WO1999054450A1 - Assay methods for modulators of hematopoietic cell phosphatase - Google Patents
Assay methods for modulators of hematopoietic cell phosphatase Download PDFInfo
- Publication number
- WO1999054450A1 WO1999054450A1 PCT/US1999/008228 US9908228W WO9954450A1 WO 1999054450 A1 WO1999054450 A1 WO 1999054450A1 US 9908228 W US9908228 W US 9908228W WO 9954450 A1 WO9954450 A1 WO 9954450A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- hcp
- protein
- activity
- modulator
- expression
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2799/00—Uses of viruses
- C12N2799/02—Uses of viruses as vector
- C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
- C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus
Definitions
- JAK2 has been shown to be associated with several growth factor receptors and involved in the JAK-STAT signal transduction pathway (Argetsinger et al., 1993, Darnell et al, 1994).
- Hematopoietic cell phosphatase (also referred to as: HCP, SH-PTP1, PTP1C, SHP1, PTPN6) is a 68 kDa. non-membrane associated protein, found primarily in hematopoietic cells and to some extent in epithelial cells (Zhao et al., 1993, Pei et al,
- HCP has been localized to human chromosome 12pl2-pl3; a region associated with chromosomal rearrangements in approximately 10% of childhood acute lymphocytic leukemia cases (Yi et al, 1992).
- the phosphatase contains two src homology 2 (SH2) domains at its amino terminus and a phosphatase domain at the C-terminal portion of the molecule.
- HCP has been shown to associate with several growth factor receptors following ligand stimulation including c-kit, IL-3 and EPO (Yi and Ihle, 1993, Yi et al., 1993, Yi et al. 1992) receptors and acts as a negative regulator of receptor activation.
- - 2 src homology 2
- HCP binds to the receptor through its SH2 domain (Yi and Ihle, 1993, Yi et al., 1993, Pei et al., 1994, Yi et al., 1995, Klingmiiller et al, 1995).
- Mutagenesis of the HCP N-terminal SH2 domain at the conserved SH2 binding site (FLVRE) eliminated binding to both the IL-3 receptor ⁇ and the EPOR.
- Antisense experiments that decrease the HCP content of IL-3 responsive cells renders those cells hypersensitive to IL-3 while over expression of HCP in IL-3 dependent cells suppresses the ability of those cells to proliferate in response to IL-3.
- the fact that HCP binds to the IL-3 receptor ⁇ suggests that HCP may also interact with IL-5 and GM-CSF since all three share a common ⁇ chain.
- HCP may also interact with IL-5 and GM-CSF since all three share a common ⁇ chain.
- HCP regulates the receptors for IL-5 or GM-CSF. (Yi et al., 1993). These data support the role of HCP as a negative regulator of hematopoietic signal transduction.
- HCP regulation of EPO receptor activity have centered upon identifying the specific region of the cytoplasmic domain that binds HCP.
- the EPOR contains 8 tyrosine residues within the C-terminal 70 amino acids. The identification of the tyrosine residues of the EPOR involved with HCP binding was first determined using short peptides containing phosphotyrosine residues of the EPOR (Yi et al., 1995). Four of the eight p-tyrosine residues were shown to interact with HCP. One of these tyrosine residues, Y455, appears to exhibit increased HCP binding relative to the other positions.
- HCP by anti-sense oligonucleotides enhanced hemoglobinization of SKT6 cells (Sharlow et al., 1997) demonstrating that HCP negatively regulates erythroid differentiation. 3 -
- HCP lacking the SH2 domains may have a higher affinity for substrates as compared to the intact molecule (Townley et al., 1993).
- HCP may be in an inactive state until the SH2 domain binds to a phosphorylated receptor, essentially eliminating the inhibition and activating the phosphatase.
- HCP has not been shown to be associated with a receptor substrate prior to ligand binding and tyrosine phosphorylation of the receptor substrate is required for HCP binding. This scenario may exist for other hematopoietic receptors, although it has only been demonstrated for the EPOR.
- mice that are genetically deficient for HCP exhibit the motheaten (me) or motheaten viable (mev) phenotype. These mice lack HCP (me) or have slight HCP activity
- mice resulting in severe immunodeficiency and death within 3 to 9 weeks of age
- Hematopoietic changes in these mice include the abnormal growth of macrophages that accumulate in the lungs of these animals.
- erythropoiesis shifts from the bone marrow to the spleen with an increase of erythroid precursors (CFU-E) found in the spleen (Van Zant and Schultz, 1989).
- CFU-Es from the motheaten mice demonstrate an increased sensitivity to EPO and may be capable of colony formation in the absence of EPO (Schultz et al., 1993).
- the HCP deficiency is the result of a single cytidine deletion that generates a splice donor site in the N-terminal SH2 domain.
- mice have impaired T and B cells exhibiting severe B cell deficiency and impaired NK cell function. Surprisingly, these mice also have a high degree of autoantibody production (Schultz et al., 1993), possibly due to HCP involvement in antigen receptor signalling and tolerance thresholds. HCP is believed to be a negative regulator of immunoglobulin antigen receptor signalling and determines the threshold of negative selection for these cells (Cyster and Goodnow, 1995).
- the motheaten mouse model exemplifies the overall involvement of HCP in hematopoiesis and the negative role that it plays in hematopoietic cell signal transduction.
- HCP human hematopoietic cell phosphatase
- SH2-containing protein tyrosine phosphatases tyrosine phosphatases
- FIG. 1 HCP Catalytic Assay: Reduction of HCP enzymatic activity by Vanadate is shown
- Figure 2- The ability of an interactive peptide to enhance the enzymatic activity of HCP, and the ability of vanadate to inhibit the activity of enhanced HCP activity is shown.
- FIG. 3- HCP Interactive Assay: Reduction of enzymatic activity, by the modulator vanadate, in the presence of a peptide known to interact with HCP is shown.
- GST-HCP protein is shown in Panel B; the nucleotide sequence of the catalytic domain of GST-HCP fusion gene is shown in Panel C; and the amino acid sequence of the catalytic domain of GST-HCP protein is shown in Panel D.
