-1β (hrlL-1β) was radiolabelled with ¹²⁵I using the Bolton-Hunter reagent (DuPont, NEN) in accordance with the manufactures instructions and stored as an approx. 50 nM solution in binding medium (RPMI-1640, 25 mM HEPES, pH 7.2, 1% (w/v) BSA, 0.1% (w/v) Na-azide) at -70ºC. The specific activity was approximately 30 μCi/μg. Displacement assays were performed as follows: cells (EL-4 or Raji) were harvested washed in Hank's balanced salt solution, resuspended in binding medium (10⁷ cells/ml) and incubated at room temperature for 1 h with increasing concentrations of hrlL-1β, ΔSND or IL-ra, and [¹²⁵I]-IL-1β (≈ 2 × 10⁵ cpm/well) in 96-well plates. Each well contained 25 μl of unlabelled ligand (final concentrations 10^-12-10^-6 M), 25 μl [¹²⁵I]-IL-1β (≈ 3 nM) and 50 μl of cell suspension (10⁵ cells/well). The cells in each well were collected by centrifugation through a phthalate oil cushion at 12 000 rpm for 0.5 minutes, the supernatants were aspirated and the radioactivity of the sedimented cells was measured in a gamma-counter. The displacement of ¹²⁵I-IL-1β from EL-4 mouse thyoma cells, which have IL-1 type I receptors, by unlabelled IL-1β, ΔSND and IL-lra, respectively, is shown in Table 4 as IC₅₀ (nM).

The displacement of ¹²⁵I-IL-1β from human Raji cells, which have IL-1 type II receptors, by unlabelled IL-1β, ΔSND and IL-lra, respectively, is shown in Table 5 as IC₅₀ (nM).

As is evident from the results presented in Tables 1 and 2, ΔSND like the endogenously occuring IL-1β and IL-lra has high affinity for both IL-1 receptor types. The relative affinity for ΔSND as compared to that of IL-1β is the same with regard to the type II receptor (IC₅₀ for ΔSND ≈ 6 nM and IC₅₀ for IL-1β ≈ 4 nM) while it is tenfold lower than that of IL-1β at the type I receptor (IC₅₀ for ΔSND ≈ 17 nM and IC₅₀ for IL-1β ≈ 1.4 nM).

The IL-lra shows lower affinity to the type II receptor

(IC₅₀ = 35 nM) than to the type I receptor. (IC₅₀ ≈ 0.5 nM) Biological effect of ΔSND was assessed using the thymocyte- ing factor (TAF) assay according to the procedure described by Rosenwasser et al. [29].

Varying amounts of IL-1β, ΔSND and IL-lra were used in the TAF assay, and the results are shown in Table 6 as ³H-thymidine incorporation (cpm) following incubation.

The results shown in Table 6 indicate that binding of IL-1β to mouse T-cells stimulates ³H-thymidine incorporation into these cells with a ED₅₀ of approx. 20 pg. ΔSND starts to stimulate H3 thymidine incorporation at a thousand fold higher concentration, while IL-lra shows no such activity in the dose range tested. When submaximal doses (100 pg) of IL-1β were used to evoke ³H-thymidine incorporation, increasing doses of ΔSND could not inhibit the response but neither did they enhance it. IL-lra in 5-100 fold excess inhibited the submaximal IL-1β dose-caused ³H-thymidine incorporation.

The finding that ΔSND does not stimulate ³H-thymidine incorporation into T-cells in concentrations which saturated all IL-1β receptors suggests that it has no or very low intrinsic activity. However, it has to be commented that ΔSND applied in 10-100 fold excess to IL-1β did not fully block the T-cell stimulating action of the former. It is unlikely that this should depend on a much faster off rate for ΔSND than for IL-1β, since the equilibrium binding constants are very close, and the off rates could not differ without an inverse difference in the on-rates, thus kinetic arguments are unlikely.

The very low or no intrinsic activity makes the ΔSND most interesting as an adjuvant or carrier in association with biologically active molecules e.g for production of vaccines.

