WO2007015886A2

WO2007015886A2 - Phosphorylated protein markers of gleevec-resistant chronic myelogenous leukemia

Info

Publication number: WO2007015886A2
Application number: PCT/US2006/027936
Authority: WO
Inventors: Valerie Goss; Kimberly Lee; Roberto Polakiewicz; Brian Druker; Michael Deninger; Ian Griswold
Original assignee: Cell Signaling Technology, Inc.
Priority date: 2005-07-21
Filing date: 2006-07-19
Publication date: 2007-02-08
Also published as: WO2007015886A8

Abstract

The invention discloses ten (10) phosphorylated protein markers that are only detected in Chronic Myelogenous Leukemia (CML) upon activation of Gleevec®-resistant BCR-ABL kinases, and provides reagents, including phosphorylation-site specific antibodies and AQUA peptides, for the selective detection and quantification of these marker phosphoproteins/sites. Also provided are methods for identifying a CML patient that has, or is likely to develop, resistance to Gleevec® (Imanitib mesylate, STI-571) by detecting and/or quantifying one or more of the disclosed phosphorylated sites/proteins in a biological sample from the patient. Methods for identifying a compound that inhibits a Gleevec®- resistant BCR-ABL mutant by determining the effect of the compound on one or more of the disclosed phosphorylated marker proteins are also provided.

Description

PHOSPHORYLATED PROTEIN MARKERS OF GLEEVEC-RESISTANT CHRONIC MYELOGENOUS LEUKEMIA

RELATED APPLICATIONS

This application claims priority to USSN 60/701 ,268, filed July 21 , 2005 (presently pending), USSN 60/651 ,583, filed February 10, 2005 (now abandoned), and PCT/US2004/26199, filed August 12, 2004, (presently pending) which itself claims priority to USSN 10/777,893, filed February 12, 2004 (presently pending).

JOINT RESEARCH AGREEMENT

This application describes and claims certain subject matter that was developed under a written joint collaborative research agreement between CELL SIGNALING TECHNOLOGY, INC., and THE OREGON HEALTH & SCIENCE UNIVERSITY, having an effective date of May 4, 2004, pertaining to markers of cancer drug resistance.

FIELD OF THE INVENTION

The invention relates generally to cancer, to cancer markers, and to antibodies and peptide reagents for the characterization of cancer.

BACKGROUND OF THE INVENTION

Many cancers are characterized by disruptions in cellular signaling pathways that lead to aberrant control of cellular processes, or to uncontrolled growth and proliferation of cells. These disruptions are often caused by changes in the phosphorylation state, and thus the activity of, particular signaling proteins. Among these cancers are hematopoietic diseases, such as chronic myelogenous leukemia (CML). There are about 4,600 new cases of CML in the United States annually, and it is estimated that almost 1 ,000 patients will die annually from the disease in the United States alone. See "Cancer Facts and Figures 2005," American Cancer Society.

It has been directly demonstrated that the BCR-ABL oncoprotein, a protein tyrosine kinase, is the causative agent in human chronic myelogenous leukemia (CML). See, e.g. Skorski et al., J. CHn Invest. 92: 194-202 (1993). The BCR-ABL oncoprotein is generated by the translocation of gene sequences from the c-ABL protein tyrosine kinase on chromosome 9 into BCR sequences on chromosome 22, producing the so-called Philadelphia chromosome. See, e.g. Kurzock et al., N. Engl. J. Med. 319: 990-998 (1988). The BCR-ABL oncogene has been found in at least 90-95% of cases of CML. See, e.g. Fialkow et a/., Am. J. Med. 63: 125-130 (1977). The translocation is also observed in approximately 20% of adults with acute lymphocytic leukemia (ALL), 5% of children with ALL, and 2% of adults with acute myelogenous leukemia (AML). See, e.g. Whang-Peng et al., Blood 36: 448-457 (1970).

The BCR-ABL gene produces three alternative chimeric proteins, P230 BCR-ABL, P210 BCR-ABL, and P190 BCR-ABL. Of these, P210 BCR-ABL is characteristic of CML while P190 BCR-ABL is characteristic of ALL. BCR-ABL proteins exhibit heightened tyrosine kinase and transforming capabilities compared to the normal c-ABL protein. See, e.g. Konopka et a/., Cell 37: 1035-1042 (1984). Many reports have indicated that BCR-ABL indeed acts as an oncogene and causes a variety of hematological malignancies, including granulocytic hyperplasia resembling human CML, myelomonocytic leukemia, ALL, lymphomas, and erythroid leukemia, in vivo. See, e.g. Honda, Blood 91: 2067-2075 (1998).

As a result, BCR-ABL has become a target for the development of therapeutics to treat leukemia. Most recently, Gleevec® (Imanitib mesylate, STI571), a small molecule inhibitor of the ABL kinase, has been approved for the treatment of CML. This drug is the first of a new class of anti- proliferative agents designed to interfere with the signaling pathways that drive the growth of tumor cells. The development of this drug represents a significant advance over the conventional therapies for CML and ALL, chemotherapy and radiation, which are plagued by well known side-effects and are often of limited effect since they fail to specifically target the underlying causes of the malignancies. However, Gleevec®, like many other therapeutics in development, only targets a single signaling protein among several implicated in the progression of the disease.

Clinical results since the introduction of Gleevec® have shown that patients often develop resistance to Gleevec®. See, e.g. Sawyers, Science 294(5548): 1834 (2001). The mechanism of resistance may vary from patient to patient, but is most often a result of mutations in the BCR- ABL DNA that results in an activated mutant kinase that is not affected by the inhibitor. In such patients, the BCR-ABL kinase is reactivated mostly due to specific mutations in the kinase domain that prevent Gleevec® binding without abolishing the kinase activity. Among such mutations are those affecting amino acid residues Y253, E255, T315 and M351 , which represent approximately 60% of all reported mutations. See Cowan-Jacob et al., Mini Rev. Med Chem 4: 285-299 (2004).

Improved BCR-ABL kinase inhibitors are now being developed that will target the mutant forms of BCR-ABL kinase. See Weisberg, Cancer Ceil (7): 129-141 (2005) O'Hare, Cancer Research (65): 4500- 4505 (2005). However, one particular mutation, T351 I, remains resistant to even the new generation of BCR-ABL inhibitors. It is therefore important to identify as early as possible during the course of Gleevec® treatment if resistance starts to arise, and whether a patient having or developing resistance may be switched to other BCR-ABL inhibitors or combinations of them, to increased doses of Gleevec®, or to alternative treatments such as bone marrow transplantation in the case of T315I mutations. Accordingly, in order to most effectively treat CML patients, it will be crucial to develop suitable assays for detecting specific markers of mutant BCR-ABL kinase activity capable of distinguishing among the different types of Gleevec®-resistant BCR-ABL variants (T315I in particular) in patients undergoing Gleevec® treatment. Most desirable would be assays that would reveal the appearance of resistance at the earliest possible stage.

Presently, tumor burden or residual disease and BCR-ABL expression in CML patients is detected by genetic tests such as FISH. CML cells are also identified using flow cytometry through the use of a number of cell-surface markers, but this assay is not precise and may result in misidentifying normal cells as CML cells.

Accordingly, there remains a need for the discovery of substrates of mutant BCR-ABL kinases that are uniquely phosphorylated by a given mutant. The development of new reagents that specifically detect these substrates of mutant, but not wild-type, BCR-ABL would enable more direct and reliable identification of Gleevec®-resistant CML cells, and would be well suited to the clinical analysis of BCR-ABL kinase activity using sensitive, reliable, and widely-used techniques such as immuno- histochemistry (IHC) and flow cytometry (FC). The development of such new reagents and methods would greatly assist in optimally treating each CML patient as resistance to Gleevec® or other BCR-ABL targeted therapies develops. These reagents and methods would also greatly assist in the staging of a CML patient's disease, where the detection of the transition from chronic phase to acute or blast crisis is critical for patient care.

SUMMARY OF THE INVENTION

The invention discloses ten (10) phosphorylated protein markers that are only detected in Chronic Myelogenous Leukemia (CML) upon activation of Gleevec®-resistant BCR-ABL kinases, and provides reagents, including phosphorylation-site specific antibodies and AQUA peptides, for the selective detection and quantification of these phosphorylated markers/sites. Also provided are methods for identifying a CML patient that has, or is likely to develop, resistance to Gleevec® (Imanitib mesylate, STI-571) by detecting and/or quantifying one or more of the disclosed phosphorylated sites/proteins in a biological sample from the patient. Methods for identifying a compound that inhibits a Gleevec®- resistant BCR-ABL mutant by determining the effect of the compound on one or more of the disclosed phosphorylated marker proteins are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 - Is the amino acid sequence of human BCR-ABL fusion protein (GenBank accession # AAB60393) (SEQ ID NO: 11).

FIG. 2 - Is a table (corresponding to Table 2) enumerating the ten BCR-ABL phosphorylated signaling proteins that are unique to CML cells having various BCR-ABL Gleevec®-resistant mutants. Column A = the name of the parent protein; Column B = the tyrosine residue (in the parent protein amino acid sequence) at which phosphorylation occurs; Column C = the phosphorylation site sequence encompassing the phosphorylated residue (Y* = phosphotyrosine, corresponding to Column B); Column D = the NCBI accession number (human sequence); and Columns E-H = the particular BCR-ABL-mutant(s) in which the protein is specifically phosphorylated.

FIG. 3 - is an exemplary mass spectrograph depicting the detection of the tyrosine 105 phosphorylation site in murine Nek (see Row 6 in Fig.2/Table 2), as further described in Example 1 (red and blue indicate ions detected in MS/MS spectrum); Y* indicates the phosphorylated tyrosine). FIG. 4 - is a is a Western blot analysis of five different cell lines demonstrating that an exemplary SHIP-2 (TyM 020) phospho-specific antibody detects phosphorylated SHIP2 only in Baf3 cell lines expressing the Gleevec®-resistant mutants T315I, Y235F, E255K and M351T but not in cell lines expressing wild-type BCR-ABL fusion protein kinase.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, ten (10) phosphorylated protein markers that are only detected in Chronic Myelogenous Leukemia (CML) upon activation of Gleevec®-resistant BCR-ABL kinases have now been identified. These ten tyrosine phosphorylation sites occur in signaling proteins and pathways downstream to Gleevec®-resistant BCR- ABL, and were identified by employing the techniques described in "Immunoaffinity Isolation of Modified Peptides From Complex Mixtures," U.S. Patent Publication No. 20030044848, Rush et al., using cellular extracts from a collection of Ba/F3 cells expressing wild-type or kinase domain mutants of BCR-ABL known to be resistant to Gleevec®. The tyrosine phosphorylation sites, and their corresponding parent proteins, disclosed herein are listed in Table 2/Figure 2 (the full sequences (human and mouse) of these proteins are publicly available in the NCBI database and their Accession numbers are listed in Column D of Table 2/Figure 2).

Although seven of these protein phosphorylation sites (Cbl-b (Tyr889 (= Tyr1014 in mouse), Vinculin (Tyr822), PI3K p100 delta (Tyr936 (= Tyr935 in mouse), FYN (Tyr571), Nek (Tyr105), and Dok2 (Tyr139 (= TyM 42 in mouse)) have recently been identified by the present inventors (see co-pending USSN 60/651 ,583, filed February 10, 2005, PCT/US2004/26199, filed August 12, 2004), their differential phosphorylation in Gleevec®-resistant CML and usefulness as markers of such resistance was not previously known. Three of the phosphorylation sites, CbI (Tyr700 (=Tyr 698 in mouse), Btk (Tyr 223), and SHIP1 (TyM 022 (= Tyr1020 in mouse), have been previously published, but their differential phosphorylation in Gleevec®-resistant CML and usefulness as a marker of such resistance was not previously known or described.

The discovery of the ten protein phosphorylation sites differentially regulated between wild-type and mutant BCR-ABL kinases described herein enables the production, by standard methods, of new reagents, such as phosphorylation site-specific antibodies and AQUA peptides (heavy-isotope labeled peptides), capable of specifically detecting and/or quantifying these phosphorylated sites/proteins, which serve as markers of Gleevec®-resistant CML. Such reagents are highly useful, inter alia, for studying signal transduction events underlying the progression of CML patients from Gleevec®-sensitive to Gleevec®-resistant.

Accordingly, the invention provides, in part, a method for identifying a CML patient that has, or is likely to develop, resistance to Gleevec® (Imanitib mesylate, STI-571), the method comprising the step of examining a biological sample from the patient for the presence of one or more of the phosphorylated marker proteins/sites disclosed herein (see Table 2/Figure 2). The ten phosphorylated markers/sites are: CbI (Tyr700 (=Tyr 698 in mouse)), Btk (Tyr 223), Cbl-b (Tyr889 (= TyM 014 in mouse)), FYN (Tyr571), SKAPP55 homologue (Tyr261 (= Tyr260 in mouse), Nek (TyM 05), Vinculin (Tyr822), Dok2 (Tyr139 (= Tyr142 in mouse)), PI3K p100 delta (Tyr936 (= Tyr935 in mouse)), and SHIP1 (TyM 022 (= TyM 020 in mouse)) (see SEQ ID NOs: 1-10; Table 2/Figure 2). In some preferred embodiments, the biological sample is a blood sample or bone marrow sample. In one preferred embodiment, the presence of one or more of the markers identifies the CML patient has having one of the following BCR-ABL kinase mutants: a T315I mutant, a M351T mutant, a Y235F mutant, and an E255K mutant (all in human BCR-ABL fusion protein sequence (see Figure 1)). In an other preferred embodiment, multiple of the markers are detected, while in other preferred embodiments, antibodies and AQUA peptides of the invention (as described below) are utilized to detect the presence of the resistance markers. In some preferred embodiments, the method of the invention utilizes a whole-cell assay, such as immunohistochemistry (IHC), flow cytometry (FC), or immunofluorescence (IF), as in certain preferred embodiments.

The invention also provides, in part, reagents, e.g. phospho- specific antibodies and AQUA peptides, for the specific detection and/or quantification of the CML Gleevec®-resistance marker proteins only when phosphorylated (or only when not phosphorylated) at a particular phosphorylation site as disclosed herein. Methods of detecting and/or quantifying one or more of the phosphorylated marker proteins/sites using the phosphorylation-site specific antibodies and AQUA peptides of the invention are also provided.

