WO1999054450A1 - Assay methods for modulators of hematopoietic cell phosphatase - Google Patents

Abstract

DNA encoding HCP has been cloned and characterized. The recombinant protein is capable of forming biological active HCP protein. The cDNA has been expressed in recombinant host cells that produce active recombinant protein. The recombinant protein is also purified from the recombinant host cells. In addition, the recombinant protein is utilized to establish a method for identifying modulators of the HCP activity.

Description

TITLE OF THE INVENTION

ASSAY METHODS FOR MODULATORS OF HEMATOPOIETIC CELL PHOSPHATASE

BACKGROUND OF THE INVENTION

In recent years, the importance of protein phosphorylation, specifically on tyrosine residues, and their role in signal transduction has become increasingly evident. The activation of protein tyrosine kinases has been associated with many growth factor receptor signal transduction pathways. For example, the binding of EPO with its receptor induces the phosphorylation of several proteins, including the EPO receptor (EPOR) itself

(Dusanter-Fourt et al, 1992). Tyrosine phosphorylation of the EPOR was later shown to be the result of the association of JAK2 with the EPOR (Witthuhn et al, 1993). The activation of JAK2 causes stimulation of the signal transduction pathway through the phosphorylation of one or more intracellular substrates. JAK2 has been shown to be associated with several growth factor receptors and involved in the JAK-STAT signal transduction pathway (Argetsinger et al., 1993, Darnell et al, 1994).

Hematopoietic cell phosphatase (also referred to as: HCP, SH-PTP1, PTP1C, SHP1, PTPN6) is a 68 kDa. non-membrane associated protein, found primarily in hematopoietic cells and to some extent in epithelial cells (Zhao et al., 1993, Pei et al,

1993, Plutzky et al., 1992). The gene for HCP has been localized to human chromosome 12pl2-pl3; a region associated with chromosomal rearrangements in approximately 10% of childhood acute lymphocytic leukemia cases (Yi et al, 1992). The phosphatase contains two src homology 2 (SH2) domains at its amino terminus and a phosphatase domain at the C-terminal portion of the molecule. HCP has been shown to associate with several growth factor receptors following ligand stimulation including c-kit, IL-3 and EPO (Yi and Ihle, 1993, Yi et al., 1993, Yi et al. 1992) receptors and acts as a negative regulator of receptor activation. - 2

Following stimulation by ligand, HCP binds to the receptor through its SH2 domain (Yi and Ihle, 1993, Yi et al., 1993, Pei et al., 1994, Yi et al., 1995, Klingmiiller et al, 1995). Mutagenesis of the HCP N-terminal SH2 domain at the conserved SH2 binding site (FLVRE) eliminated binding to both the IL-3 receptor β and the EPOR. Antisense experiments that decrease the HCP content of IL-3 responsive cells renders those cells hypersensitive to IL-3 while over expression of HCP in IL-3 dependent cells suppresses the ability of those cells to proliferate in response to IL-3. The fact that HCP binds to the IL-3 receptor β suggests that HCP may also interact with IL-5 and GM-CSF since all three share a common β chain. However, there have been no published reports indicating that

HCP regulates the receptors for IL-5 or GM-CSF. (Yi et al., 1993). These data support the role of HCP as a negative regulator of hematopoietic signal transduction.

Studies of HCP regulation of EPO receptor activity have centered upon identifying the specific region of the cytoplasmic domain that binds HCP. The EPOR contains 8 tyrosine residues within the C-terminal 70 amino acids. The identification of the tyrosine residues of the EPOR involved with HCP binding was first determined using short peptides containing phosphotyrosine residues of the EPOR (Yi et al., 1995). Four of the eight p-tyrosine residues were shown to interact with HCP. One of these tyrosine residues, Y455, appears to exhibit increased HCP binding relative to the other positions. Additional studies using mutant EPO receptors also indicate that HCP binds to the receptor at tyrosine residue 455 (referred to as Y429 by Klingmiiller et al) through the amino terminal SH2 domain (Klingmiiller et al, 1995). Once HCP is recruited to the EPOR, it dephosphorylates JAK2, effectively down regulating the kinase. Cells transfected with a mutant EPOR unable to bind HCP were found to have increased sensitivity to EPO, requiring ten fold less for maximal stimulation (Klingmiiller et al., 1995). Inhibition of

HCP by anti-sense oligonucleotides enhanced hemoglobinization of SKT6 cells (Sharlow et al., 1997) demonstrating that HCP negatively regulates erythroid differentiation. 3 -

Collectively, these results have led to the following model: upon ligand stimulation, the EPOR becomes phosphorylated on tyrosine residues by the JAK2 kinase that is already associated with the receptor or is recruited to the receptor following the binding of EPO. HCP, through its SH2 domains (specifically the N-terminal SH2) now associates with the EPOR at amino acid Y455, and possibly other residues. Once HCP binds to the EPOR, a conformational change occurs which allows the phosphatase domain of HCP to dephosphorylate JAK2 and down regulate the signal transduction cascade. The N-terminal SH2 domain of HCP may inhibit the phosphatase activity of HCP by acting through an autoinhibitory binding site. In vitro biochemical data suggest that HCP lacking the SH2 domains may have a higher affinity for substrates as compared to the intact molecule (Townley et al., 1993). HCP may be in an inactive state until the SH2 domain binds to a phosphorylated receptor, essentially eliminating the inhibition and activating the phosphatase. HCP has not been shown to be associated with a receptor substrate prior to ligand binding and tyrosine phosphorylation of the receptor substrate is required for HCP binding. This scenario may exist for other hematopoietic receptors, although it has only been demonstrated for the EPOR.

Mice that are genetically deficient for HCP exhibit the motheaten (me) or motheaten viable (mev) phenotype. These mice lack HCP (me) or have slight HCP activity

(mev), resulting in severe immunodeficiency and death within 3 to 9 weeks of age (Schultz, 1988). Hematopoietic changes in these mice include the abnormal growth of macrophages that accumulate in the lungs of these animals. Also, erythropoiesis shifts from the bone marrow to the spleen with an increase of erythroid precursors (CFU-E) found in the spleen (Van Zant and Schultz, 1989). The CFU-Es from the motheaten mice demonstrate an increased sensitivity to EPO and may be capable of colony formation in the absence of EPO (Schultz et al., 1993). The HCP deficiency is the result of a single cytidine deletion that generates a splice donor site in the N-terminal SH2 domain. The 4 -

additional splice site causes a 101 base pair deletion and a frameshift resulting in a truncated protein. The me mice have impaired T and B cells exhibiting severe B cell deficiency and impaired NK cell function. Surprisingly, these mice also have a high degree of autoantibody production (Schultz et al., 1993), possibly due to HCP involvement in antigen receptor signalling and tolerance thresholds. HCP is believed to be a negative regulator of immunoglobulin antigen receptor signalling and determines the threshold of negative selection for these cells (Cyster and Goodnow, 1995). The motheaten mouse model exemplifies the overall involvement of HCP in hematopoiesis and the negative role that it plays in hematopoietic cell signal transduction.

