Provisional Application No. 60/030,418, filed November 1, 1996. The teachings of both of the referenced Applications are expressly incorporated herein by reference.

BACKGROUND OF THE INVENTION Chronic myelogenous leukemia (CML) is a disease having clinical and pathological features distinct from those of other forms of leukemia. It is widely accepted that the cause of CML is a specific chromosomal translocation between human chromosome 9 and human chromosome 22. The chromosome resulting from this translocation is commonly referred to as the Philadelphia chromosome. Darnell, J. et al., Molecular Cell Biology, 2nd Ed., W.H. Freeman and Co., New York (1990), p. 992. The gene for c-abl (ABL), a tyrosine kinase thought to be involved in growth control, resides on the distal arm of human chromosome 9, while the gene for c-bcr (BCR) resides on human chromosome 22. The translocation places the promoter distal three exons of ABL, including those elements which encode the tyrosine kinase domain, downstream of either the first or second exon of BCR. Chung, S. and Wong, P.M.C., Oncoaene, 10:1261-1268 (1995). The product of the translocation between human chromosome 9 and human chromosome 22 is a

chimeric gene, BCR-ABL, which encodes a fusion protein, often referred to as pl85bcr-abl or p210bcrab1, depending upon the inclusion of the second exon of BCR. Bartram, C.R., et al., Nature, 306:277-280 (1983). p185bCr-abl causes acute leukemia, typically lymphoblastic; p210bCr-abl usually causes CML, but can occasionally also cause acute leukemia.

Compared to normal c-abl, bcr-abl has increased tyrosine kinase activity. Konopka, J., et al., Cell, 37:1035-1042 (1984). Additionally, c-abl, as a non- receptor tyrosine kinase, functions both in the nucleus and the cytoplasm and bcr-abl functions exclusively in the cytoplasm. These two characteristics of bcr-abl are essential elements of its transforming abilities.

McWhirter, J.R., et al., Mol. Cell Bio., 11:1553-1565 (1991) Following this chromosomal translocation within a single, primitive myeloid stem cell, the progeny of the affected cell gradually populate the entire intermediate and late hematopoietic maturational compartments. Despite the presence of the Philadelphia chromosome, these progeny, referred to as Ph+ cells, are able to differentiate and mature along the various myeloid lineages while retaining the capacity to function as their normal, unaffected counterparts. Invariably, in an average span of three to five years, the disease progresses into a malignant stage known as blast crisis. The affected cells acquire additional chromosomal abnormalities and lose their ability to differentiate and mature, resulting in the functional breakdown of the hematopoietic system. Clarkson, B. and Strife, A., Leukemia, 7:1683-1721 (1993). Daley, G.Q. and Ben Neriah, Y., Adv. Cancer Res., 57:151-184 (1991).

Deisseroth, A.B. and Arlinghaus, R.B., eds. Chronic Myelogenous Leukemia-Molecular Approaches to Research and Therapy, New York, Marcel Dekker (1991). Sawyers, C.L. et al., Cell 64:337-350 (1991).

Clinical treatment of CML has remained essentially unchanged for many years. To date, with the exception of marrow ablative chemotherapy and/or total body irradiation followed by allogeneic bone marrow transplantation, no effective cure has been developed for the disease. Only a minority of CML patients have been cured by complete bone marrow transplantation. Treatment with alpha interferon has led to durable remissions in about 10-20% of chronic phase patients, but longer follow-ups are necessary to determine if these patients will have late relapses. In light of this, the need for better treatment methods is apparent.

SUMMARY OF THE INVENTION As described herein, Applicants have identified and characterized a protein which is constitutively tyrosine phosphorylated in mammalian, such as human, cells in which an oncogenic tyrosine kinase is active. This protein, referred to as p62 protein downstream of tyrosine kinases, or p62d°k, is tyrosine phosphorylated in hematopoietic cells, or cells which are progeny thereof, after stimulation with a ligand for a receptor tyrosine kinase and, in its tyrosine phosphorylated form, but not in its non-tyrosine phosphorylated form, binds p120 ras GTPase- activating protein (GAP). This protein has also been referred to (e.g. in U.S. Provisional Application No.

60/030,418, filed November 1, 1996) as GAT62. The terms

p62dok and GAT62 are used interchangeably to refer to the same protein. The p62dok protein is involved in a signal transduction pathway initiated by a receptor tyrosine kinase and is rapidly tyrosine phosphorylated after the receptor tyrosine kinase is activated. The p62d°k protein of the present invention is the 62 kDa GAP-associating protein which is constitutively tyrosine phosphorylated in hematopoietic cells, or cells which are progeny thereof, of individuals with CML.

The p62d°k protein of the present invention is present in normal cells (cells in which an oncogenic tyrosine kinase is not present or is inactive) and, in one embodiment comprises the amino acid sequence of SEQ ID NO.: 2. In a further embodiment, p62dok protein of the present invention is encoded by DNA which hybridizes, as described herein, to DNA comprising the complement of the nucleotide sequence of SEQ ID NO.: 1. p62dok protein obtained from normal hematopoietic cells, or cells which are progeny thereof, is not constitutively tyrosine phosphorylated. In normal cells, p62dok protein may be tyrosine phosphorylated in the absence of an oncogenic tyrosine kinase. However, in normal cells, it will not be in a state of constitutive (constant) phosphorylation. Alternatively, p62dok protein may exist in a non-tyrosine phosphorylated state in the absence of an oncogenic tyrosine kinase in normal cells.

Non-tyrosine phosphorylated p62d°k protein does not bind GAP; tyrosine phosphorylated p62dok protein, regardless of whether the phosphorylation is constitutive, binds GAP. In normal cells, stimulation of specific cell surface receptors results in tyrosine phosphorylation of p62dok protein. Tyrosine phosphorylation in the absence of an

oncogenic tyrosine kinase in normal cells is referred to herein as normal tyrosine phosphorylation. The resulting p62d°k protein is referred to as normal or wild type tyrosine phosphorylated p62dok. In contrast, p62dok protein obtained from cells, such as human hematopoietic progenitor cells, in which tyrosine kinase activity is altered (e.g., as to level, timing, cellular location) is constitutively tyrosine phosphorylated. This is referred to herein as aberrant or constitutive tyrosine phosphorylation. The resulting p62dok protein is referred to as aberrantly or constitutively tyrosine phosphorylated p62d°k. As described herein, p62d°k protein is present in hematopoietic progenitor cells, or cells which are progeny thereof, from CML patients in which a chimeric bcr-abl gene is expressed and the protein is constitutively tyrosine phosphorylated.

Constitutively tyrosine phosphorylated p62dok protein purified from a hematopoietic cell line expressing an oncogenic tyrosine kinase is also the subject of this invention. One embodiment of such a protein is constitutively tyrosine phosphorylated p62dok protein, isolated from Mo7/p210 cells and which binds GAP. This protein consists of two components having molecular weights of approximately 62 and 64 kDa (based on migration by SDS- PAGE) i.e. a doublet. The present invention also relates to p62d°k protein, purified from a hematopoietic cell line lacking an oncogenic tyrosine kinase or containing an inactive oncogenic tyrosine kinase, which exhibits normal tyrosine phosphorylation. One embodiment of such a protein is normal tyrosine phosphorylated p62dok protein, isolated from Mo7 cells, which migrates as a singlet with a

molecular weight of approximately 61 kDa (based on migration by SDS-PAGE) and does not bind p120 ras GAP.

The present invention also relates to isolated nucleic acids (DNA, RNA), referred to as p62d°k-encoding nucleic acids, which encode p62dok protein, or fragments or portions thereof, as described herein. The term Dok is used to refer to the gene encoding the complete p62d°kprotein.

In one embodiment, the p62d°k-encoding nucleic acid of the present invention is DNA which comprises the nucleic acid sequence of SEQ ID NO:1. In another embodiment, p62d°k-encoding nucleic acid is DNA which hybridizes under highly stringent conditions to a nucleic acid comprising the complement of the nucleotide sequence of SEQ ID NO:1 and which encodes a protein which is involved in a signal transduction pathway initiated by a receptor tyrosine kinase, such as a pathway initiated by a receptor tyrosine kinase which also initiates a pathway in which p62dok protein encoded by SEQ ID NO.: 1 participates. In a further embodiment, the p62dok-encoding nucleic acid of the present invention is DNA, which due to the degeneracy of the genetic code, encodes the amino acid sequence encoded by DNA having the nucleotide sequence of SEQ ID NO.: 1.

The present invention also relates to mRNA encoded by (transcribed from) p62d°k-encoding DNA.

The present invention also relates to nucleic acids, useful as probes and primers, which comprise p62d°k-encoding DNA or fragments or portions of DNA encoding p62d°k (e.g., portions of SEQ ID NO.: 1). The probes and primers are useful in identifying, isolating and/or amplifying p62dok encoding DNA (e.g., for diagnostic, prognostic, or research purposes) . For example, the probes are useful to identify

cells which contain p62d°k-encoding DNA and, thus, to diagnose a condition in which an oncogenic tyrosine kinase is active, such as in CML; to assess the likelihood that an individual will develop the condition (e.g., CML); and to monitor the progression of the condition in an individual.

Portions of DNA encoding p62dok are also useful as primers, e.g., in amplification methods.

The present invention also relates to host cells which contain p62d°k-encoding nucleic acid (DNA, RNA) and express the p62dok protein. The host cell may, optionally, express an oncogenic tyrosine kinase at levels which are elevated, the same as or decreased, relative to activity of a normal (non-oncogenic) tyrosine kinase. In a particular embodiment, the host cell comprises nucleic acid encoding the p62dok protein of the present invention operably linked to an expression control sequence; the encoded p62dok protein is expressed when the host cell is maintained under conditions suitable for expression.

The present invention also relates to antibodies or functional portions thereof (e.g., an antigen binding portion such as an Fv, Fab, Fab', or F(ab')2 fragment) which bind the p62dok protein of the present invention. In one embodiment, antibodies of the present invention recognize (bind) aberrantly tyrosine phosphorylated p62d°k, but do not recognize (bind) normal tyrosine phosphorylated p62d"k Such antibodies are useful, for example, in detecting or identifying cells which contain aberrantly tyrosine phosphorylated p62dok and, thus, for detecting or identifying cells in which an oncogenic tyrosine kinase is active. Tyrosine phosphorylation of p62dok occurs in cells, such as chronic phase hematopoietic progenitor cells from

CML patients. Therefore, tyrosine phosphorylated p62dok can be detected and, optionally, quantitated, to diagnose or aid in the diagnosis of CML and to monitor its progression.

