OF BCR-ABL ASSOCIATED LEUKEMIAS AND OTHER CELL PROLIFERATIVE DISORDERS

This application is a continuation-in-part of United States Application Serial No. 08/246,441, filed May 20, 1994, which is incorporated by reference herein in its entirety. U.S. Application Serial No.08/246,441 is a continuation-in-part of U.S. Application Serial No. 08/127,922, filed September 28, 1993.

1. INTRODUCTION The present invention relates to compositions and methods for the prevention and treatment of cell proliferative disorders wherein a protein tyrosine kinase or protein tyrosine phosphatase capable of complexing with a member of the SH2-and/or SH3 domain- containing family of adaptor proteins is involved. This invention is based, in part, on the surprising discovery that the adaptor protein, GRB-2, binds the intracellular BCR-ABL tyrosine kinase product in vivo and is necessary for the activation of the oncogenic potential of the BCR-ABL product and that disruption of the signaling capability of GRB-2 can reverse the transformed phenotype of cells and reduce tumor growth in animals. The present invention further relates to protein tyrosine kinase/adaptor protein complexes and the uses of these complexes for the identification of agents capable of disrupting the interaction between the members of such complexes.

2. BACKGROUND 2.1. Protein Phosphor lation and Signal Transduction

Cells rely, to a great extent, on extracellular molecules as a means by which to receive stimuli from

their immediate environment. These extracellular signals are essential for the correct regulation of such diverse cellular processes as differentiation, contractility, secretion, cell division, contact inhibition, and metabolism. The extracellular molecules, which can include, for example, hormones, growth factors, lymphokines, or neurotransmitters, act as ligands that bind specific cell surface receptors. The binding of these ligands to their receptors triggers a cascade of reactions that brings about both the amplification of the original stimulus and the coordinate regulation of the separate cellular processes mentioned above. In addition to normal cellular processes, receptors and their extracellular ligands may be involved in abnormal or potentially deleterious processes such as virus-receptor interaction, inflammation, and cellular transformation to a cancerous state.

A central feature of this process, referred to as signal transduction (for recent reviews, see Posada et al . , 1992, Mol. Biol . Cell 3;583-592; Hardie, D.G., 1990, Syπrp. Soc. Exp. Biol. 44:241-255) , is the reversible phosphorylation of certain proteins.

The phosphorylation or dephosphorylation of amino acid residues triggers conformational changes in regulated proteins that alter their biological properties. Proteins are phosphorylated by protein kinases and are dephosphorylated by protein phosphatases. Protein kinases and phosphatases are classified according to the amino acid residues they act on, with one class being serine-threonine kinases and phosphatases (reviewed in Scott et al . , 1992, 2 :289-295) , which act on serine and threonine residues, and the other class being the tyrosine kinases and phosphatases (reviewed in Fischer et al . ,

1991, Science 2_53_:401-406; Schlessinger et al _* , 1992, Neuron 1:383-391; Ullrich et al _* , 1990, Cell 61:203- 212) , which act on tyrosine residues. Phosphorylation is a dynamic process involving competing phosphorylation and dephosphorylation reactions, and the level of phosphorylation at any given instant reflects the relative activities, at that instant, of the protein kinases and phosphatases that catalyze these reactions. While the majority of protein phosphorylation occurs at serine and threonine amino acid residues, phosphorylation at tyrosine residues also occurs, and has begun to attract a great deal of interest since the discovery that many oncogene products and growth factor receptors possess intrinsic protein tyrosine kinase activity. The importance of protein tyrosine phosphorylation in growth factor signal transduction, cell cycle progression and neoplastic transformation is now well established (Cantley et al _* , 1991, Cell £4:281-302; Hunter T. , 1991, Cell 64:249-270; Nurse, 1990, Nature 3_4_4:503-508; Schlessinger et al _* , 1992, Neuron 1:383-391; Ullrich et al _* , 1990, Cell £1:203- 212) . Subversion of normal growth control pathways leading to oncogenesis has been shown to be caused by activation or overexpression of protein tyrosine kinases which constitute a large group of dominant oncogenic proteins (reviewed in Hunter, T., 1991, Cell £4:249-270) .

2.2. Protein Tyrosine Kinases

Protein tyrosine kinases comprise a large family of proteins, including many growth factor receptors and potential oncogenes, which share ancestry with, but nonetheless differ from, serine/threonine-specific protein kinases (Hanks et al _* , 1988, Science 241:42-

52) . The protein kinases may further be defined as being receptors or non-receptors.

Receptor-type protein tyrosine kinases, which have a transmembrane topology have been studied extensively. The binding of a specific ligand to the extracellular domain of a receptor protein tyrosine kinase is thought to induce receptor dimerization and phosphorylation of their own tyrosine residues. Individual phosphotyrosine residues of the cytoplasmic domains of receptors may serve as specific binding sites that interact with a host of cytoplasmic signalling molecules, thereby activating various signal transduction pathways (Ullrich et al _* , 1990, Cell £1:203-212) . The intracellular, cytoplasmic, non-receptor protein tyrosine kinases may be broadly defined as those protein tyrosine kinases which do not contain a hydrophobic, transmembrane domain. Within this broad classification, one can divide the known cytoplasmic protein tyrosine kinases into four distinct morphotypes: the SRC family (Martinez et al _* , 1987, Science 237:411-414; Sukegawa et al _* , 1987, Mol . Cell . Biol. 7:41-47; Yamanishi et al _* , 1987, 7 _. :237-243; Marth et al . , 1985, Cell 42:393-404; Dymecki et al . , 1990, Science 247:332-336) , the FES family (Ruebroek et al . , 1985, EMBO J. 4:2897-2903: Hao et al . , 1989, Mol. Cell. Biol. 9:1587-1593) , the ABL family (Shtivelman et al . , 1986, Cell 47:277-284; Kruh et al . , 1986, Science 234..:1545-1548) , and the JAK family. While distinct in their overall molecular structure, each of the members of these morphotypic families of cytoplasmic protein tyrosine kinases share non- catalytic domains in addition to sharing their catalytic kinase domains. Such non-catalytic domains are the SH2 (SRC homology domain 2; Sadowski et al . ,

Mol. Cell. Biol. £: 4396-4408; Koch et al _* , 1991, Science 252 : 668- 674) domains and SH3 domains (Mayer et al., 1988, Nature 332:269-272) . Non-catalytic domains are thought to be important in the regulation of protein-protein interactions during signal transduction (Pawson et al . , 1992, Cell 71:359-362) .

While the metabolic roles of cytoplasmic protein tyrosine kinases are less well understood than that of the receptor-type protein tyrosine kinases, significant progress has been made in elucidating some of the processes in which this class of molecules is involved. For example, lck and fyn, members of the src family, have been shown to interact with CD4/CD8 and the T cell receptor complex, and are thus implicated in T cell activation, (Veillette et al . , 1992, TIG 8.:61-66) . Certain cytoplasmic protein tyrosine kinases have been linked to certain phases of the cell cycle (Morgan et al . , 1989, Cell 57:775-786: Kipreos et al * , 1990, Science 248 : 217-220; Weaver et al . , 1991, Mol. Cell. Biol. 11:4415-4422) .

Cytoplasmic protein tyrosine kinases have been implicated in neuronal development (Maness, P., 1992, Dev. Neurosci . 14 _. :257-270) . Deregulation of kinase activity through mutation or overexpression is a well- established mechanism underlying cell transformation (Hunter et al . , 1985, supra ; Ullrich et al . , supra) .

2.3. G-Proteins and Signal Transduction Guanine-nucleotide-binding proteins, (G-proteins; Simon et al * , 1991, Science 252 :802-808 ; Kaziro et al . , 1991, Ann. Rev. Biochem. £0:349-400) such as Ras (for review, see Lowy et al . , 1993, Ann Rev. Biochem. £2:851-891) , play an essential role in the transmission of mitogenic signals from receptor tyrosine kinases. Taking Ras as an example, the

activation of receptor tyrosine kinases by ligand binding results in the accumulation of the active GTP bound form of the Ras molecule (Gibbs et al . , 1990, J. Biol. Chem. 265:20437-2044; Satoh et al _* , 1990, Proc. Natl. Acad. Sci ■ USA 87:5993-5997; Li et al _* , 1992, Science 2_5£:1456-1459; Buday et al _* , 1993, Mol. Cell. Biol. 12:1903-1910; Medema et al * , 1993, Mol. Cell. Biol. 12 155-162) . Ras activation is also required for transformation by viral oncogenic tyrosine kinases (Smith et al _* , 1986, Nature 320:540-43) .

Ras activity is regulated by the opposing actions of the GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors, with GAPs stimulating the slow intrinsic rate of GTP hydrolysis on Ras and exchange factors stimulating the basal rate of exchange of GDP for GTP on Ras. Thus, GAPs act as negative regulators of Ras function, while exchange factors act as Ras activators.

Recently, a direct link between activated receptor tyrosine kinases and Ras was established with the finding that the mammalian GRB-2 protein, a 26 kilodalton protein comprised of a single SH2 and two SH3 domains (Lowenstein et al . , 1992, Cell 70 :431- 442) , directly couples receptor tyrosine kinases to the Ras exchange factor Sos in mammals and Drosophila (Buday et al . , 1993, Cell 2 :611-620; Egan et al . , 1993, Nature 363:45-51; Li et al . , 1993, Nature 363:85-87; Gale et al . , 1993, Nature 363:88-92: Rozakis-Adcock et al . , 1993, Nature 363:83-85; Chardin et al . , 1993, Science 2£0:1338-1343 ; Oliver et al . ,

Cell 73:179-191; Simon et al _* , 1993, Cell 73:169-177) . The GRB-2 SH2 domain binds to specific tyrosine phosphorylated sequences in receptor tyrosine kinases while the GRB-2 SH3 domains bind to proline-rich sequences present in the Sos exchange factor. Binding

of GRB-2 to the receptor kinases, therefore, allows for the recruitment of Sos to the plasma membrane, where Ras is located (Schlessinger, J. , 1993, TIBS 18:273-275) .

2.4. BCR-ABL in the Development of Leukemias Activation of the oncogenic potential of normal cellular proteins such as protein tyrosine kinases may occur by alteration of the proteins' corresponding enzymatic activities, their inappropriate binding to other cellular components, such as those mentioned above in Section 2.3, or both.

For example, the BCR-ABL protein tyrosine kinase oncoprotein may transform cells via changes in enzyme activity and/or altering of noncovalent protein- protein interactions. The gene encoding the BCR-ABL oncoprotein is a chimeric oncogene generated by the translocation of sequences from the cABL protein tyrosine kinase on chromosome 9 into BCR sequences on chromosome 22 (reviewed in Kurzock et al . , 1988, N. Engl. J. Med. 319:990-998, and Rosenberg et al . , 1988, Adv. in Virus Res. 35:39-81). The BCR- ABL oncogene has been implicated in the pathenogenesis of Philadelphia chromosome (Ph ¹ ) positive human leukemias. Namely, the Ph ¹ chromosome is found in at least 90 to 95 percent of cases of chronic myelogenous leukemia (CML) , which is a clonal cancer arising from the neoplastic transformation of hematopoietic stem cells (Fialkow et al . , 1977, Am. J. Med. 63:125-130), and is also observed in approximately 20 percent of adults with acute lymphocytic leukemia (ALL) , 5 percent of children with ALL, and 2 percent of adults with acute myelogenous leukemia (AMD (Whang-Peng et al., 1970, Blood 36:448-457; Look, A.T., 1985, Semin. Oncol. 12.:92-104) . The BCR-ABL gene produces two

alternative chimeric proteins, P210 BCR-ABL,. and P185 BCR-ABL, which are characteristic of CML and ALL, respectively. Further, it has recently been directly demonstrated that the BCR-ABL gene product is the causative agent in CML (Skorski et al . , 1993, J. Clin Invest. 92:194-202; Snyder et al . , 1993, Blood £2:600- 605) .

Clinically, CML is characterized by a biphasic course. The disease begins with a chronic phase marked by a greatly increased pool of uncommitted myeloid progenitor cells. Because terminal differentiation is maintained, this results in greatly increased pools of circulating mature granulocytes. After a period of several weeks to many years, a state of accelerated myeloproliferation develops wherein the myeloid cells progressively lose their capacity for terminal differentiation. During this time, thrombocytosis, basophilia, and clonal cytogenetic abnormalities often appear. These abnormalities signal the terminal, blast-crisis stage, during which immature blast cells rapidly proliferate and the patient inevitably dies.

It has previously been shown that the BCR-ABL proteins exhibit heightened tyrosine kinase and transforming capabilities compared to the normal c-ABL protein (Konopka et al . , 1984, Cell 37:1035-1042) . BCR first exon sequences specifically activate the tyrosine kinase and transforming potential of BCR-ABL (Muller et al . , 1991, Mol. Cell. Biol. 11:1785-1792; McWhirter et al . , 1991, Mol. Cell. Biol. 11:1553-

1565) . The BCR first exon is capable of binding to the ABL SH2 domain in a phosphotyrosine-independent manner (Pendergast et al . , 1991, Cell £6:161-171) , and deletion of BCR sequences essential for ABL SH2- binding render BCR-ABL nontransforming (Pendergast et

al . , 1991, Cell £6:161-171). In addition, it has been demonstrated that BCR binds, in vi tro, to some other SH2 domains encoded by other proteins (Muller et al . , 1992, Mol. Cell. Biol. 12:5087-5093) . While one may infer from these results that some aspect of SH2 domain-binding to BCR is involved in the oncogenicity of the BCR-ABL oncoprotein, the mechanism by which such BCR-ABL oncogenesis occurs is still obscure. For example, given the myriad of SH2 domain-containing proteins which are known to exist, the identification of a BCR-ABL effector(s) will necessitate much further study.

3. SUMMARY OF THE INVENTION The present invention relates to compositions and methods for the prevention and treatment of cell proliferative disorders wherein a protein tyrosine kinase or a protein tyrosine phosphatase capable of complexing with a member of the SH2-and/or SH3 domain- containing family of adaptor proteins is involved. The present invention further relates to protein tyrosine kinase/adaptor protein complexes, protein tyrosine phosphatase/adaptor protein complexes, and the uses of these complexes for the identification of agents capable of disrupting the interaction between the components of such complexes.

"Protein tyrosine kinase" will, herein, be abbreviated "PTK", and "protein tyrosine phosphatase" will, herein be abbreviated "PTP". It is to be understood that "PTK" may refer to either a transmembrane, receptor- ype protein tyrosine kinase or a cytoplasmic protein tyrosine kinase, unless otherwise indicated, and, likewise, "PTP" may refer to either a transmembrane, receptor-type protein tyrosine

phosphatase or a cytoplasmic protein tyrosine phosphatase, unless otherwise indicated.

This invention is based, in part, on the surprising discovery that the adaptor protein, GRB-2, binds the intracellular BCR-ABL PTK product in vivo and is necessary for the activation of the oncogenic potential of the BCR-ABL product. The inventors are the first to demonstrate the physiological relevance of the interactions between the members of a signal transduction pathway, in part by showing that disruption of this signal transduction can result in the reversal of the transformed phenotype of cells and inhibit tumor growth in animals. The data representing this discovery is presented in the Working Examples in Sections 6 through 14, below. The invention, therefore, represents the first instance whereby the SH2- and/or SH3 domain-containing adaptor family of proteins, especially the GRB-2 member of the GRB subfamily of proteins, are implicated in the development and maintenance of cell proliferation/activation-- herein demonstrated for the abnormal cellular proliferation involved in oncogenesis, the transformation process, and the development of human cancer. Still further, with respect to BCR-ABL transformation, the present invention discloses the first effector (i.e., GRB-2) for the BCR-ABL product.

3.1. ABBREVIATIONS The following table lists the single-letter and triple-letter abbreviations for amino acids that are in common use among protein chemists and that are used herein.

Amino Acid One Letter Code Three Letter Code

Alanine A Ala

Arginine R Arg

Asparagine N Asn

Aspartic Acid D Asp

Cysteine C Cys

Glutamic Acid E Glu

Glutamine Q Gin

Glycine G Gly

Histidine H His

Isoleucine I He

Leucine L Leu

Lysine K Lys

Methionine M Met

Phenylalanine F Phe

Proline P Pro

Serine S Ser

Threonine T Thr

Tryptophan W Trp

Tyrosine Y Tyr

Valine V Val

Not Specified X

4. DESCRIPTION OF THE FIGURES Figure 1. Stimulation of transcriptional activation by BCR-ABL through Ras requires the presence of tyrosine 177 in the BCR first exon. NIH 3T3 cells were transfected with 0.5 μg of the indicated BCR-ABL cDNA +/- 5 μg H-Ras (17N) cDNA,

along with the 1 μg pB4X-CAT reporter plasmid. At 48 hr. post-transfection, cells were harvested and assayed for CAT activity as outlined in Section 9.1.1, below. Data were recorded in arbitrary units; the basal signal due to activity of the reporter.

