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WO1996000586A9 - Methodes de traitement d'affections a proliferation cellulaire par modulation de la transduction du signal - Google Patents

Methodes de traitement d'affections a proliferation cellulaire par modulation de la transduction du signal

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Publication number
WO1996000586A9
WO1996000586A9 PCT/EP1995/002532 EP9502532W WO9600586A9 WO 1996000586 A9 WO1996000586 A9 WO 1996000586A9 EP 9502532 W EP9502532 W EP 9502532W WO 9600586 A9 WO9600586 A9 WO 9600586A9
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glu
val
receptor
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PCT/EP1995/002532
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WO1996000586A3 (fr
WO1996000586A2 (fr
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Publication of WO1996000586A2 publication Critical patent/WO1996000586A2/fr
Publication of WO1996000586A3 publication Critical patent/WO1996000586A3/fr
Publication of WO1996000586A9 publication Critical patent/WO1996000586A9/fr

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  • the present invention relates to the use of proteins, peptides and organic molecules capable of modulating inositol 1, ,5-triphosphate (IP 3 ) receptor signal transduction in order to inhibit or reverse inappropriate growth of cells associated with
  • the present invention also relates to the use of IP 3 receptor mutants in the treatment of proliferative disorders associated with abnormalities of signal transduction associated with tyrosine m - kinases, including cancer.
  • the present invention also relates to the use of IP 3 receptor and genetically engineered host cells that express the IP 3 receptor to evaluate and screen for substances and compounds that modulate IP 3 receptor
  • 2S diverse cellular processes are relayed to the interior of cells. These processes include, but are not limited to, cell proliferation, differentiation and survival.
  • a central feature of signal transduction is the reversible phosphorylation of certain proteins.
  • TKs tyrosine kinases
  • TPs tyrosine phosphatases
  • Receptor tyrosine kinases comprise a large f mily of trans embrane receptors for polypeptide growth factors with diverse biological activities and are composed of at least three domains: an extracellular ligand binding domain, a transmembrane domain and a cytoplasmic catalytic domain that can phosphorylate tyrosine residues.
  • Their intrinsic tyrosine kinase function is activated upon ligand binding, which results in phosphorylation of the receptor and multiple cellular substrates, and subsequently in a variety of cellular responses (Ullrich et al . , 1990, Cell 11:203-212) .
  • the secondary signal transducer molecules generated by activated receptors result in a signal cascade that regulates cell functions.
  • phosphorylation of phospholipase c activates this target molecule to hydrolyze phosphatidylinositol 4,5- bisphosphate, generating two secondary signal transducing molecules: inositol 1,4,5-triphosphate, which causes release of stored intracellular calcium, and diacylglycerol, which is the endogenous activator of a serine/threonine kinase, protein kinase C.
  • Reviews describing intracellular signal transduction include Aaronson, S.A., 1991, Science 254:1146-1153; Schlessinger, J., 1988, Trends Bioche ⁇ t. Sci. 13:443- 447; and Ullrich and Schlessinger, 1990, Cell 61:203- 212.
  • cell proliferative disorders including cancer, atherosclerosis, immune deficiency, neurodegenerative disease and hyperproliferative disease, such as psoriasis
  • receptor tyrosine kinases include platelet derived growth factor receptor (PDGFR) , epidermal growth factor receptor (EGFR) , and HER2.
  • PDGFR platelet derived growth factor receptor
  • EGFR epidermal growth factor receptor
  • HER2 his-2
  • neu c-erbB-2
  • Platelet-derived growth factors (PDGFs) and PDGF- receptors are expressed in cells of a variety of neoplasms including gliomas, lung carcinomas, ovarian tumors, and melanomas.
  • PDGFs Platelet-derived growth factors
  • PDGF-S-receptors have been identified in primary human lung carcinomas
  • PDGF receptors have been detected in several other cancers of epithelial origin, including human thyroid carcinoma cells (Heldin et al . , 1991, Endocrinology 129:2187-2193: Heldin et al . , 1988, Proc. Natl. Acad. Sci.
  • HER2/neu gene amplification has been linked by some investigators to neoplastic transformation.
  • the HER2/neu gene has been shown to be amplified in human breast cancer cells (Slamon et al. , 1987, supra) .
  • Amplification and/or overexpression of HER2/neu has been detected in gastrointestinal, non- small cell lung, and ovarian adenocarcinomas and occurs in a significant fraction of primary human breast cancers where it correlates with regionally advanced disease, increased probability of tumor recurrence, and reduced patient survival (Slamon et al . , 1987, supra and Slamon, 1989, Science 244:707- 712) .
  • EGF-R EGF receptor
  • IP 3 inositol 1,4 ,5-trisphosphate
  • IP 3 Rs IP 3 receptors
  • IP 3 receptors contains the second messenger binding site and the C- terminal domain, with the transmembrane sequences, is necessary for membrane insertion and assembly of the subunits to yield the tetrameric organization of the IP 3 Rs (Mignery et al., 1990, supra ; Mignery et al . ,
  • the present invention relates to the use of proteins, peptides and organic molecules capable of modulating inositol 1,4,5-triphosphate (IP 3 ) receptor signal transduction in order to inhibit or reverse inappropriate growth of cells associated with abnormalities of signal transduction associated with tyrosine kinases.
  • IP 3 inositol 1,4,5-triphosphate
  • the present invention also relates to the use of IP 3 receptor mutants in the treatment of proliferative disorders associated with abnormalities of signal transduction associated with tyrosine kinases, including cancer.
  • the present invention also relates to the use of IP 3 receptor and genetically engineered host cells that express the IP 3 receptor to evaluate and screen for substances or compounds that modulate IP 3 receptor activities.
  • the present invention is based, in part, upon the discovery that IP 3 receptors and Ca 2 + stores play an important role in intracellular signalling pathways.
  • the present invention is also based, in part, on the discovery that modulation of IP 3 receptor signal transduction affects cell growth and a transformed phenotype.
  • the present invention is based, in part, upon the unexpected discovery that introduction of signalling incompetent IP 3 receptor mutants to transformed fibroblast cells suppresses the level of transformation in those cells and the discovery that the suppression of transforming activities is not oncogene specific.
  • the inventors have discovered that introduction of signalling incompetent IP 3 receptor mutants to normal cells does not have a negative effect on cell growth or survival.
  • the present invention is therefore based, in part, on the unexpected discovery that by introducing signalling incompetent IP 3 receptor mutant gene sequences to cells, gene therapy may be used to inhibit or reverse tyrosine kinase induced cell transformation without affecting signalling properties of normal cells.
  • the present invention also relates, in part, to the use of genetically engineered host cells expressing IP 3 receptor or receptor mutants to screen and identify IP 3 receptor agonists and antagonists.
  • Soluble IP 3 receptor mutants which retain the capability to bind IP 3 may be used to screen, for example, peptide libraries or organic molecules capable of modulating the IP 3 receptor signal transduction.
  • IP 3 /IP 3 receptor interaction or other molecular interactions which activate or are activated by the IP 3 signalling pathway may be useful in inhibition of innapropriate cell growth associated with abnormalities in signal transduction associated with receptor tyrosine kinases including cancer, atherosclerosis and psoriasis.
  • Figures 1A-1B Biogenesis and expression levels of the IP receptor and mutants s-IP 3 R and ⁇ -IP 3 R, of the major lumenal Ca 2 * binding protein, calreticulin (CR),and of the SERCA ATPases in stable NIH 3T3 cell transfectant clones.
