ZAP 70 INHIBITORS AND METHODS FOR TREATMENT OF

ZAP 70 SIGNAL TRANSDUCTION DISORDERS

Field of the Invention

The present invention relates generally to the field of cellular signal transduction and more specifically to products and methods related to the treatment of various diseases and conditions associated with abnormal cellular signal transduction pathways.

Background of the Invention The following discussion of background art is not admitted to be prior art to the invention. Cellular signal transduction is a fundamental mechanism whereby external stimuli that regulate diverse cellular processes are relayed to the interior of cells. One of the key biochemical mechanisms of signal trans¬ duction involves the reversible phosphorylation of tyro- sine residues on proteins. The phosphorylation state of a protein is modified through the reciprocal actions of tyrosine kinases (TKs) and tyrosine phosphatases (TPs) . Receptor tyrosine kinases (RTKs) belong to a family of transmembrane proteins and have been impli- cated in cellular signaling pathways. The predominant biological activity of some RTKs is the stimulation of cell growth and proliferation, while other RTKs are involved in arresting growth and promoting differentia¬ tion. Ligand binding to membrane-bound receptors induces the formation of receptor dimers and allosteric

changes that activate the intracellular kinase domains and result in the self-phosphorylation

(autophosphorylation and/or transphosphorylation) of the receptor on tyrosine residues. Their intrinsic tyrosine kinase is activated upon ligand binding, thereby initiating a complex signal transduction pathway that begins with receptor autophosphorylation and culminates in the tyrosine phosphorylation of a variety of cellular substrates and ultimately in the initiation of nuclear events necessary for the overall cell response

(Schlessinger and Ullrich, Neuron 9:383-391, 1992) . Individual phosphotyrosine residues of the cytoplasmic domains of receptors may serve as specific binding sites that interact with a host of cytoplasmic signaling mole- cules, thereby activating various signal transduction pathways (Ullrich and Schlessinger, Cell 61:203-212, 1990) .

The intracellular, cytoplasmic, non-receptor protein tyrosine kinases do not contain a hydrophobic transmembrane domain or an extracellular domain and share non-catalytic domains in addition to sharing their catalytic kinase domains. Such non-catalytic domains include the SH2 domains (SRC homology domain 2; Sadowski et al., Mol . Cell . Biol . 6:4396-4408; Koch et al . , Science 252:668-674, 1991) and SH3 domains (SRC homology domain 3; Mayer et al . , Na ture 332:269-272, 1988) . The non-catalytic domains are thought to be important in the regulation of protein-protein interactions during signal transduction (Pawson and Gish, Cell 71:359-362, 1992) . A central feature of signal transduction (for reviews, see Posada and Cooper, Λol . Biol . Cell 3:583-

392, 1992; Hardie, Symp . Soc . Exp . Biol . 44:241-255, 1990) , is the reversible phosphorylation of certain proteins. Receptor phosphorylation stimulates a physi¬ cal association of the activated receptor with target molecules. Some of the target molecules such as phos- pholipase Cy are in turn phosphorylated and activated. Margolis et al . , Cell 57:1101-1107, 1989; Margolis et al., Science 248:607-610, 1990; Nishibe et al . , Science 250:1253-1255, 1990; and Kim et al., Cell 65:435-411, 1991. Such phosphorylation transmits a signal to the cytoplasm. Other target molecules are not phosphorylated, but assist in signal transmission by acting as adapter molecules for secondary signal trans¬ ducer proteins. For example, receptor phosphorylation and the subsequent allosteric changes in the receptor recruit the GrJb-2/SOS complex to the catalytic domain of the receptor where its proximity to the membrane allows it to activate ras . Pawson and Schlessinger, Current Biol . 13:434, 1993. The secondary signal transducer molecules gen¬ erated by activated receptors result in a signal cascade that regulates cell functions such as cell division or differentiation. Reviews describing intracellular sig¬ nal transduction include Aaronson, Sci ence , 254:1146- 1153, 1991; Schlessinger, Trends Biochem. Sci . , 13:443- 447, 1988; and Ullrich and Schlessinger, Cell , 61:203- 212, 1990.

The differentiation of T cells is characterized by a series of phenotypic changes (e.g.. double negative to double positive to single positive) as thymocytes progress from the outer cortex of the

thy us to the medulla. During this process of T cell development (reviewed in Sites, D. et al . , Basic & Clinical Immunology, 8th ed., chapter 8 by Imbouden, J. Jr., pages 94-101) cortical thymocyte precursor cells that lack surface expression of the CD4 or CD8 coreceptors (double-negative cells) rearrange their αβ T cell antigen receptor(TCRαβ) genes and coexpress the CD4 and CD8 coreceptor molecules (double-positive cells) that reside in the thymic cortex. The double-positive cells are the immediate precursors of the more mature CD4 and CD8 single-positive cells that populate the thymic medulla.

The transition from double-positive cells to single-positive cells is thought to require the interaction of the TCRαβ molecules with major histocompatibility complex (MHC) in the process of positive selection. Thus, TCRα and TCRβ genes are rearranged and expressed so that the mature CD4* cells and CD8 ⁺ cells express TCR molecules that interact with self-MHC class II and class I molecules, respectively.

This positive selection process requires the transduction of specific signals from the TCR and CD4 or CD8 coreceptors to the cell nucleus. The CD4 and CD8 coreceptor molecules enhance TCR recognition by binding to MHC class II or class I at different sites than the TCR-binding site. However, it has remained unclear which of the various molecules that can potentially participate in the transduction of TCR signal are critical for the various individual steps of intra- thymic T cell differentiation and the molecular

identities of the signals mediated during these selection events are still largely obscure.

A novel type of human immunodeficiency is characterized by a selective T cell defect (STD) . Peripheral circulating T cells from these patients exclusively express CD4 , CD3 , and TCRαβ but not CD8 molecules on their surface. The inability to produce peripheral CD8 single-positive cells was traced to an intrathymic development defect. While CD4* and CD8 double-positive cells were present in the thymic cortex of these patients, only CD4, not CD8, single-positive cells could be detected in the thymic medulla, suggesting a selective block of positive selection of CD8* cells. Peripheral CD4' T cells from STD patients failed to proliferate in response to mitogens or to treatment with anti-CD3 antibody. However, bypassing the TCR signaling system by stimulation with phorbol esters and ionomycin restored a normal mitogenic response as measured by thymidine incorporation. These observations raised the possibility that STD results from defective TCR signaling at a step proximal to the TCR itself.

The earliest biochemical event following ligand binding of the TCR complex is the rapid activation of protein-tyrosine kinases (PTKs) , which results in phosphorylation of multiple cellular substrates. Current studies implicate three PTKs in TCR function, Fyn and Lck of the Src family and a recently identified cytoplasmic PTK, Zap-70. All three PTKs can associate with various components of the TCR system. It

is beleived that both Fyn and Lck play important roles in TCR-mediated signal transduction.

Zap-70 kinase, which is present in T cells but not in B cells, is associated with the tyrosine- phosphorylated ζ chain of the TCR complex. The function of this kinase seems to be dependant upon the coexpression of Lck or Fyn. The isolation of a cDNA clone encoding ZAP-70 is described in Chan et al . , Cell . 71:649-662 (1992) , incorporated herein by reference in its entirety, including any drawings. T cell activation by clustered tyrosine kinases is described in Kolanus et al . , Cell. 74:171-183 (1993) , incorporated herein by reference in its entirety, including any drawings. Recent papers discussing Zap 70 include Arpaia et al, Cell. 76:947-958 (1994) ; Chan et al . , Science. 264:1599- 1601 (1994) ; Elder et al . , Science. 264:1596-1599 (1994) , and Howe L and Weiss A., TIBS. 20(2) :59-64 (1995) all of which are incorporated herein by reference in their entirety, including any drawings. Various cell proliferative disorders, including cancer (e.g., nonsmall cell lung cancer,- primary human breast cancer; carcinomas of the cervix, ovaries, esophagus, and stomach) and neoplastic transformation, have been associated with defects in different signaling pathways mediated by receptor tyrosine kinases. Zeillinger et al . , Clin. Biochem. 26:221-227, 1993. Examples of specific receptor tyrosine kinases associated with cell proliferative disorders include, platelet derived growth factor receptor (PDGFR) , epidermal growth factor receptor (EGFR) , and HER2 (HER2) .

A review of immunosuppressive therapy is provided in Sites, D., et al . , Basic & Clinical Immunology, 8th ed., chapter 58 by Winkelstein, A., pages 765-780. One novel immunosuppressant described therein is cyclosporin which acts by blocking mitogenic signal transduction and that has become the standard drug for inhibition of allogeneic transplant rejection. Compounds able to inhibit the activity of receptor tyrosine kinases have been mentioned in various publications. For example, Gazit et al . , J. Med. Chem. 34:1896-1907 (1991) , examined the receptor tyrosine kinase inhibitory effect of different tyrphostins. In a later publication Gazit et al . , J. Med. Chem. 36:3556- 3564 (1993) describe tyrphostins having a 5-aryl substituent in the 5 position. Osherov et al., Journal of Biological Chemistry 268:11134, 1993, mentions the development of two groups of tyrphostins. Additional methods and compositions for inhibiting cell proliferative disorders are described in U.S. Patent Application Serial No. 08/207,933, filed March 7, 1994, (hereby incorporated by reference herein in its entirety including any drawings) .

SUMMARY OF THE INVENTION

The present invention relates to methods of assaying for agents useful for inhibiting immune disorders. The described methods are particularly useful for detecting agents which inhibit autoimmune disorders characterized by over-activity and/or inappro¬ priate expression of a tyrosine kinase such as Zap 70.

Methods to screen for agents or compounds which can be used to inhibit Zap 70 activity are also provided.

The present invention also relates to products and methods related to Zap 70 kinases. It has been determined that Zap 70 kinases are involved in a protein-protein interaction of therapeutic importance. This interaction is associated with the basic signalling function of proteins associated with various disorders and signal transduction pathways. Thus the present invention provides several agents and methods useful for diagnosing, treating, and preventing various disorders associated with abnormalities in these pathways.

In particular, this invention relates to methods for diagnosis and treatment of a disorder, most preferably a disorder characterized by an abnormality in a signal transduction pathway, wherein the signal transduction pathway involves the interaction between a Zap 70 kinase and a Zap 70 binding partner. This invention thus relates to methods for promoting and/or inhibiting the interaction between a Zap 70 kinase and a Zap 70 binding partner. In addition, it has been determined that disruption or promotion of the interaction between a Zap 70 kinase and a Zap 70 binding partner is useful in therapeutic procedures. In addition to use as therapeutics, additional uses of the agents include use for in vi tro studies to determine the mechanism of action of tyrosine kinases, preferably Zap 70; use as lead compounds to design and screen for additional compounds having tyrosine kinase inhibitory activity; and use to help diagnose the role of a tyrosine kinase in a disorder, preferably an immune or

autoimmune disorder. For example, using standard assays, the active site of the kinase acted upon by any one of the compounds described herein may be determined, and other compounds active at the same site determined. Recombinant cell lines for selective screening for Zap 70 specific inhibitors are also described herein.

Thus, in a first aspect the invention features a method for identifying an agent capable of inhibiting transduction of a Zap 70 kinase signal. The method includes exposing a potential agent to a Zap 70 kinase and detecting any change in a level of transduction of the Zap 70 kinase signal.

