EP0904374A1 - Processed polypeptides with il-16 activity, process for preparing the same and their use - Google Patents

Processed polypeptides with il-16 activity, process for preparing the same and their use

Info

Publication number
EP0904374A1
EP0904374A1 EP97921834A EP97921834A EP0904374A1 EP 0904374 A1 EP0904374 A1 EP 0904374A1 EP 97921834 A EP97921834 A EP 97921834A EP 97921834 A EP97921834 A EP 97921834A EP 0904374 A1 EP0904374 A1 EP 0904374A1
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
polypeptide
seq
interleukin
activity
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP97921834A
Other languages
German (de)
French (fr)
Inventor
Reinhard Kurth
Michael Baier
Norbert Bannert
Albrecht Werner
Kurt Lang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Roche Diagnostics GmbH
Bundesrepublik Deutschland
Bundesministerium fuer Gesundheit
Original Assignee
Roche Diagnostics GmbH
Bundesrepublik Deutschland
Bundesministerium fuer Gesundheit
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
Priority claimed from DE1996117203 external-priority patent/DE19617203A1/en
Priority claimed from DE19617202A external-priority patent/DE19617202A1/en
Application filed by Roche Diagnostics GmbH, Bundesrepublik Deutschland, Bundesministerium fuer Gesundheit filed Critical Roche Diagnostics GmbH
Publication of EP0904374A1 publication Critical patent/EP0904374A1/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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 relates to polypeptides with LL-16 activity, processes for their preparation and their use.
  • the invention describes processed LL-16 with high activity
  • LL-16 Interleukin-16 is a lymphokine, which is also referred to as "lymphocyte chemoattracting factor” (LCF) or “immunodeficiency virus suppressing lymphokine” (ISL). Its properties are described in WO 94/28134 and in WO 96/31607 as well as by Cruikshank, WW, et al, Proc Natl Acad Sei USA 91 (1994) 5109-5113 and by Baier, M, et al, Nature 378 (1995) 563, which also describes the recombinant production of LL- 16. LL-16 is then a protein with a molecular mass of 13.385 D.
  • LCF lymphocyte chemoattracting factor
  • ISL immunodeficiency virus suppressing lymphokine
  • the object of the present invention is to improve the activity of LL-16 and to provide LL-16 forms which show low immunogenicity and are advantageously suitable for therapeutic use
  • the object of the invention is achieved by a nucleic acid with which the expression of a polypeptide with interleukin-16 activity in a prokaryotic or eukaryotic host cell can be achieved, said nucleic acid
  • nucleic acid preferably codes for a polypeptide with the amino acid sequence SEQ LD NO.2 or for a polypeptide with a sequence which is extended by an aspartic acid codon N-terminally compared to SEQ LD NO.2.
  • nucleic acid codes for a polypeptide with LL-16 activity, which is shortened by up to 8 amino acids at the C-terminus
  • Such a nucleic acid encodes a processed polypeptide with IL-16 activity, particularly preferably natural LL-16 from primates, such as human LL-16 or LL-16 from a monkey species or another mammal such as a mouse
  • FIG. 2 of WO 94/28134 does not describe the correctly processed LL-16.
  • the start codon "ATG" of the precursor form of the protein does not begin with nucleotide 783, but with nucleotide 54 or 174 if an A is inserted after nucleotide 156, a C after nucleotide 398 and a G after nucleotide 780.
  • the sequence also shows further differences from FIG. 2 of WO 94/28134. These are, for example, nucleotide exchanges (313 G in A, 717 C in A) In expression in eukaryotic cells, LL-16 is processed.
  • the sequence of LL-16 can differ to a certain extent from the protein sequences encoded by such DNA sequences. Such sequence variations can be amino acid exchanges, deletions or additions. However, the amino acid sequence of LL-16 is at least 75%, Particularly preferably at least 90% identical to the amino acid sequence of SEQ ID NO 2 Variants of parts of the amino and nucleic acid sequence SEQ LD NO 1 / SEQ LD NO 2 are for example in WO 96/31607 and the international patent applications PCT / EP96 / 05662 and PCT / EP96 / 05661 it is essential, however, that the polypeptides have a correct N-terminus. Preference is therefore given to proteins in which the first three to ten amino acids of the N-terminus are unchanged.
  • nucleic acids are understood to mean, for example, DNA, RNA and nucleic acid derivatives and analogs.
  • Preferred nucleic acid analogs are those compounds in which the sugar phosphate skeleton is replaced by other units, such as amino acids.
  • Such compounds are referred to as PNA and are described in WO 92/20702.
  • PNA-DNA bonds are stronger than DNA DNA bonds, the stringent conditions described below for PNA-DNA hybridization are not applicable. Suitable hybridization conditions are, however, described in WO 92/20703
  • LL-16 is to be understood as meaning a polypeptide with the activity of IL-16.
  • LL-16 preferably shows the stated effect in the test method described in WO 96/31607 or stimulates cell division according to WO 94 / 28134.
  • LL-16 binds to CD4 + lymphocytes and can suppress the replication of viruses such as HTV-1, HIV-2 and SIV.
  • the function of LL-16 is not limited by its presentation in the MHC complex
  • LL-16 exhibits one or more of the following properties
  • viruses preferably HIV-1, HIV-2 or
  • nucleic acids which hybridize under stringent conditions with nucleic acids of the sequence SEQ LD NO 1.
  • hybridize under stringent conditions means that two nucleic acid fragments under standardized hybridization Hybridize conditions with one another, 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 FIG. 6 , 0 x SSC at about 45 ° C, followed by a washing step at 2 x SSC at 50 ° C.
  • the salt concentration in the washing step can be, for example, between 2.0 x SSC at 50 ° C for low stringency and 0.2 x SSC at 50 ° C can be selected for high stringency.
  • the temperature of the wash step between room temperature, about 22 ° C, for narrow stringency and 65 ° C for high stringency can be varied
  • LL-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 the subject matter of the disclosure of the present invention for this purpose. However, the forms of LL-16 according to the invention by recombinant production To be defined and reproducible, additional measures must be taken via the recombinant production methods familiar to those skilled in the art
  • Recombinant LL-16 can be produced according to the methods familiar to the person skilled in the art as heterologous expression or as homologous expression (after homologous recombination of the LL16 nucleic acid into the genome of the host organism).
  • a DNA is produced which is able to To produce protein which has the activity of LL-16.
  • the DNA is cloned into a vector which can be transferred into a host cell and can be replicated there.
  • a vector contains, in addition to the IL-16 sequence, regulatory elements which are necessary for the expression of the DNA sequence is required.
  • This vector which contains the LL-16 sequence and the regulatory elements, is transferred into a vector which is capable of expressing the DNA of LL-16 Amplification of the vector are suitable, cultivated and LL-16 won. Suitable measures ensure that the protein is an active tertiary str structure in which it shows LL-16 properties
  • the 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 there in E. coli. Expression in prokaryotes leads to an unglycosylated polypeptide.
  • the expression vectors must contain a promoter which allows expression of the protein in the host organism.
  • promoters are known to the person skilled in the art and are, for example, lac promoter (Chang et al., Nature 198 (1977) 1056), trp promoter (Goeddel et al., Nuc. Acids Res. 8 (1980) 4057), ⁇ pj_ 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 Pat. No. 4,551,433) are also suitable.
  • Coupled promoter systems such as, for example, T7 RNA polymerase / promoter system (Studier et al., J. Mol. Biol. 189 (1986) 13) are also suitable.
  • Hybrid promoters consisting of a bacteriophage promoter and the operator region of the microorganism (EP-A 0267 851) are also suitable.
  • An effective ribosome binding site is required in addition to the promoter.
  • 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.
  • SD Shine-Dalgarno
  • a DNA sequence which codes for the N-terminal part of an endogenous bacterial protein or another stable protein is usually attached to the 5'- End of the sequence coding for LL-16 fused. Examples include lacZ (Phillips and Silhavy, Nature 344 (1990) 882-884), trpE (Yansura, Meth. Enzymol. 185
  • the fusion proteins are preferably cleaved with enzymes (eg factor Xa) (Nagai et al “Nature 309 (1984) 810).
  • enzymes eg factor Xa
  • Further examples of cleavage sites are the IgA protease cleavage site (WO 91/11520, EP-A 0495 398), the ubiquitin cleavage site (Miller et al., Bio / Technology 7 (1989) 698) and the enterokinase cleavage site.
  • the proteins expressed in this way in bacteria are obtained in a conventional manner by digesting the bacteria and isolating the proteins
  • a fusion product which consists of the signal sequence which is suitable for the secretion of proteins in the host organisms used and the nucleic acid which encoded for the protein.
  • the protein is either secreted into the medium (in the case of gram-positive bacteria) or in the periplasmic space (in the case of gram-negative bacteria).
  • a cleavage site is expediently located, either during processing or in an additional step allows the protein to be split off.
  • signal sequences originate, 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 also contain terminators.
  • Terminators are DNA sequences that signal the end of a transcription process.They are usually characterized by two structural characteristics: a region that is rich in reversed G / C, which can form a double helix intramolecularly, and a number of U ( or T) residues Examples are the main terminator in the DNA of phage fd (Beck and Zink, Gene 16 (1981) 35-58) 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 tetracycline (Davies et al., Ann Rev Microbiol 32 (1978) 469)
  • Selectable markers are the genes for essential substances of the biosynthesis of substances necessary for the cell, such as, for example, histidine, tryptophan and leucine
  • Suitable bacterial vectors are known.
  • vectors are described for the following bacteria: Bacillus subtilis (Palva et al., Proc Natl Acad Sei. 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, Appl Environ Microbiol 54 (1988) 655), Streptococcus lividans and Streptomyces lividans (U.S. Patent No. 4,747,056) Further genetic engineering processes for the production and expression of suitable vectors are described in J. Sambrook et al., Molecular Cloning a laboratory manual (1989), Cold Spring Harbor Laboratory Press, New York, NY.
  • yeast In addition to prokaryotic microorganisms, expression of recombinant LL-16 is also possible in eukaryotes (such as, for example, CHO cells, yeast or insect cells).
  • the yeast system or insect cells is preferred as the eukaryotic expression system.
  • Expression in yeast can be via three types of yeast vectors (integrating YIp (yeast integrating plasmids) - vectors, replicating YRp (yeast replicon plasmids) - vectors and episomal YEp (yeast episomal plasmids) - vectors). Kingsman et al, Tibtech 5 (1987) 53-57
  • the invention further relates to a prokaryotic or eukaryotic host cell which has been transformed or transfected with a nucleic acid which codes for an LL-16 polypeptide according to the invention in such a way that the host cell expresses the said polypeptide.
  • a host cell usually contains a biological one functional nucleic acid vector, preferably a DNA vector, a plasmid DNA, which contains this nucleic acid
  • a monomeric LL-16 polypeptide which cannot be split into further subunits is further preferred
  • nucleic acid and protein sequence of LL-16 described in WO 94/28134 do not correspond to the natural human sequences. It is merely a non-natural LL-16 analog. It is for therapeutic use however, it is preferred to use a protein which is either identical to the natural protein or differs only slightly from the natural protein and / or shows at least comparable activity and immunogenicity.
  • the sequence of the protein is described in SEQ LD NO.2 (optionally with an N-terminal extension by an aspartic acid residue and / or shortening at the C-terminus by up to 8 amino acids)
  • the nucleic acid sequence of LL-16 can receive deletions, mutations and additions within the scope of the invention.
  • the monomeric form of LL-16 can be multimerized. This allows the activity of LL-16 to be increased.
  • Such multimeric forms are preferably dimeric, tetrameric or octameric forms
  • the polypeptides of the invention can additionally contain a defined content of metal ions, the number of metal ions per subunit preferably being 0.5 to 2.
  • metal ions are suitable as metal ions in the sense of the invention. As has been shown, both alkaline earth metals and elements of the subgroups are suitable. Alkaline earth metals, cobalt, zinc, selenium, manganese, nickel, copper, iron, magnesium, calcium, molybdenum and silver are particularly suitable.
  • the ions can be one, two, three or four-valued. Divalent ions are particularly preferred.
  • the ions are preferably added as solutions of MgCl2, CaC ⁇ , MnCl2, BaCl2, LiC ⁇ , Sr (N03) 2, Na2Mo ⁇ 4, AgCi2.
  • the polypeptide according to the invention can be prepared by culturing a prokaryotic or eukaryotic host cell which has been transformed or transfected with a nucleic acid sequence according to claims 1 or 2 under suitable nutrient conditions and, if appropriate, isolating the desired polypeptide. If the preparation of the polypeptide is to take place in vivo as part of a gene therapy treatment, the polypeptide is of course not isolated from the cell.
  • the invention further relates to a pharmaceutical composition which contains a polypeptide according to the invention in an amount and / or specific activity sufficient for therapeutic use and, if appropriate, a pharmaceutically suitable diluent, adjuvant and / or carrier.
  • polypeptides according to the invention are particularly suitable for the treatment of pathological conditions which are caused by viral replication, in particular retroviral replication, and for immunomodulation. Such therapeutic applications are described in WO 96/31607. Diagnostic test methods are also described therein.
  • the polypeptides according to the invention can preferably be used for immunosuppression. This immunosuppression is preferably carried out by inhibiting the helper function of the THo and / or THi and / or TH2 cells.
  • the polypeptides according to the invention are accordingly of therapeutic value in all diseases in which an immunodisregulated O 97/41231 PC ⁇ YEP97 / 02216
  • ⁇ component in the pathogenesis is postulated, in particular hyperimmunity.
  • Diseases such as myocarditis, endocarditis and pericarditis can be considered as LL-16-treatable diseases in cardiology / angiology, in bronchitis, for example, bronchitis, asthma in hematology, autoimmune Europe and transplant rejection, and chronic gastritis in gastroenterology in endocrinology diabetes mellitus type I, in nephrology glomerulonephritis, diseases in the rheumatic range, diseases in ophthalmology, in neurology, such as multiple sclerosis, in dermatology eczema.
  • the polypeptides according to the invention can be used in autoimmune diseases, allergies and to avoid transplant rejections.
  • Another object of the invention is the use of the nucleic acids according to the invention in the context of gene therapy.
  • Suitable vector systems for this are, for example, retroviral or non-viral vector systems.
  • Another object of the invention is a polyclonal or monoclonal anti-LL-16 antibody or an immunoactive fragment thereof which SEQ ID NO is extended to the first 3-20 amino acids of SEQ ID NO.2 or an N-terminal by an aspartic acid residue: 2 binds, as well as methods for the production of such antibodies and their use for the determination of LL-16 and for the determination of viral infections in eukaryotic cells and in particular in mammalian cell material.
  • Virus-activated mammalian cells, in particular T cells can also be determined with LL-16.
  • Such antibodies are produced by immunization with a polypeptide according to the invention.
  • an antibody is carried out according to the methods familiar to the person skilled in the art, by immunization with an immunogen which extends the first 3-20 amino acids of SEQ ID NO: 2 or an N-terminally SEQ ID NO: Contains 2 as hapten.
  • the antibody can then be obtained from the immunized mammal in a conventional manner and, if appropriate, a monoclonal antibody can be produced.
  • RNA isolation Kit 5 ml denaturation solution (RNA isolation Kit, Stratagene) lysed. After the addition of 1 ml of Na acetate, 5 ml of phenol and 1 ml of chloroform / isoamyl alcohol (24 l), the lysate was kept on ice for 15 min. The aqueous phase was then washed with
  • the mixture for the cDNA synthesis contained 10 ⁇ g RNA, 0.2 ⁇ g Oiigo-dT, 13 mM DTT and 5 ⁇ l “bulk first strand reaction mix” (first-strand cDNA synthesis kit, Pharmacia) in an amount of 15 ⁇ l the mixture was incubated for 1 hour at 37 ° C and then stored at -20 ° C for later use
  • Amplification, cloning of LL-16 cDNA and production of an expression clone are carried out as described in WO 94/28134 or WO 96/31607, taking into account the changed sequences
  • Pre-cultures are prepared from stock cultures (plate smear or ampoules stored at -20 ° C), which are shaken and incubated at 37 ° C.
  • the inoculation volume into the next higher dimension is 1-10% by volume.
  • Ampicillin 50-100 mg / 1
  • the nutrients used are enzymatically digested protein and / or yeast extract as an N and C source, and glycerol and / or glucose as an additional C source.
  • the medium is buffered to pH 7 and metal salts are added to stabilize the fermentation process in physiologically acceptable concentrations.
  • the fermentation is carried out as a feed batch with a mixed yeast extract / C source dosage.
  • the fermentation temperature is 25-37 ° C.
  • the dissolved oxygen partial pressure (p ⁇ 2) is kept below about 20% via aeration rate, speed regulation and dosage speed.
  • the growth is determined by determining the optical density (OD) at 528 nm. Expression of the LL-16 is induced by means of IPTG. After a fermentation period of 10 to 20 hours, the biomass is harvested by centrifugation when the OD is at a standstill.
  • the biomass is taken up in 50 mM sodium phosphate, 5 mM EDTA, 100 mM sodium chloride, pH 7 and digested using a continuous high-pressure press at 1000 bar.
  • the suspension thus obtained is centrifuged again and the supernatant, which contains the dissolved LL-16, is processed further.
  • the column was then rinsed with 300 ml of 50 mM sodium phosphate, 0.5 M NaCl, pH 7.0 and the LL-16 fusion protein then 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 (2x 0.5 I gradient volume) eluted.
  • Fractions containing LL-16 were identified by SDS-PAGE, pooled and dialyzed against 20 mM sodium phosphate, pH 7.0.
  • 300 mg of the fusion protein thus obtained were dialyzed at 4 ° C. against 20 1 20 mM imidazole, pH 5.5 and then for 30 minutes to remove turbidity. centrifuged at 20,000 g. The supernatant from the centrifugation was then adjusted to pH 8.5 with NaOH, 0.3 mg of thrombin (Boehringer Mannheim GmbH) was added and the mixture was incubated at 37 ° C. for 4 hours. The gap mixture was then adjusted to pH 6.5 with HC1 and the conductivity to 1.7 mS by dilution with H2O.
  • LL-16 was eluted with a gradient of 0 to 0.3 M NaCl in 20 mM imidazole, pH 6.5. Fractions containing LL-16 were identified by SDS-PAGE and pooled. The identity of LL-16 was confirmed by mass analysis (molecular weight 13,566 + 3 D) and automated N-terminal sequence analysis.
  • an aminopeptidase for example ⁇ -aminoacylpeptide hydrolase
  • dipeptidyl peptidase for example catepsin CCATH
  • the LL-16 obtained in this way had a purity of more than 95% in SDS-PAGE under reducing conditions
  • a Vydac, Protein & Peptide Cl 8, 4x180 mm column was used for purity analysis using RP-HPLC. Elution is carried out by a linear gradient from 0% to 80% B (solvent B 90% acetonitrile in 0.1% TFA, solvent A 0 , 1% TFA in H2O) within 30 min with a flow rate of 1 ml / min. The detection was carried out at 220 nm
  • Amplification, cloning of LL-16 cDNA and production of an expression clone are carried out as described in WO 94/28134 or WO 96/31607, taking into account the changed sequences
  • An oligonucleotide of the sequence SEQ LD NO: 3 is used as the forward primer, which contains an EcoRI site, 6 His and an enterokinase cleavage site (D 4 K).
  • Cccgaattc tatg cat cac cac cac cac cac gatgacgacgacaaa-tctgcaqcctcaqcctctqc EcoRI H6 D4K
  • an oligonucleotide of the sequence SEQ ID NO: 6 is used as the forward primer, which also contains an EcoRI site, 6 His and an enterokinase cleavage site (D 4 K) cccgaattc tatg cat cac cac cac cac gatgacgacgacaaa-gactctqcaqcctcaqcctc EcoRI Hß D4K
  • Either oligonucleotide LL-16-Rj (SEQ ID NO: 4) is used as the reverse primer:
  • ILI6-R 1 qcq gat cca agc tta gga gtc tcc agc agc tgt g
  • oligonucleotide LL-16-R 2 SEQ LD NO: 5
  • ILI6-R 2 qcq qat cca agc tta ttc ctt gga ctg gag gct tttt tc
  • the two reverse primers contain BamHI and HindIII cloning sites
  • the PCR reaction, cloning and production of the expression clone are carried out according to standard conditions.
  • the column was then washed with equilibration buffer until the baseline (UV detection at 280 nm) was almost reached.
  • the column was then rinsed with 1 1 50 mM sodium phosphate, 0.5 M NaCl, pH 7.0 and with 1 1 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 ⁇ 1.6 l).
  • Fractions containing IL 16 were identified by SDS-PAGE and pooled. This LL-16 pool was concentrated in the provario (Filtron, membrane Omega 5 K) to a concentration of 5 mg protein / ml and dialyzed against 50 mM Tris, pH 8 0
  • lymphocytes isolated from a "buffy coat" using Ficoll gradients are put into 500 ⁇ l PBS azide / 1 ⁇ 10 8 cells (phosphate buffered saline without Ca 2+ and Mg 2+ , 0.01% sodium azide, 5 mM EDTA , pH 7.2) resuspended After adding 20 ml CD8-Microbeads / lx 10? cells to be expected (mouse anti-human CD8 antibodies, conjugated with magnetic particles, Miltenyi Biotec GmbH) are incubated at 4 ° C. for 15 min.
  • the CD8 + cells to which the CD8 microbeads are coupled are retained in the column, the flow fraction accordingly contains all lymphocytes (approx. 80% CD4 + cells) with the exception of the CD8 + cells.
  • the column is removed from the holder and the CD8 + cell fraction eluted with PBS azide / 1% BSA.
  • the flow-through and the CD8 + cell fraction are centrifuged, resuspended in cell culture medium (RPMI 1640, 20% FCS, 2 mM glutamine, 180 U / ml LL-2), the cell number adjusted to 3 ⁇ 10 ⁇ cells / ml and the cells stimulated with PHA (9 mg / ml).
  • the quality of the lymphocyte subpopulation separation is checked by means of FACS.
  • the masses of the fragments are determined by mass spectrography and the size of the N-terminal fragment is thus determined.
  • a naturally processed IL-16 fragment was identified which begins with the N-terminus described in SEQ ID NO.1 / 2 or which is extended by an aspartic codon.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • Communicable Diseases (AREA)
  • Pulmonology (AREA)
  • Neurosurgery (AREA)
  • Rheumatology (AREA)
  • Cell Biology (AREA)
  • Oncology (AREA)
  • Neurology (AREA)
  • Pain & Pain Management (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Physics & Mathematics (AREA)

