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  Use of cytokines and mitogens to inhibit pathological immune responsesUSPTO Application #: 20060286079Title: Use of cytokines and mitogens to inhibit pathological immune responses Abstract: The invention is generally related to methods of treating autoimmune diseases, including both antibody-mediated and cell-mediated disorders. (end of abstract) Agent: Dorsey & Whitney LLP - San Francisco, CA, US Inventor: David A. Horwitz USPTO Applicaton #: 20060286079 - Class: 424093700 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Whole Live Micro-organism, Cell, Or Virus Containing, Animal Or Plant Cell The Patent Description & Claims data below is from USPTO Patent Application 20060286079. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE [0001] This application is a continuation of U.S. Ser. No. 10/650,157, filed Aug. 27, 2003, which is a continuation of U.S. Ser. No. 10/028,944, filed Dec. 12, 2001, now U.S. Pat. No. 6,797,267, which is a continuation of 09/564,436, filed May 4, 2000, now U.S. Pat. No. 6,358,506, which claims the benefit of the filing date of U.S. Ser. No. 60/132,616, filed May 5, 1999, and is a continuation in part of U.S. Ser. No. 09/186,771, filed Nov. 5, 1998, now U.S. Pat. No. 6,228,359, which claims the benefit of the filing date of U.S. Ser. No. 60/064,507, filed Nov. 5, 1997. FIELD OF THE INVENTION [0002] The field of the invention is generally related to methods of treating autoimmune diseases, including both antibody-mediated and cell-mediated disorders. BACKGROUND OF THE INVENTION [0003] Autoimmune diseases are caused by the failure of the immune system to distinguish self from non-self. In these diseases, the immune system reacts against self tissues and this response ultimately causes inflammation and tissue injury. Autoimmune diseases can be classified into two basic categories: antibody-mediated diseases such as systemic lupus erythematosus (SLE), pemphigus vulgaris, myasthenia gravis, hemolytic anemia, thrombocytopenia purpura, Grave's disease, Sjogren's disease and dermatomyositis; and cell-mediated diseases such as Hashimoto's disease, polymyositis, disease inflammatory bowel disease, multiple sclerosis, diabetes mellitus, rheumatoid arthritis, and scleroderma. [0004] In many autoimmune diseases, tissue injury is caused by the production of antibodies to native tissue. These antibodies are called autoantibodies, in that they are produced by a mammal and have binding sites to the mammal's own tissue. Some of these disorders have characteristic waxing and waning of the amount of circulating autoantibodies causing varying symptoms over time. [0005] Of the different types of antibody-mediated autoimmune disorders, SLE is a disorder that has been well studied and documented. SLE is a disorder of generalized autoimmunity characterized by B cell hyperactivity with numerous autoantibodies against nuclear, cytoplasmic and cell surface antigens. This autoimmune disease has a multifactorial pathogenesis with genetic and environmental precipitating factors (reviewed in Hahn, B. H., Dubois' Lupus Erythematosus, 5th Ed. (1997), pp. 69-76 (D. J. Wallace et al. eds., Williams and Wilkins, Baltimore)). Among the numerous lymphocyte defects described in SLE is a failure of regulatory T cells to inhibit B cell function (Horwitz, D. A., Dubois' Lupus Erythematosus, 5th Ed. (1997), pp. 155-194 (D. J. Wallace et al. eds., Williams and Wilkins, Baltimore)). Sustained production of polyclonal IgG and autoantibodies in vitro requires T cell help (Shivakumar, S. et al. (1989), J Immunol 143:103-112). Regulatory T cells can down-regulate antibody synthesis by lytic or cytokine-mediated mechanisms. The latter involve transforming growth factor-beta (TGF-.beta.) and other inhibitory cytokines (Wahl, S. M. (1994), J Exp Med 180:1587-190). Circulating B lymphocytes spontaneously secreting antibodies are increased in patients with active SLE (Klinman, D. M. et al. (1991), Arthritis Rheum 34:1404-1410). Clinical manifestations of SLE include a rash (especially on the face in a "butterfly" distribution), glomerulonephritis, pleurisy, pericarditis and central nervous system involvement. Most patients are women, and are relatively young (average age at diagnosis is 29). [0006] The treatment of SLE depends on the clinical manifestations. Some patients with mild clinical symptoms respond to simple measures such as nonsteroidal anti-inflammatory agents. However, more severe symptoms usually require steroids with potent anti-inflammatory and immunosuppressive action such as prednisone. Other strong immunosuppressive drugs which can be used are azathioprine and cyclophosphamide. The steroids and other immunosuppressive drugs have side effects due to the global reduction of the mammal's immune system. There is presently no ideal treatment