The present invention relates to methods for identifying compounds that can reduce immune rejection, for example, transplant- or autoimmune disorder-related immune rejection. The present invention is based, in part, on the discovery, demonstrated herein, that immune rejection can be monitored by determining the amount of particular members of the Jak/Stat signal transduction pathway present within an affected tissue. The present invention is further based, in part, on the discovery, demonstrated herein, that immune rejection can be reduced and tolerance can be induced by modulating the amount of these particular members of the Jak/Stat signal transduction pathway present, expressed or active within an affected tissue. In particular, the results demonstrate that immune rejection can be monitored by determining the amount of mRNA or protein of Stat1, Stat3, Stat4, Stat6, SOCS1, or SOCS3 present, e.g., in an affected tissue.

Description

117 parts

›This application is a continuation of U.S. application…

This application is a continuation of U.S. application Ser. No. 09/549,654, filed Apr. 14, 2000, now abandoned, which application is incorporated by reference in its entirety.

1. INTRODUCTION

The present invention relates to methods and compositions for reducing immune rejection, for example, transplant- or autoimmune disorder-related immune rejection. The present invention also relates to methods and compositions for monitoring transplant acceptance and for monitoring an autoimmune disorder in a subject mammal. The present invention still further relates to methods for identifying compounds that can reduce immune rejection.

The present invention is based, in part, on the discovery, demonstrated herein, that immune rejection can be monitored by determining the amount of particular members of the Jak/Stat signal transduction pathway present within an affected tissue (that is, a transplant cell, tissue, organ, or organ system, or a cell, tissue, organ, or organ system that is, or is suspected of, being affected by an autoimmune disorder). The present invention is further based, in part, on the discovery, demonstrated herein, that immune rejection can be reduced and tolerance can be induced by modulating the amount of these particular members of the Jak/Stat signal transduction pathway present, expressed or active within an affected tissue. In particular, the results presented herein demonstrate that immune rejection can be monitored by determining the amount of Stat1 mRNA or protein, Stat2 mRNA or protein, Stat3 mRNA, protein Stat4 mRNA or protein, Stat6 mRNA or protein, SOCS1 mRNA or protein, or SOCS3 mRNA or protein present, e.g., present in an affected tissue.

2. BACKGROUND OF THE INVENTION

Ongoing advances in transplantation, including new immunosuppressive agents and improvements in histocompatibility matching, organ procurement, and surgical techniques, are gradually improving the outcome of clinical transplantation (Hariharan et al, 2000. N Engl J Med 342:605-12). However, chronic allograft rejection remains the prime determinant of long-term graft survival (Paul. L. C., 1999, Kidney International 56:783-793).

Tissue transplantation between genetically nonidentical individuals results in immunological rejection of the tissue through T cell-dependent mechanisms. To prevent allograft rejection, immunosuppressive agents such as calcineurin phosphatase inhibitors and glucocorticosteroids which directly or indirectly interfere with IL-2 signaling are administered to transplant recipients (see, e.g., Borel, J. F., 1989, Pharmacol. Rev. 42:260-372; Morris, P. J., 1991, Curr. Opin. Immunol. 3:748-751; Sigal et al., 1992, Ann. Rev. Immunol. 10:519-560; and L'Azou et al., 1999, Arch. Toxicol. 73:337-345). The most commonly used immunosuppressive agents today are cyclosporin A, FK506, and rapamycin. These immunosuppressive agents act indiscriminately on all T cells by impairing T cell receptor (“TCR”) signal transduction. Further, since the effect of the immunosuppressive agents is short-lasting, transplant recipients normally require life-long treatment of immunosuppressive agents to prevent transplant rejection. As a result of the long-term nonspecific immunosuppression, these immunosuppressive agents have many serious adverse effects. For example, the administration of cyclosporin A or FK506 to a transplant recipient results in degenerative changes in renal tubules. Transplant recipients receiving long-term immunosuppressive treatment have a high risk of developing infections and tumors. For example, patients receiving immunotherapy are at higher risk of developing lymphomas, skin tumors and brain tumors (see, e.g., Fellstrom et al., 1993, Immunol. Rev. 134:83-98).

An alternative to immunosuppressive agents for the prevention of allograft rejection is the blockage of specific receptors involved in T cell costimulation. T cell activation requires both TCR-mediated signal transduction and simultaneously delivered costimulatory signals. These costimulatory signals are contributed, in part, by the activation of the costimulatory molecule CD28, which is expressed on resting T cells, by CD80 (B7-1) or CD86 (B7-2) expressed on antigen presenting cells (APCs). The activation of the costimulatory molecule CD40, which is expression on antigen presenting cells (i.e., B cells, dendritic cells, and macrophages), by CD40 ligand (“CD40L”), which is expressed on activated T cells, contributes to the upregulation of T cell activation by inducing the expression of B7-1 and B7-2 on antigen presenting cells and the production of certain chemokines and cytokines such as IL-8, MIP-1α, TNF-α, and IL-12 (Cella et al., 1996, J. Exp. Med. 184:747-752: and Caux et al., 1994, J. Exp. Med. 180:1263-1272). The CD40/CD40L interaction also results in the differentiation of T cells to T helper (“TH”) type 1 cells in part due to the expression of cytokines such as IL-12 by dendritic cells and macrophages.

CTLA-4 is normally expressed as a membrane-bound receptor on T cells and has been shown to downregulate T cell activation by competing with CD28 for B7-1 and B7-2. The administration of soluble CTLA-4Ig is believed to prevent allograft rejection by competing with CD28 for B7-1 and B7-2. Soluble CTLA-4Ig has been administered to transplant recipients to disrupt the CD28/B7 interaction so that T cell costimulation is blocked and allograft rejection does not occur (Zheng et al., 1999, J. Immunol. 162:4983-4990; Lenschow et al., 1996, Ann. Rev. Immunol. 14:233-258). Unfortunately, CTLA-4Ig has variable efficacy, and typically does not prevent development of chronic rejection.

Anti-CD40L (anti-CD154) monoclonal antibodies have also been administered to transplant recipients to prevent allogaft rejection. These antibodies function by blocking the interaction of CD40 on antigen presenting cells (APC) and CD40L on activated T cells. It has recently been shown that graft survival achieved through the use of anti-CD40L monoclonal antibodies results in a significant inhibition of TH1 type cytokines (i.e., IL-2, IL-12, TNFα, and IFNγ), and an increase in the levels of the TH2 type cytokines (i.e., IL-4, and IL-10) in the graft sections (Hancock et al., 1996, Proc. Natl. Acad. Sci. USA 93:13967-13972). Although the administration of anti-CD40L monoclonal antibodies has been shown to result in permanent graft survival when given to mice in combination with donor-specific spleen cells, adverse side effects such as coagulation have also been shown to be associated with the administration of anti-CD40L monoclonal antibodies. Initial clinical trials in adult renal transplant recipients receiving anti-CD40L monoclonal antibody plus glucocorticoids were halted because of thromboembolic complications (Vincent, J., Biogen News, press release, Nov. 2, 1999, www.prnewswire.com), though the extent to which thromoboembolism was attributable to monoclonal antibodies versus non-specific factors in the antibody formulation is unclear (Kawai et al., 2000, Nature Med. 6:114; and Kirk et al., 2000, Nature Med. 6:114). Further, in the primate renal allograft study, concomitant use of mainstream immunosuppressive agents such as FK-506, methylprednisolone and mycophenolate mofetil diminished the efficacy of CD40L (CD154) mAb, though the exact contribution of each of the individual drugs to this reduction in efficacy was not determined (Kirk, A. D., 1999, Nature Medicine 5:686-693.). The results presented herein demonstrate that some, but not all, combinations of CD154 mAb and immunosuppressive agents are antagonistic, and that strategies for design of clinical trials based on use of CD154 mAb can be logically developed by taking into account the extent to which a given drug inhibits induction of CD154.

›In addition, no satisfactory methods presently exist for…

In addition, no satisfactory methods presently exist for monitoring whether a transplant graft is being accepted or rejected by a recipient. In general, signs of cellular damage within the transplant tissue can be assayed. Alternatively, for tissues such as kidney or liver, physiological function of the transplant tissue can be assayed. Often, however, by the time overt signs of either cellular damage or a decrease in physiological function are detected, the tissue graft is already beyond rescue. This is particularly true in the case of such organ transplants as heart transplants, with which the first overt signs of rejection are often complete failure of the heart's function.

Accordingly, there is a need for improved, safer immunomodulatory treatments that have long-lasting effects for the prevention of transplant rejection. In particular, there is a need for treatments that are more specific and less toxic than the currently available therapeutic agents. Further, there is also a great need for an improved method for monitoring acceptance of transplant tissue in subject mammals that have undergone a transplant.

2.1. Jak/STAT Signal Transduction

Signal transduction pathways represent molecular solutions to the fact that such molecules as polypeptide hormones, growth factors and cytokines cannot cross the cell membrane, but must activate intracellular signaling molecules to elicit a response in target cells. Among such signal transduction pathways is the Jak/Stat signal transduction pathway. See, e.g., Heim, M. H., 1999, J. Recept. & Sig. Trans. Res. 19:75-120; and Leonard, W. J. & O'Shea, J. J., 1998, Ann. Rev. Immunol. 16:293-322.

While the pathway was originally discovered as part of a study of interferon-induced intracellular signalling, to date, several dozen polypeptide ligands have been identified that activate the Jak/Stat pathway. Defects in the Jak/Stat pathway have been identified in a number of diseases, including leukemias, lymphomas, inherited immunodeficiency syndromes, breast cancer and a form of dwarfism caused by constitutively activation of a Stat by a mutant fibroblast growth factor-receptor.

Stats (Signal transducers and activators of transcription) are phosphoproteins that are transcription factors, and that are activated in response to cytokines, growth factors and interferons. Stats are activated by receptor-associated Janus kinases (“Jaks”), which include Jak1, Jak2, Tyk2, and Jak3. Specifically, a ligand-induced receptor aggregation results in the transphorphorylation and activation of the catalytic activity of the associated Jak. The activated Jak phosphorylates the receptors at multiple sites. Stats are recruited to the multimeric complex consisting of the phosphorylated receptor and catalytically active Jak. The catalytically active Jak phosphorylates tyrosine residues in the carboxy-terminus of the Stats. The phosphorylated Stats form homodimers and heterodimers (Darnell, J. E., 1997, Science 277:1630-1635; and Leonard et al., 1998, Ann. Rev. Immunol. 16:293-322; and Darnell et al., 1994, Science 264:1415-1421). The dimerization of Stats is believed to trigger the dissociation of Stats from the receptor complex and their translocation to the nucleus. In the nucleus, Stat dimers bind to their cognate DNA regulatory elements, which binding results in increased transcription, i.e., transactivation. Thus, the Jak/Stat system provides a method of both signal amplification and transduction.

Seven Stat genes (Stat1, Stat2, Stat3, Stat4, Stat5A, Stat5B, and Stat6) and several Stat isoforms have been discovered, the isoforms resulting from alternative splicing or posttranslational processing (for review see, e.g., Leonard et al., 1998, Ann. Rev. Immunol. 16:293-322). Different Stats are activated in response to different cytokines and growth factors. For example, Stat4 has been shown be activated in response to IL-12 induced signal transduction (Thierfelder et al., 1996, Nature 382:171-174; and Kaplan et al., 1996, Nature 382:174-177). Stat6 has been shown to be activated in response to IL-4 and IL-13 induced signal transduction (Takeda et al., 1996, Nature 380:627-630). Certain transcription factors activated in response to a given cytokine have been shown to be important in TH1 and/or TH2 differentiation. Stat4 has been shown to be important in TH1 differentiation and Stat6 has been shown to be important in TH2 differentiation (see, e.g., Romagnani, S., 1997, Immunology Today 18:263-266; Ray, A. and Cohn, L., 1999, J. Clin. Invest. 104(8):985-993).

With respect to TH1 and TH2, the majority of mature T lymphocytes can be divided into two distinct phenotypes: CD8 + cytotoxic T lymphocytes (CTLs), which display the CD8 marker on their cell surface, and CD4 + helper T lymphocytes (T helper or TH cells), which display the CD4 marker on their cell surface. TH cells are involved in both humoral (i.e., antibody) and cell-mediated forms of immune response. TH cells have been further categorized into two distinct subpopulations, termed TH1 and TH2 cell subpopulations. These two subpopulations of TH cells have been categorized on the basis of their restricted cytokine profiles and different functions. For example, TH1 cells are known to produce IL-2, IL-12, tumor necrosis factor β (“TNF-β”), and interferon-α (“IFN-α”). TH2 cells are known to produce IL-4, IL-5, IL-10 and IL-13. Inappropriate immune responses have been shown to be associated with various diseases and disorders. For example, an inappropriate TH2-like response has shown to be associated with atopic conditions, such as asthma and allergy (see, e.g. , Holgate, S. T., 1997, Lancet 350(suppl. II):5-9; Ray, A. and Cohn, L, supra; Oettgen, H. C. and Geha, R. S., 1999, J. Clin. Invest. 104(7):829-835). Further, an inappropriate TH 1-like response has been shown to associated with the pathogenesis of autoimmune diseases such multiple sclerosis, pancreases of insulin-dependent diabetes patients, thyroid glands of Hashimoto's thyroiditis, and gut of Crohn's disease patients.

›2.2. Negative Regulators of the Jak/STAT Signaling Pathway…

2.2. Negative Regulators of the Jak/STAT Signaling Pathway

Three protein families have been discovered that negatively regulate cytokine-induced Jak/Stat signaling, tyrosine phosphatases SHP1 and SHP2, the suppressors of cytokine signaling (“SOCS”), and protein inhibitors of activated Stats (PIAS). SHP1 and SHP2 bind to phosphorylated tyrosine residues on receptors or Jaks, and inactivate signaling by dephosphorylating them (Haque et al., 1998, J. Biol. Chem. 273:33898-33896; and You et al., 1999, Mol. Cell. Biol. 19:2416-2424).

The SOCS family of proteins have been shown to inhibit the Jak/Stat pathway by inhibiting the activity of the Jaks (Hilton et al., Proc. Natl. Acad. Sci. USA 95:114-119; and Hilton, 1999, Cell. and Mol. Life Sci. 55:1658-1577). The nature of the interaction between the different receptors, Jaks, and the SOCS is unclear (Hilton, D. J., 1999, Cell. Mol. Sci. 55:1568-1577). SOCS1 have been shown to directly interact with all the Jaks and Tyk2. CIS (Cytokine inducible SH2 containing protein), a member of the SOCS family, on the other hand, was shown to interact with the EPO receptor or the β chain of the IL-3 receptor in a phosphorylation dependent manner, indicating it may act by competing with Stat molecules for binding to receptors (Yoshimura et al., 1995, EMBO J. 14:2816-2826). SOCS1 expression inhibits IL-6, LIF, oncostatin M, IFN-γ, IFN-β, IFN-α, thrombopoeitin, and growth hormone (GH) induced Jak/Stat signaling. SOCS3 expression inhibits IFN-γ, IFN-β, IFN-α, GH and leptin.

Four members of the PIAS family have been identified, PIAS1, PIAS3, PIASxα, and PIASxβ. PIAS1 was found to bind only to activated Stat1, and PIAS3 to only activated Stat3 (Liu et al., 1998, Proc. Natl. Acad. Sci. USA 95:10626-10631; and Chung et al., 1997, Science 278:1803-1805). PIAS-mediated inhibition of the Jak/Stat signaling pathway, unlike SOCS-mediated inhibition of the Jak/Stat signaling pathway, is very specific. However, unlike some of the SOCS which are elevated rapidly in response to cytokines, the PIAS levels in the cells are more or less constant.

3. SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for reducing immune rejection, for example, transplant- or autoimmune disorder-related immune injury or rejection. The present invention also relates to methods and compositions for monitoring transplant acceptance and for monitoring an autoimmune disorder in a subject mammal. The present invention still further relates to methods for identifying compounds that can reduce immune injury.

The present invention is based, in part, on the discovery, demonstrated herein, that immune rejection can be monitored by determining the amount of particular members of the Jak/Stat signal transduction pathway present within an affected tissue (that is, a transplant cell, tissue, organ, or organ system, or a cell, tissue, organ, or organ system that is, or is suspected of, being affected by an autoimmune disorder). In particular, the results presented herein demonstrate that immune rejection can be monitored by determining the amount of Stat4 mRNA or protein, Stat6 mRNA or protein, SOCS1 mRNA or protein, or SOCS3 mRNA or protein, present in an affected tissue. The results presented herein also demonstrate that immune rejection can be monitored by determining the amount of Stat1 mRNA or protein, Stat2 mRNA or protein, or Stat3 mRNA or protein present, e.g., present in an affected tissue. The present invention is further based, in part, on the discovery, demonstrated herein, that immune rejection can be reduced and tolerance can be induced by modulating the amount of these particular members of the Jak/Stat signal transduction pathway present, expressed or active within an affected tissue.

Thus, in one aspect, the invention relates to methods for monitoring acceptance of a transplant in a subject mammal that has undergone a transplant, wherein said method comprises: determining the amount of at least one of the following: (i) Stat4 mRNA or Stat4 protein, (ii) Stat6 mRNA or Stat6 protein, (iii) SOCS1 mRNA or SOCS1 protein, or (iv) SOCS3 mRNA or SOCS3 protein, present in a transplant sample from the subject. In alternate embodiments, such methods comprise determining the amount of at least two, at least three, or each of (i) to (iv) present in the transplant sample. In certain embodiments, the amount of mRNA is determined, and can, for example, be determined via use of nucleic acid microarrays. In other embodiments, the amount of protein is determined, while in still other embodiments, the amount of mRNA and protein is determined. With respect to Stat6, when the amount of Stat6 is being determined, it is preferable that the amount of Stat6 protein be determined. In any such embodiment wherein a Stat protein amount is determined, the amount determined can be the total amount of the Stat protein present in a sample or, alternatively, can be the amount of phosphorylated Stat protein present in the sample.

In a preferred embodiment, a method for monitoring acceptance of a transplant in a subject mammal that has undergone a transplant comprises determining the amount of Stat4 and Stat6 mRNA or Stat4 and Stat6 protein present in a transplant sample from the subject. Such an embodiment can further comprise determining the ratio of Stat4 to Stat6 amounts.

The methods for monitoring acceptance of a transplant in a subject mammal that has undergone a transplant can further comprise assaying the transplant sample for evidence of lymphocyte infiltration or tissue damage (cell injury) using standard techniques. For example, histological techniques well known to those of skill in the art can be utilized to evaluate internationally recognized and used diagnostic criteria for the evaluation of graft rejection, which include features specific for each organ involved. For example, immunohistologic evaluation of such tissues, via, e.g., use of labeled antibody techniques to localize and quantitate gene expression. The evaluation of such criteria can, therefore, be enhanced by, for example, localization of Stat4, Stat6, SOCS1 and/or SOCS3 proteins, and/or detection of corresponding mRNAs via, e.g., in situ hybridization.

›Such methods can also further comprise comparing the…

Such methods can also further comprise comparing the amount or ratio determined to that present in a control sample, for example, a corresponding pre-transplant subject sample or a subject blood sample. In instances wherein the amount of Stat4, SOCS1, or SOCS3 mRNA or protein in the transplant sample is greater than, or the amount of Stat6 mRNA or protein in the transplant sample is less than, that of the control sample, such a result indicates that acceptance of the transplant has not occurred, has not been induced or is not being maintained. In instances wherein the amount of Stat4, SOCS1, or SOCS3 mRNA or protein in the transplant sample is less than, or the amount of Stat6 mRNA or protein in the transplant sample is equal to or greater than that of the control sample, such a result indicates that acceptance of the transplant has occurred, is being induced or is being maintained. In instances wherein the ratio of Stat4 to Stat6 in the transplant sample is greater than or equal to that in the control sample, such a result indicates that acceptance of the transplant has not occurred, has not been induced or is not being maintained. In instances wherein the ratio of Stat4 to Stat6 in the transplant sample is less than that in the control sample, such a result indicates that acceptance of the transplant has occurred, has been induced or is being maintained.

In another aspect, the invention relates to methods for monitoring acceptance of a transplant in a subject mammal that has undergone a transplant, wherein said method comprises: determining the amount of at least one of the following: (i) Stat1 mRNA or Stat1 protein, (ii) Stat2 mRNA or Stat2 protein, or (iii) Stat3 mRNA or Stat3 protein, present in a cell sample from the subject. In alternate embodiments, such methods comprise determining the amount of at least two or each of (i) to (iii) present in the sample. In certain embodiments, the amount of mRNA is determined, and can, for example, be determined via use of nucleic acid microarrays. In other embodiments, the amount of protein is determined, while in still other embodiments, the amount of mRNA and protein is determined. In any such embodiment wherein a Stat protein amount is determined, the amount determined can be the total amount of the Stat protein present in a sample or, alternatively, can be the amount of phosphorylated Stat protein present in the sample.

In a particular embodiment of such Stat 1-, Stat 2-, and/or Stat 3-related methods, the cell sample is a transplant sample obtained within 2 to 3 days post-transplantation. In an alternative embodiment of such Stat 1-, Stat 2-, and/or Stat 3-related methods, the cell sample is a subject blood sample.

Such Stat 1-, Stat-2, and/or Stat 3-related methods can also further comprise comparing the amount determined to that present in a control sample, for example, a corresponding pre-transplant subject sample or, in the case of embodiments wherein the cell sample is a transplant sample obtained within 2-3 days post-transplantation, a subject blood sample. In instances wherein the amount of Stat1, Stat2, or Stat3 mRNA or protein in the cell sample is greater than that of the control sample, such a result indicates that acceptance of the transplant has not occurred, has not been induced or is not being maintained. In instances wherein the amount of Stat1, Stat2, or Stat3 mRNA or protein in the transplant sample is less than that of the control sample, such a result indicates that acceptance of the transplant has occurred, is being induced or is being maintained.

In another aspect, the invention relates to methods for monitoring an autoimmune disorder in a subject mammal, wherein said method comprises: determining the amount of at least one of the following: (i) Stat4 mRNA or Stat4 protein, (ii) Stat6 mRNA or Stat6 protein, (iii) SOCS1 mRNA or SOCS1 protein, or (iv) SOCS3 mRNA or SOCS3 protein, present in a sample from a subject mammal being treated for or suspected of exhibiting the autoimmune disorder, wherein the sample is obtained from a tissue affected by the disorder. In alternate embodiments, such methods comprise determining the amount of at least two, at least three, or each of (i) to (iv) present in the sample. In certain embodiments, the amount of mRNA is determined, and can, for example, be determined via use of nucleic acid microarrays. In other embodiments, the amount of protein is determined, while in still other embodiments, the amount of mRNA and protein is determined. With respect to Stat6, when the amount of Stat6 is being determined, it is preferable that the amount of Stat6 protein be determined. In any such embodiment wherein a Stat protein amount is determined, the amount determined can be the total amount of the Stat protein present in a sample or, alternatively, can be the amount of phosphorylated Stat protein present in the sample.

In a preferred embodiment, a method for monitoring an autoimmune disorder in a subject mammal comprises determining the amount of Stat4 and Stat6 mRNA or Stat4 and Stat6 protein present in a sample from the subject mammal being treated for or suspected of exhibiting the autoimmune disorder, wherein the sample is obtained from a tissue affected by the disorder. Such an embodiment can further comprise determining the ratio of Stat4 to Stat6 amounts.

The methods for monitoring an autoimmune disorder in a subject mammal can further comprise assaying the sample for evidence of leukocyte infiltration or tissue damage (cell injury) using standard techniques. For example, histological techniques well known to those of skill in the art can be utilized. Alternatively, standard techniques can be utilized to assay (e.g., in serum) for the presence of autoimmune antibodies associated with the particular autoimmune disorder of interest. There are internationally used diagnostic criteria for evaluation of graft rejection, with features specific for each organ. The immunohistologic evaluation of such tissues, i.e., use of unlabeled-antibody techniques to localize and quantitate gene expression, can be enhanced by localization of Stat4 and Stat6 proteins, or detection of corresponding mRNAs by in situ hybridization.

›Such methods for monitoring an autoimmune disorder in…

Such methods for monitoring an autoimmune disorder in a subject mammal can further comprise comparing the amount or ratio determined to that present in a control sample, for example, a corresponding tissue not affected by the disorder or a subject blood sample. In instances wherein the amount of Stat4, SOCS1, or SOCS3 mRNA or protein in the sample is greater than, or the amount of Stat6 mRNA or protein in the sample is less than, that of the control sample, such a result indicates that the subject mammal exhibits or continues to exhibit the disorder. In instances wherein the amount of Stat4, SOCS1, or SOCS3 mRNA or protein in the sample is less than, or the amount of Stat6 mRNA or protein in the sample is equal to or greater than that of the control sample, such a result indicates that the subject mammal does not exhibit the disorder or that treatment for the disorder is effective. In instances wherein the ratio of Stat4 to Stat6 in the sample is greater than or equal to that in the control sample, such a result indicates that the subject mammal exhibits or continues to exhibit the disorder. In instances wherein the ratio of Stat4 to Stat6 in the transplant sample is less than that in the sample, such a result indicates that the subject mammal does not exhibit the disorder or that treatment for the disorder is effective.

The methods for monitoring transplant acceptance or monitoring an autoimmune disorder can be performed with kits designed for carrying out such methods. As such, the present invention also relates to kits for monitoring transplant acceptance and autoimmune disorders.

In yet another aspect, the present invention relates to a method for identifying a compound to be tested for an ability to reduce immune rejection, said method comprises: (a) contacting an activated T cell sample with a test compound; (b) determining the amount of at least one of the following: (i) Stat1 mRNA or Stat1 protein, (ii) Stat2 mRNA or Stat2 protein, (iii) Stat3 mRNA or Stat3 protein, (iv) Stat4 mRNA or Stat4 protein, (v) Stat6 mRNA or Stat6 protein; (vi) SOCS1 mRNA or SOCS1 protein, or (vii) SOCS3 mRNA or SOCS3 protein, present in (a); and (c) comparing the amount(s) in (a) to that/those present in a corresponding control activated T cell sample that has not been contacted with the test compound, so that if the amount of (i), (ii), (iii), (iv), (vi), or (vii) is decreased, or the amount of (v) is increased, relative to the amount in the control sample, a compound to be tested for an ability to reduce immune rejection is identified. In alternate embodiments, such methods comprise determining the amount of at least two, at least three, at least four, at least five, at least six, or each of (i) to (vii) present in the activated T cell sample and comparing the amounts to those present in the control sample.

In certain embodiments, the amount of mRNA is determined, in other embodiments, the amount of protein is determined, while in still other embodiments, the amount of mRNA and protein is determined. With respect to Stat6, when the amount of Stat6 is being determined, it is preferable that the amount of Stat6 protein be determined. In any such embodiment wherein a Stat protein amount is determined, the amount determined can be the total amount of the Stat protein present in a sample or, alternatively, can be the amount of phosphorylated Stat protein present in the sample.

In a preferred embodiment of a method for identifying a compound to be tested for an ability to reduce immune rejection, said method comprises: (a) contacting an activated T cell sample with a test compound; (b) determining the amount of Stat4 mRNA and Stat6 mRNA or Stat4 protein and Stat6 protein present in the sample; and (c) comparing the amounts in (b) to those present in a corresponding control activated T cell sample that has not been contacted with the test compound, so that if the amount of Stat4 is decreased or the amount of Stat6 is increased relative to the amount in the control sample, a compound to be tested for an ability to reduce immune rejection is identified.

In another preferred embodiment of a method for identifying a compound to be tested for an ability to reduce immune rejection, said method comprises: (a) contacting an activated T cell sample with a test compound; (b) determining the ratio of Stat4 mRNA to Stat6 mRNA or Stat4 protein to Stat6 protein present in the sample; and (c) comparing the ratio in (b) to that present in a corresponding control activated T cell sample that has not been contacted with the test compound, so that if the ratio in the sample is decreased relative to that in the control sample, a compound to be tested for an ability to reduce immune rejection is identified.

In another aspect, the present invention relates to a method for identifying a compound to be tested for an ability to reduce immune rejection, said method comprises: (a) contacting a resting T cell sample, a T cell activator and a test compound; (b) determining the amount of at least one of the following: (i) Stat1 mRNA or Stat1 protein, (ii) Stat2 mRNA or Stat2 protein, (iii) Stat3 mRNA or Stat3 protein, (iv) Stat4 mRNA or Stat4 protein, (v) Stat6 mRNA or Stat6 protein; (vi) SOCS1 mRNA or SOCS1 protein, or (vii) SOCS3 mRNA or SOCS3 protein, present in (a); and (c) comparing the amount(s) in (a) to that/those present in a corresponding resting T cell sample that has been contacted with the T cell activator, but has not been contacted with the test compound, so that if the amount of (i), (ii), (iii), (iv), (vi), or (vii) is decreased, or the amount of (v) is increased, relative to the amount in the control sample, a compound to be tested for an ability to reduce immune rejection is identified. In alternate embodiments, such methods comprise determining the amount of at least two, at least three, at least four, at least five, at least six, or each of (i) to (vii) present in the activated T cell sample and comparing the amounts to those present in the control sample.

›In certain embodiments of such methods, the amount…

In certain embodiments of such methods, the amount of mRNA is determined, in other embodiments, the amount of protein is determined, while in still other embodiments, the amount of mRNA and protein is determined. With respect to Stat6, when the amount of Stat6 is being determined, it is preferable that the amount of Stat6 protein be determined. In any such embodiment wherein a Stat protein amount is determined, the amount determined can be the total amount of the Stat protein present in a sample or, alternatively, can be the amount of phosphorylated Stat protein present in the sample. Further, in certain embodiments, the resting T cell is a primary T cell, and in other embodiments, the resting T cell is a T cell line.

In a preferred embodiment of a method for identifying a compound to be tested for an ability to reduce immune rejection, said method comprises: (a) contacting a resting T cell sample, a T cell activator and a test compound; (b) determining the amount of Stat4 mRNA and Stat6 mRNA or Stat4 protein and Stat6 protein present in the sample; and (c) comparing the amounts in (b) to those present in a corresponding control resting T cell sample that has been contacted with the T cell activator, but has not been contacted with the test compound, so that if the amount of Stat4 is decreased or the amount of Stat6 is increased relative to the amount in the control sample, a compound to be tested for an ability to reduce immune rejection is identified.

In another preferred embodiment of a method for identifying a compound to be tested for an ability to reduce immune rejection, said method comprises: (a) contacting a resting T cell sample, a T cell activator and a test compound; (b) determining the ratio of Stat4 mRNA to Stat6 mRNA or Stat4 protein to Stat6 protein present in the sample; and (c) comparing the ratio in (b) to that present in a corresponding control resting T cell sample that has been contacted with a T cell activator, but has not been contacted with the test compound, so that if the ratio in (a) is decreased relative to that in the control sample, a compound to be tested for an ability to reduce immune rejection is identified.

In another aspect, the present invention relates to a method for identifying a compound to be tested for an ability to reduce immune rejection, comprising: (a) contacting a T cell sample, a cytokine and a test compound, wherein the T cell sample is responsive to the cytokine; (b) determining the amount of at least one of the following: (i) Stat1 mRNA or Stat1 protein, (ii) Stat2 mRNA or Stat2 protein, (iii) Stat3 mRNA or Stat3 protein, (iv) Stat4 mRNA or Stat4 protein, (v) Stat6 mRNA or Stat6 protein; (vi) SOCS1 mRNA or SOCS1 protein, or (vii) SOCS3 mRNA or SOCS3 protein, present in (a); and (c) comparing the amount(s) in (a) to that/those present in a corresponding control T cell sample that has been contacted with the cytokine, but has not been contacted with the test compound, so that if the amount of (i), (ii), (iii), (iv), (vi), or (vii) is decreased, or the amount of (v) is increased, relative to the amount in the control sample, a compound to be tested for an ability to reduce immune rejection is identified. In preferred embodiments, the cytokine is IL-2, IL-4, IL-12, or IL-13.

In a preferred embodiment of such a method for identifying a compound to be tested for an ability to reduce immune rejection, said method comprises: (a) contacting a T cell sample, a cytokine and a test compound, wherein the T cell sample is responsive to the cytokine; (b) determining the amount of Stat4 and Stat6 mRNA or Stat4 and Stat6 protein present in the sample; and (c) comparing the amounts in (a) to those present in a corresponding control T cell sample that has been contacted with the cytokine, but has not been contacted with the test compound, so that if the amount of Stat4 is decreased or the amount of Stat6 is increased relative to the amounts in the control sample, a compound to be tested for an ability to reduce immune rejection is identified.

In another preferred embodiment of such a method for identifying a compound to be tested for an ability to reduce immune rejection, said method comprises: (a) contacting a T cell sample, a cytokine and a test compound, wherein the T cell sample is responsive to the cytokine; (b) determining the ratio of Stat4 mRNA to Stat6 mRNA or Stat4 mRNA to Stat6 protein present in the sample; and (c) comparing the ratio to in (a) to that present in a corresponding control T cell sample that has been contacted with the cytokine, but has not been contacted with the test compound, so that if the ratio in the sample is decreased relative to that in the control sample, a compound to be tested for an ability to reduce immune rejection is identified.

In yet another aspect, the present invention relates to methods for reducing immune rejection in a subject mammal, said methods comprising: administering to a subject mammal in need of such a reduction a concentration of a compound sufficient to decrease the level of Stat4 mRNA or protein in the subject relative to that observed in the subject in the absence of the compound, wherein said compound does not induce platelet aggregation and does not affect NF-κB activation in CD40L + cells.

Alternatively, such methods for reducing immune rejection in a subject mammal can comprise: administering to a subject mammal in need of such a reduction a concentration of a compound sufficient to increase the level of Stat6 mRNA or protein in the subject relative to that observed in the subject in the absence of the compound, wherein said compound does not induce platelet aggregation and does not affect NF-κB activation in CD40L + cells.

›Such methods for reducing immune rejection in a…

Such methods for reducing immune rejection in a subject mammal can also comprise: administering to a subject mammal in need of such a reduction a concentration of a compound sufficient to decrease the level of Stat4 mRNA or protein and maintain or increase the level of Stat6 mRNA or protein in the subject relative to that observed in the subject in the absence of the compound, wherein said compound does not induce platelet aggregation and does not affect NF-κB activation in CD40L + cells.

3.1. Definitions

As used herein, the term “transplant” includes any cell, organ, organ system or tissue which can elicit an immune response in a recipient subject mammal. In general, therefore, a transplant includes an allograft or a xenograft cell, organ, organ system or tissue. An allograft refers to a graft (cell, organ, organ system or tissue) obtained from a member of the same species as the recipient. A xenograft refers to a graft (cell, organ, organ system or tissue) obtained from a member of a different species as the recipient.

The term “immune rejection,” as used herein, is intended to refer to immune responses involved in transplant rejection, as well as to the concomitant physiological result of such immune responses, such as for example, interstitial fibrosis, chronic graft artheriosclerosis, or vasculitis. The term “immune rejection,” as used herein, is also intended to refer to immune responses involved in autoimmune disorders, and the concomitant physiological result of such immune responses, including T cell-dependent infiltration and direct tissue injury; T cell-dependent recruitment and activation of macrophages and other effector cells; and T cell-dependent B cell responses leading to autoantibody production.

The term “transplant rejection,” as used herein, refers to T cell-mediated rejection of transplant cells, organs, organ systems or tissue. In general, such transplant rejection generally includes accelerated, acute and chronic rejection. It is intended that the term, as used herein, also refer to graft versus host disease, and the physiological results of such a disorder.

The term “reducing immune rejection,” is meant to encompass prevention or inhibition of immune rejection, as well as delaying the onset or the progression of immune rejection. The term is also meant to encompass prolonging survival of a transplant in a subject mammal, or reversing failure of a transplant in a subject. Further, the term is meant to encompass ameliorating a symptom of an immune rejection, including, for example, ameliorating an immunological complication associated with immune rejection, such as for example, interstitial fibrosis, chronic graft atherosclerosis, or vasculitis. The term is also meant to encompass induction of tolerance in a subject mammal that has undergone a transplant.

The term “tolerance,” as used herein, refers to a state wherein the immune system of a transplant recipient subject mammal is non-responsive to the transplant. This state is considered donor transplant-specific, and, as such, is distinguished from nonspecific immunosuppression. Operatively, the term as used herein, refers to permanent acceptance of a graft without ongoing immunosuppression, wherein, for example, challenge with a second graft of donor origin (especially when the second graft is of the same tissue as the first graft) should be accepted, and challenge with a third party graft should be rejected.

The term “autoimmune rejection,” as used herein, refers to immune responses involved in autoimmune disorders, and the concomitant physiological result of such immune responses.

The term “activated T cell,” as used herein, refers to a T cell that expresses antigens indicative of T-cell activation (that is, T cell activation markers). Examples of T cell activation markers include, but are not limited to, CD25, CD26, CD30, CD38, CD69, CD70, CD71, ICOS, OX-40 and 4-1BB. The expression of activation markers can be measured by techniques known to those of skill in the art, including, for example, western blot analysis, northern blot analysis, RT-PCR, immunofluorescence assays, and fluorescence activated cell sorter (FACS) analysis.

The term “resting T cell,” as used herein, refers to a T cell that does not express T-cell activation markers. Resting T cells include, but are not limited to, T cells which are CD25 31 , CD69 − , ICOS − , SLAM − , and 4-1BB − . The expression of these markers can be measured by techniques known to those of skill in the art, including, for example, western blot analysis, northern blot analysis, RT-PCR, immunofluorescence assays, and fluorescence activated cell sorter (FACS) analysis.

The term “T cell activator,” as used herein, refers to any compound or factor that is a T cell receptor stimulatory factor, that is, induces T cell receptor signalling. Preferably, the compound or factor also induces co-stimulatory pathways. Non-limiting examples of T cell activators include, but are not limited to, anti-CD3, antibodies (preferably monoclonal antibodies) either alone or in conjuntion with anti-CD28 antibodies (preferably monoclonal antibodies), or mitogens such as, for example, phorbol 12-myristate 13-acetate (PMA), phytohemagglutinin (PHA) or concanavalin-A (Con-A).

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Human Stat1 nucleic acid and amino acid sequences (SEQ ID NOs:1, 2, respectively).

FIG. 2 . Human Stat2 nucleic acid and amino acid sequences (SEQ ID NOs:3, 4, respectively).

FIG. 3 . Human Stat3 nucleic acid and amino acid sequences (SEQ ID NOs:5, 6, respectively).

›FIG. 4 . Human Stat4 nucleic acid and…

FIG. 4 . Human Stat4 nucleic acid and amino acid sequences (SEQ ID NOs:7, 8, respectively).

FIG. 5 . Human Stat6 nucleic acid and amino acid sequences (SEQ ID NOs:9, 10, respectively).

FIG. 6 . Human SOCS1 nucleic acid and amino acid sequences (SEQ ID NOs:11, 12, respectively).

FIG. 7 . Human SOCS3 nucleic acid and amino acid sequences (SEQ ID NOs:13, 14, respectively).

FIG. 8 . Human Jak2 nucleic acid and amino acid sequences (SEQ ID NOs:15, 16 respectively).

FIG. 9 . Human Tyk2 nucleic acid and amino acid sequences (SEQ ID NOs:17, 18 respectively).

FIG. 10 . Effects of immunosuppressants on CD154 mAb-induced cardiac allograft survival. Murine recipients were followed for up to 100 days post-transplant, and mean (±SD) cardiac allograft survival are shown (n=6/group). Statistical analysis (Mann-Whitney U test) showed that CD154 mAb or combined CD154 and rapamycin (CD154RPM) induced highly significant prolongation of allograft survival (p<0.001) compared to recipients treated with IgG, combined CD154 and cyclosporin A (CD154/CsA), or CD154 plus methylprednisolone (CD154/MP).

FIGS. 11A-11B. Contrasting effects of immunosuppressive agents on activation-induced CD154 expression by CD4+ T cells, as determined by dual color flow cytometry after 7 hr of culture. Except for the control sample depicted in the uppermost section of Panel A, all culture plates were pre-coated with CD3 mAb. Representative individual samples are shown in panel A, in which the histograms depict CD154 expression by gated CD4+ T cells. The percentage of CD154+ cells and the mean channel fluorescence of CD154 staining are indicated. Panel B shows the average activation-induced CD154 expression determined from 3 independent experiments. Percent CD154 expression (mean±SD) was calculated relative to the percentage of CD4+ CD154+ cells observed in the absence of immunosuppressive drugs. Cyclosporin A (CsA) and methylprednisolone (MP) significantly inhibited activation-induced CD154 expression (p<0.01).

FIGS. 12A-12B. Critical role for NF-κB in activation-induced CD154 expression by CD4+ T cells in vitro. The experimental design and data presentation arc identical to that shown in FIG. 10 : splenic cells were derived from either NFκB/p50 KO or control wild-type (WT) mice: Genetic deletion of NFκB/p50 inhibited activation-induced CD154 expression (p<0.02). Inhibition of NF-κB p50 in WT mice by the proteasome antagonists lactacystin (LC) and MG-273 (MG) also significantly blocked activation-induced CD154 expression (p<0.04).

FIG. 13 . Permanent cardiac allograft survival using CD154 mAb is NF-κB-dependent. Mice were followed for up to 100 days post-transplant and mean (±SD) cardiac allograft survival are shown (n=6/group). Statistical analysis (Mann-Whitney U test) showed that use of NF-κB/p50 KO mice as recipients, or administration of a lactacystin-derived (LC) proteasome inhibitor to wild-type mice, significantly impaired the efficacy of CD154 mAb therapy (p<0.001).

FIG. 14 A. Kinetics of Stat RNA expression in heart allografts. Post-transplantation expression of Stats at day 1, day 2, day 3, day 5 were studied using RNA obtained from transplanted (Balb/c), native (B6/129), and control (B6/129) hearts of 8-10 weeks old female mice. Equal amounts of heart RNA (25 μg) were loaded onto each lane of three 1.2% agarose-fornaldehyde gels. Hybridizations were done with probes specific to the transcriptional activation domains and 3′-untranslated regions of the Stats. The Stat probes described in the Materials and Methods Section of Section 8, below, were used for all the hybridizations. The locations of probes are shown underneath the cDNA line drawings next to the hybridization patterns. Murine GAPDH cDNA fragment was used as a control. The Stats and SOCS listed on the right of the GAPDH hybridization patterns indicate the probes used with the particular membranes. Designations: C, control heart (B6/129); N, native heart; T, transplanted heart. These data demonstrate that in the allografts Stats 1 and 2 increase progresssively, peaking at day 5, whereas Stat3 rises to a plateau level by day 1. All 3 Stats increase within native control hearts by day 5.

FIGS. 14B-14G. Kinetics of Stat RNA expression in heart allografts. The Stat levels were normalized against the GAPDH values. Normalized Stat levels are graphed as relative optical density (relative to other Stats). Closed, open, and gray bars indicate Stat RNA levels in transplant, native, and control hearts, respectively. Designations: D1, day 1; D2, day 2; D3, day 3; D5, day5; NAT, native; TRA, transplant; CONT, control.

FIG. 15 . Kinetics of SOCSCIS RNA expression in heart allografts. Post-transplantation expression of SOCS/CIS RNA at day 1, day 2, day 3, and day 5 post-transplantation were studied by deprobing and rehybridizing the same membranes initially used for studying the Stat RNA expression studies described above in FIG. 14 A. Hybridizations were done with probes specific to the 3′-untranslated regions of the SOCS. The SOCS and CIS probes described in the Materials and Methods Section of Section 8, below, were used for all the hybridizations. The locations of probes are shown underneath the cDNA line drawings next to the hybridization patterns. Murine GAPDH cDNA fragment was used as a control. The SOCS/CIS list on the right of the GAPDH hybridization patterns indicate the probes used with the individual membranes. Designations: N, native hearts; T, transplanted hearts; C, control hearts (B6/129). These data demonstrate that SOCS1 is densely expressed in allografts at day 5, with only minor expression in native heart samples. SOCS3 is well-expressed from day 1 in allografts, with a further increase at day 5; no expression was seen in native hearts. SOCS5 is expressed predominantly as a 4.4 kb species, with a minor species at 3.8 kb, in control and native heart samples. However, in allografts, the 2 SOCS5 species are expressed in approximately equal amounts. By contrast, CIS expression remained unchanged in native hearts but decreased in allografts from day 1 onwards.

›FIG. 16 . The similarity in the expression…

FIG. 16 . The similarity in the expression patterns of Stat4 and SOCS3 RNA in heart allografts. Stat4 and SOCS3 RNA expression in the days that follow the surgery show a high degree of similarity between the patterns of Stat4 and SOCS3 RNAs, albeit differences in their abundance (using Kodak Biomax MR film at 80° C., with intensifying screen, the exposure time for the Stat4 blot was approximately 4 days, and for SOCS3 only 15 hrs).

FIG. 17 . Stat protein levels following cardiac transplantation. Total proteins from the native and transplant hearts of untreated mice were extracted following transplantation, electrophoresed, transferred onto Immobilon-P membranes and Stat levels analyzed by Western blots as described in Materials and Methods section of Section 8, below. To identify Stats correctly, cell extracts recommended as positive controls by the antibody suppliers were used in the Western blots, together with Precision Protein standards. Designations: N, native hearts; T, transplant hearts; C, control hearts (Balb/c or B6/129), HSC 70, constitutive heat-shock protein. Molecular weights of the Stats are shown on the right. The list on the right of the HSC70 patterns indicates which Stat antibody was used with the individual membranes. These data demonstrate a general agreement with the mRNA data, and show that all of the Stats except Stat5A increase in allografts just prior to rejection (i.e. at day 5), with the greatest and most allograft-specific expression being found for Stat4.

FIG. 18 . Phosphorylated Stat levels following cardiac transplantation. Proteins were prepared and Stat levels analyzed by Western blotting as described in Materials and Methods section of Section 8, below. Designations: N, native hearts; T, transplant hearts; C, control hearts (Balb/c or B6/129), HSC 70, constitutive heat-shock protein. Molecular weights of the Stats are shown on the right. These data demonstrate an increase in pStat1 which parallels the rise in Stat1 levels.

FIG. 19 . Stat RNA expression in day 5 post-transplant heart allografts and the effect of anti-CD40L antibody MR-1 on Stat expression. Expression of Stat1, Stat2, Stat3, Stat4, Stat5A and Stat6 RNA was studied using RNA from transplanted (Balb/c) native (B6/129), and control (B6/129) hearts of 8-10 wks old female mice. Equal amounts of heart RNA (25 μg) were loaded onto each lane of four 1.2% agarose-formaldehyde gels, blotted and analyzed as explained in the Materials and Methods section presented in Section 8, below. The Stat probes described in the Materials and Methods section presented in Section 8 were used for all the hybridizations. Murine GADPH cDNA fragment was used as a control. The size of the Stat RNAs are shown on the right. The Stat and SOCS list on the right of the GAPDH hybridization patterns indicate the probes used with the particular membranes. Designations: C, control heart (B6/129); N, native heart; T, transplanted heart. These data demonstrate that CD40L mAb suppresses expression of each of the Stat mRNA.

FIG. 20 . SOCS/CIS RNA expression in day 5 post-transplant heart allografts and the effect of anti-CD40L antibody MR-1 on SOCS/CIS expression. Expression of SOCS1, SOCS2, SOCS3, and CIS RNA was studied by deprobing and rehybridizing the same membranes initially used for studying the Stat RNA expression. Hybridizations were done with probes specific 3′-untranslated regions of the SOCS. The locations of probes are shown on the right. Designations: C, control heart (B6/129); N, native heart; T, transplanted heart. These data demonstrate that SOCS1 and SOCS3 levels increased during allograft rejection, but CD40L mAb prevented this induction. SOCS2 levels remained constant. CIS levels were decreased in transplants, regardless of CD40L mAb therapy.

FIG. 21 . The effect of treatment with the anti-CD40L monoclonal antibody MR-1 on the levels of Stats. Mice were given a single dose injection (DST plus IgG or DST plus MR-1) the day of the transplant surgery. Proteins were extracted from the native and transplanted hearts of both the IgG and MR-1 treated groups (2 mice/group) 5 days and 7 days following transplant surgery. Designations: C, control heart (B6/129); N, native heart; T, transplanted heart. Molecular weights of the Stats are shown on the right. These data demonstrate that CD40L mAb suppresses intragraft levels of each of the Stat proteins except that of Stat6, which at day 7 in this series was increased post-CD40L mAb therapy.

