MXPA98008964A - Polypeptides processed with acativity il-16, processes for its production and its - Google Patents

Abstract

A nucleic acid, which can be used to express a polypeptide with interleukin-16 activity in a prokaryotic or eukaryotic host cell wherein the nucleic acid encodes a polypeptide with the amino acid sequence SEQ ID NO: 2 or a SEQ ID NO: 2 N-terminally elongated by an aspartic acid residue or a C-terminally successful form for up to 8 amino acids, it is convenient for the production of an IL-16 polypeptide.

Description

POLYPEPTIDES PROCESSED WITH ACTIVITY IL-16, PROCESSES FOR YOUR PRODUCTION AND ITS USE.

The invention concerns polypeptides with IL-16 activity, processes for their production and their use. The invention describes IL-16 processed with high activity.

IL-16 (interleukin-16) is a lymphocyte which is also referred to as chemo attraction or lymphocyte chemical attraction factor (LCF) or immunodeficiency virus suppression lymphocyte (ISL). IL-16 and its properties are described in WO 94/28134 and WO 96/31607 and by Crui Shank, W.W. et al., Proc. Nati Acad. Sci. USA 91 (1994) 5109-5113 and by Baier, M., et al., Nature 378 (1995) 563. The recombinant production of IL-16 is also described in these references. In accordance with this IL-16 is a protein with a molecular mass of 13,385 D. Cruikshank also finds that the LSI elutes in a molecular-mesh chromatography as a multimeric form with a molecular weight of 50-60 and 55-60 kD. The activity of chemo attraction or chemical attraction has been attributed to this multimeric form which is a cationic homotetramer (product information AMS Biotechnology Ltd., Europe, Cat. No. 11177186). It is described EF: 28535 by Baier, a homodimeric form of IL-16 with a molecular weight of 28 kü. However, the chemo attraction or chemical attraction activity described by Cruikshank et al. in J. Immunol. 146 (1991) 2989-2934 and the activity of the human recombinant IL-16 described by Baire is very small.

The object of the present invention is to improve the activity of IL-lβ and to provide IL-16 forms which have a low immunogenicity and are advantageously suitable for a therapeutic application.

The object of the invention is achieved by a nucleic acid which can be used to express a polypeptide with interleukin-16 activity in a prokaryotic or eukaryotic host cell wherein said nucleic acid a) corresponds to the DNA sequence of SEC. ID NO: l or DNA elongated to the 5 'end by an aspartic acid codon (GAC), or its complementary strands. b) generate hybridization under stringent conditions with the DNA of the SEC. ID No: l or with a DNA which is elongated to the 5 'end by an aspartic acid codon. c) or is a nucleic acid sequence which could be hybridized under stringent conditions to the nucleic acid sequences defined by a) or b) without the degeneracy of the genetic code. d) and to the 5 'end codes by one of the amino acid sequences SEQ. ID NO: 7 to 10 or by the analogous sequences which are N-terminally elongated by an aspartic acid.

Such a nucleic acid code preferably codes for a polypeptide with the amino acid sequence SEQ ID NO: 2 or for a polypeptide with a sequence which, compared to SEQ ID NO: 2, is N-terminally elongated by an aspartic acid codon. In a further preferred embodiment, the nucleic acid codes for a polypeptide with IL-16 activity are shortened with up to 8 amino acids to the C-terminus.

The nucleic acid code for a polypeptide processed with IL-16 activity, particularly and preferably natural IL-16 from primates such as human IL-16 or IL-16 from a species of monkey or other mammal such as the mouse.

It has surprisingly been found that Figure 2 of WO 94/28134 does not disclose properly processed IL-16. The initial codon "ATG" of the precursor form of the protein is not with nucleotide 783 but preferably with nucleotides 54 or 174. This reading structure results when an A is inserted after nucleotide 156, a C is inserted after nucleotide 398 and a G is inserted after nucleotide 780. The sequences also show additional differences to Figure 2 of WO 94/28134. These are, for example, nucleotide substitutions (313 G in A, 717 C in A). IL-16 is processed during expression in eukaryotic cells. In this way, a polypeptide according to SEQ ID NO: 2 and / or a polypeptide with a sequence that is N-terminally elongated is compared to SEQ ID NO: 2 by an aspartic acid codon. The knowledge of the processed IL-16 increases the production of IL-16 and its derivatives with high activity and low immunodeficiency.

The sequence of IL-16 may differ by a certain extent from the sequences of proteins encoded by such DNA sequences. Such sequence variations can be amino acid substitutions, deletions or additions. However, the amino acid sequence of IL-16 is preferably at least 75% and particularly preferably at least 90% identical to the amino acid sequence of SEQ ID NO: 2. Variants of the amino and amino acid sequence of SEQ ID NO: 1 / SEQ ID NO: 2 are for example described in WO 96/31607 and in International Patent Applications PCT / EP96 / 05662 and PCT / EP96 / 05661. However, it is essential that the polypeptides have a correct N-term. Consequently, proteins are preferred in which the first three to ten amino acids of the N-terminus are unchanged or are unchanged and furthermore are N-terminally with the amino acid sequences SEQ ID NO: 6 to 8 or with analogous sequences which they are N-terminally extended by a residue of aspartic acid. The also preferred proteins are those which are shortened to the C-terminus by up to 8 amino acids.

