MX2007004094A - Enhancement of b cell proliferation by il-15 - Google Patents

Abstract

Compositions and methods for modulating the growth, proliferation, and/or differentiation of B-cells in the germinal center are disclosed, and include use of IL-15 inhibitors, antagonists, and agonists. The compositions and methods find use in treating B-cell-related disorders, including neoplasms of the B-cell lineage.

Description

INCREMENT OF PROLIFERATION OF CELL B BY IL-15 FIELD OF THE INVENTION The present invention is in the field of modulation related to IL-15 of growth and / or proliferation of B cell.

BACKGROUND OF THE INVENTION The B cells activated by antigen proliferate and differentiate in the germinal center ("GC"). B cells provide protection through the production of antibodies with optimal affinity against invading micro-organisms (MacLennan, ICM 1994. Annu., Rev. Immunology 12: 117, Liu, Y.-J., et al., 1997. Immunology Rev 156: 111; Manser, T. 2004. J Immunology 172: 3369). However, B cells are also involved in numerous neoplastic conditions characterized by uncontrolled growth and multiplication of B-cell precursors. The GC provides a specialized micro-environment. The factors that control the vigorous proliferation of GC B cells in this micro-environment are crucial for the expansion of a few initial cXLons as well as somatic hypermutation, a process by which a sufficient mixture of diverse B-cell receptors is obtained ( At the same time, to ensure that immune responses are not directed toward self-antigens, the factors that control the selection process within the CG are also critical (Lindhout, E., et al., 1997). Immunology Today 18: 573; Pulendran, B., et al., 1997. Immunology Today 18:27; Choe, J., et al., 1996. J. Immunology 157: 1006.) The signals received through a BCR that is known to be important for these GC reactions (Liu, Y.-J., et al., 1989. Nature 342: 929, Kelsoe, G. 1996. Immunity 4: 107, Haberman, A.M., et al. 2003. Nat Rev Immunology 3: 757; Hande, S., et al., 1998. Immunity 8: 189). coming from the micro-environment of the GC, they are not understood so clearly. An important producer of micro-environmental factors of GC is the follicular dendritic cell (FDC), which is present in the lymphoid follicles and belongs to the stromal cells of these organs (Haberman, A. M., Et al., 2003. Nat Rev Immunology 3: 757; Li, L., et al., 2002. Semin Immunology 14: 259; van Nierop, K., et al., 2002. Semin Immunology 14: 251; Lindhout, E., et al., 1995. Histochem J 27: 167; Tew, JG, et al., 1964. Immunology Rev 156: 39). Initially it was known that FDCs retain antigens on their surface for a prolonged time, and present said original antigens to the B cells of the GC (Nossal, GJ et al., 1964. Aust. J. Exp. Biol. 42: 311; Vilbois, MH, et al., 1995. Current Topics of Microbiology in Immunology 201: 69). FDCs are essential for GC B cells to survive and proliferate in vitro after stimulation with cytokines such as IL-2, IL-4 and IL-10 (Choe, J., et al., 1996. J. Immunology 157 : 1006; Zhang, X., et al., 2001. J. Immunology 167: 49). Despite research on FDCs that have focused on their extraordinary capabilities to support the survival and proliferation of GC cell B through both direct cell-cell contact and secreted soluble factors (Tew, JG, et al., 1990). Immunology Rev. 117: 185; Grouard, G., et al., 1995. Journal of Immunology 155: 3345; Kim, H.-S., et al., 1995. J. Immunology 155: 1101; Kosco-Vilbois, MH. 2003. Nat Rev Immunology 3: 764), it has not been shown that the factors identified to date completely replace the effect of FDC (Lindhout, E., et al., 1995. Histochem J 27: 167; Kim, H.-S ., et al., 1995. J. Immunology 155: 1101; van Eijk, M., et al., 1999. J Immunology 163: 2478). Therefore, there is a need in the art with respect to novel compositions and methods for modulating GC cell B survival and proliferation to treat B cell-related conditions including neoplasms derived from B cell, autoimmune disease, and cell deficiencies. B.

SUMMARY OF THE INVENTION The invention is directed to IL-15 antagonists and to a method for using the antagonists for the treatment of human B cell-related disease., said treatment includes inhibiting the proliferation of neoplastic cells of the B-cell lineage. IL-15 antagonists are effective in preventing IL-15 from transducing a signal to a cell through any of the β or β subunits. of the IL-15 receptor complex, thereby antagonizing the biological activity of IL-15 towards B cells in the germinal centers. The invention encompasses monoclonal antibodies that react immunologically with native IL-15 and prevent signal transduction to the IL-15 receptor complex. The invention also encompasses humanized antibodies and human antibodies that can inhibit or prevent the binding of IL-15 to the β or β subunit. of the IL-15 receptor complex. The invention also encompasses antagonists that block IL-15Ra, including antibodies to this receptor subunit.

Antagonists according to the invention include soluble IL-15, and mature, or original, IL-15 muteins, in which IL-15 has undergone mutagenesis at one or more residues or amino acid regions that play a role in binding to the ß subunit? of the IL-15 receptor complex. Said muteins prevent IL-15 from transducing a signal to the cells through any of the β or β subunits. of the IL-15 receptor complex, while maintaining the high affinity of IL-15 towards IL-15R. Typically, said muteins are created by additions, deletions or substitutions at key positions, for example, Asp56 or Gln156 of simian and human IL-15 as shown in SEQ ID NOS: 1 and 2, respectively. It is believed that Asp56 affects the union with the β subunit and that Gln156 affects the union with the subunit? of the IL-15 receptor complex. Also included in the field of the invention are modified IL-15 molecules that retain the ability to bind to IL-15Rα, but that have substantially decreased affinity or have no affinity to the β and / or β subunits. of the IL-15 receptor complex. The modified IL-15 molecules can take any form as long as the modifications are made in such a way that they interfere with, or prevent binding, normally by modification at or near the target binding site. Examples of such modified IL-15 molecules include natural IL-15 or an IL-15 mutein that is covalently conjugated to one or more chemical groups that spherically interfere with the IL-15 / IL-15 receptor binding. For example, natural IL-15 may contain site-specific glycosylation or may be covalently attached to groups such as polyethylene glycol (PEG), monomethoxy-PEG (mPEG), dextran, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), poly -amino acids such as poly-L-lysine or poly-histidine, albumin, gelatin at specific sites in the IL-15 molecule that can interfere with the binding of IL-15 to the β or β chains? of the IL-15 receptor complex, while maintaining the high affinity of IL-15 towards IL-15Ra. Taking advantage of the spherical hindrance properties of the group, binding to specific receptor subunits can be antagonized. Other advantages of conjugating PEG chains to proteins such as IL-2, GM-CSF, asparagine, immunoglobulins, hemoglobin, and others are known in the art. For example, it is known that PEG prolongs circulating half-lives in vivo (see, Delgado, et al., Crit. Rev. Ther, Drug Carr. Syst., 9: 249 (1992)), increases solubility ( see, Katre, et al., Proc. Nati, Acad. Sci, 84: 1487 (1987)) and reduces immunogenicity (see, Katre, NV, Immunology 144: 209 (1990)). The invention is also directed to the use of the antagonists in a method for treating a disease or condition in which a reduction in the activity of IL-15 on B cells is desired. Such diseases include leukemias and B-cell lymphomas. , it is an object of the present invention to provide a method for treating malignant B cell tumors using anti-IL-15 antibodies. It is a further object of this invention to provide multimodal methods for the treatment of malignant B-cell tumors in which doses of anti-IL-15 antibodies are supplemented by the administration of a therapeutic protein, such as an immuno-conjugate or fusion protein. to antibody, or by a chemotherapeutic regimen. These and other objects are achieved, in accordance with one embodiment of the present invention, by the provision of a method for treating a malignant B-cell tumor, comprising the step of administering to an individual having a malignant B-cell tumor a anti-IL-15 antibody and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. IL-15 is expressed in CDF of human tonsils, but not in B cells. Preparations are stained by cyto-centrifugation of FDC groups of human tonsils with goat polyclonal anti-IL-15 antibody (FIG. A and B: green), mouse anti-IL-15 monoclonal antibody (D: green), corresponding control antibodies (C and D-box: green). The preparations are co-stained with FDC-specific DRC-1 monoclonal antibody for FDC (A and C: red), anti-CD20 monoclonal antibody for B cells (B: red) and DAPI for core (D: blue). Original increase x400. Figure 2. FDC / HK cells express IL-15 on its surface bound to IL-15Ro1. (A) Surface expression of IL-15 by FACS. Surface FACS staining with specific monoclonal or control antibody is amplified with Flow-Amp equipment (thick line and dotted line, respectively). Competition experiments are performed to confirm the specificity by incubating specific monoclonal antibody with IL-15 (300 ng) for 30 minutes on ice before staining the cells (thin line). (B) Change of surface IL-15 after acid removal. FDC / HK cells are incubated in cold glycine buffer (pH 3.0) for 10 minutes on ice and then stained with specific antibody or isotype control antibody. (acid treatment: thick line; without treatment: thin line; isotype control: dotted line). (C) Expression of IL-15Ra mRNA in FDC / HK cells. RT-PCR is performed for IL-15Ra and IL-2Ra (an internal control) with the same amount of FDC / HK cell mRNA under the same conditions. Figure 3. IL-15 bound to membrane on the surface of FDC / HK is biologically active. Different numbers of FDC / HK cells (double dilution from 2 x 104 to no cells / well) are cultured in 96-well plates for 1 day and fixed with 1% paraformaldehyde. CTLL-2 cells (5 x 10 3 cells / well) are cultured for 1 day in 96-well plates coated with FDC / HK cells in triplicate in RPMI medium containing 10% FCS, 1 U / ml IL-2 and 2 -ME. The cells are pulsed with 0.5 μCi of [3 H] TdR (20 Ci / mM) during the last 4 hours. The incorporation of [3 H] TdR is measured using a liquid scintillation counter. The results are expressed as average cpm ± SEM of cultures in triplicate. (A) Proliferation of CTLL-2 cells in various numbers of FDC / HK cells added to the fixed number of CTLL-2 cells (None: control of RPMI medium with 10% FCS without FDC / HK cell coating; spn: supernatant cultivation of FDC / HK). (B) Inhibition of increased proliferation of CTLL-2 cells by specific anti-IL-15 monoclonal antibody (10 μg / ml). The dotted line represents the cpm value of cultured CTLL-2 cells without FDC / HK cells or antibody. These results are reproduced in two independent experiments. Figure 4. Expression in B cell of the GC of the IL-15 and IL-2 receptors. (A) RT-PCR is performed with the mRNA molecules from freshly isolated or cultured GC B cells as described in the Materials and Methods section. ((+) control: plasmid containing the respective genes; GCB dO: freshly isolated GC B cells; GC-B d4: GC B cells are cultured for 4 days; D: distilled water which serves as a negative control). (B) FACS profiles of the IL-15 binding test. Freshly isolated GC and FDC / HK B cells are incubated with a saturating dose of IL-15 (100 ng) for 30 minutes on ice, and then stained with anti-IL-15 mAb. Figure 5. IL-15 in FDC / HK cells increases GC B cell recovery when cultured with FDC / HK cells and cytokines. (A) The recovery of viable cells corresponding to the aggregate amount of anti-IL-15 mAb is reduced. GC B cells (2 x 10 5 cells / well) are grown in 24-well plates with FDC / HK cells (2 x 10 4 cells / well, 5,000 Rad), CD40L (100 ng / ml), IL-2 (30 U / ml) and IL-4 (50 U / ml) with the indicated amount of specific mAb for 10 days. The cells are harvested on day 10 and counted by trypan blue exclusion. (B) The numbers of viable cells increase proportionally with the added amount of IL-15. The indicated amount of IL-15 is added to the GC B-cell cultures. IL-2 is not included in this experiment. Representative results are presented from four separate experiments. Figure 6. IL-15 increases the proliferation of GC cell B in vitro. GC B cells are labeled with CFSE (5 μl / ml) and then cultured for 6 days with IL-15 (100 ng / ml), anti-IL-15 (10 μg / ml) or control mAb in the presence of combinations of FDC / HK cells and cytokine. The collected cells are counted and subjected to FACS analysis to measure the intensity of CFSE. The results are analyzed with the ModFit software. (A) Comparison of viable cell numbers. (B) Comparison of CFSE profiles of cells recovered in percent in each division. (D: division). Figure 7. Levels of IL-15 on the surface of FDC / HK are increased by B cells of GC or TNFa. FDC / HK cells are incubated for 3 days in IMDM medium with 10% FCS with various induction conditions as follows: Medium only (Medium), IL-2, IL-4 and CD40L (24L); IL-2, IL-4 and CD40L with GC B cells (24L + GC-B); TNF-a (10 ng / ml). The harvested cells are stained for FACS analysis. The numbers in parentheses represent MFI of each sample, which is calculated by subtracting the value of the control from the specific mAb (dotted line and solid line, respectively).

