WO2007126913A1

WO2007126913A1 - Methods for prevention or treatment of viral disease

Info

Publication number: WO2007126913A1
Application number: PCT/US2007/007711
Authority: WO
Inventors: Julie A. READINGER; Pamela L. Schwartzberg; Andrew J. Henderson; Gillian M. Schiralli; Avery August
Original assignee: Penn State Research Foundation; US Department of Health and Human Services
Current assignee: Penn State Research Foundation; US Department of Health and Human Services
Priority date: 2006-03-27
Filing date: 2007-03-27
Publication date: 2007-11-08
Anticipated expiration: 2008-09-27

Abstract

Compositions and methods are provided for preventing or treating viral disease and other related diseases in a mammalian subject. The method provides administering to the mammalian subject a compound capable of inhibiting a TEC tyrosine kinase, wherein the compound is administered in an amount effective to reduce or eliminate the viral disease or to prevent its occurrence or recurrence. A method for identifying a compound capable of inhibiting viral infection of a cell is provided wherein the compound is a TEC tyrosine kinase inhibitor.

Description

METHODS FOR PREVENTION OR TREATMENT OF VIRAL DISEASE

CROSS REFERENCE TO RELATED APPLICATION

[[0001] This application is related to U.S. Provisional Application No. 60/786,245, filed March 27, 2006, the disclosure of which is incorporated by reference in its entirety.

FIELD

[0002] The present invention relates generally to a method for preventing or treating viral disease in a mammalian subject. The method provides administering to the mammalian subject a compound capable of inhibiting a TEC tyrosine kinase, wherein the compound is administered in an amount effective to reduce or eliminate the viral disease or to prevent its occurrence or recurrence. A method for identifying compounds capable of inhibiting viral infection in a cell is provided wherein the compound is a TEC tyrosine kinase inhibitor.

BACKGROUND

[0003] The TEC family nonreceptor tyrosine kinases have recently emerged as key regulators of signaling pathways in T lymphocytes. The importance of this family was first established in 1993 when mutations affecting BTK were found to be associated with the human genetic disorder X-linked agammaglobulinemia (XLA) and the murine mutant X-linked immunodeficiency (pad), immunodeficiencies associated .with decreased serum immunoglobulins and impaired B cell development. XLA provided the first example of mutations affecting a tyrosine kinase linked to a primary human immunodeficiency, highlighting the importance of tyrosine kinases for antigen receptor signaling pathways and their roles in lymphocyte development and function.

[0004] The TEC family of kinases now consists of five family members, which are expressed primarily in hematopoietic cells: TEC, BTK, ITK (also known as TSK and EMT), RLK (also known as TXK), and BMX (also known as ETK). Additional related TEC family kinases have been found in Drosophila melanogaster, zebrafϊsh (Danio reriό), skate (Raja eglanteria), and sea urchin (Anthocidaris crassispina). Although the TEC family kinases resemble Src family kinases, having tyrosine kinase catalytic and Src homology protein interaction domains, they are notable in that most family members possess a pleckstrin homology (PH) domain that binds to the products of phosphoinositide 3-kinase (PI3K). The TEC family kinases are the only tyrosine kinases that possess PH domains and that can be regulated by PI3K. In T lymphocytes, three major TEC kinases are expressed: ITK, RLK, and TEC, all of which are tyrosine phosphorylated upon T cell receptor (TCR) stimulation.

[0004] ITK is a TEC family tyrosine kinase expressed in a limited number of cell types including T cells, NK cells, mast cells, and eosinophils. ITK has been shown to be important in the activation of T cells through the T cell Receptor (TCR), where it participates in regulation of PLC γl , Ca⁺* mobilization, and activation of the downstream transcription factors NFAT and AP-I. T cells from ITK deficient mice show decreases in IL-2 production and proliferation as well as defects in T_H2 differentiation and cytokine production (as reviewed in Finkelstein, et al. Trends Cell Biol 14: 443-451, 2004; Lucas, et al. Immunol Rev 191: 119-138, 2003; Schwartzberg, et al. Nat Rev Immunol 5: 284-295, 2005). Nonetheless, ITK-deficient mice are still able to mount some immune responses to and clear viral infections, suggesting that some immune functions are intact in the absence of ITK. Bachmann, et al. J Virol 71: 7253-7257, 1997.

[0005] Infection of T cells with the human immunodeficiency virus (HIV), a major health threat in both the United States and in the developing world, requires T cell activation, as well as function of chemokine receptors, which serve as co-receptors for viral infection. Currently, anti-HFV therapy relies on the use of inhibitors of viral replication which unfortunately leads to the rapid selection of resistant viruses. A need exists in the art for alternative treatments for HIV infection, and in particular, for treatments that target additional mechanisms relating to HIV infection.

SUMMARY

[0006] A method for preventing or treating viral disease in a mammalian subject is provided comprising administering to the mammalian subject a TEC tyrosine kinase inhibitor in an amount effective to reduce or eliminate the viral disease. The target of the inhibitor is a TEC tyrosine kinase, including but not limited to, TEC, BTK, ITK, RLK, or BMX. The inhibitor can be a small chemical compound, siRNA, shRNA, ribozyme, antisense, antibody, peptide, or peptide mimetic. [0007] The use of inhibitors against cellular proteins required for viral entry or replication provides a potential alternative therapeutic approach to treatment of viral disease, for example, HIV disease or human herpesviral disease. The implication of ITK in these processes suggests that the defects associated with ITK-deficiency may affect HIV infection and raise the possibility that ITK is a potential therapeutic target for the treatment of HIV.

[0008] The use of inhibitors directed against cellular proteins required for viral entry or replication provides an alternative therapeutic approach to treatment of infectious disease, e.g., viral disease. ITK is a member of the TEC family of tyrosine kinases that is expressed in T lymphocytes, NK cells, eosinophils and mast cells. ITK is required for full T cell activation through the T cell receptor, as well as T cell responses to chemokines. Nonetheless, ITK- deficient mice can still mount responses to many viral infections suggesting that many immune functions in these mice are preserved. Since productive infection of T cells by HIV involves both cellular activation and chemokine receptor function, the present study examined whether ITK inhibition can affect HIV replication. In a similar manner, a number of viruses make viral homologs of chemokine receptors and chemokines, including lentivirus, human immunodeficiency virus, feline immunodeficiency virus, maedi-visna, herpesvirus, cytomegalovirus, Kaposi's sarcoma herpesvirus, poxvirus, or myxoma virus, have been suggested to use chemokine receptors for entry, and ITK inhibition may affect replication of these viruses. The results show that blocking ITK, either by reducing its expression using short- interfering RNA (siRNA) or by blocking its function with a small molecule chemical inhibitor, or by expression of ITK mutants, interferes with HIV infection of T lymphocytes at multiple stages of infection. These results provide evidence for the use of ITK as a cellular therapeutic target for blocking HIV infection. Furthermore, other TEC family kinases play similar roles in other cells of the immune system. Other TEC tyrosine kinases, e.g., TEC, BTK, ITK, RLK, or BMX tyrosine kinase, are potential therapeutic targets for blocking HIV infection in other cell types (i.e., TEC and BTK are expressed in macrophages). Inhibitors of TEC family kinases can be a cellular therapeutic target to prevent infection by lentivirus, human immunodeficiency virus, feline immunodeficiency virus, human T cell leukemia virus I and π, maedi-visna, herpesvirus, cytomegalovirus, Kaposi's sarcoma herpesvirus, poxvirus, or myxoma virus.

[0009] A method for preventing or treating viral disease in a mammalian subject is provided which comprises administering to the mammalian subject a TEC tyrosine kinase inhibitor, wherein the inhibitor is administered in an amount effective to reduce or eliminate the viral disease or to prevent its occurrence or recurrence. The TEC tyrosine kinase includes, but not limited, TEC, BTK, ITK, RLK, or BMX tyrosine kinase. In one aspect, the inhibitor can be a small chemical compound, short interfering RNA, dominant-negative molecule, short hairpin RNA, ribozyme, antisense oligonucleotide, antibody, peptide or peptidomimetic. The dominant- negative molecule includes, but is not limited to, a dominant-negative peptide or peptidomimetic. In a detailed aspect, the small chemical compound is BMS509744, or another chemical inhibitor of TEC tyrosine kinase.

[0010] The method for preventing or treating viral disease in a mammalian subject is provided wherein the viral disease is an infection by viruses including, but not limited to, lentivirus, herpes virus, pox virus, human immunodeficiency virus, human T cell leukemia virus type I, human T cell leukemia virus type II, human herpesvirus, human herpesvirus 6, human herpesvirus 7, human cytomegalovirus, Kaposi's sarcoma herpesvirus, feline immunodeficiency virus, maedivisna virus, or myxoma virus.

[0011] An in vitro method of screening inhibitors of TEC tyrosine kinase activity is provided which comprises contacting a cell line with a test compound that inhibits TEC tyrosine kinase activity, and detecting an increase or a decrease in susceptibility of the cell line to viral infection, wherein effectiveness of the test compound in the assay is indicative that TEC tyrosine kinase inhibitor modulates viral infection in the cell. The TEC family tyrosine kinase includes, but is not limited to, a TEC, BTK, ITK, RLK, or BMX tyrosine kinase. The effectiveness of the test compound in the assay can be indicative of the inhibition of viral infection of the cell. The TEC tyrosine kinase inhibitor can increase or decrease viral replication in the cell. The TEC tyrosine kinase inhibitor can increase or decrease viral entry of the cell. The TEC tyrosine kinase inhibitors that decrease viral replication or decrease viral entry in the cell are useful as therapeutics to treat viral infection in a mammalian subject. The TEC tyrosine kinase inhibitors that may increase viral replication or increase viral entry in the cell can be useful to identify other targets of the TEC tyrosine kinase pathway that may be a target for therapeutic inhibitors to treat viral infection in a mammalian subject. In one aspect, the cell line is a T cell, NK cell, mast cell, or eosinophil cell. In a detailed aspect, the cell line is a Jurkat cell line or a human CDA⁺ cell.

[0012] The in vitro method of screening inhibitors of TEC tyrosine kinase activity further comprises detecting an increase or a decrease in susceptibility of the cell line to viral infection using an HIV-I vector or a pseudotyped HIV-I vector capable of signaling viral infection in the cell line. In a further aspect a compound identified according to the in vitro method is provided.

[0013] A method for identifying a compound capable of modulating viral infection of a cell is provided which comprises contacting a test compound with a cell-based assay system comprising a cell expressing TEC tyrosine kinase and capable of signaling responsiveness to TEC tyrosine kinase, detecting an effect of the test compound as an inhibitor of TEC tyrosine kinase in the assay system, and detecting an effect of the test compound to modulate susceptibility of the cell to viral infection, effectiveness of the test compound in the assay being indicative that the test compound modulates viral infection in the cell. The TEC family tyrosine kinase includes, but is not limited to, a TEC, BTK, ITK, RLK, or BMX tyrosine kinase. The TEC tyrosine kinase inhibitor can increase or decrease viral infection as measured by an increase or decrease viral replication in the cell, respectively, or by an increase or decrease viral entry of the cell, respectively. The TEC tyrosine kinase inhibitors that decrease viral replication or decrease viral entry in the cell are useful as therapeutics to treat viral infection in a mammalian subject. The TEC tyrosine kinase inhibitors that may increase viral replication or increase viral entry in the cell can be useful to identify other targets of the TEC tyrosine kinase pathway that may be a target for therapeutic inhibitors to treat viral infection in a mammalian subject. In one aspect, the test compound is a small chemical molecule, interfering RNA, dominant-negative molecule, short hairpin RNA, ribozyme, antisense oligonucleotide, protein inhibitor, monoclonal antibody, polyclonal antibody, peptide, peptidomimetic, or a nucleic acid. In a detailed aspect, the dominant-negative molecule is a dominant-negative peptide or peptidomimetic.

[0014] The method is further provided wherein the viral infection is an infection by viruses including, but not limited to, lentivirus, herpes virus, pox virus, human immunodeficiency virus, human T cell leukemia virus type I, human T cell leukemia virus type II, human herpesvirus, human herpesvirus 6, human herpesvirus 7, human cytomegalovirus, Kaposi's sarcoma herpesvirus, feline immunodeficiency virus, maedivisna virus, or myxoma virus. In a further aspect, the method comprises detecting an increase or a decrease in susceptibility of the cell line to viral infection using an HIV-I vector or a pseudotyped HIV-I vector capable of signaling viral infection in the cell-based assay system. In one aspect, the cell- based assay system utilizes a T cell, NK cell, mast cell, or eosinophil cell. In a detailed aspect, the cell-based assay system is a Jurkat cell or a human CD4⁺ cell -based assay system. The TEC tyrosine kinase includes, but is not limited to, TEC, BTK, ITK, RLK, or BMX tyrosine kinase. A compound is provided which is identified according to the method.

[0015] A pharmaceutical composition is provided comprising a TEC family kinase inhibitor for treatment of viral infection in a mammalian subject. The virus to be treated includes, but is not limited to, lentivirus, herpes virus, pox virus, human immunodeficiency virus, human T cell leukemia virus type I, human T cell leukemia virus type π, human herpesvirus, human herpesvirus 6, human herpesvirus 7, human cytomegalovirus, Kaposi's sarcoma herpesvirus, feline immunodeficiency virus, maedivisna virus, or myxoma virus. In one aspect, the inhibitor is interfering RNA, short hairpin RNA, ribozyme, antisense oligonucleotide, or protein inhibitor, a monoclonal antibody, polyclonal antibody, dominant-negative molecule, peptide, peptidomimetic, or a small chemical molecule. In a detailed aspect, the dominant- negative molecule is a dominant-negative peptide or peptidomimetic.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 shows that loss of ITK by siRNA does not alter CD4, CXCR4 or CCR5 expression.

[0017] Figures 2 A, 2B, 2C show that loss of ITK by siRNA leads to a decrease in HIV infection.

[0018] Figure 3 shows that ITK inhibition in human CD4⁺ T cells alters virus entry but not binding.

[0019] Figure 4 shows that loss of ITK by siRNA reduces actin polarization to SDFl α and gpl20.

[0020] Figures 5A and 5B show that reverse transcription and integration are not affected during receptor independent infection.

[0021] Figures 6A and 6B show that loss of ITK decreases HIV transcription.

[0022] Figure 7 shows that ITK is a positive regulator of HIV transcription in Jurkat cells.

[0023] Figure 8 shows that a decrease in intracellular P24 occurs with loss of ITK.

[0024] Figure 9 shows that loss of ITK post infection also inhibits HIV replication.

[0025] Figure 10 shows that inhibition of ITK in human CD4⁺ T cells decreases HIV replication.

[0026] Figure 11 shows loss of ITK reduces HIV replication post infection.

[0027] Figure 12 shows cell death is unaffected by loss of ITK.

[0028] Figure 13 shows cell death after BMS509744 treatment does not account for changes in HIV infection.

[0029] Figure 14 shows expression of TTK increases the release of Gag- Virus like particles.

DETAILED DESCRIPTION

[0030] A method for preventing or treating viral disease in a mammalian subject is provided comprising administering to the mammalian subject an inhibitor of a TEC tyrosine kinase in an amount effective to reduce or eliminate the viral disease. The target of the inhibitor is a TEC tyrosine kinase, including but not limited to, TEC, BTK, ITK, RLK, or BMX tyrosine kinase. The inhibitor can be a small chemical compound, siRNA, shRNA, ribozyme, antisense, antibody, peptide, or peptide mimetic.

[0031] ITK is a TEC family tyrosine kinase that is an important mediator of T cell function including activation, differentiation, and chemotaxis. Biochemical and cellular studies have demonstrated that ITK regulates TCR-induced activation of PLC γl , Ca⁺⁺ mobilization and NFAT activation, as well as actin rearrangement downstream from both the T cell receptor and chemokine receptors. Since productive infection of T cells by HIV involves both actin rearrangement and cellular activation, it was hypothesized that ITK may be a therapeutic target for HIV infection. To evaluate this question ITK function was blocked using ITK-specific siRNA (silTK) or an ITK inhibitor, BMS509774. See U.S. Patent Application No. 2004/0077695. The results show that inhibition of ITK partially blocks HIV viral entry. Nonetheless, treatment of cells with siRNA to knockdown ITK did not affect CD4 receptor or CXCR4 and CCR5 coreceptor expression. Additionally, silTK treatment of Jurkat cells decreased transcription of a transiently transfected HIV-luciferase vector. Finally, preliminary data suggests that a loss of ITK by either chemical inhibition in primary human CD4+ T cells or siRNA in Jurkat cells resulted in a marked defect in intracellular p24 expression. The data suggest that ITK is a potential cellular target for therapeutic development against AIDS that affects HIV replication at multiple steps.

[0032] Recent data has implicated ITK in the regulation of actin rearrangement downstream from both the T cell receptor and chemokine receptors. Dombroski, et al. J Immunol YlA: 1385-1392, 2005; Grasis, et al. J Immunol 170: 3971-3976, 2003; Takesono, et al. Curr Biol 14: 917-922, 2004; Tsoukas, et al. Trends Immunol 22: 17-20, 2001. In particular, ITK is involved in chemokine receptor signaling through CXCR4 and other chemokine receptors. ITK deficient cells have defects in migration, actin polarization, and activation of Racl in response to SDFlα and other chemokines. Takesono, et al. Curr Biol 14: 917-922, 2004. The CXCR4 and CCR5 chemokine receptors are coreceptors for HIV binding and entry into T cells. Moreover, changes in actin rearrangement and activation of Racl have been shown to regulate the entry of HIV virus into the host cell (as reviewed in Matarrese, et al. Cell Death Differ 12 Suppl 1: 932-941, 2005; Pontow, et al. J Virol 78: 7138-7147, 2004).

[0033] ITK can be a drug target for inhibitors of ITK activity to treat or prevent infectious disease, e.g., viral disease. Deficiency of ITK results in a reduction of CXCR4 dependent T cell activity. Pharmaceutical groups are already targeting ITK for potential drugs against allergic responses, e.g., allergic asthma. ITK is expressed in limited cell types: T cells, NK cells, mast cells, and eosinophils. Deficiency of ITK results in reduction in T_H2 but not all T_HI responses.

• The results presented herein show that drugs that inhibit ITK activity have antiviral activity, in particular, against HIV.

• CXCR4, a coreceptor for HIV, requires ITK for T cell dependent responses (Takesono, et al. Curr Biol 14: 917-922, 2004; Fischer, et al. J. Biol. Chem. 279: 29816-29820, 2004). The results presented herein show that HIV infection is related to ITK activity.

• CXCR4 is a coreceptor for HIV entry. ITK is activated downstream of CXCR4. (Takesono, et al. CurrBiol 14: 917-922, 2004; Fischer, et al. J. Biol. Chem. 279: 29816- 29820, 2004)

• Actin rearrangement is important for HIV virus infection (Bukrinskaya, J Exp Med., 188: 2113-2125, 1998). F-actin accumulation to SDFlα is blocked by a dominant negative ITK mutant or siRNA to TTK. (Takesono, et al. Curr Biol 14: 917-922, 2004) The results presented herein show that F-actin accumulation to HIV gpl20 is blocked by siRNA to ITK.

• HIV viral entry activates Rac 1 downstream CXCR4 (Pontow, et al. J Virol 78: 7138- 7147, 2004). Dominant negative ITK mutation blocks Racl activation downstream of CXCR4. (Takesono, et al. Curr Biol 14: 917-922, 2004) The results presented herein show that inhibition of ITK can decrease HIV viral entry into cells.

• Inhibition of PI3 kinase downstream of CXCR4 inhibits infection (Francois, J. Virol. 11: 2539-2549, 2003). PI3 kinase is an upstream activator of ITK. (Berg et al., Annu. Rev. Immunol, 23: 549-600, 2005)

• Itk is required for efficient activation of NFAT in response to TCR stimulation (Schaeffer, et al. Nat Immunol 2: 1183-1188, 2001). The results presented herein demonstrate that ITK is required for efficient transcription from the HIV LTR (long- terminal repeat) and efficient viral production.

[0034] Small chemical compounds that are useful as an inhibitor of TEC family kinase include, but are not limited to BMS509774, an inhibitor of ITK. See Lin, et al. Biochemistry 43: 11056-11062, 2004 and U.S. Patent Application No. 2004/0077695. Compositions and methods for synthesis of thiazolyl inhibitors of Tec family tyrosine kinase are found in U.S. Patent Application Nos. 2006/0030598; 2004/0110752; 2004/0077695; 2004/0067990; 2004/0067989; and 2003/0069238; and U.S. Patent Nos. 6,306,897 and 6,221,900. EXPRESSION OF TEC TYROSINE KINASES IN LYMPHOCYTES

[0035] T lymphocytes express at least three TEC family kinases: ITK, RLK, and TEC. Additionally, BMX/ETK expression can be detected in the Jurkat lymphoma cell line. Each of the three major T cell TEC family kinases exhibits distinct patterns and levels of expression that may reflect their functional importance in different T cell subsets and stages of development. Berg et al., Annu. Rev. Immunol, 23: 549-600, 2005.

[0036] ITK (inducible T cell kinase; also known as EMT, expressed in mast cells and T lymphocytes, and TSK, T cell-specific kinase) is the predominant TEC kinase in T cells and was first cloned by degenerate PCR of T cell-specific tyrosine kinases. At least two forms of ITK have been cloned from mouse, which vary by inclusion or deletion of a sequence encoding six amino acids. The longer version, which appears to be a splice variant, is not detected in human cells. ITK expression is found in thymocytes and mature T cells, mast cells, natural killer cells, and NKT cells, with the highest levels of expression in the mature adult thymus. Expression of ITK is induced upon T cell activation and treatment with IL-2. Two recent reports demonstrate higher levels of ITK mRNA and protein in T_H2 cells relative to T_HI cells, perhaps reflecting ITK's importance in the development of T_H2 responses. Mature T cells and thymocytes from mice deficient in ITK show impaired activation of phospholipase C-γ (PLC-γ), defective actin reorganization, and multiple functional defects in response to TCR stimulation.