Figure 2. Stimulation of transcriptional activation by BCR-ABL through Ras requires the presence of a GRB2 SH3 domain. Ratl cells expressing p210 BCR/ABL cells were transfected with either of the GRB2 deletion mutants, along with the 1 μg pB4X-CAT reporter plasmid. At 48 hr. post-transfection, cells were harvested and assayed for CAT activity as outlined in Section 9.1.1, below. LANE 1: RAT 1; LANE 2: RAT l/p210/pCEN Vector; LANE 3: RAT l/p210/pCEN

GRB2 ΔN' ; LANE 4; RAT l/p210/pCEN GRB2 ΔC . Data were recorded in arbitrary units with RAT 1 normalized to 1. The basal signal is due to activity of the reporter. Values are means of the duplicates +/- range.

Figure 3. Hemoglobin content in K562 cells expressing wild type and mutant GRB2 proteins. Cell lysates were prepared and hemoglobin content determined using a benzidine stain as described (Feinstein et al. , 1992, Oncogene 7 _. : 1853-1857). Triplicate samples were analyzed for each cell population. Data represents the amount of hemoglobin per one microgram of total cellular protein transfected with the indicated constructs.

5. DETAILED DESCRIPTION OF THE INVENTION Described below are compositions and methods for the prevention and treatment of cell proliferative disorders wherein a protein tyrosine kinase or a

protein tyrosine phosphatase capable of complexing with an SH2- and/or SH3 domain-containing member of the adaptor family of proteins is involved. Further, protein tyrosine kinase/adaptor protein complexes, 5 protein tyrosine phosphatase/adaptor protein complexes, methods for the production of such complexes, and uses of these complexes are described herein. Such uses may include, but are not limited to, the identification of agents capable disrupting 0 the interaction between the components of such complexes.

This invention is based, in part, on the surprising discovery that the adaptor protein, GRB-2, binds the intracellular BCR-ABL product in vivo, is 5 necessary for the activation of the oncogenic potential of the BCR-ABL product and that disruption of the GRB-2 signal transduction can reverse the transformed phenotype of cells and reduce tumor growth in animals. The data representing this discovery is 0 presented in the Working Examples of Sections 6 through 14, below.

5.1. PROTEIN TYROSINE KINASE/ ADAPTOR PROTEIN COMPLEXES

₂₅ The PTK/adaptor protein complexes of the invention comprise at least one member of the PTK family of proteins and at least one member of the adaptor family of proteins, as described below. Under standard physiological conditions, the components of

₃₀ such complexes are capable of forming stable, non- covalent attachments with one or more of the other PTK/adaptor protein complex components.

The PTK components of the PTK/adaptor protein complexes of the invention are either intracellular,

_,. cytoplasmic non-receptor PTKs, intracellular, nuclear non-receptor PTKs, or transmembrane, receptor-type

PTKs, each of which comprises one or more characteristic peptide domains . Such domains may include one or more catalytic domains which may include, but are not limited to, a tyrosine kinase domain. A tyrosine kinase catalytic domain generally ranges in length from about 250 to about 300 amino acids, corresponding to a molecular weight of approximately 30kDa. The location of the tyrosine kinase catalytic domain, while not fixed, is generally near the carboxyl terminus of the protein. Short, conserved, stretches of amino acid residues may be present within the tyrosine kinase domain, which alternate in sequence with variable-length stretches of amino acid residues which do not exhibit a high level of conservation. The consensus sequences, corresponding to the most highly conserved of the tyrosine kinase catalytic domain amino acid residues have been compiled and are well known to those of ordinary skill in the art. See, for example, Hanks et al . (Hanks et al . , 1991, Science 241:42-52) , and Wilks (Wilks, A.F., 1990, Prog. Growth Factor Res. 2:97-111) which are incorporated herein, by reference, in their entirety. Among such consensus sequences are the PTK-specific sequences D-L-R-A-A-N or D-L-A-A-R-N, and P-I/V-K/R-W-T/M-A-P-E. Moreover, see Wilks, A.F., 1990, Progress in Growth Factor Res. 2:97-111, for additional examples of such sequence motifs.

The PTK component of the PTK/adaptor protein complexes of the invention may further include one or more non-catalytic domains, which may include, but are not limited to, one or more SH2 and/or one or more SH3 domains, one or more SH2-binding, and/or one or more SH3-binding peptide domains, and/or (in the case of receptor PTKs) a hydrophobic transmembrane domain. An SH2 (i.e., src homology 2) non-catalytic domain is

generally approximately 100 amino acid residues in length. Such SH2 domains may contain a number of highly conserved or invariant amino acid residues within several, preferably five, well-conserved amino acid sequence motifs, which are well known to those of ordinary skill in the art. See, for example Koch et al . (Koch et al . , 1991, Science 252:668-674) , which is incorporated herein, by reference, in its entirety. For example, the amino acid consensus sequences may include, but are not limited to, F-L-I-R-E-S and F-L- V-R-E-S. The R residue of these consensus sequences is invariant among SH2 domains. Such well-conserved amino acid sequences motifs are separated by stretches of more variable amino acid sequence elements, which, while variable, generally contain one or more G or P residues.

An SH3 (i.e., src homology 3) non-catalytic domain is approximately 50 amino acids residues in length. While the amino acid sequence within an SH3 domain may be variable, the 3-dimensional, tertiary, structure of the domain is well conserved. Such an SH3 tertiary structure is well known to those of ordinary skill in the art. See, for example, Koyama et al . (Koyama et al . , 1993, Cell 72:945-952) which is incorporated herein, by reference, in its entirety.

SH2-binding peptide domains are well known in the art. See, for example, Songyang et al . (Songyang et al . , 1993, Cell Tl :767-778) , Rotin et al . (Rotin et al . , EMBO J. 11:559-567), and Skolnick et al . (Skolnick et al . , 1993, EMBO J. 12:1929-1936) . which are incorporated herein, by reference, in its entirety. SH2 domains may exhibit a specificity for certain SH2-binding domains. For example, SH2-binding peptide domains may include, but are not limited to a phosphotyr-hydrophilic-hydrophilic-Ile/Pro amino acid

sequence motif (generally, such a sequence motif is preferred for SH2 domains of the type found in, for example, the src, fyn, lck, fgr, abl, crk, and nek proteins) , and phosphoTyr-hydrophobic-X-hydrophobic, and/or phosphotyr-Met-X-Met (generally, such sequence motifs are preferred for SH2 domains of the type found in, for example, p85, phospholipase C-γ, and SHPTP2 proteins) . Further, a consensus sequence developed from the analysis of the domains of several proteins that bind the SH2 domains of the GRB-2 protein has been determined to be X-P-X-Y-V/I-N-V/I . In addition, SH2-binding peptide domains may comprise regions rich in Ser and Thr residues some or all of which are phosphorylated (Pendergast et al . , 1991, Cell 66:161- 171) .

SH3-binding peptide domains are also well known to those of ordinary skill in the art. See, for example, Ren et al . (Ren et al . , 1993, Science 259:1157-1161) and Cicchetti et al . (Cicchetti et al . , 1992, Science 257:803-806) , which are incorporated herein, by reference, in their entirety. Such SH3- binding peptide domains are generally rich in Pro amino acid residues, although amino acid residues in addition to solely Pro are also critical for SH3 binding. One possible consensus sequence for a SH3- binding domain is: X-P-X-X-P-P-P-hydrophobic residue- X-P. Further, the SH3 domains of GRB-2 have been determined to be P-P-P-V-P-P-R-R, an amino acid sequence motif found in the SOS protein (Li et al . , 1993, Nature 363 :8588; Schlessinger, 1993, TIBS, 18:273-275.

Intracellular, cytoplasmic PTK components of the PTK/adaptor protein complex of the invention may include, for example, members of the Src family, such molecules as src, yes, fgr, fyn, lyn, hck, lck, and

blk; members of the Fes family, such as fes and fer; members of the Abl family, such as abl and arg; and members of the Jak family, such as jakl and jak2. In a preferred embodiment of the invention, the PTK component of the PTK/adaptor protein complex is the intracellular PTK product of the BCR-ABL gene. Transmembrane, receptor PTK components of the PTK/adaptor protein complex of the invention may include, for example, such molecules as members of the FGF receptor, Sevenless/ROS, Insulin receptor, PDGF receptor, EGF receptor family of growth factor receptors or any other molecule that associates with he adaptor protein (see for example Lowenstein et al . , 1992, Cell 7 :431-442) . The adaptor protein components of the PTK/adaptor protein complexes of the invention comprise one or more SH2 and/or one or more SH3 non-catalytic domains. The SH2 and SH3 domains which may be a part of the adaptor proteins are as described, above, for the PTK components. Adaptor proteins which may be components of the PTK/adaptor protein complexes of the invention, may include, for example, p85, c-Crk, SHC, Nek, ISGF3α, guanine triphosphatase activator protein (GAP) , and members of the GRB subfamily of proteins, such as GRB1, GRB-2, GRB-3, GRB-4, GRB-7, and GRB-10. In a preferred embodiment, the PTK/adaptor protein complex of the invention comprises the PTK product of the BCR-ABL gene and GRB-2 protein.

The complexes of the invention, and/or the individual components of the complexes of the invention may be substantially purified utilizing methods which are described below, in Section 5.3.1. Further, the PTK and/or adaptor components of the complexes of the invention may be produced by utilizing a variety of methods which include, but are

not limited to, chemical synthesis or recombinant DNA techniques, as described in Section 5.3.2, below.

5.2. Protein Tyrosine Phosphatase/

Adaptor Protein Complexes

The PTP/adaptor protein complexes of the invention comprise at least one member of the PTP family of proteins and at least one member of the adaptor family of proteins. Under standard physiological conditions, the components of such complexes are capable of forming stable, non-covalent attachments with one or more of the other PTP/adaptor protein complex components.

The PTP components of the PTP/adaptor protein complexes of the invention are either cytoplasmic, intracellular, non-receptor PTPs or transmembrane, receptor-type PTPs, each of which comprises one or more characteristic peptide domains. Such domains may include one or more catalytic domains which may include, but are not limited to, a tyrosine phosphatase domain. Generally, a tyrosine phosphatase catalytic domain is approximately about 230 amino acids in length. Approximately 40 of the amino acid residues of the catalytic phosphatase domain are highly conserved, and of these, a very highly conserved segment of 11 amino acid residues with the consensus sequence I/V-H-C-X-A-G-X-X-R-S/T-G is generally present. Non-receptor PTPs generally contain a single such catalytic domain, while the transmembrane receptor PTPs generally contain two such catalytic domains separated by a peptide segment approximately 58 amino acid residues in length.

The PTP component of the PTP/adaptor protein complexes of the invention may further include one or more non-catalytic domains, which may include, but are not limited to one or more SH2 domains, one or more SH3 domains, one or more SH2-binding domains, and/or one or more SH3-binding domains. Each of these non- catalytic domains may be as described, above, in Section 5.2.

Transmembrane, receptor PTP components of the PTP/adaptor protein complexes of the invention may include, for example, CD45, LAR, RPTPα, RPTPS, RPTPχ, and RPTP/ . Intracellular, cytoplasmic PTP components of the PTP/adaptor protein complexes of the invention may include, for example, PTPIC, PTPID, and corkscrew.

5.3. PURIFICATION AND PRODUCTION OF

PTK/ADAPTOR AND PTP/ADAPTOR COMPLEXES

5.3.1. PURIFICATION METHODS

The PTK/adaptor and PTP/adaptor complexes of the invention may be substantially purified, i.e., may be purified away from at least 90% (on a weight basis) , and from at least 99%, if desired, of other proteins, glycoproteins, and other macromolecules with which it is associated. Such purification can be achieved by utilizing a variety of procedures well known to those of skill in the art, such as subjecting cells, tissue or fluid containing the PTK/adaptor or PTP/adaptor complex to a combination of standard methods, for example, ammonium sulfate precipitation, molecular sieve chromatography, and/or ion exchange chromatography. Alternatively, or additionally, a PTK/adaptor or PTP/adaptor complex may be purified by immunoaffinity chromatography using an immunoadsorbent column to which an antibody is immobilized which is

capable of binding to one or more components of the PTK/adaptor or PTP/adaptor complex. Such an antibody may be of monoclonal or polyclonal in origin. Other useful types of affinity purification for a PTK/adaptor or PTP/adaptor complex may utilize, for example, a solid-phase substrate which binds the catalytic domain (i.e., kinase domain of PTK or phosphatase domain of PTP) , or an immobilized binding site for noncatalytic domains of the PTK, PTP, and/or adaptor components of the complex, which bind in such a manner as to not disrupt the complex.

The PTK/adaptor or PTP/adaptor complexes of the present invention may be biochemically purified from a variety of cell or tissue sources. For purification of a naturally occurring PTK/adaptor complex, cellular sources may include, for example, baculovirus-infected SF9 cells, A-431, CHO, and/or 3T3 cells. In a preferred embodiment of the present invention, the PTK/adaptor complex comprises a BCR-ABL PTK and a GRB2 protein. Sources for the purification of such a

PTK/GRB-2 complex may include, but are not limited to K562, NMG-01, and ALL-1 cell lines.

5.3.2. SYNTHESIS METHODS Methods for the synthesis of polypeptides or fragments thereof, which are capable of acting as components of the PTK/adaptor or PTP/adaptor complexes of the present invention, are well-known to those of ordinary skill in the art. See, for example, Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman and Co., NY, which is incorporated herein, by reference, in its entirety. Components of a PTK/adaptor or PTP/adaptor complexes which have been separately synthesized or recombinantly produced, may be reconstituted to form a

complex by standard biochemical techniques well known to those skilled in the art. For example, samples containing the components of the PTK/adaptor complex may be combined in a solution buffered with greater than about 150mM NaCl, at a physiological pH in the range of 7, at room temperature. For example, a buffer comprising 20mM Tris-HCl, pH 7.4, 137mM NaCl, 10% glycerol, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate and 2mM EDTA could be used. Such a procedure may also be utilized for the reconstitution of a PTP/adaptor complex.

Methods for preparing the components of PTK/adaptor complexes of the invention by expressing nucleic acid encoding PTK, PTP, and/or adaptor proteins are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing PTK, PTP, and/or adaptor protein coding sequences and appropriate transcriptional/translational control signals. These methods include, for example, in vi tro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. DNA and RNA synthesis may, additionally, be performed using an automated synthesizers. See, for example, the techniques described in Maniatis et al . , 1989,

Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. and Ausubel et al . , 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y. A variety of host-expression vector systems may be utilized to express the coding sequences of the components of the PTK/adaptor PTP/adaptor complexes of the invention. These include but are not limited to microorganisms such as bacteria (e.g. E. coli , B. subtilis) transformed with recombinant bacteriophage

DNA, plasmid DNA or cosmid DNA expression vectors containing PTK, PTP, or adaptor protein coding sequences; yeast (e.g. Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the PTK, PTP, and/or adaptor protein coding sequences; insect cell systems infected with recombinant virus expression vectors { e . g. baculovirus) containing the PTK, PTP, and/or adaptor protein coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g. cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g. Ti plasmid) containing the PTK, PTP, and/or adaptor protein coding sequences coding sequence; or mammalian cell systems { e . g. COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g. metallothionein promoter) or from mammalian viruses (e.g. the adenovirus late promoter; the vaccinia virus 7.5K promoter) .

In bacterial systems a number of expression vectors may be advantageously selected depending upon the use intended for the PTK/adaptor or PTP/adaptor complex being expressed. For example, when large quantities of PTK/adaptor or PTP/adaptor complex proteins are to be produced for the generation of antibodies or to screen peptide libraries, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include but are not limited to the E. coli expression vector pUR278 (Ruther et al . , 1983, EMBO J. 2:1791) , in which the PTK, PTP, and/or adaptor protein coding sequence may be ligated individually into the vector in frame with the lac Z

coding region so that a fusion protein is produced; pIN vectors (Inouye et al . , 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke et al . , 1989, J. Biol. Chem. 264:5503-5509) ; and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST) . In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned PTK and/or adaptor protein can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The PTK/adaptor or PTP/adaptor complex component coding sequences may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter) . Successful insertion of the coding sequences will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene) . These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. { e . g. see Smith et al . , 1983, J. Viol. 4£:584; Smith, U.S. Patent No. 4,215,051).