In particular, the invention provides, in part, an isolated phosphorylation site-specific antibody that specifically binds one of the previously unpublished Gleevec®-resistance marker protein phosphorylation sites disclosed herein (see Column A, Rows 3-9 of Table 2/Fig.2 (Cbl-b (Tyr889 (= Tyr1014 in mouse)), FYN binding protein (Tyr571), SKAPP55 homologue (Tyr261 (= Tyr260 in mouse)), Nek (Tyr105), Vinculin (Tyr822), Dok2 (Tyr139 (=Tyr142 in mouse)), and PI3K p100 delta (Tyr936 (=Tyr935 in mouse))) only when phosphorylated (or not phosphorylated, respectively) at the respective tyrosine residue enumerated in corresponding Column B of Table 2/Fig. 2, which residue is comprised within the phosphorylatable peptide site sequence enumerated in corresponding Column C.

In further part, the invention provides a heavy-isotope labeled peptide (AQUA peptide) for the detection and quantification of a given Gleevec®-resistance marker phosphoprotein, the labeled peptide comprising eight or more consecutive residues (including the phosphorylatable residue) of a particular phosphorylatable peptide site/sequence enumerated in Column C, Rows 3-9, of Table 2/Fig. 2 herein (Cbl-b (Tyr889 (= Tyr1014 in mouse)), FYN binding protein (Tyr571), SKAPP55 homologue (Tyr261 (= Tyr260 in mouse)), Nek (TyM 05), Vinculin (Tyr822), Dok2 (Tyr139 (=Tyr142 in mouse)), and PI3K p100 delta (Tyr936 (=Tyr935 in mouse)) (SEQ ID NOs: 3-9). In certain preferred embodiments, the phosphorylatable tyrosine within the labeled peptide is phosphorylated, while in other preferred embodiments, the phosphorylatable residue within the labeled peptide is not phosphorylated.

In one preferred embodiment, the antibody is a phosphorylation- site specific antibody, as further described below. In other preferred embodiments, the antibody is a monoclonal or polyclonal antibody. Also provided are immortalized cell lines producing such monoclonal antibodies. The invention also provides a kit for the identification of a chronic myelogenous leukemia (CML) patient that has, or is likely to develop, resistance to Gleevec® (Imanitib mesylate (STI-571)), the kit comprising at least one detectable antibody or heavy isotope-labeled (AQUA) peptide of the invention. In some preferred embodiments, the AQUA peptides provided by the invention consist of the phosphorylation site sequences enumerated in Column C of Table 2/Figure 2 (SEQ ID NOs: 3-9).

Antibodies and AQUA peptides provided by the invention are described in further detail in Sections A and B below. For example, among the novel reagents provided by the invention is an isolated phosphorylation site-specific antibody that specifically binds Nek protein only when phosphorylated (or only when not phosphorylated) at tyrosine 105 (see Row 2 (and Columns B and C) of Table 2/Fig. 2 (see SEQ ID NO: 6). By way of further example, among the group of reagents provided by the invention is an AQUA peptide for the quantification of phosphorylated Nek protein, the AQUA peptide consisting of the phosphorylatable peptide sequence listed in Column C, Row 6, of Table 2/Figure 1 (which encompasses the phosphorylatable tyrosine at position 105 (see SEQ ID NO: 6)).

In yet another embodiment, the invention provides a method for identifying a compound that inhibits a BCR-ABL kinase mutant that is resistant to lmanitib mesylate (STI-571) (Gleevec®), said method comprising the steps of: (a) contacting a sample comprising said BCR- ABL kinase mutant with said compound; and (b) utilizing at least one isolated antibody and/or at least one heavy isotope labeled peptide of the invention to determine the effect of the compound on the level of one or more of the ten phosphorylated resistance marker proteins disclosed herein, wherein a decrease in the level of one or more of said phosphorylated marker proteins following contact with said compound identifies said compound as inhibiting a BCR-ABL kinase mutant that is resistant to lmanitib mesylate (STI-571). In a preferred embodiment of the method, the BCR-ABL kinase mutant is selected from the group consisting of a T315I mutant, a M351T mutant, a Y235F mutant, and a E255K mutant.

Definitions.

As used herein, the following terms have the meanings indicated:

"Antibody" or "antibodies" refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE, including F_ab or antigen-recognition fragments thereof, including chimeric, polyclonal, and monoclonal antibodies. The term "does not bind" with respect to an antibody's binding to one phospho-form of a sequence means does not substantially react with as compared to the antibody's binding to the other phospho-form of the sequence for which the antibody is specific. "Heavy-isotope labeled peptide" (used interchangeably with AQUA peptide) means a peptide comprising at least one heavy-isotope label, which is suitable for absolute quantification or detection of a protein as described in WO/03016861 , "Absolute Quantification of Proteins and Modified Forms Thereof by Multistage Mass Spectrometry" (Gygi et al.), further discussed below.

"Protein" is used interchangeably with polypeptide, and includes protein fragments and domains as well as whole protein.

"Phosphoprotein" means a protein comprising at least one phosphorylated amino acid.

"Phosphorylatable amino acid" means any amino acid that is capable of being modified by addition of a phosphate group, and includes both forms of such amino acid.

"Phosphorylatable peptide sequence" means a peptide sequence comprising a phosphorylatable amino acid.

"Phosphorylation site-specific antibody" means an antibody that specifically binds a phosphorylatable peptide sequence/epitope only when phosphorylated, or only when not phosphorylated, respectively. The term is used interchangeably with "phospho-specific" antibody.

The further aspects and advantages of invention are described in detail below.

A. Antibodies and Cell Lines

Phospho-specific antibodies of the invention specifically bind to one of the GleevecO-resistance marker phospho-proteins indicated in Table 2/Fig. 2 only when phosphorylated at the tyrosine sites indicated and do not substantially bind to the non-phosphorylated versions of the proteins. More particularly, the invention provides an isolated antibody that specifically binds one of the following lmanitib mesylate (STI-571)- resistance marker proteins only when phosphorylated at the indicated tyrosine residue, but does not bind said marker protein when not phosphorylated at said tyrosine residue:

(i) Cbl-b (phosphorylated at tyrosine 889 (SEQ ID NO: 3) (= tyrosine

1014 in mouse);

(ii) FYN binding protein (phosphorylated at tyrosine 571 (SEQ ID

NO: 4);

(iii) SKAPP55 homologue (phosphorylated at tyrosine 261 (SEQ ID

NO: 5) (=tyrosine 261 in mouse);

(iv) Nek (phosphorylated at tyrosine105 (SEQ ID NO: 6);

(v) Vinculin (phosphorylated at tyrosine 822 (SEQ ID NO: 7);

(vi) Dok2 (phosphorylated at tyrosine 139 (SEQ ID NO: 8) (= tyrosine

142 in mouse); and

(vii) PI3K p100 delta (phosphorylated at tyrosine 936) (SEQ ID NO: 9)

(= tyrosine 935 in mouse).

The phospho-specific antibodies of the invention include (a) monoclonal antibodies, (b) purified polyclonal antibodies, (c) antibodies as described in (a)-(c) above that bind equivalent and highly phosphorylation sites in other non-human species proteins (e.g. mouse, rat), as disclosed herein, and (d) fragments of (a)-(c) above that bind to the antigen (or more preferably the epitope) bound by the exemplary antibodies disclosed herein.

Such antibodies and antibody fragments that are within the scope of the present invention may be produced by a variety of techniques well known in the art, as further discussed below. Antibodies that bind to the phosphorylated proteins can be identified in accordance with known techniques.

The term "antibody" or "antibodies" as used herein refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibodies may be monoclonal or polyclonal and may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. See, e.g., M. Walker et al., Molec. Immunol. 26: 403-11 (1989); Morrision et al., Proc. Natl Acad. ScL 81: 6851 (1984); Neuberger et al., Nature 312: 604 (1984)). The antibodies may be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Pat. No. 4,474,893 (Reading) or U.S. Pat. No. 4,816,567 (Cabilly et al.) The antibodies may also be chemically constructed specific antibodies made according to the method disclosed in U.S. Pat. No. 4,676,980 (Segel et al.)

Polyclonal antibodies of the invention may be produced according to standard techniques by immunizing a suitable animal (e.g., rabbit, goat, etc.) with an antigen encompassing the phosphorylation sites of the proteins as listed in Table 2/Figure 2, collecting immune serum from the animal, and separating the polyclonal antibodies from the immune serum, and purifying polyclonal antibodies having the desired specificity, in accordance with known procedures. In a preferred embodiment, the antigen is a synthetic phosphopeptide antigen comprising the sequence surrounding and including the phosphorylation, as described above, the antigen being selected and constructed in accordance with well-known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL, Chapter 5, p. 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology, 201: 264-283 (1991); Merrifield, J. Am. Chem. Soc. 85/ 21-49 (1962)).

Monoclonal antibodies of the invention may be produced in a hybridoma cell line according to the well-known technique of Kohler and Milstein. Nature 265: 495-97 (1975); Kohler and Milstein, Eur. J. Immunol. 6: 511 (1976); see also, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al. Eds. (1989). Monoclonal antibodies so produced are highly specific, and improve the selectivity and specificity of diagnostic assay methods provided by the invention. For example, a solution containing the appropriate antigen may be injected into a mouse and, after a sufficient time (in keeping with conventional techniques), the mouse sacrificed and spleen cells obtained. The spleen cells are then immortalized by fusing them with myeloma cells, typically in the presence of polyethylene glycol, to produce hybridoma cells. Rabbit fusion hybridomas, for example, may be produced as described in U. S Patent No. 5,675,063, C. Knight, Issued October 7, 1997. The hybridoma cells are then grown in a suitable selection media, such as hypoxanthine- aminopterin-thymidine (HAT), and the supernatant screened for monoclonal antibodies having the desired specificity, as described below. The secreted antibody may be recovered from tissue culture supernatant by conventional methods such as precipitation, ion exchange or affinity chromatography, or the like.

Alternatively, immortalized monoclonal antibody producing cell lines may be produced without fusion hybridomas, for example, by using transgenic spleen cells that are conditionally immortal. See, e.g. Pasqualini et al., PNAS 101(1): 257-259 (2004); Jat et al., U.S. Patent No. 5,866,759 (Issued February 2, 1999).

Monoclonal Fab fragments may also be produced in Escherichia coli by recombinant techniques known to those skilled in the art. See, e.g., W. Huse, Science 246: 1275-81 (1989); Mullinax et al., Proc. Nat'l Acad. Sci. 87: 8095 (1990). If monoclonal antibodies of one isotype are preferred for a particular application, particular isotypes can be prepared directly, by selecting from the initial fusion, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of different isotype by using the sib selection technique to isolate class-switch variants (Steplewski, et al., Proc. Nat'l. Acad. Sci., 82: 8653 (1985); Spira et al., J. Immunol. Methods, 74: 307 (1984)).

The invention also provides immortalized cell lines, such as hybridoma clones, constructed as described above, that produce monoclonal antibodies of the invention. Similarly, the invention includes recombinant cells producing antibodies as disclosed herein, which cells may be constructed by well known techniques; for example the antigen combining site of the monoclonal antibody can be cloned by PCR and single-chain antibodies produced as phage-displayed recombinant antibodies or soluble antibodies in E. coli (see, e.g., ANTIBODY ENGINEERING PROTOCOLS, 1995, Humana Press, Sudhir Paul editor.)

Antibodies of the invention, whether polyclonal or monoclonal, may be screened for phospho- specificity according to standard techniques. See, e.g. Czernik et al., Methods in Enzymology, 201: 264-283 (1991). For example, the antibodies may be screened against a peptide library by ELISA to ensure specificity for both the desired antigen and for reactivity only with the fusion form of the antigen. The antibodies may also be tested by Western blotting against cell preparations containing the phosphorylated proteins, in cell lines expressing BCR-ABL, and Gleevec®- resistant BCR-ABL mutants. Specificity against the desired phosphorylated epitope may also be examined by constructing mutants lacking phosphorylatable residues at positions outside the desired epitope that are known to be phosphorylated, or by mutating the desired phospho- epitope and confirming lack of reactivity.

Phosphorylation-site specific antibodies of the invention may exhibit some limited cross-reactivity to related epitopes in non-target proteins. This is not unexpected as most antibodies exhibit some degree of cross-reactivity, and anti-peptide antibodies will often cross-react with epitopes having high homology to the immunizing peptide. See, e.g., Czernik, supra. Cross-reactivity with non-target proteins is readily characterized by Western blotting alongside markers of known molecular weight. Amino acid sequences of cross-reacting proteins may be examined to identify sites highly homologous to the carcinoma-related signaling protein epitope for which the antibody of the invention is specific.

In certain cases, polyclonal antisera may exhibit some undesirable general cross-reactivity to phosphotyrosine itself, which may be removed by further purification of antisera, e.g. over a phosphotyramine column. Antibodies of the invention specifically bind their target protein (i.e. one of the ten resistance-marker proteins listed in Column A of Table 2) only when phosphorylated (or only when not phosphorylated, as the case may be) at the tyrosine phosphorylation site disclosed herein (see corresponding Columns B and C), and do not (substantially) bind to the other form (as compared to the form for which the antibody is specific).

The antibodies of this invention may be further characterized by flow cytometry using normal and Philadelphia chromosome-positive CML patient samples (blood or marrow), and Gleevec®-resistant CML patients. Flow cytometry may be carried out according to standard methods. See Chow etal., Cytometry (Communications in Clinical Cytometry) 46: 72-78 (2001 ). Briefly and by way of example, the following protocol for cytometric analysis may be employed: samples may be centrifuged on Ficoll gradients to remove erythrocytes, and cells may then be fixed with 2% paraformaldehyde for 10 minutes at 37 ⁰C followed by permeabilization in 90% methanol for 30 minutes on ice. Cells may then be stained with the primary phospho-specific antibody, washed and labeled with a fluorescent-labeled secondary antibody. Additional fluorochrome-conjugated marker antibodies (e.g. CD45, CD34) may also be added at this time to aid in the subsequent identification of specific hematopoietic cell types. The cells would then be analyzed on a flow cytometer (e.g. a Beckman Coulter FC500) according to the specific protocols of the instrument used. Such an analysis would identify the presence of the GleevecO-resistant CML cells. Antibodies of the invention may also be advantageously conjugated to fluorescent dyes {e.g. Alexa 488, PE) for use in multi- parametric analyses along with other signal transduction (phospho-CrkL, phospho-Erk 1/2) and/or cell marker (CD34) antibodies. They may also be desirably employed in a kit for the identification of a chronic myelogenous leukemia (CML) patient that has, or is likely to develop, resistance to lmanitib mesylate (STI-571), the kit comprising at least one detectable antibody of the invention (and/or at least one detectable heavy isotope-labeled (AQUA) peptide of the invention).