DETAILED DESCRIPTION OF THE INVENTION Introduction In accordance with the invention, IL-15 is produced by follicular dendritic cells (FDCs) and is presented on the surface of FDC / HK cells, captured by IL-15Ra and trans-presented to B cells of GC. The function of IL-15 in B cells of GC and FDCs is studied using an in vitro experimental model that mimics the GC reaction in vivo. GC B cells do not express IL-15Ra but express the signal transduction complex IL-2/15 Rβ and Ry. IL-15 presented in the membrane of FDC / HK cells is biologically active and co-stimulates the proliferation of GC B cells after stimulation with CD40L. By identifying this mechanism, the invention provides new means to modulate the normal and aberrant proliferation of GC B cells.

Stimulation with GC B-cell IL-15 The discovery of GC B-cell stimulation mechanism by IL-15 indicates that B-cell tumors of GC origin are particularly susceptible to treatment using a mediated B-cell stimulation inhibitor by IL-15. Such inhibitors are discussed in greater detail in the present invention. Examples of such tumors include acute precursor B-cell lymphoblastic leukemia ("ALL") and lymphoma. The data presented in the present invention are important because it has been difficult to clarify the role of B cells in some disease states. For example, the previous study of the function of IL-15 in B cells has been hampered because in genetically modified mice, either by elimination of IL-15 or forced expression model reveals no obvious differences in cell responses B compared to wild-type mice (Kennedy, MK, et al 2000. J Exp Med 191: 771; Lodolce, JP, et al., 1998. Immunity 9: 669; Marks-Konczalik, J., et al., 2000 Proc Nati Acad Sci USA 97: 11445). IL-15 increases the proliferation and secretion of Ig from peripheral B cells of human (Armitage, RJ, et al., 1995. J Immunology 154: 483, Bernasconi, NL, et al., 2002. Science 298: 2199. Litinskiy, MB , et al., 2002. Nat Immunology 3: 822), inhibits anti-IgM-induced apoptosis (Bulfone-Paus, S., et al., 1997. Wat Med 3: 1124), and induces proliferation of malignant B cells (Tinhofer , I., et al., 2000. Blood 95: 610. Trentin, L., et al., 1997. Leuk Lymphoma 27:35). However, the biological function of IL-15 in the GC reaction has not been demonstrated. In order to clarify the role of IL-15 and thus develop compositions and methods to modulate this effect of IL-15, several studies are described in the present invention. These studies reveal for the first time that follicular dendritic cells produce IL-15, and that IL-15 occurs on the surface of follicular dendritic cells. In this form of cell surface presentation, IL-15 increases the proliferation of B lymphocytes by cell contact. In contrast, the soluble form of IL-15 does not have a detectable effect on target B lymphocytes. These discoveries are achieved through a series of experiments described below and in more detail in the examples. First, the cellular source of IL-15 within the GC is examined. Although it has been reported that IL-15 mRNA and small amounts of soluble IL-15 are produced by FDC cultured in vitro (Husson, H., et al., 2000. Cell Immunology 203: 134), it has not been previously shown the production of IL-15 by CDF at the protein level. IL-15 mRNA is expressed almost ubiquitously, and protein production and secretion are mainly controlled by complex and inefficient post-translational mechanisms (Aldmann, TA, et al., 1999. Annu Rev Immunology 17:19. , TA, et al., 2001. Blood 97: 14.33, 34). The data described in the present invention reveal that FDCs produce IL-15 as demonstrated by immuno-fluorescent ("IF") staining of freshly isolated FDC groups. This in vivo observation is confirmed by the data in the present invention which show that a FDC cell line, FDC / HK cells, produces IL-15. The IL-15 protein is detected on the surface of FDC / HK cells. The specificity of membrane bound IL-15 is confirmed by competition FACS analysis and by the blocking experiment of the biological test of CTLL-2. However, IL-15 is not detected by ELISA in the supernatant of the FDC / HK culture and this is subsequently confirmed with the CTLL-2 test. Without being limited to a mechanism by which surface expression of IL-15 is achieved, complete loss of IL-15 staining after acid treatment, and increased binding after incubation with exogenous IL-15, strongly suggests a mechanism anchored to receptor instead of the presence of an alternative membrane form of IL-15. Although the possibility that failure to detect IL-15 after treatment resulting from denaturation of the transmembrane form can not be completely eliminated, the expression of IL-15Ra-specific mRNA in FDC / HK cells also supports this mechanism. In the present invention, the biological relevance of IL-15 signaling in the GC is demonstrated, measuring the effect of IL-15 on the proliferation of GC B cell by the removal or addition of IL-15. As shown in FIG. Figure 5, GC B cell growth is significantly reduced in the presence of anti-IL-15 blocking mAb and is increased when IL-15 is added. The recovery of GC B cells in the culture containing a saturating dose of IL-15 (100 ng / ml) is four times higher than that in the culture in which the endogenous IL-15 activity is depleted by the blocking mAb . IL-15 is present in FDC in GC in vivo and endogenous IL-15 from FDC / HK cells supports GC B cell proliferation in vitro at levels comparable with, or greater than, exogenous IL-2 alone when removed Endogenous IL-15 by the Ab blocker (4.2 x 105 in the first bar on the left of Figure 5A vs. 2.9 x 105 in the final bar on the right in Figure 5B). Likewise, GC B cells proliferate in the presence of IL-15, dividing faster than cultured cells 1 without IL-15. Together, these results indicate that IL-15 signaling may be one of the mechanisms responsible for the rapid proliferation of centroblasts in the GC in vivo. Because the presentation of IL-15 by FDC can be an important trigger in the initiation of lymphomagenesis, immune modulation can be achieved by targeting the activity of IL-15 on the proliferation of GC B cell. This mechanism also indicates that the inhibition of IL-15 signaling in germinal centers provides adequate treatment for B-cell lymphomas.

Conditions that can be treated by modulation of stimulation with B-cell IL-15 B cells stimulated in the germinal centers can take a variety of developmental pathways, some of which are normal, and some others are pathological. The route selected for modulation by the methods of the invention, and the related medical condition, are those that determine whether or not to use an antagonist, or a stimulator or agonist of IL-15. The conditions and disorders suitable for modulation in accordance with the methods described in the present invention are listed below, and discussed in more detail below: B-cell lymphomas; leukemias of origin in B cell; antibody immunodeficiency disorders; combined disorders of antibody-mediated immunodeficiency (B cell) and cell-mediated immunodeficiency (T cells); and auto-immune disease. In addition to these disorders, the invention also provides for the treatment of any other disorder in which the modulation of B-cell stimulation through IL-15 at the germinal center plays a role.

