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WO1996001838A1 - Tyrosine-kinase proteique jak3 et adn la codant - Google Patents

Tyrosine-kinase proteique jak3 et adn la codant Download PDF

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WO1996001838A1
WO1996001838A1 PCT/US1995/008354 US9508354W WO9601838A1 WO 1996001838 A1 WO1996001838 A1 WO 1996001838A1 US 9508354 W US9508354 W US 9508354W WO 9601838 A1 WO9601838 A1 WO 9601838A1
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jak3
leu
gly
arg
cells
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Sushil G. Rane
Premkumar E. Reddy
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Temple Univ School of Medicine
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Temple Univ School of Medicine
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    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a novel protein tyrosine kinase and recombinant DNA encoding therefor.
  • cytokine receptors bind to their cognate receptors and mediate intracellular signal transduction events, which result in the modulation of gene expression (Kishimoto et. al.. , supra) .
  • cytokine receptors bind to their cognate receptors and mediate intracellular signal transduction events, which result in the modulation of gene expression (Kishimoto et. al.. , supra) .
  • cytokine receptors lack a cytoplasmic kinase domain.
  • the JAK family of PTKs differs markedly from other classes of PTKs by the presence of an additional kinase " domain (Harpur et al. , Oncogene, 7, 1347-1353, 1992) .
  • the JAK family consists of three members, JAKl, JAK2 and TYK2.
  • These tyrosine kinases have a unique structure in that they contain two kinase domains at the carboxy-terminus, each of which has highly conserved stretches of sequence within them. The two kinase domains are separated by a variable stretch of amino acids which are divergent among the various JAK kinases .
  • the JAK kinases do not contain the SH2 and SH3 domains that characterize the SRC family tyrosine kinases.
  • the JAK family constitutes an important group of kinases which transduce both growth and differentiation signals from cytokine receptors following their interaction with cytokines (Stahl and Yancopoulous, supra) .
  • Current models suggest that upon interaction of cytokines with their receptors, oligo- merization of the receptors is induced which in turn permits the interaction of these receptors with JAK kinases. This results in the activation of the JAK kinases through an event associated with tyrosine phosphorylation.
  • the cDNA nucleotide sequence and predicted amino acid sequence of human JAKl is disclosed in ilks et. a_l . , Mol . Cell. Biol. 11, 2057-2065 (1991) , and also appear in WO 92/10519 (1992) .
  • the cDNA nucleotide sequence and predicted amino acid sequence of murine JAK2 is disclosed in Harpur et al. , Oncogene, 7, 1347-1353 (1992) and in Silvennoinen et al., Proc. Natl. Acad. Sci. 90, 8429-8433 (1992) .
  • the JAK2 nucleotide and amino acid sequences also appear in WO 92/10519 (1992) .
  • the JAK kinases appear to be responsible for effects mediated by several cytokines and neurokines including the interleukins, the interferons, erythropoietin, prolactin, growth hormone, oncostatin M (OSM) , and ciliary neurotrophic factor (CNTF) , (Argetsinger et. al. , Cell 74, 237-244, 1993; Hunter, ' supra; Muller et. aJL. , supra,- Silvennoinen et . al . . , supra,- Witthuhn et al. , supra; Lutticken et . al . .
  • JAK kinases are a common denominator involved in a variety of signals which culminate in diverse responses, suggesting that the kinases isolated and characterized thus far may be members of a large multigene family.
  • uncontrolled cell division lies at the heart of neoplastic phenotypes.
  • the fundamental change inneoplas- tic cells is generally considered to be their departure from normal mechanisms of growth regulation.
  • One way that oncogenes may disturb the normal mechanism of growth regula ⁇ tion is by preventing a cell's normal differentiation into a cell with a limited growth potential.
  • many tumor cells are less differentiated than their normal counterparts. This is an especially prominent feature of leukemias (cancers of blood cells) and has led to the suggestion that these tumors occur because cells in a specific lineage fail to complete their maturation. Since blood cell precursors appear to be capable of incessant division, a continuously expanding population, i.e., a tumor, results.
  • one type of retrovirus causes cancers of red blood cell precursors. If the viral oncogene is inactivated by a mutation, the previously immature cells begin to differentiate and lose their neoplastic properties. Alberts et al . , Molecular Biology of the Cell, Garland Publishing, Inc., New York, NY, 1983, p.626. What is needed is a factor which is capable of fostering hematopoietic cell differentiation of immature cells to counteract expansion of a leukemic phenotype in an afflicted individual.
  • the invention is directed to an isolated nucleic,acid molecule coding for mammalian JAK3.
  • the nucleic acidmolecule may be a DNA, cDNA, or RNA molecule.
  • the mammal in which the nucleic acid molecule exists may be a mammal, such as a mouse, a rat, or a human.
  • the nucleic acid molecule may comprise, for example, the DNA sequence SEQ ID NO:l, more particularly the coding portion thereof commencing at the initiation codon ATG (nucleotides 7-9) and concluding with the termination codon TGA (nucleotides 4111-4113) .
