Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/54—Interleukins [IL]
  • C07K14/5446—IL-16
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the invention concerns new IL-16 mutants with a high activity, processes for their production, and their use.
• IL-16 is a lymphokine which is also referred to as lymphocyte chemo- attracting factor (LCF) or immunodeficiency virus suppressing lymphokine (ISL).
• IL-16 and its properties are described in WO 94/28134 and in WO 96/31607 and by Cruikshank, W.W., et al., Proc. Natl. 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. According to these IL-16 is a protein with a molecular mass of 13,385 D.
• Cruikshank also found that ISL elutes in a molecular sieve chromatography as a multimeric form with a molecular weight of 50-60 and 55-60 kD.
• the chemoattractant activity has been attributed to this multimeric form which is a cationic homotetramer (product information AMS Biotechnology Ltd., Europe, Cat. No. 11177186).
• a homodimeric form of IL-16 with a molecular weight of 28 kD is described by Baier.
• the chemoattractant activity described by Cruikshank et al. in J. Immunol. 146 (1991) 2928-2934 and the activity of recombinant human IL-16 described by Baier are very small.
• the object of the present invention is to improve the activity of IL-16 and to provide IL-16 mutants which have, in addition, a low immunogenicity and are advantageously suitable for a therapeutic application.
• nucleic acid which can be used to express a polypeptide with interleukin-16 activity in a prokaryotic or eukaryotic host cell wherein the said nucleic acid codes for a polypeptide starting with amino acids 1 -5 and ending with amino acids 93-107 of the sequence SEQ ID NO:2.
• the IL-16 mutant consists of amino acids 1-100, 5-107, or, most preferably, of amino acids 5-93 of of SEQ ID NO:2. - 9 -
• Such a nucleic acid codes for an IL-16 mutant with improved activity. Its sequence is preferably based on the sequence of natural IL-16 from primates such as human IL-16 or IL-16 of an ape species or of another mammal such as the mouse.
• NMR and proteolytic data indicated that the N-terminal residues up to Glu 4 and the C- terminal residues starting from Lys 94 were disordered in solution and that these residues did not belong to the proper IL-16 structure.
• these residues lacked long- and medium-range NOEs and showed low values of heteronuclear 15N NOEs for backbone amides; features characteristic of highly flexible residues.
• Two shorter fragments, starting as SI to El 00 and from A5 to SI 07, show full chemoattractant activity and no change in structural properties of the 89 residue folded part in the four constructs. Therefore, the shortest fully folded and cytokine active IL-16 has a size of 89 amino acids; this corresponds to residues A5 - R93 of the IL-16 amino acid sequence SEQ ID NO:2.
• IL-16 The structure of IL-16 was primarily determined from the 3D lH- 13 C NOESY-HSQC spectra acquired on a double 13C/15N labelled IL-16. 1H, ⁇ N and ⁇ C chemical shift assignments were obtained using standard triple resonance NMR techniques using uniformly 15 N, 13 C/ 15 N labelled and selectively ⁇ N-Ala, 15 N-Gly/Ser, 15 N-Leu labelled samples of IL-16l"l j ⁇ ⁇ The tertiary structure of IL-16 was calculated by simulated annealing calculations from 689 experimental constraints, containing 588 interresidue distance constraints, 68 backbone dihedral angles and 21 ⁇ j side chain angles for the 89 residue folded part of the protein.
• the structure of IL-16 consists of a central up-and-down ⁇ -sandwich (formed by ⁇ -strands ⁇ l-B5) which is flanked by an ⁇ -helix (amino acids R71-A81).
• the N-terminal ⁇ -strand ( ⁇ l, T6-E13) makes an antiparallel ⁇ -sheet with the C-terminal ⁇ -strand B5 (V87-R93) and forms, together with the short ⁇ -strand B4 (I59-L62), one side of the ⁇ -barrel.
• the second antiparallel ⁇ -sheet B2- ⁇ 3, which is formed by the residues F21-24 and F37-141, contains a ⁇ -bulge in ⁇ - strand ⁇ 3 at residue 138.
• the two ⁇ -sheets, which build the ⁇ -sandwich, are packed against each other in a parallel manner, with a rotation of 39° degrees between a central axis of ⁇ - sheet B2 ⁇ 3 and a central axis of ⁇ -sheet ⁇ l B5.
