WO2010068923A2 - Agents that selectively inhibit cd25 on dendritic cells or t cells and their use - Google Patents

Abstract

Agent that selectively inhibit CD25 on dendritic cells on T cells are disclosed herein. Methods are provided for treating a subject with an immune mediated disease. These methods include administering to the subject a therapeutically effective amount of an agent that selectively inhibits CD25 on dendritic cells, thereby treating the immune mediated disorder in the subject. In other embodiments, methods are provided for treating a subject with a tumor. The methods include administering to the subject a therapeutically effective amount of an agent that selectively inhibits CD25 on T cells, thereby treating the tumor in the subject. Compositions are disclosed that include a composite molecule and a carrier, wherein the composite molecule comprises an agent that selectively binds dendritic cells or an agent that selectively binds T cells, wherein the agent is covalently bound to a CD25 antagonist. Methods for selecting an agent of use in treating an immune mediated disorder are also disclosed.

Description

AGENTS THAT SELECTIVELY INHIBIT CD25 ON DENDRITIC CELLS OR T CELLS AND THEIR USE

PRIORITY CLAIM This claims the benefit of U.S. Provisional Application No. 61/201,589, filed

December 12, 2008, which is incorporated by reference herein in its entirety.

FIELD

This application relates to the field of autoimmune diseases and tumor therapy, specifically to CD25 antagonists that selectively inhibit CD25 on dendritic cells or T cells and their use.

BACKGROUND

The high affinity interleukin-2 receptor (IL- 2R) is a heterotrimeric cell surface receptor composed of alpha (α), beta (β) and gamma (γ)_c-polypeptide chains (K_D 10^"Π M). The 55 kDa α-chain, also known as IL-2R.alpha., CD25, p55, and Tac (T cell activation) antigen, is unique to the IL-2R. The beta (CD122; P75) and γ_c (CD 132) chains are part of a cytokine receptor superfamily (hematopoietin receptors), and are functional components of other cytokine receptors, such as IL- 15R (Waldmann (1993) Immunol. Today 14(6):264-70; Ellery et al. (2002)

Cytokine Growth Factor Rev. 13(1): 27-40). The intermediate affinity receptor is a dimer composed of a β- and a γ c-chain (K_D lO^"9 M) while the low affinity receptor consists of a monomeric α-subunit that has no signal transduction capacity (K_D 10^" M) (Waldmann (1993) Immunol. Today 14(6):264-70). CD25 lacks a signaling component and binds IL-2 only with low affinity (Kdi_S ~ 10^" M). However, when it associates with CD122 and CD132 it stabilizes the receptor complex and increases its affinity for IL-2 by at least 10-fold (K_dls ~ 10^"π M).

Resting T cells, B cells, and monocytes express few CD25 molecules. However, the receptor is rapidly transcribed and expressed upon activation (Ellery et al. (2002) Cytokine Growth Factor Rev. 13(1): 27-40; Morris et al. (2000) Ann.

Rheum. Dis. 59 (Suppl. l):ilO9-14). T cells, B cells and monocytes that express the high affinity IL- 2R express CD25 (the CD25-subunit) in excess. There are high and low affinity IL-2 binding profiles (Waldmann et al. (1993) Blood 82(6): 1701-12; de Jong et al. (1996) J. Immunol. 156(4): 1339-48).

CD25 is highly expressed by T cells in some autoimmune diseases, such as rheumatoid arthritis, scleroderma, and uveitis, as well as skin disorders, such as psoriasis and atopic dermatitis, and a variety of lymphoid neoplasms, such as T cell leukemia, and Hodgkin's disease (Waldmann (1993) Immunol. Today 14(6):264-70; Kuttler et al. (1999) J. MoI. Med. 77(l):226-9). In addition, CD25 expression is associated with allograft rejection and graft- versus-host responses (Jones et al. (2002) J. Immunol. 168(3): 1123-1130; Anasetti et al. (1994) Blood 84(4): 1320-7).

Antibodies that specifically bind CD25 have been used in therapy, such as to reduce inflammation in autoimmune diseases, treat tumors, and prevent transplant rejection. Antibodies that specifically bind CD25 have also been used for the treatment of multiple sclerosis. However, while it has been established that CD25 as a useful target for immunotherapy, a need exists for CD25 antagonists that are more specific, such that they have a higher therapeutic efficacy and fewer side effects.

SUMMARY

It is disclosed herein that mature dendritic cells (mDC) use CD25 for targeted trans-presentation of IL-2 via immune synapse to primed antigen-specific T cells. It is also disclosed that blockade of CD25 on the surface of mDCs abrogates T cell proliferation. CD25 expression on T cells contributes to IL-2 signaling with resultant enhancement of T cell entry into proliferation cycle and it can prime activated T cells to apoptosis. In addition, CD25 expression on regulatory T cells (such as T cells expressing FoxP3) is important for the inhibitory function of these cells. As a consequence, CD25 antagonists that do not selectively inhibit CD25 on one specific cell type have apposing effects on the immune system: they can inhibit T cell proliferation (such as by blocking CD25 on mDC present in lymph nodes and tissues) and they can promote survival of expanded T cells. Therefore, CD25 antagonists selective for dendritic cells or T cells can be utilized in therapeutics.

CD25 antagonists specific for dendritic cells inhibit T cell responses and can be used to treat immune-mediated disorders. CD25 antagonists specific for T cells would enhance T cell responses. In some embodiments these CD25 antagonists can be used in cancer therapy.

In some embodiments, methods are provided for treating a subject with an immune mediated disease. These methods include administering to the subject a therapeutically effective amount of an agent that selectively inhibits CD25 on dendritic cells, thereby treating the immune mediated disorder in the subject. In some non-limiting examples, the immune mediated disorder is an autoimmune disorder such as multiple sclerosis. In other embodiments, methods are provided for treating a subject with a tumor. The methods include administering to the subject a therapeutically effective amount of an agent that selectively inhibits CD25 on T cells, thereby treating the tumor in the subject.

Compositions are also provided herein that include a composite molecule and a carrier, wherein the composite molecule comprises an agent that selectively binds dendritic cells or an agent that selectively binds T cells, wherein the agent is covalently bound to a CD25 antagonist. Methods for selecting an agent of use in treating an immune mediated disorder are also disclosed herein.

The foregoing and other features and advantages will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES FIGS. 1A-1C are a set of graphs showing antigen (Ag)-specific T cell proliferation in DC:T cell co-culture is profoundly inhibited by daclizumab. (a) CFSE proliferation assay: mDCs loaded with FIuHA (0.5μg/ml) or Brain Protein Medley (BPM; lOμg/ml) were co-cultured with autologous CFSE-stained T cells in the presence or absence of CD25 blocking- Ab control MA-251 (lOμg/ml), or daclizumab (Dae; lOμg/ml). After 7-10 days, T cell proliferation was assessed by CFSE dilution assay after gating on CD4⁺ (light grey) and CD8⁺ T cells (dark grey). Data are representative of five independent donors/experiments, (b) Box plots represent group data on Ag-specific CD4⁺ T cell proliferation with marked group medians (black horizontal line) and means (light horizontal line), (c) CFSE proliferation assay after polyclonal T cell activation with CD3/CD28 Dynabeads (0.3:1 bead:T cell ratio) in the presence or absence of daclizumab. Proliferation was measured by CFSE dilution after 5 days using the same gating strategy (i.e. CD4⁺ T cells in light grey and CD8⁺ T cells in dark grey).

FIGS. 2A-C are a set of graphs showing mDCs do not express a complete IL-2 receptor and they do not phosphorylate Stat5 in response to IL-2. (a) iDCs and mDCs were cultured as indicated in methods and then stained with directly- conjugated Abs against CD25, CD80, CD83, CD86 and CDl Ic (open histograms) or appropriate isotype controls (gray filled histograms) and analyzed by flow cytometry, (b) iDCs and mDCs were stained for surface expression of CD25, CD122 and CD132 (open histograms) or appropriate isotype controls (gray filled histograms), (c) IL-2 signaling and (d) IL-4 signaling experiments: lymphocytes (upper panels) or mDC (lower panels) were cultured with IL-2, (100 IU/ml; right panel) or diluent control (left panel) and stained intracellularly for pStat5 (open histograms) or (d) lymphocytes (upper panels) or mDC (lower panels) were cultured with IL-4, (20ng/ml; right panel) or diluent control (left panel) and stained for pStatό (open histograms). Filled histograms represent isotype controls. Data are representative of at least three independent donors/experiments.

FIGS. 3A-3B are graphs, plots and schematic diagrams illustrating that blockade of CD25 on mDC by pre-incubation of mDC with daclizumab is sufficient to abrogate T cell proliferation, (a) Histogram of the CD3⁺ gate of CFSE stained T cells cultured with Ag-loaded mDCs (left panel); Ag-loaded mDCs pre-treated with daclizumab (Dae; middle panel) or T cells pre-treated with daclizumab cultured with Ag-loaded mDCs (right panel). Corresponding dot-plots are depicted below. Data are representative of at least five independent donors/experiments and (b) represents box plots with analyzed group data (** P < 0.002, *** P < 0.001).

FIGS. 4A-4B are schematic diagrams, plots and digital images showing trans-presentation of IL-2 to T cells by mDCs. (a) Flow cytometric analysis for pStat5 (open histogram) on cells cultured in the presence of exogenous IL-2 (50 IU/ml) and IL- 15 -blocking Ab for 20 min. Conditions (from left to right) are untreated T cells, T cells pre-treated with daclizumab, T cells pre-treated with daclizumab and incubated with mDCs, T cells pre-treated with daclizumab and incubated with mDCs pre-treated with daclizumab, untreated T cells incubated with mDCs, and T cells pre-treated with daclizumab and incubated with iDCs. Filled histograms represent appropriate isotype controls. Data are representative of at least three independent donors/experiments, (b) Florescent microscopy of mDCs and T cells co-cultured in a glass bottom dish and stained with florescent antibodies against CD25 (white), CD3 (light grey) and nuclei are visualized by DAPI staining (dark grey). Pictures show mDCs not in contact with T cells (left) and in different stages of immune synapse formation with T cells.

FIGS. 5A-5D are plots and graphs showing enhanced numbers of Ag- specific effector T cells following CD25 inhibition on T cells by siRNA methodology, (a) Efficacy of siRNA technology in suppressing activation-induced upregulation of CD25 24h and 48h after polyclonal T cell activation by CD3/CD28 Dynabeads. Functional assessment of Ag- specific T cell activation 7-10 days post- stimulation by Ag-loaded mDC using identical siRNA nucleofected T cells: (b) Proliferation and quantification of absolute numbers of proliferating T cells nucleofected with control or CD25 siRNA as measured by CFSE dilution following culture with Ag-loaded mDCs pre-treated with daclizumab (Dae or D) or control Ab; cytokine production by ICCS: (c) %-cytokine producing proliferating CD4⁺ T cells and (d) quantification of absolute numbers of cytokine -producing proliferating CD4⁺ T cells. Gating was set from isotype controls. One out of six independent donors/experiments; representative of subjects with increased absolute numbers of proliferating T cells in CD25 siRNA cultures as compared to control siRNA cultures.

FIGS. 6A-6D are graphs and parts of results from experiments on T cells derived from a CD25 negative patient, (a) T cells from a CD25 negative subject were polyclonally activated and stained several times during the proliferation cycle for surface expression of CD25, CD122, and CD132 (open histograms) or appropriate isotype control (gray filled histograms) (b) Flow cytometric analysis for pStat5 (open histogram) on T cells from CD25 negative subject cultured in the presence of exogenous IL-2 for 20 min. Conditions (from left to right) are T cells no IL-2, T cells with IL-2, T cells incubated with mDCs and no IL-2, T cells incubated with mDCs and IL-2, T cells incubated with mDCs pre-treated with daclizumab (all five conditions gated on T cells), (c) Proliferation of CD25^" CD4⁺ (left) and CD8⁺ (right) T cells following co-incubation with FIuHA- specific HLA-matched CD25⁺ mDCs measured by CFSE dilution after 7 days. Proliferation was inhibited when mDCs were treated with daclizumab prior to co-incubation. No exogenous IL-2 was added to the culture, (d) Cytokine production (IL-2, IFN-γ and IL- 17) by proliferating CD25^" CD4⁺ and CD8⁺ T cells following coculture with CD25⁺ mDCs. Cells were restimulated briefly with PMA/Ionomycin before staining for intracellular cytokines. Analogous results were obtained using two different Ag: KLH and copolymer- 1.

FIGS. 7A-7B are plots and a graph showing exogenous IL-7 partially restores proliferation inhibited by blockade of IL-2 trans-presentation, (a)

Representative histogram plots of CFSE dilution gated on CD4⁺ (light gray) and CD8⁺ (dark grey) T cells 7 day and 15 days after Ag-specific stimulation by Ag- loaded mDC. (b) Quantification of the data of T cell:mDC co-cultures following pretreatment of mDCs with MA-251 control or daclizumab and incubation with or without IL-7 (5ng/ml). One of two independent donors/experiments.