SUMMARY OF THE INVENTION

A DNA molecule encoding a human hematopoietic cell phosphatase (HCP), has been cloned and characterized and it represents a member of a subclass of tyrosine phosphatases known as SH2-containing protein tyrosine phosphatases. Using a recombinant expression system, functional DNA molecules encoding full length HCP and its phosphatase domain have been expressed. The biological and structural properties of these proteins are disclosed. The recombinant proteins are useful to identify modulators of the HCP enzymatic activity and/or HCP -receptor interaction activity. Modulators identified in the assay disclosed herein are useful as therapeutic agents. The recombinant DNA molecules and portions thereof, are useful for isolating homologues of the DNA molecules, identifying and isolating genomic equivalents of the DNA molecules, and identifying, detecting or isolating mutant forms of the DNA molecules.

BRIEF DESCRIPTION OF THE DRAWING

Figure 1- HCP Catalytic Assay: Reduction of HCP enzymatic activity by Vanadate is shown Figure 2- The ability of an interactive peptide to enhance the enzymatic activity of HCP, and the ability of vanadate to inhibit the activity of enhanced HCP activity is shown.

Figure 3- HCP Interactive Assay: Reduction of enzymatic activity, by the modulator vanadate, in the presence of a peptide known to interact with HCP is shown.

Figure 4, Panels A, B, C, and D - The nucleotide sequence of the full-length

GST-HCP fusion gene is shown in Panel A; the amino acid sequence of the full-length

GST-HCP protein is shown in Panel B; the nucleotide sequence of the catalytic domain of GST-HCP fusion gene is shown in Panel C; and the amino acid sequence of the catalytic domain of GST-HCP protein is shown in Panel D.

DETAILED DESCRIPTION

The present invention relates to DNA encoding human HCP that was isolated from hematopoietic cells. HCP, as used herein, refers to protein that can specifically function as a hematopoietic cell phosphatase.

The purpose of the invention is to utilize HCP phosphatase domain, which retains its enzymatic activity, in an assay to screen compounds that specifically inhibit the protein's enzymatic activity. In addition, full length HCP is utilized in an assay to screen for compounds that inhibit the HCP phosphatase activity by blocking the interaction of

HCP with the cytoplasmic domain of the erythropoietin receptor (EPOR). A description of the two assays can be found in the text of the patent document. Small molecules that either inhibit the HCP phosphatase domain or prevent HCP from binding to a receptor would effectively inhibit this enzyme's functions. The advantage of small molecules that inhibit the action of HCP includes augmentation of the effect of EPO. If HCP is inhibited, then the negative effect of HCP would be reduced and the cells would become more sensitive to EPO. Therefore, a lower dose of therapeutic EPO may be utilized. Since HCP has been known to interact with other growth factor receptors including, but not limited to IL-3, IL-5, GM-CSF and c-kit (Yi and Ihle, 1993, Yi, et al., 1993), these receptors may also be affected by HCP inhibitors.

The complete amino acid sequence of human HCP was previously known, as 5 was the complete nucleotide sequence encoding human HCP. It is expected that a wide variety of cells and cell types will contain the described HCP. Vertebrate cells capable of producing HCP include, but are not limited to hematopoietic cells isolated from mammals, that show HCP activity. Other cells and cell lines may also be suitable for use to isolate HCP cDNA. Cells that possess HCP expression may be suitable for o the isolation of HCP DNA or mRNA.

Any of a variety of procedures known in the art may be used to molecularly clone HCP DNA. These methods include, but are not limited to, direct functional expression of the HCP genes following the construction of a HCP containing cDNA library in an appropriate expression vector system. Another method is to screen HCP- 5 containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a labelled oligonucleotide probe designed from the amino acid sequence of the HCP subunits. An additional method consists of screening a HCP-containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA encoding the HCP protein. This partial cDNA is obtained by the specific PCR amplification of o HCP DNA fragments through the design of degenerate oligonucleotide primers from the amino acid sequence of the purified HCP protein.

Another method is to isolate RNA from HCP producing cells and translate the RNA into protein via an in vitro or an in vivo translation system. The translation of the RNA into a peptide a protein will result in the production of at least a portion of the 5 HCP protein which can be identified by, for example, immunological reactivity with an anti-HCP antibody or by biological activity of HCP protein. In this method, pools of RNA isolated from HCP-producing cells can be analyzed for the presence of an RNA that encodes at least a portion of the HCP protein. Further fractionation of the RNA pool can be done to purify the HCP RNA from non-HCP RNA. The peptide or protein produced by this method may be analyzed to provide amino acid sequences, which in turn are used to provide primers for production of HCP cDNA, or the RNA used for translation can be analyzed to provide nucleotide sequences encoding HCP and produce probes for the production of HCP cDNA. This method is known in the art and can be found in, for example, Maniatis, T., Fritsch, E.F., Sambrook, J. in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 1989.

It is readily apparent to those skilled in the art that other types of libraries, as well as libraries constructed from other cells or cell types, may be useful for isolating

HCP-encoding DNA. Other types of libraries include, but are not limited to, cDNA libraries derived from other cells, from organisms other than humans, and genomic DNA libraries that include YAC (yeast artificial chromosome) and cosmid libraries.

It is readily apparent to those skilled in the art that suitable cDNA libraries may be prepared from cells or cell lines which have HCP activity. The selection of cells or cell lines for use in preparing a cDNA library to isolate HCP cDNA may be done by first measuring cell associated HCP activity.

Preparation of cDNA libraries can be performed by standard techniques well known in the art. Well known cDNA library construction techniques can be found for example, in Maniatis, T., Fritsch, E.F., Sambrook, J., Molecular Cloning: A

Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989).

It is also readily apparent to those skilled in the art that DNA encoding HCP may also be isolated from a suitable genomic DNA library. Construction of genomic DNA libraries can be performed by standard techniques well known in the art. Well known genomic DNA library construction techniques can be found in Maniatis, T., Fritsch, E.F., Sambrook, J. in Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989). In order to clone the HCP gene by the above methods, the amino acid sequence of HCP may be necessary. To accomplish this, HCP protein may be purified and partial amino acid sequence determined by automated sequencers. It is not necessary to determine the entire amino acid sequence, but the linear sequence of two regions of 6 to 8 amino acids from the protein is determined for the production of primers for

PCR amplification of a partial HCP DNA fragment.

Once suitable amino acid sequences have been identified, the DNA sequences capable of encoding them are synthesized. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and therefore, the amino acid sequence can be encoded by any of a set of similar DNA oligonucleotides.

Only one member of the set will be identical to the HCP sequence but will be capable of hybridizing to HCP DNA even in the presence of DNA oligonucleotides with mismatches. The mismatched DNA oligonucleotides may still sufficiently hybridize to the HCP DNA to permit identification and isolation of HCP encoding DNA. DNA isolated by these methods can be used to screen DNA libraries from a variety of cell types, from invertebrate and vertebrate sources, and to isolate homologous genes.

