WO1999037781A1 - Mutants of il-16, processes for their production, and their use - Google Patents

Mutants of il-16, processes for their production, and their use Download PDF

Info

Publication number
WO1999037781A1
WO1999037781A1 PCT/EP1999/000428 EP9900428W WO9937781A1 WO 1999037781 A1 WO1999037781 A1 WO 1999037781A1 EP 9900428 W EP9900428 W EP 9900428W WO 9937781 A1 WO9937781 A1 WO 9937781A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
mutant
amino acids
host cell
sequence
Prior art date
Application number
PCT/EP1999/000428
Other languages
French (fr)
Inventor
Reinhard Kurth
Michael Baier
Albrecht Werner
Dorothee Ambrosius
Kurt Lang
Martin LANZENDÖRFER
Original Assignee
Bundesrepublik Deutschland
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bundesrepublik Deutschland filed Critical Bundesrepublik Deutschland
Priority to MXPA00006059A priority Critical patent/MXPA00006059A/en
Priority to BR9907216-5A priority patent/BR9907216A/en
Priority to CA002320624A priority patent/CA2320624A1/en
Priority to JP2000528688A priority patent/JP2002500889A/en
Priority to EP99903675A priority patent/EP1049782A1/en
Priority to EA200000769A priority patent/EA200000769A1/en
Priority to AU24243/99A priority patent/AU2424399A/en
Publication of WO1999037781A1 publication Critical patent/WO1999037781A1/en

Links

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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Public Health (AREA)
  • Toxicology (AREA)
  • Genetics & Genomics (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Transplantation (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pulmonology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

A nucleic acid, which can be used to express an IL-16 mutant in a prokaryotic or eukaryotic host cell, wherein the said nucleic acid codes for a polypeptide with an amino acid sequence beginning with amino acids 1-5 and ending with amino acids 93-107 of SEQ ID NO:2, is suitable for producing IL-16 mutants with improved activity.