FIG. 8 is a schematic diagram of a model of diverse IL-2 functions in T cell activation. DCs upregulate CD25 and IL-2 only after activation. Upon formation of the immune synapse (IS) with an Ag-specific T cell, a mDC polarizes CD25 to the site of T cell contact and presumably releases its IL-2 to the IS. This limits diffusion of secreted IL-2 and allows efficient capture of released IL-2 by CD25 on the mDC surface for its trans-presentation to intermediate IL- 2R expressed on resting T cell, providing an efficient IL-2 signal. This "signal 3" together with Ag-MHC-TCR "signal 1" and co-stimulatory "signal 2" leads to effective T cell activation, which leads to upregulation of CD25 on the T cell, entry to the proliferation cycle and production of cytokines, the earliest being IL-2. This T cell secreted IL-2 then enhances T cell proliferation and further upregulation of CD25. However, at the same time, CD25 expression on T cells and the subsequent abundance of the high affinity IL-2 signal primes activated T cell to later cell death, assuring effective contraction of activated T cells at the end of the immune response.

FIG, 9 is a set of plots showing a comparison of IL-2 receptor expression on activated T cells versus mDCs. Activated T cells (CD3/CD28 Dynabeads x 48h) and mDCs were stained for surface expression of CD25, CD122 and CD132 (open histograms), and analyzed by flow cytometry. Filled histograms represent isotype controls.

FIG. 10 is a set of plots showing a comparison of maturation markers and cytokine production on DCs matured in the presence or absence of daclizumab and IL-2. Flow cytometry of iDCs loaded with Ag matured by addition of maturation cocktail only (control; 1^st row), or in the presence of IL-2 (IL-2; 2^nd row), daclizumab (DAC; 3^rd row), or a combination of both (IL-2+DAC; 4^th row) for 48h, stained for surface expression of CD25, CD83 and CD80, and stained by ICCS for IL-2, and IL-6 (black open histograms). Gray filled histograms represent isotype controls. Data are representative of at least three independent donors/experiments. FIGS. 11A-11D are schematic diagrams and plots illustrating that mDC do not express FcR and consequently, murine anti-Tac Ab, which does not bind human FcR, has an inhibitory effect on IL-2 trans-presentation and T cell proliferation analogous to daclizumab. (a) iDCs (upper panel) and mDCs (lower panel) were stained for surface expression of CD 16, CD32 and CD64. (b) iDCs (upper panel) and mDCs (lower panel) were stained for murine anti-Tac binding to the surface, without FcR blocking with IVIg (left column) and with IVIg blocking (right column), (c) Flow cytometric analysis for pStat5 (open histogram) on cells cultured in the presence of exogenous IL-2 for 20 min. Conditions (from left to right) are untreated T cells, T cells pre-treated with murine anti-Tac, T cells pre-treated with murine anti-Tac and incubated with mDCs, T cells pre-treated with murine anti-Tac and incubated with mDCs pre-treated with murine anti-Tac, and T cells pre-treated with murine anti-Tac and incubated with iDCs (all five conditions gated on T cells). (d) Histogram of CFSE stained T cells cultured with FcR blocked Ag-loaded mDCs pre-treated with MA251 (left plot), and FcR blocked Ag-loaded mDCs pre-treated with anti-Tac (right plot).

FIG. 12 is two plots showing that blocking of CD25 on the surface of T cells and ICCS for pStat5 on mDCs. Flow cytometry of CD25 expression on T cells surface without daclizumab (left plot) and with daclizumab (right plot). Data are representative of at least three independent donors/experiments.

FIGS. 13A-13D are plots and graphs illustrating that enhanced numbers of Ag-specific effector T cells following CD25 inhibition on T cells by siRNA methodology. In this proliferation assay paralleling Fig 6, CD25 knockdown was 27.8% effective, (a) Ag-specific proliferation data: Histograms of CFSE dilution gated on CD4⁺ (light gray) and CD8⁺ (dark gray) T cells. Quantification of percentage and absolute numbers of proliferating CD4⁺ T cells nucleofected with control or CD25 siRNA as measured by CFSE dilution following culture for 7-10 days with Ag-loaded mDCs pre-treated with daclizumab or control Ab. (b) Cytokine production by proliferating CD4⁺ T cells: ICCS for IL-2, IFN-g and IL-17⁺ effector T cells for the same co-cultures. Gating was set from isotype controls. Quantification of the (c) percentage and (d) absolute numbers of Ag-specific proliferating CD4⁺ T cells. One out of four independent subjects/experiments, representative of subjects with decreased absolute numbers of proliferating T cells in CD25 siRNA cultures as compared to control siRNA cultures.

FIG. 14 is a set of plots showing the monocyte isolation purity. Flow cytometry of CD14 and CD25 expression on the cell surface of monocytes (right plot) and isotype control (left plot). Data are representative of at least three independent donors/experiments. FIGS. 15A-15C are a set of plots showing that daclizumab or original murine anti-Tac Ab bind to mDC via F_ab fragment, (a) iDCs (upper panel) and mDCs (lower panel) were stained for surface expression of human F_cRs CD 16, CD32 and CD64. (b) iDCs (upper panel) and mDCs (lower panel) were stained for murine anti-Tac binding to the surface, without F_cRs blocking with IVIg (left column) and with IVIg F_cRs block (right column), (c) Histogram of CFSE stained T cells cultured with F_cRs blocked Ag-loaded mDCs pre-treated with MA251 (upper panel), and F_cRs blocked Ag-loaded mDCs pre-treated with anti-Tac (lower panel) for 7 days.

FIGS. 16A-16E are FACS plots and a digital image showing that mDC do not express β-chain of IL-2R and therefore do not signal to IL-2. (a) iDCs and mDCs were cultured as indicated in methods and then stained with directly- conjugated Abs against CD25, CD80, CD83, CD86 and CDl Ic (open histograms) or appropriate isotype controls (gray filled histograms) and analyzed by flow cytometry, (b) iDCs and mDCs were stained for surface expression of CD25, CD122 and CD132 (open histograms) or appropriate isotype controls (gray filled histograms), (c) Analysis of CD 122 mRNA levels in iDCs and mDCs was compared to levels in the T cell line (TCL) Kit225-K6. These data are representative of three separate experiments on three healthy human subjects, (d) IL- 2 signaling and (e) IL-4 signaling experiments: lymphocytes (upper panels) or mDC (lower panels) were cultured with IL-2, (right panel) or diluent control (left panel) and stained intracellularly for pStat5 (open histograms) or (e) lymphocytes (upper panels) or mDC (lower panels) were cultured with IL-4 (right panel) or diluent control (left panel) and stained for pStatό (open histograms). Filled histograms represent isotype controls. Data are representative of at least three independent donors/experiments . FIGS. 17A-17B are FACS plots showing the kinetics of CD25 and IL-2 expression on activated T cells: T cells do not upregulate CD25 during first 10 hours post-activation. Purified T cells were analyzed for the expression of CD25 and IL-2 before- or 10 hours, 24hours, 48hours and 72hours after polyclonal stimulation (CD3/CD28 beads at 0.3:1 bead:T cell ratio). During last 5 hours before harvesting, cells were incubated in the presence of brefeldin A. Data are presented after gating on (a) CD4+ T cells (top panels, grey) and (b) CD8+ T cells (bottom panels, dark grey). T cells were cultured in the absence (top panels) or presence (bottom panels) of IL-2 neutralizing Ab used in saturating concentration. SEQUENCE LISTING

The nucleic and amino acid sequences are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand:

SEQ ID NO: 1 is the amino acid sequence of apa-Il-2. SEQ ID NO: 2 is the amino acid sequence of apamin. SEQ ID NO: 3 is the amino acid sequence of an exemplary peptide linker.

DETAILED DESCRIPTION

/. Abbreviations Ab: antibody

Ag: antigen

BPM: Brain Protein Medley

CSFE: carboxyfluorescein diacetate succinimidyl diester d: days DAC: daclizumab

DC: dendritic cell

GFP: green fluorescent protein

GM-CSF: granulocyte macrophage colony stimulating factor

ICCS: intracellular cytokine staining iDCs: immature dendritic cells

IFN: interferon

IL: interleukin mDC: mature dendritic cells

PBMC: peripheral blood mononuclear cells Rh: recombinant human

RT: room temperature siRNA: small inhibitory ribonucleic acid TNF: tumor necrosis factor

//. Terms Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19- 854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

Allogeneic: Organisms, cells, tissues, organs, and the like Ifom, or derived from, individuals o1 the same species, but wheiein the organisms, coils, tissues, organs, and the like arc genetically different one from another "'transplant rejection^" refers to a partial or complete destruction of a transplanted cell, tissue, organ, or the like on or in a recipient of said transplant due to an immune response to an allogeneic graft. Adverse Effects: Any undesirable signs, including the clinical manifestations of abnormal laboratory results, or medical diagnoses noted by medical personnel, or symptoms reported by the subject that have worsened. Adverse events include, but are not limited to, life-threatening events, an event that prolongs hospitalization, or an event that results in medical or surgical intervention to prevent an undesirable outcome.

Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non- human mammals. Similarly, the term "subject" includes both human and veterinary subjects. Antagonist of CD25: An agent that specifically bind to the CD25 component of IL- 2R, or a component thereof, and/or inhibits a biological function of the IL-2 receptor or this component of the IL- 2R. Functions that can be inhibited are the binding of IL-2 to the IL-2R, the intracellular transmission of a signal from binding of IL-2, antigen presentation by dendritic cells, and proliferation and/or activation of lymphocytes such as T cells in response to IL-2. In one embodiment, IL-2R antagonists of use in the methods disclosed herein inhibit at least one of these functions, can inhibit more than one of these function, or can inhibit or all of these functions.

An antagonist of CD25 that selectively inhibits CD25 on dendritic cells affects antigen presentation by mature dendritic cells, and thus inhibits differentiation of effector T cells and/or expansion of NK cells. This antagonist selectively inhibits CD25 on dendritic cells (and affects dendritic cell activity) as compared to the inhibitory effect of the antagonist on T cells. An antagonist of CD25 that selectively inhibits CD25 on T cells affects at least one function of CD25 specific to T cells, and has a substantially less effect on CD25 on dendritic cells (such that it does not affect antigen presentation by mature dendritic cells to T cells). These antagonists that selectively inhibit CD25 on T cell or dendritic cells can be composite molecules, such as an agent (e.g., an antibody) that specifically binds dendritic cells (but not T cells) covalently linked to a CD25 antagonist, or an agent (e.g., and antibody) that specifically binds T cells (but not dendritic cells) covalently linked to a CD25 antagonist.

In one example, an IL-2 receptor antagonist is an antibody that specifically binds Tac (p55), such as ZEN AP AX® (see below). Other anti-p55 agents include the chimeric antibody basiliximab (SIMULECT®), BT563 (see Baan et al., Transplant. Proc. 33:224-2246, 2001), and 7G8. Basiliximab has been reported to be beneficial in preventing allograft rejection (Kahan et al., Transplantation 67:276- 84,1999), and treating psoriasis (Owen & Harrison, Clin. Exp. Dermatol. 25:195-7, 2000). An exemplary human anti-p55 antibody of use in the methods of the invention is HUMAX-T AC®, being developed by Genmab. In another example, an IL-2 receptor antagonist is an antibody that specifically binds the p75 or β subunit of the IL-2R. Additional antibodies that specifically bind the IL-2 receptor are known in the art. For example, see U.S. Patent No. 5,011,684; U.S. Patent No.5152,980; U.S. Patent No. 5,336,489; U.S. Patent No. 5,510,105; U.S. Patent No. 5,571,507; U.S. Patent No. 5,587,162; U.S. Patent No. 5,607,675; U.S. Patent No. 5,674,494; U.S. Patent No. 5,916,559. The mik-βl antibody is an antagonist that specifically binds the beta chain of human IL- 2R.

In another example, an IL-2 receptor antagonist is a peptide antagonist that is not an antibody. Peptide antagonists of the IL-2 receptor, including antagonists of Tac (p55) and p75 (IL-2Rβ) are also known. For example, peptide antagonists for p55 and p75 are disclosed in U.S. Patent No. 5,635,597. Nonpeptidic inhibitors include acylphenyalanine derivatives (see Emerson et al., Protein Science 12: 811- 82 (2003), herein incorporated by reference). These peptides, which include apa-Il- 2 (C-N-C-K-A-P-E-T-K-L-C-R-M-L-C-F-K-F-Y-M (SEQ ID NO: I)) and analogs thereof with an IC50 value of 20 to 70 μM, are also of use in the methods disclosed herein.

In a further example, an IL-2 receptor antagonist is a chemical compound or small molecule that specifically binds to the IL-2 receptor and inhibits a biological function of the receptor.

Antibody fragment (fragment with specific antigen binding): Various fragments of antibodies have been defined, including Fab, (Fab')₂, Fv, and single- chain Fv (scFv). These antibody fragments are defined as follows: (1) Fab, the fragment that contains a monovalent antigen-binding fragment of an antibody molecule produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain or equivalently by genetic engineering; (2) Fab', the fragment of an antibody molecule obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')₂, the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction or equivalently by genetic engineering; (4) F(Ab')₂, a dimer of two FAb' fragments held together by disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Methods of making these fragments are routine in the art.

Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T-cell response in an animal, including compositions that are injected or absorbed into an animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous immunogens. The term "antigen" includes all related antigenic epitopes.

Autoimmune disorder: A disorder in which the immune system produces an immune response (e.g. a B cell or a T cell response) against an endogenous antigen, with consequent injury to tissues.

Chemotherapy; chemotherapeutic agents: As used herein, any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer as well as diseases characterized by hyperplastic growth such as psoriasis. In one embodiment, a chemotherapeutic agent is an agent of use in treating neoplasms such as solid tumors. In one embodiment, a chemotherapeutic agent is radioactive molecule. One of skill in the art can readily identify a chemotherapeutic agent of use (e.g. see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2^nd ed., © 2000 Churchill Livingstone, Inc; Baltzer L, Berkery R (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer DS, Knobf MF, Durivage HJ (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993).