The present invention also relates to a method of detecting p62dok protein, particularly aberrantly tyrosine phosphorylated p62d°k protein, in a sample of cells, such as cancer cells or cells thought to be progressing toward a cancerous state. As described herein, the appearance of aberrantly tyrosine phosphorylated p62d°k in cells, such as hematopoietic progenitor cells, is correlated with progression toward the transformed phenotype. Thus, it is possible to assess progression of cells toward transformation by, for example, quantitative or qualitative assessment of phosphorylation of p62d°k. Increased tyrosine phosphorylated p62dok (relative to the amount of tyrosine phosphorylated p62d°k present in normal cells) is indicative of unregulated or increased tyrosine kinase activity and, thus, assessment of tyrosine phosphorylated p62dok provides a method of diagnosing, aiding in the diagnosis of and monitoring the progression of diseases, particularly CML and other human cancers, in which aberrant (e.g., unregulated or increased) tyrosine kinase activity plays a causative role. Alternatively, presence of tyrosine phosphorylated p62dok at a different time in the cell cycle from that at which normal tyrosine phosphorylation of p62dok occurs can also be assessed in diagnosing, aiding in the diagnosis of or monitoring the progression of a condition (e.g. cancer) in which tyrosine kinase activity plays a causative role.

In one embodiment of the present method of detecting a tyrosine phosphorylated p62dok protein, a sample of cells to

be assessed (e.g., cells obtained from an individual, such as a human) is combined with an agent, such as an antibody or a small organic molecule, which recognizes (binds) aberrantly tyrosine phosphorylated p62d°k, but not normal tyrosine phosphorylated p62dok. Binding of the agent is assessed, using known methods. If binding occurs (e.g., if a complex of the agent and aberrantly tyrosine phosphorylated p62dok is formed), then aberrantly tyrosine phosphorylated p62dok is present, indicating that the cell is progressing toward or has acquired the transformed phenotype. Alternatively, an antibody or other agent which binds both normal and aberrantly tyrosine phosphorylated p62dok is used and the extent and/or timing of tyrosine phosphorylation of p62dok is assessed in the cells being analyzed and compared with the extent and/or timing of p62d°k tyrosine phosphorylation in control cells (e.g., corresponding normal cells). Abnormal tyrosine phosphorylation in the cells being analyzed, as indicated, for example, by tyrosine phosphorylation of p62dok to a greater extent, at a different point in the life cycle of the cells or at different tyrosine residue(s) than occurs in the normal cells, indicates that the cells being assessed are progressing toward or have acquired the transformed phenotype.

In one embodiment of the present method, an antibody which binds aberrantly tyrosine phosphorylated p62dok but does not bind the normal tyrosine phosphorylated p62d°k, is combined with cells to be assessed, under conditions suitable for specific binding of the antibody to the aberrantly tyrosine phosphorylated p62dak protein. The presence of antibody-aberrantly-tyrosine phosphorylated

p62dok protein complex is assessed, such as by detecting a label present on the antibody in the complex or by addition of an antibody which binds the antibody specific for aberrantly tyrosine phosphorylated p62d°k. Presence of the complex indicates that aberrantly tyrosine phosphorylated p62dok is present and, thus, that the cell is progressing toward or has acquired the transformed phenotype.

The present method of detecting aberrantly tyrosine phosphorylated p62dok in cells also provides a method of assessing the progression of CML or other types of cancer in which p62dok is constitutively tyrosine phosphorylated and, thus, enables a method of providing a prognosis of the cancer.

In one embodiment, cells which contain an oncogenic tyrosine kinase, which aberrantly phosphorylates a protein which, in its phosphorylated form, forms a complex with p120 ras GAP, progress to a transformed phenotype. A sample of cells to be assessed is obtained from the individual. This sample is treated to render aberrantly tyrosine phosphorylated p62dok available for binding by antibodies which bind aberrantly tyrosine phosphorylated p62dok but do not bind normal tyrosine phosphorylated p62d°k, thereby producing a treated sample. The treated sample is combined with antibodies which bind aberrantly tyrosine phosphorylated p62dok but do not bind normal tyrosine phosphorylated p62dok, under conditions appropriate for binding of the antibodies to aberrantly tyrosine phosphorylated p62dok The extent to which the antibody binds aberrantly tyrosine phosphorylated p62dok is determined, wherein if binding of the antibody with aberrantly tyrosine phosphorylated p62dok occurs, the sample

contains transformed cells or cells progressing to the transformed phenotype. The aforementioned steps are repeated at intervals, thereby producing sequential determinations of the extent to which the antibody binds aberrantly tyrosine phosphorylated p62d°k. Finally, the sequential determinations of the extent to which the antibody binds aberrantly tyrosine phosphorylated p62dok are determined wherein if the extent determined at an interval is greater than the extent determined at the previous interval, the condition is progressing and if the extent determined at an interval is less than the extent determined at the previous interval, the condition is not progressing.

Further, the present invention relates to methods of therapy for CML and other types of cancers and other conditions in which an oncogenic tyrosine kinase acts to constitutively tyrosine phosphorylate p62d°k protein. In the present method, a drug which interferes with tyrosine phosphorylation of p62dok and/or tyrosine phosphorylated p62dok function, directly or indirectly, is introduced into cells in which p62dok is undergoing constitutive tyrosine phosphorylation or has been constitutively tyrosine phosphorylated. In the method, the drug can be an antibody (e.g., an antibody which selectively binds tyrosine phosphorylated p62dok), a nucleic acid (e.g., antisense DNA, DNA or RNA encoding a protein which degrades or otherwise disables tyrosine phosphorylated p62dok or a protein or peptide which mimics pl20 ras GAP by binding tyrosine phosphorylated p62d°k), the GAP "mimic" itself, or a small organic molecule. The drug is introduced into an individual, such as a human or other mammal, in need of

therapy (prevention, slowing or inhibition of progression toward or reversal of the transformed phenotype) for CML or other cancer in which an oncogenic tyrosine kinase which constitutively tyrosine phosphorylated p62dok is present.

The drug can be administered systemically (e.g., orally, intramuscularly, intravenously, rectally) or introduced into a specific site (e.g., into bone marrow or a tumor) by known methods (e.g., injection, microparticle projectile bombardment). An antibody which selectively binds tyrosine phosphorylated p62dok will prevent its further function, such as by preventing it from binding p120 ras GAP.

Antisense DNA will bind DNA encoding p62dok and prevent expression of p62d°k. DNA encoding a protein which degrades or otherwise disables tyrosine phosphorylated p62d°k and DNA encoding a GAP "mimic" will be expressed in cells and the resulting product will act to inhibit p62d°k. Similarly, a protein or peptide which mimics GAP will interfere with p62dok function by binding p62dok and preventing it from forming a complex with GAP and/or interacting with other molecule(s) with which it would otherwise interact.

In another embodiment, a transgenic, non-human vertebrate, such as a transgenic, non-human mammal, in which cells have been modified to express, or modified to be able to express upon induction, an oncogenic tyrosine kinase is used as a model for the study of CML. For instance, the animal may be used to identify drugs useful in treating or preventing CML. In another example, a transgenic animal, which has restricted expression of the Dok gene in hematopoietic cells, could be used to study BCR-ABL signaling. In another example, a transgenic animal, in which expression of the Dok gene is reduced or

eliminated (e.g., by removal or disablement of the gene), may be used to study the ability of BCR-ABL to transform hematopoietic cells.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a scheme for p62dok purification, in which cytoplasmic lysate of Mo7/p210 cells is passed sequentially over two ion exchange columns and subsequently a-phosphotyrosine immuno-affinity chromatography is used to capture tyrosine phosphorylated p62d°k.

Figure 2A-2C shows the nucleotide sequence (SEQ ID NO.: 1) and amino acid sequence (SEQ ID NO.: 2) of human p62dok cDNA Figure 3 shows amino acid sequences (SEQ ID NO.: 3-9) of individual peptides purified by HPLC. Residues which were unreadable are indicated with a '?,' and those whose identity was uncertain are marked with parentheses.

Figure 4 shows the design of successful oligonucleotide primers, which were designed based on the amino acid sequence of the indicated peptides (SEQ ID NO.: 7-8) and were degenerate at the indicated positions.

Inosine bases (I) were included at positions of greatest degeneracy in the nucleotide sequence SEQ ID No.: 10-15.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to isolated nucleic acids (DNA, RNA) referred to as p62dok-encoding nucleic acids and portions or fragments of p62d°k-encoding nucleic acids, such as fragments of SEQ ID NO.: 1, which do not encode p62d"k. Such fragments are useful as nucleic acid probes for use in hybridization assays and as primers for

use, e.g., in amplification reactions. The present invention also relates to isolated p62dok proteins and polypeptides, as well as to methods for obtaining isolated p62dok proteins and polypeptides (normal and aberrantly phosphorylated p62d°k) and for producing recombinant p62dok proteins in cells in which tyrosine kinase activity is altered, resulting in aberrant tyrosine phosphorylation of cellular proteins. The present invention also relates to antibodies which bind p62d°k proteins and polypeptides, methods of detecting p62d°k in cells, and methods of therapy for CML and other types of cancer and other conditions in which an oncogenic tyrosine kinase aberrantly tyrosine phosphorylates a cellular protein, such as p62d"k.

Following is a description of the compositions and methods of the present invention.

Nucleic Acids, Constructs and Vectors As described herein, Applicants have isolated and sequenced DNA, referred to as p62d°k-encoding DNA, from a human teratocarcinoma cDNA library and have shown that it encodes a protein (p62d"k) which is constitutively tyrosine phosphorylated in mammalian, specifically human, cells in which tyrosine kinase activity is altered.

p62d°k-encoding DNA, as defined herein, includes: a) DNA comprising the nucleotide sequence of, or a nucleotide sequence substantially the same as, SEQ ID NO. : 1 or a portion of SEQ ID NO.: 1 sufficient to encode functional p62dok protein (e.g., a portion comprising the open reading frame); b) DNA which hybridizes to the complement of DNA of a) and encodes p62d"k; c) DNA which encodes the amino acid sequence of SEQ ID NO.: 2 or a sufficient number of amino

acid residues of SEQ ID NO. : 2 for the encoded protein to be a functional p62dok protein; and d) DNA which, due to the degeneracy of the genetic code, encodes the amino acid sequence encoded by DNA having the nucleotide sequence of SEQ ID NO.: 1. p62d°k-encoding DNA can be genomic DNA or cDNA and can be obtained from sources in which it occurs in nature (e.g., tissue or cell samples), from a DNA library, by means of recombinant technology or amplification procedures, by synthetic techniques. p62dok-encoding DNA is mammalian, particularly human, DNA. It is useful to produce p62dok protein or polypeptide, which can be carried out using available expression systems.

Alternatively, DNA of the present invention can be antisense DNA. Antisense nucleic acid is complementary, in whole or in part, to a sense strand and can hybridize with the sense strand. The target can be DNA, or its RNA counterpart (i.e., wherein T residues of the DNA are U residues in the RNA counterpart). When introduced into a cell, antisense nucleic acid can inhibit the expression of the gene encoded by the sense strand. Antisense nucleic acids can be produced by standard techniques.