[0037] RLK (resting lymphocyte kinase; also known as TXK) was also cloned in degenerate PCR screens for novel tyrosine kinases expressed in T lymphocytes. Like ITK, RLK expression is found in thymocytes, mature resting T cells, and mast cells. Real-time RT-PCR demonstrates that RLK mRNA is expressed at 3- to 10-fold lower levels than ITK in resting mature T cells. Expression of RLK drops dramatically upon T cell receptor stimulation. RLK expression increases again in primary differentiated T_HI cells and T_HI cell clones, but it remains very low in differentiated T_H2 cells and cell clones, suggesting that RLK is functionally more important for T_HI cell function. Consistent with these observations, IL-12 was shown to increase and IL-4 to decrease expression of RLK in peripheral blood CD4+ cells. Although two forms of RLK have been detected, there are no clear differences in the ratio of these isoforms in different T cell populations. Mutation of RLK leads to minimal T cell defects, yet it can exacerbate defects observed in TTK-deficient T cells.

[0038] TEC (tyrosine kinase expressed in hepatocellular carcinoma), the founding member of this family, was originally cloned from a mouse liver cDNA library. TEC has a more broad pattern of expression both in hematopoietic and other cell types. TEC is expressed at relatively low levels in resting T cells. Real-time RT-PCR studies suggest its mRNA is 100-fold lower than that of ITK. However, TEC expression is induced after stimulation of T cells for 2-3 days, suggesting that it may be more important for effector cell function or upon restimulation of preactivated cells. Like ITK, TEC is expressed at slightly higher levels (twofold) in T_H2 cells than in T_HI cells. No functional defects have been reported to date in TEC-deficient T cells. However, in mouse B cells, mutation of TEC worsens defects associated with BTK deficiency, suggesting functional redundancy with other family members. Multiple splice forms of TEC have been reported, but their functional differences in T cells remain unknown. BTK, a TEC family kinase, is broadly expressed in most hematopoietic cells, but it is not expressed in T cells.

[0039] The TEC-family kinases are characterized by a common domain organization: they have an amino-terminal phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3)- binding pleckstrin-homology domain, which is followed by a TEC-homology domain that contains one or two proline-rich regions (PRRs), then SRC homology 3 (SH3) and SH2 protein-interaction domains, and a carboxyterminal kinase domain. The TEC kinases are the only tyrosine kinases that have pleckstrin-homology domains, which inducibly recruit TEC-family members to the plasma membrane by binding the phosphatidylinositol 3-kinase (PI3K) product PtdIns(3,4,5)P3, thereby promoting their activation. Membrane localization and activation of the pleckstrin- homology domain- containing TEC kinases are therefore regulated by PI3K and the lipid phosphatases PTEN (phosphatase and tensin homologue) and SHIP (SH₂-domain containing inositol-5-phosphatase), which catalyze the breakdown of PtdIns(3,4,5)P₃. The atypical TEC kinase RLK lacks a pleckstrin-homology domain and, instead, has a palmitoylated string of cysteine residues, which leads to constitutive membrane association of RLK, independent of PI3K activity. Schwartzberg, et ah, Nature Reviews Immunology, 5: 284-295, 2005.

[0040] Activation of TEC-family kinases requires several interrelated steps: first, recruitment to the plasma membrane through interactions between their pleckstrin homology domains and the products of PI3K and/or other proteins; second, phosphorylation by SRC-family kinases; and third, interactions with other proteins that bring the TEC-family kinases into antigen-receptor signaling complexes. In addition, TEC-family kinases are thought to be regulated by conformational changes directed by intra- and intermolecular interactions involving their SH2 domains, SH3 domains and PRRs. These have been best characterized for ITK, for which nuclear-magnetic-resonance studies have shown that the PRR binds the SH3 domain within the same molecule. An additional interaction between the SH2 domain of one ITK molecule and the SH3 domain of another ITK molecule can also be detected. These interactions are thought to be inhibitory and to prevent interactions with other proteins. The peptidylprolyl isomerase cyclophilin A contributes to UK activation through isomerization of a proline residue in the SH₂ domain of ITK, which alters the specificity of the protein interactions of the SH2 domain so that the cis form favors intramolecular interactions with the SH3 domain and the trans form favors interactions with other proteins in TCR-signaling complexes and activation of its own kinase activity.

[0041] Although ITK, RLK and TEC are all found in T cells, they are expressed at different levels and by different subpopulations. Evaluation of mRNA levels by realtime reverse- transcriptase PCR showed that ITK is the main TEC kinase expressed by naive mouse T cells, with RLK mRNA expressed at 3-10-fold lower levels and TEC mRNA at <100-fold lower levels. On T-cell activation, ITK expression is increased, particularly in T_H2 cells, whereas RLK expression rapidly drops and is re-established only in T_HI cells. TEC expression increases after several days of T-cell stimulation. There is evidence indicating that all three of these kinases function downstream of TCR signaling.

[0042] It is to be understood that this invention is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0044] As the TEC tyrosine kinase molecules of the present invention share structural features with protein kinases and can modulate protein kinase-mediated activities, the TEC tyrosine kinase compositions of embodiments of the invention {e.g., nucleic acids, short interfering RNA, short hairpin RNA ribozyme, antisense oligonucleotide polypeptides, proteins, peptide mimetics, small chemical compound, or antibodies) which are inhibitors of TEC tyrosine kinase protein sequences, e.g., TEC, BTK, ITK, RLK, or BMX, gene expression or biological activity) are useful for developing novel diagnostic and therapeutic agents for protein kinase associated disorders, e.g., in this case, for treatment to prevent or ameliorate viral disease. A "protein kinase associated disorder" includes a disorder, disease, or condition which is caused by, characterized by, or associated with a misregulation (e.g., an aberrant downregulation or upregulation) of a protein kinase mediated activity. Protein kinase associated disorders can result in, e.g., upregulated or downregulated, cell growth and/or proliferation.

[0045] To determine whether a polypeptide or protein of interest has a conserved sequence or domain common to members of a protein family, the amino acid sequence of the protein can be searched against a database of profile hidden Markov models (profile HMMs), which uses statistical descriptions of a sequence family's consensus (e.g., HMMER, version 2.1.1) and PFAM, a collection of multiple sequence alignments and hidden Markov models covering many common protein domains (e.g., PFAM, version 5.5) using the default parameters (http://www.sanger.ac.uk/Software/Pfam_- HMM search). For example, the hmmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for MILPAT0063 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g., to 8 bits). A description of the PFAM database can be found in Sonhammer et al., (1997) Proteins 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al., (1990) Meth. Enzymol. 183:146-159; Gribskov et al., (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al., (1994) J. MoI. Biol. 235:1501-1531; and Stultz et al., (1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference. See also, for example, http://hmmer.wustl.edu/. For general information regarding PFAM identifiers, PS prefix and PF prefix domain identification numbers, refer to Sonnhammer et al. (1997) Protein 28:405-420 and http://pfam.wustl.edu/. See also, for example, http://www.expasy.ch/prosi- te and http://smart.embl-heidelberg.de/.

[0046] Using such search tools, the TEC tyrosine kinase protein sequences, e.g., TEC, BTK, ITK, RLK, or BMX, were found to contain significant structural characteristics in common with members of the protein kinase family of molecules. Some of these structural characteristics include, for example, a protein kinase catalytic domain (e.g., PFAM Accession No. PF00069), a pleckstrin homology domain consensus sequence (e.g., PFAM Accession No. PF00169), a fibronectin type m domain (e.g., PFAM Accession No. PF00041), a RhoGEF domain (e.g., PFAM Accession No. PF00621), and a IQ calmodulin-binding motif (e.g., PFAM Accession No. PF00612), a Src Homology 2 (SH2) protein interaction domain and a Src Homology 3 (SH3) protein interaction domain.

[0047] "Protein kinase" refers to a protein or polypeptide which is capable of modulating its own phosphorylation state or the phosphorylation state of another molecule, e.g., protein or polypeptide. Protein kinases can have a specificity for (Le., a specificity to phosphorylate) serine/threonine residues, tyrosine residues, or both serine/threonine and tyrosine residues, e.g., the dual specificity kinases.

[0048] "Family" when referring to the protein and nucleic acid molecules of embodiments of the invention is intended to mean two or more proteins or nucleic acid molecules having a common structural domain or signature sequence and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin, as well as other distinct proteins of human origin, or alternatively, can contain homologues of non-human origin, e.g., rat or mouse proteins. Members of a family can also have common functional characteristics.

[0049] With regard to common structural characteristics described above, the protein kinases of the present invention include a protein kinase catalytic core or domain and can include at least one of the following signature sequences or motifs within the catalytic core: a protein kinase ATP-binding region signature sequence, a serine/threonine protein kinase active site signature sequence, and a tyrosine kinase active site signature sequence (see Hanks et al. Science 241: 42-52, 1988

[0050] "Protein kinase catalytic core or domain" includes a consensus sequence, e.g., PFAM Accession No. PF00069, that includes the catalytic domain of the enzyme. The catalytic domain can be characterized by the presence of an ATP binding signature sequence (e.g., Prosite Accession No. PSOO 107) and/or a serine/threonine or tyrosine kinase active-site signature sequence (e.g., Prosite Accession No. PS00108 or Prosite Accession No. PS00109). The protein kinase catalytic domain of the present invention preferably includes a catalytic domain of about 150-400 amino acid residues in length, preferably about 200-300 amino acid residues in length, or more preferably about 225-300 amino acid residues in length, which includes at least one of the signature sequences or motifs described herein.

[0051] Accordingly, protein kinase polypeptides having at least 50-60% homology, preferably about 60-70%, more preferably about 70-80%, or about 80-90% homology with a protein kinase catalytic domain of TEC tyrosine kinase are within the scope of the invention.

[0052] The protein kinase ATP-binding region signature sequence is located in the N- terminal extremity of the catalytic domain and typically includes a glycine-rich stretch of residues in the vicinity of a lysine residue. A consensus sequence (Prosite Accession No. PS00107; SEQ ID NO:7) for this region is [LIV]-G-{P}-G-{P}-[FYWMGSTNH]-[SGA]-{PW}- [LIVCAT- ]-{PD}-x-[GSTACLIVMFY]-x(5,18)-[LIVMFYWCSTAR]-[AIVP]-[LIV MFAGCKR]-K. In the above consensus sequence pattern, lysine (K) binds ATP.

[0053] In this and the following consensus sequence patterns, each element in the pattern is separated by a dash (-); square brackets, [ ], indicate the particular residues that are accepted at that position; ornate brackets, { }, indicate the residues that are not accepted at that position; x indicates any residue is accepted at that position; repetition of a particular element is indicated by following the element with a numerical value or a numerical range enclosed in parentheses (i.e., above, x(5,18) indicates anywhere from 5 to 18 residues are present in the element, and any amino acid residue is accepted at each of these 5 to 18 residue positions); and the standard IUPAC one-letter code for the amino acids is used.

[0054] In one aspect, the polypeptide or peptidomimetic composition can be a dominant-negative mutant within the scope of the invention if it can inhibit an activity of a TEC tyrosine kinase polypeptide, e.g., TEC, BTK, ITK, RLK, or BMX polypeptides of embodiments of the invention, e.g., be a dominant-negative mutant or bind to an antibody. An example of a dominant negative peptide is a peptide with a mutation in a lysine residue in the ATP binding domain of the TEC tyrosine kinase, as described herein, that inhibits TEC tyrosine kinase activity. A further example of a dominant negative peptide is a peptide with a mutation in the SH2 domain or SH3 domain of the TEC tyrosine kinase as described herein, that inhibits TEC tyrosine kinase activity.

[0055] "Pleckstrin homology domain" is a domain that is characterized by the matrix profile described by PFAM Accession No. PFOO 169 or Prosite Accession No. PS50003. The pleckstrin homology domain is a domain of about 100 amino acid residues that can occur in protein kinases, e.g., serine/threonine protein kinases belonging to the Akt/Rac family, the beta- adrenergic receptor kinase family, the trypanosomal NrkA family, and the mu isoform of protein kinase C, and tyrosine protein kinases, e.g., belonging to the BTK/TTK/TEC subfamily. This domain binds phospholipids (inositol phosphates and phosphoinositol phosphates) and can also be protein interaction domains.

[0056] As used interchangeably herein, he terms, a "TEC tyrosine kinase-mediated activity", "biological activity of TEC tyrosine kinase " or "functional activity of TEC tyrosine kinase ", refer to an activity exerted by a TEC tyrosine kinase protein, polypeptide or nucleic acid molecule on, e.g., a TEC tyrosine kinase -responsive cell or tissue, or on a TEC tyrosine kinase substrate, ligand, interacting protein, or target molecule, e.g., a protein substrate or target molecule, as determined in vivo, in vitro, or in situ according to standard techniques. An "interacting protein" can be a ligand, target, or other signaling factor for the TEC tyrosine kinase. The interacting protein can be inhibited by a peptide or peptide fragment, e.g., a peptidomimetic or dominant negative molecule, that can block protein-protein interaction and can inhibit phosphorylation by TEC tyrosine kinase for example, at the kinase active site, at the ATP binding site, or at an SH2 or SH3 binding domain site of the kinase.

[0057] In one embodiment, a TEC tyrosine kinase activity is a direct activity, such as an association with a TEC tyrosine kinase ligand, binding partner, or target molecule. A "ligand", "binding partner", or "target molecule" refers to a molecule with which a TEC tyrosine kinase protein binds or interacts in nature, such that a TEC tyrosine kinase -mediated function is achieved. A TEC tyrosine kinase target molecule can be a TEC tyrosine kinase protein or polypeptide, e.g., TEC, BTK, ITK, RLK, or BMX, of the present invention or a non- TEC tyrosine kinase protein molecule. In an exemplary embodiment, a TEC tyrosine kinase target molecule is a the TEC tyrosine kinase ligand or an interacting molecule, e.g., ligand of TEC, BTK, ITK, RLK, or BMX, e.g., a. protein kinase ligand, e.g., serine, threonine, or tyrosine containing polypeptide.

[0058] Protein kinases play a role in signaling pathways associated with cellular growth. For example, protein kinases are involved in the regulation of signal transmission from cellular receptors, e.g., growth-factor receptors; entry of cells into mitosis; and the regulation of cytoskeleton function, e.g., actin bundling. Thus, the TEC tyrosine kinase molecules of the present invention can be involved in: 1) the regulation of transmission of signals from cellular receptors, e.g., cell growth factor receptors, antigen receptors, or chemokine receptors; 2) the modulation of the entry of cells, e.g., precursor cells, into mitosis; 3) the modulation of cellular differentiation; 4) the modulation of cell death; and 5) the regulation of cytoskeleton function, e.g., actin bundling. These kinases can function in these biological activities because of their ability to phosphorylate themselves or other substrate molecules.

[0059] Substrates of tyrosine protein kinases are generally characterized by a lysine or an arginine seven residues to the N-terminal side of the phosphorylated tyrosine. An acidic residue (aspartic acid or glutatmic acid) is often found at either three or four residues to the N- terminal side of the tyrosine (see Patschinsky et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79:973- 977; Hunter T. (1982) J. Biol. Chem. 257:4843-4848; Cooper et al. (1984) J. Biol. Chem. 259:7835-7841).

[0060] A TEC tyrosine kinase activity can also be an indirect activity, such as an activity mediated by interaction of the TEC tyrosine kinase protein with a target molecule such that the target molecule modulates a downstream cellular activity, e.g., a cellular signaling activity modulated indirectly by an interaction of the TEC tyrosine kinase protein with a target molecule.

[0061] The TEC tyrosine kinase protein sequences, e.g., TEC, BTK, ITK, RLK, or BMX, molecules of embodiments of the invention can modulate the activities of cells in tissues where they are expressed. For example, the TEC tyrosine kinase ITK mRNA is expressed in T cells, NK cells, mast cells, and eosinophil cells. Accordingly, the TEC tyrosine kinase molecules of embodiments of the invention can act as therapeutic or diagnostic agents for treatment of immune disorders.

[0062] Protein kinase associated disorders can detrimentally affect cellular functions such as cellular proliferation, growth, differentiation, and cellular regulation of homeostasis, e.g., glucose homeostasis; inter- or intra-cellular communication; tissue function, such as cardiac function or musculoskeletal function; systemic responses in an organism, such as nervous system responses, hormonal responses (e.g., insulin response), or immune responses; and protection of cells from toxic compounds (e.g., carcinogens, toxins, mutagens, and toxic byproducts of metabolic activity, e.g., reactive oxygen species). Accordingly, the TEC tyrosine kinase molecules of embodiments of the invention, as protein kinases, can mediate various protein kinase associated disorders, including cellular proliferative and/or differentiative disorders, hormonal disorders, immune and inflammatory disorders, neurological disorders, cardiovascular disorders, blood vessel disorders, and platelet disorders.

[0063] The TEC tyrosine kinase protein sequences, e.g., TEC, BTK, ITK, RLK, or BMX, proteins, fragments thereof, and derivatives and other variants thereof, are collectively referred to as "polypeptides or proteins of the invention" or " TEC tyrosine kinase polypeptides or proteins" or "TEC, BTK, ITK, RLK, or BMX polypeptides or proteins". Nucleic acid molecules encoding such polypeptides or proteins are collectively referred to as "nucleic acids of the invention" or " TEC tyrosine kinase nucleic acids."

[0064] "Nucleic acid molecule" includes DNA molecules {e.g., a cDNA or genomic DNA) and RNA molecules (e.g., an mRNA) and analogs of the DNA or RNA generated, e.g., by the use of nucleotide analogs. The nucleic acid molecule can be single-stranded or double- stranded, but preferably is double-stranded DNA.

[0065] "Isolated or purified nucleic acid molecule" includes nucleic acid molecules which are separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term "isolated" includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (Le., sequences located at the 5' and/or 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5^* and/or 3' nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0066] "Hybridizes under stringent conditions" describes conditions for hybridization and washing. Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (19S9), 6.3.1-6.3.6. Aqueous and nonaqueous methods are described in that reference and either can be used. A preferred, example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45° C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 50° C. Another example of stringent hybridization conditions are hybridization in 6X SSC at about 45° C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 55° C. A further example of stringent hybridization conditions are hybridization in 6X SSC at about 45° C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60° C. Preferably, stringent hybridization conditions are hybridization in 6X SSC at about 45° C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 65° C. Particularly preferred stringency conditions (and the conditions that should be used if the practitioner is uncertain about what conditions should be applied to determine if a molecule is within a hybridization limitation of embodiments of the invention) are 0.5 M sodium phosphate, 7% SDS at 65° C, followed by one or more washes at 0.2X SSC, 1% SDS at 65° C. Preferably, an isolated nucleic acid molecule of embodiments of the invention that hybridizes under stringent conditions to the nucleic acid sequence of TEC tyrosine kinase protein, e.g., TEC, BTK, ITK, RLK, or BMX, corresponds to a naturally-occurring nucleic acid molecule.

[0067] A "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).

[0068] "Gene" and "recombinant gene" refer to nucleic acid molecules which include an open reading frame encoding a TEC tyrosine kinase protein, e.g., TEC, BTK, ITK, RLK, or BMX, protein, preferably a mammalian TEC tyrosine kinase protein, and can further include non-coding regulatory sequences, and introns.

[0069] An "isolated" or "purified" polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. In one embodiment, the language "substantially free" means a preparation of TEC tyrosine kinase protein having less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non- TEC tyrosine kinase protein (also referred to herein as a "contaminating protein"), or of chemical precursors or non- TEC tyrosine kinase chemicals. When the TEC tyrosine kinase protein, or biologically active portion thereof, is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. Embodiments of the invention include isolated or purified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry weight.

[0070] A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of the TEC tyrosine kinase protein sequences, e.g., TEC, BTK, ITK, RLK, or BMX, without abolishing or more preferably, without substantially altering a biological activity, whereas an "essential" amino acid residue results in such a change. For example, amino acid residues that are conserved among the polypeptides of the present invention, e.g., those present in the protein kinase domain, are predicted to be particularly not amenable to alteration.

[0071] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a TEC tyrosine kinase protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a TEC tyrosine kinase coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for TEC tyrosine kinase biological activity to identify mutants that retain activity. Following mutagenesis of TEC tyrosine kinase protein sequences, e.g., TEC, BTK, ITK, RLK, or BMX, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

[0072] A "biologically active portion" of a TEC tyrosine kinase protein includes a fragment of a TEC tyrosine kinase protein which participates in an interaction between a TEC tyrosine kinase molecule and an effector molecule. Biologically active portions of a TEC tyrosine kinase protein include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the TEC tyrosine kinase protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the TEC tyrosine kinase protein, e.g., TEC, BTK, TTK, RLK, or BMX, including, e.g., the ability to show a protein kinase activity, activate a protein kinase activity, or to interact with another protein.

[0073] A biologically active portion of a TEC tyrosine kinase protein can be a polypeptide which is, for example, 10, 25, 50, 100, 200, or more, amino acids in length. Biologically active portions of a TEC tyrosine kinase protein can be used as targets for developing agents which modulate a TEC tyrosine kinase -mediated activity as described herein.

[0074] Calculations of homology or sequence identity (the terms are used interchangeably herein) between sequences are performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence (e.g., when aligning a second sequence to the TEC tyrosine kinase amino acid sequence, at least 789, preferably at least 1052, more preferably at least 1315, even more preferably at least 1578, and even more preferably at least 1841, 2104, 2367, or 2630 amino acid residues of the two sequences are aligned. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0075] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((197O) J. MoI. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of embodiments of the invention) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0076] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0077] The nucleic acid and protein sequences described herein can be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. MoI. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to TEC tyrosine kinase nucleic acid molecules of embodiments of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to TEC tyrosine kinase protein molecules of embodiments of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[0078] Particular TEC tyrosine kinase polypeptides of the present invention have an amino acid sequence sufficiently identical or substantially identical to the amino acid sequence of the TEC tyrosine kinase protein sequences, e.g., TEC, BTK, ITK, RLK, or BMX. "Sufficiently identical" or "substantially identical" is used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain or common functional activity. For example, amino acid or nucleotide sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are defined herein as sufficiently or substantially identical.

[0079] "Misexpression or aberrant expression", as used herein, refers to a non-wild type pattern of gene expression, at the RNA or protein level. It includes: expression at non-wild type levels, i.e., over- or under-expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus.

[0080] "Subject" refers to a mammal, e.g., a human, or to an experimental animal or disease model. The subject can also be a non-human primate or animal animal, e.g., a horse, cow, goat, or other domestic animal.

[0081] A "purified preparation of cells", as used herein, refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and more preferably 50% of the subject cells.