Complementarity-determining region (CDR): The CDRs are three hypervariable regions within each of the variable light (VL) and variable heavy (VH) regions of an antibody molecule that form the antigen-binding surface that is complementary to the three-dimensional structure of the bound antigen. Proceeding from the N-terminus of a heavy or light chain, these complementarity-determining regions are denoted as "CDRl", "CDR2," and "CDR3," respectively. CDRs are involved in antigen- antibody binding, and the CDR3 comprises a unique region specific for antigen- antibody binding. An antigen-binding site, therefore, may include six CDRs, comprising the CDR regions from each of a heavy and a light chain V region. Alteration of a single amino acid within a CDR region can destroy the affinity of an antibody for a specific antigen (see Abbas et al., Cellular and Molecular Immunology, 4th ed. 143-5, 2000). The locations of the CDRs have been precisely defined, e.g., by Kabat et al., Sequences of Proteins of Immunologic Interest, U.S. Department of Health and Human Services, 1983. Cytokine: One type of proteins made by cells that affect the behavior of other cells, such as lymphocytes. Cytokines include the interleukins. In one embodiment, a cytokine is a chemokine, a molecule that affects cellular trafficking.

Dendritic cell (DC): Dendritic cells are the principle antigen presenting cells (APCs) involved in primary immune responses. Dendritic cells include plasmacytoid dendritic cells and myeloid dendritic cells. Their major function is to obtain antigen in tissues, migrate to lymphoid organs and present the antigen in order to activate T cells. Immature dendritic cells originate in the bone marrow and reside in the periphery as immature cells. In one embodiment, a dendritic cell is a plasmacytoid dendritic cell. Plasmacytoid dendritic cells differentiate from precursors called "DC2" while myeloid dendritic cells differentiate from precursors termed "DCl." An agent that specifically binds dendritic cells binds substantially only to dendritic cells and does not substantially bind T cells. In one example, an agent that specifically binds dendritic cells binds mature dendritic cells and does not significantly bind T cells. DCs are capable of evolving from immature, antigen-capturing cells to mature, antigen-presenting, T cell-priming cells; converting antigens into immunogens and expressing molecules such as cytokines, chemokines, costimulatory molecules and proteases to initiate an immune response.

DCs are derived from hematopoietic stem cells in the bone marrow and are widely distributed as immature cells within all tissues, particularly those that interface with the environment (e.g. skin, mucosal surfaces) and in lymphoid organs. Immature DCs are recruited to sites of inflammation in peripheral tissues following pathogen invasion. Chemokine responsiveness and chemokine receptor expression are essential components of the DC recruitment process to sites of inflammation and migration to lymphoid organs. "Immature" DCs may express the chemokine receptors CCRl, CCR2, CCR5, CCR6 and CXCRl. Immature DCs capture antigens by phagocytosis, macropinocytosis or via interaction with a variety of cell surface receptors and endocytosis. Internalization of foreign antigens can subsequently trigger their maturation and migration from peripheral tissues to lymphoid organs. Immature cells that can differentiate into mature dendritic cells. In one embodiment a dendritic cell precursor is a DCl cell that differentiates into myeloid cells (e.g. monocytes). In another embodiment, a dendritic cell precursor is a DC2 cell that differentiates into a plasmacytoid dendritic cell. Plasmacytoid dendritic cells and monocytes are also dendritic cell precursors as they differentiate into mature dendritic cells. The ability of DCs to regulate immunity is dependent on DC differentiation, as it depends on their maturation state. A variety of factors can induce differentiation following antigen uptake and processing within DCs, including: whole bacteria or bacterial-derived antigens (e.g. lipopolysaccharide, LPS), inflammatory cytokines, ligation of select cell surface receptors (e.g. CD40) and viral products (e.g. double- stranded RNA). During their conversion from immature to mature cells, DCs undergo a number of phenotypical and functional changes. The process of DC maturation, in general, involves a redistribution of major histocompatibility complex (MHC) molecules from intracellular endocytic compartments to the DC surface, down-regulation of antigen internalization, an increase in the surface expression of costimulatory molecules, morphological changes (e.g. formation of dendrites), cytoskeleton re-organization, secretion of chemokines, cytokines and proteases, and surface expression of adhesion molecules and chemokine receptors.

Differentiation: The process by which cells become more specialized to perform biological functions, and differentiation is a property that is totally or partially lost by cells that have undergone malignant transformation. For example, dendritic cell precursors such as monocytes or plasmacytoid dendritic cells can differentiate into dendritic cells under the influence of certain cytokines and growth factors.

Epitope: The site on an antigen recognized by an antibody as determined by the specificity of the amino acid sequence. Two antibodies are said to bind to the same epitope if each competitively inhibits (blocks) binding of the other to the antigen as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50:1495-1502, 1990). Alternatively, two antibodies have the same epitope if most amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies are said to have overlapping epitopes if each partially inhibits binding of the other to the antigen, and/or if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

Framework region (FR): Relatively conserved sequences flanking the three highly divergent complementarity-determining regions (CDRs) within the variable regions of the heavy and light chains of an antibody. Hence, the variable region of an antibody heavy or light chain consists of a FR and three CDRs. Some FR residues may contact bound antigen; however, FRs are primarily responsible for folding the variable region into the antigen-binding site, particularly the FR residues directly adjacent to the CDRs. Without being bound by theory, the framework region of an antibody serves to position and align the CDRs. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. A "human" framework region is a framework region that is substantially identical (about 85% or more, usually 90-95% or more) to the framework region of a naturally occurring human immunoglobulin.

Hybridization: The ability of complementary single-stranded DNA, RNA, or DNA/RNA hybrids to form a duplex molecule (also referred to as a hybridization complex). Nucleic acid hybridization techniques can be used to form hybridization complexes between an inhibitory RNA, such as a siRNA, and the gene it is designed to target. In particular examples, the siRNAs have been optimized to target the IL-2 receptor, such as CD25. Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (such as the Na+ concentration) of the hybridization buffer will determine the stringency of hybridization. Calculations regarding hybridization conditions for attaining particular degrees of stringency are discussed in Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, NY (chapters 9 and 11). The following is an exemplary set of hybridization conditions and is not limiting: Very High Stringency (detects sequences that share at least 90% identity)

Hybridization: 5x SSC at 65°C for 16 hours

Wash twice: 2x SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5x SSC at 65°C for 20 minutes each

High Stringency (detects sequences that share at least 80% identity) Hybridization: 5x-6x SSC at 65°C-70°C for 16-20 hours

Wash twice: 2x SSC at RT for 5-20 minutes each

Wash twice: Ix SSC at 55°C-70°C for 30 minutes each

Low Stringency (detects sequences that share at least 50% identity) Hybridization: 6x SSC at RT to 55°C for 16-20 hours Wash at least twice: 2x-3x SSC at RT to 55°C for 20-30 minutes each.

Immune-Mediated Disorder: A disorder in which the immune response plays a key role in the development or progression of the disease. Immune-mediated disorders include autoimmune disorders, allograft rejection and inflammatory conditions. Immune response: A response of a cell of the immune system, such as a B cell, or a T cell, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen- specific response").

A "parameter of an immune response" is any particular measurable aspect of an immune response, including, but not limited to, cytokine secretion (IL-6, IL-10, IFN-γ, etc.), immunoglobulin production, dendritic cell maturation, and proliferation of a cell of the immune system. One of skill in the art can readily determine an increase in any one of these parameters, using known laboratory assays. In one specific non-limiting example, to assess cell proliferation, incorporation of ³H- thymidine can be assessed. A "substantial" increase in a parameter of the immune response is a significant increase in this parameter as compared to a control. Specific, non-limiting examples of a substantial increase are at least about a 50% increase, at least about a 75% increase, at least about a 90% increase, at least about a 100% increase, at least about a 200% increase, at least about a 300% increase, and at least about a 500% increase. Similarly, an inhibition or decrease in a parameter of the immune response is a significant decrease in this parameter as compared to a control. Specific, non-limiting examples of a substantial decrease are at least about a 50% decrease, at least about a 75% decrease, at least about a 90% decrease, at least about a 100% decrease, at least about a 200% decrease, at least about a 300% decrease, and at least about a 500% decrease. A non-paramentric ANOVA can be used to compare differences in the magnitude of the response induced by one agent as compared to the percent of samples that respond using a second agent. In some examples, p <_0.05 is significant, and indicates a substantial increase or decrease in the parameter of the immune response. One of skill in the art can readily identify other statistical assays of use.

Immunoglobulin: A protein including one or more polypeptides substantially encoded by immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha (IgA), gamma (IgG], IgG₂, IgG₃, IgG₄), delta (IgD), epsilon (IgE) and mu (IgM) constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin light chains are generally about 25 Kd or 214 amino acids in length. Full-length immunoglobulin heavy chains are generally about 50 Kd or 446 amino acid in length. Light chains are encoded by a variable region gene at the NH2-terminus (about 110 amino acids in length) and a kappa or lambda constant region gene at the COOH- terminus. Heavy chains are similarly encoded by a variable region gene (about 116 amino acids in length) and one of the other constant region genes.

The basic structural unit of an antibody is generally a tetramer that consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions bind to an antigen, and the constant regions mediate effector functions. Immunoglobulins also exist in a variety of other forms including, for example, Fv, Fab, and (Fab')₂, as well as bifunctional hybrid antibodies and single chains (e.g., Lanzavecchia et al., Eur. J. Immunol. 17:105, 1987; Huston et al., Proc. Natl. Acad. ScL U.S.A., 85:5879-5883, 1988; Bird et al., Science 242:423-426, 1988; Hood et al., Immunology, Benjamin, N.Y., 2nd ed., 1984; Hunkapiller and Hood, Nature 323: 15-16, 1986). An immunoglobulin light or heavy chain variable region includes a framework region interrupted by three hypervariable regions, also called complementarity determining regions (CDR's) (see, Sequences of Proteins of Immunological Interest, E. Kabat et al., U.S. Department of Health and Human Services, 1983). As noted above, the CDRs are primarily responsible for binding to an epitope of an antigen.

Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin variable and constant region genes belonging to different species. For example, the variable segments of the genes from a mouse monoclonal antibody can be joined to human constant segments, such as kappa and gamma 1 or gamma 3. In one example, a therapeutic chimeric antibody is thus a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody (e.g., ATCC Accession No. CRL 9688 secretes an anti-Tac chimeric antibody), although other mammalian species can be used, or the variable region can be produced by molecular techniques. Methods of making chimeric antibodies are well known in the art, e.g., see U.S. Patent No. 5,807,715, which is herein incorporated by reference.

A "humanized" immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the

CDRs is termed a "donor" and the human immunoglobulin providing the framework is termed an "acceptor." In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A "humanized antibody" is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary conservative substitutions are those such as gly, ala; val, ile, leu; asp, glu; asn, gin; ser, thr; lys, arg; and phe, tyr (see U.S. Patent No. 5,585,089, which is incorporated herein by reference). Humanized immunoglobulins can be constructed by means of genetic engineering, e.g., see U.S. Patent No. 5,225,539 and U.S. Patent No. 5,585,089, which are herein incorporated by reference. A human antibody is an antibody wherein the light and heavy chain genes are of human origin. Human antibodies can be generated using methods known in the art. Human antibodies can be produced by immortalizing a human B cell secreting the antibody of interest. Immortalization can be accomplished, for example, by EBV infection or by fusing a human B cell with a myeloma or hybridoma cell to produce a trioma cell. Human antibodies can also be produced by phage display methods (see, e.g., Dower et al., PCT Publication No. WO91/17271; McCafferty et al., PCT Publication No. WO92/001047; and Winter, PCT Publication No. WO92/20791, which are herein incorporated by reference), or selected from a human combinatorial monoclonal antibody library (see the Morphosys website). Human antibodies can also be prepared by using transgenic animals carrying a human immunoglobulin gene (e.g., see Lonberg et al., PCT Publication No. WO93/12227; and Kucherlapati, PCT Publication No. WO91/10741, which are herein incorporated by reference).

Inhibition (selective): An antagonist of CD25 that selectively inhibits CD25 activity on dendritic cells affects antigen presentation by mature dendritic cells, and thus inhibits differentiation of effector T cells and/or expansion of NK cells. An antagonist of CD25 that selectively inhibits CD25 activity on T lymphocytes affects at least one function specific to T cells, and does not affect antigen presentation by mature dendritic cells to T cells. These antagonists can be composite molecules, such as an antibody that specifically binds dendritic cells (but not T cells) covalently linked to a CD25 antagonist, or an antibody that specifically binds T cells (but not dendritic cells) covalently linked to a CD25 antagonist.

An agent that selectively inhibits CD25 on dendritic cells as compared to inhibition on T cells has a substantially greater effect on dendritic cells than T cells. In some embodiments, the agent selectively inhibits CD25 on dendritic cells, and thus the agent inhibits CD25 on dendritic cells about 2- fold, 5-fold, 10-fold, 20- fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or 100-fold or more than the agent inhibits CD25 on T cells. An agent that selectively inhibits CD25 on T cells as compared to dendritic cells has a substantially greater effect on T cells than dendtric cells. In some embodiments, the agent selectively inhibits CD25 on T cells, and thus selectively inhibits CD25 on T cells about 2-fold, 5-fold, 10-fold, 20- fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or 100-fold or more than the agent inhibits CD25 on dendritic cells.