  • Such genetic sequences include not only native JAK3 molecules, but also sequences including single or multiple nucleotide substitutions, deletions, and/or additions relative to the native nucleotide sequence, encoding derivatives, homologues and functional analogs of JAK3 molecules.
  • a range of mutants can be obtained using standard techniques such as oligonucleotide mutagenesis and chemical mutagenesis.
  • the invention is further directed to recombinant cloning vehicle comprising a DNA segment comprising a mammalian JAK3 DNA sequence.
  • the invention is also a host transformed by the aforesaid cloning vehicle, and a process for preparing JAK3 which comprises culturing prokaryotic or eukaryotic host cells hosting a cloning vehicle comprising DNA encoding mammalian JAK3.
  • the invention is also an isolated protein comprising mammalian JAK3, such as the protein having the amino acid sequence SEQ ID NO:2.
  • the invention is also a method of stimulating the differentiation of hematopoietic cells comprising contacting the cells with a differentiation-stimulating effective amount of an isolated mammalian JAK3 protein.
  • Fig. 1A is a diagram of the seven homology domains of JAK family members.
  • Fig. IB shows oligonucleotide primers used ' in the cloning of JAK3.
  • Fig. 1C is a schematic representation of the cloning strategy for JAK3.
  • Fig. 2 shows the gel electrophoresis of amplified JAK- related sequences from 32Dcl3 total RNA (1 ⁇ g, lane 1) ; 0.5 ⁇ g, lane 2) .
  • ⁇ l74 DNA is used as a size standard (lane 3) .
  • the size of the DNA standards is shown on the right.
  • Fig. 3 is a Northern blot of total RNA from 32Dcl3 cells growing in the presence of G-CSF for 0, 1, 2, 4, 6, 8 and 10 days. Total RNA was hybridized with 32 P-labeled 950 bp JAK3 probe and photoimaged. The position of JAK3 mRNA (4.3 kb) is indicated by the arrow.
  • Fig. 4 is a comparison of the expression of JAKl, JAK2 and JAK3 RNAs in 32Dcl3 cells at different stages of differ ⁇ entiation by ribonuclease (RNase) protection analysis of total 32Dcl3 RNA.
  • RNase ribonuclease
  • Invitro transcribed JAKl, JAK2 and JAK3 antisense cRNA was labeled with 32 -P-dCTP and used as probes in the RNase protection analysis.
  • Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) probe served as a control for RNA quantitation.
  • Fig. 5 is a Northern blot of total RNA extracted from 32Dcl3 cells growing in the presence of G-CSF for 0, 5, and 10 days, and total RNA extracted from the following cell types: 70Z/3B (a pre-B cell type) ; A20.2J (a mature B-cell type) ; MOPC 31C (a terminally differentiated plasma cell type) ; Friend virus-induced erythroleukemic treated with (FMEL +DMS0) or without (FMEL -DMSO) dimethylsulfoxide.
  • Total RNA was hybridized with 32 P-labeled 950 bp JAK3 probe and photoimaged. The position of JAK3 mRNA (4.3 kb) is indicated by the arrow.
  • Fig. 6 comprises the nucleotide sequence and deduced amino acid sequence of murine JAK3 cDNA.
  • the two kinase domains and the two unique domains are boxed.
  • FIG. 1A-1C The approach used in the discovery and cloning of JAK3 is outlined in Figures 1A-1C. This approach takes advantage of two conserved tyrosine kinase (TK) domains present in the JAK family of kinases. The seven homology domains of JAK family members, designated domains JH1-JH7, are shown in Fig. 1A. Oligonucleotide primers designated J3 and J4 were designed from Pro-Phe-Ile-Lys-Leu-Ser (SEQ ID NO: 9) and Phe- Trp-Tyr-Ala-Pro-Glu (SEQ ID NO:3) motifs in the JH2 and JH1 domains, respectively.
  • TK tyrosine kinase
  • Fig. IB The nucleotide sequences of the primers are indicated in Fig. IB. Both primers contained an EcoRI restriction enzyme-specific linker to facilitate cloning of fragments. Reverse transcriptase-polymerase chain reaction (RT-PCR) amplification using these primers was expected to result in the generation of a 950 bp fragment which contains the C-terminal end of the first TK domain followed by the junction sequence and the N-terminal end of the second TK domain. Since the junction sequence between the two kinase domains is highly divergent in all known JAK kinases, sequence analysis of this region was expected to reveal the presence of new JAK kinases. The overall strategy for cloning new JAK family members is diagramed in Fig. 1C.
  • RT-PCR amplifi ⁇ cation studies were performed on total RNA extracted from 32Dcl3 cells either growing in IL-3 or in G-CSF (Valteri et al . , J. Immunol. 138, 3829-3835, 1987; Rovera etal., Oncogene 1, 29-35, 1987) .
  • the results of this analysis are shown in Figure 2.