Nucleic acids within the meaning of the invention are understood, for example, with DNA, RNA and nucleic acid analogues and derivatives. Preferred nucleic acid analogs are those compounds in which the main column of sugar phosphate is replaced by other units such as for example amino acids. Such compounds are preferred as PNA and are described in WO 92/20702. Since the PNA-DNA bonds are for example stronger than the DNA-DNA bonds, the stringent conditions described above are not applicable for PNA-DNA hybridization. However, suitable hybridization conditions are described in WO 92/20703.

The term "IL-16" is understood within the meaning of the invention as a polypeptide with the activity of IL-16. IL-16 preferably exhibits the action established in the test procedure described in WO 96/31607 or stimulates cell division according to WO 94/28134.

IL-16 binds to CD4 + lymphocytes and can suppress the replication of viruses such as for example HIV-1, HIV-2 and SIV. The function of IL-16 is not limited by its presentation in the MHC complex.

In particular, IL-16 exhibits one or more of the following properties: - binding to T cells via the CD4 receptor - stimulation of IL-2 receptor expression and / or antigen HLA-DR on CD + 4 lymphocytes, stimulation of helper cell proliferation or T-helpers in the presence of IL-2, - the suppression of the proliferation of helper or assistant T cells with anti-CD3 antibodies - the suppression of the replication of the viruses preferably of HIV-1, HIV-2 or SIV.

Preferred nucleic acids are those that generate hybrids with nucleic acids of SEQ ID NO: 1 under stringent conditions. The term "generate hybrids under stringent conditions" suggests that two nucleic acid fragments generate hybrids with others under standardized hybridization conditions as described for example in Sa brook et al., "Expression of genes cloned in E. coli" in Molecular Cloning : A laboratory manual (1989), Cold Spring Harbor Laboratory Press, New York, USA. Such conditions are for example hybridization in 6.0 x SSC at about 45 ° C followed by a washing step with 2 x SSC at 50 ° C. In order to select stringency, the concentration of the salt in the washing step can for example be chosen or selected from 2.0 x SSC at 50 ° C for low stringency and 0.2 x SSC at 50 ° C for high stringency. In addition, the temperature of the washing step can be varied between the ambient temperature, approximately 22 ° C, for low stringency and 65 ° C for high stringency.

IL-16 is preferably recombinantly produced in prokaryotic or eukaryotic host cells. Such production processes are described for example in WO 94/28134 and WO 96/31607 which are also for this purpose a subject of the description of the present invention. Nevertheless, in order to obtain the forms of conformance with the invention of IL-1β by recombinant production in a defined and reproducible manner, additional measures have been taken beyond the procedures for recombinant production, familiar to one skilled in the art.

The recombinant IL-16 can be produced by familiar methods by a person skilled in the art as a heterologous expression or as an homologous expression (after homologous recombination of the IL-16 nucleic acid in the genome of the host organism). For this a DNA is produced first, which is capable of producing a protein which has the activity of IL-16. The DNA is cloned into a vector which can be transferred into a host cell and can be replicated there. Such a vector contains regulatory elements additional to the sequence 1L-16 which are necessary for the expression of the DNA sequence. This vector which contains the IL-16 sequence and the regulatory elements is transferred into a vector which is capable of expressing IL-16 DNA. The host cell is cultured under conditions which are convenient for amplification of the vector and then isolate IL-16. In this process, adequate measures allow the protein to adopt an active tertiary structure in which it exhibits IL-16 properties.

The nucleic acid sequence of the protein can also be modified. Such modifications are for example: - modification of the nucleic acid in order to introduce several recognition sequences of the restriction enzymes to facilitate the steps of ligation, cloning and mutagenesis. - modification of the nucleic acid to incorporate the preferred codons for the host cell - extension of the nucleic acid by the additional operator elements in order to optimize the expression in the host cell.

The protein is preferably expressed in microorganisms in particular in prokaryotes and in this case in E. Coli. Expression in prokaryotes produces a non-glycosylated polypeptide.

The expression vectors may contain a promoter which allows the expression of the protein in the host organism. Such promoters are known to one skilled in the art and are for example the lac promoter (Chang et al., Nature 198 (1977) 1056), trp promoter (Goeddel et al., Nuc Acids Res. 8 (19'80) 4057), PL promoter (Shimatake et al., Nature 292 (1981) 128) and T5 promoter (U.S. Patent No. 4,689,406), Synthetic promoters such as for example the tac promoter (US Patent No., 4, 551, 433) are also suitable. Paired or coupled promoter systems are equally convenient as for example the T7-RNA polymerase / promoter system (Studier et al., J. Mol. Biol. 189 (1986) 113). Hybrid promoters composed of a bacteriophage promoter and the operator region of the microorganism are also convenient (EP-A 0 267 851). An effective binding site for the ribosome is necessary in addition to the promoter. In the case of E. Coli this ribosome binding site is referred to as the Shine-üalgarno (Sü) sequence (Sambrook et al., "Expression of cloned genes in E. coli" in Molecular Cloning: A laboratory manual (1989) Cold Spring Harbor Laboratory Press, New York, USA).

In order to improve expression, it is possible to express the protein as a fusion protein. In this case, a DNA sequence which encodes the N-terminal part of an endogenous bacterial protein or other stable protein is usually fused to the 5-terminus of the coding sequence for IL-16. Examples of these are for example lacZ (Phillips and Silhavy, Nature 344 (1990) 882-884), trpE (Yansura, Meth. Enzymol 185 (1990) 161-166).