Inhibitory RNA: An RNA molecule or multiple RNA molecules that can inhibit the expression of a target gene in a cell, such as when introduced in a cell, for example, a white blood cell or bone marrow cell of a subject. Generally, inhibitory RNAs hybridize to a target nucleic acid or the complement thereof and decrease of the target gene expression. Examples of inhibitory RNAs that can be used in the methods provided herein are siRNAs, miRNAs, shRNAs and ribozymes.

Interfering with or inhibiting (expression of a target gene): This phrase refers to the ability of a molecule, such as an inhibitory RNA (for example a siRNA) to measurably reduce the expression of a target gene, for example the interleukin-2 receptor. It contemplates reduction of the end-product of the gene, for example the expression or function of the encoded protein, and thus includes reduction in the amount or longevity of the mRNA transcript. It is understood that the phrase is relative, and does not require absolute suppression of the gene. Thus, in certain embodiments, interfering with or inhibiting gene expression of a target gene requires that, following contact with an inhibitory RNA that targets the gene that the gene is expressed at least 5% less than prior to application, such as at least 10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at least 35% less, at least 40% less, at least 45% less, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less or even more reduced. Thus, in some particular embodiments, application of an inhibitory RNA reduces expression of the target tyrosine kinase by about 30%, about 40%, about 50%, about 60%, or more. In specific examples, where the inhibitory RNA is particularly effective, expression is reduced by about 70%, about 80%, about 85%, about 90%, about 95%, or even more.

Interleukin 2 (IL-2): A protein of 133 amino acids (15.4 kDa) with a slightly basic pi that does not display sequence homology to any other factors and is a cytokine. Murine and human IL-2 display a homology of approximately 65%. IL- 2 is synthesized as a precursor protein of 153 amino acids with the first 20 amino terminal amino acids functioning as a hydrophobic secretory signal sequence. The protein contains a single disulfide bond (positions Cys58/105) essential for biological activity. The human IL-2 gene contains four exons and maps to human chromosome 4q26-28 (murine chromosome 3). The biological activities of IL-2 are mediated by a membrane receptor that is expressed on activated, but not on resting, T cells and natural killer (NK) cells. Activated B cells and resting mononuclear leukocytes also rarely express this receptor.

Interleukin-2 (IL-2) receptor: A cellular receptor that binds IL-2 and mediates its biological effects. Three different types of IL-2 receptors are distinguished that are expressed differentially and independently. The high affinity IL-2 receptor (K_d -10 pM) constitutes approximately 10% of all IL-2 receptors expressed by cells. This receptor is a membrane receptor complex consisting of the two subunits: IL-2R-alpha (also known as T cell activation (TAC) antigen or p55) and IL-2R-beta (also known as p75 or CD122). An intermediate affinity IL-2 receptor (K_d =100 pM) consists of the p75 subunit and a gamma chain, while a low affinity receptor (K_d =10 nM) is formed by p55 alone. p75 is 525 amino acids in length. It has an extracellular domain of 214 amino acids and a cytoplasmic domain of 286 amino acids. The p75 gene maps to human chromosome 22qll. 2-ql2, contains 10 exons and has a length of approximately 24 kb. p55 is 251 amino acids in length with an extracellular domain of 219 amino acids and a very short cytoplasmic domain of 13 amino acids. The gene encoding p55 maps to human chromosome 10pl4-pl5. p75 is expressed constitutively on resting T-lymphocytes, NK cells, and a number of other cell types while the expression of p55 is usually observed only after activation. Activated lymphocytes continuously secrete a 42 kDa fragment of p55 (TAC antigen). This fragment circulates in the serum and plasma and functions as a soluble IL2 receptor (see Smith, Ann. Rev. Cell Biol. 5:397-425, 1989; Taniguchi and Minami, Cell 73:5-8, 1993). p55 (also known as CD25) has a length of 251 amino acids with an extracellular domain of 219 amino acids an a very short cytoplasmic domain of 13 amino acids. The p55 gene maps to human chromosome 10pl4-pl5. The expression of p55 is regulated by a nuclear protein called RPT-I.

A third 64 kDa subunit of the IL2 receptor, designated gamma, has been described. This subunit is required for the generation of high and intermediate affinity IL-2 receptors but does not bind IL-2 by itself. The gene encoding the gamma subunit of the IL2 receptor maps to human chromosome XqI 3, spans approximately 4.2 kb and contains eight exons.

Isolated: An "isolated" biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins which have been "isolated" thus include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

Leukocyte: Cells in the blood, also termed "white cells," that are involved in defending the body against infective organisms and foreign substances. Leukocytes are produced in the bone marrow. There are 5 main types of white blood cell, subdivided between 2 main groups: polymorphomnuclear leukocytes (neutrophils, eosinophils, basophils) and mononuclear leukocytes (monocytes and lymphocytes). When an infection is present, the production of leukocytes increases.

Mammal: This term includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects.

Maturation: The process in which an immature cell, such as dendritic cell precursor, changes in form or function to become a functionally mature dendritic cell, such as an antigen presenting cell (APC).

Mobilization Agent: A compound such as a naturally occurring protein or a derivative thereof, that acts on hematopoietic progenitor or stem cells to mobilize precursor cells. A mobilizing agent causes DC precursors to migrate from their tissue of origin such as the bone marrow, and move into other tissues and the peripheral blood. Mobilization agents include, but are not limited to, FLT-3 ligand or GM-CSF. Monoclonal antibody: An antibody produced by a single clone of B- lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. Multiple sclerosis: An autoimmune disease classically described as a central nervous system white matter disorder disseminated in time and space that presents as relapsing-remitting illness in 80-85% of patients. Diagnosis can be made by brain and spinal cord magnetic resonance imaging (MRI), analysis of somatosensory evoked potentials, and analysis of cerebrospinal fluid to detect increased amounts of immunoglobulin or oligoclonal bands. MRI is a particularly sensitive diagnostic tool. MRI abnormalities indicating the presence or progression of MS include hyperintense white matter signals on T2-weighted and fluid attenuated inversion recovery images, gadolinium enhancement of active lesions, hypointensive "black holes" (representing gliosis and axonal pathology), and brain atrophy on Tl-weighted studies. Serial MRI studies can be used to indicate disease progression.

Relapsing-remitting multiple sclerosis is a clinical course of MS that is characterized by clearly defined, acute attacks with full or partial recovery and no disease progression between attacks.

Secondary-progressive multiple sclerosis is a clinical course of MS that initially is relapsing-remitting, and then becomes progressive at a variable rate, possibly with an occasional relapse and minor remission.

Primary progressive multiple sclerosis presents initially in the progressive form.

Neoplasm: An abnormal cellular proliferation, which includes benign and malignant tumors, as well as other proliferative disorders.

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs, such as, for example and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term

"oligonucleotide" typically refers to short polynucleotides, generally no greater than about 100 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T." An RNA/DNA hybrid can have any combination of ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. The term polynucleotide or nucleic acid sequence refers to a polymeric form of nucleotide at least 10 bases in length. A recombinant polynucleotide includes a polynucleotide that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (such as a cDNA) independent of other sequences. The nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single- and double- stranded forms of DNA.

The nucleic acid molecule can be double stranded (ds) or single stranded (ss). Where single stranded, the nucleic acid molecule can be the sense strand or the antisense strand. Nucleic acid molecules can include natural nucleotides (such as A, T/U, C, and G), and can also include analogs of natural nucleotides. In some examples a nucleic acid molecule is an inhibitory RNA, such as a siRNA molecule, that has been optimized to target a gene encoding CD25.

Nucleotide: The fundamental unit of nucleic acid molecules. A nucleotide includes a nitrogen-containing base attached to a pentose monosaccharide with one, two, or three phosphate groups attached by ester linkages to the saccharide moiety.

The major nucleotides of DNA are deoxyadenosine 5 '-triphosphate (dATP or A), deoxyguanosine 5'-triphosphate (dGTP or G), deoxycytidine 5'-triphosphate (dCTP or C) and deoxythymidine 5 '-triphosphate (dTTP or T). The major nucleotides of RNA are adenosine 5 '-triphosphate (ATP or A), guanosine 5'- triphosphate (GTP or G), cytidine 5 '-triphosphate (CTP or C) and uridine 5'- triphosphate (UTP or U).

Nucleotides include those nucleotides containing modified bases, modified sugar moieties and modified phosphate backbones, for example as described in U.S. Patent No. 5,866,336 to Nazarenko et al. Examples of modified base moieties which can be used to modify nucleotides at any position on its structure include, but are not limited to: 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N~6-sopentenyladenine, 1-methylguanine, 1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3- methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, methoxyarninomethyl-2-thiouracil, beta-D- mannosylqueosine, S'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio- N6-isopentenyladenine, uracil-5-oxyacetic acid, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil- 5-oxyacetic acid methylester, uracil-S-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3- amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine amongst others. Examples of modified sugar moieties, which may be used to modify nucleotides at any position on its structure, include, but are not limited to arabinose, 2-fluoroarabinose, xylose, and hexose, or a modified component of the phosphate backbone, such as phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, or an alkyl phosphotriester or analog thereof.

Oligonucleotide or "oligo": Multiple nucleotides (i.e. molecules comprising a sugar (e.g. ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (Py) (e.g. cytosine (C), thymine (T) or uracil (U)) or a substituted purine (Pu) (e.g. adenine (A) or guanine (G)). The term "oligonucleotide" as used herein refers to both oligoribonucleotides (ORNs) and oligodeoxyribonucleotides (ODNs). The term "oligonucleotide" also includes oligonucleosides (i.e. an oligonucleotide minus the phosphate) and any other organic base polymer. Oligonucleotides can be obtained from existing nucleic acid sources (e.g. genomic or cDNA), but are preferably synthetic (e.g. produced by oligonucleotide synthesis).

A "stabilized oligonucleotide" is an oligonucleotide that is relatively resistant to in vivo degradation (for example via an exo- or endo-nuclease). In one embodiment, a stabilized oligonucleotide has a modified phosphate backbone. One specific, non-limiting example of a stabilized oligonucleotide has a phophorothioate modified phosphate backbone (wherein at least one of the phosphate oxygens is replaced by sulfur). Other stabilized oligonucleotides include: nonionic DNA analogs, such as alkyl- and aryl- phophonates (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), phophodiester and alkylphosphotriesters, in which the charged oxygen moiety is alkylated. Oligonucleotides which contain a diol, such as tetraethyleneglycol or hexaethyleneglycol, at either or both termini have also been shown to be substantially resistant to nuclease degradation. Polypeptide: A polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha- amino acids, either the L-optical isomer or the D-optical isomer can be used, the L- isomers being preferred. The terms "polypeptide" or "protein" as used herein is intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term "polypeptide" is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.

The term "fragment" refers to a portion of a polypeptide that is at least 8, 10, 15, 20 or 25 amino acids in length. The term "functional fragments of a polypeptide" refers to all fragments of a polypeptide that retain an activity of the polypeptide (e.g., the binding of an antigen). Biologically functional fragments, for example, can vary in size from a polypeptide fragment as small as an epitope capable of binding an antibody molecule to a large polypeptide capable of participating in the characteristic induction or programming of phenotypic changes within a cell. The term "soluble" refers to a form of a polypeptide that is not inserted into a cell membrane.

Pharmaceutical agent or drug: A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject. Pharmaceutical agents include, but are not limited to, chemotherapeutic agents and anti-infective agents. Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers useful in this invention are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the fusion proteins herein disclosed.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Preventing or treating a disease: "Preventing" a disease refers to inhibiting the full development of a disease, for example in a person who is known to have a predisposition to a disease such as an autoimmune disorder. An example of a person with a known predisposition is someone with a history of multiple sclerosis in the family, or who has been exposed to factors that predispose the subject to a condition. "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.

Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its natural environment within a cell. Preferably, a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation. Small inhibitory RNA (siRNA): A short sequence of RNA molecule capable of RNA interference or "RNAi." (See, for example, Bass Nature 411: 428- 429, 2001; Elbashir et al, Nature 411: 494-498, 2001; and Kreutzer et al, PCT Publication No. WO 00/44895; Zernicka-Goetz et al, PCT Publication No. WO 01/36646; Fire, PCT Publication No. WO 99/32619; Plaetinck et al, PCT Publication No. WO 00/01846; Mello and Fire, PCT Publication No. WO 01/29058; Deschamps-Depaillette, PCT Publication No. WO 99/07409; and Li et al , PCT Publication No. WO 00/44914.) As used herein, siRNA molecules need not be limited to those molecules containing only RNA, but further encompasses chemically modified nucleotides and non-nucleotides having RNAi capacity or activity. In some embodiments, and siRNA is used to silence gene expression, for example the expression of CD25.

Specific binding agent: An agent that binds substantially only to a defined target. Thus an IL-2 receptor- specific binding agent binds substantially only the IL- 2 receptor, or a component thereof. As used herein, the term "IL-2 receptor-specific binding agent" includes anti-IL-2 receptor antibodies and other agents that bind substantially only to an IL-2 receptor or a component thereof (e.g., p55, p75).

Anti-IL-2 receptor antibodies may be produced using standard procedures described in a number of texts, including Harlow and Lane {Using Antibodies, A Laboratory Manual, CSHL, New York, 1999, ISBN 0-87969-544-7). In addition, certain techniques may enhance the production of neutralizing antibodies (U.S. Patent No. 5,843,454; U.S. Patent No. 5,695,927; U.S. Patent No. 5,643,756; and U.S. Patent No. 5,013,548). The determination that a particular agent binds substantially only to an IL-2 receptor component may readily be made by using or adapting routine procedures. One suitable in vitro assay makes use of the Western blotting procedure (described in many standard texts, including Harlow and Lane, 1999). Western blotting may be used to determine that a given protein binding agent, such as an anti-IL-2 receptor monoclonal antibody, binds substantially only to the IL-2 receptor. Antibodies to the IL-2 receptor are well known in the art.