In mammalian host cells, a number of viral based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the PTK/adaptor or PTP/adaptor complex component coding sequences may be ligated to an adenovirus

transcription/translation control complex, e . g. the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vi tro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g. region El or E3) will result in a recombinant virus that is viable and capable of expressing PTK, PTP, and/or adaptor proteins in infected hosts, { e . g. see Logan et al . , 1984, Proc. Natl. Acad. Sci. USA 81:3655-3659) . Specific initiation signals may also be required for efficient translation of inserted PTK, PTP, and/or adaptor coding sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire PTK, PTP, or adaptor protein gene, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of the PTK, PTP, or adaptor coding sequence is inserted, exogenous translational control signals, including the ^' ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al . , 1987, Methods in Enzvmol . 152:516-544) .

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product

in the specific fashion desired. Such modifications { e . g. glycosylation) and processing (e.g. cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cells lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc. For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably coexpress both the PTK and adaptor protein or PTP and adaptor protein may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with the PTK and adaptor protein DNA or PTP and adaptor protein DNA independently or coordinately controlled by appropriate expression control elements (e.g. promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.) , and a selectable marker. Following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to

engineer cell lines which coexpress both the PTK and adaptor protein or PTP and adaptor protein. Such engineered cell lines are particularly useful in screening and evaluation of compounds that affect signals mediated by the complexes.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al . , 1971 , Cell 11:223) , hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al . , 1962, Proc. Natl. Acad. Sci. USA

4_8:2026) , and adenine phosphoribosyltransferase (Lowy, et al . , 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt- cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler et al . , 1980, Natl. Acad. Sci. USA 77:3567; O'Hare et al . , 1981, Proc. Natl. Acad. Sci . USA 7£:1527); gpt, which confers resistance to mycophenolic acid (Mulligan et al . , 1981, Proc. Natl. Acad. Sci. USA 78:2072) ; neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al . , 1981, J. Mol. Biol. 150 :1) ; and hygro, which confers resistance to hygromycin (Santerre et al . , 1984, Gene 20:147) genes. New members of the PTK, PTP, and/or adaptor protein families capable of forming the complexes of the invention may be identified and isolated by molecular biological techniques well known in the art. For example, a previously unknown PTK, PTP, or adaptor protein gene may be isolated by performing a polymerase chain reaction (PCR) using two degenerate oligonucleotide primer pools designed on the basis of highly conserved sequences within domains common to members of the PTK, PTP, or adaptor protein family. The template for the reaction may be cDNA obtained by

reverse transcription of mRNA prepared from cell lines or tissue known to express PTK/adaptor and/or PTP/adaptor complexes. The PCR product may be subcloned and sequenced to insure that the amplified 5 sequences represent the sequences of a member of the PTK, PTP, or adaptor subfamily. The PCR fragment may then be used to isolate a full length PTK, PTP, or adaptor protein cDNA clone by radioactively labeling the amplified fragment and screening a bacteriophage

10 cDNA library. Alternatively, the labeled fragment may be used to screen a genomic library. For a review of cloning strategies which may be used, see e . g. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y. ; and Ausubel

15 et al . , 1989, Current Protocols in Molecular Biology, (Green Publishing Associates and Wiley Interscience, N.Y.) .

A general method for cloning previously unknown adaptor proteins has been described by Skolnick

20 (Skolnick, E.Y., 1991, Cell 65:75) and Skolnick et al . , (U.S. Patent Application Serial No. 07/643,237) which are incorporated herein, by reference, in their entirety. Briefly, new members of the adaptor family of proteins may be identified by their ability to

25 specifically bind to at least a portion of a tyrosine- phosphorylated peptide comprising an adaptor-protein- binding region. Such a region may include, but is not limited to an SH2-binding domain.

30 5.4 DERIVATIVES OF PTK/ADAPTOR

AND PTP/ADAPTOR COMPLEXES

Also provided herein are functional derivatives of a PTK/adaptor and PTP/adaptor complexes. By

"functional derivative" is meant a "chemical

-.. derivative," "fragment," "variant," "chimera," or

"hybrid" of the PTK/adaptor and PTP/adaptor complex,

which terms are defined below. A functional derivative retains at least a portion of the function of the PTK, PTP, or adaptor protein, for example reactivity with an antibody specific for the PTK/adaptor or PTP/adaptor complex, PTK or PTP enzymatic activity or binding activity mediated through noncatalytic domains, which permits its utility in accordance with the present invention. A "chemical derivative" of the PTK/adaptor or PTP/adaptor complex contains additional chemical moieties not normally a part of the protein. Covalent modifications of the protein complex or peptides are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues, as described below.

Cysteinyl residues most commonly are reacted with alpha-haloacetates (and corresponding amines) , such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, α.-bromo-β(5- imidozoyDpropionic acid, chloroacetyl phosphate, N- alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2- chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2- oxa-1,3-diazole. Histidyl residues are derivatized by reaction with diethylprocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect or reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing α-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O- methylisourea; 2,4 pentanedione; and transaminase- catalyzed reaction with glyoxylate.

Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2, 3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK _a of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine e-amino group. Tyrosyl residues are well-known targets of modification for introduction of spectral labels by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizol and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction carbodiimide (R' -N-C- N-R' ) such as l-cyclohexyl-3- (2-morpholinyl (4-ethyl) carbodiimide or l-ethyl-3- (4-azonia-4,4- dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residue are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues

are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.

Derivatization with bifunctional agents is useful, for example, for cross-linking the component peptides of the PTK/adaptor or PTP/adaptor complexes to each other or the PTK/adaptor receptor complex to a water-insoluble support matrix or to other macromolecular carriers. Commonly used cross-linking agents include, for example, 1, 1-bis (diazoacetyl) -2- phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'- dithiobis (succinimidylpropionate) , and bifunctional maleimides such as bis-N-maleimido-1, 8-octane. Derivatizing agents such as methyl-3- [p-azidophenyl) dithiolpropioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide- activated carbohydrates and the reactive substrates described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.

Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains (Creighton, T.E., Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)) , acetylation of the N- terminal amine, and, in some instances, amidation of the C-terminal carboxyl groups.

Such derivatized moieties may improve the stability, solubility, absorption, biological half life, and the like. The moieties may alternatively eliminate or attenuate any undesirable side effect of the protein complex and the like. Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 1990, 18th ed. , Mack Publishing Co., Easton, PA.

The term "fragment" is used to indicate a polypeptide derived from the amino acid sequence of the PTK, PTP, or adaptor proteins, of the PTK/adaptor or PTP/adaptor complexes having a length less than the full-length polypeptide from which it has been derived. Such a fragment may, for example, be produced by proteolytic cleavage of the full-length protein. Preferably, the fragment is obtained recombinantly by appropriately modifying the DNA sequence encoding the PTK, PTP, or adaptor proteins to delete one or more amino acids at one or more sites of the C-terminus, N-terminus, and/or within the native sequence. Fragments of a PTK, PTP, or adaptor protein, when present in a complex resembling the naturally occurring PTK/adaptor or PTP/adaptor complex, are useful for screening for compounds that act to modulate signal transduction, as described below. It is understood that such fragments, when present in a complex may retain one or more characterizing portions of the native PTK/adaptor or PTP/adaptor complex. Examples of such retained characteristics include: catalytic activity; substrate specificity; interaction with other molecules in the intact cell; regulatory functions; or binding with an antibody specific for the native PTK/adaptor or PTP/adaptor complex, or an epitope thereof.

Another functional derivative intended to be within the scope of the present invention is a PTK/adaptor or PTP/adaptor complex comprising at least one "variant" polypeptide (e.g. PTK, PTP, or adaptor) which either lack one or more amino acids or contain additional or substituted amino acids relative to the native polypeptide. The variant may be derived from a naturally occurring PTK/adaptor or PTP/adaptor complex component by appropriately modifying the PTK, PTP, and/or adaptor protein DNA coding sequence to add, remove, and/or to modify codons for one or more amino acids at one or more sites of the C-terminus, N- terminus, and/or within the native sequence. It is understood that such variants having added, substituted and/or additional amino acids retain one or more characterizing portions of the native PTK/adaptor or PTP/adaptor complex, as described above.

A functional derivative of PTK/adaptor or PTP/adaptor complexes comprising PTK, PTP, and/or adaptor proteins with deleted, inserted and/or substituted amino acid residues may be prepared using standard techniques well-known to those of ordinary skill in the art. For example, the modified components of the functional derivatives may be produced using site-directed mutagenesis techniques (as exemplified by Adelman et al . , 1983, DNA 2:183) wherein nucleotides in the DNA coding the sequence are modified such that a modified coding sequence is modified, and thereafter expressing this recombinant DNA in a prokaryotic or eukaryotic host cell, using techniques such as those described above. Alternatively, components of functional derivatives of PTK/adaptor or PTP/adaptor complexes with amino acid deletions, insertions and/or substitutions may be

conveniently prepared by direct chemical synthesis, using methods well-known in the art. The functional derivatives of the PTK/adaptor or PTP/adaptor complexes typically exhibit the same qualitative 5 biological activity as the native complexes.

5.5. .ANTIBODIES TO PTK/ADAPTOR OR PTP/ADAPTOR COMPLEXES

10 The present invention further relates to antibodies which are capable of specifically recognizing a PTK/adaptor complex or PTP/adaptor complex or an epitope thereof, or of specifically recognizing an epitope on either the PTK, PTP, or

- .. adaptor components of the complex which would not be recognized by the antibody when the PTK, PTP, and/or adaptor component is present separate and apart from the PTK/adaptor or PTP/adaptor complex. Such antibodies may include, but are not limited to

₂₀ polyclonal antibodies, monoclonal antibodies ( Abs) , humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') ₂ fragments, fragments produced by a FAb expression library, anti-idotypic (anti-Id) antibodies, and epitope-binding fragments of

25 any of the above. Such antibodies may be used, for example, in the detection of a PTK/adaptor complex in a biological sample, or, alternatively, as a method for the inhibition of PTK/adaptor complex formation, thus, inhibiting the development of a cell

30 proliferative disorder.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, such as PTK/adaptor complex, or an antigenic functional derivative thereof. For the production of polyclonal

35 antibodies, various host animals may be immunized by

injection with the PTK/adaptor or PTP/adaptor complex including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete) , mineral gels such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. A monoclonal antibody, which is a substantially homogeneous population of antibodies to a particular antigen, may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to the hybridoma technique of Kohler et al . (Kohler et al . , Nature 256:495-497 (1975) and U.S. Patent No. 4,376,110) , the human B-cell hybridoma technique (Kosbor et al . , 1983, Immunology Today 4 _. :72; Cole et al . , 1983, Proc. Natl. Acad. Sci. USA 80:2026- 2030), and the EBV-hybridoma technique (Cole et al . , 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) . Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vi tro or in vivo . Production of high titers of mAbs in vivo production makes this the presently preferred method of production. In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al . , 1984, Proc. Natl. Acad. Sci.. £1:6851-6855; Neuberger et al . , 1984, Nature 312:604-608; Takeda et al . , 1985, Nature 314 :452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity

together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.

Alternatively, techniques described for the production of single chain antibodies (U.S. Patent 4,946,778; Bird, 1988, Science 242:423-426; Huston et al . , 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883: and Ward et al . , 1989, Nature 334:544-546) can be adapted to produce PTK/adaptor complex-specific or PTP/adaptor complex-specific single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragment of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.

Antibody fragments which contain specific binding sites of a PTK/adaptor or PTP/adaptor complex may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab') ₂ fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') ₂ fragments. Alternatively, Fab expression libraries may be constructed (Huse et al . , 1989, Science 246 :1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity to the PTK/adaptor or PTP/Adaptor complex.

5.6. TREATMENT OF PTK/ADAPTOR PROTEIN- AND PTP/ADAPTOR PROTEIN-RELATED CELL PROLIFERATIVE DISORDERS

The present invention demonstrates, for the first time, that the binding of a member of the SH2- and/or

SH3 domain-containing family of adaptor proteins can represent an essential step in oncogenesis and the transformation process. More specifically, the data presented in the Working Examples in Section 6 through 12, below, detail the binding of the GRB2 member of the GRB subfamily of adaptor proteins to the intracellular PTK product of the human BCR-ABL gene.

Described in this Section are some of the variety of uses to which the binding of such PTKs and adaptor proteins and/or PTPs and adaptor proteins can be put for the treatment of cell proliferative disorders involving such complexes. The uses described herein focus on, but are not limited to, the identification of agents capable of disrupting such complexes (i.e., decreasing or inhibiting the interaction between the component PTK or PTP, and adaptor members of the complexes) , and the utilization of such compounds for the treatment of cell proliferative disorders involving a PTK or a PTP capable of complexing with a member of the SH2- and/or SH3- containing family of adaptor proteins. "Disrupting", s used herein, is meant to refer not only to a physical separation of protein complex components, but also refers to a perturbation of the activity of the complexes, regardless of whether or not such complexes remain able, physically, to form. "Activity", as used here, refers to the function of the protein complex in the signal transduction cascade of the cell in which such a complex is formed, i . e . , refers to the function of the complex in effecting or inhibiting transduction of an extracellular signal into a cell . Examples of such cell proliferative disorders, include, but are not limited to, oncogenic disorders such as, for example chronic myelogenous and acute lymphocytic leukemias as well as psoriasis and atherosclerosis. The complexes

of the present invention may also be involved in such cellular processes such as activation, differentiation, and survival.

Depending on the individual PTK/adaptor protein complex or PTP/adaptor protein complex, disrupting the interaction between component members of such complexes may have differing modulatory effects on the signal transduction event involved, i.e., the effect of the complex disruption may activate, reduce, or block the signal normally transduced into the cell.

Likewise, depending on the cell proliferative disorder involved, either activation, reduction, or blockage of the signal normally transduced into the cell will be desirable for the treatment of the disorder. For example, one effect of the complexing of the BCR-ABL PTK with the GRB-2 adaptor protein causes the activation of the Ras signalling pathway (see the Working Example presented, below, in Section 8) . Thus, the disruption of such a BCR-ABL PTK/GRB-2 adaptor protein complex would inhibit the transduction of the abnormal signal and prevent activation of the Ras pathway. Alternatively, a cell proliferative disorder involving a PTK/adaptor or PTP/adaptor complex may, for example, develop because the presence of such complexes brings about the aberrant inhibition of a normal signal transduction event. In such a case, the disruption of the complex would allow the restoration of the usual signal transduction event. Further, an aberrant complex may bring about an altered subcellular adaptor protein localization, which may result in, for example, such dysfunctional cellular events as a cytoskeletal reorganization, as can be the case for the GRB-2 member of the GRB subfamily of adaptor proteins. An inhibition of the PTK/adaptor or PTP/adaptor complex in this case would

allow for restoration or maintenance of a normal cellular architecture. Still further, an agent or agents that cause(s) disruption of the PTK/adaptor or PTP/adaptor complex may bring about the disruption of the interactions among other potential components of such a complex, which may include, but are not limited to an SOS protein.

When considering PTK/adaptor and PTP/adaptor protein complexes wherein the PTK or the PTP component of the complex is a transmembrane, receptor-type PTK or PTP molecule, the receptors or their ligands may be used directly to modulate signal transduction events which may lead to the development of cell proliferative disorders. For example, taking the case of PTKs, soluble PTKs, peptides representing extracellular PTK domains, or peptides representing those portions of extracellular PTK domains which are known to bind ligands may be administered, using techniques well known to those skilled in the art, that, when exposed to the PTK-expressing cells of interest could act to compete with endogenous transmembrane PTK receptor molecules for available ligands, thus reducing or inhibiting ligand binding to endogenous PTKs. The effect of such a procedure could bring about a reduction or inhibition of the interaction between the PTK and the adaptor protein, possibly by blocking the autophosphorylation of the PTK which could, in turn, reduce the affinity of the adaptor protein for the PTK molecule. An analogous situation would hold in the case of PTPs.