The antibodies may be further characterized via immuno- histochemical (IHC) staining using normal and diseased tissues to examine the presence of these markers in diseased tissue. IHC may be carried out according to well-known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL, Chapter 10, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988). Briefly, paraffin-embedded tissue (e.g. tumor tissue) is prepared for immunohistochemical staining by deparaffinizing tissue sections with xylene followed by ethanol; hydrating in water then PBS; unmasking antigen by heating slide in sodium citrate buffer; incubating sections in hydrogen peroxide; blocking in blocking solution; incubating slide in primary antibody and secondary antibody; and finally detecting using ABC avidin/biotin method according to manufacturer's instructions.

The preferred epitope of an isolated phospho-specific antibody of the invention is a peptide fragment consisting essentially of about 4 to 17 amino acids including the phosphorylatable tyrosine, wherein about 2 to 8 amino acids are positioned on each side of the phosphorylatable tyrosine (for example, Dok2 (tyrosine 139 (= tyrosine 142 in mouse) phosphorylation site sequence disclosed in Row 8, Column C of Table 2), and antibodies of the invention thus specifically bind a target Gleevec®- resistance marker phosphoprotein comprising such epitopic sequence. Particularly preferred epitopes bound by the antibodies of the invention comprise all or part of a phosphorylatable site sequence listed in Column C of Table 2/Figure 1 , including the phosphorylatable amino acid. In one preferred embodiment, antibodies of the invention bind an epitope on a disclosed resistance marker protein (see Table 2, Column A) that comprises at least four amino acids encompassing and including the phosphorylated residue within the phosphorylation site sequence (see Table 2, Columns B, C).

Included in the scope of the invention are equivalent non-antibody molecules, such as protein binding domains or nucleic acid aptamers, which bind, in a phospho-specific manner, to essentially the same phosphorylatable epitope to which the phospho-specific antibodies of the invention bind. See, e.g., Neuberger et ai, Nature 312: 604 (1984). Such equivalent non-antibody reagents may be suitably employed in the methods of the invention further described below.

The antibodies of the invention specifically bind to human phosphoproteins, but are not limited only to binding the human species, per se. The invention includes antibodies that may also bind conserved and highly homologous or identical sites in other species (e.g. mouse, rat, monkey, yeast). Highly homologous or identical sites conserved in other species can readily be identified by standard sequence comparisons, such as using BLAST, with the human carcinoma-related signal transduction protein phosphorylation sites disclosed herein.

B. Heavy-Isotope Labeled Peptides (AQUA Peptides).

The novel CML Gleevec®-resistance marker phosphoproteins/ sites disclosed herein now enable the production of corresponding heavy- isotope labeled peptides for the absolute quantification of such marker proteins (both phosphorylated and not phosphorylated at a disclosed site) in biological samples. The production and use of AQUA peptides for the absolute quantification of proteins (AQUA) in complex mixtures has been described. See WO/03016861 , "Absolute Quantification of Proteins and Modified Forms Thereof by Multistage Mass Spectrometry," Gygi et al. and also Gerber et al. Proc. Natl. Acad. Sci. U.S.A. 100: 6940-5 (2003) (the teachings of which are hereby incorporated herein by reference, in their entirety).

The AQUA methodology employs the introduction of a known quantity of at least one heavy-isotope labeled peptide standard (which has a unique signature detectable by LC-SRM chromatography) into a digested biological sample in order to determine, by comparison to the peptide standard, the absolute quantity of a peptide with the same sequence and protein modification in the biological sample. Briefly, the AQUA methodology has two stages: peptide internal standard selection and validation and method development; and implementation using validated peptide internal standards to detect and quantify a target protein in sample. The method is a powerful technique for detecting and quantifying a given peptide/protein within a complex biological mixture, such as a cell lysate, and may be employed, e.g., to quantify change in protein phosphorylation as a result of drug treatment, or to quantify differences in the level of a protein in different biological states.

Generally, to develop a suitable internal standard, a particular peptide (or modified peptide) within a target protein sequence is chosen based on its amino acid sequence and the particular protease to be used to digest. The peptide is then generated by solid-phase peptide synthesis such that one residue is replaced with that same residue containing stable isotopes (¹³C, ¹⁵N). The result is a peptide that is chemically identical to its native counterpart formed by proteolysis, but is easily distinguishable by MS via a 7-Da mass shift. A newly synthesized AQUA internal standard peptide is then evaluated by LC-MS/MS. This process provides qualitative information about peptide retention by reverse-phase chromatography, ionization efficiency, and fragmentation via collision- induced dissociation. Informative and abundant fragment ions for sets of native and internal standard peptides are chosen and then specifically monitored in rapid succession as a function of chromatographic retention to form a selected reaction monitoring (LC-SRM) method based on the unique profile of the peptide standard.

The second stage of the AQUA strategy is its implementation to measure the amount of a protein or modified protein from complex mixtures. Whole cell lysates are typically fractionated by SDS-PAGE gel electrophoresis, and regions of the gel consistent with protein migration are excised. This process is followed by in-gel proteolysis in the presence of the AQUA peptides and LC-SRM analysis. (See Gerber et al. supra.) AQUA peptides are spiked in to the complex peptide mixture obtained by digestion of the whole cell lysate with a proteolytic enzyme and subjected to immunoaffinity purification as described above. The retention time and fragmentation pattern of the native peptide formed by digestion (e.g. trypsinization) is identical to that of the AQUA internal standard peptide determined previously; thus, LC-MS/MS analysis using an SRM experiment results in the highly specific and sensitive measurement of both internal standard and analyte directly from extremely complex peptide mixtures. Because an absolute amount of the AQUA peptide is added (e.g. 250 fmol), the ratio of the areas under the curve can be used to determine the precise expression levels of a protein or phosphorylated form of a protein in the original cell lysate. In addition, the internal standard is present during in-gel digestion as native peptides are formed, such that peptide extraction efficiency from gel pieces, absolute losses during sample handling (including vacuum centrifugation), and variability during introduction into the LC-MS system do not affect the determined ratio of native and AQUA peptide abundances. An AQUA peptide standard is developed for a known phosphorylation site sequence previously identified by the IAP-LC-MS/MS method within a target protein. One AQUA peptide incorporating the phosphorylated form of the particular residue within the site may be developed, and a second AQUA peptide incorporating the non- phosphorylated form of the residue developed. In this way, the two standards may be used to detect and quantify both the phosphorylated and non-phosphorylated forms of the site in a biological sample.

Peptide internal standards may also be generated by examining the primary amino acid sequence of a protein and determining the boundaries of peptides produced by protease cleavage. Alternatively, a protein may actually be digested with a protease and a particular peptide fragment produced can then sequenced. Suitable proteases include, but are not limited to, serine proteases (e.g. trypsin, hepsin), metallo proteases (e.g. PUMP1), chymotrypsin, cathepsin, pepsin, thermolysin, carboxypeptidases, etc.

A peptide sequence within a target protein is selected according to one or more criteria to optimize the use of the peptide as an internal standard. Preferably, the size of the peptide is selected to minimize the chances that the peptide sequence will be repeated elsewhere in other non-target proteins. Thus, a peptide is preferably at least about 6 amino acids. The size of the peptide is also optimized to maximize ionization frequency. Thus, peptides longer than about 20 amino acids are not preferred. The preferred ranged is about 7 to 15 amino acids. A peptide sequence is also selected that is not likely to be chemically reactive during mass spectrometry, thus sequences comprising cysteine, tryptophan, or methionine are avoided.

A peptide sequence that does not include a modified region of the target region may be selected so that the peptide internal standard can be used to determine the quantity of all forms of the protein. Alternatively, a peptide internal standard encompassing a modified amino acid may be desirable to detect and quantify only the modified form of the target protein. Peptide standards for both modified and unmodified regions can be used together, to determine the extent of a modification in a particular sample (i.e. to determine what fraction of the total amount of protein is represented by the modified form). For example, peptide standards for both the phosphorylated and unphosphorylated form of a protein known to be phosphorylated at a particular site can be used to quantify the amount of phosphorylated form in a sample.

The peptide is labeled using one or more labeled amino acids (i.e. the label is an actual part of the peptide) or less preferably, labels may be attached after synthesis according to standard methods. Preferably, the label is a mass-altering label selected based on the following considerations: The mass should be unique to shift fragment masses produced by MS analysis to regions of the spectrum with low background; the ion mass signature component is the portion of the labeling moiety that preferably exhibits a unique ion mass signature in MS analysis; the sum of the masses of the constituent atoms of the label is preferably uniquely different than the fragments of all the possible amino acids. As a result, the labeled amino acids and peptides are readily distinguished from unlabeled ones by the ion/mass pattern in the resulting mass spectrum. Preferably, the ion mass signature component imparts a mass to a protein fragment that does not match the residue mass for any of the 20 natural amino acids.

The label should be robust under the fragmentation conditions of MS and not undergo unfavorable fragmentation. Labeling chemistry should be efficient under a range of conditions, particularly denaturing conditions, and the labeled tag preferably remains soluble in the MS buffer system of choice. The label preferably does not suppress the ionization efficiency of the protein and is not chemically reactive. The label may contain a mixture of two or more isotopically distinct species to generate a unique mass spectrometric pattern at each labeled fragment position. Stable isotopes, such as ²H, ¹³C, ¹⁵N, ¹⁷O, ¹⁸O, or ³⁴S, are among preferred labels. Pairs of peptide internal standards that incorporate a different isotope label may also be prepared. Preferred amino acid residues into which a heavy isotope label may be incorporated include leucine, proline, valine, and phenylalanine.

Peptide internal standards are characterized according to their mass-to-charge (m/z) ratio, and preferably, also according to their retention time on a chromatographic column (e.g. an HPLC column). Internal standards that co-elute with unlabeled peptides of identical sequence are selected as optimal internal standards. The internal standard is then analyzed by fragmenting the peptide by any suitable means, for example by collision-induced dissociation (CID) using, e.g., argon or helium as a collision gas. The fragments are then analyzed, for example by multi-stage mass spectrometry (MSⁿ) to obtain a fragment ion spectrum, to obtain a peptide fragmentation signature. Preferably, peptide fragments have significant differences in m/z ratios to enable peaks corresponding to each fragment to be well separated, and a signature that is unique for the target peptide is obtained. If a suitable fragment signature is not obtained at the first stage, additional stages of MS are performed until a unique signature is obtained.

Fragment ions in the MS/MS and MS³ spectra are typically highly specific for the peptide of interest, and, in conjunction with LC methods, allow a highly selective means of detecting and quantifying a target peptide/protein in a complex protein mixture, such as a cell lysate, containing many thousands or tens of thousands of proteins. Any biological sample potentially containing a target protein/peptide of interest may be assayed. Crude or partially purified cell extracts are preferably employed. Generally, the sample has at least 0.01 mg of protein, typically a concentration of 0.1-10 mg/mL, and may be adjusted to a desired buffer concentration and pH.

A known amount of a labeled peptide internal standard, preferably about 10 femtomoles, corresponding to a target protein to be detected/quantified is then added to a biological sample, such as a cell lysate. The spiked sample is then digested with one or more protease(s) for a suitable time period to allow digestion. A separation is then performed (e.g. by HPLC, reverse-phase HPLC, capillary electrophoresis, ion exchange chromatography, etc.) to isolate the labeled internal standard and its corresponding target peptide from other peptides in the sample. Microcapillary LC is a preferred method.

Each isolated peptide is then examined by monitoring of a selected reaction in the MS. This involves using the prior knowledge gained by the characterization of the peptide internal standard and then requiring the MS to continuously monitor a specific ion in the MS/MS or MS" spectrum for both the peptide of interest and the internal standard. After elution, the area under the curve (AUC) for both peptide standard and target peptide peaks are calculated. The ratio of the two areas provides the absolute quantification that can be normalized for the number of cells used in the analysis and the protein's molecular weight, to provide the precise number of copies of the protein per cell. Further details of the AQUA methodology are described in Gygi et al., and Gerber et a/, supra.

In accordance with the present invention, AQUA internal peptide standards (heavy-isotope labeled peptides) may now be produced, as described above, for any of the ten Gleevec®-resistance marker phosphoproteins/sites disclosed herein (see Table 2/Figure 2). Peptide standards for a given phosphorylation site (e.g. the tyrosine 822 site in Vinculin - see Row 7 of Table 2) may be produced for both the phosphorylated and non-phosphorylated forms of the site (e.g. see Vinculin site sequence in Column C, Row 7 of Table 2 (SFLDSGY*R) (SEQ ID NO: 7)) and such standards employed in the AQUA methodology to detect and quantify both forms of such phosphorylation site in a biological sample.

AQUA peptides of the invention may comprise all, or part of, a phosphorylation site peptide sequence disclosed herein (see Column C of Table 2/Figure 2). In a preferred embodiment, an AQUA peptide of the invention comprises eight or more consecutive residues encompassing the phosphorylatable residue of one the six previously unpublished phosphorylation site sequences disclosed herein in Table 2/Figure 2, Rows 3-9, Column C. For example, an AQUA peptide of the invention for detection/ quantification of Cbl-b protein when phosphorylated at tyrosine Y889 (= tyrosine 1014 in mouse) may consist of, or comprise, the sequence SFLDSGY*R (y=phosphotyrosine), which comprises phosphorylatable tyrosine 889 (see Table 2, Row 7, Column C; (SEQ ID NO: 7)). Heavy-isotope labeled equivalents of the peptides enumerated in Table 2/Figure 2 (both in phosphorylated and unphosphorylated form) can be readily synthesized and their unique MS and LC-SRM signature determined, so that the peptides are validated as AQUA peptides and ready for use in quantification experiments.