The cloned HCP DNA obtained through the methods described herein may be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant HCP protein. Techniques for such manipulations are fully described in Maniatis, T, et al., supra, and are well known in the art.

Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria including E. coli, blue-green algae, plant cells, insect cells, fungal cells including yeast cells, and animal cells. 9 -

Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells or bacteria-fungal cells or bacteria- invertebrate cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one that causes mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.

A variety of mammalian expression vectors may be used to express recombinant HCP in mammalian cells. Commercially available mammalian expression vectors which may be suitable for recombinant HCP expression, include but are not limited to, pGZT-4T-l (Pharmacia), pMAMneo (Clontech), pcDNA3 (InVitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO- pSV2-neo (ATCC 37593) pBPV-1 (8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and 1ZD35 (ATCC 37565).

A variety of bacterial expression vectors may be used to express recombinant HCP in bacterial cells. Commercially available bacterial expression vectors which may be suitable for recombinant HCP expression include, but are not limited to pET vectors (Novagen) and pQE vectors (Qiagen).

A variety of fungal cell expression vectors may be used to express recombinant HCP in fungal cells such as yeast. Commercially available fungal cell expression vectors which may be suitable for recombinant HCP expression include but are not limited to pYES2 (InVitrogen) and Pichia expression vector (InVitrogen).

A variety of insect cell expression vectors may be used to express recombinant HCP in insect cells. Commercially available insect cell expression vectors that may be 10

suitable for recombinant expression of HCP include but are not limited to pBlueBacII (InVitrogen).

DNA encoding HCP may be cloned into an expression vector for expression in a recombinant host cell. Recombinant host cells may be prokaryotic or eukaryotic, 5 including but not limited to bacteria such as E. coh, fungal cells such as yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to drosophila and silkworm derived cell lines. Cell lines derived from mammalian species that may be suitable and that are commercially available, include but are not limited to, CV-1 o (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1

(ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171), L-cells, and HEK-293 (ATCC CRL1573).

The expression vector may be introduced into host cells via any one of a 5 number of techniques including but not limited to transformation, transfection, protoplast fusion, lipofection, and electroporation. The expression vector-containing cells are clonally propagated and individually analyzed to determine whether they produce HCP protein. Identification of HCP expressing host cell clones may be done by several means, including but not limited to immunological reactivity with anti-HCP o antibodies, and the presence of host cell-associated HCP activity.

Expression of HCP DNA may also be performed using in vitro produced synthetic mRNA. Synthetic mRNA or mRNA isolated from HCP producing cells can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based 5 systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocytes being generally preferred.

To determine the HCP DNA sequence(s) that yields optimal levels of HCP activity and/or HCP protein, HCP DNA molecules including, but not limited to, the 11

following can be constructed: the full-length open reading frame of the HCP cDNA encoding the entire HCP protein, and several constructs containing portions of the cDNA encoding portions of the HCP protein. All constructs can be designed to contain none, all or portions of the 5' or the 3' untranslated region of HCP cDNA. HCP activity and levels of protein expression can be determined following the introduction, both singly and in combination, of these constructs into appropriate host cells. Following determination of the HCP DNA cassette yielding optimal expression in transient assays, this HCP DNA construct is transferred to a variety of expression vectors, for expression in host cells including, but not limited to, mammalian cells, baculovirus-infected insect cells, K coli, and the yeast S. cerevisiae.

Host cell transfectants and microinjected oocytes may be used to assay both the levels of HCP activity and levels of HCP protein by the following methods. In the case of recombinant host cells, this involves the co-transfection of one or possibly two or more plasmids, containing the HCP DNA encoding one or more fragments or subunits. In the case of oocytes, this involves the co-injection of synthetic RNAs for

HCP protein. Following an appropriate period of time to allow for expression, cellular protein is metabolically labelled with, for example -"S-methionine for 24 hours, after which cell lysates and cell culture supernatants are harvested and subjected to immunoprecipitation with polyclonal antibodies directed against the HCP protein. Levels of HCP protein in host cells are quantitated by immuno affinity and/or ligand affinity techniques. Cells expressing HCP can be assayed for the number of HCP molecules expressed by measuring the amount of radioactive ligand and/or substrate binding. HCP-specific affinity beads or HCP-specific antibodies are used to isolate for example -^S-methionine labelled or unlabelled HCP protein. Labelled HCP protein is analyzed by SDS-PAGE. Unlabelled HCP protein is detected by

Western blotting, ELISA or RIA assays employing HCP specific antibodies.

It is known that DNA sequences coding for a peptide may be altered so as to code for a peptide having properties that are different than those of the natural peptide. - 12

Methods of altering the DNA sequences include, but are not limited to site directed mutagenesis. Examples of altered properties include but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand.

As used herein, a "functional derivative" of HCP is a compound that possesses a biological activity (either functional or structural) that is substantially similar to the biological activity of HCP. The term "functional derivatives" is intended to include the "fragments," "variants," "degenerate variants," "analogs" and "homologues" or to "chemical derivatives" of HCP. The term "fragment" is meant to refer to any polypeptide subset of HCP. The term "variant" is meant to refer to a molecule substantially similar in structure and function to either the entire HCP molecule or to a fragment thereof. A molecule is "substantially similar" to HCP if both molecules have substantially similar structures or if both molecules possess similar biological activity.

Therefore, if the two molecules possess substantially similar activity, they are considered to be variants even if the structure of one of the molecules is not found in the other or even if the two amino acid sequences are not identical. The term "analog" refers to a molecule substantially similar in function to either the entire HCP molecule or to a fragment thereof.

Following expression of HCP in a recombinant host cell, HCP protein may be recovered to provide HCP in active form. Several HCP purification procedures are available and suitable for use. As described above for purification of HCP from natural sources, recombinant HCP may be purified from cell lysates and extracts, or from conditioned culture medium, by various combinations of, or individual application of salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography and hydrophobic interaction chromatography.

In addition, recombinant HCP can be separated from other cellular proteins by use of an immunoaffmity column made with monoclonal or polyclonal antibodies specific for full length nascent HCP, polypeptide fragments of HCP or HCP subunits. 13

Monospecific antibodies to HCP are purified from mammalian antisera containing antibodies reactive against HCP or are prepared as monoclonal antibodies reactive with HCP using the technique of Kohler and Milstein, Nature 256: 495-497 (1975). Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogenous binding characteristics for HCP.

Homogenous binding as used herein refers to the ability of the antibody species to bind to a specific antigen or epitope, such as those associated with the HCP, as described above. HCP specific antibodies are raised by immunizing animals such as mice, rats, guinea pigs, rabbits, goats, horses and the like, with rabbits being preferred, with an appropriate concentration of HCP either with or without an immune adjuvant.