By "identifying" is meant one or more chemical agents are tested in the method of the invention to determine their activity in inhibition of signal transduction. Those agents which inhibit by at least 50% (preferably 90%, more preferably 95%) the level of signal transduction in the absence of such an agent are potentially useful for treatment of a proliferative disorder. Thus, the identifying may encompass a single test of a single agent or a plurality of tests of a plurality of agents. The level of signal transduction can be measured in a variety of ways well known to those skilled in the art. For example, the presence or amount of phosphorylation of a given protein (such as a Zap 70 protein) can be used to measure signal transduction. By "inhibiting" is meant that the level of signal transduction is reduced at least 50% (preferably at least 90%, more preferably at least 95%) by the agent in the assay performed.

By "transduction of a Zap 70 kinase signal" is meant the passage of a molecular signal to or from the Zap 70 kinase to or from one or more molecules in the signal cascade within the cell. For example, such a signal may be transduced dependent upon the level of phosphorylation of the Zap 70 kinase. As noted above, other molecules which may assist in signal transmission may be blocked by agents of this invention and thus reduce signal transduction. By "Zap 70 kinase" is meant two (preferably 7, more preferably 13, most preferably 25) or more contiguous amino acids set forth in the full length amino acid sequence of Zap 70 as described in Figure 3 of Chan et al . , Cell, 71:649-662 (1992) , incorporated herein by reference in its entirety, including any drawings. The Zap 70 kinase can be encoded by a full- length nucleic acid sequence or any portion of the full- length nucleic acid sequence, so long as a functional activity of the kinase is retained. The term "Zap 70 kinase" is meant to include the functional derivatives described herein. In preferred embodiments the "Zap 70 kinase" has an amino acid sequence substantially similar to the sequence of Chan et al . , supra, or active fragments thereof . A sequence that is substantially similar will have at least 70% identity (preferably at least 80% and most preferably 90-100%) to the sequence of Chan et al . , supra. By "identity" is meant a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100. Thus, two

copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved and have deletions, additions, or replacements may have a lower degree of identity. By "signal transduction pathway" is meant the sequence of events that involves the transmission of a message from an extracellular protein to the cytoplasm through a cell membrane. The signal ultimately will cause the cell to perform a particular function, for example, to uncontrollably proliferate and therefore cause cancer. Various mechanisms for the signal trans¬ duction pathway (Fry et al . , Protein Science, 2:1785- 1797, 1993) provide possible methods for measuring the amount or intensity of a given signal . Depending upon the particular disease associated with the abnormality in a signal transduction pathway, various symptoms may be detected. Those skilled in the art recognize those symptoms that are associated with the various other diseases described herein. Furthermore, since some adapter molecules recruit secondary signal transducer proteins towards the membrane, one measure of signal transduction is the concentration and localization of various proteins and complexes. In addition, conforma- tional changes that are involved in the transmission of a signal may be observed using circular dichrois and fluorescence studies.

In preferred embodiments, the inhibition of the signal transduction is by inhibition of the enzymatic activity of Zap 70, for example, by agents with molecular weight less than 3000, preferably less than 1500 such as quinazolines, tyrphostins,

quinoxalines, and extracts from natural sources. Other agents may act by inhibiting the interaction of the components of a Zap 70-binding partner complex.

The quinazolines, tyrphostins, quinolines, and quinoxalines referred to above include well known compounds such as those described in the literature. For example, representative publications describing quinazoline include Barker et al . , EPO Publication No. 0 520 722 Al; Jones et al . , U.S. Patent No. 4,447,608; Kabbe et al . , U.S. Patent No. 4,757,072; Kaul and Vougioukas, U.S. Patent No. 5, 316,553; Kreighbaum and Comer, U.S. Patent No. 4,343,940; Pegg and Wardleworth, EPO Publication No. 0 562 734 Al; Barker et al . , Proc. of Am. Assoc. for Cancer Research 32:327 (1991) ; Bertino, J.R., Cancer Research 3:293-304 (1979) ; Bertino, J.R., Cancer Research 9(2 part 1) :293- 304 (1979) ; Curtin et al . , Br. J. Cancer 53:361-368 (1986) ; Fernandes et al . , Cancer Research 43:1117-1123 (1983) ; Ferris et al . J. Orα. Chem. 44 (2) :173-178 ; Fry et al., Science 265:1093-1095 (1994) ; Jackman et al., Cancer Research 51:5579-5586 (1981) ; Jones et al . J. Med. Chem. 29 (6) :1114-1118; Lee and Skibo, Biochemistry 26 (23) :7355-7362 (1987) ; Lemus et al . , J. Orα. Chem. 54:3511-3518 (1989) ; Ley and Seng, Synthesis 1975:415-522 (1975) ; Maxwell et al . , Magnetic Resonance in Medicine 17:189-196 (1991) ; Mini et al., Cancer Research 45:325-330 (1985) ; Phillips and Castle, J. Heterncycl i c Chem. 17 (19) :1489-1596 (1980) ; Reece et al., Cancer Research 47 (11) :2996-2999 (1977) ; Sculier et al., Cancer Immunol, and Immunother. 23:A65 (1986) ; Sikora et al . , Cancer Letters 23:289-295 (1984) ; Sikora

13 et al., Analytical Biochem. 172:344-355 (1988) ; all of which are incorporated herein by reference in their entirety, including any drawings.

Quinoxaline is described in Kaul and Vougioukas, U.S. Patent No. 5,316,553, incorporated herein by reference in its entirety, including any drawings .

Quinolines are described in Dolle et al . , J. Med. Chem. 37:2627-2629 (1994) ; MaGuire, J. Med. Chem. 37:2129-2131 (1994) ; Burke et al . , J. Med.

Chem. 36:425-432 (1993) ; and Burke et al . BioOrganic Med. Chem. Letters 2:1771-1774 (1992) , all of which are incorporated by reference in their entirety, including any drawings . Tyrphostins are described in Allen et al . ,

Clin. Exp. Immunol. 91:141-156 (1993) ; Anafi et al . , Blood 82:12:3524-3529 (1993) ; Baker et al . , J. Cell Sci . 102:543-555 (1992) ; Bilder et al . , Amer. Physioi. Soc. pp. 6363-6143:C721-C730 (1991) ; Brunton et al . , Proceedings of Amer. Assoc. Cancer Rsch. 33:558 (1992) ; Bryckaert et al. , Experimental Cell Research 199:255-261 (1992) ; Dong et al . , J. Leukocyte Biology 53:53-60 (1993) ; Dong et al . , J. Immunol. 151 (5) :2717-2724 (1993) ; Gazit et al . , J. Med. Chem. 32:2344-2352 (1989) ; Gazit et al., " J. Med. Chem. 36:3556-3564 (1993) ; Kaur et al., Anti-Cancer Druαs 5:213-222 (1994) ; Kaur et al . , King et al., Biochem. J. 275:413-418 (1991) ; Kuo et al . , Cancer Letters 74:197-202 (1993) ; Levitzki, A., The FASEB J. 6:3275-3282 (1992) ; Lyall et al. , J. Biol. Chem. 264:14503-14509 (1989) ; Peterson et al . , The

Prostate 22:335-345 (1993) ; Pillemer et al . , Int. J.

Cancer 50:80-85 (1992) ; Posner et al. , Molecular Pharmacology 45:673-683 (1993) ; Rendu et al . , Biol. Pharmacology 44 (5) : 881-888 (1992) ; Sauro and Thomas, Life Sciences 53:371-376 (1993) ; Sauro and Thomas, _∑__ Pharm. and Experimental Therapeutics 267 (3) : 1 19-1125

(1993) ; Wolbring et al . , J. Biol. Chem. 269 (36) :22470- 22472 (1994) ; and Yoneda et al . , Cancer Research 51:4430-4435 (1991) ; all of which are incorporated herein by reference in their entirety, including any drawings.

The above-noted groups of compounds are believed to be particularly useful for screening in this assay and to be potentially useful in treatment of the noted immune disorders . Those in the art can use the assays described or equivalents of such assays for routine screening of such molecules to find those which are active in inhibition of the signal transduction pathway discussed above and thus useful for treatment of those immune disorders, such that the life expectancy of an individual affected with such a disorder will be increased or that one or more symptoms of the disease will be reduced.

In other preferred embodiments, the kinase may be provided within a cell, preferably a cell having a recombinant nucleic acid encoding a Zap 70 kinase, in which case the method includes providing a ligand which initiates the signal transduction pathway. It is the action of the ligand which is preferably inhibited by the agent and thus the agent is preferably contacted with the cell prior to contact of the cell with the ligand. Potential cells for use in the method include

standard immune cells all of which are well known in the art and can be obtained from recognized sources or equivalents from such sources. The detecting step may involve measuring any change in a level of interaction between a Zap 70 kinase and a Zap 70 binding partner.

Alternatively, the detecting step may involve measuring any change in a level of phosphorylation of a Zap 70 kinase or the level of phosphorylation at any step of the pathway. The recombinant nucleic acid noted above is available to those of ordinary skill in the art and thus the cells can be readily constructed using routine experimentation and molecular biology techniques. In addition, the method for detecting levels of phosphorylation as one example of measurement of the signal transduction by the heterodimers are routine in the art.

By "interaction" is meant any physical associ¬ ation between proteins, whether covalent or non- covalent . Examples of non-covalent bonds include electrostatic bonds, hydrogen bonds, and Van der Waals bonds. Stryer, Biochemistry. 1988, pages 7-8. Furthermore, the interactions between proteins may either be direct or indirect. Thus, the association between two given proteins may be achieved with an intermediary agent, or several such agents, that connects the two proteins of interest. Another example of an indirect interaction is the independent production, stimulation, or inhibition of both Zap 70 kinase and a Zap 70 binding partner by a regulatory agent. Depending upon the type of interaction present, various methods may be used to measure the level of

interaction. For example, the strengths of covalent bonds are often measured in terms of the energy required to break a certain number of bonds (i.e., kcal/mol) Non-covalent interactions are often described as above, and also in terms of the distance between the interact¬ ing molecules. Indirect interactions may be described in a number of ways, including the number of interme¬ diary agents involved, or the degree of control exercised over the Zap 70 kinase relative to the control exercised over the Zap 70 binding partner.

By "Zap 70 binding partner" is meant an amino acid sequence that interacts with a Zap 70 kinase. A Zap 70 binding partner is preferably able to bind to a Zap 70 kinase with a dissassociation constant of at least 100 nM, more preferably 10 nM, most preferably 10 pM. The binding agent is preferably a purified antibody which recognizes an epitope present on a Zap 70 kinase. Other binding agents include molecules which bind to the Zap 70 kinase and analogous molecules which bind to a Zap 70 kinase. By "purified" in reference to an antibody is meant that the antibody is distinct from naturally occurring antibody, such as in a purified form. Preferably, the antibody is provided as a homogeneous preparation by standard techniques. Uses of antibodies to the cloned polypeptide include those to be used as therapeutics, or as diagnostic tools. The Zap 70 binding partner will preferably inhibit one or more activities of the Zap 70 kinase. In eome preferred embodiments the Zap 70 binding partner is molecule with a tyrosine residue important for a Zap 70 enzymatic or signalling function. For example, the binding partner

may be a molecule that binds Zap 70 when phosphorylated and then in turn activates Zap 70.

In another aspect the invention features a method of screening for an agent that inhibits an activity of a Zap 70 kinase by assaying the agent for the ability to inhibit the activity.