After expression of the vector which is preferably a biologically functional plasmid or a viral vector, the fusion proteins are preferably split with enzymes (for example factor Xa) (Nagai et al., Nature 309 (1984) 810). Additional examples of the cleavage sites are the cleavage sites of the IgA protease (WO 91/11520, EP-A or 495 398), the ubiquitous cleavage site (Miller et al., Bio / Technology 7 (1989) 698). and the cleavage site of enterokinase.

The proteins expressed in this manner in the bacteria are obtained in the usual manner by disruption or disjunctive disruption of the bacteria and isolation of the protein.

In a further embodiment it is possible to secrete the proteins from the microorganisms as active proteins. For this a fusion product is preferably used which is composed of the sequence signal which is convenient for the secretion of the proteins in the host organisms used and the nucleic acid encoding the protein. In this case the protein is either secreted in the medium (in a gram-positive bacterium) or in the periplasmic space (in a gram-negative bacterium). It is appropriate to insert a cleavage site between the signal sequence and the sequence coding for IL-16 which allows the unfolding of the protein either during processing or in an additional step. Such signal sequences are derived from for example ompA (Ghrayeb et al., EMBO J. 3 (1984) 2437), phoA (Oka et al., Proc. Nati. Acad. Sci. USA 82 (1985) 7212) .

The vectors additionally contain terminators. Terminators are DNA sequences that signal the end of a transcription process. They are usually characterized by two structural features: a reversibly repeating C / G rich region which can form a double helix intramolecularly as well as a number of U (or T) residues. Examples are the main terminators in the DNA of the fd phases (Beck and Zink, Gene 16 (1981) 35-38) and rrnB (Brosius et al., J. Mol. Biol. 148 (1981) 107-127).

In addition to the expression vectors, they usually contain a selective marker in order to select the transformed cells. Such selective markers are, for example, the resistance genes for ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline (Davies et al., Ann.Rev. Microbiol., 32 (1978) 469). The selective markers which are equally convenient are the genes for the substances essential for the biosynthesis of substances necessary for the cell, such as, for example, histidine, tryptophan and leucine.

Numerous convenient bacterial vectors are known. Vectors, for example, have been described for the following bacteria: Bacillus subtilis (Palva et al., Proc. Nati, Acad. Sci. USA 79 (1982) 5582), E. coli (Aman et al., Gene 40 (1985 ) 183); Studier et al., J. Mol. Biol. 189 (1986) 113), Streptococcus cremoris (Powell et al., Environ. Microbiol. 54 (988) 655), Streptococcus lividans and Streptomyces lividans (U.S. Patent No. 4,747,056).

Additional genetic engineering methods for the production and expression of convenient vectors are described in J. Sambrook et al., Molecular cloning: a laboratory manual (1989), Cold Spring Harbor Laboratory Press, New York, N.Y.

In addition to prokaryotic microorganisms, it is also possible to express recombinant IL-16 in eukaryotes (such as for example CHO cells, yeast or insect cells). Yeast systems or insect cells are preferred as a system of eukaryotic expression. The expression in the yeasts can be carried out by means of three types of yeast vectors: (integrating YIP vectors (integrating plasmids of the yeasts), YRP replicating vectors (plasmids of yeast replication) and episomal YEP vectors (episomal plasmids of yeasts) More details of these are described in for example SM Kingsman et al. Tibtech 5 (1987) 53-57.

The invention further concerns a prokaryotic or eukaryotic host cell which is transformed or transfected with a nucleic acid which encodes an IL-16 polypeptide according to the invention in a form such that the host cell expresses the polypeptide. Such a host cell usually contains a biologically functional nucleic acid vector, preferably a DNA vector, a plasmid DNA, which contains this nucleic acid.

Further preferred is a monomeric IL-16 polypeptide which can not be split into additional subunits.

It has surprisingly been found that the nucleic acid and the IL-16 protein sequence described in WO 94/28134 does not correspond to the natural human sequences. It is known that in these is an unnatural IL-16 analogue. However, a protein preferably used for a therapeutic application which either identical to the natural protein or only differs slightly from the natural protein and at least exhibits comparable activity and immunogenicity. The sequence of the protein is described in SEQ ID NO: 2 (optionally with the N-terminal elongation by a residue of aspartic acid and / or the shortening to the C-term by up to 8 amino acids).

The nucleic acid sequence of IL-16 can, within the scope of the invention, contain deletions, mutations and additions. The monomeric form of IL-16 can be multimerized in a preferred embodiment. The activity of IL-16 can be increased in this way. Such multimeric forms are preferably dimeric, tetrameric or octameric forms.

In a further embodiment the polypeptides of the invention may additionally contain a defined amount of metal ions, the number of metal ions per subunits are preferably 0.5 to 2.

The numerous metal ions are suitable as metal ions within the meaning of the invention. It has been found that the ferrous alkali metals as well as the elements of the side groups are convenient. The ferrous alkaline metals, cobalt, zinc, selenium, manganese, nickel, copper, iron, magnesium, calcium, molybdenum and silver are particularly convenient. The ions can be monovalent, divalent, trivalent or tetravalent. The divalent ions are particularly preferred. The ions are preferably added as solutions of MgCl2, CaCl2, MnCl2, BaCl2, LiCl2, Sr (N03) 2, Na2Mo04, AgCl2.

Such multimeric forms and forms of IL-16 containing metal ions are described in International Patent Application PCT / EP96 / 05661.

The polypeptide according to the invention can be produced by the culture of eukaryotic or prokaryotic host cells, which have been transformed or transfected with a nucleic acid sequence as claimed in claims 1 or 2 under the conditions of suitable nutrients and optionally isolated to the desired polypeptide. If one tries to produce the polypeptide in vivo in the context of a gene therapy treatment, the polypeptide is of course not isolated from the cell.