- the present invention relates to DNA encoding human HCP that was isolated from hematopoietic cells.
- HCP refers to protein that can specifically function as a hematopoietic cell phosphatase.
- the purpose of the invention is to utilize HCP phosphatase domain, which retains its enzymatic activity, in an assay to screen compounds that specifically inhibit the protein's enzymatic activity.
- full length HCP is utilized in an assay to screen for compounds that inhibit the HCP phosphatase activity by blocking the interaction of
- HCP with the cytoplasmic domain of the erythropoietin receptor EPOR.
- EPO erythropoietin receptor
- HCP has been known to interact with other growth factor receptors including, but not limited to IL-3, IL-5, GM-CSF and c-kit (Yi and Ihle, 1993, Yi, et al., 1993), these receptors may also be affected by HCP inhibitors.
- Vertebrate cells capable of producing HCP include, but are not limited to hematopoietic cells isolated from mammals, that show HCP activity. Other cells and cell lines may also be suitable for use to isolate HCP cDNA. Cells that possess HCP expression may be suitable for o the isolation of HCP DNA or mRNA.
- HCP DNA Any of a variety of procedures known in the art may be used to molecularly clone HCP DNA. These methods include, but are not limited to, direct functional expression of the HCP genes following the construction of a HCP containing cDNA library in an appropriate expression vector system. Another method is to screen HCP- 5 containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a labelled oligonucleotide probe designed from the amino acid sequence of the HCP subunits. An additional method consists of screening a HCP-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA encoding the HCP protein. This partial cDNA is obtained by the specific PCR amplification of o HCP DNA fragments through the design of degenerate oligonucleotide primers from the amino acid sequence of the purified HCP protein.
- Another method is to isolate RNA from HCP producing cells and translate the RNA into protein via an in vitro or an in vivo translation system.
- the translation of the RNA into a peptide a protein will result in the production of at least a portion of the 5 HCP protein which can be identified by, for example, immunological reactivity with an anti-HCP antibody or by biological activity of HCP protein.
- pools of RNA isolated from HCP-producing cells can be analyzed for the presence of an RNA that encodes at least a portion of the HCP protein. Further fractionation of the RNA pool can be done to purify the HCP RNA from non-HCP RNA.
- the peptide or protein produced by this method may be analyzed to provide amino acid sequences, which in turn are used to provide primers for production of HCP cDNA, or the RNA used for translation can be analyzed to provide nucleotide sequences encoding HCP and produce probes for the production of HCP cDNA.
- This method is known in the art and can be found in, for example, Maniatis, T., Fritsch, E.F., Sambrook, J. in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 1989.
- HCP-encoding DNA Other types of libraries include, but are not limited to, cDNA libraries derived from other cells, from organisms other than humans, and genomic DNA libraries that include YAC (yeast artificial chromosome) and cosmid libraries.
- cDNA libraries derived from other cells from organisms other than humans
- genomic DNA libraries that include YAC (yeast artificial chromosome) and cosmid libraries.
- cDNA libraries may be prepared from cells or cell lines which have HCP activity.
- the selection of cells or cell lines for use in preparing a cDNA library to isolate HCP cDNA may be done by first measuring cell associated HCP activity.
- DNA encoding HCP may also be isolated from a suitable genomic DNA library. Construction of genomic DNA libraries can be performed by standard techniques well known in the art. Well known genomic DNA library construction techniques can be found in Maniatis, T., Fritsch, E.F., Sambrook, J. in Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989).
- the amino acid sequence of HCP may be necessary. To accomplish this, HCP protein may be purified and partial amino acid sequence determined by automated sequencers. It is not necessary to determine the entire amino acid sequence, but the linear sequence of two regions of 6 to 8 amino acids from the protein is determined for the production of primers for
- the DNA sequences capable of encoding them are synthesized. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and therefore, the amino acid sequence can be encoded by any of a set of similar DNA oligonucleotides.
- DNA isolated by these methods can be used to screen DNA libraries from a variety of cell types, from invertebrate and vertebrate sources, and to isolate homologous genes.
- the cloned HCP DNA obtained through the methods described herein may be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant HCP protein. Techniques for such manipulations are fully described in Maniatis, T, et al., supra, and are well known in the art.
- Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria including E. coli, blue-green algae, plant cells, insect cells, fungal cells including yeast cells, and animal cells. 9 -
- Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells or bacteria-fungal cells or bacteria- invertebrate cells.
- An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters.
- a promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis.
- a strong promoter is one that causes mRNAs to be initiated at high frequency.
- Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
- mammalian expression vectors may be used to express recombinant HCP in mammalian cells.
- Commercially available mammalian expression vectors which may be suitable for recombinant HCP expression, include but are not limited to, pGZT-4T-l (Pharmacia), pMAMneo (Clontech), pcDNA3 (InVitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO- pSV2-neo (ATCC 37593) pBPV-1 (8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and 1ZD35 (ATCC 37565).
- bacterial expression vectors may be used to express recombinant HCP in bacterial cells.
- Commercially available bacterial expression vectors which may be suitable for recombinant HCP expression include, but are not limited to pET vectors (Novagen) and pQE vectors (Qiagen).
- a variety of fungal cell expression vectors may be used to express recombinant HCP in fungal cells such as yeast.
- Commercially available fungal cell expression vectors which may be suitable for recombinant HCP expression include but are not limited to pYES2 (InVitrogen) and Pichia expression vector (InVitrogen).
- insect cell expression vectors may be used to express recombinant HCP in insect cells.
- Commercially available insect cell expression vectors that may be 10
- HCP suitable for recombinant expression of HCP include but are not limited to pBlueBacII (InVitrogen).
- DNA encoding HCP may be cloned into an expression vector for expression in a recombinant host cell.
- Recombinant host cells may be prokaryotic or eukaryotic, 5 including but not limited to bacteria such as E. coh, fungal cells such as yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to drosophila and silkworm derived cell lines.