  • Figure 1A shows audoradiographs of immunoprecipitates obtained with the anti-type I IP 3 R-specific T210 Ab.
  • Figure IB shows Western blotting of the transfected clones. Size marker positions in kDa are indicated to the left.
  • Figures 2A, 2B and 2C Ca 2 * responses and IP 3 production induced by ATP and EGF in stable NIH 3 3 transfectant clones challenged in Ca 2 *-poor incubation medium.
  • Figure 2A shows the concentration-dependence of the [Ca 2 * i responses induced by EGF or ATP in stable cell clones infected with NTK-HERc virus (M0I:5) and expressing endogenous IP 3 receptors.
  • FIG. 2A shows the IP 3 production in the control and ⁇ -IP 3 R expressing clones, labeled as in Figure 2A.
  • the resting average [Ca 2 *]i was 130 nM and the average radioactivity of IP 3 was 7800 cpm. Results shown are means +/- SD of five-ten separate experiments.
  • FIG. 4 EGF-sti ulated [ 3 H] thymidine incorporation.
  • Cells were infected with the NTK-HERc virus (M0I:5), expanded, seeded in 12-well plates, grown to confluence, and starved for 18 hours before stimulation. Error bars indicate the range of two independent experiments.
  • FIGS 5A, 5B, 5C and 5D Differential effects of IP 3 receptor mutant expression of long-term growth of NIH 3T3 cells stimulated by autocrine TGF ⁇ and EGF- R overexpression.
  • Cells were infected with NTK-HERc virus (MOI:5), superinfected with ⁇ 2-TGF ⁇ virus (MOI:3), and seeded in 96-well plates.
  • Cells were infected with NTK-HERc virus (M0I:5) together with ⁇ 2-N2 virus (M0I:5) (lanes 1, 2, 4, 5) or *2-TGF ⁇ virus (M0I:3) (lanes 3,6).
  • Cells were grown to confluence, starved for 24 hours in DMEM containing 0.5% FCS, and stimulated for 10 min with lOOng/ml EGF or by the autocrine loop with TGF ⁇ . The cells were solubilized, and for each lane 30 ⁇ g total protein separated by SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting with the antiphosphotyrosine antibody 5E2 (Fendly et al.
  • Figures 7A-7F The nucleic acid (SEQ ID N0:1) and deduced amino acid sequence (SEQ ID NO: 2) of rat IP3 receptor.
  • Figures 8A-8E The shared amino acid sequence homology of rat IP3 receptor and human IP3 receptor.
  • the present invention relates to the use of proteins, peptides and organic molecules capable of modulating inositol 1,4,5-triphosphate (IP 3 ) receptor signal transduction in order to inhibit or prevent inappropriate growth of cells associated with abnormalities in signal transduction associated with tyrosine kinases.
  • IP 3 inositol 1,4,5-triphosphate
  • Such modulators of IP 3 may be used therapeutically.
  • antagonists of IP 3 receptor signal transduction may find application in the treatment of proliferative disorders associated with abnormalities in signal transduction associated with tyrosine kinase, such as deregulation of tyrosine kinase function, including for example, cancer, atherosclerosis and psoriasis.
  • the present invention also relates to the use of IP 3 receptor_mutants in the treatment of proliferative disorders associated with abnormalities of signal transduction associated with tyrosine kinases.
  • the present invention also relates to the use of IP 3 receptor and genetically engineered host cells that express the IP 3 receptor to evaluate and screen for substances and compounds that modulate IP 3 receptor activities.
  • the invention is based, in part, on the unexpected discovery that introduction of signalling incompetent IP 3 receptor mutants to transformed fibroblast cells suppresses the level of transformation in those cells without corresponding adverse effects in normal cells, and the discovery that the suppression of transforming activities is not oncogene specific.
  • NIH 3T3 cells were transformed by coinfection of TGF ⁇ -expressing virus and the human EGF receptor, NTK-HERc.
  • the level of transformation was suppressed to a level of approximately 10%.
  • the level of transformation was reduced significantly by about 35% in comparison to control cells.
  • the present invention is also based, in part, on the discovery that while the introduction of signalling incompetent IP 3 receptor mutants to normal cells may modulate signal transduction and cellular Ca 2 * homeostasis, other cellular mechanisms act to keep near normal cellular signalling properties.
  • the phrase, "inappropriate growth of cells” includes abnormal, uncontrolled or deregulated cellular proliferation or differentiation, e .g. , under or over production of mature differentiated cells and inappropriate proliferation of immature cells, growth of abnormal cells and untimely cell death.
  • the IPj Receptor/Receptor Mutant Coding Sequences include abnormal, uncontrolled or deregulated cellular proliferation or differentiation, e .g. , under or over production of mature differentiated cells and inappropriate proliferation of immature cells, growth of abnormal cells and untimely cell death.
  • Figures 7A-7F are the nucleic acid and deduced amino acid sequence of rat IP3 receptor.
  • Figures 8A-8E demonstrate the shared amino acid sequence homology of rat IP3
  • any nucleotide sequence which encodes the amino acid sequence of an IP 3 receptor gene product can be used to generate recombinant molecules which direct the expression of
  • the invention contemplates, in addition to the DNA sequences disclosed herein, 1) any DNA sequence that encodes the same amino acid sequence as encoded by the DNA sequences shown in Figures 7A- 7F; 2) any DNA sequence that hybridizes to the
  • the invention also encompasses 1) DNA vectors
  • __ that contain any of the coding sequences disclosed herein; and/or their complements (i.e., antisense) ; 2) DNA expression vectors that contain any of the coding sequences disclosed herein, and/or their complements (i.e., antisense) , operatively associated with a regulatory element that directs the expression of the coding and/or antisense sequences; and 3) genetically engineered host cells that contain any of the coding sequences disclosed herein, and/or their complements (i.e., antisense), operatively associated with a regulatory element that directs the expression of the coding and/or antisense sequences in the host cell.
  • Regulatory element includes but is not limited to inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression.
  • the invention includes fragments of any of the DNA sequences disclosed herein.
  • IP 3 receptor is a term which refers to any member of the IP 3 receptor family from any species, including, bovine, ovine, porcine, equine, and preferably human, in naturally occurring- sequence or in variant form, or from any source, whether natural, synthetic, or recombinant.
  • IP 3 receptor mutant refers to a non-naturally occurring IP 3 receptor. Preferred IP 3 receptor mutants are those which lack the N-terminal IP 3 binding domain and/or those which lack the C-terminal transmembrane domain.
  • the phrase "signalling incompetent IP 3 receptor mutant” refers to an IP 3 receptor mutant that is not capable of transducing a signal or that transduces a signal to a lesser extent than the naturally occurring IP 3 receptor.
  • a particularly preferred IP 3 receptor mutant of the present invention is ⁇ -IP 3 R lacking 418 amino acids in the IP 3 receptor N-terminal sequence, resulting in an IP 3 receptor which lacks the IP 3 binding domain and which is expected to be signalling incompetent, i.e., unable to transduce a signal (Mignery and S ⁇ dhof et al . , 1990, supra ; Mignery et al., 1990, supra) .
  • ⁇ -IP 3 R is expected to retain the ability to assemble with naturally occurring IP 3 receptor subunits to form a tetrameric Ca 2 * channel across the endoplasmic reticulum membrane.
