WO1992006195A1 - Dna and amino acid sequence specific for natural killer cells and t cells - Google Patents

Dna and amino acid sequence specific for natural killer cells and t cells Download PDF

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Publication number
WO1992006195A1
WO1992006195A1 PCT/EP1991/001824 EP9101824W WO9206195A1 WO 1992006195 A1 WO1992006195 A1 WO 1992006195A1 EP 9101824 W EP9101824 W EP 9101824W WO 9206195 A1 WO9206195 A1 WO 9206195A1
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Prior art keywords
leu
protein
arg
thr
ala
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PCT/EP1991/001824
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French (fr)
Inventor
J. P. Houchins
Toshio Yabe
Cynthia Mcsherry
Fritz H. Bach
Samuel Nwafor Chujor
Franz Kricek
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University Of Minnesota
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Publication of WO1992006195A1 publication Critical patent/WO1992006195A1/en

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to a gene derived from a Natural Killer (NK) cell line or T cell line, as well as to the protein encoded by the gene and to the use of the protein as a medicament.
  • the gene is detected by employing a combination of differential and subtractive hybridisation methodologies, which can successfully identify cell-specific cDNA clones representing medium to high abundance transcripts, to identify genes that are expressed in NK cells but not in lymphoblastoid cell lines (LCL).
  • NK Natural Killer
  • NK cells are present in the peripheral blood in a state capable of lysing NK sensitive targets in a reaction that is not restricted by proteins of the major histocompatibility complex. These cells typically account for about 5% of peripheral blood lymphocytes and they usually possess the large granular lymphocyte (LGL) morphology.
  • LGL large granular lymphocyte
  • NK activity is usually defined as the ability to lyse cells of the promyeloid leukaemia cell line, K562.
  • IL-2 interleukin 2
  • LAK lymphokine activated killer
  • Many ongoing studies are attempting to use cells with LAK activity in anti - tumour therapy.
  • Natural killer (NK) cells are cytotoxic lymphocytes that share numerous cell surface antigens and functional components with T cells.
  • Jongstra et al. have previously described a gene, designated 519, that is expressed in functional cytotoxic T cells and helper T cells but not in tumour cells or other hematopoietic cell lines.
  • the gene is expressed in functional T cell lines but not in established tumour cell lines.
  • the 519 gene product seems to be involved in the activation and subsequent proliferation and / or differentiation of resting T cells.
  • the translation of 519 RNA is restricted to nontransformed, IL-2 dependent, antigen-driven functional T cell lines.
  • the restricted expression pattern of 519 mRNA indicates that the expression of the 519 gene is regulated by the periodic addition of IL-2 and antigen to the functional T cell lines.
  • the expression of the 519 gene is regulated in normal T cells, but not in normal B cells, by mitogenic or antigenic stimulation.
  • the 519 protein acts intra-cellularly, since the predicted amino acid sequence of the 519 protein does not support the hypothesis that the 519 protein is secreted or membrane bound to function either as a lymphokine or as a membrane receptor molecule.
  • the present invention provides a new cDNA or genomic DNA sequence that encodes a secreted protein which has the function- of a lymphokine.
  • the gene is isolated from human natural killer cells.
  • the protein encoded by the gene is normally synthesised by natural killer cells and by T cells.
  • the present invention provides a vector comprising a gene coding for a secreted protein having the following amino acid sequence
  • the amino acid sequence is also indicated in Sequence Identifier No. 1.
  • the genetic code is degenerate; that is, most amino acids are coded by more than one codon of three nucleotides. Accordingly, allelic variations in the nucleotide sequence may or may not alter the amino acid sequence. Therefore, allelic variations are primarily on the DNA level and may also exist secondarily on the level of the amino acid sequence.
  • DNA sequence coding for the protein of the invention can be modified by conventional techniques to produce variations in the final protein of the invention which still has substantially the same activity as the protein with the amino acid sequence mentioned under a) or Seq Id No 1.
  • one or more amino acids for example up to fourteen amino acids
  • one or more amino acids can be added, substituted or removed without substantially affecting the activity of the protein of the invention.
  • Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table I, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule, or (c) the bulk of the side chains.
  • deletions and insertions, and substitutions in particular are not expected to produce radical changes in the characteristics of the protein of the invention.
  • the comparison of the functions of the mutated protein with the characteristic functions of the protein of the invention clarify whether the altered protein has a comparable activity.
  • the protein has a sequence of amino acids having a homology of 97 to 100 % of the sequence of Seq Id No 1 and more preferably only one or two amino acids are exchanged.
  • the cDNA coding for the amino acid sequence indicated in a) above is a human gene; corresponding DNA sequences from other species preferably mammals, more preferably primates, may be used.
  • DNA of the gene is to be transferred to a procaryotic or eucaryotic cell, it is advantageous to combine the DNA sequence of the invention with a suitable promoter which allows the expression of the protein in sufficient quantity.
  • the host cell is a non-mammalian cell, for example a micro-organism such as a bacterium or yeast.
  • the cDNA coding for the protein indicated in the amino acid sequence in a) is preferably inserted in the fragment EcoRI- Xba I which is introduced in the lambda vector GEM 2 (supplied by Promega Biotec).
  • GEM 2 lambda vector
  • a suitable transformation vector can rely on the transient introduction of DNA into host cells.
  • Vectors used for the protein of the invention typically contain various elements such as enhancers, promoters, polyadenylation sites and translation activators.
  • the present invention comprises a method of producing the protein of the invention which has the amino acid sequence of Seq Id No 1.
  • Procaryotic or eucaryotic host cells e.g. E. coli, yeast, mammalian cells (Chinese Hamster Ovary cells) transformed with the cDNA sequence of the invention are cultivated in a suitable nutrient medium.
  • the protein is isolated and purified by standard methods.
  • the invention further provides a new cDNA sequence that encodes secreted protein, wherein the cDNA coding and non-coding sequence is selected from the group consisting of: aa) the following cDNA sequence:
  • the invention comprises cDNA sequences that hybridise under stringent conditions to the DNA sequence defined in aa) or Seq Id No 2 and have a homology of 90 to 100 X.
  • a homology of 95 to 100 X is preferred, more preferred is a homology of 97 to 100%, particularly preferred 99 to 100%.
  • the full genomic DNA of the invention correlating to the cDNA sequence is indicated in SEQ ID NO 3 and normally transcribed and translated by natural killer cells or T cells.
  • gene 519 contains an insert of 242 base pairs that is not present in the DNA of the invention.
  • the polypeptide encoded by the DNA of the invention has a strongly hydrophobic N-terminal amino acid sequence which is characteristic for signal peptides.
  • the lack of additional hydrophobic regions in the rest of the protein shows that the protein of the invention is a secreted protein while gene 519 was speculated to encode an intracellular polypeptide possibly involved in some aspect of growth and/or differentiation of functional T cells.
  • the gene encoding for the protein of the invention is specifically transcribed in NK and T cells but not in a variety of other hematopoietic and non hematopoietic cell lines.
  • ⁇ .
  • the protein encoded by the DNA according to the invention is secreted. It is a cytokine either involved in differentiation and / or proliferation of target cells or related to the killing function of natural killer cells or T cells.
  • the invention further comprises a protein having the following amino acid sequence:
  • post-translational variations as mentioned in ccc) above is meant variations during or after translation, such as glycosylation, formation of disulfide bridges and chemical modifications of amino acids.
  • Glycosylation is one of the major bio-synthetic functions of the endoplas ic reticulum and/or Golgi apparatus.
  • the sequence and branching of the oligosaccharides formed in the reticulum can be altered in the Golgi apparatus, the lysosomes or plasma membrane.
  • the oligosaccharides can be N-linked oligosaccharides (asparagine linked) or 0-linked oligosaccharides (serine, threonine or hydroxylysine linked).
  • the glycosylation is dependent on the producing cell type and the species from which the cell type derives.
  • the amount and form of glycosylation can be influenced by compounds as described in European Patent Application EP 0 222 313. Changes in glycosylation may affect the function of the protein.
  • Proteins often form covalent intrachain bonds. These disulfide bonds are formed between cysteine-SH amino acids in the folded protein or in the protein which folds during translation. The bonds stabilise the three-dimensional structure of the protein. Such disulfide bonds are rarely formed in protein molecules that are still in the cell cytosol because the high intracellular concentration of the -SH reducing agent glutathione breaks most of such bonds. Once the proteins are outside the cytoplasm, are secreted or are on the cell surface, they often form additional covalent intrachain bounds. Furthermore, the amino acids may be altered as described in PCT Application WO 91/10684. Other alterations of the side chains of the amino acids are possible.
  • a preferred homology is 97 to 100% and more preferably only one or two amino acids are exchanged.
  • the protein of the invention has at least a purity of 40%, preferably 60%, more preferably 80% and most preferably 90%.
  • the purity is defined by the amount of the protein of the invention in relation to the total amount of protein.
  • One method to purify the protein is to use a fusion protein comprising the protein of the invention and a carrier protein, e.g. galactosidase or alkaline phosphatase. Fusion-proteins can be detected during purification because of the presence of the carrier protein (galactosidase or alkaline phosphatase reaction). Fusion-proteins can be enriched by antibody coated columns (anti-galactosidase or anti-phosphatase monoclonal antibodies). The fusion-protein can be separated in two pieces by a kinase forming a separated carrier protein and a separated protein of the invention.
  • a carrier protein e.g. galactosidase or alkaline phosphatase reaction.
  • Fusion-proteins can be enriched by antibody coated columns (anti-galactosidase or anti-phosphatase monoclonal antibodies).
  • the fusion-protein can be separated in two pieces by a kinase forming a separated carrier protein and a
  • the protein of the invention can be isolated from the mixture consisting substantially of the carrier protein, the kinase and protein of the invention by using a column with anti-carrier protein and anti-kinase monoclonal antibodies.
  • a column with anti-carrier protein and anti-kinase monoclonal antibodies See C. MAN0IL et al. (1990) J. of Bacteriology, Vol. 172, 515 - 518)
  • the protein of the invention is a cytokine preferably a human cytokine.
  • a cytokine is a protein produced and secreted from one or more cell types. It recognises specific receptors on the membrane of one or more target cell types, possibly also including the producer cells. Binding of cytokines to their receptor usually initiates regulatory changes in the target cells which may relate to effector functions, differentiation or proliferation.
  • the function of the cytokine can be measured by proliferation tests (e.g. 3 [H]-desoxythymidine-incorporation) or by cytotoxicity tests ( 51 [Cr]-release-test) or by colony forming assays or by production of an activity that affects other cells.
  • the DNA and protein of the invention have advantageous properties, in particular they exhibit pharmacological activity and are, therefore, useful as pharmaceuticals.
  • the protein of the invention causes increased activity of natural killer cells and cytotoxic T cells against human or gibbon tumour cells.
  • the activity of the system is tested by the 51 [Cr] release test described by Brunner et al. (BRUNNER et al. (1981) J Exp Med 154: 362 - 373) Tumour cells as target cells which are labelled by 51 [Cr] are incubated in the presence with effector cells for 4 to 6 hours.
  • the effectiveness of the effector cells is expressed by the release of 51 [Cr] material into the medium.
  • the 51 [Cr] release is increased in the presence of the protein according to the invention.
  • the protein of the invention can be used as an active therapeutic substance.
  • the invention provides a pharmaceutical composition comprising a protein of the invention in association with a pharmaceutically acceptable diluent or carrier. Further it comprises a pharmaceutical composition comprising a therapeutically effective protein of the invention and a pharmaceutically acceptable salt or a pharmaceutically acceptable carrier or diluent.
  • the protein of the invention can be used for the manufacture of a medicament for treatment of autoimmune deficiency by enhancing the immune response, or for treatment of cancer by killing cancer cells.