Abstract

A nucleic acid makes it possible to express a polypeptide with interleukin-16 activity in a procaryotic or eucaryotic host cell. Said nucleic acid codes for a polypeptide with the amino acid sequence SEQ ID NO:2 or for a SEQ ID NO:2 elongated at the N-terminus by an aspartic acid radical or shortened at the C-terminus by up to 8 amino acids. This nucleic acid is useful for producing an active IL-16 polypeptide.

Description

Prozessierte Polypeptide mit IL-16-Aktivität, Verfahren zu ihrer Herstellung und Ver¬ wendung Processed polypeptides with IL-16 activity, process for their preparation and use
Gegenstand der Erfindung sind Polypeptide mit LL- 16- Aktivität, Verfahren zu ihrer Herstel¬ lung und ihre Verwendung. Die Erfindung beschreibt prozessiertes LL- 16 mit hoher AktivitätThe invention relates to polypeptides with LL-16 activity, processes for their preparation and their use. The invention describes processed LL-16 with high activity
LL-16 (Interleukin- 16) ist ein Lymphokin, welches auch als "lymphocyte chemoattracting factor" (LCF) oder "immunodeficiency virus suppressing lymphokine" (ISL) bezeichnet wird LL-16 und seine Eigenschaften sind in der WO 94/28134 und der WO 96/31607 sowie von Cruikshank, W W , et al , Proc Natl Acad Sei USA 91 (1994) 5109-5113 und von Baier, M , et al , Nature 378 (1995) 563 beschrieben Dort ist auch die rekombinante Herstellung von LL-16 beschrieben Danach ist LL-16 ein Protein mit einer molekularen Masse von 13,385 D Von Cruikshank wurde ebenfalls gefunden, daß ISL in einer Molekularsieb¬ chromatographie als multimere Form mit einem Molekulargewicht von 50-60 bzw 55-60 kD eluiert Dieser multimeren Form, welche ein kationisches Homotetramer ist (Produktinformation AMS Biotechnology Ltd , Europe, Cat No 11177186), wird die "chemoattraetant activity" zugeschrieben Von Baier wird eine homodimere Form von LL-16 mit einem Molekulargewicht von 28 kD beschrieben Die von Cruikshank et al in J Immunol 146 (1991) 2928-2934 beschriebene "chemoattraetant activity" und die von Baier beschrie¬ bene Aktivität von rekombinantem humanem LL-16 sind jedoch sehr geringLL-16 (Interleukin-16) is a lymphokine, which is also referred to as "lymphocyte chemoattracting factor" (LCF) or "immunodeficiency virus suppressing lymphokine" (ISL). Its properties are described in WO 94/28134 and in WO 96/31607 as well as by Cruikshank, WW, et al, Proc Natl Acad Sei USA 91 (1994) 5109-5113 and by Baier, M, et al, Nature 378 (1995) 563, which also describes the recombinant production of LL- 16. LL-16 is then a protein with a molecular mass of 13.385 D. Cruikshank also found that ISL in molecular sieve chromatography as a multimeric form with a molecular weight of 50-60 or 55-60 kD elutes. This multimeric form, which is a cationic homotetramer (product information AMS Biotechnology Ltd, Europe, Cat No 11177186), the "chemoattraetant activity" is attributed. Baier describes a homodimeric form of LL-16 with a molecular weight of 28 kD. The method described by Cruikshank et al i n J Immunol 146 (1991) 2928-2934 described "chemoattraetant activity" and the activity of recombinant human LL-16 described by Baier are however very low
Aufgabe der vorliegenden Erfindung ist es, die Aktivität von LL-16 zu verbessern und LL-16- Formen bereitzustellen, die geringe Immunogenitat zeigen und vorteilhaft für eine therapeuti¬ sche Anwendung geeignet sindThe object of the present invention is to improve the activity of LL-16 and to provide LL-16 forms which show low immunogenicity and are advantageously suitable for therapeutic use
Die Aufgabe der Erfindung wird gelost durch eine Nukleinsäure, mit welcher die Expression eines Polypeptids mit Interleukin- 16- Aktivität in einer prokaryontischen oder eukaryontischen Wirtszelle erreicht werden kann, wobei die genannte NukleinsäureThe object of the invention is achieved by a nucleic acid with which the expression of a polypeptide with interleukin-16 activity in a prokaryotic or eukaryotic host cell can be achieved, said nucleic acid
a) der DNA-Sequenz SEQ ID NO 1 oder einer DNA, die am 5'-Ende um ein Asparagin- saurecodon (GAC) verlängert ist oder ihrem komplementären Strang entspricht, b) mit der DNA der Sequenz SEQ ID NO.l oder einer DNA, die am 5'-Ende um ein Asparaginsaurecodon verlängert ist, unter stringenten Bedingungen hybridisiert, c) oder eine Nukleinsauresequenz, die ohne die Degeneration des genetischen Codes mit den durch a) und b) definierten Nukleinsauresequenzen unter stringenten Bedingungen hybri¬ disieren würde d) und am 5'-Ende für eine der Aminosäuresequenzen SEQ LD NO 7 bis 10 oder für analoge Sequenzen, die N-terminal um eine Asparaginsaure verlängert sind, codierta) the DNA sequence SEQ ID NO 1 or a DNA which is extended at the 5 'end by an aspartic acid codon (GAC) or corresponds to its complementary strand, b) with the DNA of the sequence SEQ ID NO.l or one DNA, which is extended at the 5 'end by an aspartic acid codon, hybridizes under stringent conditions, c) or a nucleic acid sequence which would hybridize under stringent conditions without the degeneration of the genetic code with the nucleic acid sequences defined by a) and b) d) and at the 5 'end for one of the amino acid sequences SEQ LD NO 7 to 10 or for analogous sequences that are N-terminally extended by an aspartic acid are encoded
Vorzugsweise codiert eine solche Nukleinsäure für ein Polypeptid mit der Aminosäuresequenz SEQ LD NO.2 oder für ein Polypeptid mit einer Sequenz, die gegenüber SEQ LD NO.2 N- terminal um ein Asparaginsäurecodon verlängert ist In einer weiteren bevorzugten Ausfuh¬ rungsform codiert die Nukleinsäure für ein Polypeptid mit LL-16- Aktivität, welches am C- Terminus um bis zu 8 Aminosäuren verkürzt istSuch a nucleic acid preferably codes for a polypeptide with the amino acid sequence SEQ LD NO.2 or for a polypeptide with a sequence which is extended by an aspartic acid codon N-terminally compared to SEQ LD NO.2. In a further preferred embodiment, the nucleic acid codes for a polypeptide with LL-16 activity, which is shortened by up to 8 amino acids at the C-terminus
Eine solche Nukleinsäure codiert ein prozessiertes Polypeptid mit IL- 16- Aktivität, besonders bevorzugt naturliches LL-16 von Primaten, wie humanes LL-16 oder LL-16 einer Affenart oder eines anderen Säugetiers, wie z B MausSuch a nucleic acid encodes a processed polypeptide with IL-16 activity, particularly preferably natural LL-16 from primates, such as human LL-16 or LL-16 from a monkey species or another mammal such as a mouse
Es hat sich überraschenderweise gezeigt, daß Fig 2 der WO 94/28134 nicht das korrekt pro¬ zessierte LL-16 beschreibt Das Startcodon "ATG" der precursor-Form des Proteins beginnt nicht mit Nukleotid 783, sondern mit Nukleotid 54 oder 174 Dieser Leserahmen ergibt sich, wenn nach Nukleotid 156 ein A, nach Nukleotid 398 ein C und nach Nukleotid 780 ein G ein¬ gefügt wird Die Sequenz zeigt noch weitere Unterschiede zu Fig 2 der WO 94/28134 Dabei handelt es sich z B um Nukleotidaustausche (313 G in A, 717 C in A) Bei der Expression in eukaryontischen Zellen wird LL-16 prozessiert Dabei entsteht ein Polypeptid gemäß SEQ ID NO.2 und/oder ein Polypeptid mit einer Sequenz, die gegenüber SEQ LD NO:2 N- terminal um eine Asparaginsaure verlängert ist. Die Kenntnis des prozessierten LL-16 erlaubt die Herstellung von LL-16 und Derivaten mit hoher Aktivität und geringer ImmunogenitatIt has surprisingly been found that FIG. 2 of WO 94/28134 does not describe the correctly processed LL-16. The start codon "ATG" of the precursor form of the protein does not begin with nucleotide 783, but with nucleotide 54 or 174 if an A is inserted after nucleotide 156, a C after nucleotide 398 and a G after nucleotide 780. The sequence also shows further differences from FIG. 2 of WO 94/28134. These are, for example, nucleotide exchanges (313 G in A, 717 C in A) In expression in eukaryotic cells, LL-16 is processed. This results in a polypeptide according to SEQ ID NO.2 and / or a polypeptide with a sequence which, compared to SEQ LD NO: 2, is N-terminal aspartic acid is extended. Knowledge of the processed LL-16 allows the production of LL-16 and derivatives with high activity and low immunogenicity
LL-16 kann sich in seiner Sequenz von den von solchen DNA-Sequenzen codierten Protein¬ sequenzen in gewissem Umfang unterscheiden Solche Sequenzvariationen können Amino- saureaustausche, -deletionen oder -additionen sein Vorzugsweise ist die Aminosauresequenz von LL-16 jedoch zu wenigstens 75%, besonders bevorzugt zu wenigstens 90% identisch mit der Aminosauresequenz von SEQ ID NO 2 Varianten von Teilen der Amino- und Nuklein¬ sauresequenz SEQ LD NO 1/SEQ LD NO 2 sind beispielsweise in der WO 96/31607 und den internationalen Patentanmeldungen PCT/EP96/05662 und PCT/EP96/05661 beschrieben Wesentlich ist jedoch, daß die Polypeptide einen korrekten N-Terminus besitzen Bevorzugt sind demnach Proteine, in denen die ersten drei bis zehn Aminosäuren des N-Terminus unver- andert sind und damit N-terminal mit den Aminosauresequenzen SEQ LD NO.6 bis 8 bzw. mit analogen Sequenzen, die N-terminal noch um einen Asparaginsäurerest verlängert sind, begin¬ nen. Ebenfalls bevorzugt sind Proteine, die am C-Terminus um bis zu 8 Aminosäuren verkürzt sindThe sequence of LL-16 can differ to a certain extent from the protein sequences encoded by such DNA sequences. Such sequence variations can be amino acid exchanges, deletions or additions. However, the amino acid sequence of LL-16 is at least 75%, Particularly preferably at least 90% identical to the amino acid sequence of SEQ ID NO 2 Variants of parts of the amino and nucleic acid sequence SEQ LD NO 1 / SEQ LD NO 2 are for example in WO 96/31607 and the international patent applications PCT / EP96 / 05662 and PCT / EP96 / 05661 it is essential, however, that the polypeptides have a correct N-terminus. Preference is therefore given to proteins in which the first three to ten amino acids of the N-terminus are unchanged. are changed and thus begin N-terminally with the amino acid sequences SEQ LD NO.6 to 8 or with analogous sequences which are extended N-terminally by an aspartic acid residue. Proteins that are shortened at the C-terminus by up to 8 amino acids are also preferred
Unter Nukleinsäuren im Sinne der Erfindung sind beispielsweise DNA, RNA und Nukleinsäu- rederivate und -Analoga zu verstehen. Bevorzugte Nukleinsäure-Analoga sind solche Verbin¬ dungen, bei denen das Zuckerphosphatgerüst durch andere Einheiten, wie z.B Aminosäuren, ersetzt ist Solche Verbindungen werden als PNA bezeichnet und sind in der WO 92/20702 beschrieben Da beispielsweise PNA-DNA-Bindungen starker als DNA-DNA-Bindungen sind, sind die nachstehend beschriebenen stringenten Bedingungen für PNA-DNA-Hybridisierung nicht anwendbar Geeignete Hybridisierungsbedingungen sind jedoch in der WO 92/20703 beschriebenFor the purposes of the invention, nucleic acids are understood to mean, for example, DNA, RNA and nucleic acid derivatives and analogs. Preferred nucleic acid analogs are those compounds in which the sugar phosphate skeleton is replaced by other units, such as amino acids. Such compounds are referred to as PNA and are described in WO 92/20702. For example, PNA-DNA bonds are stronger than DNA DNA bonds, the stringent conditions described below for PNA-DNA hybridization are not applicable. Suitable hybridization conditions are, however, described in WO 92/20703
Unter dem Ausdruck "LL- 16" ist im Sinne der Erfindung ein Polypeptid mit der Aktivität von IL-16 zu verstehen Vorzugsweise zeigt LL-16 in dem in der WO 96/31607 beschriebenen Testverfahren die genannte Wirkung oder stimuliert die Zellteilung gemäß WO 94/28134.For the purposes of the invention, the expression “LL-16” is to be understood as meaning a polypeptide with the activity of IL-16. LL-16 preferably shows the stated effect in the test method described in WO 96/31607 or stimulates cell division according to WO 94 / 28134.
LL-16 bindet an CD4+ Lymphozyten und kann die Replikation von Viren, wie beispielsweise HTV-1, HIV-2 und SIV supprimieren Die Funktion von LL-16 ist nicht durch seine Präsenta¬ tion im MHC-Komplex limitiertLL-16 binds to CD4 + lymphocytes and can suppress the replication of viruses such as HTV-1, HIV-2 and SIV. The function of LL-16 is not limited by its presentation in the MHC complex
Insbesondere zeigt LL-16 eine oder mehrere der folgenden EigenschaftenIn particular, LL-16 exhibits one or more of the following properties
Bindung an T-Zellen über den CD4-Rezeptor,Binding to T cells via the CD4 receptor,
Stimulierung der Expression von LL-2-Rezeptor und/oder HLA-DR-Antigen auf CD4+-Lymphozyten,Stimulation of the expression of LL-2 receptor and / or HLA-DR antigen on CD4 + lymphocytes,