  • IP 3 receptor mutant a deletion of 379 amino acids is made in the C-terminal IP 3 receptor sequence resulting in IP 3 receptors which are expected to retain the IP 3 binding domain but which lack the transmembrane domain necessary for transmembrane insertion (Mignery and S ⁇ dhof et al., 1990, supra ; Mignery et al . , 1990, supra) .
  • s-IP 3 R is expected to remain as a soluble monomer in the cytosol.
  • IP 3 receptor polynucleotide sequences which encode naturally occurring IP 3 receptors, peptide fragments of IP 3 receptors, IP 3 receptor fusion proteins or functional equivalents thereof, or IP 3 receptor mutants, for example, signalling incompetent IP 3 receptor mutants, may be used to generate recombinant DNA molecules that direct the expression of IP 3 receptor protein, IP 3 receptor peptide fragment, fusion proteins or a functional equivalent thereof or IP 3 receptor mutants, in appropriate host cells.
  • IP 3 receptor polynucleotide sequences as well as other polynucleotides which selectively hybridize to at least a part of such IP 3 receptor polynucleotides or their complements, may also be used in nucleic acid hybridization assays, Southern and Northern blot analyses, etc. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence, may be used in the practice of the invention for the cloning and expression of IP 3 receptor protein. Such DNA sequences include those which are capable of hybridizing to the human IP 3 receptor sequence under stringent conditions.
  • stringent conditions refers to those hybridizing conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50 C C; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M Sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 X SSC and 0.1% SDS.
  • formamide for example, 50% (vol/vol) form
  • Altered DNA sequences which may be used in accor ⁇ dance with the invention include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent gene product.
  • the gene product itself may contain deletions, additions or substitutions of amino acid residues within an IP 3 receptor sequence, which result in a silent change thus producing a functionally equivalent IP 3 receptor.
  • Such amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipatic nature of the residues involved.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine; glycine, alanine; asparagine, gluta ine; serine, threonine; phenylalanine , tyrosine.
  • the DNA sequences of the invention may be engi ⁇ neered in order to alter an IP 3 receptor coding sequence for a variety of ends including but not limited to alterations which modify processing and expression of the gene product.
  • mutations may be introduced using techniques which are well known in the art, e .g. , site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, phosphorylation, etc.
  • an IP 3 receptor or receptor mutant may be ligated to a heterologous sequence to encode a fusion protein.
  • a fusion protein may also be engineered to contain a cleavage site located between an IP 3 receptor or receptor mutant sequence and the heterologous protein sequence, so that the IP 3 receptor or receptor mutant may be cleaved away from the heterologous moiety.
  • the coding sequence of an IP 3 receptor or receptor mutant could be synthesized in whole or in part, using chemical methods well known in the art. See, for example, Caruthers et al . , 1980, Nuc. Acids Res. Sv p. Ser. 2:215-233; Crea et al . , 1980, Nuc. Acids Res. 9X101:2331; Matteucci et al . , 1980, Tetrahedron Letters 2_1:719; and Chow et al . , 1981, Nuc. Acids Res. 9(12) :2807-2817.
  • the protein itself could be produced using chemical methods to synthesize an IP 3 receptor or receptor mutant amino acid sequence in whole or in part.
  • peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high perform ⁇ ance liquid chromatography (e.g., see Creighton, 1983, Proteins Structures And Molecular Principles. W.H.
  • IP 3 receptor In order to express a biologically active IP 3 receptor or IP 3 receptor mutant, the nucleotide sequence coding for IP 3 receptor, receptor mutant or a functional equivalent, is inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • an appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • the IP 3 receptor gene products as well as host cells or cell lines transfected or transformed with recombinant IP 3 receptor expression vectors can be used for a variety of purposes. These include but are not limited to generating antibodies (i.e., monoclonal or polyclonal) that competitively inhibit activity of an IP 3 receptor and neutralize its activity. Anti-IP 3 receptor antibodies may be used in detecting and quantifying expression of an IP 3 receptor in cells and tissues. 5.3. Expression Systems
  • a variety of host-expression vector systems may be utilized to express an IP 3 receptor or receptor mutant coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing an IP 3 receptor or receptor mutant coding sequence; yeast transformed with recombinant yeast expression vectors containing an IP 3 receptor or receptor mutant coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g.
  • baculovirus containing an IP 3 receptor or receptor mutant coding sequence
  • plant cell systems infected with recombinant virus expression vectors e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV
  • recombinant plasmid expres- sion vectors e.g., Ti plasmid
  • any of a number of suitable transcription and translation elements may be used in the expression vector.
  • inducible promoters such as pL of bacteriophage ⁇ , plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used; when cloning in insect cell systems, promoters such as the baculovirus polyhedrin promoter may be used; when cloning in plant cell systems, promoters derived from the genome of plant cells (e.g., heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll a/b binding protein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; the coat protein promoter of TMV) may be used; when cloning in mamm
  • a number of expression vectors may be advantageously selected depending upon the use intended for the IP 3 receptor or receptor mutant expressed * For example, when large quantities of IP 3 "receptor are to be produced for the generation of antibodies, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • Such vectors include but are not limited to the E . coli expression vector pUR278 (Ruther et al . , 1983, EMBO J. 2:1791), in which the IP 3 receptor coding sequence may be ligated into the vector in frame with the lac Z coding region so that a hybrid lac Z protein is produced; pIN vectors (Inouye et al . , 1985, Nucleic acids Res.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST) .
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety.
  • yeast a number of vectors containing constitutive or inducible promoters may be used.
  • an IP 3 receptor or receptor mutant coding sequence may be driven by any of a number of promoters.
  • viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al . , 1984, Nature 310:511-5141. or the coat protein promoter of TMV (Taka atsu et al . , 1987, EMBO J. 1:307-311) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al . , 1984, EMBO J. 2:1671-1680; Broglie et al .
  • IP 3 receptor or receptor mutant An alternative expression system which could be used to express an IP 3 receptor or receptor mutant is an insect system.
  • Autographa californlca nuclear polyhidrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells.
  • An IP 3 receptor or receptor mutant coding sequence may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example, the polyhedrin promoter) .
  • IP 3 receptor or receptor mutant coding sequence Successful insertion of an IP 3 receptor or receptor mutant coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene) . These recombinant viruses are then used to infect Spodoptera fpigiperda cells in which the inserted gene is expressed. (e.g., see Smith et al . , 1983, J. Viol. 4_6:584; Smith, U.S. Patent No. 4,215,051).
  • an IP 3 receptor or receptor mutant coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or In vivo recombination. Insertion in a non- essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing an IP 3 receptor or receptor mutant in infected hosts (e.g.. See Logan et al .
  • the vaccinia 7.5 K promoter may be used. (See, e .g. , Mackett et al . , 1982, Proc. Natl. Acad. Sci. (USA) 79:7415-7419; Mackett et al . , 1984, J. Virol. 49.:857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. 79:4927-4931) .
  • Specific initiation signals may also be required for efficient translation of inserted IP 3 receptor or receptor mutant coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire IP 3 receptor or receptor mutant gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of an IP 3 receptor or receptor mutant coding sequence is inserted, exogenous translational control signals, including the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of an IP 3 receptor or receptor mutant coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
  • telomeres may be shortened by the telomeres.