  • the invention provides a method of treating an immune deficiency in humans or animals in need of such treatment comprising administering a therapeutically effective amount of the protein according to the invention. Further the invention provides a method of treating cancer in human or animals in need of such treatment comprising administering a therapeutically effective amount of the protein according to the invention.
  • the compound of the invention shows this activity in the gibbon when administered by injection at daily dosages of from about 10--* 1 M to about 5x10" 10 M per kg or about 0,2 ⁇ g to 13 ⁇ g per kg-
  • the appropriate dosage will vary depending upon, for example, the host, the mode of administration and the nature and severity of the condition being treated. In general, satisfactory results in animals are indicated to be obtained at daily dosages from about 3x10"-* 1 M to 10 -10 M per kg body weight or 0,5 ⁇ g to 2,5 ⁇ g per kg. The dosages are administered by one to four injections per day.
  • the protein of the invention shows activity in a system in which isolated cytotoxic T cells kill virus infected human or gibbon target cells.
  • the compound of the invention shows this activity in the gibbon when administered by injection at daily dosages of from about 10" 10 M to about 5x10" 10 M per kg or 1,5 ⁇ g to 13 ⁇ g per kg.
  • the compound of the invention is therefore indicated for the treatment of viral diseases.
  • the appropriate dosage will vary depending upon, for example, the host, the mode of administration and the nature and severity of the condition being treated. In general, satisfactory results in animals are indicated to be obtained at daily dosages from about 2x10" 10 M to 5x10" 10 M per kg body weight or 3 ⁇ g to 13 ⁇ g per kg. The dosages are administered by one to four injections per day.
  • the compound of the invention may be administered by any conventional route, in particular by injection in the form of a solution or suspension in a pharmaceutically acceptable diluent.
  • Such compositions may be manufactured in conventional manner.
  • the compound with the amino acid sequence of SEQ ID NO 1 is the preferred compound for the treatment of cancer and viral infections.
  • a cell of the immune system transformed with the DNA of the invention can stimulate the immune response against different diseases, for example cancer, or diseases caused by viruses or eucaryotic parasites, e.g. protozoa or multicellular organisms.
  • Lymphocytes are taken from one individual, transformed with the DNA of the invention and the transformed cells replaced in the same individual.
  • the potency of the natural immune system is increased by this method.
  • the natural functions of the body are augmented without introducing foreign cells or foreign proteins which might induce a immune response against the newly introduced protein.
  • the isolated DNA sequence can be introduced into patient-derived lymphocytes.
  • the protein encoded by such introduced DNA does not differ from person to person, unlike other proteins, for example Major Histocompatibility Complex antigens.
  • the re-injected cell had learnt during the time of maturation to distinguish between self and non-self. Therefore an uncontrolled attack of the transformed cells against self antigens can be excluded.
  • the protein of the invention can be used for treatment of autoimmune disease by blocking the action of the cytokine by antagonists or down regulation of gene expression.
  • Figure 1 shows the pAX-vector which is supplied by MEDAC.
  • the lacZ gene includes the lac operator.
  • the hinge region of 187 bp codes for a collagen fragment.
  • the vector comprises an endoproteinase Xa recognition site and a multicloning site including Eco RI in all translational reading frames.
  • the fl-origin is used for replication for isolation of single-stranded DNA.
  • the pBR322 fragment harbours the origin of replication and finally the vector has the ampicillin resistance marker.
  • lac lacZ gene promotor
  • lacZ lacZ gene
  • CS collagen fragment
  • Xa Xa cleavage site
  • MCS multi cloning site
  • t 0 lambda t 0 terminator
  • fl ori phage fl intergenic region
  • bla ampicillin resistance gene
  • the cloned human NK cell, B22 (CD3-, CD16-, CD56+), and the NK cell populations EDF (CD3-, CD16+, CD56-) and 221707 (CD3-,
  • CD16+, CD56+ derived from NK cell leukocytosis patients are cultured in RPMI 1640 medium containing 15 % pooled human serum, 20 X T cell growth factor (TCGF) (Biotest, FRG), 2 mM
  • L-glutamine supplemented with streptomycin and penicillin and stimulated weekly with irradiated (10,000 Rad) LCL feeder cells
  • the EDF culture is treated with LCL feeder cells at two week intervals and cells are harvested after 3 weeks of culture.
  • 221707 is not treated with feeder cells and is harvested after 2 weeks of culture. After complement depletion of CD3+ cells and growth for 2 to 3 weeks in TCGF-containing medium, virtually 100
  • % of the cells in the population have the NK phenotype.
  • Allogeneic cytotoxic T cell clones are cultured in the same medium as the NK cell lines and are stimulated weekly with LCL feeder cells. Tel and TC3 are CD4+ and Tc2 is CD8+.
  • the lymphoblastoid cell line, FJ0, and the leukaemic T cell line, Jurkat, are cultured in RPMI 1640 containing 10 % foetal calf serum, glutamine and antibiotics.
  • the CD4-positive allogeneic T cell clones, KD15, .3-78, KD33, and a CD4-positive cytomegalovirus-specific helper T cell clone, WRC-16, are cultured in the same medium as the NK clone.
  • the chronic myelogenous leukaemia line, K562, the histocytic lymphoma line, U937, the leukaemic T cell line, Jurkat, and the Epstein-Barr virus-transformed lymphoblastoid cell line, FJO are cultured in RPMI 1640 containing 10 % fetal calf serum, glutamine and antibiotics.
  • the T cell lymphoma line, Hut78 is cultured in the same medium as above, with the addition of 10 % TCGF.
  • the promyelocytic leukaemia line, HL60 is cultured in the same medium as above except it contains 20 % fetal calf serum.
  • One half of the HL60 is stimulated with 1.25 % DMS0 at days 1 and 3 and harvested at day 7.
  • the monocyte line, THP-1 is cultured in RPMI 1640 containing 10 % fetal calf serum and 2 x 10" 5 M ⁇ -mercaptoethanol.
  • B22 normally maintained in 20 % TCGF, is resuspended in medium containing 5 % TCGF, in which cells remain viable but do not proliferate. After two days, the cells are returned to medium containing 20 % TCGF. Total cytoplasmic RNA is extracted at various time points and is subjected to Northern blot analysis.
  • RNA used for preparation of cDNA libraries, Northern blots, or cDNA probe is extracted and eluted from oligo dT cellulose using the method of J.E. BADLEY et al. (1988): BioTechniques 6, 114.
  • cDNA libraries of B22 and FJO are prepared using the method of PALAZZOLO & MEYEROWITZ (1987): Gene 52, 197.
  • poly(A) RNA is converted to double-stranded cDNA using the Bethesda Research Laboratories cDNA Synthesis System, except that first-strand synthesis is primed with an oligonucleotide having an Xbal cloning site at its 5' end and a homopolymeric T tail at its 3' end.
  • the double-stranded cDNA is methylated with EcoRI methylase, ligated to EcoRI linkers, and digested with EcoRI and Xbal.
  • the cDNA is then size fractionated on a Biogel A50M column and all fractions with a minimum size of 400 bp are combined, ligated to EcoRI-Xbal arms of the Lambda ( ⁇ ) GEM-2 vector (Promega Biotech), and packaged using Gigapack Gold (Stratagene).
  • a library of 10 6 primary plaques is amplified.
  • the final libraries consist of cDNA inserts with an Xbal site adjacent to an SP6 RNA polymerase promoter at the 3' end of the message and an EcoRI site next to a T7 RNA polymerase promoter at the 5' end.
  • ⁇ -DNA from libraries or clones is prepared from plate lysates using the polyethylene glycol/NaCl precipitation method described by T. MANIATIS et al. (1982) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. DNA preparation from the B22 and FJO libraries are subsequently used to prepare a subtracted cDNA library following the method of MJ PALAZZ0L0 et al. (1989): Neurone 3, 527.
  • T7 RNA polymerase is used to synthesise poly(A) RNA (hereafter referred to as synthetic RNA) which is immediately loaded onto an oligo (dT) cellulose column, and bound fractions containing approximately 80 % of the newly synthesised RNA are recovered.
  • 32 [P]-labelled first-strand cDNA is prepared from the B22 synthetic RNA using the same priming oligomer described above, and the RNA is subsequently base hydrolyzed. The annealing procedure and subsequent steps are carried out twice starting either with 2 or 6 ⁇ g of B22 first-strand cDNA.
  • the sample is applied to a BioRad HPHT HPLC column equilibrated with 10 mM sodium phosphate (pH 6.8) at 60°.
  • Non-annealed, single-stranded cDNA is eluted with 154 mM phosphate buffer, and fractions containing cDNA are combined.
  • NK cDNA After two cycles of subtraction approximately 0.2 ⁇ g single-stranded NK cDNA is recovered. An oligonucleotide homologous to a short vector encoded stretch at the 3' end of the subtracted first-strand cDNA is used to prime second-strand synthesis. The double-stranded cDNA is then cleaved with EcoRI and Xbal, size fractionated on a Biogel A50M column and ligated into EcoRI-Xbal ⁇ GEM-2 arms. A total of 1100 plaques are obtained from the two trials after in vitro packaging and plating. Each plaque is picked with a glass capillary tube and the plug transferred to a well of a 96-well' plate containing 100 ⁇ l of SM buffer.
  • Differential hybridisation is performed on plaque lifts that are prepared on nitro-cellulose membranes (MANIATIS et al.). During the first differential screening of the total library, lifts are prepared from 150 mm plates containing 500-800 plaques. All subsequent screenings are performed on ordered plaques that are prepared using a sterile stainless steel transfer device that duplicates the ordered array of a 96-well plate.
  • First strand 32 [P]-labelled cDNA probe is prepared from mRNA using the same conditions of cDNA synthesis described above except that dCTP is present at 25 ⁇ M and ( ⁇ - 32 [P])dCTP is present at 3.5 ⁇ Ci/ ⁇ l.
  • RNA is base hydrolyzed and cDNA is separated from unincorporated label on a small Sephadex G50 column.
  • Previously described gene probes are labelled using either the Nick Translation Kit or the Multiprime Labelling System from Amersham. Probes prepared from individual clones for cross-hybridization studies are of several types.
  • DNA inserts are excised from individual clones, electrophoresed on a 1.3 % low 5 melting point agarose gel and the insert band recovered from the gel and labelled using the multiprime labelling system of Amersham.
  • one ng of ⁇ -DNA from a clone is amplified by the polymerase chain reaction using the GeneAmp system from Perkin Elmer Cetus. The amplified product is again 0 purified on a 1.3 % low melting point agarose gel and labelled using the Multiprime system. Plaque hybridisations with all DNA probes are carried out as in MANIATIS et al. Asymmetric polymerase chain reaction is used to amplify inserts and the 5'-end sequence (up to 450 nucleotides in length) is determined
  • a full length cDNA sequence is determined by subcloning Pstl fragments encompassing the entire NKG5 insert into M13mpl9.
  • Oligomer 1 5'-ATCCAGAGGCCCAGCCAAATCCACTCTCCCTTCCA-3' , is complementary to a portion of the 242-base insert.
  • Oligomer 2 25 5'-CTGGGCCAGGCACGGGCAGGATTTCTCCTCATCA-3', is complementary to NKG5.
  • Oligonucleotides are end-labelled with ⁇ - 32 [P]-ATP (>5000Ci/mmol, Amersham). Labelled oligonucleotides are used as probes in Northern blot hybridisation and plaque hybridisation.
  • RNA samples are isolated as described above. At appropriate time points after exposure to 20 % TCGF, cells are lysed with a low concentration of NP40, nuclei are pelleted and the aqueous phase extracted with phenol/chloroform. Northern analysis is performed as described by P. THOMAS (1980) Proc. Natl. Acad. Sci. USA 77, 5201. Briefly, RNA samples are denatured and applied to formaldehyde-agarose denaturing gels. The RNA is transferred from the gel to GeneScreen Plus (DuPont) by capillary blotting.
