AU616461B2 - Recombinant human interleukin-1 alpha polypeptides - Google Patents

Description

14 ii p FORM COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 COMPLETE SPECIFICATION S F Ref: 82633 616461

(ORIGINAL)

FOR OFFICE USE: Class Int Class

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Complete Specification Lodged: Accepted: Published: a Priority: Related Art: Name and Address of Applicant: F Hoffmann-La Roche Co Aktiengesellschaft Grenzacherstrasse 124-184 CH-4002 Basle

SWITZERLAND

Address for Service: Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Complete Specification for the invention entitled: Recomblnant Human Interleukin-I Alpha Polypeptldes The following statement is a full description of this invention, including the best method of performing it known to me/us j, i i I iI i i 5845/3

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RAN 4105/86-010

ABSTRACT

€4I4 e o o a o S 0e *t 0 0 0 40 I Cl CI C 1 c This invention provides homogeneous recombinant human interleukin-lo polypeptides, DNA sequences coding for such polypeptides, recombinant vectors containing such DNA 15 sequences, unicellular host organisms containing such recombinant vectors, and methods for producing the polypeptides. The human interleukin-la polypeptides and pharmaceutical compositions containing them may be used for the stimulation of the immune system of a host subject, for the promotion of wound healing and for improving the recovery of critically ill, protein-malnourished patients.

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S- RAN 4105/86-010 S Two forms of human interleukin-1 are known, the so-called a- and the B-form (March et al., Nature 315, 641-647 [1985]). The analysis of the nucleotide sequence of a DNA encoding human interleukin-la (hIL-la) revealed a large open reading frame of 271 amino acids, corresponding 4 0 to a hIL-la precursor polypeptide having a molecular 1 weight of 30'606 Dalton. The nucleotide sequence and the S o amino acid sequence of the hIL-la precursor polypeptide as 15 determined by Gubler et al., J. Immunol. 136, 2492-2497 (1986) is shown in Fig. la/lb.

C CO European Patent Application, Publication No. 188 864 discloses that the C-terminal portion of the hIL-la S 20 precursor polypeptide extending from Ser at position 113 to a 00 j Ala at position 271 (Fig. la/lb) is biologically active.

i I European Patent Application, Publication No. 188 920 discloses a hIL-la precursor polypeptide which differs in the amino acid sequence from the amino acid sequence shown 040 00 25 in Fig. la/lb by having a Ser at position 114 instead of a Ala, which Ser is encoded by the codon TCA. European Patent S 0 Application No. 188 920 discloses further the preparation of various hIL-la polypeptides, namely the hIL-la polypeptides having an amino acid sequence extending from positions 63 to 271, 113 to 271, 115 to 271, 123 to 271, 127 to 271, 128 to 271, 129 to 271, 113 to 267, 113 to 264, 113 to 266, 113 to 265 and 128 to 267 of the amino acid sequence shown in Fig. la/lb, but wherein, as mentioned above, the alanine residue at position 114, where present, is replaced by a serine residue. European Patent Appliction, Publication Wa/22.11.88 2 No. 200 986 describes the preparation of a recombinant hIL-la polypeptide having an amino acid sequence extending from position 118 to 271 and having a methionine residue at position 117, which methionine is derived from the ATG codou used for expressing a DNA fragment encoding the amino acid sequence from position 118 to 271. Since the natural hIL-la polypeptide has a serine residue at position 117 the recombinant hIL-la polypeptide prepared as described in European Patent Appliction, Publication No. 200 986 differs at the N-terminus from a corresponding natural oooo mature hIL-la polypeptide isolated from a human body fluid o or from a supernatant of a cultured human cell. The presence of the modified N-terminus in the recombinant hIL-la eo o polypeptide might give rise to side effects in a patient 15 treated with said recombinant polypeptide. In order to avoid o, g such side effects ways and means were searched for methods S 00 for the preparation of recombinant mature hIL-la polypeptides which have an amino acid sequence which is 0000 O identical to the amino acid sequence of the natural mature 0foo 20 hIL-la. It was found that surprisingly the recombinant 0 00 mature hIL-la polypeptide having the amino acid sequence 0o0000 0 0 Ser Phe Leu Ser Asn Val Lys Tyr Asn Phe MET Arg Ile Ile Lys Tyr Glu Phe Ile Leu Asn Asp Ala Leu Asn Gln Ser Ile Ile Arg Ala Asn Asp Gin Tyr Leu Thr Ala Ala Ala Leu His Asn Leu Asp Glu Ala Val °o Lys Phe Asp MET Gly Ala Tyr Lys Ser Ser Lys Asp Asp Ala Lys Ile o o 25 Thr Val Ile Leu Arg Ile Ser Lys Thr Gln Leu Tyr Val Thr Ala Gln .aooS Asp Glu Asp Gln Pro Val Leu Leu Lys Glu MET Pro Glu Ile Pro Lys 0 Thr Ile Thr Gly Ser Glu Thr Asn Leu Leu Phe Phe Trp Glu Thr His Gly Thr Lys Asn Tyr Phe Thr Ser Val Ala His Pro Asn Leu Phe l e Ala Thr Lys Gln Asp Tyr Trp Val Cys Leu Ala Gly Glv Pro Pro Ser Ile Thr Asp Phe Gin Ile Leu Glu Asn Gln Ala (1) which amino acid sequence corresponds to the amino acid sequerce extending from position 117 to 271 of the amino acid sequence shown in Fig. la/lb and the recombinant mature hIL-la polypeptide having the amino acid sequence -3- Ser Asn Val Lys Tyr Asn Phe MET Arg Ile Ile Lys Tyr Glu Phe Ile Leu Asn Asp Ala Leu Asn Gin Ser Ile Ile Arg Ala Asn Asp Gin Tyr Leu Thr Ala Ala Ala Leu His Asn Leu Asp Glu Ala Val Lys Phe Asp MET Gly Ala Tyr Lys Ser Ser Lys Asp Asp Ala Lys Ile Thr Val Ile Leu Arg Ile Ser Lys Thr Gin Leu Tyr Val Thr Ala Gin Asp Glu Asp Gin Pro Val Leu Leu Lys Glu MET Pro Glu Ile Pro Lys Thr Ile ,T4, Gly Ser Glu Thr Asn Leu Leu Phe Phe Trp Glu Thr His Gly Thr Lys Asn Tyr Phe Thr Ser Val Ala His Pro Asn Leu Phe Ile Ala Thr Lys Gin Asp Tyr Trp Val Cys Leu Ala Gly Gly Pro Pro Ser Ile Thr Asp Phe Gin Ile Leu Glu Asn Gin Ala (II) which amino acid sequence corresponds to the amino acid sequence extending from position 120 to 271 of the amino acid sequence shown in Fig. la/lb can be produced in a unicellular organism without the N-terminal methionine residue.