A further subject of the invention is a pharmaceutical composition which contains a polypeptide according to the invention in a specific amount and / or activity which is sufficient for a therapeutic application as well as optionally a pharmaceutically convenient adjuvant and / or carrier diluent.

The polypeptides according to the invention are especially suitable for the treatment of disease states which are caused by viral replication, in particular by retroviral replication and by in unomodulation. Such therapeutic applications are also described in WO 96/31607. It also describes the diagnosis of test procedures.

The polypeptides according to the invention can also be preferably used for immunosuppression. This immunosuppression is preferably achieved or carried out by an inhibition of the helper or helper function of the TH0 and / or TKi and / or TH2 cells. Since the polypeptides according to the invention are of therapeutic value in all diseases in which an immuno-regulatory component is postulated in the pathogenesis and in particular a hyperimmunity. The conditions can be treated by IL-16 in cardiology / angiology are for example conditions such as myocarditis, endocarditis and pericarditis, in pulmonology eg bronchitis, asthma, in autoimmune neuropenia hematologies and in transplant rejection, in gastroenterology of chronic gastritis, in endocrinology of diabetes mellitus type I, in nephrology glomerulonephritis, rheumatic diseases, diseases in ophthalmology, in neurology such as multiple sclerosis and eczema in dermatology. The polypeptides according to the invention can be used in particular for autoimmune diseases, allergies and to avoid rejection of transplants.

The invention also concerns the use of nucleic acids according to the invention within the context of gene therapy. Retroviral or non-viral vector systems are for example suitable vector systems for these.

In addition to the invention, a polyclonal antibody or anti-IL-16 monoclonal antibody or an immunoactive fragment thereof which binds the first 3-20 amino acids of SEQ ID NO: 2 or SEQ ID NO: 2 N- is concerned. terminally elongated by a residue of aspartic acid, as well as the processes for the production of such antibodies and their use for the determination of IL-16 and for the determination of viral infections in eukaryotic cells and in particular in mammalian cell material . Mammalian cells of viruses activated in particular with T cells can also be determined with IL-16. The production of such antibodies is carried out by immunization with a polypeptide according to the invention. The production of such an antibody is carried out in accordance with familiar procedures by one skilled in the art by immunization with an immunogen which contains the first 3-20 amino acids of SEQ ID NO: 2 or a SEQ ID NO: 2 N-terminally elongated by a residue of aspartic acid such as hapten. Subsequently the antibody can be isolated in a usual manner from the immunized mammal and optionally a monoclonal antibody can be produced.

The following examples and publications as well as the sequence protocol further clarify the protective scope of the invention which results from the patent claims. The methods described are thus well understood as the examples that still describe the object of the invention even after the modifications.

EXAMPLE 1 Cloning, expression and purification of IL-16 1. 1 RNA isolation 5 x 107 PMBC (from humans or monkeys) were cultured for 48 hours with 10 μg / ml concanavalin A and 180 U / ml IL-2. In order to prepare the RNA, the cells were washed once with PBS and subsequently the lysis or dissolution of the cells with 5 ml of denaturation solution (RNA isolation kit, estratagen). The crippling or dissolution of the cells was kept on ice for 15 minutes after the addition of 1 ml of NA acetate, 5 ml of phenol and 1 ml of chloroform / isoamyl alcohol (24: 1). The aqueous phase was subsequently mixed with 6 ml of isopropanol in order to precipitate the RNA and stored for 2 hours at -20 ° C. The precipitate was finally washed once with pure ethanol and dissolved in 150 μl of H20. The yield was determined photometrically and was 120 μg. 1. 2 cDNA Synthesis The mixture for the cDNA synthesis contained 10 μg of RNA, 0.2 μg of oligo-dT, 13 mM of DTT and 5 μl of volume of the first strand of the reaction mixture (cDNA synthesis kit). the first strand, Pharmacia) in an amount of 15 μl. The mixture was incubated for 1 hour at 37 ° C and subsequently stored at -20 ° C for later use.

Amplification, cloning of cDNA IL-16 and production of an expression clone was carried out or performed as described in WO 94/28134 or WO 96/31607 taking into account the modified sequences. 1. 3 10 1 fermentation of an E. coli expression clone for IL-16 and high-pressure disjunctive disruption or disruption The precultures were taken from stacks of cultures (greased substance in a plate or ampules stored at -20 ° C) which are incubated at 37 ° C while shaking. The volume of inoculation in the following higher dimensions is 1-10% volume in each case). Ampicillin (50-100 mg / l) is used in preculture and main culture for selection against less plasmid.

The enzymatically digested proteins and / or yeast extracts as a source N and C as well as glycerol and / or glucose as an additional C source are used as nutrients. The medium is buffered to pH 7 and the metal salts are added at physiologically tolerated concentrations to stabilize the fermentation process. The fermentation is carried out as a batch feeder with a mixed yeast extract / C source dosages. The fermentation temperature is 25-37 ° C. The partially dissolved oxygen pressure (p02) is maintained approximately below 20% by means of the proportion of aeration, r.p.m, regulation and proportion of dosages. The growth is determined by the determination of the optical density (OD) at 528 nm. The expression of IL-16 is induced by means of IPTG. After a fermentation period of 10 to 20 hours the biomass is harvested or harvested by centrifugation to OD still stopped.

