WO1997004091A1 - Nouveaux ligands de recepteurs et sequences genetiques codant ces ligands - Google Patents

Nouveaux ligands de recepteurs et sequences genetiques codant ces ligands Download PDF

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Publication number
WO1997004091A1
WO1997004091A1 PCT/AU1996/000460 AU9600460W WO9704091A1 WO 1997004091 A1 WO1997004091 A1 WO 1997004091A1 AU 9600460 W AU9600460 W AU 9600460W WO 9704091 A1 WO9704091 A1 WO 9704091A1
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WIPO (PCT)
Prior art keywords
seq
sequence
nucleic acid
nlerk2
set forth
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PCT/AU1996/000460
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English (en)
Inventor
Nicos Anthony Nicola
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Amrad Operations Pty. Ltd.
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Publication date
Priority claimed from AUPN4263A external-priority patent/AUPN426395A0/en
Priority claimed from AUPN6847A external-priority patent/AUPN684795A0/en
Priority claimed from AUPN7299A external-priority patent/AUPN729995A0/en
Priority claimed from AUPN7890A external-priority patent/AUPN789096A0/en
Application filed by Amrad Operations Pty. Ltd. filed Critical Amrad Operations Pty. Ltd.
Priority to EP96923786A priority Critical patent/EP0842272A4/fr
Priority to AU64098/96A priority patent/AU711646B2/en
Publication of WO1997004091A1 publication Critical patent/WO1997004091A1/fr

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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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates generally to novel receptor ligands and to genetic sequences encoding same.
  • the novel receptor ligands and their encoding genetic sequences are useful in the development of a wide range of agonist, antagonist, therapeutic and diagnostic reagents based on ligand-receptor interaction.
  • the present invention further provides a means for identifying ligands without knowledge of their receptor and a means for identifying receptors.
  • RPTKs cell surface receptor protein tyrosine kinases
  • RPTKs can be subdivided into several classes dependent primarily on the predicted structural 5 features of their extracellular domains and whether or not they contain an uninterrupted tyrosine kinase domain in the cytoplasmic portion ofthe receptor (Hilton In "Guidebook to Cytokines and Their Receptors" N.A. Nicola ed, Oxford University Press, Oxford pp 8-16, 1994).
  • the largest class of RPTKs is known as the eph-related kinases (ERKs) and include the tyrosine kinases eph, elk, eck, eek, erk, cek 4-10, mek 4, hek, tk2, tyro 5 and sek. They are 10 characterised by an extracellular sequence that includes a single cysteine-rich repeat domain followed by two fibronectin type III domains and an uninterrupted tyrosine kinase domain in the cytoplasmic region.
  • ERKs comprise the largest subclass of RPTKs
  • most of the ERKs were 15 identified without prior knowledge of their biological function nor of the ligands with which they interact. Nevertheless, the expression patterns ofthe ERKs suggest that they might have important roles in early vertebrate development and in brain function and hemopoietic cell function as well as functions on other organs in the adult (Cheng and Flanagan Cell 79: 157- 168, 1994; Shao et al J. Biol. Chem. 270: 3467-3470, 1995; Beckman et al EMBO J. 13: 20 3757-3762, 1994).
  • LERKs Ugands for ERKs
  • B61 B61 gene product previously identified as an interleukin- 1 - or tumour necrosis factor- inducible product of human umbilical vein endothelial cells
  • LERK2 appears to be identical to human ELK ligand (ELK-L) and the human homologue ofthe mouse proteins elg 2 (eph-ligand 2) and cek5 L (cek5-ligand) (Beckmann et al EMBO J. 13: 3757-3762, 1994; Fletcher et al Genomics 24: 127-132, 1994; Shao et al J. Biol. Chem. 269: 26602-26609, 1994; Davis etal Science 266: 816-819, 1994).
  • LERK3 is nearly identical to EHK1-L (ehkl- ligand) Kozlosky et al Oncogene 10: 299-306, 1995; Davis et al Science 266: 816-819, 1994), LERK4 is unique (Kozlosky et al Oncogene 10: 299-306, 1995) and the cek7 ligand is identical to elf-1 (eph ligand family- 1) (Shao et al J. Biol. Chem. 270: 3467-3470, 1995); Cheng and Flanagan Cell 79: 157-168, 1994).
  • ligands appear to be cell surface-associated either through a C-terminal glycosyl-phosphatidyl inositol (GPI)-linkage (LERKs 1, 3 and 4) or through a traditional transmembrane domain (LERK2). Most appear to require cell surface expression or soluble dimer forms in order to activate the tyrosine kinase activity of the appropriate ERKs (Davis et al Science 266: 816-819, 1994). Moreover, none of the LERKs described so far show an absolute specificity for one ERK.