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have charac ⁇ teristic and specific mechanisms for the post-transla- tional processing and modification of proteins. Appropriate cells lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, WI38, etc.
  • IP 3 receptor or receptor mutant For long-term, high-yield production of recombi ⁇ nant proteins, stable expression is preferred.
  • cell lines which stably express an IP 3 receptor or receptor mutant may be engineered.
  • host cells can be transformed with IP 3 receptor or receptor mutant DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription termina ⁇ tors, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription termina ⁇ tors, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may be used advantageously to engineer cell lines which express an IP 3 receptor or receptor mutant.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al . , 1977, Cell 11:223) . hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al . , 1962, Proc. Natl. Acad. Sci. USA 48.2026), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 2 . 2:817) genes can be employed in tk, hgprt" or aprt" cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to ethotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 27:3567; O'Hare et al . , 1981, Proc. Natl. Acad. Sci. USA 2 ⁇ .1527); gpt, which confers resistance to mycophenolic acid (Mulligan et al., 1981, Proc. Natl. Acad. Sci. USA 78.:2072) ; neo, which confers resistance to the a inoglycoside G-418 (Colberre-Garapin et al . , 1981, J. Mol. Biol.
  • trpB which allows cells to utilize indole in place of tryptophan
  • hisD which allows cells to utilize histinol in place of histidine
  • ODC ornithine decarboxylase
  • the host cells which contain the coding sequence and which express the biologically active gene product may be identified by at least four general approaches; (a) DNA-DNA or DNA-RNA hybridization; (b) the presence or absence of "marker" gene functions; (c) assessing the level of transcription as measured by the expres ⁇ sion of IP 3 receptor or receptor mutant mRNA transcripts in the host cell; and (d) detection of the gene product as measured by immunoassay or by its biological activity.
  • the presence of the IP 3 receptor or receptor mutant coding sequence inserted in the expression vector can be detected by DNA-DNA or DNA-RNA hybridization using probes comprising nucleotide sequences that are homologous to the IP 3 receptor or receptor mutant coding sequence, respectively, or portions or derivatives thereof.
  • the recombinant expres ⁇ sion vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.).
  • certain "marker" gene functions e.g., thymidine kinase activity, resistance to antibiotics, resistance to methotrexate, transformation phenotype, occlusion body formation in baculovirus, etc.
  • a marker gene can be placed in tandem with an IP 3 receptor or receptor mutant sequence under the control of the same or different promoter used to control the expression of the IP 3 receptor or receptor mutant coding sequence. Expression of the marker in response to induction or selection indicates expression of the IP 3 receptor or receptor mutant coding sequence.
  • transcriptional activity for an IP 3 receptor or receptor mutant coding region can be assessed by hybridization assays.
  • RNA can be isolated and analyzed by Northern blot using a probe homologous to an IP 3 receptor or receptor coding sequence or particular portions thereof.
  • total nucleic acids of the host cell may be extracted and assayed for hybridization to such probes.
  • the expression of an IP 3 receptor or receptor mutant protein product can be assessed immunologically, for example by Western blots, immunoassays such as radioimmuno-precipitation, enzyme-linked immunoassays and the like.
  • gliomas including gliomas, lung carcinomas, ovarian tumors, thyroid carcinomas, human breast cancer, gastric carcinomas and melanomas; gastrointestinal, non-small cell lung and ovarian adenocarcinomas; cervical, ovarian, esophagal and stomach carcinomas; and atherosclerosis and psoriasis.
  • Modulation of receptor-generated signal transduction events can be mediated through secondary signal transducer molecules, such as IP 3 .
  • secondary signal transduction molecules refers to any component or product found in the cascade of signal transduction events.
  • IP 3 and its binding to and activation of IP 3 receptor which in tetrameric organization forms intracellular Ca 2 *- permeable channels, plays a role in Ca 2 * homeostasis and intracellular signalling events. It has been observed that IP 3 signal transduction affects cell growth and oncogenesis. Therefore, modulators of IP 3 receptor signal transduction may be used therapeutically for the treatment of disorders and diseases states resulting from defects in different signal transduction pathways associated with receptor tyrosine kinases.
  • an IP 3 receptor or receptor mutant and/or cell line that expresses an IP 3 receptor or receptor mutant may be used to screen for antibodies, peptides, or other molecules that act as agonists or antagonists of IP 3 receptor through modulation of signal transduction pathways.
  • anti-IP 3 receptor antibodies capable of neutralizing the activity of IP 3 receptor may be used to inhibit an IP 3 receptor associated signal transduction pathway.
  • Such antibodies can act intracellularly utilizing the techniques described in Marasco et al., 1993 fPNAS 90:7889-7893. for example or through delivery by liposomes.
  • screening of organic or peptide libraries with recombinantly expressed IP 3 receptor or receptor mutant protein or cell lines expressing IP 3 receptor or receptor mutant protein may be useful for identification of therapeutic molecules that function by modulating IP 3 receptor signal transduction or Ca 2 * homeostasis.
  • Synthetic compounds, natural products, and other sources of potentially biologically active materials can be screened in a number of ways deemed to be routine to those of skill in the art.
  • IP 3 5 receptor signal transduction responses may be measured. For example, responses such as modulation of Ca 2* may be monitored.
  • responses such as modulation of Ca 2* may be monitored.
  • Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression
  • Neutralizing antibodies i.e., those which inhibit the signal transducing activity of an IP 3 receptor are especially preferred for diagnostics and therapeutics.
  • 2S animals may be immunized by injection with an IP 3 receptor protein including but not limited to rabbits, mice, rats, etc.
  • IP 3 receptor protein including but not limited to rabbits, mice, rats, etc.
  • adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's
  • mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants
  • Monoclonal antibodies to an IP 3 receptor or receptor mutant may be prepared by using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler et al., 1975 (Nature 256:495-497) . the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today. 1:72; and Cote et al., 1983, Proc. Natl. Acad. Sci.. 80:2026-2030) and the EBV-hybridoma technique (Cole et al .
  • Antibody fragments which contain specific binding sites of an IP 3 receptor may be generated by known techniques.
  • fragments include but are not limited to: the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab .fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries may be constructed (Huse et al. , 1989, Science 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity o the IP 3 receptor.
  • Random peptide libraries consisting of all possible combinations of amino acids attached to a solid phase support may be used to identify peptides that are able to bind to the ligand binding site of a given receptor or other functional domains of a receptor such as kinase domains (Lam, K.S. et al . , 1991, Nature 354: 82-84) .
  • the screening of peptide libraries may have therapeutic value in the discovery of pharmaceutical agents that act to modulate the biological activity of receptors through their interactions with the given receptor.
  • Identification of molecules that are able to bind to an IP 3 receptor may be accomplished by screening a peptide library with a recombinant soluble IP 3 receptor mutant. Soluble IP 3 receptor mutant is described in Mignery and S ⁇ dhof et al . , 1990, supra and Mignery et al., 1990, supra . Methods for expression of IP 3 receptor or IP 3 receptor mutants are described in Section 5.2 and may be used to express recombinant IP 3 receptor or receptor mutants or fragments of IP 3 receptors depending on the functional domains of interest. For example, the extracellular ligand binding domains of IP 3 receptor may be separately expressed and used to screen peptide libraries.