The phosphorylation site peptide sequences disclosed herein (see Column E of Table 2/Figure 2) are particularly well suited for development of corresponding AQUA peptides, since the IAP method by which they were identified (see Part A above and Example 1) inherently confirmed that such peptides are in fact produced by enzymatic digestion (trypsinization) and are in fact suitably fractionated/ionized in MS/MS. Thus, heavy-isotope labeled equivalents of these peptides (both in phosphorylated and unphosphorylated form) can be readily synthesized and their unique MS and LC-SRM signature determined, so that the peptides are validated as AQUA peptides and ready for use in quantification experiments. Accordingly, in a preferred embodiment, the invention provides a heavy isotope-labeled (AQUA) peptide for the quantification of one of the following lmanitib mesylate (STI-571)-resistance marker proteins only when phosphorylated at the indicated tyrosine residue:

(i) Cbl-b (phosphorylated at tyrosine 889 (SEQ ID NO: 3) (= tyrosine

1014 in mouse);

(ii) FYN binding protein (phosphorylated at tyrosine 571 (SEQ ID

NO: 4);

(iii) SKAPP55 homologue (phosphorylated at tyrosine 261 (SEQ ID

NO: 5) (=tyrosine 261 in mouse);

(iv) Nek (phosphorylated at tyrosinei 05 (SEQ ID NO: 6);

(v) Vinculin (phosphorylated at tyrosine 822 (SEQ ID NO: 7);

(vi) Dok2 (phosphorylated at tyrosine 139 (SEQ ID NO: 8) (= tyrosine

142 in mouse); and

(vii) PI3K p100 delta (phosphorylated at tyrosine 936) (SEQ ID NO: 9)

(= tyrosine 935 in mouse).

In some preferred embodiments, the heavy isotope-labeled peptides provided by the invention comprise of eight or more contiguous amino acids (including the phosphorylatable residue) of a sequence selected from the group consisting of SEQ ID NOs: 3-9.

For example, an AQUA peptide consisting of the sequence SFLDSGY*R (see SEQ ID NO: 7) (where Y* may be either phosphotyrosine or tyrosine, and where L = labeled leucine (e.g. ¹⁴C)) is provided for the quantification of phosphorylated (or non-phosphorylated) Vinculin (Tyr 822) in a biological sample (see Row 7 of Table 2/Fig. 2, tyrosine 822 being the phosphorylatable residue within the site). However, it will be appreciated that a larger AQUA peptide comprising a disclosed phosphorylation site sequence (and additional residues downstream or upstream of it) may also be constructed. Similarly, a smaller AQUA peptide comprising less than all of the residues of a disclosed phosphorylation site sequence (but still comprising the phosphorylatable residue enumerated in Column C of Table 2/Figure 2) may alternatively be constructed. Such larger or shorter AQUA peptides are within the scope of the present invention, and the selection and production of preferred AQUA peptides may be carried out as described above (see Gygi et al., Gerber et al. supra.).

Example 5 is provided to further illustrate the construction and use, by standard methods described above, of exemplary AQUA peptides provided by the invention. For example, the above-described AQUA peptides corresponding to the both the phosphorylated and non- phosphorylated forms of the disclosed Vinculin tyrosine 822 phosphorylation site may be used to quantify the amount of phosphorylated Vinculin (Tyr 822) in a biological sample from a CML patient, e.g. a bone marrow sample, either before or after treatment with a drug such as Gleevec®.

AQUA peptides (as well as antibodies) of the invention may also be employed within a kit that comprises one or multiple AQUA peptide(s) provided herein (for the quantification of a Gleevec®-resistance marker phosphoprotein disclosed in Table 2/Figure 2). Such reagent is preferably provided in a detectable form. Optionally, a second detecting reagent conjugated to a detectable group may be employed. For example, a kit may include AQUA peptides for both the phosphorylated and non-phosphorylated form of a phosphorylation site disclosed herein. The reagents may also include ancillary agents such as buffering agents and protein stabilizing agents, e.g., polysaccharides and the like. The kit may further include, where necessary, other members of the signal- producing system of which system the detectable group is a member (e.g., enzyme substrates), agents for reducing background interference in a test, control reagents, apparatus for conducting a test, and the like. The test kit may be packaged in any suitable manner, typically with all elements in a single container along with a sheet of printed instructions for carrying out the test.

C. Detection Methods & Compound Screening.

The phospho-specific antibodies provided by the invention enable powerful and previously unavailable immunological methods for the rapid and simple detection of Gleevec®-resistant BCR-ABL mutant expression and/or activity in a biological sample, such as a blood or bone marrow from a CML patient sample. The methods disclosed herein may be employed with any biological sample potentially containing, or suspected of containing, Gleevec®-resistant mutant BCR-ABL proteins (in particular, a T315I mutant, a M351T mutant, a Y235F mutant, and a E255K mutant protein). Biological samples taken from human CML subjects for use in the methods disclosed herein are generally biological fluids such as serum, blood plasma, or bone marrow, and may comprise whole cells or a cell lysate, whether or not purified. In a preferred embodiment, the biological sample comprises whole cells.

In part, the invention provides a method for identifying a chronic myelogenous leukemia (CML) patient that has, or is likely to develop, resistance to lmanitib mesylate (STI-571), said method comprising the step of examining a biological sample from said patient for the presence of one or more of the following phosphorylated marker proteins:

(i) CbI (phosphorylated at tyrosine 700 (=Tyr 698 in mouse))

(SEQ ID NO: 1);

(ii) Btk kinase (phosphorylated at tyrosine 223 (SEQ ID NO: 2); (iii) Cbl-b (phosphorylated at tyrosine 889 (= Tyr1014 in mouse))

(SEQ ID NO: 3); (iv) FYN binding protein (phosphorylated at tyrosine 571)

(SEQ ID NO: 4); (v) SKAPP55 homologue (phosphorylated at tyrosine 261 (= Tyr260 in mouse)) (SEQ ID NO: 5);

(vi) Nek (phosphorylated at tyrosine105 (SEQ ID NO: 6); (vii) Vinculin (phosphorylated at tyrosine 822 (SEQ ID NO: 7); (viii) Dok2 (phosphorylated at tyrosine 139 (= Tyr142 in mouse))

(SEQ ID NO: 8); (ix) PI3K p100 delta (phosphorylated at tyrosine 936 (= Tyr935 in mouse)) (SEQ ID NO: 9); and/or (x) SHIP1 (phosphorylated at tyrosine 1022 (= Tyr1020 in mouse))

(SEQ ID NO: 10); wherein, the presence of one or more of said phosphorylated marker proteins identifies said CML patient as having, or likely to develop, resistance to lmanitib mesylate (STI-571).

In some preferred embodiments, the method employs one or more phospho-specific antibodies and/or AQUA peptides of the invention to detect the presence of one or more of the above Gleevec®-resistance marker phosphoproteins. In one preferred embodiment of the method, the presence of one or more of said phosphorylated marker proteins identifies said CML patient as having a BCR-ABL kinase mutant selected from the group consisting of a T315I mutant, a M351T mutant, a Y235F mutant, and a E255K mutant. In another preferred embodiment, the presence of multiple of said phosphorylated marker proteins is examined.

The method may be employed with a biological sample that has been contacted with at least one BCR-ABL inhibitor or is obtained from a CML subject treated with such inhibitor. Accordingly, changes in mutant BCR-ABL activity and/or expression resulting from contacting a biological sample with a test compound, such as a BCR-ABL inhibitor, may be examined to determine the effect of such compound. Accordingly, in one embodiment, the invention provides a method for identifying a compound that inhibits a BCR-ABL kinase mutant that is resistant to lmanitib mesylate (STI-571), said method comprising the steps of:

(a) contacting a sample comprising said BCR-ABL kinase mutant with said compound; and

(b) utilizing at least one isolated antibody of the invention and/or at least one heavy isotope labeled peptide of the invention to determine the effect of said compound on the level of one or more of the following phosphorylated marker proteins:

(i) CbI (phosphorylated at tyrosine 700 (=Tyr 698 in mouse))

(SEQ ID NO: 1);

(SEQ ID NO: 3); (iv) FYN binding protein (phosphorylated at tyrosine 571)

(SEQ ID NO: 8); (ix) PI3K p100 delta (phosphorylated at tyrosine 936 (= Tyr935 in mouse)) (SEQ ID NO: 9); and/or (x) SHIP1 (phosphorylated at tyrosine 1022 (= TyM 020 in mouse))

(SEQ ID NO: 10); wherein a decrease in the level of one or more of said phosphorylated marker proteins following contact with said compound identifies said compound as inhibiting a BCR-ABL kinase mutant that is resistant to lmanitib mesylate (STI-571). In one preferred embodiment of the method, the BCR-ABL kinase mutant is selected from the group consisting of a T315I mutant, a M351T mutant, a Y235F mutant, and a E255K mutant. Exemplary inhibitors of BCR-ABL include, but are not limited to Gleevec® (STI-571), AMN 107 and BMS-354825 and its analogues. Inhibitory compounds may be targeted inhibitors that modulate post-kinase activity of BCR-ABL, or may be upstream expression inhibitors, such as siRNA or anti-sense inhibitors. In another preferred embodiment, the compound is being tested for inhibition of BCR-ABL activity or expression. Such compound may, for example, directly inhibit BCR-ABL activity, or may indirectly inhibit its activity by, e.g., inhibiting another kinase that phosphorylates and thus activates BCR-ABL.

Biological samples may be obtained from a subject having, or at risk of having, a disease or condition involving BCR-ABL mutant kinase expression or activity (e.g., CML). For example, samples may be analyzed to monitor subjects who have been previously diagnosed as having CML, to screen subjects who have not been previously diagnosed as having CML, or to monitor the desirability or efficacy of therapeutics targeted at BCR-ABL. In the case of CML, for example, the subjects will most frequently be adult patients undergoing or candidates for Gleevec® treatment, who are at risk of developing Gleevec®-resistance.

The invention also provides a method for profiling mutant BCR- ABL kinase activity in a biological sample potentially having (or suspected of involving) mutant BCR-ABL, by (a) contacting the biological sample with at least one phospho-specific antibody of the invention under conditions suitable for formation of an antibody-antigen complex, (b) detecting the presence of the complex in the biological sample, wherein the presence of the complex indicates the presence of one or more of the marker phosphoproteins disclosed herein in the biological sample, thereby indicating the presence of a Gleevec®-resistant BCR-ABL mutant in the biological sample. In some preferred embodiments, the test tissue is bone marrow or blood samples from a CML patient potentially having (or suspected of involving) Gleevec®-resistant BCR-ABL fusion protein expression.

Conditions suitable for the formation of antibody-antigen complexes are well known in the art (see part (D) below and references cited therein). It will be understood that more than one antibody may be used in the practice of the above-described methods. For example, a wild type BCR-ABL-specific antibody and one of the phospho-specific antibodies of the invention separately or simultaneously employed to detect GleevecO-resistant BCR-ABL activity in one step.

The methods described above are applicable to examining tissues or biological samples from any disease or condition involving or characterized by the activity or expression of a mutant BCR-ABL kinase that is Gleevec®-resistant, particularly CML, in which the presence of such variants of BCR-ABL has predictive value as to the outcome of the disease or the response of the disease to therapy. It is anticipated that the antibodies, AQUA peptides, and assays of this invention will have diagnostic utility in a disease characterized by, or involving, mutant BCR- ABL expression or activity. The methods are applicable, for example, where samples are taken from a subject previously diagnosed as having CML, and undergoing Gleevec® treatment for the disease, and the method is employed to help diagnose early the development of resistance to Gleevec®, or AMN 107 or BMS-354825 (in the case of the T315I mutation) and/or monitor the possible progression of the condition.

Such diagnostic assay may be carried out prior to preliminary blood evaluation or surgical surveillance procedures. Such a diagnostic assay may be advantageously employed to identify patients with BCR- ABL mutant kinase expression/activity, who relapse on Gleevec® treatment, but would be likely to respond to other therapeutics targeted at inhibiting mutant BCR-ABL activity, such as AMN107 or BMS-354825, or future therapeutic compounds and its analogues. Such a selection of patients would be useful in the clinical evaluation of efficacy of future BCR-ABL-targeted therapeutics as well as in the future prescription of such novel drugs to patients.

D. Immunoassay Formats & Kits

Assays carried out in accordance with methods of the present invention may be homogeneous assays or heterogeneous assays. In a homogeneous assay the immunological reaction usually involves a phosphospecific antibody of the invention as a reagent, a labeled analyte, and the sample of interest. The signal arising from the label is modified, directly or indirectly, upon the binding of the antibody to the labeled analyte. Both the immunological reaction and detection of the extent thereof are carried out in a homogeneous solution. Immunochemical labels that may be employed include free radicals, radioisotopes, fluorescent dyes, enzymes, bacteriophages, coenzymes, and so forth.

In a heterogeneous assay approach, the reagents are usually the specimen, a phospho-specific reagent, and suitable means for producing a detectable signal. Similar specimens as described above may be used. The antibody is generally immobilized on a support, such as a bead, plate or slide, and contacted with the specimen suspected of containing the antigen in a liquid phase. The support is then separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means for producing such signal. The signal is related to the presence of the analyte in the specimen. Means for producing a detectable signal include the use of radioactive labels, fluorescent labels, enzyme labels, and so forth. For example, if the antigen to be detected contains a second binding site, an antibody which binds to that site can be conjugated to a detectable group and added to the liquid phase reaction solution before the separation step. The presence of the detectable group on the solid support indicates the presence of the antigen in the test sample. Examples of suitable immunoassays are the radioimmunoassay, immunofluorescence methods, enzyme-linked immunoassays, and the like.