  • GPI glycosyl-phosphatidyl inositol
  • LERK2 traditional transmembrane domain
  • LERK1 binds to hek, elk and eck; LERKs 2, 3 and 4 bind to hek and elk and cek 7-L binds to sek and mek 4 with equilibrium dissociation constants varying from 1 nM to 500 nM.
  • the inventors have identified a new LERK which will assist in the development of a range of new therapeutics and diagnostics and in the identification of new ERKs.
  • one aspect ofthe present invention contemplates an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a LERK or part thereof, said nucleotide sequence or its complementary form encoding at least one of the following amino acid sequences: (i) VXWXSXN [SEQ ID NO: 1];
  • nucleotide sequence or its complementary form encodes a product comprising at least two of, more preferably at least three of and even more preferably all four of amino acid sequences SEQ ID NO: 1 to SEQ ID NO:4.
  • the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding a LERK or part thereof, said nucleotide sequence or its complementary form encoding an amino acid sequence selected from the list consisting of:
  • the nucleic acid molecule of the present invention encodes a LERK referred to herein as "NLERK2", said nucleic acid molecule selected from the list consisting of:
  • nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ED NO:5;
  • nucleic acid molecule comprising a sequence of nucleotides having at least about 70% similarity to the nucleotide sequence set forth in SEQ ED NO: 5;
  • nucleic acid molecule capable of hybridising under low stringency conditions to the nucleotide sequence set forth in SEQ ID NO: 5.
  • the nucleotide molecule is preferably derivable from the human genome but genomes and nucleotide sequences from non-human animals are also encompassed by the present invention.
  • Non-human animals contemplated by the present invention include livestock animals (e.g. sheep, cows, pigs, goats, horses, donkeys), laboratory test animals (e.g. mice, rats, guinea pigs, hamsters, rabbits), domestic companion animals (e.g. dogs, cats), birds (e.g. chickens, geese, ducks and other poultry birds, game birds, emus, ostriches) and captive wild or tamed animals (e.g. foxes, kangaroos, dingoes).
  • livestock animals e.g. sheep, cows, pigs, goats, horses, donkeys
  • laboratory test animals e.g. mice, rats, guinea pigs, hamsters, rabbits
  • domestic companion animals e.g. dogs
  • Preferred human genomic sequences include sequences from brain, liver, kidney, neonatal, embryonic, cancer or tumour-derived tissues.
  • oligonucleotides may be designed which bind cDNA clones with high stringency. Direct colony hybridisation may be employed or PCR amplication may be used. The use of oligonucleotide primers which bind under conditions of high stringency ensures rapid cloning of a molecule encoding the novel LERK and less time is required in screening out cloning artefacts. However, depending on the primers used, low or medium stringency conditions may also be employed.
  • Reference herein to a low stringency at 42 ⁇ C includes and encompasses from at least about 1% v/v to at least about 15% v/v formamide and from at least about IM to at least about 2M salt for hybridisation, and at least about IM to at least about 2M salt for washing conditions.
  • Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.0 IM to at least about 0.15M salt for hybridisation, and at least about 0.0 IM to at least about 0.15M salt for washing conditions.
  • medium stringency which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions
  • high stringency which includes and encompasses from at least about 31% v/v to at least about 50% v/
  • another aspect of the present invention contemplates a method for cloning a nucleotide sequence encoding a novel LERK, said method comprising searching a nucleotide data base for a sequence which encodes at least one of amino acid sequences SEQ ID NO: 1 to SEQ ID NO:4, designing one or more oligonucleotide primers based on a nucleotide sequence located in the search, screening a nucleic acid library with said one or more oligonucleotides and obtaining a clone therefrom which encodes said novel LERK or part thereof.
  • the nucleic acid library is a cDNA, genomic or mRNA library.
  • the nucleic acid library is a cDNA expression library.
  • the nucleotide data base is of human origin such as from brain, liver, kidney, neo ⁇ natal tissue, embryonic tissue, tumour or cancer tissue.
  • the oligonucleotide primers are selected from SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l 1, SEQ ID NO: 12 and SEQ ID NO: 13.
  • the LERK is selected from the list consisting of: (i) a nucleotide sequence substantially as set forth in SEQ ID NO:5; (ii) a nucleotide sequence having at least about 75% similarity to the nucleotide sequence set forth in SEQ ID NO:5; and
  • nucleotide sequence capable of hybridising under low stringency conditions to the nucleotide sequence as set forth in SEQ ID NO:5.
  • medium stringency conditions are employed.
  • high stringency conditions are employed.
  • Another aspect ofthe invention is directed to the genomic gene and to 3 ' and 5 ' regions thereof.
  • the 3 ' region ofthe NLERK2 gene is represented in SEQ ID NO: 14 and the 5 ' region is shown in SEQ ID NO: 15.