In addition, again when considering receptor-type PTKs, extracellular molecules which bind to such PTKs may be administered, using techniques well known to those in the art, which, while binding the PTK do not activate the molecule. Extracellular molecules of

this type may be composed, for example, of modified forms of a native ligand for the PTK of interest, such that receptor binding may still occur, but activation of the kinase does not. A molecule with such a design could act in much the same way that administration of soluble PTK would, in that both procedures could have the final effect of reducing or inhibiting the formation of the PTK/adaptor protein complexes. Once again, an analogous situation would occur in the case of the PTPs.

Still further, molecules which are capable of binding native ligands of the receptor PTKs of the PTK/adaptor complexes or the receptor PTPs of the PTP/adaptor complexes of the invention may be administered, using techniques well known to those of skill in the art. Molecules in this class would act to inhibit the ligands' ability to bind it's respective receptor, and thus would have the final effect of reducing or inhibiting the formation of PTK/adaptor or PTP/adaptor protein complexes.

Depending on the specific conditions being treated, such agents may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in "Remington's Pharmaceutical Sciences," 1990, 18th ed. , Mack Publishing Co., Easton, PA. Suitable routes may include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, just to name a few. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's

solution, Ringer's solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

Agents which act intracellularly to directly interfere with the formation of the PTK/adaptor and/or PTP/adaptor complexes of the invention may be administered for the treatment of cell proliferative disorders. Such agents may include, but are not limited to, peptides and/or phosphopeptides comprising SH2 and or SH3 domains, or SH2 and/or SH3-binding domains, small organic molecules or extracts of natural products which would act to compete with the components of the complexes for binding, thus reducing or inhibiting the formation of complexes, which would, in turn, reduce or inhibit the development of the cell proliferative disorder of interest. SH2 and SH3 peptide domains, and SH2-binding and SH3-binding peptide domains are as described, above, in Section 5.1. Such agents may also disrupt the complex by interfering with down stream signaling capability instead of or in addition to complex formation. Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external microenvironment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell

cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be directly administered intracellularly.

Nucleotide sequences encoding the peptide agents which are to be utilized intracellularly may be expressed in the cells of interest, using techniques which are well known to those of ordinary skill in the art. For example, expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno- associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery and expression of such nucleotide sequences into the targeted cell population. Methods for the construction of such vectors are well known. See, for example, the techniques described in Maniatis et al . , 1989,

Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. and Ausubel et al . , 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y. Alternatively, antibodies capable of interfering with PTK/adaptor and/or PTP/adaptor complex formation may be administered for the treatment of cell proliferative disorders involving a PTK or PTP capable of forming a complex with an adaptor protein. For example, neutralizing antibodies which are capable of interfering with ligand binding to receptor type PTKs or PTPs may be administered using techniques such as those described above. The effect of such an administration would be similar to that described, above, for the administration of soluble PTKs or PTPs. Additionally, neutralizing antibodies which bind to intracellular epitopes to effect a disruption of PTK/adaptor or PTP/adaptor complex formation may also be administered. Such antibodies may be administered, for example, by utilizing the techniques described

above for the administration of agents intended to act intracellularly. Alternatively, nucleotide sequences encoding single-chain antibodies may be expressed within the target cell population by utilizing, for example, techniques such as those described in Marasco et al . (Marasco et al . , 1993, Proc. Natl. Acad. Sci. USA 20:7889-7893) .

The PTK/adaptor complexes and/or PTP/adaptor complexes of the invention may be used to screen for additional molecules that can act to disrupt the activity of the component members of such complexes, and thus may be capable of modulating the signal transduction event such complexes effect. Such compounds may include, but are not limited to, peptides made of D- and/or L-configuration amino acids (in, for example, the form of random peptide libraries; see Lam et al . , 1991, Nature 354:82-84) , phosphopeptides (in, for example, the form of random or partially degenerate, directed phosphopeptide libraries, see Songyang et al . , 1993, Cell 767-778) . antibodies, and small organic molecules.

For example, compounds that bind to individual components, or functional portions of the individual components of the PTK/adaptor or PTP/adaptor complexes (and may additionally be capable of disrupting complex formation) may be identified. A functional portion of an individual component of the complexes may be defined here as a peptide portion of an individual component of a complex still capable of forming a stable complex with another member of the complex. For example, a peptide portion of the SH2-binding domain of a PTK which is still capable of stably binding an SH2 domain of an adaptor protein, and thus is still capable of forming a complex with that adaptor protein. Further, in the case of the

catalytic domains of the individual PTK or PTP components of the invention, a functional portion of a catalytic domain may refer to a peptide still capable of stably binding a substrate molecule. One method utilizing this approach that may be pursued in the isolation of such PTK/adaptor or PTP/adaptor complex component-binding molecules would include the attachment of a component molecule, or a functional portion thereof, to a solid matrix, such as agarose or plastic beads, microtiter wells, petri dishes, or membranes composed of, for example, nylon or nitrocellulose, and the subsequent incubation of the attached component molecule in the presence of a potential component-binding compound or compounds. After incubation, unbound compounds are washed away, component-bound compounds are recovered. By utilizing this procedure, large numbers of types of molecules may be simultaneously screened for PTK/adaptor or PTP/adaptor complex component-binding activity. The PTK/adaptor complex components which may be utilized in the above screening method may include, but are not limited to, PTK molecules or functional portions thereof, such as PTK catalytic domains, phosphorylation domains, SH2 domains, SH3 domains, SH2-binding domains, or SH3-binding domains, and adaptor proteins, or functional portions thereof, such as SH2 domains and SH3 domains. The peptides used may be phosphorylated, e . g. may contain at least one phosphorylated amino acid residue, preferably a phosphorylated Tyr amino acid residue, or may be unphosphorylated. A phosphorylation domain may be defined as a peptide region that is specifically phosphorylated at certain amino acid residues. A functional portion of such a phosphorylation domain may be defined as a peptide capable of being

specifically phosphorylated at certain amino acids by a specific PTK. A functional portion of an SH2 domain may be defined as a peptide comprising at least a portion of an SH2 domain which is capable of specifically binding an SH2-binding domain. Likewise, a functional portion of an SH3 domain may be defined as a peptide comprising at least a portion of an SH3 domain which is capable of specifically binding an SH3-binding domain. A functional portion of an SH2- binding domain may be defined as a peptide capable of binding an SH2 domain, and may be at least about 4 amino acid residues in length. A functional portion of an SH3-binding domain may be defined as a peptide capable of binding an SH3 domain, and may be at least about 4 amino acids in length, with a length of about 10 amino acid residues being preferred.

The PTP/adaptor complex components which may be utilized in the above screening method may include, but are not limited to, PTP molecules or functional portions thereof, such as PTP catalytic domains, phosphorylation domains, SH2 domains, SH3 domains, SH2-binding domains, or SH3-binding domains, and adaptor proteins, or functional portions thereof, such as SH2 domains and SH3 domains. The peptides used may be phosphorylated, e . g. may contain at least one phosphorylated amino acid residue, preferably a phosphorylated Tyr amino acid residue, or may be unphosphorylated. A phosphorylation domain may be defined as a peptide region that is specifically phosphorylated at certain amino acid residues. A functional portion of such a phosphorylation domain may be defined as a peptide capable of being specifically phosphorylated at certain amino acids by a specific PTK.Functional portions of SH2, SH3, SH2-

binding, and SH3-binding domains may be as described above.

Molecules exhibiting binding activity may be further screened for an ability to disrupt PTK/adaptor or PTP/adaptor complexes. Alternatively, molecules may be directly screened for an ability to disrupt PTK/adaptor or PTP/adaptor complexes. For example, in vi tro complex formation may be assayed by, first, immobilizing one component, or a functional portion thereof, of the complex of interest to a solid support. Second, the immobilized complex component may be exposed to a compound such as one identified as above, and to the second component, or a functional portion thereof, of the complex of interest. Third, it may be determined whether or not the second component is still capable of forming a complex with the immobilized component in the presence of the compound.

Additionally, in vivo complex formation may be assayed by utilizing co-immunoprecipitation techniques well known to those of skill in the art. Briefly, a cell line capable of forming a PTK/adaptor or PTP/adaptor complex of interest may be exposed to a compound such as one identified as above, and a cell lysate may be prepared from this exposed cell line. An antibody raised against one of the components of the complex of interest may be added to the cell lysate, and subjected to standard immunoprecipitation techniques. In cases where a complex is still formed, the immunoprecipitation will precipitate the complex, whereas in cases where the complex has been disrupted, only the complex component to which the antibody is raised will be precipitated.

The effect of an agent on the transformation capability of the PTK/adaptor or PTP/adaptor complex

of interest may be directly assayed. Such agents may, but are not required to, include those agents identified by utilizing the above screening technique. For example, an agent or agents may be administered of a cell such as a fibroblast or hematopoietic cell capable of forming a PTK/adaptor complex which, in the absence of any inhibitory agent, would lead to the cell's transformation (Muller et al . , 1991, Mol. Cell. Biol. 11:1785-1792; McLaughlin et al . , 1987, Proc. Natl. Acad. Sci. USA 84:6558-6562) . The transformation state of the cell may then be measured in vi tro, by monitoring, for example, its ability to form colonies in soft agar (Lugo et al . , 1989, Mol. Cell. Biol. 2:1263-1270; Gishizky et al . , 1992, Science 256:836-839) . Alternatively, a cell's transformation state may be monitored in vivo by determining its ability to form tumors in immunodeficient nude on severe combined immunodeficiency (SCID) mice (Sawyers et al . , 1992, Blood 22:2089-2098) . Further, in the case of BCR-ABL, an agent or agents may be administered to animal models of ALL and/or CML which are well known to those of ordinary skill in the art (Gishizky et al . , 1993, Proc. Natl. Acad. Sci. USA 22:3755-3759) and/or reverse the progress of this oncogenic disorder.

Agents capable of disrupting PTK/adaptor and/or PTP/adaptor complex formation and capable of reducing or inhibiting cell proliferative disorders which arise from the formation of such complexes may be used in the treatment of patients exhibiting or at risk for such disorders. A sufficient concentration of an agent or agents such as those described above may be administered to a patient so that the cell proliferative capability of cells which, in the absence of such agents, would contain PTK/adaptor

and/or PTP/adaptor protein complexes, is reduced or eliminated.

Alternatively, in the case of hematopoietic cell proliferative disorders, such as leukemias, rather than direct administration to the patient, the agent or agents may be used in conjunction with autologous bone marrow transplantation and chemoradiotherapy techniques, which are well known to those of skill in the art. Briefly, an aliquot of bone marrow cells, generally taken from the pelvis, are removed from the patient. The cells are then cultured in the presence of a concentration of agent or agents which is capable of effectively disrupting PTK/adaptor or PTP/adaptor complex. By blocking the signal transduction pathway of those bone marrow cells capable of forming such complexes, one selects against the presence of clonal descendants of these cells, thus effectively purging the cultures of those cells responsible for the hematopoietic cell proliferative disorder being treated. While the bone marrow cells are being cultured and purged of cells with a high oncogenic capacity, the patient is treated with chemoradiotherapy appropriate for the disease involved, using techniques and doses well known to those of skill in the art. Upon completion of such chemoradiotherapy treatment, the patient receives an autologous infusion of the cultured bone marrow cells, which have been purged of oncogenic cells.

In a preferred embodiment of the invention, a PTK/adaptor complex is disrupted or prevented and is one in which the PTK component is an intracellular PTK product of the human BCR-ABL gene, and the adaptor protein component is a GRB-2 member of the GRB subfamily of adaptor proteins. Further, the cell proliferative disorders which administration of such

agents treats, in this preferred embodiment include, but are not limited to, chronic myelogenous leukemia and acute lymphocytic leukemia.

5.7 IDENTIFICATION OF COMPOUNDS CAPABLE OF DISRUPTING BINDING PARTNER INTERACTIONS

Any of a number of assay systems may be utilized to test compounds for their ability to interfere with (i.e. , disrupt or inhibit) the

10 interaction of the activated tyrosine kinase molecule and an adaptor protein molecule, which will be referred to herein herein as "binding partners." In addition to activated tyrosine kinase molecules, it is to be understood that other molecules which bind to

15 and form a complex with adaptor proteins are to be included within this definition of binding partners.

An additional binding partner may, therefore, for example, include SHC, an SH2 domain-containing protein which has been shown to bind the GRB-2 adaptor protein

20 (Tauchi, T. et al. , 1994, J. Exp. Med. 179:167-175;

Rozakis-Adcock,M. et al . , 1992, Nature 360:689-692;

Pelicci, G. et al . , 1992, Cell 22:93-104) .

However,rapid high throughput assays for screening large numbers of compounds, including but not limited

, ₅ to ligands (natural or synthetic) , peptides, or small organic molecules are preferred. Compounds that are so identified to interfere with the interaction of the binding partners can be further evaluated for inhibitory activity as described herein.

₃₀ The basic principle of the assay systems used to identify compounds that interfere with the interaction between the binding partners involves preparing a reaction mixture containing the binding partners under conditions and for a time sufficient to allow the two

₃₅ proteins to interact and bind, thus forming a complex. In order to test a compound for inhibitory activity,

the reaction is conducted in the presence and absence of the test compound, i.e., the test compound may be initially included in the reaction mixture, or added at a time subsequent to the addition of the binding partners; controls are incubated without the test compound or with a placebo. The formation of any complexes between the binding partners protein is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, or a decrease in the level of complex formation in the reaction mixture relative to the control reaction, indicates that the compound interferes with the interaction of the binding partners. The assay components and various formats that may be utilized are described below.

The binding partners used as components in the assay may be derived from natural sources, e.g. , purified from cells using protein separation techniques well known in the art; produced by recombinant DNA technology using techniques known in the art (see e.g. , Sambrook et al. , 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories Press, Cold Spring Harbor, N.Y.); and/or chemically synthesized in whole or in part using techniques known in the art; e.g. , peptides can be synthesized by solid phase techniques, cleaved from the resin and purified by preparative high performance liquid chromatography (see, e.g. , Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y. , pp. 50-60). The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing; e.g. , using the Edman degradation procedure (see e.g. , Creighton, 1983, supra) .

Peptide fragments may be produced to correspond to the binding domains of the respective proteins. For example, such fragments may include, but are not limited to SH2 domains, SH3 domains, SH2 domain- binding, and/or SH3 domain-binding peptide fragments. Any number of methods routinely practiced in the art can be used to identify and isolate the proteins' binding site. These methods include but are not limited to mutagenesis of one of the genes encoding the protein and screening for disruption of binding in a co-immunoprecipitation assay. Sequence analysis of the genes encoding the respective proteins will reveal the mutations that correspond to the region of the protein involved in interactive binding. Alternatively, one protein can be anchored to a solid surface and allowed to interact with and bind to its labeled binding partner, which has been treated with a proteolytic enzyme, such as trypsin. After washing, a short, labeled peptide comprising the binding domain may remain associated with the solid material, which can be isolated and identified by amino acid sequencing. Also, once the gene for the protein is obtained, short gene segments can be engineered to express peptide fragments of the protein, which can then be tested for binding activity and purified or synthesized.

Whether produced by molecular cloning methods or by chemical synthetic methods, the amino acid sequence of the binding partners which may be used in the assays of the invention need not be identical to the reported sequence of the genes encoding them. The binding partners may comprise altered sequences in which amino acid residues are deleted, added, or substituted resulting in a functionally equivalent product.

For example, functionally equivalent amino acid residues may be substituted for residues within the sequence resulting in a change of sequence. Such substitutes may be selected from other members of the class to which the amino acid belongs; e.g. , the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; the positively charged (basic) amino acids include arginine, lysine, and histidine; the negatively charged (acidic) amino acids include aspartic and glutamic acid.

One of the binding partners used in the assay system should be labeled, either directly or indirectly, to facilitate detection of a complex formed between the binding partners. Any of a variety of suitable labeling systems may be used including but not limited to radioisotopes such as ¹²⁵ I; enzyme labelling systems that generate a detectable colorimetric signal or light when exposed to substrate; and fluorescent labels.

Where recombinant DNA technology is used to produce the binding partners of the assay, it may be advantageous to engineer fusion proteins that can facilitate labeling, immobilization and/or detection. For example, the coding sequence of a tyrosine kinase or adaptor protein can be fused to that of a heterologous protein that has enzyme activity or serves as an enzyme substrate in order to facilitate labeling and detection. The fusion constructs should be designed so that the heterologous component of the fusion product does not interfere with binding of the binding partners.