Immunoassay formats and variations thereof, which may be useful for carrying out the methods disclosed herein, are well known in the art. See generally E. Maggio, Enzyme-lmmunoassay, (1980) (CRC Press, Inc., Boca Raton, FIa.); see also, e.g., U.S. Pat. No. 4,727,022 (Skold et ai, "Methods for Modulating Ligand-Receptor Interactions and their Application"); U.S. Pat. No. 4,659,678 (Forrest et ai, "Immunoassay of Antigens"); U.S. Pat. No. 4,376,110 (David et ai, "Immunometric Assays Using Monoclonal Antibodies"). Conditions suitable for the formation of reagent-antibody complexes are well described. See id. Monoclonal antibodies of the invention may be used in a "two-site" or "sandwich" assay, with a single cell line serving as a source for both the labeled monoclonal antibody and the bound monoclonal antibody. Such assays are described in U.S. Pat. No. 4,376,110. The concentration of detectable reagent should be sufficient such that the binding of mutant BCR-ABL is detectable compared to background.

The phospho-specific antibodies disclosed herein may be conjugated to a solid support suitable for a diagnostic assay (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as precipitation. Antibodies of the invention, may likewise be conjugated to detectable groups such as radiolabels (e.g., ³⁵S, ¹²⁵1, ¹³¹I), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein) in accordance with known techniques.

Reagents provided by the invention for the specific detection and/or quantification of one of the ten Gleevec®-resistance marker phosphoproteins/sites disclosed herein may be advantageously employed in whole-cell assays to detect the presence of such markers in a biological sample from a CML patient. Certain preferred whole-cell assays are described below.

Phospho-specific antibodies of the invention may be advantageously employed in a flow cytometry (FC) assay to determine the presence of Gleevec®-resistant BCR-ABL mutants in patients before, during, and after treatment with a drug targeted at inhibiting BCR-ABL kinase activity. For example, bone marrow cells or peripheral blood cells or smears from patients may be analyzed by flow cytometry for mutant BCR-ABL expression, as well as for the phosphoprotein markers disclosed herein. In this manner, the presence of a Gleevec®-resistant variant of BCR-ABL may be specifically characterized, using this clinically suitable assay format. Flow cytometry may be carried out according to standard methods. See, e.g. Chow ef a/., Cytometry (Communications in Clinical Cytometry) 46: 72-78 (2001). Briefly and by way of example, the following protocol for cytometric analysis may be employed: fixation of the cells with 2% paraformaldehyde for 10 minutes at 37 ⁰C followed by permeabilization in 90% methanol for 30 minutes on ice. Cells may then be stained with the primary BCR-ABL antibody (or phosphoprotein marker antibody), washed and labeled with a fluorescent-labeled secondary antibody. The cells would then be analyzed on a flow cytometer (e.g. a Beckman Coulter FC500) according to the specific protocols of the instrument used. Such an analysis would identify the presence of the phosphorylated marker proteins of this invention in a cell of interest and reveal the drug response on the targeted BCR-ABL kinase. lmmunohistochemical (IHC) staining may be also employed to determine the expression and/or activation status of one or more GleevecΘ-resistance marker phosphoprotein(s) in a biological sample from a CML patient before, during, and after treatment with Gleevec® or its analogues. IHC may be carried out according to well-known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL, Chapter 10, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988). Briefly, and by way of example, paraffin-embedded tissue (e.g. bone marrow from a biopsy) is prepared for immunohistochemical staining by deparaffinizing tissue sections with xylene followed by ethanol; hydrating in water then PBS; unmasking antigen by heating slide in sodium citrate buffer; incubating sections in hydrogen peroxide; blocking in blocking solution; incubating slide in primary anti-marker phosphoprotein antibody (i.e. against any of the ten resistance marker phosphoproteins/sites listed in Table 2/Figure 2) and secondary antibody; and finally detecting using ABC avidin/biotin method according to manufacturer's instructions.

Immunofluorescence (IF) assays may be also employed to determine the expression and/or activation status of one or more GleevecΘ-resistance marker phosphoprotein(s) in a biological sample from a CML patient before, during, and after treatment with Gleevec® or its analogues. IF may be carried out according to well-known techniques. See, e.g., J. M. Polak and S. Van Noorden (1997) INTRODUCTION TO IMMUNOCYTOCHEMISTRY, 2nd Ed.; ROYAL MICROSCOPY SOCIETY MICROSCOPY HANDBOOK 37, BioScientific/Springer-Verlag. Briefly, and by way of example, patient samples may be fixed in paraformaldehyde followed by methanol, blocked with a blocking solution such as horse serum, incubated with the primary antibody against the marker phosphoprotein(s) followed by a secondary antibody labeled with a fluorescent dye such as Alexa 488 and analyzed with an epifluorescent microscope.

Antibodies employed in the above-described assays may be advantageously conjugated to fluorescent dyes (e.g. Alexa 488, PE), or other labels, such as quantum dots, for use, in multi-parametric analyses along with other signal transduction (EGFR, phospho-AKT, phospho-Erk 1/2) and/or cell marker (cytokeratin) antibodies, as described earlier.

Kits for carrying out the methods disclosed above are also provided by the invention. Such kits comprise one or more of the phospho-specific antibodies of this invention alone or together with other antibodies to determine the presence of BCR-ABL. In one embodiment, the invention provides a kit for the identification of a chronic myelogenous leukemia (CML) patient that has, or is likely to develop, resistance to lmanitib mesylate (STΪ-571), said kit comprising at least one detectable antibody of the invention and/or at least one detectable heavy isotope- labeled peptide of the invention (for the detection/quantification of one or more of the ten Gleevec®-resistance marker phosphoproteins disclosed herein.

The kits may also include ancillary agents such as buffering agents and protein stabilizing agents, e.g., polysaccharides and the like. The diagnostic kit may further include, where necessary, other members of the signal-producing system of which system the detectable group is a member (e.g., enzyme substrates), agents for reducing background interference in a test, control reagents, apparatus for conducting a test, and the like. The test kit may be packaged in any suitable manner, typically with all elements in a single container along with a sheet of printed instructions for carrying out the test.

The following Examples are provided only to further illustrate the invention, and are not intended to limit its scope, except as provided in the claims appended hereto. The present invention encompasses modifications and variations of the methods taught herein which would be obvious to one of ordinary skill in the art. EXAMPLE 1

Isolation of Phosphotyrosine-Containing Peptides from Extracts of

BA/F3 Cell Lines Expressing Gleevec®-resistant Variants of BCR-

ABL and Identification of Phosphoprotein Markers/Sites.

Most newly diagnosed patients with treated with lmanitib (Gleevec®) respond well during the chronic phase of the disease. However, some of these patients and others with advanced disease develop resistance Gleevec® therapy. In such patients, the BCR-ABL kinase is re-activated mostly due to specific mutations in the kinase domain that prevent Gleevec® binding without substantially abolishing the kinase activity. Among such mutations are those affecting amino acid residues Y253, E255, T315 and M351 , which represent approximately 60% of all reported mutations. See Cowan-Jacob et al., supra.

Crystallographic studies have shown that Gleevec® binds to BCR- ABL by filling a pocket created in the ATP binding site by the DFG motif on the activation loop, displacing it from a position that it occupies in the catalytically active conformation of the enzyme. See Schindler et al. Science 289:1938-1942 (2000); Nagar et al., Cancer Res. 62/ 4236-4243 (2002). Point mutations of BCR-ABL have been classified as those that destabilize this inactive contact between the kinase and Gleevec® or those that sterically impede direct contact between the kinase and the drug. See Cowan-Jacob SW et al., supra. It is possible that such mutations would not only affect the binding of the drug to the kinase, but also change intrinsic properties of the kinase affecting how it interacts with its cellular substrates and downstream signaling pathways. This possibility has not previously been explored. If correct, it implies that it would be possible to identify substrates or protein phosphorylation events that would specifically occur in the presence of a particular Gleevec®- resistant Bcr-Abl mutant, but not wild-type Gleevec®-sensitive BCR-ABL. Therefore, in order to discover such potential phosphoprotein markers/sites that are detectable only when GleevecΘ-resistant (but not wild-type) BCR-ABL kinases are expressed, IAP isolation techniques (as described above) were employed to identify phosphotyrosine-containing peptides in cell extracts from the following cell lines expressing activated BCR-AbI wild-type and mutant kinases such as: BaF/3, Ba/F3-p210 BCR- AbI, Ba/F3-M351 T-BCR-ABL, Ba/F3-E255K-BCR-Abl, Ba/F3-Y253F- BCR-AbI, BaF3-T315l-BCR-Abl. These mutant BCR-AbI kinases and the cell Ba/F3 cell lines expressing them have been constructed as described elsewhere. See La Rosee et al., Cancer Res. 62: 7149-7153 (2002).

Tryptic phosphotyrosine- and phosphoserine- containing peptides were purified and analyzed from extracts of each of the 7 cell lines mentioned above, as follows. Cells were cultured in DMEM medium or RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cells were harvested by low speed centrifugation. After complete aspiration of medium, cells were resuspended in 1 mL lysis buffer per 1.25 x 10⁸ cells (20 mM HEPES pH 8.0, 9 M urea, 1 mM sodium vanadate, supplemented or not with 2.5 mM sodium pyrophosphate, 1 mM β-glycerol-phosphate) and sonicated.

Sonicated cell lysates were cleared by centrifugation at 20,000 x g, and proteins were reduced with DTT at a final concentration of 4.1 mM and alkylated with iodoacetamide at 8.3 mM. For digestion with trypsin, protein extracts were diluted in 20 mM HEPES pH 8.0 to a final concentration of 2 M urea and soluble TLCK-trypsin (Worthington) was added at 10-20 μg/mL. Digestion was performed for 1-2 days at room temperature.

Trifluoroacetic acid (TFA) was added to protein digests to a final concentration of 1%, precipitate was removed by centrifugation, and digests were loaded onto Sep-Pak Ci₈ columns (Waters) equilibrated with 0.1 % TFA. A column volume of 0.7-1.0 ml was used per 2 x 10⁸ cells. Columns were washed with 15 volumes of 0.1% TFA, followed by 4 volumes of 5% acetonitrile (MeCN) in 0.1 % TFA. Peptide fraction I was obtained by eluting columns with 2 volumes each of 8, 12, and 15% MeCN in 0.1% TFA and combining the eluates. Fractions Il and III were a combination of eluates after eluting columns with 18, 22, 25% MeCN in 0.1% TFA and with 30, 35, 40% MeCN in 0.1% TFA, respectively. All peptide fractions were lyophilized.

Peptides from each fraction corresponding to 2 x 10⁸ cells were dissolved in 1 ml of IAP buffer (20 mM Tris/HCI or 50 mM MOPS pH 7.2, 10 mM sodium phosphate, 50 mM NaCI) and insoluble matter (mainly in peptide fractions III) was removed by centrifugation. IAP was performed on each peptide fraction separately. The phosphotyrosine monoclonal antibody P-Tyr-100 (Cell Signaling Technology, Inc., catalog number 9411) was coupled at 4 mg/ml beads to protein G or protein A agarose (Roche), respectively. Immobilized antibody (15 μl, 60 μg) was added as 1 :1 slurry in IAP buffer to 1 ml of each peptide fraction, and the mixture was incubated overnight at 4° C with gentle rotation. The immobilized antibody beads were washed three times with 1 ml IAP buffer and twice with 1 ml water, all at 4° C. Peptides were eluted from beads by incubation with 75 μl of 0.1% TFA at room temperature for 10 minutes.

Alternatively, one single peptide fraction was obtained from Sep-Pak C18 columns by elution with 2 volumes each of 10%, 15%, 20 %, 25 %, 30 %, 35 % and 40 % acetonitirile in 0.1% TFA and combination of all eluates. IAP on this peptide fraction was performed as follows: After lyophilization, peptide was dissolved in 1.4 ml IAP buffer (MOPS pH 7.2, 10 mM sodium phosphate, 50 mM NaCI) and insoluble matter was removed by centrifugation. Immobilized antibody (40 μl, 160 μg) was added as 1 :1 slurry in IAP buffer, and the mixture was incubated overnight at 4° C with gentle shaking. The immobilized antibody beads were washed three times with 1 ml IAP buffer and twice with 1 ml water, all at 4° C. Peptides were eluted from beads by incubation with 55 μl of 0.15% TFA at room temperature for 10 min (eluate 1), followed by a wash of the beads (eluate 2) with 45 μl of 0.15% TFA. Both eluates were combined.

Analysis bv LC-MS/MS Mass Spectrometry.

40 μl or more of IAP eluate were purified by 0.2 μl StageTips or ZipTips. Peptides were eluted from the microcolumns with 1 μl of 40% MeCN, 0.1 % TFA (fractions I and II) or 1 μl of 60% MeCN, 0.1 % TFA (fraction III) into 7.6 μl of 0.4% acetic acid/0.005% heptafluorobutyric acid. This sample was loaded onto a 10 cm x 75 μm PicoFrit capillary column (New Objective) packed with Magic C18 AQ reversed-phase resin (Michrom Bioresources) using a Famos autosampler with an inert sample injection valve (Dionex). The column was then developed with a 45-min linear gradient of acetonitrile delivered at 200 nl/min (Ultimate, Dionex), and tandem mass spectra were collected in a data-dependent manner with an LCQ Deca XP Plus ion trap mass spectrometer essentially as described by Gygi et al., supra.

Database Analysis & Assignments.

MS/MS spectra were evaluated using TurboSequest in the Sequest Browser package (v. 27, rev. 12) supplied as part of BioWorks 3.0 (ThermoFinnigan). Individual MS/MS spectra were extracted from the raw data file using the Sequest Browser program CreateDta, with the following settings: bottom MW, 700; top MW, 4,500; minimum number of ions, 20; minimum TIC, 4 x 10⁵; and precursor charge state, unspecified. Spectra were extracted from the beginning of the raw data file before sample injection to the end of the eluting gradient. The IonQuest and VuDta programs were not used to further select MS/MS spectra for Sequest analysis. MS/MS spectra were evaluated with the following TurboSequest parameters: peptide mass tolerance, 2.5; fragment ion tolerance, 0.0; maximum number of differential amino acids per modification, 4; mass type parent, average; mass type fragment, average; maximum number of internal cleavage sites, 10; neutral losses of water and ammonia from b and y ions were considered in the correlation analysis. Proteolytic enzyme was specified except for spectra collected from elastase digests.