Description

Mutants of IL-16, processes for their production, and their use
The invention concerns new IL-16 mutants with a high activity, processes for their production, and their use.
IL-16 (interleukin-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. However, 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.
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 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.
In a preferred embodiment of the invention, 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. For example, 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.
The structure of IL-16 was primarily determined from the 3D lH-13C 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 15N, 13C/15N labelled and selectively ^N-Ala, 15N-Gly/Ser, 15N-Leu labelled samples of IL-16l"ljυ\ 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 γ 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 β-sandwich (formed by β-strands βl-B5) which is flanked by an α-helix (amino acids R71-A81). The N-terminal β-strand (βl, T6-E13) makes an antiparallel β-sheet with the C-terminal β-strand B5 (V87-R93) and forms, together with the short β-strand B4 (I59-L62), one side of the β-barrel. The second antiparallel β-sheet B2-β3, which is formed by the residues F21-24 and F37-141, contains a β-bulge in β- strand β3 at residue 138. The two β-sheets, which build the β-sandwich, are packed against each other in a parallel manner, with a rotation of 39° degrees between a central axis of β- sheet B2 β3 and a central axis of β-sheet βl 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 α-helix and β-strand β5, β-strand β4 and the α-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.
The term "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 -
measured suitably as the ratio of CD4+/CD25+ cells which describes the inhibition of T cell stimulation.
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 several of the following properties:
- binding to T cells via the CD4 receptor,
- stimulation of the expression of the IL-2 receptor and/or HLA-DR antigen on CD4+ lymphocytes,
- stimulation of the proliferation of T helper cells in the presence of IL-2,
- suppression of the proliferation of T helper cells stimulated with anti-CD3 antibodies,
- suppression of the replication of 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. The term "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. In order to select the stringency 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. In addition 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). For this 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. However, a monomeric IL-16 polypeptide which cannot be cleaved into further subunits is preferred.
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 various recognition sequences of restriction enzymes to facilitate the steps of ligation, cloning and mutagenesis
- modification of the nucleic acid to incorporate preferred codons for the host cell
- extension of the nucleic acid by additional operator elements in order to optimize expression in the host cell.
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. Such 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), λPL 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 -
promoter (US Patent No. 4,551,433) are also suitable. Coupled promoter systems are equally suitable such as for example the T7-RNA polymerase/ promoter system (Studier et al., J. Mol. Biol. 189 (1986) 1 13). Hybrid promoters composed of a bacteriophage promoter and the operator region of the microorganism (EP-A 0 267 851) are also suitable. An effective ribosome binding site is necessary in addition to the promoter. In the case of E. coli this ribosome binding site is referred to as the Shine-Dalgarno (SD) 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 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).
After expression of the vector which is preferably a biologically functional plasmid or a viral vector, the fusion proteins are preferably cleaved with enzymes (e.g. enterokinase or factor Xa) (Nagai et al., Nature 309 (1984) 810). Further examples of 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.
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 signal sequence that is suitable for secretion of proteins in the host organisms used and of the nucleic acid that codes for the protein. In this case the protein is either secreted into the medium (in gram-positive bacteria) or into the periplasmatic space (in gram-negative bacteria). It is expedient to insert 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. Such 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).
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 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).
In addition the expression vectors usually contain a selectable marker in order to select transformed cells. Such 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.
Numerous suitable bacterial vectors are known. 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).
Further genetic engineering methods for the production and expression of suitable vectors are described 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.
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). 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. In a particular embodiment of the invention it is possible to use a 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. According to the invention it is then necessary to ensure the desired processing (cleavage) in the recombinant production. This can be done for example by treating the expression product (before or after separation from the fermentation broth) with an endonuclease which cleaves at the desired N-terminus of the IL- 16 mutant. - 8 -