By "activity" is meant any function regulated or modulated by the polypeptide. For example, the func¬ tion 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 a transduction of an extracellular signal into a cell. For example, the effect of complex disrup¬ tion may augment, reduce, or block a signal normally transduced into the cell. Likewise, depending on the disorder involved, either augmentation, reduction, or blockage of a signal normally transduced into the cell will be desirable for the treatment of the disorder. In preferred embodiments the activity is binding to a Zap 70 kinase activity, a phosphorylation activity; a proliferation activity; a differention activity; an IL-2 production activity; a calcium ion production activity; a tyrosine phosphorylation of peripheral CD4 ⁺ T cell activity; or a mitogen or antigen stimulated T cell receptor proliferation activity.

Other activities of a Zap 70 kinase are well-known in the art and may be measured using conventional techniques as demostrated by the exemplary techniques described herein. By "proliferation activity" is meant the ability of a cell to grow or extend by multiplation of

cells and includes cell division. The rate of cell proliferation may be measured by counting the number of cells produced in a given unit of time.

By "differentiation activity" is meant the ability of a cell to undergo the complex of changes whereby cells change to mature or specialized forms. The process can include specializations in cell morphology and physiology, and involves differential transcription. The process can include both reversible and irreversible steps. The observable changes may differ between cell lines and at various stages of development; those skilled in the art would know and recognize the cellular changes characteristic of other cell lines and development stages. The term "assaying" is used in its well recognized form to mean performing of a protocol on an agent to determine the activity of that agent in inhibition of signal transduction.

In preferred embodiments the activity is binding to a Zap 70 kinase and the assaying involves the steps of exposing the agent to the Zap 70 kinase and detecting the amount of the agent bound to the polypeptide. In another aspect the invention features a method of screening for an agent that selectively inhibits a Zap 70 kinase activity. The method involves assaying a potential agent for the ability to inhibit the Zap 70 kinase activity but not inhibit an activity of another kinase.

In another aspect the invention features a method of screening for an agent useful in treating a disorder characterized by an abnormality in a signal

transduction pathway. The signal transduction pathway involves the interaction between a Zap 70 kinase and a Zap 70 binding partner and the method involves the step of assaying potential agents for those able to disrupt or promote said interaction as an indication of a useful said agent . The screening may also involve assaying potential agents for the ability to remove or reduce the effect of an abnormality in a signal transduction path¬ way, wherein the signal transduction pathway contains a Zap 70 kinase and a Zap 70 binding partner.

By "screening" is meant investigating for the presence or absence of a property. The process may include measuring or detecting various properties, including the level of signal transduction and/or the level of interaction between a Zap 70 kinase and a Zap 70 binding partner. In preferred embodiments the screening involves looking for agonists or antagonists of a protein of interest, for example a Zap 70 kinase or Zap 70 binding partner. The term agonist refers to agents that bind the protein and that maintain the activity of the protein to which they bind. An antagonist competes with the natural ligand for binding the protein, but does not maintain the activity of the protein to which it binds. By "disorder" is meant is meant a state in an organism, e.g.. a human, which is recognized as abnormal by members of the medical community. The disorder may be characterized by an abnormality in one or more signal transduction pathways in a cell (preferably an immune cell, more preferably a T cell) wherein one of the components of the signal transduction pathway is a Zap

20

70 kinase. Examples of disorders to be prevented, treated or diagnosed by the present invention include organ transplant rejection and immune disorders, including autoimmune disorders, such as multiple sclerosis or lupus include Addison's disease, autoimmune hemolytic anemia, Crohne' s disease, Goodpasture' s syndrome, Graves' disease, Hashimoto's thyroiditis, idiopathic thrombocytopenic purpura, insulin-dependent diabetes mellitus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, poststreptococcal glo rulonephritis, psoriasis, rheumatoid arthritis, scleroderma, Sjόgren's syndrome, spontaneous infertility, and systematic lupus erythematosus . By "abnormality" is meant an a level which is statistically different from the level observed in organisms not suffering from such a disorder and may be characterized as either an excess amount, intensity or duration of signal or a deficient amount, intensity or duration of signal. The abnormality in signal transduction may be realized as an abnormality in cell function, viability or differentiation state. It has been determined that such abnormal interaction in a pathway can be alleviated by action at the polypeptide- binding partner interaction site in the pathway. An abnormal interaction level may also either be greater or less than the normal level and may impair the normal performance or function of the organism. Thus, it is also possible to screen for agents that will be useful for treating a disorder, characterized by an abnormality in the signal transduction pathway, by testing compounds

for their ability to affect the interaction between a Zap 70 kinase and a Zap 70 binding partner, since the complex formed by such interaction is part of the signal transduction pathway. However, the disorder may be characterized by an abnormality in the signal trans¬ duction pathway even if the level of interaction between a Zap 70 kinase and a Zap 70 binding partner is normal.

By "disrupt" is meant that the interaction between the Zap 70 kinase and a Zap 70 binding partner is reduced either by preventing expression of the Zap 70 kinase, or by preventing expression of the Zap 70 binding partner, or by specifically preventing interaction of the naturally synthesized proteins having these domains or by interfering with the interaction of the proteins. The term "disrupt" 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. By "promote" is meant that the interaction between a Zap 70 kinase and a Zap 70 binding partner is increased either by increasing expression of a Zap 70 kinase, or by increasing expression of a Zap 70 binding partner, or by decreasing the dephosphorylating activity of the corresponding regulatory TP (or other phosphatase acting on other phosphorylated signalling components) by promoting interaction of the Zap 70 kinase and a Zap 70 binding partner or by prolonging the duration of the interaction. Many bivalent or polyvalent linking agents are useful in coupling polypeptides, such as an antibody, to other molecules. For example,

representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehydes, diazobenzenes and hexamethylene diamines . This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents. (See Killen and Lindstrom 1984, J. Immunol. 13_3:1335-2549; Jansen, F.K. et al . , 1982, Immunological Rev. 62:185- 216; and Vitetta ___ ____, supra) .

In another aspect the invention features a method of treating a disorder. The method involves administering to an organism in need of treatment a therapeutically effective amount of a compound identified by a screening assay described herein in a pharmaceutical composition.

By "organism" is meant any living creature. The term includes mammals, and specifically humans. Preferred organisms include mice, as the ability to treat or diagnose mice is often predictive of the abil¬ ity to function in other organisms such as humans.

By "therapeutically effective amount" is meant agents of this invention have a "therapeutic effect" which generally refers to either the inhibition, to some extent, of proliferation or differentiation of cells causing or contributing to a disorder; or the inhibition, to some extent, of the production of causes or contributors to such a disorder. A "therapeutic effect" relieves to some extent one or more of the symptoms of the disorder. The doses of Zap 70 and antagonist (s) thereof which are useful as a treatment

are "therapeutically effective" amounts. Thus, as used herein, a "therapeutically effective amount" means an amount of the antigen, fragment or antagonist thereof, which produces the desired therapeutic effect. This amount can be routinely determined by one of skill in the art and will vary depending upon several factors such as the particular illness from which the patient suffers and the severity thereof, as well as the patient's height, weight, sex, age, and medical history. Generally, Zap 70 of the present invention is preferably provided at a dose of between about 5 to about 5000 mg/dose/week/patient . More specifically, one preferable dose range is from 50 to 500 mg/dose/week/patient and another is from 200 to 300 mg/dose/week/patient. By "in need of the treatment" is meant an organism having a disorder that is effectively treated by administration of a Zap 70 kinase or inhibitor. Organisms in need of treatment may be identified using routine diagnostic methodologies, for example by detecting the symptoms characteristic of the particular disorder to be treated.

In preferred embodiments the agent is has an EC ₅₀ or IC ₅₀ as described below. An EC ₅₀ or IC ₅₀ of less than or equal to 5 μM is preferable, and even more preferably less than or equal to 1 μM, 100 nmolar, 10 nmolar, or 1 nmolar. Such lower EC ₅₀ ' s or IC ₅₀ 's are advantageous since they allow lower concentrations of molecules to be used in vivo or in vi tro for therapy or diagnosis. The discovery of molecules with such low EC ₅₀ 's and IC ₅₀ 's enables the design and synthesis of additional molecules having similar potency and

24 effectiveness. In addition, the molecule may have an EC ₅₀ or IC ₅₀ less than or equal to 5 μM at one or more, but not all cells chosen from the group consisting of parathyroid cell, bone osteoclast, juxtaglomerular kidney cell, proximal tubule kidney cell, distal tubule kidney cell, cell of the thick ascending limb of Henle' s loop and/or collecting duct, central nervous system cell, keratinocyte in the epidermis, parafollicular cell in the thyroid (C-cell) , intestinal cell, trophoblast in the placenta, platelet, vascular smooth muscle cell, cardiac atrial cell, gastrin-secreting cell, glucagon- secreting cell, kidney mesangial cell, mammary cell, beta cell, fat/adipose cell, immune cell and GI tract cell. In other preferred embodiments the agent is a dominant negative mutant protein provided by gene therapy or other equivalent methods as described below. That is, the agent is a peptide which blocks or promotes interaction of the Zap 70 kinase and a Zap 70 binding partner. The peptide may be recombinant, purified, or placed in a pharmaceutically acceptable carrier or diluent. In preferred embodiments Zap 70 kinase may be either a truncated or non-truncated form of Zap 70.

By "dominant negative mutant protein" is meant a mutant protein that interferes with the normal signal transduction pathway. The dominant negative mutant protein contains the domain of interest (e.g.. the binding domains Zap 70 kinase or a Zap 70 binding partner) , but has a mutation preventing proper signaling, for example by preventing binding of a second domain from the same protein. One example of a dominant

negative protein is described in Millauer et al . , Na ture February 10, 1994.

Agents of this invention thus have a "thera¬ peutic effect" which generally refers to either the inhibition, to some extent, of growth of cells causing or contributing to a disorder; or the inhibition, to some extent, of the production of causes or contributors to such a disorder. A therapeutic effect relieves to some extent one or more of the symptoms of the disorder. In reference to the treatment of a cancer, a therapeutic effect refers to one or more or the following: 1) reduction in tumor size; 2) inhibition (i.e.. slowing to some extent, preferably stopping) of tumor metastasis; 3) inhibition, to some extent, of tumor growth; and/or 4) relieving to some extent one more or the symptoms associated with the disorder. Compounds with efficiency for treating leukemia can be identified as above, but rather than inhibiting metastasis, they may instead slow or decrease cell proliferation or growth. When used as a therapeutic the compounds described herein are preferably administered with a physiological acceptable carrier. A physiological acceptable carrier is a formulation to which the compound can be added to dissolve it or otherwise facilitate its administration. Examples of physio¬ logical acceptable carriers include water, saline, physiologically buffered saline, cyclodextrins and PBTE:D5W (described below) . Hydrophobic compounds are preferably administered using a carrier such as PBTE:D5W. An important factor in choosing an appropri¬ ate physiological acceptable carrier is choosing a

carrier in which the compound remains active or the combination of the carrier and the compound produces an active compound. The compound may also be administered in a continuous fashion using a slow release formulation or a pump to maintain a constant or varying drug level in a patient.

In yet another aspect the invention features a method of preventing or treating a disorder characterized by an abnormality in a signal transduction pathway. The signal transduction pathway involves the interaction between a Zap 70 kinase and a Zap 70 binding partner and the method involves the step of disrupting or promoting said interaction in vivo.