The biomass is taken up to 50 mM sodium phosphate, 5 mM EDTA, 100 mM NaCl, pH 7 and is disrupt or has disjunctive disruption at 1000 bar by means of a continuous pressure at high pressure. The suspension obtained in this manner is centrifuged again and the supernatant which contains the IL-16 is further processed. 1. 4 Purification of human recombinant IL-16 550 ml of the lysis supernatant or dissolution of the cell in 50 mM sodium phosphate, 5 mM EDTA, 100 mM NaCl, pH 7.2 are mixed with 55 ml of 5 M NaCl, 60 mM MgCl 2, pH 8.0, stirred for 30 minutes and subsequently centrifuged for 30 minutes at 20,000 g. 400 ml of the supernatant are taken in a nickel chelate Sepharose column (V = 60 ml, Pharmacia), which has been previously loaded with 30 μMol NiCl 2 / ml gel and equilibrated with 50 mM sodium phosphate, 0.2 M NaCl, pH 8.0. The column is subsequently washed with 300 ml of 50 mM sodium phosphate, 0.5 M NaCl, pH 7.0 and the IL-16 fusion protein is then eluted with a gradient from 0 M to 300 mM imidazole, pH 7.0 in 50 mM sodium phosphate, 0.1 M NaCl, pH 7.0 (volumes of 2 x 0.5 1 gradients). The fractions containing IL-16 were identified by means of SDS-PAGE, discharged and dialysed against 20 mM sodium phosphate, pH 7.0. 300 mg of the fusion protein obtained in this way was dialyzed or dissolved, at 4 ° C against 20 1 of 20 mM imidazole, pH 5.5 and subsequently centrifuged for 30 minutes at 20,000 g in order to eliminate turbosities. The supernatant from the centrifugation was subsequently adjusted to pH 8.5 with NaOH, mixed with 0.3 mg of thrombin (Boehringer Mannheim GmbH) and incubated for 4 hours at 37 ° C.

Subsequently, the split mixture was adjusted to pll 6.5 with HCl and the conductivity was set at 1.7 S by dilution with H20. The samples were applied to a FF column of Q-Sepharose (45 ml, Pharmacia) which had previously been equilibrated with 20 M of i idazole, pH 6.5. IL-16 was identified by SDS-PAGE and poured. The identity of IL-16 was confirmed by means of mass analysis (molecular weight 13.566 ± 3D) and automated analysis of the N-terminal sequence. The UV absorption of IL-16 at 280 nm and a calculated molar extinction coefficient of 5540 M "1 c" 1 at its wavelength (Mack et al. (1992) Analyt, Biochem 200, 74-80) used to determine the concentration.

In order to obtain the desired N-terminus it is optionally re-split with an aminopeptidase (for example hydrolase of a-aminoacyl peptide) or dipeptidyl peptidase (for example cathepsin CCATH).

IL-16 obtained in this manner has a purity of more than 95% in SDS-PAGE under reduced conditions.

A 4 x 180 mm column of Vidac, Protein & Petptido C18, to analyze the purity by means of RP-HPLC. It was eluted with a linear gradient from 0% to 80% B (solvent B: 90% acetonitrile in 0.1% TFA, solvent A: 0.1% TFA in H20) within 30 minutes at a flow rate of 1 ml / min . The detection was at 220 nm.

Example 2 Production of shortened IL-16 using an enterokinase cleavage site 2. 1 Expression Clone The amplification and cloning of IL-16 cDNA and the preparation of an expression clone were performed as described in WO 94/28134 or WO 96/31607 taking into account the modified sequences.

An oligonucleotide having the sequence SEQ ID NO: 3 is used as a first principle which contains the EcoRI site, 6 His and an enterokinase cleavage site (D4K): cccqaattc tatg cat falls falls falls falls qatqacqac acaaa-tctqcaqcctcaqcctctqc EcoRI H6 D4K An oligonucleotide that has the sequence SEQ ID NO: 6 which also contains an EcoRi site, 6His and an enterokinase cleavage site (D4K) is used as a first principle for the sequence elongated to end 5 by an aspartic acid codon: cccgaattc tatg cat cae cae cae falls cae falls gatgacqac acaaa-tctqcaqccteaqcctctqc EcoRI H6 D4K Any of the oligonucleotides IL-16-R? (SEQ ID NO: 4): IL-16-Rj .: gcg gat cea age tta gga gtc tec age age tgt g or the oligonucleotides IL-16-R2 (SEQ ID NO: 5) are used as the first reverse: IL-16-R2: gcg gat cea age tta ttc ctt gga ctg gag gct ttt te The first two backs contain BamHI and HindIII cleavage sites for cloning.

A sequence is obtained with IL-16-R ?, which codes for a protein in accordance with SEC ID NO: 2 A sequence is obtained with IL-I6-R1 which codes for an IL-16 shortened by 8 amino acids to the C-terminus. This C-term IL-16 is also active.