  • another aspect of the present invention is directed to a nucleic acid molecule corresponding to or derived from the 3' region ofthe genomic gene encoding NLERK2 or its derivatives, said nucleic acid molecule or its complementary form having a nucleotide sequence substantially as set forth in SEQ ID NO: 14 or having at least 75% similarity thereto or capable of hybridising thereto under low stringency conditions.
  • Other levels of stringency such as medium and high may also be used.
  • a further embodiment is directed to a nucleic acid molecule corresponding to or derived from the 5 ' region of the genomic gene encoding NLERK2 or its derivatives, said nucleic acid molecule or its complementary form having a nucleotide sequence substantially as set forth in SEQ ID NO: 15 or having at least 75% similarity thereto or capable of hybridising thereto under low stringency conditions. Other levels of stringency such as medium and high may also be used.
  • the present invention also extends to hybrid genetic sequences comprising a coding region of a structural gene flanked or fused to one or both of SEQ ID NO: 14 and/or SEQ ID NO: 15 or derivatives thereof.
  • Still another embodiment contemplates the promoter or a functional part thereof ofthe genomic gene encoding NLERK2.
  • the promoter may readily be obtained by, for example, "chromosome walking".
  • NLERK2 including a recombinant polypeptide having an ability to interact with an ERK wherein said NLERK2 or said polypeptide is encoded by a nucleotide sequence translatable to at least one of amino acid sequences SEQ ID NO:l to SEQ ID NO:4 and subsequently cloned by designing at least one oligonucleotide probe based on said nucleotide sequence and using same to clone a nucleotide sequence encoding said LERK or polypeptide from a DNA or RNA library.
  • NLERK2 which comprises an amino acid sequence substantially as set forth in SEQ ID NO:6 or having at least about 10% similarity, preferably at least about 80% similarity, more preferably at least about 90% similarity or most preferably at least about 95 to 100% similarity to all or part of the sequence set forth in SEQ ID NO:6.
  • This aspect encompasses NLERK2 and derivatives thereof such as fragments, parts, portions, mutants and homologues.
  • the present invention contemplates a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding the amino acid sequence set forth in SEQ ID NO:6 or to an amino acid sequence having at least about 70% or above similarity to all or part of the amino acid sequence set forth in SEQ ID NO:6.
  • the present invention further contemplates a range of derivatives of NLERK2.
  • Derivatives include fragments, parts, portions, mutants, homologues and analogues of the NLERK2 polypeptide and corresponding genetic sequence. Derivatives also include single or multiple amino acid substitutions, deletions and/or additions to NLERK2 or single or multiple nucleotide substitutions, deletions and or additions to the genetic sequence encoding NLERK2.
  • NLERK2 immunologically interactive derivatives.
  • Analogues of NLERK2 contemplated herein include, but are not Umited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogues.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde foUowed by reduction with NaBE ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5- phosphate followed by reduction with N-1BH4.
  • modifications of amino groups such as by reductive alkylation by reaction with an aldehyde foUowed by reduction with NaBE ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TN
  • the guanidine group of arginine residues may be modified by the foimation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4- chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
  • Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5- phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and or D- isomers of amino acids.
  • a Ust of unnatural amino acid, contemplated herein is shown in Table 1.
  • peptides can be conformationally constrained by, for example, incorporation of C Intel and N-.-methylamino acids, introduction of double bonds between C ⁇ and C p atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
  • the present invention further contemplates chemical analogues of NLERK2 capable of acting as antagonists or agonists of NLERK2 or which can act as functional analogues of NLERK2.
  • Chemical analogues may not necessarily be derived from NLERK2 but may share certain conformational similarities.
  • chemical analogues may be specifically designed to mimic certain physiochemical properties of NLERK2.
  • Chemical analogues may be chemically synthesised or may be detected following, for example, natural product screening.
  • NLERK2 permits the generation of a range of therapeutic molecules capable of modulating expression of NLERK2 or modulating the activity of NLERK2.
  • Modulators contemplated by the present invention includes agonists and antagonists of NLERK2 expression.
  • Antagonists of NLERK2 expression include antisense molecules, ribozymes and co-suppression molecules.
  • Agonists include molecules which increase promoter ability or interfere with negative regulatory mechanisms.
  • Agonists of NLERK2 include molecules which overcome any negative regulatory mechanism.
  • Antagonists of NLERK2 include antibodies and inhibitor peptide fragments. TABLE 1
  • Non-conventional Code Non-conventional Code amino acid amino acid
  • D-N-methyllysine Dnmlys N-methyl- ⁇ -aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen
  • glycosylation variants from a completely unglycosylated molecule to a modified glycosylated molecule. Altered glycosylation patterns may result from expression of recombinant molecules in different host cells.