SUMMARY OF THE INVENTION According to a first embodiment, the present invention provides homogeneous recombinant human interleukin-la polypeptides having the amino acid sequence or (II).

According to a second embodiment of this invention, there is provided a DNA coding for a human interleukin-la polypeptide as defined in the first embodiment.

According to a third embodiment of this invention, there is provided a recombinant vector containing a DNA according to the second 15 embodiment, which recombinant vector is capable of directing expression of said DNA in a compatible unicellular host organism.

According to a fourth embodiment of this invention, there is S' provided a unicellular organism containing a recombinant vector according to the third embodiment which unicellular organism is capable of expressing the DNA ancoding the human interleukin-la.

According to a fifth embodiment of this invention, there is provided a process for producing a human interleukin-la polypeptide as defined in the first embodiment comprising: culturing a unicellular organism according to the fourth embodiment under conditions suitable for the expression of the DNA encoding the human in";erleukin-la polypeptide; and isolating 'Ihe human Interleukin-la polypeptide from the culture and purifying it to homogeneity.

According to a sixth embodiment of this invention, there is provided a pharmaceutical composition comprising a human Interleukin-la r 'A4 SMM/583Z Y U1 3A polypeptide according to the first embodiment and a pharmaceutically acceptable carrier.

According to a seventh embodiment of this invention, there is provided use of a human interleukin-lo polypeptide according to the first embodiment for stimulating the immune system of a host subject, for the promotion of wound healing and for improving the recovery of critically ill, protein-malnourished patients.

According to an eighth embodiment of this invention, there is provided use of a human interleukin-la polypeptide according to the first embodiment for the preparation of a pharmaceutical composition according to the seventh embodiment.

DESCRIPTION OF THE INVENTION The polypeptides of the present invention may be produced by conventional methods of peptide synthesis in the liquid phase or, preferably, on the solid phase, such as the methods of Merrifield Am.

Chem. Soc. B3, 2149-2154 [1963]) or by other equivalent methods of the state of the art.

000 1 0 0 0 0 0 0 100 0 1 0* S 00 r o *0 LMM(883Z "'02r 4 Alternatively and preferably, the polypeptides of the present invention are produced using methods of the recombinant DNA technology. Thereby a unicellular organism containing a recombinant vector containing a DNA sequence coding for a mature human interleukin-la polypeptide of the present invention is cultured under conditions suitable for the expression of said DNA sequence and the human interleukin-la polypeptide produced by the unicellular organism is isolated from the culture.