for SLE and the disease cannot be cured. [0007] Currently, considerable attention has been focused on the identity of genes which enhance the susceptibility or resistance to SLE, the identification of antigenic determinants that trigger the disease, the molecular mechanisms of T cell activation which results in survival or apoptosis, cytokines which determine T cell function, and the properties of the autoantibody-forming B cells. Many examples of T cell dysregulation in SLE have been described (reviewed in Horwitz, D. A. et al., Dubois' Lupus Erythematosus, 5th Ed. (1997), pp. 83-96 (D. J. Wallace et al. eds., Williams and Wilkins, Baltimore). Although it is well recognized that the primary role of certain lymphocytes is to down-regulate immune responses, progress in elucidating the identity and mechanisms required for generation of these cells has been slow. [0008] Interleukin-2 (IL-2) has previously been considered to have an important role in the generation of antigen non-specific T suppressor cells. Anti-IL-2 antibodies given to mice coincident with the induction of graft-versus-host-disease resulted in several features of SLE (Via, C. S. et al. (1993), International Immunol. 5:565-572). Whether IL-2 directly or indirectly is important in the generation of suppression has been controversial (Fast, L. D. (1992), J. Immunol. 149:1510-1515; Hirohata, S. et al. (1989), J. Immunol. 142:3104-3112; Baylor, C. E. (1992), Advances Exp. Med. Biol. 319:125-135). Recently, IL-2 has been shown to induce CD8+ cells to suppress HIV replication in CD4+ T cells by a non-lytic mechanism. This effect is cytokine mediated, but the specific cytokine has not been identified (Kinter, A. L. et al. Proc. Natl. Acad. Sci. USA 92:10985-10989; Barker, T. D. et al. (1996), J. Immunol. 156:4478-4483). T cell production of IL-2 is decreased in SLE (Horwitz, D. A. et al. (1997), Dubois' Lupus Erythematosus, 5th Ed. (1997), pp. 83-96, D. J. Wallace et al. eds., Williams and Wilkins, Baltimore). [0009] CD8+ T cells from subjects with SLE sustain rather than suppress polyclonal IgG production (Linker-Israeli, M. et al. (1990), Arthritis Rheum. 33:1216-1225). CD8+ T cells from healthy donors can be stimulated to enhance antibody production (Takahashi, T. et al. (1991), Clin. Immunol. Immunopath. 58:352-365). However, neither IL-2 nor CD4+ T cells, by themselves, were found to induce CD8+ T cells to develop strong suppressive activity. When NK cells were included in the cultures, strong suppressive activity appeared (Gray, J. D. et al. (1994) J. Exp. Med. 180:1937-1942). It is believed that the contribution of NK cells in the culture was to produce transforming growth factor beta (TGF-.beta.) in its active form. It was then discovered that non-immunosuppressive (2-10 pg/ml) concentrations of this cytokine served as a co-factor for the generation of strong suppressive effects on IgG and IgM production (Gray, J. D. et al. (1994) J. Exp. Med. 180:1937-1942). In addition, it is believed that NK cells are the principal source of TGF-.beta. in unstimulated lymphocytes (Gray, J. D. et al. (1998), J. Immunol. 160:2248-2254). TGF-.beta.s are a multifunctional family of cytokines important in tissue repair, inflammation and immunoregulation (Massague, J. (1980), Ann. Rev. Cell Biol. 6:597). TGF-.beta. is unlike most other cytokines in that the protein released is biologically inactive and unable to bind to specific receptors (Sporn, M. B. et al. (1987) J. Cell Biol. 105:1039-1045). The latent complex is cleaved extracelluarly to release active cytokine as discussed below. The response to TGF-.beta. requires the interaction of two surface receptors (TGF-.beta.-R1) and TGF-.beta.-R2) which are ubiquitously found on mononuclear cells (Massague, J. (1992), Cell 69:1067-1070). Thus, the conversion of latent to active TGF-.beta. is the critical step which determines the biological effects of this cytokine. [0010] It was found that SLE patients have decreased production of TGF-.beta.1 by NK cells. Defects in constitutive TGF-.beta. produced by NK cells, as well induced TGF-.beta. were documented in a study of 38 SLE patients (Ohtsuka, K. et al. (1998), J. Immunol. 