  • Another embodiment of the present invention contemplates a method for modulating expression of NLERK2 in a human, said method comprising contacting the NLERK2 gene encoding NLERK2 with an effective amount of a modulator of NLERK2 expression for a time and under conditions sufficient to up-regulate or down-regulate or otherwise modulate expression of NLERK2.
  • a nucleic acid molecule encoding NLERK2 or a derivative thereof may be introduced into a cell to enhance the ability of that cell to survive, conversely, NLERK2 antisense sequences such as oligonucleotides may be introduced to decrease the survival capacity of any cell expressing the endogenous NLERK2 gene.
  • Another aspect of the present invention contemplates a method of modulating activity of NLERK2 in a human, said method comprising administering to said mammal a modulating effective amount of a molecule for a time and under conditions sufficient to increase or decrease NLERK2 activity.
  • the molecule may be a proteinaceous molecule or a chemical entity and may also be a derivative of NLERK2 or its receptor or a chemical analogue or truncation mutant of NLERK2 or its receptor.
  • the present invention contemplates a pharmaceutical composition comprising NLERK2 or a derivative thereof or a modulator of NLERK2 expression or NLERK2 activity and one or more pharmaceuticaUy acceptable carriers and/or diluents. These components are referred to as the active ingredients.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion or may be in the form of a cream or other form suitable for topical application. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the Uke), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as licithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants.
  • the preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying abso ⁇ tion, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
  • the active ingredients When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food ofthe diet.
  • the active compound For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 1% by weight of active compound.
  • the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% ofthe weight ofthe unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained.
  • Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 ug and
  • the tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: A binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • a binder such as gum, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen,
  • tablets, pills, or capsules may be coated with sheUac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compound(s) may be incorporated into sustained-release preparations and formulations.
  • the present invention also extends to forms suitable for topical application such as creams, lotions and gels.
  • Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics ofthe active material and the particular therapeutic effect to be achieved, and (b) the timitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
  • the principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed.
  • a unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 ⁇ g to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 ⁇ g to about 2000 mg/ml of carrier.
  • the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
  • the pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of modulating NLERK2 expression or NLERK2 activity.
  • the vector may, for example, be a viral vector.
  • Still another aspect of the present invention is directed to antibodies to NLERK2 and its derivatives.
  • Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to NLERK2 or may be specifically raised to NLERK2 or derivatives thereof. In the case of the latter, NLERK2 or its derivatives may first need to be associated with a carrier molecule.
  • the antibodies and/or recombinant NLERK2 or its derivatives ofthe present invention are particularly useful as therapeutic or diagnostic agents.
  • NLERK2 and its derivatives can be used to screen for naturally occurring antibodies to NLERK2. These may occur, for example in some autoimmune diseases. Alternatively, specific antibodies can be used to screen for NLERK2. Techniques for such assays are well known in the art and include, for example, sandwich assays and ELISA. Knowledge of NLERK2 levels may be important for diagnosis of certain cancers or a predisposition to cancers or for monitoring certain therapeutic protocols.
  • Antibodies to NLERK2 of the present invention may be monoclonal or polyclonal.
  • fragments of antibodies may be used such as Fab fragments.
  • the present invention extends to recombinant and synthetic antibodies and to antibody hybrids.
  • a “synthetic antibody” is considered herein to include fragments and hybrids of antibodies.
  • the antibodies of this aspect of the present invention are particularly useful for immunotherapy and may also be used as a diagnostic tool for assessing apoptosis or monitoring the program of a therapeutic regimen.
  • NLERK2 proteins can be used to screen for NLERK2 proteins.
  • the latter would be important, for example, as a means for screening for levels of NLERK2 in a cell extract or other biological fluid or purifying NLERK2 made by recombinant means from culture supernatant fluid.
  • Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.
  • second antibodies (monoclonal, polyclonal or fragments of antibodies or synthetic antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody.
  • An antibody as contemplated herein includes any antibody specific to any region ofNLERK2.
  • Both polyclonal and monoclonal antibodies are obtainable by immunization with the enzyme or protein and either type is utiUzable for immunoassays.
  • the methods of obtaining both types of sera are well known in the art.
  • Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of NLERK2, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any ofthe known immunoadsorbent techniques.
  • antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.
  • the use of monoclonal antibodies in an immunoassay is particularly preferred because ofthe ability to produce them in large quantities and the homogeneity of the product.
  • the preparation of hybridoma ceU lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.
  • Another aspect of the present invention contemplates a method for detecting NLERK2 in a biological sample from a subject said method comprising contacting said biological sample with an antibody specific for NLERK2 or its derivatives or homologues for a time and under conditions sufficient for an antibody-NLERK2 complex to form, and then detecting said complex.
  • the presence of NLERK2 may be accomplished in a number of ways such as by Western blotting and ELISA procedures.
  • a wide range of immunoassay techniques are available as can be seen by reference to US Patent Nos. 4,016,043, 4, 424,279 and 4,018,653.
  • Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention.
  • an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule.
  • a second antibody specific to the antigen is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody.
  • the presence of the antigen is determined by observation of a signal produced by the reporter molecule.
  • the results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten.
  • Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent.
  • the sample is one which might contain NLERK2 including cell extract, tissue biopsy or possibly serum, saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid.
  • the sample is, therefore, generally a biological sample comprising biological fluid but also extends to fermentation fluid and supernatant fluid such as from a cell culture.
  • a first antibody having specificity for the NLERK2 or antigenic parts thereof is either covalently or passively bound to a solid surface.
  • the solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the solid supports 5 may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay.
  • binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-
  • the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hapten.
  • the second antibody is linked to a reporter molecule which is used to indicate the binding ofthe second antibody to the hapten.
  • An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength ofthe reporter molecule signal, a bound target may be detectable by direct labelling with the antibody. 0 Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target- first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
  • reporter molecule as used in the present specification, is meant a molecule which, by its 5 chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative.
  • the most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
  • an enzyme immunoassay an enzyme is conjugated to the second antibody, 0 generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody.
  • the substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication ofthe amount of hapten which was present in the sample.
  • Reporter molecule also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
  • fluorescent compounds such as fluorescein and rhodamine
  • fluorescein and rhodamine may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome-labelled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission ofthe light at a characteristic colour visually detectable with a light microscope.
  • the fluorescent labelled antibody is allowed to bind to the first antibody-hapten complex. After washing of the unbound reagent, the remaining tertiary complex is then exposed to the light ofthe appropriate wavelength the fluorescence observed indicates the presence ofthe hapten of interest.
  • Immunofluorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
  • the present invention also contemplates genetic assays such as involving PCR analysis to detect NLERK2 gene or its derivatives.
  • Alternative methods or methods used in conjunction include direct nucleotide sequencing or mutation scanning such as single stranded conformation polymorphoms analysis (SSCP) as specific oligonucleotide hybridisation, as methods such as direct protein truncation tests.
  • SSCP single stranded conformation polymorphoms analysis
  • the nucleic acid molecules ofthe present invention may be DNA or RNA.
  • the nucleic acid molecule When the nucleic acid molecule is in DNA form, it may be genomic DNA or cDNA.
  • RNA forms of the nucleic acid molecules of the present invention are generally mRNA.
  • nucleic acid molecules of the present invention are generally in isolated form, they may be integrated into or ligated to or otherwise fused or associated with other genetic molecules such as vector molecules and in particular expression vector molecules.
  • Vectors and expression vectors are generally capable of replication and, if applicable, expression in one or both of a prokaryotic cell or a eukaryotic cell.
  • prokaryotic cells include E. coli, Bacillus sp and Pseudomonas sp.
  • Preferred eukaryotic cells include yeast, fungal, mammalian and insect cells.
  • another aspect of the present invention contemplates a genetic construct comprising a vector portion and a mammalian and more particularly a human NLERK2 gene portion, which NLERK2 gene portion is capable of encoding an NLERK2 polypeptide or a functional or immunologically interactive derivative thereof.
  • the NLERK2 gene portion ofthe genetic construct is operably linked to a promoter on the vector such that said promoter is capable of directing expression of said NLERK2 gene portion in an appropriate cell.
  • the NLERK2 gene portion ofthe genetic construct may comprise all or part of the gene fused to another genetic sequence such as a nucleotide sequence encoding glutathione-S- transferase or part thereof.
  • the present invention extends to such genetic constructs and to prokaryotic or eukaryotic cells comprising same.
  • the present invention also extends to any or all derivatives of NLERK2 including mutants, part, fragments, portions, homologues and analogues or their encoding genetic sequence including single or multiple nucleotide or amino acid substitutions, additions and/or deletions to the naturally occurring nucleotide or amino acid sequence.
  • the NLERK2 and its genetic sequence ofthe present invention will be useful in the generation of a range of therapeutic and diagnostic reagents and will be especially useful in the detection of a corresponding ERK.
  • recombinant NLERK2 may be bound or fused to a reporter molecule capable of producing an identifiable signal, contacted with a cell or group of cells putatively carrying ERKs and screening for binding of the labelled LERK to the ERK.
  • labelled NLERK2 may be used to screen expression libraries of putative ERK genes or functional parts thereof.
  • LERKs are important for the proliferation, differentiation and survival of a diverse array of cell types. Accordingly, it is proposed that NLERK2 or its functional derivatives be used to regulate development, maintenance or regeneration in an array of different cells and tissues in vitro and in vivo. For example, NLERK2 is contemplated to be useful in modulating neuronal proliferation, differentation and survival.