B-cell lymphomas Lymphomas that are suitable for treatment by inhibition of IL-15-mediated proliferation of GC B cells include non-Hodgkin's lymphoma, which is derived from B cells of the germinal center with non-productive transpositions of the immunoglobulin gene; B-cell lymphomas (the most common non-Hodgkin lymphomas in the United States of America); Hodgkin's lymphoma; small lymphocytic lymphoma (SLL / CLL); mantle cell lymphoma (MCL); follicular lymphoma; marginal cell lymphoma, which includes extranodal lymphoma, or MALT; nodal lymphoma, or monocytoid B cell; spleen lymphoma; diffuse large cell lymphoma; Burkitt's lymphoma; high-grade Burkitt-type lymphoma; and lymphoblastic lymphoma. Also included is diffuse large cell lymphoma, which may exist as one of at least six morphological variants (centroblastic, immunoblastic, cell-histocyte-rich I, lymphomatoid granulomatosis, anaplastic, and plasma blastic), and one of at least three subtypes (mediastinum, or thymus, primary effusion lymphoma, and intravascular (previously known as malignant angioendotheliomatosis) Hodgkin's lymphoma (Hodgkin's disease) itself is classified into several categories according to the system of WHO classification: nodular lymphocyte-predominant Hodgkin lymphomas, and classic Hodgkin's lymphomas, including nodular sclerosis Hodgkin's lymphoma, lymphocyte-rich Hodgkin lymphoma, mixed-cell Hodgkin lymphoma, and depleted Hodgkin's lymphoma lymphocyte B Cell Proliferative Disorders B cell proliferative disorders suitable for treatment described in the present invention include post-transplant lymphoproliferative disorders (PTLD's). Early lesions of this disorder include plasmacytic hyperplasia, atypical lymphoid hyperplasia, and infectious mononucleosis-type PTLD. Other categories include polymorphic PTLD and monomorphic PTLD. Although these conditions often return spontaneously or with reduced immunosuppression after transplantation, they can be fatal.

Antibody immunodeficiency disorders (cell B) Antibody disorders associated with deficient B-cell differentiation and proliferation can be treated by increasing GC B cell proliferation induced by IL-15. These disorders include: X-linked hypogammaglobulinemia (congenital hypogammaglobulinemia); transient hypogammaglobulinemia of childhood; common, variable, non-classifiable immunodeficiency (acquired hypogammaglobulinemia); immunodeficiency with hyper-IgM; Neutropenia with hypogammaglobulinemia; lack of response to polysaccharide-type antigen; selective IgA deficiency; selective IgM deficiency; selective deficiency of IgG subclasses; secondary B cell immunodeficiency associated with drug, protein loss conditions; and lymphoproliferative disease linked to X. 1 Combined disorders of antibody-mediated immunodeficiency (B cell) and cell-mediated immunodeficiency (T cell) The increase of the B-cell component of these diseases can be achieved as discussed above for B-cell immunodeficiency disorders. Such diseases include: of severe combined immunodeficiency (autosomal recessive, X-linked, sporadic); cellular immunodeficiency with abnormal synthesis of immunoglobulin (Nezelof's syndrome); immunodeficiency with ataxia-telangiectasia; immunodeficiency with eczema and thrombocytopenia (Wiskott-Aldrich syndrome); immunodeficiency with thymoma; immunodeficiency with dwarfism of short extremities; immunodeficiency with adenosine deaminase deficiency; immunodeficiency with nucleoside phosphorylase deficiency; biotin-dependent multiple carboxylase deficiency; graft versus host disease (GVH); and acquired immunodeficiency syndrome (AIDS).

Auto-immune disorders B cells produce immunoglobulins, and they play a critical role in antibody-mediated self-immunity. B-cell deficient mice, which are produced by administration of anti-μ antibodies starting from birth, are resistant to some autoimmune diseases, including experimental autoimmune encephalitis, and insulin-dependent spontaneous diabetes. (Looney, Ann, Rheum, Dis. 61: 863). Mice genetically deficient in B cells may also have a lower tendency to develop autoimmune disease. For example, in B cell deficient mice, autoantibodies are absent, and the increase in T cells in lymphoid organs is avoided, as described by Chan et al., J. Immunol. 160: 51-59 (1998). Depletion of B cells using anti-CD-20 antibodies may be of therapeutic benefit in the treatment of autoimmune diseases such as autoimmune cytopenias. In addition to reducing the potentially pathogenic antibodies, the reduction in B cells can modulate T cell activity, further reducing the immune response towards auto-antigens. (Gorozny et al., Arthritis Res. Ther.5: 131-135, 2003). There is substantial evidence of a critical function for B cells in the induction and progression of autoimmune disease. Therefore, the methods of the invention find use in the treatment of autoimmune disease by inhibiting the development of B cell, and therefore reduce or completely avoid the levels of pathological autoantigen in the patient. The autoimmune diseases that can be treated with such treatment include diseases of the nervous system such as multiple sclerosis, myasthenia gravis, autoimmune neuropathies including Guillain-Barre, and auto-immune uveitis. Diseases of the gastrointestinal system include Crohn's disease, ulcerative colitis, primary biliary cirrhosis, and autoimmune hepatitis. Diseases that affect the blood include autoimmune hemolytic anemia, pernicious anemia, and auto-immune thrombocytopenia; Diseases that affect blood vessels include temporal arteritis, anti-phospholipid syndrome, vasculitis including egener's granulomatosis, and Bechet's disease. Diseases of the endocrine glands include diabetes mellitus type I or mediated by the immune system, Grave's disease, Hashimoto's thyroiditis, oophoritis and auto-immune orchitis, and auto-immune disease of the adrenal gland. Skin diseases include psoriasis, dermatitis herpetiformis, pemphigus vulgaris, and vitiligo. Finally, diseases affecting multiple organs, also called connective tissue diseases, include rheumatoid arthritis, systemic lupus erythematosus, scleroderma, polymyositis and dermatomyositis, spondyloarthropathies including ankylosing spondylitis, and Sjogren's syndrome.

B cell leukemias Acute lymphocytic leukemia (ALL) can also be treated with inhibitors of B-cell IL-15 stimulation. ALL is a malignant cellular disorder caused by. clonal proliferation of lymphoid precursor cells with suspended maturation. ALL can originate in cells of lineage B or T, causing B cell leukemias, T cell leukemia, and mixed cell lineage leukemias. Both B-cell leukemia and mixed cell line leukemia are suitable for treatment using the methods of the present invention. In adults, ALL constitutes approximately 20% of the leukemias (Brincker, H., Scand, J. Maematol, 29: 241-249, 1982), and approximately 1-2% of all cancers (Boring, CC et al. al., Cancer J. Clin 44: 7-16, 1994). ALL classifications related to B cell include early ALL of pre-cell B; ALL of pre-cell B; ALL of transition pre-cell B; and mature B cell ALL. B cell mature ALL represents a leukemic phase of Burkitt's lymphoma (Magrath, LT et al., Leukemia Res. 4: 33-59, 1979).