- Cell lines derived from mammalian species that may be suitable and that are commercially available, include but are not limited to, CV-1 o (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1
- the expression vector may be introduced into host cells via any one of a 5 number of techniques including but not limited to transformation, transfection, protoplast fusion, lipofection, and electroporation.
- the expression vector-containing cells are clonally propagated and individually analyzed to determine whether they produce HCP protein. Identification of HCP expressing host cell clones may be done by several means, including but not limited to immunological reactivity with anti-HCP o antibodies, and the presence of host cell-associated HCP activity.
- HCP DNA may also be performed using in vitro produced synthetic mRNA.
- Synthetic mRNA or mRNA isolated from HCP producing cells can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based 5 systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocytes being generally preferred.
- HCP DNA molecules including, but not limited to, the 11
- HCP DNA construct is transferred to a variety of expression vectors, for expression in host cells including, but not limited to, mammalian cells, baculovirus-infected insect cells, K coli, and the yeast S. cerevisiae.
- Host cell transfectants and microinjected oocytes may be used to assay both the levels of HCP activity and levels of HCP protein by the following methods. In the case of recombinant host cells, this involves the co-transfection of one or possibly two or more plasmids, containing the HCP DNA encoding one or more fragments or subunits. In the case of oocytes, this involves the co-injection of synthetic RNAs for
- HCP protein Following an appropriate period of time to allow for expression, cellular protein is metabolically labelled with, for example -"S-methionine for 24 hours, after which cell lysates and cell culture supernatants are harvested and subjected to immunoprecipitation with polyclonal antibodies directed against the HCP protein. Levels of HCP protein in host cells are quantitated by immuno affinity and/or ligand affinity techniques. Cells expressing HCP can be assayed for the number of HCP molecules expressed by measuring the amount of radioactive ligand and/or substrate binding. HCP-specific affinity beads or HCP-specific antibodies are used to isolate for example - ⁇ S-methionine labelled or unlabelled HCP protein. Labelled HCP protein is analyzed by SDS-PAGE. Unlabelled HCP protein is detected by
- DNA sequences coding for a peptide may be altered so as to code for a peptide having properties that are different than those of the natural peptide.
- Methods of altering the DNA sequences include, but are not limited to site directed mutagenesis.
- altered properties include but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand.
- a “functional derivative” of HCP is a compound that possesses a biological activity (either functional or structural) that is substantially similar to the biological activity of HCP.
- the term “functional derivatives” is intended to include the “fragments,” “variants,” “degenerate variants,” “analogs” and “homologues” or to “chemical derivatives” of HCP.
- fragment is meant to refer to any polypeptide subset of HCP.
- variant is meant to refer to a molecule substantially similar in structure and function to either the entire HCP molecule or to a fragment thereof.
- a molecule is "substantially similar” to HCP if both molecules have substantially similar structures or if both molecules possess similar biological activity.
- analog refers to a molecule substantially similar in function to either the entire HCP molecule or to a fragment thereof.
- HCP protein may be recovered to provide HCP in active form.
- HCP purification procedures are available and suitable for use. As described above for purification of HCP from natural sources, recombinant HCP may be purified from cell lysates and extracts, or from conditioned culture medium, by various combinations of, or individual application of salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography and hydrophobic interaction chromatography.
- recombinant HCP can be separated from other cellular proteins by use of an immunoaffmity column made with monoclonal or polyclonal antibodies specific for full length nascent HCP, polypeptide fragments of HCP or HCP subunits. 13
- Monospecific antibodies to HCP are purified from mammalian antisera containing antibodies reactive against HCP or are prepared as monoclonal antibodies reactive with HCP using the technique of Kohler and Milstein, Nature 256: 495-497 (1975). Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogenous binding characteristics for HCP.
- Homogenous binding refers to the ability of the antibody species to bind to a specific antigen or epitope, such as those associated with the HCP, as described above.
- HCP specific antibodies are raised by immunizing animals such as mice, rats, guinea pigs, rabbits, goats, horses and the like, with rabbits being preferred, with an appropriate concentration of HCP either with or without an immune adjuvant.
- HCP antibody affinity columns are made by adding the antibodies to Affigel- 10 (Bio-Rad), a gel support which is activated with N-hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support.
- the antibodies are then coupled to the gel via amide bonds with the spacer arm.
- the remaining activated esters are then quenched with 1M ethanolamine HC1 (pH 8).
- the column is washed with water followed by 0.23 M glycine HC1 (pH 2.6) to remove any non-conjugated antibody or extraneous protein.
- the column is then equilibrated in phosphate buffered saline (pH 7.3) and the cell culture supernatants or cell extracts containing HCP or HCP subunits are slowly passed through the column.
- the column is then washed with phosphate buffered saline until the optical density (A280) ⁇ s to background, then the protein is eluted using 0.23 M glycine-HCl (pH 2.6).
- the purified HCP protein is then dialyzed against phosphate buffered saline.
- HCP is found primarily in hematopoietic cells and has been shown to be a negative regulator of one or more signal transduction pathways in these cells.
- the identification of an HCP inhibitor would provide a synthetic stimulator to the hematopoietic system that could be used in conjunction with other cytokine or cytokine mimetic therapy; for example, the administration of 14
- erythropoietin Modulation of HCP provides the potential for multiple points of intervention with small molecule therapeutics. Screening assays can target the HCP phosphatase domain and/or the interaction of HCP with receptor complexes, and intracellular substrates of the phosphatase such as the Janus kinase 2 (JAK2) molecule. This multi-faceted approach should increase the probability of discovering an HCP inhibitor. Since HCP has been implicated in several hematopoietic cell-signaling pathways, a single inhibitor may provide a potent boost to multiple cell lineages.
- HCP histone deacetylase
- hematopoietic cells i.e., EPO, IL-3, c-kit, and potentially others
- an HCP inhibitor could enhance the overall effect of hematopoietic cell growth factors, thereby decreasing the necessary dose required for that exogenous factor or potentially eliminating the need for administering EPO by enhancing the activity of the endogenous growth factor.
- HCP cannot bind to the receptor and exert its negative effect.