  • GeneScreen Plus DuPont
  • Labelled oligonucleotide probes are mixed with denatured salmon sperm DNA (35 ⁇ g/ml hybridisation solution) and then added to the , 5 blot at 1-5 x 10 6 cpm/ml hybridisation buffer. Following overnight hybridisation at 42°C, blots are washed two times in 2 x SSC for 5 min at room temperature, then twice in 2 x SSC and 1 % SDS for 30 min at 50°C.
  • the B22 cDNA library is screened with the oligomer 1 probe.
  • Plaque lifts prepared on Colony/Plaque Screen nylon membrane (NEN), are hybridised using the same condition as for Northern blots. Positive clones are picked and their insert sizes are determined. The longest clone is subjected to DNA sequencing.
  • Genomic DNA is digested with the indicated restriction enzyme and 10 ⁇ g/lane of digested DNA is electrophoresed on a 0.7 % agarose gel. The DNA is transferred to nitro-cellulose membrane by capillary blotting using 20 x SSC. Hybridisation and washes are performed as in J.A. NICKLAS, et al. (1985) Human Immunol. 13, 95.
  • samples are purified using Millipore Ultrafree-MC low binding filter units (10,000 mol weight cut-off).
  • Genomic clones carrying the NKG5 gene are isolated from a placental genomic DNA library.
  • the entire NKG5 gene is found to be located on two BamHI fragments of 4.4 and 5.6 kb in length which are inserted into the pIRC77 vector, a T7 promoter carrying pUC19 derivative.
  • a "genome walking" strategy is followed, i.e. after starting from a chemically synthesized oligonucleotide complementary to a region within the known NKG5 cDNA sequence, the genomic sequence is determined which sequence is proceeded from primers complementary to the 3' sequence information obtained from each previous sequencing run.
  • Organization of the gene analysis of sequencing data
  • the gene of the invention consists of five exons and four introns.
  • the shortest intron has a length of 534 nucleotides (intron 2) whereas the longest intron (intron 3) is 1448 nucleotides long.
  • the positions of the four introns can be defined unambigously if the GT/AG rule (S.M. MOUNT (1982) Nucleic Acids Res. 10, 459 - 474) is followed.
  • intron 1 and intron 4 Two of the introns (intron 1 and intron 4) are of class 1 (P.A. SHARP (1981) Cell 23, 643 - 646) and therefore interrupt the coding sequence between the first and second nucleotide of a codon. Introns 2 and 3 belong to class 0 thus interrupting the coding sequence between codons.
  • Intron - exon boundaries (numbering according to positions in Fig. SEQ ID NO 3): exon 1 / intron 1 2261/2262 intron 1 / exon 2 3110/3111 exon 2 / intron 2 3214/3215 intron 2 / exon 3 3748/3749 exon 3 / intron 3 3847/3848 intron 3 / exon 4 5295/5296 exon 4 / intron 4 5467/5468 intron 4 / exon 5 6352/6353
  • the introns interrupt the coding sequence at gly-18, after gln-52, after gln-85, and at gly-143.
  • homologous regions longer than 40 base pairs and homologies higher than 70% can be identified.
  • the homologous regions are: a) 2297 - 2339 (intron 1) 5194 - 5239 (intron 3)
  • Intron 2 does not contain a DNA sequence homologous to this repeat regions.
  • the region of intron 1 which comprises the "Jongstra exon" contains one of those repeats. It is known that such homologous repeats (e.g. Alu repeats) were also found in intron sequences of various other genes including those encoding cytokines.
  • the cDNA of the invention is inserted into the plasmid vector pcDNA I (Invitrogen), which is a derivative of pCDM 8 (B. SEED, (1987) Nature 329, 840 - 842) to give the expression construct pSFI 1003.
  • pcDNA I Invitrogen
  • pCDM 8 B. SEED, (1987) Nature 329, 840 - 842
  • the cDNA of the invention is under the control of the CMV promoter.
  • BB Construct for transient and/or stable expression in COS or CHO cells
  • cDNA of the invention is inserted into the plasmid vector pMAMneo-luc (delivered by Clontech) to give the expression construct pSFI 1004.
  • the cDNA is under the control of the MMTV-LTR promoter which allows induction of expression by steroid hormones (dexamthasone).
  • the polycistronic mRNA encodes firefly luciferase which can be used as reporter system for gene expression.
  • the neomycin resistance gene allows for selection of stably transfected cell clones
  • cDNA of the invention is inserted into a retroviral double expression vector derived from Moloney murine leukaemia virus (D. ARMENTANO et al. , (1987) J. Virol. 61, 1647 - 1650; P.A. HANTZ0P0UL0S et al. (1989) Proc. Natl. Acad. Sci. USA 86, 3519 - 3523) to give the expression construct pSFI 1009 and pSFI 1010.
  • Moloney murine leukaemia virus D. ARMENTANO et al. , (1987) J. Virol. 61, 1647 - 1650; P.A. HANTZ0P0UL0S et al. (1989) Proc. Natl. Acad. Sci. USA 86, 3519 - 3523
  • the 5' end of the cDNA of the invention is removed and reconstructed in a way that a) the sequence encoding the putative eucaryotic signal peptide is absent in the bacterial expression constructs and b) the fusion proteins synthesised in the bacterial cell should be, after purification from E. coli extracts, specifically cleavable either by blood coagulation factor Xa or by the protease kallikrein thus releasing the inventive protein moiety from the fusion protein.
  • a series of fusion constructs with different fusion partners is designed and prepared (See Table III).
  • the construct in which the cDNA or the invention is N-terminally fused to a gene encoding E. coli ⁇ -galactosidase (pAX-vector; delivered by Medac) is the preferred construct used for the purification process for the recombinant protein of the invention.
  • the pAX-vector is shown in Figure 1. With this preferred construct (pSFI 1014), expression rates are found to be high.
  • the recombinant fusion protein remains soluble in the cytoplasm and can be purified by affinity chromatography on APTG columns (supplied by MEDAC). Further purification is done by standard methods known to the man skilled in the art.
  • ADDRESSEE SANDOZ LTD.
  • STREET Lichtstrasse 35 CITY: CH-4002 BASEL COUNTRY: Switzerland
  • LENGTH 746 nucleotides
  • TYPE nucleotide sequence
  • STRANDEDNESS single-stranded TOPOLOGY: linear
  • CTCTGTGGGC ACCCAGGTGC CCCTGCCTCC CCCCGGCCTT CTCCCCCGTC 2390 ATGGAGGCCT GGCCCTCCCC TCAGAGCCAG GCTTAGTCCG GTGTGCTGCC 2440
  • CAGCCTGTCA CTGGCCTGGC CAAGGAGGAG AGACAGGCCA GGGATTCTGG 2490
  • AAGACAAGCA AAAGGGTGGG TAGGGGCCAG GCAGCCAGTT CAGGCCTTCA 3614
  • TAATAGTCCC TATCTTGCAA GTTGTCAAAC CCITTTTTTTTT TTTTTTCCTT 4347
  • CAGTACTCCC ATTGCTAGGG CTGCTGGAAC CTGCAGGGTT GGCAGAGCTG 5247
  • AGA AGT GTT TCC AAT GCT GCG ACC CGG GTG TGT AGG ACG GGG 5337 Arg Ser Val Ser Asn Ala Ala Thr Arg Val Cys Arg Thr Gly
  • CTGTGTAGTC CTTCAATAAA TGTCTGTCGT GTGTCCCATA CACTGTTGTA 6653

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Abstract

The invention relates to a gene derived from a Natural Killer cell line as well as to the protein encoded by the gene and to the use of the protein as a medicament. The genomic DNA of the invention having a sequence of 6746 nucleotides comprises a promoter region, 5 exons and 4 introns. The protein having a sequence of 145 amino acids is a secreted cytokine. The protein can be used as a medicament in order to support the immune reactions of Natural Killer cells and cytotoxic T cells against cancer and virus infected cells.

Description

DNA AND AMINO ACID SEQUENCE SPECIFIC FOR NATURAL KILLER CELLS AND T CELLS
This invention relates to a gene derived from a Natural Killer (NK) cell line or T cell line, as well as to the protein encoded by the gene and to the use of the protein as a medicament. The gene is detected by employing a combination of differential and subtractive hybridisation methodologies, which can successfully identify cell-specific cDNA clones representing medium to high abundance transcripts, to identify genes that are expressed in NK cells but not in lymphoblastoid cell lines (LCL).
This invention was made with government support under R01 Al 19007 awarded by National Institutes of Health. The government has certain rights in the invention.
Natural killer (NK) cells are present in the peripheral blood in a state capable of lysing NK sensitive targets in a reaction that is not restricted by proteins of the major histocompatibility complex. These cells typically account for about 5% of peripheral blood lymphocytes and they usually possess the large granular lymphocyte (LGL) morphology. In human systems NK activity is usually defined as the ability to lyse cells of the promyeloid leukaemia cell line, K562. In many cases stimulation of NK cells with interleukin 2 (IL-2) will increase the lymphokine activated killer (LAK) activity, the ability to lyse a broader range of tumour target cells. Many ongoing studies are attempting to use cells with LAK activity in anti - tumour therapy.
Natural killer (NK) cells are cytotoxic lymphocytes that share numerous cell surface antigens and functional components with T cells.
Jongstra et al. have previously described a gene, designated 519, that is expressed in functional cytotoxic T cells and helper T cells but not in tumour cells or other hematopoietic cell lines. (J. JONGSTRA, et al. (1987) J Exp Med 165, 601 - 614) The cDNA was subtracted with RNA from the EBV-transformed B cell line LB (EBV = Epstein-Barr Virus). The gene is expressed in functional T cell lines but not in established tumour cell lines.
The 519 gene product seems to be involved in the activation and subsequent proliferation and / or differentiation of resting T cells. The translation of 519 RNA is restricted to nontransformed, IL-2 dependent, antigen-driven functional T cell lines. The restricted expression pattern of 519 mRNA indicates that the expression of the 519 gene is regulated by the periodic addition of IL-2 and antigen to the functional T cell lines. The expression of the 519 gene is regulated in normal T cells, but not in normal B cells, by mitogenic or antigenic stimulation.
The 519 protein acts intra-cellularly, since the predicted amino acid sequence of the 519 protein does not support the hypothesis that the 519 protein is secreted or membrane bound to function either as a lymphokine or as a membrane receptor molecule.
The present invention provides a new cDNA or genomic DNA sequence that encodes a secreted protein which has the function- of a lymphokine. The gene is isolated from human natural killer cells. The protein encoded by the gene is normally synthesised by natural killer cells and by T cells.
The present invention provides a vector comprising a gene coding for a secreted protein having the following amino acid sequence
a)
MET Ala Thr Trp Ala Leu Leu Leu Leu Ala Ala Met Leu Leu Gly
5 10 15
Asn Pro Gly Leu Val Phe Ser Arg Leu Ser Pro Glu Tyr Tyr Asp
20 25 30
Leu Ala Arg Ala His Leu Arg Asp Glu Glu Lys Ser Cys Pro Cys
35 40 45 Leu Ala Gin Glu Gly Pro Gin Gly Asp Leu' Leu Thr Lys Thr Gin
50 55 60
Glu Leu Gly Arg Asp Tyr Arg Thr Cys Leu Thr He Val Gin Lys
65 70 75
Leu Lys Lys Met Val Asp Lys Pro Thr Gin Arg Ser Val Ser Asn
80 85 90
Ala Ala Thr Arg Val Cys Arg Thr Gly Arg Ser Arg Trp Arg Asp
95 100 105
Val Cys Arg Asn Phe Met Arg Arg Thr Gin Ser Arg Val Thr Gin
110 115 120
Gly Leu Val Ala Gly Glu Thr Ala Gin Gin He Cys Glu Asp Leu
125 130 135
Arg Leu Cys He Pro Ser Thr Glu Pro Leu
140 145
or b) allelic variations thereof in which one or more amino acids have been added, substituted or removed without substantially affecting the activity of the protein mentioned under a) . The amino acid sequence is also indicated in Sequence Identifier No. 1.