IL-15 Antagonists The IL-15 antagonists of the invention that can modulate the effects of IL-15 at the germinal center include (a) soluble IL-15, in which soluble IL-15 is expected to block the binding of IL-15 bound to IL-15-R to the β or β subunits? of the B-cell IL-15 receptor of the germinal center; (b) a mature, or natural, IL-15 mutein that can bind to the IL-15 receptor subunit and that is unable to transduce a signal through the β and / or subunits. of the IL-15 receptor complex; (c) a monoclonal antibody against IL-15 that prevents IL-15 from effecting signal transduction through the β and / or subunits. of the IL-15 receptor complex; Y (d) an IL-15 molecule that is covalently linked to a chemical group that interferes with the ability of IL-15 to effect signal transduction through any of the β or β subunits? of the IL-15 receptor complex, but that does not interfere with the binding of IL-15 to IL-15Ra. The polynucleotides encoding the muteins described above are also included in the field of the present invention. "IL-15 mutein" or "IL-15 muteins" refer to the mature, or original, simian IL-15 molecules having the amino acid sequence 49-162 of SEQ ID NO: the IL-15 molecules of human having amino acid sequence 49-162 of SEQ ID NO: 2, which have been mutated according to the invention to produce an IL-15 antagonist. Such IL-15 muteins can bind to the IL-15 Ro subunit, and can not transduce a signal through the β or β subunits. of the IL-15 receptor complex. Simian or human L-15 can be obtained in accordance with the procedures described by Grabstein et al., Science, 264: 965 (1994), which has been incorporated in the present invention for reference, or by conventional methods such as polymerase chain reaction (PCR). There are many possible mutations of IL-15 that can produce antagonists. Such mutations can be made at specific amino acid sites that are believed to be responsible for β or β subunit signaling; or the mutations can be effected through entire regions of IL-15 that are considered necessary for the β or β subunit signaling. Typically, the mutations can be effected as additions, substitutions or deletions of amino acid residues. Preferably, the muteins are preferred by substitution and deletion, with muteins being most preferred by substitution. It is believed that Asp56 affects the union with the β subunit and that Gln156 affects the union with the subunit? of the IL-15 receptor complex. Adding or substituting other amino acid residues present in nature near or at the Asp56 and Gln156 sites may affect the binding of IL-15 to either or both β or β subunits. of the IL-15 receptor complex. For example, removing the negatively charged aspartic acid residue and replacing it with another negatively charged residue might not be as effective in blocking the receptor binding as if the aspartic acid were replaced with a positively charged amino acid such as arginine, or uncharged waste. such as serine or cysteine. Recombinant production of an IL-15 mutein requires first the isolation of a DNA clone (i.e., cDNA) that codes for an IL-15 mutein. The cDNA clones are obtained from primary cells or cell lines expressing mammalian IL-15 polypeptides. First, the whole cell mRNA is isolated, then a cDNA library is made from the mRNA by reverse transcription. A cDNA clone can be isolated and identified using the DNA sequence information provided in the present invention to design a hybridization probe or cross-type PCR primer as described above. Such cDNA clones have the sequence of SEQ ID NO: 1 and SEQ ID NO: 2. The recombinant production of muteins of IL-15 is described in the patent E.U.A. No. No. 6, 177.079, incorporated in the present invention for reference. Equivalent DNA constructs that code for various additions or substitutions of amino acid residues or sequences, or deletions of residues or terminal or internal sequences not necessary for activity, are useful for the methods of the invention. For ele, N-glycosylation sites in IL-15 can be modified to exclude glycosylation, allowing the expression of a reduced carbohydrate analog in mammalian and yeast expression systems. The N-glycosylation sites in eukaryotic polypeptides are characterized by an amino acid triplet Asn-X-Y, in which X is any amino acid except Pro and Y is Ser or Thr. The IL-15 simian protein comprises two of said triplets, at amino acids 127-129 and 160-162 of SEQ ID NO: l. The human IL-15 protein comprises three of said triplets, at amino acids 119-121, 127-129 and 160-162 of SEQ ID NO: 2. The appropriate substitutions, additions or deletions to the nucleotide sequence coding for these triplets results in the binding of carbohydrate residues in the side chain of Asn being avoided. The alteration of a single nucleotide, chosen so that Asn is replaced with a different amino acid, for ele, is sufficient to inactivate an N-glycosylation site. Known procedures for inactivating N-glycosylation sites in proteins include those described in the U.S.A. No. 5,071,972 and in EP 276,846, incorporated in the present invention for reference. Recombinant expression vectors include synthetic DNA fragments or cDNA derivatives that encode an IL-15 mutein. The DNA encoding an IL-15 mutein is operably linked to an appropriate transcriptional or translational regulatory or transcriptional nucleotide sequence, such as one that is obtained from mammalian, microbe, viral or other genes. insect. Eles of regulatory sequences include, for ele, a genetic sequence that has a regulatory function in gene expression (e.g., transcription promoters or enhancers), an optional operator sequence to control transcription, a sequence encoding sites of ribosomal binding of appropriate mRNAs, and appropriate sequences that control the initiation and termination of transcription and translation. The nucleotide sequences are operably linked when the regulatory sequence is functionally related to the structural gene. For ele, a DNA sequence for a signal peptide (secretory leader) can be operably linked to a structural gene DNA sequence for an IL-15 mutein if the signal peptide is expressed as part of a precursor sequence of amino acid and participates in the secretion of an IL-15 mutein. In addition, a promoter nucleotide sequence is operably linked to a coding sequence (eg, structural gene DNA) if the promoter nucleotide sequence controls the transcription of the structural gene nucleotide sequence. Even, a ribosome binding site can be operably linked to a structural gene nucleotide coding sequence (eg, IL-15 mutein) if the ribosome binding site is positioned within the vector to promote translation. Host cells suitable for expression of an IL-15 mutein include prokaryotic, yeast or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include Gram-negative or Gram-positive organisms, for ele E. coli or bacilli. Prokaryotic host cells suitable for transformation include, for ele, E. coli, Bacillus subtilis, Salmonella typhimurium, and some other species within the genera Pseudomonas, Streptomyces, and Staphylococcus. Eles of suitable host cells also include yeasts such as S. cerevisiae, a mammalian cell line such as Chinese hamster ovary (CHO) cells, or insect cells. Cell-free translation systems can also be used to produce an IL-15 mutein using RNA molecules obtained from the DNA constructs described in the present invention. Suitable cloning and expression vectors for use with bacterial, insect, yeast, and mammalian cell hosts are described, for example, in Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985. When an IL-15 mutein is expressed in a yeast host cell, the nucleotide sequence (eg, structural gene) that encodes an IL-15 mutein may Include a leader sequence. The leader sequence may allow for enhanced extracellular secretion of the translated polypeptide by a yeast host cell. Methods for preparing and purifying IL-15 muteins are described in US Pat. No. 6,177,079, incorporated in the present invention for reference. Preferably, an IL-15 mutein is used in cases where at least one of the amino acid residues Asp56 or Gln156 of IL-15 (simian IL-15 having the sequence of amino acid residues 49-162 shown) in SEQ ID NO: the human IL-15 having the sequence of amino acid residues 49-162 shown in SEQ ID NO: 2) is deleted or substituted with a different amino acid residue present in nature. Any combination of substitutions and / or deletions can be made. For example, Asp56 can be deleted while Asp56 is substituted with any other amino acid, or both Asp56 and Gln156 are each substituted with the same amino acid residue or different amino acid residue. In addition, Asp56 can be substituted with any amino acid while Gln166 is deleted. Generally speaking, muteins are preferred by substitution, and those that do not severely affect the natural folding of the IL-15 molecule are more preferred. Muteins by preference substitution include those in which Asp56 is substituted with serine or cysteine; or in which Gln156 is substituted with serine or cysteine, or in which both Asp56 and Gln156 are each substituted with a serine or cysteine. Examples of deletion muteins include those in which Asp5 &'and Gln156 of mature IL-15 are both deleted; in which only Asp56 is deleted; or in which only Gln156 is deleted. It is possible that other amino acid residues may be substituted or deleted in the region of either Asp56 and Gln156 and still have an effect of preventing signal transduction through either or both β or β subunits. of the IL-15 receptor complex. Therefore, the invention also utilizes muteins in which amino acid residues within the region of Asp56 and Gln156 can be substituted or deleted, and which possess IL-15 antagonist activity. Said muteins can be made using the methods described in the present invention and can be analyzed for IL-15 antagonist activity using conventional methods. Further description of a method that can be used to create the IL-15 muteins used in the invention is provided in the patent E.U.A. No. 6,177,079. The mature IL-15 polypeptides used in the present invention (mature simian IL-15 comprising amino acid sequence 49-162 of SEQ ID NO: mature human IL-15 having the sequence of amino acid residues 49- 162 shown in SEQ ID NO: 2), as well as IL-15 muteins, can be modified by formation of covalent conjugates or aggregation with other chemical moieties. Said portions may include PEG, mPEG, dextran, PVP, PVA, polyamino acids such as poly-L-lysine or polyhistidine, albumin and gelatin at specific sites in the IL-15 molecule that may interfere with the binding of IL-15 to the ß or chains? of the IL-15 receptor complex, while maintaining the high affinity of IL-15 towards IL-15Ra. Additionally, IL-15 can be specifically glycosylated at sites that can interfere with the binding of IL-15 to the β or β chains. of the IL-15 receptor complex, while maintaining the high affinity of IL-15 towards IL-15Ra. The preferred groups for conjugation are PEG, dextran and PVP. Most preferred for use in the invention is PEG, in which the molecular weight of the PEG is preferably from about 1,000 to about 20,000. A molecular weight of about 5000 is preferred for use in the conjugation of IL-15, although other weight PEG molecules could also be suitable. A variety of PEG forms are suitable for use in the invention. For example, PEG can be used in the form of succinimidyl-succinate-PEG (SS-PEG) which provides an ester-type bond that is susceptible to hydrolytic cleavage in vivo, succinimidyl-carbonate-PEG (SC-PEG) which provides a urethane-type bond and is stable against hydrolytic cleavage in vivo, succinimidyl-propionate-PEG (SPA-PEG) provides an ether-type bond that is stable in vivo, vinylsulfone-PEG (VS-PEG) and maleimide-PEG (Mal-PEG) ) of which all can be obtained commercially from Shearwater Polymers, Inc. (Huntsville, Ala.). In general, SS-PEG, SC-PEG and SPA-PEG react specifically with lysine residues in the polypeptide, while VS-PEG and Mal-PEG each react with free cysteine residues. However, Mal-PEG is prone to react with lysine residues at alkaline pH. Preferably, SC-PEG and VS-PEG are preferred, and SC-PEG is more preferred due to its in vivo stability and specificity towards lysine residues. PEG portions can be linked to IL-15 at strategic sites to take advantage of the large molecular size of PEG. As described in the patent E.U.A. No. 6,177,079, the PEG portions can be bound to IL-15 using lysine or cysteine residues of natural origin in the protein or by substitution with site-specific PEG. A method of substitution with site-specific PEG is through protein design methods in which cysteine or lysine residues are introduced into IL-15 at specific amino acid locations. It is believed that the large molecular size of the PEG chain or chains conjugated to IL-15 blocks the region of IL-15 that binds the β and / or subunits. but not to the a subunit of the IL-15 receptor complex. The conjugations can be carried out by addition reaction in which PEG is added to a basic solution containing IL-15. Typically, the PEG substitution is carried out either at (1) approximately pH 9.0 and at molar ratios of SC-PEG to lysine residue from 1: 1 to approximately 100: 1, or higher, or (2) at pH 7.0 approximately and to ratios molar VS-PEG to cysteine residue from 1: 1 to approximately 100: 1, or greater. Alternatively, an antagonist according to the invention can take the form of a monoclonal antibody against IL-15 that interferes with the binding of IL-15 to any of the β or β subunits. of the IL-15 receptor complex. Within one aspect of the invention, IL-15, including derivatives thereof, as well as portions or fragments of these proteins such as IL-15 peptides, can be used to prepare antibodies that bind specifically to IL-15. Within the context of the invention, it is to be understood that the term "antibodies" includes polyclonal antibodies, monoclonal antibodies, fragments thereof such as F (ab ') 2 and Fab fragments, as well as binding partners that are produced in recombinant form . The affinity of a monoclonal antibody or binding partner can be readily determined by the person skilled in the art (see Scatchard, Ann. N. Y. Acad. Sci, 51: 660-672 (1949)). Specific examples of said monoclonal antibodies include antibodies produced by the clones designated M110, Mili and M112, which are IgGl monoclonal antibodies. Hybridomas producing the monoclonal antibodies MI 10, Mill and MI 12 are deposited in the American Type Culture Reservoir, Rockville, Md. , E.U.A. (ATCC) on March 13, 1996 and assigned access numbers HB-12061, HB-12062, and HB-12063, respectively. All deposits are made in accordance with the terms of the Budapest Treaty. In general, monoclonal antibodies against IL-15 can be generated as described in the patent E.U.A. No. 6,177,079, using the following procedure. Briefly, purified IL-15, a fragment thereof, synthetic peptides or cells expressing IL-15 can be used to generate monoclonal antibodies against IL-15 using techniques known per se, for example, the techniques described in US Pat. No. 4,411,993. Mice are immunized with IL-15 as an immunogen emulsified in complete Freund's adjuvant or RIBI adjuvant (RIBI Corp., Hamilton, ont.), And injected in amounts ranging from 10 to 100 pg subcutaneously or intraperitoneally. Ten to twelve days later, the immunized animals are boosted with additional IL-15 emulsified in incomplete Freund's adjuvant. The mice are periodically reinforced after this in a weekly to biweekly immunization program. Serum samples are taken periodically by retro-orbital bleeding or cutting at the tip of the tail to analyze for antibodies against IL-15 by dot blot analysis, ELISA (enzyme-linked immunosorbent assay) or inhibition of activity of IL-15 in CTLL-2 cells.