Shorter fragments of antibodies can also serve as specific binding agents. For instance, Fabs, Fvs, and single-chain Fvs (SCFvs) that bind to an IL-2 receptor would be IL-2 receptor-specific binding agents. Symptom and sign: Any subjective evidence of disease or of a subject's condition, i.e., such evidence as perceived by the subject; a noticeable change in a subject's condition indicative of some bodily or mental state. A "sign" is any abnormality indicative of disease, discoverable on examination or assessment of a subject. A sign is generally an objective indication of disease. Signs include, but are not limited to any measurable parameters such as tests for immunological status or the presence of lesions in a subject with multiple sclerosis.

T Cell: A white blood cell critical to the immune response. T cells include, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ T lymphocyte is an immune cell that carries a marker on its surface known as cluster of differentiation 4 (CD4). These cells, classically known as helper T cells (Th cells), help orchestrate the immune response, including antibody responses as well as killer T cell responses. CD8⁺ T cells carry the cluster of differentiation 8 (CD8) marker. In one embodiment, CD8 T cells are cytotoxic T lymphocytes (Tc cells) which are capable of lysing target cells by direct cell contact. These cells play a role in the elimination of virus-infected cells and tumor cells, and are involved in transplant rejection processes. In another embodiment, a CD8 cell is a suppressor T cell. Mature T cells express CD3. An agent that specifically binds T cells binds substantially only to T cells and does not substantially bind dendrictic cells. Therapeutically effective dose: A dose sufficient to prevent advancement, or to cause regression of the disease, or which is capable of relieving symptoms caused by the disease, such as pain or swelling.

Tumor: An abnormal growth of cells, which can be benign or malignant. Cancer is a malignant tumor, which is characterized by abnormal or uncontrolled cell growth. Other features often associated with malignancy include metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels and suppression or aggravation of inflammatory or immunological response, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc. "Metastatic disease" refers to cancer cells that have left the original tumor site and migrate to other parts of the body for example via the bloodstream or lymph system. The amount of a tumor in an individual is the "tumor burden" which can be measured as the number, volume, or weight of the tumor. A tumor that does not metastasize is referred to as "benign." A tumor that invades the surrounding tissue and/or can metastasize is referred to as "malignant." Examples of hematological tumors include leukemias, including acute leukemias (such as 1 Iq23-positive acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblasts, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular breast carcinoma), lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyrgioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma).

Tumor antigens (TAs): A antigen expressed on a tumor which can stimulate tumor- specific T-cell-defined immune responses. Exemplary TAs include, but are not limited to, RAGE-I, tyrosinase, MAGE-I, MAGE-2, NY-ESO-I, Melan- A/MART-1, glycoprotein (gp) 75, gplOO, beta-catenin, PRAME, MUM-I, WT-I, CEA, and PR-I. Additional TAs are known in the art (for example see Novellino et al., Cancer Immunol. Immunother. 2004 Aug 7 [Epub ahead of print]) and includes TAAs not yet identified. Uveal tract: The uveal tract is composed of three parts, the iris, the ciliary body, and the choroid. It is the middle, vascular layer of the eye, protected externally by the cornea and the sclera. It contributes to the blood supply of the retina.

The iris is the anterior section of the ciliary body. It has a relatively flat surface with an aperture in the middle called the pupil. The iris lies in contact with the lens and divides the anterior chamber from the posterior chamber. The function of the iris is to control the amount of light that enters the eye.

The ciliary body extends forward from the anterior termination of the choroid to the root of the iris. It is composed of two zones, the pars plicata and the pars plana. There are two layers of epithelium in the ciliary body, the external pigmented and an internal non-pigmented layer. The ciliary body forms the root of the iris and governs the size of the lens. Aqueous humor is secreted by the ciliary processes into the posterior chamber of the eye.

The choroid is the posterior portion of the uveal tract and the middle part of the eye, which lies between the retina and the sclera. It is largely composed of blood vessels. The function of the choroid is to nourish the outer portion of the underlying retina.

Uveitus: An intraocular inflammatory disease that includes iritis, cyclitis, panuveits, posterior uveitis and anterior uveitis. Iritis is inflammation of the iris. Cyclitis is inflammation of the ciliary body. Panuveitis refers to inflammation of the entire uveal (vascular) layer of the eye. Intermediate uveitis, also called peripheral uveitis, is centered in the area immediately behind the iris and lens in the region of the ciliary body and pars plana, and is also termed "cyclitis" and "pars planitis." "Posterior" uveitis generally refers to chorioretinitis (inflammation of the choroid and retina). Posterior uveitis can give rise to diverse symptoms but most commonly causes floaters and decreased vision similar to intermediate uveitis. Signs include cells in the vitreous humor, white or yellow-white lesions in the retina and/or underlying choroid, exudative retinal detachments, retinal vasculitis, and optic nerve edema.

Anterior uveitis refers to iridocyclitis (inflammation of the iris and the ciliary body) and/or iritis. Anterior uveitis tends to be the most symptomatic, typically presenting with pain, redness, photophobia, and decreased vision. Signs of anterior uveitis include pupillary miosis and injections of the conjunctiva adjacent to the cornea, so-called perilimbal flush. Biomicroscopic, or slit lamp, findings include cells and flare in the aqueous humor as well as keratic precipitates, which are clumps of cells and proteinaceous material adherent to the corneal endothelium. "Diffuse" uveitis implies inflammation involving all parts of the eye, including anterior, intermediate, and posterior structures.

"Acute" uveitis is a form of uveitis in which signs and symptoms occur suddenly and last for up to about six weeks. "Chronic" uveitis is a form in which onset is gradual and lasts longer than about six weeks.

The inflammatory products (i.e., cells, fibrin, excess proteins) of ocular inflammation are commonly found in the fluid spaces of the eye, i.e., anterior chamber, posterior chamber and vitreous space as well as infiltrating the tissue imminently involved in the inflammatory response. Uveitis may occur following surgical or traumatic injury to the eye; as a component of an autoimmune disorder (such as rheumatoid arthritis, Bechet's disease, ankylosing spondylitis, sarcoidosis), as an isolated immune mediated ocular disorder (such as pars planitis or iridocyclitis), as a disease unassociated with known etiologies, and following certain systemic diseases which cause antibody-antigen complexes to be deposited in the uveal tissues. Uveitis includes ocular inflammation associated with Bechet's disease, sarcoidosis, Vogt-Koyanagi-Harada syndrome, birdshot chorioretinopathy and sympathetic ophthalmia. Thus, non-infectious uveitis occurs in the absence of an infectious agent.

A wide variety of infective agents can also cause uveitis. When an infective etiology has been diagnosed, an appropriate antimicrobial drug can be given to cure the disease. However, the etiology of uveitis remains elusive in the majority of cases.

ZENAPAX® (daclizumab): A particular recombinant, humanized monoclonal antibody of the human IgGl isotype that specifically binds Tac (p55). The recombinant genes encoding ZENAPAX® are a composite of human (about 90%) and murine (about 10%) antibody sequences. The donor murine anti-Tac antibody is an IgG2a monoclonal antibody that specifically binds the IL-2R Tac protein and inhibits IL-2-mediated biologic responses of lymphoid cells. The murine anti-Tac antibody was "humanized" by combining the complementarity- determining regions and other selected residues of the murine anti-TAC antibody with the framework and constant regions of the human IgGl antibody. The humanized anti-Tac antibody daclizumab is described and its sequence is set forth in U.S. Patent No. 5,530,101, see SEQ ID NO: 5 and SEQ ID NO: 7 for the heavy and light chain variable regions respectively. U.S. Patent No. 5,530,101 and Queen et al., Proc. Natl. Acad. ScL 86:1029-1033, 1989 are both incorporated by reference herein in their entirety. Daclizumab inhibits IL-2-dependent antigen-induced T cell proliferation and the mixed lymphocyte response (MLR) (Junghans et al., Cancer Research 50:1495-1502, 1990), as can other antibodies of use in the methods disclosed herein. ZENAPAX® has been approved by the U.S. Food and Drug Administration

(FDA) for the prophylaxis of acute organ rejection in subjects receiving renal transplants, as part of an immunosuppressive regimen that includes cyclosporine and coritcosteroids. ZENAPAX® has been shown to be active in the treatment of human T cell lymphotrophic virus type 1 associated myelopathy/topical spastic paraparesis (HAM/TSP, see Lehky et al., Ann. Neuro., 44:942-947, 1998). The use of ZENAPAX® to treat posterior uveitis has also been described (see Nussenblatt et al., Proc. Natl. Acad. ScL, 96:7462-7466, 1999).

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Compositions Compositions are provided herein that selectively inhibit CD25 on T cells or dendritic cells. In some embodiments, the composition that selectively inhibits CD25 on T cells or dendritic cells is a composite molecule including an agent that specifically binds either dendritic cells or T cells, respectively, and a CD25 receptor antagonist. A composition that includes an agent that selective inhibits CD25 on dendtric cells or T cells are of use for treating an immune-mediated disorder or a tumor, respectively.

CD25 Antagonists

A variety of CD25 antagonists are of use in the compositions and methods disclosed herein. In one specific non-limiting example, the IL-2 receptor antagonist is an antibody, such as a monoclonal antibody, for example, a chimeric, humanized or human monoclonal antibody. A specific example of a humanized monoclonal antibody that specifically binds CD25 (p55) is daclizumab, which is described and its sequence is set forth in U.S. Patent No. 5,530,101, which is incorporated by reference herein, and in Queen et al., Proc. Natl. Acad. ScL 86:1029-1033, 1989. Thus, the antibody can be a humanized immunoglobulin having complementarity determining regions (CDRs) from a donor immunoglobulin and heavy and light chain variable region frameworks from human acceptor immunoglobulin heavy and light chain frameworks, wherein the humanized immunoglobulin specifically binds to a human interleukin-2 receptor with an affinity constant of at least 10⁸ M^"1. The sequence of the humanized immunoglobulin heavy chain variable region framework can be at least 65% identical to the sequence of the donor immunoglobulin heavy chain variable region framework. A specific example of the variable region of the anti-Tac antibody is set forth as SEQ ID NO: 1 and SEQ ID NO: 3 of U.S. Patent No. 5,520,101 (light and heavy chain, respectively), and the variable region of the humanized anti-Tac antibody daclizumab is set forth as SEQ ID NO: 5 and SEQ ID NO: 7 (heavy and light chain, respectively) of U.S. Patent No. 5,530,101, which is herein incorporated by reference. The antibody can be a functional fragment of an antibody, such as a Fab, Fab', (Fab')₂, Fv, or scFv that specifically binds CD25. The antibody can also be a single chain antibody (SCA). The antibody can include two light chain/heavy chain dimers, and specifically binds to either p55 (such as the anti-Tac antibody). I1-2R antagonists of use include agents that bind specifically to p55 (also known as the alpha chain or Tac subunit) of the human IL- 2R. In one example, the agent is a monoclonal antibody, such as daclizumab, basiliximab, BT563, and 7G8 or their chimeric or humanized forms. The agent can also be a human antibody, or a humanized antibody with synthetic CDRs that specifically binds p55. Antibodies that bind the same (or overlapping) epitope as daclizumab or basiliximab can also be used in the methods disclosed herein. In other embodiments, the antibody will have high sequence identity with daclizumab or basiliximab, at least 90 or 95%, such as at least 98% or 99% sequence identity, while retaining the functional properties of the antibody, i.e., its antagonist properties to the IL-2R. The antibody may be of any isotype, but in several embodiment that antibody is an IgG, including but not limited to, IgGi, IgG₂, IgG₃ and IgG₄.

In other embodiments the antibody is basiliximab, marketed as SIMULECT® by Novartis Pharma AG. SIMULECT® is a chimeric

(murine/human) monoclonal antibody (IgG]K), produced by recombinant DNA technology, that functions as an immunosuppressive agent, specifically binding to and blocking the alpha chain of the IL-2R on the surface of activated T- lymphocytes. SIMULECT® is a glycoprotein obtained from fermentation of an established mouse myeloma cell line genetically engineered to express plasmids containing the human heavy and light chain constant region genes and mouse heavy and light chain variable region genes encoding the RFT5 antibody that binds selectively to the IL-2R(alpha). Based on the amino acid sequence, the calculated molecular weight of the protein is 144 kilodaltons. The antibody also can be HUMAX-TAC®. The CD25 antagonist may also be a functional fragment of an antibody (e.g., a chimeric, humanized, or human antibody) such as an Fab, (Fab')₂, Fv, or scFv that specifically bind p55. Further, the fragment may be pegylated to increase its half-life.

The CD25 antagonist can also be an inhibitory RNA that targets CD25, and reduces the expression of CD25 in cells contacted with the inhibitory RNAs. Generally, the principle behind inhibitory RNA technology is that an inhibitory RNA hybridizes to a target nucleic acid and effects the modulation of gene expression activity, or function, such as transcription, translation or splicing. The modulation of gene expression can be achieved by, for example, target RNA degradation or occupancy-based inhibition. An example of modulation of target RNA function by degradation is RNase H-based degradation of the target RNA upon hybridization with a DNA-like inhibitory RNA. Inhibitory RNA can also be used to modulate gene expression, such as splicing, by occupancy-based inhibition, such as by blocking access to splice sites.