• the main body core residues in IL-16 are all hydrophobic (138, F21, F73, W76, 189, 179, L23). Poorly defined regions in structures are localized at loops K14 to A 17, G28 to D32 and G44 to T52, where no NOEs to the core of the protein were found. This was also in agreement with 1 ⁇ N relaxation measurements which showed low values of the heteronuclear ⁇ N NOEs for these regions indicating increased flexibility of these fragments. Not all loops are flexible, however, the connecting loops between the ⁇ -helix and ⁇ -strand ⁇ 5, ⁇ -strand ⁇ 4 and the ⁇ -helix are well defined in the IL-16 structure.
• PDZ domains are intracellular protein modules that mediate clustering of ion channels, receptors and other membrane proteins and connect them to their appropriate signal transduction complexes (Ponting and Phillips, Trends in Biochem. Sci. 20 (1995) 102-103). PDZ domains are identified by the presence of a conserved GLGF sequence that is responsible for binding of defined peptide consensus sequences. For example, PDZ domains were found to bind carboxyl termini of several membrane proteins that possess a consensus sequence (Ser/Thr)Xaa-Val(COOH) (Songyang et al., Science 275 (1997) 73-77). IL-16 sequence also contains the GLGF motif.
• IL-16 might belong to the family of PDZ domains where the peptide binding capability is important for autoaggregation of tetrameric IL-16 or for clustering of receptors on the CD4 + surface (Rumsaeng et al., J. Immunol. 159 (1997) 2904-2910).
• the sequence of IL-16 can differ to a certain extent from protein sequences coded by such DNA sequences. Such sequence variations may preferably be amino acid substitutions. 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 parts of the amino and of the nucleic acid sequences SEQ ID NO: 1/SEQ ID NO: 2 are for example described in WO 96/31607 and the International Patent Applications PCT/EP96/05662 and PCT/EP96/05661. Proteins are also preferred which are shortened by up to 14 amino acids at the C-terminus of SEQ ID NO:2.
• Nucleic acids within the sense of the invention are understood for example as DNA, RNA and nucleic acid derivatives and analogues.
• Preferred nucleic acid analogues are those compounds in which the sugar phosphate backbone is replaced by other units such as e.g. amino acids.
• Such compounds are referred to as PNA and are described in WO 92/20702. Since PNA-DNA bonds are for example stronger than DNA-DNA bonds, the stringent conditions described below are not applicable to PNA-DNA hybridization. However, suitable hybridization conditions are described in WO 92/20703.
• IL-16 is understood within the sense of the invention as a polypeptide with the activity of IL-16. IL-16 preferably exhibits the stated action in the test procedure described in WO 96/31607 or stimulates cell division according to WO 94/28134. The activity of IL-16 is - 4 -
• 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.
• IL-16 exhibits one or several of the following properties:
• viruses preferably of HIV-1 , HIV-2 or SIV.
• Nucleic acids are preferred which hybridize under stringent conditions with nucleic acids which are complementary to nucleic acids of sequence SEQ ID NO:l and code for an IL-16 mutant according to the invention.
• hybridize under stringent conditions means that two nucleic acid fragments hybridize with one another under standardized hybridization conditions as described for example in 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. 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.
• the salt concentration in the washing step can for example be chosen between 2.0 x SSC at 50°C for low stringency and 0.2 x SSC at 50°C for high stringency.
• the temperature of the washing step can be varied between room temperature, ca. 22°C, for low stringency and 65 °C for high stringency.
• IL-16 is preferably produced recombinantly 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 matter of the disclosure of the present invention. However, in order to obtain the IL-16 mutants according to the invention by recombinant production in a defined and reproducible manner, additional measures have to be taken beyond the processes for recombinant production familiar to a person skilled in the art.
• Recombinant IL-16 which is essentially free of other human proteins, can be produced by methods familiar to a person skilled in the art as a heterologous expression or homologous expression (after homologous recombination of the IL-16 nucleic acid into the genome of the host organism).
• a DNA is firstly produced which is able to produce 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 in addition to the IL-16 sequence 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 able to express the DNA of IL-16.
• the host cell is cultured under conditions which are suitable for the amplification of the vector and IL-16 is isolated. In this process suitable measures ensure that the protein can adopt an active tertiary structure in which it exhibits IL- 16 properties.
• the IL-16 mutant according to the invention can occur in monomeric or oligomeric (e.g., tetrameric) form.
• a monomeric IL-16 polypeptide which cannot be cleaved into further subunits is preferred.
• nucleic acid sequence of the protein can also be modified. Such modifications are for example:
• the protein is preferably expressed in microorganisms in particular in prokaryotes and in this case in E. coli.
• the expression in prokaryotes yields an unglycosylated polypeptide.
• the expression vectors must contain a promoter which allows expression of the protein in the host organism.