  • Lanes 1 and 2 reveal specific amplification of a 950 bp fragment. The amplified fragment was agarose-gel purified, digested with EcoRI and used for cloning into pBluescript II KS(-) vector (Stratagene) .
  • the 950 bp PCR fragment was used as a probe to screen a cDNA library derived from 32Dcl3 cells treated with G-CSF for 5 days. Screening of 400,000 recombinants yielded fourteen positive clones which were further analyzed for the presence of inserts which hybridized with the 950 bp PCR fragment. This analysis revealed the presence of inserts in all clones, the size of which varied from 2.0 kb to 4.3 kb. Of these, the largest clone with a 4.3 kb insert was used for further analysis.
  • JAK kinases exhibit a unique structure with two adjacent kinase domains (Harpur et al . , Oncogene 7 , 1347-1353, 1992) .
  • the C-terminal kinase domain appears to contain all the functional motifs required for tyrosine kinase activity and is expected to encode for the active domain.
  • the N-terminal kinase domain though well conserved among the JAK kinases, lacks several amino acid residues considered to be essential for an active kinase domain and is therefore unlikely to carry an associated kinase activity.
  • JAK3 amino acid sequence A comparison of the JAK3 amino acid sequence with that of the other three known JAK kinases reveals in JAK3 the presence of two stretches of additional amino acid sequences of 147 and 28 residues which span between positions 322 to 469 and 632 to 660, respectively ' .
  • the presence of these additional stretches of amino acids makes JAK3 protein the largest among the JAK kinase family members known to date.
  • JAK3 sequence exhibits the seven domains of sequence homology that have been shown to exist among the other JAK kinases.
  • the two kinase domains have been named JH1 and JH2 domains.
  • the other five upstream regions have been termed as JH3-JH7.
  • JAK3 Compared with JAK2 , JAK3 exhibits 76% homology in the JH1 domain, 74% homology in JH2 the domain, 47% homolo ⁇ gy in the JH3 domain, 74% homology in the JH4 domain, 30% homology in the JH5 domain, 60% homology in the JH6 domain and 57% homology in the JH7 domain.
  • JAK3 appears to exhibit highest sequence homology with JAK2 in JH1 and JH2 domains, with decreasing levels of sequence conservation in JH4 , JH5, JH3 , JH7, JH6.
  • the first stretch of 147 amino acids which is unique to JAK3 lies between the JH5 and JH6 domains.
  • the 28 amino acid unique stretch lies within the JH4 domain of JAK3.
  • the JH1 domain contains the sequence Phe-Trp-Tyr-Ala-Pro-Glu (SEQ ID NO:3) , which distinguishes JAK kinases from the other tyrosine kinases.
  • the JH4 domain of JAK3 bears the sequence Gly-Thr-Tyr-Ile-Leu-Arg-Arg-Ser (SEQ ID NO:4) , close to its C-terminal boundary which is homologous to the Gly-Leu- Tyr-Val-Leu-Arg-Trp-Ser (SEQ ID NO:5) sequence present in human JAKl .
  • JAK3 contained the most highly conserved sequence of this region, Val-Asp-Gly-Tyr-Phe-Arg-Leu (SEQ ID NO:6) which has been proposed to be the potential phosphorylation site in JAK kinase.
  • SEQ ID NO:6 Val-Asp-Gly-Tyr-Phe-Arg-Leu
  • JAK3 probe hybridizes to a single transcript of 4.3 kb which is either not expressed or expressed at very low levels in 32Dcl3 cells growing in the presence of IL-3.
  • its synthesis was rapidly induced in 32Dcl3 cells by G-CSF.
  • the levels of JAK3 RNA reached a peak by day 5 and remained relatively stable until day 10, by which time the entire population of cells had differentiated into metamyelocytes and granulocytes. JAK3 appears to be preferentially induced during the terminal differentiation of myeloid, erythroid and lymphoid cells.
  • ribonuclease protection analysis was performed with JAK3 specific probe. Since JAKl and JAK 2 are known to be expressed in myeloid cells, similar RNase protection assays were carried out with JAKl and JAK2 specific probes under identical conditions. As control for RNA quantitation, similar assays were carried out with glyceraldehyde 3-phosphate dehydrogenase (G3PDH) probe. The results of RNase protection assays show that JAKl
  • RNA is expressed equally well in 32Dcl3 cells proliferating in IL-3 as well as those undergoing terminal differentiation in the presence of G-CSF (Fig. 4) . Similar to JAKl, JAK2 was also expressed in cells proliferating in the presence of IL-3. Removal of IL-3 from the medium and addition of G-CSF, seemed to result in 2-3 fold elevation of mRNA levels of this RNA which continued to be expressed until day 10. In contrast, JAK3 RNA was either not expressed or expressed at very low levels in 32Dcl3 cells proliferating in the presence of IL-3. Addition of G-CSF to the culture medium resulted in a rapid increase in the levels of this mRNA. As was seen with the Northern assay (Fig.