Another example of inhibitory RNA modulation of gene expression by target degradation is RNA interference (RNAi) using small interfering RNAs (siRNAs). RNAi is a form of antisense-mediated gene silencing involving the introduction of RNA-like oligonucleotides leading to the sequence-specific reduction of targeted endogenous mRNA levels. Another type of inhibitory RNA that utilizes the RNAi pathway is a microRNA. MicroRNAs are naturally occurring RNAs involved in the regulation of gene expression. However, these compounds can be synthesized to regulate gene expression via the RNAi pathway. Similarly, shRNAs are RNA molecules that form a tight hairpin turn and can be used to silence gene expression via the RNAi pathway. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA.

Other compounds that are often classified as inhibitory RNAs are ribozymes. Ribozymes are catalytic RNA molecules that can bind to specific sites on other RNA molecules and catalyze the hydrolysis of phosphodiester bonds in the RNA molecules. Ribozymes modulate gene expression by direct cleavage of a target nucleic acid, such as a messenger RNA.

Each of the above-described inhibitory RNAs provides sequence-specific target gene regulation. This sequence-specificity makes inhibitory RNAs effective tools for the selective modulation of a target nucleic acid of interest, such as human CD25 and can be used in the disclosed methods. To target CD25 any type of inhibitory RNAs that specifically target and regulate expression are contemplated for use with the disclosed methods. Such inhibitory RNAs include siRNAs, miRNAs, shRNAs and ribozymes. Methods of designing, preparing and using inhibitory RNAs that specifically target CD25 are within the abilities of one of skill in the art, and are commercially available. In some embodiments of the methods disclosed herein, the subject is human and the siRNA is specific for human CD25. Inhibitory RNAs can be prepared by designing compounds that are complementary to the CD25 nucleotide sequences, for example the nucleic acid sequences given by the GENB ANK® Accession No. NM_000417 (October 12, 2008), which is incorporated herein by reference.

Inhibitory RNAs targeting CD25 need not be 100% complementary to specifically hybridize and regulate expression the target gene. For example, the inhibitory RNA, or antisense strand of the compound if a double- stranded compound, can be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% complementary to the CD25 nucleic acid sequence are a portion thereof, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the nucleic acid sequences or a portion thereof, such as between about 10 and about 100 nucleotides, given by the GENBANK® Accession No. NM 000417. In some examples, the inhibitory RNA is between about 10 and about 100 nucleotides in length for example , such as about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 nucleotides in length, for example about 10 to about 30, about 20 to about 50, about 40 to about 70, about 50 to about 90, or about 70 to about 100 nucleotides in length.

The CD 25 antagonist can also be a small molecule inhibitor. Small molecule inhibitors include those disclosed in Braisted et al., J. Am. Chem. Soc. 125: 3714-3715, 2003, incorporated herein by reference. For example, the CD25 inhibitor can have one of the structures set forth as:

4

For example, the small molecule inhibitor can be Ro26-4550 (compound 1 above). In additional examples, the small molecule inhibitor is related to Compound 2 above, for example (IC50 indicated):

Additional small molecule inhibitors of CD25 are known in the art (see Tilley et al., J. Am. Chem. Soc. 119: 7589-7590, 1997, incorporated by reference herein). For example, there are a number of acylphenylalanine derivatives that were designed to complex with the IL-2 receptor by emulating residues R38 and F42 of the IL-2 ligand. In one embodiment, these acylphenylalanine derivatives have an IC₅₀ for the IL-2 receptor of at least about 3 μM, such as about 3 μM to about 500 μM, such as about 30 μM to about 400 μM, or at least about 40 μM. Exemplary small molecule inhibitors are apamin (CNCKPET ALCARRCQQH (SEQ ID NO: 2)) and apa-IL-2 (CNCKAPETKLCRMLCFKFYM (SEQ ID NO: I)). There are a number of known derivatives of apa-IL-2 that are effective as CD25 antagonists, including a 2,6-dichlorobenzyl-tyrosine derivative and an alanine; 2, 6- dichlorobenzyl-tyrosine derivative (see Emerson et al., Protein Science 12: 811-822, 2003, herein incorporated by reference, which describes small molecule CD25 antagonists and derivatives of use). Other derivatives and stabilized polypeptides can also be of use.

Peptides can be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified peptides, and optionally having other desirable properties. For example, carboxylic acid groups of the protein, whether carboxyl-terminal or side chain, may be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form a C₁-C₁₆ ester, or converted to an amide of formula NR]R₂ wherein R] and R₂ are each independently H or C₁-C₁₆ alkyl, or combined to form a heterocyclic ring, such as a 5- or 6- membered ring. Amino groups of the peptide, whether amino-terminal or side chain, may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or may be modified to Ci-Ci₆ alkyl or dialkyl amino or further converted to an amide.

Hydroxyl groups of the peptide side chains can be converted to Ci-Ci₆ alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl and phenolic rings of the peptide side chains can be substituted with one or more halogen atoms, such as F, Cl, Br or I, or with Ci-Ci₆ alkyl, Ci-Ci₆ alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids. Methylene groups of the peptide side chains can be extended to homologous C₂-C₄ alkylenes. Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups. Those skilled in the art will also recognize alternative methods for introducing a cyclic structures into the peptides of this disclosure to select and provide conformational constraints to the structure that result in enhanced stability. For example, a C- or N-terminal cysteine can be added to the peptide, so that when oxidized the peptide will contain a disulfide bond, generating a cyclic peptide. Other peptide cyclizing methods include the formation of thioethers and carboxyl- and amino-terminal amides and esters. Peptidomimetic and organomimetic embodiments are also within the scope of the present disclosure, whereby the three-dimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three- dimensional arrangement of the peptide backbone and component amino acid side chains, resulting in such peptido- and organomimetics of the proteins of this disclosure. For computer modeling applications, a pharmacophore is an idealized, three-dimensional definition of the structural requirements for biological activity. Peptido- and organomimetics can be designed to fit each pharmacophore with current computer modeling software (using computer assisted drug design or CADD). See Walters, "Computer-Assisted Modeling of Drugs", in Klegerman & Groves, eds., 1993, Pharmaceutical Biotechnology, Interpharm Press: Buffalo Grove, IL, pp. 165-174 and Principles of Pharmacology Munson (ed.) 1995, Ch. 102, for descriptions of techniques used in CADD.

Agents that Selectively Inhibit CD25 on Dendritic Cells or T Cells

In one embodiment, an agent that selectively inhibits CD25 on T cells or dendritic cells has this property inherently. For example, the agent can specifically bind CD25 on T cells and not specifically bind CD25 on dendritic cells. The agent can specifically bind CD25 on dendritic cells, and not specifically bind CD25 on T cells. In other embodiments, agents that selectively inhibit CD25 on dendritic cells or T cells can be composite molecules of a CD25 antagonist and an agent.

In some embodiments, the agent selectively inhibits CD25 on dendritic cells, and thus the agent inhibits CD25 on dendritic cells at least about 2-fold, 5-fold, 10- fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or 100-fold more than the agent inhibits CD25 on T cells. In other embodiments, the agent selectively inhibits CD25 on T cells, and thus selectively inhibits CD25 on T cells at least about 2-fold 5 -fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or 100-fold more than the agent binds dendritic cells.

In one example, an agent that selectively inhibits CD25 on dendritic cells inhibits CD25 on dendritic cells, but does not inhibit CD25 on T cells. This agent can selectively bind CD25 on dendritic cells, but does not bind CD25 on T cells. In another example, an agent that selectively inhibits CD25 on T cells inhibits CD25 on T cells, but does not inhibit CD25 on dendritic cells. This agent can selectively bind CD25 on T cells, but does not bind CD25 on dendritic cells.

Composite molecule of use can include an agent that specifically binds dendritic cells or T cells and a separate CD25 antagonist. Suitable agents to target either T cells or dendritic cells are well known in the art. These agents include antibodies and other molecules, such as lectins, that selectively inhibit CD25 on either dendritic cells or T cells In some embodiments, the composite molecule includes an agent that specifically binds dendritic cells. In some examples, an agent that binds dendritic cells is an antibody that specifically binds an antigen expressed on dendritic cells. For example, the antigen can be expressed on dendritic cells but not on T cells. Suitable exemplary antibodies that specifically bind dendritic cells include DEC205, anti-BDCA-3, anti-BDCA-1 (specific for myeloid DC) or anti-BDCA-2 (specific for plasmacytoid DC), which are commercially available. In other embodiments, the agent that specifically binds dendritic cells binds a DC-specific C-type lectin, such as DC-SIGN. Suitable agents include ICAM-3, which specifically binds DC-SIGN. In other embodiments, the composite molecule includes an agent that specifically binds T cells. In one example, an agent that binds T cells is an antibody that specifically binds an antigen expressed on T cells. For example, the antigen can be expressed on T cells but not on dendritic cells. Suitable antibodies that specifically bind T cells include antibodies that specifically bind a T cell antigen. Exemplary antibodies specifically bind CD28, OX40 or ICOS. The agent that selectively binds T cells or dendritic cells can be covalently bound to the CD25 antagonist. Alternatively a linker can be included between the agent that selectively binds T cell or dendritic cells and the CD25 antagonist. The linker can be a polypeptide linker, such as a linker of about 2 to about 20 amino acids in length, such as about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 10, about 12, about 15, about 20, about 30 or about 40 amino acids in length. Suitable peptide linkers are well known in the art, and include, for example, six histidines. Suitable linkers also include those comprised of alanine and proline or glycine and leucine. Another suitable linker is (GIy₄SCr)₃ (SEQ ID NO: 3).

There are procedures known in the art that can be used to couple small molecules to proteins, and for the analysis of the resulting conjugates. For example, small molecules can be coupled to a protein (including antibodies) using carboxyl, hydroxyl, and amine residues to amine and sulfhydryl residues on the proteins using known linkage techniques. These techniques are described, for example, in Thompson, "Small-Molecule-Protein Conjugation Procedures," Molecular Diagnosis of Infectious Diseases, Second Edition (Jochen Decler and Udo Reischl, eds.), Volume 94, pp. 255-265, 2004, incorporated herein by reference. Commonly used cross-linking reagents include:glutaraldehyde, carbodiimide (EDC, - attaches carrier to C-terminus of peptide), succinimide esters (binds free amino group and Cys residues, benzidine (links to Tyr residues), periodate (attaches to carbohydrate groups), and isothiocyanate.

Pharmaceutical Compositions and Administration

Pharmaceutical compositions are thus provided for both local (such as topical or inhalational) use and for systemic (such as oral or intravenous) use. Therefore, the disclosure includes within its scope pharmaceutical compositions comprising a CD25 antagonist that selectively inhibits CD25 on dendritic cells or T cells formulated for use in human or veterinary medicine. While the agent that selectively inhibits CD25 on dendritic cells or T cells will typically be used to treat human subjects they may also be used to treat similar or identical diseases in other vertebrates, such as other primates, dogs, cats, horses, and cows. A suitable administration format may best be determined by a medical practitioner for each subject individually. Various pharmaceutically acceptable carriers and their formulations are described in standard formulation treatises, e.g., Remington' s Pharmaceutical Sciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A., Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42: 2S, 1988. The dosage form of the pharmaceutical composition will be determined by the mode of administration chosen. The compositions or pharmaceutical compositions also can be administered by any route, including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intraperitoneal, intrasternal, or intraarticular injection or infusion, or by sublingual, oral, topical, intranasal, or transmucosal administration, or by pulmonary inhalation. When an agent that selectively inhibits CD25 dendritic cells or T cells is provided as parenteral compositions, e.g. for injection or infusion, they are generally suspended in an aqueous carrier, for example, in an isotonic buffer solution at a pH of about 3.0 to about 8.0, preferably at a pH of about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0. Useful buffers include sodium citrate-citric acid and sodium phosphate-phosphoric acid, and sodium acetate-acetic acid buffers. A form of repository or "depot" slow release preparation may be used so that therapeutically effective amounts of the preparation are delivered into the bloodstream over many hours or days following transdermal injection or delivery. Agents that selectively inhibit CD25 on dendritic cells or T cells are also suitably administered by sustained-release systems. Suitable examples of sustained- release formulations include suitable polymeric materials (such as, for example, semi-permeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules), suitable hydrophobic materials (such as, for example, an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt). Sustained-release compositions may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. Preparations for administration can be suitably formulated to give controlled release of the agent that selectively inhibits CD25 on dendritic cells or T cells over an extended period of time. For example, the pharmaceutical compositions may be in the form of particles comprising a biodegradable polymer and/or a polysaccharide jellifying and/or bioadhesive polymer, an amphiphilic polymer, an agent modifying the interface properties of the particles and a pharmacologically active substance. These compositions exhibit certain biocompatibility features which allow a controlled release of the active substance. See U.S. Patent No. 5,700,486.

For oral administration, the pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (for example, pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (for example, lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (for example, magnesium stearate, talc or silica); disintegrants (for example, potato starch or sodium starch glycolate); or wetting agents (for example, sodium lauryl sulphate). The tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g. , almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. For solid compositions, conventional non-toxic solid carriers can include pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. Actual methods of preparing such dosage forms are known, or will be apparent, to those of ordinary skill in the art.

The pharmaceutically acceptable carriers and excipients are conventional. For instance, parenteral formulations usually comprise injectable fluids that are pharmaceutically and physiologically acceptable fluid vehicles such as water, physiological saline, other balanced salt solutions, aqueous dextrose, glycerol or the like. Excipients that can be included are, for instance, proteins, such as human serum albumin or plasma preparations. If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art.