Such unicellular organism may be a prokaryotic or a 0 eukaryotic cell. A large number of such unicellular organisms 0 0 0°o are commercially available or freely available from o° a depositories such as the American Type Culture Collection *o 15 (ATCC) located at 12301 Parklawn Drive, Rockville, Maryland, USA. Examples of prokaryotic cells are bacteria such as E.coli E.coli M15 described as DZ291 by Villarejo et al. in J. Bacteriol. 120, 466-474 [1974], E.coli 294 (ATCC No. 31446). E.coli RR1 (ATCC No. 31343) or E.coli W3110 (ATCC No. 27325)], bacilli such as B.subtilis and enterobactericeae among which can be mentioned as examples Salmonella oo' typhimurium and Serratia marcescens. Especially preferred is E.coli s.train MC1061 containing the plasmid pRK248cIts (see Example). Alternatively yeast cells such as Saccharomyces 25 cerevisiae may be used as host organisms. A large number of eukaryotic cells suitable as host organisms for recombinant vectors are known to the man skilled in the art. Mammalian cells such as CV-1 (ATCC No. CCL 70) and derivatives thereof such as COS-1 (ATCC No. CRL 1650) or COS-7 (ATCC No. CRL 1651) are preferably used. In addition, it is possible to use insect cells such as described by Smith et al. (Mol. Cell.

Biol. 2, 2156-2165 (1983]).

Various methods for introducing recombinant vectors into unicellular organisms a.a known. Examples for such methods i are microinjection, transfectioti or transduction. The man skilled in the art has no difficulty to select the most .4 5 suitable method for the specific host organism used.

The recombinant vectors used in the present invention are vectors containing a DNA coding for the hIL-lo polypeptide of the present invention such as the DNAs having the nucleotide sequence from position 385 to position 852 or from position 394 to position 852 of the nucleotide sequence shown in Fig. la/lb. Such a DNA may be synthesized by conventional chemical methods, e.g. by the phosphotriester method which is described by Narang et al. in Meth. Enzymol. 68, 90-108 woCo [1979], or by the phosphodiester method (Brown et al., Moth.

o 0 Enzymol. 68, 109-151 [1979]). In both methods long oO 9 doo l oligonucleotides are first synthesized and then joined o together in a predetermined way. The nucleotide sequence c2 S° 0 15 the DNA may be identical to the nucleotide sequences g mentioned above or may be partially or completely different.

0 0 This is due to the fact that the genetic code is degenerate, that means that one amino acid may be coded by several codons. The todons selected may be adapted to the preferred 20 codon usage of the host organism used to express the hIL-la polypeptide (Grosjean et al., Gene 18, 199-209 [1982]). Care omust be taken that the DNA obtained in this way does not contain partial sequences which make the construction of the recombinant vector difficult, e.g. by introducing an C 25 undesired restriction enzyme cleavage site, or which prevents the expression of the polypeptide.

The DNA coding for the hIL-la polypeptide of the present invention may also be prepared by isolating a DNA coding for the hIL-lI precursor polypeptide ftom a human cDNA library or a human genomic library according to procedures known in the art March et al., supra; Gubler et al., supra) and then tailoring the DNA to the desired size and nucleotide sequence using restriction endonucleases and possibly small synthetic oligonucleotides according to methods well known in the art of recombinant DNA technology.

1 64 A large numher of vectors such as those mentioned in European Patent Application, Publication No. 200 986 may be used for constructing the recombinant vectors mentioned above. These vectors contain elements necessary for transcription and translation of the DNA coding for the hIL-la polypeptide as well as elements needed for the maintenance and replication of the vector in the host. The selection o'f a suitable vector for constructing the recombinant vectors of the present invention and the selection of a unicellular host organism compatible with said recombinant vectors is within the skills of an artisan in the field.

0 00 O"e 00 tto e Alternatively and preferably the recombinant vectors of 15 the present invention are prepared by modifying recombinant 0 Ott vectors containing a DNA coding for a hlL-lct polypeptide using the oligonucleotide directed site specific mutagenesis method described by Morinaga et al.. BIO/TECHNOLOGY 2.

636-639 (1984). An example for such a recombinant vector containing a vector and a DNA coding for a hlL-1Ic polypeptide is the plasmid phil #1-154*. The construction of this plasmid is described in European Patent Application.

Publication No. 200 986.

The manner in which the expression of the polypeptides in accordance with the invention is effected depends on the €1 expression vector and on the host organism used. Usually, the host organisms which contain the expression vector are grown up under conditions which are optimal for the growth of the host organism. Towards the end of the exponential growth, when the increase in the number of cells per unit time decreases, the expression of the polypeptide of the present invention is induced, i.e. the DNA coding for the polypeptide is transcribed and the transcribed mRNA is translated. The induction can be effected by adding an inducer or a derepressor to the growth medium or by altering a physical 44 it -7- 7 parameter, e.g. by a temperature change.

The polypeptide produced in the host organisms can be secreted from the cell by special transport mechanisms or can be isolated by breaking open the cell. The cell can be broken open by mechanical (Charm et al., Meth. Enzymol. 22, 476-556 [1971]), enzymatic (lysozyme treatment) or chemical (detergent treatment, urea or guanidine-HCl treatment, etc.) means or by a combination thereof.