FIG. 22 . Stat levels in isografts versus allografts following cardiac transplantation. Stat levels were studied in protein extracts from isografts (both donor and recipient mice were Balb/c) and allografts (donor mice were Balb/c and recipient mice were B6/129). Proteins were prepared and Stat, Bax, and HSC70 levels were analyzed by Western blotting as described in Materials and Methods section of Section 8, below. Designations: N, native hearts; T, transplant hearts; C, control hearts (Balb/c or B6/129), HSC 70, constitutive heat-shock protein. Molecular weights of the Stats are shown on the right. The list on the right of the HSC70 patterns indicates which Stat antibody was used with the individual membranes. These data demonstrate that isografts showed only low levels of Stats, whereas allografts had progressive increases in Stat proteins, peaking at day 5. This pattern of induction was allo-specific and differed from that of an unrelated protein, Bax, which increased in both isografts and allografts.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1. Methods and Compositions for Monitoring Transplant Acceptance and Autoimmune Disorders

As demonstrated below, immune rejection can be monitored by determining the amount of particular members of the Jak/Stat signal transduction pathway present within an affected tissue. Specifically, the results presented herein demonstrate that immune rejection can be monitored by determining the amount of Stat4 mRNA or protein, Stat6 mRNA or protein, SOCS1 mRNA or protein, or SOCS3 mRNA or protein present in an affected tissue. In particular, the results presented herein demonstrate that immune rejection can be monitored by determining the amount of Stat4 mRNA or protein, Stat6 mRNA or protein, SOCS1 mRNA or protein, or SOCS3 mRNA or protein, present in an affected tissue. The results presented herein also demonstrate that immune rejection can be monitored by determining the amount of Stat1 mRNA or protein, Stat2 mRNA or protein, or Stat3 mRNA or protein present, e.g., present in an affected tissue.

›The term “affected tissue,” as used herein, refers…

The term “affected tissue,” as used herein, refers to a transplant cell, tissue, organ, or organ system, or a cell, tissue, organ, or organ system. For example, such an affected tissue can include, but is not limited to, heart, liver, kidney, lung, bone marrow, skin, muscle, pancreatic islet, or intestine transplant cells, tissues, organs or organ system. The term “affected tissue,” as used herein, also refers to a cell, tissue, organ or organ system that is, or is suspected of, being affected by an autoimmune disorder. For example, such an affected tissue can include, but is not limited to, a cell, tissue, organ, or organ system involved in systemic lupus erythematosus, glomerulonephritis, rheumatoid arthritis, Wegener's granulomatosis, chronic active hepatitis, or vasculitis.

Thus, aspects of the present invention relate to methods and compositions for monitoring such immune rejection. In particular, such methods and compositions can relate, for example, to methods for monitoring acceptance of a transplant in a subject mammal that has undergone a transplant, and can also relate, for example, to methods for monitoring an autoimmune disorder in a subject mammal being treated for or suspected of exhibiting an autoimmune disorder. Such methods and compositions are discussed in detail herein.

In one aspect, the invention relates to methods for monitoring acceptance of a transplant in a subject mammal that has undergone a transplant, wherein said method comprises: determining the amount of at least one of the following: (i) Stat4 mRNA or Stat4 protein, (ii) Stat6 mRNA or Stat6 protein, (iii) SOCS1 mRNA or SOCS1 protein, or (iv) SOCS3 mRNA or SOCS3 protein, present in a transplant sample from the subject. In alternate embodiments, such methods comprise determining the amount of at least two, at least three, or each of (i) to (iv) present in the transplant sample. In certain embodiments, the amount of mRNA is determined, and can, for example, be determined via use of nucleic acid microarrays. In other embodiments, the amount of protein is determined, while in still other embodiments, the amount of mRNA and protein is determined. With respect to Stat6, when the amount of Stat6 is being determined, it is preferable that the amount of Stat6 protein be determined. In any such embodiment wherein a Stat protein amount is determined, the amount determined can be the total amount of the Stat protein present in a sample or, alternatively, can be the amount of phosphorylated Stat protein present in the sample.

In instances wherein the amount of Stat4, SOCS1, or SOCS3 mRNA or protein in the transplant sample is greater than, or the amount of Stat6 mRNA or protein in the transplant sample is less than, that of the control sample, such a result indicates that acceptance of the transplant has not been induced or is not being maintained. Likewise, in instances wherein the ratio of Stat4 to Stat6 in the transplant sample is greater than or equal to that in the control sample, such a result indicates that acceptance of the transplant has not occurred, has not been induced or is not being maintained. Such results suggest a course of action that can include, for example, administration of a high dose of immunosuppressive drugs (e.g., a high dose of corticosteroids, in, for example, the form of a single bolus intravenous injection) and /or administration of compounds to effectuate T cell depletion, including but not limited to administration of anti-CD3 antibodies.

In instances wherein the amount of Stat4, SOCS1, or SOCS3 mRNA or protein in the transplant sample is less than, or the amount of Stat6 mRNA or protein in the transplant sample is equal to or greater than that of the control sample, such a result indicates that acceptance of the transplant has occurred, is being induced or is being maintained. Likewise, in instances wherein the ratio of Stat4 to Stat6 in the transplant sample is less than that in the control sample, such a result indicates that acceptance of the transplant has occurred, has been induced or is being maintained.

The methods for monitoring acceptance of a transplant in a subject mammal that has undergone a transplant can further comprise assaying the transplant sample for evidence of lymphocyte infiltration or tissue damage (cell injury) using standard techniques. For example, histological techniques well known to those of skill in the art can be utilized to evaluate internationally recognized and used diagnostic criteria for the evaluation of graft rejection, which include features specific for each organ involved. For example, for evaluation of heart allograft transplants see, e.g., Billingham, M. E., 1990, J. Heart Transplant. 9(3 Pt 2):272-6. For evaluation of renal allografts see, e.g., Racusen et al., 1999, Kidney Int. 55(2):713-23. In one non-limiting embodiment, immunohistologic evaluation of transplant tissues (such as heart or kidney) can be performed via, e.g., use of labeled antibody techniques to localize and quantitate gene expression. The evaluation of such criteria can, therefore, be enhanced by, for example, localization of Stat4, Stat6, SOCS1 and/or SOCS3 proteins, and/or detection of corresponding mRNAs via, e.g., in situ hybridization.

Additionally, methods for monitoring acceptance of a transplant in a subject mammal that has undergone a transplant can comprise: determining the amount of at least one of the following: (i) Stat1 mRNA or Stat1 protein, (ii) Stat2 mRNA or Stat2 protein, or (iii) Stat3 mRNA or Stat3 protein, present in a cell sample from the subject. In alternate embodiments, such methods comprise determining the amount of at least two or each of (i) to (iii) present in the sample. In certain embodiments, the amount of mRNA is determined, and can, for example, be determined via use of nucleic acid microarrays. In other embodiments, the amount of protein is determined, while in still other embodiments, the amount of mRNA and protein is determined. In any such embodiment wherein a Stat protein amount is determined, the amount determined can be the total amount of the Stat protein present in a sample or, alternatively, can be the amount of phosphorylated Stat protein present in the sample.

›In a particular embodiment of such Stat 1-…

Methods for monitoring acceptance of a transplant can be performed at any point post-transplantation. In a preferred embodiment, monitoring is performed daily during the first week post-transplant, followed by weekly monitoring until approximately one month post-transplant, followed by monthly monitoring until approximately one year post-transplant. It is understood, of course, that the frequency of monitoring can, at least in part, depend upon the particular situation (e.g., the nature of the graft, overall health of the recipient subject mammal, the particular immunotherapeutic, immunosuppressive, or immunomodulatory treatment being administered, etc.).

In instances wherein the amount of Stat4, SOCS1, or SOCS3 mRNA or protein in the sample is greater than, or the amount of Stat6 mRNA or protein in the sample is less than, that of the control sample, such a result indicates that the subject mammal exhibits or continues to exhibit the disorder. Likewise, in instances wherein the ratio of Stat4 to Stat6 in the sample is greater than or equal to that in the control sample, such a result indicates that the subject mammal exhibits or continues to exhibit the disorder. Such results suggest a course of action that can include, for example, the need to increase immunosuppression, as might be undertaken by bolus intravenous steroids (e.g., methylprednisolone) or use of a CD3 mAb such as OKT3.

In instances wherein the amount of Stat4, SOCS1, or SOCS3 mRNA or protein in the sample is less than, or the amount of Stat6 mRNA or protein in the sample is equal to or greater than that of the control sample, such a result indicates that the subject mammal does not exhibit the disorder or that treatment for the disorder is effective. Likewise, in instances wherein the ratio of Stat4 to Stat6 in the transplant sample is less than that in the sample, such a result indicates that the subject mammal does not exhibit the disorder or that treatment for the disorder is effective.

The methods for monitoring an autoimmune disorder in a subject mammal can further comprise assaying the sample for evidence of leukocyte infiltration or tissue damage (cell injury) using standard techniques. For example, histological techniques well known to those of skill in the art can be utilized. Alternatively, standard techniques can be utilized to assay (e.g., in serum) for the presence of autoimmune antibodies associated with the particular autoimmune disorder of interest. For example, there are standard immunohistology methods for detection of autoantibodies directed against a particular tissue (e.g., anti-glomerular basement membrane, anti-parietal cell, anti-thyroid and anti-islet etc.), as well as assays for their detection in serum (e.g. rheumatoid factor assay and anti-double-stranded DNA antibodies). See, e.g., Manual of Clinical Laboratory Immunology (N R Rose, H Friedman, J L Fahey eds. 1986, Am Soc Microbiol, Washington, D.C.; Diagnostic Immunopathology (R B Colvin, et al., eds., 1995, Raven Press, New York).

›The methods described herein can be performed using…

The methods described herein can be performed using a sample from any subject mammal that has undergone a transplant or either exhibits or is suspected of exhibiting an autoimmune disorder. Preferably, the mammal is a human, however, such subject mammals can also include, but are not limited to, pigs, dogs, cats, horses, cattle, sheep, mice, rats, and rabbits.

It is noted that such methods for monitoring transplant acceptance and for monitoring autoimmunde disorders can be used to determine whether a subject can be effectively treated with a specific agent or class of agents intended to promote transplant acceptance or to treat the autoimmune disorder. Thus, in one embodiment, the present invention provides such methods for determining whether a subject can be effectively treated with an agent for an autoimmune disorder or for reducing immune rejection. Monitoring the influence of agents (e.g., drugs and compounds) on the expression or activity of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1 or SOCS3 mRNA or polypeptide can, therefore, be applied in basic drug screening, preclinical studies, clinical trials and during therapeutic treatment regimens designed to reduce immune rejection or to ameliorate a symptom of an autoimmune disorder.

The methods described herein comprise determining the amount of Stat1 mRNA or protein, Stat2 mRNA or protein, Stat3 mRNA or protein, Stat4 mRNA or protein, Stat6 mRNA or protein, SOCS1 mRNA or protein, and/or SOCS3 mRNA or protein present in a sample. Standard techniques, as described below, can routinely be utilized to determine these amounts. In general, such methods of the invention can routinely be performed using standard techniques for detecting the presence or absence of a polypeptide or nucleic acid of the invention in a biological sample. This involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting the polypeptide or mRNA such that the presence of a polypeptide or nucleic acid of the invention is detected in the biological sample.

When comparing levels, such comparisons can be either quantitative or qualitative. Thus, in qualitative instances, for example, in instances wherein a control sample is determined to contain none of a given molecule (that is, Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1, SOCS3 mRNA or protein) and the molecule is determined to be present in the test sample (that is, either a transplant or autoimmune sample), the amount of the molecule in the test sample is greater than that present in the control sample. In quantitative instances wherein both the control and test samples are determined to contain a given molecule, using standard techniques, the amount in the test sample can routinely be determined to be greater than, equal to, or less than that of the control sample. Likewise, using standard techniques, the ratio of Stat4 to Stat6 mRNA or protein present in test and control samples can routinely be determined. In general, the amount of a given molecule in test and control samples will differ by at least 2-fold, and in certain instances, 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold.

With respect to methods for monitoring acceptance of a transplant, such methods can be performed on any transplant from which a sample to be analyzed can be obtained. Such transplants include, but are not limited to, heart, liver, kidney, lung, bone marrow, skin, muscle, pancreatic islet, or intestine transplants.

Likewise, with respect to methods for monitoring autoimmune disorders, such methods can be performed for any autoimmune disorder (or suspected autoimmune disorder) for which a sample of an affected tissue (or a tissue suspected of being affected) can obtained. Such autoimmune disorders include, but are not limited to, systemic lupus erythematosus, glomerulonephritis, rheumatoid arthritis, Wegener's granulomatosis, chronic active hepatitis, and vasculitis

Methods for obtaining samples from a recipient transplant subject mammal or from a subject mammal exhibiting or suspected of exhibiting an autoimmune disorder are well known to those of skill in the art. Such methods can include biopsy methods, such as, for example, standard needle or punch biopsy methods. In certain embodiments, a particular subset of the sample can be isolated for the analysis. For example, a particular subset of a transplant or autoimmune disorder sample containing cell types of interest (e.g. leukocyte cell types) can be isolated. Such isolation can performed utilizing standard techniques such as, for example, laser microdissection (see, e.g., Fend et al., 1999, Am. J. Pathol. 154(1):61-6; Schutze et al., 1998, Nat. Biotechnol. 16(8):737-42; and Simone et al., 1998, Trends Genet. 14(7):272-6).

Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1 and SOCS3 nucleic acid and amino acid sequences are well known to those of skill in the art. For Stat1, see, for example, Schindler et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:7836-7839 and Zhong et al., 1994, Science 264:95-98. For Stat2, see, for example, Yan et al., 1995, Nucleic Acids Res. 23(3):459-463, Bluyssen and Levy, 1997, J. Biol. Chem. 272(7):4600-4605, and Paulson et al., 1999, J. Biol. Chem. 274(36):25343-25349. For Stat3, see, for example, Ripperger, 1995, J. Biol. Chem. 270(50):29998-30006, Akira et al., 1994, Cell 77(1):63-71, Zhong et al., 1994, Science 264:95-98, Zhong et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91:4806-4810. For Stat4, see, for example, Quelle et al., 1995, Mol. Cell. Biol. 15:3336-3343; and Hou et al., 1994, Science 265:1701-1706. For Stat6, see, for example, Yamamoto et al., 1994, Mol. Cell. Biol. 17:4342-4349; Zhang et al., 1994, 91:4806-4810; and Yamamoto et al., 1997, ytogenet. Cell. 77:207-210. For SOCS1 and 3 sequences, see, for example, Starr et al., 1997, Nature, 387:917-921; Minamoto et al., 1997, Biochem., Biophys. Res. Commun. 237:79-83; Masuhara et al., 1997, Biochem, Biophys. Res. Commun. 239:439-446; Naka et al., 1997, Nature 387:924-929; and Endo et al., 1997, Nature 387:921-924. Representative examples of human Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1 and SOCS3 sequences are shown in FIGS. 1-7 (SEQ ID NOs:1-14), respectively.

›Further, additional forms, e.g., alleles or species homologs…

Further, additional forms, e.g., alleles or species homologs of such sequences can routinely be obtained and detected using the sequences described above in conjunction with standard cloning and hybridization techniques such as those find in Sambrook et al., eds., Molecular Cloning: A Laboratory Manual , 2 nd . ed., Cold Spring Harbor Laboratory , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

Such kits can be utilized for determining the amount of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1, and/or SOCS3 mRNA present within a sample (e.g., a transplant sample or a sample obtained from an autoimmune tissue or a tissue suspected of being effected by an autoimmune disorder). Alternatively, such kits can be utilized for determining the amount of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1, and/or SOCS3 polypeptide present within a sample (e.g., a transplant sample or a sample obtained from an autoimmune tissue or a tissue suspected of being effected by an autoimmune disorder). A kit can be capable of being used to determine the amount of any one, two, three, four, five, six, or seven of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1, and/or SOCS3 sequences (mRNA or polypeptide).

The kit, for example, can comprise a microarray for determining such amounts, wherein the microarray comprises one or more nucleic acid sequences immobilized onto a solid surface, said nucleic acid sequence or sequences exhibiting complementarity to at least one of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1, and/or SOCS3 mRNA. The kit can, in addition, comprise a labeled compound or agent capable of detecting the of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1, and/or SOCS3 polypeptide or mRNA encoding the polypeptide in a biological sample and means for determining the amount of the polypeptide or mRNA in the sample (e.g., an antibody which binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA encoding the polypeptide). Kits can also include instructions for use in determining the amount or amounts of mRNA or polypeptide, and can also include directions for monitoring and diagnosis.

For antibody-based kits, the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) which binds to a of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1, and/or SOCS3 polypeptide; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.

For oligonucleotide-based kits, the kit can comprise, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1, and/or SOCS3 nucleic acid sequence; or (2) a pair of primers useful for amplifying a of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1, and/or SOCS3 nucleic acid molecule. The kit can also comprise, e.g., a buffering agent, a preservative, or a protein stabilizing agent. The kit can also comprise components necessary for detecting the detectable agent (e.g., an enzyme or a substrate).

For microarray-based kits, such kits can comprise a nucleotide sequence, e.g., an oligonucleotide sequence, immobilized onto the surface of a solid support (e.g., a glass or porous solid support).

The kits can also contain a control sample or a series of control samples (postive control, negative control, or both) which can be assayed and compared to the test sample contained.

Each component of the kit is usually enclosed within an individual container and all of the various containers are within a single package along with instructions for observing and monitoring transplant acceptance or monitoring an autoimmune disorder.

›NUCLEIC ACID DETECTION · 1 of 2

Preferred agents for detecting an mRNA of interest (that is Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1 or SOCS3 mRNA) are primers or labeled nucleic acid probes capable of hybridizing to the mRNA under stringent hybridization conditions. Nucleic acid probes can be, for example, full-length sequences, such as the nucleic acid sequences depicted in FIGS. 1-7 (SEQ ID NOs:1, 3, 5, 7, 9, 11 and 13), or complements thereof, or portions of such sequences (or complements thereof, such as oligonucleotides of at least about 12, 15, 25 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to the mRNA.

In certain embodiments, determination of the amount of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1 and/or SOCS3 mRNA involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as, for example, anchor PCR, RACE PCR or RT-PCR. Such methods can include the steps of collecting a cell sample, isolating mRNA from the cells of the sample, reverse transcribing the mRNA, contacting the sample with one or more primers which specifically hybridize to the selected sequence under conditions such that hybridization and amplification of the sequence (if present) occurs, and determining the amount of product that is present.

Alternative amplification methods can also routinely be utilized. Such methods can include, for example, self sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology 6:1197), or any other nucleic acid amplification method, followed by the detection/quantitation of the amplified molecules using techniques well known to those of skill in the art. These schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (preferably at least 75%, more preferably at least 85%, most preferably at least 95%) identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology , John Wiley & Sons, NY (1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C. (preferably 65° C.).

Probes can comprise any readily detectable label moiety. For example, probes utilized herein comprise a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor as a label moiety.

In alternate embodiments, the Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1 and/or SOCS3 mRNA sequences can be detected “in situ” directly upon the sample, e.g., the biopsy sample. Techniques for such procedures are well known to those of skill in the art. See, e.g., Nuovo, G. J., 1992, “PCR In Situ Hybridization: Protocols and Applications,” Raven Press, NY.

In other embodiments, the amount of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1 and/or SOCS3 mRNA can be determined by hybridizing nucleic acid arrays, e.g., microarrays. In a specific embodiment of the invention, the expression of one or more of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1 and SOCS3 is measured or detected using a DNA microarray. A DNA microarray or chip is a microscopic array of DNA fragments or synthetic oligonucleotides, disposed in a defined pattern on a solid support, wherein they are amenable to analysis by standard hybridization methods (see, e.g., Schena, 1996, BioEssays 18: 427).

Microarrays share certain preferred characteristics: The arrays are reproducible, allowing multiple copies of a given array to be produced and easily compared with each other. Preferably the microarrays are small, usually smaller than 5 cm 2 , and they are made from materials that are stable under binding (e.g., nucleic acid hybridization) conditions. Microarrays contain a surface to which sequences corresponding to gene products (e.g., mRNA, cDNA, cRNA, or complements thereof), can be specifically hybridized or bound at a known position. For practicing the methods of the present invention, the binding sites of the microarray are polynucleotides, preferably DNA polynucleotides, that specifically hybridize to at least a portion of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1, or SOCS3 mRNA or cDNA, or any combination of such mRNA or cDNA molecules, produced by a subject mammal. That is, a given binding site or unique set of binding sites in the microarray will specifically bind the product (e.g., mRNA or cDNA) of a single gene, e.g., Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1 or SOCS3.

Preferably, the nucleotide sequence of each of the different polynucleotide bound to the surface is in the range of about 15 to about 100 nucleotides in length. Polynucleotides can be synthesized using conventional methods, such as phosphoramidite-based synthesis methods. Alternatively, the binding site polynucleotide sequences can be derived from cDNA or genomic clones.

DNA microarrays can be probed using mRNA, extracted and, optionally, reverse-transcribed and amplified from a sample (e.g., a transplant, autoimmune or control sample). Nucleic acid hybridization and wash conditions are optimally chosen so that the probe “specifically binds” or “specifically hybridizes” to a specific array site, i.e., the probe hybridizes, duplexes or binds to a sequence array site with a complementary nucleic acid sequence but does not hybridize to a site with a non-complementary nucleic acid sequence. As used herein, one polynucleotide sequence is considered complementary to another when, if the shorter of the polynucleotides is less than or equal to 25 bases, there are no mismatches using standard base-pairing rules or, if the shorter of the polynucleotides is longer than 25 bases, there is no more than a 5% mismatch. Preferably, the polynucleotides are perfectly complementary (no mismatches). It can easily be demonstrated that specific hybridization conditions result in specific hybridization by carrying out a hybridization assay including negative controls (see, e.g., Shalon et al., 1996, Genome Research 6:639-645, and Chee et al., 1996, Science 274:610-614) or positive controls. Thus, in a preferred embodiment, a microarray of the invention further comprises a binding site designed to act as a negative control and/or a binding site designed to act as a positive control. For example, a positive control can relate to a constitutively expressed gene sequence, e.g., a ubiquitin sequence, HSC70, or GADPH. A negative control can relate to a gene sequence not expressed in the test cell or tissue being assayed.

›NUCLEIC ACID DETECTION · 2 of 2

Exemplary, non-limiting examples of hybridization conditions that can be utilized with DNA microarrays are as follows: hybridization in 5×SSC plus 0.2% SDS at 65° C. for 4 hours followed by washes at 25° C. in low stringency wash buffer (1×SSC plus 0.2% SDS) followed by 10 minutes at 25° C. in high stringency wash buffer (0.1×SSC plus 0.2% SDS) (Shena et al., 1996, Proc. Natl. Acad. Sci. USA, 93:10614-19).

The use of a two-color fluorescence labeling and detection scheme to define alterations in gene expression has been described, e.g., in Shena et al., 1995, Science 270:467-470. An advantage of using mRNA, cRNA, or cDNA labeled with two different fluorophores is that a direct and internally controlled comparison of the mRNA levels corresponding to each arrayed gene in two cell states (e.g., control and transplant) can be made, and variations due to minor differences in experimental conditions (e.g., hybridization conditions) will not affect subsequent analyses. However, it will be recognized that it is also possible to use cDNA from a single cell, and compare, for example, the absolute amount of a particular mRNA in, e.g., a transplant or autoimmune sample cell.

To facilitate detection the mRNA or cDNA are typically labeled with fluorescent dyes that emit at different wavelengths. Examples of fluorescent dyes include, but are not limited to, rhodamine, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. The fluorescence emissions at each site of a DNA array can be, preferably, detected by scanning confocal laser microscopy. In one embodiment, a separate scan, using the appropriate excitation line, is carried out for each of two fluorophores used. Alternatively, a laser can be used that allows simultaneous specimen illumination at wavelengths specific to the two fluorophores and emissions from the two fluorophores can be analyzed simultaneously (see, e.g., Shalon et al., 1996, Genome Research 6:639-645).

Signals are recorded and, in a preferred embodiment, analyzed by computer, e.g., using a 12 bit analog to digital board. In one embodiment the scanned image is despeckled using a graphics program (e.g., Hijaak Graphics Suite) and then analyzed using an image gridding program that creates a spreadsheet of the average hybridization at each wavelength at each site.

It will be appreciated that when mRNA or cRNA is hybridized to a microarray under suitable hybridization conditions, the level of hybridization to the site in the array corresponding to any particular gene will reflect the prevalence in the cell of mRNA transcribed from that gene. For example, when detectably labeled (e.g., with a fluorophore) cRNA complementary to the total cellular mRNA is hybridized to a microarray, the site on the array corresponding to a gene (i.e., capable of specifically binding the product of the gene) that is not transcribed in the cell will have little or no signal (e.g., fluorescent signal), and a gene for which the encoded mRNA is prevalent will have a relatively strong signal.

Microarrays can be made in a number of ways well known to those of skill in the art. With respect to the nucleic acids of the binding sites, the nucleic acid for the microarray can be generated by synthesis of synthetic polynucleotides or oligonucleotides, e.g., using N-phosphonate or phosphoramidite chemistries (e.g., Froehler et al., 1986, Nucleic Acid Res 14:5399-5407). In some embodiments, synthetic nucleic acids include non-natural bases, e.g., inosine. Additionally, it is possible to vary the charge on the phosphate backbone of the oligonucleotide, for example, by thiolation or methylation, or even to use a peptide rather than a phosphate backbone. The making of such modifications is within the skill of one trained in the art. Further, nucleic acid analogues may be used as binding sites for hybridization. An example of a suitable nucleic acid analogue is peptide nucleic acid (see, e.g., Egholm et al., 1993, Nature 365:566-568; see also U.S. Pat. No. 5,539,083, Cook et al., entitled “Peptide nucleic acid combinatorial libraries and improved methods of synthesis,” issued Jul. 23, 1996). In addition, binding (hybridization) sites can also be made from plasmid or phage clones of genes, cDNAs (e.g., expressed sequence tags), or inserts therefrom (Nguyen et al., 1995, Genomics 29:207-209). In yet another embodiment, the polynucleotide of the binding sites is RNA.

The nucleic acid or analogue is attached to a solid support to produce the binding site. Solid supports may be made from glass, silicon, plastic (e.g., polypropylene, nylon, polyester), polyacrylamide, nitrocellulose, cellulose acetate or other materials. In general, non-porous supports, and glass in particular, are preferred. The solid support may also be treated in such a way as to enhance binding of oligonucleotides thereto, or to reduce non-specific binding of unwanted substances thereto. Preferably, the glass support is treated with polylysine or silane to facilitate attachment of oligonucleotides to the slide.

Methods of immobilizing DNA on the solid support may include direct touch, micropipetting (Yershov et al., Proc. Natl. Acad. Sci. USA, 1996, 93:4913-4918), or the use of controlled electric fields to direct a given oligonucleotide to a specific spot in the array (U.S. Pat. No. 5,605,662). In principal, any type of array, for example, dot blots on a nylon hybridization membrane (see Sambrook et al., 1989 , Molecular Cloning—A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), can used, although, as will be recognized by those of skill in the art, very small arrays are be preferred because hybridization volumes will be smaller. DNA can typically be immobilized at a density of 50, 75, 100, up to 10,000 oligonucleotides per cm 2 and preferably at a density of about 1000 oligonucleotides per cm 2 .

In addition, nucleic acids can be attached to a surface by printing on glass plates (Schena et al., 1995, Science 270:467-470; DeRisi et al., 1996, Nature Genetics 14:457-460; Shalon et al., 1996, Genome Res. 6:639-645; and Schena et al., Proc. Natl. Acad. Sci. USA, 1996, 93(20):10614-19.) As an alternative to immobilizing pre-fabricated oligonucleotides onto a solid support, it is possible to synthesize oligonucleotides directly on the support (Maskos et al., 1993, Nucl. Acids Res. 21: 2269-70; Fodor et al., 1991, Science 251: 767-73 ; Lipshutz et al., 1999, Nat. Genet. 21(1 Suppl):20-4; McGall et al., Proc. Natl. Acad. Sci. USA 93: 13555-60, 1996). Other methods for making microarrays, e.g., by masking (Maskos and Southern, 1992, Nuc. Acids Res. 20:1679-1684), may also be used.

›PROTEIN DETECTION · 1 of 11

Standard techniques can also be utilized for determining the amount of the protein or proteins of interest (that is, Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1 and/or SOCS3 protein) present in a sample. It is to be understood, that such a determination of the amount of a protein present includes determining the total amount of a protein present, and also includes, especially with respect to determining the amount of a Stat protein present, determining the amount of a phosphorylated form of the protein present.

For example, standard techniques can be employed using, e.g., immunoassays such as, for example, Western blot, immunoprecipitation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), immunocytochemistry, and the like to determine the amount of the protein or proteins of interest present in a sample. A preferred agent for detecting a protein of interest is an antibody capable of binding to a protein of interest, preferably an antibody with a detectable label.

With respect to determining the amount of a phosphorylated form of a protein of interest that is present in a sample, such a determination can also be performed using standard techniques well known to those of skill in the art. For example, such a determination can include, first, immunoprecipitation with an antibody that is specific for a phosphorylated amino acid residue, e.g., an anti-phosphotyrosine antibody, such that all exhibiting such a phosphorylated residue in a sample will be inmmunoprecipitated. Second, the immunoprecipitated proteins can be contacted with a second antibody that is specific for the particular protein of interest, e.g., Stat1, Stat2, Stat3, Stat4, or Stat6. Alternatively, a phosphorylated protein of interest can be identified and quantitated using an antibody specific for the phosphorylated form of the particular protein itself, e.g, an antibody specific for phosphorylated Stat1 that does not recognize non-phosphorylated Stat1. Such antibodies exist, and are well known to those of skill in the art.

For such detection methods, protein from the sample to be analyzed can easily be isolated using techniques which are well known to those of skill in the art. Protein isolation methods can, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D., 1988, “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Preferred methods for the detection of the protein or proteins of interest involve their detection via interaction with a protein-specific antibody. For example, antibodies directed a protein of interest can be utilized as described herein. Antibodies directed against Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1 or SOCS3 protein are well known to those of skill in the art. For example, antibodies directed against Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1 or SOCS3 can be obtained from such companies as Zymed Laboratories, Inc. (South San Francisco, Calif.), Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.), and Research Diagnostics, Inc., (Flanders, N.J.). Alternatively, such antibodies can be generated utilizing standard techniques well known to those of skill in the art. See, e.g., Section 5.3, below, for a more detailed discussion of such antibody generation techniques. Briefly, such antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab′) 2 ) can, for example, be used.

For example, antibodies, or fragments of antibodies, specific for a protein of interest can be used to quantitatively or qualitatively detect the presence of the protein. This can be accomplished, for example, by immunofluorescence techniques. Antibodies (or fragments thereof) can, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of a protein of interest. In situ detection can be accomplished by removing a histological specimen (e.g., a biopsy specimen) from a patient, and applying thereto a labeled antibody thereto that is directed to a Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1 or SOCS3 protein. The antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the protein of interest, but also its distribution, its presence in lymphocytes within the sample. A wide variety of well-known histological methods (such as staining procedures) can be utilized in order to achieve such in situ detection.

Immunoassays for a protein of interest typically comprise incubating a biological sample, e.g., a biopsy or subject blood sample, of a detectably labeled antibody capable of identifying a protein of interest, and detecting the bound antibody by any of a number of techniques well-known in the art. As discussed in more detail, below, the term “labeled” can refer to direct labeling of the antibody via, e.g., coupling (i.e., physically linking) a detectable substance to the antibody, and can also refer to indirect labeling of the antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody.

The biological sample can be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble proteins. The support can then be washed with suitable buffers followed by treatment with the detectably labeled fingerprint gene-specific antibody. The solid phase support can then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on solid support can then be detected by conventional means.

By “solid phase support or carrier” is intended any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material can have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration can be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface can be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.

›PROTEIN DETECTION · 2 of 11

One of the ways in which a Stat1-, Stat2-, Stat3-, Stat4-, Stat6-, SOCS1- or SOCS3-specific antibody can be detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA) (Voller, A., “The Enzyme Linked Immunosorbent Assay (ELISA)”, 1978, Diagnostic Horizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, Md.); Voller, A. et al., 1978, J. Clin. Pathol. 31:507-520; Butler, J. E., 1981, Meth. Enzymol. 73:482-523; Maggio, E. (ed.), 1980, ENZYME IMMUNOASSAY, CRC Press, Boca Raton, Fla.; Ishikawa, E. et al., (eds.), 1981, ENZYME IMMUNOASSAY, Kgaku Shoin, Tokyo). The enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be accomplished by calorimetric methods which employ a chromogenic substrate for the enzyme. Detection can also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

Detection can also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect a protein of interest through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope (e.g. 125 I, 131 I, 35 S or 3 H) can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound can be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

5.2. Methods and Compositions for Identifying Compounds That Reduce Immune Rejection

As demonstrated below, immune rejection can be reduced and tolerance can be induced by modulating the amount of particular members of the Jak/Stat pathway present, expressed or active within an affected tissue. Specifically, the results presented herein demonstrate that modulation of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1 and/or SOCS3 levels can reduce immune rejection.

The methods described herein identify compounds that modulate the expression and/or activity of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1 or SOCS3 sequences in a manner that can reduce immune rejection (either, for example, in a transplant situation or in an autoimmune situation). The compounds identified via such methods are, therefore, useful as lead compounds in the development of therapeutic compositions for the reduction of immune rejection. Such methods are particularly useful in that the effort and great expense involved in testing potential therapeutics in vivo is efficiently focused on those compounds identified via the in vitro and ex vivo methods described herein.

Thus, the present invention relates to a method for identifying a compound to be tested for an ability to reduce immune rejection, said method comprises: (a) contacting an activated T cell sample with a test compound; (b) determining the amount of at least one of the following: (i) Stat1 mRNA or Stat1 protein, (ii) Stat2 mRNA or Stat2 protein, (iii) Stat3 mRNA or Stat3 protein, (iv) Stat4 mRNA or Stat4 protein, (v) Stat6 mRNA or Stat6 protein; (vi) SOCS1 mRNA or SOCS1 protein, or (vii) SOCS3 mRNA or SOCS3 protein, present in (a); and (c) comparing the amount(s) in (a) to that/those present in a corresponding control activated T cell sample that has not been contacted with the test compound, so that if the amount of (i), (ii), (iii), (iv), (vi), or (vii) is decreased, or the amount of (v) is increased, relative to the amount in the control sample, a compound to be tested for an ability to reduce immune rejection is identified. In alternate embodiments, such methods comprise determining the amount of at least two, at least three, at least four, at least five, at least six, or each of (i) to (vii) present in the activated T cell sample and comparing the amounts to those present in the control sample.

›PROTEIN DETECTION · 3 of 11

In a preferred embodiment of a method for identifying a compound to be tested for an ability to reduce immune rejection, said method comprises: (a) contacting an activated T cell sample with a test compound; (b) determining the amount of Stat4 mRNA and Stat6 mRNA or Stat4 protein and Stat6 protein present in the sample; and (c) comparing the amounts in (a) to those present in a corresponding control activated T cell sample that has not been contacted with the test compound, so that if the amount of Stat4 is decreased or the amount of Stat6 is increased relative to the amount in the control sample, a compound to be tested for an ability to reduce immune rejection is identified.

In another preferred embodiment of a method for identifying a compound to be tested for an ability to reduce immune rejection, said method comprises: (a) contacting an activated T cell sample with a test compound; (b) determining the ratio of Stat4 mRNA to Stat6 mRNA or Stat4 protein to Stat6 protein present in the sample; and (c) comparing the ratio in (a) to that present in a corresponding control activated T cell sample that has not been contacted with the test compound, so that if the ratio in (a) is decreased relative to that in the control sample, a compound to be tested for an ability to reduce immune rejection is identified.

Optionally, such methods can further include comparing the amount or amounts in (a) to a control resting T cell sample that has not been contacted with the T cell activator or with the test compound and/or with a control resting T cell sample that has been contacted with the test compound, but has not been contacted with a T cell activator (and, therefore, remains in the resting state). Such controls provide evidence regarding the specificity and toxicity of the test compound.

In a preferred embodiment of a method for identifying a compound to be tested for an ability to reduce immune rejection, said method comprises: (a) contacting a resting T cell sample, a T cell activator and a test compound; (b) determining the amount of Stat4 mRNA and Stat6 mRNA or Stat4 protein and Stat6 protein present in the sample; and (c) comparing the amounts in (a) to those present in a corresponding control resting T cell sample that has been contacted with the T cell activator, but has not been contacted with the test compound, so that if the amount of Stat4 is decreased or the amount of Stat6 is increased relative to the amount in the control sample, a compound to be tested for an ability to reduce immune rejection is identified.

In another preferred embodiment of a method for identifying a compound to be tested for an ability to reduce immune rejection, said method comprises: (a) contacting a resting T cell sample, a T cell activator and a test compound; (b) determining the ratio of Stat4 mRNA to Stat6 mRNA or Stat4 protein to Stat6 protein present in the sample; and (c) comparing the ratio in (a) to that present in a corresponding control resting T cell sample that has been contacted with a T cell activator, but has not been contacted with the test compound, so that if the ratio in the sample is decreased relative to that in the control samples, a compound to be tested for an ability to reduce immune rejection is identified.

›PROTEIN DETECTION · 4 of 11

Cytokines that can be used as part of these methods include, but are not limited to, IL-2, IL-4, IL-12, or IL-13.

Standard methods and compositions for determining the amount of Stat1 mRNA or protein, Stat2 mRNA or protein, Stat3 mRNA or protein, Stat4 mRNA or protein, Stat6 mRNA or protein, SOCS1 mRNA or protein, and SOCS3 mRNA or protein can be utilized. Such methods and compositions are described in detail, above, in Section 5.1.

In addition to the ability to modulate Stat and/or SOCS levels as described herein, it may be desirable, at least in certain instances, that compounds that reduce immune rejection also modulate the expression or activity of such molecules as IL-4, interferon-γ (IFN-γ), IL-12, or IL-13. Thus, the methods described herein for identifying compounds to be tested for an ability to reduce immune rejection can further comprise determining the level of IL-4, IFN-γ or IL-13 in the T cell sample that has been contacted with the test compound, and comparing this level with that of the control T cell sample that has not been contacted with the test compound. Preferred compounds are ones wherein: the level of IL-12 or IFN-γ in the test sample is decreased relative to the corresponding level in the control sample, or wherein the level of IL-4 or IL-13 in the test sample is equal to or greater than the corresponding level in the control sample.

The present methods of identifying compounds that to be tested for an ability to reduce immune rejection, can comprise methods for identifying compounds that modulate the activity of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1, or SOCS3. Thus, such methods can comprise: (a) contacting a T cell sample with a test compound; (b) determining the activity of at least one of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1, or SOCS3; and (c) comparing the activity level or levels to that/those in a corresponding control T cell sample that has not been contacted with the test compound, so that if the level of Stat1, Stat2, Stat3, Stat4, SOCS1 or SOCS3 activity in (a) is decreased, or the level of Stat6 activity in (a) is increased, relative to the level of activity in the control sample, a compound to be tested for an ability to reduce immune rejection is identified.

In one preferred embodiment, the activity of Stat4 and Stat6 is determined. Such a preferred embodiment can further include determining the ratio of Stat4 activity to Stat6 activity so that if the ratio in the test sample is decreased relative to that in the control sample, a compound to be tested for an ability to reduce immune rejection is identified.

The T cell sample utilized herein can be an activated T cell sample, a resting T cell sample, or a cytokine-responsive T cell sample, as discussed above. In instances wherein the T cell sample is a resting T cell sample, the T cell sample is contacted with a T cell activator and the test compound. In instances wherein the T cell sample is a cytokine-responsive T cell sample, the T cell sample is contacted with the cytokine and the test compound.

Standard techniques can be utilized to determine the level of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1 or SOCS3 activity. For example, the activity of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1 or SOCS3 can be determined by detecting the binding of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1 or SOCS3 to its cognate DNA binding element, via, for example, an electromobility shift assay (“EMSA”), detecting the expression of a gene whose expression is controlled by a promoter that is responsive to Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1 or SOCS3, detecting the induction of a reporter gene that comprises a regulatory element that is responsive to Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1 or SOCS3, wherein the element is operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase.

Genes whose expression is controlled by a Stat 1-responsive promoter are well known, and include, for example GBP-1, inducible NO synthase (iNOS), ICAM, IRF-1, major histocompatibility complex (MHC) class II transactivator (CIITA). See, e.g., Lew et al., 1991, Mol. Cell. Biol. 11:182-191, Gao et al., 1997, J. Biol. Chem. 272:1226-1230, Caldenhoven et al., 1994, J. Biol. Chem. 269:21146-21154, Sims et al., 1993, Mol. Cell. Biol. 13:690-702, Pine et al., 1994, EMBO J. 13:158-167, Harada et al., 1994, Mol. Cell. Biol. 14:1500-1509, and Piskurich et al., 1999, Mol. Cell. Biol. 19(1):431-40. Thus, expression of such genes in the T cell sample in the presence and absence of a test compound can routinely be determined using standard techniques. Likewise, the structure of Stat 1-responsive promoters are well known (see, e.g., Sims et al., 1993, Mol. Cell. Biol. 13:690-702, Pine et al., 1994, EMBO J. 13:158-167, and Piskurich et al., 1999, Mol. Cell. Biol. 19(1):431-40), making the construction and assay of Stat 1-reporter genes routine.

›PROTEIN DETECTION · 5 of 11

Genes whose expression is controlled by a Stat 2-responsive promoter are well known, and include, for example IRF-1. See, e.g., Li et al., 1996, J. Biol. Chem. 271(10):5790-5794. Thus, expression of such genes in the T cell sample in the presence and absence of a test compound can routinely be determined using standard techniques. Likewise, the structure of Stat 2-responsive promoters are well known (see, e.g., Ghislain et al., 1996, J. Biol. Chem. 271(21):12408-12413 and Li et al., 1996, J. Biol. Chem. 271(10):5790-5794), making the construction and assay of Stat 2-reporter genes routine.

Genes whose expression is controlled by a Stat 3-responsive promoter are well known, and include, for example alpha-2-macroglobulin, fibrinogen, junb, haptoglobin, matrix metalloproteinase (MMP-1), TIMP-1, and p21 WAF/CIPI See, e.g., Wegenka et al., 1993, Mol. Cell. Biol. 13:276-288, Fujitani et al., 1994, Biochem. Bioph. Res. Co. 202:1181-1187, Coffer et al., 1995, Oncogene 10:985-994, Akira et al., 1994, Cell 77:63-71, and Chin et al., 1996, Science 272:719-722. Thus, expression of such genes in the T cell sample in the presence and absence of a test compound can routinely be determined using standard techniques. Likewise, the structure of Stat 3-responsive promoters are well known (see, e.g., Wegenka et al., 1993, Mol. Cell. Biol. 13:276-288 and Chin et al., 1996, Science 272:719-722), making the construction and assay of Stat 3-reporter genes routine.

Genes whose expression is controlled by a Stat 4-responsive promoter are well known, and include, for example interferon-γ and IL-12. See, e.g., Grigorieva et al., 2000, J Biol. Chem. 275(10):7343-7350 and Naeger, L. K. et al., 1999, J. Biol. Chem. 274:1875-1878). Thus, expression of such genes in the T cell sample in the presence and absence of a test compound can routinely be determined using standard techniques. Likewise, the structure of Stat 4-responsive promoters are well known (see, e.g., Grigorieva et al., 2000, J Biol. Chem. 275(10):7343-7350 and Naeger, L. K. et al., 1999, J. Biol. Chem. 274:1875-1878), making the construction and assay of Stat 4-reporter genes routine.

Genes whose expression is controlled by a Stat 6-responsive promoter are well known, and include, for example IL-4, CD23, IL-4 receptor, MHC class II. See, e.g., Tinnell et al., 1998, Int. Immunol. 10(10):1529-38, Linehan et al., 1998, J. Immunol. 161(1):302-10, and Kotanides et al., 1996, J. Biol. Chem. 271(41):25555-25561. Thus, expression of such genes in the T cell sample in the presence and absence of a test compound can routinely be determined using standard techniques. Likewise, the structure of Stat 6-responsive promoters are well known (see, e.g., Curiel, R. E. et al., 1997, Eur. J. Imm. 27:1982-1987, Linehan et al., 1998, J. Immunol. 161(1):302-10, and Kotanides et al., 1996, J. Biol. Chem. 271(41):25555-25561), making the construction and assay of Stat 6-reporter genes routine.

EMSAs can also routinely be utilized to assess Stat1, Stat2, Stat3, Stat4 or Stat6 activity. Such techniques are well known to those of skill in the art. See, e.g., Amici et al., 1995, Cancer Research 55: 14452-4457. Briefly, in a representative, non-limiting example, extracts of cells treated with a test compound are mixed with 32 P-Stat 4 (or Stat1, 2, 3, or 6) element or a control oligonucleotide and poly(dI-dC) (Pharmacia Biotech Inc.) in binding buffer (e.g., Tris-Cl, pH 7.8, 50 mM NaCl, 1 mM EDTA, 0.5 mM dithiothreitol, 5% glycerol). After an approximately 20 minute incubation at room temperature, Stat4 (or Stat1, 2, 3, or 6)-DNA-complexes or control oligonucleotide complexes are analyzed by nondenaturing 4% polyacrylamide gel electrophoresis and autoradiography. The amount of shifted Stat4 (or Stat 1, 2, 3, or 6) probe, an indicator Stat4 (or Stat1, 2, 3, or 6) activity, respectively, can be quantitated by Molecular Dynamics Phospholmager (MDP) analysis.

The activity of SOCS1 or SOCS3 activity can be determined by, e.g., detecting the expression of a gene whose expression is controlled by SOCS1 or SOCS3. For example, SOCS1 expression inhibits IL-6, LIF, oncostatin M, IFN-γ, IFN-β, IFN-α, thrombopoeitin, and growth hormone (GH) induced Jak/Stat signaling. SOCS3 expression inhibits IFN-γ, IFN-β, IFN-α, GH and leptin. Thus, expression of such genes in the T cell sample in the presence and absence of a test compound can routinely be determined using standard techniques.

The activity of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1 or SOCS3 can also be assessed by detecting the proliferation of the T cell sample, detecting the effector function of the sample or detecting differentiation of the sample. Techniques known to those of skill in the art can be used for measuring these activities. For example, cellular proliferation can be assayed by 3 H-thymidine incorporation assays and trypan blue cell counts. The effector function of T-cells can be measured, for example, by a 51 Cr-release assay (see, e.g., Palladino et al., 1987, Cancer Res. 47:5074-5079 and Blachere et al., 1993, J. Immunotherapy 14:352-356).

As set forth above, the methods described herein for identifying compounds to be tested for an ability to reduce immune rejection assay whether a test compound has an effect on the expression and/or activity of Stat 1 mRNA or protein, Stat2 mRNA or protein, Stat3 mRNA or protein, Stat4 mRNA or protein, Stat6 mRNA or protein, SOCS1 mRNA or protein, and/or SOCS3 mRNA or protein produced by a T cell, in particular, an activated T cell, or at a minimum, a T cell that has the ability to respond to exogenous cytokines.

The T cell used as part of the methods can be one that is constitutively activated (e.g., a constitutively activated T cell line), one that has or has gained the ability to respond to cytokines, one that is activated prior to performing the method, or one that is activated concurrently with the method. A T cell to be used as part of the methods described herein can be activated either prior to or simultaneously with contacting the cell with a test compound. With respect to activated, including constitutively activated T cells, activation of such T cells can, in certain instances be further enhanced by addition and contact with a T cell activator.

›PROTEIN DETECTION · 6 of 11

An activated T cell is one that expresses antigens indicative of T-cell activation (that is, T cell activation markers). Examples of T cell activation markers include, but are not limited to, CD25, CD26, CD30, CD38, CD69, CD70, CD71, ICOS, OX-40 and 4-1BB. The expression of activation markers can be measured by techniques known to those of skill in the art, including, for example, western blot analysis, northern blot analysis, RT-PCR, immunofluorescence assays, and fluorescence activated cell sorter (FACS) analysis. The activated T cell used as part of the present methods can be an activated T cell line or can be a primary cell that has been activated.

Activated T cell lines are well known to those of skill in the art. Examples of activated T cell lines include TH1 cell lines such as AE7, PL17, and OF6, and TH2 cell lines such as D10 and CDC35.

T cell lines that do not express the T cell activation markers required to constitute activation, but nonetheless have the ability to respond to cytokines are also well known to those of skill in the art. Examples of such T cell lines include CTLL-2 and HT-2.

