INTERLEUKIN-1 beta DELETION MUTANT

Abstract: An Interleukin-1 beta deletion mutant, which has a quasi conserved sequence of three amino acids deleted from the amino acid sequence of an endogenous mammalian Interleukin-1 beta is disclosed. The deletion is Ser Asn Asp (numbers 52-54 of mature human IL-1 beta ) in the case of human, mouse, rat and rabbit, and Arg Asp Asn in the case of bovine and sheep Interleukin-1 beta . The Interleukin-1 beta 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-1 beta deletion mutants may be used as carriers and adjuvants for biologically active molecules as well as in therapeutic and/or prophylactic agent.

Description: INTERLEUKIN-lβ DELETION MUTANT The present invention relates to an Interleukin-lβ deletion mutant which has a quasi conserved sequence of three amino acids deleted from the amino-acid sequence of an endogenous mammalian Interleukin-lβ. In the case of a human Interleukin- lβ deletion mutant, said three deleted amino acids are Ser Asn Asp (numbers 52-54 of mature human IL-lβ), which mutant is also called ΔSND. Background The cytokines Interleukin-lβ and β (IL-lα and IL-lβ), are mediators of a multitude of processes involving the immune endocrine and nervous system (cf for review [1]). The role of interleukin-lα and β as inflammatory mediators, as lymphocyte activating factors, endogenous pyrogens and radiation-protec¬ tive agents render these two small proteins of approximately 153 amino acids extremely interesting in both physiological and pharmacological studies. However, widespread use of IL-lα and IL-lβ in therapy has been hampered by their high pyroge- nicity. Since the molecular identification and cloning of the cDNA coding for the urine IL-lα by Lo edico et al in 1984, [2] the interleukin-lα and β have been in the focus of interest by molecular biologists, protein chemists and crystallo- graphers. The cDNA sequences for IL-lα and IL-lβ are known from several species. Two IL-1 receptors have been cloned. Type I from T-lymphocytes [3] and type II from B-lymphocyteε [4]. Discovery and cloning of an endogenously occuring IL-1 receptor antagonist (IL-1 ra), which belongs to the same protein family as IL-lα and β [e.g. 5], and which antagonizes the actions of these agonists by binding to the same recep¬ tor, has accelerated studies on the IL-1 receptor-ligand interactions. A large number of modifications, such as point-mutations, of the human IL-lβ have been examined with respect to their effect on biological and receptor binding properties of the IL-lβ molecule [e.g.6, 7, 8]. Important residues in both the N-terminal portion of IL-lβ [9, 10, 11] and in its C-terminal region [12] have been identified. However, to our knowledge no interesting internal deletion mutants of IL-lβ have been reported. Description of the invention The amino acid sequences of IL-lβ 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 struc- tural 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 sequ¬ ences from rat and mouse, and both these also lack said motif [5]. Deletion of Ser Asn Asp from said motif from the human IL-lβ (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-lβ. ΔSND binds with a tenfold less affinity to Interleukin-1 receptors (type I) on mouse El-4 thyoma cells. In comparison with interleukin-lβ, ΔSND exhibits a signifi¬ cantly reduced biological activity in a mouse thymocyte proliferation assay. ΔSND is shown to have a lower pyrogenic activity than IL-lβ 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-lβ 