HCP antibody affinity columns are made by adding the antibodies to Affigel- 10 (Bio-Rad), a gel support which is activated with N-hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the spacer arm. The remaining activated esters are then quenched with 1M ethanolamine HC1 (pH 8). The column is washed with water followed by 0.23 M glycine HC1 (pH 2.6) to remove any non-conjugated antibody or extraneous protein. The column is then equilibrated in phosphate buffered saline (pH 7.3) and the cell culture supernatants or cell extracts containing HCP or HCP subunits are slowly passed through the column. The column is then washed with phosphate buffered saline until the optical density (A280) ^^{s to} background, then the protein is eluted using 0.23 M glycine-HCl (pH 2.6). The purified HCP protein is then dialyzed against phosphate buffered saline.

The discovery of inhibitors of HCP will lead to a method of modulating the activity of hematopoietic receptors. HCP is found primarily in hematopoietic cells and has been shown to be a negative regulator of one or more signal transduction pathways in these cells. The identification of an HCP inhibitor would provide a synthetic stimulator to the hematopoietic system that could be used in conjunction with other cytokine or cytokine mimetic therapy; for example, the administration of 14

erythropoietin (EPO). Modulation of HCP provides the potential for multiple points of intervention with small molecule therapeutics. Screening assays can target the HCP phosphatase domain and/or the interaction of HCP with receptor complexes, and intracellular substrates of the phosphatase such as the Janus kinase 2 (JAK2) molecule. This multi-faceted approach should increase the probability of discovering an HCP inhibitor. Since HCP has been implicated in several hematopoietic cell-signaling pathways, a single inhibitor may provide a potent boost to multiple cell lineages.

The association of HCP with several growth factor receptors found in hematopoietic cells (i.e., EPO, IL-3, c-kit, and potentially others) and the negative regulatory function that HCP appears to serve with regard to these receptors suggests that the discovery of an HCP inhibitor would provide a hematopoietic cell potentiator. By supplementing existing therapies such as EPO, an HCP inhibitor could enhance the overall effect of hematopoietic cell growth factors, thereby decreasing the necessary dose required for that exogenous factor or potentially eliminating the need for administering EPO by enhancing the activity of the endogenous growth factor. This hypothesis is supported by the recently reported finding that 32D cells expressing a mutant EPOR (i.e., EPOR unable to bind HCP) are more sensitive to EPO as assessed by cellular proliferation. Moreover, the increased sensitivity to IL-3 in cells depleted of HCP supports the argument of stimulating the hematopoietic system through the use of an HCP inhibitor. Discovery of an inhibitor of the HCP-EPOR interaction could prove to be a specific EPO enhancer, potentially reducing the chances of unwanted side effects. Clinically, there exists a Finnish family with autosomal dominant benign erythrocytosis, believed to be the result of a truncated EPOR. The truncated EPOR contains a premature stop codon that eliminates the C-terminal 70 amino acids (de la Chapelle et al, 1993). This 70 amino acid portion of the EPOR contains the binding site for HCP, (specifically Y455).

Therefore, HCP cannot bind to the receptor and exert its negative effect. In this case, the clinical manifestations are minor. Patients have increased hemoglobin levels and higher hematocrits, and cultured erythroid progenitors from these individuals are more sensitive - 15

to EPO. Therefore, modifying the negative regulatory mechanism (without completely eliminating it) could prove useful as a powerful supplemental therapy.

The present invention is also directed to methods for screening for compounds that modulate the expression of DNA or RNA encoding HCP as well as the function of HCP protein in vivo. Compounds that modulate these activities may be DNA, RNA, peptides, proteins, or non-proteinaceous organic molecules. Compounds may modulate by increasing or attenuating the expression of DNA or RNA encoding HCP, or the function of HCP protein. Compounds that modulate the expression of DNA or RNA encoding HCP or the function of HCP protein may be detected by a variety of assays. The assay may be a simple "yes/no" assay to determine whether there is a change in expression or function.

The assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression or function in a standard sample. Modulators identified in this process are useful as therapeutic agents.

The present invention is directed to two methods to screen for compounds that modulate the activity of the HCP protein. For ease of discussion, the assays are named

HCP Catalytic assay, and HCP Interactive assay. The HCP Catalytic assay may be used to detect compounds that modulate enzymatic activity associated with the phosphatase domain of the HCP protein. The HCP Interactive assay was developed to assay compounds that may modulate the enzymatic activity of the full-length molecule by modulating the carboxyl terminal catalytic domain or by modulating the amino terminal scaffolding domains important for receptor binding. Since it had previously been shown that the region imperative for receptor binding also negatively regulates HCP by an intramolecular interaction (Pei et al., 1994), the full-length protein assay is performed in the presence of a phosphotyrosine peptide denoted as the interactive peptide that unmasks the carboxyl terminal phosphatase domain. The interactive peptide is derived from the

ITIM peptide motif found in the Fc and T cell receptors.

Kits containing HCP DNA or RNA, antibodies to HCP, or HCP protein may be prepared. Such kits are used to detect DNA that hybridizes to HCP DNA or to detect 16

the presence of HCP protein or peptide fragments in a sample. Such characterization is useful for a variety of purposes including but not limited to forensic analyses, diagnostic applications, and epidemiological studies.

The DNA molecules, RNA molecules, recombinant proteins and antibodies of the present invention may be used to screen and measure levels of HCP DNA, HCP

RNA or HCP protein. The recombinant proteins, DNA molecules, RNA molecules and antibodies lend themselves to the formulation of kits suitable for the detection and typing of HCP. Such a kit would comprise a compartmentalized carrier suitable to hold in close confinement at least one container. The carrier would further comprise reagents such as recombinant HCP proteins or anti-HCP antibodies suitable for detecting HCP. The carrier may also contain a means for detection such as labeled antigen or enzyme substrates or the like.

Nucleotide sequences that are complementary to the HCP encoding DNA sequence can be synthesized for antisense therapy. These antisense molecules may be DNA, stable derivatives of DNA such as phosphorothioates or methylphosphonates,

RNA, stable derivatives of RNA such as 2'-O-alkylRNA, or other HCP antisense oligonucleotide mimetics. HCP antisense molecules may be introduced into cells by standard methods including, but not limited to, microinjection, liposome encapsulation or by expression from vectors harboring the antisense sequence. HCP antisense therapy may be particularly useful for the treatment of diseases where it is beneficial to reduce HCP activity.

HCP gene therapy may be used to introduce HCP into the cells of target organisms. The HCP gene can be ligated into viral vectors that mediate transfer of the HCP DNA by infection of recipient host cells. Suitable viral vectors include retro virus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poliovirus and the like. Alternatively, HCP DNA can be transferred into cells for gene therapy by non- viral techniques including receptor-mediated targeted DNA transfer using ligand- DNA conjugates or adenovirus-ligand-DNA conjugates, lipofection membrane fusion - 17

or direct microinjection. These procedures and variations thereof are suitable for ex vivo as well as in vivo HCP gene therapy. HCP gene therapy may be particularly useful for the treatment of diseases where it is beneficial to elevate HCP activity.

Pharmaceutically useful compositions comprising HCP DNA, HCP RNA, or HCP protein, or modulators of HCP receptor activity, may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the protein, DNA, RNA, or modulator.