In a particular embodiment, the antisense nucleic acid is wholly or partially complementary to and hybridizes with a nucleic acid having a sequence shown in Figure 1 (SEQ ID NO:1). For example, antisense nucleic acid can be complementary to a target nucleic acid having the sequence shown as the open reading frame in Figure 1 (SEQ ID NO:1) or to a portion thereof sufficient to allow hybridization.

In another embodiment, the antisense nucleic acid is wholly or partially complementary to and hybridizes with a target nucleic acid which encodes p62dok protein.

Nucleic acids referred to herein as "isolated" are nucleic acids separated away from the nucleic acids of the genomic DNA or cellular RNA of their source of origin (e.g., as it exists in cells or in a mixture of nucleic acids such as a library), and may have undergone further processing. "Isolated" nucleic acids include nucleic acids obtained by methods described herein, similar methods or other suitable methods, including essentially pure nucleic acids, nucleic acids produced by chemical synthesis, by combinations of biological and chemical methods, and recombinant nucleic acids which are isolated.

Nucleic acids referred to herein as "recombinant" are nucleic acids which have been produced by recombinant DNA methodology, including those nucleic acids that are generated by procedures which rely upon a method of artificial recombination, such as the polymerase chain reaction (PCR) and/or cloning into a vector using restriction enzymes.

A nucleic acid encoding p62d°k protein or a variant (e.g, portion) thereof can be incorporated into a vector, operably linked to one or more expression control elements, and the resulting construct introduced into host cells, which are maintained under conditions suitable for expression of p62dok protein, whereby the encoded protein is produced. The construct can be introduced into cells by a method appropriate to the host cell selected (e.g., transformation, transfection, electroporation, infection).

The encoded p62dok protein can be isolated from the host cells or medium. p62dok RNA can be obtained by these means as well.

Fragments of p62dok-encoding DNA, such as fragments of DNA of SEQ ID NO.: 1, which do not encode p62dok are useful as probes and primers for assays, amplification procedures, diagnostic and therapeutic methods. For example, p62dok DNA fragments characteristic of p62d°k-encoding DNA can be used as probes in standard hybridization methods to identify cells which contain p62d°k-encoding DNA. DNA which encodes p62d°k (full-length p62d°k DNA) can also be used as a probe.

The nucleic acids can be used as probes to detect and/or isolate (e.g., by hybridization with RNA or DNA) p62d°k-encoding DNA comprising SEQ ID NO.: 1 or variants, for example, in a sample (e.g., tissue) obtained from a host (e.g. human). Moreover, the presence or frequency of p62dok-encoding DNA or a particular variant in a sample(s) obtained from one or more affected hosts, as compared with a sample(s) from normal host(s), can be indicative of an association between a disease and a particular variant, which in turn can be used in the diagnosis of the condition.

DNA fragments are also useful for therapeutic purposes. For example, sense or anti-sense p62dok DNA fragments can be introduced into cells in which p62d°k expression is to be reduced (e.g., to decrease progression toward the transformed phenotype, by preventing or reducing expression of p62d°k and, thus, reducing its availability for tyrosine phosphorylation). The sense or anti-sense p62d°k DNA fragments hybridize with their complements, rendering them unavailable for further processing in the cells; as a result, p62dok expression is reduced (totally or partially).

Pharmaceutical compositions which comprise nucleic acids of the present invention and a suitable carrier (e.g., a buffer) are also the subject of this invention.

The compositions can include additional components, such as stabilizers.

Proteins and Peptides The present invention relates to p62dok proteins and polypeptides which can be obtained (isolated) from sources (e.g., cells) in which they occur in nature, produced using recombinant or genetic engineering methods or synthesized chemically. It also relates to pharmaceutical compositions which comprise p62d°k protein (constitutively tyrosine phosphorylated or normal tyrosine phosphorylated) and an appropriate carrier, such as a buffer. They may also comprise other components, such as stabilizers and other drugs.

As described herein, Applicants have obtained p62dok from human cells, determined its amino acid sequence and characterized it as to certain of its biochemical features and functions. As described in detail in the examples, p62dok was obtained from a human hematopoietic cell line which expresses an oncogenic tyrosine kinase (specifically, bcr-abl) and a corresponding human hematopoietic cell line which does not express the oncogenic tyrosine kinase (Mo7/p210 and Mo7 cell lines, respectively) by immunoprecipitating GAP from cytoplasmic lysates and analyzing the precipitated complexes by antiphosphotyrosine immunoblotting. Results showed that the corresponding cells in which an oncogenic tyrosine kinase was expressed contained tyrosine phosphorylated p62dok and that cells in

which the oncogenic tyrosine kinase was not expressed did not. (Example 1) p62dok was purified, as described in Example 1, by precipitating GAP from cytoplasmic lysates of 32P-labeled cells, expressing p210bCr-abl, i.e. Mo7/p210 cells, and analyzing the immune complexes by two-dimensional gel electrophoresis. The pattern of spots migrating at 62 kDa on the gel suggested different phosphorylation state isoforms existed. In vitro binding experiments using glutathione-5-transferase fused in frame to the GAP SH2- SH3-SH2 region resulted in the identical two-dimensional pattern of phosphorylated proteins migrating at 62 kDa as that obtained from GAP immunoprecipitations. Purified p62dok was produced using conventional and immuno-affinity chromatography; the final step in purification was separation by two-dimensional gel electrophoresis.

Characterization of p62d°k is described in Examples 2 and 3. As can be seen, p62dok is a novel protein with features of a signaling molecule. Tyrosine phosphorylated p62dok (e.g., from cells in which an oncogenic tyrosine kinase is active) associates with GAP; p62d°k from cells which lack or do not express an oncogenic tyrosine kinase does not associate with GAP. As is also described in the Examples, p62dok is the 62 kDa GAP-associating protein which is constitutively tyrosine phosphorylated in the hematopoietic progenitor cell population of CML patients.

Anti-p62d°k antibodies immunoprecipitated a pTyr-containing protein that co-migrated during SDS-PAGE with the tyrosine phosphorylated p62 that co-immunoprecipitated with GAP antibodies from lysates of CML progenitor cells. GAP was present in anti-p62d°k immunoprecipitates and p62dok

antibodies quantitatively depleted the lysates of a pTyr- containing p62, which provided further evidence that p62dok is the constitutively tyrosine-phosphorylated protein that migrated at 62 kDa in CML progenitor cells. (Example 3) In one embodiment, the subject protein is normal tyrosine phosphorylated p62d°k, which is present in normal cells and is not constitutively tyrosine phosphorylated and does not bind GAP in the assay described herein. In an additional embodiment, the protein is aberrantly tyrosine phosphorylated p62d°k, which is constitutively tyrosine phosphorylated in cells containing active oncogenic tyrosine kinases; it binds GAP. It is possible that tyrosine phosphorylation occurs in normal cells (i.e., it is possible that tyrosine phosphorylation occurs to some extent in normal cells). However, the extent and/or timing of tyrosine phosphorylation of p62dok are characteristically different in cells containing active oncogenic tyrosine kinases from the extent and/or timing in normal cells. One of the biochemical difference identified between p62dok produced in normal cells (p62dok produced in Mo7 cells) and p62dok produced in cells expressing an oncogenic tyrosine kinase (p62dok produced in Mo7/p210 cells) is the presence of phosphotyrosine residues in the latter. Results of a combined immunoprecipitation/ immunoblot assay showed that Mo7 p62dok migrates by SDS-PAGE as a singlet of approximately 61 kDa and that two slower migrating forms of p62dok (62 and 64 kDa) were produced in Mo7/p210 cells.

Both forms were tyrosine phosphorylated in Mo7/p210 cells and were detected in anti-GAP immunoprecipitates. It appears, however, that only a small proportion of total tyrosine phosphorylated p62dok complexes with GAP. The non-

phosphorylated form of p62dok in Mo7 cells was not found associated with GAP.

Proteins or polypeptides referred to herein as "isolated" are proteins or polypeptides purified to a state beyond that in which they exist in cells in which they are produced. "Isolated" proteins or polypeptides include proteins or polypeptides obtained by methods described herein, similar methods or other suitable methods, including essentially pure proteins or polypeptides isolated from the source in which they occur, proteins or polypeptides produced by chemical synthesis (e.g., synthetic peptides) , or by combinations of biological and chemical methods, and recombinant proteins or polypeptides which are isolated.

The term "p62dok" or rrp62dok protein" includes proteins and polypeptides obtained by any of the previously described methods. As used herein, "p62d°k protein" refers to naturally occurring or endogenous p62dok protein, proteins having an amino acid sequence which is the same as that of a naturally occurring or endogenous p62d°k protein (e.g., recombinant proteins), and functional variants of each of the foregoing (e.g., functional fragments and/or mutants produced via mutagenesis and/or recombinant techniques). Accordingly, as defined herein, the term includes p62d°k protein and functional fragments of p62d°k.

Both p62dok protein produced in mammalian (e.g., human) cells which lack an active oncogenic tyrosine kinase (referred to as normal tyrosine phosphorylated p62dok) and phosphorylated p62dok produced in mammalian (e.g., human) cells which contain an active oncogenic tyrosine kinase (referred to as constitutively or aberrantly tyrosine

phosphorylated p62d°k) are encompassed in these definitions.

Examples of "p62d°k protein" of the present invention include proteins having an amino acid sequence as set forth or substantially as set forth in Figure 1 (SEQ ID NO:2), proteins encoded by p62dok-encoding DNA as defined herein and functional portions thereof. The proteins can be obtained in an isolated state of at least about 50% by weight, preferably at least about 75% by weight, and more preferably, in essentially pure form.

Proteins or polypeptides referred to herein as "recombinant" or "recombinantly produced" are proteins or polypeptides produced by the expression of nucleic acids encoding the proteins in a host cell which is modified to contain the nucleic acids encoding the protein (e.g., by transfection with exogenous DNA which encodes p62dok protein) or is modified to express a gene present, but silent or expressed at low levels, in the host cell as obtained (e.g., by transfection with appropriate regulatory sequences which enhance or turn on expression of an endogenous gene). The term also refers to recombinantly produced or recombinant tyrosine phosphorylated p62d°k, which can be produced in cells which, as obtained or as modified, express p62dok and in which an oncogenic tyrosine kinase is also produced (either in the cells as obtained or as they are modified).

Suitable fragments or mutants of p62dok protein can be identified by screening. For example, the N-terminal, C-terminal, or internal regions of the protein can be deleted in a step-wise fashion and the resulting protein or polypeptide can be screened using a suitable binding assay.

A "functional fragment or portion" of p62d"k is an isolated

protein or polypeptide which has at least one property, activity and/or function characteristic of a p62dok protein, such as migrating at 62 kDa by SDS-PAGE, associating with GAP or binding an anti-GAP antibody.