Searches were performed against the NCBI human protein database (either as released on April 29, 2003 and containing 37,490 protein sequences or as released on February 23, 2004 and containing 27,175 protein sequences). Cysteine carboxamidomethylation was specified as a static modification, and phosphorylation was allowed as a variable modification on serine, threonine, and tyrosine residues or on tyrosine residues alone. It was determined that restricting phosphorylation to tyrosine residues had little effect on the number of phosphorylation sites assigned.

In proteomics research, it is desirable to validate protein identifications based solely on the observation of a single peptide in one experimental result, in order to indicate that the protein is, in fact, present in a sample. This has led to the development of statistical methods for validating peptide assignments, which are not yet universally accepted, . and guidelines for the publication of protein and peptide identification results (see Carr et a/., MoI. Cell Proteomics 3: 531-533 (2004)), which were followed in this Example. However, because the immunoaffinity strategy separates phosphorylated peptides from unphosphorylated peptides, observing just one phosphopeptide from a protein is a common result, since many phosphorylated proteins have only one tyrosine- phosphorylated site.

For this reason, it is appropriate to use additional criteria to validate phosphopeptide assignments. Assignments are likely to be correct if any of these additional criteria are met: (i) the same sequence is assigned to co-eluting ions with different charge states, since the MS/MS spectrum changes markedly with charge state; (ii) the site is found in more than one peptide sequence context due to sequence overlaps from incomplete proteolysis or use of proteases other than trypsin; (iii) the site is found in more than one peptide sequence context due to homologous but not identical protein isoforms; (iv) the site is found in more than one peptide sequence context due to homologous but not identical proteins among species; and (v) sites validated by MS/MS analysis of synthetic phosphopeptides corresponding to assigned sequences, since the ion trap mass spectrometer produces highly reproducible MS/MS spectra. The last criterion is routinely employed to confirm novel site assignments of particular interest.

All spectra and all sequence assignments made by Sequest were imported into a relational database. Assigned sequences were accepted or rejected following a conservative, two-step process. In the first step, a subset of high-scoring sequence assignments was selected by filtering for XCorr values of at least 1.5 for a charge state of +1 , 2.2 for +2, and 3.3 for +3, allowing a maximum RSp value of 10. Assignments in this subset were rejected if any of the following criteria were satisfied: (i) the spectrum contained at least one major peak (at least 10% as intense as the most intense ion in the spectrum) that could not be mapped to the assigned sequence as an a, b, or y ion, as an ion arising from neutral-loss of water or ammonia from a b or y ion, or as a multiply protonated ion; (ii) the spectrum did not contain a series of b or y ions equivalent to at least six uninterrupted residues; or (iii) the sequence was not observed at least five times in all the studies we have conducted (except for overlapping sequences due to incomplete proteolysis or use of proteases other than trypsin).

In the second step, assignments with below-threshold scores were accepted if the low-scoring spectrum showed a high degree of similarity to a high-scoring spectrum collected in another study, which simulates a true reference library-searching strategy. All spectra supporting the final list of 120 sequences assigned to individual phosphorylation sites enumerated in Table 1 below were reviewed by at least three people to establish their credibility. The Table lists all tyrosine phosphorylation sites/peptides identified in the five cell lines (human and murine sequences). The type of BCR-ABL protein (mutant or normal) with which the phosphopeptide(s) is/are associated is indicated in the final six columns.

Table 1.

Protein

Gene Name/ pTyr Baf

Symbol Description NGBI Acc# Site Phosphopeptides Detected 3 p?1 f)

Adaptor/scaffold

AbM

AbM protein AAH04657U 23 ALIESyQNLTR

AbM

AbM protein NP_031406 108 NTPyKTLEPVKPPTVPNDyMTSPAR

NTPyKTLEPVKPPTVPNDy MTSPAR,

AbM NTPYKTLEPVKPPTVPNDyMTSPAR,

AbM protein NP_031406 123 TLEPVKPPTVPNDyMTSPAR abl- interactor

Abi2 2 NP_937760 207 TLEPVRPPVVPNDyVPSPTR

Casitas B- lineage •

CbI lymphoma NP_031645 655 IKPSSSANAIySLAARPLPMPK

Casitas B- lineage

CbI lymphoma NP_031645 681 LPPGEQGESEEDTEyMTPTSRPVGVQKPEPK

Casitas B- lineage

CbI lymphoma NP_031645 828 KASSyQQGGGATANPVATAPSPQLSSElER

CD2- associated

Cd2ap protein NP_033977fl 88 ISTyGLPAGGIQPHPQTK docking

Dok1 protein 1 NP_034200 336 KPLyWDLYGHVQQQLLK

LTDSKEDPIyDEPEGLAPAPPR,

Docking TKLTDSKEDPIyDEPEGLAPAPPR,

Dok1 protein 1 NP_034200 361 TKLTDSKEDPIyDEPEGLAPAPPRGLYDLPQEPR docking GFSSDTALySQVQK,

Dok1 protein 1 NP_034200 450 GSGSKGFSSDTALySQVQK

Docking

Dok2 protein 2 NP_034201 142 SGSPCMEENELySSSTTGLCK FYN binding

Fyb protein NP_035945 559 TTAVEIDyDSLK non-catalytic region of KPSVPDTASPADDSFVDPGERLyDLNMPAFVK, tyrosine kinas PSVPDTASPADDSFVDPGERLyDLNMPAFVK, adaptor RKPSVPDTASPADDSFVDPGERLyDLNMPAFVK,

Nck1 protein 1 NP .035008 105 RKPSVPDTASPADDSFVDPGERLyDLNMPAFVK PDZ and LIM domain

Pdlimδ 5 NP. _062782U 251 NTEFyHIPTHSDASK phosphoinosϊ e-3-kinase adaptor

Pik3ap1 protein 1 NP_113553 694 HSQHLPEKVEFGVyESGPR similar to Casitas B- lineage CbIb lymphoma b XP_ .15625711 845 ASQDyDQLPSSSDGSQAPARPPKPRPR

SKAP55 SQPIDDEIyEELPEEEEDTASVK,

Scap2 homologue NP_ .061243 260 SQPIDDEIyEELPEEEEDTASVKMDEQGKGSR src homology domain- KQMLPPPPCPGRELFDDPSyVNIQNLDKAR, containing QMLPPPPCPGRELFDDPSyVNIQNLDKAR, transforming ELFDDPSyVNIQNLDK,

Shd protein C NP_ 035498 313 ELFDDPSyVNIQNLDKAR v-crk sarcoms virus CT10 oncogene RVPNAyDKTALALEVGELVK,

Crk homolog NP .598417 251 VPNAyDKTAULEVGELVK Wiskott-Aldric syndrome AGISEAQLTDAETSKLIyDFIEDQGGLEAVR,

Was homolog NP_ .033541 293 AGISEAQLTDAETSKLIyDFIEDQGGLEAVRQEMR

Adhesion/ cytoskeleton a disintegrin

Scmetalloprot Adam ease domain 18 18 NP_034214 46 VTyVITIDGKPYSLHLR actin, beta, Actb cytoplasmic NP_031419fl 166 TTGIVMDSGDGVTHTVPIyEGYALPHAILR actin, beta, ELCyVALDFEQEMATAASSSSLEK, Actb cytoplasmic NP_031419fl 218 DIKEKLCyVALDFEQEMATAASSSSLEK

KDLyANTVLSGGTTMYPGIADR, CDVDIRKDLyANTVLSGGTTMYPGIADR, actin, beta, CDVDIRKDLyANTVLSGGTTMYPGLADR, Actb cytoplasmic NP_0314191J 294 KDLyANTVLSGGTTMYPGLADR actinin alpha AlMTYVSSFyHAFSGAQK, Actn4 4 NP_068695fl 266 AIMTYVSSFyHAFSGAQKAETAANR actin-related protein 3- NP_0010043 Actr3b beta 65fl 202 DITyFIQQLLR cofilin 1 , non- GfII muscle NP_031713 68 NIILEEGKEILVGDVGQTVDDPyTTFVK laminin receptor 1

(ribosomal Lamrl protein SA) NP_0351591f 139 ADHQPLTEASyVNLPTIALCNTDSPLR lymphocyte cytosolic Lcp1 protein 1 NP_032905 124 EGICAIGGTSEQSSVGTQHSYSEEEKyAFVNWINK lymphocyte cytosolic

Lcp1 protein 1 NP_032905 598 VyALPEDLVEVNPK myosin, heavy polypeptide HEMPPHIyAITDTAYR, 9, non- KRHEMPPHIyAITDTAYR,

Myh9 muscle NP_0718551J 151 RHEMPPHIyAITDTAYR myosin, heavy polypeptide 9, non-

Myh9 muscle NP_071855 754 ALELDSNLyRIGQSK vasodilator- stimulated phospho-

Vasp protein NP .033525 39 VQIyHNPTANSFR

Vim vimentin NP..035831 53 SLySSSPGGAYVTR

VcI vinculin NP 033528 822 SFLDSGyR

Enzyme, misc glutamate oxaloacetate transaminase Got1 1 , soluble NP_034454 71 IANDNSLNHEyLPILGLAEFR

Calm2 calmodulin 2 NP_031615fl 100 VFDKDGNGyISAAELR

GNPTVEVDLyTAK, enolase 2, GNPTVEVDLyTAKGLFR, gamma EIFDSRGNPTVEVDLyTAK, Eno2 neuronal NP .038537U 25 EIFDSRGNPTVEVDLyTAKGLFR enolase 3, AAVPSGASTGIyEALELR, Eno3 beta muscleNP .031959H 44 AAVPSGASTGIyEALELRDNDKTR glucose-6- phosphate dehydrogen- G6pdx ase X-linked NP_032088 401 VQPNEAVyTK glyceraldehy de-3- phosphate dehydrogen- NPJD010013 LISWYDNEyGYSNR,

Gapdh ase 03 316 LISWYDNEyGYSNRVVDLMAYMASKE

Phospho- glycerate

Pgam2 mutase 2 NP .061358H 92 HyGGLTGLNK proteasome (prosome, macropain) subunit,

Psma2 alpha type 2 NP_032970fl 24 LVQIEyALAAVAGGAPSVGIK proteasome (prosome, macropain) _• • subunit,

Psma2 alpha type 2 NP_032970 76 HIGLVySGMGPDYR proteasome (prosome, macropain) subunit,

Psma2 alpha type 2 NP _032970 101 KLAQQYYLVyQEPIPTAQLVQR Psmb4 proteasome NP_032971 102 VNDSTMLGASGDyADFQYLK beta 4 subunit pyruvate EATESFASDPILyRPVAVALDTK,

Pkm2 kinase 3 NP. .035229 105 EATESFASDPILyRPVAVALDTKGPEIR pyruvate Pkm2 kinase 3 NP. _035229 148 ITLDNAyMEKCDENILWLDYK pyruvate

Pkm2 kinase 3 NP 035229 370 AEGSDVANAVLDGADCIMLSGETAKGDyPLEAVR serine hydroxy- methyl transferase 1

Shmti (soluble) NP_033197 28 MLSQPLKDSDAEVySIIKK Transglutaminase 2, C

Tgm2 polypeptide NP_033399 369 SEGTyCCGPVSVR Transglutaminase 2, C

Tgm2 polypeptide NP_033399 617 KLVAEVSLKNPLSDPLyDCIFTVEGAGLTKEQK

G proteins and regulators

IQ motif containing

GTPase activating Iqgapi protein 1 NP_057930 1510 LQQTySALNSK

RAN, member

RAS oncogene Ran family NP_033417fl 147 NLQYyDISAK

ILNKKGQQGWWRGEIyGR, vav 1 KGQQGWWRGEIyGR,

Vav1 oncogene NP_035821 826 GQQGWWRGEIyGR

Lipid kinase phosphatidy

(inositol 3- kinase catalytic delta Pik3cd polypeptide NPJD32866 484 GNPNTESAAALVIYLPEVAPHPVyFPALEK phosphatidy linositol 3- kinase catalytic delta

Pik3cd polypeptide NP_032866 935 ERVPFILTyDFVHVIQQGK phosphatidyl! nositol 3- kinase, regulatory subunit, polypeptide 1 Pik3r1 (p85 alpha) NP_035215 197 SREYDRLyEEYTR Lipid Phosphatase inositol polyphosphate phosphatase

InppH -like 1 NP_034697 987 NSFNNPAyYVLEGVPHQLLPLEPPSLAR inositol polyphosphate phosphatase

InppH -like 1 NP_034697 1136 TLSEVDyAPGPGR inositol polyphosphate phosphatase

InppH -like 1 NP_034697 1161 SALLPNPLELQPPRGPSDyGRPLSFPPPR inositol polyphos- phate-5- phosphatase

Inppδd D , NP_034696 1021 VEALLQEDLLLTKPEMFENPLyGSVSSFPK

Other

CD84 Cd84 antigen NP_ .038517 280 NAQPTESRIyDEIPQSK centaurin,

Centd2 delta 2 NP. .081456If 729 AAASLGDTLSEQQLGDSDlPVIVyR chaperonin subunit 2

Cct2 (beta) NP. .031662 297 QLIyNYPEQLFGAAGVMAIEHADFAGVER chaperonin subunit δ

Cct8 (theta) NP. .033970 30 HFSGLEEAVyR eukaryotic translation elongation factor 1 STTTGHLIyKCGGIDKR, Eef1a2 alpha 2 NP_031932ff 29 STTTGHLIyK

Fc receptor, IgE, high affinity I, gamma

Fcerig polypeptide NP .034315 65 EKADAVyTGLNTR heat shock protein 1 ,

Hspcb beta NP .032328Tf 484 SIyYITGESK heat shock protein 1 , Hspcb beta NP 032328 596 LVSSPCCIVTSTyGWTANMER heat shock NP_ 0010020 Hspa2 protein 2 121J 42 TTPSyVAFTDTER poly A bindinς protein, Pabpd cytoplasmic 1NP_032800fl 116 ALyDTFSAFGNILSCK poly A bindinς protein, Pabpd cytoplasmic 1NP_032800fl 364 IVATKPLyVALAQR polypyrimidi Ptbpi ne tract NP_032982fl 126 GQPIyIQFSNHK binding protein 1 valosin containing GFGSFRFPSGNQGGAGPSQGSGGGTGGSVYT