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-lβ deletion mutant, which has a quasi conserved sequence of three amino acids deleted from the amino-acid sequence of an endogenous mammalian Interleukin-lβ, said sequence of amino acids being Ser Asn Asp (numbers 52-54 of mature human IL-lβ) in the case of human, mouse, rat and rabbit, and Arg Asp Asn in the case of bovine and sheep Interleukin-lβ. In one embodiment of this aspect of the invention the endoge- nous mammalian Interleukin-lβ is an endogenous human Inter¬ leukin-lβ. In another embodiment of this aspect of the invention the Interleukin-lβ 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-lβ deletion mutant according to the last mentioned embodiment may have additional amino-acid dele¬ tion^) 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-lβ deletion mutant according to the invention. Yet another aspect of the invention is directed to an adju¬ vant for biologically active molecules which is an Interleu¬ kin-lβ 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-lβ 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-lβ 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-lβ 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-lβ from rat was taken from the literature [13]. Multiple alignments of the sequence of human IL-lra with IL-lβ 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-lβ 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 ASND 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 Ml3 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-[ 35 S]-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-lβ gene was purchased from British Bio-tech¬ nology (BBG25). [The nucleotide and amino acid sequences are shown in Table 2.] It was subcloned as an EcoRI-Hindlll frag¬ ment 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 oligonucleo¬ tides 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. Reco binants 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 AGJ3A£GAAATAACCATGG sequence, which contains the consensus Shine-Dalgarno sequen- ce 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 frag¬ ment of pMC9 [26] containing the lac I q gene, the lac Z con- trol 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-lβ (hrlL-lβ) or its analogs were cloned between these sites, using JM109 as E. coli host for trans¬ formation and for recombinant phagemid isolation. The recom- binants 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-lβ genes. Expression and purification of ASND JM101 E. coli cells transformed with the expression construc- tions were grown in LB medium containing 100 mg/1 ampicillin at 37°C and the exponentially growing cultures (A 600 =0.3-0.4) were induced by adding iεopropyl-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 hydropho- bic interaction chromatography over phenyl Sepharose C1-4B column [27]. Further purification on Mono S column (Pharma¬ cia) 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-lβ (hrlL-lβ) was radiolabelled with 125 I 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 -70 e C. 