Also provided is a method for preventing or treating a disorder. The method involves administering a therapeutically effective amount of a Zap 70 inhibitor to an organism in need of the treatment . In preferred embodiments, the disorder is an autoimmune disorder or a cancer. By an "inhibitor" of a receptor tyrosine kinase is meant that the compound reduces to some extent the activity of a Zap 70 kinase. By "significantly inhibit" is meant the compound has an IC ₅₀ less than 50 μm in an assay as described below. The present invention also features a pharmaceutical composition comprising a compound identified by a screening assay described herein and a physiologically acceptable carrier or diluent. In addition, the invention features novel compounds of the groups noted above which are identified by use of these methods. It also includes molecules which are derived

by standard methodology from such agents when such agents are used as lead compounds.

The present invention also provides a pharmaceutical composition comprising, consisting, or consisting essentially of a therapeutically effective amount of a Zap 70 inhibitor and a physiologically acceptable carrier or diluent.

By "comprising" it is meant including, but not limited to, whatever follows the word "comprising" . Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be pre¬ sent .

By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present .

By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates .that the listed elements are required or mandatory, but that other ele¬ ments are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

In other aspects, products and methods useful for Zap 70 related gene therapy and gene transfer techniques are provided. The genomic gene of Zap 70 has

been altered in mouse and homologous recombination has been achieved in a cell line. Thus, in preferred embodiments the invention provides cell lines and "knock-out" mice for performing such techniques. The choice of transfected lineages, vectors, and targets may all be confirmed in the mouse animal model. For example, potential targets for gene transfer include peripheral T cells, stem cells (mixture of bone marrow projenitors) and CD34 positive cells. In particular, the invention provides a vector comprising nucleic acid encoding a human or mouse Zap-70, the vector being adapted to cause expression of the Zap- 70. Expression of the human or mouse Zap-70 may result in the production of functional human or mouse Zap-70 proteins. The vector may comprise a retroviral vector. In addition, the invention provides a vector comprising nucleic acid encoding a Zap-70, the vector being adapted to cause expression of the Zap-70 only in specific tissue.

Also provided is a transfected cell line containing a vector comprising nucleic acid encoding a human or mouse Zap-70. Zap-70 may be expressed as a secreted protein. A transformed cell line containing a vector comprising nucleic acid encoding a human or mouse Zap-70 is also encompassed by the present invention. Again, the human or mouse Zap-70 may be expressed as a secreted protein.

A transgenic non-human animal containing a Zap- 70 is also provided. The transgenic animal may be a mammal, in particular a mouse. Also provided is a method for introducing a continuous supply of Zap-70 into an

animal or tissue culture, comprising the step of administering an effective amount of a vector described above to an animal or into the tissue culture. The step of administration to an animal may comprise injection into a skeletal muscle of the animal.

In addition, a method of gene replacement, comprising the step of administering an effective amount of a vector described above to an animal, wherein the Zap- 70 nucleic acid sequence will correct a genetic condition characterized by a defective or nonexistent Zap-70 is provided.

Further provided is a method of screening compounds for their pharmacological effects on biological activities such as tyrosine phosphorylation comprising the steps of administering a compound to a transgenic animal expressing a Zap-70 and measuring the activity in the transgenic animals .

The invention also features a method of administering a nucleic acid sequence encoding a Zap-70 to an animal comprising the steps of removing cells from the animal, transducing the cells with the Zap-70 nucleic acid sequence, and reimplanting the transduced cells into the animal. The nucleic acid sequence may encode a human or mouse Zap-70. Also featured is a method of administering a

Zap-70 nucleic acid sequence utilizing an in vivo approach comprising the steps of administering directly to an animal the Zap-70 nucleic acid sequence selected from the group of methods of administration consisting of intravenous injection, intramuscular injection, or by catheterization and direct delivery of the Zap-70 nucleic

acid sequence via the blood vessels supplying a target organism. The Zap-70 nucleic acid sequence may encode a human Zap-70 and the animal to which the Zap-70 is administered may be a human. The target organ can be selected from the group consisting of heart, skeletal muscle, adipose tissues, spleen, lung, brain, kidney, testis, adrenal or small intestine. The Zap-70 nucleic acid sequence may be administered as naked DNA or may be contained in a viral vector, for example one selected from the group consisting of papovaviruses, adenovirus, vaccinia virus, adeno-associated virus, herpesviruses and retroviruses of avian, murine or human origin.

Featured herein is a method of administering a Zap-70 nucleic acid sequence in a two-component system comprising the steps of administering a packaging cell, wherein the packaging cell produces a viral vector. The packaging cell can be administered to cells in vi tro .

Also provided is a method of administering a Zap-70 nucleic acid sequence comprising the step of administering a retroviral vector containing the Zap-70 nucleic acid sequence, wherein a retroviral envelope glycoprotein is replaced with the G glycoprotein of vesicular stomatitis virus.

The invention also features a method of administering a Zap-70 nucleic acid sequence comprising the step of administering to an animal an adenovirus vector, wherein an El region of the adenovirus vector is replaced with the Zap-70 nucleic acid sequence and administering the adenovirus vector by a method of administration selected from the group consisting of

intravenous injection, intramuscular injection, intraportal injection or intra-arterial injection.

The summary of the invention described above is non-limiting and other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present disclosure describes compounds and methods which can be used to inhibit tyrosine kinase activity. The compounds and methods are preferably used in the treatment of immune disorders characterized by over-activity or inappropriate activity of a tyrosine kinase.

The compounds described herein can differ in their selectivity. Selectivity, or selective inhibition refers to the ability of a compound to significantly inhibit the activity of a first tyrosine kinase or complex (e.g.. Zap 70: binding partner complex) but not inhibit a second tyrosine kinase or complex. In general, it is preferred that a therapeutic compound be selective for a particular tyrosine kinase. Tyrosine kinases are important in many biological processes including immune response cell growth, differentiation, aggregation, chemotaxis, cytokine release, and muscle contraction. Many of these events are mediated through different tyrosine kinases. In addition, different tyrosine kinases may be important for a particu¬ lar biological function in different cell types. By developing selective inhibitors for a particular tyrosine kinases, the potential for side effects of the compound is

decreased. In those conditions where more than one tyrosine kinase plays a role a compound which can inhibit both of these activities, but not other receptor kinases would be preferred. The present disclosure relates to the identifi¬ cation of specific compounds including to the classes and groups described herein which are useful in the present invention. Identification can be carried out by assaying the ability of a compound to inhibit tyrosine kinase activity, and preferably, the ability of the compound to inhibit growth of cells having a tyrosine kinase driven disorder. Such assays can be preformed as described in the art, or as described in the examples below. Those in the art will recognize these assays are not limiting in this invention, and that equivalent assays are readily devised. After identification of agents from cellular kinase-type assays, in vivo tests can be used to ensure utility for human therapy, e.g.. as described below, for example, in vivo soft agar assays. Examples of cell lines which can be used to study the effect of a compound, for example in vi tro or in animal models, include immune cells, and others well known in the art or which can be generated by standard methodology. One skilled in the can choose other suitable cell lines using standard techniques and the present application as a guide. For example, the diagnostic section described infra can be used to help determine whether a cell line is driven by a tyrosine receptor kinase . Animal model systems can also be used to further measure the therapeutic effect of a compound. Examples of

suitable animal models include subcutaneous xenograft model and in si tu mammary fat pad model.

1_ Xenograft Model

The ability of human tumors to grow as xenografts in athymic mice (e.g.. Balb/c, nu/nu) provides a useful in vivo model for studying the biological response to therapies for human tumors. Since the first successful xenotransplantation of human tumors into athymic mice by Ryaard and Povlsen (Rygaard, J. and Polvsen, CO., Acta Pa ol • Microbial. Scand..

77:758-760, 1969.) , many different human tumor cell lines

(i.e.. mammary, lung, genitourinary, gastrointestinal, head and neck, glioblastoma, bone, and malignant melanomas) have been transplanted and successfully grown in nude mice. Human mammary tumor cell lines, including MCF-7, ZR75-1, and MDA-MB-231, have been established as subcutaneous xenografts in nude mice (Warri, A.M., et al . , Int. J. Cancer. 49:616-623, 1991; Ozzello, L. and Sordat, M., Eur. J. Cancer, 16:553-559, 1980; Osborne, C.K., et al., Cancer Res. , 45:584-590, 1985; Seibert, K. , et al., Cancer Res .. 43:2223-2239, 1983) .

The following type of xenograft protocol can be used: 1) implant tumor cells (subcutaneously) into the hindflank of five- to six-week-old female Balb/c nu/nu athymic mice; 2) administer the anti-tumor compound; 3) measure tumor growth by measuring tumor volume. The tumors can also be analyzed for the presence of a kinases, such as Zap 70, by Western and immunohistochemical analyses. Using techniques known in the art, one skilled

in the art can vary the above procedures, for example through the use of different treatment regimes.

2_ Mammary Fat Pad Model The mammary fat pad model is particularly useful for measuring the efficacy of compounds which inhibit Zap 70, because of the role Zap 70 plays in cancer. By implanting tumor cells directly into the location of interest, in si tu models more accurately reflect the biology of tumor development than do subcutaneous models. Human mammary cell lines, including MCF-7, have been grown in the mammary fat pad of athymic mice (Shafie, S.M. and Grantham, F.H., J. Natl. Cancer Instit.. 67:51-56, 1981; Gottardis, M.M. , ^' et al. , J. Steroid Biochem.. 30:311-314, 1988) .

For example, the following procedure can be used: 1) MDA-MB-231 and MCF-7 cells transfected with Zap 70 are implanted at various concentrations into the axillary mammary fat pads of female athymic mice; 2) the compound is administered; and 3) tumor growth is measured at various time points. The tumors can also be analyzed for the presence of a kinase such as Zap 70, by Western and immunohistochemical analyses . Using techniques known in the art, one skilled in the arc can vary the above procedures, for example through the use of different treatment regimes .

3. Further Analysis

Therapeutic compounds should be more potent in inhibiting receptor tyrosine kinase activity than in exerting a cytotoxic effect. A measure of the effective-

ness and cell toxicity of a compound can be obtained by determining the therapeutic index: IC ₅₀ /LD ₅₀ . IC ₅₀ , the dose required to achieve 50% inhibition, can be measured using standard techniques such as those described herein. LD _50; the dosage which results in 50% toxicity, can also be mea¬ sured by standard techniques, such as using an MTT assay as described by Mossman J. Immunol . Methods 65 : 55-63 (1983) , by measuring the amount of LDH released (Korzeniewski and Callewaert, J. Immunol . Methods 64:313 (1983) ; Decker and Lohmann-Mattheε, J. Immunol . Methods 115 : 61 (1988) , or by measuring the lethal dose in animal models. Compounds with a large therapeutic index are preferred. The therapeutic index should be greater than 2, preferably at least 10, more preferably at least 50. In addition to measuring tumor growth in the animal models, plasma half-life and biodistribution of the drug and metabolites in plasma, tumors, and major organs can be determined to facilitate the selection of drugs most appropriate for the inhibition of a disorder. Such measurements can be carried out, for example, using HPLC analysis. Compounds that show potent inhibitory activity in the screening assays but have poor pharmacokinetic characteristics can be optimized by altering the chemical structure and retesting. In this regard, compounds displaying good pharmacokinetic characteristics can be used as model.

Toxicity studies can also be carried out by measuring the blood cell composition. For example, toxicity studies can be carried out as follows: 1) the compound is administered to mice (an untreated control mouse should also be used) ; 2) blood samples are

periodically obtained via the tail vein from one mouse in each treatment group; and 3) the samples are analyzed for red and white blood cell counts, blood cell composition, and the percent of lymphocytes versus polymorphonuclear cells. A comparison of results for each dosing regime with the controls indicates if toxicity is present.