The PCR reaction, cloning and preparation of the expression clone (fusion protein with a poly-His N-terminal part for purification) are carried out in accordance with the standard conditions: a mixture of 0.2 mM dNTP, 1 mol / μl each of the first beginnings and reverse, 1 x high fidelity buffer (Boehringer Mannheim, D), 1.5 Mm MgCl2, 2.6 U of high fidelity enzyme mixture (Boehringer Mannheim, D ). μl final instrument volume: Perkin Elmer GeneAmp 9600 Course of the reaction: 3 minutes 94 ° C, 1 min 56 ° C, 2 min 72 ° C, then 25 cycles (20 sec 94 ° C, 20 sec 56 ° C, 1 min 72 ° C). 2. 2 Fermentation The fermentation is carried out analogously to example 1.3. 2. 3 Puri ication and splitting. 700 ML of lysis of the supernatant was mixed in 50 mM of 5 nM sodium phosphate EDTA, 100 mM NaCl, pH 7.2, with 70 ml of 5 M NaCl, 60 mM MgCl2, pH 8.0, agitated for 30 minutes and subsequently centrifuged for 30 minutes at 20,000 g. The centrifuged supernatant was applied to a nickel chelate column (V = 200 ml, Pharmacia) which was previously loaded with a solution of NiS04 (c = 10 mg / ml) and equilibrated with 50 mM sodium phosphate, 0.5 NaCl. M, pH. 8.0. The column was subsequently washed with equilibrium of the buffer to the base of the line (UV detection at 280 nm) reaching more. Next or later the column was rinsed with 1 1 of 50 mM sodium phosphate, 0.5 M NaCl, pH 7.0 and with 1 1 of 50 mM sodium phosphate, 1 M NaCl, pH 7.0. The fusion protein was eluted with a gradient of 0-300 mM imidazole, pH 7.0 in a solution of 50 mM sodium phosphate, 0.1 M NaCl, pH 7.0 (1 1 2 x 1.6 = fractions containing IL-16). were identified by means of SDS-PAGE and poured in. This IL-16 poured was concentrated in a Provario (Filtron, omega 5K membrane) at a concentration of 5 mg protein / ml and dialyzed against 50 M Tris, pH 8.0.

An equivalent of the spill containing 100 mg of the fusion protein was diluted with 50 mM Tris, pH 8.0 at a protein concentration of c = 1 mg / ml for the cleavage of the enterokinase. After the addition of 33 μg of enterokinase (Boehringer Mannheim; 1: 3000 p / p) the split mixture was incubated overnight (14 hours) at 37 ° C. Subsequently, the pH value was adjusted to pH 6.5 with HCl. Unfolded IL-16 was removed by stirring in 20 ml of nickel chelate sepharose (for the preparation see above, bonding time 2 hours) and subsequent centrifugation (10,000 g) or by suction filtration of the supernatant on a filter melted. The identity of the cleaved IL-16 contained in the supernatant was confirmed by the N-terminal sequence and the mass analysis. The purity was verified by SDS-PAGE and EP-HPLC (Vidac, diphenyl, 4.6 x 150 mm, linear gradient from 20% to 95% B in 45 minutes, solution A: 20 mM potassium phosphate in H20, pH 7.5; B: 100% acetonitrile.

EXAMPLE 3 Cleavage of recombinant IL-16 with cells diluted by lysis. 3. 1 Separation of CD8 + and CD4 + lymphocytes via MACS Lymphocytes isolated from a smooth envelope by means of Ficoll gradients are resuspended in 500 μl of PBS-azide / 1 x 108 cells (phosphate buffered saline without Ca2 + and Mg2 +, 0.01% sodium azide, 5 mM EDTA, pH 7.2 ). After the addition of 20 ml of CD8 micro cells / expected 1 x 107 cells (antibodies from anti-human CD8 mice, conjugated with magnetic particles, Miltenyi Biotec GmbH), they are incubated for 15 minutes at 4 ° C. 2 mg of DTAF / 1 x 107 of expected cells (anti-mouse conjugated FITC IgG, Dianova Company) are added for 5 more minutes at 4 ° C. After dilution with 25 ml of PBS-azide / 1% BSA it is again centrifuged (10 min, 1200 rpm, 4 ° C). The supernatant is discharged, the cells are resuspended in 2 ml of PBS / 1% BSA and the suspension of the cells is applied to a column which is located in a magnetic separator (Miltenyi Biotec GmbH). The CD8 + cells to which the CD8 microcars are coupled are retained in the column, in addition, the fraction of the flow contains all the lymphocytes (approximately 80% of CD4 + cells) except the CD8 + cells. After washing the column is taken from the hole and the fraction of the CD8 + cell is eluted with PBS-azide / 1% BSA. The fraction of the flow and the fraction of the CD8 + cell are centrifuged, resuspended in a cell culture medium (RPMI 1649, 20% FCS, 2 mM glutamine, 180 U / ml IL-2), the cell count is adjusted at 3 x 10 6 cells / ml and the cells are stimulated with PHA (9 mg / ml). The quality of the separation of the lymphocyte subpopulation is verified by means of FACS. 3. 2 Preparation of lysis or dilution of cells and digestion of IL-16 The 1 x 108 CD8 + lymphocytes which have been stimulated for three days with PHA are crippled or diluted for 10 minutes on ice in 2 ml of PBS which is mixed with 1% Triton x 100. Then, the remains of cells and nuclei Cells are eliminated by centrifugation. 45 μl of the dissolution or lysis cell obtained in this manner is incubated for 16 hours at room temperature with 10 μg of recombinant IL-16 which is expressed either to the amino-terminal or carboxy-terminal in fusion with a target or histidine blank (HIS blank, for example six histidines with cleavage sites from the example of WO 94/28134). Then the fragments taken from the HiS objective are purified with the help of a nickel agarose matrix. After further purification of the fragments that are formed in the HPLC, the masses of the fragments are determined by mass spectrography and the size of the N-terminal fragment is determined thereby. In this way, a naturally-identified processed IL-16 fragment is that which starts with the N-terminus described in SEQ ID NO: 1/2 or with an N-terminus elongated by an aspartic acid codon.