The pyrogenic effects of ΔSND was tested in comparison with IL-1β in male Shinshilla rabbits (weight 2.5-2.9 kg). Samples (10 and 30 ng/kg) were injected into the ear vein in a volume of 2.5-3 ml. Rectal temperature (T_re) was measured before injection and then at times indicated in Table 7. Heat treated ΔSND and IL-1β (90°C, 30 min) served as controls. From Table 7 is evident that the fever reactions evoked by ΔSND were significantly lower than those caused by the same doses of IL-1β. Heat treated ΔSND and IL-1β had no pyrogenic activity. Due to the low or no pyrogenic activity of the ΔSND, the

IL-1β deletion mutants of the invention will probably be used instead of IL-1β in therapeutic and/or prophylactic agents. Additional testing of biological activity:

IL-1 (recombinant human IL-1β, specific activity 1 × 10⁷ U/mg) was a kind gift from Sclavo, Siena, Italy.

Animals and in vivo treatments

Male, adult (25-30 g) CD-1 mice (Charles River, Calco, Italy) were used. Animals were housed five per cage in air-conditioned quarters (60% relative humidity, 22°C) with a 12-h light/dark cycle, and were given standard laboratory chow (Altromin, Rieper, Bolzano, Italy).

Food intake and body weight were measured at day 0 and at day 1. IL-1β and ΔSND was dissolved in 0.15 M Sodium Phosphate buffer, pH 5.7, 0.1 M NaCl.

Animals were treated with IL-1β or ΔSND at the doses indicated i.p. in a final volume of 0.2 ml. Control mice received buffer alone. All treatments were performed between 9.00 am and 11.00 am.

Blood was obtained from the retroorbital plexus under light ether anestesia and serum preparated for IL-6, glucose, corticosterone and SAA determination. Blood was collected at 2 h for IL-6, corticosterone and glucose determinations and at 8 h for SAA determination, since previous experiments indicated that, at these times, peak levels were observed. Serum IL-6 levels were measured as hybridoma growth factor using 7TD1 cells (a kind gift from Dr. van Snick, Bruxelles, Belgium) as previously described [30]. IL-6 is expressed as costimulatory units/ml using a standard curve with human recombinant IL-6 (Immunex Corp., Seattle, WA; specific activity 10⁷ U/ml). Serum corticosterone was measured by a RIA using a polyclonal antibody to corticosterone from Sigma, following the manufacturer's instructions. (³H)corticosterone was purchased from Amersham.

Serum glucose was measured by the glucose oxidase/peroxidase method with a commercially available kit (Boehringer, Mannheim). Serum amyloid-A (SAA) was measured in sera by an ELISA [31] using a polyclonal rabbit anti mouse SAA (kind gift from Dr. J. Sipe, Boston). icv. injection

Male rats (CD-COBS, Charles River, Italy, 250-300 g) were used. IL-1 was dissolved in pyrogen-free saline containing 0.1% bovine serum albumin or phosphate buffer. ΔSND was dissolved in phosphate buffer.

Six μl of solution were injected through one polyethylene cannula permanently implanted in the lateral ventricle three days before the experiment. Control rats were given the same volume of vehicle.

Table 1

Alignment of human IL-lra and the sequences of mature IL-lβ proteins from different species