RNA AlNfD DNA INTERFERENCE METHODS

A. Short Interfering RNAs (RNAi)

[0082] RNA interference (RNAi) is a mechanism of post-transcriptional gene silencing mediated by double-stranded RNA (dsRNA), which is distinct from antisense and ribozyme- based approaches (see Jain, Pharmacogenomics 5: 239-42, 2004 for a review of RNAi and siRNA). RNA interference is useful in a method for treating a viral disease in a mammal by administering to the mammal a nucleic acid molecule (e.g., dsRNA) that hybridizes under stringent conditions to a TEC tyrosine kinase target gene, and attenuates expression of said target gene. dsRNA molecules are believed to direct sequence-specific degradation of mRNA in cells of various types after first undergoing processing by an RNase Hi-like enzyme called DICER (Bernstein et al., Nature 409: 363, 2001) into smaller dsRNA molecules comprised of two 21 nt strands, each of which has a 5' phosphate group and a 3' hydroxyl, and includes a 19 nt region precisely complementary with the other strand, so that there is a 19 nt duplex region flanked by 2 nt-3' overhangs. RNAi is thus mediated by short interfering RNAs (siRNA), which typically comprise a double-stranded region approximately 19 nucleotides in length with 1-2 nucleotide 3' overhangs on each strand, resulting in a total length of between approximately 21 and 23 nucleotides. In mammalian cells, dsRNA longer than approximately 30 nucleotides typically induces nonspecific mRNA degradation via the interferon response. However, the presence of siRNA in mammalian cells, rather than inducing the interferon response, results in sequence- specific gene silencing.

[0083] In general, a short, interfering RNA (siRNA) comprises an RNA duplex that is preferably approximately 19 basepairs long and optionally further comprises one or two single- stranded overhangs or loops. An siRNA may comprise two RNA strands hybridized together, or may alternatively comprise a single RNA strand that includes a self-hybridizing portion. siRNAs may include one or more free strand ends, which may include phosphate and/or hydroxyl groups. siRNAs typically include a portion that hybridizes under stringent conditions with a target transcript. One strand of the siRNA (or, the self-hybridizing portion of the siRNA) is typically precisely complementary with a region of the target transcript, meaning that the siRNA hybridizes to the target transcript without a single mismatch. In certain embodiments of the invention in which perfect complementarity is not achieved, it is generally preferred that any mismatches be located at or near the siRNA termini.

[0084] siRNAs have been shown to downregulate gene expression when transferred into mammalian cells by such methods as transfection, electroporation, or microinjection, or when expressed in cells via any of a variety of plasmid-based approaches. RNA interference using siRNA is reviewed in, e.g., Tuschl, Nat. Biotechnol. 20: 446-448, 2002; See also Yu, J., et al, Proc. Natl. Acad. ScL, 99: 6047-6052, 2002; Sui, et al, Proc. Natl. Acad. Sci USA. 99: 5515- 5520, 2002; Paddison, et al., Genes andDev. 16: 948-958, 2002; Brummelkamp, et al, Science 296: 550-553, 2002; Miyagashi, et al, Nat. Biotech. 20: 497-500, 2002; Paul, et al, Nat. Biotech. 20: 505-508, 2002. As described in these and other references, the siRNA may consist of two individual nucleic acid strands or of a single strand with a self-complementary region capable of forming a hairpin (stem-loop) structure. A number of variations in structure, length, number of mismatches, size of loop, identity of nucleotides in overhangs, etc., are consistent with effective siRNA-triggered gene silencing. While not wishing to be bound by any theory, it is thought that intracellular processing {e.g., by DICER) of a variety of different precursors results in production of siRNA capable of effectively mediating gene silencing. Generally it is preferred to target exons rather than introns, and it may also be preferable to select sequences complementary to regions within the 3' portion of the target transcript. Generally it is preferred to select sequences that contain approximately equimolar ratio of the different nucleotides and to avoid stretches in which a single residue is repeated multiple times.

[0085] siRNAs may thus comprise RNA molecules having a double-stranded region approximately 19 nucleotides in length with 1-2 nucleotide 3' overhangs on each strand, resulting in a total length of between approximately 21 and 23 nucleotides. As used herein, siRNAs also include various RNA structures that may be processed in vivo to generate such molecules. Such structures include RNA strands containing two complementary elements that hybridize to one another to form a stem, a loop, and optionally an overhang, preferably a 3' overhang. Preferably, the stem is approximately 19 bp long, the loop is about 1-20, more preferably about 4-10, and most preferably about 6-8 nt long and/or the overhang is about 1-20, and more preferably about 2-15 nt long. In certain embodiments of the invention the stem is minimally 19 nucleotides in length and may be up to approximately 29 nucleotides in length. Loops of 4 nucleotides or greater are less likely subject to steric constraints than are shorter loops and therefore may be preferred. The overhang may include a 5' phosphate and a 3' hydroxyl. The overhang may but need not comprise a plurality of U residues, e.g., between 1 and 5 U residues. Classical siRNAs as described above trigger degradation of mRNAs to which they are targeted, thereby also reducing the rate of protein synthesis. In addition to siRNAs that act via the classical pathway, certain siRNAs that bind to the 3' UTR of a template transcript may inhibit expression of a protein encoded by the template transcript by a mechanism related to but distinct from classic RNA interference, e.g., by reducing translation of the transcript rather than decreasing its stability. Such RNAs are referred to as microRNAs (mRNAs) and are typically between approximately 20 and 26 nucleotides in length, e.g., 22 nt in length. It is believed that they are derived from larger precursors known as small temporal RNAs (stRNAs) or mRNA precursors, which are typically approximately 70 nt long with an approximately 4-15 nt loop. (See Grishok, et al., Cell 106: 23-24, 2001; Hutvagner, etal., Science 293: 834-838, 2001; Ketting, et al., Genes Dev., 15: 2654-2659, 2001). Endogenous RNAs of this type have been identified in a number of organisms including mammals, suggesting that this mechanism of post- transcriptional gene silencing may be widespread (Lagos-Quintana, et al., Science 294: 853-858, 2001; Pasquinelli, Trends in Genetics 18: 171-173, 2002, and references in the foregoing two articles). MicroRNAs have been shown to block translation of target transcripts containing target sites in mammalian cells (Zeng, et al., Molecular Cell 9: 1-20, 2002). 11

[0086] siRNAs such as naturally occurring or artificial (i.e., designed by humans) mRNAs that bind within the 3' UTR (or elsewhere in a target transcript) and inhibit translation may tolerate a larger number of mismatches in the siRN A/template duplex, and particularly may tolerate mismatches within the central region of the duplex. In fact, there is evidence that some mismatches may be desirable or required as naturally occurring stRNAs frequently exhibit such mismatches as do mRNAs that have been shown to inhibit translation in vitro. For example, when hybridized with the target transcript such siRNAs frequently include two stretches of perfect complementarity separated by a region of mismatch. A variety of structures are possible. For example, the mRNA may include multiple areas of nonidentity (mismatch). The areas of nonidentity (mismatch) need not be symmetrical in the sense that both the target and the mRNA include nonpaired nucleotides. Typically the stretches of perfect complementarity are at least 5 nucleotides in length, e.g., 6, 7, or more nucleotides in length, while the regions of mismatch may be, for example, 1, 2, 3, or 4 nucleotides in length.

[0087] Hairpin structures designed to mimic siRNAs and mRNA precursors are processed intracellular^ into molecules capable of reducing or inhibiting expression of target transcripts (McManus, et al., RNA 8: 842-850, 2002). These hairpin structures, which are based on classical siRNAs consisting of two RNA strands forming a 19 bp duplex structure are classified as class I or class π hairpins. Class I hairpins incorporate a loop at the 5' or 3' end of the antisense siRNA strand (i.e., the strand complementary to the target transcript whose inhibition is desired) but are otherwise identical to classical siRNAs. Class II hairpins resemble mRNA precursors in that they include a 19 nt duplex region and a loop at either the 3' or 5' end of the antisense strand of the duplex in addition to one or more nucleotide mismatches in the stem. These molecules are processed intracellularly into small RNA duplex structures capable of mediating silencing. They appear to exert their effects through degradation of the target mRNA rather than through translational repression as is thought to be the case for naturally occurring mRNAs and stRNAs.

[0088] Thus it is evident that a diverse set of RNA molecules containing duplex structures is able to mediate silencing through various mechanisms. For the purposes of the present invention, any such RNA, one portion of which binds to a target transcript and reduces its expression, whether by triggering degradation, by inhibiting translation, or by other means, is considered to be an siRNA, and any structure that generates such an siRNA (Le., serves as a precursor to the RNA) is useful in the practice of the present invention.

[0089] In the context of the present invention, siRNAs are useful both for therapeutic purposes, e.g., to modulate the expression of a TEC tyrosine kinase protein in a subject at risk of or suffering from a viral disease and for various of the inventive methods for the identification of compounds for treatment of a viral disease that modulate the activity or level of the molecules described herein. In a preferred embodiment, the therapeutic treatment of viral disease, e.g., HIV or herpesvirus infection, with an antibody, antisense vector, or double stranded RNA vector.

[0090] Embodiments of the invention therefore provide a method of inhibiting expression of a gene encoding a TEC tyrosine kinase protein comprising the step of (i) providing a biological system in which expression of a gene encoding TEC tyrosine kinase protein is to be inhibited; and (ii) contacting the system with an siRNA targeted to a transcript encoding the TEC tyrosine kinase protein. According to certain embodiments of the invention the TEC tyrosine kinase protein is encoded by a gene within or linked to a viral disease susceptibility locus, or within which a functional mutation causing or contributing to susceptibility or development of a viral disease (e.g., HIV, herpesvirus, T cell leukemia virus) may exist. In other embodiments, TEC tyrosine kinase proteins are inhibited. According to certain embodiments of the invention the biological system comprises a cell, and the contacting step comprises expressing the siRNA in the cell. According to certain embodiments of the invention the biological system comprises a subject, e.g., a mammalian subject such as a mouse or human, and the contacting step comprises administering the siRNA to the subject or comprises expressing the siRNA in the subject. According to certain embodiments of the invention the siRNA is expressed inducibly and/or in a cell-type or tissue specific manner.

[0091] By "biological system" is meant any vessel, well, or container in which biomolecules {e.g., nucleic acids, polypeptides, polysaccharides, lipids, and the like) are placed; a cell or population of cells; a tissue; an organ; an organism, and the like. Typically the biological system is a cell or population of cells, but the method can also be performed in a vessel using purified or recombinant proteins.

[0092] Embodiments of the invention provide siRNA molecules targeted to a transcript encoding any TEC tyrosine kinase protein. In particular, embodiments of the invention provides siRNA molecules selectively or specifically targeted to a transcript encoding a polymorphic variant of such a transcript, wherein existence of the polymorphic variant in a subject is indicative of susceptibility to or presence of a viral disease {e.g., HIV, human herpesvirus, or human T cell leukemia virus I or II). The terms "selectively" or "specifically targeted to", in this context, are intended to indicate that the siRNA causes greater reduction in expression of the variant than of other variants {i.e., variants whose existence in a subject is not indicative of susceptibility to or presence of a viral disease). The siRNA, or collections of siRNAs, may be provided in the form of kits with additional components as appropriate. 07711

B. Short hairpin RNAs (shRNA)

[0093] RNA interference (RNAi),a mechanism of post-transcriptional gene silencing mediated by double-stranded RNA (dsRNA), is useful in a method for treating a viral disease in a mammal by administering to the mammal a nucleic acid molecule (e.g., dsRNA) that hybridizes under stringent conditions to a TEC tyrosine kinase target gene, and attenuates expression of said target gene. See Jain, Pharmacogenomics 5: 239-42, 2004 for a review of RNAi and siRNA. A further method of RNA interference in the present invention is the use of short hairpin RNAs (shRNA). A plasmid containing a DNA sequence encoding for a particular desired siRNA sequence is delivered into a target cell via transfection or virally-mediated infection. Once in the cell, the DNA sequence is continuously transcribed into RNA molecules that loop back on themselves and form hairpin structures through intramolecular base pairing. These hairpin structures, once processed by the cell, are equivalent to transfected siRNA molecules and are used by the cell to mediate RNAi of the desired protein. The use of shRNA has an advantage over siRNA transfection as the former can lead to stable, long-term inhibition of protein expression. Inhibition of protein expression by transfected siRNAs is a transient phenomenon that does not occur for times periods longer than several days. In some cases, this may be preferable and desired. In cases where longer periods of protein inhibition are necessary, shRNA mediated inhibition is preferable.

C. Full and Partial Length Antisense RNA Transcripts

[0094] Antisense RNA transcripts have a base sequence complementary to part or all of any other RNA transcript in the same cell. Such transcripts have been shown to modulate gene expression through a variety of mechanisms including the modulation of RNA splicing, the modulation of RNA transport and the modulation of the translation of mRNA (Denhardt, Ann N Y Acad. ScL 660: 70, 1992; Nellen, Trends Biochem. ScL 18: 419, 1993; Baker et al, Biochim. Biophys. Acta, 1489: 3, 1999; Xu, et al., Gene Therapy 7: 438, 2000; French et al, Curr. Opin. Microbiol. 3: 159, 2000; Terryn et al, Trends Plant ScL 5: 1360, 2000).

D. Antisense RNA and DNA Oligonucleotides

[0095] Antisense nucleic acids are generally single-stranded nucleic acids (DNA, RNA, modified DNA, or modified RNA) complementary to a portion of a target nucleic acid (e.g., an mRNA transcript) and therefore able to bind to the target to form a duplex. Typically they are oligonucleotides that .range from 15 to 35 nucleotides in length but may range from 10 up to approximately 50 nucleotides in length. Binding typically reduces or inhibits the function of the target nucleic acid. For example, antisense oligonucleotides may block transcription when bound 11

to genomic DNA, inhibit translation when bound to mRNA, and/or lead to degradation of the nucleic acid. Reduction in expression of a TEC tyrosine kinase polypeptide may be achieved by the administration of antisense nucleic acids or peptide nucleic acids comprising sequences complementary to those of the mRNA that encodes the polypeptide. Antisense technology and its applications are well known in the art and are described in Phillips, M. I. (ed.) Antisense Technology, Methods Enzymol., 2000, Volumes 313 and 314, Academic Press, San Diego, and references mentioned therein. See also Crooke, S. (ed.) "ANTISENSE DRUG TECHNOLOGY: PRINCIPLES, STRATEGIES, AND APPLICATIONS" (1^st Edition) Marcel Dekker; and references cited therein.

[0096] Antisense oligonucleotides can be synthesized with a base sequence that is complementary to a portion of any RNA transcript in the cell. Antisense oligonucleotides may modulate gene expression through a variety of mechanisms including the modulation of RNA splicing, the modulation of RNA transport and the modulation of the translation of mRNA (Denhardt, 1992). Various properties of antisense oligonucleotides including stability, toxicity, tissue distribution, and cellular uptake and binding affinity may be altered through chemical modifications including (i) replacement of the phosphodiester backbone (e.g., peptide nucleic acid, phosphorothioate oligonucleotides, and phosphoramidate oligonucleotides), (ii) modification of the sugar base (e.g., 2'-O-propylribose and 2'-methoxyethoxyribose), and (iii) modification of the nucleoside (e.g., C-5 propynyl U, C-5 thiazole U, and phenoxazine C) (Wagner, Nat. Medicine 1: 1116, 1995; Varga, et al., Immun. Lett. 69: 217, 1999; Neilsen, Curr. Opin. Biotech. 10: 71, 1999; Woolf, Nucleic Acids Res. 18: 1763, 1990).

[0097] Embodiments of the invention provide a method of inhibiting expression of a gene encoding a TEC tyrosine kinase protein comprising the step of (i) providing a biological system in which expression of a gene encoding a TEC tyrosine kinase protein is to be inhibited; and (ii) contacting the system with an antisense molecule that hybridizes to a transcript encoding the TEC tyrosine kinase protein. According to certain embodiments of the invention the TEC tyrosine kinase protein is encoded by a gene within or linked to a viral disease susceptibility locus, or within which a functional mutation causing or contributing to a viral disease or development of a viral disease (e.g., HIV, human herpesvirus, or human T cell leukemia virus) may exist. In other embodiments, TEC tyrosine kinase proteins are inhibited. According to certain embodiments of the invention the biological system comprises a cell, and the contacting step comprises expressing the antisense molecule in the cell. According to certain embodiments of the invention the biological system comprises a subject, e.g., a mammalian subject such as a mouse or human, and the contacting step comprises administering the antisense molecule to the 7 007711

subject or comprises expressing the antisense molecule in the subject. The expression may be inducible and/or tissue or cell type-specific. The antisense molecule may be an oligonucleotide or a longer nucleic acid molecule. Embodiments of the invention provide such antisense molecules.

E. Ribozymes

[0098] Certain nucleic acid molecules referred to as ribozymes or deoxyribozymes have been shown to catalyze the sequence-specific cleavage of RNA molecules. The cleavage site is determined by complementary pairing of nucleotides in the RNA or DNA enzyme with nucleotides in the target RNA. Thus, RNA and DNA enzymes can be designed to cleave to any RNA molecule, thereby increasing its rate of degradation (Cotten et al, EMBO J. 8: 3861-3866, 1989; Usman et al, Nucl. Acids MoI Biol. 10: 243, 1996; Usman, et al, Curr. Opin. Struct. Biol. 1: 527, 1996; Sun, et al, Pharmacol. Rev., 52: 325, 2000. See also e.g., Cotten et al, EMBO J. 8: 3861-3866, 1989).

[0099] Embodiments of the invention provide a method of inhibiting expression of a gene encoding a TEC tyrosine kinase protein comprising the step of (i) providing a biological system in which expression of a gene encoding a TEC tyrosine kinase protein is to be inhibited; and (ii) contacting the system with a ribozyme that hybridizes to a transcript encoding the TEC tyrosine kinase protein and directs cleavage of the transcript. According to certain embodiments of the invention the TEC tyrosine kinase protein is encoded by a gene within or linked to a viral disease susceptibility locus, or within which a functional mutation causing or contributing to susceptibility or development of a viral disease {e.g., HIV, human herpesvirus, or human T cell leukemia virus) may exist. In other embodiments, TEC tyrosine kinase proteins are inhibited. According to certain embodiments of the invention the biological system comprises a cell, and the contacting step comprises expressing the ribozyme in the cell. According to certain embodiments of the invention the biological system comprises a subject, e.g., a mammalian subject such as a mouse or human, and the contacting step comprises administering the ribozyme to the subject or comprises expressing the ribozyme in the subject. The expression may be inducible and/or tissue or cell-type specific according to certain embodiments of the invention. Embodiments of the invention provide ribozymes designed to cleave transcripts encoding TEC tyrosine kinase proteins, or polymorphic variants thereof, as described above.

SCREENING METHODOLOGIES

[0100] Methods for identifying compounds that inhibit a function of Tec tyrosine kinase protein, e.g., TEC, BTK, ITK, RLK, or BMX Tec kinases can identify compounds that 7 007711

can inhibit viral disease. A function of TEC tyrosine kinase can include activation of key components of T cell receptor signaling that contribute to the regulation of phospholipase C-γ, the mobilization of Ca^+"*^", and the activation of mitogen activated protein kinases, as well as regulation of the actin cytoskeleton.

[0101] In some embodiments, the test compounds bind to an TEC tyrosine kinase polypeptide or nucleic acid, e.g., mRNA, and cause a decrease in levels of TEC tyrosine kinase polypeptide.

[0102] These methods can be used to identify test compounds that inhibit TEC tyrosine kinase function. In some embodiments, the methods include determining whether a compound can bind to TEC tyrosine kinase and cause the inhibition of HIV binding, entry, or replication in cells.

[0103] In some embodiments, the methods include determining whether a compound that is known to bind to TEC tyrosine kinase also inhibits TEC tyrosine kinase role in viral disease, e.g., inhibition of HIV binding, entry, or replication in cells.

[0104] In some embodiments, the methods include providing one or more samples that include both TEC tyrosine kinase and one or more test compounds. An "active fragment" is a fragment that retains the ability to bind the other protein, e.g., an active fragment of TEC tyrosine kinase retains the ability to activate key components of T cell receptor signaling that contribute to the regulation of phospholipase C-γ, the mobilization of Ca^+"1", and the activation of mitogen activated protein kinases.

[0105] A number of suitable assay methods to detect binding of test compounds to TEC tyrosine kinase protein, e.g., TEC, BTK, ITK, RLK, or BMX, are known in the art and described herein, and include, but are not limited to, surface plasmon resonance (SPR)/Biacore™, fluorogenic binding assays, fluid phase binding assays, affinity chromatography, size exclusion or gel filtration, ELISA, immunoprecipitation, competitive binding assays, gel shift assays, and mass spectrometry based methods.

[0106] In some embodiments, methods described herein include a first screen for compounds that bind to TEC tyrosine kinase protein, e.g., TEC, BTK, TTK, RLK, or BMX. Compounds that are identified as binding to TEC tyrosine kinase can then be used in a second screen to identify those compounds that inhibit a function of TEC tyrosine kinase. Alternatively, the first screen can be omitted and the compounds can simply be screened for their ability to inhibit a function of TEC tyrosine kinase, e.g., to inhibit HIV binding, entry, or replication in cells. T/US2007/0077H

[0107] Once a compound that inhibits an action of TEC tyrosine kinase is identified, the compound can be considered a candidate compound for the treatment of viral disease. The ability of such compounds to treat viral disease can be evaluated in a population of viable cells or in an animal, e.g., an animal model. A number of methods are known in the art and described herein for measuring HIV infection by HIV binding, entry, or replication in cells.

[0108] Such compounds are useful, e.g., as candidate therapeutic compounds for the treatment of viral disease. Thus, included herein are methods for screening for candidate therapeutic compounds for the treatment of viral disease, as described herein. The methods include administering the compound to a model of the condition, e.g., contacting a cell (in vitro) model with the compound, or administering the compound to an animal model of the condition, e.g., an animal model of a condition associated with viral disease. The model is then evaluated for an effect of the candidate compound on the rate of viral infection of a model in vitro cell line, and a candidate compound that decrease the rate of viral infection of a model in vitro cell line can be considered a candidate therapeutic compound for the treatment of the condition. Such effects can include clinically relevant effects such as decreased HIV infection as measured by HIV binding, entry, or replication in cells. Such effects can be determined on a macroscopic or microscopic scale. Methods are such as those described herein. Candidate therapeutic compounds identified by these methods can be further verified, e.g., by administration to human subjects in a clinical trial.