- the clinical manifestations are minor. Patients have increased hemoglobin levels and higher hematocrits, and cultured erythroid progenitors from these individuals are more sensitive - 15
- the present invention is also directed to methods for screening for compounds that modulate the expression of DNA or RNA encoding HCP as well as the function of HCP protein in vivo.
- Compounds that modulate these activities may be DNA, RNA, peptides, proteins, or non-proteinaceous organic molecules.
- Compounds may modulate by increasing or attenuating the expression of DNA or RNA encoding HCP, or the function of HCP protein.
- Compounds that modulate the expression of DNA or RNA encoding HCP or the function of HCP protein may be detected by a variety of assays.
- the assay may be a simple "yes/no" assay to determine whether there is a change in expression or function.
- the assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression or function in a standard sample. Modulators identified in this process are useful as therapeutic agents.
- the present invention is directed to two methods to screen for compounds that modulate the activity of the HCP protein.
- the assays are named
- the HCP Catalytic assay may be used to detect compounds that modulate enzymatic activity associated with the phosphatase domain of the HCP protein.
- the HCP Interactive assay was developed to assay compounds that may modulate the enzymatic activity of the full-length molecule by modulating the carboxyl terminal catalytic domain or by modulating the amino terminal scaffolding domains important for receptor binding.
- the full-length protein assay is performed in the presence of a phosphotyrosine peptide denoted as the interactive peptide that unmasks the carboxyl terminal phosphatase domain.
- the interactive peptide is derived from the
- ITIM peptide motif found in the Fc and T cell receptors.
- Kits containing HCP DNA or RNA, antibodies to HCP, or HCP protein may be prepared. Such kits are used to detect DNA that hybridizes to HCP DNA or to detect 16
- HCP protein or peptide fragments in a sample. Such characterization is useful for a variety of purposes including but not limited to forensic analyses, diagnostic applications, and epidemiological studies.
- DNA molecules, RNA molecules, recombinant proteins and antibodies of the present invention may be used to screen and measure levels of HCP DNA, HCP
- kits suitable for the detection and typing of HCP Such a kit would comprise a compartmentalized carrier suitable to hold in close confinement at least one container.
- the carrier would further comprise reagents such as recombinant HCP proteins or anti-HCP antibodies suitable for detecting HCP.
- the carrier may also contain a means for detection such as labeled antigen or enzyme substrates or the like.
- Nucleotide sequences that are complementary to the HCP encoding DNA sequence can be synthesized for antisense therapy.
- These antisense molecules may be DNA, stable derivatives of DNA such as phosphorothioates or methylphosphonates,
- RNA stable derivatives of RNA such as 2'-O-alkylRNA, or other HCP antisense oligonucleotide mimetics.
- HCP antisense molecules may be introduced into cells by standard methods including, but not limited to, microinjection, liposome encapsulation or by expression from vectors harboring the antisense sequence. HCP antisense therapy may be particularly useful for the treatment of diseases where it is beneficial to reduce HCP activity.
- HCP gene therapy may be used to introduce HCP into the cells of target organisms.
- the HCP gene can be ligated into viral vectors that mediate transfer of the HCP DNA by infection of recipient host cells. Suitable viral vectors include retro virus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poliovirus and the like.
- HCP DNA can be transferred into cells for gene therapy by non- viral techniques including receptor-mediated targeted DNA transfer using ligand- DNA conjugates or adenovirus-ligand-DNA conjugates, lipofection membrane fusion - 17
- HCP gene therapy may be particularly useful for the treatment of diseases where it is beneficial to elevate HCP activity.
- compositions comprising HCP DNA, HCP RNA, or HCP protein, or modulators of HCP receptor activity, may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the protein, DNA, RNA, or modulator.
- Therapeutic or diagnostic compositions of the invention are administered to an individual in amounts sufficient to treat or diagnose disorders in which modulation of HCP -related activity is indicated.
- the effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration.
- the pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular.
- chemical derivative describes a molecule that contains additional chemical moieties that are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule.
- the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.
- compositions containing compounds or modulators identified according to this invention as the active ingredient for use in the 5 modulation of HCP can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration.
- the compounds or modulators can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection.
- they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
- An effective but non-toxic amount of the compound desired can be employed as a HCP modulating agent. 5
- the daily dosage of the products may be varied over a wide range from 0.01 to
- compositions are preferably provided in the form of scored or unscored tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, and 50.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
- An effective o amount of the drug is ordinarily supplied at a dosage level of from about 0.0001 mg/kg to about 100 mg/kg of body weight per day. The range is more particularly from about 0.001 mg/kg to 10 mg/kg of body weight per day.
- the dosages of the HCP modulators are adjusted when combined to achieve desired effects. On the other hand, dosages of these various agents may be independently optimized and combined 5 to achieve a synergistic result wherein the pathology is reduced more than it would be if either agent were used alone.
- compounds or modulators of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in - 19 -
- compounds or modulators for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
- the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
- the active agents can be administered concurrently, or they each can be administered at separately staggered times.
- the dosage regimen utilizing the compounds or modulators of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound thereof employed.
- a physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
- Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.
- the compounds or modulators herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier” materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.
- carrier suitable pharmaceutical diluents, excipients or carriers
- the active drug component can be combined with an oral, non-toxic pharmaceutically 20
- suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like.
- Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
- Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.
- the active drug component can be combined in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like.
- suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like.
- Other dispersing agents include glycerin and the like.
- sterile suspensions and solutions are desired.
- Isotonic preparations which generally contain suitable preservatives, are employed when intravenous administration is desired.
- Topical preparations containing the active drug component can be admixed with a variety of carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations.
- carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations.
- the compounds or modulators of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
- Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
- Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are 21 -
- the compounds or modulators of the present invention may also be coupled with soluble polymers as targetable drug carriers.
- soluble polymers can include polyvinyl-pyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-amidephenol, polyhydroxy-ethylaspartamidephenol, or polyethyl-eneoxidepolylysine substituted with palmitoyl residues.