  • IP 3 receptor or receptor mutant protein may be conjugated to enzymes such as alkaline phosphatase or horseradish peroxidase or to other reagents such as fluorescent labels which may include fluorescein isothyiocynate (FITC) , phycoerythrin (PE) or rhoda ine. conjugation of any given label to an IP 3 receptor or receptor mutant may be performed using techniques that are routine in the art.
  • IP 3 receptor or receptor mutant expression vectors may be engineered to express a chimeric IP 3 receptor or receptor mutant protein containing an epitope for which a commercially available antibody exist.
  • the epitope-specific antibody may be tagged using methods well known in the art including labeling with enzymes, fluorescent dyes or colored or magnetic beads.
  • the "tagged" IP 3 receptor or receptor mutant conjugate is incubated with the random peptide library for 30 minutes to one hour at 22°C to allow complex formation between IP 3 receptor or receptor mutant and peptide species within the library. The library is then washed to remove any unbound IP 3 receptor or receptor mutant protein.
  • the whole library is poured into a petri dish containing a substrates for either alkaline phosphatase or peroxidase, for example, 5-bromo-4-chloro-3-indoyl phosphate (BCIP) or 3,3' ,4,4"-diamnobenzidine (DAB) , respectively.
  • a substrates for either alkaline phosphatase or peroxidase for example, 5-bromo-4-chloro-3-indoyl phosphate (BCIP) or 3,3' ,4,4"-diamnobenzidine (DAB) , respectively.
  • BCIP 5-bromo-4-chloro-3-indoyl phosphate
  • DAB 3,3' ,4,4"-diamnobenzidine
  • the peptide/solid phase-IP 3 receptor complex changes color, and can be easily identified and isolated physically under a dissecting microscope with a micromanipulator. If a fluorescent tagged IP 3 receptor molecule has been used
  • detection of the peptide/IP 3 receptor complex may be accomplished by using a labeled epitope specific antibody. Once isolated, the identity of the peptide attached to the solid phase support may be determined by peptide sequencing.
  • peptides that bind to cell surface receptors using intact cells.
  • the use of intact cells is preferred for use with receptors that are multi-subunits or labile or with receptors that require the lipid domain of the cell membrane to be functional.
  • Methods for generating cell lines expressing IP 3 receptor or receptor mutants are described in Sections 5.3 and 5.4.
  • the cells used in this technique may be either live or fixed cells. The cells will be incubated with the random peptide library and will bind to certain peptides in the library to form a "rosette" between the target cells and the relevant solid phase support/peptide. The rosette can thereafter be isolated by differential centrifugation or removed physically under a dissecting microscope.
  • the receptor molecules can be reconstituted into liposomes where label or "tag" can be attached.
  • IP 3 receptor or IP 3 receptor mutants may be used to screen for molecules that modulate IP 3 signal transduction or affect Ca 2* homeostasis.
  • molecules may include small organic or inorganic compounds, or extracts of biological materials such as plants, fungi, etc., or other molecules that modulate IP 3 receptor signal transduction or that promote or prevent the formation of IP 3 /IP 3 receptor complex.
  • Synthetic compounds, natural products, and other sources of potentially biologically active materials can be screened i a number of ways.
  • test molecule to interfere with IP 3 binding to the IP 3 receptor and/or IP 3 receptor signal transduction may be measured using standard biochemical techniques. Other responses such as phosphorylation or dephosphorylation of other proteins, activation or modulation of other molecules in the signal cascade, changes in cellular ion levels, association, dissociation or translocation of signalling molecules, or transcription or translation of specific genes may also be monitored. These assays may be performed using conventional techniques developed for these purposes in the course of screening.
  • IP 3 receptor signalling pathway may include, but are not limited to, normal cellular functions, Ca 2 * homeostasis, proliferation, differentiation, in addition to abnormal or potentially deleterious processes such as unregulated cell proliferation, loss of contact inhibition, blocking of differentiation or untimely cell death.
  • IP 3 receptor, or functional derivatives thereof, useful in identifying compounds capable of modulating signal transduction may have, for example, amino acid deletions and/or insertions and/or substitutions as long as they retain significant ability to interact with some or all relevant components of an IP 3 receptor mediated signal transduction pathway.
  • a functional derivative of an IP 3 receptor may be prepared from a naturally occurring or recombinantly expressed IP 3 receptor by proteolytic cleavage followed by conventional purification procedures known to those skilled in the art.
  • the functional derivative may be produced by recombinant DNA technology by expressing parts of an IP 3 receptor which include the functional domain, for example the ligand binding domain, in suitable cells.
  • Functional derivatives may also be chemically synthesized. Cells expressing IP 3 receptor may be used as a source of IP 3 receptor, crude or purified for testing in these assays.
  • IP 3 receptor signalling pathway Various embodiments are described below for screening, identification and evaluation of compounds that interact with the IP 3 receptor, which compounds may affect various cellular processes under the control of the IP 3 receptor signalling pathway.
  • the present invention includes a method for identifying a compound which is capable of modulating signal transduction, comprising:
  • step (b) incubating the mixture of step (a) in the presence of IP 3 , for an interval sufficient for the compound to stimulate or inhibit the signal transduction;
  • IP 3 receptor, or functional derivatives or IP 3 receptor mutant thereof useful in identifying compounds capable of modulating signal transduction may have, for example, amino acid deletions and/or insertions and/or substitutions as long as they retain significant signal transducing capacity.
  • a preferred IP 3 mutant is one lacking the C-terminal transmembrane sequences necessary for membrane insertion, i.e., a soluble IP 3 receptor, while retaining the N-terminal IP 3 binding domain.
  • a functional derivative of IP 3 receptor may be prepared from a naturally occurring or recombinantly expressed IP 3 receptor by proteolytic cleavage followed by conventional purification procedures known to those skilled in the art.
  • the functional derivative may be produced by recombinant DNA technology by expressing parts of IP 3 receptor which include the functional domain in suitable cells.
  • Functional derivatives may also be chemically synthesized.
  • Cells expressing IP 3 receptor or receptor mutants may be used as a source of IP 3 recep.tor or receptor mutants, crude or purified, or in a membrane preparation, for testing in these assays. Alternatively, whole live or fixed cells may be used directly in those assays.
  • IP 3 receptor signal transduction activity may be measured by standard biochemical techniques or by monitoring the cellular processes controlled by the signal, such as Ca 2 * levels.
  • the invention further provides for a method of screening compounds that, upon interacting with IP 3 receptor, elicit or trigger a signal mimicking the action of IP 3 binding to the IP 3 receptor.
  • Signal transduction is mimicked if the cellular processes under the control of the signalling pathway are affected in a way similar to that caused by ligand binding.
  • Such compounds may be naturally occurring or synthetically produced molecules that activate the IP 3 receptor.
  • the invention also includes a method for identifying a molecule in a chemical or biological preparation capable of binding to IP 3 receptor, comprising:
  • the above method may further include the step of:
  • Compounds capable of binding to IP 3 receptor may directly or indirectly modulate IP 3 signal transduction and may include molecules that are naturally associated with the intracellular domain of IP,.
  • Compounds capable of binding to IP 3 receptor refers to a naturally occurring or synthetically produced molecule which interacts with IP 3 . Examples of such compounds are (i) a natural substrate of IP 3 receptor; (ii) a naturally occurring molecule which is part of the signalling complex; and/or a naturally occurring si nalling molecule produced by other cell types.