Vcp protein NPJD33529 805 EDNDDDLyG

Protein kinase breakpoint cluster region BCR (human) NP_0043181f 910 LQTVHSIPLTINKEDDESPGLyGFLNVIVHSATGFK breakpoint KGPAQPGSADAEKPFyVNVEFHHER, cluster GHGQPGADAEKPFyVNVEFHHER, region IRKGHGQPGADAEKPFyVNVEFHHER, Bcr homolog XPJ 25706 95 KGHGQPGADAEKPFyVNVEFHHER breakpoint cluster _• • region Bcr homolog XP_125706 507 USQLGVyR breakpoint cluster region NSLETLLyKPVDR, Bcr homolog XP_125706 560 NSLETLLyKPVDRVTR

Bruton agammaglo bulinemia tyrosine KWALyDYIMPMNANDLQLR, Btk kinase NP 038510 223 VVALyDYMPMNANDLQLR

IEKIGEGTyGVVYK, cell IEKIGEGTyGVVYKGR, division IGEGTyGVVYKGR, cycle 2 IEKIGEGTyGVVyKGR,

Cdc2a homolog A NP_031685 15 IGEGTyGVVYK cell division cycle 2 IEKIGEGTyGVVyKGR,

Cdc2a homolog A NP_031685 19 IGEGTYGWyKGR cyclin- dependent IGEGTyGWYKAK,

Cdk2 kinase 2 NP__058036fl 1.5 VEKIGEGTyGVVYK dual-specificit tyrosine-(Y)- phosphorylati KVYNDGYDDDNyDYIVK, • • n regulated KVYNDGYDDDNyDYIVKNGEK,

Dyrki a kinase 1a NP_0319161J 145 VYNDGYDDDNyDYIVK dual- specificity tyrosine-(Y)- _• • phosphorylati on regulated

Dyrki a kinase 1a NP_031916fl 321 IYQyIQSR dual- specificity tyrosine-(Y)- phosphorylati on regulated Dyrk4 kinase 4 NP_997093fl 344 VYTyIQSR glycogen synthase GEPNVSyICSR,

Gsk3b kinase 3 betaNP_062801fl 216 QLVRGEPNVSyICSR homeodomai n-interacting _• • protein Hipki kinasel NP_034562fl 352 AVCSTyLQSR homeodomai n-interacting protein Hipk3 kinase3 NP_034564 359 TVCSTyLQSR mitogen activated • • protein Mapki kinasel NP_036079 185 VADPDHDHTGFLTEyVATR mitogen activated protein Mapk14 kinase 14 NP_036081 182 HTDDEMTGyVATR mitogen- activated protein

Mapk12 kinase 12 NP_038899 185 QADSEMTGyVVTR PRP4 pre- mRNA processing factor 4 LCDFGSASHVADNDITPyLVSR, Prpf4b homolog B NP_038858 849 TILKLCDFGSASHVADNDITPyLVSR v-abl Abelson murine • » leukemia AbH oncogene 1 NP_0337241f 115 NGQGWVPSNyITPVNSLEK v-abl Abelson murine leukemia AbM oncogene 1 NP_033724 139 NAAEyLLSSGINGSFLVR v-abl Abelson murine leukemia AbM oncogene 1 NP_033724 185 INTASDGKLyVSSESR v-abl Abelson HKLGGGQYGEVyEGVWK, murine HKLGGGQYGEVyEGVWKK, • • leukemia LGGGQYGEVyEGVWK, AbH oncogene 1 NP_033724 257 LGGGQYGEVyEGVWKK v-abl Abelson murine LMTGDTyTAHAGAK, • • • leukemia LMTGDTyTAHAGAKFPIK, AbH oncogene 1 NP_033724U 393 VADFGLSRLMTGDTyTAHAGAK v-abl Abelson murine leukemia AbH oncogene 1 NP_033724 469 MERPEGCPEKVyELMR v-abl Abelson murine leukemia viral • • oncogene NKPTVyGVSPNYDKWEMER, ABL1 homolog 1 NP_005148fl 226 RNKPTVyGVSPNYDKWEMER v-abl Abelson murine leukemia viral ABL1 oncogene NPjD05148fl 232 NKPTVYGVSPNyDKWEMER homolog 1

Protein phosphatase protein tyrosir phosphatase, • • non-receptor Ptpn18 type 18 NP_035336 62 yKDWAYDETR protein tyrosir phosphatase, non-receptor Ptpn18 type 18 NP_035336 380 APTSTDTPIySQVAPR

Transcription factor

Dpf2 requiem NP_035392 172 HEPDDFLDDLDDEDyEEDTPKRR signal transducer and activator oftranscript- Statδa ion 5A NP_035618 694 AVDGyVKPQIK signal transducer and activator oftranscript- AADGyVKPQIK, Statδb ion δB NP 035619 699 YYTPVPCEPATAKAADGyVKPQIK

Unknown carnitine deficiency- associated gene expressed in

Cdv3 ventricle 3 NP_780774fl 213 KTPQGPPEIySDTQFPSLQSTAK hypothetical

D8Ert protein CVGQAAELQPASLLRDPVQPEPIyAESAK, • • d82e LOC244418 NP_766499 196 CVGQAAELQPASLLRDPVQPEPIyAESAKR

C230 hypothetical

081 A protein

13Rik LOC244895 NP_766512 528 SSAIRyQEWVTSSTSPR

49215 hypothetical

21J11 protein

Rik LOC70885 NP_081866 458 IFLTDMIIyQGQyK

49215 hypothetical 21J11 protein

Rik LOC70885 NP 081866 462 IFLTDMIIyQGQyK

28104

57I06 RIKEN cDNA • •

Rik 2810457106 NP 789830 8 EELySKVTPR

31100

50K2 RIKEN cDNA _• •

1 Rik 3110050K21 NP '__080359Tf 284 yKVKDRIEEKPR signal- induced proliferation- associated 1 Sipai H like 1 NP_766167 1166 NSPSNLSSSSETGSGGGTyRQK similar to

BC03 BC036961

6961 protein XP_357399 261 SGAYRGCTyETQLQLSAR similar to hypothetic BC05 al protein 3440 BC008207 XP_134209H 507 INDTMyFAPSMKDFTQYIFTEK similar to

KIAA0522 protein

LOC3 [Rattus 17433 norvegicus] XP_228841 283 EAGySAAVGVGQRPPRER similar to LOC3 Pgk1 81164 protein XP_355086U 55 yAEAVGRAKRIVWNGPVGVFEWE/ similar to ras-GTPase- activating protein SH3- domain

LOC5 binding 46090 protein XP_620697 56 NSSYAHGGLDSNGKPADAVyGKK

EXAMPLE 2

Confirmation of Phosphoproteins/Sites found only in Gleevec®- Resistant BCR-ABL Mutant Kinase-Expressing Cells

Differences in expression of the above-identified phosphorylation sites between Gleevec®-resistant and -responsive cell lines were validated using a combination of targeted MS/MS and Western Blotting using phospho-specific antibodies when available. This method targets the mass spectrometer to scan for peptide masses that are desired to specifically compare.

Targeted analysis of phosphopeptides was performed by placing the m/z values most commonly observed for these peptides during qualitative analysis on the parent mass list. MS/MS spectra of the targeted mass values were collected using a top-two method, a dynamic exclusion repeat count of 3, and a repeat duration of 0.5 min. Using this approach, the mass spectrometer is not busy fragmenting so many peptides that it misses peptides of interest. This gives a higher level of confidence in the differences observed than qualitative analysis. Phosphorylation of SHIP1 (Y1022 (=Y1020 in mouse)) was observed in the M351T but not in the other Bcr-Abl mutant expressing cell lines. Western blotting confirmed that SHIP1 (Y1020) is strongly phosphorylated in the M351T expressing Baf3 cells (Figure 4). This finding is interesting since SHIP1 is a negative regulator of hematopoiesis and is normally down regulated in Bcr-Abl containing cells. Furthermore, the phosphorylation of Y1020 is involved in the reduction of cell motility and migration (Sattler et al., J.Biol. Chem 276: 2451-2458 (2001)). Interestingly, these results indicate that the M351T mutant is less transforming than wild-type Bcr-Abl. Potentially, phosphorylation of SHIP1 at tyrosine 1020 may play a role in the transforming potential of the M351 T mutation.

Table 2 below (see also Figure 2) provides a list of the ten phosphorylated peptides (and their parent proteins (human)) identified as differentially expressed in Gleevec®-resistant CML; the particular BCR- ABL mutants for which these respective phosphorylated proteins/sites serve as markers are indicated.

All sequences were identified in either human or murine cell lines. Murine sequences were normalized to homologous sites in published human sequences. Actual murine sequences as isolated are listed in Table 1 , and the equivalent murine phosphorylation sites provided in Column J.

EXAMPLE 3

Production of Phospho-specific Polyclonal Antibodies for the Detection of Phosphoprotein Markers of Gleevec®-Resistant

BCR-ABL Mutants

Polyclonal antibodies that specifically bind a Gleevec®-resistance marker protein only when phosphorylated at the respective phosphorylation site disclosed herein (see Table 2/Figure 2) are produced according to standard methods by first constructing a synthetic peptide antigen comprising the phosphorylation site sequence and then immunizing an animal to raise antibodies against the antigen, as further described below. Production of exemplary polyclonal antibodies is provided below.

A. CbI (tyrosine 700 (= Tyr698 in mouse)).

A 15 amino acid phospho-peptide antigen, EGEEDTEy*MTPSSRP (where y*= phosphotyrosine) that corresponds to the sequence encompassing the tyrosine 700 phosphorylation site in human CbI (see Row 1 of Table 2; SEQ ID NO: 1), plus cysteine on the C-terminal for coupling, is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A LABORATORY MANUAL, supra.; Merrifield, supra. This peptide is then coupled to KLH and used to immunize animals to produce (and subsequently screen) phospho-specific CbI (Tyr700) polyclonal antibodies as described in Immunization/ Screening below.

B. Nek (tyrosine 105)

A 15 amino acid phospho-peptide antigen, VDPGERLy*DLNMPAF (where y*= phosphotyrosine) that corresponds to the sequence encompassing the tyrosine 105 phosphorylation site in human Nek (see Row 6 Table 2; SEQ ID NO: 6), plus cysteine on the C-terminal for coupling, is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A LABORATORY MANUAL, supra.; Merrifield, supra. This peptide is then coupled to KLH and used to immunize animals to produce (and subsequently screen) phospho-specific Nek (tyr105) polyclonal antibodies as described in Immunization/ Screening below.

Immunization/Screening.

A synthetic phospho-peptide antigen as described in A-B above is coupled to KLH, and rabbits are injected intradermal^ (ID) on the back with antigen in complete Freunds adjuvant (500 μg antigen per rabbit). The rabbits are boosted with same antigen in incomplete Freund adjuvant (250 μg antigen per rabbit) every three weeks. After the fifth boost, bleeds are collected. The sera are purified by Protein A-affinity chromatography by standard methods (see ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor, supra.). The eluted immunoglobulins are further loaded onto a non-phosphorylated synthetic peptide antigen-resin Knotes column to pull out antibodies that bind the non-phosphorylated form of the phosphorylation site. The flow through fraction is collected and applied onto a phospho-synthetic peptide antigen-resin column to isolate antibodies that bind the phosphorylated form of the site. After washing the column extensively, the bound antibodies (i.e. antibodies that bind a phosphorylated peptide described in A-B above, but do not bind the non-phosphorylated form of the peptide) are eluted and kept in antibody storage buffer.

The isolated antibody is then tested for phospho-specificity using Western blot assay using an appropriate cell line that expresses (or overexpresses) target phospho-protein (Ae. phosphorylated CbI, etc,), for example, Baf3-T351 l BCR-AbI cells, respectively. Cells are cultured in DMEM or RPMI supplemented with 10% FCS. Cell are collected, washed with PBS and directly lysed in cell lysis buffer. The protein concentration of cell Iysates is then measured. The loading buffer is added into cell lysate and the mixture is boiled at 100 ⁰C for 5 minutes. 20 μl (10 μg protein) of sample is then added onto 7.5% SDS-PAGE gel.

A standard Western blot may be performed according to the lmmunoblotting Protocol set out in the CELL SIGNALING TECHNOLOGY, INC. 2003-04 Catalogue, p. 390. The isolated phospho-specific antibody is used at dilution 1 :1000. Phosphorylation-site specificity of the antibody will be shown by binding of only the phosphorylated form of the target protein. Isolated phospho-specific polyclonal antibody does not (substantially) recognize the target protein when not phosphorylated at the appropriate phosphorylation site in the non-stimulated cells (e.g. CbI is not bound when not phosphorylated at tyrosine 700).

In order to confirm the specificity of the isolated antibody, different cell Iysates containing various phosphorylated signal transduction proteins other than the target protein are prepared. The Western blot assay is performed again using these cell Iysates. The phospho-specific polyclonal antibody isolated as described above is used (1 :1000 dilution) to test reactivity with the different phosphorylated non-target proteins on Western blot membrane. The phospho-specific antibody does not significantly cross-react with other phosphorylated signal transduction proteins, although occasionally slight binding with a highly homologous phosphorylation-site on another protein may be observed. In such case the antibody may be further purified using affinity chromatography, or the specific immunoreactivity cloned by rabbit hybridoma technology.

EXAMPLE 4

Production of Phospho-specific Monoclonal Antibodies for the Detection of BCR-ABL mutant specific downstream proteins

Monoclonal antibodies that specifically bind a Leukemia-related signal transduction protein only when phosphorylated at the respective phosphorylation site disclosed herein (see Table 2/Figure 2) are produced according to standard methods by first constructing a synthetic peptide antigen comprising the phosphorylation site sequence and then immunizing an animal to raise antibodies against the antigen, and harvesting spleen cells from such animals to produce fusion hybridomas, as further described below. Production of exemplary monoclonal antibodies is provided below.