Indirect labeling involves the use of a third protein, such as a labeled antibody, which specifically binds to one of the binding partners. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by an Fab expression library.

The assay can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring one of the binding partners onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction between the binding partners, e.g. , by competition, can be identified by conducting the reaction in the presence of the test substance; i.e. , by adding the test substance to the reaction mixture prior to or simultaneously with the binding partners. On the other hand, test compounds that disrupt preformed complexes, e.g. compounds with higher binding constants that displace one of the binding partners from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed.

In a heterogeneous assay system, one binding partner is anchored onto a solid surface, and its binding partner, which is not anchored, is labeled, either directly or indirectly. In practice, microtiter plates are conveniently utilized. The anchored species may be immobilized by non-covalent or covalent attachments. Non-covalent attachment may be

accomplished simply by coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody specific for the protein may be used to anchor the protein to the solid surface. The surfaces may be prepared in advance and stored. In order to conduct the assay, the binding partner of the immobilized species is added to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g. , by washing) and any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the binding partner was pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the binding partner is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g. , using a labeled antibody specific for the binding partner (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody) . Depending upon the order of addition of reaction components, test compounds which inhibit complex formation or which disrupt preformed complexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g.. using an immobilized antibody specific for one binding partner to anchor any complexes formed in solution, and a labeled antibody specific for the other binding partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds which inhibit complex

or which disrupt preformed complexes can be identified.

In an alternate embodiment of the invention, a homogeneous assay can be used. In this approach, a preformed complex of the binding partners is prepared in which one of the binding partners is labeled, but the signal generated by the label is quenched due to complex formation (see, e.g. , U.S. Patent No. 4,109,496 by Rubenstein which utilizes this approach for immunoassays) . The addition of a test substance that competes with and displaces one of the binding partners from the preformed complex will result in the generation of a signal above background. In this way, test substances which disrupt the viral protein-host cell protein interaction can be identified.

EXAMPLE: BCR-ABL ASSOCIATES WITH GRB-2 BOTH IN VITRO AND IN VIVO

In the Working Example presented in this section, it is demonstrated that the GRB-2 member of the GRB subfamily of adaptor proteins binds to the BCR-ABL intracellular PTK both in vi tro and in vivo.

6 . 1 . MATERIALS AND METHODS 6.1.1 CELLS AND VIRUSES Spodoptera frugiperda (Sf9) insect cells were propagated in complete Grace's media (Smith, G.E., 1983, Mol. Cell. Biol. 3:2156-2165) . The Ph^positive leukemic cell lines K562 and NMG-01 were derived from patients with chronic myelogenous leukemia and express P210 BCR-ABL. The PH^positive cell line, ALL-1, was derived from a patient with acute lymphocytic leukemia and expresses P185 BCR-ABL. The PH^positive cell lines were cultured in RPMI 1640 medium with 10% fetal calf serum. COS African green monkey cells and Ratl

fibroblasts were grown in DMEM medium with 5% fetal calf serum.

Recombinant baculoviruses for expression of cBCR, cABL and BCR-ABL were prepared by cotransfecting the corresponding cDNAs cloned into the pAcCI2 baculovirus vector in the presence of wild type baculovirus DNA as described (Pendergast et al . , 1991, Cell £6:161-171; Pendergast et al . , 1991, Proc. Natl. Acad. Sci. USA £8:5927-5931) . Helper-free retroviral stocks were prepared by transient hyperexpression in COS cells according to methods previously described (Muller et al . , 1991, Mol. Cell. Biol. 11:1785-1792) . Retroviral stocks were characterized according to their ability to transfer wild type and mutant forms of the BCR-ABL gene product to Ratl fibroblasts using immunohistochemical methods (Muller et al . , 1991, Mol. Cell. Biol. 11:1785-1792) . Endogenous levels of rat c-abl protein were not detected in these staining procedures. The level of gene transfer was further evaluated by measuring levels of BCR-ABL protein expression (Western blot) . Only retroviral stocks showing comparable levels of gene transfer were used in these studies. Titers were in the range of 105 infectious particles per ml as determined by the frequency of G418 resistant Ratl colonies following exposure to limiting dilutions of the viral stocks.

6.1.2. ANTIBODIES Polyclonal rabbit antibodies directed against the amino terminus of cBCR, and amino- and carboxy- terminal sequences of cABL have been previously described (Pendergast et al . , 1991, Cell 66:161-171; Konopka et al . , 1984, Cell £7:1035-1042). A mouse monoclonal anti-ABL (21-63) antibody was employed for

immunoblotting (Pendergast et al . , 1991, Proc. Natl . Acad. Sci. USA 88:5927-5931) . Polyclonal rabbit antibodies to the C-terminal SH3 domain of GRB-2 (Ab50) and to a synthetic peptide derived from the N- terminal SH3 domain of GRB-2 (Ab86) were used for immunoprecipitation and immunoblotting, respectively (Lowenstein et al . , 1992, Cell 70:431-442) .

6.1.3. PLASMID CONSTRUCTIONS The 650-base pair human GRB-2 CDNA (Lowenstein et al . , 1992, Cell 22:431-442; Matuoka et al . , 1992, Proc. Natl. Acad. Sci. USA £9:9015-9019) was cloned from a human placenta cDNA library by polymerase chain reaction (PCR) as (5') SH3-SH2 and (3') SH3 fragments. A unique Kpnl site at codon 154 of GRB-2 was employed to generate the full length GRB-2 cDNA. The entire coding sequence of GRB-2 and the (3') SH3 domain of GRB-2 were subcloned in-frame into the Bam HI site of the pGEX-2T vector (Pharmacia) . The isolated (5') SH3 and SH2 domains of GRB-2 were prepared as described (Skolnik et al . , 1993, EMBO J. 12:1929-1936) .

Preparation of cDNAs for wild type P185 BCR-ABL (McLaughlin et al . , 1989, Mol. Cell. Biol. 9:1866- 1874) and P185 (Δ176-426) (Muller et al . , 1991, Mol. Cell. Biol. 11:1785-1792) and cloning of the corresponding cDNAs into pSRα (Pendergast et al . , 1991, Proc. Natl. Acad. Sci. USA £8:5927-5931) and pSRα MSVtKneo (Muller et al . , 1991, Mol. Cell. Biol. 11:1785-1792) vectors was performed as previously described. The P185 BCR-ABL (Y177F) mutant was created by oligonucleotide-site directed mutagenesis using the Muta-Gene Phagemid in vi tro mutagenesis system (Bio Rad) . Template was generated by subcloning a 1.6 Kb EcoRI-SacI fragment of cBCR from pGEM4/cBCR into the EcoRI and SacI sites of the

pBlueScript SK+ vector (Stratagene) and rescuing the single stranded DNA by coinfecting XL-1 Blue bacteria with the helper phage R408 (Stratagene) . The mutagenic oligonucleotide, 5'-AAG CCC TTC TTC GTT AAC 5 GTC GAG-3', was employed to create a phenylalanine codon in place of tyrosine, by changing nucleotide 530 from an A to a T. In addition, a silent base change at nucleotide 534 was introduced to create a unique Hpal site. Mutagenized plasmids were selected for the

10 presence of the unique Hpal site. The mutations were verified by dideoxy chain termination sequence analysis in both directions. The mutated BCR sequence was introduced into the wild type P185 BCR-ABL cDNA. P185 BCR-ABL (Y177F) was subcloned into the pSRα and

15 pSRα MSVtKneo mammalian expression vectors and the AcC12 vector for baculovirus expression. Cloning of the cDNA for wild type cABL into the pSRce vector has been previously described (Pendergast et al . , Proc. Natl. Acad. Sci. USA £8:5927-5931) . BCR (Δ872-1271)

20 was also cloned into pSRα as described (Pendergast et al . , 1991, Cell £6:161-171) .

6.1.4. EXPRESSION AND PURIFICATION OF GST-FUSION PROTEINS

, _ς GST-fusion proteins were expressed and purified using glutathione-Sepharose 4B beads (Pharmacia) as previously described (Pendergast et al . , 1991, Cell

£6:161-171) . Fusion proteins were left on the resin and stored at 4°C.

30

6.1.5. METABOLIC LABELING AND IMMUNOPRECIPITATION

Sf9 cells were infected with the indicated recombinant baculoviruses. Three days post-infection the cells were incubated with O.lmCi/ml [ ³⁵ S]

35 methionine (ICN) in methionine-free media for 4 to 6

hrs at 27°C. Labeled cells were lysed with either KLB (lOmM sodium phosphate pH 7.0, 150mM NaCl, 1% Triton X-100) or PCLB (50mM HEPES, pH 7.0, 150mm NaCl, lmM MgCl ₂ , 1% Triton X-100, 10% glycerol) supplemented with 5mM EDTA, lmM PMSF, 50μg/ml leupeptin, 25μg/ml aprotinin, 25mM NaF, lmM Na ₃ V0 ₄ , and 0.lmM Na ₂ Mo0 ₄ . The lysates were clarified by centrifugation at 100,000 x g for 1 hour. Lysates were incubated with the indicated antibodies directly or after a 3-fold dilution with Incubation Buffer (20mM HEPES, pH 7.0, 150mM NaCl, 0.1% Triton X-100, 10% glycerol, 0.5mM Na ₃ V0 ₄ , O.lmM Na ₂ Mo0 ₄ , 25mM NaF, lmM PMSF, 25μg/ml leupeptin) as indicated. Immune complexes were collected by incubation with Protein A-Sepharose beads (Pharmacia) for 120 minutes at 4°C. The beads were washed extensively with Incubation buffer to remove unbound material. Bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis and visualized by fluorography. Whenever unlabeled lysates were employed, the proteins were detected by immunoblotting with the indicated antibodies.

6.1.6. BINDING ASSAYS Protein lysates were diluted 3-fold with Incubation Buffer and incubated with GST or GST-fusion proteins attached to glutathione-Sepharose beads. After incubation for 90 min. at 4°C, the beads were washed extensively with Incubation Buffer or with RIPA buffer (20mM Tris-HCl, pH 7.4, 137mM NaCl, 10% glycerol, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate and 2mM EDTA) , as indicated. Bound proteins were analyzed by SDS-polyacrylamide followed by fluorography for radiolabelled proteins.

6.1.7. IMMUNOBLOTTING, IN VITRO

AUTOPHOSPHORYLATION AND

DEPHOSPHORYLATION REACTIONS Procedures for immunoblotting and in vi tro labeling with [γ ³² P]ATP were carried out essentially as previously described (Pendergast et al . , 1991, Cell £6:161-171; Pendergast et al . , 1991, Proc. Natl. Acad. Sci. USA £8:5927-5931) . Dephosphorylation with potato acid phosphatase was performed as described (Pendergast et al . , 1991, Cell £6:161-171; Pendergast et al . , 1991, Proc. Natl. Acad. Sci. USA 88:5927- 5931) .

6.2. RESULTS

6.2.1. BCR-ABL ASSOCIATES WITH GRB-2 IN VIVO To determine whether the BCR-ABL tyrosine kinase forms a physical complex with the GRB-2 adaptor protein in intact cells, it was examined whether the two proteins co-immunoprecipitate. Cell lysates were prepared from the P^-positive leukemic cell lines K562, MEG-01, and ALL-1 Ph ¹ hematopoietic cells and Rat 1 fibroblast cells, and were subjected to immunoprecipitation with anti-ABL pEx4, anti-GRB-2, or control antibodies. Following extensive washing, the immunoprecipitates were collected on protein A- Sepharose beads and subjected to in vitro phosphorylation in the presence of [γ ³² P] ATP and MnCl ₂ . Such conditions promote autophosphorylation of the BCR-ABL kinase. The reactions were terminated after 30 minutes at 30°C, washed and analyzed by SDS/8% polyacrylamide gel electrophoresis. The ³² P- labeled proteins were detected by autoradiography. Comparable levels of BCR-ABL protein were precipitated using either anti-GRB-2 or anti-ABL antibodies. Further, both forms of BCR-ABL (P210 and P185) associated with GRB-2 in Ratl fibroblasts expressing the corresponding BCR-ABL proteins.

Similarly, immunoprecipitation with GRB-2 protein can be precipitated by anti-ABL antibodies in cell lines where BCR-ABL is expressed. Specifically, cell lysates were prepared from MEG-01 cells and were subjected to immunoprecipitated with control (pre- immune sera), anti-ABL pEX4, and anti-GRB-2 antibodies. The immunoprecipitated proteins were separated by SDS/12% polyacrylamide gel electrophoresis, transferred to nitrocellulose and immunoblotted with anti-GRB-2 antibodies. Bound antibodies were visualized with [ ¹²⁵ I] protein A, and could be detected in both the lanes containing anti- GRB-2 and anti-ABL immunoprecipitated material.

Metabolic labeling of BCR-ABL expressing cells with ³⁵ S-methionine followed by immunoprecipitation with anti-ABL or anti-GRB-2 antibodies demonstrated that 50% to 90% of the BCR-ABL kinase available in the cell is complexed with GRB-2 in agreement with the results described above. Interestingly, no association of GRB-2 with the oncogenic v-abl kinase was observed. These experiments demonstrate that the GRB-2 adaptor protein forms a stable complex with both forms of the BCR-ABL tyrosine kinase but not the v-abl kinase and that the BCR-ABL/GRB-2 complexes remain intact following in vi tro phosphorylation of BCR-ABL.

6.2.2. BCR-ABL BINDS TO GRB-2 IN VITRO To examine the molecular basis for the association of BCR-ABL with GRB-2, the full-length GRB-2 cDNA sequence was cloned into pGEX-2T for expression in bacteria as a glutathione S-transferase (GST) fusion protein. The GST-GRB-2 protein was purified and tested for its ability to bind to BCR-ABL in vi tro . Specifically, ³⁵ S-methionine-labeled proteins from lysates of Sf9 insect cells infected

with P185 BCR-ABL recombinant baculovirus were incubated with equal amounts of immobilized GST alone, GST-GRB-2 full length, GST-amino-terminal, GRB-2 SH3 domain, GST-GRB-2 SH2 domain GST-carboxy-terminal, GRB-2 SH3 domain and anti-ABL antibodies bound to protein A-Sepharose beads. After incubation for 90 minutes at 4°C, the beads were washed four times with incubation buffer and twice with RIPA buffer to remove unbound material. Bound proteins were analyzed by SDS/7% polyacrylamide gel electrophoresis and detected by fluorography. It was found that the P185 BCR-ABL, expressed in baculovirus-infected insect cells, bound to full length GRB-2 immobilized on glutathione- Sepharose beads. No binding to GST alone was detected. The complex of BCR-ABL and GST-GRB-2 remained intact after washing with buffer containing SDS and deoxycholate detergents.

To identify which GRB-2 domain(s) bind to BCR- ABL, GST fusion proteins were prepared that contained the isolated GRB-2 SH3 and SH2 domains. BCR-ABL bound to the GST-GRB-2 SH2 fusion protein. GST fusion proteins containing the amino- and carboxy-terminal SH3 domains of GRB-2 also bound to the baculovirus- produced BCR-ABL protein in vi tro. The interaction of BCR-ABL with the SH2 and SH3 domains of GRB-2 was resistant to washing with a buffer containing SDS and deoxycholate. Treatment of BCR-ABL with potato acid phosphatase completely eliminated its ability to associate with the GRB-2 SH2 domain, but did not affect its binding to the GRB-2 SH3 domains.

7. EXAMPLE: BCR-ABL SEQUENCES NECESSARY FOR SH2-MEDIATED BINDING OF GRB-2 TO BCR-ABL

In the Working Example presented in this section, amino acid sequences necessary for BCR-ABL/GRB-2 binding via the GRB-2 SH2 domains are investigated.

Specifically, it is demonstrated that binding requires the presence of Tyr-phosphorylated amino acid residues, and further, it is shown that a BCR first exon mutation is capable of abolishing the SH2- mediated BCR/GRB-2 binding.

7.1. MATERIALS AND METHODS The materials and techniques utilized in the experiments presented in this Working Example are as those described in Section 6.1, above.