  • proteins, peptides or moleucles which bind IP 3 receptor, IP 3 receptor mutants, or a fragment containing the IP 3 binding site, or an IP 3 receptor anti-sense molecule containing a sequence complementary to at least a part of the coding sequence of IP 3 receptor and which inhibits translation of IP 3 receptor mRNA could be administered in vivo to modulate IP 3 receptor signal transduction.
  • IP 3 receptor mutant or a fragment containing the IP 3 receptor binding domain or an organic molecule capable of binding to the IP 3 receptor binding domain could competitively bind to IP 3 and inhibit its interaction with the naturally occurring IP 3 receptor in vivo to inhibit or reverse inappropriate growth of cells associated with abnormalities in signal transduction associated with tyrosine kinases.
  • ligands for IP 3 receptor including anti-IP 3 receptor antibodies or fragments thereof, may be used to 0 modulate inappropriate cell growth associated with abnormalities in signal transduction associated with tyrosine kinases.
  • Antagonists of IP 3 activity may be used to inhibit tumor growth.
  • the particular peptides, proteins, organic _ compounds, antibodies, anti-sense molecules or IP 3 receptor mutants that modulate IP, receptor signal transduction can be administered to a patient either alone, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) .
  • these agents may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition. Suitable routes may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, or, in the case of solid tumors, directly injected into a solid tumor.
  • the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks , s solution.
  • Ringer's solution or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. Determination of the effective eimounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.
  • compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • compositions for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP) .
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may be prepared, placed in an appropriate container, and labelled for treatment of an indicated condition.
  • Suitable conditions indicated on the label may include treatment of a tumor, such as a glioma or breast cancer.
  • the pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients.
  • suitable solid or gel phase carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • Many of the IP 3 receptor modulating compounds of the invention may be provided as salts with pharmaceutically compatible counterions.
  • Pharmaceutically compatible salts may be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the PTP activity) .
  • Such information can be used to more accurately determine useful doses in humans.
  • a therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e .g. , for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) .
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds which exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with. little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e . g. , Fingl et al . , 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 pi).
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the IP 3 receptor-inhibitory effects.
  • Usual patient dosages for systemic administration range from 1 - 2000 mg/day, commonly from 1 - 250 mg/day, and typically from 10 -150 mg/day.
  • usual dosages range from 0.02 - 25 mg/kg/day, commonly from 0.02 - 3 mg/kg/day, typically from 0.2 - 1.5 mg/kg/day.
  • usual dosages range from 0.5 - 1200 mg/m 2 /day, commonly from 0.5 - 150 mg/m 2 /day, typically from 5 - 100 mg/m 2 /day.
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which axe sufficient to maintain the IP 3 receptor-inhibitory effects. Usual average plasma levels should be maintained within 50 - 5000 ⁇ g/ l, commonly 50 - 1000 ⁇ g/ml, and typically 100 - 500 ⁇ g/ml. Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a tumor, often in a depot or sustained release formulation.
  • the liposomes will be targeted to and taken up selectively by the tumor.
  • the effective local concentration of the drug may not be related to plasma concentration.
  • IP 3 receptor coding sequence may be used for diagnostic purposes for detection of IP 3 receptor expression. Included in the scope of the invention are oligoribonucleotide sequences, that include antisense RNA and DNA molecules and ribozymes that function to inhibit translation of IP 3 receptor. Polynucleotides encoding an IP 3 receptor mutant, for example a soluble or a signalling incompetent mutant, having the ability to compete with the endogenous IP 3 receptor protein for access to molecules in the IP 3 signalling pathway thereby exhibiting a dominant negative effect, may be expressed in targeted cell populations to modulate the activity of naturally occurring IP 3 receptor.
  • oligoribonucleotide sequences that include anti-sense RNA and DNA molecules and ribozymes that function to inhibit the translation of IP 3 receptor mRNA.
  • Anti ⁇ sense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation.
  • antisense DNA oligodeoxyribonucleotides derived from the translation initiation site, e.g., between -10 and +10 regions of the IP 3 receptor nucleotide sequence, are preferred.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mecha ⁇ nism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complemen ⁇ tary target RNA, followed by a endonucleolytic cleav ⁇ age.
  • engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of IP 3 receptor RNA sequences.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligo- nucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complemen ⁇ tary oligonucleotides, using ribonuclease protection assays.
  • RNA molecules and DNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively,
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule.
  • DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
  • Various modifications to the DNA molecules may be introduced as a means of increasing intracellular stability and half-life.
  • Possible modifications include but are not limited to the addition of flanking sequences of ribo- or deoxy- nucleotides to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phospho- diesterase linkages within the oligodeoxyribonucleo- tide backbone.
  • IP 3 receptor is known to assemble in tetrameric organization of identical or highly homologous subunits forming Ca 2* -permeable channels.
  • IP 3 receptor mutants can function as dominant negative mutations by suppressing the activation and response of naturally occurring IP 3 receptors through formation of tetramers with naturally occurring receptors, or other mutant receptors, wherein such tetramers are signalling incompetent.
  • IP 3 receptor mutants lacking a transmembrane domain while retaining the IP 3 binding domain may lack the ability to form tetramers with naturally occurring receptors and may also be signalling incompetent.
  • IP 3 receptors can be engineered into recombinant viral vectors and used in gene therapy in individuals that inappropriately express receptor tyrosine kinase or that exhibit inappropriate growth of cells associated with abnormalities in signal transduction associated with tyrosine kinases.
  • IP 3 mutant receptors to suppress transformation of growth factor induced transformed NIH 3T3 cells is demonstrated in Section 7.
  • the therapeutic potential of an IP 3 receptor mutant used in gene therapy may be measured by examining suppression of receptor tyrosine kinase induced transforming activity.
  • mutant forms of the IP 3 receptor having a therapeutic effect may be identified by expression in selected cells. Deletion or missense mutants of IP 3 receptor that retain the ability to form tetramers with naturally occurring IP 3 receptor protein or other mutants of IP 3 receptors but which cannot function in signal transduction may be used to inhibit the biological activity of the naturally occurring IP 3 receptor.
  • the IP 3 binding domain of an IP 3 receptor may be deleted resulting in a mutant IP 3 receptor molecule that is still able to undergo formation of tetramers with naturally occurring receptors but unable to transduce a signal.
  • the invention provides a method of inhibiting the effects of IP 3 receptor mediated signal transduction by an endogenous IP 3 protein in a cell comprising delivering a DNA molecule encoding a signalling incompetent form of the IP 3 receptor protein to the cell so that the signalling incompetent IP 3 receptor protein is produced in the cell and competes with the endogenous IP 3 receptor protein for access to molecules in the IP 3 receptor protein signalling pathway which activate or are activated by the endogenous IP 3 receptor protein.
  • Recombinant vixuses may be engineered to express signalling incompetent forms of the IP 3 receptor which are capable of inhibiting the activity of naturally occurring IP 3 receptor. These viruses may be used therapeutically for treatment of diseases resulting from aberrant expression or activity of receptor tyrosine kinases or activity of the IP 3 receptor.
  • Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine pa illoma virus may be used for delivery of recombinant IP 3 receptor mutants into the targeted, cell population.
  • Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors containing IP 3 receptor mutant coding sequence. See, for example, the techniques described in Maniatis et al . , 1989, Molecular Cloning A Laboratory Manual. Cold Spring Harbor Laboratory, N.Y. and Ausubel et al . , 1989, Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley Interscience, N.Y.