A. Cbl-b (tyrosine 889 (= Tyr1014 in mouse)).

A 10 amino acid phospho-peptide antigen, TSQDy*DQLPS (where y*= phosphotyrosine) that corresponds to the sequence encompassing the tyrosine 889 phosphorylation site in human Cbl-b (see Row 3 of Table 2 (SEQ ID NO: 3)), plus cysteine on the C-terminal for coupling, is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A LABORATORY MANUAL, supra.; Merrifield, supra. This peptide is then coupled to KLH and used to immunize animals and harvest spleen cells for generation (and subsequent screening) of phospho- specific monoclonal Cbl-b (Tyr889) antibodies as described in Immunization/Fusion/Screening below.

B. Dok2 (tyrosine 139 (= TyM 42 in mouse)).

A 10 amino acid phospho-peptide antigen, MEENELy*SSAVTVG (where y*= phosphotyrosine) that corresponds to the sequence encompassing the tyrosine 139 phosphorylation site in human Dok2 (see Row 8 of Table 2 (SEQ ID NO: 8)), plus cysteine on the C-terminal for coupling, is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer. See ANTIBODIES: A LABORATORY MANUAL, supra.; Merrifield, supra. This peptide is then coupled to KLH and used to immunize animals and harvest spleen cells for generation (and subsequent screening) of phospho- specific monoclonal Dok2 (Tyr139) antibodies as described in Immunization/ Fusion/Screening below.

Immunization/Fusion/Screening.

A synthetic phospho-peptide antigen as described in A-B above is coupled to KLH, and BALB/C mice are injected intradermal^ (ID) on the back with antigen in complete Freunds adjuvant (e.g. 50 μg antigen per mouse). The mice are boosted with same antigen in incomplete Freund adjuvant (e.g. 25 μg antigen per mouse) every three weeks. After the fifth boost, the animals are sacrificed and spleens are harvested.

Harvested spleen cells are fused to SP2/0 mouse myeloma fusion partner cells according to the standard protocol of Kohler and Milstein (1975). Colonies originating from the fusion are screened by ELISA for reactivity to the phospho-peptide and non-phospho-peptide forms of the antigen and by Western blot analysis (as described in Example 1 above). Colonies found to be positive by ELISA to the phospho-peptide while negative to the non-phospho-peptide are further characterized by Western blot analysis. Colonies found to be positive by Western blot analysis are subcloned by limited dilution. Mouse ascites are produced from a single clone obtained from subcloning, and tested for phospho- specificity (against the Cbl-b phospho-peptide antigen, as the case may be) on ELISA. Clones identified as positive on Western blot analysis using cell culture supernatant as having phospho-specificity, as indicated by a strong band in the induced lane and a weak band in the uninduced lane of the blot, are isolated and subcloned as clones producing monoclonal antibodies with the desired specificity.

Ascites fluid from isolated clones may be further tested by Western blot analysis. The ascites fluid should produce similar results on Western blot analysis as observed previously with the cell culture supernatant, indicating phospho-specificity against the phosphorylated target (e.g. Cbl-b (Tyr889)).

EXAMPLE 5

Production and Use of AQUA Peptides for the Quantification of Gleevec®-Resistance Marker Phosphoproteins

Heavy-isotope labeled peptides (AQUA peptides (internal standards)) for the detection and quantification of a Leukemia-related signal transduction protein only when phosphorylated at the respective phosphorylation site disclosed herein (see Table 2/Figure 2) are produced according to the standard AQUA methodology (see Gygi et a/., Gerber et al., supra.) methods by first constructing a synthetic peptide standard corresponding to the phosphorylation site sequence and incorporating a heavy-isotope label. Subsequently, the MSⁿ and LC-SRM signature of the peptide standard is validated, and the AQUA peptide is used to quantify native peptide in a biological sample, such as a digested cell extract. Production and use of exemplary AQUA peptides is provided below.

A. Nek (tyrosine 105).

An AQUA peptide comprising the sequence,

VDPGERLy*DLNMPAF (y*= phospho- tyrosine; sequence incorporating ¹⁴C/¹⁵N-labeled leucine (indicated by bold L), which corresponds to the tyrosine 105 phosphorylation site in human Nek (see Row 6 in Table 2 (SEQ ID NO: 6)), is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer (see Merrifield, supra.) as further described below in Synthesis & MS/MS Signature. The Nek (Tyr105) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated Nek (TyM 05) in the sample, as further described below in Analysis & Quantification.

B. FYN binding protein (tyrosine 571).

An AQUA peptide comprising the sequence, TTAVEIDY*DSLK (y*= phosphotyrosine; sequence incorporating ¹⁴C/¹⁵N-labeled leucine (indicated by bold L), which corresponds to the tyrosine 105 phosphorylation site in human Fyn binding protein (see Row 4 in Table 2 (SEQ ID NO: 4)), is constructed according to standard synthesis techniques using, e.g., a Rainin/Protein Technologies, Inc., Symphony peptide synthesizer (see Merrifield, supra.) as further described below in Synthesis & MS/MS Signature. The Fyn binding protein (Tyr571) AQUA peptide is then spiked into a biological sample to quantify the amount of phosphorylated Fyn binding protein (Tyr571) in the sample, as further described below in Analysis & Quantification. Synthesis & MS/MS Spectra.

Fluorenylmethoxycarbonyl (Fmoc)-derivatized amino acid monomers may be obtained from AnaSpec (San Jose, CA). Fmoc-derivatized stable- isotope monomers containing one ¹⁵N and five to nine ¹³C atoms may be obtained from Cambridge Isotope Laboratories (Andover, MA). Preloaded Wang resins may be obtained from Applied Biosystems. Synthesis scales may vary from 5 to 25 μmol. Amino acids are activated in situ with 1-H- benzotriazolium, i-bis(dimethylamino) methylene]-hexafluorophosphate (1-),3-oxide:1-hydroxybenzotriazole hydrate and coupled at a 5-fold molar excess over peptide. Each coupling cycle is followed by capping with acetic anhydride to avoid accumulation of one-residue deletion peptide byproducts. After synthesis peptide-resins are treated with a standard scavenger-containing trifluoroacetic acid (TFA)-water cleavage solution, and the peptides are precipitated by addition to cold ether. Peptides (i.e. a desired AQUA peptide described in A-B above) are purified by reversed- phase C18 HPLC using standard TFA/acetonitrile gradients and characterized by matrix-assisted laser desorption ionization-time of flight (Biflex III, Bruker Daltonics, Billerica, MA) and ion-trap (ThermoFinnigan, LCQ DecaXP) MS.

MS/MS spectra for each AQUA peptide should exhibit a strong y-type ion peak as the most intense fragment ion that is suitable for use in an SRM monitoring/analysis. Reverse-phase microcapillary columns (0.1 A- 150-220 mm) are prepared according to standard methods. An Agilent 1100 liquid chromatograph may be used to develop and deliver a solvent gradient [0.4% acetic acid/0.005% heptafluorobutyric acid (HFBA)/7% methanol and 0.4% acetic acid/0.005% HFBA/65% methanol/35% acetonitrile] to the microcapillary column by means of a flow splitter. Samples are then directly loaded onto the microcapillary column by using a FAMOS inert capillary autosampler (LC Packings, San Francisco) after the flow split. Peptides are reconstituted in 6% acetic acid/0.01% TFA before injection.

Analysis & Quantification.

Target protein (e.g. a phosphorylated protein of A-B above) in a biological sample is quantified using a validated AQUA peptide (as described above). The IAP method is then applied to the complex mixture of peptides derived from proteolytic cleavage of crude cell extracts to which the AQUA peptides have been spiked in.

LC-SRM of the entire sample is then carried out. MS/MS may be performed by using a ThermoFinnigan (San Jose, CA) mass spectrometer (LCQ DecaXP ion trap or TSQ Quantum triple quadrupole). On the DecaXP, parent ions are isolated at 1.6 m/z width, the ion injection time being limited to 150 ms per microscan, with two microscans per peptide averaged, and with an AGC setting of 1 x 10⁸; on the Quantum, Q1 is kept at 0.4 and Q3 at 0.8 m/z with a scan time of 200 ms per peptide. On both instruments, analyte and internal standard are analyzed in alternation within a previously known reverse-phase retention window; well-resolved pairs of internal standard and analyte are analyzed in separate retention segments to improve duty cycle. Data are processed by integrating the appropriate peaks in an extracted ion chromatogram (60.15 m/z from the fragment monitored) for the native and internal standard, followed by calculation of the ratio of peak areas multiplied by the absolute amount of internal standard {e.g., 500 fmol).

Claims

WHAT IS CLAIMED IS:

1. A method for identifying a chronic myelogenous leukemia (CML) patient that has, or is likely to develop, resistance to lmanitib mesylate (STI-571), said method comprising the step of examining a biological sample from said patient for the presence of one or more of the following phosphorylated marker proteins:

(i) CbI (phosphorylated at tyrosine 700 (SEQ ID NO: 1); (ii) Btk kinase (phosphorylated at tyrosine 223 (SEQ ID NO: 2); (iii) Cbl-b (phosphorylated at tyrosine 889 (SEQ ID NO: 3); (iv) FYN binding protein (phosphorylated at tyrosine 571)

(SEQ ID NO: 4); (v) SKAPP55 homologue (phosphorylated at tyrosine 261)

(SEQ ID NO: 5);

(vi) Nek (phosphorylated at tyrosine 105 (SEQ ID NO: 6); (vii) Vinculin (phosphorylated at tyrosine 822 (SEQ ID NO: 7); (viii) Dok2 (phosphorylated at tyrosine 139 (SEQ ID NO: 8); (ix) PI3K p100 delta (phosphorylated at tyrosine 936)

(SEQ ID NO: 9); and/or (x) SHIP1 (phosphorylated at tyrosine 1022 (SEQ ID NO: 10); wherein, the presence of one or more of said phosphorylated marker proteins identifies said CML patient as having, or likely to develop, resistance to lmanitib mesylate (STI-571).

2. The method of claim 1 , wherein said biological sample comprises a blood sample or a bone marrow sample.

3. The method of claim 1 , wherein the presence of one or more of said phosphorylated marker proteins identifies said CML patient as having a BCR-ABL kinase mutant selected from the group consisting of a T315I mutant, a M351T mutant, a Y235F mutant, and a E255K mutant.

4. The method of claim 1 , wherein the presence of multiple of said phosphorylated marker proteins is examined.

5. The method of claim 1 , wherein the presence of one or more of said phosphorylated marker proteins is detected with a phospho-specific antibody.

6. The method of claim 1 , wherein the presence of one or more of said phosphorylated marker proteins is detected with a heavy isotope- labeled (AQUA) peptide corresponding to the phosphorylation site.

7. The method of claim 1 , wherein the presence of one or more of said phosphorylated marker proteins is determined in a whole cell assay.

8. The method of claim 1 , wherein said whole cell assay is selected from the group consisting of immunohistochemistry (IHC), flow cytometry (FC), or immunofluorescence (IF).

9. An isolated antibody that specifically binds one of the following lmanitib mesylate (STI-571)-resistance marker proteins only when phosphorylated at the indicated tyrosine residue, but does not bind said marker protein when not phosphorylated at said tyrosine residue:

(i) Cbl-b (phosphorylated at tyrosine 700 (SEQ ID NO: 3); (ii) FYN binding protein (phosphorylated at tyrosine 571)

(SEQ ID NO: 4); (iii) SKAPP55 homologue (phosphorylated at tyrosine 261)

(SEQ ID NO: 5);

(iv) Nek (phosphorylated at tyrosine 105 (SEQ ID NO: 6); (v) Vinculih (phosphorylated at tyrosine 822 (SEQ ID NO: 7); (vi) Dok2 (phosphorylated at tyrosine 139 (SEQ ID NO: 8); and (vii) PI3K p100 delta (phosphorylated at tyrosine 936)

(SEQ ID NO: 9).

10. The isolated antibody of claim 9, wherein said antibody binds an epitope of said phosphorylated marker protein comprising at least four amino acids comprising said phosphorylated tyrosine.

11. The isolated antibody of claim 9, wherein said antibody is a phosphorylation-site specific antibody.

12. The isolated antibody of claim 9, wherein said antibody is polyclonal.

13. The isolated antibody of claim 9, wherein said antibody is monoclonal.

14. An immortalized cell line producing the monoclonal antibody of claim 13.

15. A heavy isotope-labeled (AQUA) peptide for the quantification of one of the following lmanitib mesylate (STI-571)-resistance marker proteins only when phosphorylated at the indicated tyrosine residue:

(SEQ ID NO: 4); (iii) SKAPP55 homologue (phosphorylated at tyrosine 261)

(SEQ ID NO: 5);

(iv) Nek (phosphorylated at tyrosine 105 (SEQ ID NO: 6); (v) Vinculin (phosphorylated at tyrosine 822 (SEQ ID NO: 7); (vi) Dok2 (phosphorylated at tyrosine 139 (SEQ ID NO: 8); and (vii) PI3K p100 delta (phosphorylated at tyrosine 936)

(SEQ ID NO: 9).

16. The heavy isotope-labeled peptide of claim 15, wherein said peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3-9.

17. A kit for the identification of a chronic myelogenous leukemia (CML) patient that has, or is likely to develop, resistance to lmanitib mesylate (STI-571), said kit comprising at least one detectable antibody of claim 9 and/or at least one detectable heavy isotope-labeled peptide of claim 15.

18. A method for identifying a compound that inhibits a BCR-ABL kinase mutant that is resistant to lmanitib mesylate (STI-571), said method comprising the steps of:

(b) utilizing at least one isolated antibody of claim 9 and/or at least one heavy isotope labeled peptide of claim 15 to determine the effect of said compound on the level of one or more of the following phosphorylated marker proteins:

(SEQ ID NO: 4); (v) SKAPP55 homologue (phosphorylated at tyrosine 261)

(SEQ ID NO: 5);

(SEQ ID NO: 9); and/or (x) SHIP1 (phosphorylated at tyrosine 1022 (SEQ ID NO: 10); wherein a decrease in the level of one or more of said phosphorylated marker proteins following contact with said compound identifies said compound as inhibiting a BCR-ABL kinase mutant that is resistant to lmanitib mesylate (STI-571).

19. The method of claim 18, wherein said BCR-ABL kinase mutant is selected from the group consisting of a T315I mutant, a M351T mutant, a Y235F mutant, and a E255K mutant.