  • JAK3 The expression of JAK3 was studied in cells of other hematopoietic lineages. JAK3 expression patterns were determined by Northern blot analysis using total RNA from 70Z/3B, A20.2J and MOPC 31C cells of the B-cell lymphoid lineage and the Friend virus-induced erythroleukemic (F-MEL) cells of erythroid lineage.
  • the 70Z/3B cells represent pre-B cells
  • the A20.2J cells represent a mature B cell type
  • MOPC 31C cells represent fully differentiated plasma cells.
  • DMSO-treated F-MEL cells were used.
  • JAK3 mRNA expression was very low in the pre-B cell line 70Z/3B and was slightly higher in the mature B-cell line A20.2J.
  • the highest expression of JAK3 mRNA was seen in the MOPC 31C cell line, which represents the fully differentiated plasma cell phenotype.
  • JAK3 RNA expres ⁇ sion was also very low in undifferentiated F-MEL cells.
  • treatment of F-MEL cells for 3 days with DMSO which induces their differentiation to erythrocytes, dramatically elevated the levels of JAK3 mRNA.
  • JAK3 RNA Little or no expression of JAK3 RNA was seen in NIH 3T3 cells, which represent the fibroblast phenotype. Thus, examination of the pattern of expression of JAK3 in hematopoietic cell lines of B and ery ⁇ throid cell lineages suggests that even in these lineages, the gene is up-regulated in cells of more mature phenotype. These observations indicate that JAK3 gene expression is associated with the differentiated status of hematopoietic cells. Its transcription is induced by cytokines as well as chemical inducers of differentiation such as DMSO. The pattern of expression suggests that JAK3 may mediate specific signaling events that are associated with the terminal differentiation of hematopoietic cells. Without wishing to be bound by any theory, it is possible that the unique stretch of amino acids present in JAK3 protein allows this kinase to mediate events associated with terminal differentiation of hematopoietic cells.
  • the nucleic acid molecules that encode JAK3 protein may be inserted into known vectors for use in standard recombinant DNA techniques for producing recombinant JAK3 protein.
  • Standard recombinant DNA techniques include those techniques as described by Sambrook et. al . , Eds., Molecular Cloning: A Laboratory Manual, 2nd ed. , Cold Spring Harbor Laboratory Press, 1989 and by Ausubel et. a . , Current Protocols in Molecular Biology, J. Wiley & Sons, New York, NY (1991) .
  • the vectors may be circular or non-circular.
  • the host nay be prokaryotic or eukaryotic.
  • the preferred prokaryotic host comprises J . coli.
  • Preferred eukaryotic hosts include yeasts, insect and mammalian cells.
  • Preferred mammalian cells include, primate cells such monkey cells transformed by simian viruses (e.g., COS cells) , human cells, and Chinese hamster ovary (CHO) cells.
  • Insertion of the JAK3 gene into an appropriate expres ⁇ sion vector is easily accomplished when the requisite DNA sequences and cloning vector have been cut with the same restriction enzyme or enzymes, since complementary DNA termini are thereby produced. If this cannot be accomplished, it may be necessary to modify the cut ends that are produced by digesting back single-stranded DNA to produce blunt ends, or by achieving the same result by filling in the single-stranded termini with an appropriate DNA polymerase such as the Klenow fragment of DNA Polymerase I. In this way, blunt-end ligation with an enzyme such as T4 DNA ligase may be carried out.
  • an enzyme such as T4 DNA ligase
  • any site desired could also be produced by ligating nucleotide sequences (linkers) onto the DNA termini.
  • linkers may comprise specific oligonucleotide sequences that encode restriction site recognition sequences.
  • the cleaved vector and the modified JAK3 gene may also be modified by homopolymeric tailing, as described by Morrow, Methods in Enzvmology 68:3 (1979) .
  • recombinant JAK3 protein is obtained via bacterial expression vectors.
  • One such useful vector pDS56-6XHIS (Hochuli et al . , Biotechnology 6 , 7351-7367, 1988), contains an initiator codon followed by six histidine residues at the amino terminus to facilitate purification by chromatography on a nickel matrix.
  • Similar vectors suitable for this purpose are commercially available under the tradename "The QIA expressionist" from Quiagen Inc., Chatsworth, CA. Methods of protein purification by nickel matrix chromatography are known (Reddy et al . , Oncogene 7 , 2085-2092, 1992) .
  • Modified versions of vector pDS56-6XHIS may be substituted, such as versions using the natural methionine of the cDNA but containing six histidines at the C-terminal end of the coding region.
  • the JAK3 gene is engineered into such vectors by site-directedmutagenesis, such as according to the procedure of Higuchi et al . , Nucleic Acids Res. 16, 7351-6367 (1988) , and the recombinant vector is used to transform bacterial cells to produce recombinant JAK3 protein.
  • bacteria is transformed with an appropriate plasmid, e.g. pDS56-6XHIS, which contains JAK3 DNA and which permits subsequent purification of recombinant JAK3 protein by nickel affinity matrix chromatography. Transformation of host bacteria is carried out, for example, according to the teachings of Reddy et al., Oncogene 7, 2085- 2092 (1992) . An overnight culture of bacteria transformed with the appropriate plasmid is induced by the addition of isopropyl-S-D-thiogalactopyranoside to a final concentration of 1 mM and further grown for 2 hours .