[0109] The test compounds utilized in the assays and methods described herein can be, for example, nucleic acids, small molecules, organic or inorganic compounds, antibodies or antigen-binding fragments thereof, polynucleotides, peptides, or polypeptides. For example, TEC tyrosine kinase protein, e.g., TEC, BTK, ITK, RLK, or BMX, polypeptides or polynucleotides (e.g., TEC tyrosine kinase protein, TEC, BTK, ITK, RLK, or BMX, polypeptide variants including truncation mutants, deletion mutants, and point mutants; nucleic acids including sense, antisense, aptamers, and small inhibitory RNAs (siRNAs) including short hairpin RNAs (shRNAs) and ribozymes) can be used as test compounds in the methods described herein. Alternatively, compounds or compositions that mimic the TEC tyrosine kinase can be used. A test compound that has been screened by an in vitro method described herein and determined to have a desired activity, e.g., a TEC kinase inhbitor that causes inhibition of HIV binding, entry, or replication in cells, can be considered a candidate compound. A candidate compound that has been screened, e.g., in an in vitro or in vivo model, and determined to have a desirable effect on one or more symptoms of a disorder associated with viral disease, can be considered a candidate therapeutic agent. Candidate therapeutic agents, once screened in a clinical setting, are therapeutic agents, and both types of agents can be optionally optimized (e.g., by derivatization), and formulated with pharmaceutically acceptable excipients or carriers to form pharmaceutical compositions.

[0110] Small chemical molecule test compounds can initially be members of an organic or inorganic chemical library. As used herein, "small molecules" refers to small organic or inorganic molecules of molecular weight below about 3,000 Daltons. The small molecules can be natural products or members of a combinatorial chemistry library. A set of diverse molecules should be used to cover a variety of functions such as charge, aromaticity, hydrogen bonding, flexibility, size, length of side chain, hydrophobicity, and rigidity. Combinatorial techniques suitable for synthesizing small molecules are known in the art, e.g., as exemplified by Obrecht and Villalgordo, Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular- Weight Compound Libraries, Pergamon-Elsevier Science Limited (1998), and include those such as the "split and pool" or "parallel" synthesis techniques, solid-phase and solution-phase techniques, and encoding techniques (see, for example, Czarnik, Curr. Opin. Chem. Bio., 1: 60, 1997. In addition, a number of small molecule libraries are commercially available.

[0111] The test compound can have a structure that is based on an active fragment of TEC tyrosine kinase. For example, computer modeling methods known in the can be used to rationally design a molecule that has a structure similar to an active fragment of TEC tyrosine kinase.

[0112] In some embodiments, the compounds are optimized to improve their therapeutic index, i.e., increase therapeutic efficacy and/or decrease unwanted side effects. Thus, in some embodiments, the methods described herein include optimizing the test or candidate compound. In some embodiments, the methods include formulating a therapeutic composition including a test or candidate compound (e.g., an optimized compound) and a pharmaceutically acceptable carrier. In some embodiments, the compounds are optimized by derivatization using methods known in the art.

[0113] In some embodiments, the test compound comprises a polynucleotide that encodes TEC tyrosine kinase protein, e.g., TEC, BTK, ITK, RLK, or BMX, or an active fragment thereof. In some embodiments, the compound is a polynucleotide that encodes an active or inactive fragment of TEC tyrosine kinase protein, e.g., TEC, BTK, ITK, RLK, or BMX.

[0114] In some embodiments, the test compound comprises a polynucleotide that encodes a polypeptide that is at least about 85% identical to the amino acid sequence of TEC tyrosine kinase protein, e.g., TEC, BTK, XTK, RLK, or BMX. In some embodiments, the polynucleotide encodes a polypeptide that is at least about 90%, 95%, 99%, or 100% identical to US2007/007711

the full length sequence of a TEC tyrosine kinase or an active fragment thereof. In some embodiments, the polynucleotide encodes an active peptide fragment thereof that retains the ability to inhibit viral infection by inhibition of virus binding, entry, or replication in cells. In some embodiments, the active fragment is at least about 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80 or more amino acids long. The nucleic acid can include one or more noncoding regions of the coding strand of a nucleotide sequence encoding TEC tyrosine kinase protein, e.g., TEC, BTK, ITK, RLK, or BMX (e.g., the 5¹ and 3' untranslated regions). A number of methods are known in the art for obtaining suitable nucleic acids, see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; 3rd ed. 2001).

[0115] In practicing the methods of embodiments of the invention, a variety of apparatus and methodologies can be used to in conjunction with the polypeptides and nucleic acids of embodiments of the invention, e.g., to screen polypeptides for TEC tyrosine kinase activity, to screen compounds as potential modulators (e.g., inhibitors) of a TEC tyrosine kinase activity, for antibodies that bind to a polypeptide of embodiments of the invention, for nucleic acids that hybridize to a nucleic acid of embodiments of the invention, to screen for cells expressing a polypeptide of embodiments of the invention and the like.

[0116] In one aspect, the peptides and polypeptides of embodiments of the invention can be bound to a solid support. Solid supports can include, e.g., membranes (e.g., nitrocellulose or nylon), a microtiter dish (e.g., PVC, polypropylene, or polystyrene), a test tube (glass or plastic), a dip stick (e.g., glass, PVC, polypropylene, polystyrene, latex and the like), a microfuge tube, or a glass, silica, plastic, metallic or polymer bead or other substrate such as paper. One solid support uses a metal (e.g., cobalt or nickel)-comprising column which binds with specificity to a histidine tag engineered onto a peptide.

[0117] Adhesion of peptides to a solid support can be direct (i.e., the protein contacts the solid support) or indirect (a particular compound or compounds are bound to the support and the target protein binds to this compound rather than the solid support). Peptides can be immobilized either covalently (e.g., utilizing single reactive thiol groups of cysteine residues (see, e.g., Colliuod, Bioconjugate Chem. 4: 528-536, 1993) or non-covalently but specifically (e.g., via immobilized antibodies (see, e.g., Schuhmann, Adv. Mater. 3: 388-391, 1991; Lu, Anal. Chem. 67: 83-87, 1995; the biotin/strepavidin system (see, e.g., Iwane, Biophys. Biochem. Res. Comm. 230: 76-80, 1997); metal chelating, e.g., Langmuir-Blodgett films (see, e.g., Ng, Langmuir 11: 4048-55, 1995); metal-chelating self-assembled monolayers (see, e.g., Sigal, Anal. Chem. 68: 490-497, 1996) for binding of polyhistidine fusions. [0118] Indirect binding can be achieved using a variety of linkers which are commercially available. The reactive ends can be any of a variety of functionalities including, but not limited to: amino reacting ends such as N-hydroxysuccinimide (NHS) active esters, imidoesters, aldehydes, epoxides, sulfonyl halides, isocyanate, isothiocyanate, and nitroaryl halides; and thiol reacting ends such as pyridyl disulfides, maleimides, thiophthalimides, and active halogens. The heterobifunctional crosslinking reagents have two different reactive ends, e.g., an amino-reactive end and a thiol-reactive end, while homobifunctional reagents have two similar reactive ends, e.g., bismaleimidohexane (BMH) which permits the cross-linking of sulfhydryl-containing compounds. The spacer can be of varying length and be aliphatic or aromatic. Examples of commercially available homobifunctional cross-linking reagents include, but are not limited to, the imidoesters such as dimethyl adipimidate dihydrochloride (DMA); dimethyl pimelimidate dihydrochloride (DMP); and dimethyl suberimidate dihydrochloride (DMS). Heterobifunctional reagents include commercially available active halogen-NHS active esters coupling agents such as N-succinimidyl bromoacetate and N-succinimidyl (4- iodoacetyl)aminobenzoate (SLAB) and the sulfosuccinimidyl derivatives such as sulfosuccinimidyl(4-iodoacetyl)aminobenzoate (sulfo-SIAB) (Pierce). Another group of coupling agents is the heterobifunctional and thiol cleavable agents such as N-succinimidyl 3-(2- pyridyidithio)propiona- te (SPDP) (Pierce Chemicals, Rockford, 111.).

[0119] Antibodies can be used for binding polypeptides and peptides of embodiments of the invention to a solid support. This can be done directly by binding peptide-specific antibodies to the column or it can be done by creating fusion protein chimeras comprising motif- containing peptides linked to, e.g., a known epitope {e.g., a tag (e.g., FLAG, myc) or an appropriate immunoglobulin constant domain sequence (an "immunoadhesin," see, e.g., Capon, Nature 377: 525-531, 1989.

ARRAYS OR '⁴BIOCHIPS"

[0120] Embodiments of the invention provide methods for identifying/screening for modulators (e.g., inhibitors) of TEC tyrosine kinase activity, e.g., TEC, BTK, ITK, RLK, or BMX tyrosine kinase activity, using arrays. The modulators (e.g., inhibitors) of TEC tyrosine kinase activity can inhibit viral disease. Potential modulators, including small molecules, nucleic acids, polypeptides (including antibodies) can be immobilized to arrays. Nucleic acids or polypeptides of embodiments of the invention can be immobilized to or applied to an array. Arrays can be used to screen for or monitor libraries of compositions (e.g., small molecules, antibodies, nucleic acids, etc.) for their ability to bind to or modulate the activity of a nucleic acid or a polypeptide of embodiments of the invention, e.g., TEC tyrosine kinase activity. For 11

example, in one aspect of the invention, a monitored parameter is transcript expression of a gene comprising a nucleic acid of embodiments of the invention. One or more, or, all the transcripts of a cell can be measured by hybridization of a sample comprising transcripts of the cell, or, nucleic acids representative of or complementary to transcripts of a cell, by hybridization to immobilized nucleic acids on an array, or "biochip." By using an "array" of nucleic acids on a microchip, some or all of the transcripts of a cell can be simultaneously quantified. Alternatively, arrays comprising genomic nucleic acid can also be used to determine the genotype of a newly engineered strain made by the methods of embodiments of the invention. Polypeptide arrays can be used to simultaneously quantify a plurality of proteins. Small molecule arrays can be used to simultaneously analyze a plurality of TEC tyrosine kinase modulating or binding activities.

[0121] The present invention can be practiced with any known "array," also referred to as a "microarray" or "nucleic acid array" or "polypeptide array" or "antibody array" or "biochip," or variation thereof. Arrays are generically a plurality of "spots" or "target elements," each target element comprising a defined amount of one or more biological molecules, e.g., oligonucleotides, immobilized onto a defined area of a substrate surface for specific binding to a sample molecule, e.g., mRNA transcripts. In practicing the methods of embodiments of the invention, any known array and/or method of making and using arrays can be incorporated in whole or in part, or variations thereof, as described, for example, in U.S. Pat. Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174; 5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522; 5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g., WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g., Johnston, Curr. Biol. 8: R171-R174, 1998; Schummer, Biotechniques 23: 1087-1092, 1997 '; Kern, Biotechniques 23: 120-124, 1997; Solinas-Toldo, Genes, Chromosomes & Cancer 20: 399-407, 1997; Bowtell, Nature Genetics Supp. 21: 25-32, 1999. See also U.S. patent applications Nos. 20010018642; 20010019827; 20010016322; 20010014449; 20010014448; 20010012537; 20010008765.

[0122] The terms "array" or "microarray" or "biochip" or "chip" as used herein is a plurality of target elements, each target element comprising a defined amount of one or more polypeptides (including antibodies) or nucleic acids immobilized onto a defined area of a substrate surface.

COMBINATORIAL CHEMICAL LIBRARIES

[0123] Embodiments of the invention provide methods for identifying/screening modulators (e.g., inhibitors) of a TEC tyrosine kinase activity, e.g., TEC, BTK, ITK, RLK, or BMX tyrosine kinase activity. The modulators (e.g., inhibitors) of TEC tyrosine kinase activity T/US2007/0077H

can inhibit viral disease. In practicing the screening methods of embodiments of the invention, a test compound is provided. It can be contacted with a polypeptide of embodiments of the invention in vitro or administered to a cell of embodiments of the invention or an animal of embodiments of the invention in vivo. Compounds are also screened using the compositions, cells, non-human animals and methods of embodiments of the invention for their ability to treat or ameliorate a viral disease in an animal. Combinatorial chemical libraries are one means to assist in the generation of new chemical compound leads for, e.g., compounds that inhibit an TEC tyrosine kinase activity of embodiments of the invention, or a compound that can be used to treat or ameliorate a viral disease.

[0124] A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. For example, the systematic, combinatorial mixing of 100 interchangeable chemical building blocks results in the theoretical synthesis of 100 million tetrameric compounds or 10 billion pentameric compounds (see, e.g., Gallop et al. (1994) 37(9): 1233-1250). Preparation and screening of combinatorial chemical libraries are well known to those of skill in the art, see, e.g., U.S. Pat. No. 6,004,617; 5,985,356. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175; Furka, Int. J. Pept. Prot. Res. 37: 487-493, 1991, Houghton et al Nature, 354: 84-88, 1991). Other chemistries for generating chemical diversity libraries include, but are not limited to: peptoids (see, e.g., WO 91/19735), encoded peptides (see, e.g., WO 93/20242), random bio-oligomers (see, e.g., WO 92/00091), benzodiazepines (see, e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (see, e.g., Hobbs, Proc. Nat. Acad. ScL USA 90: 6909-6913, 1993), vinylogous polypeptides (see, e.g., Hagihara, J. Amer. Chem. Soc. 114: 6568, 1992), non-peptidal peptidomimetics with a Beta-D- Glucose scaffolding (see, e.g., Hirschmann, /. Amer. Chem. Soc. 114: 9217-9218, 1992), analogous organic syntheses of small compound libraries (see, e.g., Chen, J. Amer. Chem. Soc. 116: 2661, 1994), oligocarbamates (see, e.g., Cho, Science 261: 1303, 1993), and/or peptidyl phosphonates (see, e.g., Campbell, J. Org. Chem. 59: 658, 1994). See also Gordon, J. Med. Chem. 37: 1385, 1994; for nucleic acid libraries, peptide nucleic acid libraries, see, e.g., U.S. Pat. No. 5,539,083; for antibody libraries, see, e.g., Vaughn, Nature Biotechnology 14: 309-314, 11

1996; for carbohydrate libraries, see, e.g., Liang et al. Science 274: 1520-1522, 1996, U.S. Pat. No. 5,593,853; for small organic molecule libraries, see, e.g., for isoprenoids U.S. Pat. No. 5,569,588; for thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; for pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; for morpholino compounds, U.S. Pat. No. 5,506,337; for benzodiazepines U.S. Pat. No. 5,288,514.

[0125] Devices for the preparation of combinatorial libraries are commercially available (see, e.g., U.S. Pat. Nos. 6,045,755; 5,792,431; 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.). A number of robotic systems have also been developed for solution phase chemistries. These systems include automated workstations, e.g., like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate π, Zymark Corporation, Hopkinton, Mass; Orca, Hewlett-Packard, Palo Alto, Calif.) which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art. In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, NJ., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, Md., etc.).

ANTIBODIES AND ANTIBODY-BASED SCREENING METHODS

[0126] Embodiments of the invention provide isolated or recombinant antibodies that specifically bind to a polypeptide or nucleic acid of embodiments of the invention, e.g., TEC tyrosine kinase polypeptides, e.g., TEC, BTK, ITK, RLK, or BMX polypeptides. The antibodies can be used as modulators (e.g., inhibitors) of TEC tyrosine kinase activity to inhibit viral infection or viral disease. These antibodies can be used to isolate, identify or quantify a polypeptide of embodiments of the invention or related polypeptides. These antibodies can be used to isolate other polypeptides within the scope the invention that act as inhibitors of viral infection in pathways related to entry or replication of infectious viruses in the cells.

[0127] The term "antibody" includes a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope, see, e.g. Fundamental Immunology, Third Edition, W. E. Paul, ed., Raven Press, N.Y., 1993; Wilson, J. Immunol. Methods 175: 267-273, 1994; Yarmush, /. Biochem. Biophys. Methods 25: 85-97, 1992. The term antibody includes antigen-binding portions, i.e., "antigen binding sites," (e.g., fragments, 007/007711

subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the V_L, V_H, CL and C_HI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V_H and C_H1 domains; (iv) a Fv fragment consisting of the V_L and V_H domains of a single arm of an antibody, (v) a dAb fragment (Ward et at, Nature 341: 544-546, 1989), which consists of a V_H domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term "antibody."

[0128] The antibodies can be used in immunoprecipitation, staining (e.g., FACS), immunoaffinity columns, and the like. If desired, nucleic acid sequences encoding for specific antigens can be generated by immunization followed by isolation of polypeptide or nucleic acid, amplification or cloning and immobilization of polypeptide onto an array of embodiments of the invention. Alternatively, the methods of embodiments of the invention can be used to modify the structure of an antibody produced by a cell to be modified, e.g., an antibody's affinity can be increased or decreased. Furthermore, the ability to make or modify antibodies can be a phenotype engineered into a cell by the methods of embodiments of the invention.

[0129] Methods of immunization, producing and isolating antibodies (polyclonal and monoclonal) are known to those of skill in the art and described in the scientific and patent literature, see, e.g., Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY (1991); Stites (eds.) BASIC AND CLINICAL IMMUNOLOGY (7^th ed.) Lange Medical Publications, Los Altos, Calif. ("Stites"); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York, N. Y., 1986; Kohler, Nature 256: 495, 1975; Harlow, ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publications, New York, 1988. Antibodies also can be generated in vitro, e.g., using recombinant antibody binding site expressing phage display libraries, in addition to the traditional in vivo methods using animals. See, e.g., Hoogenboom, Trends Biotechnσl. 15: 62-70, 1997; Katz, Annu. Rev. Biophys. Biomol. Struct. 26: 27-45, 1997.

[0130] Polypeptides or peptides can be used to generate antibodies which bind specifically to the polypeptides of embodiments of the invention. The resulting antibodies can be used in immunoaffinity chromatography procedures to isolate or purify the polypeptide or to determine whether the polypeptide is present in a biological sample. In such procedures, a protein preparation, such as an extract, or a biological sample is contacted with an antibody capable of specifically binding to one of the polypeptides of embodiments of the invention. T/US2007/0077H

[0131] In immunoaffinity procedures, the antibody is attached to a solid support, such as a bead or other column matrix. The protein preparation is placed in contact with the antibody under conditions in which the antibody specifically binds to one of the polypeptides of embodiments of the invention. After a wash to remove non-specifically bound proteins, the specifically bound polypeptides are eluted.

[0132] The ability of proteins in a biological sample to bind to the antibody can be determined using any of a variety of procedures familiar to those skilled in the art. For example, binding can be determined by labeling the antibody with a detectable label such as a fluorescent agent, an enzymatic label, or a radioisotope. Alternatively, binding of the antibody to the sample can be detected using a secondary antibody having such a detectable label thereon. Particular assays include ELISA assays, sandwich assays, radioimmunoassay, and Western Blots.

[0133] Polyclonal antibodies generated against the polypeptides of embodiments of the invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to a non-human animal. The antibody so obtained will then bind the polypeptide itself. In this manner, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies which can bind to the whole native polypeptide. Such antibodies can then be used to isolate the polypeptide from cells expressing that polypeptide.

[0134] For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique, the trioma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (see, e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

[0135] Techniques described for the production of single chain antibodies (see, e.g., U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to the polypeptides of embodiments of the invention. Alternatively, transgenic mice can be used to express humanized antibodies to these polypeptides or fragments thereof.

[0136] Antibodies generated against the polypeptides of embodiments of the invention can be used in screening for similar polypeptides from other organisms and samples. In such techniques, polypeptides from the organism are contacted with the antibody and those polypeptides which specifically bind the antibody are detected. Any of the procedures described above can be used to detect antibody binding. US2007/007711

TEC TYROSINE KINASE INHIBITORS

[0137] The present invention provides a method for preventing or treating viral disease in a mammalian subject which comprises administering to the mammalian subject a compound capable of inhibiting a TEC tyrosine kinase, wherein the compound is administered in an amount effective to reduce or eliminate the viral disease or to prevent its occurrence or recurrence. The present invention further provides a method for identifying a compound capable of inhibiting viral infection of a cell comprising contacting a test compound with a cell-based assay system comprising a cell expressing TEC tyrosine kinase and capable of signaling responsiveness to TEC tyrosine kinase, and detecting an effect of the test compound in the assay system as an increase or a decrease in susceptibility of the cell line to viral infection, effectiveness of the test compound in the assay being indicative of the inhibition of viral infection of the cell.

[0138] "Susceptibility to viral infection" refers to susceptibility to an infectious virus, e.g., lentivirus, human immunodeficiency virus, human T cell leukemia virus type I human T cell leukemia virus type II, feline immunodeficiency virus, maedi-visna, herpesvirus, human herpesvirus, human herpesvirus 6, human herpesvirus 7, cytomegalovirus, Kaposi's sarcoma herpesvirus, poxvirus, or myxoma virus. Susceptibility to infection with HIV was measured as described herein using an in vitro cell assay incorporating Jurkat cells or human CD4+ cells infected with pseudotyped HIV-I.

[0139] "Immune cell response" refers to the response of immune system cells to external or internal stimuli (.e.g., antigen, cytokines, chemokines, and other cells) producing biochemical changes in the immune cells that result in immune cell migration, killing of target cells, phagocytosis, production of antibodies, other soluble effectors of the immune response, and the like.

[0140] "T lymphocyte response" and "T lymphocyte activity" are used here interchangeably to refer to the component of immune response dependent on T lymphocytes (i.e. , the proliferation and/or differentiation of T lymphocytes into helper, cytotoxic killer, or suppressor T lymphocytes, the provision of signals by helper T lymphocytes to B lymphocytes that cause or prevent antibody production, the killing of specific target cells by cytotoxic T lymphocytes, and the release of soluble factors such as cytokines that modulate the function of other immune cells).

[0141] "Immune response" refers to the concerted action of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of invading pathogens, 11

cells or tissues infected with pathogens, cancerous cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.

[0142] "Patient", "subject" or "mammal" are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals. Animals include all vertebrates, e.g., mammals and non-mammals, such as sheep, dogs, cows, chickens, amphibians, and reptiles.