Alternatively, primary T cells can be isolated, the majority of which will be in a resting state, and activated using standard techniques. For example, immune cells can be collected or isolated from blood, or secondary lymphoid organs of the subject, such as but not limited to lymph nodes, tonsils, the spleen, Peyer's patch of the intestine, and bone marrow, by any of the methods known in the art. Immune cells obtained from such sources typically comprise predominantly recirculating lymphocytes and macrophages at various stages of differentiation and maturation. Optionally, standard techniques, such as morphological observation and immunochemical staining, can be used, if desired, to verify the presence of the desired cells, that is, T cells. In a preferred aspect, the immune cells used in the methods of the invention described herein are human peripheral blood compositions lacking red blood cells, e.g., whole blood leukocytes (whole peripheral blood from which the red blood cells and serum have been substantially removed), which can be collected from a human subject by standard techniques, such as by use of a syringe to withdraw the blood, followed by subjecting the blood to Ficoll-Hypaque (Pharmacia) gradient centrifugation. Blood, anticoagulated with preservative-free heparin, usually yields 0.5 to 1×10 6 lymphocytes/ml. Separated blood cells (e.g., leukocytes) may be frozen by standard techniques prior to use in the present methods. In a specific embodiment, the immune cells used are purified white blood cells comprising lymphocytes and macrophages.

In one embodiment wherein further purification of T cells is desired, antibodies against specific surface markers can be directly labeled by conjugation of a detectable compound to such antibodies to facilitate detection and separation of T cells. Alternatively, in another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Examples of detectable compounds include, but are not limited to, biotin, photobiotin, fluorescein isothiocyanate (FITC), or phycoerythrin (PE), or other compounds known in the art. Cells retaining labeled antibodies are then separated from cells that do not bind such antibodies by techniques known in the art such as, but not limited to, various cell sorting methods (e.g., FACS), affinity chromatography, and panning.

In another embodiment wherein further purification of T cells is desired, T cells are sorted using a fluorescence activated cell sorter (FACS). Fluorescence activated cell sorting (FACS) is a well-known method for separating particles, including cells, based on the fluorescent properties of the particles (Kamarch, 1987, Methods Enzymol, 151:150-165). Laser excitation of fluorescent moieties in the individual particles results in a small electrical charge allowing electromagnetic separation of positive and negative particles from a mixture.

In another embodiment wherein further purification of T cells is desired, magnetic beads can be used to separate T cells. T cells may be sorted using a magnetic activated cell sorting (MACS) technique, a method for separating particles based on their ability to bind magnetic beads (0.5-100μ diameter; Dynal, Inc., Lake Success, N.Y.) as un dertaken according to the manufacturer's instructions. A variety of useful modifications can be performed on the magnetic microspheres, including covalent addition of antibody which specifically recognizes a cell-solid phase surface molecule or hapten. A magnetic field is then applied, to physically manipulate the selected beads. The beads are then mixed with the immune cells to allow binding. Cells are then passed through a magnetic field to separate out cells having T cell surface markers.

The isolated resting T cells can then be activated by contacting with a T cell activator. Any T cell activator can be utilized for this purpose. For example, any compound or factor that is a T cell receptor stimulatory factor, that is, induces T cell receptor signalling can be used. Preferably, the compound or factor also induces co-stimulatory pathways. Representative, non-limiting examples of T cell activators include, but are not limited to, anti-CD3 antibodies (preferably monoclonal antibodies) either alone or in conjunction with anti-CD28 antibodies (preferably monoclonal antibodies), or mitogens such as, for example, phorbol 12-myristate 13-acetate (PMA), phytohemagglutinin (PHA), or concanavalin-A (ConA).

Compounds that can be tested and identified methods described herein can include, but are not limited to, compounds obtained from any commercial source, including Aldrich (1001 West St. Paul Ave., Milwaukee, Wis. 53233), Sigma Chemical (P.O. Box 14508, St. Louis, Mo. 63178), Fluka Chemie AG (Industriestrasse 25, CH-9471 Buchs, Switzerland (Fluka Chemical Corp. 980 South 2nd Street, Ronkonkoma, N.Y. 11779)), Eastman Chemical Company, Fine Chemicals (P.O Box 431, Kingsport, Tenn. 37662), Boehringer Mannheim GmbH (Sandhofer Strasse 116, D-68298 Mannheim), Takasago (4 Volvo Drive, Rockleigh, N.J. 07647), SST Corporation (635 Brighton Road, Clifton, N.J. 07012), Ferro (111 West Irene Road, Zachary, La. 70791), Riedel-deHaen Aktiengesellschaft (P.O. Box D-30918, Seelze, Germany), PPG Industries Inc., Fine Chemicals (One PPG Place, 34th Floor, Pittsburgh, Pa. 15272). Further any kind of natural products may be screened using the methods of the invention, including microbial, fungal, plant or animal extracts.

›PROTEIN DETECTION · 7 of 11

Furthermore, diversity libraries of test compounds, including small molecule test compounds, may be utilized. For example, libraries may be commercially obtained from Specs and BioSpecs B. V. (Rijswijk, The Netherlands), Chembridge Corporation (San Diego, Calif.), Contract Service Company (Dolgoprudny, Moscow Region, Russia), Comgenex USA Inc. (Princeton, N.J.), Maybridge Chemicals Ltd. (Cornwall PL34 OHW, United Kingdom), and Asinex (Moscow, Russia).

Still further, combinatorial library methods known in the art, can be utilize, including, but not limited to: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the “one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, 1997, Anticancer Drug Des.12:145). combinatorial libraries of test compounds, including small molecule test compounds, can be utilized, and may, for example, be generated as disclosed in Eichler & Houghten, 1995, Mol. Med. Today 1:174-180; Dolle, 1997, Mol. Divers. 2:223-236; and Lam, 1997, Anticancer Drug Des. 12:145-167.

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al., 1993, Proc. Natl. Acad. Sci. USA 90:6909; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al., 1994. J. Med. Chem. 37:2678; Cho et al., 1993, Science 261:1303; Carrell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al., 1994, J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten, 1992, Bio/Techniques 13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci. USA 89:1865-1869) orphage (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; and Felici, 1991, J. Mol. Biol. 222:301-310).

Among the test compounds that can be tested are compounds, including small organic molecule compounds that act as protein tyrosine kinase inhibitors, including, but not limited to, the tyrphostin class of protein tyrosine kinase inhibitors (see, e.g., Gazit et al. 1989, J. Medicinal Chem. 32:2344-2352; and Levitski, 1992, FASEB J. 6:3275). Among such tyrphostin compounds that can be tested are A25 (available, e.g., from CalBiochem) or AG-490 (B42) (Meydan, N. et al., 1996, Nature 379:645-648) or derivatives thereof. Further, among the compounds that can be tested are compounds that interfere with SH2 domain interactions (see, e.g., U.S. Pat. No. 5,710,129; 5,776,902; and 5,580,97, or derivatives of compounds therein), e.g., SH2-mediated Stat 4/IL-12 receptor-β 2 interactions. Still further, among the compounds that can be tested are compounds that interfere with Jak 2/IL-12 receptor-β 2 interactions and/or ones that interfere with Tyk 2/IL-12 receptor-β 1 , interactions.

Upon identification of compounds to be tested for an ability to reduce immune rejection, the compounds can be further investigated. In particular, for example, the compounds identified via the present methods can be further tested in vivo in accepted animal models of transplant or autoimmune disorders. Further, the compounds identified can also be analyzed with respect to their specificity. In particular, the compounds can be tested for an effect on platelet aggregation and/or on NF-κB activation. Techniques for such additional compound investigation are described below.

Accepted animal models can be utilized to determine whether the compounds identified via the methods described herein. Such models can include both transplant-related models as well as autoimmune disorder models.

For example, the ability of a compound to reduce immune rejection, including the ability of the compound to induce tolerance in a subject mammal that has undergone a transplant can include, but are not limited to, a murine allograft model in which an allogeneic heart is transplanted into a subject mouse recipient (Hancock et al., 1998, Nature Medicine 4:1392-1396). In addition, primate models can also be tested. Such models include, for example, a primate renal allograft model (Kirk et al., 1997, Proc. Natl. Acad. Sci. USA 94:8789-8794). In addition, a graft versus host disease (GVHD) model can be used (see, e.g., Guillen et al., 1986, Laboratory Investigation 55:35-42). In such models, chronic and acute GVHD is made to result from introduction of donor cells into a host exhibiting disparate MHC alleles. The GVHD results, therefore, from the donor cells' response to such the host's disparate MHC alleles.

The ability of a compound to reduce immune rejection can also be tested in such autoimmune disorder models as, first, an experimental allergic encephalomyelitis (EAE) model. EAE is an experimental autoimmune disease of the central nervous system (CNS) (Zamvil et al., 1990, Ann. Rev, Immunol. 8:579) and is a disease model for the human autoimmune condition, multiple sclerosis (MS). EAE is an example of a cell-mediated autoimmune disorder that is mediated via T cells. No direct evidence exists for an autoantibody requirement in disease progression. EAE is readily induced in mammalian species by immunizations of myelin basic protein purified from the CNS or an encephalitogenic proteolipid (PLP). SJL/J mice are a susceptible strain of mice (H-2′) and, upon induction of EAE, these mice develop an acute paralytic disease and an acute cellular infiltrate is identifiable within the CNS.

In addition, a collagen-induced arthritis (CIA) model can be utilized to determine whether the compound of interest reduce immune rejection. CIA is an animal model for the human autoimmune disease rheumatoid arthritis (RA) (Trenthom et al., 1977, J. Exp. Med., 146:857). This disease can be induced in many species by the administration of heterologous type II collagen (Courtenay et al., 1980, Nature 283:665; Cathcart et at, 1986, Lab. Invest., 54:26). With respect to animal models of arthritis see, in addition, e.g., Holmdahl, R., 1999, Curr. Biol. 15:R528-530.

›PROTEIN DETECTION · 8 of 11

Still further, animal models for type 1 diabetes, thyroid autoimmunity or systemic lupus erythematosus, including glomerulonephritis can be utilized to determine whether the compound of interest reduces immune rejection (see, e.g., Flanders et al., 1999, Autoimmunity 29:235-246; Krogh et al., 1999, Biochimie 81:511-515; and Foster, N. H., 1999, Semin. Nephrol. 19:12-24, respectively).

In addition, it is preferred that compounds to be utilized as therapeutic according to the methods described herein not induce platelet aggregation. Therefore, it is preferable that compounds identified via the methods described herein that are to be tested for an ability to reduce immune rejection be further tested for an ability to induce platelet aggregation. In vitro and ex vivo assays for platelet aggregation are well known and compounds of interest can easily be tested via such assays.

Specifically, such assays include, but are not limited to the turbidometric method, in which aggregation is measured as an increase in transmission of visible light through a stirred or agitated platelet suspension. See, e.g., Chanarin, L., 1989, Laboratory Haematology, Chapter 30, Churchill, Livingstone, London; and Schmidt, R. M. (ed), 1979, CRC Handbook Series in Clinical Laboratory Science, CRC Press, Inc.: Boca Raton, Fla.

Platelet aggregation can also be assayed via methods such as those described in U.S. Pat. No. 5,976,532. For example, in a non-limiting example of such a method, the platelet concentration in platelet-rich plasma obtained (PRP) obtained from blood samples is adjusted to 200,000 to 300,000/mm 3 . In an in vitro assay, the PRP is aliquoted and incubated in the presence or absence of a compound of interest for a period of time (e.g., 15 minutes at 37° C.) prior to the addition of a platelet inducing agonist (e.g., ADP, thrombin, collagen, epinephrine, and ristocetin). In an ex vivo assay, the PRP obtained from individuals treated with the compound of interest or a placebo is aliquoted and incubated in the presence of a platelet inducing agonist (e.g., ADP, thrombin, collagen, epinephrine, and ristocetin). Platelet aggregation is measured by assessing an increase in the transmission of visible light through a platelet suspension using a spectrophotometer.

It is also preferred that compounds to be utilized as therapeutic according to the methods described herein not affect NF-κB activation, in particular, NF-κB activation in CD40L + cells. Therefore, it is preferable that compounds identified via the methods described herein that are to be tested for an ability to reduce immune rejection be further tested for possible effect on NF-κB activation in CD40L+ cells. In such tests, a CD40L + cell is contacted with the compound of interest, and its effect on NF-κB activation, if any is assayed, and compared to the level of NF-κB activation in a corresponding control CD40L + cell that has not been contacted with the compound.

Standard techniques can be utilized to test for NF-κB activation. For example, the activity of NF-κB can be assessed by detecting the binding of NF-κB to its cognate DNA binding element in an electromobility shift assay (EMSA), detecting the expression of a gene whose expression is controlled by a promoter that is responsive to NF-κB, detecting the induction of the expression of a reporter gene construct that comprises a regulatory element that is responsive to NF-κB is operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase.

Genes whose expression is controlled by an NF-κB-responsive promoter are well known, and include, for example granulocyte/macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), IL-2, IL-6, IL-8, tumor necrosis factor-α (TNF-α), and intercellular cell adhesion molecule 1 (ICAM-1). See, e.g., Baeuerle and Henkel, 1994, Ann. Rev. Immunol. 12:141-179. Thus, expression of such genes in CD40L + cells in the presence and absence of a compound of interest can routinely be determined using standard techniques. Expression can be determined using standard techniques. Preferably, the compound being tested will not increase such expression and, most preferably, will have no effect on such expression. Likewise, the structure of NF-κB-responsive promoters are well known (see, e.g., Baeuerle and Henkel, 1994, Ann. Rev. Immunol. 12:141-179; and Thanos et al., 1995, Cell 80:529-532), making the construction and assay of NF-κB reporter genes routine. Thus, the induction and expression of such reporter genes in CD40L + cells in the presence and absence of a compound of interest can routinely be determined using standard techniques. Preferably, the compound being tested will not induce expression of the reporter gene.

EMSAs can also routinely be utilized to assess NF-κB activity. Such techniques are well known to those of skill in the art. See, e.g., Amici et al., 1995, Cancer Research 55: 14452-4457. Briefly, in a representative, non-limiting example, extracts of cells treated with a test composition or control composition are mixed with 32 P-NF-κB element or a control oligonucleotide and poly(dI-dC) (Pharmacia Biotech Inc.) in binding buffer (e.g., Tris-Cl, pH 7.8, 50 mM NaCl, 1 mM EDTA, 0.5 mM dithiothreitol, 5% glycerol). After an approximately 20 minute incubation at room temperature, NF-κB-DNA-complexes or control oligonucleotide complexes are analyzed by non-denaturing 4% polyacrylamide gel electrophoresis and autoradiography. The amount of shifted NF-κB probe, an indicator NF-κB activity, respectively, can be quantitated by Molecular Dynamics PhosphoImager (MDP) analysis.

Further, the effect, if any, of the compound of interest on NF-κB activation can also be tested by assaying for a cellular response, for example, cellular differentiation, or cell proliferation. Cellular proliferation can be assayed by, for example, 3 H-thymidine incorporation assays and trypan blue cell counts. Preferably, the compound will have no effect on such cellular responses.

›PROTEIN DETECTION · 9 of 11

5.3. Methods and Compositions for Reducing Immune Rejection

Described herein are methods and compositions for reducing immune rejection in a subject mammal. In particular, such methods comprise administration of compounds that serve to reduce the amount or activity of Stat1 mRNA or protein, Stat2 mRNA or protein, Stat4 mRNA or protein, SOCS1 mRNA or protein or SOCS3 mRNA or protein, and/or increase the amount or activity of Stat6 mRNA or protein. The compounds utilized herein are ones that neither induce platelet aggregation nor affect NF-κB activation.

Immune rejection in any subject mammal that has undergone a transplant, or that exhibits or is suspected of exhibiting an autoimmune disorder can be reduced using the methods presented herein. Preferably, the mammal is a human, however, such subject mammals can also include, but are not limited to, other primates, including monkeys, as well as pigs, dogs, cats, horses, cattle, sheep, mice, rats, and rabbits.

Specifically, the present invention relates to methods for reducing immune rejection in a subject mammal, said methods comprising: administering to a subject mammal in need of such a reduction a concentration of a compound sufficient to reduce the level or activity of Stat4 mRNA or protein in the subject relative to that observed in the subject in the absence of the compound, wherein said compound does not induce platelet aggregation and does not affect NF-κB activation in CD40L + cells.

The present invention also relates to methods for reducing immune rejection in a subject mammal, said methods comprising: administering to a subject mammal in need of such a reduction a concentration of a compound sufficient to reduce the level or activity of Stat1 mRNA or protein, Stat2 mRNA or protein, or Stat3 mRNA or protein in the subject relative to that observed in the subject in the absence of the compound, wherein said compound does not induce platelet aggregation and does not affect NF-κB activation in CD40L + cells.

Such methods can also include methods for reducing immune rejection in a subject mammal, comprising administering to the subject mammal in need of such a reduction a concentration of a compound sufficient to decrease the level or activity of Stat4 mRNA or protein in the subject relative to that observed in the subject in the absence of the compound, and wherein the level or activity of Stat6 mRNA or protein in the subject is maintained or increased relative to that observed in the subject in the absence of the compound. Further, the compound administered is one that does not induce platelet aggregation or affect NF-κB activation in CD-40L + cells.

Alternatively, such methods for reducing immune rejection in a subject mammal can comprise: administering to a subject mammal in need of such a reduction a concentration of a compound sufficient to increase the level or activity of Stat6 mRNA or protein in the subject relative to that observed in the subject in the absence of the compound, wherein said compound does not induce platelet aggregation and does not affect NF-κB activation in CD40L + cells.

Such methods for reducing immune rejection in a subject mammal can also comprise: administering to a subject mammal in need of such a reduction a concentration of a compound sufficient to decrease the level or activity of Stat4 mRNA or protein and maintain or increase the level or activity of Stat6 mRNA or protein in the subject subject relative to that observed in the subject in the absence of the compound, wherein said compound does not induce platelet aggregation and does not affect NF-κB activation in CD40L + cells.

Generally, practice of these methods does not solely entail administration of compositions that are considered signal 2-type blockers (see, e.g., Gummert J. F., et al., 1999, J. Am. Soc. Nephrol. 10: 1366), that is, compounds (e.g., CD40L antibodies) that act to inhibit CD40/CD40L (CD154) interactions or B7/CD28 interactions.

It is noted, however, that embodiments of the present invention further include combinatorial immune reduction therapy utilizing compositions as taught herein in conjunction with immunosuppressive or immunomodulatory drug therapies, as described in detail, below.

The methods of the present invention for reducing immune rejection can be utilized, e.g., for reducing immune rejection in a subject mammal that has undergone a transplant. For example, such methods can induce tolerance in a subject mammal that has undergone a transplant. Such methods can be used to reduce immune reject in a transplant situation involving any cell, organ, organ system or tissue which can elicit an immune response in a recipient subject mammal. In general, therefore, a transplant includes an allograft, or a xenograft cell, organ, organ system or tissue. An allograft refers to a graft (cell, organ, organ system or tissue) obtained from a member of the same species as the recipient. A xenograft refers to a graft (cell, organ, organ system or tissue) obtained from a member of a different species as the recipient. In particular, the transplant can, for example, be an allograft heart, liver, kidney, lung, bone marrow, skin, muscle, pancreatic islet, intestine or cornea transplant.

The methods of the present invention for reducing immune rejection can also be utilized, e.g., for reducing immune rejection in a subject mammal exhibiting an autoimmune disorder. Thus, the present invention can treat an autoimmune disorder affecting any body cell, tissue, organ or organ system, including but not limited to cutaneous, cardiac, pericardial, endocardial, vascular lining or wall, blood, blood-forming (e.g., marrow or spleen), endocrine (e.g., pancreatic or thyroid), gastrointestinal (e.g., bowel), respiratory (e.g., lung), renal, central nervous system, peripheral nervous system, muscular or skeletal joint (e.g., articular cartilage or synovial) tissue. The methods and compositions of the present invention can, therefore, be utilized to treat any autoimmune disorder including, but not limited to atopic dermatitis, contact dermatitis, eczematous dermatitides, seborrheic dermatitis, Lichen planus, Pemphilgus, bullous pemphigus, Epidermolysis bullosa, Alopecia areata, urticaria, angioedemas, erythema, eosinophilias, migraine, lupus, including cutaneous lupus (discoid lupus erythematosus), extracutaneous lupus, including systemic lupus erythematosus, acute lupus, lupus annularis, lupus discretus, lupus lymphaticus, lupus papillomatis, lupus psoriasis, lupus vulgaris, lupus sclerosis, neonatal lupus erythematosus, and drug-induced lupus; anti-phospholipid syndrome (APS), hemolytic anemia (HA), idiopathic thrombocytopenia (ITP), thyroiditis, diabetes mellitus (DM), inflammatory bowel disease, e.g., Crohn's disease or ulcerative cholitis, rhinitis, uveitis, nephrotic syndrome, demyelinating diseases such as multiple sclerosis (MS), myasthenia gravis (MG), and arthritis, e.g., rheumatoid arthritis, non-rheumatoid inflammatory arthritis, arthritis associated with Lyme disease, or osteoarthritis.

›PROTEIN DETECTION · 10 of 11

The compounds utilized as part of these methods include, but are not limited to, ones identified via the methods described above. A number of different points along the Jak/Stat pathway can be targeted by the compounds utilized as part of the methods for reducing immune rejection described herein. Administration methods, including gene therapy methods, and pharmaceutical preparations by which such compounds can routinely be utilized as part of methods for reducing immune rejection are taught below.

For example, compounds that specifically downregulate Stat4 mRNA or protein levels or activity, while not affecting NF-κB activation or platelet aggregation can be utilized as part of these methods. In addition, compounds that specifically downregulate Stat1 mRNA or protein levels or activity, while not affecting NF-κB activation or platelet aggregation can be utilized as part of these methods. Also, compounds that specifically downregulate Stat2 mRNA or protein levels or activity, while not affecting NF-κB activation or platelet aggregation can be utilized as part of these methods. In addition, compounds that specifically downregulate Stat3 mRNA or protein levels or activity, while not affecting NF-κB activation or platelet aggregation can be utilized as part of these methods. In addition, compounds that specifically downregulate SOCS1 mRNA or protein evels or activity, while not affecting NF-κB activation or platelet aggregation can be utilized as part of these methods. Likewise, compounds that specifically downregulate SOCS3 mRNA or protein levels or activity, while not affecting NF-κB activation or platelet aggregation can be utilized as part of these methods. In addition, compounds or methods that specifically increase Stat 6 mRNA or protein levels or activity, while not affecting NF-κB activation or platelet aggregation can be utilized as part of these methods. Representative, non-limiting examples of such compounds are described in detail below.

First, such compounds can include, for example, antisense, ribozyme, or triple helix compounds that can downregulate the expression or Stat1, Stat2, Stat3, Stat4, SOCS1 or SOCS3. Such compounds are described in detail in the subsection below.

Second, such compounds can include, for example, antibody compositions that can downregulate the expression or activity of Stat1, Stat2, Stat3, Stat4, SOCS1 or SOCS3. Such compounds are described in detail in the subsection below.

Further, among such compounds are ones, including ones administered via gene therapy techniques, that serve to upregulate Stat6 expression or activity, and compounds that act in maintaining Stat6 expression or activity levels as Stat4 activity or expression levels are decreased.

Compositions can include, for example ones can be utilized that compete with Stat4 for binding to the IL-12 receptor β 2 (“IL-12Rβ 2 ”). Examples of such compounds include, but are note limited to limited to, peptide compositions as in Naeger, L. K. et al., 1999, J. Biol. Chem. 274:1875-1878. Additional examples of compounds that can be utilized include compounds, such as small organic compounds that act as inhibitors of SH2 domain-mediated interactions such as SH2-mediated Stat4/IL-12Rβ 2 interactions. One example of such an embodiment involves a composition of the invention comprises one or more peptides that bind to the Stat4 SH2 domain which prevent Stat4 from binding to the IL-12Rβ 2 , or that comprise one or more dominant-negative Stat4 polypeptides (e.g., a Stat4 polypeptide lacking its SH2 domain or a Stat4 polypeptide lacking its DNA binding domain). Examples of such polypeptides include, but are not limited to, (using the standard one-letter amino acid code) phospho-YLPSNID peptides (Naeger, L. K. et al., 1999, J. Biol. Chem. 274:1875-1878).

In specific embodiments, Stat1 antisense oligonucleotides, Stat2 antisense oligonucleotides, Stat3 antisense oligonucleotides, or any combination thereof, are administered to reduce immune rejection. In other embodiments one or more anti-Stat1 antibodies, anti-Stat2 antibodies, or anti-Stat3 antibodies are administered to reduce immune rejection. In other embodiments, one or more peptides that compete with Jak1 or Tyk2 for binding to the IFN-α receptor or IFN-β receptor are administered to reduce immune rejection. In yet another embodiment, one or more peptides that compete with Jak1 or Jak2 for binding to the IFN-γ receptor are administered to reduce immune rejection. In other embodiments, one or more peptides that compete with Stat1 or Stat2 for binding to the IFN-α receptor or IFN-β receptor are administered to reduce immune rejection. In other embodiments, one or more peptides that compete with Stat1 for binding to the IFN-γ receptor are administered to reduce immune rejection. In yet other embodiments, one or more dominant-negative Stat1 polypeptides (e.g., a Stat1 polypeptide lacking its SH2 domain or a Stat1 polypeptide lacking its DNA binding domain), dominant-negative Stat2 polypeptides (e.g., a Stat2 polypeptide lacking its SH2 domain or a Stat2 polypeptide lacking its DNA binding domain), or dominant-negative Stat3 polypeptides (e.g., a Stat3 polypeptide lacking its SH2 domain or a Stat3 polypeptide lacking its DNA binding domain) are administered to reduce immune rejection.

Jak2 is involved in activation of Stat4 protein. In view of this, another composition that can be utilized as part of the methods of the invention comprises a composition that reduces the expression or activity of Jak2, while not affecting NF-κB activation or platelet aggregation. In one embodiment, therefore, one or more peptides that compete with Jak2 for binding to the IL-12Rβ 2 can be utilized. In other embodiments, such compounds include Jak2 antisense molecules, triple helix molecules or ribozyme molecules that serve to downregulate the expression of Jak2. Representative antisense compositions are described in detail below. Such compounds also include antibodies or fragments thereof that specifically bind to and inhibit the activity of Jak2.

›PROTEIN DETECTION · 11 of 11

Tyk2 is also involved in activation of Stat4 protein. In view of this, another composition that can be utilized as part of the methods of the invention comprises a composition that reduces the expression or activity of Tyk2, while not affecting NF-κB activation or platelet aggregation. In another embodiment, a composition of the invention comprises one or more peptides that compete with Tyk2 for binding to the IL-12Rβ 1 . In other embodiments, such compounds include Tyk2 antisense molecules, triple helix molecules or ribozyme molecules that serve to downregulate the expression of Tyk2. Representative antisense compositions are described in detail below.

In yet another embodiment, a composition that can be utilized as part of these methods comprises one or more small molecules that decrease or downregulate Stat4 expression or activity, while not affecting NF-κB activation or platelet aggregation. For example, among the compounds that can be utilized as part of these methods are protein tyrosine kinase inhibitors, including, but not limited to the tyrphostin class of protein tyrosine kinase inhibitors. Preferable tyrphostin compositions are ones that inhibit or downregulate Stat4 activity by (without wishing to be bound by any particular mechanism) inhibiting Jak2 or Tyk2 protein tyrosine kinase activity without deleterious effects on normal hematopoiesis. In specific embodiments, the tyrphostin is AG-490 (B42), although it is preferred that this particular tyrphostin not be utilized for treatment of autoimmune disorders, specifically multiple sclerosis (MS).

›ANTISENSE, RIBOZYME, TRIPLE-HELIX COMPOSITIONS · 1 of 2

Representative, non-limiting examples of Stat1 antisense molecules include the following: 5′-GCT GAA GCT CGA ACC ACT GTG ACA TCC-3′ (SEQ ID NO:19); and 5′-AAG TTC GTA CCA CTG AGA CAT CCT GCC (SEQ ID NO:20).

Representative, non-limiting examples of Stat2 antisense molecules include the following: 5′-CAT CTC CCA CTG CGC CAT TTG GAC TCT TCA -3′ (SEQ ID NO:21); and 5′-CAG CAT TTC CCA CTG CGC CAT TTG GGC-3′ (SEQ ID NO:22).

Representative, non-limiting examples of Stat3 antisense molecules include the following: 5′-CTG GTT CCA CTG AGC CAT CCT GCT GCA TCAG-3′ (SEQ ID NO:23); and 5′-CTG TAG CTG ATT CCA TTG GGC CAT CCT-3′ (SEQ ID NO:24).

Representative, non-limiting examples of Stat4 antisense molecules include the following: 5′-GAT TCC ACT GAG ACA TGC TGC TCT CTC TCT C-3′ (SEQ ID NO:25); and 5′-GAC TTG ATT CCA CTG AGA CAT GCT AGC-3′ (SEQ ID NO:26).

Representative, non-limiting examples of Jak2 antisense molecules include the following: 5′-GCC AGG CCA TTC CCA TCT AGA GCT TTT TTC-3′ (SEQ ID NO:27); and 5′-CGT AAG GCA GGC CAT TCC CAT GCA GAG-3′ (SEQ ID NO:28).

Representative, non-limiting examples of Tyk2 antisense molecules include the following: 5′-CCC ACA CAG AGG CAT GGT CCC CAC CAT TCA-3′ (SEQ ID NO:29); and 5′-GGC CAT CCC CCA GTG GCG CAG AGG CAT GCT CCC-3′ (SEQ ID NO:30).

Representative, non-limiting examples of SOCS1 antisense molecules include the following: 5′-CCT GGT TGC GTG CTA CCA TCC TAC TCG AGG GGC-3′ (SEQ ID NO:31); and 5′-CAC CTG GTT GTG TGC TAC CAT CCT ACT-3′ (SEQ ID NO:32).

Representative, non-limiting examples of SOCS3 antisense molecules include the following: 5′-GCT GTG GGT GAC CAT GGC GCA CGG AGC CAG CG-3′ (SEQ ID NO:33); and 5′-GGC GGG AAA CTT GCT GTG GGT GAC CAT-3′ (SEQ ID NO:34).

In addition, standard techniques can be utilized to produce antisense, triple helix, or ribozyme molecules for use as part of the methods described herein. First, standard techniques can be utilized for the production of antisense nucleic acid molecules, i.e., molecules which are complementary to a sense nucleic acid encoding a polypeptide of interest (e.g., Stat1, Stat2, Stat3, Stat4, Jak2, Tyk2, SOCS1, or SOCS3), e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame). An antisense nucleic acid molecule can be antisense to all or part of a non-coding region of the coding strand of a nucleotide sequence encoding a polypeptide of interest. The non-coding regions (“5′ and 3′ untranslated regions”) are the 5′ and 3′ sequences which flank the coding region and are not translated into amino acids.

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides or more in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).

Antisense nucleic acid molecules administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA encoding the polypeptide of interest to thereby inhibit expression, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue, e.g., transplant or autoimmune lesion, site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell, e.g., T cell, surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using vectors, e.g., gene therapy vectors, described below. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

›ANTISENSE, RIBOZYME, TRIPLE-HELIX COMPOSITIONS · 2 of 2

An antisense nucleic acid molecule of interest can be an α-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res . 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res . 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett . 215:327-330).

Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region, and can also be generated using standard techniques. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of the protein encoded by the mRNA. A ribozyme having specificity for a nucleic acid molecule encoding a polypeptide of interest can be designed based upon the nucleotide sequence of a cDNA disclosed herein. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, an mRNA encoding a polypeptide of interest can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and Szostak (1993) Science 261:1411-1418.

Triple helical structures can also be generated using well known techniques. For example, expression of a polypeptide of interest can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gene encoding the polypeptide (e.g., the promoter and/or enhancer) to form triple helical structures that prevent transcription of the gene in target cells. See generally Helene (1991) Anticancer Drug Des . 6(6):569-84; Helene (1992) Ann. N.Y. Acad. Sci . 660:27-36; and Maher (1992) Bioassays 14(12):807-15.

In various embodiments, nucleic acid compositions can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al. (1996) Bioorganic & Medicinal Chemistry 4(1): 5-23). As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14670-675.

PNAs can, for example, be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras can be generated which may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNAse H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996), supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996), supra, and Finn et al. (1996) Nucleic Acids Res . 24(17):3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs. Compounds such as 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite can be used as a link between the PNA and the 5′ end of DNA (Mag et al. (1989) Nucleic Acids Res . 17:5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn et al. (1996) Nucleic Acids Res . 24(17):3357-63). Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment (Peterser et al. (1975) Bioorganic Med. Chem. Lett . 5:1119-11124).

In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo ), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al. (1988) Bio/Techniques 6:958-976) or intercalating agents (see, e.g., Zon (1988) Pharm. Res . 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

›ANTIBODY COMPOSITIONS · 1 of 3

In one embodiment, anti-Stat1 antibodies, anti-Stat2 antibodies, anti-Stat3 antibodies, anti-Stat4 antibodies, anti-SOCS1 antibodies or anti-SOCS3 antibodies are administered to a mammal, preferably a human, to reduce immune rejection. In another embodiment, any combination of anti-Stat1 antibodies, anti-Stat2 antibodies, anti-Stat3 antibodies, anti-Stat4, anti-SOCS1 antibodies and anti-SOCS3 antibodies are administered to a mammal, preferably a human, to reduce immune rejection. In a preferred embodiment, anti-Stat1 antibodies, anti-Stat2 antibodies, anti-Stat3 antibodies, anti-Stat4 antibodies, anti-SOCS1 antibodies or anti-SOCS3 antibodies are administered to a mammal, preferably a human, in combination with other types of treatments (e.g., immunosuppressive agents) to reduce immune rejection. In yet another preferred embodiment, any combination of anti-Stat1 antibodies, anti-Stat2 antibodies, anti-Stat3 antibodies, anti-Stat4, anti-SOCS1 antibodies and anti-SOCS3 antibodies are administered to a mammal, preferably a human, in combination with other types of treatments (e.g., immunosuppressive agents) to reduce immune rejection.

Anti-Stat1 antibodies, anti-Stat2 antibodies, anti-Stat3 antibodies, anti-Stat4 antibodies, anti-SOCS1 antibodies, anti-SOCS3 antibodies, or any combination thereof can be administered to a mammal, preferably a human, using various delivery systems are known to those of skill in the art. For example, anti-Stat1 antibodies, anti-Stat2 antibodies, anti-Stat3 antibodies, anti-Stat4 antibodies, anti-SOCS1 antibodies, anti-SOCS3 antibodies, or any combination thereof can be administered by encapsulation in liposomes, microparticles or microcapsules. See, e.g., U.S. Pat. No. 5,762,904, U.S. Pat. No. 6,004,534, and PCT Publication WO 99/52563. In addition, anti-Stat1 antibodies, anti-Stat2 antibodies, anti-Stat3 antibodies, anti-Stat4 antibodies, anti-SOCS1 antibodies, anti-SOCS3 antibodies, or any combination thereof can be administered using recombinant cells capable of expressing the antibodies, or retroviral, other viral vectors or non-viral vectors capable of expressing the antibodies.

Anti-Stat1 antibodies, anti-Stat2 antibodies, anti-Stat3 antibodies, anti-Stat4, anti-SOCS1 antibodies and anti-SOCS3 antibodies can be obtained from any known source. For example, anti-Stat1 antibodies, anti-Stat2 antibodies, anti-Stat3 antibodies, anti-Stat4, anti-SOCS1 antibodies and anti-SOCS3 antibodies can be obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.), Research Diagnostics, Inc. (Flanders, N.J.) or Zymed Laboratories (South San Francisco, Calif.). Alternatively, anti-Stat1 antibodies, anti-Stat2 antibodies, anti-Stat3 antibodies, anti-Stat4, anti-SOCS1 antibodies and anti-SOCS3 antibodies can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.

Antibodies of the invention include, but are not. limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments. The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, 1 gM, IgD, IgA and IgY), class (e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 and IgA 2 ) or subclass of immunoglobulin molecule. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab′) 2 fragments which can be generated by treating the antibody with an enzyme such as pepsin or papain.

An isolated Stat1, Stat2, Stat3, Stat4, SOCS1 or SOCS3, or a fragment thereof, can be used as an immunogen to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. The full-length Stat1, Stat2, Stat3, Stat4, SOCS1 or SOCS3 polypeptide or protein can be used or, alternatively, the invention provides antigenic peptide fragments of Stat1, Stat2, Stat3, Stat4, SOCS1 or SOCS3 for use as immunogens. An antigenic peptide comprises at least 8 (preferably 10, 15, 20, or 30) amino acid residues of the amino acid sequence of Stat1, Stat2, Stat3, Stat4, SOCS1 or SOCS3, and encompasses an epitope of Stat1, Stat2, Stat3, Stat4, SOCS1 or SOCS3 such that an antibody raised against the peptide forms a specific immune complex with Stat1, Stat2, Stat3, Stat4, SOCS1 or SOCS3.

The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide. If desired, the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography to obtain the IgG fraction. Alternatively, antibodies specific for a protein or polypeptide of the invention can be selected for (e.g., partially purified) or purified by, e.g., affinity chromatography. For example, a recombinantly expressed and purified (or partially purified) protein of the invention is produced as described herein, and covalently or non-covalently coupled to a solid support such as, for example, a chromatography column. The column can then be used to affinity purify antibodies specific for the proteins of the invention from a sample containing antibodies directed against a large number of different epitopes, thereby generating a substantially purified antibody composition, i.e., one that is substantially free of contaminating antibodies. By a substantially purified antibody composition is meant, in this context, that the antibody sample contains at most only 30% (by dry weight) of contaminating antibodies directed against epitopes other than those on the desired Stat1, Stat2, Stat3, Stat4, SOCS1 or SOCS3 protein or polypeptide, and preferably at most 20%, yet more preferably at most 10%, and most preferably at most 5% (by dry weight) of the sample is contaminating antibodies. A purified antibody composition means that at least 99% of the antibodies in the composition are directed against the desired Stat1, Stat2, Stat3, Stat4, SOCS1 or SOCS3 protein or polypeptide.

›ANTIBODY COMPOSITIONS · 2 of 3

At an appropriate time after immunization, e.g., when the specific antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497, the human B cell hybridoma technique (Kozbor et al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy , Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing hybridomas is well known (see generally Current Protocols in Immunology (1994) Coligan et al. (eds.) John Wiley & Sons, Inc., New York, N.Y.). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide of interest, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody directed against Stat 1, Stat2, Stat3, Stat4, SOCS1 or SOCS3 polypeptide can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with Stat1, Stat2, Stat3, Stat4, SOCS1 or SOCS3. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System , Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit , Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO 92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO 93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J . 12:725-734.

Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, can be made using standard recombinant DNA techniques. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397, which are incorporated herein by reference in their entirety.) Humanized antibodies are antibody molecules from non-human species having one or more complementarily determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g., Queen, U.S. Pat. No. 5,585,089, which is incorporated herein by reference in its entirety.) Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT Publication No. WO 87/02671; European Patent Application 184,187; European Patent Application 171,496; European Patent Application 173,494; PCT Publication No. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol . 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Canc. Res . 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst . 80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol . 141:4053-4060.

Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies can be produced, for example, using transgenic mice which are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995 , Int. Rev. Immunol . 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No. 5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806. In addition, companies such as Abgenix, Inc. (Fremont, Calif.), can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al. (1994) Bio/technology 12:899-903).

As described herein, anti-Stat1, anti-Stat2, anti-Stat3, anti-Stat4, anti-SOCS1 or anti-SOCS3 antibodies can be used diagnostically to monitor protein levels within affected tissue (e.g., a transplant cell, tissue, organ or organ system, or a cell, tissue, organ or organ system that is, or is suspected of being affected by an autoimmune disorder) as part of a clinical testing procedure, e.g., to, for example, determine transplant rejection or the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 I, 35 S or 3 H.

›ANTIBODY COMPOSITIONS · 3 of 3

Further, as described herein, anti-Stat1 antibodies, anti-Stat2 antibodies, anti-Stat3 antibodies, anti-Stat4 antibodies, anti-SOCS1 antibodies, anti-SOCS3 antibodies, or any combination thereof can be conjugated to a therapeutic moiety and administered to a mammal, preferably a human, to reduce or prevent immune rejection. Examples of therapeutic moieties that can be conjugated to antibodies include, but are not limited to, a cytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells such as taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

The antibodies can also be conjugated a drug moiety that modifies a given biological response. For example, a drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; and a lymphokine such as IL-4 or IL-13.

The invention also provides kits comprising an anti-Stat1 antibody, an anti-Stat2 antibody, an anti-Stat3 antibody, an anti-Stat4 antibody, an anti-SOCS1 antibody, an anti-SOCS3 antibody, or any combination thereof conjugated to a detectable substance, and instructions for use. Still another aspect of the invention is a pharmaceutical composition comprising an anti-Stat1 antibody, an anti-Stat2 antibody, an anti-Stat3 antibody, an anti-Stat4 antibody, an anti-SOCS1 antibody, an anti-SOCS3 antibody and a pharmaceutically acceptable carrier. In preferred embodiments, the pharmaceutical composition contains an anti-Stat1 antibody, an anti-Stat2 antibody, an anti-Stat3 antibody, an anti-Stat4 antibody, an anti-SOCS1 antibody, or an anti-SOCS3 antibody, a therapeutic moiety, and a pharmaceutically acceptable carrier.

In instances wherein an anti-Stat1 antibody, an anti-Stat2 antibody, an anti-Stat3 antibody, an anti-Stat4 antibody, an anti-SOCS1 antibody, an anti-SOCS3 antibody is to be utilized as a therapeutic, characterization of the antibody can routinely be assayed and ascertained via the methods presented herein. For example, the fact that lymphocytes and animal models for transplants and autoimmune disorders are readily available, coupled with the availability of multiple assays for Stat and SOCS expression and activity provide for routine testing and analysis (e.g., for in vitro and in vivo testing and analysis) of such antibodies. The antibodies described herein can be tested, for example, for their ability to modulate the expression and/or activity of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1, SOCS3, or any combination thereof, and for their specificity and toxicity.

›GENE THERAPY TECHNIOUES · 1 of 4

Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.

In one embodiment, Stat4 antisense oligonucleotides are administered to reduce immune rejection by way of gene therapy. In another embodiment, nucleic acid molecules comprising sequences encoding one or more anti-Stat4 antibodies are administered to reduce immune rejection, by way of gene therapy. In another embodiment, nucleic acid molecules comprising sequences encoding one or more peptides that compete with Jak2 for binding to the IL-12 receptor β 2 (“IL-12Rβ 2 ”) are administered to immune rejection, by way of gene therapy. In another embodiment, nucleic acid molecules comprising sequences encoding one or more peptides that compete with Tyk2 for binding to the IL-12Rβ 1 are administered to reduce immune rejection, by way of gene therapy. In another embodiment, nucleic acid molecules comprising sequences encoding one or more peptides that compete with Stat4 for binding to the IL-12Rβ 2 are administered to reduce immune rejection, by way of gene therapy. In another embodiment, nucleic acid molecules comprising sequences encoding one or more peptides that bind to the Stat4 SH2 domain which prevent Stat4 from binding to the IL-12Rβ 2 are administered to reduce immune rejection, by way of gene therapy. In yet another embodiment, nucleic acid molecules comprising sequences encoding one or more dominant-negative Stat4 polypeptides (e.g., a Stat4 polypeptide lacking its SH2 domain or a Stat4 polypeptide lacking its DNA binding domain) are administered to reduce immune rejection, by way of gene therapy.

In specific embodiments, Stat1 antisense oligonucleotides, Stat2 antisense oligonucleotides, Stat3 antisense oligonucleotides, or the combination thereof are administered to reduce immune rejection by way of gene therapy. In other embodiments, nucleic acid molecules comprising sequences encoding one or more anti-Stat1 antibodies, anti-Stat2 antibodies, or anti-Stat3 antibodies are administered to reduce immune rejection, by way of gene therapy. In other embodiments, nucleic acid molecules comprising sequences encoding one or more peptides that compete with Jak1 or Tyk2 for binding to the IFN-α receptor or IFN-β receptor are administered to reduce immune rejection, by way of gene therapy. In yet another embodiment, nucleic acid molecules comprising sequences encoding one or more peptides that compete with Jak1 or Jak2 for binding to the IFN-γ receptor are administered to reduce immune rejection, by way of gene therapy. In other embodiments, nucleic acid molecules comprising sequences encoding one or more peptides that compete with Stat1 or Stat2 for binding to the IFN-α receptor or IFN-β receptor are administered to reduce immune rejection, by way of gene therapy. In other embodiments, nucleic acid molecules comprising sequences encoding one or more peptides that compete with Stat1 for binding to the IFN-γ receptor are administered to reduce immune rejection, by way of gene therapy. In yet other embodiments, nucleic acid molecules comprising sequences encoding one or more dominant-negative Stat1 polypeptides (e.g., a Stat1 polypeptide lacking its SH2 domain or a Stat1 polypeptide lacking its DNA binding domain), dominant-negative Stat2 polypeptides (e.g., a Stat2 polypeptide lacking its SH2 domain or a Stat2 polypeptide lacking its DNA binding domain), or dominant-negative Stat3 polypeptides (e.g., a Stat3 polypeptide lacking its SH2 domain or a Stat3 polypeptide lacking its DNA binding domain) are administered to reduce immune rejection, by way of gene therapy.

For general reviews of the methods of gene therapy, see Goldspiel et al., 1993 , Clinical Pharmacy 12:488-505; Wu and Wu, 1991 , Biotherapy 3:87-95; Tolstoshev, 1993 , Ann. Rev. Pharmacol. Toxicol . 32:573-596; Mulligan, 1993 , Science 260:926-932; and Morgan and Anderson, 1993 , Ann. Rev. Biochem . 62:191-217; May, 1993, TIBTECH 11(5):155-215). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; and Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY.

In one aspect, a composition of the invention comprises nucleic acid sequences encoding one or more anti-Stat 1, anti-Stat 2, anti-Stat 3, anti-Stat 4, anti-SOCS1, or anti-SOCS 3 antibodies or fragments thereof, said nucleic acid sequences being part of expression vectors that express one or more anti-Stat 1, anti-Stat 2, anti-Stat 3, anti-Stat 4, anti-SOCS1, or anti-SOCS 3 antibodies or fragments thereof in a suitable host. In particular, such nucleic acid sequences have promoters operably linked to the anti-Stat 1, anti-Stat 2, anti-Stat 3, anti-Stat 4, anti-SOCS1, or anti-SOCS 3 antibodies or fragments thereof, said promoter being inducible or constitutive, and, optionally, tissue-specific.

In another aspect, a composition of the invention comprises nucleic acid sequences encoding dominant-negative Stat1, Stat2, Stat3,Stat4, SOCS1, or SOCS3 polypeptides, said nucleic acid sequences being part of expression vectors that express dominant-negative Stat1, Stat2, Stat3,Stat4, SOCS1, or SOCS3 polypeptides in a suitable host. In particular, such nucleic acid sequences have promoters operably linked to the dominant-negative Stat1, Stat2, Stat3,Stat4, SOCS1, or SOCS3, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, nucleic acid molecules are used in which the dominant-negative Stat 1, Stat2, Stat3,Stat4, SOCS1, or SOCS3 coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the dominant-negative Stat1, Stat2, Stat3,Stat4, SOCS1, or SOCS3 nucleic acids (Koller and Smithies, 1989 , Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989 , Nature 342:435-438).

›GENE THERAPY TECHNIOUES · 2 of 4

In another aspect of the invention, a composition of the invention comprises nucleic acid molecules comprising sequences encoding one or more peptides that compete with Tyk2 for binding to the IL-12Rβ 1 , said nucleic acid sequences being part of expression vectors that express one or more peptides in a suitable host. In another aspect of the invention, a composition of the invention comprises nucleic acid sequences nucleic acid molecules comprising sequences encoding one or more peptides that compete with Stat4 for binding to the IL-12Rβ 2 , said nucleic acid sequences being part of expression vectors that express one or more peptides in a suitable host. In yet another aspect of the invention, a composition of the invention comprises nucleic acid molecules comprising sequences encoding one or more peptides that bind to the Stat4 SH2 domain which prevent Stat4 from binding to the IL-12Rβ 2 , said nucleic acid sequences being part of expression vectors that express one or more peptides in a suitable host. In particular a embodiment of the invention, the nucleic acid sequences encoding peptides of the invention have promoters operably linked to said nucleic acid sequences, said promoter being inducible or constitutive, and, optionally, tissue-specific.

Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequence is directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing it as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987 , J. Biol. Chem . 262:4429-4432) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180 dated Apr. 16, 1992 (Wu et al.); WO 92/22635 dated Dec. 23, 1992 (Wilson et al.); WO92/20316 dated Nov. 26, 1992 (Findeis et al.); WO93/14188 dated Jul. 22, 1993 (Clarke et al.), WO 93/20221 dated Oct. 14, 1993 (Young)). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, 1989 , Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

In one embodiment, viral vectors that contain Stat4 antisense oligonucleotides are used (see Miller et al., 1993 , Meth. Enzymol . 217:581-599). In another embodiment, viral vectors that contain nucleic acids encoding one or more anti-Stat4 antibodies are used. In another embodiment, viral vectors that contain nucleic acids encoding one or more peptides that compete with Jak2 for binding to the IL-12 receptor β 2 (“IL-12Rβ 2 ”) are used. In another embodiment, viral vectors that contain nucleic acids encoding one or more peptides that compete with Tyk2 for binding to the IL-12Rβ 1 are used. In another embodiment, viral vectors that contain nucleic acids encoding one or more peptides that compete with Stat4 for binding to the IL-12Rβ 2 are used. In another embodiment, viral vectors that contain nucleic acids encoding one or more peptides that bind to the Stat4 SH2 domain which prevent Stat4 from binding to the IL-12Rβ 2 are used. In yet another embodiment, viral vectors that contain nucleic acids encoding dominant-negative Stat4 polypeptides (e.g., Stat4 lacking its SH2 domain or Stat4 lacking its DNA binding domain) are used. For example, a retroviral vector can be used. These retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA. The nucleic acid sequences encoding the Stat1, Stat2, Stat3, Stat4, SOCS1 or SOCS3 antibodies, or polypeptides or peptides of the invention to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., 1994 , Biotherapy 6:291-302, which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., 1994 , J. Clin. Invest . 93:644-651; Kiem et al., 1994 , Blood 83:1467-1473; Salmons and Gunzberg, 1993 , Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993 , Curr. Opin. in Genetics and Devel . 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, 1993 , Current Opinion in Genetics and Development 3:499-503 present a review of adenovirus-based gene therapy. Bout et al., 1994 , Human Gene Therapy 5:3-10 demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., 1991 , Science 252:431-434; Rosenfeld et al., 1992 , Cell 68:143-155; Mastrangeli et al., 1993 , J. Clin. Invest . 91:225-234; PCT Publication WO94/12649; and Wang, et al., 1995 , Gene Therapy 2:775-783. In a preferred embodiment, adenovirus vectors are used.

›GENE THERAPY TECHNIOUES · 3 of 4

Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., 1993 , Proc. Soc. Exp. Biol. Med . 204:289-300; U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, 1993 , Meth. Enzymol . 217:599-618; Cohen et al., 1993 , Meth. Enzymol . 217:618-644; Cline, 1985 , Pharmac. Ther . 29:69-92) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.

In a preferred embodiment, the cell used for gene therapy is autologous to the patient.

In one embodiment in which recombinant cells are used in gene therapy, nucleic acid sequences encoding Stat1, Stat2, Stat3, Stat4, SOCS1 or SOCS3 antibodies, or polypeptides or peptides of the invention are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598, dated Apr. 28, 1994; Stemple and Anderson, 1992 , Cell 71:973-985; Rheinwald, 1980 , Meth. Cell Bio . 21A:229; and Pittelkow and Scott, 1986 , Mayo Clinic Proc . 61:771).

Promoters that may be used to control the expression of nucleic acid sequences encoding Stat1, Stat2, Stat3, Stat4, SOCS1 or SOCS3 antibodies, or polypeptides or peptides of the invention include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, 1981 , Nature 290:304-310), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42); prokaryotic expression vectors such as the β-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731), or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25); see also “Useful proteins from recombinant bacteria” in Scientific American, 1980, 242:74-94; plant expression vectors comprising the nopaline synthetase promoter region (Herrera-Estrella et al., Nature 303:209-213) or the cauliflower mosaic virus 35S RNA promoter (Gardner et al., 1981, Nucl. Acids Res. 9:2871), and the promoter of the photosynthetic enzyme ribulose biphosphate carboxylase (Herrera-Estrella et al., 1984, Nature 310:115-120); promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlau fet al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58; alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globin gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

›GENE THERAPY TECHNIOUES · 4 of 4

In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription.

›PHARMACEUTICAL COMPOSITIONS · 1 of 12

The nucleic acid molecules, polypeptides, antibodies and small molecules (also referred to herein as “active compounds”) described herein can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the active compound and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

The invention includes methods for preparing pharmaceutical compositions for modulating the expression or activity of a polypeptide or nucleic acid of interest (e.g., Stat1, Stat2, Stat3, Stat4, Stat6, SOS 1, or SOCS3). Such methods comprise formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid of interest (e.g., Stat1, Stat2, Stat3, Stat4, Stat6, SOS 1, or SOCS3). Such compositions can further include additional active agents. Thus, the invention further includes methods for preparing a pharmaceutical composition by formulating a pharmaceutically acceptable carrier with an agent which modulates expression or activity of a polypeptide or nucleic acid of interest and one or more additional active compounds.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Intravenous administration is preferred. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

›PHARMACEUTICAL COMPOSITIONS · 2 of 12

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

For antibodies, the preferred dosage is 0.1 mg/kg to 100 mg/kg of body weight (more preferably, 0.1 to 20 mg/kg, 0.1-10 mg/kg, or 0.1 to to 1.0 mg/kg). If the antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate. Generally, partially human antibodies and fully human antibodies have a longer half-life within the human body than other antibodies. Accordingly, lower dosages and less frequent administration is often possible. Modifications such as lipidation can be used to stabilize antibodies and to enhance uptake and tissue penetration (e.g., into the brain). A method for lipidation of antibodies is described by Cruikshank et al. ((1997) J. Acquired Immune Deficiency Syndromes and Human Retrovirology 14:193).

As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 0.1 to 1.0 mg/kg, 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.

The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.

In a representative, non-limiting example, a subject is treated at the time of transplantation, or when diagnosed as exhibiting a reoccurrence of rejection, or an occurrence of a rejection (e.g., an autoimmune rejection) with one to several (for example, between 3 and 7) doses of an appropriate modulator of Stat1, Stat2, Stat3, Stat4, Stat6, SOS 1, and/or SOCS3 for a maximum of one week. In a preferred embodiment of such an example, treatment would further comprise additional administration approximately once per month for about 3 to 6 months. The preferred route of administration is intravenous bolus injection. It will also be appreciated that the effective dosage of the modulator used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.

Preferably, administration of modulator is by intravenous injection, and can also be are or near the site of the cells or tissue to be treated, e.g., administration is at or near the site of the transplant or autoimmune disorder lesion.

In addition to those compounds described above, the present invention encompasses agents and use of agents which modulate expression or activity of a nucleic acid or polypeptide of interest. An agent may, for example, be a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e,. including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. It is understood that appropriate doses of small molecule agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher. The dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention. Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

›PHARMACEUTICAL COMPOSITIONS · 3 of 12

The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

In one embodiment, one or more compositions for modulation of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1, or SOCS3 can be used combinatorially. For example, compositions for decreasing expression or activity of Stat4 can be utilized in combination (either simultaneously or serially) with compositions or techniques for increasing expression or activity of Stat6 can be utilized.

In another embodiment, one or more compositions of the present invention that modulate expression or activity of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1 or SOCS3 can be administered to a mammal, preferably a human, in combination with one or more standard immunosuppressive or immunomodulatory compounds to reduce or prevent immune rejection resulting from an autoimmune disorder or an allograft. Examples of immunosuppressive agents include, but are not limited to, azathioprine, corticosteriods (e.g., prednisone), cyclosporine, OKT3 (anti-CD3 monoclonal human antibody), mycophenolate mofetil, rapamycin (sirolimus), mizoribine, deoxyspergualin, macrolide antibiotics such as, for example, FK506 (tacrolimus), brequinar, malononitriloamindes.(e.g., leflunamide), and anti-IL-2R antibodies (e.g., anti-Tac monoclonal antibody and BT 536). See, e.g., Grummet et al., 1999, J. Am. Soc. Nephrol. 10:1366-1388; and Norman and Wadi, eds., 1998, “Primer on Transplantation,” Am. Soc. Tx. Phys, 1 st ed.).

Immunosuppressive agents may be administered at high doses initially and then tapered off over time to reduce or prevent immune rejection. For example, one or more compositions of the invention in combination with an initial dose of cyclosporine ranging from between 5 and 10 mg/kg per day, an initial dose of 10 mg/kg per day prednisone, or an initial dose of 10 mg/kg per day mycophenolate mofetil may be administered to animal to reduce or prevent immune rejection. Alternatively, one or more compositions of the invention in combination with an initial dose of cyclosporine ranging from between 5 and 10 mg/kg per day, an initial dose of 10 mg/kg per day prednisone, and an initial dose of 10 mg/kg per day mycophenolate mofetil may be administered to animal to reduce or prevent immune rejection. Preferably, corticosteroids are not administered children.

In yet another embodiment, one or more compositions of the present invention that modulate expression or activity of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1 or SOCS3 can be administered to a mammal, preferably a human, in combination with one or more standard autoimmune therapeutic agents used for treating a particular autoimmune disorder. For example, one or more compositions of the present invention may be administered in combination with one or more conventional anti-lupus therapeutic agents or drugs such as, for example, salicylates, corticosteroids, and immunosuppresants.

In another embodiment, one or more compositions of the present invention for modulating the expression or activity of Stat1, Stat2, Stat3, Stat4, Stat6, SOCS1, or SOCS3 are administered to a mammal, preferably a human, in combination with one or more T cell-targeted or B cell-targeted agents. Examples of such agents include, but are limited to, CTLA-4Ig, IL-2 antagonists (e.g., anti-IL-2 receptor antibodies and IL-2 toxin conjugates), B7 monoclonal antibodies, anti-CD40L monoclonal antibodies, CD4 antagonists (e.g., anti-CD4 monoclonal antibodies), CD3 antagonists (e.g., anti-CD3 monoclonal antibodies), and IL-12 antagonists (e.g., anti-IL-12 monoclonal antibodies and IL-12 toxin conjugates) to reduce or prevent immune rejection an autoimmune disorder or an allograft.

In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner.

6. EXAMPLE: QUANTITATIVE ANALYSIS OF NF-κB AND IκB PROTEINS IN MOUSE CARDIAC ALLOGRAFTS

NF-κB proteins are transcription factors complexed with IκB proteins in the cytoplasm but which upon cell activation are released, translocate to the nucleus and bind κB motifs in the promoters of many genes, in particular of the promoters of genes whose expression is involved the immune response. Since NF-κB plays an important role in the transcription of genes involved in immune responses, the expression levels of NF-κB and IκB proteins and their localization were determined in mouse cardiac allografts. The data generated and analyzed represents the first comprehensive analysis of NF-κB and IκB protein expression, phosphorylation, and localization as detected by Western blotting and immunohistology in serially harvested allografts (BALB/c→B6), isoftafts and native hearts from recipients treated with IgG (rejection by day 8) or CD40 ligand monoclonal antibody (“CD40L mAb”; permanent survival).

Heterotopic cardiac allografting was performed with anastomoses to the abdominal aorta and vena cava (Hancock et al., 1998, Nature Medicine 4:1392-1396), using BALB/c donors and B6/129 wild-type or other Balb/c wild-type mice as recipients. Recipients were treated with hamster IgG or hamster anti-mouse (CD40L mAb; 250 μg, administered intravenously) plus DST (5×10 6 splenic mononuclear cells) at the time of transplantation (Hancock et al., 1998, Nature Medicine 4:1392-1396). The protein expression, phosphorylation and localization of NF-κB and IκB were detected by Western blot analysis and immunohistology using serially harvested allografts, isografts and native hearts from recipients treated with IgG (rejection by day 8) or CD40L mAb (permanent survival).

The following tissue-specific expression patterns in the basal cardiac state relative to other organs were detected: low p50; low p52; low p65; low C-rel; low relB; low IkBa; high IkBb; low IkBe; low BCL-3 and high p105. The level of NF-κB proteins was significantly increased (2-4 fold) upon allografting and these levels were only modestly affected by CD40L mAb. Significant increases in the levels of IκBa (greater than 2 fold) and IκBe (greater than 6 fold) proteins were also detected in cardiac allografts. In contrast, a significant decrease in the level of IκBb protein, low to undetectable levels of p105 protein, and trace levels of BCL-3 were detected in cardiac allografts, but were only modestly affected by CD40L mAb. Thus, these results demonstrate that in cardiac allografts the NF-κB regulatory apparatus is highly activated at the protein level and is only modestly affected by CD40L mAb.

›PHARMACEUTICAL COMPOSITIONS · 4 of 12

Cardiac samples principally express p1O5 and IκBb, but these are down-regulated during rejection, presumably through the action of the proteasome. By contrast, cardiac allograft rejection is associated primarily with expression by infiltrating leukocytes of p65, p50 and c-rel NF-κB proteins, plus IκBa and IκBc proteins.

The results, therefore, suggest that monitoring of the levels of NF-κB and IκB proteins in biopsies from transplant recipients may be of diagnostic and/or prognostic significance.

7. EXAMPLE: DIFFERENTIAL EFFECTS OF IMMUNOSUPPRESSIVE AGENTS ON ANTI-CD40L ANTIBODY-MEDIATED TOLERANCE INDUCTION

The data presented herein demonstrate that concomitant use of the immunosuppressive agents cyclosporin A or methylprednisolone, but not rapamycin, blocks CD154 mAb efficacy in experimental allograft recipients. Indeed, the differential effects of these agents on CD154 mAb-induced tolerance correlates with their capacity to inhibit activation-induced CD154 expression on CD4+ T cells. Full expression of CD154 expression was found to require NF-κB activation, and CD154 mAb was ineffective in NF-κB/p50 deficient allograft recipients or control mice in which NF-κB activation was blocked by a proteasome inhibitor. Hence, these data indicate that strategies to use CD154 mAb clinically must take into account the effects of immunosuppressive agents on CD154 induction, which appears to be at least partially NF-κB dependent, and suggest that ligation of surface-expressed CD154 provides an important signal that modulates T cell activation.

Materials & Methods

Media and Reagents:

Cell culture media, serum and supplements were purchased from Gibco BRL (Rockville. Md.) and all mAbs were from PharMingen (San Diego, Calif.). Cyclosporin A (catalog C-3662, Sigma. St. Louis. Mo.) was prepared as a 5 mg/ml stock solution in 0.9% saline; rapamycin (catalog 380-004-M001, Alexis, San Diego. Calif.) as a 1 mg/ml stock solution in ethanol: 6α-methylprednisolone (catalog M-0369, Sigma) as a 5 mg/ml stock solution in 80% ethanol; mycophenolate mofetil (catalog M-5255, Sigma) as a 20 mM stock solution in DMSO; and the 3 proteasome inhibitors (Grisham, M. B., et al., 1999, Methods Enzymol 300:345-63), clasto-lactacystin β-lactone (catalog 426102, Calbiochem, San Diego, Calif.) and its derivative PS-519 (Proscript, Cambridge, Mass.), and dipeptide-boronate (MG-273. ProScript), each as a 10 mM stock solution in DMSO.

Mice:

BALB/c (H-2 d ) and B6/129 (H-2 b ) mice were obtained from Jackson Labs (Bar Harbor, Me.), and NF-κB/p50 knockout (p50 KO) B6/129 mice (H-2 b ) (Sha, W. C. et al., 1995, Cell 80:321-30.) were provided by Dr. David Baltimore (MIT, Cambridge, Mass.), and were housed under specific pathogen-free conditions.

Cardiac Transplantation:

Heterotopic cardiac allografting was performed with anastomoses to the abdominal aorta and vena cava (Hancock, W. W. et al., 1998, Nature Medicine 4:1392-1396), using BALB/c donors and B6/129 wild-type or NF-κB/p50 KO mice as recipients (n=6/group). Recipients were treated with hamster IgG or hamster anti-mouse CD154 mAb (250 μg, i.v.) plus DST (“donor specific transfusion”; 5×10 6 splenic mononuclear cells) at the time of transplantation (Hancock, W. W. et al., 1998, Nature Medicine 4:1392-1396). Additional groups of allografted wild-type mice were treated with CD154 mAb/DST plus (i) rapamycin (0.2 mg/kg/d i.p.) or (ii) cyclosporin A (10 mg/kg/d i.p.) on day 0 and every other day until day 14; (iii) methylprednisolone (1 mg/kg i.p.) on day 0, 1 and 2; and (iv) PS-519 (1 mg/kg/d i.p.) daily from the time of transplantation. Graft survival was monitored by daily palpation, and rejection was confirmed by laparotomy and histologic evaluation.

Measurement of Activation-induced CD154 Expression:

Six-well plates (Costar) were pre-coated overnight with 1 μg/ml of rat anti-mouse CD3 mAb (2C11). After washing with media (RPMI supplemented with 10% fetal bovine serum, 50 U/ml penicillin, 50 μg/ml streptomycin, 2 mM glutamine and 50 μM 2-mercaptoethanol) one ml of media containing 2× final drug concentration was added to each well. After testing of a range of concentrations of each agent, the optimal final physiologic drug concentrations for the data to be reported were 125 ng/ml cyclosporin A, 20 ng/ml rapamycin. 100 μg/ml methylprednisolone, 1 μM mycophenolate mofetil, 10 μm lactacystin and 10 μM MG-273. Cells were obtained from mechanically disrupted spleens and, after collection by centrifugation and washing once with media, one ml aliquots containing two million viable splenocytes were added to each well. Cultures were incubated at 37° C. in 5% CO 2 for 7 hr. and were then diluted two-fold with chilled media containing 0.1% sodium azide (FACS media). Cells were collected by centrifugation at 4° C., resuspended in FACS media containing 1 μg Fc Block (PharMingen) and incubated on ice for 15 min. Samples were then split and either a mixture of 1 μg each of FITC-conjugated CD4-specific mAb and PE-conjugated control hamster IgG mAb or FITC-conjugated CD4-specific mAb and PE-conjugated CD154-specific mAb were added. After a 30 min incubation on ice, cells were washed twice and analyzed on a Becton Dickinson FACScan using Cell Quest software. Viable CD4+ lymphocytes were gated using a combination of forward/side scatter and CD4 staining.

Statistics

Flow cytometry data was analyzed using student t-test and cardiac allograft survival was evaluated by the two-tailed Mann-Whitney U test using the program InStat (GraphPad Software, San Diego. Calif.); p.<0.05 indicated a significant result.

Results

Given the apparent efficacy of CD154 mAb in inducing long-term cardiac allograft survival in various mouse strains (Larsen, C. P. et. al., 1996, Nature 381:434-438; Hancock. W. W. et al., 1996 Proc. Natl. Acad. Sci. USA. 93:13967-13972), without development of transplant arteriosclerosis or other sequelae of chronic rejection (Hancock, W. W. et al., 1998, Nature Medicine 4:1392-1396), investigators have begun clinical testing. As human allograft recipients presently derive significant benefit from conventional immunosuppressant therapy, initial CD154 mAb trial designs have included pharmacologic immunosuppression. However, conventional immunosuppression with glucocorticoids or cyclosporin A has prevented the success of otherwise potent experimental approaches for tolerance induction, such as that seen with intrathymic injection of donor MHC antigen in rodents (Perico, N. et al.,1995, J. Amer. Soc. Nephrol. 5:1618-1623). Likewise, CD154 mAb-induced prolongation of renal allograft survival in the initial primate studies was diminished by concomitant use of either glucocorticoids or FK506 (Kirk, A. D. et. Al, 1999, Nature Medicine 5:686-693): FK-506, like cyclosporin A, blocks activation of the calcium-dependent serine phosphatase calcineurin.

›PHARMACEUTICAL COMPOSITIONS · 5 of 12

To understand the potential for inhibitory effects of immunosuppressive agents on the therapeutic efficacy of CD154 mAb, the effectiveness of CD154 mAb was investigated in conjunction with adjunct therapies in a completely MHC-mismatched (H2 d→N 2 b ) mouse cardiac allograft model. The results of concomitant administration of cyclosporin A, methylprednisolone or rapamycin on allograft survival in mice treated with CD154 mAb plus DST are summarized in FIG. 10 . Whereas CD154 mAb therapy induced permanent cardiac allograft survival (>100 days), the effects of CD154 mAb were blocked by addition of cyclosporin A or methylprednisolone (p<0.001 vs. CD154 mAb/DST alone), but not by rapamycin (FIG. 10 ).

Given these markedly contrasting differences in effects of standard immunosuppressive agents on the efficacy of CD154 mAb therapy, and recent evidence that CCD154 may signal to T cells (Blair, P. J. et al., 2000, J Exp Med 191:651-660), it was hypothesized that inhibition of CD154 expression was involved. Accordingly, the in vitro effects of standard immunosuppressive agents on the expression of CD154 by activated T cells were tested in vitro. It was found that whereas resting CD4+ splenic cells lacked CD154 expression, 25-30% of cells expressed the molecule within 7 hours of activation with plate-bound CD3 mAb (FIG. 11 A). This upregulation was markedly suppressed by therapeutic doses of cyclosporin A or methylprednisolone but not by rapamycin or mycophenolate mofetil (FIG. 11 a ). Analysis of three separate experiments showed >90% suppression by methylprednisolone (p<0.01) and >70% suppression by cyclosporin A (p<0.01) whereas the effects of rapamycin (<20% inhibition) and mycophenolate mofetil (<10% inhibition) were not statistically distinguishable from control activated cells (FIG. 11 B). Thus, pharmacologic immunosuppressives that reduce the efficacy of CD154 mAb in vivo also inhibit activation-induced CD154 expression in vitro.

Since the allograft response is highly T cell-dependent, immunosuppressants must modulate one or more aspects of the T cell response. As reviewed in this context (Gummert, J. F. et al., 1999, J. Am. Soc. Nephrol. 10:1366-1380), full T cell activation requires three signals: signal 1 is the triggering of the T cell antigen receptor, signal 2 is costimulation through CD28 and related molecules, and signal 3 is provided by cytokines. Cyclosporin A, like FK-506, blocks activation of calcineurin, an early event in T cell activation, just downstream of signal 1. Calcineurin dephosphorylates the transcription factor, nuclear factor of activated T cells (NFAT), and though it is well known that NFAT is essential to the transcription of IL-2 and (FN-γ (Ullman, K. S. et al., 1990, Anna Rev Immunol 8:421-52), it is less appreciated that NFATp-binding sites are also present in the CD154 promoter (Schubert, L. A.,et al., 1995, J. Biol Chem 270:29624-7). It was found here that CD154 induction on murine CD4+ T cells was markedly inhibited by cyclosporin A, as was reported for human T cells (Fuleihan. R., et al., 1994., J. Clin Invest 93:1315-20).

In contrast to cyclosporin A, the macrolide rapamycin blocks a relatively late stage of T cell activation, as the target of rapamycin (“TOR”), is downstream of signal 3, cytokine receptor activation. Notably, though rapamycin is known to block various CD28-mediated events, and costimulation though CD28 can augment CD154 expression (Klaus. S. J., et al., 1994, J. Immunol 152:5643-5652), it was found here that rapamycin had no significant inhibitory effect on CD154 induction, suggesting that the effects of CD28 costimulation on CD154 expression are not critical. Hence, our data suggest that the contrasting efficacies of rapamycin and other immunophilin-binding agents such as cyclosporin A or FK506 in trials of CD154 mAb correlate with their capacities to block CD154 induction and may reflect inhibition of late and early T cell activation events, respectively.

The immunosuppressive agent mycophenolate mofetil blocks the final stage of T cell activation. T cell proliferation, which requires de novo synthesis of purine and pyrimidine nucleotides. Mycophenolate mofetil is converted within lymphocytes to its active metabolite, mycophenolic acid, which is a reversible inhibitor of a key enzyme in the de-novo purine synthesis pathway, inosine monophosphate dehydrogenase (Gummert, J. F. et al., 1999, J. Am. Soc. Nephrol. 10:1366-1380). Mycophenolate mofetil was notable in the current studies for its complete lack of effect on CD154 expression by T cells, consistent with an antagonistic function at late stages of T cell activation. Accordingly, use of CD154 mAb with mycophenolate mofetil and/or rapamycin may be particularly efficacious in the management of transplant recipients.

Methylprednisolone resulted in almost complete inhibition of CD154 induction in mouse CD4+ T cells, consistent with a single previous report of the effects of dexamethasone on human CD4+ T cells in which activation was induced by a non-TCR-dependent mechanism (PMA/ionomycin) (Bischof, F. et al., 1998, Cell Immunol 187:3844). Glucocorticoids enter target cells, bind cytoplasmic receptors and form complexes which translocate to the nucleus and bind specific response elements in the promoters of target genes (Gummert, J. F. et al., 1999, J. Am. Soc. Nephrol. 10:1366-1380). In addition to inhibiting late events in T cell activation including proliferation and cytokine production, glucocorticoids block early events such as the activation of transcription factors AP-1 and NF-κKB. Indeed, ligand-bound glucocorticoid receptors bind Rel A and NF-κB p50 subunits in vitro (Epinat, J. C. et al., 1999, Oncogene 18:6896-6909), affecting the transactivation potential of RelA/p65, and also enhance transcription of the NF-κB antagonist IκB-α. Thus. we considered whether NF-κB activation was required for CD154 expression.

Compared with the responses in normal CD3 mAb-activated CD4+ T cells, CD154 induction was consistently inhibited by about 65% in NF-κB/p50 KG cells (FIG. 12) (p<0.02). Since activation of NF-κB requires the signal-coupled phosphorylation and proteolysis of IκB-α through the 26S proteasome (Lin, Y. C., et al., 1995, Proc Natl Acad Sci USA 92:552-6; Traenckner, E. B. et al., 1995, EMBO J 14:2876-83), we also tested the effects of 2 different types of proteasome inhibitors on CD154 induction by activated T cells (FIG. 12 ). Lactacystin irreversibly blocks proteasome activity by acylating a threonine residue in the active site of the mammalian proteasome subunit X, whereas dipeptide boronates, such as MG-273, act by irreversible inhibition of proteasomal chymotryptic activity (Grisham, M. B. et al., 1999, Methods Enzymol 300:345-63). Use of either agent in vitro significantly decreased CD154 expression by CD3-stimulated CD4+ cells (p<0.05) (FIG. 12 ). Together, these data indicate that NF-κB activation is required for optimal induction of CD154 by activated T cells.

›PHARMACEUTICAL COMPOSITIONS · 6 of 12

To assess the validity of these in vitro data as guides to the in vivo efficacy of CD154 mAb therapy, CD154 mAb-induced cardiac allograft survival in normal vs. NF-κB/p50 KO mice, as well as in wild-type mice treated with a proteasome inhibitor; all experiments involved the same H2 d →H2 b MHC disparity. As anticipated from the in vitro data using spleen cells from NF-κB/p50 KO mice was evaluated, the efficacy of CD154 mAb therapy in vivo was abrogated in NF-κB/p50 KG mice (p<0.001) (FIG. 13 ). Moreover, administration of a proteasome inhibitor to wild-type mice also blocked the effects of CD154 mAb therapy in vivo (p<0.001) (FIG. 13 ). In this light, even agents such as the calcineurin-inhibitors, cyclosporin A and FK-506, which are known to inhibit NF-κB activation (Epinat, J. C. et al., 1999, Oncogene 18:6896-6909), may exert their effects on CD154 induction and CD154 mAb therapy as consequences of NF-κB inhibition.

The first conclusion of these in vitro and in vivo studies is that CD154 mAb fails to prolong allograft survival under conditions that diminish upregulation of CD154 upon CD4+ T cell activation. Indeed, the flow cytometric assay of CD154 expression provides a ready approach to evaluating potential clinical utility of CD154 mAb in combination with other immunosuppressive agents, and predicting those combinations which are likely to be successful (e.g. CD154 mAb plus rapamycin or mycophenolate mofetil) or not (e.g. CD154 mAb plus cyclosporin A or glucocorticoids). These studies further suggest that agents blocking early stages of T cell activation, thereby suppressing CD154 induction, will not be useful adjuncts to CD154 mAb therapy.

A second conclusion from the data is that NP-κB is required for CD154 induction, a finding that has not been previously described. Interestingly, the phenotype of NF-κB/p50KO is one of moderate immunodeficiency associated with defective antibody responses (Sha, W. C. et al.,1995, Cell, 80:321-30). These data indicate, therefore, that much of this phenotype may relate to defects in CD154 induction, which is key to development of B cell responses.

In addition, these findings provide an alternate interpretation for recent experimental data emphasizing an important role for CD40/CD154 interactions in the development of apoptosis in vivo, and which have suggested that concomitant use of cyclosporin A prevents tolerance induction by impairing activation-induced cell death (Li, X. C. et al., 1999, J. Immunol 163:2500-2 507; Wells. A. D. et al., 1999 Nat Med 5:1303-7). These data are more consistent with models suggesting that CD154mAb functions by stimulating CD154-dependent events (Blair, P. J. et al., 2000, J Exp Med 191:651-660), at least transiently. Further, these data demonstrate, for the first time, that NF-κB is required for CD154 induction.

8. EXAMPLE: Post-Transplantation Stat and SOCS Levels and Their Involvement in Immune Reduction and Tolerance Induction

This Example presents the first data that has been generated on the post-transplantation levels of Stat and SOCS members post-transplantation. Using a vascularized cardiac transplant model, the levels of Stats (Stats 1-6) and SOCS (inhibitors of Stats) were measured during rejection and tolerance (via CD40L antibody treatment). During rejection, a rapid upregulation of Stat1, Stat2 and Stat3 mRNA was observed, followed by upregulation of these Stats in the animals' own hearts, indicating a systemic expression pattern. In contrast, RNA and protein analysis demonstrate that changes in Stat4 and Stat6 expression are confined to the transplant tissue. Specifically, Stat4 mRNA, however, was upregulated only later, and only in the transplant heart. Likewise, Stat6 mRNA was upregulated locally in the transplant tissue. Treatment with CD40L monoclonal antibody (MR-1) resulted in the downregulation of all the Stats, with the exception of Stat6, which was upregulated upon MR-1 treatment. Thus, reduction of immune rejection, via induction of tolerance, was accompanied by a downregulation of Stat4 and an increase in Stat6 levels. Cardiac transplants in Stat6 knockout mice were rejected despite treatment with MR-1. These results indicate that an immune deviation from TH1 (mediated by IL-12 and Stat4) to TH2 (mediated by IL-4 and Stat6) is a prerequisite in the induction of tolerance, and that Stat4 and Stat6, at a minimum, are citrical signals in graft survival. Further, these results indicate that Stat6, at a minimum, is a prerequisite in the induction of tolerance.

Materials & Methods

Cloning by RT-PCR:

5 μg of IL-6 (Pharmingen, San Diego, Calif.) was injected intravenously to a B6/129 strain female mouse. The heart, liver and spleen from the mouse was collected 1 hr after the intravenous injection of IL-6. Total RNA from these three organs were prepared using the acid-guanidine thiocyanate-phenol-chloroform method (Promega, Madison, Wis.). The RNA was then dissolved in water, quantitated, and a portion of the RNA was combined in equal amounts, and 1 μg of the combined RNA was used in a 50 μl reaction volume for the synthesis of first-strand cDNA. The ProStar Ultra HF RT-PCR System (Stratagene, La Jolla, Calif.) reagents were used both for the first-strand cDNA generation and for the subsequent steps during the amplification of the cDNA template. The following upstream and downstream primers were used:

Stat1: 5′-GAACTTTCAGCTGTTACTTTCC-3′ (SEQ ID NO:35)

5′-CTGTGCTCATCATACTGTC-3′ (SEQ ID NO:36)

Stat2: 5′-GTGTTACAGTCACTCCCACTG-3 (SEQ ID NO:37)

5′-CCTCAGGCAAATCTGACTCTG-3′ (SEQ ID NO:38)

Stat3: 5′-GAAAGTACTGTAGGCCCGAG-3′ (SEQ ID NO:39)

5′-CTGGAACCACAAAGTTAGGAG-3′ (SEQ ID NO:40)

Stat4: 5′-GAAGTGAGATTCCACTCTGTAG-3′ (SEQ ID NO:41)

5′-CACTCTCCAGTTTCATCTGC-3′ (SEQ ID NO:42)

Stat5A: 5′-CGAAAGCAGTTGACGGATACG-3′ (SEQ ID NO:43)

5′-CTCCAACTTAGTTGCCTAAACC-3′ (SEQ ID NO:44)

Stat5B: 5′-CAAGCCGTTAGAAGCAGGAG-3′ (SEQ ID NO:45)

5′CCATGGTTCACAACCTACAG-3′ (SEQ ID NO:46)

Stat6: 5′-GATGAGGCTTTCCGGAGTCAC-3′ (SEQ ID NO:47)

5′-CAGTTGTATCACATTCGAGC-3′ (SEQ ID NO:48)

›PHARMACEUTICAL COMPOSITIONS · 7 of 12

SOCS1: 5′-CTGTGCCGCAGCATTAAGTG-3′ (SEQ ID NO:49)

5′-GTTTATTACCTAAACTGGCTG-3′ (SEQ ID NO:50)

SOCS2: 5′-CCAGGTATAAGTATTTCTCTC-3′ (SEQ ID NO:51)

5′-GGCCATTTGATCTTGAGCAGC-3′ (SEQ ID NO:52)

SOCS3: 5′-GCAGATTGGCTTCTTCCTCAG-3′ (SEQ ID NO:53)

5′-GGCATTTAAGGCGAGTCTCC-3′ (SEQ ID NO:54)

SOCS5: 5′-GGAGCTTACTCGCAGTAGGCTC-3′ (SEQ ID NO:55)

5′-GTAGGAGTCTCTCCGTGCAAGC-3′ (SEQ ID NO:56)

CIS: 5′-CCAACTCTGACTGAGCCAGG-3′ (SEQ ID NO:57)

5′-CATCCATACGCAGGTGGATG-3′ (SEQ ID NO:58)

Amplification reactions included 5 μl 10× Ultra HF PCR buffer, 1 μl dNTP (40 mM) mix, 0.5 μl upstream primer (approximately 500 ng/ul), 0.5 μl downstream primer (˜500 ng/μl), 1 μl first-strand cDNA reaction, 41 μl H 2 O, 1 μl Pfu Turbo DNA polymerase (2.5 U/μl). The same PCR program was used for all the amplifications: samples were heated for 1 min at 95° C., followed by 40 cycles of 1 min at 95° C., 1 min at 58° C., 2 min at 68° C., and final extension at 68° C. for 5 min. The PCR samples were then loaded onto agarose or acrylamide gels, the cDNA fragments were isolated and cloned directly into SfrI cut PCR-Script (Stratagene). Sequence analysis of the plasmids were performed by Tufts Core Facility (Boston, Mass.). The length of the cloned fragments were as follows: Stat1 (334 bp); Stat2 (694 bp); Stat3 (373 bp); Stat4 (442 bp); stat 5A (634 bp); Stat 5B (458 bp); Stat6 (894 bp); SOCS1 (381 bp); SOCS 2 (266 bp); SOCS3 (381 bp); SOCS 5 (558 bp); and CIS (688 bp).

Cardiac Transplantation:

Heterotopic cardiac allografting was performed with anastomoses to the abdominal aorta and vena cava, using BALB/c donors and B6/129 wild-type or Stat-6 KO mice as recipients (n=6/group). See, Hancock et al., 1998, Nature Med. 4:1392-1396. Recipients were intravenously administered donor specific transfusion (“DST”; 5×10 6 splenic mononuclear cells) (“DST+IgG”) plus 250 μg hamster anti-mouse CD40L mAb (CD154 mAb ; BioExpress, West Lebanon, N.H.) and DST (“DST+MR-1”) or control hamster IgG (“DST+IgG”) at the time of transplantation. Graft survival was monitored by daily palpation and rejection was confirmed by laparotomy and histologic evaluation.

RNA Isolations and Northern Blot Analysis:

Total RNA from native or transplanted hearts of Balb/c or B6/129 mice was prepared using the acid-guanidine thiocyanate-phenol-chloroform method (Chomezynski, P. and Sacchi, N., 1987, Anal. Biochem. 162:156-159). 25 μg of RNA was loaded onto each lane of 1.2% agarose-formaldehyde gels. The 0.24 kB RNA ladder (GIBCO-BRL, Rockville, Md.) was used as a size control. After electrophoresis the RNA was blotted overnight onto Nytran Supercharge membranes (Schleicher & Schuell, Keene, N.H.) with 20×SSC and cross-linked onto the membranes by irradiation with UV light using a Stratalinker (Stratagene). 32 P-labeled probes were prepared by using the Multiprime DNA labelling system and 32 P-dCTP (both from Amersham Pharmacia Biotech, Piscataway, N.J.). Hybridizations with the 32 P-labeled probes were done at 68° C. in roller bottles using ExpressHyb Solution (Clontech Laboratories, Palo Alto, Calif.). For re-use, the membranes were deprobed in 0.5% SDS at 95-100° C. and exposed to film to assure complete removal of previous hybridization signals.

The nucleotide sequences of the probes utilized are presented below:

Stat1 (SEQ ID NO:59):

gaactttcagctgttactttcccagatattattcgcaactacaaagtcatggctgccgagaacataccagagaatcccctgaa gtatctgtaccccaatattgacaaagaccacgcctttgggaagtattattccagaccaaaggaagcaccagaaccgatgga gcttgacgaccctaagcgaactggatacatcaagactgagttgatttctgtgtctgaagtccacccttctagacttcagacca cagacaacctgcttcccatgtctccagaggagtttgatgagatgtcccggatagtgggccccgaatttgacagtatgatga gcacac

Stat2 (SEQ ID NO:60):

gtgttacagtcactcccactgacagagatcatccgccactaccaggttcttgccgaagagaacatccccgagaacccact ccgcttcctctatcccgaatccctcgggacgaagcttttgggtgttactaccaggaaaaagttaatttggaagaacaggag gaatatttgaaacataaactcattgtgatctctaacagacaggtggacgagctgcagcagcctctggagctcaaacaggat tcagagtccttagaagtgaatgcagagctcttgttagcacacgaccaggagttgccattgatgatgcagactgggctggtt tgggcacagagctgaaagtggaccccatactgagtacagccccacaagtectgctggagccagecccacaagtcctg ctggagccagccccacaagtcctgctggagccagccccacaagtcctgctggagccagcccacaagtcctgctggag cagccccacaagtcctgctggagccagcccacaagtcctgctggagccagccccacaagtccagctggagccagc cccacaagtcctgctggagctagccccacaagtcctgctggagccagccccacaagtcctgctggagctagccccacaa gtccagctggagccagcacacttgctgcagcagccatcagagtcagatttgcctgagg

Stat3 (SEQ ID NO:61):

gaaagtactgtaggcccgagagccaggagcaccccgaagccgacccaggtagtgctgccccgtacctgaagaccaag ttcatctgtgtgacaccaacgacctgcagcaataccattgacctgccgatgtccccccgcactttagattcattgatgcagttt ggaaataacggtgaaggtgctgagccctcagcaggagggcagttgagtcgctcacgtttgacatggatctgacctcgga gtgtgctaccteccccatgtgaggagctgaaaccagaagctgcagagacgtgacttgagacacctgccccgtgctccac ccctaagcagccgaaccccatatcgtctgaaactcctaactttgtggttccag

Stat4 (SEQ ID NO:62):

gaagtgagattccactctgtagaaccctacaacaaagggagactgtcggctctggccttcgctgacatcctgcgagaetac aaggttatcatggctgaaaacatccctgaaaaccctctgaagtacctctaccctgacattcccaaagacaaagcctttggca aacactacagctcccagccgtgcgaagtctcaagaccaaccgaacggggagacaagggttacgtcccctctgtttttatc cccatttaacaatccgaagcgattccacggagccacaatctccttcagaccttctccccatgtctccaagtgcatatgctgt gctgagagaaaacctgagcccaacgacaattgaaactgcaatgaattccccatattctgctgaatgacggtgcaaacgga cactttaaagaaggaagcagatgaaactggagagtg

Stat5A (SEQ ID NO:63):

cgaaagcagttgacggatacgtgaagccacagatcaagcaagtggtccctgagttcgtcaatgcatccacagatgccgg agccagcgccacctacatggaccaggctccttccccagtcgtgtgccctcaacctcactacaacatgtacccacccaacc ctgaccctgtccttgaccaagatggcgagtttgacctggatgagagcatggatgttgccaggcacgtggaagaacttttac gccggcccatggacagtctcgacgcccgcctctccccacctgctggtctcttcacctccgctagaagctccctgtcctgaa cgctggactccatgcttctcttggaaaccaccttcagtgtaaggagcccacgtcagftgtagtatctctgttcataccaacaat ggctttgcacgttcacagggctaccttgcccacacagftctgggtttgtggctaaagcggtggtgaccttfttgftcagacct caagggcccccagggcctctcgtgtaagagctgaacctatcattgctgacaaacctatttctccggtgtcctttttctgtcca atggccatttcagtgaaattctagaaaaggcagggaggcaggtttaggcaactaagttggag

Stat 5B (SEQ ID NO:64):

caagccaagccgttagaagcaggagcccctggccagtgcctggtcacggagctgagctgtgtttagatgtgttggctgct gcgtggtgaaggaagacccgtctccagaaaagcaatttaggcalaaagggattccgtttgatggcagagtcccagtgcta gaaaggtagcgaaggtggaca cttacagtctcaactcatcgtcgtaaatgtcctcgtaacgacattgattcttctacctg gataaccttttgtttgtttgtttgtttgtttttgttftgtttftcccctgtaaccatttttttttctgacaagaaaacattttaattftctaag caagaagcatttttcaaataccatgtctgtgacccaaagttaaaaatggatgataattcatgtaaatgttgcaacatagcaac ctgaacctgcacgcgattcgggctctgtaggttgtgaaccatgg

›PHARMACEUTICAL COMPOSITIONS · 8 of 12

Stat6 (SEQ ID NO:65):

gatgaggctttccggagtcactataagcccgaacagatggggaaggacgggaggggttatgtctctactactatcaagat gactgtggaaagggaccagccccttcctactccagagccccagatgcctgccatggtgccaccttatgatcttggaatgg cccctgatgcttccatgcaactcagctcagatatggggtatcctccacagtccatccactcatttcagagcctagaagagtc catgagtgtactgccatcttttcaggagcctcacctgcaaatgccccccaacatgagccagataaccatgccctttgaccag cctcacccccagggtctgctgcagtgccagtcccaggaacatgctgtgtccagccctgaacccatgctttggtcagatgtg actatggtagaggacagttgcctaactcagcctgtgggaggtttcccccaaggcacctgggtcagtgaagacatgtaccc tcccctgctgcctcccactgaacaggacctcaccaagcttctcctggagaaccaaggggagggaggagggtccttagga agccagcccctcctgaaaccatctccttatgggcaatcagggatctcactgtcccacctggacctaaggaccaaccccag ctggtgatcccagctggagaagcccagaaacaaagcctcttctgtctctatggaccagctctggacacctgctcatgcagg tgccttccgtctcaactgttccttggttaagagaaaagaactggctgggagaccatgtggtgtatggaactgctgtgctctgt cctacctgccatatcagggccccccttttccagcactgggtgcaaagggatgagtggggtgttaatgctcgaatgtgatac aactg

SOCS1 (SEQ ID NO:66):

ctgtgccgcagcattaagtgggggcgccttattatttcttattattaattattattatttttctggaaccacgtgggagccctccc cgcctgggtcggagggagtggttgtggagggtgagatgcctcccacttctggctggagacctcatcccacctctcaggg gtgggggtgctcccctcctggtgctccctccgggtcccccctggttgtagcagcttgtgtctggggccaggacctgaattc cactcctacctctccatgtttacatattcccagtatctttgcacaaaccaggggtcggggagggtctctggcttcatttttctgc tgtgcagaatatcctattttatatttttacagccagtttaggtaataaac

SOCS2 (SEQ ID NO:67):

ccaggtataagtatttctctctctttttcgtttttttttaaaaaaaaaaaaacacatgcctcatatagactatctccgaatgcagct atgtgaaagagaacccagaggccctcctctggataactgcgcagaattctctcttaaggacagttgggctcagtctaactta aaggtgtgaagatgtagctaggtattttaaagttccccttaggtagttttagctgaatgatgctttctttcctatggctgctcaag atcaaatggcc

SOCS3 (SEQ ID NO:68):

gcagattggcttcttcctcaggccctccactcccgcagagtagagctggcaggacctggaattcgtctgaggggagggg gagctgccacctgctttcccccctcccccagctccagcttctttcaagtggagccagccggcctggcctggtgggacaata cctttgacaagcggactctcccctccccttcctccacaccccctctgcttcccaagggaggtggggacacctccaagtgtt gaacttagaactgcaaggggaatcttcaaactttcccgctggaacttgtttgcgctttgatttggtttgatcaagagcaggca cctgggggaaggatggaagagaaaagggtgtgtgaagggtttttatgctggccaaagaaataaccactcccactgccca acctaggtgaggagtggtggctcctggctctggggagagtggcaaggggtgacctgaagagagctatactggtgccag gctcctctccatggggcagctaatgaaacctcgcagatcccttgcaccccagaaccctccccgttgtgaagaggcagtag catttagaagggagacagatgaggctggtgagctggccgccttttccaacaccgaagggaggcagatcaacagatgag ccatcttggagcccaggtttcccctggagcagatggagggttctgctttgtctctcctatgtggggctaggagactcgcctt aaatgcc

SOCS5 (SEQ ID NO:69):

ggagcttactcgcagtaggctctcgctcttctaatcaatggataaagtggggaaaatgtggaacaacttaaaatacagatgc cagaatctcttcagccacgagggaggaagccgtaatgagaacgtggagatgaaccccaacagatgtccgtctgtcaaag agaaaagcatcagtctgggagaggcagctccccagcaagagagcagtcccttaagagaaaatgttgccttacagctggg actgagcccttccaagaccttttccaggcggaaccaaaactgtgccgcagagatccctcaagtggttgaaatcagcatcg agaaagacagtgactcgggtgccaccccaggaacgaggcttgcacggagagactcctac

CIS (SEQ ID NO:70):

ccaactctgactgagccaggcaccctgctctgcctcacacagtcacatcctggagggaacacagtccccagctggacttg gggttctgctgtcctttcttcagtcatcctggtgcctgcatgcatgtgacagctggaccagagaatgccagcaagaacaag gcaggtggaggagggattgtcacacaactctgaggtcaacgcctctaggtacaatatggctctttgtggtgagccatgtat cagagcgagacaggcaggacctcgtctctccacagaggctggacctaggtctccactcacttgcctgcccttgccacctg asctgtgtctattctcccagccctggtttctcagtctgctgagtagggcaggccccctacccatgtatagaatagcgagcct gtttctgggagaatatcagccagaggttgatcatgccaaggccccttatggggacgcagactgggctaggggactacac agttatacagtatttatttatttattctccttgcaggggttgggggtggaatgatggcgtgagccatcccacttctctgccctgt gctctgggtggtccagagacccccaggtctggttcttccctgtggagacccccatcccaaaacattgttgggcccaaagta gtctcgaatgtcctgggcccatccacctgcgtatggatg

Western Blotting:

Hearts obtained from mice were frozen in liquid nitrogen and homogenized with Tissue Tearor (model 985370, Biospec Products, Inc.) in 800 μl lysis buffer containing 4% SDS, 125 mM Tris-HCl, pH 6.8, and protease inhibitors Antipain, Benzamidin, BeStatin, Chymostatin, Leupeptin, Pefabloc C, Pepstatin A, PMSF, TLCK, TPCK. The protease inhibitors were prepared and used as suggested by the manufacturer, Roche Molecular Biochemicals, Indianapolis, IN. Following homogenization, the DNA in the samples was sheared by sonication for 30 sec at 5 Watts (RMS) output power, with a Virtis sonicator (model Virsonic 60, Virtis Company, Gardiner, N.Y.). Samples were then heated at 95° C. for minutes and centrifuged at 14,000×g for 30 minutes, to get rid of particulate material. Protein concentrations in the extracts were determined using DC Protein Assay (Bio-Rad Laboratories, Hercules, Calif.). The protein concentration in all the samples was equalized to 10 mg/ml by adding lysis buffer, and 1 vol. 2×Sample buffer (20% Glycerol, 0.005% Bromophenol blue) was added to yield samples with a protein concentration of 5 mg/ml. The samples were kept at − 80° C. until use. Prior to loading onto 10% SDS-polyacrylamide gels, 2.5% β-mercaptoethanol was added to every sample (100 μg) followed by heating to 95° C. for 10 min. After electrophoresis, proteins were transferred onto Immobilon-P membranes (Millipore Corporation, Bedford, Mass. 01730) for 2.5 hrs at 100 V, using a Hoefer Transphor Electrophoresis unit. The Transfer buffer consisted of 12 mM Tris base, 96 mM Glycine, and 200 ml Methanol in 1 liter. After the transfer, the membranes were placed in roller bottles and rotated for 1 hr (at 4° C.) in a Blocking solution (1×PBS +0.1% Tween-20+5% non-fat dry milk+0.02% sodium azide), the membranes were rinsed once with the Rinse solution (1×PBS+0.1% Tween-20) and rolled overnight with the primary antibody in the Blocking solution. After washing times in the Rinse solution (15, 10, 5 minutes), the blots were incubated for 1 hour with the secondary antibody conjugated to horseradish peroxidase, followed by another set of washes with the Rinse solution and one final wash in 1×TBS (10 mM Tris-HCl, pH 8.0, 150 mM NaCl). Chemiluminescence was performed by using the Luminol Reagent (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) and detection was with Biomax MR film (Eastman Kodak Company, Rochester, N.Y.). All the antibodies were used at 1:1000 dilution. Stat1, phospo-Stat1, Stat3, phospho-Stat3, Stat4, and Stat5A were from UpState Biotechnology (Lake Placid, N.Y.). Stat2, Stat6, HSC70, and the secondary antibodies were from Santa Cruz Biotechnology.