At the termination of each study, further studies can be carried out by sacrificing the animals (preferably, in accordance with the American Veterinary Medical Association guidelines Report of the American Veterinary Medical Assoc. Panel on Euthanasia. Journal of American Veterinary Medical Assoc , 202:229-249, 1993) . Representative animals from each treatment group can then be examined by gross necropsy for immediate evidence of metastasis, unusual illness, or toxicity. Gross abnormal¬ ities in tissue are noted, and tissues are examined histologically. Compounds causing a reduction in body weight or blood components less preferred, as are compounds having an adverse effect on major organs. In general, the greater the adverse effect the less preferred the compound.

Zap 70 driven disorders are characterized by over-activity or hyperphosphorylation of Zap 70. Over- activity of Zap 70 refers to either an amplification of the gene encoding Zap 70 or the production of a level of Zap 70 activity which can be correlated with a cell proliferative disorder (i.e. , as the level of Zap 70 increases the severity of one or more of the symptoms of the cell proliferative disorder increases) . Activation of Zap 70 activity can result from several different events including: 1) ligand binding; 2)

Zap 70 stimulation through transphosphorylation; and 3) overexpression of the gene encoding a Zap 70 protein. Zap 70 activity can be assayed by measuring one or more of the following activities: (1) phosphorylation of Zap 70; (2) phosphorylation of a Zap 70 substrate; (3) activation of a Zap 70 adapter molecule; and (4) increased cell division. These activities can be measured using tech¬ niques described below and known in the art.

Treatment of patients suffering from a Zap 70 disorder is facilitated by first determining whether the disorder is characterized by an over-activity of Zap 70. After the disorder is identified, patients suffering from such a disorder can be identified by analysis of their symptoms by procedures well known to medical doctors. Such identified patients can then be treated as described herein.

4. Diagnostic uses

Another use of the compounds described herein is to help diagnose whether a disorder is driven, to some extent, by a particular receptor tyrosine kinase.

A diagnostic assay to determine whether a parti¬ cular disorder is driven by a specific receptor can be carried out using the following steps: 1) culturing test cells or tissues; 2) administering a compound which can inhibit one or more receptor tyrosine kinase; and

3) measuring the degree of growth inhibition of the test cells .

These steps can be carried out using standard techniques in light of the present disclosure. For example, standard techniques can be used to isolate cells

or tissues and culturing in vi tro or in vivo . An example of an in vi tro assay is a cellular kinase assay as described below. An example of an in vivo assay is a xenograft experiment where the cells or tissues are implanted into another host such as a mouse.

Compounds of varying degree of selectivity are useful for diagnosing the role of a receptor tyrosine kinase. For example, compounds which inhibit more than one type of receptor tyrosine kinase can be used as an initial test compound to determine if one of several receptor tyrosine kinases drive the disorder. More selective compounds can then be used to further eliminate the possible role of different receptor tyrosine kinases in driving the disorder. Test compounds should be more potent in inhibiting receptor tyrosine kinase activity than in exerting a cytotoxic effect (e.g.. an IC ₅₀ /LD ₅₀ of greater than one) . As noted above, IC ₅₀ and LD ₅₀ can be measured by standard techniques, such as described in the present application and using an MTT assay as described by Mossman supra, or by measuring the amount of LDH released (Korzeniewski and Callewaert, J. supra.- Decker and Lohmann-Matthes, supra) . The degree of IC ₅₀ /LD ₅₀ of a compound should be taken into account in evaluating the diagnostic assay. Generally, the larger the ratio the more reliable the information. Appropriate controls to take into account the possible cytotoxic effect of a compound, such as treating cells not associated with a cell proliferative disorder (e.g.. control cells) with a test compound, can also be used as part of the diagnostic assay.

Sulforhodamine B assays for measuring effects of rest compounds on cell growth may be based on procedures described by Skehan et al . J. Natl . Cancer Inst . 82:1107, 1990. The soft agar assay is well known in the art as a method for measuring the effects of substances on cell growth.

The present invention relates to methods of diagnosing and treating various disorders characterized by an abnormality in a signal transduction pathway that involves an interaction between a Zap 70 kinase and a Zap 70 binding partner. Methods of screening for agents useful in such methods and pharmaceutical compositions for use in such methods are also provided.

I. Isolation of Compounds Which Interact With Zap 70.

The present invention relates to a method of detecting a compound capable of binding to a Zap 70 kinase comprising incubating the compound with a Zap 70 kinase and detecting the presence of the compound bound to the Zap 70 polpypeptide.

The present invention also relates to a method of detecting an agonist or antagonist of Zap 70 activity comprising incubating cells that produce a Zap 70 kinase in the presence of a compound and detecting changes in the level of Zap 70 activity. The compounds thus identified would produce a change in activity indicative of the presence of the compound. The compound may be present within a complex mixture, for example, serum, body fluid, or cell extracts. Once the compound is identified it can be isolated using techniques well known in the art.

The present invention also encompasses a method of agonizing (stimulating) or antagonizing Zap 70 associated activity in a mammal comprising administering to said mammal an agonist or antagonist to Zap 70 in an amount sufficient to effect said agonism or antagonism. A method of treating the disorders of the present invention in a mammal with an agonist or antagonist of Zap 70 activity comprising administering the agonist or antagonist to a mammal in an amount sufficient to agonize or antagonize Zap 70 associated functions is also encompassed in the present application.

II . Compositions

The present invention relates to removing or reducing an abnormality in a signal transduction pathway, wherein the signal transduction pathway contains a Zap 70 kinase and a Zap 70 binding partner. The present invention also relates to compositions and methods for the treatment of disorders which involve modulating the activity and/or level of individual components, and relates to methods for the identification of agents for such treatments. Additionally, the present invention relates to methods and compositions for prognostic evaluation of such disorders.

Described herein are compositions and methods for the prevention, prognostic evaluation, and treatment of disorders in which a Zap 70 kinase may be involved. Also described are compositions and methods for the prevention, prognostic evaluation and treatment of cell proliferative disorders, especially cancer, in which a Zap 70 kinase is involved.

First, methods and compositions for the treat¬ ment of such disorders are described. Such methods and compositions may include, but are not limited to the agents capable of decreasing or inhibiting the interaction between a Zap 70 kinase and a Zap 70 binding partner and agents capable of inhibiting or decreasing the activity of such complexes, agents capable of modulating the activity and/or level of individual components of the proteins, and the use and administration of such agents. Agents capable of modulating the activity and/or level of interaction between Zap 70 kinase and a Zap 70 binding partner include those agents that inhibit or decrease the dephosphorylat- ing activity of tyrosine phosphatases.

Second, methods are described for the identifi- cation of such agents. These methods may include, for example, assays to identify agents capable of disrupting or inhibiting or promoting the interaction between compo¬ nents of the complexes (e.g., Zap 70:binding partner complexes) , and may also include paradigms and strategies for the rational design of drugs capable of disruption and/or inhibition and/or promotion of such complexes.

III. Binding Partner/Receptor Complexes

The complexes involved in the invention include a Zap 70 kinase and a Zap 70 binding partner or deri- vatives thereof, as described below. Under standard physiological conditions, the components of such complexes are capable of forming stable, non-covalent attachments with one or more of the other complex components . Methods for the purification and production of such protein

complexes, and of cells that exhibit such complexes are described below.

The complexes involved in the invention also include tyrosine phosphatases responsible for dephospho- rylating activated Zap 70 receptors, thus modulating the ability to bind to a ligand and other signal transduction components. Identification of such tyrosine phospha- tase(s) may be accomplished using techniques known to one skilled in the art.

IV. Disruption of Protein Complexes

Disruption of complexes (e.g., Zap 70:binding partner complexes) , for example by decreasing or inhibiting or promoting the interactions between component members of such a complex may have differing modulatory effects on the event involved, depending on the individual protein complex.

A disorder involving a complex may, for example, develop because the presence of such a complex brings about the aberrant inhibition of a normal signal transduc- tion event. In such a case, the disruption of the complex would allow the restoration of the usual signal transduc¬ tion event. Further, an aberrant complex may bring about an altered subcellular adapter protein localization, which may result in, for example, dysfunctional cellular events. An inhibition of the complex in this case would allow for restoration or maintenance of a normal cellular architec¬ ture. Still further, an agent or agents that cause (s) disruption of the complex may bring about the disruption of the interactions among other potential components of a complex.

Nucleotide sequences encoding 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 retrovi- ruses, vaccinia virus, adenoviruses, 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 in Ausubel et al . , Current Protocols in Molecular Biology. Greene Publishing Associ- ates and Wiley Interscience, N.Y, 1989. Complex-binding domains can be identified using, for example, techniques such as those described in Rotin et al . (Rotin et al . , EMBO J. 11:559-567, 1992) , Songyang et al . (Songyang et al., Cell 72:767-778, 1993) , Felder et al . , Mol . Cell . Biol . 13:1449-1455, 1993) , Fantl et al . ( Cell 69:413-422, 1992) , and Domchek et al . (Biochemistry 31:9865-9870, 1992) .

Alternatively, antibodies capable of interfering with complex formation may be produced as described below and administered for the treatment of disorders involving a component capable of forming a complex with another protein. For example, neutralizing antibodies which are capable of interfering with ligand binding may be adminis¬ tered using standard techniques. Alternatively, nucleo- tide 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., Proc . Na tl . Acad . Sci . USA 90:7889-7893, 1993) .

Agents which act intracellularly to interfere with the formation and/or activity of the protein complex¬ es of the invention may also be small organic or inorganic compounds. A method for identifying these and other intracellular agents is described below.

V. Antibodies to Complexes Described herein are methods for the production of antibodies which are capable of specifically recogniz¬ ing a complex or an epitope thereof, or of specifically recognizing an epitope on either of the components of the complex, especially those epitopes which would not be recognized by the antibody when the component is present separate and apart from the complex. Such antibodies may include, but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs) , humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') ₂ fragments, fragments produced by a FAb expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. Such antibodies may be used, for example, in the detection of a complex in a biological sample, or, alternatively, as a method for the inhibition of a complex formation, thus inhibiting the development of a disorder.

Polyclonal antibodies are heterogeneous popula¬ tions of antibody molecules derived from the sera of animals immunized with an antigen, such as a complex, or an antigenic functional derivative thereof. For the pro-

duction of polyclonal antibodies, various host animals may be immunized by injection with the 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 parvu •

A monoclonal antibody, which is a substantially homogeneous population of antibodies to a particular anti¬ gen, 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 and Milstein (Na ture 256:495-497, 1975) and U.S. Patent No. 4,376,110) , the human B-cell hybridoma technique (Kosbor et al . , Immunology Today 4:72, 1983; Cole et al . , Proc . Na tl . Acad. Sci . USA 80:2026-2030, 1983) , and the EBV-hybridoma technique (Cole et al . , Monoclonal Antibodies And Cancer Therapy. Alan R. Liss, Inc., 1985, 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 vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production. In addition, techniques developed for the pro¬ duction of "chimeric antibodies" (Morrison et al., Proc .

Na tl . Acad . Sci . , 81:6851-6855, 1984; Νeuberger et al . , Na ture, 312:604-608, 1984; Takeda et al. , Nature, 314:452- 454, 1985) 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 immunoglob- ulin constant region.

Alternatively, techniques described for the production of single chain antibodies (U.S. Patent 4,946,- 778; Bird, Science 242:423-426, 1988; Huston et al . , Proc. Na tl . Acad . Sci . USA 85:5879-5883, 1988; and Ward et al . , Na ture 334:544-546, 1989) can be adapted to produce 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 bind¬ ing sites of a complex may be generated by known tech¬ niques. 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/adapter complex.