Stimulierung der Proliferation von T-Helferzellen in Gegenwart von IL-2,Stimulating the proliferation of T helper cells in the presence of IL-2,
Suppression der Proliferation von mit Anti-CD3 -Antikörpern stimulierten T-Suppression of proliferation of T- stimulated with anti-CD3 antibodies
Helferzellen,Helper cells,
Suppression der Replikation von Viren, vorzugsweise von HIV- 1 , HIV-2 oderSuppression of replication of viruses, preferably HIV-1, HIV-2 or
SIVSIV
Bevorzugt sind Nukleinsäuren, die unter stringenten Bedingungen mit Nukleinsäuren der Sequenz SEQ LD NO 1 hybridisieren Der Ausdruck "unter stringenten Bedingungen hybridi¬ sieren" bedeutet, daß zwei Nukleinsaurefragmente unter standardisierten Hybridisierungs- bedingungen miteinander hybridisieren, wie beispielsweise beschrieben 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 Solche Bedingungen sind beispiels¬ weise Hybridisierung in 6,0 x SSC bei etwa 45°C, gefolgt durch einen Waschschritt bei 2 x SSC bei 50°C Zur Auswahl der Stringenz kann die Salzkonzentration im Waschschritt beispielsweise zwischen 2,0 x SSC bei 50°C für geringe Stringenz und 0,2 x SSC bei 50°C für hohe Stringenz gewählt werden Zusatzlich kann die Temperatur des im Waschschritt zwi¬ schen Raumtemperatur, ca 22°C, für gennge Stringenz und 65°C bei hoher Stringenz variiert werdenPreferred are nucleic acids which hybridize under stringent conditions with nucleic acids of the sequence SEQ LD NO 1. The expression "hybridize under stringent conditions" means that two nucleic acid fragments under standardized hybridization Hybridize conditions with one another, 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 FIG. 6 , 0 x SSC at about 45 ° C, followed by a washing step at 2 x SSC at 50 ° C. To select the stringency, the salt concentration in the washing step can be, for example, between 2.0 x SSC at 50 ° C for low stringency and 0.2 x SSC at 50 ° C can be selected for high stringency. In addition, the temperature of the wash step between room temperature, about 22 ° C, for narrow stringency and 65 ° C for high stringency can be varied
LL-16 wird vorzugsweise rekombinant in prokaryontischen oder eukaryontischen Wirtszellen hergestellt Derartige Herstellverfahren sind beispielsweise beschrieben in WO 94/28134 und WO 96/31607, die auch hierfür Gegenstand der Offenbarung der vorliegenden Erfindung sind Um allerdings die erfindungsgemaßen Formen von LL-16 durch rekombinante Herstellung definiert und reproduzierbar zu erhalten, müssen über die dem Fachmann gelaufigen Verfahren zur rekombmanten Herstellung zusatzliche Maßnahmen ergriffen werdenLL-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 the subject matter of the disclosure of the present invention for this purpose. However, the forms of LL-16 according to the invention by recombinant production To be defined and reproducible, additional measures must be taken via the recombinant production methods familiar to those skilled in the art
Die Herstellung von rekombmantem LL-16 kann nach den dem Fachmann gelaufigen Metho¬ den als heterologe Expression oder als homologe Expression (nach homologer Rekombination der LL16 Nukleinsäure ins Genom des Wirtsorganismus) erfolgen Dazu wird zunächst eine DNA hergestellt, welche in der Lage ist, ein Protein zu produzieren, welches die Aktivität von LL-16 besitzt Die DNA wird in einen Vektor kloniert, der in eine Wirtszelle transferiert werden kann und dort replizierbar ist Ein derartiger Vektor enthalt zusatzlich zur IL-16- Sequenz Regulator-Elemente, die zur Expression der DNA-Sequenz erforderlich sind Dieser Vektor, der die LL-16-Sequenz und die Regulator-Elemente enthalt, wird in einen Vektor transferiert, der in der Lage ist, die DNA von LL-16 zu exprimieren Die Wirtszelle wird unter Bedingungen, die zur Amplifikation des Vektors geeignet sind, kultiviert und LL-16 gewon¬ nen Dabei wird durch geeignete Maßnahmen sichergestellt, daß das Protein eine aktive tertiä¬ re Struktur einnehmen kann, in der es LL- 16-Eιgenschaften zeigtRecombinant LL-16 can be produced according to the methods familiar to the person skilled in the art as heterologous expression or as homologous expression (after homologous recombination of the LL16 nucleic acid into the genome of the host organism). First of all, a DNA is produced which is able to To produce protein which has the activity of LL-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, in addition to the IL-16 sequence, regulatory elements which are necessary for the expression of the DNA sequence is required. This vector, which contains the LL-16 sequence and the regulatory elements, is transferred into a vector which is capable of expressing the DNA of LL-16 Amplification of the vector are suitable, cultivated and LL-16 won. Suitable measures ensure that the protein is an active tertiary str structure in which it shows LL-16 properties
Auch die Nukleinsauresequenz des Proteins kann modifiziert sein Derartige Modifikationen sind beispielsweiseThe nucleic acid sequence of the protein can also be modified. Such modifications are, for example
- Veränderung der Nukleinsäure, um verschiedene Erkennungssequenzen von Restπktionsenzymen zur Erleichterung der Schritte der Ligation, Klonierung und Mutagenese einzuführen - Veränderung der Nukleinsäure zum Einbau von bevorzugten Codons für die Wirtszelle- Modification of the nucleic acid in order to introduce different recognition sequences of residual enzymes to facilitate the steps of ligation, cloning and mutagenesis - Change the nucleic acid to incorporate preferred codons for the host cell
- Ergänzung der Nukleinsäure um zusätzliche Operator-Elemente, um die Expres¬ sion in der Wirtszelle zu optimieren.Supplementing the nucleic acid with additional operator elements in order to optimize the expression in the host cell.
Die Expression des Proteins erfolgt vorzugsweise in Mikroorganismen, insbesondere in Pro- karyonten, und dort in E. coli. Die Expression in Prokaryonten führt zu einem unglycosylier- ten Polypeptid.The protein is preferably expressed in microorganisms, in particular in prokaryotes, and there in E. coli. Expression in prokaryotes leads to an unglycosylated polypeptide.
Die Expressionsvektoren müssen einen Promotor enthalten, der die Expression des Proteins im Wirtsorganismus erlaubt. Derartige Promotoren sind dem Fachmann bekannt und sind bei¬ spielsweise lac Promotor (Chang et al., Nature 198 (1977) 1056), trp Promotor (Goeddel et al., Nuc. Acids Res. 8 (1980) 4057), λpj_ Promotor (Shimatake et al., Nature 292 (1981) 128) und T5 Promotor (US-Patent Nr. 4,689,406). Ebenfalls geeignet sind synthetische Promoto¬ ren wie beispielsweise tac Promotor (US-Patent Nr. 4,551,433). Ebenso geeignet sind ge¬ koppelte Promotorsysteme wie beispielsweise T7-RNA-Polymerase/Promotorsystem (Studier et al., J. Mol. Biol. 189 (1986) 1 13). Ebenso geeignet sind hybride Promotoren aus einem Bacteriophagen-Promotor und der Operator-Region des Mikroorganismus (EP-A 0267 851). Zusätzlich zum Promotor ist eine effektive Ribosomenbindungsstelle erforderlich. Für E. coli wird diese Ribosomenbindungsstelle als Shine-Dalgarno (SD)-Sequenz bezeichnet (Sambrook et al., "Expression of cloned genes in E. coli" in Molecular Cloning. A laboratory manualThe expression vectors must contain a promoter which allows expression of the protein in the host organism. Such promoters are known to the person skilled in the art and are, for example, lac promoter (Chang et al., Nature 198 (1977) 1056), trp promoter (Goeddel et al., Nuc. Acids Res. 8 (1980) 4057), λpj_ 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 Pat. No. 4,551,433) are also suitable. Coupled promoter systems such as, for example, T7 RNA polymerase / promoter system (Studier et al., J. Mol. Biol. 189 (1986) 13) are also suitable. Hybrid promoters consisting of a bacteriophage promoter and the operator region of the microorganism (EP-A 0267 851) are also suitable. An effective ribosome binding site is required in addition to the promoter. For 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).(1989) Cold Spring Harbor Laboratory Press, New York, USA).
Zur Verbesserung der Expression ist es möglich, das Protein als Fusionsprotein zu exprimie¬ ren. In diesem Fall wird üblicherweise eine DNA-Sequenz, welche für den N-terminalen Teil eines endogenen bakteriellen Proteins oder ein anderes stabiles Protein codiert, an das 5'-Ende der für LL-16 codierenden Sequenz fusioniert. Beispiele hierfür sind beispielsweise lacZ (Phillips and Silhavy, Nature 344 (1990) 882-884), trpE (Yansura, Meth. Enzymol. 185To improve the 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 attached to the 5'- End of the sequence coding for LL-16 fused. Examples include lacZ (Phillips and Silhavy, Nature 344 (1990) 882-884), trpE (Yansura, Meth. Enzymol. 185
(1990) 161 - 166).(1990) 161-166).
Nach Expression des Vektors, vorzugsweise eines biologisch fünktionellen Plasmids oder eines viralen Vektors, werden die Fusionsproteine vorzugsweise mit Enzymen (z.B. Faktor Xa) gespalten (Nagai et al„ Nature 309 (1984) 810). Weitere Beispiele für Spaltstellen sind die IgA-Protease-Spaltstelle (WO 91/11520, EP-A 0495 398), die Ubiquitin-Spaltstelle (Miller et al., Bio/Technology 7 (1989) 698) und die Enterokinase-Spaltstelle. Die auf diese Weise in Bakterien exprimierten Proteine werden durch Aufschluß der Bakterien und Proteinisolierung in üblicher Weise gewonnenAfter expression of the vector, preferably a biologically functional plasmid or a viral vector, the fusion proteins are preferably cleaved with enzymes (eg 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 0495 398), the ubiquitin cleavage site (Miller et al., Bio / Technology 7 (1989) 698) and the enterokinase cleavage site. The proteins expressed in this way in bacteria are obtained in a conventional manner by digesting the bacteria and isolating the proteins
In einer weiteren Ausführungsform ist es möglich, die Proteine als aktive Proteine aus den Mikroorganismen zu sezernieren Hierzu wird vorzugsweise ein Fusionsprodukt verwendet, welches aus der Signalsequenz, die für die Sekretion von Proteinen in den verwendeten Wirts¬ organismen geeignet ist, und der Nukleinsäure, welche für das Protein codiert, besteht. Das Protein wird dabei entweder in das Medium (bei grampositiven Bakterien) oder in den peri- plasmatischen Raum (bei gramnegativen Bakterien) sezerniert Zwischen der Signalsequenz und der für LL-16 codierenden Sequenz ist zweckmäßig eine Spaltstelle angebracht, die ent¬ weder bei der Prozessierung oder in einem zusätzlichen Schritt die Abspaltung des Proteins erlaubt. Derartige Signalsequenzen stammen beispielsweise von ompA (Ghrayeb et al , EMBO J. 3 (1984) 2437), phoA (Oka et al., Proc Natl Acad. Sei. USA 82 (1985) 7212).In a further embodiment it is possible to secrete the proteins as active proteins from the microorganisms. For this purpose, a fusion product is preferably used which consists of the signal sequence which is suitable for the secretion of proteins in the host organisms used and the nucleic acid which encoded for the protein. The protein is either secreted into the medium (in the case of gram-positive bacteria) or in the periplasmic space (in the case of gram-negative bacteria). Between the signal sequence and the sequence coding for LL-16, a cleavage site is expediently located, either during processing or in an additional step allows the protein to be split off. Such signal sequences originate, for example, from ompA (Ghrayeb et al, EMBO J. 3 (1984) 2437), phoA (Oka et al., Proc Natl Acad. Sci. USA 82 (1985) 7212).
Zusatzlich enthalten die Vektoren noch Terminatoren Terminatoren sind DNA-Sequenzen, die das Ende eines Transkriptionsvorganges signalisieren Sie zeichnen sich meist durch zwei strukturelle Eigenarten aus eine umgekehrt repetitive G/C-reiche Region, die intramolekular eine Doppelhelix bilden kann, sowie eine Anzahl von U(bzw T)-Resten Beispiele sind der Haupt-Terminator in der DNA des Phagen fd (Beck und Zink, Gene 16 (1981) 35-58) sowie rrnB (Brosius et al , J Mol. Biol 148 (1981) 107 - 127)In addition, the vectors also contain terminators. Terminators are DNA sequences that signal the end of a transcription process.They are usually characterized by two structural characteristics: a region that is rich in reversed G / C, which can form a double helix intramolecularly, and a number of U ( or T) residues Examples are the main terminator in the DNA of phage fd (Beck and Zink, Gene 16 (1981) 35-58) and rrnB (Brosius et al, J Mol. Biol 148 (1981) 107-127)