S In eukaryotes, polypeptides which are secreted from the I cell are synthesized in the form of a precursor molecule. The mature polypeptide results by cleaving off the so-called j 00 signal peptide. As prokaryotic host organisms are not capable 15 of cleaving eukaryotic signal peptides from precursor *0 molecules, eukaryotic polypeptides must be expressed directly c a° in their mature form in prokaryotic host organisms. The translation start signal AUG, which corresponds to the codon 0,c ATG on the level of the DNA, causes that all polypeptides are c .0 20 synthesized in a prokaryotic host organism with a methionine a 0 0 S11 0 residue at the N-terminus. In certain cases, depending on the ooo expression system used and possibly depending on the I0 S polypeptide to be expressed this N-teminal methionine residue is cleaved off.

ac oo 0o The polypeptides in accordance with the present invention O000 0 0 Scan be purified to homogeneity by known methods such as, for f example, by centrifugation ac different velocities, by j~ precipitation with ammonium sulphate, by dialysis (at normal pressure or at reduced pressure), by preparative isoelectric focusing, by preparative gel electrophoresis or by various chromatographic methods such as gel filtration, high performance liquid chromatography (HPLC), ion exchange chromatography, reverse phase chromatography znd affinity chromatography on Sepharose" Blue CL.6B or on carrier-bound monoclonal antibodies directed against a i hIL-la polypeptide).

The purified recombinant hIL-la polypeptides of tihe present invention can be employed in a manner known per se to stimulate the immune system of a host subject, such as, for example, by improving host defense response to pathogens, by acting as a vaccine adjuvant and by enhancing host defense against neoplastic diseases. Other clinical uses identified for hIL-la in the art include promotion of wound healing via stimulation of fibroblast proliferation and improvement o. of the recovery of critically ill, protein-malnourished 0 0 0 0 0 patients.

000 0 0 S00 00 0° Homogeneous hIL-l polypeptides prepared in accordance 0 00 "o 15 with this invention may be administered to warm blooded o0 0 mammals for the clinical uses indicated above. The 0 administration may be by any conventional method such as by parenteral application either intravenously, subcutaneously 0Gt or intramuscularly. Obviously, the required dosage will vary a'0 20 with the particular condition being treated, the severity of 0 .a the condition, the duration of the treatment and the method 0°oo for administration. A suitable dosage form for pharmaceutical o 0 use may be obtained from sterile filtered, lyophilized hIL-1c polypeptide reconstituted prior to use in a S° 25 conventional manner. It is also within the skill of' the oa, artisan in the field to prepare pharmaceutical compositions containing a homogeneous human interleukin la of the present invention by mixing said interleukin-la with compatible pharmaceutically acceptable carrier materials such as buffers, stabilizers, bacteriostats and other excipients and additives conventionally employed in pharmaceutical parenteral dosage forms.

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r 9 Example Thle oligonucleotide-directed site specific mutagenesis method described by Morinaga et al., BIO/TECHNOLOGY 2, 636-639 (1984) was used to mutate plasmid phil 1-154* (European Patent Application, Publication No. 200 986) to produce genes encoding hIL-la polypeptides having an amino acid sequence extending from position 117 to 271, 119 to 271 and 120 to 271 of Fig. la/lb. To carry out these procedures, synthetic oligonucleotides having the nucleotide sequences ooo o GGA ATT AAT ATG AGC TTC CTG AGC 117), 9o 0 0 S, GGA ATT AAT ATO CTG AGC AAT GTG 119) and V00 9 ooo* GGA ATT AAT ATG AGC AAT GTG AAA 120) at t were produced by the phosphoramidita solid support method of Matteucci et al., J. Am. Chom. Soc. 103, 3185-3191 (1981).

Plasmid phil *l-154* was converted to the gapped form using BglII and Sall and to the linearized form using Pstl.

Gapped heteroduplexos were croated. In three separate reactions, the above oligonucleotides were annealed, converted into a double-stranded form and ligated, and transformed into E.coli K-12 strain MC1061 (Casadaban et al., J. Mol. Biol. 138, 179-207 E1980]) containing the plasmid pRK248cIts (Bernard et al., Meth. Enzym. 68, 482-492 [1979]) using the CaC12-Method (Maniatis et al., in "Molecular Cloning: A Laboratory Manual", pp. 250-251, Cold Spring Harbor Laboratory [1982]). E.coli MC1061 and the plasmid pRK248clts are available from the American Type Culture Collection, 12301 Packlawn Drive, Rockville, Maryland, USA under the accession numbers ATCC No. 53338 and ATCC No. 33766, respectively.

c- 1 10 Colonies containing the desired mutation were identified by hybridization as described by Maniatis et al. (supra, pp, 312-315). The same oligonucleotides used for primer-directed mutagenesis wern used as probes for the hybridizations after 32 5' end labeling with y- P-ATP using polynucleotide kinase according to the procedure of Maniatis et al., supra, p. 396.