In one embodiment, p62dok protein or a variant has an amino acid sequence which has at least about 50% identity, more preferably at least about 75% identity, and still more preferably at least about 90% identity, to the amino acid sequence shown in Figure 1 (SEQ ID NO:2).

In one particular application utilizing proteins and peptides, a drug containing a protein or peptide which mimics p120 ras GAP by binding tyrosine phosphorylated p62dok is introduced into an individual in need of therapy for a condition (e.g., CML) in which a protein (e.g., p62d°k)is aberrantly tyrosine phosphorylated as the result of the presence of an active oncogenic tyrosine kinase.

In this application, the aberrantly tyrosine phosphorylated protein is bound by the "mimic" and is therefore unavailable to bind pl20 ras GAP.

Method of Producina Recombinant Proteins Another aspect of the invention relates to a method of producing p62dok protein or a variant (e.g., portion) thereof. Recombinant p62dok protein can be obtained, for example, by the expression of a recombinant DNA molecule encoding p62dok protein or a variant thereof in a suitable host cell. Alternatively, recombinantly produced p62dok is expressed in a suitable host cell by turning on or enhancing expression of a p62d°k-encoding gene present in (endogenous to) the host cell.

Constructs suitable for the expression of p62dok protein or a variant thereof are also provided. The constructs can be introduced into a suitable host cell.

Cells which express a recombinantly-produced p62dok protein or variant thereof, can be produced and maintained in culture. Such cells are useful for a variety of purposes and can be used in the production of p62dok protein for characterization, isolation and/or purification, (e.g., affinity purification), and as immunogens, for instance.

Suitable host cells can be procaryotic, including bacterial cells such as E. coli, B. subtilis and other suitable bacteria (e.g., Streptococci) or eucaryotic, such as fungal or yeast cells (e.g., Pichia pastoris, Aspergillus species, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa), or other lower eucaryotic cells, and cells of higher eucaryotes, such as those from insects (e.g., Sf9 insect cells) or mammals, including humans (e.g., Chinese hamster ovary cells (CHO), COS cells, HuT 78 cells, 293 cells). See, e.g., Ausubel, F.M. et al., eds.

Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons Inc., (1993).

Host cells which produce p62dok protein or a variant thereof can be produced as follows. A nucleic acid (e.g., DNA) encoding p62dok is inserted into a nucleic acid vector, e.g., a DNA vector, such as a plasmid, virus or other suitable replicon for expression. The resulting vector is introduced into a host cell, using known methods, and the host cell is maintained under conditions appropriate for growth of the host cell and expression of the p62dok encoding DNA. For example, a nucleic acid encoding p62dok protein or a variant thereof can be incorporated into a

vector, operably linked to one or more expression control elements, and the construct can be introduced into host cells, which are maintained under conditions suitable for expression of p62dok protein whereby the encoded protein is produced. The construct can be introduced into cells by a method appropriate to the host cell selected (e.g., transformation, transfection, electroporation, infection).

The encoded p62dok protein can be isolated from the host cells or medium.

A variety of vectors is available, including vectors which are maintained in single copy or multiple copy, or which become integrated into the host cell chromosome.

Suitable expression vectors can contain a number of components, including, but not limited to one or more of the following: an origin of replication; a selectable marker gene; one or more expression control elements, such as a transcriptional control element (e.g., a promoter, an enhancer, terminator), and/or one or more translation signals; a signal sequence or leader sequence for membrane targeting or secretion (of mammalian origin or from a heterologous mammal or non-mammalian species). For example, nucleic acid encoding p62dok protein or a variant thereof could be inserted into any one of the multicloning sites of the pcDNA3 vector (Invitrogen, San Diego). Other acceptable vectors will be readily apparent to one skilled in the art.

Antibodies The present invention also relates to antibodies, both polyclonal and monoclonal, which bind p62dok in vitro and/or in vivo and, optionally, inhibit an activity or function

characteristic of p62d°k. In one embodiment, the antibodies bind constitutively tyrosine phosphorylated p62dok or normal tyrosine phosphorylated p62dok but not both, thus making them useful, for example, in assays in which the two forms of p62dok are to be distinguished (e.g., in an immuno-assay carried out to determine if cells are progressing toward a transformed state). In another embodiment, the types of antibodies that act as the inhibitors of p62dok are monoclonal antibodies, chimeric antibodies, or humanized antibodies. The present invention further relates to pharmaceutical compositions which comprise p62dok antibodies and a suitable carrier, such as a buffer; they can also include further components, such as stabilizers.

Preferably, the antibodies can bind mammalian (e.g.

human) p62dok with high affinity (for example, a Ka in the range of about 1 - 10 nM, or a Kd in the range of about 1 X 10-8 to 1 X 10-10 model).

The antibodies of the present invention are useful in a variety of applications, including separation techniques, research and diagnostic and therapeutic applications. For instance, they can be used to isolate and/or purify p62d°k or variants thereof (e.g., by affinity purification or other suitable methods), and to study p62dok structure (e.g., conformation) and function.

In addition, antibodies of the present invention can be used to detect and/or measure the level of p62dok in a sample (e.g., tissue or body fluid) obtained from an individual (e.g., a human). For example, a sample (e.g., tissue and/or fluid) can be obtained from an individual and a suitable immunological method can be used to detect and/or measure p62dok levels of constitutively tyrosine

phosphorylated p62dok or normal tyrosine phosphorylated p62d°k. In one embodiment, a method of detecting one or both forms of p62dok in a sample is carried out by obtaining a sample of tissue or cells from an individual; treating the sample to render p62dok available for binding by antibodies; contacting the treated sample with a p62dok antibody (e.g., an antibody which binds aberrantly tyrosine phosphorylated p62dok, normal tyrosine phosphorylated p62dok or both forms) under conditions suitable for binding of the antibody to p62dok and formation of p62d°k/antibody complexes; and detecting p62d°k/antibody complexes which are formed. The presence of complexes indicates that p62dok is present in the sample; the antibody used will determine which form(s) of p62dok is/are present in the complexes.

In an application of the method, antibodies which bind p62dok are used to analyze tissues or cells in mammals for p62dok reactivity and/or expression (e.g., immunohistolo- gically) . Thus, the antibodies of the present invention are useful in immunological diagnostic methods of assessing expression of p62dok (especially tyrosine-phosphorylated p62d°k) in normal tissues or cells and cancerous tissues or cells.

p62dok antibodies also have therapeutic uses. A p62dok antibody can be administered in an amount effective to inhibit p62dok activity. For therapy, an effective amount is sufficient to achieve the desired therapeutic and/or prophylactic effect (such as an amount sufficient to reduce or prevent tyrosine phosphorylated p62dok function or activity, such as binding to GAP). The antibody can be administered in a single dose or multiple doses. The dosage can be determined by methods known in the art and is

dependent, for example, upon the individual's age, sensitivity, tolerance and overall well-being. Suitable dosages for antibodies can be from 0.1-1.0 mg/kg body weight per treatment.

According to the method, an antibody can be administered to an individual (e.g., a human) alone or in conjunction with another agent, which is administered before, along with or subsequent to administration of the antibody.

Administration of Compositions of the Present Invention Compositions of the present invention can be administered by a variety of routes, including, but not limited to, parenteral (e.g., injection, including but not limited to, intravenous, intraarterial, intramuscular, subcutaneous; inhalation, including but not limited to, intrabronchial, intranasal or oral inhalation, intranasal drops; topical) and non-parenteral (e.g., oral, including but not limited to, dietary; rectal).

The formulation used will vary according to the route of administration selected (e.g., solution, emulsion, capsule). An appropriate composition comprising the nucleic acids, proteins or antibodies to be administered can be prepared in a physiologically acceptable vehicle or carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles can include various additives, preservatives, or

fluid, nutrient or electrolyte replenishers. See, generally, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. (1980). For inhalation, the compound can be solubilized and loaded into a suitable dispenser for administration (e.g., an atomizer, nebulizer or pressurized aerosol dispenser). Nucleic acids, proteins and antibodies can be administered individually, together or in combination with other drugs or agents (e.g., other chemotherapeutic agents, immune system enhancers).

The present invention is further illustrated by the following examples, which are not intended to be limiting in any way.

Methods and Materials The methods and materials described below were used in carrying out the work described in the examples which follow.

Immunoprecipitation and Western Blot Analysis The Mo7 cell line, and a derivative of Mo7 which expresses p210bcraM, Mo7/p210 (provided by Dr. Brian Druker, Portland, OR), were maintained in RPMI (Gibco) + 10% Fetal Clone III (Hyclone) at 37"C and 5% CO2. Avanzi, G.C. et al., Brit. J. Hemat., 69:359-366 (1988); Matsuguchi,T. et al., J. Biol. Chem., 269:5016-5021 (1994).

The Mo7 growth media contained 10 ng/ml recombinant GM-CSF (provided by Genetics Institute, Boston, MA).

For immunoprecipitations, cells were washed twice in PBS containing 1 mM Na3VO4 and 100uM phenylarsine oxide (PAO), and lysed. Lysis buffer (LB) was 50 mM Tris-HCI, pH 7.6, 150 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1% Triton X-100,

0.5 ug/ml leupeptin, 50 ug/ml soybean trypsin inhibitor, 0.1 ug/ml aprotinin, 100 ug/ml phenymethyl sulfonyl fluoride, 1 mM Na3VO4, and 100 uM PAO. Prior to immunoprecipitations, cell lysates were clarified by centrifugation at 14,000 rpm at 40C. All experiments were carried out with equal protein concentration (750 pg protein/0.5 ml LB). Lysates were precleared by 15 min rotation at 40C with Protein A-Sepharose (Pierce).

Following addition of antibody, immune complexes were allowed to form during a 90 minute rotation at 40C. After Protein A-Sepharose was added, rotation at 40C was continued for 1 hr; then, the beads were washed five times in buffer containing 20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 100 ug/ml PMSF, and 1 mM Na3VO4. SDS-PAGE and western transfer were done according to standard protocols. Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988). Following transfer, the nitrocellulose was blocked for 1 hour in TBST containing 3% BSA (Sigma), incubated for 1 hour with blocking buffer containing primary antibody, washed with TBST, incubated for 1 hour with secondary antibody, again washed with TBST, and developed by the ECL method (Amersham). The following antibodies were used for the precipitations: anti-GAP mAb B4F8 (Santa Cruz); anti-phosphotyrosine mAb PY20 (ICN); anti-p62 polyclonal antibody SC-108 (Santa Cruz); and anti- p62dok polyclonal antibody (see below). The following antibodies were used for immunoblotting: anti- phosphotyrosine polyclonal antibody B5 (Kozma, L.M. et al., Methods in Enz., 201:28-43 (1991)), anti-PTyr mAb 4G10

(UBI), anti-GAP polyclonal antibody RH6-2A, and antip62dok polyclonal antibody.