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. Such 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. Hence 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. 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.
The following examples and publications as well as the sequence listing further elucidate the invention the protective scope of which results from the patent claims. The processes described are to be understood as examples that still describe the object of the invention even after modifications.
Example 1
Production of an IL-16 mutant (amino acids 1-100 of SEQ ID NO:2) using an enterokinase cleavage site
1.1 Expression clone
The 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 (fusion protein with N-terminal poly-His part for purification) is carried out according to standard conditions:
0.2 mM dNTP mix, 1 pmol/μl each forward and reverse primer, 1 x high fidelity buffer (Boehringer Mannheim GmbH, DE) 1.5 mM MgCl2, 2.6 U high fidelity enzyme mix (Boehringer Mannheim GmbH, DE).
20 μl final volume
Instrument: Perkin Elmer GeneAmp 9600
Reaction course:
3 min 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) - 10 -
1.2 Fermentation
10 1 fermentation of an E. coli expression clone for IL-16 and high pressure disruption
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 (pθ2) 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.
1.3 Purification and cleavage
700 ml lysis supernatant in 50 mM sodium phosphate, 5 mM EDTA, 100 mM NaCl, pH 7.2 was admixed with 70 ml 5 M NaCl, 60 mM MgCl2, pH 8.0, stirred for 30 min and subsequently centrifuged for 30 min at 20,000 g. The centrifuged supernatant was applied to a nickel-chelate column (V = 200 ml, Pharmacia) which had previously been loaded with a NiSO-j. solution (c = 10 mg/ml) and equilibrated with 50 mM sodium phosphate, 0.5 NaCl, pH 8.0. 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 -
were identified by means of SDS-PAGE and pooled. This 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.
An equivalent of the pool containing 100 mg fusion protein was diluted to a protein concentration of c = 1 mg/ml with 50 mM Tris, pH 8.0 for the enterokinase cleavage. After adding 33 μg enterokinase (Boehringer Mannheim GmbH; 1 :3000 w/w) the cleavage preparation was incubated overnight (14 h) at 37°C. Subsequently the pH value was adjusted to pH 6.5 with HC1.
Uncleaved IL-16 was removed by stirring in 20 ml nickel-chelate sepharose (prepared as above; binding time 2 h) and subsequent centrifugation (10,000 g) or filtering the supernatant over a nutsch filter. The indentity of the cleaved IL-16 contained in the supernatant was confirmed by N-terminal sequencing and mass analysis. The purity was checked by SDS- PAGE and RP-HPLC (Vydac, Diphenyl, 4.6 x 150 mm, linear gradient from 20 % to 95 % B within 45 minutes; solution A: 20 mM potassium phosphate in H2O, pH 7.5; solution B: 100 % acetonitrile).
Example 2
Determination of the activity of the IL-16 mutant
3 x 10^ peripheral blood monocytes (PBMC) were in each case placed in 200 μl of medium (RPMI/10% FCS) and incubated with the IL-16 polypeptide for 1 h at 37°C. The IL-16 concentration was 1 μg/ml in each case. Then the samples were transferred to a 96-well microtiter plate which had been pretreated as follows.
100 μl of anti-CD3 -antibodies (UCHT obtained from Pharmingen, San Diego, USA, order No. 30100D) at a concentration of 2.5 μg/ml were placed in PBS. Then the plate was incubated for 90 min. at 37°C and washed twice with PBS prior to the addition of the cells. After four days the cells were in each case labeled with 5 μl of an anti-CD4-antibody PE conjugate (13B.8.2, order No. 0449, Coulter Coop., Miami, USA) and anti-CD25-antibody FITC conjugate (B 1.49.9, order No. 0478, Coulter Coop., Miami, USA) and analyzed in a FACS (Becton Dickinson). The table below shows the percentage of CD4+/CD25+ double- positive cells. 12
The expression of CD25 is a measure of T cell activation. Thus, the decrease in CD25 expression after incubation with IL-16 is a measure of IL-16-induced inhibition of stimulation by the anti-CD3-antibody.
Table 1
IL-16 type CD4+/CD25+ control (PBS)1) 46 (100%)
IL-162) 39 (84%)
Figure imgf000014_0001
IL- 16 mutant3) 24 (52%)
) only phosphate buffered saline
2) recombinant IL-16 according to WO 94/28138
3) IL-16 mutant (amino acids 1-100 of SEQ ID NO:2)
The percentage of CD4/CD25-positive cells decreases by about 20%) for recombinant IL-162) and about 50% for the IL-16 mutant according to the invention3). Thus, the IL-16 mutant according to the invention exhibits a distinctly higher activity compared to IL-16 according to the state of the art.
List of references
Aman et al, Gene 40 (1985) 183
Baier, M., et al, Nature 378 (1995) 563
Beck and Zink, Gene 16 (1981) 35-58
Brosius et al, J. Mol. Biol. 148 (1981) 107 - 127
Chang et al., Nature 198 (1977) 1056
Cruikshank, W.W., et al., J. Immunol. 146 (1991) 2928-2934
Cruikshank, W.W., et al., Proc. Natl. Acad. Sci. USA 91 (1994) 5109-5113
Davies et al., Ann. Rev. Microbiol. 32 (1978) 469
EP-A 0 267 851
EP-A 0 495 398
Ghrayeb et al., EMBO J. 3 (1984) 2437
Goeddel et al., Nuc. Acids Res. 8 (1980) 4057
International Patent Application PCT/EP96/05661
International Patent Application PCT/EP96/05662 - 13 -
Kingsman, S.M., et al, Tibtech 5 (1987) 53-57
Mack et al., Analyt. Biochem. 200 (1992) 74-80
Miller et al., Bio/Technology 7 (1989) 698
Nagai et al., Nature 309 (1984) 810
Oka et al., Proc. Natl. Acad. Sci. USA 82 (1985) 7212
Palva et al., Proc. Natl. Acad. Sci. USA 79 (1982) 5582
Phillips and Silhavy, Nature 344 (1990) 882-884
Ponting and Phillips, Trends in Biochem. Sci. 20 (1995) 102-103
Powell et al., Appl. Environ. Microbiol. 54 (1988) 655
Rumsaeng et al., J. Immunol. 159 (1997) 2904-2910
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
Songyang et al., Science 275 (1997) 73-77
Studier et al., J. Mol. Biol. 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
WO 94/28134
WO 96/31607
Yansura, Meth. Enzymol. 185 (1990) 161-166