Therapeutic or diagnostic compositions of the invention are administered to an individual in amounts sufficient to treat or diagnose disorders in which modulation of HCP -related activity is indicated. The effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration. The pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular.

The term "chemical derivative" describes a molecule that contains additional chemical moieties that are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule.

Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.

Compounds identified according to the methods disclosed herein may be used alone at appropriate dosages defined by routine testing in order to obtain optimal inhibition of the HCP receptor or its activity while minimizing any potential toxicity. In addition, co-administration or sequential administration of other agents may be desirable. The present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention. The compositions containing compounds or modulators identified according to this invention as the active ingredient for use in the 5 modulation of HCP can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration. For example, the compounds or modulators can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection. 0 Likewise, they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts. An effective but non-toxic amount of the compound desired can be employed as a HCP modulating agent. 5 The daily dosage of the products may be varied over a wide range from 0.01 to

1,000 mg per patient, per day. For oral administration, the compositions are preferably provided in the form of scored or unscored tablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, and 50.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. An effective o amount of the drug is ordinarily supplied at a dosage level of from about 0.0001 mg/kg to about 100 mg/kg of body weight per day. The range is more particularly from about 0.001 mg/kg to 10 mg/kg of body weight per day. The dosages of the HCP modulators are adjusted when combined to achieve desired effects. On the other hand, dosages of these various agents may be independently optimized and combined 5 to achieve a synergistic result wherein the pathology is reduced more than it would be if either agent were used alone.

Advantageously, compounds or modulators of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in - 19 -

divided doses of two, three or four times daily. Furthermore, compounds or modulators for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.

For combination treatment with more than one active agent, where the active agents are in separate dosage formulations, the active agents can be administered concurrently, or they each can be administered at separately staggered times. The dosage regimen utilizing the compounds or modulators of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound thereof employed. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.

In the methods of the present invention, the compounds or modulators herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier" materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically 20

acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

For liquid forms the active drug component can be combined in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like. Other dispersing agents that may be employed include glycerin and the like. For parenteral administration, sterile suspensions and solutions are desired. Isotonic preparations, which generally contain suitable preservatives, are employed when intravenous administration is desired.

Topical preparations containing the active drug component can be admixed with a variety of carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations.

The compounds or modulators of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.

Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are 21 -

coupled. The compounds or modulators of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinyl-pyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-amidephenol, polyhydroxy-ethylaspartamidephenol, or polyethyl-eneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds or modulators of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels. For oral administration, the compounds or modulators may be administered in capsule, tablet, or bolus form or alternatively they can be mixed in the animals feed. The capsules, tablets, and boluses are comprised of the active ingredient in combination with an appropriate carrier vehicle such as starch, talc, magnesium stearate, or di-calcium phosphate. These unit dosage forms are prepared by intimately mixing the active ingredient with suitable finely-powdered inert ingredients including diluents, fillers, disintegrating agents, and/or binders such that a uniform mixture is obtained. An inert ingredient is one that will not react with the compounds or modulators and which is non-toxic to the animal being treated. Suitable inert ingredients include starch, lactose, talc, magnesium stearate, vegetable gums and oils, and the like. These formulations may contain a widely variable amount of the active and inactive ingredients depending on numerous factors such as the size and type of the animal species to be treated and the type and severity of the infection. The active ingredient may also be administered as an additive to the feed by simply mixing the compound with the feedstuff or by applying the compound to the surface of the feed. Alternatively the active ingredient may be mixed with an inert carrier and the resulting composition may then either be mixed with the feed or fed directly to the animal. Suitable inert carriers include corn meal, citrus meal, fermentation residues, soya grits, dried 22

grains and the like. The active ingredients are intimately mixed with these inert carriers by grinding, stirring, milling, or tumbling such that the final composition contains from 0.001 to 5% by weight of the active ingredient.

The compounds or modulators may alternatively be administered 5 parenterally via injection of a formulation consisting of the active ingredient dissolved in an inert liquid carrier. Injection may be either intramuscular, intraruminal, intratracheal, or subcutaneous. The mjectable formulation consists of the active ingredient mixed with an appropriate inert liquid carrier. Acceptable liquid carriers include the vegetable oils such as peanut oil, cottonseed oil, sesame o oil and the like as well as organic solvents such as solketal, glycerol formal and the like. As an alternative, aqueous parenteral formulations may also be used. The vegetable oils are the preferred liquid carriers. The formulations are prepared by dissolving or suspending the active ingredient in the liquid carrier such that the final formulation contains from 0.005 to 10% by weight of the active ingredient. 5 Topical application of the compounds or modulators is possible through the use of a liquid drench or a shampoo containing the instant compounds or modulators as an aqueous solution or suspension. These formulations generally contain a suspending agent such as bentonite and normally will also contain an antifoaming agent. Formulations containing from 0.005 to 10% by weight of the o active ingredient are acceptable. Preferred formulations are those containing from

0.01 to 5% by weight of the instant compounds or modulators.

The following examples illustrate the present invention without, however, limiting the same thereto.

5 EXAMPLE 1

Cloning of the HCP cDNA into E. coli Expression Vectors

Recombinant human HCP was produced in E. coli following the transfer of the HCP expression cassette into R, coli expression vectors, including but not limited to, - 23

the pET series (Novagen). The pET vectors place HCP expression under control of the tightly regulated bacteriophage T7 promoter. Following transfer of this construct into an R coli host that contains a chromosomal copy of the T7 RNA polymerase gene driven by the inducible lac promoter, expression of HCP is induced when an appropriate lac substrate (IPTG) is added to the culture. The levels of expressed HCP are determined by the assays described herein.

The cDNA encoding the entire open reading frame for HCP is inserted into the EcoRI-NotI sites of pET28a. Constructs are identified by sequence analysis and used to transform the expression host strain BL21pLys. Transformants are then used to inoculate cultures for the production of HCP protein. Cultures may be grown in M9 or

LB media, whose formulation is known to those skilled in the art. After growth to an OD₆₀₀ between 0.6-0.9, expression of HCP is induced with 1 mM IPTG for 4 hours at

33°C.

EXAMPLE 2

Human HCP catalytic domain cDNA cloning, expression and purification:

Messenger RNA from Jurkat cells was purified from total cellular RNA using a Dynal mRNA kit. RT-PCR was performed to obtain full-length human HCP using 5'- primer which consists of a sequence recognized by EcoRI and the sequence of the first 8 amino acids of HCP catalytic domain, and 3 '-primer which consists of the DNA sequence of the last 9 amino acids of HCP and a sequence recognized by Notl. The full length HCP catalytic domain PCR fragment was then cloned into EcoRI and Notl sites of pGEX-4T-l vector.

The HCP-GST fusion gene was transformed into BL21 cells for fusion protein expression. 10 ml of overnight culture of transformed BL21 cells were inoculated into 1 liter of LB medium in the presence of 100 ug/ml of ampicillin. The culture was incubated at 37°C with shaking until the OD₆oo reached between 0.6 to 0.9. The cells were induced for HCP-GST expression by adding IPTG to final concentration of 1 mM and incubated - 24 -

with shaking at 33°C. The cells were then harvested after 4 hours of culture and stored at - 20°C for later purification.

