CA2203706A1 - Human jak2 kinase - Google Patents

Description

CA 02203706 1997-04-2~

HUMAN Jak2 KINASE

Field of the Invention This invention relates to the human Jak kinase family of genes and 5 their protein products. More particularly, it relates to the cDNA sequence of human Jak2 kinase and to the expression and role of that kinase in humans.

Background of the Invention Because cytokine receptors do not possess a kinase domain, great 10 efforts have been made to identify the cytoplasmic protein tyrosine kinases (PTK's) which are responsible for the rapid increase in protein tyrosine kinase activity seen upon ligand-receptor interaction (3).
A commonly used technique has involved PCR with degenerate oligodeoxynucleotide primers directed to conserved tyrosine kinase catalytic 15 domain motifs. Many new PTK's have been discovered by this method, including members of the Jak family (4,5). The Jak kinase family is unusual in possessing two distinct kinase domains (5,6). The kinase domain proximal to the carboxyl terminal contains all of the recognized essential PTK motifs (7) and is therefore likely to be catalytically active. However, the function of the20 second kinase domain remains unknown as it lacks residues believed to be essential for catalytic activity. Members of the Jak family such as Jak1, Jak2, Jak3, and Tyk2 strikingly lack the src homology 2 (SH2) and the src homology 3 (SH3) domains, but they show among them other homology regions of yet unknown significance (5,8). Although Jak1, Jak2, and Tyk2 have been 25 reported to be widely expressed, Jak3 expression appears limited to the myeloid and Iymphoid lineages (6,9). Expression of Jak3 was found originally in natural killer (NK) cells but not in resting T cells or in other tissues.
Furthermore, Jak3 expression can be induced in stimulated T cells (9).
It has been shown recently that in addition to some src PTK's, cytokine 30 receptors activate one or more Jak kinases. Cytokines utilizing receptors consisting of a single chain such as eyrthropoietin (EPO), prolactin and growth hormone (GH), primarily activate Jak2 [14, 15], whereas the CA 02203706 1997-04-2~

interferons activate two other members of the Jak family [18,19, 40]. The receptors for IL-2, IL-4, IL-7,1L-9 and IL-15 are functionally coupled to Jak1 and Jak3 [11, 12] whereas the common r-chain (~c) selectively recruits Jak2.
The cytokines, ciliary neurotropic factor (CNTF), leukemia inhibiting factor 5 (LIF), oncostatin M (OSM) and IL-6 make up a unique cytokine sub-family on the basis of their predicted structural similarities and shared ~ signal-transducing receptor component (gp 130). This family is capable of recruiting three different members of the Jak family (Jak1, Jak2, Tyk2), in different cell types [29, 30].
It is of interest that a mutation in the Drosophila Tumorous-lethal gene of the hopscotch locus, which has been identified as a Jak homolog, causes fly leukemia [34]. Recently, it has been shown that some human pre-B
leukemia cells express an elevated level of Jak2 which appears to be constitutively activated (21). Inhibition of Jak2 with a specific tyrphostin blocker resulted in programmed cell death in these leukemic cells (21).
Until the work of the present inventor, the human Jak2 kinase gene had not been isolated and its sequence was unknown.
Furthermore, the exact chromosome localisation of the human Jak2 gene was in some doubt, since studies using murine Jak2 cDNA localised the human gene to chromosome 9p24 (10) whereas the murine Jak2 gene is genetically linked to Fas on mouse chromosome 19, which corresponds to human 1 Oq23-124(6).

Summary of the Invention A new human gene, Jak2, has been identified and located on chromosome 9p23-24. Its cDNA has been cloned and sequenced and has been successfully expressed to give human Jak2 kinase protein. Antibodies specific for human Jak2 kinase have been prepared and used to demonstrate the pattern of expression of the protein in human tissues. The protein is ubiquitously expressed but is expressed most highly in spleen, Iymph nodes and peripheral blood Iymphocytes. Expression of the protein has been shown CA 02203706 1997-04-2~

to increase modestly on activation of the mature T Iymphocytes and dramatically on activation of mature B Iymphocytes.
In accordance with one embodiment of the invention, an isolated nucleic acid is provided comprising a nucleotide sequence encoding human 5 Jak2 kinase protein. The polynucleotide may be in the form of DNA, genomic DNA, cDNA, mRNA and various fragments and portions of the gene sequence encoding human Jak2 kinase.
In accordance with a further embodiment of the invention, a purified nucleotide sequence is provided comprising genomic DNA, cDNA, mRNA, 10 anti-sense DNA or homologous DNA corresponding to the cDNA sequence of Sequence ID No.1.
In accordance with a further embodiment of the invention, is a purified nucleotide sequence sharing 95% homology to Sequence ID No.1 and encoding for human Jak2 kinase protein.
In accordance with a further embodiment of the invention is an amino acid sequence corresponding to the amino acid sequence of Sequence ID
No.2.
In accordance with another embodiment of the invention are protein fragments comprising at least 12 contiguous amino acids of Sequence ID
20 No.2.
In accordance with a further embodiment of the invention is a purified protein sharing 95% homology to Sequence ID No.2 and having human Jak2 kinase activity.
In accordance with a further embodiment of the invention, substantially 25 pure human Jak2 kinase protein is provided.
In accordance with a further embodiment of the invention, a substantially pure polypeptide is provided comprising at least one functional domain of a human Jak2 kinase protein.
In accordance with a further embodiment of the invention, a 30 substantially pure polypeptide is provided comprising an antigenic determinant of a human Jak2 kinase protein.

CA 02203706 1997-04-2~

In accordance with a further embodiment of the invention, a method is provided for suppressing the neoplastic phenotype of a cell or groups of cells in which Jak2 kinase is active comprising administering to the cell an agent selected from the group consisting of;
(a) monoclonal or polyclonal antibodies recognizing human Jak2 kinase protein;
(b) monoclonal or polyclonal antibodies recognizing mutant human Jak2 kinase protein;
(c) a polynucleotide antisense strand capable of hybridizing to human Jak2 gene or gene transcripts;
(d) a polynucleotide antisense strand capable of hybridizing to human mutant Jak2 gene or mutant gene transcripts;
(e) an agent to inhibit phosphorylation activity of human Jak2 kinase protein or mutant protein;
(f) an agent to stimulate dephosphorylation of human Jak2 kinase protein or mutant protein; and (g) a composition comprising a mixture of (a) to (f).
In accordance with a further embodiment of the invention, a method is provided for suppressing autoimmune disorders involving activated Iymphocytes comprising administering to a patient an agent selected from the group consisting of:
(a) a composition comprising monoclonal or polyclonal antibodies recognizing human Jak2 kinase protein or mutant protein;
(b) a composition containing an agent to inhibit phosphorylation activity of human Jak2 kinase protein or mutant protein;
(c) a composition containing an agent to stimulate dephosphorylation of human Jak2 kinase protein or mutant protein;
and (d) a composition comprising a mixture of (a) to (c).
In accordance with a further embodiment of the invention, a diagnostic method is provided for determining if a subject carries a mutant Jak2 gene CA 02203706 1997-04-2~

producing a mutant Jak2 kinase protein possessing increased kinase activity, comprising the steps of (a) providing a biological sample from the subject; and (b) detecting in the sample a mutant Jak2 nucleic acid, a mutant 5 Jak2 kinase protein, elevated Jak2 kinase activity or intracellular downstream phosphorylated proteins.
In accordance with a further embodiment of the invention, a method is provided for identifying allelic variants or heterospecific homologues of a Jak2gene comprising (a) choosing a nucleic acid probe or primer capable of hybridizing to a human Jak2 kinase gene sequence under stringent hybridization conditions;
(b) mixing said probe or primer with a sample of nucleic acids which may contain a nucleic acid corresponding to the variant or homologue;
(c) detecting hybridization of the probe or primer to the nucleic acid corresponding to the variant of homologue.
In accordance with a further embodiment of the invention, a method is provided for producing antibodies which selectively bind to a human Jak2 kinase protein comprising the steps of administering an immunogenically effective amount of a human Jak2 kinase immunogen to an animal;
allowing the animal to produce antibodies to the immunogen; and obtaining the antibodies from the animal or from a cell culture derived therefrom.
In accordance with a further embodiment of the invention, a method is provided for producing antibodies which selectively bind to a mutant human hyperactive Jak2 kinase protein comprising the steps of administering an immunogenically effective amount of a mutant human Jak2 kinase immunogen to an animal;
allowing the animal to produce antibodies to the immunogen; and obtaining the antibodies from the animal or from a cell culture derived therefrom.