1 45 ... ..APVRS LNCTLRDSQQ KSLVMSGPYE LKALHLQGQD MBQQWFSMS

... .. APVQS IKCKLQDRBQ KSLVLASPCV LKALHLLSQE MNREWFCMS

.... .. AAVQS VKCKLQDREQ KSLVLDSPCV LKALHLLSQE MSREWFCMS

... .. VPIRQ LHYRLRDEQQ KSLVLSDPYE LKALHLNGQN INQQVIFSMS

... .. VPIRQ LHC-RI--RDEJQQ KCLVLSDPCE LKALHLNGQN ISQQWFSMS

... .. AVRS LHCRLQDAQQ KSLVLSGTYE LKALHLNAEN LNQQWFSMS

RPSGRKSSKM CAFRIWDVNQ KTFYLRNN.Q LVAGYLQGFN VNLEEKIDVV

* * * * * *

46 94

FVQGEESNEK IPVALGLKEK NLYLSCVLKD EKPTLQLESV DPKNYP.KKK

FVQGEERENK IPVALGIKEK NLYLSCVKKG DTPTLQLEEV DPKVYP.KRN

FVQGEERENK IPVALGIRDK NLYLSCVKKG DTPTLQLEEV DPKVYP.KRN

FVQGEPSNCK IPVALGLKGK NLYLSCVMKD GTPTLQLESV DPKQYP.KKK

FVQGETSNDK IPVALGLKGK NLYLSCVMKD GTPTLQLESV DPKQYP.KKK

FVQGEESNDK IPVALGLRGK NLYLSCVMKD EKPTLQLESV DPNRYP.KKK

PIEPH ... .... . ALFLGIH3G KMCLSCVKSG DEIRLQLEAV NITDLSE-MRK

** **** **** *

95 143

MEK RFVFNKI EINNKI-EFES AQFFNWYIST SQAENMPVFL GGTKG.GQDI

MEKRFVFYKT EIKNIVEFES VLYFNWTCST SQIEERPVFL GHFTV .GQDI

MEKRFVFYKT EIXNIVEFES VLYANCYIST SQIEEKPVFL GRFEG.GQDI

MEKRFVFNKI EVKSKVEFES AEFPNWHST SQM-HKPVFL GNNS. .GQOI

MEKRFVFNKI EVKTKVEFES AQFRWYIST SQW-HRPVFL GNSN. .GRDI

MEXRFVFNKI EIKDKLEFT-S AQFFNWYIST SQTEYMPVFL GNNSG.GQΩL

QDKRFAFIRS DSGPTTSFES AACPGWFLCT AMEADQPVSL TNMPDBGVMV

***** *** * * ** * *

144 153

TDFTMQFVSS Human IL-1β

TDFRMETLSP Bovine IL-1β

TDFRMETLSP Sheep IL-1β

IDFTMESVSS Mouse IL-1β

VDFTMESS. . Rat IL-1β

IDFSMEFVSS Rabbit IL-1β

TKFYPQEDE. Human IL-1ra

*

The numbering is for human IL-1β and stars indicate identical residues in all sequences . Table 2

Amino-acid and nucleotide sequence of Interleukin 1β

1 Ala Pro Val Arg Ser Leu Asn Cys Thr Leu Arg Asp Ser Gln Gl n Lys 16

1 GCA CCT GTA CGA TCA CTG AAC TGC ACG CTC CGG GAC TCA CAG CAA AAG 48

17 Ser Leu Val Het Ser Gl y Pro Tyr Glu Leu Lys Ala Leu His Leu Gln 32

49 AGC TTG GTG ATG TCT GGT CCA TAT GAA CTG AAA GCT CTC CAC CTC CAG 96

33 Gly Gl n Asp Het Gl u Gl n Gl n Val Val Pht Ser Het Ser Pht Val Gl n 48

97 GGA CAG GAT ATG GAG CAA CAA GTG GTG TTC TCC ATG TCC TTT GTA CAA 144

49 Gl y Glu Gll Ser Asn Asp Lys Ile Pro Val Ala Leu Gl y Leu Lys Glu 64 145 GGA GAA GAA AGT AAT GAC AAA ATA CCT GTG GCC TTG GGC CTC AAG GAA 192 65 Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp Lys Pro Thr Leu 80 193 AAG AAT CTG TAC CTG TCC TGC GTG TTG AAA GAT GAT AAG CCC ACT CTA 240

81 Gl n Leu Glu Ser Val Asp Pro Lys Asn Tyr Pro Lys Lys Lys Het Gl u 96 241 CAG CTG GAG AGT GTA GAT CCC AAA AAT TAC CCA AAG AAG AAG ATG GAA 288

97 Lys Arg Pht Val Phe Asn Lys Ile Gl u Ile Asn Asn Lys Leu Gl u Pht 112 289 AAG CGA TTT GTC TTC AAC AAG ATA GAA ATC AAT AAC AAG CTG GAA TTT 336 113 Glu Str Ala Gln Pht Pro Asn Trp Tyr Ile Ser Thr Ser Gl n Ala Gl u 128 337 GAG TCT GCC CAG TTC CCC AAC TGG TAC ATC AGC ACC TCT CAA GCA GAA 384 129 Asn net Pro Val Pht Leu Gl y Gl y Thr Lys Gl y Gl y Gl n Asp lit Thr 144 385 AAC ATG CCC GTC TTC CTG GGA GGG ACC AAA GGC GGC CAG SAT ATA ACT 432 145 Asp Pht Thr Met Gl n Pht Val Ser Ser

433 GAC TTC ACC ATG CAA TTT GTG TCT TCC

Table 3

Amino-acid and nucleotide sequence of ΔSND

1 Al a Pro Val Arg Ser Leu Asn Cys Thr Leu Arg Asp Ser Gl n Gl n Lys 16

1 GCA CCT GTA CGA TCA CTG AAC TGC ACG CTC CGG GAC TCA CAG CAA AAG 48

17 Ser Leu Val Het Ser Gl y Pro Tyr Gl u Leu Lys Al a Leu Hi s Leu Gl n 32

49 AGC TTG GTG ATG TCT GGT CCA TAT GAA CTG AAA GCT CTC CAC CTC CAG 96

33 Gl y Gl n Asp net Gl u Gln Gl n Val Val Pht Ser het Ser Pht Val Gln 48 97 GGA CAG GAT ATG GAG CAA CAA GTG GTG TTC TCC ATG TCC TTT GTA CAA 144