Four liters of harvested cell pellets were resuspended in 100 ml of lysing buffer consisting of 50 mM Tris-HCl pH7.4 with 1 mM final concentration of MgCl , 0.2 mM final concentration of AEBSF (Calbiotech), 7500 units of Benzonase (EM Science) and

100 mg of lysozyme (Sigma). The cell suspension was then briefly sonicated and the cell lysate was incubated at room temperature with slow rocking for about 45 min. Triton X- 100 was then added to final concentration of 0.1% and the lysate was incubated at room temperature with slow rocking for another 45 min. The lysate was then spun at 14,000 φm for 10 min to remove cell debris. The supernatant was saved for further HCP-GST fusion protein purification.

Five milliliters of glutathione Sepharose 4B beads (Pharmacia) were first washed with 50 mM Tris-HCl pH 7.4 thoroughly to remove ethanol in storing solution. The beads were then mixed with the cell lysate supernatant at 4°C with slow rocking overnight. The beads were then washed with 50 mM Tris-HCl pH 7.4 and incubated with 5 ml of 10 mM reduced Glutathione dissolved in 50 mM Tris-HCl pH 8.0 at room temperature for ten minutes to elute the HCP-GST fusion protein. The beads were pelleted and the supernatant was collected. The elution process was repeated two more times. The collected supernatant containing HCP-GST fusion protein were pooled and the enzyme activity was measured in HCP catalytic assay described in Example 4.

Primer pair sequences used to clone the catalytic domain of human HCP: SEQ.ID.NO.4: 5 '-primer: GTCAGAATTCGGCGCCTTTGTCTACCTGCGGCAG SEQ.ID.NO.l : 3'-ρrimer: GTCAGCGGCCGCTCACTTCCTCTTGAGGGAACCCTTGCTC

EXAMPLE 3

Human full-length HCP cDNA cloning, expression and purification: - 25

Messenger RNA from Jurkat cells was purified from total cellular RNA using a Dynal mRNA kit. RT-PCR was performed to obtain full-length human HCP using 5'- primer which consists of a sequence recognized by EcoRI and the sequence of the first 8 amino acids of HCP, and 3 '-primer which consists of the DNA sequence of the last 9 amino acids of HCP and a sequence recognized by Notl. The full length HCP PCR fragment was then cloned into EcoRI and Notl sites of pGEX-4T-l vector.

The HCP-GST fusion gene was transformed into BL21 cells for fusion protein expression. 10 ml of overnight culture of transformed BL21 cells were inoculated into 1 liter of LB medium in the presence of 100 ug/ml of ampicillin. The culture was incubated at 37°C with shaking until the OD₆₀o reached between 0.6 to 0.9. The cells were induced for HCP-GST expression by adding IPTG to final concentration of 1 mM and incubated with shaking at 33°C. The cells were then harvested after 4 hours of culture and stored at - 20°C for later purification.

Four liters of harvested cell pellets were resuspended in 100 ml of lysing buffer consisting of 50 mM Tris-HCl pH7.4 with 1 mM final concentration of MgCl , 0.2 mM final concentration of AEBSF (Calbiotech), 7500 units of Benzonase (EM Science) and 100 mg of lysozyme (Sigma). The cell suspension was then briefly sonicated and the cell lysate was incubated at room temperature with slow rocking for about 45 min. Triton X- 100 was then added to final concentration of 0.1% and the lysate was incubated at room temperature with slow rocking for another 45 min. The lysate was then spun at 14,000 φm for 10 min to remove cell debris. The supernatant was saved for further HCP-GST fusion protein purification.

Five milliliters of glutathione Sepharose 4B beads (Pharmacia) were first washed with 50 mM Tris-HCl pH 7.4 thoroughly to remove ethanol in storing solution. The beads were then mixed with the cell lysate supernatant at 4°C with slow rocking overnight. The beads were then washed with 50 mM Tris-HCl pH 7.4 and incubated with 5 ml of 10 mM reduced Glutathione dissolved in 50 mM Tris-HCl pH 8.0 at room temperature for ten minutes to elute the HCP-GST fusion protein. The beads were pelleted and the 26 -

supernatant was collected. The elution process was repeated two more times. The collected supernatant containing HCP-GST fusion protein was pooled and the enzyme activity was measured in the HCP interactive assay described in Example 5.

Primer pair sequences used to clone full-length human HCP:

SEQ.ID.NO.2: 5'-primer: ACGTGAATTCATGGTGAGGTGGTTTCACCGAGAC

SEQ.ID.NO.3: 3'-primer: GTCAGCGGCCGCTCACTTCCTCTTGAGGGAACCCTTGCTC

EXAMPLE 4

HCP Catalytic Assay

The GST-HCP phosphatase domain fusion protein (rhuHCPcat) was resuspended in assay buffer (50 mM imidazole, 1 mM EDTA, 45 mM BME, 0.1% ovalbumin, pH 6.8).

The amount of rhuHCPcat used in the assay was determined upon its enzymatic activity required to produce a final readout of approximately 2.0 at OD₆₅₀. Compounds at a concentration of approximately 1 mM in 100% DMSO were diluted 1 :40 in assay buffer for determination of enzyme inhibition activity at a final compound concentration of approximately 25 μM in 2.5% DMSO. Thus, fifty-eight microliters of rhuHCPcat enzyme in assay buffer are mixed with 2 μl of the ImM test compound in 100% DMSO and incubated at room temperature for 30 minutes. Then 20 μl of 0.8 mM phosphorylated

JAK2 peptide substrate (diluted in the assay buffer) was added to the mixture to yield a final concentration of 200 μM in the test well. The reaction was incubated for an additional 60 minutes. The reaction was then terminated by the addition of 70 μl of malachite green solution containing ammonium molybdate (Fisher and Higgins. Pharm Res. 11(5), 1994). Following a final 30-minute incubation, the optical density was read at

650 nm. A known tyrosine phosphatase inhibitor, sodium ortho vanadate, would be expected to inhibit the rhuHCPcat enzymatic activity and cause a reduction of free phosphate production, therefore producing a reduced OD₆₅o absorbance. The inhibitory 27 -

effect of vanadate on HCP enzymatic activity is shown in Figure 1, and demonstrates that the assay can measure the effects of a HCP modulator.