›PHARMACEUTICAL COMPOSITIONS · 9 of 12

Quantification of RNA and Protein

Quantification of the RNA and protein bands on the Kodak Biomax MR film was performed on a Macintosh Performa 6300CD computer using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet).

Results

STAT and SOCS RNA Levels in Heart Allografts During the 5 Days Following Transplantation

Hearts from Balb/c mice were transplanted into B6/129 female mice using a vascularized mouse cardiac allograft model. The heart transplant resulted with animals having two hearts: their own hearts and a second functional heart grafted adjacent to the kidneys. To determine the kinetics of Stat and SOCS RNA expression following cardiac allograft surgery, total RNA was prepared from the animals' own hearts (native), the transplanted hearts and from the hearts of healthy controls (designated as “N”, “T”, and “C”, respectively) one, two, three, and five days post-transplantation. RNA (25 μg/lane) was then electrophoresed on three identical gels, transferred to Nytran Supercharge membranes and the membranes were hybridized with Stat probes, SOCS probes, and a GAPDH probe as a control (FIGS. 14 - 16 ).

As shown in FIGS. 14 A and 14 B-G, increases of approximately 5-fold in Stat1, Stat2 and Stat3 RNA levels were detected in the transplanted hearts relative to the control hearts as early as one day post-transplantation, and were more evident by the second day. During the first two days, the increase appeared to be localized to only the transplanted tissue. By the third day, and much more strongly by the fifth day, higher levels of Stat1, 2, and 3 RNA were also detected in the native hearts.

Stat4 RNA levels, almost undetectable the first three days post-transplantation, increased significantly (approximately 60-fold) in the transplanted hearts at around day five post-transplantation, but not earlier. Stat5A RNA levels did not change in the native and transplanted hearts during the five days following the transplant surgery. An increase in Stat6 RNA expression was detected in transplanted hearts as early as day one post-transplantation (approximately 2-fold), and remained at approximately the same level during the five days following transplantation. The fact that increases in Stat4 and Stat6 RNA expression were only detected in the transplanted hearts by day five post-transplantation suggests that the up-regulation of the expression of these two Stats was localized to the transplanted hearts (FIGS. 14A, 14 B-G).

An increase in SOCS1 RNA expression levels in the transplanted hearts began to increase by day two post-transplantation and by day five post-transplantation SOCS1 RNA expression in the transplanted hearts had increased approximately 300-fold (FIG. 15 ). Between days 1 and 3, there was an approximately 6-fold difference in such SOCS1 RNA levels, and between days 3 and 5, there was an approximately 25-fold difference in such SOCS1 RNA levels. At approximately 38-fold, the SOCS1 RNA increase in native hearts was much less pronounced (FIG. 15 ).

A considerably high level of SOCS3 RNA expression was detected in the transplanted hearts as early as one day post-transplantation (approximately 100-fold increase), and reached an approximately 230-fold increase at five days post-transplantation. (FIG. 15 ). However, SOCS3 RNA expression remained low in native hearts, and was hardly detectable in the native hearts five days following the transplant surgery. As shown in FIG. 16, Stat4 and SOCS3 RNA expression patterns were found to be quite similar to each other, both of them being expressed only in the transplanted hearts, and both of them being significantly up-regulated at approximately day 5 post-transplantation.

Two species of SOCS5 RNA were detected (4.4 kb and 3.8 kb) in the naive and transplanted hearts. The shorter species 3.8 kb SOCS5 RNA species was much more abundant in the transplanted hearts than in naive hearts or in control hearts. The level of CIS RNA expression detected in naive hearts remained almost unchanged relative to the control hearts, while a lower level of CIS RNA expression was detected in the transplanted hearts relative to the naive hearts or control hearts (FIG. 15 ).

STAT Protein Levels in Heart Allografts and Native Hearts During the 5 Days Post-transplantation

To determine the effect of cardiac allografts on Stat and SOCS protein expression, protein extracts were prepared from transplanted and native hearts at day one, day two, day three, and day five, post-transplantation. In the transplanted hearts an increase in Stat1 protein levels relative to the control hearts was detected as early as one day post-transplantation, increased steadily, and by day five post-transplantation Stat1 protein levels were approximately 17-fold higher (FIG. 17 ). In the native hearts an increase in Stat1 protein levels began increasing at day two post-transplantation and by day five post-transplantation Stat1 levels were 15-fold higher.

An increase in Stat2 protein expression levels in the transplanted hearts was initially detected three days post-transplantation, and by day five post-transplantation had increased sharply by approximately 27-fold. In native hearts an increase in Stat2 protein levels was detected by day three post-transplantation and by day five post-transplantation a 10-fold increase in the levels of Stat2 protein was detected.

An increase in Stat3 protein levels in the transplanted hearts was detected as early as day two post-transplantation and by day five post-transplantation the levels of Stat3 protein in the transplanted hearts exhibited an approximately 3.5-fold increase. In the native hearts a 2-fold increase in the levels of Stat3 protein was detectable only by day five post-transplantation.

Stat4 levels began increasing around one day post-transplantation in the transplanted heart, with a dramatic increase (approximately 16-fold) between day 2 and day 5 post-transplantation. Stat4 was detected only in the transplant tissue, not in the native heart tissue (FIG. 17 ).

›PHARMACEUTICAL COMPOSITIONS · 10 of 12

Stat5A protein levels did not change in the transplanted or native hearts relative to the control hearts.

Stat6 is normally made at a low level in hearts (see, e.g., FIG. 17, control lanes). As was observed with Stat4, Stat6 expression, however, only increased (aproximately 1.5-3.5-fold) in transplanted hearts.

Phosphorylated Forms of Stat1 and Stat3 During Graft Rejection

To determine whether the levels of phosphorylated Stat1 and phosphorylated Stat3 protein parallel that of the newly synthesized Stat1 and Stat3 in the native and transplanted hearts following cardiac allograft surgery, the levels of these Stats and their phosphorylated forms were analyzed (FIG. 18 ). As shown in FIG. 18, an increase in phosphorylated Stat1 protein levels paralleled the increase in the total Stat1 protein levels detected both in native and transplanted hearts on days three and five post-transplantation. In contrast, the level of phosphorylated Stat3 protein detected in native and transplanted hearts over the five day post-transplantation period did not parallel the increase in the total Stat3 protein levels detected during this period. Ijn particular, although there was a steady increase of total Stat3 over the five day period, there was no major change in the levels of phosphorylated Stat3 protein detected over this same period, with the exception of the appearance of a slightly higher molecular weight form of phosphorylated Stat3 protein.

The Effect of Anti-CD40L Monoclonal Antibody MR-1 on the Stat and SOCS RNA Levels in 5-day Post-Transplant Hearts

To determine how Stat and SOCS RNA expression levels are affected in cardiac allografts when tolerance has been induced, six mice having undergone cardiac transplant surgery were divided into three groups. One group of mice were intravenously administered DST (“donor specific transfusion”) +MR-1 anti-CD40L antibody which has been shown to induce tolerance), on the day of the transplant surgery. The second group of mice were intravenously administered DST+IgG as control on the day of the transplant surgery and the third group received no treatment. The animals were sacrificed five days post-transplantation and total RNA was prepared from the animals' own hearts (native), the transplanted hearts and from the hearts of healthy controls (designated as “N”, “T”, and “C”, respectively). The RNA (25 μg /lane) was electrophoresed on four identical gels and the Northern blots were hybridized to Stat probes, SOCS probes and a GAPDH probe as a control. (FIGS. 19, 20 )

Stat1, Stat2, and Stat3 mRNA were found to be expressed at some level in the normal heart (see control lanes of the no treatment group). Five days post-transplantation, the transplant allograft tissue exhibited a dramatic upregulation of Stat1, Stat2 (approximately 45-fold), and Stat3 (approximately 6-fold) mRNAs. This upregulation was found to be systemic in nature in that the increases were also observed in the native heart tissue (approximately 43-fold, 48-fold, and 5.5-fold, respectively). Treatment with DST+IgG caused minimal changes n the transplant Stat RNA levels, but the single does of DST+MR-1 resulted in lower levels of Stat1, Stat2, and Stat3 mRNAs (62%, 76%, and 50% lower than the animals that received no treatment).

Stat4 mRNA was found only in the transplanted heart tissue, as a local signal, and was not detected in the control or native hearts (FIG. 19; see the control and native lanes of the no treatment group). Stat4 mRNA was significantly reduced (approximately 6-fold) in transplants obtained from MR-1-treated animals.

Only minimal differences in Stat 5A mRNA expression in transplant tissue of the group that received no treatment and the controls (approximately 1.4-fold increase) were observed. MR-1 treatment, however, lowered the Stat 5 A expression level by 70%. MR-1 treatment resulted in Stat 5A levels that were 60% lower than control heart levels. Stat 5B mRNA levels were too low to be detected (data not shown).

Stat6 mRNA is normally expressed at some level in the heart, and five days post-transplantation, Stat6 mRNA levels doubled only in the transplanted, not native, hearts. Upon MR-1 treatment, however, Stat6 mRNA levels were downregulated to pre-transplantation (control) levels.

The same membranes were used to determine the mRNA expression levels of SOCS1, SOCS 2, SOC, and CIS (FIG. 20 ). SOCS1 and SOCS3 mRNA levels were almost undetectable in control hearts. SOCS1 and SOCS3 mRNA levels were increased five days post-transplantation in transplant tissue in both DST+IgG treated mice and untreated mice (some low level of SOCS1 expression was also detected in the native hearts of the untreated groups). Treatment with DST+MR-1 resulted in a 93% and 73% reduction in the level of SOCS1 and SOCS3 mRNA expression, respectively, relative to that in transplanted hearts of untreated mice.

SOCS 2 RNA levels remained unchanged in the control, native, and transplanted hearts in the three experimental groups. Interestingly, CIS levels were 50% higher in the transplanted hearts of mice, regardless of treatment.

The Effect of Anti-CD40L Monoclonal Antibody MR-1 on Stat Protein Levels in 5-day and 7-day Post-Transplant Hearts

To determine how Stat protein levels are affected in cardiac allografts when tolerance has been induced, the day of the surgery mice were intravenously administered a single dose of either DST+IgG or DST+MR-1, and the Stat protein levels were analyzed five and seven days post-transplantation. As shown in FIG. 21, at day 5 post-transplantation thee were minimal changes in the Stat levels between IgG and MR-1 treated animals, but a day 7 post-transplantation, there was significant changes in such levels.

Stat1 and Stat2 levels were reduced 64% and 52%, respectively, in native hearts of MR-1 treated animals. Stat3 levels were reduced in the transplant tissue by 29%. The most dramatic change at day 7 post-transplantation was the amount of Stat4 reduction (approximately 63%) in the transplanted hearts. Stat6, on the other hand, tended to increase about 2-fold upon MR-1 treatment.

›PHARMACEUTICAL COMPOSITIONS · 11 of 12

Stat and Bax Protein Levels in Isografts and Allografts

The level of Stat1, Stat2, Stat3, Stat4, Stat5A, and Stat6 were compared in isografts and allografts to exclude the possibility that the observed changes in Stats were due to the surgery and the healing process that follows the surgery. As shown in FIG. 22, between day 1 and day 2 post-transplantation, the isografts showed a small increase in different Stats. The low levels, however, of Stat1, Stat2, and Stat5A remained constant between days 2 and 7 post-transplantation, and Stat3 and Stat6 levels declined between these days. In allografts, on the other hand, all of the Stats reached high levels by day 5. Bax, an unrelated protein, showed a very different profile, with its levels increasing in both isografts and allografts.

Transplantation in Stat6 Knockout Mice

Stat6 is activated by IL-4 and IL-13, and Stat6 knock-out animals have been shown to be deective in TH2 differentiation (Kaplan, M. H. et al., 1996, Imm. 4:313-319; Takeda, K. et al., 1996, Nature 380:627-630; and Shimoda, K. et al., 1996, Nature 380:630-633). The results presented herein indicate that induction of tolerance, by causing lower Stat4 and higher Stat6 levels, shifts the differentiation of T cells toward the TH2 lineage.

To assess the importance of Stat6 and the TH2 response in MR-1 mediated tolerance induction, hearts from B6/129 background mice were transplanted into three Stat6 (−/−) mice with a Balb/c background. The day of the surgery the animals were injected with MR-1 and splenocytes from the donor animals. In normal animals, such a transplant results in indefinite graft survival. Stat6 (−/−) animals, however, readily rejected the transplanted hearts at day 11. This result points out the importance of Stat6 in the induction of tolerance, e.g., the induction of tolerance via MR-1 administration.

The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference.