CA 02203706 1997-04-2~

In accordance with a further embodiment of the invention, a substantially pure antibody is provided which binds selectively to an antigenic determinant of a human Jak2 kinase protein selected from the group 5 consisting of a normal human Jak2 kinase protein and mutant human Jak2 kinase protein.
In accordance with a further embodiment of the invention, a method is provided for identifying compounds modulating expression of a human Jak2 kinase gene comprising contacting a cell with a test candidate wherein the cell includes a regulatory region of a human Jak2 kinase gene operably joined to a coding region; and detecting a change in expression of the coding region.
In accordance with a further embodiment of the invention, a method is provided for treating a subjecting having a Jak2 kinase gene encoding a Jak2 kinase protein possessing hyperactivated kinase activity, comprising administering to the subject a therapeutically effective amount of an agent selected from the group consisting of:
(a) an isolated antisense nucleotide sequence which hybridizes with normal human Jak2 kinase gene sequence;
(b) an isolated antisense nucleotide sequence which hybridizes with a mutant human Jak2 kinase gene sequence;
(c) a substantially pure monoclonal antibody which recognizes normal Jak2 kinase protein; and (d) a substantially pure monoclonal antibody which recognizes mutant Jak2 kinase protein.
In accordance with a further embodiment of the invention, a pharmaceutical composition is provided comprising an active ingredient selected from the group consisting of:
(a) an antisense sequence which hybridizes to a human Jak2 kinase nucleotide sequence or to a transcript of the sequence;

CA 02203706 1997-04-2~

(b) an antisense sequence which hybridizes to a mutant human Jak2 kinase nucleotide sequence or to a transcript of the mutant sequence;
(c) a substantially pure antibody which binds selectively to human Jak2 kinase protein and a pharmaceutically acceptable carrier;
(d) a mimetic of human Jak2 kinase protein;
(e) a functional analog of human Jak2 kinase protein;
(f) a mimetic of human mutant Jak2 kinase protein;
(g) a functional analog of human mutant Jak2 kinase protein;
(h) an inhibitor of human Jak2 kinase protein activity; and (i) an agent capable of dephosphorylating human Jak2 kinase protein.
In accordance with a further embodiment of the invention, a method is provided of screening for an agent useful in treating a disorder characterized by an abnormality in a cytokine phosphorylation signaling pathway of haematopoietic and Iymphoid cells, wherein the pathway involves an interaction between a human Jak2 kinase protein and a human Jak2 kinase activator, comprising screening potential agents for ability to disrupt or promote the interaction as an indication of a useful agent.
In accordance with a further aspect of the invention, a method is provided of preventing or treating a disorder in a mammal characterized by an abnormality in a cytokine intracellular phosphorylation signaling pathway of haematopoietic and Iymphoid cells, wherein the pathway involves an interaction between a human Jak2 kinase protein and a human Jak2 kinase substrate, comprising the step of disrupting or promoting said interaction in VIVO.
In accordance with another aspect of the invention, a transgenic animal model for malignancy comprising an animal which has in its genome a human Jak2 kinase gene sequence with at least one mutation which when expressed results in mutant Jak2 kinase activity in the animal cells and thereby manifests a malignant phenotype. The animal may be a rodent and is CA 02203706 1997-04-2~

preferably a mouse.
In accordance with another aspect of the invention, a transgenic mouse model for various immune diseases involving activated Iymphocytes which comprises an animal which has in its genome a human gene encoding Jak2 kinase mutated to manifest increased kinase activation. The transgenic mouse may exhibit symptoms indicative of autoimmune disorders. In addition or alternatively, the symptoms may appear as inappropriate cellular growth resulting in tissue malignancy.
In accordance with another aspect of the invention, the Jak2 kinase 10 protein or mutant protein can be used as a starting point for rationale drug design to provide ligands, therapeutic drugs inhibitors or other types of small chemical molecules.
Summary of the Drawings Certain embodiments of the invention are described, reference being 15 made to the accompanying drawings, wherein:
Figure 1 illustrates diagrammatically the strategy used to clone human Jak2. A combination of library screening using as a probe the mouse Jak2 cDNA and RT-PCR was used to obtain the full length cDNA of human Jak2.
The screening yielded a series of overlapping fragments combining to a 1.8 20 Kb ORF (nucleotides 1398-3207). An RT-PCR, utilizing primers to the published murine sequence and to the 1.8 Kb ORF obtained from the library screening, was used to complete the cloning of the 5' (nucleotides 1-1416) and 3' (nucleotides 3055-3503) end of the human Jak2. A stop codon was found in position 3402 as indicated by the arrow.
Figure 2 shows chromosomal localization of the human Jak2 gene.
Positional mapping was performed by FISH to normal human Iymphocyte metaphase chromosomes. Both a 1.5 Kb Jak2 cDNA probe and a corresponding genomic probe obtained by screening a PAC library, showed positive signal in ~95% of the cells on both chromatides of each homologue 30 of the chromosome 9p23-24, as indicated by the arrows.

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Figure 3 shows a Northern blot of human Jak2 expression in various human tissues. Approximately 2 ~lg per lane of Poly A+ RNA from the indicated human tissues (purchased from Clontech) was run on a denaturing formaldehyde 1.2% agarose gel, transferred to a charge-modified nylon 5 membrane and probed with a 400 bp cDNA corresponding to the 3' end of Jak2. Actin was used for comparison of RNA loading.
Figure 4 shows a Northern blot of human Jak2 expression in various immune-relevant tissues, prepared under the conditions described for Figure 3.
Figure 5 shows in vitro translation and detection of Jak2. (A).
Approximately 1~19 of Luciferase, Jak3 and Jak2 cDNA's were translated in a combined transcription and translation reticulocyte Iysate system (Invitrogen) including (35S) methionine. Aliquots of each product were run on SDS-PAGE
gel followed by transfer to nitrocellulose and visualized by autoradiography.
15 The reactions yielded proteins of 60, 120 and 130 kDa, corresponding to Luciferase, Jak3 and Jak2, respectively (lanes 1, 2, 3). (B). The remaining Iysate of each reaction was immunoprecipitated with anti-human Jak2 antibody, resolved by SDS-PAGE gel, transferred to nitrocellulose and visualized by autoradiography. Only Jak2 was immunoprecipitated by the 20 anti-human Jak2 antibody with no cross reactivity to Jak3 or to irrelevant protein detected. (C). Only the immunoprecipitated Jak2 (lane 3) was detected by ECL after the same membrane was probed with a anti-mouse Jak2 antibody (Pharmingen).
Figure 6 shows expression of Jak2 protein. Immunoprecipitates were 25 prepared from Iysates of 2 x 107 cells with anti-sera to human Jak2 and subjected to 6% SDS-PAGE. After transfer to nitrocellulose membrane, immunoblotting was performed with anti-Jak2 antibody (Pharmingen) detecting by ECL.
Figure 7 shows induction of Jak2 expression in activated T and B
30 Iymphocytes. (Left panel). Human T cells (2 x 107 cells per lane) were incubated for the indicated time with 10 ~lg/ml PHA. Cell Iysates were CA 02203706 l997-04-2~

prepared and Jak2 immunoprecipitated before anti-Jak2 immunoblotting.
(Right panel); Human B cells (2 x 107 cells per lane) were incubated for the indicated time with 2 mg/ml SAC. Cell Iysates were prepared, followed by immunoprecipitation with anti-human Jak2 and immunoblotting with anti-5 mouse Jak2 antibody (Pharmingen).

Detailed Description of the Invention The present invention relates to the identification and sequencing of the human Jak2 kinase gene. The gene has been identified, cDNA isolated and cloned and its transcripts and gene products identified and sequenced.
10 With the identification and sequencing of the gene and the gene product, probes and antibodies raised to the gene product can be used in a variety of hybridization and immunological assays to screen for and detect the presence of either a normal or mutated gene or gene product. Knowledge of the gene sequence also enables for the identification of mutant forms of the human 15 Jak2 kinase gene which are involved in hyperactivation of the protein and which contributes to leukemogenesis and other immune system disorders.
The gene sequence for human Jak2 kinase also provides for the study of structure and functional relationships of the protein and allows for the use of site-directed mutagenesis of the gene sequence to provide for mutant 20 forms of the protein to study its activity and intracellular signalling effects which manifests as malignant transformation of cells or which may lead to other immune disorders.