One or more components of a protein complex may be present at a higher than normal cellular level (i.e.. higher than the concentration known to usually be present in the cell type exhibiting the protein complex of inter- est) and/or may exhibit an abnormally increased level of cellular activity (i.e.. greater than the activity known to usually be present in the cell type exhibiting the protein complex of interest) .

For example, the gene encoding a protein complex component may begin to be overexpressed, or may be ampli¬ fied (i.e.. its gene copy number may be increased) in certain cells, leading to an increased number of component molecules within these cells. Additionally, a gene encoding a protein complex component may begin to express a modified protein product that exhibits a greater than normal level of activity. "Activity", here, refers to the normal cellular function of the component, either enzymat¬ ic or structural whose function may include, for example, bringing two or more cellular molecules into the appropri- ate proximity.

Such an increase in the cellular level and/or activity of a protein complex may lead to the development of a disorder. Treatment of such disorders may, there¬ fore, be effectuated by the administration of agents which decrease the cellular level and/or the activity of the overexpressed and/or overactive protein complex component.

Techniques for decreasing the cellular level and/or the activity of one or more of the protein complex components of interest may include, but are not limited to antisense or ribozyme approaches, and/or gene therapy

approaches, each of which is well known to those of skill in the art .

VI. Antisense and Ribozyme Approaches

Included in the scope of the invention are oligoribonucleotides, including antisense RNA and DNA molecules and ribozy es that function to inhibit transla¬ tion of one or more components of a protein complex. Anti-sense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted RNA and preventing protein translation. With respect to antisense DNA, oligodeoxyribonucleotides derived from the transla¬ tion initiation site, e.g.. between -10 and +10 regions of the relevant nucleotide sequence, are preferred.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific interaction of the ribozyme molecule to complementary target RNA, fol¬ lowed by a endonucleolytic cleavage. Within the scope of the invention are engineered hammerhead or other motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of RNA sequences encod¬ ing protein complex components.

Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features, such as secondary struc-

ture, that may render the oligonucleotide sequence unsuit¬ able. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays. See, Draper PCT WO 93/23569.

Both anti-sense RNA and DNA molecules and ribo¬ zymes of the invention may be prepared by any method known in the art for the synthesis of RNA molecules. See, Draper, j__ . hereby incorporated by reference herein. These include techniques for chemically synthesizing oligodeoxyribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthe¬ sis. Alternatively, RNA molecules may be generated by in vi tro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitu- tively or inducibly, depending on the promoter used, can be introduced stably into cell lines.

Various modifications to the DNA molecules may be introduced as a means of increasing intracellular sta¬ bility and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribo- or deoxy- nucleotides to the 5 ' and/or 3 ' ends of the molecule or the use of phosphorothioate or 2 ' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone .

VII. Gene Therapy

Target cell populations may be modified by introducing altered forms of one or more components of the protein complexes in order to modulate the activity of such complexes. For example, by reducing or inhibiting a complex component activity within target cells, an abnor¬ mal signal transduction event (s) leading to a condition may be decreased, inhibited, or reversed. Deletion or missense mutants of a component, that retain the ability to interact with other components of the protein complexes but cannot function in signal transduction may be used to inhibit an abnormal, deleterious signal transduction event .

Expression vectors derived from viruses such as retroviruses, vaccinia virus, adenovirus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of nucleotide sequences encoding recom- binant protein complex components into the targeted cell population. Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors containing coding sequences. See, for example, the techniques described in Maniatis et al . , Molecular

Cloning: A Laboratory Manual . Cold Spring Harbor

Laboratory, N.Y. (1989) , and in Ausubel et al . , Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley Interscience, N.Y. (1989) . Alter¬ natively, recombinant nucleic acid molecules encoding protein sequences can be used as naked DNA or in reconstituted system e.g.. liposomes or other lipid systems for delivery to target cells (See e.g.. Feigner et al., Na ture 337:387-8, 1989) .

By "vector" is meant a nucleic acid, e . g . , DNA derived from a plasmid, cosmid, phagemid or bacteriophage, into which fragments of nucleic acid may be inserted or cloned. The vector can contain one or more unique restriction sites for this purpose, and may be capable of autonomous replication in a defined host or organism such that the cloned sequence is reproduced. The vector molecule can confer some well-defined phenotype on the host organism which is either selectable or readily detected. Some components of a vector may be a DNA molecule further incorporating a DNA sequence encoding a therapeutic or desired product, and regulatory elements for transcription, translation, RNA stability and replication. A viral vector in this sense is one that contains a portion of a viral genome, e . g. , a packaging signal, and is not merely DNA or a located gene within a viral article.

A "cell surface receptor" is a specific chemical grouping on the surface of a cell to which a ligand can attach. Cell surface receptors which may be used in the present invention include the folate receptor, the biotin receptor, the lipoic acid receptor, the low density lipoprotein receptor, the asialoglycoprotein receptor, IgG antigenic sites, insulin-like growth factor type II/cation-independent mannose-6-phosphate receptor, calcitonin gene-related peptide receptor, insulin-like growth factor I receptor, nicotinic acetylcholine receptor, hepatocyte growth factor receptor, endothelin receptor, bile acid receptor. Further, incorporating DNA into macromolecular complexes that undergo endocytosis increases the range of cell types that will take up

foreign genes from the extracellular space. Such complexes may include lipids, polylysine, viral particles, ligands for specific cell-surface receptors or nuclear proteins. The term "DNA transporter" refers to a molecular complex which is capable of non-covalently binding to DNA and efficiently transporting the DNA through the cell membrane. Although not necessary, it is preferable that the transporter also transport the DNA through the nuclear membrane. The methods and material set forth in International Publication No. WO 93/18759, filed March 19, 1993 and published September 30, 1993 are hereby incorporated by reference.

In preferred embodiments the target organ is selected from the group consisting of heart, skeletal muscle, adipose tissues, spleen, lung, brain, kidney, testis, adrenal or small intestine; the nucleic acid sequence is administered as naked DNA; the nucleic acid sequence is contained in a viral vector such as papovaviruses, adenovirus, vaccinia virus, adeno- associated virus, herpesviruses and retroviruses of avian, murine or human origin.

In its simplest form, gene transfer can be performed by simply injecting minute amounts of DNA into the nucleus of a cell, through a process of microinjection. Capecchi MR, Cell 22:479-88 (1980) . Once recombinant genes are introduced into a cell, they can be recognized by the cells normal mechanisms for transcription and translation, and a gene product will be expressed. Other methods have also been attempted for introducing DNA into larger numbers of cells. These

methods include: transfection, wherein DNA is precipitated with CaPO„ and taken into cells by pinocytosis (Chen C. and Okaya a H, Mol . Cell Biol. 7:2745-52 (1987)) ; electroporation, wherein cells are exposed to large voltage pulses to introduce holes into the membrane (Chu G. et al., Nucleic Acids Res., 15:1311-26 (1987)) ; lipofection/liposome fusion, wherein DNA is packaged into lipophilic vesicles which fuse with a target cell (Feigner PL., et al., Proc. Natl. Acad. Sci. USA. 84:7413-7 (1987) ) ; and particle bombardment using DNA bound to small projectiles (Yang NS. et al . , Proc. Natl. Acad. Sci. 87:9568-72 (1990)) . Another method for introducing DNA into cells is to couple the DNA to chemically modified proteins. It has also been shown that adenovirus proteins are capable of destabilizing endosomes and enhancing the uptake of DNA into cells. The admixture of adenovirus to solutions containing DNA complexes, or the binding of DNA to polylysine covalently attached to adenovirus using protein crosslinking agents substantially improves the uptake and expression of the recombinant gene . Curiel DT et al., Am. J. Respir. Cell. Mol. Biol., 6:247-52 (1992) .

As used herein "gene transfer" means the process of introducing a foreign nucleic acid molecule into a cell. Gene transfer is commonly performed to enable the expression of a particular product encoded by the gene. The product may include a protein, polypeptide, anti-sense DNA or RNA, or enzymatically active RNA. Gene transfer can be performed in cultured cells or by direct administration into animals. Generally gene transfer involves the process of nucleic acid contact with a target

cell by non-specific or receptor mediated interactions, uptake of nucleic acid into the cell through the membrane or by endocytosis, and release of nucleic acid into the cytoplasm from the plasma membrane or endosome . Expression may require, in addition, movement of the nucleic acid into the nucleus of the cell and binding to appropriate nuclear factors for transcription.

As used herein "gene therapy" is a form of gene transfer and is included within the definition of gene transfer as used herein and specifically refers to gene transfer to express a therapeutic product from a cell in vivo or in vi tro . Gene transfer can be performed ex vivo on cells which are then transplanted into a patient, or can be performed by direct administration of the nucleic acid or nucleic acid-protein complex into the patient.

In another preferred embodiment, a vector having nucleic acid sequences encoding Zap 70 is provided in which the nucleic acid sequence is expressed only in specific tissue. Methods of achieving tissue-specific gene expression as set forth in International Publication No. WO 93/09236, filed November 3, 1992 and published May 13, 1993.

In all of the preceding vectors set forth above, a further aspect of the invention is that the nucleic acid sequence contained in the vector may include additions, deletions or modifications to some or all of the sequence of the nucleic acid, as defined above.

In another preferred embodiment, a method of gene replacement is set forth. "Gene replacement" as used herein means supplying a nucleic acid sequence which is capable of being expressed in vivo in an animal and

thereby providing or augmenting the function of an endogenous gene which is missing or defective in the animal .

Some methods of delivery that may be used include: a. encapsulation in liposomes, b. transduction by retroviral vectors, c. localization to nuclear compartment utilizing nuclear targeting site found on most nuclear proteins, d. transfection of cells ex vivo with subsequent reimplantation or administration of the transfected cells, e. a DNA transporter system. Many nonviral techniques for the delivery of a

IR-95 nucleic acid sequence into a cell can be used, including direct naked DNA uptake (e.g., Wolff et al . , Science 247: 1465-1468, 1990) , receptor-mediated DNA uptake, e . g. , using DNA coupled to asialoorosomucoid which is taken up by the asialoglycoprotein receptor in the liver (Wu and Wu, J. Biol. Chem. 262: 4429-4432, 1987; Wu et al., J. Biol. Chem. 26-6: 14338-14342, 1991) , and liposome-mediated delivery ( e . g. , Kaneda et al . , Expt . Cell Res. 173: 56-69, 1987; Kaneda et al . , Science 243: 375-378, 1989; Zhu et al . , Science 261: 209-211, 1993) . Many of these physical methods can be combined with one another and with viral techniques; enhancement of receptor-mediated DNA uptake can be effected, for example, by combining its use with adenovirus (Curiel et al . , Proc. Natl. Acad. Sci. USA 88: 8850-8854, 1991; Cristiano et al., Proc. Natl. Acad. Sci. USA 90: 2122-2126, 1993) .

VIII. Pharmaceu ic l Formulations and Modes of

Administration

The particular compound, antibody, antisense or ribozyme molecule that affects the protein complexes and the disorder of interest can be administered to a patient either by themselves, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) .

In treating a patient exhibiting a disorder of interest, a therapeutically effective amount of a agent or agents such as these is administered. A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient .

Toxicity and therapeutic efficacy of such com- pounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. , for determining the LD ₅₀ (the dose lethal to 50% of the population) and the ED ₅₀ (the dose therapeutically effec¬ tive in 50% of the population) . The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD ₅₀ /ED ₅₀ . Compounds which exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be

estimated initially from cell culture assays. For exam¬ ple, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC ₅₀ as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal disrup¬ tion of the protein complex, or a half-maximal inhibition of the cellular level and/or activity of a complex compo¬ nent) . Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by HPLC.