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USA 79 (1982) 5582 Phillips and Silhavy, Nature 344 (1990) 882-884 Powell et al., Appl. Environ. Microbiol. 54 (1988) 655 Sambrook et al., "Expression of Cloned Genes in E. coli 'in Molecular Cloning: A Laboratory Manual (1989), Cold Spring Harbor Laboratory Press, New York, USA Shimatake et al., Nature 292 (1981) ) 128 Studier et al., J. Mol. Bio. 189 (1986) 113 US-Patent No. 4,551,433 US-Patent No. 4,689,406 US-Patent No. 4,747,056 WO 91/11520 WO 92/20702 WO 92/20703 Yansura, Meth, Enzymol 18b (1990) 191-166 LIST OF SEQUENCES fl) GENERAL INFORMATION: (i) APPLICANT (A) NAME: Federal Republic of Germany, represented by the Ministry of Health (B) DIRECC. - (C) CITY: Bonn (E) COUNTRY: Germany (F) POSTAL CODE (2IP); D-53108 (A) OMBRE: BOEHRINGER MANNHEIM GMBH () ADDRESS: Sand ofer Str. 116 (C) CITY: Mannheim (E * COUNTRY: Germany (F> POSTAL CODE (ZIP): D-68305 (G) TELEPHONE: 08856 / 60-3446 (H) TELEFAX: 08856 / 60-3451 (ii) TITLE OF THE INVENTION: Processed polypeptides with IL-16 activity, processes for their production and use (iii) SEQUENCE NUMBER: 10 (lv) LEGIBLE FORMAT IN COMPUTER (A) TYPE OF MEDIUM: Hard Disk (Bj COMPUTER- IBM pC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Fatentln Relay # 1.0, Version # 1.30B (EPO) (vi) DATE OF PREVIOUS APPLICATION: (A) APPLICATION NUMBER: OF 196 17 202.0 (B) DATE OF SUBMISSION 30-A BR "" 1996 (v: ¡_) DATE OF PREVIOUS APPLICATION: (A) APPLICATION NUMBER: DE 196 17 203.9 (B) DATE OF PRESENTATION 30-APR-1996 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 366 base pairs (B) TYPE: nucleotide O (C) TYPE OF HEBRA: Double strand r (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: cDMA (ix) CHARACTERISTIC: (A) NAME / KEY: CDS (B) LOCATION: 1.. 366PTION OF THE SEQUENCE: SEQ ID NO: 1: TCT GCA GCC TCA GCC TCT GCA GCC AGT GAT GTT TCT GTA GAA TCT ACÁ 48 Ser Ala Ala Ser Ala Ser Ala Ala Ser Asp Val ser Val Glu Ser Thr 1 5 10 15 GCA GAG GCC ACA GTC TGC ACG GTG ACA CTG GAG AAG ATG TCG GCA GGG 96 Wing Glu Wing Thr Val Cys Thr Val Thr Leu Glu Lys Met Ser Wing Gly 20 25 30 G GGC TTC AGC CTG GAA GGA GGG AAG GGC TCC CTA CAC GGA GAC AAG 144 eu Gly Phe Ser Leu Glu Gly Gly Lys Gly Ser Leu His Gly Asp Lys 35 40 45 CCT CTC ACC ATT AAC AGG ATT TTC AAA GGA GCC GCC TCA GAA CAA AGT 192 Pro Leu Thr He Asn Arg He Phe Lys Gly Ala Ala Ser Glu Gln Ser 50 55 60 GAG ACÁ GTC CAG CCT GGA GAT GAA ATC TTG CAG CTG GGT GGC ACT GCC 240 Glu Thr Val Glp Pro Gly Asp Glu He Leu Gln Leu Gly Gly Thr Wing 65 70 75 30 ATG CAG GGC CTC ACA CGG TTT GAA GCC TGG AAC ATC ATC AAG GCA CTG 288 Met Gln Gly Leu Thr Arg Phe Glu Wing Trp Asn lie He Lys Ala Leu 85 90 95"CCT GAT GGA CCT GTC ACG ATT GTC ATC AGG AGA AAA AGC CTC CAG TCC 336 Pro Asp Gly Pro Val Thr He Val He Arg Arg Lys Ser Leu Gln Ser 100 105 110 AAG GAA ACC AC GCT GCT GGA GAC TCC TAG_366_Lys Glu Thr Thr Ala Wing Gly Asp Ser * 115 120 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 122 amino acids (B) UFO: amino acids (D) TOPOLOGY: linear (Ü) TYPE OF MOLECULE: protinin (XÍ) DESCRIPTION OF SEQUENCE: SEQ ID NO: 2 * Ser Ala Ala Ser Ala Ala Ala Ser Asp Val Ser Val Glu Ser Thr 1 5 10 15 Illa Glu Ala Thr Val Cys Thr Val Thr Leu Glu Lys Met Ser Ala Gly 20 25 30 Leu Gly Phß Ser Leu Glu Gly Gly Lys Gly Ser Leu His Gly Asp Lys 35 40 45 Pro Leu Thr He Asn Arg He Phe Lys Gly Wing Wing Ser Glu Gln Ser 50 55 60 Glu Thr Val Gln Pro Gly Asp Glu He Leu Gln Leu Gly Gly Thr Wing 65 70 75 80 Met Gln Gly Leu Thr Arg Phe Glu Wing Trp Asn He He Lys Wing Leu 85 90 95 Pro Asp Gly Pro Val Thr He Val He Arg Arg Lys Ser Leu Gln Ser 100 105 110 Lys Glu Thr Thr Ala Ala Gly Asp Ser * 115 120 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: () LENGTH: 66 base pairs (B) TYPE: nucleotide (C) TYPE OF FLEX: a single hebr a () TOPOLOGY: 1 ineal (ii) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc = -primary end " (Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: CCCGAATTCT ATGCATCACC ACCACCACCA CGATGACGAC GACAAATCTG CAGCCTCAGC 60 CTCTGC 66 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 base pairs (B) TYPE: nusleot id o (C) TYPE OF FLEECE: a single strand (D) TOPOLOGY linear (Ü) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / dess = "first back IL-16-R1" (xi) DESCRIPTION OF THE SEQUENCE SEQ ID NO: 4: GCGGATCCAA GCTTAGGAGT CTCCAGCAGC TGTG 34 (2) INFORMATION FOR THE S? Q ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 38 base pairs (B) TYPE: nucleotide (C) TYPE OF HEBRA: single strand (°) TOPOLOGY : linear (ii) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / d? SC »" first reverse I L- 16 -R2" (XÍ) DESCRIPTION OF THE SEQUENCE SEQ ID NO: 5: GCGGATCCAA GCTTATTCCT TGGACTGGAG GCTTTTTC 38 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 66 base pairs Cß) TYPE: nucleotide (C) TYPE OF HEBRA: single strand (D) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc m -primary end " (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: CCCGAATTCT ATGCATCACC ACCACCACCA CGATGACGAC GACAAAGACT CTGCAGCCTC 60 AGCCTC 66 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: single strand (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (XÍ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: Be Ala Ala Be Ala Be Ala 1 5 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 6 amino acids (B) TYPE: amino acids (C) TYPE OF HEBRA: single strand (D) TOPOLOGY: Linear ( ü) TYPE OF COLECULA: peptide (XÍ) DESCRIPTION OF THE SEQUENCE SEQ 10 NO: 8: Be Ala Ala Be Ala be i 5 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: single strand (D) TOPOLOGY: linear ( ü) TYPE OF MOLECULE: pepfldo (xi) SEQ ID NO: 9: Be Ala Ala Be Ala 1 5 (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: one single strand (D) TOPOLOGY: linear (Ü) MOLECULE TYPE: peptide (i) SEQ ID NO: 10; Ser Ala Ala Ser 1 It is noted that, in relation to this date, the best method known by the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following is claimed as property.