After detection of an appropriate antibody titer, positive animals are given an additional intravenous injection of IL-15 in saline. Three to four days later, the animals are sacrificed, the spleen cells are harvested, and the spleen cells are fused to a murine myeloma cell line, for example, NS1 or P3x63Ag8.653 (ATCC CRL 1580). The fusions generate hybridoma cells, which are seeded in multiple microtiter plates in a selective medium HAT (hypoxanthine, aminopterin and thymidine) to inhibit the proliferation of unfused myeloma cells and myeloma hybrids. Hybridoma cells are selected by ELISA for reactivity against purified IL-15 by adaptations of the techniques described in Engvall et al., Immunochem. 8: 871, 1971 and in the patent E.U.A. No. 4,703,004. A preferred selection technique is the antibody capture technique described in Beckmann et al., (J. Immunology 144: 4212, 1990). Hybridoma positive cells can be injected intraperitoneally into syngeneic Balb / c mice to produce ascites containing high concentrations of anti-IL-15 monoclonal antibodies. Alternatively, the hybridoma cells can be cultured in vitro in flasks or spinner bottles using various techniques. Monoclonal antibodies that are produced in mouse ascites can be purified by precipitation with ammonium sulfate, followed by gel exclusion chromatography. Alternatively, affinity chromatography based on the binding of antibody to protein A or protein G, as well as affinity chromatography based on binding to IL-15, can also be used. Other antibodies can be prepared using the description and incorporated material for reference provided in the present invention, and are useful for the present invention. The methods used to generate humanized antibodies can be found in US Patents Nos. 4,816,567, 6,500,931 and in WO 94/10332, all of which are incorporated for reference in the present invention. Methods for generating human antibodies, such as the use of mice or other mammals that express polynucleotides that encode human antibody proteins, are described in, for example, US Patents Nos. 6,075,181; 6,111,166; 6,673,986; 6,680,209; and 6,682,726, all of which are incorporated by reference in the present invention. Methods for generating micro-bodies can be found in WO 94/09817; and additional procedures for generating transgenic antibodies can be found in GB 2 272 440, all of which are incorporated in the present invention for reference. Additional antagonists for use in the methods of the invention include antagonists for the IL-15Ra subunit. Such antagonists are described in, for example, US Pat. No. 5,591,630, which is incorporated for reference in the present invention. To determine which monoclonal antibodies are antagonists, the use of a selection test is preferred. For this purpose, a proliferation test of CTLL-2 is preferred. See, Gillis and Smith, Nature 268: 154 (1977), which is incorporated in the present invention for reference. Briefly, monoclonal antibody antagonist activity, PEG-modified IL-15 and IL-15 muteins can be evaluated using a modified 3H-Thymidine incorporation test of CTLL-2 cell (Gillis, et al., Id.). Serial dilutions of antagonist can be prepared in 96-well flat bottom tissue culture plates (Costar, Cambridge, Mass.) In DMEM medium (supplemented with 5% FCS, NEAA, sodium pyruvate, HEPES pH 7.4, 2 -me, PSG) to a final volume of 50 μ ?. A sub-optimal amount of IL-15 (final concentration of 20-40 pg / ml) is then added to all test cavities (5 μ? / Well) after serial dilution of the samples and before addition of the cells. Wash washed CTLL-2 cells (approximately 2000 per cavity in 50 μm) are added and the plates are incubated for 24 hours at 37 ° C in a humidified atmosphere of 10% C02 in air. This is followed by a five hour incubation with 0.5 μ? of 3 H-Thymidine (25 Ci / mMol, Amersham, Arlington Heights, 111.). The cultures are then harvested on glass fiber filters and counted by avalanche gas ionization either in a direct multiple detector beta counter (Matrix 96, Packard Instrument Company, eridien, Conn.) Or in a beta scintillation counter . The per minute counts (CPM) generated by the test are converted to inhibition in percent and the inhibition values are used in percent of each titrated antagonist sample used to calculate the antagonist activity in units / ml. The data showing the concentration necessary to neutralize 40 pg / ml of IL-15 in a CTLL inhibition test are given in the following Table I. The following table II shows the activity of IL-15 (agonist activity) and IL-15 antagonists in CTLL and CTLL inhibition tests.

TABLE I Specific activity of IL-15 antagonists The concentration of antagonist required to neutralize 40 pg / ml of IL-15 in the CTLL inhibition test Antagonist Concentration Method of protein determination huIL-15 minutes 848-2560 pg / ml ELISA / calculated from AAA M110, Million 5 ng / ml DO PGEGhuIL-15 D56C 7.7 ng / ml Calculated from AAA M112 40 ng / ml DO PEGf-s-IL-15 140-196 ng / ml AAA D0 = optical density absorbance at 280 nm; extinction coefficient of 1.35 AAA = analysis of amino acid PEGf-s-IL-15 + simulated IL-15 with mark substituted with PEG TABLE II Activity of IL-15 and IL-15 antagonists in CTLL and CTLL inhibition tests Sample CTLL test Units / ml inhibition of CTLL (activity units / ml agonist) (antagonist activity) IL-15 7.09 x 10 279 IL-15-Q156C - 3 x 106 IL-15-Q156S 1.5 x 106 IL-15-D56C - 2 x 106 IL-15-D56C- - 7 x 106 Q156C IL-15-D56C - - 7.2 x 105 TABLE II (cont.) Sample CTLL test Units / ml inhibition of CTLL (activity units / ml agonist) (antagonist activity) Q156S IL-15-D56S 2.2 x 10- IL-15-D56S- 7.2 x 10f Q156S Vector control 1141 IL-15 3.7 x 10 'PEG-IL-15 3 x 106 PEG-IL-15- 96 x 106 D56C IL -15-D56C 5 x 106 IL-15 5.6 x 10s NA PEG-IL-15 NA 7 x 105 Q156C = Glnl b substituted with Cys Q156S = Gln b substituted with Ser D56C = Asp substituted with Cys D56S = Asp56 substituted with Ser NA : not analyzed Antagonists according to the invention find use, as described above and how described in more detail later in the treatment of B cell tumors of origin in GC, and conditions in which the inhibition of B cell proliferation in the germinal center is desired.

As described above, another modality of the invention uses the nucleic acids encoding for the IL-15 muteins of the invention. Said nucleic acids comprise either RNA or the cDNA having the nucleotide sequence of 144 to 486 of SEQ D N0: 1 and 144 to 486 of SEQ ID NO: 2. Also within the scope of the invention are the expression vectors which comprise a cDNA encoding an IL-15 mutein and host cells transformed or transfected with said expression vector. Transformed host cells are cells that have been transformed or transfected with a recombinant expression vector using standard procedures. Expressed mammalian IL-15 can be located within the host cell and / or secreted into the culture supernatant, depending on the nature of the host cell and the gene construct inserted into the host cell. Pharmaceutical compositions comprising any of the IL-15 antagonists described above are also encompassed by this invention.