  • recombinant IP 3 receptor molecules can 5 be reconstituted into liposomes for delivery to target cells.
  • IP 3 receptor mutants ⁇ -IP 3 R lacking the N-terminal IP 3 binding domain and s-IP 3 R, lacking the 0 C-terminal transmembrane sequences necessary for membrane insertion, were shown to suppress the level of transformation in NIH T3 cells induced to transform by receptor tyrosine kinase.
  • This example describes characterization of the expression and functioning of the IP 3 receptor and the regulation of IP 3 generation and Ca 2 * storage systems - in transfected NIH 3T3 clones.
  • NIH 3T3 cells were grown in Dulbecco's modified 5 Eagle's medium (DMEM, GIIBCO) containing 10% fetal calf serum (FCS, GIBCO) and 2 mM glutamine.
  • DMEM Dulbecco's modified 5 Eagle's medium
  • FCS fetal calf serum
  • FCS fetal calf serum
  • Purified expression plasmid cDNAs were introduced into subconfluent cells by cotransfeetion with a plasmid conferring resistance to the antibiotic geneticin S (G418), at a molar ratio of 10:1, using the calcium phosphate technique (Graham, F.L. and van der Eb, A.J., 1973, Virology 12:456-467). Control cells were transfected in parallel with the vector and the resistance plasmid. One day after transfection, cells were split into DMEM/10% FCS/2 mM glutamine supplemented with 1 mg/ml G418. Expression of the wt and mutated IP 3 R was established at day 11-14. G418 r colonies were picked and tested by in vivo labeling with [ 3S S] methionine (Amersham, U.K.) and subsequent immunoprecipitation, and by Western blotting.
  • Lysates were centrifuged for 10 min at 4 ⁇ C in an Eppendorf centrifuge (13000 x g) .
  • the supernatants were incubated overnight at 4°C with an excess of the anti-IP 3 receptor polyclonal antibody (Ab) T 210/ (raised against the 19°C terminus amino acids of the receptor, as described by Mignery et al., 1990, supra) and protein A-Sepharose.
  • the immunoprecipitates were washed three times with washing buffer (20 mM HEPES pH
  • anti-SERCA sarcoplasmicendoplasmic reticulu Ca 2* ATPase
  • PLC phospholipase C
  • EGF receptor human EGF receptor
  • HERc subconfluent cells (1 x 10 s cells per 6-cm dish) were incubated with supernatants of GP+E-86 cells releasing high-titer NTK-HERc virus
  • G418 r CFU/ml kindly provided by David Lee
  • polybrene 8 ⁇ g/ml; Aldrich
  • FIG. 1 A control, parallel cell monolayers were superinfected with *2 cell supernatants of 2 cells releasing ⁇ 2-N2 control virus (5xlO s G418 2 CFU/ml) in the same experimental conditions.
  • the retroviral expression vectors pN2 and pNTK-HERc have been described previously (Keller et al., 1985, Nature 318:149-154: von Ruden et al., 1988, EMBO J. 2:2749- 2756) .
  • Virus titers were determined by infecting NIH 3T3 cells with serial dilutions of retrovirus containing cell-free conditioned media of the individual virus-producer lines and determining the number of the G418 resistant colonies.
  • Radioactive inositol phosphates were separated by either stepwise elution ion-exchange chromatography or HPLC, and radioactivity counted in a Beckman ⁇ counter. Note that all IP 3 , measurements were performed together with [C 2* ⁇ measurements, employing parallel aliquots of the same batches of cells processed concomitantly.
  • NIH-3T3 microsomal fractions prepared as described by Alderson et al., 1989, (Arch. Biochem. Biophvs. 272:162-174) were resuspended in a medium containing: 100 mM KC1; 10 mM NaCl; 10 mM Tris-HCl buffer; pH 8.4. Binding to the fractions (1 mg protein/ml) was measured in the same buffer, supplemented with 0.1 mM EGTA and 5 nM [ 3 H]IP 3 (44 Ci/mmol, Amersham) mixed with non-radioactive IP 3 in the 0-150 nM range. To measure unspecific binding, parallel samples were prepared with excess IP 3 (5 ⁇ M) added. After 10 min incubation at 4 ⁇ C, bound and free counts were separated by filtration through glass fiber filters (GF/C, Whatman) , which were washed twice with cold buffer.
  • GF/C glass fiber filters
  • Thymidine Incorporation Cells were seeded on 12-well Costax plates at a density of 5x10* cells per well in DMEM containing 10% FCS/2 mM glutamine and grown for three days. After washing with PBS, the confluent cell layers were starved for 24 hrs in DMEM containing 0.5% FCS/2 mM glutamine, stimulated with EGF for 18 hrs in DMEM containing 0.5% bovine serum albumin (BSA) , 2 mM glutamine, and pulsed for 4 hrs with 0.5 ⁇ Ci of [methyl- 3 H]thymidine (Amersham, U.K.).
  • BSA bovine serum albumin
  • radioactive thymidine was stopped on ice, the cells were washed twice with PBS, and soluble radioactivity was extracted with 0.5 ml of 10% trichloroacetic acid for 20 min on ice, followed by another wash with the same solution. Precipitates were then lysed in 0.2 ml 0.2 N NaOH/0.2% SDS and later neutralized with 0.2 N HC1. The incorporated radioactivity was quantitated by scintillation counting.
  • MTT stock solution 2.5 mg/ml in PBS, filtered
  • PBS PBS
  • filtered PBS
  • plates were incubated for another 2 hrs at 37 ⁇ C.
  • Cells were subsequently lysed and the dye-crystals solubilized in acid/isopropanol (0.04 N HCI in isopropanol) .
  • the plates were read within one hour on a Dynatech MR5000 Microelisa Reader, using a test wavelength of 570 nm and a reference wavelength of 690 nm.
  • nitrocellulose filters were stripped with stripping buffer (100 mM 0-mercaptoethanol, 62.5 mM Tris- (hydroxymethyl)aminoethane, 2% SDS) for 30 min at 50 ⁇ C, and reprobed with the anti-EGF-R Ab RK2 (Kris et al . , 1985, Cell 4 ⁇ :619-625) as an EGF-R control.
  • the second antibody was a peroxidase-coupled goat anti- rabbit Ab followed by the ECL substrate reaction.
  • Figures 1A and IB illustrates the data obtained with isolated 3T3 clones transfected with expression plasmids under cytomegalovirus promoter control, either alone (3T3/T10) or containing; rat type I IP 3 R cDNA, both wild type (wt) and deletion mutants, s-IP 3 R and ⁇ -IP 3 R. Two independently isolated clones for each receptor construct were used in the transfections.
  • Figure 1A shows immunoprecipitation results of lysates obtained from cells prelabeled with [ 3S S] methionine for 16 hours * .
  • ⁇ -IP 3 R clone In one ⁇ -IP 3 R clone, ⁇ -IP 3 R5, shown in Figure IB and in the other clone investigated, ⁇ -IP 3 R6 the truncated forms accounted for 30 and 21.7% of the wt values, respectively; in the sIP 3 R clone shown in IB, and in two other sIP 3 R clones, the corresponding values ranged from 15 to 23%.
  • Two additional proteins of intracellular Ca 2* stores were investigated by Western blotting, the intracellular Ca2+ ATPases, the SERCAs, and calreticulin (CR) .