Claims

- 14 -Patent Claims
1. Nucleic acid which can be used to express an IL-16 mutant in a prokaryotic or eukaryotic host cell, wherein the said nucleic acid codes for a polypeptide beginning with amino acids 1-5 and ending with amino acids 93-107 of the sequence SEQ ID NO:2.
2. Nucleic acid as claimed in claim 1, which codes for IL-16 mutants of amino acids 1-100, 5-107 or 5-93 of SEQ ID NO:2.
3. Prokaryotic or eukaryotic host cell which is transformed or transfected with a nucleic acid as claimed in claim 1 or 2 in such a way that the host cell expresses the said IL-16 mutant.
4. Biologically functional nucleic acid vector that contains a nucleic acid as claimed in claim 1 or 2.
5. IL-16 mutant as it can be obtained as the product of a eukaryotic or prokaryotic expression of a nucleic acid as claimed in claim 1 or 2 which is essentially free of other human proteins.
6. IL-16 mutant as claimed in claim 5, consisting of amino acids 1-107 of SEQ ID NO:2.
7. Process for the production of an IL-16 mutant as claimed in claim 5 or 6, wherein a prokaryotic or eukaryotic host cell which is transformed or transfected with a nucleic acid sequence as claimed in claim 1 or 2 is cultured under suitable nutrient conditions and the desired polypeptide is optionally isolated.
8. Pharmaceutical composition which contains an IL-16 mutant as claimed in claim 5 or 6 as well as a pharmaceutically suitable diluent, adjuvant and/or carrier.
9. Pharmaceutical composition containing an IL-16 mutant as claimed in claim 5 or 6 in an amount adequate for a therapeutic application.
PCT/EP1999/000428 1998-01-24 1999-01-22 Mutants of il-16, processes for their production, and their use WO1999037781A1 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
MXPA00006059A MXPA00006059A (en) 1998-01-24 1999-01-22 Mutants of il-16, processes for their production, and their use.
BR9907216-5A BR9907216A (en) 1998-01-24 1999-01-22 Il-16 mutants, processes for their production and use
CA002320624A CA2320624A1 (en) 1998-01-24 1999-01-22 Mutants of il-16, processes for their production, and their use
JP2000528688A JP2002500889A (en) 1998-01-24 1999-01-22 Mutants of IL-16, methods for their production and uses thereof
EP99903675A EP1049782A1 (en) 1998-01-24 1999-01-22 Mutants of il-16, processes for their production, and their use
EA200000769A EA200000769A1 (en) 1998-01-24 1999-01-22 IL-16 MUTANTS, METHODS OF THEIR PRODUCTION AND THEIR APPLICATION
AU24243/99A AU2424399A (en) 1998-01-24 1999-01-22 Mutants of il-16, processes for their production, and their use

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP98101232 1998-01-24
EP98101232.1 1998-01-24

Publications (1)

Publication Number Publication Date
WO1999037781A1 true WO1999037781A1 (en) 1999-07-29

Family

ID=8231307

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1999/000428 WO1999037781A1 (en) 1998-01-24 1999-01-22 Mutants of il-16, processes for their production, and their use

Country Status (8)

Country Link
EP (1) EP1049782A1 (en)
JP (1) JP2002500889A (en)
AU (1) AU2424399A (en)
BR (1) BR9907216A (en)
CA (1) CA2320624A1 (en)
EA (1) EA200000769A1 (en)
MX (1) MXPA00006059A (en)
WO (1) WO1999037781A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001010893A2 (en) 1999-08-05 2001-02-15 Research Corporation Technologies, Inc. Il-16 antagonists

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994028134A1 (en) * 1993-05-21 1994-12-08 The Trustees Of Boston University Lymphocyte chemoattractant factor and uses thereof
WO1997023616A1 (en) * 1995-12-22 1997-07-03 BUNDESREPUBLIK DEUTSCHLAND, vertreten durch den BUNDESMINISTER FÜR GESUNDHEIT Polypeptides with interleukin 16 activity, process for the preparation and use thereof
DE19617202A1 (en) * 1996-04-30 1997-11-06 Boehringer Mannheim Gmbh Recombinant interleukin 16
WO1998023751A1 (en) * 1996-11-25 1998-06-04 BUNDESREPUBLIK DEUTSCHLAND, vertreten durch den BUNDESMINISTER FÜR GESUNDHEIT Processed polypeptides with il-16 activity, processes for their production and their use

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994028134A1 (en) * 1993-05-21 1994-12-08 The Trustees Of Boston University Lymphocyte chemoattractant factor and uses thereof
WO1997023616A1 (en) * 1995-12-22 1997-07-03 BUNDESREPUBLIK DEUTSCHLAND, vertreten durch den BUNDESMINISTER FÜR GESUNDHEIT Polypeptides with interleukin 16 activity, process for the preparation and use thereof
DE19617202A1 (en) * 1996-04-30 1997-11-06 Boehringer Mannheim Gmbh Recombinant interleukin 16
WO1998023751A1 (en) * 1996-11-25 1998-06-04 BUNDESREPUBLIK DEUTSCHLAND, vertreten durch den BUNDESMINISTER FÜR GESUNDHEIT Processed polypeptides with il-16 activity, processes for their production and their use