The genetic code is degenerate; that is, most amino acids are coded by more than one codon of three nucleotides. Accordingly, allelic variations in the nucleotide sequence may or may not alter the amino acid sequence. Therefore, allelic variations are primarily on the DNA level and may also exist secondarily on the level of the amino acid sequence.
The DNA sequence coding for the protein of the invention can be modified by conventional techniques to produce variations in the final protein of the invention which still has substantially the same activity as the protein with the amino acid sequence mentioned under a) or Seq Id No 1. Thus, one or more amino acids (for example up to fourteen amino acids) can be added, substituted or removed without substantially affecting the activity of the protein of the invention.
Belgian Patent No. 898,015 describes a typical such technique for replacing cysteine by, e.g., serine.
Substitutions can generally be made in accordance with the following Table 1 when it is desired to modulate finely the amino acid sequence of the protein of the invention.
Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table I, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule, or (c) the bulk of the side chains.
Most deletions and insertions, and substitutions in particular, are not expected to produce radical changes in the characteristics of the protein of the invention. As it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance, the comparison of the functions of the mutated protein with the characteristic functions of the protein of the invention clarify whether the altered protein has a comparable activity.
Preferably the protein has a sequence of amino acids having a homology of 97 to 100 % of the sequence of Seq Id No 1 and more preferably only one or two amino acids are exchanged.
TABLE I NORMAL SUBSTITUTIONS OF AMINO ACIDS IN A PROTEIN
Original Residue Exemplary
Substitutions
Ala Gly, Ser
Arg Lys
Asn Gin, His
Asp Glu
Cys Ser
Gin Asn
Glu Asp Gly Ala, Pro
His Asn, Gin
He Leu, Val
Leu He, Val
Lys Arg, Gin, Glu
Met Leu, Tyr, He
Phe Met, Leu, Tyr Ser Thr
Thr Ser
Trp Tyr
Tyr Trp, Phe
Val He, Leu The cDNA coding for the amino acid sequence indicated in a) above is a human gene; corresponding DNA sequences from other species preferably mammals, more preferably primates, may be used.
If the DNA of the gene is to be transferred to a procaryotic or eucaryotic cell, it is advantageous to combine the DNA sequence of the invention with a suitable promoter which allows the expression of the protein in sufficient quantity.
Using a vector will be helpful if the host cell is a non-mammalian cell, for example a micro-organism such as a bacterium or yeast.
The cDNA coding for the protein indicated in the amino acid sequence in a) is preferably inserted in the fragment EcoRI- Xba I which is introduced in the lambda vector GEM 2 (supplied by Promega Biotec). A suitable transformation vector can rely on the transient introduction of DNA into host cells. (P. MELLON; V. PARKER; Y. GLUTZMAN; T. MANIATIS (1981) Cell 27 279 - 288) Vectors used for the protein of the invention typically contain various elements such as enhancers, promoters, polyadenylation sites and translation activators.
Furthermore, the present invention comprises a method of producing the protein of the invention which has the amino acid sequence of Seq Id No 1. Procaryotic or eucaryotic host cells, e.g. E. coli, yeast, mammalian cells (Chinese Hamster Ovary cells) transformed with the cDNA sequence of the invention are cultivated in a suitable nutrient medium. The protein is isolated and purified by standard methods.
The invention further provides a new cDNA sequence that encodes secreted protein, wherein the cDNA coding and non-coding sequence is selected from the group consisting of: aa) the following cDNA sequence:
10 20 30 40 50
GTATCTGTGG TAAACCCAGT GACACGGGGG AGATGACATA CAAAAAGGGC
60 70 80 90 100
AGGACCTGAG AAAGATTAAG CTGCAGGCTC CCTGCCCATA AAACAGGGTG
110 120 130 TGAAAGGCAT CTCAGCGGCT GCCCCACC ATG GCT ACC 140 170
TGG GCC CTC CTG CTC CTT GCA GCC ATG CTC CTG GGC AAC
200 CCA GGT CTG GTC TTC TCT CGT CTG AGC CCT GAG TAG TAC
• 230 GAC CTC GCA AGA GCC CAC CTC CGT GAT GAG GAG AAA TCC 260 290
TGC CCG TGC CTG GCC CAG GAG GGC CCC CAG GGT GAC CTG 320
TTG ACC AAA ACA CAG GAG CTG GGC CGT GAC TAC AGG ACC 350
TGT CTG ACG ATA GTC CAA AAA CTG AAG AAG ATG GTG GAT 380 410
AAG CCC ACC CAG AGA AGT GTT TCC AAT GCT GCG ACC CGG 440 GTG TGT AGG ACG GGG AGG TCA CGA TGG CGC GAC GTC TGC
470 AGA AAT TTC ATG AGG AGG TAT CAG TCT AGA GTT ACC CAG 500 530
GGC CTC GTG GCC GGA GAA ACT GCC CAG CAG ATC TGT GAG 560 570
GAC CTC AGG TTG TGT ATA CCT TCT ACA GGT CCC CTC TGA G
580 590 600 610 620
CCCTCTCACC TTGTCCTGTG GAAGAAGCAC AGGCTCCTGT CCTCAGATCC
630 640 650 660 670
CGGGAACCTC AGCAACCTCT GCCGGCTCCT CGCTTCCTCG ATCCAGAATC 680 690 700 710 720 CACTCTCCAG TCTCCCTCCC CTGACTCCCT CTGCTGTCCT CCCCTCTCAC
730 740 GAGAATAAAG TGTCAAGCAA GAAAAAAA
or
bb) cDNA sequences that encode alleles or minor modifications which do not substantially change the activity.
Additionally the invention comprises cDNA sequences that hybridise under stringent conditions to the DNA sequence defined in aa) or Seq Id No 2 and have a homology of 90 to 100 X. A homology of 95 to 100 X is preferred, more preferred is a homology of 97 to 100%, particularly preferred 99 to 100%.
The full genomic DNA of the invention correlating to the cDNA sequence is indicated in SEQ ID NO 3 and normally transcribed and translated by natural killer cells or T cells.
A comparison of the full-length cDNA sequence of the gene of the invention and the published sequence of gene 519 shows that gene 519 contains an insert of 242 base pairs that is not present in the DNA of the invention.
This deletion leads to the use of a different translational start codon. As a consequence, unlike 519, the polypeptide encoded by the DNA of the invention has a strongly hydrophobic N-terminal amino acid sequence which is characteristic for signal peptides. The lack of additional hydrophobic regions in the rest of the protein shows that the protein of the invention is a secreted protein while gene 519 was speculated to encode an intracellular polypeptide possibly involved in some aspect of growth and/or differentiation of functional T cells. The gene encoding for the protein of the invention is specifically transcribed in NK and T cells but not in a variety of other hematopoietic and non hematopoietic cell lines.
<=. In contrast the protein encoded by the DNA according to the invention is secreted. It is a cytokine either involved in differentiation and / or proliferation of target cells or related to the killing function of natural killer cells or T cells.
0 The invention further comprises a protein having the following amino acid sequence:
aaa)
MET Ala Thr Trp Ala Leu Leu Leu Leu Ala Ala Met Leu Leu Gly 5
5 10 15
Asn Pro Gly Leu Val Phe Ser Arg Leu Ser Pro Glu Tyr Tyr Asp
20 25 30
Leu Ala Arg Ala His Leu Arg Asp Glu Glu Lys Ser Cys Pro Cys
35 40 45 0
Leu Ala Gin Glu Gly Pro Gin Gly Asp Leu Leu Thr Lys Thr Gin
50 55 60
Glu Leu Gly Arg Asp Tyr Arg Thr Cys Leu Thr He Val Gin Lys
65 70 75
Leu Lys Lys Met Val Asp Lys Pro Thr Gin Arg Ser Val Ser Asn 5
80 85 90
Ala Ala Thr Arg Val Cys Arg Thr Gly Arg Ser Arg Trp Arg Asp
95 100 105
Val Cys Arg Asn Phe Met Arg Arg Thr Gin Ser Arg Val Thr Gin
110 115 120 0
Gly Leu Val Ala Gly Glu Thr Ala Gin Gin He Cys Glu Asp Leu
125 130 135
Arg Leu Cys He Pro Ser Thr Glu Pro Leu
140 145 or bbb) allelic variations thereof in which one or more amino acids have been added, substituted or removed without substantially affecting the activity of the protein; and/or ccc) post-translational variations thereof which do not substantially affect the activity of the protein.
By "post-translational variations" as mentioned in ccc) above is meant variations during or after translation, such as glycosylation, formation of disulfide bridges and chemical modifications of amino acids.
Glycosylation is one of the major bio-synthetic functions of the endoplas ic reticulum and/or Golgi apparatus. The sequence and branching of the oligosaccharides formed in the reticulum can be altered in the Golgi apparatus, the lysosomes or plasma membrane. The oligosaccharides can be N-linked oligosaccharides (asparagine linked) or 0-linked oligosaccharides (serine, threonine or hydroxylysine linked). The glycosylation is dependent on the producing cell type and the species from which the cell type derives. The amount and form of glycosylation can be influenced by compounds as described in European Patent Application EP 0 222 313. Changes in glycosylation may affect the function of the protein.
Proteins often form covalent intrachain bonds. These disulfide bonds are formed between cysteine-SH amino acids in the folded protein or in the protein which folds during translation. The bonds stabilise the three-dimensional structure of the protein. Such disulfide bonds are rarely formed in protein molecules that are still in the cell cytosol because the high intracellular concentration of the -SH reducing agent glutathione breaks most of such bonds. Once the proteins are outside the cytoplasm, are secreted or are on the cell surface, they often form additional covalent intrachain bounds. Furthermore, the amino acids may be altered as described in PCT Application WO 91/10684. Other alterations of the side chains of the amino acids are possible.
A preferred homology is 97 to 100% and more preferably only one or two amino acids are exchanged.
The protein of the invention has at least a purity of 40%, preferably 60%, more preferably 80% and most preferably 90%. The purity is defined by the amount of the protein of the invention in relation to the total amount of protein.
One method to purify the protein is to use a fusion protein comprising the protein of the invention and a carrier protein, e.g. galactosidase or alkaline phosphatase. Fusion-proteins can be detected during purification because of the presence of the carrier protein (galactosidase or alkaline phosphatase reaction). Fusion-proteins can be enriched by antibody coated columns (anti-galactosidase or anti-phosphatase monoclonal antibodies). The fusion-protein can be separated in two pieces by a kinase forming a separated carrier protein and a separated protein of the invention. Then the protein of the invention can be isolated from the mixture consisting substantially of the carrier protein, the kinase and protein of the invention by using a column with anti-carrier protein and anti-kinase monoclonal antibodies. (See C. MAN0IL et al. (1990) J. of Bacteriology, Vol. 172, 515 - 518)
The protein of the invention is a cytokine preferably a human cytokine.
A cytokine is a protein produced and secreted from one or more cell types. It recognises specific receptors on the membrane of one or more target cell types, possibly also including the producer cells. Binding of cytokines to their receptor usually initiates regulatory changes in the target cells which may relate to effector functions, differentiation or proliferation. The function of the cytokine can be measured by proliferation tests (e.g. 3 [H]-desoxythymidine-incorporation) or by cytotoxicity tests (51 [Cr]-release-test) or by colony forming assays or by production of an activity that affects other cells.
The DNA and protein of the invention have advantageous properties, in particular they exhibit pharmacological activity and are, therefore, useful as pharmaceuticals.