Generally, the formulations are prepared by contacting the agent that selectively inhibits CD25 on dendritic cells or T cells uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Optionally, the carrier is a parenteral carrier, and in some embodiments it is a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.

The pharmaceutical compositions that comprise the agent that selectively inhibits CD25 on T cells or dendritic cells, in some embodiments, will be formulated in unit dosage form, suitable for individual administration of precise dosages. The amount of active compound(s) administered will be dependent on the subject being treated, the severity of the affliction, and the manner of administration, and is best left to the judgment of the prescribing clinician. Within these bounds, the formulation to be administered will contain a quantity of the active component(s) in amounts effective to achieve the desired effect in the subject being treated. Multiple treatments are envisioned, such as over defined intervals of time, such as daily, biweekly, weekly, bi-monthly or monthly, such that chronic administration is achieved. As disclosed herein, therapeutically effective amounts of the agent that selectively inhibits CD25 on dendritic cells or T cells are of use in treating an immune-mediated disorder or treating a tumor. Administration may begin whenever the suppression or prevention of disease is desired.

The therapeutically effective amount of the agent that selectively inhibits CD25 on dendritic cells or T cells will be dependent on the CD25 antagonist utilized, the subject being treated, the severity and type of the affliction, and the manner of administration. The exact dose is readily determined by one of skill in the art based on the potency of the specific compound (such as the CD25 antagonist utilized), the age, weight, sex and physiological condition of the subject. A therapeutically effective amount of an agent that selectively inhibits CD25 on dendritic cells or T cells can be administered with a therapeutically effective amount of another agent, such as a chemotherapeutic agent or an immunosuppressive agent.

Methods for Treating an Immune-Mediated Disorder and Pharmaceutical

Compositions

Methods are provided herein for treating an immune-mediated disease, such as, but not limited to an autoimmune disease, uveitis or transplant rejection. The methods include administering to the subject with the immune-mediated disease a therapeutically effective amount of an agent that selectively inhibits CD25 on dendritic cells, thereby treating the immune-mediated disease.

In one example, the immune-mediated disease is an autoimmune disease. In an autoimmune disease, an immune response is generated that is directed to the subjects own constituents, resulting in an undesirable and often terribly debilitating condition. An "autoantigen" is a subject's self-produced epitope, which is perceived to be foreign or undesirable, thus triggering an autoimmune response in the subject. This can lead to a chain of events, including the synthesis of other autoantigens or autoantibodies. An "autoantibody" is an antibody produced by an autoimmune patient to one or more of his own constituents which are perceived to be antigenic. For example, in systemic lupus erythematosus (SLE) autoantibodies are produced to DNA.

The methods disclosed herein can be used to treat autoimmune disease, including, but not limited to, rheumatoid arthritis, insulin dependent diabetes mellitus, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, multiple sclerosis (MS), systemic lupus erythematosus and others. The treatment of subjects with a therapeutically effective amount of an agent that selectively inhibits CD25 on dendritic cells can alleviate the clinical manifestations of the disease and/or minimize or prevent further deterioration or worsening of the subject's condition. Treatment of a subject at an early stage of an autoimmune disease including, for example, rheumatoid arthritis, insulin-dependent diabetes mellitus, clinically isolated demyelinating syndrome, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, or others, will minimize or eliminate deterioration of the disease state into a more serious condition.

For example, insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease which is believed to result from the autoimmune response directed against the beta cells of the islets of Langerhans which secrete insulin. Treatment of a subject suffering from an early stage of IDDM prior to the complete destruction of the beta cells of the islets of Langerhans would be particularly useful in preventing further progression of the disease, since it would prevent or inhibit further destruction of the remaining insulin-secreting beta cells, and could prevent or inhibit the natural progression of the disease state to more serious stages.

Methods are provided herein for the treatment of subjects that have multiple sclerosis using agents that selectively inhibit CD25 on dendritic cells. In one embodiment the subject has relapsing-remitting multiple sclerosis. However, the methods disclosed herein can also be used for the treatment of subjects with other forms of multiple sclerosis, such as secondary or primary progressive multiple sclerosis. Current therapies that target CD25 on both T cells and dendritic cells result in side effects, such as brain and spinal cord vasculitis and skin reactions, including alopecia. The presently disclosed methods provide an effective therapy wherein the subject does not develop brain or spinal cord vasculitis, and/or wherein in the subject does not develop skin reactions, or wherein these side effects are reduced as compared to treatment with a non-selective CD25 antagonist.

Methods are also disclosed herein for the treatment of uveitis. The methods include administering to the subject with the immune-mediated disease a therapeutically effective amount of an agent that selectively inhibits CD25 on dendritic cells. Any form of uveitis can be treated the methods disclosed herein. For example, iritis, cyclitis, panuveits, iridocyclitis, posterior uveitis, anterior uveitis and diffuse uveitis can be treated using the methods disclosed herein. Anterior and/or posterior uveitis can be treated. Both acute onset uveitis and chronic uveitis also can be treated. In one embodiment, a method is provided for treating anterior uveitis in a subject. Subjects can be treated that are affected with idiopathic iridocyclitis, HLA- B27 positive iridocyclitis, uveitis associated with juvenile rheumatoid arthritis, Fuch's heterochromatice iridocyclitis, herpes simplex keatovueitis, ankylosing spondylitis, intraocular lens related uveitis, Reiter's syndrome, Herpes zoster keratouveitis, uveitis associated with syphilis, traumatic iridocyclitis, uveitis associated with inflammatory bowel disease, tuberculosis iridocyclitis.

In another embodiment, a method is provided for treating posterior uveitis in a subject. Thus subjects can be treated that are affected with toxoplasma retinochroiditis, retinal vasculitis, idiopathic posterior uveitis, ocular histoplasmosis, toxocariasis, cytomegalovirus retinitis, idiopathic retinitis, serpinous choroidopathy, acute multifocal placoid, pigment eptiheliopathy, acute retinal necrosis, bird shot choroidopathy, uveitis associated with a leukemia or a lymphoma, reticulum cell sarcoma, ocular candidiasis, tuberculous uveitis, lupus retinitis. In a further embodiment, a method is provided for treating diffuse uveitis.

Thus, subjects can be treated that are affected with sarcoidosis, syphilis, Vogt- Koyanagi-Harada syndrome, or Bechet's disease.

In one embodiment, a sign or a symptom of the uveitis is decreased or alleviated. Ocular signs include ciliary injection, aqueous flare, the accumulation of cells visible on ophthalmic examination, such as aqueous cells, retrolental cells, and vitreous cells, keratic precipitates, and hypema. Symptoms include pain (such as ciliary spasm), redness, photophobia, increased lacrimation, and decreased vision. One of skill in the art can readily diagnose uveitis. In one embodiment, biomicroscopy (for example, a "slit lamp") is used to diagnose uveitis, to evaluate the clinical course of the disease or to verify that a treatment protocol has been successful.

In a further embodiment, the methods disclosed herein can be used to treat transplant rejection, such as allograft rejection. The methods include administering to the subject with the immune-mediated disease a therapeutically effective amount of an agent that selectively inhibits CD25 on dendritic cells. Techniques are well known to one of ordinary skill in the art for the transplantation of numerous cell, tissue, and organ types including, but not limited to: pancreatic islet transplantation, corneal transplantation, bone marrow transplantation, stem cell transplantation, skin graft transplantation, skeletal muscle transplantation, aortic and aortic valve transplantation, and vascularized organ transplantation including, but not limited to: heart, lung, heart and lung, kidney, liver, pancreas, and small bowel transplantation (see, e.g., Experimental Transplantation Models in Small Animals (1995) Publisher T&F STM, 494 pages). The present methods are not limited by the particular variety of transplantation. In general, any allogeneic transplantat between a non-syngeneic donor and recipient, in the absence of a transplant rejection inhibitor results in transplant rejection characterized by the partial or complete, typically progressive, destruction of the transplanted cells, tissue, or organ(s). The present compositions can be used to inhibit transplant rejection or graft versus host disease. The methods disclosed herein can include administering other immunosuppressants, such as cyclosporin (see Mathiesen, in: "Prolonged Survival and Vascularization of Xenografted Human Glioblastoma Cells in the Central Nervous System of Cyclosporin A-Treated Rats" (1989) Cancer Lett., 44:151-156), prednisone, azathioprine, and methotrexate (R. Handschumacher "Chapter 53: Drugs Used for Immunosuppression" pages 1264-1276). In some examples, such as for the treatment of rheumatoid arthritis, an agent that selectively inhibits CD25 on dendritic cells can be administered with pharmaceuticals including, but not limited to, corticosteroids, nonsteroidal anti-inflammatory drugs/Cox-2 inhibitors, methotrexate, hydroxychloroquine, sulphasalazopryine, gold salts, etanercept, infliximab, anakinra, azathioprine, and/or other biologies like anti-TNF. For the treatment of systemic lupus eryhtemathosus, an agent that selectively inhibits CD25 on dendritic cells can be administered with pharmaceuticals including, but not limited to, corticosteroids, Cytoxan, azathioprine, hydroxychloroquine, mycophenolate mofetil, and/or other biologies. Further, for the treatment of multiple sclerosis, an agent that selectively inhibits CD25 on dendritic cells can be administered with pharmaceuticals including, but not limited to, corticosteroids, interferon beta- Ia, interferon beta- Ib, glatiramer acetate, mitoxantrone hydrochloride, and/or other biologies. The agent that selectively inhibits CD25 on dendritic cells can also be used in combination with one or more of the following agents to regulate an immune response: soluble gp39 (also known as CD40 ligand (CD40L), CD154, T-BAM, TRAP), soluble CD29, soluble CD40, soluble CD80, soluble CD86, soluble CD28, soluble CD56, soluble Thy-1, soluble CD3, soluble TCR, soluble VLA-4, soluble VCAM-I, soluble LECAM-I, soluble ELAM-I, soluble CD44, antibodies reactive with gp39, antibodies reactive with CD40, antibodies reactive with B7, antibodies reactive with CD28, antibodies reactive with LFA-I, antibodies reactive with LFA-2, antibodies reactive with IL-2, antibodies reactive with IL- 12, antibodies reactive with IFN-gamma, antibodies reactive with CD2, antibodies reactive with CD48, antibodies reactive with any ICAM (e.g., ICAM-2), antibodies reactive with CTLA4, antibodies reactive with Thy-1, antibodies reactive with CD56, antibodies reactive with CD3, antibodies reactive with CD29, antibodies reactive with TCR, antibodies reactive with VLA-4, antibodies reactive with VCAM-I, antibodies reactive with LECAM-I, antibodies reactive with ELAM-I, antibodies reactive with CD44. In certain embodiments, monoclonal antibodies, including human antibodies humanized antibodies and chimeric antibodies, or fragments thereof, are utilized in the present methods. Thus, an agent that selectively inhibits CD25 on dendritic cells can be used and or formulated with and at least one other immunosuppressive agent. The determination of the optimal combination and dosages can be determined and optimized using methods well known in the art.

Methods of Treating a Tumor

Methods of treating a tumor in a subject are disclosed herein. The methods include administering to the subject a therapeutically effective amount of an agent that selectively inhibits CD25 on T cells, thereby treating the subject. In several examples, the treatment reduces tumor burden, reduces the number of metastasis, or decreases the likelihood that a benign tumor will be malignant. The tumor can be benign or malignant. In addition, the tumor can be either a hematological tumor or a solid tumor. Examples of hematological tumors include leukemias, including acute leukemias (such as Ilq23-positive acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia. Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular breast carcinoma), lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyrgioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma). In one specific non-limiting example, the tumor is a glioma.

An agent that selectively inhibits CD25 on T cells can be used in conjunction with another agent, such as a chemotherapeutic agent. Many chemotherapeutic agents are presently known in the art. In one embodiment, the chemotherapeutic agents is selected from the group consisting of mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones, e.g. anti-androgens, and anti-angiogenesis agents.

An agent that selectively inhibits CD25 on T cells can also be used in conjuction with a cytokine. For example, the agent can be used in conjunction with IL-2 in cancer in order to suppress T-reg cells, promote T cell survival and enhance natural killer (NK) cell function.