Oligonucleotide 117) directed the conversion of phil #1-154* to pHuIl-la (117-271) with the following sequence modification: ~code for extra amino acids 118 119 120 121 *o 15 phil 41-154* ATGAATAGAATTCGGATCCGC TTC CTG AGC AAT Met Phe Leu Ser Asn 117 118 119 120 pHuIL--la (117-271) GGAATTAATATG AGC TTC CTG AGC 20 Met Ser Phe Leu Ser Similarly, oligonucleotide 119) created pHuIL-la (119-271) with the following sequence: 25 119 120 121 122 GGAATTAATATG CTG AGC AAT GTG Met Leu Ser Asn Val; and oligonucleotide 120) produced pHul-la (120-271) 4 30 with the following sequence: 120 121 122 123 GGAATTAATATG AGC AAT GTG AAA Met Set Asn Val Lys.

DNA sequence analysis confirmed the above mutations.

1..4 12. E. coli MC1061/pRK248cIts containing phil #1-154 (see European Patent Application, Publication Nr. 200 986, capable of expressing hIL-la (118-271)), pHuIL-la (117-271), pHuI-la (119-271) and prIulL-lcL (120-271) were grown in M9 media (Maniatis et al., supra, pp. 68-69) containing ampicillin at 30 0 C until the absorbance at 550 nm (A 55 0 550 reached 0,7, at which time the cultures were shifted to 42 0

C

for 3 hours. The bacterial cells from 1 ml of culture were collected by centrifugation and solubilized in 50 vl 7 M guanidine*HC1. The crude bacterial extracts were assayed for interleukin-1 bioactlvity using the murine thymocyte proliferation assay of Mizel et al., 3. Immunol. 1.20, 1497-1508 (1978). All four extracts were active.

Insoluble cytoplasmic inclusion bodies wore isolated from all four induced cultures, purified as described above, colubilized and subjocld to SDS polyacrylamide gel electrophoresis (Laemmli, Nature 227, 680-685 [1970]). The major Intocleukin-la band was eluted from each gel by the method of Aobersold at al., J. Biol. Cham. 261, 4229-4238 (1986) and subjected to N-terminal amino acid soquence analysis by the method of Hewick et al., J. Biol. Chem. 256.

7990-7997 (1981), with the following results after 10 cycles: hIL-la (1.17-271): Se Phe Leu Ser Asn Val Lys Tyr Aon Pho hIL-la (118-271): Met Phe Lou Ser Asn Val Lys Tyc Asn Phe hIL-la (119-271): Met Leu Ser Asn Val Lys Tyr Asn Phe Met hIt-la (120-271): Set Asn Val Lys Tyr Asn Phe Met Arg Ile 1-2

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12 It can readily be seen that the initiating methionine was cleaved from hIL-la (117-271) and hIL-la (120-271), but not from hIL-l (119-271) or hIL-la (118-271).

To further investigate the hIL-la polypeptides hIL-la (117-271), hIL-l (118-271) and hIL-l (120-271), purification of the supernatant fractions of lysates of MC1061/pRK248cIts tran.formants harboring pHulL-la (117-271), phil #1-154 and pHuIL-lc (120-271) was carried out as follows: 1. Cultures containing 5 kg of each transformant were adjusted to pH 1.8 with H2S04 and maintained at room s temperature for 30 minutes to I hour, and the acid-killed cells were recovered by centrifugation.

2. The acid-.killed cells were suspended in 18 liters of water and stirred for 1 hour at room temperature with a mechanical blade stirrer.

3. Suspended material was filtered through 2 e a double-layered cheese cloth sections to remove cell debris.

4. The filtered material was passed twice through a Manton-Gaulin cll homogenizer (Meth. Enzymol. 22, 482-484 ii7 [1971]) operated at 8,000 psi (approx. 5,5 x 107 Pa), first at 15 0 C, then at about 30 0

C.

The material was centrifuged in a Sorvall centrifuge (Du Pont, Wilmington, Delaware, USA) at 4 0 C for 1 hour at 8,000 rpm, using a GS-3 rotor (Du Pont). About 16.6 liters of supernatant fluid were recovered, and the pellet was discarded.

6. The supernatant fluid was filtered through a 0.8/0.2 micron Sartorius filter (available from Sartorius Filters Inc., Hayward, California, USA or from Sactorius-Membranfilter 13 13 GmbH, D-3400 Gottingen), after which the pH was adjusted to with a 50% sodium hydroxide solution.

7. The supernatant fluid was applied to a 10 x 40 cm SNugel P-SP silica column (a sulfopropyl cation exchanger; 40-60 micron, 200 Angstrom; Separation Industries, Metuchen.

New Jersey, USA) which had been equilibrated with 0.02 M acetic acid. After sample application, the column was washed with 31 liters of 0.02 M acetic acid and then eluted with 30 mM Tris-HCl, pH 8.0. Column effluent was monitored spectrophotometrically at 280 nm. Two peaks of protein were thus obtained a 4.5 liter volume first peak and a liter main peak.