[0143] "Treating" or "treatment" includes the administration of the compositions, compounds or agents of the present invention to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease, alleviating or ameliorating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder (e.g. , a viral disease). "Treating" further refers to any indicia of success in the treatment or amelioration or prevention of the disease, condition, or disorder (e.g., a viral disease), including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term "treating" includes the administration of the compounds or agents of the present invention to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with a viral disease. The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject. "Treating" or "treatment" using the methods of the present invention includes preventing the onset of symptoms in a subject that can be at increased risk of a viral disease but does not yet experience or exhibit symptoms, inhibiting the symptoms of a viral disease (slowing or arresting its development), providing relief from the symptoms or side-effects of viral disease (including palliative treatment), and relieving the symptoms of viral disease (causing regression). Treatment can be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease or condition.

[0144] The ability of a molecule to bind to TEC tyrosine kinase can be determined, for example, by the ability of the putative ligand to bind to TEC tyrosine kinase immunoadhesin coated on an assay plate. Specificity of binding can be determined by comparing binding to non- TEC tyrosine kinase immunoadhesin. 11

[0145] In one embodiment, antibody binding to TEC tyrosine kinase can be assayed by either immobilizing the ligand or the receptor. For example, the assay can include immobilizing TEC tyrosine kinase fused to a His tag onto Ni-activated NTA resin beads. Antibody can be added in an appropriate buffer and the beads incubated for a period of time at a given temperature. After washes to remove unbound material, the bound protein can be released with, for example, SDS, buffers with a high pH, and the like and analyzed.

[0146] "Modulator" includes inhibitors and activators. Inhibitors are agents that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of TEC tyrosine kinase, e.g., antagonists. Activators are agents that, e.g., bind to, stimulate, increase, open, activate, facilitate, enhance activation, sensitize or up regulate the activity of TEC tyrosine kinase, e.g., agonists. Modulators include agents that, e.g., alter the interaction of TEC tyrosine kinase with proteins that bind activators or inhibitors, receptors, including proteins, peptides, lipids, carbohydrates, polysaccharides, or combinations of the above, e.g., lipoproteins, glycoproteins, and the like. Modulators include genetically modified versions of naturally-occurring TEC tyrosine kinase ligands, e.g., with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like. Such assays for inhibitors and activators include, e.g., applying putative modulator compounds to a cell expressing a TEC tyrosine kinase and then determining the functional effects on TEC tyrosine kinase activity, as described herein. Samples or assays comprising TEC tyrosine kinase that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition. Control samples (untreated with inhibitors) can be assigned a relative TEC tyrosine kinase is activity value of 100%. Inhibition of TEC tyrosine kinase is achieved when the TEC tyrosine kinase activity value relative to the control is about 80%, optionally 50% or 25- 0%. Activation of TEC tyrosine kinase is achieved when the TEC tyrosine kinase activity value relative to the control is 110%, optionally 150%, optionally 200-500%, or 1000-3000% higher.

[0147] "Inhibitors," "activators," and "modulators" of TEC tyrosine kinase protein, e.g., TEC, BTK, ITK, RLK, or BMX, activity are used to refer to inhibitory, activating, or modulating molecules, respectively, identified using in vitro and in vivo assays for TEC tyrosine kinase activity, e.g., ligands, binding partners, agonists, antagonists, and their homologs and mimetics. "Modulator" includes inhibitors and activators. Inhibitors are agents that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of TEC tyrosine kinase, e.g., antagonists. Activators are agents that, e.g., bind to, stimulate, increase, open, activate, facilitate, enhance activation, sensitize or up regulate the activity of TEC tyrosine kinase, e.g., agonists. Modulators include agents that, e.g., alter the interaction of TEC tyrosine kinase with proteins that bind activators or inhibitors, receptors, including proteins, peptides, lipids, carbohydrates, polysaccharides, or combinations of the above, e.g., lipoproteins, glycoproteins, and the like. Modulators include genetically modified versions of naturally-occurring TEC tyrosine kinase protein, e.g., TEC, BTK, ITK, RLK, or BMX, with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like. Such assays for inhibitors and activators include, e.g., applying putative modulator compounds to a cell expressing TEC tyrosine kinase protein, e.g., TEC, BTK, ITK, RLK, or BMX, and then determining the functional effects on viral infection in the cell, as described herein. Samples or assays comprising TEC tyrosine kinase protein, e.g., TEC, BTK, ITK, RLK, or BMX, that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition. Control samples (untreated with inhibitors) can be assigned a relative TEC tyrosine kinase activity value of 100%. Inhibition of viral infection is achieved when the TEC tyrosine kinase activity value relative to the control is about 80%, optionally 50% or 25-0%.

[0148] "Antagonist" is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of TEC tyrosine kinase protein, e.g., TEC, BTK, ITK, RLK, or BMX, activity. In a similar manner, the term "agonist" is used in the broadest sense and includes any molecule that mimics or enhances a biological activity of TEC tyrosine kinase protein, e.g., TEC, BTK, ITK, RLK, or BMX, activity. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native TEC tyrosine kinase polypeptides, peptides, antisense oligonucleotides, small organic molecules, and the like. Methods for identifying agonists or antagonists of TEC tyrosine kinase polypeptides can comprise contacting an TEC tyrosine kinase polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the TEC tyrosine kinase.

[0149] "Test compound" refers to a nucleic acid, DNA, RNA, protein, polypeptide, or small chemical entity that is determined to effect an increase or decrease in a gene expression or actin cytoskeleton rearrangement as a result of signaling through TEC tyrosine kinase protein, e.g., TEC, BTK, ΓTK, RLK, or BMX. The test compound can be an antisense RNA, ribozyme, polypeptide, or small molecular chemical entity. The term "test compound" can be any small chemical compound, or a biological entity, such as a protein, sugar, nucleic acid or lipid. 2007/007711

Typically, test compounds will be small chemical molecules and polypeptides. A "test compound specific for signaling through TEC tyrosine kinase" is determined to be a modulator of TEC tyrosine kinase activity.

[0150] "Cell-based assays" include TEC tyrosine kinase binding assays, for example, radioligand or fluorescent ligand binding assays for TEC tyrosine kinase activity or binding of the protein, e.g., TEC, BTK, ITK, RLK, or BMX protein, in cells, plasma membranes, detergent- solubilized plasma membrane proteins, immobilized collagen (Alberdi, J Biol Chem. 274:31605- 12, 1999; Meyer et al, 2002); TEC tyrosine kinase -affinity column chromatography (Alberdi, J Biol Chem. 274:31605-12, 1999; Aymerich et al, Invest Ophthalmol Vis ScL 42:3287-93, 2001); TEC tyrosine kinase ligand blot using a radio- or fluorosceinated-ligand (Aymerich et al., Invest Ophthalmol Vis ScL 42:3287-93, 2001; Meyer et al., 2002); Size-exclusion ultrafiltration (Alberdi et al, Biochem., 1998; Meyer et al, 2002); or ELISA. Exemplary TEC tyrosine kinase binding activity assays of the present invention are: a TEC tyrosine kinase ligand blot assay (Aymerich et al, Invest Ophthalmol Vis ScL 42:3287-93, 2001); a TEC tyrosine kinase affinity column chromatography assay (Alberdi, J Biol Chem. 274:31605-12, 1999) and a TEC tyrosine kinase ligand binding assay (Alberdi et al., J Biol Chem. 274:31605-12, 1999). Each incorporated by reference in their entirety.

[0151] In one embodiment, TEC tyrosine kinase protein, e.g. , TEC, BTK, ITK, RLK, or BMX, can be assayed by either immobilizing the ligand/interacting protein or the kinase. For example, the assay can include immobilizing TEC tyrosine kinase fused to a His tag onto Ni- activated NTA resin beads. Inhibitors of TEC tyrosine kinase can be added in an appropriate buffer and the beads incubated for a period of time at a given temperature. After washes to remove unbound material, the bound protein can be released with, for example, SDS, buffers with a high pH, and the like and analyzed.

[0152] "Contacting" refers to mixing a test compound in a soluble form into an assay system, for example, a cell-based assay system, such that an effect upon receptor-mediated signaling can be measured.

[0153] "Signaling in cells" refers to the interaction of a ligand with a kinase, such as TEC tyrosine kinase protein, e.g., TEC, BTK, ITK, RLK, or BMX, to produce a response, for example, an T cell mediated immune response, or a response to prevent or alleviate viral disease. "Signaling responsiveness" or "effective to activate signaling" or "stimulating a cell-based assay system" refers to the ability of inhibitors of TEC tyrosine kinase activity to stimulate an immune response, and to prevent or alleviate a viral disease. [0154] "Detecting an effect" refers to an effect measured in a cell-based assay system. For example, the effect detected can be TEC tyrosine kinase protein activity, e.g., TEC, BTK, ITK, RLK, or BMX, in an assay system, for example, a Jurkat cell in vitro assay or a human CD4⁺ T cell in vitro assay.

[0155] "Assay being indicative of modulation" refers to results of a cell-based assay system indicating that cell activation by TEC tyrosine kinase protein, e.g., TEC, BTK, TTK, RLK, or BMX, induces a protective response in cells against a viral disease.

[0156] "Biological activity" and "biologically active" with regard to an inhibitor of TEC tyrosine kinase protein, e.g., TEC, BTK, ITK, RLK, or BMX, of the present invention refer to the ability of the inhibitor molecule to specifically bind to and signal through a native or recombinant TEC tyrosine kinase, or to block the ability of a native or recombinant TEC tyrosine kinase to participate in signal transduction. Thus, the (native and variant) ligands of TEC tyrosine kinase of the present invention include agonists and antagonists of a native or recombinant TEC tyrosine kinase. P referred biological activities of the ligands of TEC tyrosine kinase protein, e.g. , TEC, BTK, ITK, RLK, or BMX, of the present invention include the ability to enhance an immune response, or treat a viral disease. Accordingly, the administration of the compounds or agents of the present invention can prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with a viral disease, or other disorders.

[0157] "Concomitant administration" of a known drug with a compound of the present invention means administration of the drug and the compound at such time that both the known drug and the compound will have a therapeutic effect or diagnostic effect. Such concomitant administration can involve concurrent (i.e. at the same time), prior, or subsequent administration of the drug with respect to the administration of a compound of the present invention. A person of ordinary skill in the art, would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compounds of the present invention.

[0158] In general, the phrase "well tolerated" refers to the absence of adverse changes in health status that occur as a result of the treatment and would affect treatment decisions.

[0159] "Lymphocyte" as used herein has the normal meaning in the art, and refers to any of the mononuclear, nonphagocytic leukocytes, found in the blood, lymph, and lymphoid tissues, i.e., B and T lymphocytes.

[0160] "Subpopulations of T lymphocytes" or "T cell subset(s)" refers to T lymphocytes or T cells characterized by the expression of particular cell surface markers (see Barclay, A. N. et al., (eds.), THE LEUKOCYTE ANTIGEN FACTS BOOK, 2^ND. EDITION, Academic Press, London, United Kingdom, 1997; this reference is herein incorporated by reference for all purposes).

[0161] "Epitope" means a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0162] An intact "antibody" comprises at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V_H) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, C_HI, C_H2 and C_H3. Each light chain is comprised of a light chain variable region (abbreviated herein as V_L) and a light chain constant region. The light chain constant region is comprised of one domain, C_L. The V_H and V_L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V_H and V_L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl- terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) through cellular receptors such as Fc receptors (e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIH, and FcRη) and the first component (CIq) of the classical complement system. The term antibody includes antigen-binding portions of an intact antibody that retain capacity to bind the antigen. Examples of antigen binding portions include (i) a Fab fragment, a monovalent fragment consisting of the V_L, V_H, CL and C_HI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V_H and CHI domains; (iv) a Fv fragment consisting of the V_L and V_H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341: 544-546, 1989), which consists of a V_H domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, V_L and V_H, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); See, e.g., Bird et al., Science IAl: 423-426, 1988; and Huston et al., Proc. Natl. Acad. ScL U.S.A. 85: 5879-5883, 1988). Such single chain antibodies are included by reference to the term "antibody" Fragments can be prepared by recombinant techniques or enzymatic or chemical cleavage of intact antibodies.

[0163] "Human sequence antibody" includes antibodies having variable and constant regions (if present) derived from human immunoglobulin sequences. The human sequence antibodies of embodiments of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site- specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human sequence antibody", as used herein, is not intended to include antibodies in which entire CDR sequences sufficient to confer antigen specificity and derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., humanized antibodies).

[0164] "Monoclonal antibody" or "monoclonal antibody composition" refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions (if present) derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0165] "Diclonal antibody" refers to a preparation of at least two antibodies to an antigen. Typically, the different antibodies bind different epitopes.

[0166] "Oligoclonal antibody" refers to a preparation of 3 to 100 different antibodies to an antigen. Typically, the antibodies in such a preparation bind to a range of different epitopes.

[0167] "Polyclonal antibody" refers to a preparation of more than 1 (two or more) different antibodies to an antigen. Such a preparation includes antibodies binding to a range of different epitopes.

[0168] "Recombinant human antibody" includes all human sequence antibodies of embodiments of the invention that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (described further below); antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions (if present) derived from human germline immunoglobulin sequences. Such antibodies can, however, be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the V_H and V_L regions of the recombinant antibodies are sequences that, while derived from and related to human germline V_H and V_L sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0169] A "heterologous antibody" is defined in relation to the transgenic non-human organism producing such an antibody. This term refers to an antibody having an amino acid sequence or an encoding nucleic acid sequence corresponding to that found in an organism not consisting of the transgenic non-human animal, and generally from a species other than that of the transgenic non-human animal.

[0170] A "heterohybrid antibody" refers to an antibody having a light and heavy chains of different organismal origins. For example, an antibody having a human heavy chain associated with a murine light chain is a heterohybrid antibody.

[0171] "Substantially pure" or "isolated" means an object species (e.g., an antibody of embodiments of the invention) has been identified and separated and/or recovered from a component of its natural environment such that the object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition); a "substantially pure" or "isolated" composition also means where the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. A substantially pure or isolated composition can also comprise more than about 80 to 90 percent by weight of all macromolecular species present in the composition. An isolated object species (e.g., antibodies of embodiments of the invention) can also be purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of derivatives of a single macromolecular species. For example, an isolated antibody to TEC tyrosine kinase can be substantially free of other antibodies that lack binding to human TEC tyrosine kinase and bind to a different antigen. Further, an isolated antibody that specifically binds to an epitope, isoform or variant of human TEC tyrosine kinase may, however, have cross-reactivity to other related antigens, e.g., from other species (e.g., TEC tyrosine kinase species homologs). Moreover, an isolated antibody of embodiments of the invention be substantially free of other cellular material (e.g., non-immunoglobulin associated proteins) and/or chemicals. [0172] "Specific binding' ' refers to preferential binding of an antibody to a specified antigen relative to other non-specified antigens. The phrase "specifically (or selectively) binds" to an antibody refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies. Typically, the antibody binds with an association constant (K_a) of at least about 1 x 10⁶ M^"1 or 10⁷ M^"1, or about 10⁸ M^"1 to 10⁹ M^'1, or about 10¹⁰ M^"1 to 10ⁿ M^"1 or higher, and binds to the specified antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the specified antigen or a closely-related antigen. The phrases "an antibody recognizing an antigen" and " an antibody specific for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen". A predetermined antigen is an antigen that is chosen prior to the selection of an antibody that binds to that antigen.

[0173] "Specifically bind(s)" or "bind(s) specifically" when referring to a peptide refers to a peptide molecule which has intermediate or high binding affinity, exclusively or predominately, to a target molecule. The phrases "specifically binds to" refers to a binding reaction which is determinative of the presence of a target protein in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated assay conditions, the specified binding moieties bind preferentially to a particular target protein and do not bind in a significant amount to other components present in a test sample. Specific binding to a target protein under such conditions can require a binding moiety that is selected for its specificity for a particular target antigen. A variety of assay formats can be used to select ligands that are specifically reactive with a particular protein. For example, solid-phase ELISA immunoassays, immunoprecipitation, Biacore™ and Western blot are used to identify peptides that specifically react with the antigen. Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 times background.

[0174] "High affinity" for an antibody refers to an equilibrium association constant (K_a) of at least about 10⁷M^"1, at least about 10⁸M^"1, at least about 10⁹M^'1, at least about 10¹⁰M^'1, at least about 10* ¹M^"1, or at least about 10¹²M^"1 or greater, e.g., up to 10¹³M^'1 or 10¹⁴M^"1 or greater. However, "high affinity" binding can vary for other antibody isotypes.

[0175] The term "K_a", as used herein, is intended to refer to the equilibrium association constant of a particular antibody-antigen interaction. This constant has units of 1/M.

[0176] The term "Kj", as used herein, is intended to refer to the equilibrium dissociation constant of a particular antibody-antigen interaction. This constant has units of M.

[0177] The term "k_a", as used herein, is intended to refer to the kinetic association constant of a particular antibody-antigen interaction. This constant has units of I/Ms. [0178] The term "ka", as used herein, is intended to refer to the kinetic dissociation constant of a particular antibody-antigen interaction. This constant has units of 1/s.

[0179] "Particular antibody-antigen interactions" refers to the experimental conditions under which the equilibrium and kinetic constants are measured.

[0180] "Isotype" refers to the antibody class that is encoded by heavy chain constant region genes. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Additional structural variations characterize distinct subtypes of IgG (e.g., IgGi, IgG₂, IgG₃ and IgG₄) and IgA (e.g., IgAj and IgA₂)

[0181] "Isotype switching" refers to the phenomenon by which the class, or isotype, of an antibody changes from one Ig class to one of the other Ig classes.

[0182] "Nonswitched isotype" refers to the isotypic class of heavy chain that is produced when no isotype switching has taken place; the C_H gene encoding the nonswitched isotype is typically the first C_H gene immediately downstream from the functionally rearranged VDJ gene. Isotype switching has been classified as classical or non-classical isotype switching. Classical isotype switching occurs by recombination events which involve at least one switch sequence region in the transgene. Non-classical isotype switching can occur by, for example, homologous recombination between human σ_μ and human ∑_μ (δ-associated deletion). Alternative non-classical switching mechanisms, such as intertransgene and/or interchromosomal recombination, among others, can occur and effectuate isotype switching.

[0183] "Switch sequence" refers to those DNA sequences responsible for switch recombination. A "switch donor" sequence, typically a μ switch region, are 5' (Le., upstream) of the construct region to be deleted during the switch recombination. The "switch acceptor" region are between the construct region to be deleted and the replacement constant region (e.g., γ, ε, and alike). As there is no specific site where recombination always occurs, the final gene sequence is not typically predictable from the construct.

[0184] "Glycosylation pattern" is defined as the pattern of carbohydrate units that are covalently attached to a protein, more specifically to an immunoglobulin protein. A glycosylation pattern of a heterologous antibody can be characterized as being substantially similar to glycosylation patterns which occur naturally on antibodies produced by the species of the non-human transgenic animal, when one of ordinary skill in the art would recognize the glycosylation pattern of the heterologous antibody as being more similar to said pattern of glycosylation in the species of the non-human transgenic animal than to the species from which the C_H genes of the transgene were derived. 11

[0185] "Naturally-occurring" as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.

[0186] "Immunoglobulin locus" refers to a genetic element or set of linked genetic elements that comprise information that can be used by a B cell or B cell precursor to express an immunoglobulin peptide. This peptide can be a heavy chain peptide, a light chain peptide, or the fusion of a heavy and a light chain peptide. In the case of an unrearranged locus, the genetic elements are assembled by a B cell precursor to form the gene encoding an immunoglobulin peptide. In the case of a rearranged locus, a gene encoding an immunoglobulin peptide is contained within the locus.

[0187] "Rearranged" refers to a configuration of a heavy chain or light chain immunoglobulin locus wherein a V segment is positioned immediately adjacent to a D-J or J segment in a conformation encoding essentially a complete V_H or V_L domain, respectively. A rearranged immunoglobulin gene locus can be identified by comparison to germline DNA; a rearranged locus has at least one recombined heptamer/nonamer homology element.

[0188] "Unrearranged" or "germline configuration" in reference to a V segment refers to the configuration wherein the V segment is not recombined so as to be immediately adjacent to a D or J segment.

[0189] "Nucleic acid" or "nucleic acid molecule" refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, can encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.

[0190] "Isolated nucleic acid" in reference to nucleic acids encoding antibodies or antibody portions (e.g., V_H, V_L, CDR3) that bind to the antigen, is intended to refer to a nucleic acid in which the nucleotide sequences encoding the antibody or antibody portion are free of other nucleotide sequences encoding antibodies or antibody portions that bind antigens other than, for example, TEC tyrosine kinase, which other sequences can naturally flank the nucleic acid in human genomic DNA.

[0191] "Substantially identical," in the context of two nucleic acids or polypeptides refers to two or more sequences or subsequences that have at least about 80%, about 90%, about 95% or higher nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using the following sequence comparison method and/or by visual inspection. Such "substantially identical" sequences are typically considered to be homologous. The "substantial identity" can exist over a region of sequence that is at least about 50 residues in length, over a region of at least about 100 residues, or over a region at least about 150 residues, or over the full length of the two sequences to be compared. As described below, any two antibody sequences can only be aligned in one way, by using the numbering scheme in Kabat. Therefore, for antibodies, percent identity has a unique and well-defined meaning.

[0192] Amino acids from the variable regions of the mature heavy and light chains of immunoglobulins are designated Hx and Lx respectively, where x is a number designating the position of an amino acid according to the scheme of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991). Kabat lists many amino acid sequences for antibodies for each subgroup, and lists the most commonly occurring amino acid for each residue position in that subgroup to generate a consensus sequence. Kabat uses a method for assigning a residue number to each amino acid in a listed sequence, and this method for assigning residue numbers has become standard in the field. Rabat's scheme is extendible to other antibodies not included in his compendium by aligning the antibody in question with one of the consensus sequences in Kabat by reference to conserved amino acids. The use of the Kabat numbering system readily identifies amino acids at equivalent positions in different antibodies. For example, an amino acid at the L50 position of a human antibody occupies the equivalent position to an amino acid position L50 of a mouse antibody. Likewise, nucleic acids encoding antibody chains are aligned when the amino acid sequences encoded by the respective nucleic acids are aligned according to the Kabat numbering convention. An alternative structural definition has been proposed by Chothia, et al., J. MoI. Biol. 196:901-917, 1987; Chothia, et al., Nature 342:878-883, 1989; and Chothia, et al., J. MoI. Biol. 186:651-663, 1989, which are herein incorporated by reference for all purposes.