  • Soluble NLERK2 polypeptides are also contemplated to be useful in the treatment of disease, injury or abnormality in the nervous system, e.g. in relation to central or peripheral nervous system to treat Cerebral Palsy, trauma induced paralysis, vascular ischaemia associated with stroke, neuronal tumours, motoneurone disease, Parkinson's disease, Huntington's disease, Alzheimer's disease, Multiple Sclerosis, peripheral neuropathies associated with diabetes, heavy metal or alcohol toxicity, renal failure and infectious diseases such as herpes, rubella, measles, chicken pox, HIV or HTLV-1.
  • Membrane bound NLERK2 may be used in vitro on nerve cells or tissues to modulate proliferation, differentiation or survival, for example, in grafting procedures or transplantation.
  • the NLERK2 of the present invention or its functional derivatives may be provided in a pharmaceutical composition comprising the LERK together with one or more pharmaceutically acceptable carriers and/or diluents.
  • the present invention contemplates a method of treatment comprising the administration of an effective amount of a LERK of the present invention.
  • the present invention also extends to antagonists and agonists of LERKs and their use in therapeutic compositions and methodologies.
  • a further aspect of the present invention contemplates the use of NLERK2 or its functional derivatives in the manufacture of a medicament for the treatment of NLERK2 mediated conditions defective or deficient.
  • Figure 1 is a representation ofthe amino acid sequence of NLERK2 [SEQ ID NO: 6] ofthe present invention in comparison to known LERKs.
  • LERK-1/B61 is described in International Patent Pubhcation No. WO 95/06085; MCEK7/ELF1 in WO 96/10911 and NLERK1/ELF2 in WO 96/01839.
  • the putative leader sequences, transmembrane sequences and GPI anchor sequences are shaded and boxed. Areas of amino acid sequence conservation are boxed, dashes signify gaps introduced to produce the alignment and potential N-glycosylation sites are underlined.
  • Figure 2 is a photographic representation showing expression of NLERK2.
  • Panel A represents SDS-PAGE of extracellular medium from COS cells transfected with pEF sol NLERK2 cDNA (tracks a and b) or pEF fl NLERK2 cDNA (tracks c and d).
  • Panel B represent SDS-PAGE of whole ceU lysates from COS ceUs transfected with pEF sol NLERK2 cDNA (tracks a and b) or pEF fl NLERK2 cDNA (tracks c and d). (a, b are from two independent pEF sol NLERK2 clones and c, d are from two independent pEF fl NLERK2 clones).
  • SDS-PAGE gels were transferred to PVDF filters and blotted with M2 anti-FLAGTM antibody (IBI/Kodak, CT, USA) followed by HRP-conjugated rabbit anti-mouse immunoglobulin (Dako code PO260, Denmark) and binding detected by enhanced chemiluminescence as described for Amersham ECL reagent (Amersham, Buckinghamshire, England).
  • Figure 3 is a schematic representation showing structure of NLERK2 cDNA and location of NLERK2 clones.
  • Figure 4 is a representation of the nucleotide sequence [SEQ ID NO:5] and corresponding amino acid sequence [SEQ ID NO: 14] of full length NLERK2 cDNA.
  • the leader sequence is lightly underlined, the transmembrane domain is heavily underlined and the potential N- glycosylation site is indicated by a bold N.
  • Figure 5 is a representation ofthe N-terminal amino acid sequence of pEF solNLERK2.
  • the underlined portion is the C-terminal portion of the IL-3 signal sequence.
  • the dotted underlined sequence is the sequence ofthe FLAG tag used to detect the expressed molecule.
  • the double underlined sequence is the 5' cloning site for the NLERK2 cDNA.
  • the non- underlined residues are the mature 5' coding region.
  • FLAG epitope 17 Single and triple letter abbreviations for amino acid residues are used in the subject specification, as defined in Table 3.
  • VXWXSXN [SEQ ID NO: 1];
  • DXXDIXCP [SEQ ID NO:2];
  • EXYXLYXVXXXXXXXC [SEQ ID NO:3];
  • RSU07560 human ELK-L (Gene Bank Code No. HUMEFL3), human EHK1-L (Gene Bank Code No. HUMEFL2), mouse LERK2 (Gene Bank Code No. MLERK2), human B61 (Gene Bank Code No. HUMB61), mouse ERF-1 (Gene Bank Code No. MMU14941), mouse cek 5-L (Gene Bank Code No. MMU12983).
  • Gene Bank Code No. 269886NCBI represents a human-expressed sequence tag (EST) derived from a 73 day post-natal female whole brain cDNA library. Alignment and translation of an amino acid sequence predicted to be encoded by the EST (Gene Bank Code No. 269886NCBI) demonstrates it to be related to the known LERKs ( Figure 1). The sequence does diverge, however, and hence encodes a novel human LERK, referred to herein as "NLERK2" ( Figures 1 and 4).