Administration of IL-15 antagonists The present invention provides methods for using pharmaceutical compositions comprising an effective amount of IL-15 antagonist in an appropriate diluent or vehicle. An IL-15 antagonist of the invention can be formulated in accordance with known methods used to prepare pharmaceutically useful compositions. An IL-15 antagonist may be combined in admixture, either as the sole active material or with other known active materials, with diluents (e.g., Tris-HCl, acetate, phosphate), preservatives (e.g., thimerosal, benzyl alcohol , parabens), emulsifiers, solubilizers, adjuvants and / or pharmaceutically suitable vehicles. Suitable vehicles and their formulations are described in Remington's Pharmaceutical Sciences, 16th ed. In addition, such compositions may contain an IL-15 antagonist complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, etc., or incorporated into liposomes., microemulsions, micelles, unilamellar or multilamellar vesicles, empty erythrocytes or sphenoblasts. Such compositions can influence the physical state, solubility, stability, release rate in vivo, and in vivo clearance rate of an IL-15 antagonist. An IL-15 antagonist can also be conjugated with antibodies against tissue-specific receptors, ligands or antigens, or coupled to ligands of tissue-specific receptors. The IL-15 antagonist of the invention can be administered topically, parenterally, rectally or by inhalation. The term "parenteral" includes subcutaneous, intravenous, intramuscular injections, injection 4 intracisternal, or infusion techniques. These compositions typically contain an effective amount of an IL-15 antagonist, alone or in combination with an effective amount of any other active material. Said desired doses and drug concentrations contained in the compositions may vary depending on many factors, including the intended use, the body weight and age of the patient, and the route of administration. Preliminary doses can be determined in accordance with animal tests, and scaling of doses for human administration can be effected in accordance with accepted practices in the art. Preferably, anti-IL-15 antibodies are administered at low doses of protein, such as 20 to 100 milligrams of protein per dose, administered once, or repeatedly, parenterally. Alternatively, anti-IL-15 antibodies are administered in doses of 30 to 90 milligrams of protein per dose, or 40 to 80 milligrams of protein per dose, or 50 to 70 milligrams of protein per dose. The immuno-conjugated components, and anti-IL-15 antibody fusion proteins of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, in which the therapeutic proteins are combined in a mixture with a carrier pharmaceutically acceptable. A composition is said to be a "pharmaceutically acceptable vehicle" if its administration can be tolerated by a recipient patient. Phosphate-regulated sterile saline is an example of a pharmaceutically acceptable vehicle. Other suitable vehicles are well known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, 19th Ed. (1995). For therapy purposes, the antibody (or immuno-ugly / fusion protein) components and a pharmaceutically acceptable carrier are administered to a patient in a therapeutically effective amount. It is said that a combination of an antibody component, optionally with an immuno-conjugate / fusion protein, and a pharmaceutically acceptable carrier is administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient. In the present context, an agent is physiologically significant if its presence results in the inhibition of the growth of the target tumor cells. For treatment of lymphoma, inhibition of IL-15 stimulation of GC B cells can be done in conjunction with currently used anti-lymphoma therapy, including radiation therapy, chemotherapy, and / or biological therapy. Biologic therapy is usually comprised of therapy with interferon and monoclonal antibodies. Interferon therapy is the first biological treatment studied in NHL. It is widely used in Europe for the treatment of indolent lymphomas, but it is rarely used in the United States of America. Data for the use of maintenance therapy with interferon suggest prolonged disease-free survival but not the benefit of consistent overall survival (Hagenbeek, et al., Blood 92 (Suppl 1: 315a, 1998). for interferon therapy in patients with indolent lymphomas remains under clinical evaluation.Therefore, the therapy with IL-15 described in the present invention can be used as an auxiliary for interferon therapy.Monoclonal antibodies are also used to treat lymphoma of the B cell Some monoclonal antibodies currently in use or under investigation in the treatment of B-cell lymphoma include Rituximab (Rituxan), CAMPATH-1 H (humanized IgGl), Tositumomab (Bexxarr), Ibritumomab tiuxetan (Zevalin), Epratuzumab, Bevacizumab; and Lym-1 (Oncolym). These therapies target mainly CD20, CD22, CD52, and VEGF (vascular endothelial growth factor). None of these specifically targets IL-15 or B-cell growth stimulated by IL-15 in GCs. Therefore, the present invention described in general terms will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.

EXAMPLES Material and methods for the examples Antibodies The anti-IL-15 mAb (M110 and Mili: IgGi; M112: IgG2b) is generated as generally described in the patent E.U.A. No. 5,795,966. Briefly, Balb / c mice are boosted twice with 10 pg of (h) IL-15-human marker in RIBI adjuvant (Ribi Corp, Hamilton, MT). Three months after the last boost, an animal is boosted intravenously with 3 g of hIL-15 in PBS. Three days later, the spleen is removed and fused with Ag8.653 using 50% PEG (Sigma, St. Louis, MO). The fused cells are seeded in 96-well plates in DMEM containing HAT complement (Sigma). Hybridoma supernatants are selected by antibody capture test. Briefly, 96-well plates are coated with 10 pg / ml goat anti-mouse Ig, overnight. After blocking with 3% BSA, 50 μ? of the supernatant of the cells to each cavity. After one hour, the plates are washed with PBS containing 0.05% Tween 20. Iodine hIL-15 is added to the plates for 1 hour. After washing, the plates are exposed to phosphoimage-forming plates for three hours. The positive cells are cloned twice, using a similar selection test to detect the positives. A CTLL-2 cell proliferation test is also performed to determine the blocking activity of IL-15. The specificity of these monoclonal antibodies has been previously evaluated and used (U.S. Patent No. 5,795,966, Tinhofer, I., et al., 2000. Blood 95: 610. Musso, T., et al., 1999. Blood 93: 3531). Mouse IgGx (MOPC 21) and IgG2b (MOPC 141) for isotype control are obtained from Sigma. Anti-IL-15 monoclonal antibody (MAB247, mouse Igd), goat polyclonal anti-IL-15, and normal goat control Ig are obtained from R &D systems (Minneapolis, MN). The anti-CD20 mAb conjugated with PEG and the goat anti-Ab of goat, conjugated with FITC are obtained from BD Pharmingen (San Diego, CA). The DRC1 mAb (mouse IgGi) is obtained from DAKO (Carpintería, CA). The mouse anti-Ab, goat, conjugated to Alexa 594 is obtained from Molecular Probes (Eugene, OR). The goat, donkey anti-Ab conjugate with FITC is obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).

Cytokines and reagents The culture media used are IMDM (Irvine Scientific, Santa Ana, CA) and RPMI 1640 (Sigma) supplemented with 10% FCS (Life Technologies, Inc., Grand Island, NY), 2 mM glutamine, 100 U / ml of penicillin G, and 100 μ? / P ?? of streptomycin (Irvine Scientific). The cytokines used are IL-2 (Hoffman-La Roche, Inc., Nutley, NJ), and IL-4 (Schering-Plow Schering Corporation, Union, NJ). Ligand (L) of human recombinant trimeric CD40 and IL-15 are prepared as previously described (Grabstein, KH, et al., 1994. Science 264: 965. Armitage, RJ, et al., 1995. J Immunology 154: 483 Morris, AE, et al., 1999. J Biol Chem 274: 418). Percoll and Ficoll are obtained from Pharmacia LKB Biotechnology (Uppsala, Sweden) and BSA from Sigma.

Immuno-fluorescent staining of FDC groups FDCs are isolated from human tonsils as previously described (Kim, H.-S., et al., 1994. J.

Immunology 153: 2951). The isolated cells are cyto-centrifuged on glass slides at 700 rpm for 5 minutes (Cytosine 2®, Shandon, Pittsburgh, PA). The slides subjected to cyto-centrifugation are fixed in cold acetone (-20 ° C) for 5 minutes and stored at -70 ° C until required. The slides are hydrated with PBS for 10 minutes at room temperature and then incubated with blocking solution (DAKO) for 1 hour at 25 ° C in a humid chamber. The slides are stained with the optimal amount of goat anti-IL-15 antibody or goat control Ig overnight at 4 ° C. The slides are then washed three times and incubated with goat anti-Ig conjugated with FITC for 1 hour at room temperature. For co-staining, mAb DRC-1 (FIGS. 1A and C) or anti-CD20 mAb conjugated with PE (for FIG. IB) is added together with primary antibodies. DRC-1 staining is visualized by secondary staining with mouse antiantibody conjugated with Alexa-594. For individual FDC staining (figure ID), slides are incubated in DAPI solution (Molecular Probes) for nuclear counter staining, then stained with mouse anti-IL-15 monoclonal antibody or control mAb followed by anti-Ab from mouse, goat, conjugated with FITC. The slides are washed and mounted with fluorescent anti-attenuation mounting medium (Molecular Probes). The images are collected in a deconvolution microscope (Axiovert 200M; Cari Zeiss Microimaging, Inc., Thornwood, NY). Images are processed using slidebook software (version 1.6.587, Intelligent Imaging Innovations, Denver, CO) and Adobe Photoshop 7.0 (Adobe systems, Inc., San Jose, CA).

Flow cytometry analysis FDC / HK cells are grown in RPMI medium with 10% FCS RPMI as previously described (Kim, H.-S., et al., 1994. J. Immunology 153: 2951). The FDC / HK cells from passages 4-9 are used for the experiments. For FACS analysis, FDC / HK cells are harvested with solution for enzyme-free cell dissociation (Specialty Media, Philipsburg, NJ). All FACS staining for detection of surface IL-15 is carried out with modification to previously described procedures for amplification (Jung, J., et al., 2000. Eur. J. Immunology 30: 2437). Briefly, cells are washed in buffer for cold FACS (0.05% FCS, 0.01% NaN3 in PBS) and subsequently incubated with the appropriate concentration of anti-IL-15 monoclonal antibody (B247) for 15 minutes at 4 ° C. After flushing with buffer solution for cold FACS, amplification procedures are followed using Flow-Amp® equipment (Flow-Amp systems, Cleveland, OH) in accordance with the manufacturer's instructions. For the competition study, anti-IL-15 antibody was incubated with 300 ng / ml of recombinant IL-15 for 30 minutes at 4 ° C before FACS staining. Samples are analyzed with the FACSCalibur® (Becton Dickinson, San Jose, CA) and CellQuest-Pro® programs. The average fluorescence intensity (MFI) is obtained by subtracting the fluorescence value at the corresponding control value.

Removal with acid and binding of IL-15 The acid removal of IL-15 previously bound in the manner described is carried out (Dubois, S., et al., 2002. Immunity 17: 537. Kumaki, S., et al., 1996 Sur J Immunology 26: 1235). Briefly, FDC / HK cells are washed twice with cold PBS, then incubated with glycine buffer (25 mM glycine, 150 mM NaCl, pH 3) for 10 minutes at 4 ° C. The cells are then harvested and washed twice with cold PBS and subjected to FACS staining. For binding experiments, FDC / HK cells or GC B cells are harvested and washed twice with cold PBS, and then incubated with a saturating dose of IL-15 (100 ng / ml) for 30 minutes at 4 °. C, washed with cold PBS, and then stained for FACS analysis.