The invention in another embodiment enables the development of treatments and therapies for several diseases involving cytokine mediated 25 activation of growth factor receptors in haematopoietic and Iymphoid cells which leads to increased activation or hyperactivation of Jak2 kinase. This stimulates a host of intracellular protein-protein interactions leading to increased transcription and translation of products leading to malignant transformation or to disorders of the immune system. The invention is not 30 limited to the development and prevention of malignant diseases such as CA 02203706 1997-04-2~

leukemia, but also extends to other autoimmune disorders involving activated leukocytes, allergies, eosinophilic fascitis, arthritis and gastroenteritis.
Patient therapy through removal or blocking of the mutant gene product though varies strategies can now be achieved. Correction or modification of the defective gene product by protein treatment immunotherapy (using antibodies to the defective protein) or knock-out of the mutated gene function using antisense strategies is now also possible.
Hyperactivated Jak2 kinase activity as a result of a mutation in the Jak2 kinase gene can also be controlled by gene therapy in which the gene defect 10 is corrected in situ or by the use of recombinant or other vehicles to deliver a DNA sequence capable of expressing the normal gene product, or a deliberately mutated version of the gene product whose effect counter balances the deleterious consequences of the disease mutation to the affected cells of the patient.

15 Identification of human Jak2 kinase gene Murine Jak2 cDNA (Sugen) was used to screen a human thymus cDNA library. This screening yielded a series of overlapping fragments combining to make up a 1.8kb open reading frame (ORF). This sequence was found to be 89% homologous to the kinase domain of the translated 20 murine Jak2 cDNA from nucleotide 1398 to nucleotide 3207.

Using RT-PCR and primers to the murine sequence, the cloning of the 5' and 3' end of the human cDNA sequence was completed.

Figure 1 summarizes the cloning strategy used. To obtain the 5' end of human Jak2, a degenerate oligonuc!eotide primer to the murine sequence 25 was used, beginning at the start codon, and a downstream non-degenerate primer to sequence from human Jak2 in a PCR reaction on human PBL. First strand cDNA produced a fragment of approximately 1.4kb extending from the ATG and overlapping the 5' end of our original 1.8kb ORF.

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To obtain the 3' end of Jak2, RNA extracted from human PBL and reverse transcribed with oligo(dT),2GC, was used as a substrate for PCR. A
non-degenerate primer was designed on the 1.8kb ORF from nucleotide 3207 and an antisense non-degenerate primer to the murine Jak2 untranslated 5 sequence. This amplification generated a sequence of approximately 450bp containing a stop codon and overlapping the 3' end of the 1.8kb ORF. The RT-PCR-generated fragments and the 1.8kb ORF combined to form a 3.4kb ORF corresponding to the complete gene. The cDNA sequence of Jak2 (Sequence ID NO:1) is shown in Tables 1A,1B and 1C and contains the 10 entire open reading frame of Jak2. The coding region encodes a predicted gene product of 1134 amino acids (Sequence ID NO:2; Table 2), with a predicted molecular weight of 130 kDa.

The human Jak2 cDNA shows 87% homology with murine Jak2 cDNA
and the predicted amino acid sequence 91 % overall homology with the 15 mouse protein. There is, however, a significant difference in the carboxy terminus amino acid sequence of the two proteins. An insertion of 1 base pair in position 3363 of the mouse sequence causes a frameshift alteration that alters the last 13 amino acids with an addition of 3 amino acids. Using oligodeoxynucleotides to the 5' and 3' ends of the human sequence, a full 20 length Jak2 cDNA was cloned by RT-PCR from both human thymocytes and human G2 cells (pre-B leukemia cell line). These were found to be identical to human Jak2 sequence of Tables 1A,1B and 1C.

Tables 3A and 3B show a comparison between the predicted amino acid sequences of human Jak1, Jak3, Tyk2, and the human Jak2 sequence.
25 Alignment was performed with the PILEUP program. Identical residues are shown on a black background, while related residues are shaded. Gaps were introduced for optimal alignment and are indicated by hyphens. The boundaries of the Jak homology (JH) domains are denoted by arrows. The alignment shows that the human Jak2 contains a kinase and kinase-like 30 domains (JH1 and JH2 respectively) at its C terminus, along with the other CA 02203706 1997-04-2~

five conserved domains (JH3-JH7) that have been shown to exist among other members of the Jak family. The homology between the predicted amino acid sequence of the JakZ clone and that of the murine Jak2 indicates that the clone represents the human homologue of the murine Jak2 gene.
The identification of the cDNA Jak2 kinase human gene sequence allows for the isolation and production of the Jak2 kinase protein for various uses. The Jak2 protein may be isolated and purified by methods selected on the basis of properties revealed by its sequence. Since the protein possesses properties of an intracellular protein tyrosine kinase, an 10 intracellular fraction of cells in which the protein is highly expressed (eg.spleen, Iymph nodes and peripheral blood Iymphocytes) would be isolated and the proteins removed by extraction and the proteins solubilized using a detergent.
Purification can be achieved using protein purification procedures such 15 as chromatography methods (gel-filtration, ion-exchange and immunoaffinity), by high-performance liquid chromatography (RP-HPLC, ion-exchange HPLC, size-exclusion HPLC, high-performance chromatofocusing and hydrophobic interaction chromatography) or by precipitation (immunoprecipitation).
Polyacrylamide gel electrophoresis can also be used to isolate the Jak2 20 kinase protein based on its molecular weight, charge properties and hydrophobicity.
Similar procedures to those just mentioned could be used to purify the protein from cells transfected with vectors containing the Jak2 kinase gene.
Purified protein can be used in further biochemical analyses to 25 establish secondary and tertiary structure which may aid in the design of pharmaceuticals to interact with the protein, alter protein charge configurationor charge interaction with other proteins, and in cells as an enzyme and to treat disease.

In recent years, it has been shown that members of the Jak family play 30 a central role in growth and differentiation of hematopoietic cells (1, 2). The Jak kinases have been shown to be involved in signaling through both CA 02203706 1997-04-2~

cytokine and hormone receptors (6) leading to activation of different cellular events including Ras pathway, Pl-3 kinase, and activation of c-fos and c-myc promoters (4, 13, 28). Based on its role as an intracellular cell signal mediator, various cellular assays can be developed and used in order to 5 identify activators of Jak2 kinase as well as inhibitors of Jak2 kinase activity.
Moreover, suppressors of Jak2 kinase activity can also be identified in cell culture assays.

Chromo~c...al localization A 1.5 kb DNA fragment corresponding to the 5' end of the human Jak2 10 cDNA was used as a probe. To localize the chromosomal position of the human Jak2 gene, this probe was chosen in a region of relatively low homology to other members of the Jak family. This probe was used in both FISH analysis and in obtaining a corresponding genomic PAC probe. Images of 20 well-spread metaphase preparations were captured by a 15 thermoelectrically cooled charge coupled camera, and separate images of DAPI banded chromosomes and of FITC targeted chromosomes were obtained. Both probes mapped to chromosome 9p23-24 in >95% of the cells.
The human Jak2 gene was previously localized on chromosome 9p24 using the murine Jak2 cDNA as a probe (9). However, the fact that the murine Jak2 20 gene itself was localized to chromosome 19, which corresponds to the human 1 Oq23-24, had cast some doubt upon the accuracy of the human localization.

The present data, obtained with a human sequence probe, confirm the localization of the human Jak2 gene to chromosome 9 (Fig. 2).

Human Jak2 mRNA expression in various tissues In order to assess the distribution of human Jak2 expression, a 400 bp fragment from the human Jak2 cDNA was used to probe a series of membranes containing polyA' RNA from different tissues. This Northern analysis demonstrated the presence of three transcripts of approximately 7.0, 5.4 and 4.8 kb. These transcripts were found to be expressed ubiquitously CA 02203706 1997-04-2~

(Fig. 3). While the relative level of 5.4 to 4.8 kb transcript remained constantin the different tissues, the expression of the 7.0 kb transcript appeared to belower in both testis and PBL. The three transcripts may represent the products of alternative splicing or alternatively represent use of different poly 5 adenylation sites.

In addition, the expression of Jak2 was investigated in Iymphoid tissues. Jak2 appears to be expressed preferentially in spleen, Iymph nodes and PBL and to a lesser degree in fetal liver, thymus and bone marrow (Fig.
4). This suggests that the expression of Jak2 is generally higher in mature 10 Iymphocytes than in immature stages.

Jak2 protein expression in hematopoietic cells A thirteen amino acid peptide, VLRVDQVRDNMAG (Sequence ID
NO:3) was selected from the carboxy terminal of the human Jak2 protein and used to raise polyclonal antibodies. This region of the human Jak2 protein is 15 unique to the Jak2 protein and does not exhibit homology with other members of the human Jak family, as seen in Tables 3A and 3B.