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, resuspen- ded in binding medium (10 7 cells/ml) and incubated at room temperature for 1 h with increasing concentrations of hrlL-lβ, ΔSND or IL-ra, and [ 125 I]-IL-lβ (« 2 x 10 5 cpm/well) in 96-well plates. Each well contained 25 μl of unlabelled ligand (final concentrations 10- 12 -10 "6 M) , 25 μl [ 125 I]-IL-lβ (« 3 nM) and 50 μl of cell suspension (10 5 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 15 I-IL-lβ from EL-4 mouse thyoma cells, which have IL-1 type I receptors, by unlabelled IL-lβ, ΔSND and IL-lra, respectively, is shown in Table 4 as IC 50 (nM) . The displacement of 125 I-IL-lβ from human Raji cells, which have IL-1 type II receptors, by unlabelled IL-lβ, ΔSND and IL-lra, respectively, is shown in Table 5 as IC 50 (nM) . As is evident from the results presented in Tables 1 and 2, ΔSND like the endogenously occuring IL-lβ and IL-lra has high affinity for both IL-1 receptor types. The relative affinity for ΔSND as compared to that of IL-lβ is the same with regard to the type II receptor (IC 50 for ΔSND * 6 nM and IC 50 for IL-lβ » 4 nM) while it is tenfold lower than that of IL-lβ at the type I receptor (IC 50 for ΔSND « 17 nM and IC 50 for IL-lβ « 1.4 nM). The IL-lra shows lower affinity to the type II receptor (IC 50 = 35 nM) than to the type I receptor. (IC 50 » 0.5 nM) Biological effect of ΔSND was assessed using the thymocyte- activating factor (TAF) assay according to the procedure described by Rosenwasser et al. [29]. Varying amounts of IL-lβ, ΔSND and IL-lra were used in the TAF assay, and the results are shown in Table 6 as ^-thymidi- ne incorporation (cpm) following incubation. The results shown in Table 6 indicate that binding of IL-lβ to mouse T-cells stimulates ^-thymidine incorporation into these cells with a ED 50 of approx. 20 pg. ΔSND starts to stimulate H3 thy idine 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-lβ were used to evoke 3 H-thymidine incorporation, increa¬ sing 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-lβ dose-caused 3 H-thymidine incorporation. The finding that ΔSND does not stimulate 3 H-thymidine incorpo¬ ration into T-cells in concentrations which saturated all IL-lβ 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-lβ 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-lβ, 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-lβ 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 trea¬ ted ΔSND and IL-lβ (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-lβ. Heat treated ΔSND and IL-lβ had no pyrogenic activity. Due to the low or no pyrogenic activity of the ΔSND, the IL-lβ deletion mutants of the invention will probably be used instead of IL-lβ in therapeutic and/or prophylactic agents. Additional testing of biological activity: IL-1 (recombinant human IL-lβ, specific activity 1 x 10 7 U/mg) was a kind gift from Sclavo, Siena, Italy. Animals and in vivo treatments Male, adult (25-30 g) CD-I mice (Charles River, Calco, Italy) were used. Animals were housed five per cage in air-conditio- ned 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-lβ and ΔSND was dissolved in 0.15 M Sodium Phosphate buffer, pH 5.7, 0.1 M NaCl. Animals were treated with IL-lβ or ΔSND at the doses indica¬ ted 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 acti- vity 10 7 U/ml). Serum corticosterone was measured by a RIA using a polyclonal antibody to corticosterone from Sigma, following the manufac¬ turer's instructions. ( 3 H)corticosterone was purchased from Amersham. Serum glucose was measured by the glucose oxidase/peroxidase method with a commercially available kit (Boehringer, Mann¬ heim) . 