49 Gly Gl u Gl u Lys Ile Pro Val Ala Leu Gl y Leu Lys Gl u Lys Asn Leu 64 145 GGA GAA GAA AAA ATA CCT GTG GCC TTG GGC CTC AAG GAA AAG AAT CTG 192 65 Tyr Leu Ser Cys Val Leu Lys Asp Asp Lys Pro Thr Leu Gl n Leu Glu 80 193 TAC CTG TCC TGC GTG TTG AAA GAT GAT AAG CCC ACT CTA CAG CTG GAG 240

81 Str Val Asp Pro Lys Asn Tyr Pro Lys Lys Lys net Gl u Lys Arg Pht 96

241 AGT GTA GAT CCC AAA AAT TAC CCA AAG AAG AAG ATG GAA AAG CGA TTT 288

97 Val Pht Asn Lys Ile Gl u Ile Asn Asn Lys Leu Gl u Phe Gl u Ser Ala 112

289 GTC TTC AAC AAG ATA GAA ATC AAT AAC AAG CTGGAA TTT GAG TCT GCC 336

113 Gl n Pht Pro Asn Trp Tyr Ile Ser Thr Ser Gln Ala Gl u Asn Het Pro 128

337 CAG TTC CCC AAC TGG TAC ATC AGC ACC TCT CAA GCA GAA AAC ATG CCC 384

129 Val Pht Leu Gly Gl y Thr Lys Gl y Gl y Gln Asp Ile Thr Asp Pht Thr 144 85 GTC TTC CTG GGA GGG ACC AAA GGC GGC CAG GAT ATA ACT GAC TTC ACC 432

145 met Gl n Phe Val Ser Ser

33 ATG CAA TTT GTG TCT TCC

Table 4

IL-1β, ΔSND and IL-lra as ligands to Interleukin-1 type I receptor - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Ligand IC₅₀ (nM)

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

IL-1β 1.4

ΔSND 17

IL-1RA 0.5

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Table 5

IL-1β. ΔSND and IL-lra as ligands to Interleukin-1 type II receptor - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Ligand IC₅₀ (nM)

IL-1β 4

ΔSND 6

IL-1RA 35

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Table 6

Biological effect measured as ³H-thymidine incorporation (cpm)

Amount used

(gram) IL-1β ΔSND IL-lra

10^-11 1815±290 307 ±105 426 ±105

₁₀-10 3356±631 265 ±94 585 ±55

10^-9 5316±1211 490 ±152 538 ±242

10^-8 4680±352 670 ±232 463 ±103

10^-7 4788±1300 1978±602 652 ±276

10^-6 4960±431 2974±473 464 ±275

Table 7

Changes in rectal temoerature of rabbits following hrlL-Iβ injections

substance baseline time after injection (min)

injected

T_re 15 30 45 60 90 120 180 240 hrlL-1β

100 ng/kg 38,7ºC 0,2 0,9 1,1 1,1 0,9 0,7 0,6 0,3 (n=3)

hrlL-Iβ 38,6'C 0,2 0,8 1,1 0,9 0,7 0,5 0,3 0,1 30 ng/kg (0,04) (0,05) (0,08) (0,04) (0,06) (0,08) (0,06) (0,04) (0,04) (n=8)

hrlL-1β 38,6ºC 0,1 0,5 0,7 0,5 0,4 0,3 0,2 0,1 10 ng/kg (0,03) (0,04) (0,06) (0,08) (0,05) (0,04) (0,06) (0,04) (0,05) (n=9)

hrlL-1β

heated 38,7ºC 0 0 0 0 -0,1 0 0 0

30',90ºC (0,04) (0) (0) (0) (0) (0,01) (0,01) (0) (0)

30 ng/kg

(n=5)

Changes in rectal temperature of rabbits following ΔSND injections

substance baseline time after injection (min)

injected

T_re 15 30 45 60 90 120 180 240

ΔSND 38,6ºC 0 0,5 0,7 0,4 0,3 0,1 0,1 0 30 ng/kg (0,04) (0,02) (0,05) (0,08) (0,06) (0,07) (0,04) (0,04) (0) (n=7)