  • EXAMPLE 2 CLONING FULL LENGTH NLERK2 cDNA Pooled oUgos were used to probe library 53 which is a human fetal brain cDNA library.
  • the oligos were synthesized according to the cDNA sequence ofthe expressed sequence tag [EST tag no. 269886NCBIJ.
  • the oligonucleotide probes comprise the following sequences:
  • dupUcate positive hybridizing plaques were picked and purified on CsCl gradient and sequenced on both strands. Overlapping cDNA sequences from 5 independent clones were obtained encompassing the entire coding region and were sequenced on both strands (see Figure 4).
  • EXAMPLE 3 GENERATION OF EXPRESSION CONSTRUCTS Constmcts were generated to express full length and soluble forms ofthe protein.
  • a derivative ofthe mammalian expression vector pEF-BOS (Mizushima et al, Nucl. Acids. Res. 18: 5322, 1990) was engineered to contain DNA encoding the signal sequence of murine IL-3 (MVLASSTTSIHTMLLLLLMLFHLGLQASIS [SEQ ID NO: 16]) and the FLAG epitope (DYKDDDDK [SEQ ID NO: 17]) followed by a unique Xba 1 cloning site.
  • This vector was named pEF/IL3SIG/FLAG.
  • PCR was performed using oligos to amplify DNA fragments encoding the entire mature coding region from L28 to V340 using clones 53.1 and 53.8.
  • the oligonucleotide probes comprise the following sequences:
  • PCR was used to amplify the extracellular domain without the transmembrane or cytoplasmic region.
  • sequences ofthe oligonucleotides used in this PCR are as follows:
  • COS cells were transiently transfected with these constructs. Briefly, COS cells from a confluent 175 cm 2 tissue culture flask were resuspended in PBS and electroporated (BioRad Gene pulser; 500 ⁇ F, 300 V) with 20 ⁇ g of uncut expression vector in a 0.4 cm cuvette (BioRad). After 2 to 3 days at 37°C in a fully humidified incubator containing 10% v/v CO 2 in air, cells were used for analyses of protein expression.
  • Conditioned medium was collected by centrifugation, concentrated ten ⁇ fold, and stored sterile at 4°C. Cells were also harvested and lysed for 5 min in 500 ⁇ l of 50 mM Tris Hcl pH7.4 containing 150 mM NaCl, 2 mM EDTA and 1% v/v Triton X-100. The intact nuclei were removed by centrifugation at 10,000g for 5 min. The cell lysate and conditioned medium were electrophoresed on pre-cast 12% w/v polyacrylamide gels (BioRad).
  • the resolved proteins were then electroblotted onto PVDF membranes, which were then blocked with 5% skim milk, 0.1% v/v Tween 20 in PBS, rinsed and incubated with 5 ⁇ l of anti-FLAG M2 antibody (TBI) in 2.5 ml of PBS containing 5% skim milk and 0.1% v/v Tween 20.
  • TBI anti-FLAG M2 antibody
  • the membrane was then rinksed and incubated with peroxidase-conjugated human anti ⁇ mouse Ig in 5% skim milk, 0.1% v/v Tween in PBS, rinsed and incubated with ECL reagent (Amersham, Buckinghamshire, UK) or 1 min. Filters were then blotted dry and exposed to autoradiographic film for 1 min.
  • Figure 2 shows expression of NLERK2 in COS cells.
  • the apparent molecular mass of soluble and full length NLERK2 were 32 and 50 kDa, respectively, suggesting glycosylation.
  • Figure 5 shows the N-terminal amino acid sequence of anti-FLAG M2 antibody purified soluble NLERK2 from pEF solNLERK2. This protein is transiently expressed in COS cells ( Figure 2).
  • Single underlined sequence is the C-terminal portion of the IL-3 signal sequence which was cleaved before the Alanine.
  • the dotted underlined sequence is sequence of the FLAG tag used to detect the expressed molecule.
  • the double underlined sequence is the 5' cloning site for the NLERK2 cDNA.
  • the non-underlined residues are the mature 5' coding region (lacking the leader sequence).
  • the italicised Asparagine (N) was modified for cloning reasons from the Serine residue ofthe sequence given in Figure 4.
  • the 5 ' and 3 ' portions ofthe NLERK2 genomic gene were determined and are shown in SEQ ID NO: 15 and SEQ ID NO: 14, respectively.