160:2539-2545). Neither addition of recombinant IL-2 or TNF-alpha, or antagonism of IL-10 normalized the TGF-.beta. defect in SLE. Decreased production of TGF-.beta. in SLE did not correlate with activity of disease and, therefore, may be a primary defect. [0011] Systemic administration of TGF-.beta., IL-2, or a combination of both can lead to serious side effects. These cytokines have numerous effects on different body tissues and are not very safe to deliver to a patient systemically. It is, therefore, an object of the invention to provide methods and kits for treating mammalian cells that are responsible for controlling the regulation of autoantibodies to increase the population of cells that down regulate auto-antibody production. SUMMARY OF THE INVENTION [0012] In accordance with the objects outlined herein, the present invention provides methods for inhibiting immune responses in a sample of ex vivo peripheral blood mononuclear cells (PBMCs) comprising adding an regulatory composition to the cell population. [0013] In an additional aspect, the present invention provides methods for treating an autoimmune disorder in a patient. The methods comprise removing peripheral blood mononuclear cells (PBMC) from the patient and treating the cells with an regulatory composition for a time sufficient to suppress inflamation and tissue injury. In particular, the methods of the present invention suppress antibody production or induce cells to down regulate antibody production and enhance cell mediated immune responses in patients with antibody mediated autoimmune diseases. The treated cells are then reintroduced to the patient, with a resulting amelioration of the autoimmune symptoms. The regulatory composition preferably comprises TGF-.beta. and agents which enable T cells to respond to TGF-.beta.. [0014] In an additional aspect, the present invention provides methods for treating cell-mediated autoimmune diseases. The methods comprise removing peripheral blood mononuclear cells (PBMC) from the patient and treating the cells with an regulatory composition for a time sufficient to suppress tissue injury by immune cells. The treated cells are then reintroduced to the patient, with a resulting amelioration of the autoimmune symptoms. The regulatory composition preferably comprises TGF-.beta. and agents which enable T cells to respond to TGF-.beta.. [0015] In an additional aspect, the invention provides kits for the treatment of an autoimmune disorder in a patient. The kits comprise a cell treatment container adapted to receive cells from a patient with an antibody-mediated autoimmune disorder or a cell-mediated disorder and at least one dose of an regulatory composition. BRIEF DESCRIPTION OF THE DRAWINGS [0016] FIG. 1 shows that incubation of SLE patients PBMC with IL-2 and TGF-.beta. decreases spontaneous immunoglobulin production. PBMC (2.times.10.sup.5/well) were cultured in AIM-V serum free medium with or without IL-2 (10 U/ml) and TGF-.beta. (10 pg/ml). After 3 days, the wells were washed three times and fresh AIM-V medium added. Supernatants were collected from the wells after a further 7 days and IgG content determined by an ELISA. [0017] FIG. 2 shows that both IL-2 and TGF-.beta. significantly decrease spontaneous IgG production. The values represent the mean .+-.SEM of IgG (pg/ml) produced by the 12 SLE patients PBMC cultured as described in legend to FIG. 1 except some cells were also incubated with IL-2 (10 U/ml) or TGF-.beta. (10 pg/ml) only. [0018] FIGS. 3A and 3B show that anti-TGF-.beta. can reverse the effects of IL-2. SLE patients PBMC was cultured for three days in the presence (solid bars) or absence (spotted bars) of IL-2 (10 U/ml). Included in these cultures was medium, anti-TGF-.beta. (10 pg/ml) or control mouse IgG1 (10 .mu.g/ml). After 3 days the wells were washed and fresh AIM-V medium added. Supernatants were collected after a further seven days and assayed for IgG (FIG. 3A) or anti-nucleoprotein (NP) (FIG. 3B) content by an ELISA. [0019] FIGS. 4A, 4B and 4C depict regulatory effects of CD8+ T cells on antibody production. (A) Synergism between NK cells and CD8+ cells in the suppression of IgG production in a healthy subject. CD4+ cells and B cells were stimulated with anti-CD2 and the effects of CD8+ cells and NK cells were examined. The combination of NK and CD8+ cells markedly inhibited anti-CD2 induced IgG production we previously reported (Gray, J. D. et al. (1998), J Immunol 160:2248-2254; Gray, J. D. et al. (1994), J Exp Med 180:1937-1942). (B) NK cells and CD8+ cells enhance IgG synthesis in SLE. CD4+ cells from a patient with active SLE and resting B cells from a healthy subject were stimulated with anti-CD2. Enhancement of IgG production by SLE CD8+ cells was markedly increased by the addition of NK cells. (C) Cytokine normalization of CD8+ T cell function in SLE. In parallel with the study shown in FIG. 4B, CD4+ T cells from this patient were stimulated with anti-CD2 in the presence or absence of CD8+ T cells. IL-2 (10 U/ml) and/or TGF-.beta. (2 pg/ml) was added where indicated. These cytokines abolished the helper effects of these CD8+ cells and enabled them to inhibit IgG production by 75%. Continue reading... Full patent description for Use of cytokines and mitogens to inhibit pathological immune responses Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Use of cytokines and mitogens to inhibit pathological immune responses patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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