Equivalents

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

attaaacctc tcgccgagcc cctccgcaga ctctgcgccg gaaagtttca tttgctgtat 60

gccatcctcg agagctgtct aggttaacgt tcgcactctg tgtatataac ctcgacagtc 120

ttggcaccta acgtgctgtg cgtagctgct cctttggttg aatccccagg cccttgttgg 180

ggcacaaggt ggcagg atg tct cag tgg tac gaa ctt cag cag ctt gac tca 232

Met Ser Gln Trp Tyr Glu Leu Gln Gln Leu Asp Ser

1 5 10

aaa ttc ctg gag cag gtt cac cag ctt tat gat gac agt ttt ccc atg 280

Lys Phe Leu Glu Gln Val His Gln Leu Tyr Asp Asp Ser Phe Pro Met

15 20 25

gaa atc aga cag tac ctg gca cag tgg tta gaa aag caa gac tgg gag 328

Glu Ile Arg Gln Tyr Leu Ala Gln Trp Leu Glu Lys Gln Asp Trp Glu

30 35 40

cac gct gcc aat gat gtt tca ttt gcc acc atc cgt ttt cat gac ctc 376

His Ala Ala Asn Asp Val Ser Phe Ala Thr Ile Arg Phe His Asp Leu

45 50 55 60

ctg tca cag ctg gat gat caa tat agt cgc ttt tct ttg gag aat aac 424

Leu Ser Gln Leu Asp Asp Gln Tyr Ser Arg Phe Ser Leu Glu Asn Asn

65 70 75

ttc ttg cta cag cat aac ata agg aaa agc aag cgt aat ctt cag gat 472

Phe Leu Leu Gln His Asn Ile Arg Lys Ser Lys Arg Asn Leu Gln Asp

80 85 90

aat ttt cag gaa gac cca atc cag atg tct atg atc att tac agc tgt 520

Asn Phe Gln Glu Asp Pro Ile Gln Met Ser Met Ile Ile Tyr Ser Cys

95 100 105

ctg aag gaa gaa agg aaa att ctg gaa aac gcc cag aga ttt aat cag 568

Leu Lys Glu Glu Arg Lys Ile Leu Glu Asn Ala Gln Arg Phe Asn Gln

110 115 120

gct cag tcg ggg aat att cag agc aca gtg atg tta gac aaa cag aaa 616

Ala Gln Ser Gly Asn Ile Gln Ser Thr Val Met Leu Asp Lys Gln Lys

125 130 135 140

gag ctt gac agt aaa gtc aga aat gtg aag gac aag gtt atg tgt ata 664

Glu Leu Asp Ser Lys Val Arg Asn Val Lys Asp Lys Val Met Cys Ile

145 150 155

gag cat gaa atc aag agc ctg gaa gat tta caa gat gaa tat gac ttc 712

Glu His Glu Ile Lys Ser Leu Glu Asp Leu Gln Asp Glu Tyr Asp Phe

160 165 170

aaa tgc aaa acc ttg cag aac aga gaa cac gag acc aat ggt gtg gca 760

Lys Cys Lys Thr Leu Gln Asn Arg Glu His Glu Thr Asn Gly Val Ala

175 180 185

aag agt gat cag aaa caa gaa cag ctg tta ctc aag aag atg tat tta 808

Lys Ser Asp Gln Lys Gln Glu Gln Leu Leu Leu Lys Lys Met Tyr Leu

190 195 200

atg ctt gac aat aag aga aag gaa gta gtt cac aaa ata ata gag ttg 856

Met Leu Asp Asn Lys Arg Lys Glu Val Val His Lys Ile Ile Glu Leu

205 210 215 220

ctg aat gtc act gaa ctt acc cag aat gcc ctg att aat gat gaa cta 904

Leu Asn Val Thr Glu Leu Thr Gln Asn Ala Leu Ile Asn Asp Glu Leu

225 230 235

gtg gag tgg aag cgg aga cag cag agc gcc tgt att ggg ggg ccg ccc 952

Val Glu Trp Lys Arg Arg Gln Gln Ser Ala Cys Ile Gly Gly Pro Pro

240 245 250

aat gct tgc ttg gat cag ctg cag aac tgg ttc act ata gtt gcg gag 1000

Asn Ala Cys Leu Asp Gln Leu Gln Asn Trp Phe Thr Ile Val Ala Glu

255 260 265

agt ctg cag caa gtt cgg cag cag ctt aaa aag ttg gag gaa ttg gaa 1048

Ser Leu Gln Gln Val Arg Gln Gln Leu Lys Lys Leu Glu Glu Leu Glu

270 275 280

cag aaa tac acc tac gaa cat gac cct atc aca aaa aac aaa caa gtg 1096

Gln Lys Tyr Thr Tyr Glu His Asp Pro Ile Thr Lys Asn Lys Gln Val

285 290 295 300

tta tgg gac cgc acc ttc agt ctt ttc cag cag ctc att cag agc tcg 1144

Leu Trp Asp Arg Thr Phe Ser Leu Phe Gln Gln Leu Ile Gln Ser Ser

305 310 315

ttt gtg gtg gaa aga cag ccc tgc atg cca acg cac cct cag agg ccg 1192

Phe Val Val Glu Arg Gln Pro Cys Met Pro Thr His Pro Gln Arg Pro

320 325 330

ctg gtc ttg aag aca ggg gtc cag ttc act gtg aag ttg aga ctg ttg 1240

Leu Val Leu Lys Thr Gly Val Gln Phe Thr Val Lys Leu Arg Leu Leu

335 340 345

gtg aaa ttg caa gag ctg aat tat aat ttg aaa gtc aaa gtc tta ttt 1288

Val Lys Leu Gln Glu Leu Asn Tyr Asn Leu Lys Val Lys Val Leu Phe

350 355 360

gat aaa gat gtg aat gag aga aat aca gta aaa gga ttt agg aag ttc 1336

Asp Lys Asp Val Asn Glu Arg Asn Thr Val Lys Gly Phe Arg Lys Phe

365 370 375 380

aac att ttg ggc acg cac aca aaa gtg atg aac atg gag gag tcc acc 1384

Asn Ile Leu Gly Thr His Thr Lys Val Met Asn Met Glu Glu Ser Thr

385 390 395

aat ggc agt ctg gcg gct gaa ttt cgg cac ctg caa ttg aaa gaa cag 1432

›PHARMACEUTICAL COMPOSITIONS · 12 of 12

Asn Gly Ser Leu Ala Ala Glu Phe Arg His Leu Gln Leu Lys Glu Gln

400 405 410

aaa aat gct ggc acc aga acg aat gag ggt cct ctc atc gtt act gaa 1480

Lys Asn Ala Gly Thr Arg Thr Asn Glu Gly Pro Leu Ile Val Thr Glu

415 420 425

gag ctt cac tcc ctt agt ttt gaa acc caa ttg tgc cag cct ggt ttg 1528

Glu Leu His Ser Leu Ser Phe Glu Thr Gln Leu Cys Gln Pro Gly Leu

430 435 440

gta att gac ctc gag acg acc tct ctg ccc gtt gtg gtg atc tcc aac 1576

Val Ile Asp Leu Glu Thr Thr Ser Leu Pro Val Val Val Ile Ser Asn

445 450 455 460

gtc agc cag ctc ccg agc ggt tgg gcc tcc atc ctt tgg tac aac atg 1624

Val Ser Gln Leu Pro Ser Gly Trp Ala Ser Ile Leu Trp Tyr Asn Met

465 470 475

ctg gtg gcg gaa ccc agg aat ctg tcc ttc ttc ctg act cca cca tgt 1672

Leu Val Ala Glu Pro Arg Asn Leu Ser Phe Phe Leu Thr Pro Pro Cys

480 485 490

gca cga tgg gct cag ctt tca gaa gtg ctg agt tgg cag ttt tct tct 1720

Ala Arg Trp Ala Gln Leu Ser Glu Val Leu Ser Trp Gln Phe Ser Ser

495 500 505

gtc acc aaa aga ggt ctc aat gtg gac cag ctg aac atg ttg gga gag 1768

Val Thr Lys Arg Gly Leu Asn Val Asp Gln Leu Asn Met Leu Gly Glu

510 515 520

aag ctt ctt ggt cct aac gcc agc ccc gat ggt ctc att ccg tgg acg 1816

Lys Leu Leu Gly Pro Asn Ala Ser Pro Asp Gly Leu Ile Pro Trp Thr

525 530 535 540

agg ttt tgt aag gaa aat ata aat gat aaa aat ttt ccc ttc tgg ctt 1864

Arg Phe Cys Lys Glu Asn Ile Asn Asp Lys Asn Phe Pro Phe Trp Leu

545 550 555

tgg att gaa agc atc cta gaa ctc att aaa aaa cac ctg ctc cct ctc 1912

Trp Ile Glu Ser Ile Leu Glu Leu Ile Lys Lys His Leu Leu Pro Leu

560 565 570

tgg aat gat ggg tgc atc atg ggc ttc atc agc aag gag cga gag cgt 1960

Trp Asn Asp Gly Cys Ile Met Gly Phe Ile Ser Lys Glu Arg Glu Arg

575 580 585

gcc ctg ttg aag gac cag cag ccg ggg acc ttc ctg ctg cgg ttc agt 2008

Ala Leu Leu Lys Asp Gln Gln Pro Gly Thr Phe Leu Leu Arg Phe Ser

590 595 600

gag agc tcc cgg gaa ggg gcc atc aca ttc aca tgg gtg gag cgg tcc 2056

Glu Ser Ser Arg Glu Gly Ala Ile Thr Phe Thr Trp Val Glu Arg Ser

605 610 615 620

cag aac gga ggc gaa cct gac ttc cat gcg gtt gaa ccc tac acg aag 2104

Gln Asn Gly Gly Glu Pro Asp Phe His Ala Val Glu Pro Tyr Thr Lys

625 630 635

aaa gaa ctt tct gct gtt act ttc cct gac atc att cgc aat tac aaa 2152

Lys Glu Leu Ser Ala Val Thr Phe Pro Asp Ile Ile Arg Asn Tyr Lys

640 645 650

gtc atg gct gct gag aat att cct gag aat ccc ctg aag tat ctg tat 2200

Val Met Ala Ala Glu Asn Ile Pro Glu Asn Pro Leu Lys Tyr Leu Tyr

655 660 665

cca aat att gac aaa gac cat gcc ttt gga aag tat tac tcc agg cca 2248

Pro Asn Ile Asp Lys Asp His Ala Phe Gly Lys Tyr Tyr Ser Arg Pro

670 675 680

aag gaa gca cca gag cca atg gaa ctt gat ggc cct aaa gga act gga 2296

Lys Glu Ala Pro Glu Pro Met Glu Leu Asp Gly Pro Lys Gly Thr Gly

685 690 695 700

tat atc aag act gag ttg att tct gtg tct gaa gtt cac cct tct aga 2344

Tyr Ile Lys Thr Glu Leu Ile Ser Val Ser Glu Val His Pro Ser Arg

705 710 715

ctt cag acc aca gac aac ctg ctc ccc atg tct cct gag gag ttt gac 2392

Leu Gln Thr Thr Asp Asn Leu Leu Pro Met Ser Pro Glu Glu Phe Asp

720 725 730

gag gtg tct cgg ata gtg ggc tct gta gaa ttc gac agt atg atg aac 2440

Glu Val Ser Arg Ile Val Gly Ser Val Glu Phe Asp Ser Met Met Asn

735 740 745

aca gta tag agcatgaatt tttttcatct tctctggcga cagttttcct 2489

Thr Val *

750

tctcatctgt gattccctcc tgctactctg ttccttcaca tcctgtgttt ctagggaaat 2549

gaaagaaagg ccagcaaatt cgctgcaacc tgttgatagc aagtgaattt ttctctaact 2609

cagaaacatc agttactctg aagggcatca tgcatcttac tgaaggtaaa attgaaaggc 2669

attctctgaa gagtgggttt cacaagtgaa aaacatccag atacacccaa agtatcagga 2729

cgagaatgag ggtcctttgg gaaaggagaa gttaagcaac atctagcaaa tgttatgcat 2789

aaagtcagtg cccaactgtt ataggttgtt ggataaatca gtggttattt agggaactgc 2849

ttgacgtagg aacggtaaat ttctgtggga gaattcttac atgttttctt tgctttaagt 2909

gtaactggca gttttccatt ggtttacctg tgaaatagtt caaagccaag tttatataca 2969

attatatcag tcctctttca aaggtagcca tcatggatct ggtaggggga aaatgtgtat 3029

tttattacat ctttcacatt ggctatttaa agacaaagac aaattctgtt tcttgagaag 3089

agaatattag ctttactgtt tgttatggct taatgacact agctaatatc aatagaagga 3149

tgtacatttc caaattcaca agttgtgttt gatatccaaa gctgaataca ttctgctttc 3209

atcttggtca catacaatta tttttacagt tctcccaagg gagttaggct attcacaacc 3269

actcattcaa aagttgaaat taaccataga tgtagataaa ctcagaaatt taattcatgt 3329

ttcttaaatg ggctactttg tcctttttgt tattagggtg gtatttagtc tattagccac 3389

aaaattggga aaggagtaga aaaagcagta actgacaact tgaataatac accagagata 3449

atatgagaat cagatcattt caaaactcat ttcctatgta actgcattga gaactgcata 3509

tgtttcgctg atatatgtgt ttttcacatt tgcgaatggt tccattctct ctcctgtact 3569

ttttccagac acttttttga gtggatgatg tttcgtgaag tatactgtat ttttaccttt 3629

ttccttcctt atcactgaca caaaaagtag attaagagat gggtttgaca aggttcttcc 3689

cttttacata ctgctgtcta tgtggctgta tcttgttttt ccactactgc taccacaact 3749

atattatcat gcaaatgctg tattcttctt tggtggagat aaagatttct tgagttttgt 3809

tttaaaatta aagctaaagt atctgtattg cattaaatat aatatcgaca cagtgctttc 3869

cgtggcactg catacaatct gaggcctcct ctctcagttt ttatatagat ggcgagaacc 3929

taagtttcag ttgattttac aattgaaatg actaaaaaac aaagaagaca acattaaaaa 3989

caatattgtt tcta 4003

750

›PRT · 1 of 2

Homo sapiens

Met Ser Gln Trp Tyr Glu Leu Gln Gln Leu Asp Ser Lys Phe Leu Glu

1 5 10 15

Gln Val His Gln Leu Tyr Asp Asp Ser Phe Pro Met Glu Ile Arg Gln

20 25 30

Tyr Leu Ala Gln Trp Leu Glu Lys Gln Asp Trp Glu His Ala Ala Asn

35 40 45

Asp Val Ser Phe Ala Thr Ile Arg Phe His Asp Leu Leu Ser Gln Leu

50 55 60

Asp Asp Gln Tyr Ser Arg Phe Ser Leu Glu Asn Asn Phe Leu Leu Gln

65 70 75 80

His Asn Ile Arg Lys Ser Lys Arg Asn Leu Gln Asp Asn Phe Gln Glu

85 90 95

Asp Pro Ile Gln Met Ser Met Ile Ile Tyr Ser Cys Leu Lys Glu Glu

100 105 110

Arg Lys Ile Leu Glu Asn Ala Gln Arg Phe Asn Gln Ala Gln Ser Gly

115 120 125

Asn Ile Gln Ser Thr Val Met Leu Asp Lys Gln Lys Glu Leu Asp Ser

130 135 140

Lys Val Arg Asn Val Lys Asp Lys Val Met Cys Ile Glu His Glu Ile

145 150 155 160

Lys Ser Leu Glu Asp Leu Gln Asp Glu Tyr Asp Phe Lys Cys Lys Thr

165 170 175

Leu Gln Asn Arg Glu His Glu Thr Asn Gly Val Ala Lys Ser Asp Gln

180 185 190

Lys Gln Glu Gln Leu Leu Leu Lys Lys Met Tyr Leu Met Leu Asp Asn

195 200 205

Lys Arg Lys Glu Val Val His Lys Ile Ile Glu Leu Leu Asn Val Thr

210 215 220

Glu Leu Thr Gln Asn Ala Leu Ile Asn Asp Glu Leu Val Glu Trp Lys

225 230 235 240

Arg Arg Gln Gln Ser Ala Cys Ile Gly Gly Pro Pro Asn Ala Cys Leu

245 250 255

Asp Gln Leu Gln Asn Trp Phe Thr Ile Val Ala Glu Ser Leu Gln Gln

260 265 270

Val Arg Gln Gln Leu Lys Lys Leu Glu Glu Leu Glu Gln Lys Tyr Thr

275 280 285

Tyr Glu His Asp Pro Ile Thr Lys Asn Lys Gln Val Leu Trp Asp Arg

290 295 300

Thr Phe Ser Leu Phe Gln Gln Leu Ile Gln Ser Ser Phe Val Val Glu

305 310 315 320

Arg Gln Pro Cys Met Pro Thr His Pro Gln Arg Pro Leu Val Leu Lys

325 330 335

Thr Gly Val Gln Phe Thr Val Lys Leu Arg Leu Leu Val Lys Leu Gln

340 345 350

Glu Leu Asn Tyr Asn Leu Lys Val Lys Val Leu Phe Asp Lys Asp Val

355 360 365

Asn Glu Arg Asn Thr Val Lys Gly Phe Arg Lys Phe Asn Ile Leu Gly

370 375 380

Thr His Thr Lys Val Met Asn Met Glu Glu Ser Thr Asn Gly Ser Leu

385 390 395 400

Ala Ala Glu Phe Arg His Leu Gln Leu Lys Glu Gln Lys Asn Ala Gly

405 410 415

Thr Arg Thr Asn Glu Gly Pro Leu Ile Val Thr Glu Glu Leu His Ser

420 425 430

Leu Ser Phe Glu Thr Gln Leu Cys Gln Pro Gly Leu Val Ile Asp Leu

435 440 445

Glu Thr Thr Ser Leu Pro Val Val Val Ile Ser Asn Val Ser Gln Leu

450 455 460

Pro Ser Gly Trp Ala Ser Ile Leu Trp Tyr Asn Met Leu Val Ala Glu

465 470 475 480

Pro Arg Asn Leu Ser Phe Phe Leu Thr Pro Pro Cys Ala Arg Trp Ala

485 490 495

Gln Leu Ser Glu Val Leu Ser Trp Gln Phe Ser Ser Val Thr Lys Arg

500 505 510

Gly Leu Asn Val Asp Gln Leu Asn Met Leu Gly Glu Lys Leu Leu Gly

515 520 525

Pro Asn Ala Ser Pro Asp Gly Leu Ile Pro Trp Thr Arg Phe Cys Lys

530 535 540

Glu Asn Ile Asn Asp Lys Asn Phe Pro Phe Trp Leu Trp Ile Glu Ser

545 550 555 560

Ile Leu Glu Leu Ile Lys Lys His Leu Leu Pro Leu Trp Asn Asp Gly

565 570 575

Cys Ile Met Gly Phe Ile Ser Lys Glu Arg Glu Arg Ala Leu Leu Lys

580 585 590

Asp Gln Gln Pro Gly Thr Phe Leu Leu Arg Phe Ser Glu Ser Ser Arg

595 600 605

Glu Gly Ala Ile Thr Phe Thr Trp Val Glu Arg Ser Gln Asn Gly Gly

610 615 620

Glu Pro Asp Phe His Ala Val Glu Pro Tyr Thr Lys Lys Glu Leu Ser

625 630 635 640

Ala Val Thr Phe Pro Asp Ile Ile Arg Asn Tyr Lys Val Met Ala Ala

645 650 655

Glu Asn Ile Pro Glu Asn Pro Leu Lys Tyr Leu Tyr Pro Asn Ile Asp

660 665 670

Lys Asp His Ala Phe Gly Lys Tyr Tyr Ser Arg Pro Lys Glu Ala Pro

675 680 685

Glu Pro Met Glu Leu Asp Gly Pro Lys Gly Thr Gly Tyr Ile Lys Thr

690 695 700

Glu Leu Ile Ser Val Ser Glu Val His Pro Ser Arg Leu Gln Thr Thr

705 710 715 720

Asp Asn Leu Leu Pro Met Ser Pro Glu Glu Phe Asp Glu Val Ser Arg

725 730 735

Ile Val Gly Ser Val Glu Phe Asp Ser Met Met Asn Thr Val

740 745 750

aagtcgcgac cagagccatt ggagggcgcg gggactgcaa ccctaatcag agcccaa atg 60

Met

gcg cag tgg gaa atg ctg cag aat ctt gac agc ccc ttt cag gat cag 108

Ala Gln Trp Glu Met Leu Gln Asn Leu Asp Ser Pro Phe Gln Asp Gln

5 10 15

ctg cac cag ctt tac tcg cac agc ctc ctg cct gtg gac att cga cag 156

Leu His Gln Leu Tyr Ser His Ser Leu Leu Pro Val Asp Ile Arg Gln

20 25 30

tac ttg gct gtc tgg att gaa gac cag aac tgg cag gaa gct gca ctt 204

Tyr Leu Ala Val Trp Ile Glu Asp Gln Asn Trp Gln Glu Ala Ala Leu

35 40 45

ggg agt gat gat tcc aag gct acc atg cta ttc ttc cac ttc ttg gat 252

Gly Ser Asp Asp Ser Lys Ala Thr Met Leu Phe Phe His Phe Leu Asp

50 55 60 65

cag ctg aac tat gag tgt ggc cgt tgc agc cag gac cca gag tcc ttg 300

Gln Leu Asn Tyr Glu Cys Gly Arg Cys Ser Gln Asp Pro Glu Ser Leu

70 75 80

ttg ctg cag cac aat ttg cgg aaa ttc tgc cgg gac att cag ccc ttt 348

Leu Leu Gln His Asn Leu Arg Lys Phe Cys Arg Asp Ile Gln Pro Phe

85 90 95

tcc cag gat cct acc cag ttg gct gag atg atc ttt aac ctc ctt ctg 396

Ser Gln Asp Pro Thr Gln Leu Ala Glu Met Ile Phe Asn Leu Leu Leu

100 105 110

gaa gaa aaa aga att ttg atc cag gct cag agg gcc caa ttg gaa caa 444

Glu Glu Lys Arg Ile Leu Ile Gln Ala Gln Arg Ala Gln Leu Glu Gln

115 120 125

gga gag cca gtt ctc gaa aca cct gtg gag agc cag caa cat gag att 492

Gly Glu Pro Val Leu Glu Thr Pro Val Glu Ser Gln Gln His Glu Ile

130 135 140 145

gaa tcc cgg atc ctg gat tta agg gct atg atg gag aag ctg gta aaa 540

Glu Ser Arg Ile Leu Asp Leu Arg Ala Met Met Glu Lys Leu Val Lys

150 155 160

tcc atc agc caa ctg aaa gac cag cag gat gtc ttc tgc ttc cga tat 588

Ser Ile Ser Gln Leu Lys Asp Gln Gln Asp Val Phe Cys Phe Arg Tyr

165 170 175

aag atc cag gcc aaa ggg aag aca ccc tct ctg gac ccc cat cag acc 636

Lys Ile Gln Ala Lys Gly Lys Thr Pro Ser Leu Asp Pro His Gln Thr

180 185 190

aaa gag cag aag att ctg cag gaa act ctc aat gaa ctg gac aaa agg 684

Lys Glu Gln Lys Ile Leu Gln Glu Thr Leu Asn Glu Leu Asp Lys Arg

195 200 205

aga aag gag gtg ctg gat gcc tcc aaa gca ctg cta ggc cga tta act 732

Arg Lys Glu Val Leu Asp Ala Ser Lys Ala Leu Leu Gly Arg Leu Thr

210 215 220 225

acc cta atc gag cta ctg ctg cca aag ttg gag gag tgg aag gcc cag 780

Thr Leu Ile Glu Leu Leu Leu Pro Lys Leu Glu Glu Trp Lys Ala Gln

230 235 240

cag caa aaa gcc tgc atc aga gct ccc att gac cac ggg ttg gaa cag 828

Gln Gln Lys Ala Cys Ile Arg Ala Pro Ile Asp His Gly Leu Glu Gln

245 250 255

ctg gag aca tgg ttc aca gct gga gca aag ctg ttg ttt cac ctg agg 876

Leu Glu Thr Trp Phe Thr Ala Gly Ala Lys Leu Leu Phe His Leu Arg

260 265 270

cag ctg ctg aag gag ctg aag gga ctg agt tgc ctg gtt agc tat cag 924

›PRT · 2 of 2

Gln Leu Leu Lys Glu Leu Lys Gly Leu Ser Cys Leu Val Ser Tyr Gln

275 280 285

gat gac cct ctg acc aaa ggg gtg gac cta cgc aac gcc cag gtc aca 972

Asp Asp Pro Leu Thr Lys Gly Val Asp Leu Arg Asn Ala Gln Val Thr

290 295 300 305

gag ttg cta cag cgt ctg ctc cac aga gcc ttt gtg gta gaa acc cag 1020

Glu Leu Leu Gln Arg Leu Leu His Arg Ala Phe Val Val Glu Thr Gln

310 315 320

ccc tgc atg ccc caa act ccc cat cga ccc ctc atc ctc aag act ggc 1068

Pro Cys Met Pro Gln Thr Pro His Arg Pro Leu Ile Leu Lys Thr Gly

325 330 335

agc aag ttc acc gtc cga aca agg ctg ctg gtg aga ctc cag gaa ggc 1116

Ser Lys Phe Thr Val Arg Thr Arg Leu Leu Val Arg Leu Gln Glu Gly

340 345 350

aat gag tca ctg act gtg gaa gtc tcc att gac agg aat cct cct caa 1164

Asn Glu Ser Leu Thr Val Glu Val Ser Ile Asp Arg Asn Pro Pro Gln

355 360 365

tta caa ggc ttc cgg aag ttc aac att ctg act tca aac cag aaa act 1212

Leu Gln Gly Phe Arg Lys Phe Asn Ile Leu Thr Ser Asn Gln Lys Thr

370 375 380 385

ttg acc ccc gag aag ggg cag agt cag ggt ttg att tgg gac ttt ggt 1260

Leu Thr Pro Glu Lys Gly Gln Ser Gln Gly Leu Ile Trp Asp Phe Gly

390 395 400

tac ctg act ctg gtg gag caa cgt tca ggt ggt tca gga aag ggc agc 1308

Tyr Leu Thr Leu Val Glu Gln Arg Ser Gly Gly Ser Gly Lys Gly Ser

405 410 415

aat aag ggg cca cta ggt gtg aca gag gaa ctg cac atc atc agc ttc 1356

Asn Lys Gly Pro Leu Gly Val Thr Glu Glu Leu His Ile Ile Ser Phe

420 425 430

acg gtc aaa tat acc tac cag ggt ctg aag cag gag ctg aaa acg gac 1404

Thr Val Lys Tyr Thr Tyr Gln Gly Leu Lys Gln Glu Leu Lys Thr Asp

435 440 445

acc ctc cct gtg gtg att att tcc aac atg aac cag ctc tca att gcc 1452

Thr Leu Pro Val Val Ile Ile Ser Asn Met Asn Gln Leu Ser Ile Ala

450 455 460 465

tgg gct tca gtt ctc tgg ttc aat ttg ctc agc cca aac ctt cag aac 1500

Trp Ala Ser Val Leu Trp Phe Asn Leu Leu Ser Pro Asn Leu Gln Asn

470 475 480

cag cag ttc ttc tcc aac ccc ccc aag gcc ccc tgg agc ttg ctg ggc 1548

Gln Gln Phe Phe Ser Asn Pro Pro Lys Ala Pro Trp Ser Leu Leu Gly

485 490 495

cct gct ctc agt tgg cag ttc tcc tcc tat gtt ggc cga ggc ctc aac 1596

Pro Ala Leu Ser Trp Gln Phe Ser Ser Tyr Val Gly Arg Gly Leu Asn

500 505 510

tca gac cag ctg agc atg ctg aga aac aag ctg ttc ggg cag aac tgt 1644

Ser Asp Gln Leu Ser Met Leu Arg Asn Lys Leu Phe Gly Gln Asn Cys

515 520 525

agg act gag gat cca tta ttg tcc tgg gct gac ttc act aag cga gag 1692

Arg Thr Glu Asp Pro Leu Leu Ser Trp Ala Asp Phe Thr Lys Arg Glu

530 535 540 545

agc cct cct ggc aag tta cca ttc tgg aca tgg ctg gac aaa att ctg 1740

Ser Pro Pro Gly Lys Leu Pro Phe Trp Thr Trp Leu Asp Lys Ile Leu

550 555 560

gag ttg gta cat gac cac ctg aag gat ctc tgg aat gat gga cgc atc 1788

Glu Leu Val His Asp His Leu Lys Asp Leu Trp Asn Asp Gly Arg Ile

565 570 575

atg ggc ttt gtg agt cgg agc cag gag cgc cgg ctg ctg aag aag acc 1836

Met Gly Phe Val Ser Arg Ser Gln Glu Arg Arg Leu Leu Lys Lys Thr

580 585 590

atg tct ggc acc ttt cta ctg cgc ttc agt gaa tcg tca gaa ggg ggc 1884

Met Ser Gly Thr Phe Leu Leu Arg Phe Ser Glu Ser Ser Glu Gly Gly

595 600 605

att acc tgc tcc tgg gtg gag cac cag gat gat gac aag gtg ctc atc 1932

Ile Thr Cys Ser Trp Val Glu His Gln Asp Asp Asp Lys Val Leu Ile

610 615 620 625

tac tct gtg caa ccg tac acg aag gag gtg ctg cag tca ctc ccg ctg 1980

Tyr Ser Val Gln Pro Tyr Thr Lys Glu Val Leu Gln Ser Leu Pro Leu

630 635 640

act gaa atc atc cgc cat tac cag ttg ctc act gag gag aat ata cct 2028

Thr Glu Ile Ile Arg His Tyr Gln Leu Leu Thr Glu Glu Asn Ile Pro

645 650 655

gaa aac cca ctg cgc ttc ctc tat ccc cga atc ccc cgg gat gaa gct 2076

Glu Asn Pro Leu Arg Phe Leu Tyr Pro Arg Ile Pro Arg Asp Glu Ala

660 665 670

ttt ggg tgc tac tac cag gag aaa gtt aat ctc cag gaa cgg agg aaa 2124

Phe Gly Cys Tyr Tyr Gln Glu Lys Val Asn Leu Gln Glu Arg Arg Lys

675 680 685

tac ctg aaa cac agg ctc att gtg gtc tct aat aga cag gtg gat gaa 2172

Tyr Leu Lys His Arg Leu Ile Val Val Ser Asn Arg Gln Val Asp Glu

690 695 700 705

ctg caa caa ccg ctg gag ctt aag cca gag cca gag ctg gag tca tta 2220

Leu Gln Gln Pro Leu Glu Leu Lys Pro Glu Pro Glu Leu Glu Ser Leu

710 715 720

gag ctg gaa cta ggg ctg gtg cca gag cca gag ctc agc ctg gac tta 2268

Glu Leu Glu Leu Gly Leu Val Pro Glu Pro Glu Leu Ser Leu Asp Leu

725 730 735

gag cca ctg ctg aag gca ggg ctg gat ctg ggg cca gag cta gag tct 2316

Glu Pro Leu Leu Lys Ala Gly Leu Asp Leu Gly Pro Glu Leu Glu Ser

740 745 750

gtg ctg gag tcc act ctg gag cct gtg ata gag ccc aca cta tgc atg 2364

Val Leu Glu Ser Thr Leu Glu Pro Val Ile Glu Pro Thr Leu Cys Met

755 760 765

gta tca caa aca gtg cca gag cca gac caa gga cct gta tca cag cca 2412

Val Ser Gln Thr Val Pro Glu Pro Asp Gln Gly Pro Val Ser Gln Pro

770 775 780 785

gtg cca gag cca gat ttg ccc tgt gat ctg aga cat ttg aac act gag 2460

Val Pro Glu Pro Asp Leu Pro Cys Asp Leu Arg His Leu Asn Thr Glu

790 795 800

cca atg gaa atc ttc aga aac tgt gta aag att gaa gaa atc atg ccg 2508

Pro Met Glu Ile Phe Arg Asn Cys Val Lys Ile Glu Glu Ile Met Pro

805 810 815

aat ggt gac cca ctg ttg gct ggc cag aac acc gtg gat gag gtt tac 2556

Asn Gly Asp Pro Leu Leu Ala Gly Gln Asn Thr Val Asp Glu Val Tyr

820 825 830

gtc tcc cgc ccc agc cac ttc tac act gat gga ccc ttg atg cct tct 2604

Val Ser Arg Pro Ser His Phe Tyr Thr Asp Gly Pro Leu Met Pro Ser

835 840 845

gac ttc tag gaaccacatt tcctctgttc ttttcatatc tctttgccct 2653

Asp Phe *

850

tcctactcct catagcatga tattgttctc caaggatggg aatcaggcat gtgtcccttc 2713

caagctgtgt taactgttca aactcaggcc tgtgtgactc cattggggtg agaggtgaaa 2773

gcataacatg ggtacagagg ggacaacaat gaatcagaac agatgctgag ccataggtct 2833

aaataggatc ctggaggctg cctgctgtgc tgggaggtat aggggtcctg ggggcaggcc 2893

agggcagttg acaggtactt ggagggctca gggcagtggc ttctttccag tatggaagga 2953

tttcaacatt ttaatagttg gttaggctaa actggtgcat actggcattg gccttggtgg 3013

ggagcacaga cacaggatag gactccattt ctttcttcca ttccttcatg tctaggataa 3073

cttgctttct tctttccttt actcctggct caagccctga atttcttctt ttcctgcagg 3133

ggttgagagc tttctgcctt agcctaccat gtgaaactct accctgaaga aagggatgga 3193

taggaagtag acctcttttt cttaccagtc tcctccccta ctctgccccc taagctggct 3253

gtacctgttc ctcccccata aaatgatcct gccaatct 3291

851

›PRT · 1 of 2

Homo sapiens

Met Ala Gln Trp Glu Met Leu Gln Asn Leu Asp Ser Pro Phe Gln Asp

1 5 10 15

Gln Leu His Gln Leu Tyr Ser His Ser Leu Leu Pro Val Asp Ile Arg

20 25 30

Gln Tyr Leu Ala Val Trp Ile Glu Asp Gln Asn Trp Gln Glu Ala Ala

35 40 45

Leu Gly Ser Asp Asp Ser Lys Ala Thr Met Leu Phe Phe His Phe Leu

50 55 60

Asp Gln Leu Asn Tyr Glu Cys Gly Arg Cys Ser Gln Asp Pro Glu Ser

65 70 75 80

Leu Leu Leu Gln His Asn Leu Arg Lys Phe Cys Arg Asp Ile Gln Pro

85 90 95

Phe Ser Gln Asp Pro Thr Gln Leu Ala Glu Met Ile Phe Asn Leu Leu

100 105 110

Leu Glu Glu Lys Arg Ile Leu Ile Gln Ala Gln Arg Ala Gln Leu Glu

115 120 125

Gln Gly Glu Pro Val Leu Glu Thr Pro Val Glu Ser Gln Gln His Glu

130 135 140

Ile Glu Ser Arg Ile Leu Asp Leu Arg Ala Met Met Glu Lys Leu Val

145 150 155 160

Lys Ser Ile Ser Gln Leu Lys Asp Gln Gln Asp Val Phe Cys Phe Arg

165 170 175

Tyr Lys Ile Gln Ala Lys Gly Lys Thr Pro Ser Leu Asp Pro His Gln

180 185 190

Thr Lys Glu Gln Lys Ile Leu Gln Glu Thr Leu Asn Glu Leu Asp Lys

195 200 205

Arg Arg Lys Glu Val Leu Asp Ala Ser Lys Ala Leu Leu Gly Arg Leu

210 215 220

Thr Thr Leu Ile Glu Leu Leu Leu Pro Lys Leu Glu Glu Trp Lys Ala

225 230 235 240

Gln Gln Gln Lys Ala Cys Ile Arg Ala Pro Ile Asp His Gly Leu Glu

245 250 255

Gln Leu Glu Thr Trp Phe Thr Ala Gly Ala Lys Leu Leu Phe His Leu

260 265 270

Arg Gln Leu Leu Lys Glu Leu Lys Gly Leu Ser Cys Leu Val Ser Tyr

275 280 285

Gln Asp Asp Pro Leu Thr Lys Gly Val Asp Leu Arg Asn Ala Gln Val

290 295 300

Thr Glu Leu Leu Gln Arg Leu Leu His Arg Ala Phe Val Val Glu Thr

305 310 315 320

Gln Pro Cys Met Pro Gln Thr Pro His Arg Pro Leu Ile Leu Lys Thr

325 330 335

Gly Ser Lys Phe Thr Val Arg Thr Arg Leu Leu Val Arg Leu Gln Glu

340 345 350

Gly Asn Glu Ser Leu Thr Val Glu Val Ser Ile Asp Arg Asn Pro Pro

355 360 365

Gln Leu Gln Gly Phe Arg Lys Phe Asn Ile Leu Thr Ser Asn Gln Lys

370 375 380

Thr Leu Thr Pro Glu Lys Gly Gln Ser Gln Gly Leu Ile Trp Asp Phe

385 390 395 400

Gly Tyr Leu Thr Leu Val Glu Gln Arg Ser Gly Gly Ser Gly Lys Gly

405 410 415

Ser Asn Lys Gly Pro Leu Gly Val Thr Glu Glu Leu His Ile Ile Ser

420 425 430

Phe Thr Val Lys Tyr Thr Tyr Gln Gly Leu Lys Gln Glu Leu Lys Thr

435 440 445

Asp Thr Leu Pro Val Val Ile Ile Ser Asn Met Asn Gln Leu Ser Ile

450 455 460

Ala Trp Ala Ser Val Leu Trp Phe Asn Leu Leu Ser Pro Asn Leu Gln

465 470 475 480

Asn Gln Gln Phe Phe Ser Asn Pro Pro Lys Ala Pro Trp Ser Leu Leu

485 490 495

Gly Pro Ala Leu Ser Trp Gln Phe Ser Ser Tyr Val Gly Arg Gly Leu

500 505 510

Asn Ser Asp Gln Leu Ser Met Leu Arg Asn Lys Leu Phe Gly Gln Asn

515 520 525

Cys Arg Thr Glu Asp Pro Leu Leu Ser Trp Ala Asp Phe Thr Lys Arg

530 535 540

Glu Ser Pro Pro Gly Lys Leu Pro Phe Trp Thr Trp Leu Asp Lys Ile

545 550 555 560

Leu Glu Leu Val His Asp His Leu Lys Asp Leu Trp Asn Asp Gly Arg

565 570 575

Ile Met Gly Phe Val Ser Arg Ser Gln Glu Arg Arg Leu Leu Lys Lys

580 585 590

Thr Met Ser Gly Thr Phe Leu Leu Arg Phe Ser Glu Ser Ser Glu Gly

595 600 605

Gly Ile Thr Cys Ser Trp Val Glu His Gln Asp Asp Asp Lys Val Leu

610 615 620

Ile Tyr Ser Val Gln Pro Tyr Thr Lys Glu Val Leu Gln Ser Leu Pro

625 630 635 640

Leu Thr Glu Ile Ile Arg His Tyr Gln Leu Leu Thr Glu Glu Asn Ile

645 650 655

Pro Glu Asn Pro Leu Arg Phe Leu Tyr Pro Arg Ile Pro Arg Asp Glu

660 665 670

Ala Phe Gly Cys Tyr Tyr Gln Glu Lys Val Asn Leu Gln Glu Arg Arg

675 680 685

Lys Tyr Leu Lys His Arg Leu Ile Val Val Ser Asn Arg Gln Val Asp

690 695 700

Glu Leu Gln Gln Pro Leu Glu Leu Lys Pro Glu Pro Glu Leu Glu Ser

705 710 715 720

Leu Glu Leu Glu Leu Gly Leu Val Pro Glu Pro Glu Leu Ser Leu Asp

725 730 735

Leu Glu Pro Leu Leu Lys Ala Gly Leu Asp Leu Gly Pro Glu Leu Glu

740 745 750

Ser Val Leu Glu Ser Thr Leu Glu Pro Val Ile Glu Pro Thr Leu Cys

755 760 765

Met Val Ser Gln Thr Val Pro Glu Pro Asp Gln Gly Pro Val Ser Gln

770 775 780

Pro Val Pro Glu Pro Asp Leu Pro Cys Asp Leu Arg His Leu Asn Thr

785 790 795 800

Glu Pro Met Glu Ile Phe Arg Asn Cys Val Lys Ile Glu Glu Ile Met

805 810 815

Pro Asn Gly Asp Pro Leu Leu Ala Gly Gln Asn Thr Val Asp Glu Val

820 825 830

Tyr Val Ser Arg Pro Ser His Phe Tyr Thr Asp Gly Pro Leu Met Pro

835 840 845

Ser Asp Phe

850

cagctggaat tcggggcggc ggcgcagact gggaggggga gccgggggtt ccgacgtcgc 60

agccgaggga acaagcccca accggatcct ggacaggcac cccggcttgg cgctgtctct 120

ccccctcggc tcggagaggc ccttcggcct gagggagcct cgccgcccgt ccccggcaca 180

cgcgcagccc cggcctctcg gcctctgccg gagaaacagg atg gcc caa tgg aat 235

Met Ala Gln Trp Asn

1 5

cag cta cag cag ctt gac aca cgg tac ctg gag cag ctc cat cag ctc 283

Gln Leu Gln Gln Leu Asp Thr Arg Tyr Leu Glu Gln Leu His Gln Leu

10 15 20

tac agt gac agc ttc cca atg gag ctg cgg cag ttt ctg gcc cct tgg 331

Tyr Ser Asp Ser Phe Pro Met Glu Leu Arg Gln Phe Leu Ala Pro Trp

25 30 35

att gag agt caa gat tgg gca tat gcg gcc agc aaa gaa tca cat gcc 379

Ile Glu Ser Gln Asp Trp Ala Tyr Ala Ala Ser Lys Glu Ser His Ala

40 45 50

act ttg gtg ttt cat aat ctc ctg gga gag att gac cag cag tat agc 427

Thr Leu Val Phe His Asn Leu Leu Gly Glu Ile Asp Gln Gln Tyr Ser

55 60 65

cgc ttc ctg caa gag tcg aat gtt ctc tat cag cac aat cta cga aga 475

Arg Phe Leu Gln Glu Ser Asn Val Leu Tyr Gln His Asn Leu Arg Arg

70 75 80 85

atc aag cag ttt ctt cag agc agg tat ctt gag aag cca atg gag att 523

Ile Lys Gln Phe Leu Gln Ser Arg Tyr Leu Glu Lys Pro Met Glu Ile

90 95 100

gcc cgg att gtg gcc cgg tgc ctg tgg gaa gaa tca cgc ctt cta cag 571

Ala Arg Ile Val Ala Arg Cys Leu Trp Glu Glu Ser Arg Leu Leu Gln

105 110 115

act gca gcc act gcg gcc cag caa ggg ggc cag gcc aac cac ccc aca 619

Thr Ala Ala Thr Ala Ala Gln Gln Gly Gly Gln Ala Asn His Pro Thr

120 125 130

gca gcc gtg gtg acg gag aag cag cag atg ctg gag cag cac ctt cag 667

Ala Ala Val Val Thr Glu Lys Gln Gln Met Leu Glu Gln His Leu Gln

135 140 145

gat gtc cgg aag aga gtg cag gat cta gaa cag aaa atg aaa gtg gta 715

Asp Val Arg Lys Arg Val Gln Asp Leu Glu Gln Lys Met Lys Val Val

150 155 160 165

gag aat ctc cag gat gac ttt gat ttc aac tat aaa acc ctc aag agt 763

Glu Asn Leu Gln Asp Asp Phe Asp Phe Asn Tyr Lys Thr Leu Lys Ser

170 175 180

caa gga gac atg caa gat ctg aat gga aac aac cag tca gtg acc agg 811

Gln Gly Asp Met Gln Asp Leu Asn Gly Asn Asn Gln Ser Val Thr Arg

185 190 195

cag aag atg cag cag ctg gaa cag atg ctc act gcg ctg gac cag atg 859

›PRT · 2 of 2

Gln Lys Met Gln Gln Leu Glu Gln Met Leu Thr Ala Leu Asp Gln Met

200 205 210

cgg aga agc atc gtg agt gag ctg gcg ggg ctt ttg tca gcg atg gag 907

Arg Arg Ser Ile Val Ser Glu Leu Ala Gly Leu Leu Ser Ala Met Glu

215 220 225

tac gtg cag aaa act ctc acg gac gag gag ctg gct gac tgg aag agg 955

Tyr Val Gln Lys Thr Leu Thr Asp Glu Glu Leu Ala Asp Trp Lys Arg

230 235 240 245

cgg caa cag att gcc tgc att gga ggc ccg ccc aac atc tgc cta gat 1003

Arg Gln Gln Ile Ala Cys Ile Gly Gly Pro Pro Asn Ile Cys Leu Asp

250 255 260

cgg cta gaa aac tgg ata acg tca tta gca gaa tct caa ctt cag acc 1051

Arg Leu Glu Asn Trp Ile Thr Ser Leu Ala Glu Ser Gln Leu Gln Thr

265 270 275

cgt caa caa att aag aaa ctg gag gag ttg cac caa aaa gtt tcc tac 1099

Arg Gln Gln Ile Lys Lys Leu Glu Glu Leu His Gln Lys Val Ser Tyr

280 285 290

aaa ggg gac ccc att gta cag cac cgg ccg atg ctg gag gag agg atc 1147

Lys Gly Asp Pro Ile Val Gln His Arg Pro Met Leu Glu Glu Arg Ile

295 300 305

gtg gag ctg ttc aga aac tta atg aaa agt gcc ttt gtg gtg gag cgg 1195

Val Glu Leu Phe Arg Asn Leu Met Lys Ser Ala Phe Val Val Glu Arg

310 315 320 325

cag ccc tgc atg ccc atg cat cct gac cgg ccc ctc gtc atc aag acc 1243

Gln Pro Cys Met Pro Met His Pro Asp Arg Pro Leu Val Ile Lys Thr

330 335 340

ggc gtc cag ttc act act aaa gtc agg ttg ctg gtc aag ttc cct gag 1291

Gly Val Gln Phe Thr Thr Lys Val Arg Leu Leu Val Lys Phe Pro Glu

345 350 355

ttg aat tat cag ctt aaa att aaa gtg tgc att gac aaa gac tct ggg 1339

Leu Asn Tyr Gln Leu Lys Ile Lys Val Cys Ile Asp Lys Asp Ser Gly

360 365 370

gac gtt gca gct ctc aga gga tcc cgg aaa ttt aac att ctg ggc aca 1387

Asp Val Ala Ala Leu Arg Gly Ser Arg Lys Phe Asn Ile Leu Gly Thr

375 380 385

aac aca aaa gtg atg aac atg gaa gaa tcc aac aac ggc agc ctc tct 1435

Asn Thr Lys Val Met Asn Met Glu Glu Ser Asn Asn Gly Ser Leu Ser

390 395 400 405

gca gaa ttc aaa cac ttg acc ctg agg gag cag aga tgt ggg aat ggg 1483

Ala Glu Phe Lys His Leu Thr Leu Arg Glu Gln Arg Cys Gly Asn Gly

410 415 420

ggc cga gcc aat tgt gat gct tcc ctg att gtg act gag gag ctg cac 1531

Gly Arg Ala Asn Cys Asp Ala Ser Leu Ile Val Thr Glu Glu Leu His

425 430 435

ctg atc acc ttt gag acc gag gtg tat cac caa ggt ctc aag att gac 1579

Leu Ile Thr Phe Glu Thr Glu Val Tyr His Gln Gly Leu Lys Ile Asp

440 445 450

cta gag acc cac tcc ttg tca gtt gtg gtg atc tcc aac atc tgt cag 1627

Leu Glu Thr His Ser Leu Ser Val Val Val Ile Ser Asn Ile Cys Gln

455 460 465

atg cca aat gcc tgg gcg tcc atc ctg tgg tac aac atg ctg acc aac 1675

Met Pro Asn Ala Trp Ala Ser Ile Leu Trp Tyr Asn Met Leu Thr Asn

470 475 480 485

aat ccc aag aat gtg aac ttc ttc act aag ccg cca att gga acc tgg 1723

Asn Pro Lys Asn Val Asn Phe Phe Thr Lys Pro Pro Ile Gly Thr Trp

490 495 500

gac caa gtg gcc gag gtg ctc agc tgg cag ttc tcg tcc acc acc aag 1771

Asp Gln Val Ala Glu Val Leu Ser Trp Gln Phe Ser Ser Thr Thr Lys

505 510 515

cgg ggg ctg agc atc gag cag ctg aca acg ctg gct gag aag ctc cta 1819

Arg Gly Leu Ser Ile Glu Gln Leu Thr Thr Leu Ala Glu Lys Leu Leu

520 525 530

ggg cct ggt gtg aac tac tca ggg tgt cag atc aca tgg gct aac ttc 1867

Gly Pro Gly Val Asn Tyr Ser Gly Cys Gln Ile Thr Trp Ala Asn Phe

535 540 545

tgc aaa gaa aac atg gct ggc aag ggc ttc tcc tac tgg gtc tgg cta 1915

Cys Lys Glu Asn Met Ala Gly Lys Gly Phe Ser Tyr Trp Val Trp Leu

550 555 560 565

gac aat atc atc gac ctt gtg aaa aag tat atc ttg gcc ctt tgg aat 1963

Asp Asn Ile Ile Asp Leu Val Lys Lys Tyr Ile Leu Ala Leu Trp Asn

570 575 580

gaa ggg tac atc atg ggt ttc atc agc aag gag cgg gag cgg gcc atc 2011

Glu Gly Tyr Ile Met Gly Phe Ile Ser Lys Glu Arg Glu Arg Ala Ile

585 590 595

ttg agc act aag ccc cca ggc acc ttc ctg ctg cgc ttc agt gaa agc 2059

Leu Ser Thr Lys Pro Pro Gly Thr Phe Leu Leu Arg Phe Ser Glu Ser

600 605 610

agc aaa gaa gga ggc gtc act ttc act tgg gtg gag aag gac atc agc 2107

Ser Lys Glu Gly Gly Val Thr Phe Thr Trp Val Glu Lys Asp Ile Ser

615 620 625

ggt aag acc cag atc cag tcc gtg gaa cca tac aca aag cag cag ctg 2155

Gly Lys Thr Gln Ile Gln Ser Val Glu Pro Tyr Thr Lys Gln Gln Leu

630 635 640 645

aac aac atg tca ttt gct gaa atc atc atg ggc tat aag atc atg gat 2203

Asn Asn Met Ser Phe Ala Glu Ile Ile Met Gly Tyr Lys Ile Met Asp

650 655 660

gct acc aat atc ctg ttg tct cca ctt gtc tat ctc tat cct gac att 2251

Ala Thr Asn Ile Leu Leu Ser Pro Leu Val Tyr Leu Tyr Pro Asp Ile

665 670 675

ccc aag gag gag gca ttc ggg aag tat tgt cgg cca gag agc cag gag 2299

Pro Lys Glu Glu Ala Phe Gly Lys Tyr Cys Arg Pro Glu Ser Gln Glu

680 685 690

cat cct gaa gct gac cca ggt agc gct gcc cca tac ctg aag acc aag 2347

His Pro Glu Ala Asp Pro Gly Ser Ala Ala Pro Tyr Leu Lys Thr Lys

695 700 705

ttt atc tgt gtg aca cca acg acc tgc agc aat acc att gac ctg ccg 2395

Phe Ile Cys Val Thr Pro Thr Thr Cys Ser Asn Thr Ile Asp Leu Pro

710 715 720 725

atg tcc ccc cgc gct tta gat tca ttg atg cag ttt gga aat aat ggt 2443

Met Ser Pro Arg Ala Leu Asp Ser Leu Met Gln Phe Gly Asn Asn Gly

730 735 740

gaa ggt gct gaa ccc tca gca gga ggg cag ttt gag tcc ctc acc ttt 2491

Glu Gly Ala Glu Pro Ser Ala Gly Gly Gln Phe Glu Ser Leu Thr Phe

745 750 755

gac atg gag ttg acc tcg gag tgc gct acc tcc ccc atg tga 2533

Asp Met Glu Leu Thr Ser Glu Cys Ala Thr Ser Pro Met *

760 765 770

ggagctgaga acggaagctg cagaaagata cgactgaggc gcctacctgc attctgccac 2593

ccctcacaca gccaaacccc agatcatctg aaactactaa ctttgtggtt ccagattttt 2653

tttaatctcc tacttctgct atctttgagc aatctgggca cttttaaaaa tagagaaatg 2713

agtgaatgtg ggtgatctgc ttttatctaa atgcaaataa ggatgtgttc tctgagaccc 2773

atgatcaggg gatg 2787

770

›PRT · 1 of 2

Homo sapiens

Met Ala Gln Trp Asn Gln Leu Gln Gln Leu Asp Thr Arg Tyr Leu Glu

1 5 10 15

Gln Leu His Gln Leu Tyr Ser Asp Ser Phe Pro Met Glu Leu Arg Gln

20 25 30

Phe Leu Ala Pro Trp Ile Glu Ser Gln Asp Trp Ala Tyr Ala Ala Ser

35 40 45

Lys Glu Ser His Ala Thr Leu Val Phe His Asn Leu Leu Gly Glu Ile

50 55 60

Asp Gln Gln Tyr Ser Arg Phe Leu Gln Glu Ser Asn Val Leu Tyr Gln

65 70 75 80

His Asn Leu Arg Arg Ile Lys Gln Phe Leu Gln Ser Arg Tyr Leu Glu

85 90 95

Lys Pro Met Glu Ile Ala Arg Ile Val Ala Arg Cys Leu Trp Glu Glu

100 105 110

Ser Arg Leu Leu Gln Thr Ala Ala Thr Ala Ala Gln Gln Gly Gly Gln

115 120 125

Ala Asn His Pro Thr Ala Ala Val Val Thr Glu Lys Gln Gln Met Leu

130 135 140

Glu Gln His Leu Gln Asp Val Arg Lys Arg Val Gln Asp Leu Glu Gln

145 150 155 160

Lys Met Lys Val Val Glu Asn Leu Gln Asp Asp Phe Asp Phe Asn Tyr

165 170 175

Lys Thr Leu Lys Ser Gln Gly Asp Met Gln Asp Leu Asn Gly Asn Asn

180 185 190

Gln Ser Val Thr Arg Gln Lys Met Gln Gln Leu Glu Gln Met Leu Thr

195 200 205

Ala Leu Asp Gln Met Arg Arg Ser Ile Val Ser Glu Leu Ala Gly Leu

210 215 220

Leu Ser Ala Met Glu Tyr Val Gln Lys Thr Leu Thr Asp Glu Glu Leu

225 230 235 240

Ala Asp Trp Lys Arg Arg Gln Gln Ile Ala Cys Ile Gly Gly Pro Pro

245 250 255

Asn Ile Cys Leu Asp Arg Leu Glu Asn Trp Ile Thr Ser Leu Ala Glu

260 265 270

Ser Gln Leu Gln Thr Arg Gln Gln Ile Lys Lys Leu Glu Glu Leu His

275 280 285

Gln Lys Val Ser Tyr Lys Gly Asp Pro Ile Val Gln His Arg Pro Met

290 295 300

Leu Glu Glu Arg Ile Val Glu Leu Phe Arg Asn Leu Met Lys Ser Ala

305 310 315 320

Phe Val Val Glu Arg Gln Pro Cys Met Pro Met His Pro Asp Arg Pro

325 330 335

Leu Val Ile Lys Thr Gly Val Gln Phe Thr Thr Lys Val Arg Leu Leu

340 345 350

Val Lys Phe Pro Glu Leu Asn Tyr Gln Leu Lys Ile Lys Val Cys Ile

355 360 365

Asp Lys Asp Ser Gly Asp Val Ala Ala Leu Arg Gly Ser Arg Lys Phe

370 375 380

Asn Ile Leu Gly Thr Asn Thr Lys Val Met Asn Met Glu Glu Ser Asn

385 390 395 400

Asn Gly Ser Leu Ser Ala Glu Phe Lys His Leu Thr Leu Arg Glu Gln

405 410 415

Arg Cys Gly Asn Gly Gly Arg Ala Asn Cys Asp Ala Ser Leu Ile Val

420 425 430

Thr Glu Glu Leu His Leu Ile Thr Phe Glu Thr Glu Val Tyr His Gln

435 440 445

Gly Leu Lys Ile Asp Leu Glu Thr His Ser Leu Ser Val Val Val Ile

450 455 460

Ser Asn Ile Cys Gln Met Pro Asn Ala Trp Ala Ser Ile Leu Trp Tyr

465 470 475 480

Asn Met Leu Thr Asn Asn Pro Lys Asn Val Asn Phe Phe Thr Lys Pro

485 490 495

Pro Ile Gly Thr Trp Asp Gln Val Ala Glu Val Leu Ser Trp Gln Phe

500 505 510

Ser Ser Thr Thr Lys Arg Gly Leu Ser Ile Glu Gln Leu Thr Thr Leu

515 520 525

Ala Glu Lys Leu Leu Gly Pro Gly Val Asn Tyr Ser Gly Cys Gln Ile

530 535 540

Thr Trp Ala Asn Phe Cys Lys Glu Asn Met Ala Gly Lys Gly Phe Ser

545 550 555 560

Tyr Trp Val Trp Leu Asp Asn Ile Ile Asp Leu Val Lys Lys Tyr Ile

565 570 575

Leu Ala Leu Trp Asn Glu Gly Tyr Ile Met Gly Phe Ile Ser Lys Glu

580 585 590

Arg Glu Arg Ala Ile Leu Ser Thr Lys Pro Pro Gly Thr Phe Leu Leu

595 600 605

Arg Phe Ser Glu Ser Ser Lys Glu Gly Gly Val Thr Phe Thr Trp Val

610 615 620

Glu Lys Asp Ile Ser Gly Lys Thr Gln Ile Gln Ser Val Glu Pro Tyr

625 630 635 640

Thr Lys Gln Gln Leu Asn Asn Met Ser Phe Ala Glu Ile Ile Met Gly

645 650 655

Tyr Lys Ile Met Asp Ala Thr Asn Ile Leu Leu Ser Pro Leu Val Tyr

660 665 670

Leu Tyr Pro Asp Ile Pro Lys Glu Glu Ala Phe Gly Lys Tyr Cys Arg

675 680 685

Pro Glu Ser Gln Glu His Pro Glu Ala Asp Pro Gly Ser Ala Ala Pro

690 695 700

Tyr Leu Lys Thr Lys Phe Ile Cys Val Thr Pro Thr Thr Cys Ser Asn

705 710 715 720

Thr Ile Asp Leu Pro Met Ser Pro Arg Ala Leu Asp Ser Leu Met Gln

725 730 735

Phe Gly Asn Asn Gly Glu Gly Ala Glu Pro Ser Ala Gly Gly Gln Phe

740 745 750

Glu Ser Leu Thr Phe Asp Met Glu Leu Thr Ser Glu Cys Ala Thr Ser

755 760 765

Pro Met

770

gctttctcct agggactgtg aggggcgctt ctgactttgg acttgagcac tgcctgggac 60

ctgtgctgag agagcgctag c atg tct cag tgg aat caa gtc caa cag tta 111

Met Ser Gln Trp Asn Gln Val Gln Gln Leu

1 5 10

gaa atc aag ttt ttg gag cag gtg gat caa ttc tat gat gac aac ttt 159

Glu Ile Lys Phe Leu Glu Gln Val Asp Gln Phe Tyr Asp Asp Asn Phe

15 20 25

ccc atg gaa att cgg cat ctg ttg gcc caa tgg att gaa aat caa gac 207

Pro Met Glu Ile Arg His Leu Leu Ala Gln Trp Ile Glu Asn Gln Asp

30 35 40

tgg gag gca gct tct aac aat gaa acc atg gca acg att ctt ctt caa 255

Trp Glu Ala Ala Ser Asn Asn Glu Thr Met Ala Thr Ile Leu Leu Gln

45 50 55

aac ttg tta ata caa ctg gat gaa cag tta ggt cgt gtt tcc aaa gag 303

Asn Leu Leu Ile Gln Leu Asp Glu Gln Leu Gly Arg Val Ser Lys Glu

60 65 70

aaa aac cta ctc ttg ata cac aat cta aaa aga att agg aag gtc ctt 351

Lys Asn Leu Leu Leu Ile His Asn Leu Lys Arg Ile Arg Lys Val Leu

75 80 85 90