To radiolabel proteins, cells were washed twice with PBS, and resuspended in phosphate-free media containing 10% dialyzed fetal calf serum (Gibco) and 1 mCi/ml 32p~ orthophosphate (ICN). Following a two hour labeling period, cells were washed twice as above, and resuspended in LB at 107 cells/IP. Immunoprecipitations were conducted as described above. For phosphoamino acid analysis, labeled GAT 62 was transferred to PVDF and processed exactly as described. van der Geer, P. et al., Protein Phosphorylation, A Practical Approach, D. Grahame Hardie, ed., IRL Press, pp. 31-59 (1993). Two dimensional electrophoresis was conducted exactly as described.

O'Farrell, Patrick H., J. Biol. Chem., 250:4007-4021 (1975); Garrels, J., "Quantitative two-diemensional gel electrophoresis of proteins." In Methods in Enzymology 100: Recombinant DNA(b), R. Wu et al., eds., Academic Press, San Diego (1983), pp. 411-423.

Peptide competition experiments were conducted by incubating lmg of either antigenic peptide or unrelated peptide (CLIGEGTYGVVYK), SEQ ID NO.:14, with cell lysate prior to immunoprecipitation, or with nitrocellulose prior to western blotting.

Hematopoietic progenitor cells from CML patients in the chronic phase were isolated and processed for immunoprecipitation exactly as described. Wisniewski, D.

et al., Leukemia, 10:229-237 (1996).

Purification of p62dok Fifty liters of Mo7/p210 cells were grown to a density of 2 x 106/ml in RPMI containing 20 mM Hepes (Gibco) and 5% Fetal Clone III (Hyclone), harvested by centrifugation and treated for 10 min at 37" with 100 pM PAO in growth media.

The cells were then washed as above and lysed in LB containing 50 mM NaCl. The lysate was adjusted to 3M urea by the addition of buffer containing 6M urea, 50 mM Tris- HCl, pH 8.3, 5 mM EDTA, 1 mM EGTA, following which it was loaded onto a Q Sepharose HP (Pharmacia) column (19 X 5 cm). Bound proteins were eluted from the column with an increasing linear gradient of Nail, from 25 mM NaCl to 600 mM Nazi. Alternatively, cytoplasmic proteins were batch absorbed to Q Sepharose HP and step-eluted with buffer containing 350 mM NaCl. Fractions containing p62dok were pooled, adjusted to 50 mM acetate, pH 4.6, by the addition of 2 volumes of 75 mM acetic acid, 5 mM EDTA, 3M urea, and loaded onto an SP column (5 X 2.5 cm, TosoHaas). Bound proteins were eluted with an increasing linear gradient of NaCl, from 100 mM NaCl to 800 mM Nazi. Alternatively, pooled fractions were batch absorbed to SP ToyoPearl and step-eluted with buffer containing 300 mM NaCl. Fractions containing p62dok were pooled and total protein was precipitated with ice-cold acetone. The precipitate was resuspended in 8M urea, dialyzed against 50 mM Tris-HCl, pH 7.6, 150 mM Nail, 5 mM EDTA, 0.1% Triton X-100, 0.5 mM Na3VO4 and loaded onto an anti-pTyr antibody column, 4G10- Sepharose (UBI). The column was washed with load buffer and distilled water, and bound proteins were eluted by the addition of 4% acetic acid. Following removal of the solvent by lyophilization, the eluted material was resolved

by two-dimensional gel electrophoresis. The gel was stained with 0.05% Coomassie Brilliant Blue G (Sigma) in 5% AcOH-10% MeOH, and destained with the same solution lacking Coomassie dye.

Peptide Sequenceing The acrylamide gel containing the spots of Coomassie- stained p62d°k was excised from several gels, and washed sequentially with distilled water and 50% MeOH. The protein in the gel was digested with 500ng Achromobacter protease I (Wako) in 0.05M Tris-HCl, pH 9.0, 0.1% Tween-20 for 20 hours at 300C, then the resulting peptide fragments were extracted from the gel with a solution containing 50% acetonitrile and 0.064% TFA, and separated by HPLC using a Vydac C18 column (1.0 x 250 mm, 10 pm, 300Å). Peptides were eluted with an increasing gradient of acetonitrile.

HPLC fractions containing various peaks of eluted peptides were individually applied to an automated protein sequencer (ABI model 494).

cDNA Clonina Total RNA, extracted from Mo7/p210 cells with RNAzol (Tel-Test, Inc.), was used to make random primed cDNA for the RT-PCR. Reverse transcription was carried out at 500C with Superscript II (Gibco BRL) according to the instructions of the manufacturer. Three rounds of nested PCR were conducted with the primers listed below from 5' to 3' of the cDNA, with (S) indicating sequence of the sense strand, (C) indicating sequence of the complementary strand and I indicating inosine. Round 1: K29N1, TGGGCIGTIT/CTITAT/CCC(S) [SEQ ID NO.:10] and K31C1,

TTIAG/AIAG/AIAG/AT/CT/CTGT/CTGT/CTGA/G/C/TGC(C) [SEQ ID NO.:12]; round 2: K29N2, GTIT/CTITAT/CCCT/AGCIT/AC/GCCICATGG(S) [SEQ ID NO.: 11] and K31C2, T/CTGT/CTGT/CTGIGCATGT/CTCA/GTA (C) [SEQ ID NO.:13]; round 3: K29N3, CATGGIGTIGCIA/CGIT/CTIGAA/GTT(S) [SEQ ID NO.:14] and K31C3, T/CTGIGCATGT/CTCA/GTAIAG/AA/GTC(C) [SEQ ID NO.:15].

The resulting PCR was radiolabeled and used as a probe to screen an oligo(dT)-primed human teratocarcinoma cDNA library. Skowronski, J. et al., Mol. and Cell Biology, 8: 1385-1397 (1988). Hybridization was performed in buffer containing 6 x SSC, 0.1% SDS, 5 x Denhardt's solution, and 20 ug/ml sonicated salmon sperm DNA (Sigma) at 670C. The following washes were performed at the same temperature: 30 min, 2 x SSC, 0.1% SDS; 30 min, 1 x SSC, 0.1% SDS; 30 min, 0.2 x SSC, 0.1% SDS. Positive clones were isolated, phage DNA was purified, cDNA inserts were excised by restriction enzyme digest, and the longest insert was cloned into a plasmid vector (pBluescript, SK-) for DNA sequencing, all using standard methodologies. Sambrook, J.

et al., Molecular Clonina: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). For Northern analysis, blots of poly A+ RNA from different human tissues (Clontech) were probed with a radiolabeled 1526 bp fragment of Dok cDNA obtained by PCR, according to the instructions of the manufacturer. The cDNA sequence has been submitted to Genbank (Accession number U70987).

Anti-p62d°k Antibodies A peptide with a C-terminal cysteine was synthesized (PQGPAFPEPGTATGSC [SEQ ID NO.:16]; BioSynthesis, Inc.),

conjugated to maleimide activated KLH (Pierce), and used to immunize rabbits (Hazelton). To affinity purify anti- peptide antibodies, ammonium sulfate to 50% saturation was added to sera. Precipitated material was taken up in PBS, dialyzed against a large volume of PBS, and passed over a resin to which peptide had been coupled (Sulfo-link Coupling Gel, Pierce). antibodies were released from the column by addition of 0.1 M glycine, pH 2.5.

Stimulation with c-Kit Licand Exponentially growing Mo7 cells (107/IP) were washed free of growth factor and incubated for 16 hours at 37° in IMDM (Gibco) containing 1% fetal calf serum (Gibco). Cells were then pelleted by centrifugation, resuspended in IMDM + 1% serum and exposed to c-kit ligand (100ng/ml) for 5 minutes at 37°. Cells were then washed once with PBS and processed for immunoprecipitation/western blot analysis exactly as described. Wisniewski, D., et al., Leukemia, 10: 229-237 (1996). Human recombinant c-kit ligand was from Immunex Corp., Seattle.

EXAMPLE 1 Identification and Purification of p62dok Following the initial observation of tyrosine phosphorylated p62 in primary CM,L progenitor cells (Wisniewski, D., et al., Leukemia, 8:688-693 (1994)), the Mo7 cell line and a derivative of Mo7 expressing p210bCr-abl, Mo7/p210 (Avanzi, G.C., et al., British Journal of Haematology, 69:359-366 (1988); Matsuguchi, T., et al., J.

Biol. Chem. 269:5016-5021 (1994)), was utilized to determine whether or not the p62 protein was similar to the molecule heretofore known as the GAP-associated p62. GAP

was immunoprecipitated from cytoplasmic lysates of Mo7 cells and cells expressing p210bCr-abl, i.e. Mo7/p210 cells.

The precipitated complexes were then analyzed by anti- phosphotyrosine immunoblotting. Ellis, C., et al., Nature 343:377-381 (1990). Anti-GAP immunoprecipitates from Mo7/p210 lysates, but not from Mo7 lysates, contained tyrosine phosphorylated p62 which migrated as a doublet.

The GAP-associated doublet was also observed in anti- phosphotyrosine immunoprecipitates from lysates of Mo7/p210 cells, but not from Mo7 cells. However, a similar protein in complexes precipitated by rabbit polyclonal antibodies against the molecule previously reported as the GAP- associated p62 (Wong, G., et al., Cell 69: 551-558 (1992)) were not detectable. The same polyclonal antibodies were unable to immunoblot a specific polypeptide in anti-GAP immunoprecipitates, nor could they precipitate a phosphorylated 62 kDa polypeptide from lysates of 32p~ labeled Mo7/p210 cells. As a result of these immunological discrepancies the p62 protein, henceforth called p62 protein downstream of tyrosine kinases (p62dok), was purified.

To purify p62dok, an assay for the identity of the protein was developed. GAP was precipitated from cytoplasmic lysates of 32P-labeled cells expressing p210bCr- abi and the immune complexes were analyzed by two- dimensional gel electrophoresis. A characteristic pattern of spots migrating at 62 kDa resulted, suggestive of different phosphorylation state isoforms. Because p62 previously had been shown to bind to the SH2-SH3-SH2 region of GAP (Marengere, L.E.M. and Pawson, T., J. Biol. Chem., 267:22779-22786 (1992)), in vitro binding experiments were

conducted, utilizing GST fused in frame to the GAP SH2-SH3- SH2 region. These experiments yielded the identical two- dimensional pattern of phosphorylated proteins migrating at 62 kDa as that obtained from GAP immunoprecipitations. A combination of conventional and immuno-affinity chromatography was used to purify p62dok. Lysates of Mo7/p210 cells were passed sequentially over two ion- exchange columns, following which an anti-pTyr antibody column was used to capture remaining tyrosine phosphorylated proteins. As a final step in the purification, proteins eluted from the antibody column were separated by two-dimensional gel electrophoresis.