  • an appropriate plasmid e.g. pDS56-6XHIS, which contains JAK3 DNA and which permits subsequent purification of recombinant JAK3 protein by nickel affinity matrix chromatography. Transformation of host bacteria is carried out, for example, according to the teachings of Reddy et al., Oncogene 7, 2085- 2092 (1992) .
  • the bacteria are pelleted and lysed in 15 ml of 6M GuHCl, pH 8.0.
  • the clarified supernatants are then passed through a " nickel chelate column which has been equilibrated with 6M GuHCl.
  • the recombi ⁇ nant protein is eluted with 6M GuHCl (pH 5.0) , and the eluate dialyzed stepwise in 1.0M and 0. IM GuHCl (pH 7.6) .
  • the final dialysis is performed against a buffer containing 20mM Hepes (pH 7.6), 7% glycerol, 70mM NaCI and 1 mM dithiothreitol.
  • the purity of the recombinant protein may be determined by SDS-PAGE according to the method of Lammeli, Nature 227, 680- 685 (1970) .
  • a baculovirus expression system may be used to prepare recombinant protein in insect host cells.
  • JAK3 cDNA is introduced into one of the baculovirus vectors described by Luckow & Summers, Biotechnology 6, 47-55
  • pVL1392 or pVL1393 Transformation using these vectors in the production of other proteins has achieved yields of up to 500 mg of protein per liter of cells.
  • Other suitable baculovirus vectors are commercially available from Invitrogen Corp., San Diego, CA.
  • the plasmid is transfected into insect cells, plaqued, and the appropriate recombinants are screened as described by Luckow & Summers, supra.
  • the plasmid portion of the vector is designed to place the gene of interest under the control of the promoter for polyhedron protein which is expressed normally at such high levels that it forms crystals in the nucleus of infected cells.
  • JAK3 may be expressed in mammalian cells, e.g., African green monkey kidney cells (COS) , using an appropriate vector.
  • CMV vectors are preferred. Appropriate CMV vectors for this purpose, which contain an SV40 ori sequence, a strong CMV promoter followed by a poly-linker for the insertion of foreign genes, and a polyadenylation site for efficient termination of transcription are described in U.S. Patent 4,992,367.
  • CMV vectors include pcDNA3 and pRc/CMV (Invitrogen) . JAK3 cDNA is inserted into the vector according to conven ⁇ tional techniques. Essentially, the CMV vector is cut with EcoRI . JAK3 cDNA, released from its phage or plasmid cloning vectors by partial digestion with EcoRI, is ligated to the CMV vector.
  • the DNA is transfected into COS cells according to the procedure described by Cullen, supra, and as also described in U.S. Pat. 4,992,367.
  • a COS cell line which has been transformed by an origin-minus SV40 viral genome (Glutzman, Cell 23:175, 1981) is available from the ATCC a CRL 1651.
  • the resulting purified proteins are purified according to conventional chromatographic techniques. For example, recombinant protein may be recovered using immunoaf- finity chromatography using appropriate antibodies to JAK3 protein.
  • JAK3 protein is obtained by transfection of Chinese hamster ovary (CHO) cells using a technique analogous to the procedure of Curran et. al , Cell 36, 259-268 (1984) .
  • the transfected vector DNA is allowed to co-amplify along with a plasmid carrying the dhfr gene under the control of an SV-40 promoter.
  • the vector DNA is transfected into CHOdrhf- cells along with pSVdhfr DNA and 20 ⁇ g of carrier DNA using the standard calcium phosphate-DNA technique for transfecting mammalian cells (Sambrook et al . , Eds., Molecular Cloning: A Laboratory Manual, 2nd ed.
  • CHO cell line lacking the dihydrofo- late reductase gene was originally isolated by Urlaub e_t al. , Proc. Natl. Acad. Sci. USA 77:4216 (1980) , ATCC CRL 9096. Twenty-four hours after transfection, cells are trypsinized and seeded at 1:3 dilutions in DMEM supplemented with dialyzed fetal bovine serum and 10 "7 M methotrexate.
  • amplifi ⁇ cation of the transfected genes is accomplished by stepwise selection of transfected CHO cells in increasing concentra- tions of methotrexate.
  • Transfected cells selected for resistance to 4 x 10 "4 M methotrexate have been found to contain highly amplified levels of the CMV vector and have been shown to yield milligram quantities of IL-2 and IL-2 receptor.
  • the foregoing technique provides for the isolation of permanent cell lines that continuously produce large amounts of the desired gene product.
  • the mammalian JAK3 is a biologically pure or isolated preparation meaning that it has undergone some purification away from other proteins and/or non- proteinaceous materials.