CTLL-2 cell test CTLL-2 cells (ATCC, Manasas, VA) are maintained in RPMI 1640 medium containing 10% FCS, IL-2 (30 U / ml) and 2-ME (5 x 10"5 M, Sigma) Numbers of serially diluted FDC / HK cells (from 2 x 10 4 cells / well to no cells / well) are grown in 96-well plates for 1 day in a 5% C02 incubator. Then wash and fix in 1% paraformaldehyde in PBS for 1 hour at 4 ° C followed by thorough washing in cold PBS.CTLL-2 cells (5 x 103 cells / well) in maintenance medium are added in triplicate to the 96-well plates coated with fixed FDC / HK cells and cultured with monoclonal anti-IL-15 antibody or monoclonal antibody for isotype control After 20 hours of culture, the cells are pulsed with 0.5 Ci of [3H] TdR ( 20 Ci / mM; PerkinElmer Life Sciences, Boston, MA) for an additional 4 hours.The cultures are harvested on a glass fiber filter and the poration of [3H] TdR using a liquid scintillation counter (Rackbeta; LKB instrument, Houston, TX). The results are expressed as average cpm ± SEM of the cultures in triplicate.

RT-PCR To examine the expression of mRNA for IL-15Ra, 5 IL-2Ra, IL-2R, and IL-2Ry, the total RNA is extracted from the cells using the RNeasy kit (Qiagen, Valencia, CA). An aliquot of one yg of RNA is transcribed using random oligo-dT and M-MLV RT (Invitrogen-Gibco, Carlsbad, CA). The complementary DNA is amplified in a 25μ1 reaction mixture containing 200 μ? of each of dNTP, 500 nM of primers, 2.5 U of Taq polymerase. The amplification of each cDNA sample is carried out under conditions such as the following: denaturation at 94 ° C for 50 seconds, fixation at 57 ° C for 50 seconds, and extension at 72 ° C for 50 seconds. Human GAPDH is used to ensure an equal load of sample. An imitation PCR is performed to serve as a negative control. The amplified PCR products are separated on a 1.5% agarose gel and visualized by staining with ethidium bromide. The primers used are the following: For IL-15Ra, 5'-GTCAAGAGCTACAGCTTGTAC-3 '(SEQ ID NO: 3) and 5' CATAGGTGGTGAGAGCAGTTTTC-3 '(SEQ ID NO: 4); For IL-2Ra, 5 '-AAGCTCTGCCACTCGGAACACAAC-3' (SEQ ID NO: 5) and 5 '-TGATCAGCAGGAAAACACAGC-3' (SEQ ID NO: 6); For IL-2Rβ, 5 '-ACCTCTTGGGCATCTGCAGC-3' (SEQ ID NO: 7) and 5 '-CTCTCCAGCACTTCTAGTGG-3' (SEQ ID NO: 8); For IL-2Ry, 5 '-CCAGAAGTGCAGCCACTATC-3' (SEQ ID NO: 9) and 5 '-GTGGATTGGGTGGCTCCAT-3' (SEQ ID NO: 10); and For GAPDH, 5 '-CCCTCCAAAATCAAGTGGGG-3' (SEQ ID N0: 11) and 5 '-CGCCACAGTTTCCCGGAGGG-3' (SEQ ID NO: 12).

Preparation and culture of human amygdala GC B cells GC B cells are purified from amygdala B cells by MACS (Miltenyi Biotec Inc., Auburn, CA) as described (Choe, J., et al., 1996). J. Immunology 157: 1006). The purity is greater than 95%, as assessed by the expression of CD20 and CD38. GC B cells (2 x 10 5 cells / well) are grown in 24-well plates in the presence of irradiated FDC / HK cells (2 x 10 4 cells / well, 5,000 Rad), CD40L (100 ng / ml), IL-2 (30 U / ml), and IL-4 (50 U / ml). IL-2 is included to increase sensitivity except for the experiment for Figure 5B, because total crop recoveries are very low without IL-2 (Choe, J., et al., 1996. J. Immunology 151: 1006). For blocking experiments, anti-IL-15 mAb or mAb is incubated for isotype control (10 g / ml, unless otherwise indicated) for 30 minutes before adding GC B cells. Some of the corresponding blocking and control mAbs contain less than 0.00002% sodium azide at the working concentration, which is 100 times lower than the sodium azide concentration that begins to show toxicity in the in vitro culture system . For the addition experiments (Fig. 5B), IL-15 (1-100 ng / ml) is added 30 minutes before adding GC B cells. For cell division experiments, GC B cells are labeled with CFSE (Sigma, 5 μg / ml in PBS) at 37 ° C for 10 minutes. FCS is added to stop the staining, and then the labeled cells are washed with culture medium. After culturing, the intensity of CFSE is measured by FACSCalibur® and analyzed using ModFit LT® 3.0 software (Verity Software House, Inc. Topsham, ME). The recovered viable cells are counted by trypan blue exclusion.

EXAMPLE 1 IL-15 is produced by FDC but not by B cells To identify the cellular source of IL-15 in the germinal centers, the in vivo expression of IL-15 is examined by staining freshly isolated FDC cell B groups with antibodies specific for IL-15 (Figure 1). The FDC groups are cellular aggregates consisting of a typical FDC with large cytoplasm and more than 10 B cells (Li, L., et al., 2000. Journal of Experimental Medicine 191: 1077) (Figures 1A-C). IL-15 is expressed in the FDC groups, suggesting the presence of IL-15 in vivo (Figures 1A and B). To determine the cellular source of IL-15 in the FDC groups, the monoclonal antibody DRC-1 specific marker of FDC or the monoclonal antibody anti-CD20 specific marker of cell B are stained together with goat anti-IL-15 antibody respectively (Li, L., et al., 2000. Journal of Experimental Medicine 191: 1077. Naiem, M., Et al., 1983, J. Clin. Pathol. 36: 167). The anti-IL-15 antibody (green) is stained together with mAb DRC-1 (red, co-staining: yellow, Fig 1A) but not with anti-CD20 mAb (red, Fig. IB), suggesting that FDC DRC-l-positive, not B cells, produce IL-15. The specific staining for IL-15 because there is no co-staining in the samples co-stained with the control antibodies and goat DRC-1 (Fig. 1C). Some of the CDF (10-20%) do not cluster with B cells, but can be identified by their abundant cytoplasm and frequent double nucleus (van Nierop, K., et al., 2002. Semin Immunology 14: 251) (figure ID). These individual FDCs also express IL-15 because they are stained with a murine anti-IL-15 mAb (MAB247), confirming the above result. Similarly, there is no green staining but only blue-colored nuclear staining in samples stained with mouse control mAb and DAPI (lD-box).

EXAMPLE 2 IL-15 is present on the surface of FDC / HK cells bound to IL-15Ra The production of IL-15 is investigated by a primary FDC cell line, FDC / HK, which demonstrates sharing many of the characteristics of FDC including the ability to support the survival and proliferation of GC cell B (Li, L et al., Semin. Immunol., 14: 259, 2002; Kim, H.-S., et al., J. Immunol., 155: 1101, 1995). Since IL-15 is not detected in the culture supernatant of FDC / HK cells (2 x 10 5 cells / ml) by ELISA (test sensitivity> 19 pg / ml), surface expression of IL is studied -15 using methods as reported (Morris, AE, et al., 1999. J Biol Chem 274: 418; Kim, H.-S., et al., 1994. J. Immunology 153: 2951; Naiem, M., et. al 1983. J. Clin. Pathol 36: 167; Bulfone-Paus, S., et al., 1997. Nat Med 3: 1124). A highly sensitive surface FACS staining method is used using the tyramine amplification method (Flow-Amp®) to detect IL-15. As shown in Figure 2A, IL-15 is detected in FDC / HK cells while GC B cells are negative (Figure 2A). These results are consistent with the previous IF staining data in FDC cell B groups. The specific staining of IL-15 in FDC / HK is confirmed by competition with soluble IL-15. When anti-IL-15 mAb or an excess amount of IL-15 is pre-incubated, the IL-15 staining on the surface of FDC / HK cells is completely reduced until isotype control staining. These results are reproduced in 3 separate experiments. Surface IL-15 could be due to the presence of an alternative membrane type IL-15 molecule (Musso, T., et al., 1999. Blood 93: 3531), or by re-binding secreted IL-15 ( Dubois, S., et al., 2002. Immunity 17: 537. Schluns, KS, et al., 2004. Blood 103: 988). Using acid treatment as previously described (Dubois, S., et al., 2002. Immunity 27: 537), IL-15 is completely removed from the surface of FDC / HK cells after treatment with glycine buffer (pH 3.0) to the level of staining with the control mAb (Figure 2C). This result indicates the re-binding of secreted IL-15 instead of an alternative membrane-like protein. Because IL-15Ra binds to IL-15 with high affinity (Giri, J.G., et al., 1995. Embo J 14: 3654), the presence of IL-15Ra in FDC / HK cells is examined. In the RT-PCR experiments, the band specific for IL-15Ra from the FDC / HK cell cDNA as well as the positive control plasmid is amplified while the band for IL-2ROI is not amplified, which is included to serve as an internal negative control (figure 2C). This result indicates that the FDC / HK cells express the mRNA for IL-15R.