Zusatzlich enthalten die Expressionsvektoren üblicherweise einen selektierbaren Marker, um transformierte Zellen zu selektieren Derartige selektierbare Marker sind beispielsweise die Resistenzgene für Ampicillin, Chloramphenicol, Erythromycin, Kanamycin, Neomycin und Tetracyclin (Davies et al., Ann Rev Microbiol 32 (1978) 469) Ebenso geeignete selektier¬ bare Marker sind die Gene für essentielle Substanzen der Biosynthese von für die Zelle not¬ wendigen Stoffen wie z B Histidin, Tryptophan und LeucinIn 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 tetracycline (Davies et al., Ann Rev Microbiol 32 (1978) 469) Selectable markers are the genes for essential substances of the biosynthesis of substances necessary for the cell, such as, for example, histidine, tryptophan and leucine
Es sind eine Vielzahl von geeigneten bakteriellen Vektoren bekannt Beispielsweise sind für die folgenden Bakterien Vektoren beschrieben Bacillus subtilis (Palva et al., Proc Natl Acad Sei. 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 , Appl Environ Microbiol 54 (1988) 655), Streptococcus lividans und Streptomyces lividans (US-Patent Nr 4,747,056) Weitere gentechnologische Verfahren zur Herstellung und Expression von geeigneten Vekto¬ ren sind in J. Sambrook et al., Molecular Cloning a laboratory manual (1989), Cold Spring Harbor Laboratory Press, New York, N. Y. beschrieben.A variety of suitable bacterial vectors are known. For example, vectors are described for the following bacteria: Bacillus subtilis (Palva et al., Proc Natl Acad Sei. 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, Appl Environ Microbiol 54 (1988) 655), Streptococcus lividans and Streptomyces lividans (U.S. Patent No. 4,747,056) Further genetic engineering processes for the production and expression of suitable vectors are described in J. Sambrook et al., Molecular Cloning a laboratory manual (1989), Cold Spring Harbor Laboratory Press, New York, NY.
Eine Expression von rekombinantem LL-16 ist außer in prokaryontischen Mikroorganismen auch in Eukaryonten (wie beispielsweise CHO-Zellen, Hefe oder Insektenzellen) möglich Als eukaryontisches Expressionssystem wird das Hefesystem oder Insektenzellen bevorzugt. Die Expression in Hefe kann über drei Arten von Hefevektoren (integrierende YIp (yeast integra- ting plasmids)- Vektoren, replizierende YRp (yeast replicon plasmids)- Vektoren und episomale YEp (yeast episomal plasmids)- Vektoren) erfolgen Näheres hierzu ist beispielsweise in S.M. Kingsman et al, Tibtech 5 (1987) 53-57 beschriebenIn addition to prokaryotic microorganisms, expression of recombinant LL-16 is also possible in eukaryotes (such as, for example, CHO cells, yeast or insect cells). The yeast system or insect cells is preferred as the eukaryotic expression system. Expression in yeast can be via three types of yeast vectors (integrating YIp (yeast integrating plasmids) - vectors, replicating YRp (yeast replicon plasmids) - vectors and episomal YEp (yeast episomal plasmids) - vectors). Kingsman et al, Tibtech 5 (1987) 53-57
Ein weiterer Gegenstand der Erfindung ist eine prokaryontische oder eukaryontische Wirts¬ zelle, welche mit einer Nukleinsäure, welche für ein erfindungsgemäßes LL-16-Polypeptid codiert, so transformiert oder transfiziert ist, daß die Wirtszelle das genannte Polypeptid exprimiert Eine solche Wirtszelle enthalt üblicherweise einen biologisch fünktionellen NukJeinsaurevektor, vorzugsweise einen DNA-Vektor, eine Plasmid-DNA, welche diese Nukleinsäure enthaltThe invention further relates to a prokaryotic or eukaryotic host cell which has been transformed or transfected with a nucleic acid which codes for an LL-16 polypeptide according to the invention in such a way that the host cell expresses the said polypeptide. Such a host cell usually contains a biological one functional nucleic acid vector, preferably a DNA vector, a plasmid DNA, which contains this nucleic acid
Weiter bevorzugt ist ein monomeres LL- 16-Polypeptid, welches nicht in weitere Untereinhei¬ ten aufspaltbar istA monomeric LL-16 polypeptide which cannot be split into further subunits is further preferred
Es hat sich überraschenderweise gezeigt, daß die in der WO 94/28134 beschriebene Nuklein¬ säure und Proteinsequenz von LL-16 nicht den natürlichen humanen Sequenzen entsprechen Es handelt sich hier lediglich um ein nicht natürliches LL-16-Analoges Für eine therapeutische Anwendung ist es jedoch bevorzugt, ein Protein zu verwenden, welches entweder mit dem natürlichen Protein identisch ist oder sich vom natürlichen Protein nur geringfügig unterschei¬ det und/oder mindestens vergleichbare Aktivität und Immunogenität zeigt. Die Sequenz des Proteins ist in SEQ LD NO.2 (gegebenenfalls mit N-terminaler Verlängerung um einen Asparaginsaurerest und/oder Verkürzung am C-Terminus um bis zu 8 Aminosäuren) beschrie¬ benIt has surprisingly been found that the nucleic acid and protein sequence of LL-16 described in WO 94/28134 do not correspond to the natural human sequences. It is merely a non-natural LL-16 analog. It is for therapeutic use however, it is preferred to use a protein which is either identical to the natural protein or differs only slightly from the natural protein and / or shows at least comparable activity and immunogenicity. The sequence of the protein is described in SEQ LD NO.2 (optionally with an N-terminal extension by an aspartic acid residue and / or shortening at the C-terminus by up to 8 amino acids)
Die Nukleinsauresequenz von LL-16 kann im Rahmen der Erfindung Deletionen, Mutationen und Additionen erhalten. Die monomere Form von LL-16 kann in einer bevorzugten Ausfüh- rungsform multimerisiert sein Dadurch kann die Aktivität von LL-16 gesteigert werden. Vor¬ zugsweise sind solche multimeren Formen dimere, tetramere oder oktamere Formen In einer weiteren Ausführungsform können die Polypeptide der Erfindung zusätzlich einen definierten Gehalt an Metallionen enthalten, wobei die Anzahl der Metallionen pro Unterein¬ heit vorzugsweise 0,5 bis 2 beträgt.The nucleic acid sequence of LL-16 can receive deletions, mutations and additions within the scope of the invention. In a preferred embodiment, the monomeric form of LL-16 can be multimerized. This allows the activity of LL-16 to be increased. Such multimeric forms are preferably dimeric, tetrameric or octameric forms In a further embodiment, the polypeptides of the invention can additionally contain a defined content of metal ions, the number of metal ions per subunit preferably being 0.5 to 2.
Als Metallionen im Sinne der Erfindung sind eine Vielzahl von Metallionen geeignet. Wie sich gezeigt hat, sind sowohl Erdalkalimetalle als auch Elemente der Nebengruppen geeignet. Besonders geeignet sind Erdalkalimetalle, Kobalt, Zink, Selen, Mangan, Nickel, Kupfer, Eisen, Magnesium, Kalzium, Molybdän und Silber. Die Ionen können ein-, zwei-, drei- oder vierwer- tig sein. Besonders bevorzugt werden zweiwertige Ionen. Die Ionen werden vorzugsweise als Lösungen zugesetzt von MgCl2, CaC^, MnCl2, BaCl2, LiC^, Sr(N03)2, Na2Moθ4, AgCi2.A large number of metal ions are suitable as metal ions in the sense of the invention. As has been shown, both alkaline earth metals and elements of the subgroups are suitable. Alkaline earth metals, cobalt, zinc, selenium, manganese, nickel, copper, iron, magnesium, calcium, molybdenum and silver are particularly suitable. The ions can be one, two, three or four-valued. Divalent ions are particularly preferred. The ions are preferably added as solutions of MgCl2, CaC ^, MnCl2, BaCl2, LiC ^, Sr (N03) 2, Na2Moθ4, AgCi2.
Solche multimeren Formen und metallionenhaltige Formen von IL- 16 sind in der internationa¬ len Patentanmeldung PCT/EP96/05661 beschrieben.Such multimeric forms and forms of IL-16 containing metal ions are described in the international patent application PCT / EP96 / 05661.
Das erfindungsgemäße Polypeptid kann dadurch hergestellt werden, daß eine prokaryontische oder eukaryontische Wirtszelle, die mit einer Nukleinsauresequenz nach den Ansprüchen 1 oder 2 transformiert oder transfiziert ist, unter geeigneten Nährbedingungen kultiviert wird und das gewünschte Polypeptid gegebenenfalls isoliert wird. Falls die Herstellung des Poly- peptids im Rahmen einer gentherapeutischen Behandlung in vivo erfolgen soll, wird das Poly¬ peptid natürlich nicht aus der Zelle isoliert.The polypeptide according to the invention can be prepared by culturing a prokaryotic or eukaryotic host cell which has been transformed or transfected with a nucleic acid sequence according to claims 1 or 2 under suitable nutrient conditions and, if appropriate, isolating the desired polypeptide. If the preparation of the polypeptide is to take place in vivo as part of a gene therapy treatment, the polypeptide is of course not isolated from the cell.
Ein weiterer Gegenstand der Erfindung ist eine pharmazeutische Zusammensetzung, die ein erfindungsgemäßes Polypeptid in einer für eine therapeutische Anwendung ausreichenden Menge und/oder spezifische Aktivität sowie gegebenenfalls ein pharmazeutisch geeignetes Verdünnungsmittel, Adjuvans und/oder Trägermittel enthält.The invention further relates to a pharmaceutical composition which contains a polypeptide according to the invention in an amount and / or specific activity sufficient for therapeutic use and, if appropriate, a pharmaceutically suitable diluent, adjuvant and / or carrier.
Die erfindungsgemäßen Polypeptide sind insbesondere geeignet zur Behandlung von patholo¬ gischen Zuständen, die durch virale Replikation, insbesondere retrovirale Replikation, verur¬ sacht sind, und zur Immunmodulation. Derartige therapeutische Anwendungen sind in der WO 96/31607 beschrieben. Hierin sind ebenfalls auch diagnostische Testverfahren beschrie¬ ben.The polypeptides according to the invention are particularly suitable for the treatment of pathological conditions which are caused by viral replication, in particular retroviral replication, and for immunomodulation. Such therapeutic applications are described in WO 96/31607. Diagnostic test methods are also described therein.
Vorzugsweise können die erfindungsgemäßen Polypeptide zur Immunsuppression verwendet werden. Diese Immunsuppression erfolgt vorzugsweise über eine Hemmung der Helferfunk¬ tion der THo und/oder THi und/oder TH2 Zellen. Die erfindungsgemäßen Polypeptide sind demnach bei allen Erkrankungen von therapeutischem Wert, bei denen eine immundisregulato- O 97/41231 PCΪYEP97/02216The polypeptides according to the invention can preferably be used for immunosuppression. This immunosuppression is preferably carried out by inhibiting the helper function of the THo and / or THi and / or TH2 cells. The polypeptides according to the invention are accordingly of therapeutic value in all diseases in which an immunodisregulated O 97/41231 PCΪYEP97 / 02216
rische Komponente bei der Pathogenese postuiert wird, insbesondere eine Hyperimmunität. Als LL-16-therapierbare Erkrankungen können in der Kardiologie/Angiologie Erkrankungen wie Myokarditis, Endokarditis und Perikarditis in Frage kommen, in der Pulmonologie bei¬ spielsweise Bronchitis, Asthma, in der Hämatologie Autoimmunneuropenien und Transplan- tatabstoßung, in der Gastroenterologie chronische Gastritis, in der Endokrinologie Diabetes mellitus Typ I, in der Nephrologie Glomerulonephritis, Erkrankungen im rheumatischen Formenkreis, Erkrankungen in der Ophthalmologie, in der Neurologie, wie Multiple Sklerose, in der Dermatologie Ekzeme. Insbesondere können die erfindungsgemäßen Polypeptide bei Autoimmunerkrankungen, Allergien und zur Vermeidung von Transplantatabstoßungen ver¬ wendet werden.component in the pathogenesis is postulated, in particular hyperimmunity. Diseases such as myocarditis, endocarditis and pericarditis can be considered as LL-16-treatable diseases in cardiology / angiology, in bronchitis, for example, bronchitis, asthma in hematology, autoimmune Europe and transplant rejection, and chronic gastritis in gastroenterology in endocrinology diabetes mellitus type I, in nephrology glomerulonephritis, diseases in the rheumatic range, diseases in ophthalmology, in neurology, such as multiple sclerosis, in dermatology eczema. In particular, the polypeptides according to the invention can be used in autoimmune diseases, allergies and to avoid transplant rejections.
Ein weiterer Gegenstand der Erfindung ist die Verwendung der erfindungsgemäßen Nuklein¬ säuren im Rahmen der Gentherapie. Hierfür geeignete Vektorsysteme sind beispielsweise retrovirale oder nicht-virale Vektorsysteme.Another object of the invention is the use of the nucleic acids according to the invention in the context of gene therapy. Suitable vector systems for this are, for example, retroviral or non-viral vector systems.