[0193] The nucleic acids of embodiments of the invention be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "rendered substantially pure" when purified away from other cellular components or other contaminants, e.g., other cellular nucleic acids or proteins, by standard techniques, including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and others well known in the art {See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2^nd ed., 1989; Tijssen (1993); and Ausubel (1994), incorporated by reference for all purposes). The nucleic acid sequences of embodiments of the invention and other nucleic acids used to practice this invention, whether RNA, cDNA, genomic DNA, or hybrids thereof, can be isolated from a variety of sources, genetically engineered, amplified, and/or expressed recombinantly. Any recombinant expression system can be used, including, in addition to bacterial, e.g., yeast, insect or mammalian systems. Alternatively, these nucleic acids can be chemically synthesized in vitro. Techniques for the manipulation of nucleic acids, such as, e.g., subcloning into expression vectors, labeling probes, sequencing, and hybridization are well described in the scientific and patent literature, see, e.g., Sambrook, et al., 1989. Nucleic acids can be analyzed and quantified by any of a number of general means well known to those of skill in the art. These include, e.g., analytical biochemical methods such as NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography, various immunological methods, such as fluid or gel precipitin reactions, immunodiffusion (single or double), Immunoelectrophoresis, radioimmunoassay (RIAs), enzyme-linked immunosorbent assays (ELISAs), immuno-fluorescent assays, Southern analysis, Northern analysis, dot-blot analysis, gel electrophoresis (e.g., SDS-PAGE), RT-PCR, quantitative PCR, other nucleic acid or target or signal amplification methods, radiolabeling, scintillation counting, and affinity chromatography.

[0194] The nucleic acid compositions of the present invention, while often in a native sequence (except for modified restriction sites and the like), from either cDNA, genomic or mixtures can be mutated, thereof in accordance with standard techniques to provide gene sequences. For coding sequences, these mutations, can affect amino acid sequence as desired. In particular, DNA sequences substantially homologous to or derived from native V, D, J, constant, switches and other such sequences described herein are contemplated (where "derived" indicates that a sequence is identical or modified from another sequence).

[0195] "Recombinant host cell" or "host cell" refers to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0196] A "label" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include ³²P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available (e.g., the polypeptides of embodiments of the invention can be made detectable, e.g., by incorporating a radiolabel into the peptide, and used to detect antibodies specifically reactive with the peptide). [0197] "Sorting" in the context of cells as used herein to refers to both physical sorting of the cells, as can be accomplished using, e.g., a fluorescence activated cell sorter, as well as to analysis of cells based on expression of cell surface markers, e.g., FACS analysis in the absence of sorting.

[0198] Components of an immune response can be detected in vitro by various methods that are well known to those of ordinary skill in the art. For example, (1) cytotoxic T lymphocytes can be incubated with radioactively labeled target cells and the lysis of these target cells detected by the release of radioactivity, (2) helper T lymphocytes can be incubated with antigens and antigen presenting cells and the synthesis and secretion of cytokines and proliferation as measured by assays described below and measured by standard methods (Windhagen A; et al., Immunity 2:373-380, 1995), (3) antigen presenting cells can be incubated with whole protein antigen and the presentation of that antigen on MHC detected by either T lymphocyte activation assays or biophysical methods (Harding et al., P roc. Natl. Acad. Sci. U.S.A., 86:4230-4, 1989), (4) mast cells can be incubated with reagents that cross-link their Fc- epsilon receptors and histamine release measured by enzyme immunoassay (Siraganian, et al., TIPS 4:432-437, 1983).

[0199] Similarly, products of an immune response in either a model organism {e.g., mouse) or a human patient can also be detected by various methods that are well known to those of ordinary skill in the art. For example, (1) the production of antibodies in response to vaccination can be readily detected by standard methods currently used in clinical laboratories, e.g., an ELISA; (2) the migration of immune cells to sites of inflammation can be detected by scratching the surface of skin and placing a sterile container to capture the migrating cells over scratch site (Peters et al, B hod 72:1310-5, 1988); (3) the proliferation of peripheral blood mononuclear cells in response to mitogens or mixed lymphocyte reaction can be measured using ³H-thymidine; (4) the phagocitic capacity of granulocytes, macrophages, and other phagocytes in PBMCs can be measured by placing PMBCs in wells together with labeled particles (Peters et al., 1988); and (5) the radioimmunoassay of immune system cells can be measured by labeling PBMCs with antibodies to CD molecules such as CD4 and CD 8 and measuring the fraction of the PBMCs expressing these markers.

[0200] "Signal transduction pathway" or "signal transduction event" refers to at least one biochemical reaction, but more commonly a series of biochemical reactions, which result from interaction of a cell with a stimulatory compound or agent. Thus, the interaction of a stimulatory compound with a cell generates a "signal" that is transmitted through the signal transduction pathway, ultimately resulting in a cellular response, e.g., an immune response described above.

[0201] A signal transduction pathway refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. As used herein, the phrase "cell surface receptor" includes molecules and complexes of molecules capable of receiving a signal and the transmission of such a signal across the plasma membrane of a cell. An example of a "cell surface receptor" is the T cell receptor (TCR) or the B7 ligands of CTLA-4/CD28.

[0202] A signal transduction pathway in a cell can be initiated by interaction of a cell with a stimulator that is inside or outside of the cell. If an exterior (Le., outside of the cell) stimulator (e.g., an MHC-antigen complex on an antigen presenting cell) interacts with a cell surface receptor (e.g., a T cell receptor), a signal transduction pathway can transmit a signal across the cell's membrane, through the cytoplasm of the cell, and in some instances into the nucleus. If an interior (e.g., inside the cell) stimulator interacts with an intracellular signal transduction molecule, a signal transduction pathway can result in transmission of a signal through the cell's cytoplasm, and in some instances into the cell's nucleus.

[0203] Signal transduction can occur through, e.g., the phosphorylation of a molecule; non-covalent allosteric interactions; complexing of molecules; change of protein localization; the conformational change of a molecule; calcium release; inositol phosphate production; proteolytic cleavage; cyclic nucleotide production and diacylglyceride production. Typically, signal transduction occurs through phosphorylating a signal transduction molecule.

[0204] "Nonspecific T cell activation" refers to the stimulation of T cells independent of their antigenic specificity.

[0205] This invention relies on routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual, 2^nd ed., 1989; Kriegler, Gene Transfer and Expression: A Laboratory Manual, 1990; and Ausubel et al., eds., Current Protocols in Molecular Biology, 1994.

[0206] TEC tyrosine kinase, e.g., TEC, BTK, ITK, RLK, or BMX, nucleic acids, polymorphic variants, orthologs, and alleles that are substantially identical to sequences provided herein can be isolated using nucleic acid probes and oligonucleotides of TEC tyrosine kinase, e.g., TEC, BTK, ITK, RLK, or BMX, under stringent hybridization conditions, by screening libraries. Alternatively, expression libraries can be used to isolate TEC tyrosine kinase protein, or protein encoding TEC tyrosine kinase polymorphic variants, orthologs, and alleles by detecting expressed homologs immunologically with antisera or purified antibodies made against human TEC tyrosine kinase, or portions thereof.

PEPTIDES AND POLYPEPTIDES

[0207] Embodiments of the invention provide isolated or recombinant polypeptides comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2 or SEQ ID NO:5 over a region of at least about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100 or more residues, or, the full length of the polypeptide, or, a polypeptide encoded by a nucleic acid of embodiments of the invention. Embodiments of the invention provide methods for inhibiting the activity of TEC tyrosine kinase polypeptide, e.g., TEC, BTK, ITK, RLK, or BMX polypeptides. An embodiment of the invention also provides methods for screening compositions that inhibit the activity of, or bind to (e.g., bind to the active site), of TEC tyrosine kinase polypeptides. A polypeptide, in an embodiment of the invention that inhibits the activity of TEC tyrosine kinase can inhibit viral infection.

[0208] In one aspect, the invention provides TEC tyrosine kinase polypeptides (and the nucleic acids encoding them) where one, some or all of the TEC tyrosine kinase polypeptides replacement with substituted amino acids. In one aspect, the invention provides methods to disrupt the interaction of TEC tyrosine kinase polypeptides with other proteins, in pathways related to entry or replication of infectious viruses in the cells.

[0209] The peptides and polypeptides of embodiments of the invention can be expressed recombinantly in vivo after administration of nucleic acids, as described above, or, they can be administered directly, e.g., as a pharmaceutical composition. They can be expressed in vitro or in vivo to screen for modulators of a TEC tyrosine kinase activity and for agents that can treat or ameliorate a viral disease.

[0210] Polypeptides and peptides of embodiments of the invention can be isolated from natural sources, be synthetic, or be recombinantly generated polypeptides. Peptides and proteins can be recombinantly expressed in vitro or in vivo. The peptides and polypeptides of embodiments of the invention can be made and isolated using any method known in the art. Polypeptide and peptides of embodiments of the invention can also be synthesized, whole or in part, using chemical methods well known in the art. See e.g., Caruthers, Nucleic Acids Res. Symp. Ser. 215-223, 1980; Horn, Nucleic Acids Res. Symp. Ser. 225-232, 1980; Banga, A. K., Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems Technomic Publishing Co., Lancaster, PA, 1995. For example, peptide synthesis can be performed using various solid-phase techniques (see e.g., Roberge, Science 269: 202, 1995; Merrifield, Methods Enzymol. 289: 3-13, 1997) and automated synthesis can be achieved, e.g., using the ABI 43 IA Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.

[0211] The peptides and polypeptides of embodiments of the invention, as defined above, include all "mimetic" and "peptidomimetic" forms. The terms "mimetic" and "peptidomimetic" refer to a synthetic chemical compound which has substantially the same structural and/or functional characteristics of the polypeptides of embodiments of the invention. The mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic' s structure and/or activity. As with polypeptides of embodiments of the invention which are conservative variants, routine experimentation will determine whether a mimetic is within the scope of the invention, i.e., that its structure and/or function is not substantially altered. Thus, a mimetic composition is within the scope of the invention if, when administered to or expressed in a cell, it has a TEC tyrosine kinase activity.

[0212] In one aspect, the polypeptide or peptidomimetic composition can be a dominant-negative mutant within the scope of the invention if it can inhibit an activity of a TEC tyrosine kinase polypeptide, e.g., TEC, BTK, ITK, RLK, or BMX polypeptides of embodiments of the invention, e.g., be a dominant-negative mutant or bind to an antibody. The dominant negative mutant can be a peptide or peptide mimetic that can inhibit an activity of a TEC tyrosine kinase, or a nucleic acid composition, in the form of a DNA vector or gene therapy vector, that expresses a dominant-negative polypeptide that can inhibit an activity of a TEC tyrosine kinase. The dominant negative mutant can bind to a ligand of the kinase or a target target, affecting ligand target interaction. The dominant negative molecule can act, for example, by blocking protein protein interactions, or by blocking phosphorylation of the kinase. An example of a dominant negative peptide is a peptide with a mutation in a lysine residue in the ATP binding domain of the TEC tyrosine kinase, as described herein, that inhibits TEC tyrosine kinase activity. A further example of a dominant negative peptide is a peptide with a mutation in the SH2 domain or SH3 domain of the TEC tyrosine kinase as described herein, that inhibits TEC tyrosine kinase activity.

[0213] Polypeptide mimetic compositions can contain any combination of non-natural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like. For example, a polypeptide can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds. Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC) or N₁N'- diisopropylcarbodiimide (DIC). Linking groups that can be an alternative to the traditional amide bond ("peptide bond") linkages include, e.g., ketomethylene (e.g., ~C(=O) — CH₂ — for — C(=O) — NH-), aminomethylene (CH₂ — NH), ethylene, olefin (CH=CH), ether (CH₂ — O), thioether (CH₂ — S), tetrazole (CN₄-). thiazole, retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267- 357, "Peptide Backbone Modifications," Marcell Dekker, NY).

[0214] A polypeptide can also be characterized as a mimetic by containing all or some non-natural residues in place of naturally occurring amino acid residues. Non-natural residues are well described in the scientific and patent literature; a few exemplary non-natural compositions useful as mimetics of natural amino acid residues and guidelines are described below. Mimetics of aromatic amino acids can be generated by replacing by, e.g. , D- or L- naphylalanine; D- or L-phenylglycine; D- or L-2 thieneylalanine; D- or L-I, -2,3-, or 4- pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)- alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D- (trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; K- or L-p-methoxy-biphenylphenylalanine; D- or L-2- indole(alkyl)alanines; and, D- or L-alkylainines, where alkyl can be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl, or a non- ^■ acidic amino acids. Aromatic rings of a non-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

[0215] Mimetics of acidic amino acids can be generated by substitution by, e.g., non- carboxylate amino acids while maintaining a negative charge; (phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g., aspartyl or glutamyl) can also be selectively modified by reaction with carbodiimides (R' — N — C — N — R') such as, e.g., l-cyclohexyl-3(2-morpholin- yl- (4-ethyl) carbodiimide or l-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl or glutamyl can also be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. [0216] Mimetics of basic amino acids can be generated by substitution with, e.g., (in addition to lysine and arginine) the amino acids ornithine, citrulline, or (guanidino)-acetic acid, or (guanidino)alkyl-acetic acid, where alkyl is defined above. Nitrile derivative {e.g., containing the CN-moiety in place of COOH) can be substituted for aspargine or glutamine. Asparaginyl and glutaminyl residues can be deaminated to the corresponding aspartyl or glutamyl residues.

[0217] Arginine residue mimetics can be generated by reacting arginyl with, e.g., one or more conventional reagents, including, e.g., phenylglyoxal, 2,3-butanedione, 1,2- cyclohexanedione, or ninhydrin, preferably under alkaline conditions. Tyrosine residue mimetics can be generated by reacting tyrosyl with, e.g., aromatic diazonium compounds or tetranitromethane. N-acetylimidizol and tetranitromethane can be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Cysteine residue mimetics can be generated by reacting cysteinyl residues with, e.g., alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide and corresponding amines; to give carboxymethyl or carboxyamidomethyl derivatives. Cysteine residue mimetics can also be generated by reacting cysteinyl residues with, e.g., bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid; chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide; methyl 2-pyridyl disulfide; p- chloromercuribenzoate; 2-chloromercuri-4 nitrophenol; or, chloro-7-nitrobenzo-oxa-l,3-diazole. Lysine mimetics can be generated (and amino terminal residues can be altered) by reacting lysinyl with, e.g., succinic or other carboxylic acid anhydrides. Lysine and other alpha-amino- containing residue mimetics can also be generated by reaction with imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed reactions with glyoxylate. Mimetics of methionine can be generated by reaction with, e.g., methionine sulfoxide. Mimetics of proline include, e.g., pipecolic acid, thiazolidine carboxylic acid, 3- or 4-hydroxy guanidino, dehydroproline, 3- or 4-methylproline, or 3,3,-dimethylproline. Histidine residue mimetics can be generated by reacting histidyl with, e.g., diethylprocarbonate or para-bromophenacyl bromide. Other mimetics include, e.g., those generated by hydroxylation of guanidino and lysine; phosphorylation of the hydroxyl groups of seryl or threonyl residues; methylation of the alpha- amino groups of lysine, arginine and histidine; acetylation of the N-terminal amine; methylation of main chain amide residues or substitution with N-methyl amino acids; or arnidation of C- terminal carboxyl groups.

[0218] A component of a polypeptide of embodiments of the invention can also be replaced by an amino acid (or peptidomimetic residue) of the opposite chirality. Thus, any amino acid naturally occurring in the L-configuration (which can also be referred to as the R or S, depending upon the structure of the chemical entity) can be replaced with the amino acid of the same chemical structural type or a peptidomimetic, but of the opposite chirality, referred to as the D-amino acid, but which can additionally be referred to as the R- or S-form

[0219] Embodiments of the invention also provide polypeptides that are "substantially identical" to an exemplary polypeptide of embodiments of the invention. A "substantially identical" amino acid sequence is a sequence that differs from a reference sequence by one or more conservative or non-conservative amino acid substitutions, deletions, or insertions, particularly when such a substitution occurs at a site that is not the active site of the molecule, and provided that the polypeptide essentially retains its functional properties. A conservative amino acid substitution, for example, substitutes one amino acid for another of the same class (e.g., substitution of one hydrophobic amino acid, such as isoleucine, valine, leucine, or methionine, for another, or substitution of one polar amino acid for another, such as substitution of arginine for lysine, glutamic acid for aspartic acid or glutamine for asparagine). One or more amino acids can be deleted, for example, from a TEC tyrosine kinase polypeptide of embodiments of the invention, resulting in modification of the structure of the polypeptide, without significantly altering its biological activity. For example, amino- or carboxyl-terminal, or internal, amino acids which are not required for a TEC tyrosine kinase activity or interaction can be removed.

[0220] The skilled artisan will recognize that individual synthetic residues and polypeptides incorporating these mimetics can be synthesized using a variety of procedures and methodologies, which are well described in the scientific and patent literature, e.g., Organic Syntheses Collective Volumes, Gilman, et al. (Eds) John Wiley & Sons, Inc., NY. Peptides and peptide mimetics of embodiments of the invention can also be synthesized using combinatorial methodologies. Various techniques for generation of peptide and peptidomimetic libraries are well known, and include, e.g., multipin, tea bag, and split-couple-mix techniques; see, e.g., al- Obeidi, MoL Biotechnol. 9: 205-223, 1998; Hruby, Curr. Opin. Chem. Biol. 1: 114-119, 1997; Ostergaard, MoI. Divers. 3: 17-27, 1997; Ostresh, Methods Enzymol. 267: 220-234, 1996. Modified peptides of embodiments of the invention can be further produced by chemical modification methods, see, e.g., Belousov, Nucleic Acids Res. 25: 3440-3444, 1997; Frenkel, Free Radic. Biol. Med. 19: 373-380, 1995; Blommers, Biochemistry 33: 7886-7896, 1994.

[0221] Peptides and polypeptides of embodiments of the invention can also be synthesized and expressed as fusion proteins with one or more additional domains linked thereto for, e.g., producing a more immunogenic peptide, to more readily isolate a recombinantly synthesized peptide, to identify and isolate antibodies and antibody-expressing B cells, and the like. Detection and purification facilitating domains include, e.g., metal chelating peptides such as polyhistidine tracts and histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Amgen Inc., Seattle Wash.)- The inclusion of a cleavable linker sequences such as Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between a purification domain and the motif-comprising peptide or polypeptide to facilitate purification. For example, an expression vector can include an epitope-encoding nucleic acid sequence linked to six histidine residues followed by a thioredoxin and an enterokinase cleavage site (see e.g., Williams, Biochemistry 34: 1787-1797, 1995; Dobeli, Protein Expr. Purif. 12: 404-14, 1998). The histidine residues facilitate detection and purification while the enterokinase cleavage site provides a means for purifying the epitope from the remainder of the fusion protein. Technology pertaining to vectors encoding fusion proteins and application of fusion proteins are well described in the scientific and patent literature, see e.g., Kroll, DNA Cell. Biol., 12: 441-53, 1993.

[0222] "Polypeptide" and "protein" as used herein, refer to amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and can contain modified amino acids other than the 20 gene-encoded amino acids. The term "polypeptide" also includes peptides and polypeptide fragments, motifs and the like. The term also includes glycosylated polypeptides. The peptides and polypeptides of embodiments of the invention also include all "mimetic" and "peptidomimetic" forms, as described in further detail, below.

[0223] "Isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment. As used herein, an isolated material or composition can also be a "purified" composition, i.e., it does not require absolute purity; rather, it is intended as a relative definition. Individual nucleic acids obtained from a library can be conventionally purified to electrophoretic homogeneity. In alternative aspects, embodiments of the invention provide nucleic acids which have been purified from genomic DNA or from other sequences in a library or other environment by at least one, two, three, four, five or more orders of magnitude. THERAPEUTIC APPLICATIONS

[0224] The small chemical molecule, siRNA molecule, dominant-negative mutants, or antibody inhibitors of TEC tyrosine kinase identified by the methods of the present invention can be used in a variety of methods of treatment. Thus, the present invention provides compositions and methods for treating an infectious disease, e.g., a viral disease caused by a lentivirus, human immunodeficiency virus, human T cell leukemia virus I AND II (HTLV-I and II), feline immunodeficiency virus, maedi-visna, herpesvirus, cytomegalovirus, Kaposi's sarcoma herpesvirus, poxvirus, or myxoma virus.

[0225] Additional protein kinase associated disorders include viral diseases including, but not limited to, herpes simplex virus type 1 , herpes simplex virus type 2, varicella-zoster virus (Herpes zoster), cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency virus 1, including HIV-I meningoencephalitis (subacute encephalitis), vacuolar myelopathy, AIDS- associated myopathy, peripheral neuropathy, and AIDS in children.

[0226] Exemplary infectious disease, include but are not limited to, viral, bacterial, fungal, or parasitic diseases. The TEC tyrosine kinase inhibitors of the present invention can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases can be treated. The immune response can be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, the polypeptide or polynucleotide of the present invention can also directly inhibit the infectious agent, without necessarily eliciting an immune response.

[0227] Viruses are one example of an infectious agent that can cause disease or symptoms that can be treated or detected by TEC tyrosine kinase inhibitors, e.g. , a small chemical compound, short interfering RNA, short hairpin RNA ribozyme, antisense oligonucleotide, antibody, peptide or peptide mimetic of the present invention. Examples of viruses, include, but are not limited to the following DNA and RNA viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbilli virus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza), Papovaviridae, Parvoviridae, Picoraaviridae, Poxyiridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiolitis, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia. A polypeptide or polynucleotide of the present invention can be used to treat or detect any of these symptoms or diseases.

[0228] Similarly, bacterial or fungal agents that can cause disease or symptoms and that can be treated or detected by a polynucleotide or polypeptide of the present invention include, but not limited to, the following Gram-Negative and Gram-positive bacterial families and fingi: Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia), Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia, Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, Enterobacteriaceae (Klebsiella, Salmonella, Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal), Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus, Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, and Staphylococcal. These bacterial or fungal families can cause the following diseases or symptoms, including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis, Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections, wound infections. A polypeptide or polynucleotide of the present invention can be used to treat or detect any of these symptoms or diseases.