• promoters are known to a person skilled in the art and are for example the lac promoter (Chang et al., Nature 198 (1977) 1056), tip promoter (Goeddel et al., Nuc. Acids Res. 8 (1980) 4057), ⁇ PL promoter (Shimatake et al., Nature 292 (1981) 128) and T5 promoter (US Patent No. 4,689,406).
• Synthetic promoters such as for example the tac - 6 -
• the protein in order to improve expression it is possible to express the protein as a fusion protein.
• a DNA sequence which codes for the N-terminal part of an endogenous bacterial protein or another stable protein is usually fused to the 5' end of the sequence coding for IL-16. Examples of this are for example lacZ (Phillips and Silhavy, Nature 344 (1990) 882-884), trpE (Yansura, Meth. Enzymol. 185 (1990) 161-166).
• the fusion proteins are preferably cleaved with enzymes (e.g. enterokinase or factor Xa) (Nagai et al., Nature 309 (1984) 810).
• enzymes e.g. enterokinase or factor Xa
• cleavage sites are the IgA protease cleavage site (WO 91/11520, EP-A 0 495 398) and the ubiquitin cleavage site (Miller et al., Bio/Technology 7 (1989) 698).
• the proteins expressed in this manner in bacteria are obtained in the usual manner by disrupting the bacteria and isolating the protein.
• a fusion product is preferably used which is composed of the signal sequence that is suitable for secretion of proteins in the host organisms used and of the nucleic acid that codes for the protein.
• the protein is either secreted into the medium (in gram-positive bacteria) or into the periplasmatic space (in gram-negative bacteria).
• a cleavage site between the signal sequence and the sequence coding for IL-16 which allows cleavage of the protein either during processing or in an additional step.
• signal sequences are derived for example from ompA (Ghrayeb et al., EMBO J. 3 (1984) 2437), phoA (Oka et al., Proc. Natl. Acad. Sci. USA 82 (1985) 7212).
• Terminators are DNA sequences that signal the end of a transcription process. They are usually characterized by two structural features: a reversely repetitive G/C-rich region which can form a double helix intramolecularly as well as a number of U (or T) residues. Examples are the main terminator in the DNA of the phages fd (Beck and Zink, Gene 16 (1981) 35-38) and rrnB (Brosius et al., J. Mol. Biol. 148 (1981) 107-127).
• the expression vectors usually contain a selectable marker in order to select transformed cells.
• selectable markers are for example the resistance genes for ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracyclin (Davies et al., Ann. Rev. Microbiol. 32 (1978) 469).
• Selectable markers which are equally suitable are the genes for essential substances for the biosynthesis of substances necessary for the cell such as e.g. histidine, tryptophan and leucine.
• Vectors have for example been described for the following bacteria: Bacillus subtilis (Palva et al., Proc. Natl. Acad. Sci. USA 79 (1982) 5582), E. coli (Aman et al., Gene 40 (1985) 183; Studier et al, J. Mol. Biol. 189 (1986) 1 13), Streptococcus cremoris (Powell et al., Appl. Environ. Microbiol. 54 (1988) 655), Streptococcus lividans and Streptomyces lividans (US Patent No. 4,747,056).
• yeast 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).
• eukaryotes such as for example CHO cells, yeast or insect cells.
• the yeast system or insect cells are preferred as a eukaryotic expression system.
• Expression in yeast can be achieved by means of three types of yeast vectors: (integrating YIp (yeast integrating plasmids) vectors, replicating YRp (yeast replicon plasmids) vectors and episomal YEp (yeast episomal plasmids) vectors. More details of this are described for example in S. M. Kingsman et al. Tibtech 5 (1987) 53-57.
• nucleic acid which represents a precursor form of the processed IL-16 polypeptide according to the invention.
• Such precursor forms are N-terminally elongated by further amino acids of the sequence of the IL-16 mutant according to the invention.
• the invention in addition concerns a prokaryotic or eukaryotic host cell which is transformed or transfected with a nucleic acid which codes for an IL- 16 mutant according to the invention in such a way that the host cell expresses the said polypeptide.
• a host cell usually contains a biologically functional nucleic acid vector, preferably a DNA vector, a plasmid DNA, which contains this nucleic acid.
• the IL-16 mutant according to the invention can be produced by culturing a prokaryotic or eukaryotic host cell which has been transformed or transfected with a corresponding coding nucleic acid under suitable nutrient conditions and optionally isolating the desired polypeptide. If it is intended 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 matter of the invention is a pharmaceutical composition which contains an IL-16 mutant according to the invention in an amount and/or specific activity which is sufficient for a therapeutic application as well as optionally a pharmaceutically suitable diluent, adjuvant and/or carrier.