  • the purity of the preparation may be represented as at least 40% JAK3 , preferably at least 60% JAK3 , more preferably at least 75% JAK3 , even more preferably at least 85% JAK3 , and most preferably at least 95% JAK3, relative to non-JAK3 material as determined by weight, activity, amino acid similarity, antibody reactivity or other conventional means.
  • JAK3 proteins have utility in the phosphorylation of proteins, particularly in the insertion of appropriate labels into proteins via the incorporated phosphate group.
  • the present invention contemplates a method for phosphorylating a protein comprising contacting the protein to be phosphorylated with a phosphorylating effective amount of JAK3 for a time and under conditions sufficient for the protein to be phosphorylated.
  • JAK3 is used in this manner to phosphorylate tyrosine residues on protein substrate, such as for in vitro labelling of the substrate protein.
  • Condi ⁇ tions for the utilization of protein tyrosine kinases for phosphorylation of substrate proteins are known in the art.
  • the association of JAK3 expression in differentiated hematopoietic cells indicates a therapeutic utility for the protein in the treatment or control of leukemias.
  • JAK3 protein may be administered to stimulate differen ⁇ tiation of immature hematopoietic cells to counteract a leukemic phenotype.
  • JAK3 protein may be administered by any convenient route which will result in the delivery to the bloodstream of a hematopoietic cell differentiation-inducing effective amount.
  • Contemplated routes of administration include parenteral and oral routes.
  • Intravenous adminis- tration is presently contemplated as the preferred adminis ⁇ tration route.
  • the amount administered will depend on the activity of the particular compound administered, which may be readily determined by those of ordinary skill in the art.
  • the amount may also vary depending on the nature and extent of the disease lesion which is to be treated or controlled; the size and weight of the patient; the route of administra ⁇ tion, the age, sex and health of the patient; and other factors .
  • Therapeutic dosages based upon a 70 kg body weight, may range from about 0.1 mg to several grams per day.
  • JAK3 protein may be given in any of the known pharmaceutical carriers useful for delivering polypeptide drugs.
  • the carrier will typically comprise sterile water, although other ingredients to aid solubility or for preservation purposes may be included.
  • Injectable suspensions may also be prepared in which appro ⁇ priate liquid carriers, suspending agents and the like may be employed.
  • the parenteral routes of administration may comprise intravenous injection, intramuscular injection or subcutaneous injection, with intravenous injection being preferred.
  • the protein may be dissolved in an appropriate intravenous delivery vehicle containing physiologically compatible substances such as NaCI, glycine and the like, having a buffered pH compatible with physiologic conditions.
  • physiologically compatible substances such as NaCI, glycine and the like, having a buffered pH compatible with physiologic conditions.
  • JAK3 molecules are further useful in the design of JAK3 analogues, and in the design of agonists and antagonists of JAK3.
  • the invention includes functional equivalents of the
  • a protein is considered a functional equivalent of another protein for a specific function if the equivalent protein is immunologically cross-reactive with, and has the same function as a protein of the invention.
  • the equivalent may, for example, be a fragment of the protein, or a substitution, addition or deletionmutant of the protein.
  • Groups of amino acids known normally to be equivalent are:
  • the equivalent proteins normally have substantially the same amino acid sequence as the native protein.
  • An amino acid sequence that is substantially the same as another sequence, but that differs from the other sequence by means of one or more substitutions, additions and/or deletions is considered to be an equivalent sequence.
  • Preferably, less than 25%, more preferably less than 10%, and most preferably less than 5% of the number of amino acid residues in the amino acid sequence of the native molecule are substituted for, added to, or deleted from in generating the equivalent protein molecule.
  • Equivalent nucleic acid molecules include nucleic acid sequences that encode equivalent proteins as defined above. Equivalent nucleic acid molecules also include nucleic acid sequences that differ from native nucleic acid sequences in ways that do not affect the corresponding amino acid sequenc ⁇ es.
  • the redundancy of the genetic code i.e., more than one coding nucleotide triplet (codon) can be used for most of the amino acids used to make proteins, different nucleotide sequences can code for a particular amino acid.
  • the redundancy of the genetic code can be depicted as follows : Phenylalanine (Phe) TTK Histidine (His) CAK
  • Threonine (Thr) ACL Tryptophan (Try) TGG
  • Each 3-letter deoxynucleotide triplet corresponds to a trinucleotide of mRNA, having a 5' -end on the left and a 3'- end on the right.
  • DNA sequences given herein are those of the strand whose sequence corresponds to the mRNA sequence, with thymine substituted for uracil.
  • the letters stand for the pyrine or pyrimidine bases forming the deoxynucleotide sequence.
  • QR TC if S is A, G, C or T
  • novel amino acid sequence of JAK3 can be prepared by nucleotide sequences other than those particularly disclosed herein.
  • Functionally equivalent nucleotide sequences encoding the novel amino acid sequence of mammalian JAK3, or fragments thereof, having JAK3 activity can be prepared by known synthetic procedures. Accordingly, the subject invention includes such functionally equivalent nucleotide sequences.
  • the invention is exemplified by the murine JAK3 cDNA and corresponding murine protein.