Ein weiterer Gegenstand der Erfindung ist ein polyklonaler oder monoklonaler Anti-LL-16- Antikörper oder ein immunoaktives Fragment davon, welcher an die ersten 3-20 Aminosäuren von SEQ ID NO.2 oder einer N-terminal um einen Asparaginsäurerest verlängerten SEQ ID NO: 2 bindet, sowie Verfahren zur Herstellung von solchen Antikörpern und ihre Verwendung zur Bestimmung von LL-16 sowie zur Bestimmung von viralen Infektionen in eukaryontischen Zellen und insbesondere in Säugerzellmaterial. Mit LL-16 können auch Virus¬ aktivierte Säugerzellen, insbesondere T Zellen, bestimmt werden. Die Herstellung solcher Antikörper erfolgt durch Immunisierung mit einem erfindungsgemäßen Polypeptid. Die Her¬ stellung eines solchen Antikörpers wird nach den dem Fachmann geläufigen Verfahren durchgeführt, durch Immunisierung mit einem Immunogen, welches die ersten 3-20 Aminosäuren von SEQ ID NO: 2 oder einer N-terminal um einen Asparaginsäurerest verlän¬ gerten SEQ ID NO: 2 als Hapten enthält. Anschließend kann der Antikörper aus dem immuni¬ sierten Säugetier in üblicher Weise gewonnen werden und gegebenenfalls ein monoklonaler Antikörper hergestellt werden.Another object of the invention is a polyclonal or monoclonal anti-LL-16 antibody or an immunoactive fragment thereof which SEQ ID NO is extended to the first 3-20 amino acids of SEQ ID NO.2 or an N-terminal by an aspartic acid residue: 2 binds, as well as methods for the production of such antibodies and their use for the determination of LL-16 and for the determination of viral infections in eukaryotic cells and in particular in mammalian cell material. Virus-activated mammalian cells, in particular T cells, can also be determined with LL-16. Such antibodies are produced by immunization with a polypeptide according to the invention. The production of such an antibody is carried out according to the methods familiar to the person skilled in the art, by immunization with an immunogen which extends the first 3-20 amino acids of SEQ ID NO: 2 or an N-terminally SEQ ID NO: Contains 2 as hapten. The antibody can then be obtained from the immunized mammal in a conventional manner and, if appropriate, a monoclonal antibody can be produced.
Die folgenden Beispiele und Publikationen sowie das Sequenzprotokoll erläutern die Erfin¬ dung, deren Schutzumfang sich aus den Patentansprüchen ergibt, weiter. Die beschriebenen Verfahren sind als Beispiele zu verstehen, die auch noch nach Modifikationen den Gegenstand der Erfindung beschreiben. Beispiel 1The following examples and publications as well as the sequence listing further explain the invention, the scope of protection of which results from the patent claims. The described methods are to be understood as examples which describe the subject matter of the invention even after modifications. example 1
Klonierung, Expression und Reinigung von LL-16Cloning, Expression and Purification of LL-16
1.1 RNA Isolierung1.1 RNA isolation
5 x lO^PBMC (vom Menschen oder Affen) wurden 48 Stunden mit 10 μg/ml Concanavalin A und 180 U/ml LL-2 kultiviert Zur Herstellung der RNA wurden die Zellen einmal mit PBS gewaschen und anschließend mit 5 ml Denaturierungslosung (RNA Isolation Kit, Stratagene) lysiert. Nach Zugabe von 1 ml Na-Acetat, 5 ml Phenol und 1 ml Chloroform/Isoamyl-Alkohol (24 1) wurde das Lysat 15 min auf Eis gehalten Die wäßrige Phase wurde anschließend mit5 x 10 ^ PBMC (from humans or monkeys) were cultivated for 48 hours with 10 μg / ml concanavalin A and 180 U / ml LL-2. To prepare the RNA, the cells were washed once with PBS and then with 5 ml denaturation solution (RNA isolation Kit, Stratagene) lysed. After the addition of 1 ml of Na acetate, 5 ml of phenol and 1 ml of chloroform / isoamyl alcohol (24 l), the lysate was kept on ice for 15 min. The aqueous phase was then washed with
6 ml Isopropanol vermischt, um die RNA auszufällen, und 2 Stunden bei -20°C gelagert Das Prazipitat wurde schließlich einmal mit reinem Ethanol gewaschen und in 150 μl H2O gelost Die Ausbeute wurde photometπsch bestimmt und betrug 120 μg6 ml of isopropanol mixed 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 H2O. The yield was determined photometrically and was 120 μg
1.2 cDNA Synthese1.2 cDNA synthesis
Die Mischung für die cDNA Synthese enthielt 10 μg RNA, 0,2 μg Oiigo-dT, 13 mM DTT und 5 μl "bulk first Strand reaction mix" (First-Strand cDNA Synthesis Kit, Pharmacia) in einer Menge von 15 μl Die Mischung wurde 1 Stunde bei 37°C inkubiert und anschließend bei -20°C zur spateren Verwendung gelagertThe mixture for the cDNA synthesis contained 10 μg RNA, 0.2 μg Oiigo-dT, 13 mM DTT and 5 μl “bulk first strand reaction mix” (first-strand cDNA synthesis kit, Pharmacia) in an amount of 15 μl the mixture was incubated for 1 hour at 37 ° C and then stored at -20 ° C for later use
Amplifikation, Klonierung von LL-16 cDNA und Herstellung eines Expressionsklons erfolgen wie in der WO 94/28134 oder WO 96/31607 beschπeben, unter Berücksichtigung der verän¬ derten SequenzenAmplification, cloning of LL-16 cDNA and production of an expression clone are carried out as described in WO 94/28134 or WO 96/31607, taking into account the changed sequences
1.3 10 I Fermentation eines E. coli Expressionsklons für IL-16 und Hochdruckauf¬ schluß1.3 10 l fermentation of an E. coli expression clone for IL-16 and high pressure digestion
Aus Stammkulturen (Plattenausstrich oder bei -20°C gelagerten Ampullen) werden Vorkultu¬ ren angesetzt, die geschüttelt bei 37°C inkubiert werden Das Uberimpfvolumen in die nächst¬ höhere Dimension betragt jeweils 1-10 Vol -% Zur Selektion gegen Plasmidverlust wird in Vor- und Hauptkultur Ampicillin (50-100 mg/1) eingesetztPre-cultures are prepared from stock cultures (plate smear or ampoules stored at -20 ° C), which are shaken and incubated at 37 ° C. The inoculation volume into the next higher dimension is 1-10% by volume. For selection against plasmid loss, in advance - and main culture Ampicillin (50-100 mg / 1) used
Als Nährstoffe werden enzymatisch verdautes Eiweiß und/oder Hefeextrakt als N- und C- Quelle sowie Glycerin und/oder Glucose als zusatzliche C-Quelle verwendet Das Medium wird auf pH 7 gepuffert und Metallsalze werden zur Stabilisierung des Fermentationsprozesses in physiologisch vertraglichen Konzentrationen zugesetzt Die Fermentation wird als Feed- batch mit einer gemischten Hefeextrakt/C-Quellen-Dosage durchgeführt Die Fermentations- temperatur beträgt 25-37°C. Über Belüftungsrate, Drehzahlregulierung und Dosagege- schwindigkeit wird der gelöste Sauerstoffpartialdruck (pθ2) in etwa unter 20% gehalten. Das Wachstum wird über Ermittlung der optischen Dichte (OD) bei 528 nm bestimmt. Mittels IPTG wird die Expression des LL-16 induziert. Nach einer Fermentationsdauer von 10 bis 20 Stunden wird bei OD-Stillstand die Biomasse durch Zentrifügation geerntet.The nutrients used are enzymatically digested protein and / or yeast extract as an N and C source, and glycerol and / or glucose as an additional C source. The medium is buffered to pH 7 and metal salts are added to stabilize the fermentation process in physiologically acceptable concentrations. The fermentation is carried out as a feed batch with a mixed yeast extract / C source dosage. The fermentation temperature is 25-37 ° C. The dissolved oxygen partial pressure (pθ2) is kept below about 20% via aeration rate, speed regulation and dosage speed. The growth is determined by determining the optical density (OD) at 528 nm. Expression of the LL-16 is induced by means of IPTG. After a fermentation period of 10 to 20 hours, the biomass is harvested by centrifugation when the OD is at a standstill.
Die Biomasse wird in 50 mM Natriumphosphat, 5 mM EDTA, 100 mM Natriumchlorid, pH 7 aufgenommen und über eine kontinuierliche Hochdruckpresse bei 1000 Bar aufgeschlossen. Die so erhaltene Suspension wird erneut abzentrifügiert und der Überstand, der das gelöste LL-16 enthält, wird weiterverarbeitet.The biomass is taken up in 50 mM sodium phosphate, 5 mM EDTA, 100 mM sodium chloride, pH 7 and digested using a continuous high-pressure press at 1000 bar. The suspension thus obtained is centrifuged again and the supernatant, which contains the dissolved LL-16, is processed further.
1.4 Reinigung von rekombinantem humanem LL-161.4 Purification of Recombinant Human LL-16
550 ml Aufschlußüberstand in 50 mM Natriumphosphat, 5 mM EDTA, 100 mM NaCl, pH 7,2 wurden mit 55 ml 5 M NaCl, 60 mM MgCl2, pH 8,0 versetzt, 30 min. gerührt und an¬ schließend 30 min. bei 20.000 g zentrifügiert. 400 ml des Überstandes wurden auf eine Nickel- Chelat-Sepharose-Säule (V=60 ml; Pharmacia) aufgezogen, die vorher mit 30 μMol NiCVml Gel beladen und mit 50 mM Natriumphosphat, 0,2 M NaCl, pH 8,0 äquilibriert worden war. Die Säule wurde anschließend mit 300 ml 50 mM Natriumphosphat, 0,5 M NaCl, pH 7,0 gespült und das LL- 16-Fusionsprotein dann mit einem Gradienten von 0 M bis 300 mM Imida- zol, pH 7,0 in 50 mM Natriumphosphat, 0,1 M NaCl, pH 7,0 (2x 0,5 I Gradientenvolumen) eluiert. LL-16 enthaltende Fraktionen wurden mittels SDS-PAGE identifiziert, vereinigt und gegen 20 mM Natriumphosphat, pH 7,0 dialysiert.550 ml of digestion supernatant in 50 mM sodium phosphate, 5 mM EDTA, 100 mM NaCl, pH 7.2 were mixed with 55 ml of 5 M NaCl, 60 mM MgCl 2 , pH 8.0, 30 min. stirred and then 30 min. centrifuged at 20,000 g. 400 ml of the supernatant were applied to a nickel-chelate-Sepharose column (V = 60 ml; Pharmacia), which had previously been loaded with 30 μmol NiCVml gel and equilibrated with 50 mM sodium phosphate, 0.2 M NaCl, pH 8.0 was. The column was then rinsed with 300 ml of 50 mM sodium phosphate, 0.5 M NaCl, pH 7.0 and the LL-16 fusion protein then 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 (2x 0.5 I gradient volume) eluted. Fractions containing LL-16 were identified by SDS-PAGE, pooled and dialyzed against 20 mM sodium phosphate, pH 7.0.
300 mg des so erhaltenen Fusionsproteins wurden bei 4°C gegen 20 1 20 mM Imidazol, pH 5,5 dialysiert und anschließend zur Entfernung von Trübungen 30 min. bei 20.000 g zentrifügiert. Der Überstand der Zentrifügation wurde anschließend mit NaOH auf pH 8,5 eingestellt, mit 0,3 mg Thrombin (Boehringer Mannheim GmbH) versetzt und 4 Stunden bei 37°C inkubiert. Anschließend wurde der Spaltansatz mit HC1 auf pH 6,5 und die Leitfähigkeit durch Verdün¬ nung mit H2O auf 1,7 mS eingestellt. Die Probe wurde auf eine Q-Sepharose FF-Säule (45 ml; Pharmacia) aufgetragen, die vorher mit 20 mM Imidazol, pH 6,5 äquilibriert worden war. Die Elution von LL-16 erfolgte mit einem Gradienten von 0 bis 0,3 M NaCl in 20 mM Imidazol, pH 6,5. LL-16 enthaltende Fraktionen wurden mittels SDS-PAGE identifiziert und vereinigt. Die Identität von LL-16 wurde durch Massenanalyse (Molekulargewicht 13.566 + 3 D) und automatisierte N-terminale Sequenzanalyse bestätigt. Zur Konzentrationsbestimmung wurde die UV- Absorption von LL-16 bei 280 nm und ein berechneter molarer Extinktionskoeffizient von 5540 M^cm"1 bei dieser Wellenlänge (Mack et al , Analyt Biochem 200 (1992) 74-80) verwendet300 mg of the fusion protein thus obtained were dialyzed at 4 ° C. against 20 1 20 mM imidazole, pH 5.5 and then for 30 minutes to remove turbidity. centrifuged at 20,000 g. The supernatant from the centrifugation was then adjusted to pH 8.5 with NaOH, 0.3 mg of thrombin (Boehringer Mannheim GmbH) was added and the mixture was incubated at 37 ° C. for 4 hours. The gap mixture was then adjusted to pH 6.5 with HC1 and the conductivity to 1.7 mS by dilution with H2O. The sample was applied to a Q-Sepharose FF column (45 ml; Pharmacia), which had previously been equilibrated with 20 mM imidazole, pH 6.5. LL-16 was eluted with a gradient of 0 to 0.3 M NaCl in 20 mM imidazole, pH 6.5. Fractions containing LL-16 were identified by SDS-PAGE and pooled. The identity of LL-16 was confirmed by mass analysis (molecular weight 13,566 + 3 D) and automated N-terminal sequence analysis. The UV absorption of LL-16 at 280 nm and a calculated molar extinction coefficient were used to determine the concentration of 5540 M ^ cm " 1 at this wavelength (Mack et al, Analyt Biochem 200 (1992) 74-80)
Um den gewünschten N-Terminus zu erhalten, wird gegebenenfalls mit einer Aminopeptidase (z.B α-Aminoacylpeptidhydrolase) oder Dipeptidylpeptidase (z B Catepsin CCATH) nachge¬ spaltenIn order to obtain the desired N-terminus, an aminopeptidase (for example α-aminoacylpeptide hydrolase) or dipeptidyl peptidase (for example catepsin CCATH) is used if necessary
Das so erhaltene LL-16 wies in der SDS-PAGE unter reduzierenden Bedingungen eine Rein¬ heit von mehr als 95% aufThe LL-16 obtained in this way had a purity of more than 95% in SDS-PAGE under reducing conditions
Zur Reinheitsanalyse mittels RP-HPLC wurde eine Vydac, Protein & Peptide Cl 8, 4x180 mm Säule verwendet Die Elution erfolgt durch einen linearen Gradienten von 0% nach 80% B (Losungsmittel B 90% Acetonitril in 0, 1% TFA, Losungsmittel A 0, 1% TFA in H2O) inner¬ halb von 30 min mit einer Flußrate von 1 ml/min Die Detektion erfolgte bei 220 nmA Vydac, Protein & Peptide Cl 8, 4x180 mm column was used for purity analysis using RP-HPLC. Elution is carried out by a linear gradient from 0% to 80% B (solvent B 90% acetonitrile in 0.1% TFA, solvent A 0 , 1% TFA in H2O) within 30 min with a flow rate of 1 ml / min. The detection was carried out at 220 nm