The antibody was found to immunoprecipitate a 130 kDa protein, which was recognized by commercially available anti-mouse Jak2 antisera from Pharmingen (peptide 1067), UBI (peptide 758), and Santa-Cruz Biotech 20 (peptide 758). Because the anti-human Jak2 antibody was unable to recognize the denatured Jak2 protein in Western blots, anti-mouse Jak2 (Pharmingen) was used to detect the immunoprecipitated human protein. To further confirm the specificity of the anti-human Jak2 antibody, its ability to recognize an in vitro synthesized Jak2 protein was tested. Approximately 1 25 ~9 of Luciferase, Jak3 and Jak2 cDNA's were added to a combined transcription and translation reticulocyte Iysate system and the products labeled with (35S) methionine. Aliquots of each product were run on SDS-PAGE gel and visualized by autoradiography to check the translation. The reactions yielded 60,120 and 130 kDa proteins corresponding to Luciferase, CA 02203706 1997-04-2~

Jak3 and Jak2, respective as predicted (Fig. 5a lanes 1,2,3,). The remaining Iysate from each reaction was added to anti-human Jak2 antibody and the immunoprecipitate resolved on SDS-PAGE followed by transfer to nitrocellulose and visualisation by autoradiography. Only Jak2 was 5 immunoprecipitated by the anti-human Jak2 antibody (Fig. 5b). The immunoprecipitated human Jak2 could then be recognized by immunoblotting with the anti-mouse Jak2 antibody (Pharmingen), (Fig. 5c lane).

Using the anti-human Jak2 antibody, the Jak2 protein was found to be highly expressed in G2 and C1 (human pre-B leukemia derived cell lines) but 10 its detection is markedly lower in A1 (pre-B leukemia cell lines), resting peripheral T and B Iymphocytes, thymocytes and fibroblasts. Jak2 was essentially undetectable in U937 (monocytic leukemia cell line). In summary, the finding that Jak2 is highly expressed in pre-B leukemia cell lines suggests a possible functional role for Jak2 during pre-B Iymphocyte transformation 15 into malignant cells, as previously described (21).

Jak2 protein was found to be highly expressed in human pre-B
leukemia cell lines (Fig. 6). Dysregulation of Jak function has already been shown to occur in transformation of T Iymphocytes infected with HTLF-1, and also hematopoietic malignancy (fly leukemia) in drosophila (34). Additionally, 20 it has been shown that leukemic cells from patients in relapse have constitutively activated Jak2. Inhibition of Jak2 activity by a specific tyrosine kinase blocker selectively blocks leukemia cell growth by inducing programmed cell death (21). These findings suggests that Jak2 may have a functional role during the transformation of pre-B Iymphocytes into malignant 25 cells.

E~,re~sion of Jak2 is increased in stimulated Iymphocytes Mature peripheral T and B Iymphocytes obtained from a healthy human volunteer were stimulated in vitro with PHA or SAC respectively. Jak2 protein was found to be expressed in resting peripheral T cells and slightly CA 02203706 1997-04-2~

increased in T cells cultured with PHA for 24 hours, with no further change apparent after 48 hours (Figure 7, left panel). Resting peripheral B cells also expressed low levels of Jak2, but this was dramatically increased upon culture of B cells with SAC (Figure 7, right panel). A time course of SAC-5 induced B cell activation revealed that maximum expression of Jak2 wasreached within 24 hours (data not shown).

The increased expression of Jak2 protein in activated mature T and B
Iymphocytes suggests it may be of functional importance to these cells, perhaps in clonal expansion and functional differentiation of mature B
1 0 Iymphocytes.
In another embodiment of the present invention expression of the Jak2 kinase gene in heterologous cell systems can be used to demonstrate structure-function relationships as well as provide for cell lines for the purposes of drug screening. Ligating the Jak2 kinase DNA sequence into a 15 plasmid expression vector to transfect cells is a useful method to test the proteins influence on various cellular biochemical parameters including the identification of kinase substrates as well as activators and inhibitors of the kinase. Plasmid expression vectors containing either the entire, or portions thereof, normal or mutant human Jak2 kinase sequence can be used in in 20 vitro mutagenesis experiments which will identify portions of the protein crucial for regulatory function.
The DNA sequence can be manipulated in studies to understand the expression of the gene and its product, to achieve production of large quantities of the protein for functional analysis, for antibody production, and 25 for patient therapy. The changes in the sequence may or may not alter the expression pattern in terms of relative quantities, tissue-specificity and functional properties. Partial or full-length DNA sequences which encode for the Jak2 kinase protein, modified or unmodified, may be ligated to bacterial expression vectors. E. coli can be used using the T7 RNA
30 polymerase/promoter system using two plasmids or by labeling of plasmid-encoded proteins, or by expression by infection with M13 Phage mGPI-2. E.

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coli vectors can also be used with Phage lamba regulatory sequences, by fusion protein vectors (eg. IacZ and trpE), by maltose-binding protein fusions, and by glutathione-S-transferase fusion proteins.
Alternatively, the Jak2 kinase protein can be expressed in insect cells 5 using baculoviral vectors, or in mammalian cells using vaccinia virus. For expression in mammalian cells, the cDNA sequence may be ligated to heterologous promoters, such as the simian virus (SV40) promoter in the pSV2 vector and introduced into cells, such as COS cells to achieve transient or long-term expression. The stable integration of the chimeric gene 10 construct may be maintained in mammalian cells by biochemical selection, such as neomycin and mycophoenolic acid.
The normal Jak2 kinase DNA sequence can be altered using procedures such as restriction enzyme digestion, fill-in with DNA polymerase, deletion by exonuclease, extension by terminal deoxynucleotide transferase, 15 ligation of synthetic or cloned DNA sequences, site-directed sequence alteration with the use of specific oligonucleotides together with PCR.
The cDNA sequence or portions thereof, or a mini gene consisting of a cDNA with an intron and its own promoter, is introduced into eukaryotic expression vectors by conventional techniques. These vectors permit the 20 transcription of the cDNA in eukaryotic cells by providing regulatory sequences that initiate and enhance the transcription of the cDNA and ensure its proper splicing and polyadenylation. The endogenous Jak2 kinase gene promoter can also be used. Different promoters within vectors have different activities which alters the level of expression of the cDNA. In addition, certain 25 promoters can also modulate function such as the glucocorticoid-responsive promoter from the mouse mammary tumor virus.
Some of the vectors listed contain selectable markers or neo bacterial genes that permit isolation of cells by chemical selection. Stable long-term vectors can be maintained in cells as episomal, freely replicating entities by 30 using regulatory elements of viruses. Cell lines can also be produced which have integrated the vector into the genomic DNA. In this manner, the gene CA 02203706 1997-04-2~

product is produced on a continuous basis.
Vectors are introduced into recipient cells by various methods including calcium phosphate, strontium phosphate, electroporation, lipofection, DEAE
dextran, microinjection, or by protoplast fusion. Alternatively, the cDNA can be introduced by infection using viral vectors.
Eukaryotic expression systems can be used for many studies of the Jak2 kinase gene and gene product including determination of proper expression and post-translational modifications for full biological activity, identifying regulatory elements located in the 5' region of the Jak2 kinase 10 gene and their role in tissue regulation of protein expression, production oflarge amounts of the normal and mutant protein for isolation and purification, to use cells expressing the Jak2 kinase protein as a functional assay system for antibodies generated against the protein or to test effectiveness of pharmacological agents, or as a component of a signal transduction system, 15 to study the function of the normal complete protein, specific portions of the protein, or of naturally occurring and artificially produced mutant proteins.
Using the techniques mentioned, the expression vectors containing the Jak2 kinase gene or portions thereof can be introduced into a variety of mammalian cells from other species or into non-mammalian cells.
The recombinant cloning vector, according to this invention, comprises the selected DNA of the DNA sequences of this invention for expression in a suitable host. The DNA is operatively linked in the vector to an expression control sequence in the recombinant DNA molecule so that normal and mutant Jak2 kinase protein can be expressed. The expression control 25 sequence may be selected from the group consisting of sequences that control the expression of genes of prokaryotic or eukaryotic cells and their viruses and combinations thereof. The expression control sequence may be selected from the group consisting of the lac system, the trp system, the tac system, the trc system, major operator and promoter regions of phage 30 lambda, the control region of the fd coat protein, early and late promoters of SV40, promoters derived from polyoma, adenovirus, retrovirus, baculovirus, CA 02203706 1997-04-2~