- the compounds or modulators of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
- a drug for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
- the compounds or modulators may be administered in capsule, tablet, or bolus form or alternatively they can be mixed in the animals feed.
- the capsules, tablets, and boluses are comprised of the active ingredient in combination with an appropriate carrier vehicle such as starch, talc, magnesium stearate, or
- unit dosage forms are prepared by intimately mixing the active ingredient with suitable finely-powdered inert ingredients including diluents, fillers, disintegrating agents, and/or binders such that a uniform mixture is obtained.
- An inert ingredient is one that will not react with the compounds or modulators and which is non-toxic to the animal being treated. Suitable inert ingredients include starch, lactose, talc, magnesium stearate, vegetable gums and oils, and the like.
- These formulations may contain a widely variable amount of the active and inactive ingredients depending on numerous factors such as the size and type of the animal species to be treated and the type and severity of the infection.
- the active ingredient may also be administered as an additive to the feed by simply mixing the compound with the feedstuff or by applying the compound to the surface of the feed.
- the active ingredient may be mixed with an inert carrier and the resulting composition may then either be mixed with the feed or fed directly to the animal.
- Suitable inert carriers include corn meal, citrus meal, fermentation residues, soya grits, dried 22
- the active ingredients are intimately mixed with these inert carriers by grinding, stirring, milling, or tumbling such that the final composition contains from 0.001 to 5% by weight of the active ingredient.
- the compounds or modulators may alternatively be administered 5 parenterally via injection of a formulation consisting of the active ingredient dissolved in an inert liquid carrier. Injection may be either intramuscular, intraruminal, intratracheal, or subcutaneous.
- the mjectable formulation consists of the active ingredient mixed with an appropriate inert liquid carrier.
- Acceptable liquid carriers include the vegetable oils such as peanut oil, cottonseed oil, sesame o oil and the like as well as organic solvents such as solketal, glycerol formal and the like.
- aqueous parenteral formulations may also be used.
- the vegetable oils are the preferred liquid carriers.
- the formulations are prepared by dissolving or suspending the active ingredient in the liquid carrier such that the final formulation contains from 0.005 to 10% by weight of the active ingredient.
- Topical application of the compounds or modulators is possible through the use of a liquid drench or a shampoo containing the instant compounds or modulators as an aqueous solution or suspension.
- These formulations generally contain a suspending agent such as bentonite and normally will also contain an antifoaming agent.
- Formulations containing from 0.005 to 10% by weight of the o active ingredient are acceptable.
- Preferred formulations are those containing from
- Recombinant human HCP was produced in E. coli following the transfer of the HCP expression cassette into R, coli expression vectors, including but not limited to, - 23
- the pET vectors place HCP expression under control of the tightly regulated bacteriophage T7 promoter. Following transfer of this construct into an R coli host that contains a chromosomal copy of the T7 RNA polymerase gene driven by the inducible lac promoter, expression of HCP is induced when an appropriate lac substrate (IPTG) is added to the culture. The levels of expressed HCP are determined by the assays described herein.
- the cDNA encoding the entire open reading frame for HCP is inserted into the EcoRI-NotI sites of pET28a. Constructs are identified by sequence analysis and used to transform the expression host strain BL21pLys. Transformants are then used to inoculate cultures for the production of HCP protein. Cultures may be grown in M9 or
- RNA from Jurkat cells was purified from total cellular RNA using a Dynal mRNA kit.
- RT-PCR was performed to obtain full-length human HCP using 5'- primer which consists of a sequence recognized by EcoRI and the sequence of the first 8 amino acids of HCP catalytic domain, and 3 '-primer which consists of the DNA sequence of the last 9 amino acids of HCP and a sequence recognized by Notl.
- the full length HCP catalytic domain PCR fragment was then cloned into EcoRI and Notl sites of pGEX-4T-l vector.
- the HCP-GST fusion gene was transformed into BL21 cells for fusion protein expression. 10 ml of overnight culture of transformed BL21 cells were inoculated into 1 liter of LB medium in the presence of 100 ug/ml of ampicillin. The culture was incubated at 37°C with shaking until the OD 6 oo reached between 0.6 to 0.9. The cells were induced for HCP-GST expression by adding IPTG to final concentration of 1 mM and incubated - 24 -
- lysozyme 100 mg
- the cell suspension was then briefly sonicated and the cell lysate was incubated at room temperature with slow rocking for about 45 min.
- Triton X- 100 was then added to final concentration of 0.1% and the lysate was incubated at room temperature with slow rocking for another 45 min.
- the lysate was then spun at 14,000 ⁇ m for 10 min to remove cell debris. The supernatant was saved for further HCP-GST fusion protein purification.
- Primer pair sequences used to clone the catalytic domain of human HCP SEQ.ID.NO.4: 5 '-primer: GTCAGAATTCGGCGCCTTTGTCTACCTGCGGCAG SEQ.ID.NO.l : 3'- ⁇ rimer: GTCAGCGGCCGCTCACTTCCTCTTGAGGGAACCCTTGCTC
- RNA from Jurkat cells was purified from total cellular RNA using a Dynal mRNA kit.
- RT-PCR was performed to obtain full-length human HCP using 5'- primer which consists of a sequence recognized by EcoRI and the sequence of the first 8 amino acids of HCP, and 3 '-primer which consists of the DNA sequence of the last 9 amino acids of HCP and a sequence recognized by Notl.
- the full length HCP PCR fragment was then cloned into EcoRI and Notl sites of pGEX-4T-l vector.
- the HCP-GST fusion gene was transformed into BL21 cells for fusion protein expression. 10 ml of overnight culture of transformed BL21 cells were inoculated into 1 liter of LB medium in the presence of 100 ug/ml of ampicillin. The culture was incubated at 37°C with shaking until the OD 60 o reached between 0.6 to 0.9. The cells were induced for HCP-GST expression by adding IPTG to final concentration of 1 mM and incubated with shaking at 33°C. The cells were then harvested after 4 hours of culture and stored at - 20°C for later purification.