Beispiel 2Example 2
Herstellung von verkürztem LL-16 unter Verwendung einer EnterokinaseschnittstelleProduction of truncated LL-16 using an enterokinase interface
2.1 Expressionsklon2.1 Expression clone
Amplifikation, Klonierung von LL-16 cDNA und Herstellung eines Expressionsklons erfolgen wie in der WO 94/28134 oder der WO 96/31607 beschrieben, unter Berücksichtigung der veränderten SequenzenAmplification, cloning of LL-16 cDNA and production of an expression clone are carried out as described in WO 94/28134 or WO 96/31607, taking into account the changed sequences
Als Vorwartsprimer wird ein Oligonukleotid der Sequenz SEQ LD NO:3 verwendet, das eine EcoRI site, 6 His und eine Enterokinase-Spaltstelle (D4K) enthält cccgaattc tatg cat cac cac cac cac cac gatgacgacgacaaa-tctgcaqcctcaqcctctqc EcoRI H6 D4KAn oligonucleotide of the sequence SEQ LD NO: 3 is used as the forward primer, which contains an EcoRI site, 6 His and an enterokinase cleavage site (D 4 K). Cccgaattc tatg cat cac cac cac cac cac gatgacgacgacaaa-tctgcaqcctcaqcctctqc EcoRI H6 D4K
Für die am S'-Ende um ein Asparaginsaurecodon verlängerte Sequenz wird als Vorwarts¬ primer ein Oligonukleotid der Sequenz SEQ ID NO:6 verwendet, das ebenfalls eine EcoRI site, 6 His und eine Enterokinase-Spaltstelle (D4K) enthält cccgaattc tatg cat cac cac cac cac cac gatgacgacgacaaa-gactctqcaqcctcaqcctc EcoRI Hß D4K Als reverse primer werden entweder Oligonukleotid LL-16-Rj (SEQ ID NO:4) :For the sequence extended by an aspartic acid codon at the S 'end, an oligonucleotide of the sequence SEQ ID NO: 6 is used as the forward primer, which also contains an EcoRI site, 6 His and an enterokinase cleavage site (D 4 K) cccgaattc tatg cat cac cac cac cac gatgacgacgacaaa-gactctqcaqcctcaqcctc EcoRI Hß D4K Either oligonucleotide LL-16-Rj (SEQ ID NO: 4) is used as the reverse primer:
ILI6-R1: qcq gat cca agc tta gga gtc tcc agc agc tgt gILI6-R 1 : qcq gat cca agc tta gga gtc tcc agc agc tgt g
oder Oligonukleotid LL-16-R2 (SEQ LD NO:5) verwendet:or oligonucleotide LL-16-R 2 (SEQ LD NO: 5):
ILI6-R2: qcq qat cca agc tta ttc ctt gga ctg gag gct ttt tcILI6-R 2 : qcq qat cca agc tta ttc ctt gga ctg gag gct ttt tc
Die beiden reverse primer enthalten BamHI und Hindlll Schnittstellen zur KlonierungThe two reverse primers contain BamHI and HindIII cloning sites
Mit LL-16-R] wird eine Sequenz erhalten, welche für ein Protein gemäß SEQ LD NO:2 codiertWith LL-16-R ] a sequence is obtained which codes for a protein according to SEQ LD NO: 2
Mit LL-I6-R2 wird eine Sequenz erhalten, welche für ein um 8 Aminosäuren am C-Terminus verkürztes LL-16 codiert. Dieses C-terminale LL-16 ist ebenfalls aktivWith LL-I6-R2 a sequence is obtained which codes for an LL-16 shortened by 8 amino acids at the C-terminus. This C-terminal LL-16 is also active
Die PCR-Reaktion, Klonierung und Herstellung des Expressionsklons (Fusionsprotein mit N-terminalem Poly-His- Anteil zur Aufreinigung) erfolgen nach Standardbedingungen.The PCR reaction, cloning and production of the expression clone (fusion protein with N-terminal poly-His portion for purification) are carried out according to standard conditions.
0,2 mM dNTP Mix, je 1 pmol/μl forward und reverse Primer, 1 x high fidelity buffer (Boehringer Mannheim, D), 1,5 mM MgC12, 2.6 U high fidelity enzyme mix (Boehringer Mannheim, D)0.2 mM dNTP mix, 1 pmol / μl forward and reverse primer, 1 x high fidelity buffer (Boehringer Mannheim, D), 1.5 mM MgC12, 2.6 U high fidelity enzyme mix (Boehringer Mannheim, D)
20 μl Endvolumen20 ul final volume
Maschine Perkin Eimer GeneAmp 9600Perkin bucket machine GeneAmp 9600
ReaktionsverlaufReaction course
3 min 94°C, 1 nun 56 °C, 2 min 72 °C, dann 25 Zyklen (20 see 94°C, 20 see 56°C, 1 min 72 °C)3 min 94 ° C, 1 now 56 ° C, 2 min 72 ° C, then 25 cycles (20 see 94 ° C, 20 see 56 ° C, 1 min 72 ° C)
2.2 Fermentation2.2 fermentation
Die Fermentation wird analog Beispiel 1 3 durchgeführtThe fermentation is carried out analogously to Example 1 3
2.3 Reinigung und Spaltung2.3 Cleaning and cleavage
700 ml Aufschlußüberstand in 50 mM Natriumphosphat, 5 mM EDTA, 100 mM NaCl, pH 7,2, wurden mit 70 ml 5 M NaCl, 60 mM MgCl2, pH 8 0 versetzt, 30 min gerührt und anschließend 30 min bei 20.000 g zentrifügiert. Der abzentrifügierte Überstand wurde auf eine Nickel-Chelat-Säule (V = 200 ml, Pharmacia) aufgezogen, die vorher mit einer NiSO4-Lösung (c = 10 mg/ml) beladen und mit 50 mM Natriumphosphat, 0 5 M NaCl, pH 8 0 äquilibriert worden war. Die Säule wurde anschließend mit AquilibrierungspufFer gewaschen, bis die Basislinie (UV-Detektion bei 280 nm) fast erreicht war. Daraufhin wurde die Säule mit 1 1 50 mM Natriumphosphat, 0,5 M NaCl, pH 7,0 und mit 1 1 50 mM Natriumphosphat, 0,1 M NaCl, pH 7,0 gespült. Die Elution des Fusionsproteins erfolgte mit einem Gradienten von 0 - 300 mM Imidazol, pH 7,0 in 50 mM Natriumphosphat, 0, 1 M NaCl, pH 7,0 (2 x 1,6 1). IL 16 - enthaltende Fraktionen wurden mittels SDS-PAGE identifiziert und vereinigt. Dieser LL-16-Pool wurde im Provario (Filtron, Membran Omega 5 K) auf eine Konzentration von 5 mg Protein/ml eingeengt und gegen 50 mM Tris, pH 8 0 dialysiert700 ml of digestion supernatant in 50 mM sodium phosphate, 5 mM EDTA, 100 mM NaCl, pH 7.2 were mixed with 70 ml of 5 M NaCl, 60 mM MgCl 2 , pH 8 0, stirred for 30 min and then centrifuged at 20,000 g for 30 min . The centrifuged supernatant was applied to a nickel chelate column (V = 200 ml, Pharmacia), which was previously loaded with a NiSO 4 solution (c = 10 mg / ml) and with 50 mM sodium phosphate, 0 5 M NaCl, pH 8 0 equilibrated had been. The column was then washed with equilibration buffer until the baseline (UV detection at 280 nm) was almost reached. The column was then rinsed with 1 1 50 mM sodium phosphate, 0.5 M NaCl, pH 7.0 and with 1 1 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 × 1.6 l). Fractions containing IL 16 were identified by SDS-PAGE and pooled. This LL-16 pool was concentrated in the provario (Filtron, membrane Omega 5 K) to a concentration of 5 mg protein / ml and dialyzed against 50 mM Tris, pH 8 0
Zur Enterokinasespaltung wurde ein 100 mg Fusionsprotein enthaltendes Äquivalent des Pools mit 50 mM Tris, pH 8,0 auf eine Proteinkonzentration von c = 1 mg/ml verdünnt Nach Zugabe von 33 μg Enterokinase (Boehringer Mannheim, 1 3000 w/w) wurde der Spaltansatz über Nacht (14 h) bei 37°C inkubiert Anschließend wurde der pH-Wert mit HC1 auf pH 6,5 eingestelltFor enterokinase cleavage, an equivalent of the pool containing 100 mg of fusion protein was diluted with 50 mM Tris, pH 8.0 to a protein concentration of c = 1 mg / ml. After the addition of 33 μg enterokinase (Boehringer Mannheim, 1 3000 w / w), the cleavage was started Incubated overnight (14 h) at 37 ° C. The pH was then adjusted to pH 6.5 with HC1
Ungespaltenes LL-16 wurde durch Einruhren von 20 ml Nickel-Chelat-Sepharose (Vorbereitung s oben, Bindungszeit 2 h) und anschließender Zentrifügation (10.000 g) bzw Abnutschen des Uberstands über eine Filterfritte entfernt Die Identität des im Überstand ent¬ haltenen gespaltenen LL-16 wurde durch N-terminale Sequenzierung und Massenanalyse be¬ stätigt Die Reinheit wurde mittels SDS-PAGE und RP-HPLC (Vydac, Diphenyl, 4,6 x 150 mm, linearer Gradient von 20% auf 95% B in 45 Minuten, Losung A 20 mM Kaliumphosphat in H2O, pH 7,5, Losung B 100% Acetonitril) überprüftUncleaved LL-16 was removed by stirring in 20 ml of nickel chelate sepharose (preparation see above, binding time 2 h) and subsequent centrifugation (10,000 g) or suction filtering of the supernatant via a filter frit. The identity of the split LL- contained in the supernatant 16 was confirmed by N-terminal sequencing and mass analysis. The purity was determined by SDS-PAGE and RP-HPLC (Vydac, diphenyl, 4.6 x 150 mm, linear gradient from 20% to 95% B in 45 minutes, solution A 20 mM potassium phosphate in H 2 O, pH 7.5, solution B 100% acetonitrile) checked
Beispiel 3Example 3
Spaltung von rekombinantem LL-16 mit ZellysatCleavage of recombinant LL-16 with cell lysate
3.1 Trennung von CD8+ und CD4+-Lymphozyten über MACS3.1 Separation of CD8 + and CD4 + lymphocytes via MACS
Die mittels Ficoll-Gradienten aus einem "buffy coat" isolierten Lymphozyten werden in 500 μl PBS-Azid/1 x 108 Zellen (Phosphate Buffered Saline ohne Ca2+ und Mg2+, 0,01% Natrium- azid, 5 mM EDTA, pH 7,2) resuspendiert Nach Zugabe von 20 ml CD8-Microbeads/l x 10? zu erwartende Zellen (Maus-Anti-Human-CD8-Antikorper, konjugiert mit magnetischen Par¬ tikeln, Miltenyi Biotec GmbH) erfolgt eine Inkubation bei 4°C über 15 min. Für weitere 5 min bei 4°C werden 2 mg DTAF/1 x 10^ zu erwartende Zellen (Anti-Maus-IgG FITC konjugiert, Firma Dianova) hinzugegeben Nach Verdünnung mit 25 ml PBS-Azid/1% BSA erfolgt eine erneute Zentrifügation (10 min, 1200 rpm, 4°C). Der Überstand wird verworfen, die Zellen in 2 ml PBS/1% BSA resuspendiert und die Zellsuspension auf eine Säule, die sich in einem Magnetseparator (Miltenyi Biotec GmbH) befindet, aufgetragen. Die CD8+-Zellen, an die die CD8-Microbeads gekoppelt sind, werden in der Säule zurückgehalten, die Durchflußfraktion enthält dementsprechend alle Lymphozyten (ca. 80% CD4+-Zellen) mit Ausnahme der CD8+- Zellen. Nach Waschung wird die Säule aus der Halterung genommen und die CD8+-Zellfrak- tion mit PBS-Azid/1% BSA eluiert. Die Durchfluß- und die CD8+-Zellfraktion werden zen¬ trifügiert, in Zellkulturmedium (RPMI 1640, 20% FKS, 2 mM Glutamin, 180 U/ml LL-2) resuspendiert, die Zellzahl auf 3 x 10^ Zellen/ml eingestellt und die Zellen mit PHA stimuliert (9 mg/ml). Die Qualität der Lymphozytensubpopulationstrennung wird mittels FACS über¬ prüft.The lymphocytes isolated from a "buffy coat" using Ficoll gradients are put into 500 μl PBS azide / 1 × 10 8 cells (phosphate buffered saline without Ca 2+ and Mg 2+ , 0.01% sodium azide, 5 mM EDTA , pH 7.2) resuspended After adding 20 ml CD8-Microbeads / lx 10? cells to be expected (mouse anti-human CD8 antibodies, conjugated with magnetic particles, Miltenyi Biotec GmbH) are incubated at 4 ° C. for 15 min. For a further 5 min at 4 ° C., 2 mg of DTAF / 1 × 10 ^ cells to be expected (anti-mouse IgG FITC conjugated, Dianova company) are added. After dilution with 25 ml of PBS azide / 1% BSA, centrifugation is carried out again ( 10 min, 1200 rpm, 4 ° C). The supernatant is discarded, the cells in 2 ml PBS / 1% BSA resuspended and the cell suspension applied to a column, which is located in a magnetic separator (Miltenyi Biotec GmbH). The CD8 + cells to which the CD8 microbeads are coupled are retained in the column, the flow fraction accordingly contains all lymphocytes (approx. 80% CD4 + cells) with the exception of the CD8 + cells. After washing, the column is removed from the holder and the CD8 + cell fraction eluted with PBS azide / 1% BSA. The flow-through and the CD8 + cell fraction are centrifuged, resuspended in cell culture medium (RPMI 1640, 20% FCS, 2 mM glutamine, 180 U / ml LL-2), the cell number adjusted to 3 × 10 ^ cells / ml and the cells stimulated with PHA (9 mg / ml). The quality of the lymphocyte subpopulation separation is checked by means of FACS.
3.2 Herstellung des Zellysats und Verdauung von LL-163.2 Production of Cell Lysate and Digestion of LL-16
Es werden 1 x 108 CD8+ Lymphozyten, die drei Tage mit PHA stimuliert wurden, in 2 ml PBS, das mit 1% Triton XI 00 versetzt ist, für 10 min auf Eis lysiert. Sodann werden Zell- debris und Zellkerne durch Zentrifügation entfernt. 45 μl des so gewonnenen Zellysats werden mit 10 μg rekombinantem LL-16, das entweder am amino- oder carboxyterminalen Ende in Fusion mit einem Histidin-tag (HIS-tag, z.B. sechs Histidine mit Spaltstelle vgl. WO 94/28134) exprimiert wurde, 16 Stunden bei Raumtemperatur inkubiert. Sodann werden die HIS-tag tragenden Fragmente mit Hilfe einer Nickel-Agarosematrix gereinigt. Nach weite¬ rer Reinigung der entstandenen Fragmente in der HPLC werden die Massen der Fragmente massenspektrographisch bestimmt und so die Größe des N-terminalen Fragmentes ermittelt. Derart wurde ein natürlich prozessiertes IL- 16-Fragment identifiziert, das mit dem in SEQ ID NO.1/2 beschriebenen bzw. den um ein Asparaginsäurecodon verlängerten N- Terminus beginnt.1 x 10 8 CD8 + lymphocytes stimulated with PHA for three days in 2 ml PBS to which 1% Triton XI 00 has been added are lysed on ice for 10 min. The cell debris and cell nuclei are then removed by centrifugation. 45 μl of the cell lysate obtained in this way are expressed with 10 μg of recombinant LL-16, which was expressed either at the amino or carboxy terminal end in fusion with a histidine tag (HIS tag, for example six histidines with cleavage site, see WO 94/28134). Incubated for 16 hours at room temperature. The fragments bearing the HIS tag are then cleaned with the aid of a nickel agarose matrix. After further purification of the fragments formed in the HPLC, the masses of the fragments are determined by mass spectrography and the size of the N-terminal fragment is thus determined. In this way, a naturally processed IL-16 fragment was identified which begins with the N-terminus described in SEQ ID NO.1 / 2 or which is extended by an aspartic codon.