JAK2 peptide sequence: SEQ.ID.NO.7: H-Val-Leu-Pro-Gln-Asp-Lys-Glu-(phospho)Tyr-(phospho)Tyr-Lys-Val-

Lys-Glu-Pro-Gly-Glu-OH

EXAMPLE 5

Demonstration of Interactive peptide on HCP enzymatic activity The GST-HCP fusion protein (rhuHCP) was resuspended in assay buffer (50 mM

Imidazole pH 7.4, 10 mM 2-mercaptoethanol, 1 mM EDTA and 0.1 % ovalbumin). The enzyme working solution was diluted in assay buffer as described for the catalytic assay to yield an optical density (OD) at 650 nM wavelength of approximately 2.0 in the presence of 50 μM interactive peptide and 200 μM substrate peptide. To perform the assay, a 300 μM interactive peptide working solution and 1.2 mM phospho tyrosine peptide substrate working solution was prepared from 24 mM stock solutions of each respective peptide. A sample assay is as follows; 38.5 μl of diluted rhuHCP and 1.5 μl of a 1 mM compound solution was mixed and incubated together for 20 minutes at room temperature. Ten microliters of a 300 μM ITIM peptide solution was added, mixed and incubation continued for an additional 20 minutes. This was followed by the addition of 10 μl of the

1.2 mM substrate peptide solution and incubation was continued for an additional twenty minutes. The assay was developed by the addition of 100 μl of malachite green solution as described above for the catalytic assay. As shown in Figure 2, the enzyme is more active in the presence of the interactive peptide. The activity of the enzyme in the presence of the substrate peptide yields activity equal to that of enzyme and buffer alone. The activity is inhibited approximately 3 fold by the addition of sodium orthovanadate.

ITIM peptide sequence (peptide 1 or PI in Figure 2): 28 -

SEQ.ID.NO.5: Ac-Glu-Ala-Glu-Asn-Thr-Ile-Thr-Ile-Thr-phosphoTyr-Ser-Leu-Leu-Lys- His-OH

pTyr peptide sequence (S or substrate peptide in figure 2): SEQ.ID.NO.6: H-Asp-Ala-Asp-Glu-phosphoTyr-Leu-Ile-Pro-Gln-Gln-Gly-OH

EXAMPLE 6

HCP Interactive Assay

The full length GST-HCP fusion protein (rhuHCP) was resuspended in assay buffer (50 mM Imidazole pH 7.4, 10 mM 2-ME, 1 mM EDTA, and 0.1% ovalbumin). The amount of rhuHCP used in the assay was determined upon its enzymatic activity required to produce a final readout of approximately 2.0 to 3.0 at OD₆₅o based on the conditions established in Example 5. 300 μM interactive peptide (ITIM) working solution was prepared by dilution in assay buffer from a 24 mM ITIM stock solution in DMSO. 1200 μM phosphotyrosine (pTyr) peptide substrate working solution was prepared by dilution in assay buffer from a 24 mM peptide stock solution in dH₂O. The malachite green solution is prepared by mixing 3 ml of malachite green working stock with 7 ml of dH O. The malachite green sock consists of 2.6 mM malachite green in 7.2N of H₂SO₄, 7.5% ammonium molybdate and 11% Tween-20 mixed right before use in 10/2.5/0.2 ratio by volume.

The first step is conducted by mixing 38.5 μl of diluted rhuHCP enzyme and 1.5 μl of lmM screening compound in DMSO, and incubating for 20 minutes at room temperature. Then 10 μl of the ITIM peptide working solution, is added to the reaction, mixed, and incubated at room temperature for 20 min. Then, 10 μl of pTyr peptide working solution is added to the reaction, mixed, and incubated at room temperature for

20 minutes. Subsequently, 100 μl of malachite green solution was added to the reaction, incubated for 2 hours and the OD₆₅₀ was determined. Vanadate is expected to inhibit the - 29 -

activity of rhuHCP and results in a decrease in absorbance at 650 nM, as shown in Figure 3, thus indicating that the assay is capable of measuring modulators of HCP.

EXAMPLE 7 Cloning of HCP cDNA into a Mammalian Expression Vector

The HCP cDNAs are cloned into the mammalian expression vector pcDNA3. The HCP ρGEX-4T-l plasmid is digested with EcoRI and Notl. The inserts are purified by agarose gel electrophoresis. The pcDNA3 vector is digested with EcoRI and Notl. The linear vector can be purified on agarose gel and used to ligate to the HCP cDNA inserts. Recombinants are isolated, designated HCP, and used to transfect mammalian cells (L-cells) by CaPOzi.-DNA precipitation. Stable cell clones are selected by growth in the presence of G418. Single G418 resistant clones can be isolated and shown to contain the intact HCP gene. Clones containing the HCP cDNAs are analyzed for expression using immunological techniques, such as immuneprecipitation, Western blot, and immuno fluorescence using antibodies specific to the HCP proteins. Antibody is obtained from rabbits innoculated with peptides that are synthesized from the amino acid sequence predicted from the HCP sequences.

Cells that are expressing HCP, stably or transiently, are used to test for expression of HCP and for phosphatase activity. These cells are used to identify and examine compounds for their ability to modulate HCP activity.

Cassettes containing the HCP cDNA in the positive orientation with respect to the promoter are ligated into appropriate restriction sites 3' of the promoter and identified by restriction site mapping and/or sequencing. These cDNA expression vectors are introduced into fibroblastic host cells for example COS-7 (ATCC# CRL1651), and CV-1 tat [Sackevitz et al., Science 238: 1575 (1987)], 293, L (ATCC#

CRL6362)] by standard methods including but not limited to electroporation, or chemical procedures (cationic liposomes, DEAE dextran, calcium phosphate). 30 -

Transfected cells and cell culture supematants can be harvested and analyzed for HCP expression as described herein.

All of the vectors used for mammalian transient expression can be used to establish stable cell lines expressing HCP. Unaltered HCP cDNA constructs cloned into expression vectors are expected to program host cells to make HCP protein. In addition, HCP is expressed extracellularly as a secreted protein by ligating HCP cDNA constructs to DNA encoding the signal sequence of a secreted protein. The transfection host cells include, but are not limited to, CV-l-P [Sackevitz et al., Science 238: 1575 (1987)], tk-L [Wigler, et al Cell 11 : 223 (1977)], NS/0, and dHFr- CHO [Kaufman and Shaφ, J. Mol. Biol. 159: 601, (1982)].

Co-transfection of any vector containing HCP cDNA with a drug selection plasmid including, but not limited to G418, aminoglycoside phosphotransferase; hygromycin, hygromycin-B phospholransferase; APRT, xanthine-guanine phosphoribosyl-transferase, will allow for the selection of stably transfected clones. Levels of HCP are quantitated by the assays described herein.

HCP cDNA constructs are also ligated into vectors containing amplifiable drug-resistance markers for the production of mammalian cell clones synthesizing the highest possible levels of HCP. Following introduction of these constructs into cells, clones containing the plasmid are selected with the appropriate agent, and isolation of an over-expressing clone with a high copy number of plasmids is accomplished by selection in increasing doses of the agent.

The expression of recombinant HCP is achieved by transfection of full-length HCP cDNA into a mammalian host cell.