simian virus, 3-phosphoglycerate kinase promoter, yeast acid phosphatase promoters, yeast alpha-mating factors and combinations thereof.
The host cell which may be transfected with the vector of this invention may be selected from the group consisting of E.coli, pseudomonas, bacillus 5 subtillus, bacillus stearothermophilus, or other bacili; other bacteria, yeast, fungi, insect, mouse or other animal, plant hosts, or human tissue cells.
For the mutant Jak2 DNA sequence similar systems are employed to express and the produce the mutant protein.
In situ hybridization is a method used to detect the expression of Jak2 10 kinase protein. In situ hybridization relies upon the hybridization of a specifically labelled nucleic acid probe to the cellular RNA in individual cells or tissues. Therefore, it allows the identification of mRNA within intact tissues, such as the brain. In this method, oligonucleotides corresponding to unique portions of the Jak2 kinase gene are used to detect specific mRNA species in 15 the brain.
!n this method a rat is anesthetized and transcardially perfused with cold PBS, followed by perfusion with a formaldehyde solution. The brain or other tissues is then removed, frozen in liquid nitrogen, and cut into thin micron sections. The sections are placed on slides and incubated in 20 proteinase K. Following rinsing in DEP, water and ethanol, the slides are placed in prehybridization buffer. A radioactive probe corresponding to the primer is made by nick translation and incubated with the sectioned brain tissue. After incubation and air drying, the labeled areas are visualized by autoradiography. Dark spots on the tissue sample indicate hybridization of 25 the probe with brain mRNA which demonstrates the expression of the protein.

Transgenic Animal Models The creation of transgenic animal models for malignancy and autoimmune diseases characterized by increased Jak2 kinase activity is 30 important to the understanding of the function of the kinase in intracellularsignalling, the etiology of such disease, and for the testing of possible CA 02203706 1997-04-2~

therapies. In general, techniques of generating transgenic animals are widely accepted and practiced. A laboratory manual on the manipulation of the mouse embryo, for example, is available detailing standard laboratory techniques for the production of transgenic mice (41).
There are several ways in which to create an animal model in which the Jak2 kinase gene is expressed. One could simply generate a specific mutation in the mouse Jak2 kinase gene as one strategy. Secondly a wild type human gene and/or a humanized murine gene could be inserted into the animals genome by homologous recombination. It is also possible to insert a mutant (single or multiple) human gene as genomic or minigene construct using wild type or mutant or artificial promoter elements. More commonly, knock-out of the endogenous murine genes may be accomplished by the insertion of artificially modified fragments of the endogenous gene by homologous recombination. The modifications include insertion of mutant stop codons, the deletion of DNA sequences, or the inclusion of recombination elements (lox p sites) recognized by enzymes such as Cre recombinase.
To inactivate the mouse Jak2 kinase gene chemical or x-ray mutagenesis of mouse gametes, followed by fertilization, can be applied.
Heterozygous offspring can then be identified by Southern blotting to - demonstrate loss of one allele by dosage, or failure to inherit one parental allele using RFLP markers.
To create a transgenic mouse, a mutant or normal version of the human Jak2 kinase gene can be inserted into a mouse germ line using standard techniques of oocyte microinjection or transfection or microinjection into stem cells. Alternatively, if it is desired to inactivate or replace the endogenous Jak2 kinase gene, homologous recombination using embryonic stem cells may be applied.
For oocyte injection, one or more copies of the mutant or normal Jak2 kinase gene can be inserted into the pronucleus of a just-fertilized mouse oocyte. This oocyte is then reimplanted into a pseudo-pregnant foster CA 02203706 1997-04-2~

mother. The liveborn mice can then be screened for integrants using analysis of tail DNA for the presence of human Jak2 kinase gene sequences. The transgene can be either a complete genomic sequence injected as a YAC or chromosome fragment, a cDNA with either the natural promoter or a 5 heterologous promoter, or a minigene containing all of the coding region and other elements found to be necessary for optimum expression.
Retroviral infection of early embryos can also be done to insert the a mutant human Jak2 kinase gene. In this method, the mutant human Jak2 kinase gene is inserted into a retroviral vector which is used to directly infect 10 mouse embryos during the early stages of development to generate a chimera, some of which will lead to germline transmission.
Homologous recombination using stem cells allows for the screening of gene transfer cells to identify the rare homologous recombination events.
Once identified, these can be used to generate chimeras by injection of 15 mouse blastocysts, and a proportion of the resulting mice will show germline transmission from the recombinant line. This methodology is especially useful if inactivation of the human Jak2 kinase gene is desired. For example, inactivation of the Jak2 kinase gene can be done by designing a DNA
fragment which contains sequences from a Jak2 kinase exon flanking a 20 selectable marker. Homologous recombination leads to the insertion of the marker sequences in the middle of an exon, inactivating the Jak2 kinase gene. DNA analysis of individual clones can then be used to recognize the homologous recombination events.
It is also possible to create mutations in the mouse germline by 25 injecting oligonucleotides containing the mutation of interest and screening the resulting cells by PCR.
This embodiment of the invention has the most significant commercial value as a mouse model for malignancy and in particular for Iymphoblastic leukemia. Because of the high percentage of sequence conservation 30 between human and mouse it is contemplated that an orthologous gene will exist also in many other species. It is thus contemplated that it will be CA 02203706 1997-04-2~

possible to generate other animal models using similar technology.

Screening for Dise~ e In another embodiment of the invention the knowledge of the human 5 Jak2 kinase sequence provides for screening for various malignant diseases and in particular leukemia and autoimmune disease involving hyperactivated Jak2 kinase in which the hyperactivation is due to a mutant Jak2 kinase gene.
People with at a risk for cancer or, individuals not previously known to be at risk, or people in general may be screened routinely using probes to 10 detect the presence of a mutant Jak2 kinase gene by a variety of techniques.
Genomic DNA used for the diagnosis may be obtained from body cells, such as those present in the blood, tissue biopsy, surgical specimen, or autopsy material. The DNA may be isolated and used directly for detection of a specific sequence or may be PCR amplified prior to analysis. RNA or cDNA
15 may also be used. To detect a specific DNA sequence hybridization using specific oligonucleotides, direct DNA sequencing, restriction enzyme digest, RNase protection, chemical cleavage, and ligase-mediated detection are all methods which can be utilized. Oligonucleotides specific to mutant sequences can be chemically synthesized and labelled radioactively with 20 isotopes, or non-radioactively using biotin tags, and hybridized to individual DNA samples immobilized on membranes or other solid-supports by dot-blot or transfer from gels after electrophoresis. The presence or absence of these mutant sequences are then visu~ ed using methods such as autoradiography, fluorometry, or colorimetric reaction. Suitable PCR primers 25 can be generated which are useful for example in amplifying portions of the subject sequence containing identified mutations.
Direct DNA sequencing reveals sequence differences between normal and mutant Jak2 kinase DNA. Cloned DNA segments may be used as probes to detect specific DNA segments. PCR can be used to enhance the 30 sensitivity of this method. PCR is an enzymatic amplification directed by sequence-specific primers, and involves repeated cycles of heat denaturation CA 02203706 1997-04-2~

of the DNA, annealing of the complementary primers and extension of the annealed primer with a DNA polymerase. This results in an exponential increase of the target DNA.
Other nucleotide sequence amplification techniques may be used, such as ligation-mediated PCR, anchored PCR and enzymatic amplification as would be understood by those skilled in the art.
Sequence alterations may also generate fortuitous restriction enzyme recognition sites which are revealed by the use of appropriate enzyme digestion followed by gel-blot hybridization. DNA fragments carrying the site (normal or mutant) are detected by their increase or reduction in size, or by the increase or decrease of corresponding restriction fragment numbers.
Genomic DNA samples may also be amplified by PCR prior to treatment with the appropriate restriction enzyme and the fragments of different sizes are visualized under UV light in the presence of ethidium bromide after gel 1 5 electrophoresis.
Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels.Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. Small deletions may also be detected as changes in the migration pattern of DNA heteroduplexes in non-denaturing gel electrophoresis. Alternatively, a single base substitution mutation may be detected based on differential primer length in PCR. The PCR products of the normal and mutant gene could be differentially detected in acrylamide gels.
Nuclease protection assays (S1 or ligase-mediated) also reveal sequence changes at specific locations. Alternatively, to confirm or detect a polymorphism restriction mapping changes ligated PCR, ASO, REF-SSCP
and SSCP may be used. Both REF-SSCP and SSCP are mobility shift assays which are based upon the change in conformation due to mutations.
DNA fragments may also be visualized by methods in which the individual DNA samples are not immobilized on membranes. The probe and CA 02203706 1997-04-2~