ReferenzlisteReference list
Aman et al., Gene 40 (1985) 183Aman et al., Gene 40 (1985) 183
Baier, M., et al., Nature 378 (1995) 563Baier, M., et al., Nature 378 (1995) 563
Beck und Zink, Gene 16 (1981) 35-58Beck and Zink, Gene 16 (1981) 35-58
Brosius et al., J. Mol. Biol. 148 (1981) 107 - 127Brosius et al., J. Mol. Biol. 148 (1981) 107-127
Chang et al., Nature 198 (1977) 1056Chang et al., Nature 198 (1977) 1056
Cruikshank, W.W., et al., J. Immunol. 146 (1991) 2928-2934Cruikshank, W.W., et al., J. Immunol. 146 (1991) 2928-2934
Cruikshank, W.W., et al., Proc. Natl. Acad. Sei. USA 91 (1994) 5109-5113Cruikshank, W.W., et al., Proc. Natl. Acad. Be. USA 91 (1994) 5109-5113
Davies et al., Ann. Rev. Microbiol. 32 (1978) 469 EP-A 0 267 851Davies et al., Ann. Rev. Microbiol. 32 (1978) 469 EP-A 0 267 851
EP-A 0 495 398EP-A 0 495 398
Ghrayeb et al., EMBO J. 3 (1984) 2437Ghrayeb et al., EMBO J. 3 (1984) 2437
Goeddel et al., Nuc. Acids Res. 8 (1980) 4057Goeddel et al., Nuc. Acids Res. 8 (1980) 4057
Internationale Patentanmeldung PCT/EP96/05661International patent application PCT / EP96 / 05661
Internationale Patentanmeldung PCT/EP96/05662International patent application PCT / EP96 / 05662
Kingsman, S.M., et al, Tibtech 5 (1987) 53-57Kingsman, S.M., et al, Tibtech 5 (1987) 53-57
Mack et al., Analyt. Biochem. 200 (1992) 74-80Mack et al., Analyt. Biochem. 200 (1992) 74-80
Miller et al., Bio/Technology 7 (1989) 698Miller et al., Bio / Technology 7 (1989) 698
Nagai et al., Nature 309 (1984) 810Nagai et al., Nature 309 (1984) 810
Oka et al., Proc. Natl. Acad. Sei. USA 82 (1985) 7212Oka et al., Proc. Natl. Acad. Be. USA 82 (1985) 7212
Palva et al., Proc. Natl. Acad. Sei. USA 79 (1982) 5582Palva et al., Proc. Natl. Acad. Be. USA 79 (1982) 5582
Phillips and Silhavy, Nature 344 (1990) 882-884Phillips and Silhavy, Nature 344 (1990) 882-884
Powell et al., Appl. Environ. Microbiol. 54 (1988) 655Powell 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, USASambrook 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) 128Shimatake et al., Nature 292 (1981) 128
Studier et al., J. Mol. Biol. 189 (1986) 1 13Studier et al., J. Mol. Biol. 189 (1986) 1 13
US-Patent Nr. 4,551,433U.S. Patent No. 4,551,433
US-Patent Nr. 4,689,406U.S. Patent No. 4,689,406
US-Patent Nr. 4,747,056U.S. Patent No. 4,747,056
WO 91/11520WO 91/11520
WO 92/20702WO 92/20702
WO 92/20703WO 92/20703
WO 94/28134WO 94/28134
WO 96/31607WO 96/31607
Yansura, Meth. Enzymol. 185 (1990) 161-166 Yansura, Meth. Enzymol. 185 (1990) 161-166
SEQUENZPROTOKOLLSEQUENCE LOG
(1) ALLGEMEINE ANGABEN:(1. GENERAL INFORMATION:
(i) ANMELDER:(i) APPLICANT:
(A) NAME: Bundesrepublik Deutschland vertreten durch den Bundesminister fuer Gesundheit(A) NAME: Federal Republic of Germany represented by the Federal Minister of Health
(B) STRASSE: -(B) ROAD: -
(C) ORT: Bonn(C) LOCATION: Bonn
(E) LAND: Deutschland(E) COUNTRY: Germany
(F) POSTLEITZAHL: D-53108(F) POSTAL NUMBER: D-53108
(A) NAME: BOEHRINGER MANNHEIM GMBH(A) NAME: BOEHRINGER MANNHEIM GMBH
(B) STRASSE: Sandhofer Str. 116(B) STREET: Sandhofer Str. 116
(C) ORT: Mannheim(C) LOCATION: Mannheim
(E) LAND: Deutschland(E) COUNTRY: Germany
(F) POSTLEITZAHL: D-68305(F) POSTAL NUMBER: D-68305
(G) TELEFON: 08856/60-3446 (H) TELEFAX: 08856/60-3451(G) TELEPHONE: 08856 / 60-3446 (H) TELEFAX: 08856 / 60-3451
(ii) BEZEICHNUNG DER ERFINDUNG: Prozessierte Polypeptide mit IL-16-Aktivitaet, Verfahren zu ihrer Herstellung und Verwendung(ii) DESCRIPTION OF THE INVENTION: Processed polypeptides with IL-16 activity, process for their preparation and use
(iii) ANZAHL DER SEQUENZEN: 10(iii) NUMBER OF SEQUENCES: 10
(iv) COMPUTER-LESBARE FASSUNG:(iv) COMPUTER READABLE VERSION:
(A) DATENTRÄGER: Floppy disk(A) DISK: Floppy disk
(B) COMPUTER: IBM PC compatible(B) COMPUTER: IBM PC compatible
(C) BETRIEBSSYSTEM: PC-DOS/MS-DOS(C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30B (EPA)(D) SOFTWARE: Patentin Release # 1.0, Version # 1.30B (EPA)
(vi) DATEN DER URANMELDUNG:(vi) DATA OF THE URN REGISTRATION:
(A) ANMELDENUMMER: DE 196 17 202.0(A) REGISTRATION NUMBER: DE 196 17 202.0
(B) ANMELDETAG: 30-APR-1996(B) REGISTRATION DAY: 30-APR-1996
(vi) DATEN DER URANMELDUNG:(vi) DATA OF THE URN REGISTRATION:
(A) ANMELDENUMMER: DE 196 17 203.9(A) REGISTRATION NUMBER: DE 196 17 203.9
(B) ANMELDETAG: 30-APR-1996(B) REGISTRATION DAY: 30-APR-1996
(2) ANGABEN ZU SEQ ID NO: 1:(2) INFORMATION ON SEQ ID NO: 1:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 366 Basenpaare(A) LENGTH: 366 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Doppelstrang(C) STRAND FORM: double strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: cDNA(ii) MOLECULE TYPE: cDNA
Iix) MERKMAL:Iix) FEATURE:
(A) NAME/SCHLÜSSEL: CDS ( B ) LAGE : 1 . . 366(A) NAME / KEY: CDS (B) LOCATION: 1. , 366
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 1:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
TCT GCA GCC TCA GCC TCT GCA GCC AGT GAT GTT TCT GTA GAA TCT ACA 48 Ser Ala Ala Ser Ala Ser Ala Ala Ser Asp Val Ser Val Glu Ser Thr 1 5 10 15TCT GCA GCC TCA GCC TCT GCA GCC AGT GAT GTT TCT GTA GAA TCT ACA 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 Ala Glu Ala Thr Val Cys Thr Val Thr Leu Glu Lys Met Ser Ala Gly 20 25 30GCA GAG GCC ACA GTC TGC ACG GTG ACA CTG GAG AAG ATG TCG GCA GGG 96 Ala Glu Ala Thr Val Cys Thr Val Thr Leu Glu Lys Met Ser Ala Gly 20 25 30
CTG GGC TTC AGC CTG GAA GGA GGG AAG GGC TCC CTA CAC GGA GAC AAG 144 Leu Gly Phe Ser Leu Glu Gly Gly Lys Gly Ser Leu His Gly Asp Lys 35 40 45CTG GGC TTC AGC CTG GAA GGA GGG AAG GGC TCC CTA CAC GGA GAC AAG 144 Leu 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 GCA GCC TCA GAA CAA AGT 192 Pro Leu Thr Ile Asn Arg Ile Phe Lys Gly Ala Ala Ser Glu Gin Ser 50 55 60CCT CTC ACC ATT AAC AGG ATT TTC AAA GGA GCA GCC TCA GAA CAA AGT 192 Pro Leu Thr Ile Asn Arg Ile Phe Lys Gly Ala Ala Ser Glu Gin Ser 50 55 60
GAG ACA GTC CAG CCT GGA GAT GAA ATC TTG CAG CTG GGT GGC ACT GCC 240 Glu Thr Val Gin Pro Gly Asp Glu Ile Leu Gin Leu Gly Gly Thr Ala 65 70 75 80GAG ACA GTC CAG CCT GGA GAT GAA ATC TTG CAG CTG GGT GGC ACT GCC 240 Glu Thr Val Gin Pro Gly Asp Glu Ile Leu Gin Leu Gly Gly Thr Ala 65 70 75 80
ATG CAG GGC CTC ACA CGG TTT GAA GCC TGG AAC ATC ATC AAG GCA CTG 288 Met Gin Gly Leu Thr Arg Phe Glu Ala Trp Asn Ile Ile Lys Ala LeuATG CAG GGC CTC ACA CGG TTT GAA GCC TGG AAC ATC ATC AAG GCA CTG 288 Met Gin Gly Leu Thr Arg Phe Glu Ala Trp Asn Ile Ile Lys Ala Leu
85 90 9585 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 Ile Val Ile Arg Arg Lys Ser Leu Gin Ser 100 105 110CCT GAT GGA CCT GTC ACG ATT GTC ATC AGG AGA AAA AGC CTC CAG TCC 336 Pro Asp Gly Pro Val Thr Ile Val Ile Arg Arg Lys Ser Leu Gin Ser 100 105 110
AAG GAA ACC ACA GCT GCT GGA GAC TCC TAG 366AAG GAA ACC ACA GCT GCT GGA GAC TCC TAG 366
Lys Glu Thr Thr Ala Ala Gly Asp Ser * 115 120Lys Glu Thr Thr Ala Ala Gly Asp Ser * 115 120
(2) ANGABEN ZU SEQ ID NO: 2:(2) INFORMATION ON SEQ ID NO: 2:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 122 Aminosäuren(A) LENGTH: 122 amino acids
(B) ART: Aminosäure (D) TOPOLOGIE: linear(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: Protein(ii) MOLECULE TYPE: Protein
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 2:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Ser Ala Ala Ser Ala Ser Ala Ala Ser Asp Val Ser Val Glu Ser Thr 1 5 10 15Ser Ala Ala Ser Ala Ser Ala Ala Ser Asp Val Ser Val Glu Ser Thr 1 5 10 15
Ala Glu Ala Thr Val Cys Thr Val Thr Leu Glu Lys Met Ser Ala Gly 20 25 30 Leu Gly Phe Ser Leu Glu Gly Gly Lys Gly Ser Leu His Gly Asp Lys 35 40 45Ala Glu Ala Thr Val Cys Thr Val Thr Leu Glu Lys Met Ser Ala Gly 20 25 30 Leu Gly Phe Ser Leu Glu Gly Gly Lys Gly Ser Leu His Gly Asp Lys 35 40 45
Pro Leu Thr Ile Asn Arg Ile Phe Lys Gly Ala Ala Ser Glu Gin Ser 50 55 60Pro Leu Thr Ile Asn Arg Ile Phe Lys Gly Ala Ala Ser Glu Gin Ser 50 55 60
Glu Thr Val Gin Pro Gly Asp Glu Ile Leu Gin Leu Gly Gly Thr Ala 65 70 75 80Glu Thr Val Gin Pro Gly Asp Glu Ile Leu Gin Leu Gly Gly Thr Ala 65 70 75 80
Met Gin Gly Leu Thr Arg Phe Glu Ala Trp Asn Ile Ile Lys Ala LeuMet Gin Gly Leu Thr Arg Phe Glu Ala Trp Asn Ile Ile Lys Ala Leu
85 90 9585 90 95
Pro Asp Gly Pro Val Thr Ile Val Ile Arg Arg Lys Ser Leu Gin Ser 100 105 110Pro Asp Gly Pro Val Thr Ile Val Ile Arg Arg Lys Ser Leu Gin Ser 100 105 110
Lys Glu Thr Thr Ala Ala Gly Asp Ser * 115 120Lys Glu Thr Thr Ala Ala Gly Asp Ser * 115 120
(2) ANGABEN ZU SEQ ID NO: 3:(2) INFORMATION ON SEQ ID NO: 3:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 66 Basenpaare(A) LENGTH: 66 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "forward primer"(A) DESCRIPTION: / desc = "forward primer"
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 3: CCCGAATTCT ATGCATCACC ACCACCACCA CGATGACGAC GACAAATCTG CAGCCTCAGC 60 CTCTGC 66(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CCCGAATTCT ATGCATCACC ACCACCACCA CGATGACGAC GACAAATCTG CAGCCTCAGC 60 CTCTGC 66
(2) ANGABEN ZU SEQ ID NO: 4:(2) INFORMATION ON SEQ ID NO: 4:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 34 Basenpaare(A) LENGTH: 34 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "reverse primer IL-16-R1"(A) DESCRIPTION: / desc = "reverse primer IL-16-R1"
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 4: GCGGATCCAA GCTTAGGAGT CTCCAGCAGC TGTG 34 (2) ANGABEN ZU SEQ ID NO: 5:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GCGGATCCAA GCTTAGGAGT CTCCAGCAGC TGTG 34 (2) INFORMATION ON SEQ ID NO: 5:
(l) SEQUENZKENNZEICHEN:(l) SEQUENCE LABEL:
(A) LANGE: 38 Basenpaare(A) LONG: 38 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "reverse primer IL-16-R2"(A) DESCRIPTION: / desc = "reverse primer IL-16-R2"
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 5: GCGGATCCAA GCTTATTCCT TGGACTGGAG GCTTTTTC 38(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GCGGATCCAA GCTTATTCCT TGGACTGGAG GCTTTTTC 38
(2) ANGABEN ZU SEQ ID NO: 6:(2) INFORMATION ON SEQ ID NO: 6:
(1) SEQUENZKENNZEICHEN:(1) SEQUENCE LABEL:
(A) LANGE: 66 Basenpaare(A) LONG: 66 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(xx) ART DES MOLEKÜLS: other nucleic acxd(xx) MOLECULE TYPE: other nucleic acxd
(A) BESCHREIBUNG: /desc = "forward primer"(A) DESCRIPTION: / desc = "forward primer"
(xx) SEQUENZBESCHREIBUNG: SEQ ID NO: 6: CCCGAATTCT ATGCATCACC ACCACCACCA CGATGACGAC GACAAAGACT CTGCAGCCTC 60 AGCCTC 66(xx) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CCCGAATTCT ATGCATCACC ACCACCACCA CGATGACGAC GACAAAGACT CTGCAGCCTC 60 AGCCTC 66
(2) ANGABEN ZU SEQ ID NO: 7:(2) INFORMATION ON SEQ ID NO: 7:
(x) SEQUENZKENNZEICHEN:(x) SEQUENCE LABEL:
(A) LÄNGE: 7 Aminosäuren(A) LENGTH: 7 amino acids
(B) ART: Aminosäure(B) TYPE: amino acid
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(li) ART DES MOLEKÜLS: Peptid(left) MOLECULE TYPE: Peptide
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 7:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Ser Ala Ala Ser Ala Ser AlaSer Ala Ala Ser Ala Ser Ala
1 5 (2) ANGABEN ZU SEQ ID NO: 8:1 5 (2) INFORMATION ON SEQ ID NO: 8:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 6 Aminosäuren(A) LENGTH: 6 amino acids
(B) ART: Aminosäure(B) TYPE: amino acid
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: Peptid(ii) MOLECULE TYPE: Peptide
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 8:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Ser Ala Ala Ser Ala Ser 1 5Ser Ala Ala Ser Ala Ser 1 5
[ 2 ) ANGABEN ZU SEQ ID NO: 9:[2) INFORMATION ON SEQ ID NO: 9:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 5 Aminosäuren(A) LENGTH: 5 amino acids
(B) ART: Aminosäure(B) TYPE: amino acid
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: Peptid(ii) MOLECULE TYPE: Peptide
[xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 9:[xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Ser Ala Ala Ser Ala 1 5Ser Ala Ala Ser Ala 1 5
(2) ANGABEN ZU SEQ ID NO: 10:(2) INFORMATION ON SEQ ID NO: 10:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 4 Aminosäuren(A) LENGTH: 4 amino acids
(B) ART: Aminosäure(B) TYPE: amino acid
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: Peptid(ii) MOLECULE TYPE: Peptide
(xx) SEQUENZBESCHREIBUNG: SEQ ID NO: 10:(xx) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Ser Ala Ala Ser 1 Ser Ala Ala Ser 1