EXAMPLE 8

Cloning of HCP cDNA into a Baculovirus Expression Vector for Expression in Insect Cells 31

Baculovirus vectors, which are derived from the genome of the AcNPV virus, are designed to provide high level expression of cDNA in the Sf9 line of insect cells (ATCC CRL# 1711). Recombinant baculoviruses expressing HCP cDNA is produced by the following standard methods (InVitrogen Maxbac Manual): the HCP cDNA constructs are ligated into the polyhedrin gene in a variety of baculovirus transfer vectors, including the pAC360 and the BlueBac vector (InVitrogen). Recombinant baculoviruses are generated by homologous recombination following co-transfection of the baculovirus transfer vector and linearized AcNPV genomic DNA [Kitts, P.A., Nuc. Acid. Res. 18: 5667 (1990)] into Sf9 cells. Recombinant pAC360 viruses are identified by the absence of inclusion bodies in infected cells and recombinant pBlueBac viruses are identified on the basis of β-galactosidase expression (Summers, M. D. and Smith, G. E., Texas Agriculture Exp. Station Bulletin No. 1555). Following plaque purification, HCP expression is measured by the assays described herein.

The cDNA encoding the entire open reading frame for HCP is inserted into the BamHI site of pBlueBacII. Constructs in the positive orientation are identified by sequence analysis and used to transfect Sf9 cells in the presence of linear AcNPV mild type DNA.

Authentic, active HCP is found in the cytoplasm of infected cells. Active HCP is extracted from infected cells by hypotonic or detergent lysis.

EXAMPLE 9

Cloning of HCP cDNA into a yeast expression vector

Recombinant HCP is produced in the yeast S. cerevisiae following the insertion of the optimal HCP cDNA cistron into expression vectors designed to direct the intracellular or extracellular expression of heterologous proteins. In the case of intracellular expression, vectors such as EmBLyex4 or the like are ligated to the HCP cistron [Rinas, U. et ah, Biotechnology 8: 543-545 (1990); Horowitz B. et al, J. Biol. Chem. 265: 4189-4192 (1989)]. For extracellular expression, the HCP cistron is 32 -

ligated into yeast expression vectors which fuse a secretion signal (a yeast or mammalian peptide) to the NH2 terminus of the HCP protein [Jacobson, M. A., Gene

85: 511-516 (1989); Riett L. and Bellon N. Biochem. 28: 2941-2949 (1989)].

These vectors include, but are not limited to pAVEl>6, which fuses the human serum albumin signal to the expressed cDNA [Steep O. Biotechnology 8: 42-46

(1990)], and the vector pL8PL which fuses the human lysozyme signal to the expressed cDNA [Yamamoto, Y., Biochem. 28: 2728-2732)]. In addition, HCP is expressed in yeast as a fusion protein conjugated to ubiquitin utilizing the vector pVEP [Ecker, D. J., J. Biol. Chem. 264: 7715-7719 (1989), Sabin, E. A., Biotechnology 7: 705-709 (1989), McDonnell D. P., Mol. Cell Biol. 9: 5517-5523 (1989)]. The levels of expressed HCP are determined by the assays described herein.

EXAMPLE 10

Purification of Recombinant HCP Recombinantly produced HCP may be purified by antibody affinity chromatography.

HCP antibody affinity columns are made by adding the anti-HCP antibodies to Affigel-10 (Biorad), a gel support which is pre-activated with N-hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the spacer arm. The remaining activated esters are then quenched with 1M ethanolamine HC1 (pH 8). The column is washed with water followed by 0.23 M glycine HC1 (pH 2.6) to remove any non-conjugated antibody or extraneous protein. The column is then equilibrated in phosphate buffered saline (pH 7.3) together with appropriate membrane solubilizing agents such as detergents and the cell culture supematants or cell extracts containing solubilized HCP are slowly passed through the column. The column is then washed with phosphate- buffered saline together with detergents until the optical density (A280) falls to background, then the protein is eluted with 0.23 M glycine-HCl - 33

(pH 2.6) together with detergents. The purified HCP protein is then dialyzed against phosphate buffered saline.

34 -

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Claims

38WHAT IS CLAIMED IS:

1. An expression vector for expression of HCP protein in a recombinant host as a fusion protein, wherein said vector contains a recombinant gene encoding HCP protein, wherein said protein functions as HCP.

2. A recombinant host cell containing a recombinantly cloned gene encoding the HCP fusion protein of claim 1, wherein said HCP protein functions as

HCP.

3. A HCP fusion protein, in substantially pure form which functions as HCP protein.

4. A monospecific antibody immunologically reactive with HCP protein.

5. The antibody of Claim 4, wherein the antibody blocks activity of the

HCP.

6. A process for expression of HCP fusion protein wherein said protein functions as HCP in a recombinant host cell, comprising:

(a) Transferring the expression vector of Claim 1 into suitable host cells; and

(b) Culturing the host cells of step (a) under conditions that allow expression of the HCP fusion protein from the expression vector.

7. A method of identifying compounds that modulate HCP protein activity, comprising: 39

(a) Combining a modulator of HCP protein activity with HCP protein and HCP protein substrate, wherein said protein functions as a HCP; and

(b) Measuring an effect of the modulator on the activity of the HCP protein.

8. The method of claim 7, wherein the effect of the modulator on the activity of the HCP protein is inhibiting or enhancing binding of HCP ligands.

9. The method of claim 7, wherein the effect of the modulator on the activity of the HCP protein is stimulation or inhibition of HCP phosphatase activity.

10. The method of claim 7, wherein the effect of the modulator on the activity of the HCP protein is the stimulation or inhibition of EPOR activity.

11. A compound active in the method of Claim 7, wherein said compound i s a modulator o f a HCP .

12. A compound active in the method of Claim 7, wherein said compound is an inhibitor, agonist or antagonist of a HCP.

13. A compound active in the method of Claim 7, wherein said compound is a modulator of expression of a HCP.

14. A pharmaceutical composition comprising a compound active in the method of Claim 7, wherein said compound is a modulator of HCP activity.

15. A method of treating a patient in need of such treatment for a condition which is mediated by HCP, comprising administration of a HCP modulating compound active in the method of Claim 7. - 40

16. A method of identifying compounds that modulate HCP protein activity, comprising:

(a) Combining a modulator of HCP protein activity with full length HCP protein, HCP interactive peptide and HCP protein substrate, wherein said full length HCP protein functions as a HCP; and

(b) Measuring an effect of the modulator on the activity of the HCP protein.

17. The method of claim 16, wherein the effect of the modulator on the activity of the HCP protein is inhibiting or enhancing binding of HCP ligands.

18. The method of claim 16, wherein the effect of the modulator on the activity of the HCP protein is stimulation or inhibition of HCP phosphatase activity.

19. The method of claim 16, wherein the effect of the modulator on the activity of the HCP protein is the stimulation or inhibition of EPOR activity.

20. A compound active in the method of Claim 16, wherein said compound is a modulator of a HCP.

21. A compound active in the method of Claim 16, wherein said compound is an inhibitor, agonist or antagonist of a HCP.

22. A compound active in the method of Claim 16, wherein said compound is a modulator of expression of a HCP.

23. A pharmaceutical composition comprising a compound active in the method of Claim 16, wherein said compound is a modulator of HCP activity. 41

24. A method of treating a patient in need of such treatment for a condition which is mediated by HCP, comprising administration of a HCP modulating compound active in the method of Claim 16.