In particular, the protein of the invention causes increased activity of natural killer cells and cytotoxic T cells against human or gibbon tumour cells. The activity of the system is tested by the 51[Cr] release test described by Brunner et al. (BRUNNER et al. (1981) J Exp Med 154: 362 - 373) Tumour cells as target cells which are labelled by 51[Cr] are incubated in the presence with effector cells for 4 to 6 hours. The effectiveness of the effector cells is expressed by the release of 51[Cr] material into the medium. The 51[Cr] release is increased in the presence of the protein according to the invention.
Accordingly the protein of the invention can be used as an active therapeutic substance. The invention provides a pharmaceutical composition comprising a protein of the invention in association with a pharmaceutically acceptable diluent or carrier. Further it comprises a pharmaceutical composition comprising a therapeutically effective protein of the invention and a pharmaceutically acceptable salt or a pharmaceutically acceptable carrier or diluent.
The protein of the invention can be used for the manufacture of a medicament for treatment of autoimmune deficiency by enhancing the immune response, or for treatment of cancer by killing cancer cells. The invention provides a method of treating an immune deficiency in humans or animals in need of such treatment comprising administering a therapeutically effective amount of the protein according to the invention. Further the invention provides a method of treating cancer in human or animals in need of such treatment comprising administering a therapeutically effective amount of the protein according to the invention.
The compound of the invention shows this activity in the gibbon when administered by injection at daily dosages of from about 10--*1 M to about 5x10"10 M per kg or about 0,2 μg to 13 μg per kg-
For this indication the appropriate dosage will vary depending upon, for example, the host, the mode of administration and the nature and severity of the condition being treated. In general, satisfactory results in animals are indicated to be obtained at daily dosages from about 3x10"-*1 M to 10-10 M per kg body weight or 0,5 μg to 2,5 μg per kg. The dosages are administered by one to four injections per day.
Furthermore, the protein of the invention shows activity in a system in which isolated cytotoxic T cells kill virus infected human or gibbon target cells.
The compound of the invention shows this activity in the gibbon when administered by injection at daily dosages of from about 10"10 M to about 5x10"10 M per kg or 1,5 μg to 13 μg per kg. The compound of the invention is therefore indicated for the treatment of viral diseases.
For this indication the appropriate dosage will vary depending upon, for example, the host, the mode of administration and the nature and severity of the condition being treated. In general, satisfactory results in animals are indicated to be obtained at daily dosages from about 2x10"10 M to 5x10"10 M per kg body weight or 3 μg to 13 μg per kg. The dosages are administered by one to four injections per day.
The compound of the invention may be administered by any conventional route, in particular by injection in the form of a solution or suspension in a pharmaceutically acceptable diluent. Such compositions may be manufactured in conventional manner.
The compound with the amino acid sequence of SEQ ID NO 1 is the preferred compound for the treatment of cancer and viral infections.
Further a cell of the immune system transformed with the DNA of the invention can stimulate the immune response against different diseases, for example cancer, or diseases caused by viruses or eucaryotic parasites, e.g. protozoa or multicellular organisms.
It is possible to alter natural killer cells or cytotoxic T cells in order to treat diseases such as cancer or viral infections. Lymphocytes are taken from one individual, transformed with the DNA of the invention and the transformed cells replaced in the same individual. The potency of the natural immune system is increased by this method. The natural functions of the body are augmented without introducing foreign cells or foreign proteins which might induce a immune response against the newly introduced protein.
It may also be possible to take blood, lymph node or spleen cells from a patient, to transform these cells in vitro and to re-inject these cells in the patient. The isolated DNA sequence can be introduced into patient-derived lymphocytes. The protein encoded by such introduced DNA does not differ from person to person, unlike other proteins, for example Major Histocompatibility Complex antigens. The re-injected cell had learnt during the time of maturation to distinguish between self and non-self. Therefore an uncontrolled attack of the transformed cells against self antigens can be excluded.
Further, the protein of the invention can be used for treatment of autoimmune disease by blocking the action of the cytokine by antagonists or down regulation of gene expression.
10
Figure 1 shows the pAX-vector which is supplied by MEDAC. The lacZ gene includes the lac operator. The hinge region of 187 bp codes for a collagen fragment. The vector comprises an endoproteinase Xa recognition site and a multicloning site including Eco RI in all translational reading frames. There are
15 stop codons in all three reading frames and a long lambda t0 terminator. The fl-origin is used for replication for isolation of single-stranded DNA. The pBR322 fragment harbours the origin of replication and finally the vector has the ampicillin resistance marker.
20
(Plac: lacZ gene promotor; lacZ: lacZ gene; CS: collagen fragment; Xa: Xa cleavage site; MCS: multi cloning site; t0: lambda t0 terminator; fl ori: phage fl intergenic region; bla: ampicillin resistance gene)
■*n EXAMPLES
Cell Culture
The cloned human NK cell, B22 (CD3-, CD16-, CD56+), and the NK cell populations EDF (CD3-, CD16+, CD56-) and 221707 (CD3-,
CD16+, CD56+) derived from NK cell leukocytosis patients are cultured in RPMI 1640 medium containing 15 % pooled human serum, 20 X T cell growth factor (TCGF) (Biotest, FRG), 2 mM
L-glutamine, supplemented with streptomycin and penicillin and stimulated weekly with irradiated (10,000 Rad) LCL feeder cells
(LCL = Ly phoblastoid cell line).
Cells are harvested and RNA isolated on day 6 or 7 of the growth cycle. Frozen PBL (= peripheral blood lymphocytes) from the patients are depleted for CD3+ cells (which constituted less than 15 % of the original samples) by treatment with 0KT3 (Ortho Biotech) and rabbit complement.
The EDF culture is treated with LCL feeder cells at two week intervals and cells are harvested after 3 weeks of culture.
221707 is not treated with feeder cells and is harvested after 2 weeks of culture. After complement depletion of CD3+ cells and growth for 2 to 3 weeks in TCGF-containing medium, virtually 100
% of the cells in the population have the NK phenotype.
Allogeneic cytotoxic T cell clones (Tc) are cultured in the same medium as the NK cell lines and are stimulated weekly with LCL feeder cells. Tel and TC3 are CD4+ and Tc2 is CD8+. The lymphoblastoid cell line, FJ0, and the leukaemic T cell line, Jurkat, are cultured in RPMI 1640 containing 10 % foetal calf serum, glutamine and antibiotics.
The CD4-positive allogeneic T cell clones, KD15, .3-78, KD33, and a CD4-positive cytomegalovirus-specific helper T cell clone, WRC-16, are cultured in the same medium as the NK clone. The chronic myelogenous leukaemia line, K562, the histocytic lymphoma line, U937, the leukaemic T cell line, Jurkat, and the Epstein-Barr virus-transformed lymphoblastoid cell line, FJO, are cultured in RPMI 1640 containing 10 % fetal calf serum, glutamine and antibiotics. The T cell lymphoma line, Hut78, is cultured in the same medium as above, with the addition of 10 % TCGF. The promyelocytic leukaemia line, HL60, is cultured in the same medium as above except it contains 20 % fetal calf serum. One half of the HL60 is stimulated with 1.25 % DMS0 at days 1 and 3 and harvested at day 7. DMS0 ( = dimethylsulfoxid) stimulation induces approximately 50 % of the cells to differentiate into more mature myeloid cell forms. The monocyte line, THP-1, is cultured in RPMI 1640 containing 10 % fetal calf serum and 2 x 10"5M β-mercaptoethanol.
NK cell activation
B22, normally maintained in 20 % TCGF, is resuspended in medium containing 5 % TCGF, in which cells remain viable but do not proliferate. After two days, the cells are returned to medium containing 20 % TCGF. Total cytoplasmic RNA is extracted at various time points and is subjected to Northern blot analysis.
Isolation and sequencing of NKG5 cDNA clones
Nine independent cDNA clones are isolated from the B22 cDNA library using differential hybridisation as described below.
cDNA Library Preparation
Messenger RNA used for preparation of cDNA libraries, Northern blots, or cDNA probe is extracted and eluted from oligo dT cellulose using the method of J.E. BADLEY et al. (1988): BioTechniques 6, 114. cDNA libraries of B22 and FJO are prepared using the method of PALAZZOLO & MEYEROWITZ (1987): Gene 52, 197. Briefly, 2 μg poly(A) RNA is converted to double-stranded cDNA using the Bethesda Research Laboratories cDNA Synthesis System, except that first-strand synthesis is primed with an oligonucleotide having an Xbal cloning site at its 5' end and a homopolymeric T tail at its 3' end. The double-stranded cDNA is methylated with EcoRI methylase, ligated to EcoRI linkers, and digested with EcoRI and Xbal.
The cDNA is then size fractionated on a Biogel A50M column and all fractions with a minimum size of 400 bp are combined, ligated to EcoRI-Xbal arms of the Lambda (λ) GEM-2 vector (Promega Biotech), and packaged using Gigapack Gold (Stratagene). A library of 106 primary plaques is amplified. The final libraries consist of cDNA inserts with an Xbal site adjacent to an SP6 RNA polymerase promoter at the 3' end of the message and an EcoRI site next to a T7 RNA polymerase promoter at the 5' end.
λ-DNA from libraries or clones is prepared from plate lysates using the polyethylene glycol/NaCl precipitation method described by T. MANIATIS et al. (1982) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. DNA preparation from the B22 and FJO libraries are subsequently used to prepare a subtracted cDNA library following the method of MJ PALAZZ0L0 et al. (1989): Neurone 3, 527. After digestion of library DNA with Xbal, T7 RNA polymerase is used to synthesise poly(A) RNA (hereafter referred to as synthetic RNA) which is immediately loaded onto an oligo (dT) cellulose column, and bound fractions containing approximately 80 % of the newly synthesised RNA are recovered. 32[P]-labelled first-strand cDNA is prepared from the B22 synthetic RNA using the same priming oligomer described above, and the RNA is subsequently base hydrolyzed. The annealing procedure and subsequent steps are carried out twice starting either with 2 or 6 μg of B22 first-strand cDNA. The cDNA is twice annealed to a 10-20 fold excess of FJO synthetic RNA at 42 0 for 72 hours in the presence of 40 % formamide, 0.5 M sodium phosphate (pH 6.8), 10 mM EDTA (=ethylenediaminetetra- aceticacid) and 0.2 % SDS (=sodium dodecylsulfate). After each annealing, the sample is applied to a BioRad HPHT HPLC column equilibrated with 10 mM sodium phosphate (pH 6.8) at 60°. Non-annealed, single-stranded cDNA is eluted with 154 mM phosphate buffer, and fractions containing cDNA are combined.
After two cycles of subtraction approximately 0.2 μg single-stranded NK cDNA is recovered. An oligonucleotide homologous to a short vector encoded stretch at the 3' end of the subtracted first-strand cDNA is used to prime second-strand synthesis. The double-stranded cDNA is then cleaved with EcoRI and Xbal, size fractionated on a Biogel A50M column and ligated into EcoRI-Xbal λGEM-2 arms. A total of 1100 plaques are obtained from the two trials after in vitro packaging and plating. Each plaque is picked with a glass capillary tube and the plug transferred to a well of a 96-well' plate containing 100 μl of SM buffer.
Plaque Hybridisation Studies
Differential hybridisation is performed on plaque lifts that are prepared on nitro-cellulose membranes (MANIATIS et al.). During the first differential screening of the total library, lifts are prepared from 150 mm plates containing 500-800 plaques. All subsequent screenings are performed on ordered plaques that are prepared using a sterile stainless steel transfer device that duplicates the ordered array of a 96-well plate.