7.2. RESULTS

7.2.1 SH2-MEDIATED BINDING OF GRB-2 TO BCR-ABL REQUIRES TYROSINE PHOSPHORYLATION OF BCR SEQUENCES

To identify which regions in BCR-ABL participate in GRB-2-binding, BCR and ABL sequences were expressed separately in baculovirus-infected insect cells and tested for their ability to bind to full length GRB-2 as well as to the isolated GRB-2 SH2 and SH3 domains. Baculovirus-produced cABL bound to full length GRB-2 in vi tro but not to the GRB-2 SH2 domain alone. Specifically, Sf9 insect cells singly infected with cABL recombinant baculoviruses. Three days post- infection, the cells were labeled with ³⁵ S-methionine The labeled cells were lysed and the lysate proteins were incubated with GST alone, GST-GRB-2 full length, GST-GRB-2 amino SH3, GST-GRB-2 SH2, GST-GRB-2 carboxy SH3, and the corresponding anti-ABL antibodies in the presence of protein A-Sepharose beads. After incubation for 90 minutes at 4°C, the beads were washed four times with incubation buffer and twice with RIPA buffer. Bound proteins were analyzed by SDS/10% polyacrylamide gel electrophoresis and detected by fluorography. cABL hyperexpressed in insect cells is phosphorylated on tyrosine residues

(Pendergast et al _* , 1991, Proc. Natl. Acad. Sci. USA £8:5927-5931) . Thus, the inability of the GRB-2 SH2 domain to bind to the baculovirus-produced cABL protein was not due to lack of tyrosine phosphorylation.

The binding of cABL to GRB-2 in vi tro appears to be mediated exclusively via the amino-and carboxy- terminal SH3 domains of GRB-2. SH3 domains bind to specific proline-rich motifs (Cicchetti et al . , 1992, Science 257:803-806; Ren et al . , 1993, Science

259:1157-1161) . Recently, it has been shown that GRB- 2 binds to the Sosl guanine nucleotide exchanger through the direct interaction of the GRB-2 SH3 domains with the proline-rich sequence, PPPVPPR, present in the carboxy-terminal region of Sosl (Li et al . , 1993, Nature 363:85-87: Rozakis-Adcock et al . , 1993, Nature 363 :83-85) . The carboxy-terminus of cABL contains several proline-rich stretches. However, no PPPVPPR sequence is found in the cABL protein. Binding of the GRB-2 SH3 domains to cABL in vi tro is significantly reduced but not completely eliminated in a cABL deletion mutant protein that lacks the majority of the cABL carboxy-terminal domain. These data together with the lack of detectable association between normal cABL and GRB-2 following immunoprecipitation from cell lysates, as discussed below, suggest that the cABL/GRB-2 interaction observed in vi tro is not specific and may not occur in vivo. However, it is possible that the proline-rich domain in cABL serves as binding sites for other SH3 domains.

A similar analysis of the binding of cBCR to full length or isolated domains of GRB-2 in vi tro revealed that only the full length GRB-2 and to a lesser extent the N-terminal SH3 domain of GRB-2 bound to cBCR in

vitro. Specifically, Sf9 insect cells were singly infected with cBCR recombinant baculoviruses, and were then treated as discussed above, for the cABL recombinant baculoviruses. Interestingly, no binding of cBCR to the SH2 domain of GRB-2 was detected.

The complete lack of in vi tro binding of the GRB- 2 SH2 domain to cABL and cBCR contrasts with the strong binding of this domain to the chimeric BCR-ABL tyrosine kinase. The phosphorylation state of cBCR sequences is different in the full length cBCR protein versus the BCR-ABL chimera. The cBCR protein is phosphorylated only on serine/threonine residues in all cell types examined even following hyperexpression in insect cells (Timmons et al . , 1989, Oncogene 4 _. :559- 567; Pendergast et al . , 1991, Cell 66:161-171) . In contrast, in the BCR-ABL chimera, the activated ABL kinase phosphorylates BCR first exon sequences on tyrosine (Liu et al . , 1993, Oncogene 8:101-109) . To evaluate whether tyrosine-phosphorylation of BCR sequences could uncover binding to the isolated GRB-2 SH2 domain, insect cells were co-infected with baculoviruses coding for the full length cBCR and cABL proteins. Specifically, Sf9 insect cells were coinfected with cABL and cBCR recombinant baculoviruses, and treated as discussed, above, for the cABL recombinant baculoviruses. Trans- phosphorylation of cBCR by the cABL tyrosine kinase resulted in binding of the GRB-2 SH2 domain to cBCR. Western blotting with antiphosphotyrosine antibodies demonstrated that cBCR was tyrosine-phosphorylated in Sf9 cells co-infected with cABL and cBCR baculoviruses. A low level of binding of cBCR to the isolated amino- and carboxy-terminal SH3 domains of GRB-2 in vi tro was also detected. These results

demonstrate that binding of the GRB-2 SH2 domain to BCR sequences requires tyrosine phosphorylation.

7.2.2. GRB-2 INTERACTS WITH BCR-ABL BUT NOT CABL AND CBCR SEQUENCES IN VIVO

To examine whether the in vi tro binding of GRB-2 to cABL and cBCR also occurred in vivo, COS cells were transfected with expression constructs of the corresponding cDNAs. Transfection of these cDNAs results in an approximately 50- to 200-fold increase in the expression of cABL and cBCR over the corresponding endogenous protein levels (Pendergast et al . , 1991, Proc. Natl. Acad. Sci. USA 88:5927-5931) . Under these conditions, no appreciable levels of cABL co-immunoprecipitated with GRB-2. Similarly, no interaction of GRB-2 with BCR sequences could be detected following hyperexpression of the BCR sequences retained in the P210 BCR-ABL chimera in COS cells. In contrast, significant levels of P185 BCR- ABL were precipitated by the anti-GRB-2 antibodies in the same experiment.

Specifically, COS cells were transfected with P185 wild type and cABL cDNAS cloned into the pSRα. vector. Three days post-transfection, the cells were lysed and the lysates were incubated for 2 hours at 4°C with normal rabbit sera, anti-ABL 2/3 antibodies, and anti-GRB-2 antibodies. The immunoprecipitates were collected on protein-A-Sepharose beads and washed six times with incubation buffer. Bound proteins were analyzed by SDS/8% polyacrylamide gel electrophoresis, transferred to nitrocellulose filters and the filters were incubated with anti-ABL mouse monoclonal antibody. Immunoreactive bands were visualized with the enhanced chemiluminescense (ECL) detection system (Amersham) . In addition, COS cells were transfected

with BCR 872-1271) cDNA cloned into the pSRγ vector. The cells were lysed three days post-infection and the lysates were incubated with control sera, anti-BCR antibodies, or anti-GRB-2 antibodies for 2 hours at 4°C. The immunoprecipates were collected with protein A-Sepharose beads, washed four times with incubation buffer and twice with 50mM Tris-HCl, pH 7.0, and then subjected to in vitro phosphorylation in the presence of γ- ³² P-ATP and MnCl ₂ . Proteins were analyzed by SDS/8% polyacrylamide gel electrophoresis and autoradiography.

These data, together with the lack of detectable endogenous cABL and cBCR proteins in anti-GRB-2 immunoprecipitates from normal cells, supports the contention that no cABL/GRB-2 and cBCR/GRB-2 complexes may be found in vivo. These findings demonstrate that the BCR-ABL chimera exhibits novel protein binding properties which are distinct from those of its BCR and ABL protein sequence components.

7.2.3. A POINT MUTATION IN THE BCR

FIRST EXON ABOLISHES BINDING OF THE GRB-2 SH2 DOMAIN TO BCR

Binding of SH2 domains to specific tyrosine- phosphorylated proteins is dependent on the primary sequence C-terminal to the phosphorylated tyrosine

(Fanti et al . , 1992, Cell 69:413-423; Kashishian et al . , 1992, EMBO J. 11:1373-1382; Sonyang et al _* , 1993,

Cell 72:767-778) . Examination of the sequences surrounding the eleven potential tyrosine phosphorylation sites within the first exon of BCR revealed that tyrosine 177 is found within the sequence YVNV, which corresponds to an optimal binding site for the GRB-2 SH2 domain (Sonyang et al . , 1993,

Cell 72:767-778; Skolnik et al _* , 1993, EMBO J.

12:1929-1936) . Sequences surrounding the other ten

tyrosines in the BCR first exon do not conform to optimal binding sites for the GRB-2 SH2 domain or other SH2 domains examined (Sonyang et al . , 1993, Cell 22=767-778) . To determine whether tyrosine 177 is required for the binding of BCR-ABL to the GRB-2 SH2 domain, phenylalanine was substituted for tyrosine 177 in BCR-ABL by site-directed mutagenesis, the mutated protein was expressed in insect cells and was tested for binding to GRB-2. Comparison of the phosphopeptide map patterns of the P185 (Y177F) mutant and P185 wild type proteins following in vi tro autophosphorylation revealed the absence of at least one major phosphopeptide in P185 (Y177F) . Specifically, COS cells were transfected with P185 wild type and P185 (Y177F) cloned into the pSRαMSVtkneo vector. Three days post-transfection, the cells were lysed and the lysates were incubated with anti-ABL antibodies. The immunoprecipitates were subjected to in vitro autophosphorylation in the presence of γ- ³² P ATP and MnCl ₂ . Proteins were analyzed by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and subjected to phosphopeptide mapping.

Binding studies demonstrated that in contrast to the wild type P185 BCR-ABL, the P185 BCR-ABL (Y177F) mutant protein, in which tyrosine 177 is replaced by a phenylalanine in the BCR first exon, did not interact with the GRB-2 SH2 domain in vi tro . Specifically, Sf9 insect cells were co-transfected with wild type baculovirus DNA and wild type P185 BCR-ABL cDNAs cloned with the pAcC12 vector, or with wild type baculovirus DNA and P185 BCR-ABL (Y177F) cDNAs cloned into the pAcC12 vector. Six days post-transfection, the cells were metabolically labeled with ³⁵ S- methionine. The cells were lysed and the lysates were

incubated with GST-GRB-2 SH2 or with anti-ABL pEx4 antibodies with protein A-Sepharose beads. The bound proteins were analyzed by SDS/7% polyacrylamide gel electrophoresis and fluorography. Next, the ability of the P185 BCR-ABL (Y177F) mutant to interact with GRB-2 in vivo was evaluated. P185 (Y177F) did not interact with endogenous GRB-2 when hyperexpressed in COS cells, or in Ratl fibroblasts that stably express the mutant protein. Similarly, BCR-ABL deletion mutants which lack tyrosine 177 failed to bind GRB-2. Specifically, COS cells were transfected with P185 wild type and P185 (Y177F) cDNAs cloned into the pSRα MSVtkneo vector. Three days post-transfection, the cells were lysed and the lysates incubated with normal rabbit sera (NRS) , anti-ABL 2/3 antibodies, or anti-GRB-2 antibodies, for 2 hours at 4°C. The immunoprecipitates were collected on protein-A-Sepharose beads and subjected to in vitro phosphorylation in the presence of [γ ³² P] ATP and MnCl ₂ . Protein were analyzed by SDS 10% polyacrylamide gel electrophoresis and autoradiography. Further, lysates of G418-selected Ratl cells expressing P185 wild type and P185 (Y177F) were incubated for 2 hours at 4°C with anti-GRB-2 antibodies, anti-ABL 2/3 antibodies, or normal rabbit sera. The immunoprecipitates were collected on protein-A- sepharose beads. The bound proteins were analyzed by SDS 10% polyacrylamide gel electrophoresis followed by immunoblotting with anti-ABL mouse monoclonal antibody. Immunoreactive bands were visualized with the ECL detection system (Amersham) .

These data indicate that interaction between BCR- ABL and GRB-2 in vivo is mediated by the binding of the GRB-2 SH2 domain to the phosphorylated tyrosine 177 in BCR-ABL. The GRB-2 SH3 domains apparently do

not contribute to the in vivo binding between full length GRB-2 and BCR-ABL.

8. EXAMPLE: BCR-ABL PROTEINS DEFECTIVE IN GRB-2 BINDING EXHIBIT DECREASED TRANSFORMING CAPACITY

The data presented in this Working Example demonstrates that the transformation potential of the

BCR-ABL intracellular PTK is dependent upon the binding of this PTK to the GRB-2 member of the GRB subfamily of the adaptor family of proteins. This result represents the first case in which the binding of an adaptor protein to such a PTK is implicated as a step in a transformation/oncogenesis process.

8.1. MATERIALS AND METHODS 8.1.1. TRANSFORMATION ASSAYS

Infection of Ratl fibroblasts was carried out as previously described (Lugo et al . , 1989, Mol. Cell. Biol. 2:1263-1270) . Titers were in the range of >10 ⁵ infectious particles per ml as determined by the frequency of gene transfer to fibroblasts. Ratl colony formation in semisolid medium was measured by plating 5xl0 ⁴ cells per 6-cm ² dish in 5 ml of Iscoves supplemented with 15% fetal calf serum and 0.4% Noble agar (Lugo et al. , 1989, Mol. Cell. Biol. 9:1263- 1270) . G418-resistant populations were established by culturing the infected cells for 12-15 days in G518 (0.5 mg per ml) . Following selection, cells from G418-resistant cultures were plated in agar at a density of 10 ⁴ cells per ml . The number of colonies formed in the agar was recorded two weeks after plating the cells.

Infection and establishment of hematopoietic cell cultures from freshly isolated murine bone marrow were performed as previously described (McLaughlin et al . ,

1987, Proc. Natl. Acad. Sci. USA £4:6558-6562) . In brief, bone marrow elements from 4-6 week old female BALB/c mice were resuspended at 2xl0 ⁶ cells per ml in the appropriate retroviral stock supplemented with 4 μg of Polybrene per ml. Cells were incubated for 4 hours at 37°C. Following infection, cells were washed and resuspended in RPMI 1640 medium supplemented with 5% fetal calf serum and 50μM /3-mercaptoethanol, at a density of 10 ⁶ cells per ml 5 ml of the cell suspension was plated into each 6 cm ² dish. Cultures were maintained for up to 8 weeks and fresh media was added once a week. The cultures were considered transformed when the cell density of the nonadherent hematopoietic cells exceeded 10 ⁶ cells per ml.

8.1.2. MISCELLANEOUS PROCEDURES Techniques in addition to the transformation assays as described in Section 8.1.1 are as those described, above, in Section 6.1.

8.2. RESULTS To determine the biological relevance of GRB-2 binding to BCR-ABL-induced oncogenesis, the effect of mutating tyrosine 177 was examined, required for association with GRB-2, on the ability of BCR-ABL to transform fibroblasts and hematopoietic cells. Helper-free retroviral stocks of wild type and mutant BCR-ABL forms were used to infect Ratl fibroblasts and freshly isolated murine bone marrow cells. Both GRB-2 and hSos-I have been shown to be expressed in these cell types (Lowenstein et al . , 1992, Cell 70 :431-442 ; Bowtell et al . , 1992, Proc. Natl. Acad. Sci. USA £9:6511-6515; Chardin et al . , 1993, Science 260:1338- 1343) . Transformation of Ratl fibroblasts was assayed

by colony formation in soft agar (Lugo et al . , 1989, Mol. Cell. Biol. 2:1263-1270) . Hematopoietic cell transformation was assayed by culturing infected mouse bone marrow cells under conditions that support the growth of pre-B lymphocytes (McLaughlin et al . , 1987, Proc. Natl. Acad. Sci. USA £4:6558-6562) . In contrast to wild type BCR-ABL, the P185 (Y177F) protein did not transform hematopoietic cells and exhibited a decreased capacity to transform Ratl cells (see Table 1, below) . These results are consistent with the previous observation that the P185 (Δl76-426) mutant which deletes Y177 exhibited decreased transforming activity in both Ratl fibroblasts and hematopoietic cells (Muller et al . , 1991, Mol. Cell. Biol. 11:1785- 1792; Table 1) . P185 (Δ176-426) displays a more pronounced deficiency in the transformation of Ratl fibroblasts than P185 (Y177F) , particularly after G418 selection (Table 1) . Sequences downstream of Y177 in the BCR first exon have been shown to bind to SH2 domains in a phosphoserine/phosphothreonine-dependent manner (Pendergast et al . , 1991, Cell 66:161-171; Muller et al . , 1991, Mol. Cell. Biol. 11:1785-1792) . Two SH2-binding sites have been identified within this region (designated A and B) and removal of these sites may abolish specific protein interactions important for BCR-ABL-mediated transformation of fibroblasts. Both P185 (Y177F) and P185 (Δ176-426) showed no transformation activity in hematopoietic cells. Differences in the transforming activities of the BCR- ABL proteins were not due to different levels of protein expression.