- glutathione Sepharose 4B beads Five milliliters of glutathione Sepharose 4B beads (Pharmacia) were first washed with 50 mM Tris-HCl pH 7.4 thoroughly to remove ethanol in storing solution. The beads were then mixed with the cell lysate supernatant at 4°C with slow rocking overnight. The beads were then washed with 50 mM Tris-HCl pH 7.4 and incubated with 5 ml of 10 mM reduced Glutathione dissolved in 50 mM Tris-HCl pH 8.0 at room temperature for ten minutes to elute the HCP-GST fusion protein. The beads were pelleted and the 26 -
- SEQ.ID.NO.2 5'-primer: ACGTGAATTCATGGTGAGGTGGTTTCACCGAGAC
- SEQ.ID.NO.3 3'-primer: GTCAGCGGCCGCTCACTTCCTCTTGAGGGAACCCTTGCTC
- the GST-HCP phosphatase domain fusion protein (rhuHCPcat) was resuspended in assay buffer (50 mM imidazole, 1 mM EDTA, 45 mM BME, 0.1% ovalbumin, pH 6.8).
- the amount of rhuHCPcat used in the assay was determined upon its enzymatic activity required to produce a final readout of approximately 2.0 at OD 650 .
- Compounds at a concentration of approximately 1 mM in 100% DMSO were diluted 1 :40 in assay buffer for determination of enzyme inhibition activity at a final compound concentration of approximately 25 ⁇ M in 2.5% DMSO.
- fifty-eight microliters of rhuHCPcat enzyme in assay buffer are mixed with 2 ⁇ l of the ImM test compound in 100% DMSO and incubated at room temperature for 30 minutes. Then 20 ⁇ l of 0.8 mM phosphorylated
- JAK2 peptide substrate (diluted in the assay buffer) was added to the mixture to yield a final concentration of 200 ⁇ M in the test well. The reaction was incubated for an additional 60 minutes. The reaction was then terminated by the addition of 70 ⁇ l of malachite green solution containing ammonium molybdate (Fisher and Higgins. Pharm Res. 11(5), 1994). Following a final 30-minute incubation, the optical density was read at
- JAK2 peptide sequence SEQ.ID.NO.7: H-Val-Leu-Pro-Gln-Asp-Lys-Glu-(phospho)Tyr-(phospho)Tyr-Lys-Val-
- the enzyme working solution was diluted in assay buffer as described for the catalytic assay to yield an optical density (OD) at 650 nM wavelength of approximately 2.0 in the presence of 50 ⁇ M interactive peptide and 200 ⁇ M substrate peptide.
- OD optical density
- a sample assay is as follows; 38.5 ⁇ l of diluted rhuHCP and 1.5 ⁇ l of a 1 mM compound solution was mixed and incubated together for 20 minutes at room temperature. Ten microliters of a 300 ⁇ M ITIM peptide solution was added, mixed and incubation continued for an additional 20 minutes. This was followed by the addition of 10 ⁇ l of the
- the assay was developed by the addition of 100 ⁇ l of malachite green solution as described above for the catalytic assay. As shown in Figure 2, the enzyme is more active in the presence of the interactive peptide. The activity of the enzyme in the presence of the substrate peptide yields activity equal to that of enzyme and buffer alone. The activity is inhibited approximately 3 fold by the addition of sodium orthovanadate.
- ITIM peptide sequence (peptide 1 or PI in Figure 2): 28 -
- SEQ.ID.NO.5 Ac-Glu-Ala-Glu-Asn-Thr-Ile-Thr-Ile-Thr-phosphoTyr-Ser-Leu-Leu-Lys- His-OH
- pTyr peptide sequence (S or substrate peptide in figure 2): SEQ.ID.NO.6: H-Asp-Ala-Asp-Glu-phosphoTyr-Leu-Ile-Pro-Gln-Gln-Gly-OH
- the full length GST-HCP fusion protein (rhuHCP) was resuspended in assay buffer (50 mM Imidazole pH 7.4, 10 mM 2-ME, 1 mM EDTA, and 0.1% ovalbumin). The amount of rhuHCP used in the assay was determined upon its enzymatic activity required to produce a final readout of approximately 2.0 to 3.0 at OD 65 o based on the conditions established in Example 5. 300 ⁇ M interactive peptide (ITIM) working solution was prepared by dilution in assay buffer from a 24 mM ITIM stock solution in DMSO.
- ITIM interactive peptide
- 1200 ⁇ M phosphotyrosine (pTyr) peptide substrate working solution was prepared by dilution in assay buffer from a 24 mM peptide stock solution in dH 2 O.
- the malachite green solution is prepared by mixing 3 ml of malachite green working stock with 7 ml of dH O.
- the malachite green sock consists of 2.6 mM malachite green in 7.2N of H 2 SO 4 , 7.5% ammonium molybdate and 11% Tween-20 mixed right before use in 10/2.5/0.2 ratio by volume.
- the first step is conducted by mixing 38.5 ⁇ l of diluted rhuHCP enzyme and 1.5 ⁇ l of lmM screening compound in DMSO, and incubating for 20 minutes at room temperature. Then 10 ⁇ l of the ITIM peptide working solution, is added to the reaction, mixed, and incubated at room temperature for 20 min. Then, 10 ⁇ l of pTyr peptide working solution is added to the reaction, mixed, and incubated at room temperature for
- the HCP cDNAs are cloned into the mammalian expression vector pcDNA3.
- the HCP ⁇ GEX-4T-l plasmid is digested with EcoRI and Notl.
- the inserts are purified by agarose gel electrophoresis.
- the pcDNA3 vector is digested with EcoRI and Notl.
- the linear vector can be purified on agarose gel and used to ligate to the HCP cDNA inserts.
- Recombinants are isolated, designated HCP, and used to transfect mammalian cells (L-cells) by CaPOzi.-DNA precipitation.
- Stable cell clones are selected by growth in the presence of G418. Single G418 resistant clones can be isolated and shown to contain the intact HCP gene.
- Clones containing the HCP cDNAs are analyzed for expression using immunological techniques, such as immuneprecipitation, Western blot, and immuno fluorescence using antibodies specific to the HCP proteins.