Claims

Patentansprüche claims
1. Nukleinsäure, mit welcher die Expression eines Polypeptids mit Interleukin- 16- Aktivität in einer prokaryontischen oder eukaryontischen Wirtszelle erreicht werden kann, wobei die genannte Nukleinsäure1. Nucleic acid with which the expression of a polypeptide with interleukin-16 activity can be achieved in a prokaryotic or eukaryotic host cell, said nucleic acid
a) der DNA-Sequenz SEQ ID NO 1 oder einer DNA, die am 5'-Ende um ein Asparaginsaurecodon verlängert ist, oder ihrem komplementären Strang entspricht,a) the DNA sequence SEQ ID NO 1 or a DNA which is extended at the 5 'end by an aspartic acid codon or corresponds to its complementary strand,
b) mit der DNA der Sequenz SEQ ID NO 1 oder einer DNA, die am 5'-Ende um ein Asparaginsaurecodon verlängert ist, unter stringenten Bedingungen hybridisiert,b) hybridizing under stringent conditions with the DNA of the sequence SEQ ID NO 1 or a DNA which is extended at the 5 'end by an aspartic acid codon,
c) oder eine Nukleinsauresequenz, die ohne die Degeneration des genetischen Codes mit den durch a) und b) definierten Nukleinsauresequenzen unter stringenten Bedin¬ gungen hybridisieren wurdec) or a nucleic acid sequence which would hybridize under stringent conditions without the degeneration of the genetic code with the nucleic acid sequences defined by a) and b)
d) und am 5'-Ende für eine der Aminosauresequenzen SEQ ID NO 7 bis 10 oder für analoge Sequenzen, die N-terminal um eine Asparaginsaure verlängert sind, codiertd) and at the 5 'end for one of the amino acid sequences SEQ ID NO 7 to 10 or for analog sequences which are N-terminally extended by an aspartic acid
2 Nukleinsäure nach Anspruch 1, die Interleukin- 16 von Primaten codiert2 nucleic acid according to claim 1, which encodes interleukin-16 of primates
3 Prokaryontische oder eukaryontische Wirtszelle, welche mit einer Nukleinsäure nach den Ansprüchen 1 oder 2 so transformiert oder transfiziert ist, daß die Wirtszelle das genannte Polypeptid exprimiert3 prokaryotic or eukaryotic host cell which is transformed or transfected with a nucleic acid according to claims 1 or 2 such that the host cell expresses said polypeptide
4 Biologisch funktioneller Nukleinsaurevektor, der eine Nukleinsäure gemäß Anspruch 1 oder 2 enthalt4 Biologically functional nucleic acid vector containing a nucleic acid according to claim 1 or 2
5 Interleukin- 16 aus Primaten, wie es als Produkt einer eukaryontischen Expression einer Nukleinsäure gemäß Anspruch 1 oder 2 im wesentlichen frei von anderen Humanprotei¬ nen erhaltlich ist5 interleukin-16 from primates, as is obtainable as a product of a eukaryotic expression of a nucleic acid according to claim 1 or 2 essentially free of other human proteins
6 Humanes Interleukin- 16, wie es als Produkt einer eukaryontischen Expression einer Nukleinsäure gemäß Anspruch 1 oder 2 nach Prozessierung im wesentlichen frei von anderen Humanproteinen erhaltlich ist Polypeptid mit Interleukin- 16- Aktivität, welches codiert wird von einer Nukleinsäure nach den Ansprüchen 1 oder 26 human interleukin-16, as it is available as a product of a eukaryotic expression of a nucleic acid according to claim 1 or 2 after processing essentially free of other human proteins Polypeptide with interleukin-16 activity, which is encoded by a nucleic acid according to claims 1 or 2
Polypeptid mit Interleukin- 16- Aktivität gemäß Anspruch 7, dadurch gekennzeichnet, daß es ein Multimeres aus einer definierten Anzahl von Untereinheiten darstellt, wobei das Polypeptid von Anspruch 7 eine Untereinheit istPolypeptide with interleukin-16 activity according to claim 7, characterized in that it is a multimer of a defined number of subunits, the polypeptide of claim 7 being a subunit
Polypeptid nach Anspruch 8, dadurch gekennzeichnet, daß es aus 4 bis 32 Untereinhei¬ ten bestehtPolypeptide according to claim 8, characterized in that it consists of 4 to 32 subunits
Polypeptid nach den Ansprüchen 7 bis 9, dadurch gekennzeichnet, daß es einen definier¬ ten Gehalt an Metallionen enthalt, wobei die Anzahl der Metallionen pro Untereinheit 0,5 bis 2 betragtPolypeptide according to claims 7 to 9, characterized in that it contains a defined content of metal ions, the number of metal ions per subunit being 0.5 to 2
Verfahren zur Herstellung eines Polypeptids nach den Ansprüchen 7 bis 10, dadurch gekennzeichnet, daß eine prokaryontische oder eukaryontische Wirtszelle, die mit einer Nukleinsauresequenz nach den Ansprüchen 1 oder 2 transformiert oder transfiziert ist, unter geeigneten Nährbedingungen kultiviert wird und das gewünschte Polypeptid gege¬ benenfalls isoliert wirdA process for the preparation of a polypeptide according to claims 7 to 10, characterized in that a prokaryotic or eukaryotic host cell which is transformed or transfected with a nucleic acid sequence according to claims 1 or 2 is cultivated under suitable nutritional conditions and the desired polypeptide is isolated if necessary becomes
Pharmazeutische Zusammensetzung, die ein Polypeptid nach den Ansprüchen 5 bis 10 sowie ein pharmazeutisch geeignetes Verdünnungsmittel, Adjuvans und/oder Tragermit¬ tel enthaltPharmaceutical composition containing a polypeptide according to claims 5 to 10 and a pharmaceutically suitable diluent, adjuvant and / or carrier
Pharmazeutische Zusammensetzung enthaltend ein Polypeptid gemäß den Ansprüchen 5 - 10 in einer für eine therapeutische Anwendung ausreichenden MengePharmaceutical composition containing a polypeptide according to claims 5-10 in an amount sufficient for therapeutic use
Verfahren zur Herstellung eines Antikörpers gegen ein Polypeptid mit Interleukin- 16- Aktivitat, dadurch gekennzeichnet, daß ein Saugetier mit einem Immunogen welches die ersten 3 - 20 Aminosäuren von SEQ ID NO.2 oder einer N-terminal um einen Aspara¬ ginsäurerest verlängerten SEQ ID NO 2 als Hapten enthalt, immunisiert wird und der Antikörper anschließend aus dem Saugetier gewonnen wird A process for producing an antibody against a polypeptide with interleukin-16 activity, characterized in that a mammal with an immunogen which extends the first 3-20 amino acids of SEQ ID NO.2 or an N-terminal SEQ ID extended by an aspartic acid residue Contains NO 2 as a hapten, is immunized and the antibody is then obtained from the mammal
EP97921834A 1996-04-30 1997-04-30 Processed polypeptides with il-16 activity, process for preparing the same and their use Withdrawn EP0904374A1 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE19617202 1996-04-30
DE1996117203 DE19617203A1 (en) 1996-04-30 1996-04-30 Recombinant interleukin 16
DE19617203 1996-04-30
DE19617202A DE19617202A1 (en) 1996-04-30 1996-04-30 Recombinant interleukin 16
PCT/EP1997/002216 WO1997041231A1 (en) 1996-04-30 1997-04-30 Processed polypeptides with il-16 activity, process for preparing the same and their use

Publications (1)

Publication Number Publication Date
EP0904374A1 true EP0904374A1 (en) 1999-03-31

Family

ID=26025242

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97921834A Withdrawn EP0904374A1 (en) 1996-04-30 1997-04-30 Processed polypeptides with il-16 activity, process for preparing the same and their use

Country Status (11)

Country Link
US (1) US6506582B1 (en)
EP (1) EP0904374A1 (en)
JP (1) JP3251592B2 (en)
KR (1) KR100308441B1 (en)
CN (1) CN1217025A (en)
AU (1) AU712122B2 (en)
BR (1) BR9709205A (en)
CA (1) CA2253259A1 (en)
DE (1) DE19780377D2 (en)
TR (1) TR199802168T2 (en)
WO (1) WO1997041231A1 (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090130661A1 (en) * 2006-09-28 2009-05-21 Glass William G Method for Detecting IL-16 Activity and Modulation of IL-16 Activity Based on Phosphorylated Stat-6 Proxy Levels
EP2094305A4 (en) * 2006-10-27 2011-01-05 Centocor Ortho Biotech Inc Method for treating pathological pulmonary conditions and bleomycin associated pulmonary fibrosis
US20110217259A1 (en) * 2010-03-03 2011-09-08 Djordje Atanackovic IL-16 as a target for diagnosis and therapy of hematological malignancies and solid tumors
CN114984189B (en) * 2022-05-27 2024-06-04 昆明理工大学 New use of interleukin 16 protein

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5350836A (en) * 1989-10-12 1994-09-27 Ohio University Growth hormone antagonists
GB9214857D0 (en) * 1992-07-13 1992-08-26 Medical Res Council Human nucleic acid fragments and their use
EP0700439A1 (en) * 1993-05-21 1996-03-13 Trustees Of Boston University Lymphocyte chemoattractant factor and uses thereof
DE19513152A1 (en) 1995-04-07 1996-10-10 Bundesrep Deutschland Use of an "immunodeficiency virus suppressing lymphokine (ISL)" to inhibit virus multiplication, in particular of retroviruses

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9741231A1 *

Also Published As

Publication number Publication date
BR9709205A (en) 2000-01-11
KR20000065005A (en) 2000-11-06
JPH11508453A (en) 1999-07-27
KR100308441B1 (en) 2001-11-02
TR199802168T2 (en) 1999-02-22
JP3251592B2 (en) 2002-01-28
WO1997041231A1 (en) 1997-11-06
CA2253259A1 (en) 1997-11-06
AU712122B2 (en) 1999-10-28
CN1217025A (en) 1999-05-19
US6506582B1 (en) 2003-01-14
DE19780377D2 (en) 1999-08-05
AU2775297A (en) 1997-11-19

Similar Documents

Publication Publication Date Title
DE69233667T2 (en) Immunogenic, non-toxic mutants of cholera toxin and toxin LT, their preparation and their use in the preparation of vaccines
DE69827507T2 (en) TRIMERIZING MODULE
DE19605657B4 (en) Human proinsulin derivative, coding DNA, DNA-containing expression vector, with this transformed microorganism and use of this human Proinsulinderivats
DE69835756T2 (en) HIV-1 TAT AND / OR NEF CONTAINING FUSION PROTEINS
CA2494974A1 (en) Il-7 drug substance, composition, preparation and uses
JPH01501283A (en) Novel type of colony stimulating factor-1
DE102004058306A1 (en) Process for the preparation of carboxy-terminally amidated peptides
DE3586972T2 (en) PURIFIED INTERLEUKIN-1.
AU627477B2 (en) Interleukin ii analogs
EP0870030A1 (en) Polypeptides with interleukin 16 activity, process for the preparation and use thereof
WO1997041231A1 (en) Processed polypeptides with il-16 activity, process for preparing the same and their use
DE69324345T2 (en) EXPRESSION OF OSTEOGENIC FACTOR OP-1 IN SPODOPTERA FRUGIPERDA CELLS INFECTED WITH RECOMBINANT BACULOVIRUS
US6444202B1 (en) Processed polypeptides with IL-16 activity, processes for their production and their use
EP1307480B1 (en) Mutant trichosanthin
DE19617202A1 (en) Recombinant interleukin 16
JPH03297388A (en) New tnf variant, production thereof and antitumor agent containing the same variant as active ingredient
DE19649233A1 (en) Nucleic acid encoding interleukin-16 polypeptide
DE19648715A1 (en) DNA encoding human interleukin-16
DE19617203A1 (en) Recombinant interleukin 16
EP0870029A1 (en) Multimer forms of interleukin-16 (il-16), process for the preparation and use thereof
MXPA98008964A (en) Polypeptides processed with acativity il-16, processes for its production and its
DE19614099A1 (en) Genomic nucleic acids coding for polypeptides with IL-16 activity, process for their preparation and use
CA2320624A1 (en) Mutants of il-16, processes for their production, and their use
JP2000510684A (en) Interleukin-3 (IL-3) receptor agonist
JP2002515729A (en) Novel G-CSF receptor agonist

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19981130

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU NL PT SE

RIN1 Information on inventor provided before grant (corrected)

Inventor name: LANG, KURT

Inventor name: WERNER, ALBRECHT

Inventor name: BANNERT, NORBERT

Inventor name: BAIER, MICHAEL

Inventor name: KURTH, REINHARD

RIN1 Information on inventor provided before grant (corrected)

Inventor name: LANG, KURT

Inventor name: WERNER, ALBRECHT

Inventor name: BANNERT, NORBERT

Inventor name: BAIER, MICHAEL

Inventor name: KURTH, REINHARD

17Q First examination report despatched

Effective date: 20030910

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20040713