cag gga aaa ttt cat gga aat cca atg cat gta gct gtg gtt att tca 399

Gln Gly Lys Phe His Gly Asn Pro Met His Val Ala Val Val Ile Ser

95 100 105

aac tgt tta agg gaa gag agg aga ata ttg gct gca gcc aac atg cct 447

Asn Cys Leu Arg Glu Glu Arg Arg Ile Leu Ala Ala Ala Asn Met Pro

110 115 120

gtc cag ggg cct cta gag aaa tcc tta caa agt tct tca gtt tca gaa 495

Val Gln Gly Pro Leu Glu Lys Ser Leu Gln Ser Ser Ser Val Ser Glu

125 130 135

aga cag agg aat gtg gag cac aaa gtg gct gcc att aaa aac agt gtg 543

Arg Gln Arg Asn Val Glu His Lys Val Ala Ala Ile Lys Asn Ser Val

140 145 150

cag atg aca gaa caa gat acc aaa tac tta gaa gat ctg caa gac gaa 591

Gln Met Thr Glu Gln Asp Thr Lys Tyr Leu Glu Asp Leu Gln Asp Glu

155 160 165 170

ttt gac tac agg tat aaa aca att cag aca atg gat cag agt gac aag 639

Phe Asp Tyr Arg Tyr Lys Thr Ile Gln Thr Met Asp Gln Ser Asp Lys

175 180 185

aat agt gcc atg gtg aat cag gaa gtt ttg aca ctg cag gaa atg ctt 687

Asn Ser Ala Met Val Asn Gln Glu Val Leu Thr Leu Gln Glu Met Leu

190 195 200

aac agc ctc gat ttc aag aga aag gag gct ctc agt aaa atg acc caa 735

Asn Ser Leu Asp Phe Lys Arg Lys Glu Ala Leu Ser Lys Met Thr Gln

205 210 215

atc atc cat gag aca gac ctg tta atg aac acc atg ctc ata gaa gag 783

Ile Ile His Glu Thr Asp Leu Leu Met Asn Thr Met Leu Ile Glu Glu

220 225 230

ctg caa gac tgg aag cgg cgg cag caa atc gcc tgc atc ggg ggt cca 831

Leu Gln Asp Trp Lys Arg Arg Gln Gln Ile Ala Cys Ile Gly Gly Pro

235 240 245 250

ctc cac aat ggg ctc gac cag ctt cag aac tgc ttt aca cta ttg gca 879

Leu His Asn Gly Leu Asp Gln Leu Gln Asn Cys Phe Thr Leu Leu Ala

›PRT · 2 of 2

255 260 265

gaa agt ctt ttc caa ctg aga agg caa ttg gag aaa cta gag gag caa 927

Glu Ser Leu Phe Gln Leu Arg Arg Gln Leu Glu Lys Leu Glu Glu Gln

270 275 280

tct acc aaa atg aca tat gaa ggt gat ccc att cca atg caa aga act 975

Ser Thr Lys Met Thr Tyr Glu Gly Asp Pro Ile Pro Met Gln Arg Thr

285 290 295

cac atg cta gaa aga gtc acc ttc ttg atc tac aac ctt ttc aag aac 1023

His Met Leu Glu Arg Val Thr Phe Leu Ile Tyr Asn Leu Phe Lys Asn

300 305 310

tca ttt gtg gtt gag cga cag cca tgt atg cca acc cac cct cag agg 1071

Ser Phe Val Val Glu Arg Gln Pro Cys Met Pro Thr His Pro Gln Arg

315 320 325 330

ccg ttg gta ctt aaa acc cta att cag ttc act gta aaa cta agg cta 1119

Pro Leu Val Leu Lys Thr Leu Ile Gln Phe Thr Val Lys Leu Arg Leu

335 340 345

cta ata aaa ttg cca gaa cta aac tat cag gta aag gtt aag gca tca 1167

Leu Ile Lys Leu Pro Glu Leu Asn Tyr Gln Val Lys Val Lys Ala Ser

350 355 360

att gac aag aat gtt tca act cta agc aac cga aga ttt gta ctt tgt 1215

Ile Asp Lys Asn Val Ser Thr Leu Ser Asn Arg Arg Phe Val Leu Cys

365 370 375

gga act aat gtc aaa gcc atg tct att gaa gaa tct tcc aat ggg agt 1263

Gly Thr Asn Val Lys Ala Met Ser Ile Glu Glu Ser Ser Asn Gly Ser

380 385 390

ctc tca gta gaa ttt cga cat ttg caa cca aag gaa atg aag tcc agt 1311

Leu Ser Val Glu Phe Arg His Leu Gln Pro Lys Glu Met Lys Ser Ser

395 400 405 410

gct gga ggt aaa gga aat gag ggc tgt cac atg gtg act gaa gaa ctt 1359

Ala Gly Gly Lys Gly Asn Glu Gly Cys His Met Val Thr Glu Glu Leu

415 420 425

cat tcc ata acg ttt gaa aca cag atc tgc ctc tat ggc ctg acc ata 1407

His Ser Ile Thr Phe Glu Thr Gln Ile Cys Leu Tyr Gly Leu Thr Ile

430 435 440

gat ttg gag acc agc tca ttg cct gtg gtg atg att tcc aat gtc agt 1455

Asp Leu Glu Thr Ser Ser Leu Pro Val Val Met Ile Ser Asn Val Ser

445 450 455

cag tta cct aat gct tgg gca tcc atc att tgg tac aac gtg tca acc 1503

Gln Leu Pro Asn Ala Trp Ala Ser Ile Ile Trp Tyr Asn Val Ser Thr

460 465 470

aac gat tcc cag aac ttg gtt ttc ttt aat aat cct cca cct gcc aca 1551

Asn Asp Ser Gln Asn Leu Val Phe Phe Asn Asn Pro Pro Pro Ala Thr

475 480 485 490

ttg agt caa cta ctg gag gtg atg agc tgg cag ttt tca tcg tac gtt 1599

Leu Ser Gln Leu Leu Glu Val Met Ser Trp Gln Phe Ser Ser Tyr Val

495 500 505

ggt cgt ggt ctt aac tca gat caa ctc cat atg ctg gca gag aag ctt 1647

Gly Arg Gly Leu Asn Ser Asp Gln Leu His Met Leu Ala Glu Lys Leu

510 515 520

aca gtc caa tct agc tac agt gat ggt cac ctc acc tgg gcc aag ttc 1695

Thr Val Gln Ser Ser Tyr Ser Asp Gly His Leu Thr Trp Ala Lys Phe

525 530 535

tgc aag gaa cat tta cct ggt aaa tca ttt acc ttt tgg aca tgg ctt 1743

Cys Lys Glu His Leu Pro Gly Lys Ser Phe Thr Phe Trp Thr Trp Leu

540 545 550

gaa gca ata ttg gat cta att aag aaa cac att ctt ccc ctt tgg att 1791

Glu Ala Ile Leu Asp Leu Ile Lys Lys His Ile Leu Pro Leu Trp Ile

555 560 565 570

gat ggg tat gtc atg ggc ttt gtt agc aaa gag aag gaa cgg ctg ttg 1839

Asp Gly Tyr Val Met Gly Phe Val Ser Lys Glu Lys Glu Arg Leu Leu

575 580 585

cta aag gat aaa atg cct ggc acc ttt tta tta aga ttc agt gaa agc 1887

Leu Lys Asp Lys Met Pro Gly Thr Phe Leu Leu Arg Phe Ser Glu Ser

590 595 600

cat ctc gga gga ata act ttc acc tgg gtg gac cat tct gaa agt ggg 1935

His Leu Gly Gly Ile Thr Phe Thr Trp Val Asp His Ser Glu Ser Gly

605 610 615

gaa gtg aga ttc cac tct gta gaa ccc tac aat aaa ggc cgg ttg tct 1983

Glu Val Arg Phe His Ser Val Glu Pro Tyr Asn Lys Gly Arg Leu Ser

620 625 630

gct ctg cca ttc gct gac atc ctg cga gac tac aaa gtt att atg gct 2031

Ala Leu Pro Phe Ala Asp Ile Leu Arg Asp Tyr Lys Val Ile Met Ala

635 640 645 650

gaa aac att cct gaa aac cct ctg aag tac cta tat cct gac att ccc 2079

Glu Asn Ile Pro Glu Asn Pro Leu Lys Tyr Leu Tyr Pro Asp Ile Pro

655 660 665

aaa gac aaa gcc ttc ggt aaa cac tac agc tct cag cct tgc gaa gtt 2127

Lys Asp Lys Ala Phe Gly Lys His Tyr Ser Ser Gln Pro Cys Glu Val

670 675 680

tca aga cca aca gaa agg ggt gac aaa ggt tat gtt cct tct gtt ttt 2175

Ser Arg Pro Thr Glu Arg Gly Asp Lys Gly Tyr Val Pro Ser Val Phe

685 690 695

atc ccc atc tca aca atc cga agt gat tca aca gag cca cat tct cca 2223

Ile Pro Ile Ser Thr Ile Arg Ser Asp Ser Thr Glu Pro His Ser Pro

700 705 710

tca gac ctt ctt ccc atg tct cca agt gtg tat gcg gtg ttg aga gaa 2271

Ser Asp Leu Leu Pro Met Ser Pro Ser Val Tyr Ala Val Leu Arg Glu

715 720 725 730

aac ctg agt ccc aca aca att gaa act gca atg aag tct cct tat tct 2319

Asn Leu Ser Pro Thr Thr Ile Glu Thr Ala Met Lys Ser Pro Tyr Ser

735 740 745

gct gaa tga caggataaac tctgacgcac caagaaagga agcaaatgaa 2368

Ala Glu *

aaagtttaaa gactgttctt tgcccaataa ccacatttta tttcttcagc tttgtaaata 2428

748

›PRT · 1 of 2

Homo sapiens

Met Ser Gln Trp Asn Gln Val Gln Gln Leu Glu Ile Lys Phe Leu Glu

1 5 10 15

Gln Val Asp Gln Phe Tyr Asp Asp Asn Phe Pro Met Glu Ile Arg His

20 25 30

Leu Leu Ala Gln Trp Ile Glu Asn Gln Asp Trp Glu Ala Ala Ser Asn

35 40 45

Asn Glu Thr Met Ala Thr Ile Leu Leu Gln Asn Leu Leu Ile Gln Leu

50 55 60

Asp Glu Gln Leu Gly Arg Val Ser Lys Glu Lys Asn Leu Leu Leu Ile

65 70 75 80

His Asn Leu Lys Arg Ile Arg Lys Val Leu Gln Gly Lys Phe His Gly

85 90 95

Asn Pro Met His Val Ala Val Val Ile Ser Asn Cys Leu Arg Glu Glu

100 105 110

Arg Arg Ile Leu Ala Ala Ala Asn Met Pro Val Gln Gly Pro Leu Glu

115 120 125

Lys Ser Leu Gln Ser Ser Ser Val Ser Glu Arg Gln Arg Asn Val Glu

130 135 140

His Lys Val Ala Ala Ile Lys Asn Ser Val Gln Met Thr Glu Gln Asp

145 150 155 160

Thr Lys Tyr Leu Glu Asp Leu Gln Asp Glu Phe Asp Tyr Arg Tyr Lys

165 170 175

Thr Ile Gln Thr Met Asp Gln Ser Asp Lys Asn Ser Ala Met Val Asn

180 185 190

Gln Glu Val Leu Thr Leu Gln Glu Met Leu Asn Ser Leu Asp Phe Lys

195 200 205

Arg Lys Glu Ala Leu Ser Lys Met Thr Gln Ile Ile His Glu Thr Asp

210 215 220

Leu Leu Met Asn Thr Met Leu Ile Glu Glu Leu Gln Asp Trp Lys Arg

225 230 235 240

Arg Gln Gln Ile Ala Cys Ile Gly Gly Pro Leu His Asn Gly Leu Asp

245 250 255

Gln Leu Gln Asn Cys Phe Thr Leu Leu Ala Glu Ser Leu Phe Gln Leu

260 265 270

Arg Arg Gln Leu Glu Lys Leu Glu Glu Gln Ser Thr Lys Met Thr Tyr

275 280 285

Glu Gly Asp Pro Ile Pro Met Gln Arg Thr His Met Leu Glu Arg Val

290 295 300

Thr Phe Leu Ile Tyr Asn Leu Phe Lys Asn Ser Phe Val Val Glu Arg

305 310 315 320

Gln Pro Cys Met Pro Thr His Pro Gln Arg Pro Leu Val Leu Lys Thr

325 330 335

Leu Ile Gln Phe Thr Val Lys Leu Arg Leu Leu Ile Lys Leu Pro Glu

340 345 350

Leu Asn Tyr Gln Val Lys Val Lys Ala Ser Ile Asp Lys Asn Val Ser

355 360 365

Thr Leu Ser Asn Arg Arg Phe Val Leu Cys Gly Thr Asn Val Lys Ala

370 375 380

Met Ser Ile Glu Glu Ser Ser Asn Gly Ser Leu Ser Val Glu Phe Arg

385 390 395 400

His Leu Gln Pro Lys Glu Met Lys Ser Ser Ala Gly Gly Lys Gly Asn

405 410 415

Glu Gly Cys His Met Val Thr Glu Glu Leu His Ser Ile Thr Phe Glu

420 425 430

Thr Gln Ile Cys Leu Tyr Gly Leu Thr Ile Asp Leu Glu Thr Ser Ser

435 440 445

Leu Pro Val Val Met Ile Ser Asn Val Ser Gln Leu Pro Asn Ala Trp

450 455 460

Ala Ser Ile Ile Trp Tyr Asn Val Ser Thr Asn Asp Ser Gln Asn Leu

465 470 475 480

Val Phe Phe Asn Asn Pro Pro Pro Ala Thr Leu Ser Gln Leu Leu Glu

485 490 495

Val Met Ser Trp Gln Phe Ser Ser Tyr Val Gly Arg Gly Leu Asn Ser

500 505 510

Asp Gln Leu His Met Leu Ala Glu Lys Leu Thr Val Gln Ser Ser Tyr

515 520 525

Ser Asp Gly His Leu Thr Trp Ala Lys Phe Cys Lys Glu His Leu Pro

530 535 540

Gly Lys Ser Phe Thr Phe Trp Thr Trp Leu Glu Ala Ile Leu Asp Leu

545 550 555 560

Ile Lys Lys His Ile Leu Pro Leu Trp Ile Asp Gly Tyr Val Met Gly

565 570 575

Phe Val Ser Lys Glu Lys Glu Arg Leu Leu Leu Lys Asp Lys Met Pro

580 585 590

Gly Thr Phe Leu Leu Arg Phe Ser Glu Ser His Leu Gly Gly Ile Thr

595 600 605

Phe Thr Trp Val Asp His Ser Glu Ser Gly Glu Val Arg Phe His Ser

610 615 620

Val Glu Pro Tyr Asn Lys Gly Arg Leu Ser Ala Leu Pro Phe Ala Asp

625 630 635 640

Ile Leu Arg Asp Tyr Lys Val Ile Met Ala Glu Asn Ile Pro Glu Asn

645 650 655

Pro Leu Lys Tyr Leu Tyr Pro Asp Ile Pro Lys Asp Lys Ala Phe Gly

660 665 670

Lys His Tyr Ser Ser Gln Pro Cys Glu Val Ser Arg Pro Thr Glu Arg

675 680 685

Gly Asp Lys Gly Tyr Val Pro Ser Val Phe Ile Pro Ile Ser Thr Ile

690 695 700

Arg Ser Asp Ser Thr Glu Pro His Ser Pro Ser Asp Leu Leu Pro Met

705 710 715 720

Ser Pro Ser Val Tyr Ala Val Leu Arg Glu Asn Leu Ser Pro Thr Thr

725 730 735

Ile Glu Thr Ala Met Lys Ser Pro Tyr Ser Ala Glu

740 745

atcttatttt tctttttggt ggtggtggtg gaagggggga ggtgctagca gggccagcct 60

tgaactcgct ggacagagct acagacctat ggggcctgga agtgcccgct gagaaaggga 120

gaagacagca gaggggttgc cgaggcaacc tccaagtccc agatc atg tct ctg tgg 177

Met Ser Leu Trp

ggt ctg gtc tcc aag atg ccc cca gaa aaa gtg cag cgg ctc tat gtc 225

Gly Leu Val Ser Lys Met Pro Pro Glu Lys Val Gln Arg Leu Tyr Val

5 10 15 20

gac ttt ccc caa cac ctg cgg cat ctt ctg ggt gac tgg ctg gag agc 273

Asp Phe Pro Gln His Leu Arg His Leu Leu Gly Asp Trp Leu Glu Ser

25 30 35

cag ccc tgg gag ttc ctg gtc ggc tcc gac gcc ttc tgc tgc aac ttg 321

Gln Pro Trp Glu Phe Leu Val Gly Ser Asp Ala Phe Cys Cys Asn Leu

40 45 50

gct agt gcc cta ctt tca gac act gtc cag cac ctt cag gcc tcg gtg 369

Ala Ser Ala Leu Leu Ser Asp Thr Val Gln His Leu Gln Ala Ser Val

55 60 65

gga gag cag ggg gag ggg agc acc atc ttg caa cac atc agc acc ctt 417

Gly Glu Gln Gly Glu Gly Ser Thr Ile Leu Gln His Ile Ser Thr Leu

70 75 80

gag agc ata tat cag agg gac ccc ctg aag ctg gtg gcc act ttc aga 465

Glu Ser Ile Tyr Gln Arg Asp Pro Leu Lys Leu Val Ala Thr Phe Arg

85 90 95 100

caa ata ctt caa gga gag aaa aaa gct gtt atg gaa cag ttc cgc cac 513

Gln Ile Leu Gln Gly Glu Lys Lys Ala Val Met Glu Gln Phe Arg His

105 110 115

ttg cca atg cct ttc cac tgg aag cag gaa gaa ctc aag ttt aag aca 561

Leu Pro Met Pro Phe His Trp Lys Gln Glu Glu Leu Lys Phe Lys Thr

120 125 130

ggc ttg cgg agg ctg cag cac cga gta ggg gag atc cac ctt ctc cga 609

Gly Leu Arg Arg Leu Gln His Arg Val Gly Glu Ile His Leu Leu Arg

135 140 145

gaa gcc ctg cag aag ggg gct gag gct ggc caa gtg tct ctg cac agc 657

Glu Ala Leu Gln Lys Gly Ala Glu Ala Gly Gln Val Ser Leu His Ser

150 155 160

ttg ata gaa act cct gct aat ggg act ggg cca agt gag gcc ctg gcc 705

Leu Ile Glu Thr Pro Ala Asn Gly Thr Gly Pro Ser Glu Ala Leu Ala

165 170 175 180

atg cta ctg cag gag acc act gga gag cta gag gca gcc aaa gcc cta 753

Met Leu Leu Gln Glu Thr Thr Gly Glu Leu Glu Ala Ala Lys Ala Leu

185 190 195

gtg ctg aag agg atc cag att tgg aaa cgg cag cag cag ctg gca ggg 801

Val Leu Lys Arg Ile Gln Ile Trp Lys Arg Gln Gln Gln Leu Ala Gly

200 205 210

aat ggc gca ccg ttt gag gag agc ctg gcc cca ctc cag gag agg tgt 849

Asn Gly Ala Pro Phe Glu Glu Ser Leu Ala Pro Leu Gln Glu Arg Cys

215 220 225

gaa agc ctg gtg gac att tat tcc cag cta cag cag gag gta ggg gcg 897

Glu Ser Leu Val Asp Ile Tyr Ser Gln Leu Gln Gln Glu Val Gly Ala

230 235 240

gct ggt ggg gag ctt gag ccc aag acc cgg gca tcg ctg act ggc cgg 945

Ala Gly Gly Glu Leu Glu Pro Lys Thr Arg Ala Ser Leu Thr Gly Arg

245 250 255 260

ctg gat gaa gtc ctg aga acc ctc gtc acc agt tgc ttc ctg gtg gag 993

›PRT · 2 of 2

Leu Asp Glu Val Leu Arg Thr Leu Val Thr Ser Cys Phe Leu Val Glu

265 270 275

aag cag ccc ccc cag gta ctg aag act cag acc aag ttc cag gct gga 1041

Lys Gln Pro Pro Gln Val Leu Lys Thr Gln Thr Lys Phe Gln Ala Gly

280 285 290

gtt cga ttc ctg ttg ggc ttg agg ttc ctg ggg gcc cca gcc aag cct 1089

Val Arg Phe Leu Leu Gly Leu Arg Phe Leu Gly Ala Pro Ala Lys Pro

295 300 305

ccg ctg gtc agg gcc gac atg gtg aca gag aag cag gcg cgg gag ctg 1137

Pro Leu Val Arg Ala Asp Met Val Thr Glu Lys Gln Ala Arg Glu Leu

310 315 320

agt gtg cct cag ggt cct ggg gct gga gca gaa agc act gga gaa atc 1185

Ser Val Pro Gln Gly Pro Gly Ala Gly Ala Glu Ser Thr Gly Glu Ile

325 330 335 340

atc aac aac act gtg ccc ttg gag aac agc att cct ggg aac tgc tgc 1233

Ile Asn Asn Thr Val Pro Leu Glu Asn Ser Ile Pro Gly Asn Cys Cys

345 350 355

tct gcc ctg ttc aag aac ctg ctt ctc aag aag atc aag cgg tgt gag 1281

Ser Ala Leu Phe Lys Asn Leu Leu Leu Lys Lys Ile Lys Arg Cys Glu

360 365 370

cgg aag ggc act gag tct gtc aca gag gag aag tgc gct gtg ctc ttc 1329

Arg Lys Gly Thr Glu Ser Val Thr Glu Glu Lys Cys Ala Val Leu Phe

375 380 385

tct gcc agc ttc aca ctt ggc ccc ggc aaa ctc ccc atc cag ctc cag 1377

Ser Ala Ser Phe Thr Leu Gly Pro Gly Lys Leu Pro Ile Gln Leu Gln

390 395 400

gcc ctg tct ctg ccc ctg gtg gtc atc gtc cat ggc aac caa gac aac 1425

Ala Leu Ser Leu Pro Leu Val Val Ile Val His Gly Asn Gln Asp Asn

405 410 415 420

aat gcc aaa gcc act atc ctg tgg gac aat gcc ttc tct gag atg gac 1473

Asn Ala Lys Ala Thr Ile Leu Trp Asp Asn Ala Phe Ser Glu Met Asp

425 430 435

cgc gtg ccc ttt gtg gtg gct gag cgg gtg ccc tgg gag aag atg tgt 1521

Arg Val Pro Phe Val Val Ala Glu Arg Val Pro Trp Glu Lys Met Cys

440 445 450

gaa act ctg aac ctg aag ttc atg gct gag gtg ggg acc aac cgg ggg 1569

Glu Thr Leu Asn Leu Lys Phe Met Ala Glu Val Gly Thr Asn Arg Gly

455 460 465

ctg ctc cca gag cac ttc ctc ttc ctg gcc cag aag atc ttc aat gac 1617

Leu Leu Pro Glu His Phe Leu Phe Leu Ala Gln Lys Ile Phe Asn Asp

470 475 480

aac agc ctc agt atg gag gcc ttc cag cac cgt tct gtg tcc tgg tcg 1665

Asn Ser Leu Ser Met Glu Ala Phe Gln His Arg Ser Val Ser Trp Ser

485 490 495 500

cag ttc aac aag gag atc ctg ctg ggc cgt ggc ttc acc ttt tgg cag 1713

Gln Phe Asn Lys Glu Ile Leu Leu Gly Arg Gly Phe Thr Phe Trp Gln

505 510 515

tgg ttt gat ggt gtc ctg gac ctc acc aaa cgc tgt ctc cgg agc tac 1761

Trp Phe Asp Gly Val Leu Asp Leu Thr Lys Arg Cys Leu Arg Ser Tyr

520 525 530

tgg tct gac cgg ctg atc att ggc ttc atc agc aaa cag tac gtt act 1809

Trp Ser Asp Arg Leu Ile Ile Gly Phe Ile Ser Lys Gln Tyr Val Thr

535 540 545

agc ctt ctt ctc aat gag ccc gac gga acc ttt ctc ctc cgc ttc agc 1857

Ser Leu Leu Leu Asn Glu Pro Asp Gly Thr Phe Leu Leu Arg Phe Ser

550 555 560

gac tca gag att ggg ggc atc acc att gcc cat gtc atc cgg ggc cag 1905

Asp Ser Glu Ile Gly Gly Ile Thr Ile Ala His Val Ile Arg Gly Gln

565 570 575 580

gat ggc tct cca cag ata gag aac atc cag cca ttc tct gcc aaa gac 1953

Asp Gly Ser Pro Gln Ile Glu Asn Ile Gln Pro Phe Ser Ala Lys Asp

585 590 595

ctg tcc att cgc tca ctg ggg gac cga atc cgg gat ctt gct cag ctc 2001

Leu Ser Ile Arg Ser Leu Gly Asp Arg Ile Arg Asp Leu Ala Gln Leu

600 605 610

aaa aat ctc tat ccc aag aag ccc aag gat gag gct ttc cgg agc cac 2049

Lys Asn Leu Tyr Pro Lys Lys Pro Lys Asp Glu Ala Phe Arg Ser His

615 620 625

tac aag cct gaa cag atg ggt aag gat ggc agg ggt tat gtc cca gct 2097

Tyr Lys Pro Glu Gln Met Gly Lys Asp Gly Arg Gly Tyr Val Pro Ala

630 635 640

acc atc aag atg acc gtg gaa agg gac caa cca ctt cct acc cca gag 2145

Thr Ile Lys Met Thr Val Glu Arg Asp Gln Pro Leu Pro Thr Pro Glu

645 650 655 660

ctc cag atg cct acc atg gtg cct tct tat gac ctt gga atg gcc cct 2193

Leu Gln Met Pro Thr Met Val Pro Ser Tyr Asp Leu Gly Met Ala Pro

665 670 675

gat tcc tcc atg agc atg cag ctt ggc cca gat atg gtg ccc cag gtg 2241

Asp Ser Ser Met Ser Met Gln Leu Gly Pro Asp Met Val Pro Gln Val

680 685 690

tac cca cca cac tct cac tcc atc ccc ccg tat caa ggc ctc tcc cca 2289

Tyr Pro Pro His Ser His Ser Ile Pro Pro Tyr Gln Gly Leu Ser Pro

695 700 705

gaa gaa tca gtc aac gtg ttg tca gcc ttc cag gag cct cac ctg cag 2337

Glu Glu Ser Val Asn Val Leu Ser Ala Phe Gln Glu Pro His Leu Gln

710 715 720

atg ccc ccc agc ctg ggc cag atg agc ctg ccc ttt gac cag cct cac 2385

Met Pro Pro Ser Leu Gly Gln Met Ser Leu Pro Phe Asp Gln Pro His

725 730 735 740

ccc cag ggc ctg ctg ccg tgc cag cct cag gag cat gct gtg tcc agc 2433

Pro Gln Gly Leu Leu Pro Cys Gln Pro Gln Glu His Ala Val Ser Ser

745 750 755

cct gac ccc ctg ctc tgc tca gat gtg acc atg gtg gaa gac agc tgc 2481

Pro Asp Pro Leu Leu Cys Ser Asp Val Thr Met Val Glu Asp Ser Cys

760 765 770

ctg agc cag cca gtg aca gcg ttt cct cag ggc act tgg att ggt gaa 2529

Leu Ser Gln Pro Val Thr Ala Phe Pro Gln Gly Thr Trp Ile Gly Glu

775 780 785

gac ata ttc cct cct ctg ctg cct ccc act gaa cag gac ctc act aag 2577

Asp Ile Phe Pro Pro Leu Leu Pro Pro Thr Glu Gln Asp Leu Thr Lys

790 795 800

ctt ctc ctg gag ggg caa ggg gag tcg ggg gga ggg tcc ttg ggg gca 2625

Leu Leu Leu Glu Gly Gln Gly Glu Ser Gly Gly Gly Ser Leu Gly Ala

805 810 815 820

cag ccc ctc ctg cag ccc tcc cac tat ggg caa tct ggg atc tca atg 2673

Gln Pro Leu Leu Gln Pro Ser His Tyr Gly Gln Ser Gly Ile Ser Met

825 830 835

tcc cac atg gac cta agg gcc aac ccc agt tgg tga tcccagctgg 2719

Ser His Met Asp Leu Arg Ala Asn Pro Ser Trp *

840 845

agggagaacc caaagagaca gctcttctac tacccccaca gacctgctct ggacacttgc 2779

tcatgccctg ccaagcagca gatggggagg gtgccctcct atccccacct actcctgggt 2839

caggaggaaa agactaacag gagaatgcac agtgggtgga gccaatccac tccttccttt 2899

ctatcattcc cctgcccacc tccttccagc actgactgga agggaagttc aggctctgag 2959

acacgcccca acatgcctgc acctgcagcg cgcacacgca cgcacacaca catacagagc 3019

tctctgaggg tgatggggct gagcagg 3046

847

›PRT · 1 of 2

Homo sapiens

Met Ser Leu Trp Gly Leu Val Ser Lys Met Pro Pro Glu Lys Val Gln

1 5 10 15

Arg Leu Tyr Val Asp Phe Pro Gln His Leu Arg His Leu Leu Gly Asp

20 25 30

Trp Leu Glu Ser Gln Pro Trp Glu Phe Leu Val Gly Ser Asp Ala Phe

35 40 45

Cys Cys Asn Leu Ala Ser Ala Leu Leu Ser Asp Thr Val Gln His Leu

50 55 60

Gln Ala Ser Val Gly Glu Gln Gly Glu Gly Ser Thr Ile Leu Gln His

65 70 75 80

Ile Ser Thr Leu Glu Ser Ile Tyr Gln Arg Asp Pro Leu Lys Leu Val

85 90 95

Ala Thr Phe Arg Gln Ile Leu Gln Gly Glu Lys Lys Ala Val Met Glu

100 105 110

Gln Phe Arg His Leu Pro Met Pro Phe His Trp Lys Gln Glu Glu Leu

115 120 125

Lys Phe Lys Thr Gly Leu Arg Arg Leu Gln His Arg Val Gly Glu Ile

130 135 140

His Leu Leu Arg Glu Ala Leu Gln Lys Gly Ala Glu Ala Gly Gln Val

145 150 155 160

Ser Leu His Ser Leu Ile Glu Thr Pro Ala Asn Gly Thr Gly Pro Ser

165 170 175

Glu Ala Leu Ala Met Leu Leu Gln Glu Thr Thr Gly Glu Leu Glu Ala

180 185 190

Ala Lys Ala Leu Val Leu Lys Arg Ile Gln Ile Trp Lys Arg Gln Gln

195 200 205

Gln Leu Ala Gly Asn Gly Ala Pro Phe Glu Glu Ser Leu Ala Pro Leu

210 215 220

Gln Glu Arg Cys Glu Ser Leu Val Asp Ile Tyr Ser Gln Leu Gln Gln

225 230 235 240

Glu Val Gly Ala Ala Gly Gly Glu Leu Glu Pro Lys Thr Arg Ala Ser

245 250 255

Leu Thr Gly Arg Leu Asp Glu Val Leu Arg Thr Leu Val Thr Ser Cys

260 265 270

Phe Leu Val Glu Lys Gln Pro Pro Gln Val Leu Lys Thr Gln Thr Lys

275 280 285

Phe Gln Ala Gly Val Arg Phe Leu Leu Gly Leu Arg Phe Leu Gly Ala

290 295 300

Pro Ala Lys Pro Pro Leu Val Arg Ala Asp Met Val Thr Glu Lys Gln

305 310 315 320

Ala Arg Glu Leu Ser Val Pro Gln Gly Pro Gly Ala Gly Ala Glu Ser

325 330 335

Thr Gly Glu Ile Ile Asn Asn Thr Val Pro Leu Glu Asn Ser Ile Pro

340 345 350

Gly Asn Cys Cys Ser Ala Leu Phe Lys Asn Leu Leu Leu Lys Lys Ile

355 360 365

Lys Arg Cys Glu Arg Lys Gly Thr Glu Ser Val Thr Glu Glu Lys Cys

370 375 380

Ala Val Leu Phe Ser Ala Ser Phe Thr Leu Gly Pro Gly Lys Leu Pro

385 390 395 400

Ile Gln Leu Gln Ala Leu Ser Leu Pro Leu Val Val Ile Val His Gly

405 410 415

Asn Gln Asp Asn Asn Ala Lys Ala Thr Ile Leu Trp Asp Asn Ala Phe

420 425 430

Ser Glu Met Asp Arg Val Pro Phe Val Val Ala Glu Arg Val Pro Trp

435 440 445

Glu Lys Met Cys Glu Thr Leu Asn Leu Lys Phe Met Ala Glu Val Gly

450 455 460

Thr Asn Arg Gly Leu Leu Pro Glu His Phe Leu Phe Leu Ala Gln Lys

465 470 475 480

Ile Phe Asn Asp Asn Ser Leu Ser Met Glu Ala Phe Gln His Arg Ser

485 490 495

Val Ser Trp Ser Gln Phe Asn Lys Glu Ile Leu Leu Gly Arg Gly Phe

500 505 510

Thr Phe Trp Gln Trp Phe Asp Gly Val Leu Asp Leu Thr Lys Arg Cys

515 520 525

Leu Arg Ser Tyr Trp Ser Asp Arg Leu Ile Ile Gly Phe Ile Ser Lys

530 535 540

Gln Tyr Val Thr Ser Leu Leu Leu Asn Glu Pro Asp Gly Thr Phe Leu

545 550 555 560

Leu Arg Phe Ser Asp Ser Glu Ile Gly Gly Ile Thr Ile Ala His Val

565 570 575

Ile Arg Gly Gln Asp Gly Ser Pro Gln Ile Glu Asn Ile Gln Pro Phe

580 585 590

Ser Ala Lys Asp Leu Ser Ile Arg Ser Leu Gly Asp Arg Ile Arg Asp

595 600 605

Leu Ala Gln Leu Lys Asn Leu Tyr Pro Lys Lys Pro Lys Asp Glu Ala

610 615 620

Phe Arg Ser His Tyr Lys Pro Glu Gln Met Gly Lys Asp Gly Arg Gly

625 630 635 640

Tyr Val Pro Ala Thr Ile Lys Met Thr Val Glu Arg Asp Gln Pro Leu

645 650 655

Pro Thr Pro Glu Leu Gln Met Pro Thr Met Val Pro Ser Tyr Asp Leu

660 665 670

Gly Met Ala Pro Asp Ser Ser Met Ser Met Gln Leu Gly Pro Asp Met

675 680 685

Val Pro Gln Val Tyr Pro Pro His Ser His Ser Ile Pro Pro Tyr Gln

690 695 700

Gly Leu Ser Pro Glu Glu Ser Val Asn Val Leu Ser Ala Phe Gln Glu

705 710 715 720

Pro His Leu Gln Met Pro Pro Ser Leu Gly Gln Met Ser Leu Pro Phe

725 730 735

Asp Gln Pro His Pro Gln Gly Leu Leu Pro Cys Gln Pro Gln Glu His

740 745 750

Ala Val Ser Ser Pro Asp Pro Leu Leu Cys Ser Asp Val Thr Met Val

755 760 765

Glu Asp Ser Cys Leu Ser Gln Pro Val Thr Ala Phe Pro Gln Gly Thr

770 775 780

Trp Ile Gly Glu Asp Ile Phe Pro Pro Leu Leu Pro Pro Thr Glu Gln

785 790 795 800

Asp Leu Thr Lys Leu Leu Leu Glu Gly Gln Gly Glu Ser Gly Gly Gly

805 810 815

Ser Leu Gly Ala Gln Pro Leu Leu Gln Pro Ser His Tyr Gly Gln Ser

820 825 830

Gly Ile Ser Met Ser His Met Asp Leu Arg Ala Asn Pro Ser Trp

835 840 845

ccccttctgt agg atg gta gca cac aac cag gtg gca gcc gac aat gca 49

Met Val Ala His Asn Gln Val Ala Ala Asp Asn Ala

1 5 10

gtc tcc aca gca gca gag ccc cga cgg cgg cca gaa cct tcc tcc tct 97

Val Ser Thr Ala Ala Glu Pro Arg Arg Arg Pro Glu Pro Ser Ser Ser

15 20 25

tcc tcc tcc tcg ccc gcg gcc ccc gcg cgc ccg cgg ccg tgc ccc gcg 145

Ser Ser Ser Ser Pro Ala Ala Pro Ala Arg Pro Arg Pro Cys Pro Ala

30 35 40

gtc ccg gcc ccg gcc ccc ggc gac acg cac ttc cgc aca ttc cgt tcg 193

Val Pro Ala Pro Ala Pro Gly Asp Thr His Phe Arg Thr Phe Arg Ser

45 50 55 60

cac gcc gat tac cgg cgc atc acg cgc gcc agc gcg ctc ctg gac gcc 241

His Ala Asp Tyr Arg Arg Ile Thr Arg Ala Ser Ala Leu Leu Asp Ala

65 70 75

tgc gga ttc tac tgg ggg ccc ctg agc gtg cac ggg gcg cac gag cgg 289

Cys Gly Phe Tyr Trp Gly Pro Leu Ser Val His Gly Ala His Glu Arg

80 85 90

ctg cgc gcc gag ccc gtg ggc acc ttc ctg gtg cgc gac agc cgc cag 337

Leu Arg Ala Glu Pro Val Gly Thr Phe Leu Val Arg Asp Ser Arg Gln

95 100 105

cgg aac tgc ttt ttc gcc ctt agc gtg aag atg gcc tcg gga ccc acg 385

Arg Asn Cys Phe Phe Ala Leu Ser Val Lys Met Ala Ser Gly Pro Thr

110 115 120

agc atc cgc gtg cac ttt cag gcc ggc cgc ttt cac ctg gat ggc agc 433

Ser Ile Arg Val His Phe Gln Ala Gly Arg Phe His Leu Asp Gly Ser

125 130 135 140

cgc gag agc ttc gac tgc ctc ttc gag ctg ctg gag cac tac gtg gcg 481

Arg Glu Ser Phe Asp Cys Leu Phe Glu Leu Leu Glu His Tyr Val Ala

145 150 155

gcg ccg cgc cgc atg ctg ggg gcc ccg ctg cgc cag cgc cgc gtg cgg 529

Ala Pro Arg Arg Met Leu Gly Ala Pro Leu Arg Gln Arg Arg Val Arg

160 165 170

ccg ctg cag gag ctg tgc cgc cag cgc atc gtg gcc acc gtg ggc cgc 577

Pro Leu Gln Glu Leu Cys Arg Gln Arg Ile Val Ala Thr Val Gly Arg

175 180 185

gag aac ctg gct cgc atc ccc ctc aac ccc gtc ctc cgc gac tac ctg 625

Glu Asn Leu Ala Arg Ile Pro Leu Asn Pro Val Leu Arg Asp Tyr Leu

190 195 200

agc tcc ttc ccc ttc cag att tga ccggcagcgc ccgccgtgca cgcagcatta 679

Ser Ser Phe Pro Phe Gln Ile *

205 210

actgggatgc cgtgttattt tgttattact tgcctggaac catgtgggta ccctccccgg 739

cctgggttgg agggagcgga tgggtgtagg ggcgaggcgc ctcccgccct cggctggaga 799

cgaggccgca gaccccttct cacctcttga gggggtcctc cccctcctgg tgctccctct 859

›PRT · 2 of 2

gggtccccct ggttgttgta gcagcttaac tgtatctgga gccaggacc 908

211

›PRT

Homo sapiens

Met Val Ala His Asn Gln Val Ala Ala Asp Asn Ala Val Ser Thr Ala

1 5 10 15

Ala Glu Pro Arg Arg Arg Pro Glu Pro Ser Ser Ser Ser Ser Ser Ser

20 25 30

Pro Ala Ala Pro Ala Arg Pro Arg Pro Cys Pro Ala Val Pro Ala Pro

35 40 45

Ala Pro Gly Asp Thr His Phe Arg Thr Phe Arg Ser His Ala Asp Tyr

50 55 60

Arg Arg Ile Thr Arg Ala Ser Ala Leu Leu Asp Ala Cys Gly Phe Tyr

65 70 75 80

Trp Gly Pro Leu Ser Val His Gly Ala His Glu Arg Leu Arg Ala Glu

85 90 95

Pro Val Gly Thr Phe Leu Val Arg Asp Ser Arg Gln Arg Asn Cys Phe

100 105 110

Phe Ala Leu Ser Val Lys Met Ala Ser Gly Pro Thr Ser Ile Arg Val

115 120 125

His Phe Gln Ala Gly Arg Phe His Leu Asp Gly Ser Arg Glu Ser Phe

130 135 140

Asp Cys Leu Phe Glu Leu Leu Glu His Tyr Val Ala Ala Pro Arg Arg

145 150 155 160

Met Leu Gly Ala Pro Leu Arg Gln Arg Arg Val Arg Pro Leu Gln Glu

165 170 175

Leu Cys Arg Gln Arg Ile Val Ala Thr Val Gly Arg Glu Asn Leu Ala

180 185 190

Arg Ile Pro Leu Asn Pro Val Leu Arg Asp Tyr Leu Ser Ser Phe Pro

195 200 205

Phe Gln Ile

210

atg gtc acc cac agc aag ttt ccc gcc gcc ggg atg agc cgc ccc ctg 48

Met Val Thr His Ser Lys Phe Pro Ala Ala Gly Met Ser Arg Pro Leu

1 5 10 15

gac acc agc ctg cgc ctc aag acc ttc agc tcc aag agc gag tac cag 96

Asp Thr Ser Leu Arg Leu Lys Thr Phe Ser Ser Lys Ser Glu Tyr Gln

20 25 30

ctg gtg gtg aac gca gtg cgc aag ctg cag gag agc ggc ttc tac tgg 144

Leu Val Val Asn Ala Val Arg Lys Leu Gln Glu Ser Gly Phe Tyr Trp

35 40 45

agc gca gtg acc ggc ggc gag gcg aac ctg ctg ctc agc gcc gag ccc 192

Ser Ala Val Thr Gly Gly Glu Ala Asn Leu Leu Leu Ser Ala Glu Pro

50 55 60

gcc ggc acc ttt ctg atc cgc gac agc tcg gac cag cgc cac ttc ttc 240

Ala Gly Thr Phe Leu Ile Arg Asp Ser Ser Asp Gln Arg His Phe Phe

65 70 75 80

acg ctc agc gtc aag acc cag tct ggg acc aag aac ctg cgc atc cag 288

Thr Leu Ser Val Lys Thr Gln Ser Gly Thr Lys Asn Leu Arg Ile Gln

85 90 95

tgt gag ggg ggc agc ttc tct ctg cag agc gat ccc cgg agc acg cag 336

Cys Glu Gly Gly Ser Phe Ser Leu Gln Ser Asp Pro Arg Ser Thr Gln

100 105 110

ccc gtg ccc cgc ttc gac tgc gtg ctc aag ctg gtg cac cac tac atg 384

Pro Val Pro Arg Phe Asp Cys Val Leu Lys Leu Val His His Tyr Met

115 120 125

ccg ccc cct gga gcc ccc tcc ttc ccc tcg cca cct act gaa ccc tcc 432

Pro Pro Pro Gly Ala Pro Ser Phe Pro Ser Pro Pro Thr Glu Pro Ser

130 135 140

tcc gag gtg ccc gag cag ccg tct gcc cag cca ctc cct ggg agt ccc 480

Ser Glu Val Pro Glu Gln Pro Ser Ala Gln Pro Leu Pro Gly Ser Pro

145 150 155 160

ccc aga aga gcc tat tac atc tac tcc ggg ggc gag aag atc ccc ctg 528

Pro Arg Arg Ala Tyr Tyr Ile Tyr Ser Gly Gly Glu Lys Ile Pro Leu

165 170 175

gtg ttg agc cgg ccc ctc tcc tcc aac gtg gcc act ctt cag cat ctc 576

Val Leu Ser Arg Pro Leu Ser Ser Asn Val Ala Thr Leu Gln His Leu

180 185 190

tgt cgg aag acc gtc aac ggc cac ctg gac tcc tat gag aaa gtc acc 624

Cys Arg Lys Thr Val Asn Gly His Leu Asp Ser Tyr Glu Lys Val Thr

195 200 205

cag ctg ccg ggg ccc att cgg gag ttc ctg gac cag tac gat gcc ccg 672

Gln Leu Pro Gly Pro Ile Arg Glu Phe Leu Asp Gln Tyr Asp Ala Pro

210 215 220

ctt taa gggg 682

Leu *

225

›PRT · 1 of 3

Homo sapiens

Met Val Thr His Ser Lys Phe Pro Ala Ala Gly Met Ser Arg Pro Leu

1 5 10 15

Asp Thr Ser Leu Arg Leu Lys Thr Phe Ser Ser Lys Ser Glu Tyr Gln

20 25 30

Leu Val Val Asn Ala Val Arg Lys Leu Gln Glu Ser Gly Phe Tyr Trp

35 40 45

Ser Ala Val Thr Gly Gly Glu Ala Asn Leu Leu Leu Ser Ala Glu Pro

50 55 60

Ala Gly Thr Phe Leu Ile Arg Asp Ser Ser Asp Gln Arg His Phe Phe

65 70 75 80

Thr Leu Ser Val Lys Thr Gln Ser Gly Thr Lys Asn Leu Arg Ile Gln

85 90 95

Cys Glu Gly Gly Ser Phe Ser Leu Gln Ser Asp Pro Arg Ser Thr Gln

100 105 110

Pro Val Pro Arg Phe Asp Cys Val Leu Lys Leu Val His His Tyr Met

115 120 125

Pro Pro Pro Gly Ala Pro Ser Phe Pro Ser Pro Pro Thr Glu Pro Ser

130 135 140

Ser Glu Val Pro Glu Gln Pro Ser Ala Gln Pro Leu Pro Gly Ser Pro

145 150 155 160

Pro Arg Arg Ala Tyr Tyr Ile Tyr Ser Gly Gly Glu Lys Ile Pro Leu

165 170 175

Val Leu Ser Arg Pro Leu Ser Ser Asn Val Ala Thr Leu Gln His Leu

180 185 190

Cys Arg Lys Thr Val Asn Gly His Leu Asp Ser Tyr Glu Lys Val Thr

195 200 205

Gln Leu Pro Gly Pro Ile Arg Glu Phe Leu Asp Gln Tyr Asp Ala Pro

210 215 220

Leu

225

aattcggcac gagggggggc agcagcggac gccgctaacg gcctccctcg gcgctgacag 60

gctgggccgg cgcccggctc gcttgggtgt tcgcgtcgcc acttcggctt ctcggccggt 120

cgggcccctc ggcccgggct tgcggcgcgc gtcggggctg agggctgctg cggcgcaggg 180

agaggcctgg tcctcgctgc cgagggatgt gagtgggagc tgagcccaca ctggagggcc 240

cccgagggcc cagcctggag gtcgttcaga gccgtgcccg ccccggggct tcgcagacct 300

tgacccgccg ggtaggagcc gcccctgcgg gctcgagggc gcgctctggt cgcccgatct 360

gtgtagccgg tttcagaagc aggcaacagg aacaagatgt gaactgtttc tcttctgcag 420

aaaaagaggc tcttcctcct cctcccgcga cggcaaatgt tctgaaaaag actctgc atg 480

Met

gga atg gcc tgc ctt acg atg aca gaa atg gag gga aca tcc acc tct 528

Gly Met Ala Cys Leu Thr Met Thr Glu Met Glu Gly Thr Ser Thr Ser

5 10 15

tct ata tat cag aat ggt gat att tct gga aat gcc aat tct atg aag 576

Ser Ile Tyr Gln Asn Gly Asp Ile Ser Gly Asn Ala Asn Ser Met Lys

20 25 30

caa ata gat cca gtt ctt cag gtg tat ctt tac cat tcc ctt ggg aaa 624

Gln Ile Asp Pro Val Leu Gln Val Tyr Leu Tyr His Ser Leu Gly Lys

35 40 45

tct gag gca gat tat ctg acc ttt cca tct ggg gag tat gtt gca gaa 672

Ser Glu Ala Asp Tyr Leu Thr Phe Pro Ser Gly Glu Tyr Val Ala Glu

50 55 60 65

gaa atc tgt att gct gct tct aaa gct tgt ggt atc aca cct gtg tat 720

Glu Ile Cys Ile Ala Ala Ser Lys Ala Cys Gly Ile Thr Pro Val Tyr

70 75 80

cat aat atg ttt gct tta atg agt gaa aca gaa agg atc tgg tat cca 768

His Asn Met Phe Ala Leu Met Ser Glu Thr Glu Arg Ile Trp Tyr Pro

85 90 95

ccc aac cat gtc ttc cat ata gat gag tca acc agg cat aat gta ctc 816

Pro Asn His Val Phe His Ile Asp Glu Ser Thr Arg His Asn Val Leu

100 105 110

tac aga ata aga ttt tac ttt cct cgt tgg tat tgc agt ggc agc aac 864

Tyr Arg Ile Arg Phe Tyr Phe Pro Arg Trp Tyr Cys Ser Gly Ser Asn

115 120 125

aga gcc tat cgg cat gga ata tct cga ggt gct gaa gct cct ctt ctt 912

Arg Ala Tyr Arg His Gly Ile Ser Arg Gly Ala Glu Ala Pro Leu Leu

130 135 140 145

gat gac ttt gtc atg tct tac ctc ttt gct cag tgg cgg cat gat ttt 960

Asp Asp Phe Val Met Ser Tyr Leu Phe Ala Gln Trp Arg His Asp Phe

150 155 160

gtg cac gga tgg ata aaa gta cct gtg act cat gaa aca cag gaa gaa 1008

Val His Gly Trp Ile Lys Val Pro Val Thr His Glu Thr Gln Glu Glu

165 170 175

tgt ctt ggg atg gca gtg tta gat atg atg aga ata gcc aaa gaa aac 1056

Cys Leu Gly Met Ala Val Leu Asp Met Met Arg Ile Ala Lys Glu Asn

180 185 190

gat caa acc cca ctg gcc atc tat aac tct atc agc tac aag aca ttc 1104

Asp Gln Thr Pro Leu Ala Ile Tyr Asn Ser Ile Ser Tyr Lys Thr Phe

195 200 205

tta cca aaa tgt att cga gca aag atc caa gac tat cat att ttg aca 1152

Leu Pro Lys Cys Ile Arg Ala Lys Ile Gln Asp Tyr His Ile Leu Thr

210 215 220 225

agg aag cga ata agg tac aga ttt cgc aga ttt att cag caa ttc agc 1200

Arg Lys Arg Ile Arg Tyr Arg Phe Arg Arg Phe Ile Gln Gln Phe Ser

230 235 240

caa tgc aaa gcc act gcc aga aac ttg aaa ctt aag tat ctt ata aat 1248

Gln Cys Lys Ala Thr Ala Arg Asn Leu Lys Leu Lys Tyr Leu Ile Asn

245 250 255

ctg gaa act ctg cag tct gcc ttc tac aca gag aaa ttt gaa gta aaa 1296

Leu Glu Thr Leu Gln Ser Ala Phe Tyr Thr Glu Lys Phe Glu Val Lys

260 265 270

gaa cct gga agt ggt cct tca ggt gag gag att ttt gca acc att ata 1344

Glu Pro Gly Ser Gly Pro Ser Gly Glu Glu Ile Phe Ala Thr Ile Ile

275 280 285

ata act gga aac ggt gga att cag tgg tca aga ggg aaa cat aaa gaa 1392

Ile Thr Gly Asn Gly Gly Ile Gln Trp Ser Arg Gly Lys His Lys Glu

290 295 300 305

agt gag aca ctg aca gaa cag gat tta cag tta tat tgc gat ttt tct 1440

Ser Glu Thr Leu Thr Glu Gln Asp Leu Gln Leu Tyr Cys Asp Phe Ser

310 315 320

aat att att gat gtc agt att aag caa gca aac caa gag ggt tca aat 1488

Asn Ile Ile Asp Val Ser Ile Lys Gln Ala Asn Gln Glu Gly Ser Asn

325 330 335

gaa agc cga gtt gta act atc cat aag caa gat ggt aaa aat ctg gaa 1536

Glu Ser Arg Val Val Thr Ile His Lys Gln Asp Gly Lys Asn Leu Glu

340 345 350

att gaa ctt agc tca tta agg gaa gct ttg tct ttc gtg tca tta att 1584

Ile Glu Leu Ser Ser Leu Arg Glu Ala Leu Ser Phe Val Ser Leu Ile

355 360 365

gat gga tat tat aga tta act gca gat gca cat cat tac ctc tgt aaa 1632

Asp Gly Tyr Tyr Arg Leu Thr Ala Asp Ala His His Tyr Leu Cys Lys

370 375 380 385

gaa gta gca cct cca gcc gtg ctt gaa aat ata caa agc aac tgt cat 1680

Glu Val Ala Pro Pro Ala Val Leu Glu Asn Ile Gln Ser Asn Cys His

390 395 400

ggc cca att tcg atg gat ttt gcc att agt aaa ctg aag aaa gca ggt 1728

Gly Pro Ile Ser Met Asp Phe Ala Ile Ser Lys Leu Lys Lys Ala Gly

405 410 415

aat cag act gga ctg tat gta ctt cga tgc agt cct aag gac ttt aat 1776

Asn Gln Thr Gly Leu Tyr Val Leu Arg Cys Ser Pro Lys Asp Phe Asn

420 425 430

aaa tat ttt ttg act ttt gct gtc gag cga gaa aat gtc att gaa tat 1824

Lys Tyr Phe Leu Thr Phe Ala Val Glu Arg Glu Asn Val Ile Glu Tyr

435 440 445

aaa cac tgt ttg att aca aaa aat gag aat gaa gag tac aac ctc agt 1872

Lys His Cys Leu Ile Thr Lys Asn Glu Asn Glu Glu Tyr Asn Leu Ser

450 455 460 465

ggg aca aag aag aac ttc agc agt ctt aaa gat ctt ttg aat tgt tac 1920

Gly Thr Lys Lys Asn Phe Ser Ser Leu Lys Asp Leu Leu Asn Cys Tyr

470 475 480

cag atg gaa act gtt cgc tca gac aat ata att ttc cag ttt act aaa 1968

Gln Met Glu Thr Val Arg Ser Asp Asn Ile Ile Phe Gln Phe Thr Lys

485 490 495

›PRT · 2 of 3

tgc tgt ccc cca aag cca aaa gat aaa tca aac ctt cta gtc ttc aga 2016

Cys Cys Pro Pro Lys Pro Lys Asp Lys Ser Asn Leu Leu Val Phe Arg

500 505 510

acg aat ggt gtt tct gat gta cca acc tca cca aca tta cag agg cct 2064

Thr Asn Gly Val Ser Asp Val Pro Thr Ser Pro Thr Leu Gln Arg Pro

515 520 525

act cat atg aac caa atg gtg ttt cac aaa atc aga aat gaa gat ttg 2112

Thr His Met Asn Gln Met Val Phe His Lys Ile Arg Asn Glu Asp Leu

530 535 540 545

ata ttt aat gaa agc ctt ggc caa ggc act ttt aca aag att ttt aaa 2160

Ile Phe Asn Glu Ser Leu Gly Gln Gly Thr Phe Thr Lys Ile Phe Lys

550 555 560

ggc gta cga aga gaa gta gga gac tac ggt caa ctg cat gaa aca gaa 2208

Gly Val Arg Arg Glu Val Gly Asp Tyr Gly Gln Leu His Glu Thr Glu

565 570 575

gtt ctt tta aaa gtt ctg gat aaa gca cac aga aac tat tca gag tct 2256

Val Leu Leu Lys Val Leu Asp Lys Ala His Arg Asn Tyr Ser Glu Ser

580 585 590

ttc ttt gaa gca gca agt atg atg agc aag ctt tct cac aag cat ttg 2304

Phe Phe Glu Ala Ala Ser Met Met Ser Lys Leu Ser His Lys His Leu

595 600 605

gtt tta aat tat gga gta tgt gtc tgt gga gac gag aat att ctg gtt 2352

Val Leu Asn Tyr Gly Val Cys Val Cys Gly Asp Glu Asn Ile Leu Val

610 615 620 625

cag gag ttt gta aaa ttt gga tca cta gat aca tat ctg aaa aag aat 2400

Gln Glu Phe Val Lys Phe Gly Ser Leu Asp Thr Tyr Leu Lys Lys Asn

630 635 640

aaa aat tgt ata aat ata tta tgg aaa ctt gaa gtt gct aaa cag ttg 2448

Lys Asn Cys Ile Asn Ile Leu Trp Lys Leu Glu Val Ala Lys Gln Leu

645 650 655

gca tgg gcc atg cat ttt cta gaa gaa aac acc ctt att cat ggg aat 2496

Ala Trp Ala Met His Phe Leu Glu Glu Asn Thr Leu Ile His Gly Asn

660 665 670

gta tgt gcc aaa aat att ctg ctt atc aga gaa gaa gac agg aag aca 2544

Val Cys Ala Lys Asn Ile Leu Leu Ile Arg Glu Glu Asp Arg Lys Thr

675 680 685

gga aat cct cct ttc atc aaa ctt agt gat cct ggc att agt att aca 2592

Gly Asn Pro Pro Phe Ile Lys Leu Ser Asp Pro Gly Ile Ser Ile Thr

690 695 700 705

gtt ttg cca aag gac att ctt cag gag aga ata cca tgg gta cca cct 2640

Val Leu Pro Lys Asp Ile Leu Gln Glu Arg Ile Pro Trp Val Pro Pro

710 715 720

gaa tgc att gaa aat cct aaa aat tta aat ttg gca aca gac aaa tgg 2688

Glu Cys Ile Glu Asn Pro Lys Asn Leu Asn Leu Ala Thr Asp Lys Trp

725 730 735

agt ttt ggt acc act ttg tgg gaa atc tgc agt gga gga gat aaa cct 2736

Ser Phe Gly Thr Thr Leu Trp Glu Ile Cys Ser Gly Gly Asp Lys Pro

740 745 750

cta agt gct ctg gat tct caa aga aag cta caa ttt tat gaa gat agg 2784

Leu Ser Ala Leu Asp Ser Gln Arg Lys Leu Gln Phe Tyr Glu Asp Arg

755 760 765

cat cag ctt cct gca cca aag tgg gca gaa tta gca aac ctt ata aat 2832

His Gln Leu Pro Ala Pro Lys Trp Ala Glu Leu Ala Asn Leu Ile Asn

770 775 780 785

aat tgt atg gat tat gaa cca gat ttc agg cct tct ttc aga gcc atc 2880

Asn Cys Met Asp Tyr Glu Pro Asp Phe Arg Pro Ser Phe Arg Ala Ile

790 795 800

ata cga gat ctt aac agt ttg ttt act cca gat tat gaa cta tta aca 2928

Ile Arg Asp Leu Asn Ser Leu Phe Thr Pro Asp Tyr Glu Leu Leu Thr

805 810 815

gaa aat gac atg tta cca aat atg agg ata ggt gcc ctg ggg ttt tct 2976

Glu Asn Asp Met Leu Pro Asn Met Arg Ile Gly Ala Leu Gly Phe Ser

820 825 830

ggt gcc ttt gaa gac cgg gat cct aca cag ttt gaa gag aga cat ttg 3024

Gly Ala Phe Glu Asp Arg Asp Pro Thr Gln Phe Glu Glu Arg His Leu

835 840 845

aaa ttt cta cag caa ctt ggc aag ggt aat ttt ggg agt gtg gag atg 3072

Lys Phe Leu Gln Gln Leu Gly Lys Gly Asn Phe Gly Ser Val Glu Met

850 855 860 865

tgc cgg tat gac cct cta cag gac aac act ggg gag gtg gtc gct gta 3120

Cys Arg Tyr Asp Pro Leu Gln Asp Asn Thr Gly Glu Val Val Ala Val

870 875 880

aaa aag ctt cag cat agt act gaa gag cac cta aga gac ttt gaa agg 3168

Lys Lys Leu Gln His Ser Thr Glu Glu His Leu Arg Asp Phe Glu Arg

885 890 895

gaa att gaa atc ctg aaa tcc cta cag cat gac aac att gta aag tac 3216

Glu Ile Glu Ile Leu Lys Ser Leu Gln His Asp Asn Ile Val Lys Tyr

900 905 910

aag gga gtg tgc tac agt gct ggt cgg cgt aat cta aaa tta att atg 3264

Lys Gly Val Cys Tyr Ser Ala Gly Arg Arg Asn Leu Lys Leu Ile Met

915 920 925

gaa tat tta cca tat gga agt tta cga gac tat ctt caa aaa cat aaa 3312