Table 1

Retroviral ³ Ratl ^b Bone Marrow ⁰ Constructs #colonies/l0 ⁵ cells transformed/ total

Acutely G418 Infected ⁰ Selected ⁰

P185 wild 37 690 11/12 type

P185 (Y177F) 0 40 0/14

P185 (Δ 176- 0 10 0/9 426) vector 0 0 0/7

Superscript legend: a Names of the different P185 BCR-ABL forms. The mutated or deleted amino acids are indicated in parenthesis. b Average frequency of colony formation in agar determined from two plates per assay and four to five independent assays per construct . c Cells were plated in agar 3 days after infection with helper-free retroviral stocks. d Cells were selected for 12 to 15 days with G418 (0.5 mg/ml) starting 3 days after infection with helper-free retroviral stocks. e Number of high density bone marrow cultures exhibiting transformed lymphoid outgrowth over the total of cultures plated. Data represent three separate experiments. In each experiment, cell cultures were set up after infection with the respective retroviral stock.

Western blot analysis of lysates from cells expressing the various BCR-ABL forms revealed comparable steady-state levels of the proteins following infection of Ratl fibroblasts with the corresponding retroviruses. For the western blot

analyses, RAT1 cells were infected with retroviruses encoding wild type and mutant (Y177F) forms of P185 BCR-ABL, or vector control. Three days post- infection, the cells were lysed and subjected to western blotting with anti-ABL mouse monoclonal antibody. Immunoreactive bands were visualized with the ECL detection system (Amersham) .

9. EXAMPLE: BCR-ABL ACTIVITIES TRANSCRIPTION FROM A RAS-RESPONSIVE ELEMENT PROMOTER IN A RAS-DEPENDENT MANNER

In the Working Example presented in this section, it is demonstrated that BCR-ABL intracellular PTK/GRB-

2 binding serves to activate the Ras signalling pathway.

9.1. MATERIALS AND METHODS 9.1.1. TRANSCRIPTIONAL ACTIVATION ASSAY Transcriptional activation of expression from a Ras-responsive element (ets/AP-1) promoter was done essentially as described previously (Clark et al . , 1993, Proc. Natl. Acad. Sci. USA ££:4887-4891; Hauser et al . , 1993, Methods in Enzymology, in press) . Briefly, NIH 3T3 cells were transfected with lμg of the pB4X-CAT chloramphenicol acetyl transferase reporter plasmid (Wasylyk et al . , 1989, Mol. Cell. Biol. 2:2247-2250) , together with 0.5μg pSRαMSVTkneo containing the indicated BCR-ABL mutants in the presence or absence of 5μg pZIP H-Ras (17N) (Feig et al . , 1988, Mol. Cell. Biol. 8:3235-3243) . Transfections were performed in duplicate in 60 mm dishes and the cells harvested after 48 hr. Following lysis by freeze thaw in 100 μl of 250 mM Tris HCl (pH 7.8) , cell debris was removed by centrifugation and the supernatant heated to 62°C to denature endogenous acyl transferases. Following further centrifugation,

a 50 μl aliquot of each supernatant was assayed for CAT activity by incubation with 0.1 μCi of ¹⁴ C chloramphenicol (NEN) and 0.34 mM acetyl CoA i 250 mM Tris-HCl in a final reaction volume of 140 μl for 45 minutes. The reaction was then terminated by extraction with 500 μl of ethyl acetate, evaporated under vacuum, and the resulting pellets were redissolved and subjected to thin layer chromatography on silica gel plates, using 5% methanol/95% chloroform (v/v) as solvent. Assays were quantitated using an AMBIS beta scanner.

9.1.2. MISCELLANEOUS TECHNIQUES Techniques in addition to the transformation assays as described in Section 8.1.1 are as those described, above, in Section 6.1.

9.2. RESULTS A mechanism whereby GRB-2-binding to BCR-ABL may potentiate oncogenic transformation is through direct stimulation of Ras via the Sos-1 guanine nucleotide exchange factor. To examine whether the interaction of BCR-ABL with GRB-2 feeds directly into the Ras pathway a transcriptional activation assay has been employed. Oncogenic Ras increases the rate of transcription from Ras-responsive elements { e . g. ets-1 and AP-1 DNA motifs; Hauser et al . , 1993, Methods in Enzymology, in press) . In addition, oncogenes with a wide range of functions, including protein tyrosine kinases and serine/threonine kinases can activate transcription from promoters containing Ras-responsive elements such as the ets/AP-1 motif (Schweighoffer et al . , 1992, Science 256.-825-827) . A correlation exists between the ability of various oncogenes to activate transcription from an ets/AP-1-containing promoter and

their capacity to transform cells (Wasylyk et al . , 1988, EMBO J. 2:2475-2483) . Thus, transactivation assays may complement the cell growth and tumorigenicity studies for the analysis of oncogene function.

Wild type and mutant forms of BCR-ABL were compared for their ability to activate transcription from ets/AP-1. A CAT reporter under the control of a _/ β-globin promoter that contains four tandem Ras- responsive elements (pB4X-CAT) . Oncogenic Ras has been shown to increase the rate of transcription from this element up to 15-fold (Schweighoffer, Science 25.6:825-827) . As shown in Figure 1, wild type BCR- ABL-induced activation is abolished by co-transfection of Ras (17N) , a dominant inhibitory mutant which has been shown to neutralize Ras function (Feig et al . , 1988, Mol. Cell. Biol. £:3235-3243; Thomas et al . , 1992, Cell 68:1031-1040; Wood et al . , 1992, Cell £8:1041-1050) indicating that BCR-ABL-induced transcriptional activation is mediated by Ras.

Significantly, the BCR-ABL mutants, P185 (Y177F) and P185 (Δ176-426) , that are deficient for GRB-2 binding, produced little, if any, effect on transcriptional transactivation from this promoter. The low level of transactivation obtained with the BCR-ABL mutants correlates with their decreased transforming activities compared to wild type P185 BCR-ABL ( Table 1, above) .

Further, infection of Ratl fibroblast cells with wild type BCR-ABL-containing retrovirus produces an increase in the fraction of Ras-GTP. Specifically, Ratl cells were infected with helper-free retroviral stocks for P185 BCR-ABL wild type or vector control. Two days post-infection, the cells were labeled with ³² P-orthophosphate (0.5 mCi/ml) for 16 hours in

phosphate-free medium. The cells were lysed and Ras- immunoprecipitated with anti-Ras monoclonal antibody. Guanine nucleotides bound to Ras were dissociated and subsequently separated by thin layer chromatography on PEI-cellulose plates in 0.75 M KH ₂ P0 ₄ , pH 3.5.

Quantitation of the amount of GDP and GTP bound to Ras with an AMBIS beta scanner shows a modest but reproducible increase.

10. EXAMPLE: SIGNALING INCOMPETENT GRB2

REVERSES THE PHENOTYPE OF TRANSFORMED CELLS

The following example shows that disruption of the signal transduction pathway involving BCR/ABL and

GRB2 can reverse a transformed phenotype in cells.

Elevated expression of signaling incompetent GRB2 mutants reverses BCR/ABL induced transformed growth in

Rat 1 cells. Disruption of the signaling pathway also inhibits the growth of cells isolated from a patient with chronic myelogenous leukemia (K562 cells) that express BCR/ABL as well as transformed cells dependant on the PDGF receptor.

10.1. MATERIALS AND METHODS Mutant GRB2 genes were constructed that deleted the SH3 domain on either the amino (N' ) or carboxy (C) terminus. Full length and truncated forms of GRB2 were inserted into the Bam HI site of a modified pCGN plasmid vector (Tanaka et al . , Cell £0:375-386, 1990) as described in Pendergast et al . , Cell 75 :175- 185, 1993. This vector has the hygromycin resistance gene inserted upstream of the SV40 origin of replication. To facilitate discrimination of the endogenous GRB2 protein from the transfected constructs, a sequence coding for the influenza virus hemagglutinin epitope (Pati, U.K., Gene 114:285-288,

1992) was fused in frame at the N terminus of the mutant proteins. The same antigenic tag was fused to the wild type GRB2 gene.

BCR/ABL transformed Rat 1 fibroblasts were established as described in Example 8.1.1 above. GRB2 constructs were transfected into Ratl-BCR/ABL cell lines and the rat glioma cell line C6 (ATCC CCL 107) using the standard calcium phosphate transfection protocol (Molecular Cloning Techniques, Laboratory Manual, 2nd Ed., Ed Sambrook et al . , Cold Spring

Harbor Laboratory Press, 1989 and Muller) . Following transfection, the Ratl cells were cultured for 24 hrs, then exposed to the drugs G418 + hygromycin (Sigma) for 7-9 days. The double drug selected mass population of cells were seeded in soft agar and the levels of endogenous BCR/ABL, GRB2 and transfected GRB2 determined using Western blot. Individual clonal cell lines expressing the different transfected GRB2 constructs were also established. In the case of transfected C6 cells, cells were selected in hygromycin containing media for 10 days as described in Muller, supra . Following drug selection, cells were seeded in soft agar medium containing 0.5% FCS and PDGF (lng/ml) . Colonies >0.4 mm were counted on day 10.

Expression of the GRB2 constructs was evaluated by immunoprecipitation as described in Pendergast et al., Cell 75:175-185. 1993. Briefly, BCR/ABL-GRB2 expressing Ratl cells were lysed and immunoprecipitated with anti-ABL pEx4 antibodies and the immunoprecipitates were subjected to an in vi tro autokinase assay (Pendergast et al . , supra) . The samples were analyzed by SDS-PAGE gel and visualized by autoradiography. In a second experiment, cells were lysed in 10 mM Tris-HCl, ph 7.4, 1% SDS, 1 mM

PMSF, 15 μg of protein from each sample was separated using SDS-PAGE (15%) . The proteins were electrophoretically transferred to nitrocellulose filters and immunoblotted with an anti-GRB2 mouse monoclonal antibody (Transduction Laboratories) followed by incubation with goat anti-mouse Mab conjugated to horse-radish peroxidase (BioRad) . Proteins were visualized with the Enhanced Chemiluminescence detection system (Amersham) . Soft agar assays were performed as previously described (Lugo et al . , Mol. Cell. Bio. 2:1263-1270, 1989) . Mass populations of drug selected cells were seeded at densities ranging between 10 ³ - 5xl0 ⁴ cells/6 cm ² dish depending on the cloning efficiency of each cell type. Samples were plated in duplicate in medium containing 20% fetal calf serum. Macroscopic colonies (>0.4mm) were counted after 14 days. The data represents the average number of colonies observed in 3 to 5 independent experiments performed with mass populations of transfected and drug selected cells.

Data for the growth of Ratl-BCR/ABL was derived using three independent Ratl-BCR/ABL clonal cell lines transfected with the appropriate constructs and repeated in 2 to 3 separate experiments for each cell line. Thus the data represents the average number of colonies observed in 7 independent experiments.

K562 (ATCC CRL 243) cells were transfected with the GRB2 constructs using standard methods of electroporation. Following transfection, cells were cultured for 18 to 24 hrs, then exposed to hygromycin (500 μg/ml) for 2 days. After drug selection numbers of viable cells were determined by counting cells which exclude the dye trypan blue. Equal numbers of viable cells were seeded in soft agar.

10.2. RESULTS

10.2.1. EXPRESSION OF GRB2 IN RAT 1 CELLS

,_ Expression of the N'GRB2 mutant protein in Ratl- BCR/ABL cells causes a reversion to a normal phenotype. The mass population of N' GRB2 mutant expressing cells grew as a monolayer in liquid culture and exhibited contact inhibition when reaching m m confluence. Significantly, N'GRB2 mutant expressing Ratl-BCR/ABL cells exhibited a dramatic reduction in their ability to form colonies in soft agar when compared to cells expressing the empty vector (see Table 2) . Cells expressing the CGRB2 mutant

15 expressing cells grew as monolayers in liquid culture but did not quiesce when confluent. Growth of CGRB2 mutant expressing Ratl-BCR/ABL fibroblasts in soft agar was also suppressed, albeit to a lesser extent than that observed for the N' GRB2 mutant. Because

20 comparable ratios of mutant proteins to wild type GRB2 protein were present in both the N' and C expressing cell population, the greater potency of the N' mutant to suppress BCR/ABL induced transformation is probably not simply due to differences in the level of the two

₂₅ mutants. Specifically, Ratl cells expressing p210

BCR-ABL alone or also expressing wild type GRB-2 or a truncated GRB-2 were lysed and immunoprecipitated with anti-ABL antibody. Immunoprecipitates were subjected to an in vitro autokinase assay, and the samples were

₃₀ analyzed by SDS-PAGE (8% gel) . No change in ABL kinase activity was seen in cells with elevated GRB-2 expression compared to control. Further, Ratl cells expressing p210 BCR-ABL alone or also expressing wild type GRB-2 or a truncated GRB-2 were lysed and

.,.- proteins from the cloned cell lysates were electrophoretically transferred to nitrocellulose

filter and then immunoblotted with anti-GRB-2 MAb. All GRB-2 transformants expressed approximately the same levels of GRB-2 protein. Rather these differences may indicate that the two SH3 domains bind SOS with different affinities or bind to different substrates. Thus GRB2 SH3 deletion mutant proteins act as dominant negative inhibitors of BCR/ABL induced transformation when present in equimolar or greater amounts than the endogenous wild type GRB2 in the cell.

In contrast to the N' or C GRB2 mutants, elevated expression of wild type GRB2 in Ratl-BCR/ABL fibroblasts appeared to accentuate the transformed phenotype. The drug selected mass population of cells expressing the transfected wild type GRB2 construct consistently exhibited a 25-30% increase in the number of colonies in soft agar when compared to cells transfected with the empty vector (Table 2) . These data suggest that the GRB2 protein may be a limiting effector in BCR/ABL induced transformation of Rat 1 fibroblasts.

Table 2

Construct Ratl K562 C6 empty vector 566 187 520 wild type GRB2 734 209 577

N' truncated GRB2 14 13 2

C truncated GRB2 119 86 3

(# Colonies/lXlO ⁴ cells seeded)

10.2.2. EXPRESSION OF GRB2

MUTANTS IN K562 CELLS

The SH3 GRB2 mutants also inhibit the growth of human leukemic cells. The GRB2 constructs were introduced into K562 cells, a p210 BCR/ABL expressing cell line established from a CML patient in blast crisis. Following transfection and 48 hrs of drug selection, levels of endogenous BCR/ABL, GRB2 and transfected GRB2 were determined by Western blot analysis. At the same time, viable cells were seeded in soft agar. Cultures seeded with K562 cells expressing the N'GRB2 mutant protein showed 10 times fewer the number of soft agar colonies when compared to mock transfected controls (Table 2) . Analogous to what was observed in the Ratl-BCR/ABL fibroblasts, the

CGRB2 mutant also inhibited K562 colony formation, but to a lesser degree than the N' mutant. These data indicate that signal transduction mediated by GRB2 is an essential component in BCR/ABL induced human malignancies.

10.2.3. EXPRESSION OF GRB2 IN C6 GLIOMA CELLS

The GRB2 constructs were also introduced into rat

C6 glioma cells, which are dependant on PDGF receptor

activity for their transformed growth phenotype (Zhang et al., Neurol. Res. 1 (5) .397-401. 1992) . PDGF receptor has been shown to interact with GRB2 (Lowenstein et al _* , 1992, Cell 2^:431-442) . As shown 5 in Table 2, expression of the signaling incompetent GRB2 mutants significantly inhibited colony formation. This data demonstrates that inhibition of GRB2 signal transduction is can be used to inhibit transformed cell growth that is dependant on receptor tyrosine 10 kinase activation.

11. EXAMPLE: SIGNALING INCOMPETENT GRB2

PREVENTS TUMOR GROWTH IN AN ANIMAL MODEL

This example demonstrates that cells expressing

- m signaling incompetent GRB2 are no longer able to form tumors in animals, suggesting that disruption of the

BCR/ABL signaling pathway can be used to treat cancer in mammals.

20 11.1. MATERIALS AND METHODS GRB2 (wild type and mutants) and BCR/ABL expressing Rat 1 cells were prepared as described in Section 10.1, above. Nude mice (4 mice per group) were injected subcutaneously in the left flank with

25 Rat 1 cells co-expressing p210 BCR/ABL and either wild type GRB2, N' truncated GRB2 or C truncated GRB2. Cells expressing p210 BCR/ABL alone (2 x 10 ⁶ cells in 100 μl) were injected subcutaneously on the right hind leg of each mouse to act as an internal control . The

30 mice were sacrificed at 3 weeks post-implant and the tumor volume measured.