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 IKCKLQDRB3 KSLVLASPCV LKALHLLSQE MNREWFCMS AAVQS VKCKLQDREQ KSLVLDSPCV LKAI-HLLSQE MSREWFCMS VPIRQ LHYRLRDEQQ KSLVLSDPYE LKAI-H1-N3QN INQQVIFSMS VPIRQ LHC-RI--RDEJ3Q KCLVLSDPCE LKAI-HLN3QN ISQQWFSMS AVRS LHCRLQDAQQ KSLVLSGTYE LKALHL-NAE-N I-NQQWFSMS RPSGRKSSKM CAFRIWDVNQ KTFYLRNN.Q LVAGYLQGFN VNLEEKIDW * * * * * * 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 KMCL-SCVKSG DEIRLQLEAV NITDLSE-MRK ** **** **** * 95 143 ME3 RFVFNKI E NNKI-EFES AQFFNWYIST SQAENMPVFL GGTKG.GQDI ME-XRFVF Ϊ KT EIKNIVEFES VLYFNWTCST SQIEERPVFL GHFTV-.GQDI MEKRFVFYKT EIXNIVEFES VLYANCYIST SQIEEKPVFL GRFEG.GQDI M--XRFVFNKI EVKSKVEFES AEFrøWHST SQM-HKPVFL GNNS. .GQOI ME-KRFVFNKI EVKTKVEFES AQFRWYIST SQW-HRPVF GNSN. .GRDI MEXRFVFNKI EIKDKLEFT-S AQFFNWYIST SQTEYMPVFL GNNSG.GQΩL QDKRFAFI-RS DSGPTTSFES AACPGWFLCT AMEADQPVSL TNMPDBGVMV ***** *** * * ** * * 144 153 TDFTMQFVSS Human IL-lβ TDFTOffiTLSP Bovine IL-lβ TDFRMETLSP Sheep H^lβ IDFTMESVSS Mouse IL-lβ VDFTMESS. . Rat IL-lβ IDFSMEFVSS Rabbit IL-lβ TKFYPQEDE. Human IL-lra The numbering is for human IL-lβ and stars indicate identical residues in all sequences . Table 2 Amino-acid and nucleotide sequence of Interleukin lβ 1 Ala Pro Val Arg Ser Leu Asn Cys Thr Leu Arg Asp Ser 61n 61n Lyi 16 1 6CA CCT 6TA CGA TCA CT6 AAC TGC AC6 CTC CG6 GAC TCA CAG CAA AAG 48 17 Ser Leu Val Het Ser 61y Pro Tyr Glu Leu Lys Ala Leu His Leu 61n 32 49 AGC TTG 6TG AT6 TCT GGT CCA TAT GAA CT6 AAA 6CT CTC CAC CTC CA6 96 33 61y 61n Asp Het 61u 61n 61n Val Val Pht Ser Het Ser Pht Val 61n 48 97 6GA CAG 6AT AT66A6 CAA CAA 6T66TG TTC TCC AT6 TCC TTT 6TA CAA 144 49 61y Glu 61u Ser Asn Asp Lys lie Pro Val Ala Leu 61y Leu Lys 61u 64 45 GGA 6AA GAA AGT AAT 6AC AAA ATA CCT 6T66CC TT66GC CTC AAG 6AA 192 65 Lys Asn Leu Tyr Leu Ser Cys Val Leu Lys Asp Asp Lys Pro Thr Leu 80 93 AAG AAT CT6 TAC CTG TCC TGC GT6 TT6 AAA 6AT 6AT AAG CCC ACT CTA 240 81 61n Leu 61u Ser Val Asp Pro Lys Asn Tyr Pro Lys Lys Lys Het 61u 96 41 CAG CT66A6 AGT GTA 6AT CCC AAA AAT TAC CCA AAG AAG AAG ATG GAA 288 97 Lys Arg Pht Val Phi Asn Lys He 61u II* Asn Asn Lys Leu 61u Pht 112 89 AAG CGA TTT 6TC TTC AAC AAG ATA GAA ATC AAT AAC AAG CT66AA TTT 336 13 Glu Str Ala 61n Pht Pro Asn Trp Tyr lit Ser Thr Ser 61n Ala 61u 128 37 6AG TCT 6CC CAG TTC CCC AAC TGG TAC ATC AGC ACC TCT CAA 6CA GAA 384 29 Asn net Pro Val Pht Leu 61y 61y Thr Lys 61y 61y 61n Asp lit Thr 144 85 AAC AT6 CCC 6TC TTC CT66GA GGG ACC AAA GGC 6GC CAG SAT ATA ACT 432 45 Asp Pht Thr Htt 61n Pht Val Ser Ser 3 6AC TTC ACC AT6 CAA TTT 6T6 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 61 n 61 n Lys 16 1 6CA CCT 6TA CGA TCA CT6 AAC TGC AC6 CTC CGG GAC TCA CA6 CAA AA6 48 17 Ser Leu Val Het Ser 61 y Pro Tyr 61 u Leu Lys Al a Leu Hi s Leu 61 n 32 49 AGC TT6 6T6 AT6 TCT 6GT CCA TAT 6AA CT6 AAA 6CT CTC CAC CTC CA6 96 33 61y 61n Asp net 61u 61n 61n Val Val Pht Ser het Ser Pht Val 61 48 97 6GA CAG GAT AT66AG CAA CAA 6T66T6 TTC TCC AT6 TCC TTT 6TA CAA 144 49 61y 61u 61u Lys lit Pro Val Ala Leu 61y Leu Lys 61u Lys Asn Leu 64 145 6GA 6AA 6AA AAA ATA CCT 6T66CC TT66GC CTC AAG 6AA AA6 AAT CT6 192 65 Tyr Leu Ser Cys Val Leu Lys Asp Asp Lys Pro Thr Leu 61n Leu 61u 80 193 TAC CT6 TCC TGC 6TG TT6 AAA 6AT 6AT AA6 CCC ACT CTA CAG CT66A6 240 81 Str Val Asp Pro Lys Asn Tyr Pro Lys Lys Lys net 61u Lys Arg Pht 96 241 AGT 6TA 6AT CCC AAA AAT TAC CCA AAG AAG AAG AT6 GAA AAG CGA TTT 288 97 Val Pht Asn Lys lit 61u lit Asn Asn Lys Leu 61u Phe 61u Ser Ala 112 289 6TC TTC AAC AA6 ATA 6AA ATC AAT AAC AA6 CT66AA TTT 6A6 TCT 6CC 336 113 61n Pht Pro Asn Trp Tyr lit Ser Thr Ser 61n Ala 61u Asn Het Pro 128 337 CA6 TTC CCC AAC TGG TAC ATC AGC ACC TCT CAA 6CA GAA AAC AT6 CCC 384 129 Val Pht Leu Gly 61y Thr Lys 61y 61y 61n Asp lit Thr Asp Pht Thr 144 85 6TC TTC CT66GA 6G6 ACC AAA 6GC 6GC CAG 