EXAMPLE 2 Assessment of p62dok as a Potential Signaling Molecule Coomassie-stained p62dok was excised from a gel and digested with protease to yield peptide fragments, five of which were sequenced. Degenerate oligonucleotide primers were designed based on the amino acid sequence of two peptides, and used in RT-PCR to amplify a fragment of p62dok cDNA. After three rounds of nested PCR, a 900 bp product was obtained, which was used to screen a human teratocarcinoma cDNA library. Skowronski, J. Ae al., Mol.

Cell Biology, 8:1385-1397 (1988). A screen of 750,000 individual phage clones yielded five positives. The amino acid sequence encoded by the longest insert representing Dok cDNA is illustrated in Figure 2. The cDNA encoded a novel protein of 481 amino acids with a predicted molecular mass of 53 kDa. In vitro translated p62dok migrated at 61 kDa by SDS-PAGE, similar to p62dok immunoprecipitated from Mo7 cells by anti-p62dok antibodies. A profile search

utilizing the Prosite profile database detected a putative pleckstrin homology (PH) domain (Mussachio, A. et al., TIBS, 18:343-348 (1993)) at the extreme N terminus of p62dok. Aside from this, no significant homology between p62dok and other known proteins was found. p62d°k has 15 tyrosines, 10 of which are located within a C-terminal stretch one third the length of the molecule. Kyte- Doolittle hydrophobicity analysis indicates that this region of p62dok is more hydrophilic than other portions of the molecule. Additionally, p62dok is relatively proline- rich, with ten PXXP motifs. The PXXP motif has been demonstrated to be the most conserved motif within known SH3 domain ligands (Yu, H. et al., Cell, 76:933-945 (1994)), suggesting that p62dok might form signaling complexes with other molecules that contain SH3 domains.

Recently, the substrate specificities of a variety of tyrosine kinases were proposed. Songyang, Z. et al., Nature, 373:536-539 (1995). The optimal peptide substrate of the c-abl kinase was found to be I/V/LYAAP/F, SEQ ID NO.:17. Although none of the tyrosines of p62dok have adjacent residues exactly matching this consensus sequence, residues 295-299 (LYAEP) [SEQ ID NO.:18] are a close match.

Additionally, three tyrosines are within the context I/LYXXP, SEQ ID NO.:19. Therefore, p62dok might be a direct substrate of p2l0bcrabl In addition, the recognition specificities of the SH2 domains of a variety of known signaling molecules have been proposed. Songyang, Z. et al., Cell, 72:767-778 (1993); Songyang, Z. et al., Mol.

Cell Biol., 14:2777-2785 (1994). One of the p62dok tyrosines (residue 449) is in a context which is consistent with the proposed SH2 recognition motifs. This most C-

terminal tyrosine is located within the sequence SALYSQVQ, SEQ ID NO.:20, suggesting that this site, if phosphorylated, might associate with the SH2 domain of csk, the c-terminal src kinase. Evidence for the direct interaction between the SH2 domain of csk and the GAP- associated p62 has been presented. Neet, K. and Hunter, T., Mol. Cell Biol., 15:4908-4920 (1995). It is possible that, in addition to GAP, tyrosine phosphorylated p62dok forms a complex with csk and other yet unknown SH2 domain- containing signaling proteins.

To demonstrate that the clone isolated encoded a GAP- associated p62, rabbit polyclonal antibodies were raised against a synthetic peptide derived from the sequence of p62d"k. The antigenic peptide specifically blocked the ability of the antibody to precipitate this doublet, while an unrelated peptide had no effect. Furthermore, anti- p62dok antibodies detected a 62 kDa doublet in immunoblots of proteins that co-immunoprecipitated with antibodies directed against GAP, and the antigenic peptide specifically blocked the ability of the antibody to detect this doublet, while an unrelated peptide had no effect.

Taken together, these data demonstrated that the antibodies recognized a constitutively-tyrosine phosphorylated, GAP- associated p62 from Mo7/p210 cells.

Given the important signaling role that tyrosine kinases play in hematopoietic growth control, it is not surprising that the sudden appearance of a novel tyrosine kinase activity (e.g. p210bCr-abl) within a single primitive progenitor cell would perturb the intracellular signaling cascades that ensure orderly hematopoiesis. Bolen, J.B. et al., FASEB J., 6:3403-3409 (1992). The two fused portions

of p210bCr-abl are each derived from molecules which themselves contain some of the modular features common to signaling proteins. Cohen, G.B. et al., Cell, 80:237-248 (1995). In addition, bcr-abl is a large molecule that is extensively tyrosine phosphorylated, providing numerous potential docking sites for SH2 domain-containing proteins.

Thus, this activity could interact with various components of pre-existing signaling networks within a primitive stem cell. Pawson, T., Nature, 373:573-580 (1995).

As a potential signaling molecule, p62dok has several noteworthy features. It contains a putative pleckstrin homology (PH) domain at its extreme amino terminus, comprising residues 3 - 119. Mussachio, A. et al., TIBS, 18:343-348 (1993). A variety of proteins involved in signaling and/or cytoskeletal organization contain PH domains, among them GAP. A proposed function of the PH domain is to mediate protein-protein interactions. Pawson, T., Nature, 373:573-580 (1995). Additionally, it is thought that these conserved structural domains mediate interactions with cellular membranes, possibly by binding to different inositol phosphate components of the lipid bilayer. Lemmon, M.A. et al., Cell, 85:621-624 (1996).

For example, the PH domain of PLC51 binds specifically and with high affinity to PtdIns2. Lemmon, M.A. et al., PNAS, 92:10472-10476 (1995). Thus, the function of the N- terminal region of p62dok might be to serve as a modular binding domain, either localizing the molecule to the cell surface or directing its interaction with a variety of proteins. If the function of the PH domain of p62dok is to bind other proteins or components of the membrane, then the C-terminal portion of the molecule would likely be

positioned to interact with a variety of proteins and/or small molecules within the cytosol. Therefore, it might contain a critical functional domain of the polypeptide.

The majority of tyrosines within p62dok are located in the C-terminal half of the molecule, and it is speculated that they are available as targets for the activity of neighboring tyrosine kinases, for example p210bCr-abl itself.

It is possible that these tyrosines, when phosphorylated, serve as docking sites for proteins which contain SH2 domains. In addition to the C-terminal' tyrosines, p62dok contains ten PXXP motifs. Because PXXP -is the core conserved sequence of proline-rich regions which are recognized by SH3 domains, it is possible that p62dok can interact with signaling molecules containing SH3 domains.

Yu, H. et al., Cell, 76:933-945 (1994).

p62dok binds in vitro to the N-terminus of GAP, suggesting that tyrosine phosphorylated p62dok interacts in vivo with the SH2 domains of GAP. Originally, GAP was identified based on its ability to increase the intrinsic GTPase activity of ras. Trahey, M. and McCormick, F., Science, 238:542-545 (1987). Its catalytic domain is located in the C-terminal half of the molecule. In recent years, evidence has accumulated that the N-terminus of GAP plays a functional role in intracellular signaling, separate and distinct from the C-terminus. Valius, M. et al., Mol. Cell Biol., 15:3058-3071 (1995). For example, overexpression of the N-terminal region of GAP in Rat-2 cells resulted in disruption of the actin cytoskeleton and focal contacts, as well as decreased fibronectin binding and cell adhesion. McGlade, J. et al., EMBO J., 12:3073- 3081 (1993). Therefore, it is possible that constitutive

p62dok association with GAP might deregulate a signaling network involving the N-terminus of GAP. Currently, it is unclear whether the association of p62dok with GAP is due solely to a direct interaction between a phosphotyrosine of the former and the SH2 region(s) of the latter. In view of the PXXP motifs of p62d°k, it is possible that the SH3 domain of GAP stabilizes the association of the two molecules.

EXAMPLE 3 Analysis of p62dok At present, the only biochemical difference between Mo7 p62dok and Mo7/p210 p62dok detected is the presence of phosphotyrosine residues in the latter population. Both populations contained phosphoserine, although whether identical serine residues are phosphorylated is unknown.

Moreover, a combined immunoprecipitation/immunoblot assay indicated that Mo7 p62dok migrated by SDS-PAGE as a singlet of 61 kDa, whereas it shifted completely to two slower migrating forms of 62 and 64 kDa in Mo7/p210 cells. This quantitative shift suggests that the entire population of p62dok within the cell is altered by the presence of p210bCr- abi Both forms of p62dok in p210bcrabl containing cells were tyrosine-phosphorylated, and both forms could be detected in anti-GAP immunoprecipitates and in anti-pTyr immunoprecipitates. However, preliminary data indicates that only a small proportion of total tyrosine phosphorylated p62dok complexes with GAP. The faster migrating, non-tyrosine phosphorylated form of p62dok in Mo7 cells was not found associated with GAP.

The next step was to determine whether or not p62dok was involved in a signal transduction pathway initiated by

a receptor tyrosine kinase. Previous studies had implicated a 62 kDa protein as a downstream target of c-kit receptor kinase activity in human primary lineage negative normal hematopoietic progenitor cells. Wisniewski, D. et al., Leukemia, 10:229-237 (1996). The c-kit receptor is a type II receptor tyrosine kinase that is structurally related to the PDGF receptor. van der Geer, P. et al., In Protein Phosphorylation a Practical Approach, D. Grahame Hardie, ed. (Oxford, England: IRL Press), pp. 31-59 (1993).

Its ligand, c-kit ligand (steel factor, MGF, stem cell factor), has pleiotropic effects on the development of diverse cell types. Galli, S.J. et al., Adv. Immunol., 55:1-96 (1994). Among other influences, it promotes hematopoietic progenitor cell survival, and acts synergistically with GM-CSF, G-CSF, IL-3, and Epo to enhance in vitro colony formation by early progenitors of various lineages. McNiece, I.K. et al., Exp. Hematol, 19:226-231 (1991). p62dok was found to be rapidly tyrosine phosphorylated after stimulation of Mo7 cells with c-kit ligand. This result indicates that tyrosine phosphorylation of p62dok is a rapid biochemical event, occurring shortly after a receptor tyrosine kinase is activated.

The next step was to determine whether or not p62dok was the tyrosine-phosphorylated p62 previously identified in lysates of primary chronic phase CML progenitor cells.

Wisniewski, D. et al., Leukemia, 8:688-693 (1994). Anti- p62dok antibodies immunoprecipitated a pTyr-containing protein that co-migrated during SDS-PAGE with the tyrosine phosphorylated p62 that co-immunoprecipitated with GAP antibodies from lysates of CML progenitor cells. In

addition, GAP could be observed in anti-p62d°' immunoprecipitates. Furthermore, p62dok antibodies quantitatively depleted the lysates of a pTyr-containing p62 to make it likely that p62dok was the constitutively tyrosine-phosphorylated protein migrating at 62 kDa in CML progenitor cells. Based on this experimental data, p62dok is concluded to be the 62 kDa GAP-associating protein previously reported to be constitutively tyrosine phosphorylated in the hematopoietic progenitor cell population of CML patients.