Glu Tyr Leu Pro Tyr Gly Ser Leu Arg Asp Tyr Leu Gln Lys His Lys

930 935 940 945

gaa cgg ata gat cac ata aaa ctt ctg cag tac aca tct cag ata tgc 3360

Glu Arg Ile Asp His Ile Lys Leu Leu Gln Tyr Thr Ser Gln Ile Cys

950 955 960

aag ggt atg gag tat ctt ggt aca aaa agg tat atc cac agg gat ctg 3408

Lys Gly Met Glu Tyr Leu Gly Thr Lys Arg Tyr Ile His Arg Asp Leu

965 970 975

gca acg aga aat ata ttg gtg gag aac gag aac aga gtt aaa att gga 3456

Ala Thr Arg Asn Ile Leu Val Glu Asn Glu Asn Arg Val Lys Ile Gly

980 985 990

gat ttt ggg tta acc aaa gtc ttg cca caa gac aaa gaa tac tat aaa 3504

Asp Phe Gly Leu Thr Lys Val Leu Pro Gln Asp Lys Glu Tyr Tyr Lys

995 1000 1005

gta aaa gaa cct ggt gaa agt ccc ata ttc tgg tat gct cca gaa tca 3552

Val Lys Glu Pro Gly Glu Ser Pro Ile Phe Trp Tyr Ala Pro Glu Ser

1010 1015 1020 1025

ctg aca gag agc aag ttt tct gtg gcc tca gat gtt tgg agc ttt gga 3600

Leu Thr Glu Ser Lys Phe Ser Val Ala Ser Asp Val Trp Ser Phe Gly

1030 1035 1040

gtg gtt ctg tat gaa ctt ttc aca tac att gag aag agt aaa agt cca 3648

Val Val Leu Tyr Glu Leu Phe Thr Tyr Ile Glu Lys Ser Lys Ser Pro

1045 1050 1055

cca gcg gaa ttt atg cgt atg att ggc aat gac aaa caa gga cag atg 3696

Pro Ala Glu Phe Met Arg Met Ile Gly Asn Asp Lys Gln Gly Gln Met

1060 1065 1070

atc gtg ttc cat ttg ata gaa ctt ttg aag aat aat gga aga tta cca 3744

Ile Val Phe His Leu Ile Glu Leu Leu Lys Asn Asn Gly Arg Leu Pro

1075 1080 1085

aga cca gat gga tgc cca gat gag atc tat atg atc atg aca gaa tgc 3792

Arg Pro Asp Gly Cys Pro Asp Glu Ile Tyr Met Ile Met Thr Glu Cys

1090 1095 1100 1105

tgg aac aat aat gta aat caa cgc ccc tcc ttt agg gat cta gct ctt 3840

Trp Asn Asn Asn Val Asn Gln Arg Pro Ser Phe Arg Asp Leu Ala Leu

1110 1115 1120

cga gtg gat caa ata agg gat aac atg gct gga tga aagaaatgac 3886

Arg Val Asp Gln Ile Arg Asp Asn Met Ala Gly *

1125 1130

cttcattctg agaccaaagt agatttacag aacaaagttt tatatttcac attgctgtgg 3946

actattatta catatatcat tattatataa atcatgatgc tagccagcaa agatgtgaaa 4006

atatctgctc aaaactttca aagtttagta agtttttctt catgaggcca ccagtaaaag 4066

acattaatga gaattcctta gcaaggattt tgtaagaagt ttcttaaaca ttgtcagtta 4126

acatcactct tgtctggcaa aagaaaaaaa atagactttt tcaactcagc tttttgagac 4186

›PRT · 3 of 3

ctgaaaaaat tattatgtaa attttgcaat gttaaagatg cacagaatat gtatgtatag 4246

tttttaccac agtggatgta taataccttg gcatcttgtg tgatgtttta cacacatgag 4306

ggctggtgtt cattaatact gttttctaat ttttccatag ttaatctata attaattact 4366

tcactataca aacaaattaa gatgttcaga taattgaata agtacctttg tgtccttgtt 4426

catttatatc gctggccagc attataagca ggtgtatact tttagcttgt agttccatgt 4486

actgtaaata tttttcacat aaagggaaca aatgtctagt tttatttgta taggaaattt 4546

ccctgaccct aaataataca ttttgaaatg aaacaagctt acaaagatat aatctatttt 4606

attatggttt cccttgtatc tatttgtggt gaatgtgttt tttaaatgga actatctcca 4666

aatttttcta agactactat gaacagtttt cttttaaaat tttgagatta agaatgccag 4726

gaatattgtc atcctttgag ctgctgactg ccaataacat tcttcgatct ctgggattta 4786

tgctcatgaa ctaaatttaa gcttaagcca taaaatagat tagattgttt tttaaaaatg 4846

gatagctcat taagaagtgc agcaggttaa gaattttttc ctaaagactg tatatttgag 4906

gggtttcaga attttgcatt gcagtcatag aagagattta tttccttttt agaggggaaa 4966

tgaggtaaat aagtaaaaaa gtatgcttgt taattttatt caagaatgcc agtagaaaat 5026

tcataacgtg tatctttaag aaaaatgagc atacatctta aatcttttca attaaggtcg 5086

acgcggccgc ggtcgacgcg gccgcgaatt c 5117

1132

›PRT · 1 of 3

Homo sapiens

Met Gly Met Ala Cys Leu Thr Met Thr Glu Met Glu Gly Thr Ser Thr

1 5 10 15

Ser Ser Ile Tyr Gln Asn Gly Asp Ile Ser Gly Asn Ala Asn Ser Met

20 25 30

Lys Gln Ile Asp Pro Val Leu Gln Val Tyr Leu Tyr His Ser Leu Gly

35 40 45

Lys Ser Glu Ala Asp Tyr Leu Thr Phe Pro Ser Gly Glu Tyr Val Ala

50 55 60

Glu Glu Ile Cys Ile Ala Ala Ser Lys Ala Cys Gly Ile Thr Pro Val

65 70 75 80

Tyr His Asn Met Phe Ala Leu Met Ser Glu Thr Glu Arg Ile Trp Tyr

85 90 95

Pro Pro Asn His Val Phe His Ile Asp Glu Ser Thr Arg His Asn Val

100 105 110

Leu Tyr Arg Ile Arg Phe Tyr Phe Pro Arg Trp Tyr Cys Ser Gly Ser

115 120 125

Asn Arg Ala Tyr Arg His Gly Ile Ser Arg Gly Ala Glu Ala Pro Leu

130 135 140

Leu Asp Asp Phe Val Met Ser Tyr Leu Phe Ala Gln Trp Arg His Asp

145 150 155 160

Phe Val His Gly Trp Ile Lys Val Pro Val Thr His Glu Thr Gln Glu

165 170 175

Glu Cys Leu Gly Met Ala Val Leu Asp Met Met Arg Ile Ala Lys Glu

180 185 190

Asn Asp Gln Thr Pro Leu Ala Ile Tyr Asn Ser Ile Ser Tyr Lys Thr

195 200 205

Phe Leu Pro Lys Cys Ile Arg Ala Lys Ile Gln Asp Tyr His Ile Leu

210 215 220

Thr Arg Lys Arg Ile Arg Tyr Arg Phe Arg Arg Phe Ile Gln Gln Phe

225 230 235 240

Ser Gln Cys Lys Ala Thr Ala Arg Asn Leu Lys Leu Lys Tyr Leu Ile

245 250 255

Asn Leu Glu Thr Leu Gln Ser Ala Phe Tyr Thr Glu Lys Phe Glu Val

260 265 270

Lys Glu Pro Gly Ser Gly Pro Ser Gly Glu Glu Ile Phe Ala Thr Ile

275 280 285

Ile Ile Thr Gly Asn Gly Gly Ile Gln Trp Ser Arg Gly Lys His Lys

290 295 300

Glu Ser Glu Thr Leu Thr Glu Gln Asp Leu Gln Leu Tyr Cys Asp Phe

305 310 315 320

Ser Asn Ile Ile Asp Val Ser Ile Lys Gln Ala Asn Gln Glu Gly Ser

325 330 335

Asn Glu Ser Arg Val Val Thr Ile His Lys Gln Asp Gly Lys Asn Leu

340 345 350

Glu Ile Glu Leu Ser Ser Leu Arg Glu Ala Leu Ser Phe Val Ser Leu

355 360 365

Ile Asp Gly Tyr Tyr Arg Leu Thr Ala Asp Ala His His Tyr Leu Cys

370 375 380

Lys Glu Val Ala Pro Pro Ala Val Leu Glu Asn Ile Gln Ser Asn Cys

385 390 395 400

His Gly Pro Ile Ser Met Asp Phe Ala Ile Ser Lys Leu Lys Lys Ala

405 410 415

Gly Asn Gln Thr Gly Leu Tyr Val Leu Arg Cys Ser Pro Lys Asp Phe

420 425 430

Asn Lys Tyr Phe Leu Thr Phe Ala Val Glu Arg Glu Asn Val Ile Glu

435 440 445

Tyr Lys His Cys Leu Ile Thr Lys Asn Glu Asn Glu Glu Tyr Asn Leu

450 455 460

Ser Gly Thr Lys Lys Asn Phe Ser Ser Leu Lys Asp Leu Leu Asn Cys

465 470 475 480

Tyr Gln Met Glu Thr Val Arg Ser Asp Asn Ile Ile Phe Gln Phe Thr

485 490 495

Lys Cys Cys Pro Pro Lys Pro Lys Asp Lys Ser Asn Leu Leu Val Phe

500 505 510

Arg Thr Asn Gly Val Ser Asp Val Pro Thr Ser Pro Thr Leu Gln Arg

515 520 525

Pro Thr His Met Asn Gln Met Val Phe His Lys Ile Arg Asn Glu Asp

530 535 540

Leu Ile Phe Asn Glu Ser Leu Gly Gln Gly Thr Phe Thr Lys Ile Phe

545 550 555 560

Lys Gly Val Arg Arg Glu Val Gly Asp Tyr Gly Gln Leu His Glu Thr

565 570 575

Glu Val Leu Leu Lys Val Leu Asp Lys Ala His Arg Asn Tyr Ser Glu

580 585 590

Ser Phe Phe Glu Ala Ala Ser Met Met Ser Lys Leu Ser His Lys His

595 600 605

Leu Val Leu Asn Tyr Gly Val Cys Val Cys Gly Asp Glu Asn Ile Leu

610 615 620

Val Gln Glu Phe Val Lys Phe Gly Ser Leu Asp Thr Tyr Leu Lys Lys

625 630 635 640

Asn Lys Asn Cys Ile Asn Ile Leu Trp Lys Leu Glu Val Ala Lys Gln

645 650 655

Leu Ala Trp Ala Met His Phe Leu Glu Glu Asn Thr Leu Ile His Gly

660 665 670

Asn Val Cys Ala Lys Asn Ile Leu Leu Ile Arg Glu Glu Asp Arg Lys

675 680 685

Thr Gly Asn Pro Pro Phe Ile Lys Leu Ser Asp Pro Gly Ile Ser Ile

690 695 700

Thr Val Leu Pro Lys Asp Ile Leu Gln Glu Arg Ile Pro Trp Val Pro

705 710 715 720

Pro Glu Cys Ile Glu Asn Pro Lys Asn Leu Asn Leu Ala Thr Asp Lys

725 730 735

Trp Ser Phe Gly Thr Thr Leu Trp Glu Ile Cys Ser Gly Gly Asp Lys

740 745 750

Pro Leu Ser Ala Leu Asp Ser Gln Arg Lys Leu Gln Phe Tyr Glu Asp

755 760 765

Arg His Gln Leu Pro Ala Pro Lys Trp Ala Glu Leu Ala Asn Leu Ile

770 775 780

Asn Asn Cys Met Asp Tyr Glu Pro Asp Phe Arg Pro Ser Phe Arg Ala

785 790 795 800

Ile Ile Arg Asp Leu Asn Ser Leu Phe Thr Pro Asp Tyr Glu Leu Leu

805 810 815

Thr Glu Asn Asp Met Leu Pro Asn Met Arg Ile Gly Ala Leu Gly Phe

820 825 830

Ser Gly Ala Phe Glu Asp Arg Asp Pro Thr Gln Phe Glu Glu Arg His

835 840 845

Leu Lys Phe Leu Gln Gln Leu Gly Lys Gly Asn Phe Gly Ser Val Glu

850 855 860

Met Cys Arg Tyr Asp Pro Leu Gln Asp Asn Thr Gly Glu Val Val Ala

865 870 875 880

Val Lys Lys Leu Gln His Ser Thr Glu Glu His Leu Arg Asp Phe Glu

885 890 895

Arg Glu Ile Glu Ile Leu Lys Ser Leu Gln His Asp Asn Ile Val Lys

900 905 910

Tyr Lys Gly Val Cys Tyr Ser Ala Gly Arg Arg Asn Leu Lys Leu Ile

915 920 925

Met Glu Tyr Leu Pro Tyr Gly Ser Leu Arg Asp Tyr Leu Gln Lys His

930 935 940

Lys Glu Arg Ile Asp His Ile Lys Leu Leu Gln Tyr Thr Ser Gln Ile

945 950 955 960

Cys Lys Gly Met Glu Tyr Leu Gly Thr Lys Arg Tyr Ile His Arg Asp

965 970 975

Leu Ala Thr Arg Asn Ile Leu Val Glu Asn Glu Asn Arg Val Lys Ile

980 985 990

Gly Asp Phe Gly Leu Thr Lys Val Leu Pro Gln Asp Lys Glu Tyr Tyr

995 1000 1005

Lys Val Lys Glu Pro Gly Glu Ser Pro Ile Phe Trp Tyr Ala Pro Glu

1010 1015 1020

Ser Leu Thr Glu Ser Lys Phe Ser Val Ala Ser Asp Val Trp Ser Phe

1025 1030 1035 1040

Gly Val Val Leu Tyr Glu Leu Phe Thr Tyr Ile Glu Lys Ser Lys Ser

1045 1050 1055

Pro Pro Ala Glu Phe Met Arg Met Ile Gly Asn Asp Lys Gln Gly Gln

1060 1065 1070

Met Ile Val Phe His Leu Ile Glu Leu Leu Lys Asn Asn Gly Arg Leu

1075 1080 1085

Pro Arg Pro Asp Gly Cys Pro Asp Glu Ile Tyr Met Ile Met Thr Glu

1090 1095 1100

Cys Trp Asn Asn Asn Val Asn Gln Arg Pro Ser Phe Arg Asp Leu Ala

1105 1110 1115 1120

Leu Arg Val Asp Gln Ile Arg Asp Asn Met Ala Gly

gacgcgggcg cggaaggagc gcggccggag gtcctcagga agaagccgcg gggactggct 60

gcgcttgaca ggctgcactt ggatgggagc acctggtgcc tcgggactgc tccgatgccc 120

gggtctgtgc tgaatgtgta atatgcggaa ctatattgaa acattacaac catcttttga 180

tggcaacacc ctgaggacct cccttttcca gatggggaaa ctgaggccca gaattgctaa 240

gtggcttgct tgagttgaca cagggagctc caggactcac cctcagctga gccacctgcc 300

gggagc atg cct ctg cgc cac tgg ggg atg gcc agg ggc agt aag ccc 348

Met Pro Leu Arg His Trp Gly Met Ala Arg Gly Ser Lys Pro

1 5 10

gtt ggg gat gga gcc cag ccc atg gct gcc atg gga ggc ctg aag gtg 396

Val Gly Asp Gly Ala Gln Pro Met Ala Ala Met Gly Gly Leu Lys Val

15 20 25 30

ctt ctg cac tgg gct ggt cca ggc ggc ggg gag ccc tgg gtc act ttc 444

Leu Leu His Trp Ala Gly Pro Gly Gly Gly Glu Pro Trp Val Thr Phe

›PRT · 2 of 3

35 40 45

agt gag tca tcg ctg aca gct gag gaa gtc tgc atc cac att gca cat 492

Ser Glu Ser Ser Leu Thr Ala Glu Glu Val Cys Ile His Ile Ala His

50 55 60

aaa gtt ggt atc act cct cct tgc ttc aat ctc ttt gcc ctc ttc gat 540

Lys Val Gly Ile Thr Pro Pro Cys Phe Asn Leu Phe Ala Leu Phe Asp

65 70 75

gct cag gcc caa gtc tgg ttg ccc cca aac cac atc cta gag atc ccc 588

Ala Gln Ala Gln Val Trp Leu Pro Pro Asn His Ile Leu Glu Ile Pro

80 85 90

aga gat gca agc ctg atg cta tat ttc cgc ata agg ttt tat ttc cgg 636

Arg Asp Ala Ser Leu Met Leu Tyr Phe Arg Ile Arg Phe Tyr Phe Arg

95 100 105 110

aac tgg cat ggc atg aat cct cgg gaa ccg gct gtg tac cgt tgt ggg 684

Asn Trp His Gly Met Asn Pro Arg Glu Pro Ala Val Tyr Arg Cys Gly

115 120 125

ccc cca gga acc gag gca tcc tca gat cag aca gca cag ggg atg caa 732

Pro Pro Gly Thr Glu Ala Ser Ser Asp Gln Thr Ala Gln Gly Met Gln

130 135 140

ctc ctg gac cca gcc tca ttt gag tac ctc ttt gag cag ggc aag cat 780

Leu Leu Asp Pro Ala Ser Phe Glu Tyr Leu Phe Glu Gln Gly Lys His

145 150 155

gag ttt gtg aat gac gtg gca tca ctg tgg gag ctg tcg acc gag gag 828

Glu Phe Val Asn Asp Val Ala Ser Leu Trp Glu Leu Ser Thr Glu Glu

160 165 170

gag atc cac cac ttt aag aat gag agc ctg ggc atg gcc ttt ctg cac 876

Glu Ile His His Phe Lys Asn Glu Ser Leu Gly Met Ala Phe Leu His

175 180 185 190

ctc tgt cac ctc gct ctc cgc cat ggc atc ccc ctg gag gag gtg gcc 924

Leu Cys His Leu Ala Leu Arg His Gly Ile Pro Leu Glu Glu Val Ala

195 200 205

aag aag acc agc ttc aag gac tgc atc ccg cgc tcc ttc cgc cgg cat 972

Lys Lys Thr Ser Phe Lys Asp Cys Ile Pro Arg Ser Phe Arg Arg His

210 215 220

atc cgg cag cac agc gcc ctg acc cgg ctg cgc ctt cgg aac gtc ttc 1020

Ile Arg Gln His Ser Ala Leu Thr Arg Leu Arg Leu Arg Asn Val Phe

225 230 235

cgc agg ttc ctg cgg gac ttc cag ccg ggc cga ctc tcc cag cag atg 1068

Arg Arg Phe Leu Arg Asp Phe Gln Pro Gly Arg Leu Ser Gln Gln Met

240 245 250

gtc atg gtc aaa tac cta gcc aca ctc gag cgg ctg gca ccc cgc ttc 1116

Val Met Val Lys Tyr Leu Ala Thr Leu Glu Arg Leu Ala Pro Arg Phe

255 260 265 270

ggc aca gag cgt gtg ccc gtg tgc cac ctg agg ctg ctg gcc cag gcc 1164

Gly Thr Glu Arg Val Pro Val Cys His Leu Arg Leu Leu Ala Gln Ala

275 280 285

gag ggg gag ccc tgc tac atc cgg gac agt ggg gtg gcc cct aca gac 1212

Glu Gly Glu Pro Cys Tyr Ile Arg Asp Ser Gly Val Ala Pro Thr Asp

290 295 300

cct ggc cct gag tct gct gct ggg ccc cca acc cac gag gtg ctg gtg 1260

Pro Gly Pro Glu Ser Ala Ala Gly Pro Pro Thr His Glu Val Leu Val

305 310 315

aca ggc act ggt ggc atc cag tgg tgg cca gta gag gag gag gtg aac 1308

Thr Gly Thr Gly Gly Ile Gln Trp Trp Pro Val Glu Glu Glu Val Asn

320 325 330

aag gag gag ggt tct agt ggc agc agt ggc agg aac ccc caa gcc agc 1356

Lys Glu Glu Gly Ser Ser Gly Ser Ser Gly Arg Asn Pro Gln Ala Ser

335 340 345 350

ctg ttt ggg aag aag gcc aag gct cac aag gca ttc ggc cag ccg gca 1404

Leu Phe Gly Lys Lys Ala Lys Ala His Lys Ala Phe Gly Gln Pro Ala

355 360 365

gac agg ccg cgg gag cca ctg tgg gcc tac ttc tgt gac ttc cgg gac 1452

Asp Arg Pro Arg Glu Pro Leu Trp Ala Tyr Phe Cys Asp Phe Arg Asp

370 375 380

atc acc cac gtg gtg ctg aaa gag cac tgt gtc agc atc cac cgg cag 1500

Ile Thr His Val Val Leu Lys Glu His Cys Val Ser Ile His Arg Gln

385 390 395

gac aac aag tgc ctg gag ctg agc ttg cct tcc cgg gct gcg gcg ctg 1548

Asp Asn Lys Cys Leu Glu Leu Ser Leu Pro Ser Arg Ala Ala Ala Leu

400 405 410

tcc ttc gtg tcg ctg gtg gac ggc tat ttc cgc ctg acg gcc gac tcc 1596

Ser Phe Val Ser Leu Val Asp Gly Tyr Phe Arg Leu Thr Ala Asp Ser

415 420 425 430

agc cac tac ctg tgc cac gag gtg gct ccc cca cgg ctg gtg atg agc 1644

Ser His Tyr Leu Cys His Glu Val Ala Pro Pro Arg Leu Val Met Ser

435 440 445

atc cgg gat ggg atc cac gga ccc ctg ctg gag cca ttt gtg cag gcc 1692

Ile Arg Asp Gly Ile His Gly Pro Leu Leu Glu Pro Phe Val Gln Ala

450 455 460

aag ctg cgg ccc gag gac ggc ctg tac ctc att cac tgg agc acc agc 1740

Lys Leu Arg Pro Glu Asp Gly Leu Tyr Leu Ile His Trp Ser Thr Ser

465 470 475

cac ccc tac cgc ctg atc ctc aca gtg gcc cag cgt agc cag gca cca 1788

His Pro Tyr Arg Leu Ile Leu Thr Val Ala Gln Arg Ser Gln Ala Pro

480 485 490

gac ggc atg cag agc ttg cgg ctc cga aag ttc ccc att gag cag cag 1836

Asp Gly Met Gln Ser Leu Arg Leu Arg Lys Phe Pro Ile Glu Gln Gln

495 500 505 510

gac ggg gcc ttc gtg ctg gag ggc tgg ggc cgg tcc ttc ccc agc gtt 1884

Asp Gly Ala Phe Val Leu Glu Gly Trp Gly Arg Ser Phe Pro Ser Val

515 520 525

cgg gaa ctt ggg gct gcc ttg cag ggc tgc ttg ctg agg gcc ggg gat 1932

Arg Glu Leu Gly Ala Ala Leu Gln Gly Cys Leu Leu Arg Ala Gly Asp

530 535 540

gac tgc ttc tct ctg cgt cgc tgt tgc ctg ccc caa cca gga gaa acc 1980

Asp Cys Phe Ser Leu Arg Arg Cys Cys Leu Pro Gln Pro Gly Glu Thr

545 550 555

tcc aat ctc atc atc atg cgg ggg gct cgg gcc agc ccc agg aca ctc 2028

Ser Asn Leu Ile Ile Met Arg Gly Ala Arg Ala Ser Pro Arg Thr Leu

560 565 570

aac ctc agc cag ctc agc ttc cac cgg gtt gac cag aag gag atc acc 2076

Asn Leu Ser Gln Leu Ser Phe His Arg Val Asp Gln Lys Glu Ile Thr

575 580 585 590

cag ctg tcc cac ttg ggc cag ggc aca agg acc aac gtg tat gag ggc 2124

Gln Leu Ser His Leu Gly Gln Gly Thr Arg Thr Asn Val Tyr Glu Gly

595 600 605

cgc ctg cga gtg gag ggc agc ggg gac cct gag gag ggc aag atg gat 2172

Arg Leu Arg Val Glu Gly Ser Gly Asp Pro Glu Glu Gly Lys Met Asp

610 615 620

gac gag gac ccc ctc gtg cct ggc agg gac cgt ggg cag gag cta cga 2220

Asp Glu Asp Pro Leu Val Pro Gly Arg Asp Arg Gly Gln Glu Leu Arg

625 630 635

gtg gtg ctc aaa gtg ctg gac cct agt cac cat gac atc gcc ctg gcc 2268

Val Val Leu Lys Val Leu Asp Pro Ser His His Asp Ile Ala Leu Ala

640 645 650

ttc tac gag aca gcc agc ctc atg agc cag gtc tcc cac acg cac ctg 2316

Phe Tyr Glu Thr Ala Ser Leu Met Ser Gln Val Ser His Thr His Leu

655 660 665 670

gcc ttc gtg cat ggc gtc tgt gtg cgc ggc cct gaa aat agc atg gtg 2364

Ala Phe Val His Gly Val Cys Val Arg Gly Pro Glu Asn Ser Met Val

675 680 685

aca gag tac gtg gag cac gga ccc ctg gat gtg tgg ctg cgg agg gag 2412

Thr Glu Tyr Val Glu His Gly Pro Leu Asp Val Trp Leu Arg Arg Glu

690 695 700

cgg ggc cat gtg ccc atg gct tgg aag atg gtg gtg gcc cag cag ctg 2460

Arg Gly His Val Pro Met Ala Trp Lys Met Val Val Ala Gln Gln Leu

705 710 715

gcc agc gcc ctc agc tac ctg gag aac aag aac ctg gtt cat ggt aat 2508

›PRT · 3 of 3

Ala Ser Ala Leu Ser Tyr Leu Glu Asn Lys Asn Leu Val His Gly Asn

720 725 730

gtg tgt ggc cgg aac atc ctg ctg gcc cgg ctg ggg ttg gca gag ggc 2556

Val Cys Gly Arg Asn Ile Leu Leu Ala Arg Leu Gly Leu Ala Glu Gly

735 740 745 750

acc agc ccc ttc atc aag ctg agt gat cct ggc gtg ggc ctg ggc gcc 2604

Thr Ser Pro Phe Ile Lys Leu Ser Asp Pro Gly Val Gly Leu Gly Ala

755 760 765

ctc tcc agg gag gag cgg gtg gag agg atc ccc tgg ctg gcc ccc gaa 2652

Leu Ser Arg Glu Glu Arg Val Glu Arg Ile Pro Trp Leu Ala Pro Glu

770 775 780

tgc cta cca ggt ggg gcc aac agc cta agc acc gcc atg gac aag tgg 2700

Cys Leu Pro Gly Gly Ala Asn Ser Leu Ser Thr Ala Met Asp Lys Trp

785 790 795

ggg ttt ggc gcc acc ctc ctg gag atc tgc ttt gac gga gag gcc cct 2748

Gly Phe Gly Ala Thr Leu Leu Glu Ile Cys Phe Asp Gly Glu Ala Pro

800 805 810

ctg cag agc cgc agt ccc tcc gag aag gag cat ttc tac cag agg cag 2796

Leu Gln Ser Arg Ser Pro Ser Glu Lys Glu His Phe Tyr Gln Arg Gln

815 820 825 830

cac cgg ctg ccc gag ccc tcc tgc cca cag ctg gcc aca ctc acc agc 2844

His Arg Leu Pro Glu Pro Ser Cys Pro Gln Leu Ala Thr Leu Thr Ser

835 840 845

cag tgt ctg acc tat gag cca acc cag agg cca tca ttc cgc acc atc 2892

Gln Cys Leu Thr Tyr Glu Pro Thr Gln Arg Pro Ser Phe Arg Thr Ile

850 855 860

ctg cgt gac ctc acc cgc gtg cag ccc cac aat ctt gct gac gtc ttg 2940

Leu Arg Asp Leu Thr Arg Val Gln Pro His Asn Leu Ala Asp Val Leu

865 870 875

act gtg aac cgg gac tca ccg gcc gtc gga cct act act ttc cac aag 2988

Thr Val Asn Arg Asp Ser Pro Ala Val Gly Pro Thr Thr Phe His Lys

880 885 890

cgc tat ttg aaa aag atc cga gat ctg ggc gag ggt cac ttc ggc aag 3036

Arg Tyr Leu Lys Lys Ile Arg Asp Leu Gly Glu Gly His Phe Gly Lys

895 900 905 910

gtc agc ttg tac tgc tac gat ccg acc aac gac ggc act ggc gag atg 3084

Val Ser Leu Tyr Cys Tyr Asp Pro Thr Asn Asp Gly Thr Gly Glu Met

915 920 925

gtg gcg gtg aaa gcc ctc aag gca gac tgc ggc ccc cag cac cgc tcg 3132

Val Ala Val Lys Ala Leu Lys Ala Asp Cys Gly Pro Gln His Arg Ser

930 935 940

ggc tgg aag cag gag att gac att ctg cgc acg ctc tac cac gag cac 3180

Gly Trp Lys Gln Glu Ile Asp Ile Leu Arg Thr Leu Tyr His Glu His

945 950 955

atc atc aag tac aag ggc tgc tgc gag gac caa ggc gag aag tcg ctg 3228

Ile Ile Lys Tyr Lys Gly Cys Cys Glu Asp Gln Gly Glu Lys Ser Leu

960 965 970

cag ctg gtc atg gag tac gtg ccc ctg ggc agc ctc cga gac tac ctg 3276

Gln Leu Val Met Glu Tyr Val Pro Leu Gly Ser Leu Arg Asp Tyr Leu

975 980 985 990

ccc cgg cac agc atc ggg ctg gcc cag ctg ctg ctc ttc gcc cag cag 3324

Pro Arg His Ser Ile Gly Leu Ala Gln Leu Leu Leu Phe Ala Gln Gln

995 1000 1005

atc tgc gag ggc atg gcc tat ctg cac gcg cac gac tac atc cac cga 3372

Ile Cys Glu Gly Met Ala Tyr Leu His Ala His Asp Tyr Ile His Arg

1010 1015 1020

gac cta gcc gcg cgc aac gtg ctg ctg gac aac gac agg ctg gtc aag 3420

Asp Leu Ala Ala Arg Asn Val Leu Leu Asp Asn Asp Arg Leu Val Lys

1025 1030 1035

atc ggg gac ttt ggc cta gcc aag gcc gtg ccc gaa ggc cac gag tac 3468

Ile Gly Asp Phe Gly Leu Ala Lys Ala Val Pro Glu Gly His Glu Tyr

1040 1045 1050

tac cgc gtg cgc gag gat ggg gac agc ccc gtg ttc tgg tat gcc cca 3516

Tyr Arg Val Arg Glu Asp Gly Asp Ser Pro Val Phe Trp Tyr Ala Pro

1055 1060 1065 1070

gag tgc ctg aag gag tat aag ttc tac tat gcg tca gat gtc tgg tcc 3564

Glu Cys Leu Lys Glu Tyr Lys Phe Tyr Tyr Ala Ser Asp Val Trp Ser

1075 1080 1085

ttc ggg gtg acc ctg tat gag ctg ctg acg cac tgt gac tcc agc cag 3612

Phe Gly Val Thr Leu Tyr Glu Leu Leu Thr His Cys Asp Ser Ser Gln

1090 1095 1100

agc ccc ccc acg aaa ttc ctt gag ctc ata ggc att gct cag ggt cag 3660

Ser Pro Pro Thr Lys Phe Leu Glu Leu Ile Gly Ile Ala Gln Gly Gln

1105 1110 1115

atg aca gtt ctg aga ctc act gag ttg ctg gaa cga ggg gag agg ctg 3708

Met Thr Val Leu Arg Leu Thr Glu Leu Leu Glu Arg Gly Glu Arg Leu

1120 1125 1130

cca cgg ccc gac aaa tgt ccc tgt gag gtc tat cat ctc atg aag aac 3756

Pro Arg Pro Asp Lys Cys Pro Cys Glu Val Tyr His Leu Met Lys Asn

1135 1140 1145 1150

tgc tgg gag aca gag gcg tcc ttt cgc cca acc ttc gag aac ctc ata 3804

Cys Trp Glu Thr Glu Ala Ser Phe Arg Pro Thr Phe Glu Asn Leu Ile

1155 1160 1165

ccc att ctg aag aca gtc cat gag aag tac caa ggc cag gcc cct tca 3852

Pro Ile Leu Lys Thr Val His Glu Lys Tyr Gln Gly Gln Ala Pro Ser

1170 1175 1180

gtg ttc agc gtg tgc tga ggcacaatgg cagccctgcc tgggaggact 3900

Val Phe Ser Val Cys *

1185

ggaccaggca gtggctgcag agggagcctc ctgctccctg ctccaggatg aaaccaagag 3960

ggggatgtca gcctcaccca caccgtgtgc cttactcctg tctagagacc ccacctctgt 4020

gaacttattt ttctttcttg gccgtgagcc taaccatgat cttgagggac ccaacatttg 4080

taggggcact aatccagccc ttaaatcccc cagcttccaa acttgaggcc caccatctcc 4140

accatctggt aataaactca tgttttctct gctggg 4176

1187

›PRT

Homo sapiens

Met Pro Leu Arg His Trp Gly Met Ala Arg Gly Ser Lys Pro Val Gly

1 5 10 15

Asp Gly Ala Gln Pro Met Ala Ala Met Gly Gly Leu Lys Val Leu Leu

20 25 30

His Trp Ala Gly Pro Gly Gly Gly Glu Pro Trp Val Thr Phe Ser Glu

35 40 45

Ser Ser Leu Thr Ala Glu Glu Val Cys Ile His Ile Ala His Lys Val

50 55 60

Gly Ile Thr Pro Pro Cys Phe Asn Leu Phe Ala Leu Phe Asp Ala Gln

65 70 75 80

Ala Gln Val Trp Leu Pro Pro Asn His Ile Leu Glu Ile Pro Arg Asp

85 90 95

Ala Ser Leu Met Leu Tyr Phe Arg Ile Arg Phe Tyr Phe Arg Asn Trp

100 105 110

His Gly Met Asn Pro Arg Glu Pro Ala Val Tyr Arg Cys Gly Pro Pro

115 120 125

Gly Thr Glu Ala Ser Ser Asp Gln Thr Ala Gln Gly Met Gln Leu Leu

130 135 140

Asp Pro Ala Ser Phe Glu Tyr Leu Phe Glu Gln Gly Lys His Glu Phe

145 150 155 160

Val Asn Asp Val Ala Ser Leu Trp Glu Leu Ser Thr Glu Glu Glu Ile

165 170 175

His His Phe Lys Asn Glu Ser Leu Gly Met Ala Phe Leu His Leu Cys

180 185 190

His Leu Ala Leu Arg His Gly Ile Pro Leu Glu Glu Val Ala Lys Lys

195 200 205

Thr Ser Phe Lys Asp Cys Ile Pro Arg Ser Phe Arg Arg His Ile Arg

210 215 220

Gln His Ser Ala Leu Thr Arg Leu Arg Leu Arg Asn Val Phe Arg Arg

225 230 235 240

Phe Leu Arg Asp Phe Gln Pro Gly Arg Leu Ser Gln Gln Met Val Met

245 250 255

Val Lys Tyr Leu Ala Thr Leu Glu Arg Leu Ala Pro Arg Phe Gly Thr

260 265 270

Glu Arg Val Pro Val Cys His Leu Arg Leu Leu Ala Gln Ala Glu Gly

275 280 285

Glu Pro Cys Tyr Ile Arg Asp Ser Gly Val Ala Pro Thr Asp Pro Gly

290 295 300

Pro Glu Ser Ala Ala Gly Pro Pro Thr His Glu Val Leu Val Thr Gly

305 310 315 320

Thr Gly Gly Ile Gln Trp Trp Pro Val Glu Glu Glu Val Asn Lys Glu

325 330 335

Glu Gly Ser Ser Gly Ser Ser Gly Arg Asn Pro Gln Ala Ser Leu Phe

340 345 350

Gly Lys Lys Ala Lys Ala His Lys Ala Phe Gly Gln Pro Ala Asp Arg

355 360 365

Pro Arg Glu Pro Leu Trp Ala Tyr Phe Cys Asp Phe Arg Asp Ile Thr

370 375 380

His Val Val Leu Lys Glu His Cys Val Ser Ile His Arg Gln Asp Asn

385 390 395 400

Lys Cys Leu Glu Leu Ser Leu Pro Ser Arg Ala Ala Ala Leu Ser Phe

405 410 415

Val Ser Leu Val Asp Gly Tyr Phe Arg Leu Thr Ala Asp Ser Ser His

420 425 430

Tyr Leu Cys His Glu Val Ala Pro Pro Arg Leu Val Met Ser Ile Arg

435 440 445

Asp Gly Ile His Gly Pro Leu Leu Glu Pro Phe Val Gln Ala Lys Leu

450 455 460

Arg Pro Glu Asp Gly Leu Tyr Leu Ile His Trp Ser Thr Ser His Pro

465 470 475 480

Tyr Arg Leu Ile Leu Thr Val Ala Gln Arg Ser Gln Ala Pro Asp Gly

485 490 495

Met Gln Ser Leu Arg Leu Arg Lys Phe Pro Ile Glu Gln Gln Asp Gly

500 505 510

Ala Phe Val Leu Glu Gly Trp Gly Arg Ser Phe Pro Ser Val Arg Glu

515 520 525

Leu Gly Ala Ala Leu Gln Gly Cys Leu Leu Arg Ala Gly Asp Asp Cys

530 535 540

Phe Ser Leu Arg Arg Cys Cys Leu Pro Gln Pro Gly Glu Thr Ser Asn

545 550 555 560

Leu Ile Ile Met Arg Gly Ala Arg Ala Ser Pro Arg Thr Leu Asn Leu

565 570 575

Ser Gln Leu Ser Phe His Arg Val Asp Gln Lys Glu Ile Thr Gln Leu

580 585 590

Ser His Leu Gly Gln Gly Thr Arg Thr Asn Val Tyr Glu Gly Arg Leu

595 600 605

Arg Val Glu Gly Ser Gly Asp Pro Glu Glu Gly Lys Met Asp Asp Glu

610 615 620

Asp Pro Leu Val Pro Gly Arg Asp Arg Gly Gln Glu Leu Arg Val Val

625 630 635 640

Leu Lys Val Leu Asp Pro Ser His His Asp Ile Ala Leu Ala Phe Tyr

645 650 655

Glu Thr Ala Ser Leu Met Ser Gln Val Ser His Thr His Leu Ala Phe

660 665 670

Val His Gly Val Cys Val Arg Gly Pro Glu Asn Ser Met Val Thr Glu

675 680 685

Tyr Val Glu His Gly Pro Leu Asp Val Trp Leu Arg Arg Glu Arg Gly

690 695 700

His Val Pro Met Ala Trp Lys Met Val Val Ala Gln Gln Leu Ala Ser

705 710 715 720

Ala Leu Ser Tyr Leu Glu Asn Lys Asn Leu Val His Gly Asn Val Cys

725 730 735

Gly Arg Asn Ile Leu Leu Ala Arg Leu Gly Leu Ala Glu Gly Thr Ser

740 745 750

Pro Phe Ile Lys Leu Ser Asp Pro Gly Val Gly Leu Gly Ala Leu Ser

755 760 765

Arg Glu Glu Arg Val Glu Arg Ile Pro Trp Leu Ala Pro Glu Cys Leu

770 775 780

Pro Gly Gly Ala Asn Ser Leu Ser Thr Ala Met Asp Lys Trp Gly Phe

785 790 795 800

Gly Ala Thr Leu Leu Glu Ile Cys Phe Asp Gly Glu Ala Pro Leu Gln

805 810 815

Ser Arg Ser Pro Ser Glu Lys Glu His Phe Tyr Gln Arg Gln His Arg

820 825 830

Leu Pro Glu Pro Ser Cys Pro Gln Leu Ala Thr Leu Thr Ser Gln Cys

835 840 845

Leu Thr Tyr Glu Pro Thr Gln Arg Pro Ser Phe Arg Thr Ile Leu Arg

850 855 860

Asp Leu Thr Arg Val Gln Pro His Asn Leu Ala Asp Val Leu Thr Val

865 870 875 880

Asn Arg Asp Ser Pro Ala Val Gly Pro Thr Thr Phe His Lys Arg Tyr

885 890 895

Leu Lys Lys Ile Arg Asp Leu Gly Glu Gly His Phe Gly Lys Val Ser

900 905 910

Leu Tyr Cys Tyr Asp Pro Thr Asn Asp Gly Thr Gly Glu Met Val Ala

915 920 925

Val Lys Ala Leu Lys Ala Asp Cys Gly Pro Gln His Arg Ser Gly Trp

930 935 940

Lys Gln Glu Ile Asp Ile Leu Arg Thr Leu Tyr His Glu His Ile Ile

945 950 955 960

Lys Tyr Lys Gly Cys Cys Glu Asp Gln Gly Glu Lys Ser Leu Gln Leu

965 970 975

Val Met Glu Tyr Val Pro Leu Gly Ser Leu Arg Asp Tyr Leu Pro Arg

980 985 990

His Ser Ile Gly Leu Ala Gln Leu Leu Leu Phe Ala Gln Gln Ile Cys

995 1000 1005

Glu Gly Met Ala Tyr Leu His Ala His Asp Tyr Ile His Arg Asp Leu

1010 1015 1020

Ala Ala Arg Asn Val Leu Leu Asp Asn Asp Arg Leu Val Lys Ile Gly

1025 1030 1035 1040

Asp Phe Gly Leu Ala Lys Ala Val Pro Glu Gly His Glu Tyr Tyr Arg

1045 1050 1055

Val Arg Glu Asp Gly Asp Ser Pro Val Phe Trp Tyr Ala Pro Glu Cys

1060 1065 1070

Leu Lys Glu Tyr Lys Phe Tyr Tyr Ala Ser Asp Val Trp Ser Phe Gly

1075 1080 1085

Val Thr Leu Tyr Glu Leu Leu Thr His Cys Asp Ser Ser Gln Ser Pro

1090 1095 1100

Pro Thr Lys Phe Leu Glu Leu Ile Gly Ile Ala Gln Gly Gln Met Thr

1105 1110 1115 1120

Val Leu Arg Leu Thr Glu Leu Leu Glu Arg Gly Glu Arg Leu Pro Arg

1125 1130 1135

Pro Asp Lys Cys Pro Cys Glu Val Tyr His Leu Met Lys Asn Cys Trp

1140 1145 1150

Glu Thr Glu Ala Ser Phe Arg Pro Thr Phe Glu Asn Leu Ile Pro Ile

1155 1160 1165

Leu Lys Thr Val His Glu Lys Tyr Gln Gly Gln Ala Pro Ser Val Phe

1170 1175 1180

Ser Val Cys

1185

›DNA

Artificial Sequence

antisense molecule

gctgaagctc gaaccactgt gacatcc 27

›DNA

Artificial Sequence

antisense molecule

aagttcgtac cactgagaca tcctgcc 27

›DNA

Artificial Sequence

antisense molecule

catctcccac tgcgccattt ggactcttca 30

›DNA

Artificial Sequence

antisense molecule

cagcatttcc cactgcgcca tttgggc 27

›DNA

Artificial Sequence

antisense molecule

ctggttccac tgagccatcc tgctgcatca g 31

›DNA

Artificial Sequence

antisense molecule

ctgtagctga ttccattggg ccatcct 27

›DNA

Artificial Sequence

antisense molecule

gattccactg agacatgctg ctctctctct c 31

›DNA

Artificial Sequence

antisense molecule

gacttgattc cactgagaca tgctagc 27

›DNA

Artificial Sequence

antisense molecule

gccaggccat tcccatctag agcttttttc 30

›DNA

Artificial Sequence

antisense molecule

cgtaaggcag gccattccca tgcagag 27

›DNA

Artificial Sequence

antisense molecule

cccacacaga ggcatggtcc ccaccattca 30

›DNA

Artificial Sequence

antisense molecule

ggccatcccc cagtggcgca gaggcatgct ccc 33

›DNA

Artificial Sequence

antisense molecule

cctggttgcg tgctaccatc ctactcgagg ggc 33

›DNA

Artificial Sequence

antisense molecule

cacctggttg tgtgctacca tcctact 27

›DNA

Artificial Sequence

antisense molecule

gctgtgggtg accatggcgc acggagccag cg 32

›DNA

Artificial Sequence

antisense molecule

ggcgggaaac ttgctgtggg tgaccat 27

›DNA

Artificial Sequence

primer

gaactttcag ctgttacttt cc 22

›DNA

Artificial Sequence

primer

ctgtgctcat catactgtc 19

›DNA

Artificial Sequence

primer

gtgttacagt cactcccact g 21

›DNA

Artificial Sequence

primer

cctcaggcaa atctgactct g 21

›DNA

Artificial Sequence

primer

gaaagtactg taggcccgag 20

›DNA

Artificial Sequence

primer

ctggaaccac aaagttagga g 21

›DNA

Artificial Sequence

primer

gaagtgagat tccactctgt ag 22

›DNA

Artificial Sequence

primer

cactctccag tttcatctgc 20

›DNA

Artificial Sequence

primer

cgaaagcagt tgacggatac g 21

›DNA

Artificial Sequence

primer

ctccaactta gttgcctaaa cc 22

›DNA

Artificial Sequence

primer

caagccgtta gaagcaggag 20

›DNA

Artificial Sequence

primer

ccatggttca caacctacag 20

›DNA

Artificial Sequence

primer

gatgaggctt tccggagtca c 21

›DNA

Artificial Sequence

primer

cagttgtatc acattcgagc 20

›DNA

Artificial Sequence

primer

ctgtgccgca gcattaagtg 20

›DNA

Artificial Sequence

primer

gtttattacc taaactggct g 21

›DNA

Artificial Sequence

primer

ccaggtataa gtatttctct c 21

›DNA

Artificial Sequence

primer

ggccatttga tcttgagcag c 21

›DNA

Artificial Sequence

primer

gcagattggc ttcttcctca g 21

›DNA

Artificial Sequence

primer

ggcatttaag gcgagtctcc 20

›DNA

Artificial Sequence

primer

ggagcttact cgcagtaggc tc 22

›DNA

Artificial Sequence

primer

gtaggagtct ctccgtgcaa gc 22

›DNA

Artificial Sequence

primer

ccaactctga ctgagccagg 20

›DNA

Artificial Sequence

primer

catccatacg caggtggatg 20

334

›DNA

Artificial Sequence

probe

gaactttcag ctgttacttt cccagatatt attcgcaact acaaagtcat ggctgccgag 60

aacataccag agaatcccct gaagtatctg taccccaata ttgacaaaga ccacgccttt 120

gggaagtatt attccagacc aaaggaagca ccagaaccga tggagcttga cgaccctaag 180

cgaactggat acatcaagac tgagttgatt tctgtgtctg aagtccaccc ttctagactt 240

cagaccacag acaacctgct tcccatgtct ccagaggagt ttgatgagat gtcccggata 300

gtgggccccg aatttgacag tatgatgagc acag 334

694

›DNA

Artificial Sequence

probe

gtgttacagt cactcccact gacagagatc atccgccact accaggttct tgccgaagag 60

aacatccccg agaacccact ccgcttcctc tatccccgaa tccctcggga cgaagctttt 120

gggtgttact accaggaaaa agttaatttg gaagaacagg aggaatattt gaaacataaa 180

ctcattgtga tctctaacag acaggtggac gagctgcagc agcctctgga gctcaaacag 240

gattcagagt ccttagaagt gaatgcagag ctcttgttag cacacgacca ggagttgcca 300

ttgatgatgc agactgggct ggttctgggc acagagctga aagtggaccc catactgagt 360

acagccccac aagtcctgct ggagccagcc ccacaagtcc tgctggagcc agccccacaa 420

gtcctgctgg agccagcccc acaagtcctg ctggagccag ccccacaagt cctgctggag 480

ccagccccac aagtcctgct ggagccagcc ccacaagtcc tgctggagcc agccccacaa 540

gtccagctgg agccagcccc acaagtcctg ctggagctag ccccacaagt cctgctggag 600

ccagccccac aagtcctgct ggagctagcc ccacaagtcc agctggagcc agcacacttg 660

ctgcagcagc catcagagtc agatttgcct gagg 694

373

›DNA

Artificial Sequence

probe

gaaagtactg taggcccgag agccaggagc accccgaagc cgacccaggt agtgctgccc 60

cgtacctgaa gaccaagttc atctgtgtga caccaacgac ctgcagcaat accattgacc 120

tgccgatgtc cccccgcact ttagattcat tgatgcagtt tggaaataac ggtgaaggtg 180

ctgagccctc agcaggaggg cagtttgagt cgctcacgtt tgacatggat ctgacctcgg 240

agtgtgctac ctcccccatg tgaggagctg aaaccagaag ctgcagagac gtgacttgag 300

acacctgccc cgtgctccac ccctaagcag ccgaacccca tatcgtctga aactcctaac 360

tttgtggttc cag 373

442

›DNA

Artificial Sequence

probe

gaagtgagat tccactctgt agaaccctac aacaaaggga gactgtcggc tctggccttc 60

gctgacatcc tgcgagacta caaggttatc atggctgaaa acatccctga aaaccctctg 120

aagtacctct accctgacat tcccaaagac aaagcctttg gcaaacacta cagctcccag 180

ccgtgcgaag tctcaagacc aaccgaacgg ggagacaagg gttacgtccc ctctgttttt 240

atccccattt caacaatccg aagcgattcc acggagccac aatctccttc agaccttctc 300

cccatgtctc caagtgcata tgctgtgctg agagaaaacc tgagcccaac gacaattgaa 360

actgcaatga attccccata ttctgctgaa tgacggtgca aacggacact ttaaagaagg 420

aagcagatga aactggagag tg 442

634

›DNA

Artificial Sequence

probe

cgaaagcagt tgacggatac gtgaagccac agatcaagca agtggtccct gagttcgtca 60

atgcatccac agatgccgga gccagcgcca cctacatgga ccaggctcct tccccagtcg 120

tgtgccctca acctcactac aacatgtacc cacccaaccc tgaccctgtc cttgaccaag 180

atggcgagtt tgacctggat gagagcatgg atgttgccag gcacgtggaa gaacttttac 240

gccggcccat ggacagtctc gacgcccgcc tctccccacc tgctggtctc ttcacctccg 300

ctagaagctc cctgtcctga acgctggact ccatgcttct cttggaaacc accttcagtg 360

taaggagccc acgtcagttg tagtatctct gttcatacca acaatggctt tgcacgtttc 420

acagggctac cttgcccaca cagttctggg tttgtggcta aagcggtggt gacctttttg 480

ttcagacctc aagggccccc agggcctctc gtgtaagagc tgaacctatc attgctgaca 540

aacctatttc tccggtgtcc tttttctgtc caatggccat ttcagtgaaa ttctagaaaa 600

ggcagggagg caggtttagg caactaagtt ggag 634

463

›DNA

Artificial Sequence

probe

caagccaagc cgttagaagc aggagcccct ggccagtgcc tggtcacgga gctgagctgt 60

gtttagatgt gttggctgct gcgtggtgaa ggaagacccg tctccagaaa agcaatttag 120

gcaaaaggga ttccgtttga tggcagagtc ccagtgctag aaaggtagcg aaggtggaca 180

gcttacagtc tcaactcatt tcgtcgtaaa tgtcctcgta acgacattga ttcttctacc 240

tggataacct tttgtttgtt tgtttgtttg tttttgtttt gtttttcccc tgtaaccatt 300

tttttttctg acaagaaaac attttaattt tctaagcaag aagcattttt caaataccat 360

gtctgtgacc caaagtaaaa atggatgata attcatgtaa atgtgtgcaa catagcaacc 420

tgaacctgca cgcgattcgg gctctgtagg ttgtgaacca tgg 463

894

›DNA

Artificial Sequence

probe

gatgaggctt tccggagtca ctataagccc gaacagatgg ggaaggacgg gaggggttat 60

gtctctacta ctatcaagat gactgtggaa agggaccagc cccttcctac tccagagccc 120

cagatgcctg ccatggtgcc accttatgat cttggaatgg cccctgatgc ttccatgcaa 180

ctcagctcag atatggggta tcctccacag tccatccact catttcagag cctagaagag 240

tccatgagtg tactgccatc ttttcaggag cctcacctgc aaatgccccc caacatgagc 300

cagataacca tgccctttga ccagcctcac ccccagggtc tgctgcagtg ccagtcccag 360

gaacatgctg tgtccagccc tgaacccatg ctttggtcag atgtgactat ggtagaggac 420

agttgcctaa ctcagcctgt gggaggtttc ccccaaggca cctgggtcag tgaagacatg 480

taccctcccc tgctgcctcc cactgaacag gacctcacca agcttctcct ggagaaccaa 540

ggggagggag gagggtcctt aggaagccag cccctcctga aaccatctcc ttatgggcaa 600

tcagggatct cactgtccca cctggaccta aggaccaacc ccagctggtg atcccagctg 660

gagaagccca gaaacaaagc ctcttctgtc tctatggacc agctctggac acctgctcat 720

gcaggtgcct tccgtctcaa ctgttccttg gttaagagaa aagaactggc tgggagacca 780

tgtggtgtat ggaactgctg tgctctgtcc tacctgccat atcagggccc cccttttcca 840

gcactgggtg caaagggatg agtggggtgt taatgctcga atgtgataca actg 894

381

›DNA

Artificial Sequence

probe

ctgtgccgca gcattaagtg ggggcgcctt attatttctt attattaatt attattattt 60

ttctggaacc acgtgggagc cctccccgcc tgggtcggag ggagtggttg tggagggtga 120

gatgcctccc acttctggct ggagacctca tcccacctct caggggtggg ggtgctcccc 180

tcctggtgct ccctccgggt cccccctggt tgtagcagct tgtgtctggg gccaggacct 240

gaattccact cctacctctc catgtttaca tattcccagt atctttgcac aaaccagggg 300

tcggggaggg tctctggctt catttttctg ctgtgcagaa tatcctattt tatattttta 360

cagccagttt aggtaataaa c 381

266

›DNA

Artificial Sequence

probe

ccaggtataa gtatttctct ctctttttcg ttttttttta aaaaaaaaaa aacacatgcc 60

tcatatagac tatctccgaa tgcagctatg tgaaagagaa cccagaggcc ctcctctgga 120

taactgcgca gaattctctc ttaaggacag ttgggctcag tctaacttaa aggtgtgaag 180

atgtagctag gtattttaaa gttcccctta ggtagtttta gctgaatgat gctttctttc 240

ctatggctgc tcaagatcaa atggcc 266

728

›DNA

Artificial Sequence

probe

gcagattggc ttcttcctca ggccctccac tcccgcagag tagagctggc aggacctgga 60

attcgtctga ggggaggggg agctgccacc tgctttcccc cctcccccag ctccagcttc 120

tttcaagtgg agccagccgg cctggcctgg tgggacaata cctttgacaa gcggactctc 180

ccctcccctt cctccacacc ccctctgctt cccaagggag gtggggacac ctccaagtgt 240

tgaacttaga actgcaaggg gaatcttcaa actttcccgc tggaacttgt ttgcgctttg 300

atttggtttg atcaagagca ggcacctggg ggaaggatgg aagagaaaag ggtgtgtgaa 360

gggtttttat gctggccaaa gaaataacca ctcccactgc ccaacctagg tgaggagtgg 420

tggctcctgg ctctggggag agtggcaagg ggtgacctga agagagctat actggtgcca 480

ggctcctctc catggggcag ctaatgaaac ctcgcagatc ccttgcaccc cagaaccctc 540

cccgttgtga agaggcagta gcatttagaa gggagacaga tgaggctggt gagctggccg 600

ccttttccaa caccgaaggg aggcagatca acagatgagc catcttggag cccaggtttc 660

ccctggagca gatggagggt tctgctttgt ctctcctatg tggggctagg agactcgcct 720

taaatgcc 728

379

›DNA

Artificial Sequence

probe

ggagcttact cgcagtaggc tctcgctctt ctaatcaatg gataaagtgg ggaaaatgtg 60

gaacaactta aaatacagat gccagaatct cttcagccac gagggaggaa gccgtaatga 120

gaacgtggag atgaacccca acagatgtcc gtctgtcaaa gagaaaagca tcagtctggg 180

agaggcagct ccccagcaag agagcagtcc cttaagagaa aatgttgcct tacagctggg 240

actgagccct tccaagacct tttccaggcg gaaccaaaac tgtgccgcag agatccctca 300

agtggttgaa atcagcatcg agaaagacag tgactcgggt gccaccccag gaacgaggct 360

tgcacggaga gactcctac 379

688

›DNA

Artificial Sequence

probe

ccaactctga ctgagccagg caccctgctc tgcctcacac agtcacatcc tggagggaac 60

acagtcccca gctggacttg gggttctgct gtcctttctt cagtcatcct ggtgcctgca 120

tgcatgtgac agctggacca gagaatgcca gcaagaacaa ggcaggtgga ggagggattg 180

tcacacaact ctgaggtcaa cgcctctagg tacaatatgg ctctttgtgg tgagccatgt 240

atcagagcga gacaggcagg acctcgtctc tccacagagg ctggacctag gtctccactc 300

acttgcctgc ccttgccacc tgaactgtgt ctattctccc agccctggtt tctcagtctg 360

ctgagtaggg caggccccct acccatgtat agaatagcga gcctgtttct gggagaatat 420

cagccagagg ttgatcatgc caaggcccct tatggggacg cagactgggc taggggacta 480

cacagttata cagtatttat ttatttattc tccttgcagg ggttgggggt ggaatgatgg 540

cgtgagccat cccacttctc tgccctgtgc tctgggtggt ccagagaccc ccaggtctgg 600

ttcttccctg tggagacccc catcccaaaa cattgttggg cccaaagtag tctcgaatgt 660

cctgggccca tccacctgcg tatggatg 688

›Tables in the description — 9

1	4003	DNA	Homo sapiens
CDS	(197)...(2449)

3	3291	DNA	Homo sapiens
CDS	(58)...(2613)

5	2787	DNA	Homo sapiens
CDS	(221)...(2533)

7	2588	DNA	Homo sapiens
CDS	(82)...(2328)

9	3046	DNA	Homo sapiens
CDS	(166)...(2709)

11	908	DNA	Homo sapiens
CDS	(14)...(649)

13	682	DNA	Homo sapiens
CDS	(1)...(678)

15	5117	DNA	Homo sapiens
CDS	(478)...(3876)

1125 1130

17	4176	DNA	Homo sapiens
CDS	(307)...(3870)

13 of 117 part labels are ours — the grant heads the rest

Claims as granted

9 claims

Classifications

6 codes

IPC · International Patent Classification

Section A — Human necessities

A61K39/395

Section C — Chemistry; metallurgy

C12Q1/68
C07K16/28

USPC · US Patent Classification

435/7.1436/501435/6

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Elizabeth Kemmerer

art unit 1646 · TC 1600

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