First strand 32[P]-labelled cDNA probe is prepared from mRNA using the same conditions of cDNA synthesis described above except that dCTP is present at 25 μM and (α-32[P])dCTP is present at 3.5 μCi/μl. RNA is base hydrolyzed and cDNA is separated from unincorporated label on a small Sephadex G50 column. Previously described gene probes are labelled using either the Nick Translation Kit or the Multiprime Labelling System from Amersham. Probes prepared from individual clones for cross-hybridization studies are of several types. In some cases, DNA inserts are excised from individual clones, electrophoresed on a 1.3 % low 5 melting point agarose gel and the insert band recovered from the gel and labelled using the multiprime labelling system of Amersham. Alternatively, one ng of λ-DNA from a clone is amplified by the polymerase chain reaction using the GeneAmp system from Perkin Elmer Cetus. The amplified product is again 0 purified on a 1.3 % low melting point agarose gel and labelled using the Multiprime system. Plaque hybridisations with all DNA probes are carried out as in MANIATIS et al. Asymmetric polymerase chain reaction is used to amplify inserts and the 5'-end sequence (up to 450 nucleotides in length) is determined
-jr for seven of the clones. A full length cDNA sequence is determined by subcloning Pstl fragments encompassing the entire NKG5 insert into M13mpl9.
20 Oligonucleotide probes
Two oligonucleotide probes are used in these studies. Oligomer 1, 5'-ATCCAGAGGCCCAGCCAAATCCACTCTCCCTTCCA-3' , is complementary to a portion of the 242-base insert. Oligomer 2, 25 5'-CTGGGCCAGGCACGGGCAGGATTTCTCCTCATCA-3', is complementary to NKG5. Oligonucleotides are end-labelled with γ-32[P]-ATP (>5000Ci/mmol, Amersham). Labelled oligonucleotides are used as probes in Northern blot hybridisation and plaque hybridisation.
30 Northern Blot Studies
RNA samples are isolated as described above. At appropriate time points after exposure to 20 % TCGF, cells are lysed with a low concentration of NP40, nuclei are pelleted and the aqueous phase extracted with phenol/chloroform. Northern analysis is performed as described by P. THOMAS (1980) Proc. Natl. Acad. Sci. USA 77, 5201. Briefly, RNA samples are denatured and applied to formaldehyde-agarose denaturing gels. The RNA is transferred from the gel to GeneScreen Plus (DuPont) by capillary blotting. Blots are prehybridised (1M NaCl, 50 % formaraide, 10 % dextran sulphate, 5x Denhardts and 1 % SDS) for 12-16 hours at 42 °C. Labelled denatured DNA probe is added to the blot at 1 x 106 cpm/ml (cpm = counts per minute) hybridisation buffer, and
10 hybridisation continued for 24 hours at 42 °C. The blots are then washed and exposed for autoradiography.
Labelled oligonucleotide probes are mixed with denatured salmon sperm DNA (35 μg/ml hybridisation solution) and then added to the ,5 blot at 1-5 x 106 cpm/ml hybridisation buffer. Following overnight hybridisation at 42°C, blots are washed two times in 2 x SSC for 5 min at room temperature, then twice in 2 x SSC and 1 % SDS for 30 min at 50°C.
20
Isolation of a 519-like cDNA clone from the B22 library
-c The B22 cDNA library is screened with the oligomer 1 probe.
Plaque lifts, prepared on Colony/Plaque Screen nylon membrane (NEN), are hybridised using the same condition as for Northern blots. Positive clones are picked and their insert sizes are determined. The longest clone is subjected to DNA sequencing.
30
Southern blot hybridisation
Genomic DNA is digested with the indicated restriction enzyme and 10 μg/lane of digested DNA is electrophoresed on a 0.7 % agarose gel. The DNA is transferred to nitro-cellulose membrane by capillary blotting using 20 x SSC. Hybridisation and washes are performed as in J.A. NICKLAS, et al. (1985) Human Immunol. 13, 95.
DNA Sequencing
Polymerase chain reaction amplifications is used to prepare single-stranded DNA (Gyllensten & Erlich) for sequencing contained in 100 μl final volume: 1 ng λ-DNA, 50 mM Tris/HCl pH 8.8, 10 mM MgCl2 , 10 mM (NH4)2S04, 1.5 mM of each dNTP (= deoxynucleotide-triphosphate), 100 pmoles of SP6 polymerase promoter primer, 1 pmole T7 polymerase promoter primer (both from Promega), and 2.5 units Taq polymerase (Perkin Elmer Cetus). Samples are cycled 35 times through 1 min denaturation at 94 C°, 2 min annealing at 49 °C, and 3 min extension at 72 °C.
Following the amplification, samples are purified using Millipore Ultrafree-MC low binding filter units (10,000 mol weight cut-off).
ISOLATION AND SEQUENCING OF GENOMIC NKG5 CLONES
Genomic clones carrying the NKG5 gene are isolated from a placental genomic DNA library. The entire NKG5 gene is found to be located on two BamHI fragments of 4.4 and 5.6 kb in length which are inserted into the pIRC77 vector, a T7 promoter carrying pUC19 derivative. For the determination of the DNA sequence of these inserts from double stranded plasmid DNA a "genome walking" strategy is followed, i.e. after starting from a chemically synthesized oligonucleotide complementary to a region within the known NKG5 cDNA sequence, the genomic sequence is determined which sequence is proceeded from primers complementary to the 3' sequence information obtained from each previous sequencing run. Organization of the gene ; analysis of sequencing data
Intron - exon organization
The gene of the invention consists of five exons and four introns. The shortest intron has a length of 534 nucleotides (intron 2) whereas the longest intron (intron 3) is 1448 nucleotides long. The positions of the four introns can be defined unambigously if the GT/AG rule (S.M. MOUNT (1982) Nucleic Acids Res. 10, 459 - 474) is followed.
All of the proposed exon/intron boundaries show a good degree of homology with splice donor and acceptor consensus sequences. Two of the introns (intron 1 and intron 4) are of class 1 (P.A. SHARP (1981) Cell 23, 643 - 646) and therefore interrupt the coding sequence between the first and second nucleotide of a codon. Introns 2 and 3 belong to class 0 thus interrupting the coding sequence between codons.
Intron - exon boundaries (numbering according to positions in Fig. SEQ ID NO 3): exon 1 / intron 1 2261/2262 intron 1 / exon 2 3110/3111 exon 2 / intron 2 3214/3215 intron 2 / exon 3 3748/3749 exon 3 / intron 3 3847/3848 intron 3 / exon 4 5295/5296 exon 4 / intron 4 5467/5468 intron 4 / exon 5 6352/6353
The introns interrupt the coding sequence at gly-18, after gln-52, after gln-85, and at gly-143.
Search for repeat sequences within introns
Within introns, homologous regions longer than 40 base pairs and homologies higher than 70% can be identified. The homologous regions are: a) 2297 - 2339 (intron 1) 5194 - 5239 (intron 3)
b) 2669 - 2718 (intron 1; "Jongstra exon") 2972 - 3025 (intron 1)
5152 - 5197 (intron 3)
c) 4228 - 4299 (intron 3) 5689 - 5757 (intron 4) 6257 - 6301 (intron 4)
Intron 2 does not contain a DNA sequence homologous to this repeat regions. The region of intron 1 which comprises the "Jongstra exon" contains one of those repeats. It is known that such homologous repeats (e.g. Alu repeats) were also found in intron sequences of various other genes including those encoding cytokines.
EXPRESSION OF THE cDNA OF THE INVENTION IN EUCARYOTIC CELLS AND
E. C0LI BACTERIA
Constructs for expression in mammalian cells
AA) Construct for transient expression in COS cells
The cDNA of the invention is inserted into the plasmid vector pcDNA I (Invitrogen), which is a derivative of pCDM 8 (B. SEED, (1987) Nature 329, 840 - 842) to give the expression construct pSFI 1003. In this construct, the cDNA of the invention is under the control of the CMV promoter. BB) Construct for transient and/or stable expression in COS or CHO cells
cDNA of the invention is inserted into the plasmid vector pMAMneo-luc (delivered by Clontech) to give the expression construct pSFI 1004. In this construct, the cDNA is under the control of the MMTV-LTR promoter which allows induction of expression by steroid hormones (dexamthasone). In addition to the cDNA, the polycistronic mRNA encodes firefly luciferase which can be used as reporter system for gene expression. Furthermore, the neomycin resistance gene allows for selection of stably transfected cell clones
CC) Retroviral constructs
cDNA of the invention is inserted into a retroviral double expression vector derived from Moloney murine leukaemia virus (D. ARMENTANO et al. , (1987) J. Virol. 61, 1647 - 1650; P.A. HANTZ0P0UL0S et al. (1989) Proc. Natl. Acad. Sci. USA 86, 3519 - 3523) to give the expression construct pSFI 1009 and pSFI 1010. Using these vectors, stable genomic integration of the cDNA of the invention is achieved with COS, 3T3, HeLa and CEM cells.
In Northern hybridisation experiments, transcription of the cDNA of the invention is shown with all of these expression constructs.
Constructs for expression in E. coli bacteria
AA) cDNA expression as cleavable fusion protein
The 5' end of the cDNA of the invention is removed and reconstructed in a way that a) the sequence encoding the putative eucaryotic signal peptide is absent in the bacterial expression constructs and b) the fusion proteins synthesised in the bacterial cell should be, after purification from E. coli extracts, specifically cleavable either by blood coagulation factor Xa or by the protease kallikrein thus releasing the inventive protein moiety from the fusion protein.
A series of fusion constructs with different fusion partners is designed and prepared (See Table III). The construct in which the cDNA or the invention is N-terminally fused to a gene encoding E. coli β-galactosidase (pAX-vector; delivered by Medac) is the preferred construct used for the purification process for the recombinant protein of the invention. The pAX-vector is shown in Figure 1. With this preferred construct (pSFI 1014), expression rates are found to be high. The recombinant fusion protein remains soluble in the cytoplasm and can be purified by affinity chromatography on APTG columns (supplied by MEDAC). Further purification is done by standard methods known to the man skilled in the art.
Table III cDNA of the invention expressed in E. coli
construct fusion promoter features expr. comments
pSFH005 hIL-3 pL Xa,K,3'nt Westernblot (ahIL-3) pSFHOll hIL-8 ptrp Xa,K,stp3 ++ Westernblot
3'nt (ahIL-8) pSFI1012 hIL-8 ptrp Xa,K,stp3 ++ W-blot (ahIL-8), AS-seq. full length, -16 -50 pSFI1014 lacZ plac CS,Xa,K affinity stp3, term purification by APTG SEQUENCE IDENTIFIER:
(i) APPLICANT: University of MINNESOTA, MINNEAPOLIS MN 55415-1226, U.S.A.