target sequences may be in solution or the probe sequence may be immobilized. Autoradiography, radioactive decay, spectrophotometry, and fluorometry may also be used to identify specific individual genotypes.
According to an embodiment of the invention, the portion of the DNA
5 segment that is informative for a mutation, can be amplified using PCR. The DNA segment immediately surrounding a specific mutation acquired from peripheral blood or other tissue samples from an individual can be screened using constructed oligonucleotide primers. This region would then be amplied by PCR, the products separated by electrophoresis, and transferred to 10 membrane. Labelled probes are then hybridized to the DNA fragments and autoradiography performed.
In accordance with another embodiment of the present invention are antibodies which recognize epitopes within the Jak2 kinase protein and which can be raised to provide information on the characteristics of the protein as 15 well as for the mutant Jak2 kinase protein. Generation of antibodies would enable the visualization of the protein in cells and tissues using Western blotting. In this technique, proteins are run on polyacrylamide gel and then transferred onto nitrocellulose membranes. These membranes are then incubated in the presence of the antibody (primary), then following washing 20 are incubated to a secondary antibody which is used for detection of the protein-primary antibody complex. Following repeated washing, the entire complex is visualized using colourimetric or chemiluminescent methods.
Antibodies to the Jak2 kinase protein also allow for the use of immunocytochemistry and immunofluorescence techniques in which the 25 proteins can be visualized directly in cells and tissues. This is most helpful in order to establish the subcellular location of the protein and the tissue specificity of the protein.
In general, methods for the preparation of antibodies are well known (42). In order to prepare polyclonal antibodies, fusion proteins containing 30 defined portions or all of the Jak2 kinase protein or specific Jak2 kinase generated mutants can be synthesized in bacteria by expression of CA 02203706 1997-04-2~

corresponding DNA sequences in a suitable cloning vehicle. The protein can then be purified, coupled to a carrier protein and mixed with Freund's adjuvant (to help stimulate the antigenic response by the rabbits) and injected into rabbits or other laboratory animals. Alternatively, protein can be isolated5 from cultured cells expressing the protein. Following booster injections at bi-weekly intervals, the rabbits or other laboratory animals are then bled and the sera isolated. The sera can be used directly or purified prior to use, by afffinity chromatography. The sera can then be used to probe protein extracts run on a polyacrylamide gel to identify the Jak2 kinase protein or mutant protein.
10 Alternatively, synthetic peptides can be made to the antigenic portions of the protein and used to innoculate the animals.
To produce monoclonal Jak2 kinase antibodies, cells actively expressing the protein are cultured or isolated from tissues and the cell extracts isolated. The extracts or recombinant protein extracts, containing the 15 Jak2 protein protein, are injected in Freund's adjuvant into mice. After being injected 9 times over a three week period, the mice spleens are removed and resuspended in phosphate buffered saline (PBS). The spleen cells serve as a source of Iymphocytes, some of which are producing antibody of the appropriate specificity. These are then fused with a permanently growing 20 myeloma partner cell, and the products of the fusion are plated into a numberof tissue culture wells in the presence of a selective agent such as HAT. The wells are then screened to identify those containing cells making useful antibody by ELISA. These are then freshly plated. After a period of growth, these wells are again screened to identify antibody-producing cells. Several 25 cloning procedures are carried out until over 90% of the wells contain singleclones which are positive for antibody production. From this procedure a stable line of clones is established which produce the antibody. The monoclonal antibody can then be purified by afffinity chromatography using Protein A Sepharose.

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DrugScreening Assays In accordance with another embodiment of the present invention there is provided assays for screening upstream activators and downstream effectors of human Jak2 kinase protein. Cell culture systems in which the 5 human Jak2 kinase gene has been transfected and is being expressed can be tested with a number of agents to identify specific activators of the kinase through labelled phosphorylation studies of the protein. Such assays are also useful in identifying the downstream protein effects of such activation and their effects on transcription of various genes. Mutant forms of the human 10 Jak2 kinase can also be transfected into cell lines in order to study which specifically constructed mutations in the gene sequence which lead to hyperactivation of the kinase and eventually lead to malignant transformation of the cells. Specifically, a glutamic acid to Iysine substitution at amino acidresidue 695 which is found in the JH2 kinase domain results in 15 hyperactivation of the kinase producing hyperphosphorylation of down stream proteins and which leads to malignant phenotypes (43).
Such culture systems enable the screening for pharmacological agents which will prevent activation of both normal and mutant hyperactivated Jak2 kinase as well as agents which will inhibit downstream phosphorylation 20 effects of the activated Jak2 kinase protein. Transfected culture systems also permit for the identification of kinase inhibitors which will be useful as therapeutic compositions for treating malignancies and immune disorders involving activated Iymphocytes.

25 Therapies An important aspect of the biochemical studies using the genetic information of this invention is the development of therapies to circumvent or overcome the effect of hyperactivated Jak2 kinase gene product, and thus prevent, treat, control serious symptoms or cure malignant disease or 30 autoimmune disease. In view of the expression of the Jak2 kinase gene in a variety of tissues, especially hematopoietic tissues one has to recognize that CA 02203706 1997-04-2~

hyperactivated Jak2 kinase may lead to a variety of Iymphoblastic leukemias and autoimmune disorders involving activated Iymphocytes. Hence, in considering various therapies, it is understood that such therapies may be targeted at various hematopoietic and Iymphoid tissues where Jak2 kinase is over expressed.
In accordance with another embodiment of the present invention there is provided gene therapy as another potential therapeutic approach in which normal copies of the Jak2 kinase gene are introduced into patients to successfully code for normal Jak2 kinase protein in several different affected 10 cell types. Mutated copies of the Jak2 kinase gene in which the protein product is inactivated can also be introduced into patients. The gene must be delivered to those cells in a form in which it can be taken up and code for sufficient protein to provide effective function. Alternatively, in some mutantsit has been possible to prevent disease by introducing another copy of the 15 homologous gene bearing a second mutation in that gene or to alter the mutation, or use another gene to block its effect.
Retroviral vectors can be used for somatic cell gene therapy especially because of their high efficiency of infection and stable integration and expression. The targeted cells however must be able to divide and the 20 expression of the levels of normal protein should be high. The full length Jak2 kinase gene or a mutant inactivated gene can be cloned into a retroviral vector and driven from its endogenous promoter or from the retroviral long terminal repeat or from a promoter specific for the target cell type of interest(such as hematopoietic and Iymphoid cells).
Other viral vectors which can be used include adeno-associated virus, vaccinia virus, bovine papilloma virus, or a herpesvirus such as Epstein-Barr virus.
Gene transfer could also be achieved using non-viral means requiring infection in vitro. This would include calcium phosphate, DEAE dextran, 30 electroporation, and protoplast fusion. Liposomes may also be potentially beneficial for delivery of DNA into a cell. Although these methods are CA 02203706 1997-04-2~

available, many of these are lower effficiency.
Transplantation of normal genes or mutated genes which code for an inactive Jak2 kinase into a targetted affected area of the patient can also be useful therapy for leukemia or associated malignancies. In this procedure, a Jak2 kinase gene is transferred into a cultivatable cell type such as Iymphoid cells, either exogenously or endogenously to the patient. These cells are then injected serotologically into the disease affected tissue(s).
The invention also provides a method for reversing a transformed phenotype resulting from the over expression of the Jak2 kinase human 10 gene sequence and/or hyperactivation of a mutant Jak2 kinase protein product which is responsible for transformation of cells into a malignant phenotype. Antisense based strategies can be employed to explore gene function, inhibit gene function and as a basis for therapeutic drug design. The principle is based on the hypothesis that sequence specific suppression of 15 gene expression can be achieved by intracellular hybridization between mRNA and a complementary anti-sense species. It is possible to synthesize anti-sense strand nucleotides that bind the sense strand of RNA or DNA with a high degree of specificity. The formation of a hybrid RNA duplex may interfere with the processing/transporVtranslation and/or stability of a target 20 mRNA.
Hybridization is required for an antisense effect to occur. Antisense effects have been described using a variety of approaches including the use of antisense oligonucleotides, injection of antisense RNA, DNA and transfection of antisense RNA expression vectors.
Therapeutic antisense nucleotides can be made as oligonucleotides or expressed nucleotides. Oligonucleotides are short single strands of DNA
which are usually 15 to 20 nucleic acid bases long. Expressed nucleotides are made by an expression vector such as an adenoviral, retroviral or plasmid vector. The vector is administered to the cells in culture, or to a patient, 30 whose cells then make the antisense nucleotide. Expression vectors can be designed to produce antisense RNA, which can vary in length from a few . CA 02203706 1997-04-2~