- Antibody is obtained from rabbits innoculated with peptides that are synthesized from the amino acid sequence predicted from the HCP sequences.
- HCP HCP-phosphatase activity
- Cassettes containing the HCP cDNA in the positive orientation with respect to the promoter are ligated into appropriate restriction sites 3' of the promoter and identified by restriction site mapping and/or sequencing.
- These cDNA expression vectors are introduced into fibroblastic host cells for example COS-7 (ATCC# CRL1651), and CV-1 tat [Sackevitz et al., Science 238: 1575 (1987)], 293, L (ATCC#
- CRL6362 by standard methods including but not limited to electroporation, or chemical procedures (cationic liposomes, DEAE dextran, calcium phosphate). 30 -
- Transfected cells and cell culture supematants can be harvested and analyzed for HCP expression as described herein.
- All of the vectors used for mammalian transient expression can be used to establish stable cell lines expressing HCP.
- Unaltered HCP cDNA constructs cloned into expression vectors are expected to program host cells to make HCP protein.
- HCP is expressed extracellularly as a secreted protein by ligating HCP cDNA constructs to DNA encoding the signal sequence of a secreted protein.
- the transfection host cells include, but are not limited to, CV-l-P [Sackevitz et al., Science 238: 1575 (1987)], tk-L [Wigler, et al Cell 11 : 223 (1977)], NS/0, and dHFr- CHO [Kaufman and Sha ⁇ , J. Mol. Biol. 159: 601, (1982)].
- HCP cDNA constructs are also ligated into vectors containing amplifiable drug-resistance markers for the production of mammalian cell clones synthesizing the highest possible levels of HCP. Following introduction of these constructs into cells, clones containing the plasmid are selected with the appropriate agent, and isolation of an over-expressing clone with a high copy number of plasmids is accomplished by selection in increasing doses of the agent.
- the expression of recombinant HCP is achieved by transfection of full-length HCP cDNA into a mammalian host cell.
- Baculovirus vectors which are derived from the genome of the AcNPV virus, are designed to provide high level expression of cDNA in the Sf9 line of insect cells (ATCC CRL# 1711).
- Recombinant baculoviruses expressing HCP cDNA is produced by the following standard methods (InVitrogen Maxbac Manual): the HCP cDNA constructs are ligated into the polyhedrin gene in a variety of baculovirus transfer vectors, including the pAC360 and the BlueBac vector (InVitrogen).
- Recombinant baculoviruses are generated by homologous recombination following co-transfection of the baculovirus transfer vector and linearized AcNPV genomic DNA [Kitts, P.A., Nuc. Acid. Res. 18: 5667 (1990)] into Sf9 cells.
- Recombinant pAC360 viruses are identified by the absence of inclusion bodies in infected cells and recombinant pBlueBac viruses are identified on the basis of ⁇ -galactosidase expression (Summers, M. D. and Smith, G. E., Texas Agriculture Exp. Station Bulletin No. 1555). Following plaque purification, HCP expression is measured by the assays described herein.
- the cDNA encoding the entire open reading frame for HCP is inserted into the BamHI site of pBlueBacII. Constructs in the positive orientation are identified by sequence analysis and used to transfect Sf9 cells in the presence of linear AcNPV mild type DNA.
- Active HCP is found in the cytoplasm of infected cells. Active HCP is extracted from infected cells by hypotonic or detergent lysis.
- Recombinant HCP is produced in the yeast S. cerevisiae following the insertion of the optimal HCP cDNA cistron into expression vectors designed to direct the intracellular or extracellular expression of heterologous proteins.
- vectors such as EmBLyex4 or the like are ligated to the HCP cistron [Rinas, U. et ah, Biotechnology 8: 543-545 (1990); Horowitz B. et al, J. Biol. Chem. 265: 4189-4192 (1989)].
- the HCP cistron is 32 -
- yeast expression vectors which fuse a secretion signal (a yeast or mammalian peptide) to the NH2 terminus of the HCP protein [Jacobson, M. A., Gene
- vectors include, but are not limited to pAVEl>6, which fuses the human serum albumin signal to the expressed cDNA [Steep O. Biotechnology 8: 42-46
- HCP is expressed in yeast as a fusion protein conjugated to ubiquitin utilizing the vector pVEP [Ecker, D. J., J. Biol. Chem. 264: 7715-7719 (1989), Sabin, E. A., Biotechnology 7: 705-709 (1989), McDonnell D. P., Mol. Cell Biol. 9: 5517-5523 (1989)].
- the levels of expressed HCP are determined by the assays described herein.
- Recombinantly produced HCP may be purified by antibody affinity chromatography.
- HCP antibody affinity columns are made by adding the anti-HCP antibodies to Affigel-10 (Biorad), a gel support which is pre-activated with N-hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the spacer arm. The remaining activated esters are then quenched with 1M ethanolamine HC1 (pH 8). The column is washed with water followed by 0.23 M glycine HC1 (pH 2.6) to remove any non-conjugated antibody or extraneous protein.
- the column is then equilibrated in phosphate buffered saline (pH 7.3) together with appropriate membrane solubilizing agents such as detergents and the cell culture supematants or cell extracts containing solubilized HCP are slowly passed through the column.
- the column is then washed with phosphate- buffered saline together with detergents until the optical density (A280) falls to background, then the protein is eluted with 0.23 M glycine-HCl - 33
- HCP protein (pH 2.6) together with detergents.
- the purified HCP protein is then dialyzed against phosphate buffered saline.
- JAK2 as a growth hormone receptor-associated tyrosine kinase. Cell 74: 237-244.
- Truncated erythropoietin receptor causes dominantly inherited benign human erythrocytosis. Proc. Natl. Acad. Sci.
- JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin.
- Protein tyrosine phosphatase containing SH2 domains characterization, preferential expression in hematopoietic cells, and localization to human chromosome 12pl2-pl3. Mol. Cell. Biol. 12: 836-846.
- Hematopoietic cell phosphatase associates with the interleukin (IL-3) receptor ⁇ chain and down regulates IL-3-induced tyrosine phosphorylation and mitogenesis. Mol. Cell. Biol. 13: 7577-7586.