[0229] Moreover, parasitic agents causing disease or symptoms that can be treated or detected by a polynucleotide or polypeptide of the present invention include, but not limited to, the following families: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas. These parasites can cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), Malaria, pregnancy complications, and toxoplasmosis. A polypeptide or polynucleotide of the present invention can be used to treat or detect any of these symptoms or diseases.

[0230] Preferably, treatment using TEC tyrosine kinase inhibitors, e.g., a small chemical molecule inhibitor, a polypeptide inhibitor, or a peptidomimetic inhibitor of viral, bacterial, or parasitite replication of the present invention could either be by administering an effective amount of the small chemical molecule inhibitor, the polypeptide inhibitor, or the peptidomimetic inhibitor to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to the patient (ex vivo therapy). Moreover, the polypeptide or peptidomimetic of the present invention can be used as an antigen in a vaccine to raise an immune response against viral disease.

PHARMACEUTICAL COMPOSITIONS

[0231] Small molecule chemical inhibitors, siRNA inhibitors, or dominant negative mutants of TEC tyrosine kinase activity, e.g., TEC, BTK, ITK, RLK, or BMX tyrosine kinase activity, ligand mimetics, derivatives and analogs thereof, antibodies, or nucleic acid compositions, e.g., antisense oligonucleotides or double stranded RNA oligonucleotides (RNAi),useful in the present compositions and methods can be administered to a human patient per se, in the form of a stereoisomer, prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate, N-oxide or isomorphic crystalline form thereof, or in the form of a pharmaceutical composition where the compound is mixed with suitable carriers or excipient(s) in a therapeutically effective amount, for example, cancer or metastatic cancer.

[0232] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions for administering the antibody compositions (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA 18^th ed., 1990, incorporated herein by reference). The pharmaceutical compositions generally comprise a differentially expressed protein, agonist or antagonist in a form suitable for administration to a patient. The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

LABELS

[0233] The particular label or detectable group used in the assay is not a critical aspect of the invention, so long as it does not significantly interfere with the specific binding of the small molecule chemical inhibitors or siRNA inhibitors of TEC tyrosine kinase activity, e.g., TEC, BTK, ITK, RLK, or BMX tyrosine kinase activity, ligand mimetics, derivatives and analogs thereof, antibodies, or nucleic acid compositions, e.g., antisense oligonucleotides or double stranded RNA oligonucleotides (RNAi), used in the assay. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well-developed in the field of immunoassays and, in general, most any label useful in such methods can be applied to the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include magnetic beads {e.g. Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., ³H,¹⁴C, ³⁵S, ¹²⁵I, ¹²¹I, ^{1 12}In, ⁹⁹HiTc), other imaging agents such as microbubbles (for ultrasound imaging), ¹⁸F, ¹¹C, ¹⁵O, (for Positron emission tomography), ^99mTC, ¹¹¹In (for Single photon emission tomography), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, and the like) beads. Patents that described the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, each incorporated herein by reference in their entirety and for all purposes. See also Handbook of Fluorescent Probes and Research Chemicals (6^th Ed., Molecular Probes, Inc., Eugene OR.).

[0234] The label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.

[0235] Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to an anti- ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. A number of ligands and anti-ligands can be used. Where a ligand has a natural anti- ligand, for example, biotin, thyroxine, and Cortisol, it can be used in conjunction with the labeled, naturally occurring anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody.

[0236] The molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, and the like Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol. For a review of various labeling or signal producing systems which may be used, see, U.S. Pat. No. 4,391,904, incorporated herein by reference in its entirety and for all purposes.

[0237] Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Finally simple calorimetric labels may be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.

[0238] Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies. In this case, antigen-coated particles are agglutinated by samples comprising the target antibodies. In this format, none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.

[0239] Frequently, the TEC tyrosine kinase polypeptide, e.g., TEC, BTK, ITK, RLK, or BMX polypeptides will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. v.

TREATMENT REGIMES

[0240] Embodiments of the invention provide pharmaceutical compositions comprising one or a combination of small molecule chemical inhibitors, siRNA inhibitors, or dominant- negative mutants of TEC tyrosine kinase activity, e.g., TEC, BTK, ITK, RLK, or BMX tyrosine kinase activity .(monoclonal, polyclonal or single chain Fv; intact or binding fragments thereof) or nucleic acid compositions, e.g., antisense oligonucleotides, double stranded RNA oligonucleotides (RNAi) or DNA oligonucleotides (vectors) containing nucleotide sequences encoding for the transcription of shRNA molecules, formulated together with a pharmaceutically acceptable carrier. Some compositions include a combination of multiple (e.g., two or more) small chemical molecules, siRNA molecules, monoclonal antibodies or antigen-binding portions thereof. In some compositions, each of the antibodies or antigen-binding portions thereof of the composition is a monoclonal antibody or a human sequence antibody that binds to a distinct, preselected epitope of an antigen.

[0241] In prophylactic applications, pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of a disease or condition (i.e., an immune disease) in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the outset of the disease, including biochemical, histologic and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. In therapeutic applications, compositions or medicants are administered to a patient suspected of, or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease (biochemical, histologic and/or behavioral), including its complications and intermediate pathological phenotypes in development of the disease. An amount adequate to accomplish therapeutic or prophylactic treatment is defined as a therapeutically- or prophylactically-effective dose. In both prophylactic and therapeutic regimes, agents are usually administered in several dosages until a sufficient immune response has been achieved. Typically, the immune response is monitored and repeated dosages are given if the immune response starts to wane.

EFFECTIVE DOSAGES

[0242] Effective doses of the small molecule chemical inhibitors, siRNA inhibitors, or dominant-negative mutants of TEC tyrosine kinase activity, e.g., TEC, BTK, ITK, RLK, or BMX tyrosine kinase activity, or nucleic acid compositions, e.g., antisense oligonucleotides, double stranded RNA oligonucleotides (RNAi), or DNA oligonucleotides (vectors) containing nucleotide sequences encoding for the transcription of shRNA molecules, for the treatment of viral disease described herein vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human but nonhuman mammals including transgenic mammals can also be treated. Treatment dosages need to be titrated to optimize safety and efficacy.

[0243] For administration with a small chemical molecule, nucleic acid, siRNA, or antibody composition, the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg. An exemplary treatment regime entails administration once per every two weeks or once a month or once every 3 to 6 months. In some methods, two or more small chemical molecules or siRNA molecules with different binding specificities are administered simultaneously, in which case the dosage of each small chemical molecule, siRNA molecule, or antibody administered falls within the ranges indicated. Small chemical molecule, siRNA molecule, or antibody is usually administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of small chemical molecule, siRNA molecule, or antibody in the patient. In some methods, dosage is adjusted to achieve an antibody concentration of 1-1000 μg/ml and in some methods 25-300 μg/ml. Alternatively, antibody can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the compound in the patient. In general, human antibodies show the longest half life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patent can be administered a prophylactic regime.

[0244] Doses for small chemical molecules, siRNA molecules, or nucleic acids range from about 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per patient. Doses for infectious viral vectors vary from 10-100, or more, virions per dose.

PRODRUGS

[0245] The present invention is also related to prodrugs of the agents obtained by the methods disclosed herein. Prodrugs are agents which are converted in vivo to active forms (see, e.g., R.B. Silverman, 1992, The Organic Chemistry of Drug Design and Drug Action, Academic Press, Chp. 8). Prodrugs can be used to alter the biodistribution {e.g., to allow agents which would not typically enter the reactive site of the protease) or the pharmacokinetics for a particular agent. For example, a carboxylic acid group, can be esterified, e.g., with a methyl group or an ethyl group to yield an ester. When the ester is administered to a subject, the ester is cleaved, enzymatically or non-enzymatically, reductively, oxidatively, or hydro] ytically, to reveal the anionic group. An anionic group can be esterified with moieties {e.g., acyloxymethyl esters) which are cleaved to reveal an intermediate agent which subsequently decomposes to yield the active agent. The prodrug moieties may be metabolized in vivo by esterases or by other mechanisms to carboxylic acids. [0246] Examples of prodrugs and their uses are well known in the art (see, e.g., Berge et al., "Pharmaceutical Salts", J. Pharm. ScL 66: 1-19, 1977). The prodrugs can be prepared in situ during the final isolation and purification of the agents, or by separately reacting the purified agent in its free acid form with a suitable derivatizing agent. Carboxylic acids can be converted into esters via treatment with an alcohol in the presence of a catalyst.

[0247] Examples of cleavable carboxylic acid prodrug moieties include substituted and unsubstituted, branched or unbranched lower alkyl ester moieties, (e.g., ethyl esters, propyl esters, butyl esters, pentyl esters, cyclopentyl esters, hexyl esters, cyclohexyl esters), lower alkenyl esters, dilower alkyl-amino lower-alkyl esters (e.g., dimethylaminoethyl ester), acylamino lower alkyl esters, acyloxy lower alkyl esters (e.g. , pivaloyloxymethyl ester), aryl esters (phenyl ester), aryl -lower alkyl esters (e.g., benzyl ester), substituted (e.g., with methyl, halo, or methoxy substituents) aryl and aryl-lower alkyl esters, amides, lower-alkyl amides, dilower alkyl amides, and hydroxy amides.

ROUTES OF ADMINISTRATION

[0248] Small chemical molecule, siRNA molecule, or antibody compositions for treatment or amelioration of viral disease, or nucleic acid compositions, e.g., antisense oligonucleotides, double stranded RNA oligonucleotides (RNAi), or DNA oligonucleotides (vectors) containing nucleotide sequences encoding for the transcription of shRNA molecules, for the treatment of viral disease, e.g., HIV or herpesvirus infection, can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or intramuscular means for prophylactic as inhalants for small chemical molecule, siRNA molecule or antibody preparations targeting viral disease, and/or therapeutic treatment. The most typical route of administration of an immunogenic agent is subcutaneous although other routes can be equally effective. The next most common route is intramuscular injection. This type of injection is most typically performed in the arm or leg muscles. In some methods, agents are injected directly into a particular tissue where a tumor is found, for example intracranial injection or convection enhanced delivery. Intramuscular injection or intravenous infusion are preferred for administration of antibody. In some methods, particular therapeutic antibodies are delivered directly into the cranium. In some methods, antibodies are administered as a sustained release composition or device, such as a Medipad™ device.

[0249] Agents of the invention can optionally be administered in combination with other agents that are at least partly effective in treating various diseases including various immune-related diseases. In the case of infection in the brain, agents of the invention can also be administered in conjunction with other agents that increase passage of the agents of the invention across the blood-brain barrier (BBB).

FORMULATION

[0250] Small chemical molecule, siRNA molecule, or antibody inhibitors of TEC tyrosine kinase, nucleic acid compositions, e.g., antisense oligonucleotides, double stranded RNA oligonucleotides (RNAi), or DNA oligonucleotides (vectors) containing nucleotide sequences encoding for the transcription of shRN A molecules, for the treatment of viral disease, are often administered as pharmaceutical compositions comprising an active therapeutic agent, i.e., and a variety of other pharmaceutically acceptable components. See Remington's Pharmaceutical Science (15^th ed., Mack Publishing Company, Easton, Pa., 1980). The preferred form depends on the intended mode of administration and therapeutic application. The compositions can also include, depending on the formulation desired, pharmaceutically- acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

[0251] Pharmaceutical compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized Sepharose™, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Additionally, these carriers can function as immunostimulating agents (i.e., adjuvants).

[0252] For parenteral administration, compositions of embodiments of the invention can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier that can be a sterile liquid such as water oils, saline, glycerol, or ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions. Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Antibodies can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained release of the active ingredient. An exemplary composition comprises monoclonal antibody at 5 mg/mL, formulated in aqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted to pH 6.0 with HCl.

[0253] Typically, compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. The agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.

[0254] Additional formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, and transdermal applications.

[0255] For suppositories, binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%. Oral formulations include excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%- 95% of active ingredient, preferably 25%-70%.

[0256] Topical application can result in transdermal or intradermal delivery. Topical administration can be facilitated by co-administration of the agent with cholera toxin or detoxified derivatives or subunits thereof or other similar bacterial toxins. Glenn et al., Nature 391: 851, 1998. Co-administration can be achieved by using the components as a mixture or as linked molecules obtained by chemical crosslinking or expression as a fusion protein.

[0257] Alternatively, transdermal delivery can be achieved using a skin patch or using transferosomes. Paul et al., Eur. J. Immunol. 25: 3521-24, 1995; Cevc et al., Biochem. Biophys. Acta 1368: 201-15, 1998.

[0258] The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. TOXICITY

[0259] Preferably, a therapeutically effective dose of the small chemical molecule, siRNA molecule, antibody, or nucleic acid compositions, e.g., antisense oligonucleotides, double stranded RNA oligonucleotides (RNAi), or DNA oligonucleotides (vectors) containing nucleotide sequences encoding for the transcription of shRNA molecules, described herein will provide therapeutic benefit without causing substantial toxicity.

[0260] Toxicity of the proteins described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD₅0 (the dose lethal to 50% of the population) or the LD 1₀₀ (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human. The dosage of the proteins described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. 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., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1,

KITS

[0261] Also within the scope of the invention are kits comprising the small chemical molecule, siRNA molecule, antibody, or nucleic acid compositions, e.g., antisense oligonucleotides, double stranded RNA oligonucleotides (RNAi), or DNA oligonucleotides (vectors) containing nucleotide sequences encoding for the transcription of shRNA molecules) of embodiments of the invention and instructions for use. The kit can further contain a least one additional reagent, or one or more additional human antibodies of embodiments of the invention (e.g., a human antibody having a complementary activity which binds to an epitope in the antigen distinct from the first human antibody). Kits typically include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.

[0262] The invention will be further described with reference to the following examples; however, it is to be understood that the invention is not limited to such examples. EXEMPLARY EMBODIMENTS

EXAMPLE 1

Loss of ITK by siRNA does not Alter CD4, CXCR4, or CCR5 Expression

[0263] HIV infects cells using the CD4 receptor and either CCR5 or CXCR4 as coreceptors for viral infection. Mutations affecting these coreceptors or blocking these coreceptors with pharmacologic agents prevents virus entry. Schols, Curr Top Med Chem 4: 883-893, 2004. Since ITK affects chemokine receptor function, we wished to explore whether it could also affect early stages in HIV infection. To address the role of ITK in HIV infection, we first needed to ensure that ITK does not affect receptor expression. Jurkat E6.1 cells were transiently transfected with siRNA to either green fluorescent protein (siGFP) as a control, or ITK (silTK). Cell lysates were analyzed by immunoblotting to confirm loss of ITK expression. Cells were stained with antibodies against CD4 conjugated to APC, CXCR4 conjugated to PE, or CCR5 antibody with a secondary IgG conjugated to biotin and streptavidin conjugated to PE. As shown Fig. 1 , receptor expression in both siGFP and silTK treated cells was equivalent, demonstrating that a loss of ITK expression does not alter expression of cellular receptors required for HIV infection.

[0264] Figure 1 shows that loss of ITK by siRNA does not alter CD4, CXCR4 or CCR5 expression. Jurkat E6.1 cells were transiently transfected with lμg of siRNA to GFP or ITK. After 24hrs a loss of ITK expression was detected by immunoblotting (data not shown) and cells were stained with isotype controls or CD4-APC, CXCR4-PE, or CCR5 and secondary conjugated to biotin plus streptaviden PE. Cells were washed 3x and analyzed by flow cytometry on a FACs Caliber for receptor expression. The isotype control is shown in red, siGFP treated cells in blue and silTK treated cells in green. Data are from one representative experiment of three individual experiments.

EXAMPLE 2

Loss of ITK by siRNA Leads to a Decrease in HIV Infection

[0265] To address the role of ITK in infection, we first used the NL4/3 luciferase viral vector, which has luciferase inserted into the Nef gene, in addition to a stop codon in the envelop gene (Reagent was obtained through the NIH AIDS Research and Reference Reagent Program). Connor, et al. Virology 206: 935-944, 1995. These alterations limit replication of the viral vector to a single round of infection. We pseudotyped the vector with envelops that are CXCR4 dependent (HXB), CCR5 dependent (JR-FL), or receptor independent (VSV-G)(B artz, et al. Methods 12: 337-342, 1997)(Figure 2a). Viral stocks were generated in 293T cells that were transfected with plasmids encoding the NL4/3-luciferase vector, Rev (for increased production of virus), and the indicated envelop DNAs. Virus from these cells was collected at 48hrs and used to infect Jurkat cells in which expression of ITK had been knocked down by siITK or control cells treated with siGFP. Cells were infected with the virus and incubated for 24hrs prior to lysing the cells and measuring luciferase activity. In this assay, cells were unstimulated so that the luciferase activity reflected basal viral transcription. The data is presented as a ratio of luciferase produced by cells treated with siITK compared to cells treated with siGFP (Figure 2b). Consistent with their receptor-independent entry, viruses pseudotyped with VSV-G had a ratio of close to one, indicating that the early stages of viral infection, including reverse transcription, integration and basal transcription are approximately equivalent regardless of ITK expression. However, viral vectors pseudotyped with the HXB and JR-FL envelops, which do require specific coreceptors for entry, showed decreased luciferase expression. These data suggest that loss of ITK reduces virus replication, in a manner dependent on receptor mediated viral entry.

[0266] Figure 2 shows that loss of ITK by siRNA leads to a decrease in HIV infection. A) Schematic of HIV infection strategy. Viruses were produced in 293T cells by pseudotyping the NL4/3 luciferase vector with envelops specific for non-receptor mediated entry VSV-G, CCR5 coreceptor entry (JR-FL), or CXCR4 coreceptor entry (HXB2). Virus from each of these subsets was then equally divided to infect Jurkat cells treated with siRNAs. B-C) Jurkat cells were transiently transfected with siRNA to GFP or JTK by electroporation and 24 hrs later infected with virus pseudotyped with VSV-G, JR-FL, or HXB2 envelopes. B) Immunoblots of cell lysates from Jurkat cells analyzed for JTK and Lck expression to show loss of ITK expression. C) Luciferase activity was measured 24 hrs after infection and is shown as the ratio of JTK/GFP. Cells were not stimulated prior to assaying for luciferase activity. These data are one representative experiment of three individual experiments.

EXAMPLE 3

Inhibition of ITK in Human CD4+ T cells Alters Virus Entry but not Binding

[0267] To address the role of ITK in the early stages of viral replication, we first examined the entry of HIV virus using primary human CD4+ T cells. Cells were isolated from buffy coats using a ficoll gradient and then further purifed by negative selection for CD4⁺ T cells. Since productive infection of these cells requires prior activation, the primary human CD4+ T cells were stimulated with 10 ng/ml PMA, 2 μg/ml PHA, and 20U/ml JJL-2 for two days to activate the cells. Stevenson, et al. Embo J 9: 1551-1560, 1990. Cells were resuspended at 200,000 cells/ 200ul and plated into 96 well plates. The cells were incubated with DMSO as a vehicle control, lμM JM2987, (a known inhibitor of CXCR4 virus binding that was obtained through the NIH AIDS Research and Reference Reagent Program) (Bridger, et al. J Med Chem 42: 3971-3981, 1999), or 10μM BMS509744, (ETK inhibitor; Lin, et al. Biochemistry 43: 11056-11062, 2004) for 30min prior to infection. Cells were then infected with the replication competent NL4/3 virus, which encodes the entire viral genome including Nef, and a CXCR4- binding envelop. For examination of virus binding, cells were incubated for 3 hrs and washed extensively with PBS prior to lysing the cells and looking at p24 viral protein levels by ELISA. For examination of viral entry, cells were incubated for 5 hrs with virus and then trypsinized to remove bound virus that had not entered the cells. Cells were then washed extensively before lysing to access p24 viral protein assayed by ELISA.

[0268] As expected, JM2987 blocked both binding and entry of the virus (Figure 3). BMS509744, however, did not block the binding of the virus to CXCR4 but did block the entry of the virus into the host cell. Similar data was obtained at time points up to 12 hrs postexposure to virus, arguing that the reduction seen in virus entry by BMS509744 was not merely a delay in entry (data not shown). Consistent with a role for ITK in early stages of HIV infection, we observed a decrease in the ability of the HIV LAV GP 120 envelop protein to induce actin polarization in Jurkat cells treated with siRNA against UK (Figure 4). Nonetheless, we did not see evidence for additional defects in reverse transcription nor viral integration when using VSV- G pseudotyped virus for non-receptor mediated entry (Figure 5).

[0269] Figure 3 shows that ITK inhibition in human CD4⁺ T cells alters virus entry but not binding. Primary human CD4+ T cells were isolate from Buffy Coats by ficoll centrifugation and further negatively selected for CD4+ T cells using magnetic beads. Primary CD4+ T cells were stimulated with PMA (10 ng/ml), PHA (2 μg/ml), and IL-2 (20U/ml) 2 days prior to assay. Primary human CD4+ T cells were incubated with DMSO (vehicle control), (lμM) JM2987 (CXCR4 virus binding inhibitor), or (lOμM) BMS509744 (ITK inhibitor) for 30min prior to addition of the replication competent NL4/3, CXCR4 coreceptor dependent virus. Binding was measured 3 hrs post addition of virus by washing 10 times in PBS then lysing the cells to measure binding by p24 ELISA. Entry was measured at 5 hrs post addition of virus by trypsinizing cells and then washing an additional 5 times in PBS before lysing and measuring entry by p24 ELISA. Data are from one representative experiment of three individual experiments from three separate donors.

[0270] Figure 4 shows that loss of ITK by siRNA reduces Actin polarization to SDFlalpha and GP120 LAV. Jurkat cells were transiently trarisfected 1 μg of siRNA to GFP or ITK for 24hrs. Cells were conjugated to beads coated with Fibronectin or Fibronectin and SDF (20 nM) or GP 120 (200 nM) for 5 min at 37°C. Conjugates were stained for F-actin and scored for increased f-actin polarization to the bead:cell interface. Representative pictures are shown for fibronectin and gpl20. A total of 50 conjugates were analyzed per condition and data shown is the percent of cells where actin is polarized to the contact site of the bead.