  • AGC AAT GCA ACC TCC CGG GGT GCT GAA GGC CCC CTG CCC CCT CCC AGC 672 Ser Asn Ala Thr Ser Arg Gly Ala Glu Gly Pro Leu Pro Pro Pro Pro Ser 210 215 220 ATG CCT GCA GTG GCT GGG GCA GCA GGG GGG CTG GCG CTG CTC TTG CTG 720 Met Pro Ala Val Ala Gly Ala Ala Gly Gly Leu Ala Leu Leu Leu Leu 225 230 235 240
  • GACCATGAMC CAGGGATCCT TGTCCCCCTC AMCCACCCAG AGCTAGGGGG CGGGAACAGC 340
  • ATGTGCTCTC CCGAGTAACC CAGATGGCTG TCTTGTTCAT TCCATCCTAC ATTTCTGACT 1360

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Abstract

La présente invention concerne, de manière générale, de nouveaux ligands de récepteurs ainsi que des séquences génétiques codant ces ligands. Ces nouveaux ligands de récepteurs et les séquences génétiques qui les codent sont utiles dans le développement d'une gamme étendue de réactifs agonistes, antagonistes, thérapeutiques et diagnostiques, sur le principe de l'interaction ligand-récepteur. La présente invention concerne également un système d'identification de ligands sans que l'on connaisse leurs récepteurs, ainsi qu'un système d'identification de récepteurs.
PCT/AU1996/000460 1995-07-20 1996-07-19 Nouveaux ligands de recepteurs et sequences genetiques codant ces ligands WO1997004091A1 (fr)

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EP96923786A EP0842272A4 (fr) 1995-07-20 1996-07-19 Nouveaux ligands de recepteurs et sequences genetiques codant ces ligands
AU64098/96A AU711646B2 (en) 1995-07-20 1996-07-19 Novel receptor ligands and genetic sequences encoding same

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AUPN4263 1995-07-20
AUPN4263A AUPN426395A0 (en) 1995-07-20 1995-07-20 Novel receptor ligands and genetic sequences encoding same - II
AUPN6847 1995-11-27
AUPN6847A AUPN684795A0 (en) 1995-11-27 1995-11-27 Novel receptor ligands and genetic sequences encoding same - IIA
AUPN7299 1995-12-22
AUPN7299A AUPN729995A0 (en) 1995-12-27 1995-12-27 Novel receptor ligands and genetic sequences encoding same- IIA
AUPN7890A AUPN789096A0 (en) 1996-02-05 1996-02-05 Novel receptor ligands and genetic sequences encoding same - IIIA
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Cited By (5)

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WO1999017796A2 (fr) * 1997-10-02 1999-04-15 Leukosite, Inc. Modulation de l'adhesion cellulaire induite par la lerk-2
WO2000024413A1 (fr) * 1998-10-27 2000-05-04 The Walter And Eliza Hall Institute Of Medical Research Procede de traitement
WO2003004057A1 (fr) * 2001-07-03 2003-01-16 The Hospital For Sick Children Immunomodulation induite par les recepteurs ephrin et eph
US6602683B1 (en) * 1995-10-25 2003-08-05 Regeneron Pharmaceuticals, Inc. Biologically active Eph family ligands
AU2004201391B2 (en) * 1998-10-27 2007-08-23 The Council Of The Queensland Institute Of Medical Research A method of the treatment

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WO1996010911A1 (fr) * 1994-10-05 1996-04-18 Immunex Corporation Cytokine appelee lerk-6
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WO1996017925A1 (fr) * 1994-12-06 1996-06-13 Immunex Corporation Cytokine designee par lerk-7

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ONCOGENE, Volume 10(2), issued January 1995, KOZLOSKY et al., "Ligands for the Receptor Tyrosine Kinases HEK and EHK: Isolation of cDNA's Encoding a Family of Proteins", pages 299-306. *
SCIENCE, Volume 266, issued November 1994, DAVIES et al., "Ligands for EPH-Related Receptor Tyrine Kinases that Require Membrane Attachment or Clustering for Activity", pages 816-819. *
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6602683B1 (en) * 1995-10-25 2003-08-05 Regeneron Pharmaceuticals, Inc. Biologically active Eph family ligands
WO1999017796A2 (fr) * 1997-10-02 1999-04-15 Leukosite, Inc. Modulation de l'adhesion cellulaire induite par la lerk-2
WO1999017796A3 (fr) * 1997-10-02 1999-06-17 Leukosite Inc Modulation de l'adhesion cellulaire induite par la lerk-2
US6514497B1 (en) 1997-10-02 2003-02-04 Millennium Pharmaceuticals, Inc. Inhibition of LERK-2-mediated cell adhesion
WO2000024413A1 (fr) * 1998-10-27 2000-05-04 The Walter And Eliza Hall Institute Of Medical Research Procede de traitement
AU2004201391B2 (en) * 1998-10-27 2007-08-23 The Council Of The Queensland Institute Of Medical Research A method of the treatment
US7897570B2 (en) 1998-10-27 2011-03-01 The Walter And Eliza Hall Institute Of Medical Research Method of treatment
WO2003004057A1 (fr) * 2001-07-03 2003-01-16 The Hospital For Sick Children Immunomodulation induite par les recepteurs ephrin et eph

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