  • FIG. 2B compares the ATP concentration-dependence curves of controls and ⁇ -IP 3 R clones with and without activation of an autocrine loop sustained by infection of TGF ⁇ . In the control clones expressing the growth factor the curve was significantly shifted to the left, whereas that of the ⁇ -IP 3 R clones was not.
  • the intracellular Ca 2 * release responses induced by ATP are mediated by IP 3 .
  • Receptor-induced generation of the second messenger, IP 3 was investigated.
  • Figure 2C shows the ATP concentration dependence of IP 3 accumulation estimated at the peak, i.e., 20 sec after addition of the stimulant.
  • IP 3 generation induced by AIF 4 " was not decreased but was moderately increased in the ⁇ -IP 3 R5 clone.
  • both the ⁇ - IP 3 R5 and the ⁇ -IP 3 R6 clones exhibited levels of phospholipase C, of both the ⁇ and ⁇ types (presumably activated by the ATP and the EGF receptor, respectively) , not much different from those of the controls (see Table I) .
  • the large discrepancy between labeling and Western blot results reveals a considerable acceleration of the IP 3 R turnover in the transfected cells.
  • the transfected cells are able to activate a sort of a defense program by which the overall IP 3 R level is kept near the controls.
  • the physiological role of the IP 3 R is to mediate release of Ca 2 * from specific, rapidly exchanging Ca 2* stores.
  • the wt-overexpressing and s form-expressing subunits did not induced any appreciable changes of the Ca 2* release activity.
  • the s form at least two effects of the ⁇ form expression were visible.
  • the cells of the ⁇ clones generated less IP 3 when exposed to ATP and secondly, their IP 3 Rs were found to be distinctly more sensitive to IP 3 , in both intact and permeabilized cells. These two changes tend to compensate for each other in terms of Ca 2 * release, and the concentration dependence of the [Ca 2 * ⁇ responses induced by ATP in intact cells of the ⁇ clones was only moderately shifted to the right with respect to the controls.
  • the increased IP 3 R sensitivity of the ⁇ clone cells may be a direct consequence of the ⁇ subunit expression. Based on the expression data in the analyzed clones, and assuming the ⁇ to resemble the wt subunits in their assembly properties, a 1/3 combination of the two subunits in the receptor tetramers might be frequently expected. Since the IP 3 binding experiments failed to reveal any significant differences between ⁇ and control clones, the increased sensitivity appears to concern not binding but activation of IP 3 Rs. The latter process is still debated (see Meyer et al.. Science 240:653-656 1988; Finch et al. , Science 252:443-446. 1991; Berridge, Nature 361:315-321. 1993) .
  • the ⁇ subunit may be permanently activated, independently of IP 3 binding, thus explaining the increased sensitivity observed.
  • the results suggest it to be due to reduced expression (down regulation) of the surface ATP receptors (of the P 2u type) and not to a defect of the G protein function or PLC expression.
  • the drop of the cellular phosphatidylinositol 4,5-bisphosphate concentration may be another component by which the cell defends itself from excessive sensitivity of IP 3 R to its ligand.
  • 3T3 cell induced to transform by overexpression of growth factor receptor tyrosine kinases is induced to transform by overexpression of growth factor receptor tyrosine kinases.
  • Oncogene 1:1213-1221 the ability of the various clones to form colonies in soft agar was compared.
  • Controls for each of the cell clones infected 0 with a single virus containing either HERc or TGF ⁇ alone did not form colonies in soft agar, while the control 3T3/T10 cells were efficiently transformed by coinfection with NTK-HERc and *2-TGF ⁇ viruses and formed approximately 300 colonies per 10 s infectious S units of the EGF-R virus.
  • IP 3 R 3T3 cells reproducible and consistent changes of the EGF-R/TGF ⁇ transforming potency were observed.
  • the transfected wt IP 3 R enhanced this potency by about 40% and the s- IP 3 R reduced it significantly by about 35% in 0 comparison to control cells.
  • transformation was suppressed to a level of approximately 10%, see Table II.
  • Table II shows colony formation in soft agar.
  • Ligand binding to the EGF-R initiates dimerization and activation of its intrinsic tyrosine 0 kinase activity, in turn rapidly phosphorylating carboxy-terminal tyrosine residues of the receptor itself and tyrosine residues of substrates downstream of the receptor (Downward et al . , 1984, Nature 311:483-485: Margolis et al . , 1989, J. Biol. Chem. s 211:10667-10671; Canals, 1992, Biochemistry 31:4493- 4501) .
  • IP 3 Rs that include ⁇ subunits appear therefore to modify profoundly the fibroblasts and to make them incompetent for tumoral growth, even under conditions in which the receptors and the ligands that in the controls induce the response are certainly operative, and the [Ca 2 * ⁇ responses are largely maintained.
  • the results reveal that expression in intact cells of mutated IP 3 Rs induces a complex array of signal transduction changes developed by the cell to defend its molecular and functional identity. These changes appear to modify profoundly a basic function of the cell, its growth. Unexpectedly, the processes leading to cell transformation and oncogenesis were particularly affected, especially in the ⁇ clones, revealing a differential dependence of normal and cancer cells on Ca 2 * and IP 3 second messenger systems. These results identify the IP 3 Rs and the intracellular Ca 2 * stores as important crossroads of signalling pathways involved in growth regulation.
  • AIFI 4 "-induced IP 3 generation and phospholipase ⁇ and ⁇ expression in 3T3 fibroblasts expressing endogenous and mutated, ⁇ type IP 3 receptors.
  • Values shown are averages of the results obtained in 2-4 experiments, expressed as % of the control clone 3T3/T10-HERC.
  • CAC CAG TTC CTA CAG AAG TTC TGT GCC GGG AAC CCT GGC AAC CAG GCC 3866 His Gin Phe Leu Gin Lys Phe Cys Ala Gly Asn Pro Gly Asn Gin Ala 1230 1235 1240
  • AGC GCT GCC AAC TAC AAG ACG GCC ACG AGG ACC TTC CCT CGG GTC ATC 4874 Ser Ala Ala Asn Tyr Lys Thr Ala Thr Arg Thr Phe Pro Arg Val He 1565 1S70 1575 1580
  • GAG GAA GAC CCG CTG GCC TAC TAT GAG AAC CAC ACC TCG CAG ATT GAG 650 Glu Glu Asp Pro Leu Ala Tyr Tyr Glu Asn His Thr Ser Gin He Glu 2110 2115 2120

Abstract

L'invention concerne l'utilisation de protéines, de peptides et de molécules organiques capables de moduler la transduction du signal du récepteur de 1,4,5-triphosphate d'inositol (IP3), de manière à inhiber ou inverser la croissance inopportune de cellules associée aux anomalies de transduction de signal en rapport avec les tyrosines kinases. L'invention concerne également l'utilisation de mutants du récepteur d'IP3 dans le traitement de troubles prolifératifs associés aux anomalies de transduction de signal en rapport avec les tyrosines kinases, notamment le cancer. L'invention concerne encore l'utilisation du récepteur d'IP3 ainsi que des cellules hôtes obtenues par génie génétique qui expriment le récepteur d'IP3, afin d'évaluer et de cribler les substances et composés modulant les activités du récepteur d'IP3.
PCT/EP1995/002532 1994-06-30 1995-06-29 Methodes de traitement d'affections a proliferation cellulaire par modulation de la transduction du signal WO1996000586A2 (fr)

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