- IL-3 interleukin
- Epo erythropoietin
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP99917518A EP1071759A4 (en) | 1998-04-17 | 1999-04-15 | Assay methods for modulators of hematopoietic cell phosphatase |
AU35620/99A AU3562099A (en) | 1998-04-17 | 1999-04-15 | Assay methods for modulators of hematopoietic cell phosphatase |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US8207298P | 1998-04-17 | 1998-04-17 | |
US60/082,072 | 1998-04-17 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1999054450A1 true WO1999054450A1 (en) | 1999-10-28 |
Family
ID=22168890
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1999/008228 WO1999054450A1 (en) | 1998-04-17 | 1999-04-15 | Assay methods for modulators of hematopoietic cell phosphatase |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP1071759A4 (en) |
AU (1) | AU3562099A (en) |
WO (1) | WO1999054450A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003105884A1 (en) * | 2002-06-18 | 2003-12-24 | Oxford Glycosciences (Uk) Ltd | Use of shp-1 for the therapy of asthma |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5536636A (en) * | 1991-06-26 | 1996-07-16 | Beth Israel Hospital | Methods for identifying a tyrosine phosphatase abnormality associated with neoplastic disease |
-
1999
- 1999-04-15 EP EP99917518A patent/EP1071759A4/en not_active Withdrawn
- 1999-04-15 WO PCT/US1999/008228 patent/WO1999054450A1/en not_active Application Discontinuation
- 1999-04-15 AU AU35620/99A patent/AU3562099A/en not_active Abandoned
Non-Patent Citations (6)
Title |
---|
PEI D, WANG J, WALSH C T: "DIFFERENTIAL FUNCTIONS OF THE TWO SRC HOMOLOGY 2 DOMAINS IN PROTEINTYROSINE PHOSPHATASE SH-PTP1", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 93, 1 February 1996 (1996-02-01), US, pages 1141 - 1145, XP002919802, ISSN: 0027-8424, DOI: 10.1073/pnas.93.3.1141 * |
RAAB M, RUDD C E: "HEMATOPOIETIC CELL PHOSPHATASE (HCP) REGULATES P56LCK PHOSPHORYLATION AND ZAP-70 BINDING TO T CELL RECEPTOR ZETA CHAIN", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 222, 1 May 1996 (1996-05-01), US, pages 50 - 57, XP002919803, ISSN: 0006-291X, DOI: 10.1006/bbrc.1996.0696 * |
See also references of EP1071759A4 * |
TAPLEY P, ET AL.: "INCREASED G-CSF RESPONSIVENESS OF BONE MARROW CELLS FROM HEMATOPOIETIC CELL PHOSPHATASE DEFICIENT VIABLE MOTHEATEN MICE", EXPERIMENTAL HEMATOLOGY, ELSEVIER INC., US, vol. 25, 1 February 1997 (1997-02-01), US, pages 122 - 131, XP002919800, ISSN: 0301-472X * |
YI T, ET AL.: "HEMATOPOIETIC CELL PHOSPHATASE ASSOCIATES WITH ERYTHROPOIETIN (EPO)RECEPTOR AFTER EPO-INDUCED RECEPTOR TYROSINE PHOSPHORYLATION: IDENTIFICATION OF POTENTIAL BINDING SITES", BLOOD, AMERICAN SOCIETY OF HEMATOLOGY, US, vol. 85, no. 01, 1 January 1995 (1995-01-01), US, pages 87 - 95, XP002919801, ISSN: 0006-4971 * |
YI T, IHLE J N: "ASSOCIATION OF HEMATOPOIETIC CELL PHOSPHATASE WITH C-KIT AFTER STIMULATION WITH C-KIT LIGAND", MOLECULAR AND CELLULAR BIOLOGY., AMERICAN SOCIETY FOR MICROBIOLOGY, WASHINGTON., US, vol. 13, no. 06, 1 June 1993 (1993-06-01), US, pages 3350 - 3358, XP002919804, ISSN: 0270-7306 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003105884A1 (en) * | 2002-06-18 | 2003-12-24 | Oxford Glycosciences (Uk) Ltd | Use of shp-1 for the therapy of asthma |
Also Published As
Publication number | Publication date |
---|---|
EP1071759A4 (en) | 2002-08-07 |
AU3562099A (en) | 1999-11-08 |
EP1071759A1 (en) | 2001-01-31 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP0725800B1 (en) | cDNA ENCODING A NOVEL HUMAN PROTEIN TYROSINE PHOSPHATASE | |
US6214972B1 (en) | DNA encoding prostaglandin receptor DP | |
US6030788A (en) | Cyclin-dependent protein kinase | |
US6358706B1 (en) | DNA encoding human alpha1G-C T-Type calcium channel | |
US7049401B1 (en) | Human histamine receptor of the H3 subtype | |
EP1272514B1 (en) | Dna encoding human vanilloid receptor vr3 | |
JP3501775B2 (en) | Human vanilloid receptor-like protein | |
EP1175504A1 (en) | Dna encoding the human vanilloid receptor vr1 | |
JP4231627B2 (en) | Isolated DNA encoding human H3 histamine receptor | |
EP1015607A1 (en) | Dna encoding a ras carboxyl-terminal processing protein | |
WO1999054450A1 (en) | Assay methods for modulators of hematopoietic cell phosphatase | |
US5622842A (en) | DNA encoding canine immunoglobulin A | |
US20030166045A1 (en) | CDNA encoding the human alpha2 delta4 calcium channel subunit | |
WO1998035015A1 (en) | Cyclin-dependent protein kinase | |
US20030096348A1 (en) | DNA molecules encoding mammalian nuclear receptor protein, nNR5 | |
KR19990008320A (en) | Human neuropeptide receptor |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
NENP | Non-entry into the national phase |
Ref country code: KR |
|
WWE | Wipo information: entry into national phase |
Ref document number: 35620/99 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1999917518 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 1999917518 Country of ref document: EP |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1999917518 Country of ref document: EP |