(ii) TITLE OF INVENTION: DNA and amino acid sequence specific for natural killer cells and T cells
(iii) NUMBER OF SEQUENCES: 3
(iv) CORRESPONDENCE ADDRESS:
ADDRESSEE: SANDOZ LTD. STREET: Lichtstrasse 35 CITY: CH-4002 BASEL COUNTRY: Switzerland
(iii) NUMBER OF SEQUENCES: 3
(vii) PRIOR APPLICATION DATA:
APPLICATION NUMBER: US SN 07/590,412 FILING DATE: 28 September, 1990
INFORMATION FOR SEQ ID NO: 1
(i) SEQUENCE CHARACTERISTICS:
LENGTH: 145 amino acids TYPE: amino acid sequence
(ii) MOLECULE TYPE: protein
MET Ala Thr Trp Ala Leu Leu Leu Leu Ala Ala Met Leu Leu Gly
5 10 15
Asn Pro Gly Leu Val Phe Ser Arg Leu Ser Pro Glu Tyr Tyr Asp
20 25 30
Leu Ala Arg Ala His Leu Arg Asp Glu Glu Lys Ser Cys Pro Cys
35 40 45
Leu Ala Gin Glu Gly Pro Gin Gly Asp Leu Leu Thr Lys Thr Gin
50 55 60
Glu Leu Gly Arg Asp Tyr Arg Thr Cys Leu Thr He Val Gin Lys
65 70 75
Leu Lys Lys Met Val Asp Lys Pro Thr Gin Arg Ser Val Ser Asn
80 85 90
Ala Ala Thr Arg Val Cys Arg Thr Gly Arg Ser Arg Trp Arg Asp
95 100 105
Val Cys Arg Asn Phe Met Arg Arg Thr Gin Ser Arg Val Thr Gin
110 115 120
Gly Leu Val Ala Gly Glu Thr Ala Gin Gin He Cys Glu Asp Leu
125 130 135
Arg Leu Cys He Pro Ser Thr Glu Pro Leu
140 145
INFORMATION FOR SEQ ID NO: 2
(i) SEQUENCE CHARACTERISTICS:
LENGTH: 746 nucleotides TYPE: nucleotide sequence STRANDEDNESS: single-stranded TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
GTATCTGTGG TAAACCCAGT GACACGGGGG AGATGACATA CAAAAAGGGC 50
AGGACCTGAG AAAGATTAAG CTGCAGGCTC CCTGCCCATA AAACAGGGTG 100
TGAAAGGCAT CTCAGCGGCT GCCCCACC ATG GCT ACC TGG GCC CTC CTG 149
CTC CTT GCA GCC ATG CTC CTG GGC AAC CCA GGT CTG GTC TTC TCT 194
CGT CTG AGC CCT GAG TAC TAC GAC CTC GCA AGA GCC CAC CTC CGT 239
GAT GAG GAG AAA TCC TGC CCG TGC CTG GCC CAG GAG GGC CCC CAG 284
GGT GAC CTG TTG ACC AAA ACA CAG GAG CTG GGC CGT GAC TAC AGG 329
ACC TGT CTG ACG ATA GTC CAA AAA CTG AAG AAG ATG GTG GAT AAG 374
CCC ACC CAG AGA AGT GTT TCC AAT GCT GCG ACC CGG GTG TGT AGG 419
ACG GGG AGG TCA CGA TGG CGC GAC GTC TGC AGA AAT TTC ATG AGG 464
AGG TAT CAG TCT AGA GTT ACC CAG GGC CTC GTG GCC GGA GAA ACT 509
GCC CAG CAG ATC TGT GAG GAC CTC AGG TTG TGT ATA CCT TCT ACA 554
GGT CCC CTC TGA GCCCTCTCAC CTTGTCCTGT GGAAGAAGCA CAGGCTCCTG 606
TCCTCAGATC CCGGGAACCT CAGCAACCTC TGCCGGCTCC TCGCTTCCTC 656
GATCCAGAAT CCACTCTCCA GTCTCCCTCC TCTGACTCCC TCTGCTGTCC 706
TCCCCTCTCA CGAGAATAAA GTGTCAAGCA AGAAAAAAA 746 INFORMATION FOR SEQ ID NO: 3
(i) SEQUENCE CHARACTERISTICS:
LENGTH: 6746 nucleotides TYPE:- nucleotide sequence STRANDEDNESS: double-stranded TOPOLOGY: linear
(ii) MOLECULE TYPE: genomic DNA
Figure imgf000032_0001
CCTCATCCTC CCTAGCAGGA TATCAATTTT GTTCGAAGTG TCAATGAAGG 1050
CCAGGTGCGG TGGCTGATGC CTGTAATCCT AACACTTTGG GAGGCCGAGG 1100
CAGGCGGATC ACCTGAGGTC AGGAGTTCAA GACCAGCCTG GCCAACATGG 1150
TGAAACCCTG TCTCTACTAA AAACACACAA ATTAGCAGGG CATGGTGGCG 1200
TGCACCTGTA ATCCCAGCTA CTCAGGAGGC TGAGACAGGA GAATCACTTG 1250
AACCCGGAGT GGAGGTTGCA ATCAGCCAAG ATCACACCAC TGCACTTCAG 1300
CTTGGGTGAC AAGAGTGAAA CTCTGTCTCA AAAAAGAAAA ACAAAACAAA 1350
AACAAACAAC AACAACAAAA AGCAAAGTGT CAGTGAAGGT CCAGCAAAAG 1400
ACTCCCTTCC TATTGCCCTT TGCAGCCAGG GTCATCATGT GACACAGTTC 1450
AGATCAATGA GATGGAGGCT GAGGGTCCCT GGGAAAGATG TTTTTCCTAT 1500
ACAGGTACCA CCTCTTTCAG CTTCACTCTT TCCATTTTCC ACGTGAACAG 1550
GCCTTGTAGC CTGGAGGAGC TACAGCTGCC TTTTTGAGAT GCTGAGGCAC 1600
CCTGTCTGAA GAAGGCCCTC ACATCACTCA ACTTGACTAC TGGGTGAGCC 1650
CTTGGAGAGG CTTCCCAGCC TCTGCTCTTC AAGCCGAAGT ACCACAGGGG 1700
ACACGAGTCC CAGAGTTACA GGACCCCAGC TATGGTTCAT GTGTAAAGGG 1750
AACCATTAGG CAACCAGGGG AAATGATGAA GAAGATCTAC ATTTACAAAT 1800
GTGGAAAGAT GTTCGTGGTA TATTGTTAAA TTAAAAAGCT GTTTAAAAAT 1850
AGTTTTTGGG TCAAGTGAGA TGACTCACTT ATACTTTTAG TATAAGTATG 1900
TCCCATGCAA TATCTGGAAC GTACTTGTAC TAAGGGGTTT CTCCCTCCAT 1950
CGGCACATCC CAGGCATCCT GGCAGCTGCT GGCCTCCAGC AACCCCACAT 2000
TCTAGTTGTG TGGGAGTGGG TTGTGGCATG GACCCTGTGG GCTACCACTG 2050
CCCTGACCTG CTTCTTCACA CACTGGTATT TGTATCTGTG GTAAACCCAG 2100
TGACACGGGG GAGATGACAT ACAAAAAGGG CAGGACCTGA GAAAGATTAA 2150
GCTGCAGGCT CCCTGCCCAT AAAACAGGGT GTGAAAGGCA TCTCAGCGGC 2200
TGCCCCACC ATG GCT ACC TGG GCC CTC CTG CTC CTT GCA GCC ATG 2245 MET Ala Thr Trp Ala Leu Leu Leu Leu Ala Ala MET 5 10
CTC CTG GGC AAC CCA GGTAAGGCCT TCCCCTCGGG ATCGATCCTG 2290 Leu Leu Gly Asn Pro 15 17
ATGGCCCACC CAGCCTCGCA CTCTCAGGCT GGCTGAACCT GGAGCTTGGA 2340
CTCTGTGGGC ACCCAGGTGC CCCTGCCTCC CCCCGGCCTT CTCCCCCGTC 2390 ATGGAGGCCT GGCCCTCCCC TCAGAGCCAG GCTTAGTCCG GTGTGCTGCC 2440
CAGCCTGTCA CTGGCCTGGC CAAGGAGGAG AGACAGGCCA GGGATTCTGG 2490
TCCTAACTCT ACTGGCCACA CTGTGTGGCC TGAGACCCCC CTTTCCCTCC 2540
CAAGCCCCTG CCTCCGCATC TGCGTGGTGA AGGCCATTGG CCTCATCGGT 2590
GGATCTGCGT TTCCTCGGGC CTACACTGTC TAGGATTGTG CGGGGCTGGT 2640
GAGAGAACAA GATCTCTTCC GTGTTCAAGG CAGACTTCCT GCCCCCTGCA 2690
CCCTGCTCTC TCCCAGGCCT TGAGGTCAGT GTGAGCCCCA AGGGCAAGAA 2740
CACTTCTGGA AGGGAGAGTG GATTTGGCTG GGCCATCTGG ATGGAAGGTA 2790
AAAAAAGAAA ATCCCTTGAA AGGAGATTGA GGGAAGTTTC TAGACAAACC 2840
GACCCCCAAA TCTGTGTTGC TGGGGGAACA GAGGAGAAGA GAGAGTCTCG 2890
CCCTCCTGGC TTTCTAGAAG GAACGTGAGA ACACGTGTTT GTGCTGAGAG 2940
TGGGTCAGAG CGGCTCCAGG GCAAAGCATG TGGACAGGTA TCCTGGCCCC 2990
CTGCAAGGCC CAGCTCCTGT CCTAGGCCCT GGTCACCTCC TGGACTCCCA 3040
CCAGCCAGGA GAACGGGCTT TCCCTCTCCT TCCGCCTGCG GAGGGGAAGC 3090
TGAAGTCTGG TCTTCCTCAG GT CTG GTC TTC TCT CGT CTG AGC CCT 3136
Gly Leu Val Phe Ser Arg Leu Ser Pro 18 20 25
GAG TAC TAC GAC CTG GCA AGA GCC CAC CTG CGT GAT GAG GAG 3178 Glu Tyr Tyr Asp Leu Ala Arg Ala His Leu Arg Asp Glu Glu 30 35 40
AAA TCC TGC CCG TGC CTG GCC CAG GAG GGC CCC CAG 3214 Cys Pro Cys Leu Ala Gin Glu Gly Pro Gin Lys Ser 45 50 52
GTACGTGTTG GCTCTCTGCT CACCTGCCAC AGTCCCTCTC CTTTCCCTCC 3264
TCCCTGGTGG CTCCTGGGGT GAGGTCTGGA GCTCTCTAAT GGTCAGGAGG 3314
TGGGAGTGGA GGCTGGGCTG TTTCTGACGA TGCTGGTTTT GTTGAATTCA 3364
TGTCTGGCCA GGAGGGCTAC AGGTATCTGG CAGACTCCTC CAGGAGGATC 3414
CTCTGGGGTC TCACCCTCCA AGGAGCCTGG GGCTGCAGAA CCCAAATAGG 3464
CAGACTCCCC TGGGAGTTCC TCAATAGGAG AGGGGCAAGT GCAGGGCTGG 3514
GAAAGTACTG GGGTTGTGGG AGGCTGTTTC TGGGGTGTCT CAGAGCCTCT 3564
AAGACAAGCA AAAGGGTGGG TAGGGGCCAG GCAGCCAGTT CAGGCCTTCA 3614
GTGTATCCAC GCTCTGGGAA GAGATCACGG ACATTCCTGC CGGCCTCAGA 3664
AACACAAAGG GCCCCTTTCC TGGGCACTTT CACGCGCTCC CAGAGTGTCT 3714 GAGAGACCAT CATAAGGGCT TTCTTTCCTG ACAG GGT GAC CTG TTG ACC 3763 Gly Asp Leu Leu Thr
53 55
AAA ACA CAG GAG CTG GGC CGT GAC TAC AGG ACC TGT CTG ACG 3805 Lys Thr Gin Glu Leu Gly Arg Asp Tyr Arg Thr Cys Leu Thr
60 65 70
ATA GTC CAA AAA CTG AAG AAG ATG GTG GAT AAG CCC ACC CAG 3847 He Val Gin Lys Leu Lys Lys MET Val Asp Lys Pro Thr Gin 75 80 85
GTGAGGCCAA GGGGCTACAG AGCCTCCTGT CTGCTGCTCA ATGGAGGGGC 3897
CAGCCTGTGA CCAGGTCGGG GATCGGGGAG CCCGGGGGCA CCTTGCACAG 3947
TGATCCTGGG GGAGGGCTTC CTAGAAGGGA ATCTGTGAGT CCCCGTGTGT 3997
CTGTGGATGA ATTTCAGAGA ACTTGTGAAA TTGTGACTCT CTGGAACTGT 4047
GTAAGTCAGA CGGCAGAGTA TACA" TTT TCATCATGTA TCCTCAAAGA 4097
GGGCTTGTCC CAGAGAAGTT AGGAATCTTC CCCTAAAGCC CTAACATTTG 4147
TGTCCAAGGC AGAGTTTGAG AAGCTAGTTC CCCAAGAGGC CTGGGTCAGG 4197
ACTGATAAAT CCCAGATCTG CTACTTCCAA GCTGCATGGC CTTGGGCAAG 4247
TCACTTCCAC TTTCTGAGCC CCTGTTATCT TATCTTTGAA ATGTGATGGA 4297
TAATAGTCCC TATCTTGCAA GTTGTCAAAC CCITTTTTTT TTTTTTCCTT 4347
GAGATAGGAT CTTACTCTGA GACCCAGGCT GGAGTGCACT GGTGTGATCT 4397
TGGCTCACTG CAACCTCTGC CTCCCTGGCC CAAGCAATTC TCCTGTCTAA 4447
GCCTCCTGAG TACCTGGGGC TCCAGGTGTG CGCCACCATG CCCAGCTAAT 4497
TTTTGTACTT TTGTAGAAAC AGGGTCTCAC TGTGTTGCCC AGGCTGGTCT 4547
CCAACTTCTG AGCTGAAGCA ATCCACCTGC CTTGGCCTCC CAAAGTGTGG 4597