The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g. Fingl et al . , in The Pharmacological Basis of Therapeutics. 1975, Ch. 1 p. 1) .

It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dys¬ functions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity) . The magnitude of an administrated dose in the management of the oncogenic disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veteri¬ nary medicine.

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, 18th ed. , Mack Publishing Co., Easton, PA (1990) . Suitable routes may include oral, rectal, transdermal, vaginal, transmucosal, or intestinal admini¬ stration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intra- thecal, 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 physio- logically 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 formula¬ tion. Such penetrants are generally known in the art. Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention. With proper choice of carrier and suitable manufacturing practice, the compositions of the present invention, in particular, those formulated as solutions, may be adminis¬ tered parenterally, such as by intravenous injection. The compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the invention to be formulated as tab-

lets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.

Agents intended to be administered intracellu- larly 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 adminis¬ tered 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 microenvi- ronment 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.

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharma¬ ceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceuti¬ cally. The preparations formulated for oral administra- tion may be in the form of tablets, dragees, capsules, or solutions.

The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g. , by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspen¬ sions of the active compounds may be prepared as appropri- ate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose , sodium carboxy- methylcellulose, and/or polyvinylpyrrolidone (PVP) . If

desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate .

Dragee cores are provided with suitable coat- ings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combina¬ tions of active compound doses.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticiz- er, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, option- ally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

Pharmaceutically acceptable salts can be acid addition salts such as those containing hydrochloride, sulfate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesul- fonate, p-toluenesulfonate, cyclohexylsulfamate and quinate. (See, e.g. , supra . PCT/US92/03736) . Such salts can be derived using acids such as hydrochloric acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic

acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenes- ulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid. Pharmaceutically acceptable salts can be prepared by standard techniques. For example, the free base form of the compound is first dissolved in a suitable solvent such as an aqueous or aqueous-alcohol solution, containing the appropriate acid. The salt is then isolated by evaporating the solution. In another example, the salt is prepared by reacting the free base and acid in an organic solvent .

Carriers or excipient can be used to facilitate administration of the compound, for example, to increase the solubility of the compound. Examples of carriers and excipients include calcium carbonate, calcium phosphate, various sugars or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physio¬ logically compatible solvents. The compounds or pharma- ceutical composition can be administered by different routes including intravenously, intraperitoneally, subcutaneously, and intramuscularly; orally, topically, or transmucosally.

A preferred physiological carrier is PBTE:D5W. PBTE consists of a solution of 3% w/v benzyl alcohol, 8% w/v polysorbate 80, and 65% w/v polyethylene glycol (MW =

300 daltons) in absolute ethanol. PBTE-.D5W consists of

PBTE diluted 1:1 in a solution of 5% dextrose in water.

The use of hydrophobic compounds can be facilitated by different techniques such as combining the compound with a carrier to increase the solubility of the

compound and using frequent small daily doses rather than a few large daily doses. For example, the composition can be administered at short time intervals, such as by the methods described above or using a pump to control the time interval or achieve continuous administration. Suitable pumps are commercially available (e.g.. the ALZET ^® pump sold by Alza corporation, and the BARD ambula¬ tory PCA pump sold by Bard MedSystems) .

The proper dosage depends on various factors such as the type of disease being treated, the particular composition being used, and the size and physiological condition of the patient. The expected daily dose is between 1 to 2000 g/day, preferably 1 to 250 mg/day, and most preferably 10 to 150 mg/day. Drugs can be delivered less frequently provided plasma levels of the active moiety are sufficient to maintain therapeutic effectiveness .

A factor which can influence the drug dose is body weight. Drugs should be administered at doses ranging from 0.02 to 25 mg/kg/day, preferably 0.02 to 15 mg/kg/day, most preferably 0.2 to 15 mg/kg/day. Alterna¬ tively, drugs can be administered at 0.5 to 1200 mg/m ² /day, preferably 0.5 to 150 mg/m ² /day, most preferably 5 to 100 mg/m ² /day. The average plasma level should be 50 to 5000 μg/ml, preferably 50 to 1000 μg/ml, and most preferably 100 to 500 μg/ml. Plasma levels may be reduced if pharma¬ cological effective concentrations of the drug are achieved at the site of interest.

IX. Identification of Agents

The complexes, components of such complexes, functional equivalents thereof, and/or cell lines that express such components and exhibit such protein complexes may be used to screen for additional compounds, antibod- ies, or other molecules capable of modulating the signal transduction event such complexes are involved in. Methods for purifying and/or producing such complexes, components of the complexes, functional equivalents thereof, and/or cell lines are described herein. The compounds, antibodies, or other molecules identified may, for example, act to disrupt the protein complexes of the invention (i.e.. decrease or inhibit interactions between component members of the complexes, thereby causing physical separation of the components, and/or perturbing the activity of the complexes) or may lower the cellular level and/or decrease the activity of one or more of the components of such complexes.

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 . , Nature 354:82-84, 1991) , phosphopeptides (in, for example, the form of random or partially degenerate, directed phosphopeptide libraries, see Song¬ yang et al., Cell 767-778, 1993) , antibodies, and small organic or inorganic molecules. Synthetic compounds, natural products, and other sources of potentially biologically active materials may be screened in a variety of ways, as described herein. The compounds, antibodies, or other molecules identified may be used as oncogenic disorder treatments, as described herein. Compounds that bind to individual components, or functional portions of

the individual components of the complexes (and may additionally be capable of disrupting complex formation) may be identified.

One such method included within the scope of the invention is a method for identifying an agent to be tested for an ability to modulate a signal transduction pathway disorder. The method involves exposing at least one agent to a protein comprising a functional portion of a member of the protein complex for a time sufficient to allow binding of the agent to the functional portion of the member; removing non-bound agents; and determining the presence of the compound bound to the functional portion of the member of the protein complex, thereby identifying an agent to be tested for an ability to modulate a disorder involving a polypeptide complex.

By "signal transduction disorder" is meant any disorder associated with an abnormality in a signal transduction pathway. The protein complex referred to below is a physical association of a Zap 70 kinase and a Zap 70 binding partner. The level of interaction between the two components of the complex may be abnormal and thus cause the abnormality in the signal transduction pathway. Alternatively, the level of interaction between the complex components may be normal, but affecting that interaction may effectively treat a signal transduction pathway disorder.

The term "protein" refers to a compound formed of 5-50 or more amino acids joined together by peptide bonds. An "amino acid" is a subunit that is polymerized to form proteins and there are twenty amino acids that are universally found in proteins. The general formula for an

amino acid is H ₂ N-CHR-COOH, in which the R group can be anything from a hydrogen atom (as in the amino acid glycine) to a complex ring (as in the amino acid tryptophan) . A functional portion of an individual component of the complexes may be defined here as a protein portion of an individual component of a complex still capable of forming a stable complex with another member of the complex under standard cellular and physiological condi- tions. For example, a functional portion of a component may include, but is not limited to, a protein portion of Zap 70 which is still capable of stably binding a corresponding ligand domain of an associated protein, and thus is still capable of forming a complex with that protein. Further, in the case of the catalytic domains of the individual components of the invention, a func¬ tional portion of a catalytic domain may refer to a protein still capable of stably binding a substrate molecule under standard physiological conditions. One method utilizing this approach that may be pursued in the isolation of such 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 exam¬ ple, nylon or nitrocellulose, and the subsequent incuba¬ tion of the attached component molecule in the presence of a potential component-binding compound or compounds. Attachment to said solid support may be direct or by means of a component specific antibody bound directly to the solid support. 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 complex component-binding activity. The complex components which may be utilized in the above screening method may include, but are not lim¬ ited to, molecules or functional portions thereof, such as catalytic domains, phosphorylation domains, extracellular domains, or portions of extracellular domains, such as ligand-binding domains, and adaptor proteins, or function¬ al portions thereof. The peptides used may be phosphory¬ lated, 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 specifi¬ cally phosphorylated at certain amino acid residues. A functional portion of such a phosphorylation domain may be defined as a peptide capable of being specifically phos¬ phorylated at certain amino acids by a specific protein. Molecules exhibiting binding activity may be further screened for an ability to disrupt protein com¬ plexes. Alternatively, molecules may be directly screened for an ability to promote the complexes. For example, in vitro 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. In addition, one could look for an increase in binding.

Additionally, complex formation in a whole cell may be assayed by utilizing co-immunoprecipitation tech¬ niques well known to those of skill in the art. Briefly, a cell line capable of forming a 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 immuno¬ precipitation 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.

A preferred method for assessing modulation of complex formation within a cell utilizes a method similar to that described above. Briefly, a cell line capable of forming a complex of interest is exposed to a test com¬ pound. The cells are lysed and the lysate contacted with an antibody specific to one component of the complex, said antibody having been previously bound to a solid support. Unbound material is washed away, and the bound material is exposed to a second antibody, said second antibody binding specifically to a second component of the complex. The amount of second antibody bound is easily detected by techniques well known in the art. Cells exposed to an inhibitory test compound will have formed a lesser amount of complex compared to cells not exposed to the test

compound, as measured by the amount of second antibody bound. Cells exposed to a test compound that promotes complex formation will have an increased amount of second antibody bound. The effect of an agent on the differentiation capability of the 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 to a cell such as a neuronal cell, capable of forming a complex, for example, which, in the absence of any agent, would not lead to the cell's differ¬ entiation. The differentiation state of the cell may then be measured either in vitro or in vivo. One method of measurement may involve observing the amount of neurile growth present .

Agents capable of disrupting complex formation and capable of reducing or inhibiting disorders, which involve the formation of such complexes, or which involve the lack of formation of such complexes, may be used in the treatment of patients exhibiting or at risk for such disorders. A sufficient amount of agent or agents such as those described above may be administered to a patient so that the symptoms of the disorder are reduced or eliminated.

X. Purification and Production of Complexes

Described in this Section are methods for the synthesis or recombinant expression of components, or fragments thereof, of the protein complexes of the inven- tion. Also described herein are methods by which cells

exhibiting the protein complexes of the invention may be engineered.

xi. Pur fi tion Methods

The complexes of the invention may be substan- tially purified, i.e., may be purified away from at least 90% (on a weight basis) , and from at least 99%, if de¬ sired, of other proteins, glycoproteins, and other macro- molecules 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 complex to a combi¬ nation of standard methods, for example, ammonium sulfate precipitation, molecular sieve chromatography, and/or ion exchange chromatography. Alternatively, or additionally, a complex may be purified by immunoaffinity chromatography using an immuno- adsorbent column to which an antibody is immobilized which is capable of binding to one or more components of the complex. Such an antibody may be monoclonal or poly- clonal in origin. Other useful types of affinity purifi¬ cation for the protein complex may utilize, for example, a solid-phase substrate which binds the catalytic kinase domain of a protein, or an immobilized binding site for noncatalytic domains of the components of the complex, which bind in such a manner as to not disrupt the complex. The complex of the present invention may be biochemically purified from a variety of cell or tissue sources.

XII. Synthesis and Expression Methods

Methods for the synthesis of polypeptides or fragments thereof, which are capable of acting as compo¬ nents of the complexes of the present invention, are well- known to those of ordinary skill in the art. See, for example, Creighton, Proteins: Structures and Molecular Principles, W.H. Freeman and Co., NY (1983) , which is incorporated herein, by reference, in its entirety.