• the IL-16 mutants according to the invention are especially suitable for treating pathological states which are caused by viral replication, in particular retroviral replication, and for immunomodulation. Such therapeutic applications are also described in the WO 96/31607. This also describes diagnostic test procedures.
• the IL-16 mutants according to the invention can be preferably used for immunosuppression.
• This immunosuppression is preferably achieved by an inhibition of the helper function of the THo and/or THi and TH2 cells.
• the polypeptides according to the invention are of therapeutic value in all diseases in which an immunodysregulatory component is postulated in the pathogenesis and in particular a hyperimmunity.
• IL- 16 Diseases which can be treated by IL- 16 in cardiology/angiology are for example myocarditis, endocarditis and pericarditis, in pulmonology for example bronchitis, asthma, in haematology autoimmune neuropenias and transplant rejections, in gastroenterology chronic gastritis, in endocrinology diabetes mellitus type I, in nephrology glomerulonephritis, rheumatic diseases, diseases in ophthalmology, in neurology such as multiple sclerosis and eczemas in dermatology.
• the polypeptides according to the invention can be used in particular for autoimmune diseases, allergies and to avoid transplant rejections. - 9 -
• the invention furthermore concerns the use of the nucleic acids according to the invention within the context of gene therapy.
• Retro viral or non-viral vector systems are for example suitable vector systems for this.
• amplification, cloning of IL-16 cDNA and production of an expression clone is carried out as described in WO 94/28134 or WO 96/31607 taking the modified sequences into consideration.
• An appropriate oligonucleotide is used as a forward primer which contains an EcoRJ site, 6 His, an enterokinase cleavage site, and the 5'-end of the IL-16 mutant sequence (approximately 7 codons).
• An appropriate oligonucleotide with approximately 12 codons of the 3' end of the IL-16 mutant is used as the reverse primer.
• the two reverse primers contain BamHI and Hindlll cleavage sites for the cloning.
• a sequence is obtained using IL-16 R ⁇ which codes for a protein according to SEQ ID NO:2.
• the PCR reaction, cloning and production of the expression clone is carried out according to standard conditions:
• Precultures are set up from stock cultures (plate smear or ampoules stored at -20°C) which are incubated at 37°C while shaking.
• the inoculation volume into the next higher dimension is 1-10 vol. % in each case.
• Ampicillin 50-100 mg/1) is used in the preculture and main culture to select against plasmid loss.
• Enzymatically digested protein and/or yeast extract as a N- and C-source as well as glycerol and/or glucose as an additional C-source are used as nutrients.
• the medium is buffered to pH 7 and metal salts are added at physiologically tolerated concentrations to stabilize the fermentation process.
• the fermentation is carried out as a feed batch with a mixed yeast extract/C sources dosage.
• the fermentation temperature is 25-37°C.
• the dissolved partial oxygen pressure (p ⁇ 2) is kept approximately at ⁇ 20 % by means of the aeration rate, r.p.m. regulation and dosage rate.
• the growth is determined by determining 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 by centrifugation at OD standstill.
• the biomass is taken up in 50 mM sodium phosphate, 5 mM EDTA, 100 mM sodium chloride, pH 7 and is disrupted at 1000 bar by means of a continuous high pressure press.
• the suspension obtained in this manner is centrifuged again and the supernatant which contains the dissolved IL-16 is processed further.
• the column was subsequently washed with equilibration buffer until the base line (UV detection at 280 nm) was nearly reached. Afterwards 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, 0.1 M NaCl, pH 7.0.
• the fusion protein was eluted with a gradient of 0 - 300 mM imidazole, pH 7.0 in 50 mM sodium phosphate, 0.1 M NaCl, pH 7.0 (2 x 1.6 1). Fractions containing IL-16 - 1 1 -
• IL-16 pool was concentrated in a Provario (Filtron, membrane omega 5 K) to a concentration of 5 mg protein/ml and dialysed against 50 mM Tris, pH 8.0.
• PBMC peripheral blood monocytes
• CD25 is a measure of T cell activation.
• IL-16 is a measure of IL-16-induced inhibition of stimulation by the anti-CD3-antibody.
• IL-16 2 39 (84%) IL- 16 mutant 3) 24 (52%)
• the percentage of CD4/CD25-positive cells decreases by about 20%) for recombinant IL-16 2) and about 50% for the IL-16 mutant according to the invention 3) .
• the IL-16 mutant according to the invention exhibits a distinctly higher activity compared to IL-16 according to the state of the art.

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