  • the human and other homologues of JAK3 is isolated by a similar strategy.
  • RNA encoding JAK3 is obtained from a source of human cells, particularly human hematopoietic cells, enriched for JAK3-producing cells.
  • Combinations of murine oligonucleotide pairs are used as PCR primers to amplify the human homolog.
  • the remainder of the procedures for obtaining human JAK3 cDNA is similar to the procedure exemplified hereinafter for obtaining the murine molecule.
  • the less than perfect homology between the human JAK3 homolog and the mouse oligonucleotides is taken into account in determining the stringency of the hybridization conditions.
  • A. Cell line preparation The murine IL-3 dependent 32Dcl3 cell line was main ⁇ tained in Iscove's modified Dulbecco medium (IMDM) supple ⁇ mented with 10% heat-inactivated fetal bovine serum (FBS) and 10% WEH13B conditioned medium as a source of crude IL-3 (Patel et al. , Mol. Cell. Biol. 13, 2269-2276, 1993) .
  • IMDM Iscove's modified Dulbecco medium
  • FBS heat-inactivated fetal bovine serum
  • WEH13B conditioned medium as a source of crude IL-3 (Patel et al. , Mol. Cell. Biol. 13, 2269-2276, 1993) .
  • FBS heat-inactivated fetal bovine serum
  • WEH13B conditioned medium a source of crude IL-3
  • RNA primers for 30 cycles.
  • the PCR reaction consisted of 5 minutes of denaturation at 95°C, followed by 30 cycles of 78- second denaturation at 96°C, 96-second annealing at 53°C and 150-second elongation at 71°C.
  • the RT-PCR product was subjected to electrophoresis in a 15% agarose gel and stained with ethidium bromide. The results are shown in Fig. 2.
  • ⁇ llA DNA marker is used as a size standard (lane 3) .
  • the size of the DNA standards is shown on the right.
  • Lanes 1 and 2 reveal specific amplification of a 950 bp fragment.
  • the annealed fragment was gel purified, extracted with phenol/CHCl 3 and ethanol precipitated.
  • the purified DNA was digested with restriction enzyme EcoRI in the appropriate buffer and ligated with EcoRI-digested Pbluescript II KS (-) vector (Stratagene) and used for transformation of E. coli .
  • Individual clones were isolated and the plasmid DNA extracted according to the standardproto ⁇ col .
  • the DNA from individual clones was sequenced by the dideoxy chain termination method (Sanger e_t al.. , Proc.
  • Poly(A) + mRNA from IL-3- and G-CSF-stimulated 32Dcl3 cells was reverse transcribed using oligo dT and random hexamers with reverse transcriptase (Superscript RT, Gibco BRL) .
  • the double stranded cDNA was digested with EcoRI and ligated with ⁇ gtll (Stratagene) arms and packaged according to the manufacturer's instructions. Recombinant clones were screened using 1-24 DNA probe. The DNA was labeled with a 32 P-dCTP using Amersham' s nick translation kit . Approximately 400,000 recombinants were screened and 14 positive clones were picked.
  • Hybridization was at 42°C in 2X SSC, 0.1% SDS. The final washing stringency was 0.5% SSC, 0.1% SDS at 55°C. Filters were subjected to autoradiography using Fuji films. Phage DNA was isolated from all clones and digested with EcoRI to release cDNA inserts. Inserts were present in all clones. The largest 4.3 kb clone was sequenced from both directions as described for the PCR fragment clones, above.
  • JAK3 expression was determined at different stages of 32Dcl3 cell differentiation. Twenty ⁇ g of total RNA from cells grown in the presence of G-CSF for 0, 1, 2, 4, 6, 8 and 10 days were separated by electrophoresis on 1% formaldehyde- agarose gels and transferred to nitrocellulose (Schlecter & Schuell : BA85, catalog no. 401196) membranes. Filters were prehybridized for 4 hours in 50% formamide containing 5X SSC, IX Denhardts solution, 250 mg/ml of denatured salmon sperm DNA and 50 mM sodium pyrophosphate and hybridized to nick- translated 32 P-labeled JAK3 probe for 16 hours at 43°C. Filters were washed at a stringency of 0.
  • JAK3 probe hybridizes to a single 4.3 kb transcript which is either not expressed or expressed at very low levels in 32Dcl3 cells growing in the presence of IL-3. JAK3 synthesis was rapidly induced in 32Dcl3 cells by G-CSF. The levels of JAK3 RNA reached a peak by day 5 and remained relatively stable until day 10, by which time the entire population of cells had differentiated into metamyclocytes and granulocytes.
  • RNA Ribonuclease Protection Assay of 32Dcl3 RNA Ribonuclease protection analysis was carried out to confirm the findings of the above Northern analysis.
  • Total RNA (10 ⁇ g) from 32Dcl3 cells was subjected to ribonuclease (RNase) protection assay using a 32 P-labeled, m. vitro transcribed, 610 nucleotide long JAK3 probe derived from the catalytic domain.