ΔSND 38,6ºC 0,1 0,3 0,5 0,3 0,2 0,1 0,1 0 10 ng/kg (0,06) (0,08) (0,06) (0,05) (0,04) (0,03) (0,04) (0,03) (0,05) (n=8)

ΔSND 38,7'C 0 0 0 0 0,1 0,1 0 0 heated (0,05) (0) (0) (0) (0) (0,01) (0) (0,01) (0)

30',90°C

(n=5) n - number of rabbits; in parentheses: SE

Table 8

Effects of the ip. injectioni of IL-lβ and ΔSND (100 ng/mouse) in CD1 mice saline IL-1β ΔSND

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - glucose 238±15 187+11* 237±16

(mg/dl)

corticosterone 156.3±55 559.2+95** 176±60

(ng/ml)

IL-6 <50 1439±671 188.4+97.5° (U/ml)

Effects of IL-1β and ΔSND on different biological parameters in CD1 mice injected ip. with (100 ng/mouse).

Serum levels of glucose, corticosterone and IL-6 were measured 2 h after the injection. Data are mean + SE (5 mice per group),

At this dose ΔSND is only able to induce IL-6 in the serum;

this effect is almost ten times lower than the effect of the same dose of IL-1β (p<00.5 versus IL-1β)

** p<0.01 versus saline

* p<0.05 versus saline

° p<0.05 versus IL-1β

Table 9

Effects of the ip. injection of IL-lβ and ΔSND (100 μg/mouse) in CDl mice saline IL-1β ΔSND

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - food-intake 5.76 3.64 4.6

(g/day/mouse)

glucose 172.2±16.3 102.6±2** 119±8**

(mg/dl)

corticosterone 22.2±7.6 403±40** 252±90*

(ng/ml)

SAA <0.02 191.3±33 88.6±5ºº

(μg/ml)

IL-6 <50 150,000 16,432±6263ºº

(U/ml)

Effects of IL-1β and ΔSND on different biological parameters in CDl mice injected ip. with (1 μg/mouse). Data are mean ± SE (5 mice per group).

Serum levels of glucose, corticosterone and IL-6 were measured 2 h after the injection, whereas serum amyloid A levels were measured 8 h after the injection at the peak time. The food-intake was evaluated 1 day after the ip. injection. Data are mean ± SE (5 mice per group).

ΔSND has an effect on the serum levels of glucose, corticosterone, IL-6 and SAA, whereas only a small effect was observed on food-intake. At this dose ΔSND induces significally lower IL-6 and SAA serum levels if compared with IL-1.

** p<0.01 versus saline (Dunnet's test)

* p<0.05 versus saline

ºº p<0.01 versus IL-1β Table 10

Effects of the icv. injection of IL-lβ and ΔSND (400 ng/6 μl ) on rat serum levels of IL-6. (n=6) groups buffer ΔSND+buffer IL-1β IL-1β+buffer - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

IL-6 151±44 316±28* 2485+342* 821+229

(U/ml)

* p<0.01

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Claims

CLAIMS 1. Interleukin-1β deletion mutant,

c h a r a c t e r i s e d in that it has a quasi conserved sequence of three amino acids deleted from the amino-acid sequence of an endogenous mammalian Interleukin-1β, said sequence of amino acids being Ser Asn Asp (numbers 52-54 of mature human IL-1β) in the case of human, mouse, rat and rabbit, and Arg Asp Asn in the case of bovine and sheep

Interleukin-1β.

2. Interleukin-1β deletion mutant according to claim 1, wherein said endogenous mammalian Interleukin-1β is an endogenous human Interleukin-1β.

3. Interleukin-1β deletion mutant according to claim 1 or 2, wherein said mutant is an Interleukin-1 receptor ligand which binds to both Interleukin-1 type I receptors and Interleukin-1 type II receptors with substantially different binding-affinities.

4. Interleukin-1β deletion mutant according to claim 3, wherein the amino-acid sequence of said ligand has additional amino-acid deletion(s) and/or point mutation(s).

5. Carrier for biologically active molecules which is an Interleukin-1β deletion mutant according to any one of claims 1-4.

6. Adjuvant for biologically active molecules which is an Interleukin-1β deletion mutant according to any one of claims 1-4.

7. Therapeutic and/or prophylactic agent which comprises an Interleukin-1β deletion mutant according to any one of claims 1-4.