Current evidence suggests that p62dok is a substrate of the constitutive tyrosine kinase activity of p210bCr-abl, and that its abnormal modification within Ph+ primitive hematopoietic progenitors plays a role in the complex physiological disorder manifested in the chronic phase of CML. Constitutive tyrosine phosphorylation of p62 is detected in all primary chronic phase CML primitive blasts observed to date. Wisniewski, D. et al., Leukemia, 10:229- 237 (1996) Although at present the biological function of p62dok is unknown, several lines of evidence suggest that it plays a role in distinct signal transduction networks, most likely as a component of a signaling cascade initiated by receptor or membrane-associated tyrosine kinases. It is possible that p62dok is the human homologue of the protein widely known as the classical GAP-associated p62 (Lock, P.

et al., Cell, 84:23-24 (1996)), and that many of its properties are similar to those heretofore attributed to the latter molecule. The rapid tyrosine phosphorylation of the GAP-associated p62 in Rat-1 fibroblasts upon EGF stimulation and in Rat-2 fibroblasts upon PDGF stimulation

has been reported. Ellis, C. et al., Nature, 343:377-38 (1990). Using a different system, Filvaroff et al., demonstrated the tyrosine phosphorylation of a GAP- associated p62 in primary mouse keratinocytes as one of the initial events in calcium-induced terminal differentiation.

Filvaroff et al. (1992). Other agents, such as EGF, TGF , and phorbol ester, did not induce phosphorylation of p62, although they did result in the tyrosine phosphorylation of the signaling molecules PLCy and PI3 kinase. These latter enzymes were unaffected by calcium stimulation. Finally, it has been demonstrated that c-kit ligand induces the rapid tyrosine phosphorylation of a p62, most likely the GAP-associated p62, in primitive hematopoietic progenitors.

Wisniewski, D. et al., Leukemia, 10:229-237 (1996).

Herein, it is shown that p62dok is rapidly tyrosine phosphorylated upon activation of the c-kit receptor tyrosine kinase, suggesting that it lies on a signaling pathway downstream of the c-kit receptor. In light of the constitutive tyrosine phosphorylation of p62d°k in hematopoietic progenitors containing p210bCr-abl, it is provocative that some of the biological effects of both p210bCr-abl and c-kit ligand appear to be similar, and these effects are manifested in a relatively mature stem cell population at the time of lineage commitment rather than at the level of self-renewing stem cell. Strife, A. et al., Cancer Res., 53:401-409 (1993). Furthermore, because tyrosine phosphorylation of p62dok was observed in chronic phase progenitor cells from all CML patients tested (Wisniewski, D. et al., Leukemia, 10:229-237 (1996)), it is possible that aberrant or untimely tyrosine phosphorylation

of p62d°k is a key step in the development of the chronic phase of CML.

Numerous investigators working with a variety of transformed cell lines have observed the tyrosine phosphorylation of a p62 and its association with GAP.

Ellis, C. et al., Nature, 343:377-381 (1990); Moran, M.F.

et al., Mol. Cell Biol., 11:1804-1812 (1991). Murine p62dok has been identified and demonstrated to be directly associated with the v-abl activated tyrosine kinase.

Yamanashi, Y. and Baltimore, D., Cell, January 24, 1997.

In addition, the extent to which p62 is tyrosine phosphorylated has been shown to correlate with the transforming capabilities of a number of different oncogenes, including v-src, v-abl, v-fps, and v-mos.

Ellis, C. et al., Nature, 343:377-381 (1990); Lugo et al.

(1990); Zhao, J.F. Ae al., Oncogene, 11:161-173 (1995).

The widespread correlation between constitutive tyrosine phosphorylation of p62dok and the transformed phenotype suggests that p62dok plays an important role in mitogenic signaling, and that the aberrant phosphorylation of p62dok might contribute to the progression of different human diseases.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Cold Spring Harbor Laboratory (B) STREET: 100 Bungtown Road (C) CITY: Cold Spring Harbor (D) STATE/PROVINCE: New York (E) COUNTRY: USA (F) POSTAL CODE/ZIP: 11723 (G) TELEPHONE: (516) 367-8397 (I) TELEFAX: (516) 367-8855 (ii) TITLE OF INVENTION: A Novel Human Gene/Protein Involved in Chronic Myelogenous Leukemia (iii) NUMBER OF SEQUENCES: 20 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Hamilton, Brook, Smith & Reynolds, P.C.