Components of a complex which have been sepa¬ rately 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 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.

Methods for preparing the components of complex- es of the invention by expressing nucleic acid encoding proteins are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing protein coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro 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., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.

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

A variety of host-expression vector systems may be utilized to express the coding sequences of the compo¬ nents of the complexes of the invention. Such host- expression systems represent vehicles by which the coding sequences of interest may be produced, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, exhibit the protein complexes of the invention. These include but are not limited to microorganisms such as bacteria (e.g., E. coli , B. subtilis) transformed with recombinant bacte- riophage DNA, plasmid DNA or cosmid DNA expression vectors containing protein coding sequences; yeast (e.g., Saccha- romyces and Pichia) transformed with recombinant yeast expression vectors containing the protein coding sequenc¬ es; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the protein coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or trans¬ formed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the 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 complex being expressed. For exam¬ ple, when large quantities of 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 . , EMBO J. 2:1791, 1983) , in which the 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 and Inouye, Nucleic acids Res . 13:3101-3109, 1985; Van Heeke & Schuster, J. Biol . Chem . 264:5503-5509, 1989) ; 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 protein can be released from the GST moiety. In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The complex coding sequence 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 PTK/adaptor complex coding sequence 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., J. Biol . 46:584, 1983; 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 complex coding sequence may be ligated to an adenovirus transcrip¬ tion/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 vitro 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 proteins in infected hosts. (E.g., See Logan & Shenk, Proc . Natl . Acad . Sci . USA 81:3655-3659, 1984) Specific initiation signals may also be required for efficient translation of inserted coding sequences. These signals include the ATG initiation codon and adja- cent sequences.

In cases where an entire 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 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 initia¬ tion 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 . , Methods in Enzymol . 153:516-544, 1987)

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 recombi¬ nant proteins, stable expression is preferred. For example, cell lines which stably coexpress both the proteins may be engineered. Rather than using expression vectors which contain viral origins of replication, host

cells can be transformed with the protein encoding 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. Such engineered cell lines are particu¬ larly 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., Cell 11:223, 1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl . Acad. Sci . USA 48:2026, 1962), and adenine phosphoribosyltransferase (Lowy et al . , Cell 22:817, 1980) genes can be employed in tk ^" , hgprt ^" or aprt ^" cells, respectively. Also, anti etabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler et al . , Na tl . Acad. Sci . USA 77:3567, 1980; O'Hare et al. , Proc. Natl . Acad. Sci . USA 78:1527, 1981); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc . Natl . Acad. Sci . USA 78:2072, 1981) ; neo, which confers resistance to the

aminoglycoside G-418 (Colberre-Garapin et al . , J. Mol . Biol . 150:1, 1981) ; and hygro, which confers resistance to hygromycin (Santerre et al . Gene 30:147, 1984) genes.

New members of the 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 protein encoding 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 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 complexes. The PCR product may be subcloned and sequenced to insure that the amplified sequences represent the sequences of a member of the PTK or adaptor subfamily. The PCR fragment may then be used to isolate a full length protein cDNA clone by radioactively labeling the amplified fragment and screen¬ ing a bacteriophage 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, Molecular Cloning: A Laboratory Manual . Cold Springs Harbor Press, N.Y. (1989) ; and Ausubel et al . , Current Protocols in Molecular Biology. Green

Publishing Associates and Wiley Interscience, N.Y. (1989) . A general method for cloning previously unknown proteins has been described by Skolnik (Skolnik, E.Y., Cell 65:75, 1991) and Skolnik et al. , (U.S. Patent Application Serial

797

78

No. 07/643,237) which are incorporated herein by reference, in their entirety, including drawings.

XIII. Derivatives of Complexes

Also provided herein are functional derivatives of a complex or a component thereof. By "functional derivative" is meant a "chemical derivative," "fragment," "variant," "chimera,", "hybrid", "mutant variation", "species variation" or "allelic variation" of the complex or a component thereof, which terms are defined below. A functional derivative retains at least a portion of the function of the protein, for example reactivity with an antibody specific for the complex, enzymatic activity or binding activity mediated through noncatalytic domains, which permits its utility in accordance with the present invention.

A "chemical derivative" 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 de¬ scribed below. Cysteinyl residues most commonly are reacted with alpha-haloacetates (and corresponding amines) , such as chloroacetic acid or chloroacetamide, to give carboxy¬ methyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotri- fluoroacetone, chloroacetyl phosphate, N-alkylmaleimides,

79

3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-l,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. Deriv- atization with these agents has the effect or reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing primary amine containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloro- borohydride; trinitrobenzenesulfonic acid; O-methy- lisourea; 2,4 pentanedione,- and transaminase-catalyzed reaction with glyoxylate .

Arginyl residues are modified by reaction with one or several conventional reagents, among them phenyl- glyoxal, 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, of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine alpha-amino group.

Tyrosyl residues are well-known targets of modi¬ fication 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) carbo¬ diimide or l-ethyl-3- (4-azonia-4, 4-di ethylpentyl) carbo¬ diimide. Furthermore, aspartyl and glutamyl residue are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. Glutaminyl and asparaginyl residues are fre¬ quently 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 complexes to each other or the complex to a water-insoluble support matrix or to other macromolecu¬ lar carriers. Commonly used cross-linking agents include, for example, 1, 1-bis (diazoacetyl) -2-phenylethane, glutar- aldehyde, 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 bro- mide-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 em¬ ployed 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 Pharma¬ ceutical Sciences, 18th ed., Mack Publishing Co., Easton, PA (1990) .

The term "fragment" is used to indicate a poly- peptide derived from the amino acid sequence of a protein of the complex 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 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 protein, when present in a complex resembling the naturally occurring 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 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 complex, or an epitope thereof.

By "unique fragment," is meant an amino acid sequence present in a full-length Zap 70 kinase that is not present in any other naturally occurring polypeptide. Preferably, such a sequence comprises 6 contiguous amino acids present in the full sequence. More preferably, such a sequence comprises 12 contiguous amino acids present in the full sequence. Even more preferably, such a sequence comprises 18 contiguous amino acids present in the full sequence.

Another functional derivative intended to be within the scope of the present invention is a complex comprising at least one "variant" polypeptide which either lack one or more amino acids or contain additional or substituted amino acids relative to the native polypep¬ tide. The variant may be derived from a naturally occur¬ ring complex component by appropriately modifying the 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 complex, as described above.

A functional derivative of complexes comprising one or more 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 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 complexes typically exhibit the same qualitative biological activity as the native complexes.

By "mutant variation" is meant a nucleic acid or amino acid molecule that results from any detectable change in the genetic material which may be transmitted to daughter cells giving rise to mutant cells, including nucleic acids or polypeptides having nucleotides or amino acids that are added, deleted, substituted for, inverted, or transposed to new positions with and without inversion. The mutant variation may occur spontaneously or may be induced experimentally by application of mutagens and may result from any (or a combination of) detectable, unnatural change affecting the chemical or physical constitution, mutability, replication, phenotypic

function, or recombination of one or more deoxyribonucleotides.

By "species variation" is meant a change in the nucleic acid or amino acid sequence that occurs among species and may be determined by DNA sequencing of the molecule in question.

By "allelic variation" is meant an alternative functional derivation of the typical form of a gene in an organism occupying a given locus on a chromosome.

XIV. Evaluation of Disorders

The protein complexes of the invention involved in disorders may be utilized in developing a prognostic evaluation of the condition of a patient suspected of exhibiting such a disorder. For example, biological samples obtained from patients suspected of exhibiting a disorder involving a protein complex may be assayed for the presence of such complexes. If such a protein complex is normally present, and the development of the disorder is caused by an abnormal quantity of the complex, the assay should compare complex levels in the biological sample to the range expected in normal tissue of the same cell type.

Among the assays which may be undertaken may include, but are not limited to isolation of the protein complex of interest from the biological sample, or assay¬ ing for the presence of the complex by exposing the sample to an antibody specific for the complex, but non-reactive to any single, non-complexed component, and detecting whether antibody has specifically bound.

85

Alternatively, one or more of the components of the protein complex may be present in an abnormal level or in a modified form, relative to the level or form expected is normal, nononcogenic tissue of the same cell type. It is possible that overexpression of both components may indicate a particularly aggressive disorder. Thus, an assessment of the individual and levels of mRNA and protein in diseased tissue cells may provide valuable clues as to the course of action to be undertaken in treatment of such a disorder. Assays of this type are well known to those of skill in the art, and may include, but are not limited to, Northern blot analysis, RNAse protection assays, and PCR for determining mRNA levels. Assays determining protein levels are also well known to those of skill in the art, and may include, but are not limited to, Western blot analysis, immunoprecipitation, and ELISA analysis . Each of these techniques may also reveal potential differences in the form (e.g., the primary, secondary, or tertiary amino acid sequence, and/or post-translational modifications of the sequence) of the component (s) .

EXAMPLES The examples below are non-limiting and are merely representative of various aspects and features of the present invention. The examples below demonstrate a method of screening compounds for the ability to affect tyrosine phosphorylation of Zap 70.

The following materials were used in the examples described below.

Lysis Buffer: 20mM Tris-HCI pH 7.5 + 150mM NaCl + 5mM EDTA + 1% NP40 + ImM Na ₃ V0 ₄ . Thymocytes : 50 x 10 ⁶ /ml

____ : α Zap-70 from UBI and α Ptyr nonoclonal from UBI ECL reagent : Amersham

Tyrphostins: Compounds 4, 36, 31, 46, 32, 17, 10, 19, 51, 49, 47, and 52 from Gazit et al . , J. Med. Chem.. 32 (10) .-2344-2352 (1989) and compounds 20, 7, 12, and 42 from Gazit et al . , J. Med. Chem.. 34 (6) : 1896-1907 (199i; were used.

Example 1; Tyrosine Phosphorylation of Zap 70

Stimulation and Lysis

5 x 10 ⁶ Thymocytes were incubated overnight with 50μM of different Tyrphostin blockers while controls were incubated without the blockers in RPMI

1640 containing 1% FES. The cells were then spun and resuspended in RPMI 1640 without serum at a concentration of 50 x 10 ⁶ /ml. αCD3 at 20 μg/ml was used to stimulate the cells for 10 min at 37°C. The cells were then spun and the pellets lysed in 1ml of lysis buffer.

Immunoprecipitation

5μl of α Zap-70 Ab was added and 15 μl of a

50:50 suspension of Protein-A sepharose 4B-CL in lysis buffer. Immunoprecipitation was carried out overnight with shaking at 4°C. The precipitation was then washed

3 times with lysis buffer and taken up in 50 μl of SDS-

PAGE reducing sample buffer.

Blotting

Proteins were separated on an 8% polyacrylamide gel and tranferred onto mitrocellulose . The membrand was blocked with 5% BSA in TBS containing 0.05% Tween 20. (TBST) for 4hr at room temperature and then incubated with α PTyr Ab at 1:1000 dilution in 1% BSA in TBST for 2 hr at room temperature. After washing for 20 min with TBST the membrane was incubated with sheep anti-mouse-HRP conjugated second antibody at a 1:5000 dilution in 1% BSA in TBST for lhr at room temperature and ECL carried out as per manufacturer's instructions. RESULTS

Pre-incubation of stimulated cells for 16h with compounds 183, 294, and 217 listed above reduced tyrosine phosphorylation of Zap-70 approximately 60%, 50%, and 30%-40% respectively as estimated by analysis of the blots described above.

Other embodiments are within the following claims .