11.2. RESULTS The results shown in Table 3 clearly demonstrate that expression of signaling incompetent GRB2 proteins

35 reduces growth of tumor cells in vivo.

TABLE 3

Group left right wild type GRB2 0.5 0.5

N' truncated 0.3 0.7 GRB2

C truncated <0.1 0.7 GRB2

(data shown is average tumor size per group)

12. EXAMPLE: EFFECTS OF SIGNALING

INCOMPETENT GRB2 ON RAS ACTIVATION

This example shows that the signaling incompetent

GRB2 molecules described herein inhibit BCR/ABL induced ras activation in proportion to their transformation inhibitory potency suggesting that interaction between GRB2 and downstream signaling components that lead to ras activation is disrupted.

12.1. MATERIALS AND METHODS Transcriptional activation of expression from a Ras-responsive element (ets/AP-1) promoter was done as described in Section 9.1, above. Transformation of Ratl cells, soft agar assays and immunoblotting experiments were performed as described in Section 10.1, above.

12.2. RESULTS To determine whether the growth inhibitory effect of the GRB2 mutants was due to their ability to block ras activation, lysates prepared from Ratl-BCR/ABL cell lines hyperexpressing wild type or mutant GRB2 proteins were evaluated for their ability to activate ras. Using a ras dependent transcription activation assay, we observed that lysates from N' GRB2 expressing Ratl-BCR/ABL cells exhibited a 10-15 fold

lower ras activation activity than mock transfected controls (Figure 2) . Lysates from C GRB2 expressing cell lines also had a decrease in their ras activation activity, albeit to a lesser extent than that of the N' mutant. The difference in the inhibitory effect of the N' and C mutants is consistent with their potency to reverse the BCR/ABL transformed phenotype.

If the GRB2 mutant proteins function by blocking intermediate steps in the signal transduction pathway prior to ras, then transformation by genes which circumvent ras activation should not be effected. To test this hypothesis, the GRB2 constructs were transfected into Rat 1 cells transformed with an activated form of raf, a serine kinase which elicits its mitogenic effect downstream of ras in the same signaling pathway (Kolch et al . , Nature 349 :426. 1991) . Following drug selection with hygromycin the mass population of cells were seeded in agar. Ratl/v- raf cells expressing N' or C GRB2 mutants gave rise to the same number of colonies in soft agar as the mock transfected control cells (Table 4) . Furthermore, cell lines expressing a greater than two fold excess of SH3 mutant than endogenous wild type GRB2 protein were not inhibited in their ability to grow in soft agar. These data suggest that the GRB2 mutant proteins inhibit the BCR/ABL mitogenic signal by uncoupling the signal transduction pathway upstream of raf.

TABLE 4

Construct Ratl/v-raf empty vector 620 wild type GRB2 665

N' truncated GRB2 653

C truncated GRB2 598

(# Colonies/lXlO ⁴ cells seeded)

13. EXAMPLE: GRB2 MUTANTS INHIBIT GROWTH AND INDUCE DIFFERENTIATION OF THE HUMAN Ph ¹ - POSITIVE LEUKEMIC CELL LINE, K562.

In the Example presented below, it is demonstrated that SH3 Grb2 mutant proteins not only exhibit the ability to reverse the BCR/ABL induced oncogenic phenotype in hematopoietic cells, but also that such mutants are capable of allowing erythroid differentiation of K562 cells to occur.

13.1 MATERIALS AND METHODS Transformation Assays. Soft agar assays were, performed as described, above, in Section 8.1.1. Equal numbers of viable cells were seeded in soft agar at densities ranging between 10 ³ to 5 x 10 ⁴ cells per 6 cm ² dish depending on the cloning efficiency of each cell type. Samples were plated in duplicate in medium containing 20% fetal calf serum. Macroscopic colonies (>0.4mm) were counted after 14 days. Colony numbers were adjusted for 10 ⁴ cells total.

Transcriptional Activation Assay. Transcriptional activation of expression from a Ras- responsive element (ets/AP-1) promoter was performed as described previously (Pendergast et al. , 1993). Briefly, normal Ratl cells and transformed Ratl/BCR-

ABL cells expressing the GRB2 proteins were transfected with 1 μg of pB4x-CAT reporter plasmid. At 48 hours post-transfection cells were harvested and assayed for CAT activity as described, above, in Section 9.1.1.

Binding Assays, In Vitro Autophosphorylation and Immunoblotting. Procedures for in vitro binding assays, in vitro labeling with [γ ³² p] ATP and immunoblotting were carried out essentially as described, above, in Sections 6.1.6 and 6.1.7.

Hemoglobin Protein Determinations. The amount of hemoglobin present in mass populations of transfected K562 cells was determined essentially as described (Feinstein, E. et al. , 1992, Oncogene 2:1853-1857) . In brief, lysates were prepared from each cell population and the total amount of protein determined using the Bradford assay. For each sample, 20 μg of total cellular protein were mixed with 1 % benzidine solution in 90% acetic acid and freshly prepared hydrogen peroxide. After incubation for 20 minutes at room temperature, 10% acetic acid solution was added and the optical density determined at 515 nm. Human hemoglobin was used as a standard for comparison. Tumor Growth Assay. The tumorigenicity of Ratl/BCR-ABL fibroblasts was evaluated essentially as described (Lugo et al . , 1989, Mol. Cell. Biol. 2:1263-1270) . In brief, 2xl0 ⁶ cells from mass populations of Ratl/BCR-ABL fibroblasts transfected with wild type or mutant GRB2 constructs were inoculated into the left hind flanks of nude mice. As an internal control, each mouse was also inoculated with Ratl/BCR-ABL cells transfected with the empty vector on the right hind flank. Four mice were evaluated for each Ratl/BCR-ABL cell population. Three weeks post inoculation, the mice were sacrificed

and the size of the tumors on the left and right flanks compared and recorded as grams wet weight .

13.2 RESULTS In the experiments described below, it was evaluated whether the SH3 GRB2 mutant proteins could reverse the BCR/ABL induced oncogenic phenotype in hematopoietic cells. The GRB2 constructs were introduced into Pt^-positive K562 cells. Following transfection and 48 hours of culture in the presence of hygromycin, the levels of endogenous and transfected GRB2 proteins were determined by immunoblot analysis. At the same time, cells were harvested from liquid culture and equal numbers of viable cells were seeded in soft agar. In each experiment the level of the transfected GRB2 mutant proteins were at least five times greater than endogenous GRB2. Cultures seeded with K562 cells expressing then ΔN-GRB2 mutant protein showed 10 times fewer colonies in soft agar when compared to mock transfected controls (Table 2, above) . Analogous to what was observed in the Ratl/BCR-ABL fibroblasts, the ΔC-GRB2 mutant also inhibited K562 colony formation, but to a lesser degree than the ΔN-GRB2 (Table 2, above) . These data strongly suggest that BCR-ABL induced Ras activation mediated by GRB2 is an essential component in BCR-ABL induced human malignancy.

Although K562 cell lines which stably express elevated levels of wild type GRB2 protein can be maintained in culture, it has not been possible to establish clonal cell lines expressing ΔN- or ΔC-GRB2 proteins. K562 cells retain their ability to differentiate and synthesize hemoglobin (Anderson L.C. et al., 1979, Int. J. Cancer 2: 143-147; Rowley, P.T.

et al., 1981, Exp. Hematol. 9_ : 32-37; Feinstein, E. et al., 1992, Oncogene 1 : 1853-1857) . Previous studies have demonstrated that drugs which inhibit tyrosine kinase activity can be used to allow erythroid 5 differentiation of K562 cells (Fukazawa, H. et al. , 1991, Biochem. Pharm, 4_2: 1661-1671; Anafi, M. et al . , 1993, Blood £2: 3524-3529) .

To determine whether expression of the GRB2 mutants allow a similar differentiation process, a 0 portion of the transfected K562 cells used in the soft agar assays were lysed and the amount of hemoglobin protein was determined for each transfected cell population. A dramatic increase was observed in the level of hemoglobin protein in cells expressing either 5 the ΔN- or ΔC-GRB2 mutants. In contrast, K562 cells expressing elevated levels of wild type GRB2 protein showed no increase in hemoglobin when compared to mock transfected controls. Thus, the GRB2 mutants utilized herein are not only capable of reversing BCR/ABL 0 transformation phenotype, but also allow erythroid differentiation in K562 cells. These data suggest that BCR-ABL may transform hematopoietic cells in part by blocking normal differentiation in a Ras-dependent manner. 5

13. EXAMPLE: SCREENING ASSAY FOR THE IDENTIFICATION OF COMPOUNDS THAT DISRUPT PROTEIN/TYROSINE KINASE INTERACTIONS

The Example presented herein describes a means ₀ for assessing the potential of a test substance to inhibit the interaction between binding partner complexes. Such binding partners may, for example include adaptor proteins and molecules which bind to such adaptor proteins, such as, for example, activated _ς tyrosine kinase molecules and other proteins such as the SHC protein. Compounds identified herein may be

capable of modulating cell growth control, and may also be capable of regulating oncogenesis and cell proliferative disorders.

In the specific assay described herein, an adaptor-GST fusion protein capable of binding to a phosphorylated tyrosine kinase protein is incubated with the phosphorylated tyrosine kinase protein, which has been immobilized on a solid phase, in the presence of a test substance. When the test substance is capable of inhibiting the interaction between the adaptor protein-GST fusion protein and the tyrosine kinase molecule, it causes a detectable decrease in the amount of adaptor-GST fusion protein bound to the immobilized tyrosine kinase molecule, relative to the amount of adaptor-GST fusion protein bound to the tyrosine kinase molecule in the absence of the test substance.

This example is illustrated in detail for the screening of inhibitors of the interaction between GRB-2 and EGF-R. The same principles can be applied, however, to the detection of inhibitors of the interaction between any tyrosine kinase protein or tyrosine phosphorylated substrates of tyrosine kinases (e.g. , SHC, Phosphatase ID) and any adaptor protein with which it interacts.

Adaptor-GST fusion protein: The adaptor-GST (glutathione-S-transferase) fusion proteins used herein were GRB-2-GST fusion proteins prepared by expression in E. coli transformed with GRB-2/pGEX constructs. The GRB-2 portions of these fusion proteins consisted of only the SH2 domain of the GRB-2 protein. Transformed cells are grown in Luria broth (LB) supplemented with ampicillin. After reaching an optical density (OD) at 600 nm of 0.3, the cells are

induced for 6 hours with isopropyl β-D- thiogalactopyranoside (IPTG) in order to express the fusion protein.

After the 6 hour expression period, the cells are precipitated, pelleted at 10,000 x g for 10 minutes at 4°C, washed, and resuspended in phosphate buffered saline (PBS) . Next, the cells are lysed by sonication (6 strokes, 5 seconds per stroke) . Insoluble material is removed by centrifugation at 10,000 x g for 10 minutes at 4°C, and the supernatant is passed over a Glutathion-Sepharose column. Bound GRB-2-GST fusion protein is eluted off the column with 5 mM reduced glutathion, then dialyzed against PBS.

Immobilized tyrosine kinase molecule: The tyrosine kinase molecule used herein is the epidermal growth factor receptor tyrosine kinase (EGF-R) . EGF-R is isolated from cells overexpressing EGF-R, such as the A431 (ATCC CRL 1551), cell line. The cells are lysed in HNTG buffer (20 mM Hepes/HCl, pH 7.4, 150 mM NaCl, 1.0% Triton X-100, 5% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF) , 1 mg/L aprotonin, 1 mg/L leupeptin, 10 mg/L benzamidine) .

EGF-R protein is isolated from the cell lysates by immobilization onto microtiter plates, as described below. EGF-R is subsequently phosphorylated in vitro as explained below.

The EGF-R molecule is immobilized onto microtiter plates. Microtiter plates are prepared by first coating the wells of the plate, overnight at 4°C, with an anti-EGF-R monoclonal antibody directed against the extracellular domain of EGFR (UBI, #05-101) at a concentration of 0.5 μg (in PBS) per microtiter well, at a final volume of 150 μl per well.

After overnight coating, the coating solution is removed from the microtiter wells, and replaced with blocking buffer (5% dry milk in PBS) for 30 minutes at room temperature, after which the blocking buffer is removed and the wells are washed 4 times with TBST buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.2, 0.1% Triton X-100) .

Cell lysate from EGF-R-expressing cells is added to each well, in 150 μl of PBS, incubated 30 minutes at room temperature, with shaking. Unbound EGF-R is removed by washing wells 5 times with TBST buffer. Approximately 50-100 ng of EGF-R protein is bound per well.

It is important to use an EGF-R overexpressing cell line which exhibits a high endogenous phosphatase activity, such as, for example, the A431 cell line. This is because during lysis and incubation with the immobilized antibody, the phosphatases remove phosphate groups from the EGF-R molecules, thus prohibiting endogenous adaptor proteins, such as GRB proteins, to bind EGFR, which could potentially lead to artifactual results. Alternatively, cells may be starved before lysis, if the cell line utilized may be readily starved.

Preparation of autophophorylated EGF-R: The following in vitro kinase reaction yielded autophosphorylated EGF-R. The kinase reaction was initiated by the addition of 15 μl of ATP/Mn ²⁺ mix (in 50 mM MnCl ₂ , final concentration of 10 μM ATP, for a total volume of 150 μl. The plate was incubated for 5 minutes at room temperature, shaking, the supernatent was aspirated, and the plates were then washed 5 times with TBST.

Assay procedure: Either 30 ng GRB-2-GST fusion proteins (i.e. a 1:1 ratio of EGF-R:GRB-2 proteins) or 5 ng GRB-2-GST fusion proteins (i.e. a 4:1 ratio of EGF-R:GRB-2 proteins) are added to the phosphorylated EGF-R coated microtiter wells in incubation buffer (0.1 M potassium phosphate buffer, pH 6.5) for 30 minutes, at room temperature, in the presence of a test substance dissolved in dimethyl sulfoxide (DMSO) . Control wells are incubated with GRB-2-GST fusion proteins in the absence of test substance.

After incubation, wells are washed extensively with TBST. The amount of GRB-2-GST fusion protein bound to the immobilized EGF-R is then preferably determined by with a purified rabbit antiserum against the GST-moiety of the fusion protein (AMRAD, New Victoria, Australia; Catalog No. 00001605) . Incubations are for 30 minutes at room temperature. After incubation, antibody is removed and the wells are washed extensively with TBST. For visualization, wells are next incubated with a TAGO goat-anti-rabbit peroxidase antibody at room temperature for 30 minutes. After incubation, the antibody is removed, the wells are washed with tap water, and then with TBST. Substrate solution, ABTS (2, 2' -Azinobis (3- ethylbenzthiazolinesulfonic acid) /H ₂ 0 ₂ (1.2 μl H ₂ 0 ₂ to 10 ml ABTS) is applied to the wells, and incubated for 20 minutes at room temperature. The reaction is stopped by addition of 5NH ₂ S0 ₄ . The O.D. at 410 nm is determined for each well. Utilizing this technique, it is possible to detect as little as 2 ng GRB-2-GST over background.

Alternatively, after incubation of the test substance and the GRB-2-GST fusion protein on the EGF-R wells, biotinylated monoclonal antibodies e.g. , EL-6 or EL-12, may be utilized to assay fusion protein

binding. The epitopes recognized by such antibodies map on the SH2 domain of GRB-2, but do not interfere with GRB-2 binding to phosphorylated EGFR. Binding of these antibodies is then determined by using a streptavidin-biotinylated horseradish peroxidase reactant.

Additionally, after incubation of the test substance and the GRB-2-GST fusion protein on the EGF-R wells, binding of the fusion protein to the immobilized EGFR may be assayed by incubating with 1 mM l-chloro-2,4 dinitrobenzene (CDNB) and 1.54 mg/ml reduced glutathion in incubation buffer. The OD is then measured at 340 nm. This reaction is linear up to OD 1.0, and can be stopped with competitive GST inhibitors, as described in Mannervik and Danielson

(Mannervik, B. and Danielson, U.H., 1988, CRC Critical Reviews in Biochemistry .:238) .

It is apparent that many modifications and variations of this invention as set forth here may be made without departing from the spirit and scope thereof. The specific embodiments described hereinabove are given by way of example only and the invention is limited only by the terms of the appended claims.