(B) STREET: Two Militia Drive (C) CITY: Lexington (D) STATE: MA (E) COUNTRY: US (F) ZIP: 02173 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 08/787,091 (B) FILING DATE: 22-JAN-1997 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 60/030,418 (B) FILING DATE: 01-NOV-1996 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Granahan, Patricia (B) REGISTRATION NUMBER: 32,227 (C) REFERENCE/DOCKET NUMBER: CSHL96-05pA (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 781-861-6240 (B) TELEFAX: 781-861-9540 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1446 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1443 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ATG GAC GGA GCA GTG ATG GAA GGG CCG CTT TTT TTG CAG AGT CAG CGC 48 Met Asp Gly Ala Val Met Glu Gly Pro Leu Phe Leu Gln Ser Gln Arg 1 5 10 15 TTT GGG ACC AAG AGG TGG AGG AAG ACC TGG GCC GTG CTC TAC CCG GCC 96 Phe Gly Thr Lys Arg Trp Arg Lys Thr Trp Ala Val Leu Tyr Pro Ala 20 25 30 AGT CCC CAC GGC GTA GCG CGG CTC GAG TTC TTT GAC CAT AAG GGG TCG 144 Ser Pro His Gly Val Ala Arg Leu Glu Phe Phe Asp His Lys Gly Ser 35 40 45 AGC TCT GGG GGT GGC CGA GGG AGC TCG CGC CGC CTG GAC TGC AAA GTG 192 Ser Ser Gly Gly Gly Arg Gly Ser Ser Arg Arg Leu Asp Cys Lys Val 50 55 60 ATC CGT CTG GCT GAG TGT GTG AGT GTG GCC CCC GTC ACC GTG GAG ACC 240 Ile Arg Leu Ala Glu Cys Val Ser Val Ala Pro Val Thr Val Glu Thr 65 70 75 80 CCC CCT GAG CCC GGC GCC ACT GCC TTC CGC CTG GAC ACT GCT CAG CGC 288 Pro Pro Glu Pro Gly Ala Thr Ala Phe Arg Leu Asp Thr Ala Gln Arg 85 90 95 TCG CAC CTG CTG GCG GCC GAC GCG CCG TCC AGT GCA GCC TGG GTG CAG 336 Ser His Leu Leu Ala Ala Asp Ala Pro Ser Ser Ala Ala Trp Val Gln 100 105 110 ACG CTG TGC CGA AAC GCC TTT CCG AAA GGC AGC TGG ACT CTG GCG CCT 384 Thr Leu Cys Arg Asn Ala Phe Pro Lys Gly Ser Trp Thr Leu Ala Pro 115 120 125 ACC GAT AAC CCA CCT AAG CTT TCT GCC CTG GAG ATG CTG GAG AAC TCC 432 Thr Asp Asn Pro Pro Lys Leu Ser Ala Leu Glu Met Leu Glu Asn Ser 130 135 140 TTG TAC AGC CCT ACC TGG GAA GGA TCC CAA TTC TGG GTA ACG GTG CAG 480 Leu Tyr Ser Pro Thr Trp Glu Gly Ser Gln Phe Trp Val Thr Val Gln 145 150 155 160 AGG ACT GAG GCC GCC GAG CGC TGT GGC CTG CAT GGC TCC TAC GTG CTG 528 Arg Thr Glu Ala Ala Glu Arg Cys Gly Leu His Gly Ser Tyr Val Leu 165 170 175 AGG GTG GAG GCT GAA AGG CTG ACT CTC CTG ACC GTG GGG GCC CAG AGT 576 Arg Val Glu Ala Glu Arg Leu Thr Leu Leu Thr Val Gly Ala Gln Ser 180 185 190 CAG ATA CTG GAG CCA CTC CTG TCC TGG CCC TAC, ACT CTG TTG CGT CGC 624 Gln Ile Leu Glu Pro Leu Leu Ser Trp Pro Tyr Thr Leu Leu Arg Arg 195 200 205 TAT GGC CGG GAC AAG GTC ATG TTC TCT TTC GAG GCC GGC CGC CGC TGC 672 Tyr Gly Arg Asp Lys Val Met Phe Ser Phe Glu Ala Gly Arg Arg Cys 210 215 220 CCC TCA GGC CCT GGA ACC TTC ACC TTC CAG ACG GCA CAG GGA AAT GAC 720 Pro Ser Gly Pro Gly Thr Phe Thr Phe Gln Thr Ala Gln Gly Asn Asp 225 230 235 240 ATC TTC CAG GCA GTT GAG ACT GCC ATC CAC CGG CAG AAG GCC CAG GGA 768 Ile Phe Gln Ala Val Glu Thr Ala Ile His Arg Gln Lys Ala Gln Gly 245 250 255 AAG GCC GGA CAG GGG CAC GAT GTT CTC AGA GCT GAC TCC CAT GAA GGG 816 Lys Ala Gly Gln Gly His Asp Val Leu Arg Ala Asp Ser His Glu Gly 260 265 270 GAG GTG GCA GAG GGG AAG TTG CCT TCC CCA CCT GGC CCC CAA GAG CTC 864 Glu Val Ala Glu Gly Lys Leu Pro Ser Pro Pro Gly Pro Gln Glu Leu 275 280 285 CTC GAC AGT CCC CCA GCC CTG TAT GCT GAG CCC TTA GAC TCC CTG CGC 912 Leu Asp Ser Pro Pro Ala Leu Tyr Ala Glu Pro Leu Asp Ser Leu Arg 290 295 300 ATT GCT CCA TGC CCT TCC CAG GAC TCC CTA TAC TCA GAC CCC TTG GAC 960 Ile Ala Pro Cys Pro Ser Gln Asp Ser Leu Tyr Ser Asp Pro Leu Asp 305 310 315 320 AGC ACG TCT GCT CAG GCA GGA GAG GGA GTA CAA CGG AAG AAA CCT CTC 1008 Ser Thr Ser Ala Gln Ala Gly Glu Gly Val Gln Arg Lys Lys Pro Leu 325 330 335 TAT TGG GAC TTG TAT GAG CAT GCG CAG CAG CAG TTG CTG AAG GCC AAG 1056 Tyr Trp Asp Leu Tyr Glu His Ala Gln Gln Gln Leu Leu Lys Ala Lys 340 345 350 CTG ACA GAC CCC AAA GAG GAT CCC ATC TAT GAT GAA CCT GAG GGC CTG 1104 Leu Thr Asp Pro Lys Glu Asp Pro Ile Tyr Asp Glu Pro Glu Gly Leu 355 360 365 GCC CCA GTC CCT CCC CAG GGC CTT TAT GAT CTG CCT CGG GAG CCC AAG 1152 Ala Pro Val Pro Pro Gln Gly Leu Tyr Asp Leu Pro Arg Glu Pro Lys 370 375 380 GAT GCA TGG TGG TGC CAA GCT CGG GTG AAG GAG GAG GGC TAT GAG CTC 1200 Asp Ala Trp Trp Cys Gln Ala Arg Val Lys Glu Glu Gly Tyr Glu Leu 385 390 395 400 CCC TAC AAC CCT GCC ACT GAT GAC TAC GCT GTG CCA CCC CCT CGG AGC 1248 Pro Tyr Asn Pro Ala Thr Asp Asp Tyr Ala Val Pro Pro Pro Arg Ser 405 410 415 ACA AAG CCC CTC CTT GCT CCC AAG CCC CAG GGC CCA GCC TTC CCT GAA 1296 Thr Lys Pro Leu Leu Ala Pro Lys Pro Gln Gly Pro Ala Phe Pro Glu 420 425 430 CCT GGT ACT GCA ACT GGC AGT GGC ATC AAA AGC CAC AAC TCA GCC CTG 1344 Pro Gly Thr Ala Thr Gly Ser Gly Ile Lys Ser His Asn Ser Ala Leo 435 440 445 TAC AGC CAG GTC CAG AAG AGC GGG GCC TCA GGG AGC TGG GAC TGT GGG 1392 Tyr Ser Gln Val Gln Lys Ser Gly Ala Ser Gly Ser Trp Asp Cys Gly 450 455 460 CTC TCT AGA GTA GGG ACT GAC AAG ACT GGG GTC AAG TCA GAG GGC TCT 1440 Leu Ser Arg Val Gly Thr Asp Lys Thr Gly Val Lys Ser Glu Gly Ser 465 470 475 480 ACC TGA 1446 Thr (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 481 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Asp Gly Ala Val Met Glu Gly Pro Leu Phe Leu Gln Ser Gln Arg 1 5 10 15 Phe Gly Thr Lys Arg Trp Arg Lys Thr Trp Ala Val Leu Tyr Pro Ala 20 25 30 Ser Pro His Gly Val Ala Arg Leu Glu Phe Phe Asp His Lys Gly Ser 35 40 45 Ser Ser Gly Gly Gly Arg Gly Ser Ser Arg Arg Leu Asp Cys Lys Val 50 55 60 Ile Arg Leu Ala Glu Cys Val Ser Val Ala Pro Val Thr Val Glu Thr 65 70 75 80 Pro Pro Glu Pro Gly Ala Thr Ala Phe Arg Leu Asp Thr Ala Gln Arg 85 90 95 Ser His Leu Leu Ala Ala Asp Ala Pro Ser Ser. Ala Ala Trp Val Gln 100 105 110 Thr Leu Cys Arg Asn Ala Phe Pro Lys Gly Ser Trp Thr Leu Ala Pro 115 120 125 Thr Asp Asn Pro Pro Lys Leu Ser Ala Leu Glu Met Leu Glu Asn Ser 130 135 140 Leu Tyr Ser Pro Thr Trp Glu Gly Ser Gln Phe Trp Val Thr Val Gln 145 150 155 160 Arg Thr Glu Ala Ala Glu Arg Cys Gly Leu His Gly Ser Tyr Val Leu 165 170 175 Arg Val Glu Ala Glu Arg Leu Thr Leu Leu Thr Val Gly Ala Gln Ser 180 185 190 Gln Ile Leu Glu Pro Leu Leu Ser Trp Pro Tyr Thr Leu Leu Arg Arg 195 200 205 Tyr Gly Arg Asp Lys Val Met Phe Ser Phe Glu Ala Gly Arg Arg Cys 210 215 220 Pro Ser Gly Pro Gly Thr Phe Thr Phe Gln Thr Ala Gln Gly Asn Asp 225 230 235 240 Ile Phe Gln Ala Val Glu Thr Ala Ile His Arg Gln Lys Ala Gln Gly 245 250 255 Lys Ala Gly Gln Gly His Asp Val Leu Arg Ala Asp Ser His Glu Gly 260 265 270 Glu Val Ala Glu Gly Lys Leu Pro Ser Pro Pro Gly Pro Gln Glu Leu 275 280 285 Leu Asp Ser Pro Pro Ala Leu Tyr Ala Glu Pro Leu Asp Ser Leu Arg 290 295 300 Ile Ala Pro Cys Pro Ser Gln Asp Ser Leu Tyr Ser Asp Pro Leu Asp 305 310 315 320 Ser Thr Ser Ala Gln Ala Gly Glu Gly Val Gln Arg Lys Lys Pro Leu 325 330 335 Tyr Trp Asp Leu Tyr Glu His Ala Gln Gln Gln Leu Leu Lys Ala Lys 340 345 350 Leu Thr Asp Pro Lys Glu Asp Pro Ile Tyr Asp Glu Pro Glu Gly Leu 355 360 365 Ala Pro Val Pro Pro Gln Gly Leu Tyr Asp Leu Pro Arg Glu Pro Lys 370 375 380 Asp Ala Trp Trp Cys Gln Ala Arg Val Lys Glu Glu Gly Tyr Glu Leu 385 390 395 400 Pro Tyr Asn Pro Ala Thr Asp Asp Tyr Ala Val Pro Pro Pro Arg Ser 405 410 415 Thr Lys Pro Leu Leu Ala Pro Lys Pro Gln Gly Pro Ala Phe Pro Glu 420 425 430 Pro Gly Thr Ala Thr Gly Ser Gly Ile Lys Ser His Asn Ser Ala Leu 435 440 445 Tyr Ser Gln Val Gln Lys Ser Gly Ala Ser Gly Ser Trp Asp Cys Gly 450 455 460 Leu Ser Arg Val Gly Thr Asp Lys Thr Gly Val Lys Ser Glu Gly Ser 465 470 475 480 Thr (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Gln Gly Xaa Asp Val Leu Arg Ala Asp 1 5 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Pro Gln Gly Pro Ala Phe Pro Glu Pro Gly Thr Ala Thr Gly Ser 1 5 10 15 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: Ser Xaa Thr Leu Ala Pro Thr Asp Asn Asn 1 5 10 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Gly Gln Gly His Asp Val Leu Arg Ala Asp Ser His Glu Gly Xaa Val 1 5 10 15 Ala (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Trp Ala Val Le Tyr Pro Ala Se Pro His G1 Val Ala Arg Le Glu 1 5 10 15 Phe Phe Asp His Lys 20 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Pro Leu Tyr Xaa Asp Leu Tyr Glu His Ala Gln Gln Gln Leu Le Lys 1 5 10 15 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 38 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Met Phe Ser Phe Glu Ala Gly Arg Arg Xaa Pro Ser Giy Pro Gly Th 1 5 10 15 Phe Thr Phe Gln Thr Ala Gln Gly Asn Asp Ile Phe Gln Ala Val Glu 20 25 30 Thr Ala Ile Xaa Arg Gln 35 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer" (ix) FEATURE: (A) NAME/KEY: modified~base (B) LOCATION: 6 (D) OTHER INFORMATION: /mod~base= i (ix) FEATURE: (A) NAME/KEY: modified~base (B) LOCATION: 9 (D) OTHER INFORMATION: /mod base= (ix) FEATURE: (A) NAME/KEY: modified~base (B) LOCATION: 13 (D) OTHER INFORMATION: /mod~base= (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: TGGGCNGTNT CTNTATCCC 19 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer" (ix) FEATURE: (A) NAME/KEY: modified~base (B) LOCATION: 3 (D) OTHER INFORMATION: /mod~base= i (ix) FEATURE: (A) NAME/KEY: modified~base (B) LOCATION: 7 (D) OTHER INFORMATION: /mod~base= i (ix) FEATURE: (A) NAME/KEY: modified~base (B) LOCATION: 18 (D) OTHER INFORMATION: /mod~base= i (ix) FEATURE: (A) NAME/FEY: modified base 'B) LOCATION: 25 (D) OTHER INFORMATION: /mod base= (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GTNTCTNTAT CCCTAGCNTA CGCCNCATGG 30 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer" (ix) FEATURE: (A) NAME/KEY: modified~base (B) LOCATION: 3 (D) OTHER INFORMATION: /mod~base= i (ix) FEATURE: (A) NAME/KEY: modified~base (B) LOCATION: 7 (D) OTHER INFORMATION: /mod~base= i (ix) FEATURE: (A) NAME/KEY: modified base (B) LOCATION: 11 (D) OTHER INFORMATION: /mod~base= i (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: TTNAGANAGA NAGATCTCTG TCTGTCTGAG CTGC 34 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer" (ix) FEATURE: (A) NAME/KEY: modified base (B) LOCATION: 13 (D) OTHER INFORMATION: /mod~base= i (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: TCTGTCTGTC TGNGCATGTC TCAGTA 26 (2) INFORMAtION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer" (ix) FEATURE: (A) NAME/KEY: modified base (B) LOCATION: 6 (D) OTHER INFORMATION: /mod~base= i (ix) FEATURE: (A) NAME/KEY: modified~base (B) LOCATION: 9 (D) OTHER INFORMATION: /mod~base= i (ix) FEATURE: (A) NAME/KEY: modified~base (B) LOCATION: 12 (D) OTHER INFORMATION: /mod~base= i (ix) FEATURE: (A) NAME/KEY: modified~base (B) LOCATION: 16 (D) OTHER INFORMATION: /mod base (ix) FEATURE: (A) NAME/KEY: modified~base (B) LOCATION: 20 (D) OTHER INFORMATION: /mod~base= i (xi) SEQUENCE DESCRIPTION: SEQ S NO:14: CATGGNGTNG CNACGNTCTN GAAGTT 26 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer" (ix) FEATURE: (A) NAME/KEY: modified~base (B) LOCATION: 5 (D) OTHER INFORMATION: /mod~base= i (ix) FEATURE: (A) NAME/KEY: modified~base (B) LOCATION: 19 (D) OTHER INFORMATION: /mod~base= i (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: TCTGNGCATG TCTCAGTANA GAAGTC 26 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Pro Gln Gly Pro Ala Phe Pro Glu Pro Gly Thr Ala Thr Gly Ser Cys 1 5 10 15 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Asn Val Leu Tyr Ala Ala Pro Phe 1 5 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE LESCEIPTION: SEQ ID NO:18: Leu Tyr Ala Glu Pro 1 5 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Asn Leu Tyr Xaa Xaa Pro 1 5 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: not relevant (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: Ser Ala Leu Tyr Ser Gln Val Gln 1 5