dozen bases to several thousand.
Antisense effects can be induced by control (sense) sequences. The extent of phenotypic changes are highly variable. Phenotypic effects induced by antisense are based on changes in criteria such as biological endpoints, 5 protein levels, protein activation measurement and target mRNA levels.
Multidrug resistance is a useful model for the study of molecular events associated with phenotypic changes due to antisense effects since the MDR
phenotype can be established by expression of a single gene mdr1 (MDR
gene) encoding P-glycoprotein (a 170 kDa membrane glycoprotein, ATP-10 dependent efflux pump).
In the present invention, mammalian cells in which the Jak2 kinasehuman cDNA has been transfected and which express a malignant phenotype, can be additionally transfected with anti-sense Jak2 kinase nucleotide DNA sequences which hybridize to the Jak2 kinase gene in order 15 to inhibit the transcription of the gene and reverse or reduce the malignant phenotype. Alternatively, portions of the Jak2 kinase gene can be targeted with an anti-sense Jak2 kinase sequence specific for the kinase domains or the unique amino terminal sequence which may be responsible for the malignant phenotype. Expression vectors can be used as a model for anti-20 sense gene therapy to target the Jak2 kinase which is expressed in malignantcells. In this manner malignant cells and tissues can be targeted while allowing healthy cells to survive. This may prove to be an effective treatment for malignancies induced by Jak2 kinase.
Immunotherapy is also possible for the treatment of certain 25 autoimmune or leukemic disease. Antibodies can be raised to a mutant hyperactive Jak2 kinase protein (or portion thereofl and then be administered to bind or block the mutant protein and its deliterious effects. Simultaneously,expression of the normal protein product could be encouraged.
Administration could be in the form of a one time immunogenic preparation or 30 vaccine immunization. An immunogenic composition may be prepared as injectables, as liquid solutions or emulsions. The Jak2 kinase protein may be CA 02203706 1997-04-2~

mixed with pharmaceutically acceptable excipients compatible with the protein. Such excipients may include water, saline, dextrose, glycerol, ethanol and combinations thereof. The immunogenic composition and vaccine may further contain auxiliarry substances such as emulsifying agents 5 or adjuvants to enhance effectiveness. Immunogenic compositions and vaccines may be administered parenterally by injection subcutaneously or intramuscularly.
The immunogenic preparations and vaccines are administered in such amount as will be therapeutically effective, protective and immunogenic.
10 Dosage depends on the route of administration and will vary according to the size of the host.
The above disclosure generally describes the present invention. A
more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for purposes of 15 illustration and are not intended to limit the scope of the invention. Changes in the form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitations.

EXAMPLES
Methods of molecular genetics, protein and peptide biochemistry and immunology referred to but not explicitly described in this disclosure and examples are reported in the scientific literature and are well known to those skilled in the art.
The examples are described for the purposes of illustration and are not intended to limit the scope of the invention.

Example 1: Cloning and Characterisation of human Jak2 MATERIALS AND METHODS.

CA 02203706 1997-04-2~

Library Screening and RT-PCR cloning.
A human thymus cDNA ~gt11 library (Clontech) was screened according to standard protocols (23), using the full length murine Jak2 cDNA.
Plaques were transferred to ICN Biotrans nylon filters and screened by 5 hybridization at 65~ C in 5xSSC, 5xDenhart's solution, 0.1% SDS solution.
Filters were autoradiographed overnight using Kodak XAR-5 X-ray film.
Phage DNA was prepared from positive plaques. cDNA inserts were excised, subcloned into a PUC19 vector and sequenced.
For the RT-PCR cloning, total RNA's were extracted from relevant cell 10 types according to Chomczynski and Sacchi (24). The RNA's were reverse transcribed using standard protocols (23).The resulting cDNA's were used as substrates for PCR reactions using Elongase (GIBCO-BRL) as the thermoresistant amplyfing enzyme. The PCR products were subcloned into a pUC19 vector and sequenced with Sequenase (Amersham).
15 Cloning the 5' end of the human Jak2 DNA fragment Total RNA from peripheral blood Iymphocytes (PBL) was reverse transcribed and the product of this reaction was used as a substrate for PCR
amplification. For 5' end we used a degenerate oligonucleotide primer to the mouse sequence beginning at the start codon and an antisense non-20 degenerate primer to the human ORF obtained from the library (nucleotides 1416-1394).
Oligodeoxynucleotides: - 5' primer: ATG GG(ACGT) ATG GC(ACGT) TG(CT) CT(ACGT) A

CA 02203706 1997-04-2~

3' primer: GM GTT CTT CTT TGT CCC ACT G
5 cycles at 94~C x 30s, 50~C x 30s, 68~C x 60s 35 cycles 94~C x 30s, 62~C x 30s, 68~C x 60s Cloning the 3' end of the human Jak2 DNA fragment Total RNA from PBL was reverse transcribed using oligo (dT),2GC as a primer. The production of this action was used as a substrate for PCR
amplification. We designed a non-degenerate primer to the 1.8 Kb ORF from nucleotide 3055 to 3085 and an antisense non-degenerate primer to the murine Jak2 untranslated sequence.
10 Oligodeoxynucleotides: - 5' primer: ATA TTC TGG TAT GCT CGA CM TCA
CTG ACA
3' primer: TCA TCC AGC CAT GTT ATC CCT TAC
TTG ATC
5 cycles at 94~C x 30s, 40~C x 30s, 68~C x 60s 35 cycles at 94~C x 30s, 50~C x 30s, 68~C x 60s Cloning the full length human Jak2 cDNA from pre-B leukemia G2 cells Total RNA from G2 cells (pre-B leukemia cell line) was reverse transcribed using standard protocols (23). The product of this reaction was used as a substrate for PCR amplification. For the 5' end we used a primer starting at the start codon of human Jak2 and an antisense primer starting at the stop codon of human Jak2.
Oligodeoxynucleotides: - 5' primer: ATG GGG ATG GCT TGC CTT ACG ATG
ACA GM

CA 02203706 1997-04-2~

3' primer: TCA TCC AGC CAT GTT ATC CCT TAC
TTG ATC
30 cycles at 94~C x 30s, 60~C x 30s, 68~C x 5 min Chromosomal Localization Positional mapping of the Jak2 gene was performed by fluorescence in situ hybridization (FISH) (25) to normal human Iymphocyte chromosomes counterstained with propidium iodide and 4'6-diamidin-2-phenylindole-dihydrochloride (DAPI). Both a 1.5 kb Jak2 cDNA probe and a corresponding genomic probe obtained by screening a P1-derived artificial chromosome 10 (PAC) library were labeled with biotin and was detected with avidin-fluorescein isothiocyanate (FITC) (25). Images of 20 well-spread metaphase preparations were captured by a thermoelectrically cooled charge coupled camera (Photometrics, Tucson, AZ). Separate images of DAPI banded chromosomes and of FITC targeted chromosomes were obtained (26).
15 Hybridization signals were acquired and merged using image analysis software and pseudo colored blue (DAPI) and yellow (FITC) and overlaid electronically (27).
Example 2: Expression of Jak2 in human tiss!~es Northern Blot Analysis 20 Commercial membrane containing 2 ~g of polyadenylated mRNA per lane of various human tissues, were probed and washed according to the manufacturer specifications (Clontech). The membrane was hybridized. The DNA probes were labeled with 32P by random priming (Boehringer) to a specific activity of 2x1 o8 cpm/mg of DNA.