  • RNase ribonuclease
  • Ribonuclease A and Tl were used according to the method described by Ausubel et al . . , Current Protocols in Molecular Biology, J. Wiley & Sons, New York, NY (1991) .
  • RNase protection assays were also performed using a 548 nucleotide long probe of JAKl and a 585 nucleotide long probe of JAK2 derived from their respective catalytic domains.
  • JAKl, JAK2 and JAK3 antisense cRNA was labelled with 32 P-rUTP and used in the RNAse protection analysis.
  • similar assays were carried out with glyceraldehyde-3-phosphate dehydrogenase
  • JAK3 RNA was either not expressed or expressed at very low levels in 32Dcl3 cells proliferating in the presence of IL-3.
  • Addition of G-CSF to the culture medium resulted in a rapid increase in the levels of this mRNA.
  • the RNA levels peaked by day 4 and these levels remained steady until day 8.
  • Example 4 Northern Blot Analysis of JAK3 Expression in Lymphoid and Erythroid Cell Lines
  • Northern blot analysis was carried out on total RNA (20 ⁇ g) from the following hematopoietic cell lines, using nick- translated 32 P-labeled JAK3 probe according to the procedure of Example 2: 70Z/3B, A20.2J and MOPC 31C cells of the B-cell lymphoid lineage; Friend virus-induced erythroleukemic (F- MEL) cells of erythroid lineage; and NIH/3T3 fibroblast cells.
  • the 70Z/3B, A20.2J and F-MEL cells were maintained in RPMI 1640 medium containing fetal bovine serum.
  • NIH/3T3 cells were grown in Dulbecco's modified Eagle's medium containing 10% FBS.
  • MOPC 31C a plasmacytoma, was maintained in Leibovitz medium containing 20% FBS.
  • the F-MEL cells were incubated with or without 1.8% DMSO for 3-4 days prior to RNA extraction.
  • the results are shown in Fig. 5, constituting total RNA blotted with 32 P-labeled 950 bp JAK3 probe exposed to a phosphorimager for one hour.
  • the mobility of JAK3 is indicated by the arrow.
  • the pattern of expression indicates that JAK3 is a mediator of specific signalling events that are associated with the terminal differentiation of hematopoietic cells.
  • ADDRESSEE Seidel, Gonda, Lavorgna
  • Cys Ala lie Leu Pro Val Tyr His Ser Leu Phe Ala Leu Ala Thr
  • Arg Arg lie Arg Arg Thr Val Val Leu Ala Leu Arg Val Trp Ser
  • Gly Asp Asn Gly lie Ser Trp Ser Ser Gly Asp Gin Glu Val Leu 275 280 285
  • Gly Arg Arg Pro Ser Phe Arg Ala lie Leu Arg Asp Leu Asn Gly 980 985 990
  • Leu lie Thr Ser Asp Tyr Glu Leu Leu Gin Thr Pro His Leu Ala

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Abstract

Des molécules d'acide nucléique mammaliennes isolées codent JAK3, une nouvelle tyrosine-kinase protéique exprimée dans les cellules hématopoïétiques matures. L'invention concerne également des protéines codées par les nouvelles molécules d'acide nucléique et leurs procédés d'utilisation pour induire la différenciation des cellules hématopoïétiques.
PCT/US1995/008354 1994-07-08 1995-06-28 Tyrosine-kinase proteique jak3 et adn la codant Ceased WO1996001838A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0797581A4 (fr) * 1994-12-15 2000-03-08 Univ Johns Hopkins Med Nouvelle tyrosine kinase proteique, jak3

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BLOOD, Volume 84, No. 10, Suppl. 1, issued 15 November 1994, K.S. LAI et al., "Characterization and Expression of Human JAK3 Splice Variants", page 294a, Abstract 1161. *
FEBS LETTERS, Volume 342, No. 2, issued 04 April 1994, T. TAKAHASHI et al., "Molecular Cloning of Rat JAK3, a Novel Member of the JAK Family of Protein Tyrosine Kinases", pages 124-128. *
NATURE, Volume 366, issued 09 December 1993, O. SILVENNOINEN et al., "Interferon-Induced Nuclear Signalling by Jak Protein Tyrosine Kinases", pages 583-585. *
SCIENCE, Volume 263, issued 07 January 1994, N. STAHL et al., "Association and Activation of Jak-Tyk Kinases by CNTF-LIF-OSM-IL-6beta Receptor Components", pages 92-95. *
STEM CELLS, Volume 12, issued 1994, T. HIRANO et al., "Signal Transduction Through gp130 That is Shared Among the Receptors for the Interleukin 6 Related Cytokine Subfamily", pages 262-277. *
TIBS, Volume 19, issued May 1994, J.N. IHLE et al., "Signaling by the Cytokine Receptor Superfamily: JAKs and STATs", pages 222-227. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0797581A4 (fr) * 1994-12-15 2000-03-08 Univ Johns Hopkins Med Nouvelle tyrosine kinase proteique, jak3

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