EXAMPLE 3 IL-15 bound to membrane on the surface of FDC / HK is biologically active To examine the biological activity of surface-bound IL-15 in FDC / HK cells, the CTLL-2 cell assay dependent on IL-2 and IL-15 is used. Although soluble IL-15 can not be detected by ELISA, FDC / HK cells are fixed with 1% paraformaldehyde to exclude false positive results by soluble IL-15. The incorporation of tritiated thymidine by CTLL-2 cells increases in proportion to the number of fixed FDC / HK cells present in the cultures (Figure 3A). At the 4: 1 ratio of FDC / HK cells to responsive CTLL-2 cells, the cpm value is almost three times higher than that of the negative controls (21,000 to 7,500). The relatively higher background proliferation of CTLL-2 cells (7,500 cpm) without fixed FDC / HK cell control cavities can be attributed to the sub-optimal dose of IL-2 that is added to increase the sensitivity of the test. The result is consistent with the previous report that the reassembled IL-15 is functionally active on the cell surface (Morris, A. E., Et al., 1999. J Biol Chem 274: 418., H.-S., et al. 1994. J. Immunology 153: 2951. Naiem,. , et al. 1983. J. Clin. Pathol. 36: 167 Bulfone-Paus, S., et al. 1997. Nat Med 3: 1124). To examine the possible effect of soluble IL-15 released from the FDC / HK cells, the culture supernatant from the highest concentration of FDC / HK cell (2 x 10V cavity) is added to the same culture. There is no significant difference in the cpm values between cultures with control medium and with FDC / HK cell culture supernatant, which indicates the absence of IL-15 in the culture supernatant, which is consistent with ELISA results . To confirm that the stimulatory effect on CTLL-2 cells is mediated by IL-15, blocking mAb specific for IL-15 and mAb for isotype control are added to the culture. As shown in Figure 3B, the addition of anti-IL-15 mAbs completely blocks the proliferation of CTLL-2 cells increased by fixed FDC / HK cells while the control mAb has no effect.

EXAMPLE 4 GC B cells express receptor components for signal transduction of IL-15 but not for high affinity binding The production of IL-15 by FDC implies that IL-15 possibly has a biological function in the GC reaction, more likely in GC B cells. Therefore, the expression profile of the specific receptors required for IL-15 signaling in GC B cells is examined (Figure 4A). The expression of IL-15ROI mRNA, a receptor component for high affinity binding, is virtually insignificant in RT-PCR, which shows a faint band similar to that of IL-2Ra in freshly isolated GC B cells (a negative control ). In contrast, the expressions of the mRNA molecules of IL-2R and IL-2Ry, the major components of signal transduction, are evident in GC B cells either freshly isolated or cultured, suggesting the presence of receptor components of signaling for IL-15 or IL-2 in GC B cells both in vivo and in vitro. The absence of IL-15Ra mRNA is also confirmed by the failure to detect the IL-15Ra protein in FACS staining of GC B cells and the lack of binding to IL-15 (Figure 4B). In contrast to the FDC / HK cells that exhibit intense IL-15 binding, no significant binding of IL-15 on the surface of GC B cells is detected after incubation with excess IL-15, demonstrating the absence of IL-15Ra on the surface. Because soluble IL-15 needs IL-15a to transduce its mitogenic signal (Lu, J. et al., Clin.Cancer Res. 8: 3877, 2002), the results suggest that GC B cells can not respond to soluble IL-15. This conclusion is consistent with the observation that soluble IL-15 in the absence of FDC-HK cells does not show a noticeable difference in GC B cell recovery.

EXAMPLE 5 IL-15 increases the proliferation of GC B cell GC B cells are cultured with FDC / HK cells and cytokines as described above. When different amounts of anti-IL-15 mAb are added, GC B cell proliferation is remarkably inhibited in a dose-dependent manner (FIG. 4A), suggesting that IL-15 increases B cell proliferation. of GC. On day 10, the number of viable GC B cells in the culture containing anti-IL-15 mAb (10 pg / ml) is 17% of that of the cultures containing isotype control mAb. However, blockade of IL-15 does not affect the differentiation of cultured cells measured by the surface marker and Ig secretion. This result is reproduced in four separate experiments. Similar inhibition is also observed in the experiments using other monoclonal antibodies for IL-15 (Clone Mili, M112 and MAB247). In other experiments, IL-2 is omitted to exclude the possible indirect effect by IL-2, and to verify the effect of IL-15 in the depletion experiment. As shown in Figure 4B, the amount of surface IL-15 in FDC / HK cells is further increased by incubation with exogenous IL-15. Therefore, FDC / HK cells used as a coating with different amount of IL-15 (l-100ng) are incubated before the GC B-cell cultures to increase the effect of IL-15. The MFI of IL-15 surface by FACS is increased in proportion to the added IL-15 (for 100 ng: Figure 4B panel on the right). The cell number recovered on day 10 of culture is increased in a dose-dependent manner (Figure 5B). In the presence of 100 ng / ml of IL-15, the number of viable GC B cells is increased two and a half times more than the control culture. Since GC B cells do not express IL-15Ra, these results strongly suggest that surface IL-15 in FDC / HK increases GC B cell proliferation. This result is reproduced in four separate experiments. Throughout this application, reference is made to several publications. The descriptions of these publications are incorporated in the present invention for reference in their totalities. It is considered that the above written description is sufficient to enable the person skilled in the art to practice the invention. The present invention is not limited in scope by the examples presented therein. Indeed, various modifications of the invention in addition to those shown and described therein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Claims (25)

NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and therefore the content of the following is claimed as property: CLAIMS

1. - A method for treating a B cell tumor of origin in the germinal center, which comprises administering to a human subject having said B-cell tumor a therapeutic composition comprising a pharmaceutically acceptable carrier and at least one IL-15 antagonist .

2. The method according to claim 1, characterized in that said antagonist is an anti-IL-15 antibody.

3. The method according to claim 2, characterized in that said anti-IL-15 antibody is selected from the group consisting of non-human primate antibody, murine monoclonal antibody, chimeric antibody, human antibody, and antibody humanized

4. - The method according to claim 2, characterized in that said anti-IL-15 antibody is administered parenterally in a dose of 30-90 milligrams of protein per dose.

5. The method according to claim 2, characterized in that said individual receives the anti-IL-15 antibody as repeated parenteral doses of 50-90 milligrams of protein per dose.

6. - The method according to claim 2, characterized in that said anti-IL-15 antibody is selected from the group consisting of antibodies M110, Mili and M112.

7. - The method according to claim 1, characterized in that said antagonist is a mutein of IL-15.

8. The method according to claim 7, characterized in that said mutein of IL-15 can bind to the subunit IL-15R-a, and can not transduce a signal through the subunits β or? of the IL-15 receptor complex.

9. - The method according to claim 7, characterized in that in said mutein, at least one of the amino acid residues Asp56 or Gln156 of IL-15 of SEQ ID NO: 2 is deleted or substituted with a different amino acid residue of natural origin.

10. - The method according to claim 7, characterized in that said mutein is conjugated to a chemical portion.

11. The method according to claim 10, characterized in that said mutein is conjugated to polyethylene glycol.

12. The method according to claim 1, characterized in that said antagonist is soluble IL-15.

13. - The method according to claim 12, characterized in that said soluble IL-15 is conjugated to a chemical portion.

14. - The method according to claim 13, characterized in that said soluble IL-15 is conjugated to polyethylene glycol.

15. - The method according to claim 1, characterized in that said B-cell tumor is selected from the group consisting of Hodgkin's lymphoma; Non-Hodgkin's lymphoma; B cell lymphomas; small lymphocytic lymphoma; mantle cell lymphoma; follicular lymphoma; marginal cell lymphoma; monocytoid B cell lymphoma; spleen lymphoma; diffuse large cell lymphoma; Burkitt's lymphoma; Burkitt-type lymphoma of high degree; lymphoblastic lymphoma; and diffuse large cell lymphoma.

16. - The method according to claim 15, characterized in that said B cell tumor is a non-Hodgkin lymphoma.

17. - The method according to claim 1, which also comprises administering a therapeutic protein or chemotherapeutic treatment, characterized in that said therapeutic protein is selected from the group consisting of antibody, immuno-conjugate, antibody fusion protein- immuno-modulator and antibody-toxin fusion protein.

18. - The method according to claim 17, characterized in that said therapeutic protein or said chemotherapeutic treatment is administered before the administration of the anti-IL-15 antibody.

19. - The method according to claim 17, characterized in that said therapeutic protein or said chemotherapeutic treatment is administered concurrently with the administration of said anti-IL-15 antibody.

20. - The method according to claim 17, characterized in that said therapeutic protein or said chemotherapeutic treatment is administered after the administration of the anti-IL-15 antibody.

21. - The method according to claim 17, characterized in that said chemotherapeutic treatment consists in the administration of at least one drug that is selected from the group consisting of cyclophosphamide, etoposide, vincristine, procarbazine, prednisone, carmustine, doxorubicin , methotrexate, bleomycin, dexamethasone, phenyl butyrate, brostatin-1 and leucovorin.

22. - The method according to claim 1, characterized in that said therapeutic composition also comprises a cytokine portion, wherein said cytokine portion is selected from the group consisting of interleukin-1 (IL-1), IL -2, IL-3, IL-6, IL-10, IL-12, interferon- ?, interferon-β, and interferon-? .

23. - The method according to claim 22, characterized in that said therapeutic protein is an immuno-conjugate or antibody-toxin fusion protein comprising a toxin that is selected from the group consisting of ricin, abrin, ribonuclease, DNase. I, staphylococcal enterotoxin A, anti-viral protein of Phytolacca americana, gelonin, dipterin toxin, Pseudomonas exotoxin and Pseudomonas endotoxin.

24. - The method according to claim 23, characterized in that said immuno-conjugate or said antibody-toxin fusion protein comprises an antibody or antibody fragment that binds to an antigen that selects from the group consisting of CD19, CD20 and CD22.

25. The method according to claim 24, characterized in that said therapeutic protein is an immuno-conjugate or a fusion protein, wherein said immuno-conjugate or fusion protein comprises an immunomodulatory portion that is selected from from the group consisting of interleukin-1 (IL-1), IL-2, IL-3, IL-6 and IL-10, IL-12, interferon-a, interferon-β, and interferon-? granulocyte clolinia, granulocyte macrophage colony stimulating factor and lymphotoxin.