CA 02203706 1997-04-2~

Antibody preparation The rabbit polyclonal anti-human Jak2 antibody, directed against the synthetic peptide VLRVDQVRDNMAG (the unique C-terminus of human Jak2) was prepared by Research Genetics Inc. (Huntsville, Alabama).
5 This antibody specifically immunoprecipitated human Jak2 but was unable to recognize the denatured protein on Western blots.
Cell preparation and stimulation Peripheral blood mononuclear cells were obtained from a healthy volunteer. Mononuclear cells were isolated by Ficoll-Hypaque gradient 10 centrifugation. Adherent cells were removed by adherence to plastic dishes for 60 minutes at 37~C. Separation of T cells from B cells was performed by Ficoll-Hypaque centrifugation of cells, rosetting with neuroaminidase-treated sheep erythrocytes (28). T and B cells were cultured in RPM1 with 10% FCS
at 37~C for 24 or 48 hours in the presence of 10 llg/ml PHA and 2 mg/ml 15 staphylococcus protein A cowan (SAC) respectively. Jak2 was then immunoprecipitated from Iysates of 2 x 107 T or B cells with the anti-human Jak2 antibody and electrophoresed on 6% SDS-PAGE gels. After transfer to a nitrocellulose membrane, immunoblotting was performed with anti-murine Jak2 (Pharmingen) and then detected by ECL. [?]
20 Immunoprecipitation and immunoblotting Immunoprecipitation was performed as described before (13). Briefly, immunoprecipitates were prepared from Iysates of 2x107 cells in 1 ml 20 mM
Tris ( PH 7.5), 150 mM Nacl, 1% Triton X-100, 1 mM Na3VO4 buffer with anti-sera to human Jak2, and subjected to 6% SDS-PAGE. After transfer to CA 02203706 1997-04-2~

supported nitrocellulose membrane (Amersham), immunoblotting was performed with anti-murine Jak2 antibody from PharMingen and then detected by ECL (Amersham) .
In vitro transcription and translation Full length Jak3 and Jak2 cDNA's were inserted into the pcDNA3 vector (Invitrogen) downstream from a T7 promoter. Approximately 1 ~19 of Luciferase control, Jak3 and Jak2 cDNA's in pcDNA3 were added to a combined transcription and translation reticulocyte Iysate system (Promega) in the presence of T7 polymerase. An aliquot of each product were run on a 10 6% SDS-PAGE gel followed by transfer to nitrocellulose and visualized by autoradiography. The remaining Iysate from each reaction was diluted into 1 ml of 1% Triton X-100 Iysis buffer (see above) and were immunoprecipitated with human Jak2 antisera and then resolved by SDS-PAGE.

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Rcf~r~.,c~s 1. Arai K, Lee F, Miyajima A, Miyatake S, Arai N, Yokota T: Cytokines:
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4. Firmbach-Kraft 1, Byers M, Shows T, Dalla-Favera R, Krolewski JJ: Tyk2, 10 prototype of a novel class of non-receptor tyrosine kinase genes. Oncogene

5: 1329, 1990 5. Wilks AF, Harpur AG, Kurban RR, Ralph SJ, Zurcher G, Ziemiecki A: Two novel protein-tyrosine kinases, each with a second phosphotransferase-related catalytic domain, define a new class of protein kinase. Mol Cell Biol 15 11:2057,1991

6. Ihle JN, Witthuhn BA, Quelle FW, Yamamoto K, Silvennoinen O: Signaling through the hematopoietic cytokine receptors. Annu Rev Immunol 13: 369,

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8. Pawson T, Gish GD: SH2 and SH3 domains: from structure to function.
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9. Kawamura M, Mcvicar DW, Johnston JA, Blake TB, Chen Y-Q, Lal BK, Lloyd AR, Kelvin DJ, Staples JE, Ortaldo JR, O'Shea JJ: Molecular cloning of L-JAK, a Janus family protein-tyrosine kinase expressed in natural killer cells and activated leukocytes . Proc Natl Acad Sci USA 91: 6374, 19946. Ihle 5 JN, Witthuhn BA, Quelle FW, Yamamoto K, Silvennoinen O: Signaling through the hematopoietic cytokine receptors. Annu Rev Immunol 13: 369, 7. Hanks SK, Quinn AM, Hunter T: The protein kinase family: conserved features and deduced phylogeny of the catalytic domain. Science 241: 42, 8. Pawson T, Gish GD: SH2 and SH3 domains: from structure to function.
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10. Pritchard MA, Baker E, Callen DF, Sutherland GR, Wilks AF: Two members of the JAK family of protein tyrosine kinases map to chromosomes 1p31.3 and 9p24. Mammal Genome 3: 36,1992 20 11. Witthuhn BA, Silvennoinen O, Miura O, Lai KS, Cwik C, Liu ET, Ihle JN:
Involvement of the JAK3 Janus kinase in IL-2 and IL-4 signalling in Iymphoid and myeloid cells. Nature 370: 153, 1994 CA 02203706 1997-04-2~

12. Johnston JA, Kawamura M, Kirken R, Chen Y, Blake TB, Shibuya K, Ortaldo JR, McVicar DW, O'Shea JJ: Phosphorylation and activation of the JAK3 Janus kinase in response to IL-2. Nature 370: 151,1994 13. Sharfe N, Dadi HK, Roifman CM: JAK3 protein tyrosine kinase mediates 5 IL-7-induced activation of phosphatidylinositol-3' kinase. Blood 86: 2077, 14. Witthuhn BA, Quelle FW, Silvennoinen O, Yi T, Tang B, Miura O, Ihle JN:
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Structure of the JAK2 protein tyorsine kinase and its role in IL-3 signal transduction. Proc Natl Acad Sci 90: 8429,1993 17. Qelle FW, Sato N, Witthuhn BA, Inhorn R, Ernst TJ Miyajima A, Griffin JD, Ihle JN: JAK2 associates with the bc chain of the receptor for GM-CSF and its activation requires the membrane proximal region. Molec Cell Biol 14: 4335, 18. Velazquez L, Fellous M, Stark GR, Pellegrini S: A protein tyrosine kinase in the interferon alpha/beta signaling pathway . Cell 70: 313, 1992 19. Muller M, Briscoc J, Laxton C, Guschin D, Ziemiecki A, Silvennoinen O, Harpur AG, Barbieri G, Witthuhn BA, Schindler C, Pellegrini S, Wilks AF, Ihle CA 02203706 1997-04-2~

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41. Manipulating the Mouse Embryo. A laboratory Manual 2nd Ed., Brigid Hogan, Rosa, Beddington, Frank costantini, Elizabeth Lecy. Cold Spring Harbor Laboratory Press, 1994.
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CA 02203706 l997-04-2 TABLE lA

CA 02203706 l997-04-2 TABLE lB

TABLE lC

MGMACLTMTEMEGTSTSSIYQNGDISGNANSMKQIDPVLQVYLYHSLGKPEADYLTFPSGEYVAE
EICIAASKACGITPVYHNMFALMSETERIWYPPNHVFHIDESTRHNVLYRIRFYFPRWYCSGSNR
AYRHGISRGAEAPLLDDFVMSYLFAQWRHDLVHGWIKVPVTRETQEECLGTAVLHMMRIAKENDQ
TPLAIYNSISYKTFLPKCIRAKIQDYHILTRKRIRYRFRRFIQQFSQCKATARNLKLKYLINLET
LQSAFYTEKFEVKEPGSGPSGEEIFATIIITGNGGIQWSRGKHKESETLTEQDLQLYCDFPNIID
VSIKQANQEGSNESRVVTIHKQDGKNLEIIELSSLREALSFVSLIDGYYRLTADAHHYLCKEVAP
PAVLENIQSNCHGPISMVFAISKLKNAGNQTGLYVLRCSPKDFNKYFLTFAVERENVIEYKHCLI
TKNENEEYNLSGTKKNFSSLKDLLNCYQMETVRSDNIIFQFTKCCPPKPKDKSNLLVFRTNGVSD
VPTSPTLQRPTHMNQMVFHKIRNEDLIFNESLGQGTFTKIFKGVRREVGDYGQLHETEVLLKVLD
KAHRNYSESFFEAASMMSKLSHKHLVLNYGVCVCGDENIMVQEFVKFGSLDTYLKKNKNCINILW
KLEVAKQLAWAMHFLEENTLIHGNVCAKNIQLIREEDRKTGNPPFIKLSEPGISITVLPKDILQE
RIPWVPPECIENPKNLNLATDKWSFGTTLWEICSGGDKPLSALDSQRKLQFYEDRHQLPAPKWAE
LANLINNCMDYEPDFRPSFRAIIRDLNSLFTPDYELLTENDMLPNMRNGALGFSGAFEDRDPTQF
EERHLKFLQQLGKGNFGSVEMCRYDPLQDNTGEVVAVKKLQHSTEEHLRDFEREIEILKSLQHDN
IVKYKGVCYSAGRRNLKLIMEYLPYGSLRDYLQKHKERIDHIKLLQYTSQICKGMVYLGTKRYIH
RDLATRNILVENENRVKIGDFGLTKVLPQDKEYYKVKEPGESPIFWYAPESLTESKFSVASDVWS
FGVVLYELFTYIEKSKSPPAEFMRMIGNDKQGQMIVFHLIELLKNNGRLPRPDGCPDEIYMIMTE
CWNNNVNQRPSFRDLVLRVDQVRDNMAG*

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