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BAIER M ET AL: "MOLECULAR CLONING, SEQUENCE, EXPRESSION, AND PROCESSING OF THE INTERLEUKIN 16 PRECURSOR", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 94, no. 10, 13 May 1997 (1997-05-13), pages 5273 - 5277, XP002038013 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001010893A2 (en) 1999-08-05 2001-02-15 Research Corporation Technologies, Inc. Il-16 antagonists
WO2001010893A3 (en) * 1999-08-05 2001-08-16 Res Corp Technologies Inc Il-16 antagonists
US6699466B1 (en) 1999-08-05 2004-03-02 Research Corporation Technologies, Inc. IL-16 antagonist peptides and DNA encoding the peptides
US7208149B2 (en) 1999-08-05 2007-04-24 Trustees Of Boston University IL-16 antagonist peptides and DNA encoding the peptides
US7608691B2 (en) 1999-08-05 2009-10-27 Trustees Of Boston University Antibodies against IL-16 antagonist peptides
US7914778B2 (en) 1999-08-05 2011-03-29 Trustees Of Boston University Methods of treatment using IL-16 antagonist peptides

Also Published As

Publication number Publication date
JP2002500889A (en) 2002-01-15
EA200000769A1 (en) 2001-02-26
MXPA00006059A (en) 2003-08-01
EP1049782A1 (en) 2000-11-08
BR9907216A (en) 2000-10-24
AU2424399A (en) 1999-08-09
CA2320624A1 (en) 1999-07-29

Similar Documents

Publication Publication Date Title
KR101299417B1 (en) Method for producing carboxy-terminal amidified peptides
AU731758B2 (en) Method for secretory production of human growth hormone
US6207144B1 (en) Polypeptides with interleukin-16 activity, process for the preparation and use thereof
AU627477B2 (en) Interleukin ii analogs
CA2213512A1 (en) Method for producing bioactive fusion protein
US6165746A (en) Preventing endogenous aminopeptidase mediated n-terminal amino acid cleavage during expression of foreign genes in bacteria
Tocci et al. Expression in Escherichia coli of fully active recombinant human IL 1 beta: comparison with native human IL 1 beta.
KR100360594B1 (en) Expression and secretion vector for human interferon alpha and process for producing human interferon alpha by employing same
US6444202B1 (en) Processed polypeptides with IL-16 activity, processes for their production and their use
Altmann et al. Expression and purification of a synthetic human obese gene product
US6506582B1 (en) Processed polypeptides with IL-16 activity, process for preparing the same and their use
Dalbøge et al. Cloning and expression of an interleukin-1β precursor and its conversion to interleukin-1β
EP1049782A1 (en) Mutants of il-16, processes for their production, and their use
AU765890B2 (en) Keratinocyte derived interferon
EP1307480B1 (en) Mutant trichosanthin
Kreitman et al. Circularly permuted interleukin 4 retains proliferative and binding activity
CA2240392C (en) Multimer forms of interleukin-16 (il-16), process for the preparation and use thereof
Nittoh et al. Cloning of cDNA for rat eosinophil major basic protein
MXPA98008964A (en) Polypeptides processed with acativity il-16, processes for its production and its
Santos et al. Obtaining biologically active IL-15 in Escherichia coli
KR100204504B1 (en) Process for the production of human interleukin-6 using yeast
DE19649233A1 (en) Nucleic acid encoding interleukin-16 polypeptide
DE19648715A1 (en) DNA encoding human interleukin-16
AU2003271323A1 (en) Keratinocyte derived interferon
MXPA01000692A (en) Keratinocyte derived interferon

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 1999903675

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: PA/a/2000/006059

Country of ref document: MX

ENP Entry into the national phase

Ref document number: 2320624

Country of ref document: CA

Ref document number: 2320624

Country of ref document: CA

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: KR

WWE Wipo information: entry into national phase

Ref document number: IN/PCT/2000/206/CHE

Country of ref document: IN

WWE Wipo information: entry into national phase

Ref document number: 200000769

Country of ref document: EA

WWE Wipo information: entry into national phase

Ref document number: 09600851

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: 1999903675

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWW Wipo information: withdrawn in national office

Ref document number: 1999903675

Country of ref document: EP