GATTATAGGC ATGAGCCACT GCACCTGGCT GCTGAAGCTT TTTAAAAGAG 4647
CTGAGGGCTG GGATGTGCTT AGCTCCACGT CCAGCACTGA GTAAATGCTT 4697
AACGAATGAC TGTGTTACTA CCAAGAATTA TTGTTTCACT CTCCCTCCTT 4747
CCCTCTCCTC TGCTGCCCCA AACTACTCAG CATCCTGGCA CTGCAGGCTC 4797
GCACTTAGCC CTGGATACCC AGATTCATCC TCCTCCCCTG GGATGGCATA 4847
GAAGAGACTT TAAAACCAAA TGAGCCAAGA CTCCAAGCTC TGACCACACC 4897
TCCCACCCCC ACCAGTCTTC TCTATGCACC CCCTCTATAT CTGGAGCCCC 4947
CAGCCAGGTT CTGGACCAAG GTAGCTACAT GGCAGAGCAT TTAATGTGTG 4997 CCTGGCAGCC ATGGGCACCA TTCTCCACAC AGAAGGCAGG GACAGGTGCA 5047
CAAGGGGCTG AGACCCCAGC AGGGCTAACT GTCCTTGTCT CAGGAGCCCT 5097
ACCTGGCCAG TCTTGGGCCA GGCCTTGGGG ACTGGGAGTA GGGGCTGACC 5147
CGTCTGTACA GTCTCTGGCC CCATGGCACC AGGTGCCAGC TCCTCGCACC 5197
CAGTACTCCC ATTGCTAGGG CTGCTGGAAC CTGCAGGGTT GGCAGAGCTG 5247
GGCAGGACTC ACCCTATAAC CATGTCCACT GTGGTGCTGC TGCTGCAG 5295
AGA AGT GTT TCC AAT GCT GCG ACC CGG GTG TGT AGG ACG GGG 5337 Arg Ser Val Ser Asn Ala Ala Thr Arg Val Cys Arg Thr Gly
86 90 95
AGG TCA CGA TGG CGC GAC GTC TGC AGA AAT TTC ATG AGG AGG 5379 Arg Ser Arg Trp Arg Asp Val Cys Arg Asn Phe MET Arg Arg 100 105 110
TAT CAG TCT AGA GTT ATC CAG GGC CTC GTG GCC GGA GAA ACT 5421 Tyr Gin Ser Arg Val He Gin Gly Leu Val Ala Gly Glu Thr
115 120 125
GCC CAG CAG ATC TGT GAG GAC CTC AGG TTG TGT ATA CCT TCT 5463 Ala Gin Gin He Cys Glu Asp Leu Arg Leu Cys He Pro Ser
130 135 140
ACA GGTGAGTGCA GAGGTGACAG CAGGGATACC TCCTGAGGGT 5506 Thr 142
TGGAGACAGC TTCCCCCAGG ATATATCAAA GCTGCCTCCT TACTCCCCCA 5556
TCTCCCAGCA TGGGAAAGTG TGGAGAATTG AGCAGATGGA CTTTAGCTAG 5606
AAATGTTTGA GAAATACTGA TTAGAGCTTG GGCTTCAGAC ACAGGTGGTT 5656
GTGGAGTAAA ATCTGGTCTC CATCTCTCCC TGGCTGTGTG ACCTTAAGCA 5706
AATAACTTGA CCTCTCTGAG CTTCAGTTTC TTCATCTGTG AAGGAGAGAT 5756
AGCAATCCTG ATTTTTGAGA TTGGAATGAG AATTGAAGGA GGTCACCGTG 5806
TGTGTGGACC TGACCCTGGG GAAATGTCCT CAGACTGAGG CTATTCAAGG 5856
TCATCAGACC CTCAGTCAAA CTCCAACCCA GCCCAGCACA TGGCCCCTGG 5906
GGTCGGGAGC TGGGGCCATA TCCTCCCCCA CAATCCTGGG CCCTGAGATC 5956
TGGGCTAGGG AACCCTTCAG GCAGGGGAGC ATGAGGCCTT TCCCTCCATG 6006
GCTGCCCAGG CTGTGCTGAG AGAACAGATC TCGGCTGTAG GAAACGGGGC 6056
CAGAAAGGGG CCTCGGTGAT TGGCTCTGGC AGCTCAGCTG GCACTTGCCA 6106
ATAGCTCTGG GATTTTATGC TGGCAGATCG GGGGTCCCCA CCATTTCCTG 6156
TCATTGGAGC TTGTGGCTTT TCTATTCAAG GCCCCACAGC CTGCTCAGGC 6206 TGCCGACTGG CTTCCAGGAT GTGCCTCTGG GTGTGTTCAG TAGGGTCAGG 6256
TGGCTCTGGG ACCTTAAGCA AGTAACATTC TGAGTGCCTG CTTCTCCTTG 6306
AGGACCCACC ACATCTGCCC ACAGCTAGCT GTTCTCTCCG CTCCAG GT CCC 6357
Gly Pro 143
CTC TGA GCCCTCTCAC CTTGTCCTGT GGAAGAAGCA CAGGCTCCTG 6403
Leu
145
TCCTCAGATC CCGGGAACGT CAGCAACCTC TGCCGGCTCC TCGCTTCCTC 6453
GATCCAGAAT CCACTCTCCA GTCTCCCTCC CCTGACTCCC TCTGCTGTCC 6503
TCCCCTCTCA GGAGAATAAA GTGTCAAGCA AGATTTTAGC CGCAGCTGCT 6553
TCTTCTTTGG TGGATTTGAG GGGTGGGTGT CAGTGGCATG CTGGGGTGAG 6603
CTGTGTAGTC CTTCAATAAA TGTCTGTCGT GTGTCCCATA CACTGTTGTA 6653
GATGTTATGG ATTTAGTGGT GAACGAGACA ACCTTAACAG CATTCACACA 6703
GTTAGTCGTG AAATGCTTAC TGAGCACTCA CCACAGCCAT GCA 6746

Claims

Claims:
1. A vector comprising a gene coding for a protein having the following amino acid sequence a)
MET Ala Thr Trp Ala Leu Leu Leu Leu Ala Ala Met Leu Leu Gly
5 10 15
Asn Pro Gly Leu Val Phe Ser Arg Leu Ser Pro Glu Tyr Tyr Asp
20 25 30
Leu Ala Arg Ala His Leu Arg Asp Glu Glu Lys Ser Cys Pro Cys
35 40 45
Leu Ala Gin Glu Gly Pro Gin Gly Asp Leu Leu Thr Lys Thr Gin
50 55 60
Glu Leu Gly Arg Asp Tyr Arg Thr Cys Leu Thr He Val Gin Lys
65 70 75
Leu Lys Lys Met Val Asp Lys Pro Thr Gin Arg Ser Val Ser Asn
80 85 ' 90
Ala Ala Thr Arg Val Cys Arg Thr Gly Arg Ser Arg Trp Arg Asp
95 100 105
Val Cys Arg Asn Phe Met Arg Arg Thr Gin Ser Arg Val Thr Gin
110 115 120
Gly Leu Val Ala Gly Glu Thr Ala Gin Gin He Cys Glu Asp Leu
125 130 135
Arg Leu Cys He Pro Ser Thr Glu Pro Leu
140 145
or b) allelic variations thereof in which one or more amino acids have been added, substituted or removed without substantially affecting the activity of the protein of sequence a).
2. An eucaryotic or procaryotic host cell transformed with a vector according to claim 1.
3. A host cell according to claim 2, which is E. coli.
4. A host cell according to claim 2, which is yeast or a mammalian cell. ς
5. A host cell according to claim 4, which is a Chinese Hamster Ovary cell.
6. A method of producing the protein of claim 1 which 0 comprises culturing a cell according to any one of claims 2 to 4 and isolating the protein.
7. A cDNA corresponding to the gene set forth in claim 1.
8. A protein which has the amino acid sequence 5 aa) listed as a) in claim 1, or bb) allelic variations thereof in which one or more amino acids have been added, substituted or removed without substantially affecting the activity of the protein; 0 and/or cc) post-translational variations thereof which do not substantially affect the activity of the protein.
9. A protein according to claim 8 which is a recombinant
15 protein.
10. A protein according to claim 9 which is substantially free of glycosylation.
11. A protein according to claim 8 for use as an active therapeutic substance.
12. A pharmaceutical composition comprising a protein according to claim 8 in association with a pharmaceutically acceptable diluent or carrier.
13. Use of a protein according to claim 8 for the manufacture of a medicament for a therapeutic application for treatment of antoimmune deficiency by enhancing the immune response.
14. Use of a protein according to claim 8 for the manufacture of a medicament for a therapeutic application for treatment of cancer.
15. A method for producing a protein according to any of the claims 8 to 10 which comprises culturing a host cell transformed by a vector comprising the gene coding for the protein and isolation the secreted protein.
PCT/EP1991/001824 1990-09-28 1991-09-25 Dna and amino acid sequence specific for natural killer cells and t cells WO1992006195A1 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6485928B2 (en) 1997-11-04 2002-11-26 The Board Of Trustees Of The Leland Stanford Junior University Use of granulysin as an antimicrobial agent

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0320806A2 (en) * 1987-12-15 1989-06-21 The Board Of Trustees Of The Leland Stanford Junior University T-cell activation related gene
EP0367641A2 (en) * 1988-09-26 1990-05-09 Roussel-Uclaf Lymphokines, DNA sequences encoding these lymphokines and pharmaceutical compositions containing these lymphokines

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0320806A2 (en) * 1987-12-15 1989-06-21 The Board Of Trustees Of The Leland Stanford Junior University T-cell activation related gene
EP0367641A2 (en) * 1988-09-26 1990-05-09 Roussel-Uclaf Lymphokines, DNA sequences encoding these lymphokines and pharmaceutical compositions containing these lymphokines

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF EXPERIMENTAL MEDICINE, Vol. 165, March 1987, New York, US., J. JONGSTRA et al., "The Isolation and Sequence of a Novel Gene From a Human Functional T Cell Line", pages 601-614. *
JOURNAL OF EXPERIMENTAL MEDICINE, Vol. 172, October 1990, New York, US., T. YABE et al., "A cDNA Clone Expressed in Natural Killer and T Cells that Likely Encodes a Secreted Protein", pages 1159-1163. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6485928B2 (en) 1997-11-04 2002-11-26 The Board Of Trustees Of The Leland Stanford Junior University Use of granulysin as an antimicrobial agent

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