6AT ATA ACT 6AC TTC ACC 432 145 Het 61n Phe Val Ser Ser 33 AT6 CAA TTT 6T6 TCT TCC Table 4 IL-lβ, ΔSND and IL-lra as ligands to Interleukin-1 type I receptor Ligand IC 50 (nM) IL-lβ 1.4 ΔSND 17 IL-1RA 0.5 Table 5 IL-lβ. ΔSND and IL-lra as ligands to Interleukin-1 type II receptor Ligand IC 50 (nM) IL-lβ 4 ΔSND 6 IL-1RA 35 ng g (n=5) Changes in rectal temperature of rabbits following ΔSND injections substance baseline time after injection (min) injected ΔSND 30 ng/kg (n=7) ΔSND 10 ng/kg (n=8) ΔSND heated 30',90°C (n=5) n - number of rabbits; in parentheses: SE Effects of IL-lβ 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-lβ (p<00.5 versus IL-lβ) ** p<0.01 versus saline * p<0.05 versus saline ° p<0.05 versus IL-lβ Table 9 Effects of the ip. injection of IL-lβ and ΔSND (100 μg/mouse) in CDl mice saline IL-lβ 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±5 oe (μg/ml) IL-6 <50 150,000 16,432+6263" (ϋ/ml) Effects of IL-lβ 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, corticoste¬ rone, IL-6 and SAA, whereas only a small effect was observed on food-intake. At this dose ΔSND induces εignifically 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 " p<0.01 versus IL-lβ 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-lβ IL-lβ+buffer IL-6 151±44 316±28* 2485+342* 821+229 (U/ml) * p<0.01 List of References 1. Dinarello, C.A. (1991) Blood 77 1627-52. 2. Lomedico, P.T., Gubler, U., et al. (1984) Nature 312 458-62. 3. Sims, J.E., March, C.J., et al. (1988) Science 241 585-589. 4. Mcmahan, C.J., Slack, J.L., et al. (1991) EMBO J 10 2821- 2832. 5. Eisenberg, S.P., Brewer, M.T., et al. (1991) Proc Natl Acad Sci USA 88 5232-6 6. Labriola-Tompkins, E., Chandran, C, et al. (1991) Proc Natl Acad Sci USA 88 11182-11186. 7. Veerapandian, B., Gilliland, G.L., et al. 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Claims: CLAIMS 5 1. Interleukin-lβ 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-lβ, said sequence of amino acids being Ser Asn Asp (numbers 52-54 of 10 mature human IL-lβ) in the case of human, mouse, rat and rabbit, and Arg Asp Asn in the case of bovine and sheep Interleukin-lβ. 2. Interleukin-lβ deletion mutant according to claim 1, 15 wherein said endogenous mammalian Interleukin-lβ is an endogenous human Interleukin-lβ. 3. Interleukin-lβ deletion mutant according to claim 1 or 2, wherein said mutant is an Interleukin-1 receptor ligand which 20 binds to both Interleukin-1 type I receptors and Interleu- kin-1 type II receptors with substantially different binding- affinities. 4. Interleukin-lβ deletion mutant according to claim 3, 25 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-lβ deletion mutant according to any one 30 of claims 1-4. 6. Adjuvant for biologically active molecules which is an Interleukin-lβ deletion mutant according to any one of claims 1-4. ~ 35 7. Therapeutic and/or prophylactic agent which comprises an Interleukin-lβ deletion mutant according to any one of claims 1-4.

Inventors: BARTFAI TAMAS, BRISTULF JESPER, CSERZOE MIKLOS, PONGOR SANDOR, SIMONCSITS ANDRAS, TJOERNHAMMAR MARIE-LOUISE, BARTFAI, TAMAS, BRISTULF, JESPER, CSERZOE, MIKLOS, PONGOR, SANDOR, SIMONCSITS, ANDRAS, TJOERNHAMMAR, MARIE-LOUISE
Applicants: BARTFAI TAMAS, BRISTULF JESPER, CSERZOE MIKLOS, PONGOR SANDOR, SIMONCSITS ANDRAS, TJOERNHAMMAR MARIE LOUISE, TRION FORSKNING & UTVECKLING, TRION FORSKNING- OCH UTVECKLINGS AB
Application year: 1993
Main classification: C07K13
IPC codes: A61K38, A61K39, A61K47, A61P9, A61P29, C07K14, C12N15, C12P21