[0271] Figure 5 shows that reverse transcription and integration are not affected during receptor independent infection. A) Jurkat cells were transiently transfected with lμg of siRNA to GFP or ITK. Cells were infected with NL4/3-luciferase virus pseudotyped with VSV-G envelope (to bypass defects in viral entry) and HIRT (unintegrated) DNA isolated. DNA was extracted 4 hours post infection. Jurkat cells were analyzed by PCR for the 5R and U5 LTR reverse transcription products. DNA concentration in samples was controlled by PCR to mitochondrial DNA. Fassati, et al. J Virol 75: 3626-3635, 2001. B) Jurkat cells were transiently transfected with 1 μg siRNA to GFP or ITK and incubated 24 hours prior to infection with NL4/3 luciferase virus. Genomic DNA was prepped 48 hours post infection and analyzed for integration using a nested PCR strategy using primers against ALU sequences and the HIV LTR and a second round of PCR using two internal LTR primers. Chun, et al. Proc Natl Acad Sci U S A 94: 13193-13197, 1997. To control for DNA concentration in samples PCR to GAPDH was performed. Similar results were obtained with BMS inhibitor in primary human CD4+ T cells.

EXAMPLE 4

Loss of ITK Decreases HIV Transcription

[0272] NFAT and AP-I are transcription factors that are positive regulators of HIV transcription. Li, et al. J Biol Chem 269: 30616-30619, 1994; Pessler, et al. Genes Immun 5: 158-167, 2004; Schaeffer, et al. Nat Immunol 2: 1183-1188, 2001. ITK-deficient T cells show defects in the TcR-induced activation of these transcription factors. Schaeffer, et al. Nat Immunol 2: 1183-1188, 2001; Schwartzberg, et al. Nat Rev Immunol 5: 284-295, 2005. To address whether ITK affects transcriptional regulation of the HIV virus, we transiently transfected Jurkat cells with siITK or siGFP and the NL4/3 luciferase reporter, which bypasses the early stages of infection. Twenty-four hours later, when ITK expression had been reduced, the cells were stimulated with plate bound anti-CD3 or anti-CD3 plus anti-CD28. After an additional 24 hrs, the cells were lysed and luciferase production was measured to examine HIV transcription.

[0273] Consistent with our results with VSV-G pseudotyped viruses, basal levels of luciferase in the unstimulated cells were approximately equivalent in the siGFP and siITK treated cells (Figure 6). Cells treated with siRNA against GFP and stimulated with either anti- CD3 or anti-CD3 plus CD28 showed a marked enhancement of luciferase activity (10-20 fold). However, silTK-treated cells stimulated with either CD3 or CD3 plus CD28 exhibited reduced luciferase activity compared to cells treated with siGFP. Similarly, cells overexpressing a kinase-inactive mutant of ITK show decreased transcription of HIV. Conversely, overexpression of ITK increased luciferase activity 5-6 fold in Jurkat cells transfected with the HIV-lucif erase construct (Figure 7). Similarly, a Jurkat cell line with a constituatively active ITK results in activated HIV transcription (data not shown). These data argue that ITK is a positive regulator of HIV transcription downstream of TcR activation.

[0274] Figure 6 shows that loss of UK decreases HTV transcription. A) Schematic of HIV Long Terminal Repeat transcriptional regulation. B-C) Jurkat cells were transiently transfected with lμg of siRNA to ITK or GFP along with HIV-luciferase reporter. Cells were incubated for 24hrs and then left unstimulated or stimulated with plate bound purified anti- human CD3 (5μg/ml) and anti-human CD28 (lOμg/ml). B) Loss of ITK expression was analyzed at 24hrs post transfection by immunoblot for JTK and Lck. C) Luciferase activity was measured 24hrs after stimulation. Data shown is one representative experiment of three individual experiments with triplicate samples.

[0275] Figure 7 shows that ITK is a positive regulator of HTV transcription in Jurkat cells. Jurkat cells were transient transfected with NL4/3-luc and GFP vector or GFP tagged wild type ITK. Cells were incubated for 24hrs and then assayed for luciferase activity.

EXAMPLE 5

Decrease in Intracellular P24 with Loss of ITK

[0276] Our initial results suggest that loss of ITK affects both HIV viral entry and viral transcription. To further address the requirement for ITK in productive HIV infection, we looked at viral infection using either Jurkat E6 cells in which ITK expression has been transiently reduced by siRNA or human PBT cells treated with the ITK inhibitor. Jurkat cells treated with siITK were infected with HXB2 virus (CXCR4 dependent) and incubated for 48 hrs prior to analyzing the cells for p24 production. Cells were stained for intracellular p24 and analyzed by flow cytometry. Uninfected cells were also stained for p24 as a control.

[0277] At 48 hrs post infection cells transfected with siRNA against ITK showed a clear decrease in intracellular p24 compared to control (siGFP) treated cells (Figure 8). Furthermore, reduction of ITK expression with siRNA 24 hours post-primary infection also led to decreased intracellular p24 levels (Figure 9). These data argue that decreasing JTK expression impairs HIV infection. These effects did not reflect differences in cell viability due to knockdown or inhibition of JTK. However, due to the transient nature of the siRNA used, these data were limited to analyses up to a 48 hour timepoint. Dombroski, et al. J Immunol 114: 1385-

1392, 2005. [0278] Figure 8 shows that decrease in intracellular P24 with loss of ITK. Jurkat cells were transiently transfected with 1 μg of siRNA to GFP or ITK. Cell lysates were taken after 24 hrs and analyzed for ITK and Lck expression by immunoblot. Cells were infected 24hrs post transfection with HXB2 replication competent, CXCR4 dependent virus. Cells were analyzed 48 hrs post infection and analyzed for infection by staining for intracellular p24 and analyzed by flow cytometry. ITK expression levels are knocked down for up to 72 hrs post transfection only which correlates to 48 hrs post infection. Data is one representative experiment of three individual experiments.

[0279] Figure 9 shows that loss of ITK inhibits HIV replication. Jurkat cells were infected with VSVG pseudotyped HXB plap nef + virus (reagent was obtained from NIH AIDS Research and Reference Reagents Program) for 24 hours. Cells were then transiently transfected with siRNA specific for ITK or control RNA. P24 and whole cell lysate samples were taken at several time points post siRNA transfection. Samples were then assayed for p24 values by ELISA. Expression of ITK protein was monitored overtime by assaying whole cell lysates for ITK protein by immunoblot.

[0280] To understand the role of TTK over multiple rounds of infection primary human CD4+ T cells were incubated with DMSO as a vehicle control, lμM JM2987, or lOμM BMS509744 for 30min prior to infection. Cells were infected and analyzed for intracellular p24 over a period of up to 8 days. Inhibitors were added every two days to maintain inhibition. Uninfected cells were also stained as negative controls. As shown in Fig. 10, inhibition of TTK led to a clear block in viral replication that was maintained up to 8 days post infection. Thus, either inhibition of ITK expression via siRNA, or ITK function via a chemical inhibitor, blocks HIV infection in T cells.

[0281] Figure 10 shows that inhibition of ITK in human CD4⁺ T cells decreases HIV replication. Primary human CD4+ T cells were isolate from Buffy Coats by ficoll centrifugation and further negatively selected for CD4+ T cells using magnetic beads. Primary CD4+ T cells were stimulated with PMA (10 ng/ml), PHA (2 μg/ml), and IL-2 (20U/ml) 2 days prior to assay. Primary human CD4+ T cells were incubated with DMSO (vehicle control), (lμM) JM2987 (CXCR4 virus binding inhibitor), or (lOμM) BMS509744 (ITK inhibitor) for 30min prior to addition of the replication competent NL4/3, CXCR4 coreceptor dependent virus. Cells were incubated for the indicated number of days and virus infection was measured by staining for intracellular p24 and analyzed by flow cytometry. Data is one representative experiment of three individual experiments with three separate donors. EXAMPLE 6

ITK is a Positive Regulator of HIV Infection and Provides a Target for HIV Therapy

[0282] The data indicate that either a loss of expression or inhibition of ITK function blocks HIV virus replication. Disruption of infection occurred at two important steps of the viral life cycle: virus entry into the cell and transcriptional control of the virus during cellular activation. These data suggest that ITK is a positive regulator of HIV infection and may provide a target for HIV therapy.

[0283] Treatment for HIV has primarily relied on the use of anti-retroviral agents that have been subject to the rapid development of resistant viruses. Barbaro, et al. Curr Pharm Des 11: 1805-1843, 2005. Although use of multi-drug regimens has greatly improved this problem, the use of inhibitors directed against cellular proteins required for HIV replication has been considered a potentially attractive alternative therapeutic approach. It is therefore intriguing that ITK affects HIV infection at two stages that are not targeted by current HIV therapies. Notably, the early block on entry was dependent on the presence of HIV envelop and could be circumvented using VSV-G pseudotyped virus, which does not rely on CD4 and chemokine receptors for entry. As expected, because of the ability of ITK to decrease HIV entry, corresponding decreases in reverse transcription products and integration of provirus were observed when HXB2 or JR-FL envelopes were used to package the virus. However, there were no differences observed in reverse transcription products and provirus DNA with the VSV-G pseudotyped HIV, consistent with the conclusion that ITK is influencing early entry events and not other stages that precede the establishment of the provirus (Figure 5).

[0284] Furthermore, ITK activity was required for efficient HIV transcription. Our transcription studies using si RNA, as well as overexpression of WT and mutant versions of ITK (Figure 7), argue that ITK influences late stages of viral production, demonstrating a second stage of HIV infection affected by ITK. We propose that the profound effect of TTK on HIV replication is due to this ability to target both HIV entry and transcription.

[0285] Together, these data suggest that ITK is a positive regulator of HIV infection that may provide a target for HIV therapy, particularly in combination with therapies that affect other stages of the viral life cycle, such as reverse transcription or virion maturation. Moreover, data arguing that other TEC kinases, such as TEC and BTK, affect chemokine responses and activation in other cells suggest that therapies directed against the TEC kinases may also be useful for blocking HIV infection in other cell-types such as macrophages and monocytes. Schmidt, et al. lntArch Allergy Immunol 134: 65-78, 2004. EXAMPLE 7

Effect of siRNA to ITK in Primary Human CD4⁺ T Cells

[0286] Studies were performed which utilize siRNA to ITK in primary human CD4⁺ T cells. Figure 11 shows loss of ITK reduces HIV replication. Primary human CD4 T cells were isolated using magnetic separation for negatively selected cells. Cells were stimulated with PMA lOng/ml, PHA 2ug/ml, and IL-2 20U/ml for 4 days. Cells were transfected with siRNA to ITK or GFP using amaxa transfection system (amaxa Inc., Gaithersburg, MD). Cells were infected 24hrs post transfection and intracellular p24 was measured at 48hrs post infection. It was further confirmed that loss of ITK expression does not affect HIV receptor (CD4) and coreceptor (CXCR4) expression in primary human CD4⁺ T cells.

[0287] Studies characterized data with siRNA and the BMS509744 compound to address the issue of cell death as a result of a loss of ITK activity and whether this contributes to the loss of HIV infection. Figure 12 shows cell death is unaffected by loss of ITK. Jurkat or primary human T cells were transfected with siRNA to ITK or GFP. Cells were cultured for 72 hrs and stained for 7 AAD for necrosis or Annexin V (AV) for apoptosis. Negative controls are unstained cells. For infection samples cells were infected 24hrs post siRNA transfection. There was no difference in cell death between cells treated with siRNA to ITK or GFP.

[0288] Figure 13 shows cell death after BMS509744 treatment does not account for changes in HIV infection. Primary human CD4 T cells were isolated using magnetic separation for negatively selected cells. Cells were stimulated with PMA 10ng/ml, PHA 2ug/ml, and IL-I 20U/ml for 2 days. Cells were treated with CXCR4 entry blocker (JM) IuM, ITK inhibitor BMS509744 (BMS) 10μM, DMSO equivalent, or left untreated. Cells were stained for necrosis by 7AAD at 2 and 3 days post BMS509744 addition. Although cells treated with BMS509744 show increased cell death, cells infected with HIV give a similar pattern of 7AAD expression. The increased cell death with BMS509744 does not account for the difference in infection.

[0289] An increase in virus-like particle formation in the presence of ITK provides evidence that ITK may affect another stage of HIV replication. Figure 14 shows expression of ITK increases the release of Gag- Virus like particles. 293T human embryonic kidney cell line was cotransfected by calcium phosphate with ITK or Vector along with HIV-I cmv-Gag construct. Mock samples are untransfected cells. Supernatents were collected at 48 and 72hrs post transfection and analyzed for p24 protein by ELISA. EXAMPLE 8 Methods

[0290] Cell lines and reagents. Jurkat E6.1 cells were obtained from ATCC. Primary human CD4+ T cells were obtained from Buffy coats by histopaque/ficol 1077 (Sigma) gradient centrifugation. CD4+ cells were further isolated by negative selection using CD4+ T cell isolation kit II (Miltenyi). Primary human CD4+ T cells were expanded and activated by addition of 10 ng/ml PMA (Sigma), 2 μg/ml PHA (Sigma), and 20U/ml JJL-2 (NCI). Cells were maintained in RPMI containing 10% Fetal Calf Serum (GEM CELL), 2mM L-glutamine (Gibco), 1OmM HEPES, 50 U/ml penicillin (Gibco), and 50 μg/ml streptomycin (Gibco). Purified anti-human CD3 and CD28, anti^CD4 APC, anti-CXCR4 PE, anti-CCR5, anti- IgG biotin, and streptavidin PE were obtained from BD Pharmingen. BMS509744 (Bristol-Meyers Squibb, Inc.; U.S. Patent Application No. 2004/0077695) was synthesized by the Chemical Biology Core Facility, National Institute of Diabetes and Digestive and Kidney Diseases.

[0291] Reduction of ITK expression by siRNA. Jurkat cells were transiently transfected by electroporation at 215 volts, 65msec, single pulse (BTX 8300 electroporator) with 1 μg of siRNA directed against GFP or ITK for 24hrs. Expression was analyzed by immunoblotting using anti-ITK antibodies (2F12, cell signaling). ITK siRNA sequences: ITK-2 sense, 5¹- GGAGCCUUCAUGGUAAGGGAUU-3'; and ITK-2 antisense, 5'-

UCCCUUACCAUGAAGGCUCCUU-3' were purchased from^'Qiagen and used as described previously (Dombromski et ah, JI 2005). Sequences for siGFP (Caplen et al) were also purchased from Qiagen.

. [0292] Intracelluar p24. Jurkat cells or primary human CD4+ cells were fixed using BD cytofix/cytoperm kit (BD Biosciences). Breifly cells were washed with PBS and then fixed in lOOul cytofix reagent. Cells were incubated on ice for 20min and then washed with permawash solution. Cells were then incubated with anti-p24 antibody (KC57, Beckman Coulter) 1 :400 for 20min on ice and again washed with permawash solution. Cells were resuspended in PBS containing 1% FCS, data collected using a FACscalibur flow cytometer, and analyzed by Flow Jo (Treestar).

[0293] Generation of HlV-I infectious titers and infections. JRFL, HXB, and VSV-G pseudotyped HIV-I was generated by transfecting 293T cells with 15 μg of either T-tropic pNL4-3-Luc+ Env- Nef- (HJV-luc) DNA (obtained from National Institutes of Health AIDS Research and Reference Reagent Program), 3 μg Rev in a Rous sarcoma virus expression construct DNA, and 3 μg LTR vesicular stomatitis virus glycoprotein DNA, HXB envelop DNA, or JR-FL envelop DNA with Fugene 6 (Roche). Production of replication competent viruses NL4/3 and HXB2 were also made by transfection of DNA into 293T cells using the Fugene 6 reagent. Supernatants were collected and filtered through a 0.45-μm disc before infection. Jurkat cell lines and human CD4+ T cells were infected by culturing cells in the presence of virus stock for 5hrs before replacing with fresh media.

[0294] Each recited range includes all combinations and sub-combinations of ranges, as well as specific numerals contained therein.

[0295] All publications and patent applications cited in this specification are herein incorporated by reference in their entirety for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference for all purposes.

[0296] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

What is Claimed:

1. A method for preventing or treating viral disease in a mammalian subject comprising administering to the mammalian subject a TEC tyrosine kinase inhibitor, wherein the inhibitor is administered in an amount effective to reduce or eliminate the viral disease or to prevent its occurrence or recurrence.

2. The method of claim 1 wherein the TEC tyrosine kinase is TEC, BTK, ITK, RLK, or BMX.

3. The method of claim 1 wherein the inhibitor is a small chemical compound, short interfering RNA, dominant-negative molecule, short hairpin RNA, ribozyme, antisense oligonucleotide, antibody, peptide or peptidomimetic.

4. The method of claim 3 wherein the dominant-negative molecule is a dominant-negative peptide or peptidomimetic.

5. The method of claim 3, wherein the small chemical compound is an ITK tyrosine kinase inhibitor.

6. The method of claim 3, wherein the small chemical compound is BMS509744.

7. The method of claim 1 wherein the viral disease is a disease caused by lentivirus, herpes virus, or pox virus.

8. The method of claim 1 wherein the viral disease is a disease caused by human immunodeficiency virus, human T cell leukemia virus type I, human T cell leukemia virus type π, human herpesvirus, human herpesvirus 6, human herpesvirus 7, human cytomegalovirus, Kaposi's sarcoma herpesvirus, feline immunodeficiency virus, maedivisna virus, or myxoma virus.

9. An in vitro method of screening inhibitors of TEC tyrosine kinase activity comprising, contacting a cell line with a test compound that inhibits TEC tyrosine kinase activity, and detecting an increase or a decrease in susceptibility of the cell line to viral infection, wherein effectiveness of the test compound in the assay is indicative that TEC tyrosine kinase inhibitor modulates viral infection in the cell.

10. The method of claim 9 wherein the cell line is a T cell, NK cell, mast cell, or eosinophil cell.

11. The method of claim 10 wherein the cell line is a Jurkat cell line or a human CD4⁺ cell.

12. The method of claim 9, further comprising detecting an increase or a decrease in susceptibility of the cell line to viral infection using an HIV-I vector or a pseudotyped HIV-I vector capable of signaling viral infection in the cell line.

13. The method of claim 9 wherein the TEC family tyrosine kinase inhibitor increases viral infection of the cell.

14. The method of claim 9 wherein the TEC family tyrosine kinase inhibitor decreases viral infection of the cell.

15. The method of claim 13 wherein the TEC family tyrosine kinase inhibitor increases viral entry of the cell.

16. The method of claim 14 wherein the TEC family tyrosine kinase inhibitor decreases viral entry of the cell.

17. The method of claim 13 wherein the TEC family tyrosine kinase inhibitor increases viral replication in the cell.

18. The method of claim 14 wherein the TEC family tyrosine kinase inhibitor decreases viral replication in the cell.

19. A compound identified according to the method of claim 9.

20. A method for identifying a compound capable of modulating viral infection of a cell comprising: contacting a test compound with a cell-based assay system comprising a cell expressing TEC tyrosine kinase and capable of signaling responsiveness to TEC tyrosine kinase, detecting an effect of the test compound as an inhibitor of TEC tyrosine kinase in the assay system, and • detecting an effect of the test compound to modulate susceptibility of the cell to viral infection, effectiveness of the test compound as the TEC tyrosine kinase inhibitor in the assay being indicative that the test compound modulates viral infection in the cell.

21. The method of claim 20, wherein the test compound is a small chemical molecule, interfering RNA, dominant-negative molecule, short hairpin RNA, ribozyme, antisense oligonucleotide, protein inhibitor, monoclonal antibody, polyclonal antibody, peptide, peptidomimetic, or a nucleic acid.

22. The method of claim 21 wherein the dominant-negative molecule is a dominant-negative peptide or peptidomimetic.

23. The method of claim 20 wherein the viral infection is an infection by a lentivirus, a herpes virus, or a pox virus.

24. The method of claim 20 wherein the viral infection is an infection by human immunodeficiency virus, human T cell leukemia virus type I, human T cell leukemia virus type π, human herpesvirus, human herpesvirus 6, human herpesvirus 7, human cytomegalovirus, Kaposi's sarcoma herpesvirus, feline immunodeficiency virus, maedivisna virus, or myxoma virus.

25. The method of claim 20, further comprising detecting an increase or a decrease in susceptibility of the cell line to viral infection using an HIV-I vector or a pseudotyped HIV-I vector capable of signaling viral infection in the cell-based assay system.

26. The method of claim 20 wherein the TEC family tyrosine kinase inhibitor increases viral infection of the cell.

27. The method of claim 20 wherein the TEC family tyrosine kinase inhibitor decreases viral infection of the cell.

28. The method of claim 25 wherein the TEC family tyrosine kinase inhibitor increases viral entry of the cell.

29. The method of claim 27 wherein the TEC family tyrosine kinase inhibitor decreases viral entry of the cell.

30. The method of claim 26 wherein the TEC family tyrosine kinase inhibitor increases viral replication in the cell.

31. The method of claim 27 wherein the TEC family tyrosine kinase inhibitor decreases viral replication in the cell.

32. The method of claim 20 wherein the cell line is a T cell, NK cell, mast cell, or eosinophil cell.

33. The method of claim 20 wherein the cell-based assay system is a Jurkat cell or a human CD4⁺ cell -based assay system.

34. The method of claim 20 wherein the TEC tyrosine kinase is TEC, BTK, ITK, RLK, or BMX.

35. A compound identified according to the method of claim 20.

36. A pharmaceutical composition comprising a TEC family kinase inhibitor for treatment of viral infection in a mammalian subject.

37. The composition of claim 36 wherein the viral infection is an infection by lentivirus, herpes virus, or pox virus.

38. The composition of claim 36 wherein the viral infection is an infection by human immunodeficiency virus, human T cell leukemia virus type I, human T cell leukemia virus type II, human herpesvirus, human herpesvirus 6, human herpesvirus 7, human cytomegalovirus, Kaposi's sarcoma herpesvirus, feline immunodeficiency virus, maedivisna virus, or myxoma virus.

39. The composition of claim 36 wherein the inhibitor is interfering RNA, short hairpin RNA, ribozyme, antisense oligonucleotide, or protein inhibitor.

40. The composition of claim 36 wherein the inhibitor is a monoclonal antibody, polyclonal antibody, dominant-negative molecule, peptide, peptidomimetic, or a small chemical molecule.

41. The composition of claim 40 wherein the dominant-negative molecule is a dominant- negative peptide or peptidomimetic.