,0006 15 8. The main peak from step 7 was applied to a 10 x 1 0 cm NuGei P-DE silica column (a diethylaminoethyl anion exchanger; 40-60 micron, 200 Angstrom; Separation Industries) which had been equilibrated with 30 mM Tris-HCl, pH 8.0. The C' C S column was washed with 17 liters of 30 mM Tris-HCl and 0.075 M NaCI, pH 8.0, and then eluted with 13 liters of 30 mM Tris-.HCl and 0.25 M NaCl, pH 8.0. The effluent was monitored for the presence of proteins spectrophotometrically at an optical density of 280 nm (O.D.280). An 0,D.

280 protein peak contained in a 13 liter volume was thereby obtained.

9. The eluate from step 8 was diluted 4 fold with 3.7 mM acetic acid, pH 4.8, adjusted to pH 4.8 and applied to G a 5 x 250 cm NuGel P-SP HPLC column (Separation Industries) at a flow rate of 150 ml/min. The column was then eluted with a linear gradient of from pH 4.8 (3.75 mM acetic acid) to pH 6.8 (10 mM K 2

HPO

4 over a 120 minute period, at a flow rate of 75 ml/min. A 9.3 liter O.D.

2 peak was thus 280 obtained.

10. The eluate from the HPLC column was concentrated to about 225 ml in an Amicon spiral cartridge (1,000 molecular weight cutoff, available from Amicon, Div. of W.R. Grace

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i 1 ;i r ucirai, 14 Co., Danvers, Massachusetts, USA) and then applied to two x 100 cm Sephadex G-50 columns (Pharmacia Fine Chemicals, Piscataway, New Jersey, USA) in series which had been equilibrated with 50 mM K2HPO 4 and 0.1 M NaCI, pH 6.8.

An 800 ml volume O.D.280 peak was obtained which was depyrogenated in an Amicon spiral cartridge (30,000 molecular weight cutoff). The final product was in a volume of about 1,070 ml.

Unless otherwise noted, all of the above purification steps were carried out at 4 0 C, except for the HPLC column which was run at room temperature.

The purified hIL-la (117-271), hIL-la (118-271) and 15 hIL-la (120-271) proteins were subjected to SDS polyacrylamide gel electrophoretic analysis as described above and stained with Coomassie blue. All were found to be in homogeneous form.

Bioactivity assays were carried out on the purified proteins using the murine D10 thymocyte proliferation assay of Kaye et al., J. Exp. Med. 158, 836-856 (1983), with the results shown in Table I.

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15 Table I Activity of hTL-la PolvDeDtides Soluble hIL-la in Crude Homogenatea (mq/Kq) Specific Activity of Purified Proteins __I(unLts/mq) Protein hIL-la (117-271) hIL-la (118-271) hIL-la (120-271) 1,800 600 600 2 x 2 x 109 2 x 10 9 S0o o a 1 o0 a o o 0 0 090 0 0 0 o o 4 0 a a o a The amount of soluble hIL-la in the crude homogenate was calculated based upon an immunoassay using hIL-la specific antibodies.

The data of Table I (taken together with the data 20 2 presented in Table I of European Patent Appliction, Publication No. 200 986) show that the exact length of the hIL-la protein is not critical, as long as the minimum carboxy-terminus sequence extending from position 132 to 271 (Fig. la/lb) is present in the molecule. All of the proteins had the same specific bioactivity. The data also show that the presence or absence of an N-terminal initiation methionine does not affect activity. The polypeptide hIL-la (118-271) comprising an additional methionine at the N-terminus was as active as the other proteins, both of which lacked such methionine.

Surprisingly, the data of Table I also show that the level of expression of the soluble hIL-.l (117-271) protein was three times the level of expression of the soluble hTL-.lo (118-271) and hIL-la (120-271) proteins, although the genes coding for the proteins were all in the same vector and host cell, and the cells were cultured and 1

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il, -16 harvo,.' in the same way.

The present invention will be more readily understood when considered in connection with the accompanying Figure la/lb which shows the nucleotide sequence and predicted amino acid sequence of human interleukin-cr cDNA.

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Claims (11)