Claims (14)

1. The nucleic acid which can be used to express a polypeptide with interleukin-lβ activity in a prokaryotic or eukaryotic host cell, characterized in that said nucleic acid a) corresponds to the DNA sequence of SEC. ID N0: 1 or a DNA elongated to the 5 'end by an aspartic acid codon (GAC), or its complementary strands. b) generate hybridization under stringent conditions with the DNA of SEC. ID NO: l or with a DNA which is elongated to the 5 'end by an aspartic acid codon. c) or is a nucleic acid sequence which could generate hybridization under stringent conditions with the nucleic acid sequences defined by a) or b) without the degeneracy of the genetic code. d) and the 5 'end codes by one of the amino acid sequences SEQ. ID NO: 7 to 10 or by the analogous sequences which are N-terminally elongated by an aspartic acid.

2. The nucleic acid according to claim 1, characterized in that it is encoded for interleukin-16 from primates.

3. The prokaryotic or eukaryotic host cell characterized in that it is transformed or transfected with a nucleic acid according to claim 1 or 2 in a form such that the host cell expresses said polypeptide.

4. The biologically functional nucleic acid vector characterized in that it contains a nucleic acid according to claims 1 or 2.

5. Interleukin-16 from primates can be obtained as the product of a eukaryotic expression of a nucleic acid according to claim 1 or 2, characterized in that it is essentially free of other human proteins.

6. Human interleukin-16 can be obtained as a product of a eukaryotic expression of a nucleic acid according to claims 1 or 3 after processing, characterized in that it is free of other human proteins.

7. The polypeptide with interleukin-16 activity characterized in that it is encoded by a nucleic acid according to claims 1 or 2.

8. The polypeptide with interleukin-lβ activity according to claim 7, characterized in that it represents a multimer composed of a defined number of subunits the polypeptide of claim 7 is a subunit.

9. The polypeptide according to claim 8, characterized in that it is composed of 4 to 32 subunits.

10. The polypeptide according to claims 7 to 9, characterized in that it contains a defined amount of metal ions, the number of metal ions per subunit is 0.5 to 2.

11. Process for the production of a polypeptide according to claims 7 to 10, characterized in that a prokaryotic or eukaryotic cell is transformed or transfected with a nucleic acid sequence as claimed in claims 1 or 2 cultured under suitable nutrient conditions and in wherein the desired polypeptide is optionally isolated.

12. Pharmaceutical composition characterized in that it contains a polypeptide according to claims 5 to 10 as well as a pharmaceutically convenient diluent, adjuvant and / or carrier.

13. A pharmaceutical composition containing a polypeptide according to claims 5 to 10 in an amount suitable for a therapeutic application.

14. Process for the production of an antibody against a polypeptide with interleukin-1β activity, characterized in that a mammal is immunized with an immunogen or immune agent which contains the first 3-20 amino acids of SEQ ID NO: 2 or of a SEQ ID NO: 2 NO: 2 N-terminally elongated by a residue of aspartic acid as a hapten and wherein the antibody is subsequently isolated from the mammal.