1.7 WRAT IS Q1LAIMED- TS Th claims defining the Invention are as follows: 1. A homogeneous recombinant human interleukin-Lc polypeptide having the amino acid sequence Ser Giu As p Lys Thr Asp Thr Gly Ala Ile Phe Phe Gin Phe Val Giu Ile Thr Thr Thr Leu Ser Asn V41 Lys Tyr Ile Leu Asn Asp Ala Leu Tyr Leu Thr Ala Aia Ala Asp MET Gly Ala Tyr Lys Ile Leu Arg Ile Ser Lys Asp Gin Pro Vai Let: Leu Thr Giy Ser Giu Thr Asa Lys Asn Tyr Phe Thr Ser Lys Gin Asp Tyr Trp Val Asp Phe Gin Ile Let: Giu Asn Asn Let: Ser Thr Lys Let: Val1 Cys Asn Phe MET Gin Ser His Asn Ser Lys Gin Leu Git: MET Leu ?he Ala His Let: Ala Gin Ala Arg Ile Ile Ile Ile Arg Let: Asp Giu Asp Asp Ala Tyr Val Thr Pro Gi: Ile Phe Trp Giu Pro Asn Leu Gly Gly Pro Lys Ala Ala Lys Ala Pro Thr Phe Pro Ty r Asn Val Ile Gin Lys H is Ile Ser C IC CC Ser Leu L eu MET Let: Gin Gly Asn Gin Phe Asn Vai Lys Asn Asp Ala Thr Ala Ala Gly Ala Tyr Arg Ile Ser Pro Val Leu Ser Giu Thr Tyr P1-i Thr Asp Tyr Trp Gln'1J-te Leu Ty r Leu Ala Lys Lys Let: Asn Ser Val Giu Asn Phe MET Arg Ile Asn Gin Ser Ile Ile Leu His Asn Leu Asp Ser 5cr Lys Asp Asp Thr Gin Leu Tyr Val Lys Giu MET Pro Giu Let: Leu Phei Phei Trp Val Ala 1-is Pro Asn CYS Let: Ala Giy Gly Asn Gin Ala. Ile Lys Tyr Giu Arg Ala Asn Asp Git: Ala Vai Lys Ala Lys Ile Thr Thr Ala Gin Asp Ile Pro Lys Thr Giu Thr His Gly Let: Phe Ile Ala pro Pro ec Ile Phe Ile Gin Tyr Phe Asp Vai Ile Giu Asp Ile Thr Thr Lys Thr Lys Thr Asp

2. A homogeneous recombinant htiman interieukin-la polypeptide according to claim I. having the amino acid sequence Phe Git: Phe Asp Gin Lys Phe Thr Val Asp Giu Thr Ile Giy Thr Ala Thr Ile Thr Leu Ile Tyr As p Ile As p Thr Lys Lys Asp Ser Asa Val Let: Asa Asp Let: Thr Ala MET Gly Ala Leu Arg Ilie Gin Pro Val Gly 5cr Git: Asn Tyr Phe Gin Asp Tyr Phe Gin Ile Lys Ala Ala Ty r Ser Let: Thr Thr Trp Let: Tyr Asn Ph-e MET Arg Lcu Asn Gln cr Ile Ala Leu His Asn Let: Lys Ser Scr Lys Asp Lys Thr Gin Let: Tyr Lev Lys Git: MET Pro Asn Leu Let: Phe Phe Ser Val Ala His Pro Val Cys Lcu Ala Gly Giu Asn Gin Ala. Ile Ile Ile Arg Asp Giu Asp Ala Val Thr Gt: Ile Trp Git: Asn Let: Gly Pro Lys Tyr Ala Asn Ala Val Lys Ile Ala Gin Pro Lys Thr His Phe Ile Pro Ser

3. A DNA coding for a human intterleukin-ici polYpeptide as defined in claim 21 or 2. 18

4. A recombinant vector containing a DNA according to claim 3, which recombinant vector is capable of directing expression of said DNA in a compatible unicellular host organism. A unicellular organism containing a recombinant vector according to claim 4 which unicellular organism is capable of expressing the DNA encoding the human interleukin-la.

6. A process for producing a human interleukin-la polypeptide as defined in claim 1 or 2 comprising: culturing a unicellular organism according to claim 5 under conditions suitable for the expression of the DNA encoding the human interleukin-lc polypeptide; and isolating the human interleukin-la polypeptide from the culture and purifying it to homogeneity.

7. A pharmaceutical composition comprising a human Interleukin-la polypeptide according to claim t or 2 and a pharmaceutically acceptable carrier. Si

8. Use of a human Interleukin-la polypeptide according to claim 1 or 2 for stimulating the immune system of a host subject, for the promotion of wound healing and for improving the recovery of critically 11i, protein-malnourished patients.

9. Use of a human Interleukin-la polypeptide according to claim I or 2 for the preparation of a pharmaceutical composition according to the claim 8. A human interleukin-la polypeptide according to claim 1 or 2 S, 25 prepared by a process according to claim 6.

11. A process for producing a human interleukin-la polypeptide as defined in claim 1, which process is substantially as hereinbefore described with reference to the Example. *12. The human interleukin-la polypeptide product when produced by the method of claim 11. L /583Z T -19-

13. A phartaceutical composition for stimulating the immune system of a host subject, for the promotion of wound healing in a subject, and for improving the recovery of a critically ill, protein-malnourished patient, which composition comprises an amount effective for stimulating the immune system of the host subject, for promoting wound healing and for improving the recovery of the critically ill, protein-malnourished patient of the human interleukin-lcx polypeptide of claim 12 together with a pharmaceutically arrpntable carrier, diluent, excipient and/or adjuvant.

14. A method for stimulating the immune system of a host subject, for the promotion of wound healing in a subject, and for improving the recovery of a critically ill, protein-malnourished patient, which method comprises administering to a subject or patient in need of such treatment, an amount effective for stimulating the immune system of the host subject, for promoting wound healing and for improving the recovery of the critically ill, protein-malnourished patient of the human Interleukin-l( polypeptide of claim 12, or the composition of claim 13. 04 15. A recombinant vector for the expression of a human interleukin-la polypeptide as defined in claim 1, which vector is substantially as hereinbefore described with reference to the Example, o0 16. A unicellular organism harbouring the vector of claim 15 and capable of expressing said human interleukln-l polypeptide, which organism is substantially as hereinbefore described with reference to the Example. DATED this TWENTY-SIXTH day of JULY 1991 F Hoffmann-La Roche Co Aktiengesellschaft Patent Attorneys for the Applicant SPRUSON FERGUSON kt,~ tN/ Fig. la 1 CAT TTC ATT GGC GTT TGA GTC AGC AAA GAA GTC MAG 0 *00 ~00Q I, C 0 4 I 40 C 44 00 0 04 0 40 4 4 00 4 4 0 04 004400 o t) 00 40 0 ~0 0 0 00 o 1 00 0 0 C' ATG ME T ATT I Ie CrC Lau A Lys MAG Lys CIG Lau CCT Pro TTT Phe GAT Asp CAT His ACA Thr GTT Val GAG Glu TT T P he GAA Glu CAT H is GMA Glu TCC Sen CTG Leu GC C AlIa AGC Sen G AC Asp 1.0 CTG Leu GGC Gly MAG Lys MAG Lys AT C Ie 100 TTC Phe CTG Leu TCT Ser T GC Cys CTT Lou MAG Lys GC C Ala CTG Lau MAC Asn MAT Asn GAT Asp TTC P he CGG Arg GAC Asp AMT Asn T GT Cys CAG Gin CAA G In MAG Lys TTG L eu TCA S or GTG ValI TA C Tyr A Lys TCT Ser GAG Glu AGT Sen GAG Glu A L ys AGT Ser TCC Sen GTG Val AGC S er TTA Lou QAA Glu TAC Tyr GMA Glu TC P he T CT S er AT G MET AGO Ser GAA Glu MAC Asn AT G MET GAA Glu CAT His AGT Sen GTA Val1 TCC Sen AT C Ile ATG MET GC C Ala GMA Giu GTA Val ATC Ie GTA Val ATC Ile MAG Lys 110 AGG Arg GTT Val1 AGT Sen TAT Tyr GMA Glu ACC T hr GAT Asp AGG Ar g AT C tIe 130 CCA Pro TC Sen GGC Gly ACC Thn MAC Asn GAT Asp TCA S er A Lys GAC Asp TCC Ser CCA Prno T CT Sen GGG Gly GAC Asp GCA Ala TAC T yr Fig. lb GMA TTC ATC CTG MAT GAC GCC Giu Phe Ile Leu Asn Asp Ala CT C Leu 140 MAT CAA AGT ATA ATT CGA GCC MAT GAT Asn Gin Ser Ilie f~e Arg Ala Asn Asp C .4 4 *4 4 44 4 4444 4 4 4 4* 44 44 44 44 4444 44(444 00 42 444 (444 444 44 444 42 TAC CTC ACG GCT GCT GCA TTA CAT MAT Tyr Leu Thr Ala Aia Ala Lau His Asn GAT GAA GCA GTG AAA TTT GAC ATG Asp Giu Ala Val Lys Phe Asp MET GGT GCT Gly Ala 170 TAT MAG TCA TCA MAG GAT GAT GCT A Tyr Lys Ser Ser Lys Asp Asp Ala Lys ACC (3TG ATT CTA AGA ATC Thr Val Ilie Leu Arg Ile TCA AAA ACT Scr Lys Thr TTG TAT GTG ACT GCC CMA GAT GMA GAG Lau Tyr Val Thr Ala Gin Asp Giu Asp CCA GTd CTG CTG Pro Val Leu Leu ACC MAC CTC CTC Thr As~n Leu Leu 220 MAG GAG ATG CCT GAG Lys Glu MET Pro Glu CCC MAA ACC ATC ACA GGT AGT GAG Pro Lys Thr Ilie Thr Gly Ser Giu TTC FTC TGG GMA ACT GAG GGC Phe Phe Trp Giu Thr His Gly MAG AAC TAT TTC ACA TCA OTT GCC CAT Lys Asn Tyr Phe Thr Ser Val Ala His MAC TTG TTT ATT GCC ACA MAG CMA GAG Asn Leu Phe Ilie Ala Thr Lys Gin Asp TGG GTG TG TTG GCA GGG GGG CCA Trp Val Gys Leu Ala Giy Gly Pro CCC TCT Pro 5cr 260 ATC ACT GAG TTT GAG ATA CTG GMA MG Ile Thr Asp Phe Gin Ilie Leu Glu Asn GAG GCO TAG GTC Gin Ala 270 TOG AGT CTC