Anti-angiogenesis agents, such as MMP-2 (matrix-metalloproteinase 2) inhibitors, MMP-9 (matrix-metalloproteinase 9) inhibitors, and COX-II (cyclooxygenase II) inhibitors, can be used in conjunction with an agent that selectively inhibits CD25 on T cells. Examples of useful COX-II inhibitors include CELEB REX™(alecoxib), valdecoxib, and rofecoxib. Examples of useful matrix metalloproteinase inhibitors are described in PCT Publication No. WO 96/33172 (published Oct. 24, 1996), PCT Publication No. WO 96/27583 (published Mar. 7, 1996), European Patent Application No. 97304971.1 (filed JuI. 8, 1997), European Patent Application No. 99308617.2 (filed Oct. 29, 1999), PCT Publication No. WO 98/07697 (published Feb. 26, 1998), PCT Publication No WO 98/03516 (published Jan. 29, 1998), PCT Publication No WO 98/34918 (published Aug. 13, 1998), PCT Publication No WO 98/34915 (published Aug. 13, 1998), PCT Publication No WO 98/33768 (published Aug. 6, 1998), PCT Publication No WO 98/30566 (published JuI. 16, 1998), European Patent Publication 606,046 (published JuI. 13, 1994), European Patent Publication 931,788 (published JuI. 28, 1999), PCT Publication No WO 90/05719 (published May 31, 1990), PCT Publication No WO 99/52910 (published Oct. 21, 1999), PCT Publication No WO 99/52889 (published Oct. 21, 1999), PCT Publication No WO 99/29667 (published Jun. 17, 1999), PCT

International Application No. PCT/IB98/01113 (filed JuI. 21, 1998), European Patent Application No. 99302232.1 (filed Mar. 25, 1999), U.S. Patent No. 5,863,949 (issued January 26, 1999), United States Patent No. 5,861,510 (issued Jan. 19, 1999), and European Patent Publication 780,386 (published Jun. 25, 1997). In one example, the MMP inhibitors do not induce arthralgia upon administration. In another example, the MMP inhibitor selectively inhibits MMP-2 and/or MMP-9 relative to the other matrix- metalloproteinases (such as MMP-I, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-IO, MMP-I l, MMP-12, and MMP-13). Some specific examples of MMP inhibitors of use are AG-3340, RO 32-3555, RS 13-0830, 3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(l-hydroxycarbamoyl- cyclopentyl)-amino]-propionic acid; 3-exo-3-[4-(4-fluoro-phenoxy)- benzenesulfonylamino]-8-oxa-bicyclo[3.2.1]octane-3-carboxylic acid hydroxyamide; (2R, 3R) l-[4-(2-chloro-4-fluoro-benzyloxy)-benzenesulfonyl]-3- hydroxy-3-methyl-piperidine-2-carboxylic acid hydroxyamide; 4-[4-(4-fluoro- phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-4-carboxylic acid hydroxyamide; 3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(l-hydroxycarbamoyl- cyclobutyl)-amino]-propionic acid; 4-[4-(4-chloro-phenoxy)- benzenesulfonylamino]-tetrahydro-pyran-4-carboxylic acid hydroxyamide; (R) 3-[4- (4-chloro-phenoxy)-benzenesulfonylamino]-tetrahydro-pyran-3-carboxylic acid hydroxyamide; (2R, 3R) l-[4-(4-fluoro-2-methyl-benzyloxy)-benzenesulfonyl]-3- hydroxy-3-methyl-piperidine-2-carboxylic acid hydroxyamide; 3-[[4-(4-fluoro- phenoxy)-benzenesulfonyl]-(l-hydroxycarbamoyl-l-methyl-ethyl)-amino]-propionic acid; 3-[[4-(4-fluoro-phenoxy)-benzenesulfonyl]-(4-hydroxycarbamoyl-tetrahydro- pyran-4-yl )-amino]-propionic acid; 3-exo-3-[4-(4-chloro-phenoxy)- benzenesulfonylamino]-8-oxaicyclo[3.2.1 ]octane-3-carboxylic acid hydroxyamide; 3-endo-3-[4-(4-fluoro-phenoxy)-benzenesulfonylamino]-8-oxa-icyclo[3.2.1]octane- 3-carboxylic acid hydroxyamide; and (R) 3-[4-(4-fluoro-phenoxy)- benzenesulfonylamino]-tetrahydro-furan-3-carboxylic acid hydroxyamide; and pharmaceutically acceptable salts and solvates of said compounds.

The agent can be used with signal transduction inhibitors, such as agents that can inhibit EGF-R (epidermal growth factor receptor) responses, such as EGF-R antibodies, EGF antibodies, and molecules that are EGF-R inhibitors; VEGF (vascular endothelial growth factor) inhibitors, such as VEGF receptors and molecules that can inhibit VEGF; and erbB2 receptor inhibitors, such as organic molecules or antibodies that bind to the erbB2 receptor, for example, HERCEPTIN™ (Genentech, Inc.). EGF-R inhibitors are described in, for example in PCT Publication Nos. WO 95/19970 (published JuI. 27, 1995), WO 98/14451 (published Apr. 9, 1998), WO 98/02434 (published Jan. 22, 1998), and U.S. Patent No. 5,747,498 (issued May 5, 1998). EGFR-inhibiting agents also include, but are not limited to, the monoclonal antibodies C225 and anti-EGFR 22Mab (ImClone Systems Incorporated), ABX-EGF (Abgenix/Cell Genesys), EMD-7200 (Merck KgaA), EMD-5590 (Merck KgaA), MDX-447/H-477 (Medarex Inc. and Merck KgaA), and the compounds ZD-1834, ZD-1838 and ZD-1839 (AstraZeneca), PKI- 166 (Novartis), PKI- 166/CGP-75166 (Novartis), PTK 787 (Novartis), CP 701 (Cephalon), leflunomide (Pharmacia/Sugen), Cl- 1033 (Warner Lambert Parke Davis), C1-1033/PD 183,805 (Warner Lambert Parke Davis), CL-387,785 (Wyeth- Ayerst), BBR-1611 (Boehringer Mannheim GmbH/Roche), Naamidine A (Bristol Myers Squibb), RC-3940-II (Pharmacia), BIBX-1382 (Boehringer Ingelheim), OLX- 103 (Merck & Co.), VRCTC-310 (Ventech Research), EGF fusion toxin (Seragen Inc.), DAB-389 (Seragen/Lilgand), ZM-252808 (Imperial Cancer Research Fund), RG-50864 (INSERM), LFM-A12 (Parker Hughes Cancer Center), WHI-P97 (Parker Hughes Cancer Center), GW-282974 (Glaxo), KT-8391 (Kyowa Hakko) and EGF-R Vaccine (York Medical/Centro de Immunologia Molecular (CIM)).

VEGF inhibitors, for example SU-5416 and SU-6668 (Sugen Inc.), SH-268 (Schering), and NX- 1838 (NeXstar) can also be used in conjunction with an antibody that specifically binds HMW-MAA. VEGF inhibitors are described in, for example in PCT Publication No. WO 99/24440 (published May 20, 1999), PCT International Application PCT/IB99/00797 (filed May 3, 1999), PCT Publication No. WO 95/21613 (published Aug. 17, 1995), PCT Publication No. WO 99/61422 (published Dec. 2, 1999), U.S. Patent No. 5,834,504 (issued Nov. 10, 1998), PCT Publication No. WO 98/50356 (published Nov. 12, 1998), U.S. Patent No. 5,883,113 (issued Mar. 16, 1999), U.S. Patent No. 5,886,020 (issued Mar. 23, 1999), U.S. Patent No. 5,792,783 (issued Aug. 11, 1998), PCT Publication No. WO 99/10349 (published Mar. 4, 1999), PCT Publication No. WO 97/32856 (published Sep. 12, 1997), PCT Publication No. WO 97/22596 (published Jun. 26, 1997), PCT Publication No. WO 98/54093 (published Dec. 3, 1998), PCT Publication No. WO 98/02438 (published Jan. 22, 1998), WO 99/16755 (published Apr. 8, 1999), and PCT Publication No. WO 98/02437 (published Jan. 22, 1998). Other examples of some specific VEGF inhibitors are IM862 (Cytran Inc.); anti-VEGF monoclonal antibody of Genentech, Inc.; and angiozyme, a synthetic ribozyme from Ribozyme and Chiron. These and other VEGF inhibitors can be used in conjunction with the presently described compositions. ErbB2 receptor inhibitors, such as GW-282974 (Glaxo Wellcome), and the monoclonal antibodies AR-209 (Aronex Pharmaceuticals Inc.) and 2B-1 (Chiron), can be utilized, for example those indicated in PCT Publication No. WO 98/02434 (published Jan. 22, 1998), PCT Publication No. WO 99/35146 (published JuI. 15, 1999), PCT Publication No. WO 99/35132 (published JuI. 15, 1999), PCT Publication No. WO 98/02437 (published Jan. 22, 1998), PCT Publication No. WO 97/13760 (published Apr. 17, 1997), PCT Publication No. WO 95/19970 (published JuI. 27, 1995), U.S. Patent No. 5,587,458 (issued Dec. 24, 1996), and U.S. Patent No. 5,877,305 (issued Mar. 2, 1999). ErbB2 receptor inhibitors of use are also described in U.S. Provisional Application No. 60/117,341, filed Jan. 27, 1999, and in U.S. Provisional Application No. 60/117,346, filed Jan. 27, 1999.

In one example, for the prevention and treatment of cancer, the agent that selectively inhibits CD25 on T cells can be used with an alkylating agent. In another example, for the treatment of a melanoma, the agent can be used in conjunction with Imiquimod. In a further example, such as for treatment of prostate cancer, the agent that selective inhibits CD25 on T cells can be used in conjunction with, for example, surgery, radiation therapy, chemotherapy and hormonal therapy (such as anti- androgens or GnRH antagonists). For the treatment of HNSCC, the agent that selective inhibits CD25 on T cells can be used in conjunction with surgery, radiation therapy, chemotherapy, other antibodies (such as cetuximab and bevacizumab) or small-molecule therapeutics (such as erlotinib). The agent that selectively inhibits CD25 on T cells can be used with any other therapeutic methods, including radiation and surgery.

Antitumor immunity can be provided to a subject by producing of target antigen-specific T cells that recognize a target tumor-associated antigen. Administration of a therapeutically effective amount of a tumor antigen to a recipient will enhance the recipient' s immune response to the tumor by providing T cells that are targeted to, recognize, and immunoreact with a preselected tumor antigen. The preselected tumor antigen is chosen based on the recipient's tumor. For example, if the recipient has a breast tumor, a breast tumor- antigen is selected, and if the recipient has a prostate tumor, a prostate tumor- antigen is selected, and so forth. Shown below in Table 2 are tumors and respective tumor antigens that can be used to generate target antigen-specific T cells. These antigens can be administered to a subject having that particular tumor. However, one skilled in the art will recognize that other tumors can be treated using other tumor antigens.

Table 2: Exem lar tumors and their tumor anti ens

Administration of a therapeutic amount of target tumor antigen-specific T cells can be used to prevent recurrence of the tumor in the recipient, or to treat a relapse of the tumor.

Selection of Agents for the Treatment of an Immune-Mediated Disorder

Methods are provided herein for selecting a CD25 antagonist of use in treating an immune-mediated disorder. These methods include contacting mature dendritic cells with the CD25 antagonist. T cells are then primed with the mature dendritic cells contacted with the CD25 antagonist. It is then determined if the

CD25 antagonist has an inhibitory effect on T cell priming by the mature dendritic cells as compared to a control. An inhibitory effect of the antagonist on T cell priming by the mature dendritic cells indicates that agent is of use for treating the immune-mediated disorder. In some examples, the control is a standard value. In other examples, the control is the effect on T cell priming by mature dendritic cells contacted with a control agent, such as a carrier or buffer. Suitable assays to evaluate T cell priming are known in the art, and exemplary assays are described in the examples below. In one example, assessing T cell priming comprises assessing T cell proliferation. Generally, the determination of an inhibitory effect utilizes a statistical test. One of skill in the art can readily identify statistical tests of use. The methods can also include determining if trans -presentation of IL-2 to T cells by dendritic cells is reduced in the presence of the antagonist. A decrease in the trans -presentation of IL-2 to T cells by dendritic cells as compared to a control indicates the antagonist is of use for treating the immune-mediated disorder. Exemplary assays for trans-presentation of IL-2 to T cells by dendritic cells are provided in the examples section below. In these methods, the control can be a value for T cell priming by mature dendritic cells in the absence of the antagonist or the contral can be a standard value.

These methods can be used to identify agents of use for treating any immune mediated disorder, such as those described above. Specific, non-limiting examples of the immune mediated disorder are autoimmune diseases, including, but not limited to, rheumatoid arthritis, insulin dependent diabetes mellitus, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, multiple sclerosis (MS), systemic lupus erythematosus and others. The methods can also be used to identify agents of use for treating uveitis. The methods are further of use for identifying agent of use for treating transplant rejection.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES It is disclosed herein that mDC use CD25 for targeted trans-presentation of

IL-2 via immune synapse to primed antigen-specific T cells. Blockade of CD25 on the surface of mDCs abrogates T cell proliferation. On the other hand, CD25 expression on T cells is not only dispensable for their proliferation, but it also limits effector T cell survival. The data presented herein reveal novel mechanisms for IL-2 in DC-mediated activation of T cell responses. The IL-2 receptor (IL-2R) consists of 3 chains: α-chain (CD25), β-chain

(CD 122) and common γ-chain (CD 132). CD 122 and CD 132 are both signaling chains that together form the intermediate affinity IL- 2R (K_d18 ~ 10^~10 M). CD25 lacks a signaling component and binds IL-2 only with low affinity (K_dls ~ 10^~8 M). However, when it associates with CD122 and CD132 it stabilizes the receptor complex and increases its affinity for IL-2 by at least 10-fold (K_dls ~ 10^"π M). The high affinity IL-2 receptor is only expressed on a small percentage of resting human T cells, but it is readily upregulated upon T cell activation (Cantrell et al., Science 224, 1312-1316 (1984)). This upregulation of CD25 allows sustained IL-2 signaling, which is believed to be necessary for IL-2 mediated T cell proliferation (Van Parjis et al., Immunity 11, 281-288 (1999)), but which also mediates activation induced cell death (AICD) (see, for example, Suzki et al., Science 268, 1472-1476 (1995)).

Based on the T cell growth-promoting properties of IL-2 in-vitro, it was expected that inhibition of IL-2 signaling would result in defective T cell responses in-vivo. Contrary to expectations, mice with genetic deletion of IL-2 or its receptor components develop lymphoproliferative disorders and autoimmunity (see, for example, Willerford et al., Immunity 3, 521-530 (1995)), indicating that while IL-2 is not essential for T cell proliferation in-vivo, it plays a crucial role in immunoregulation. Indeed, it was recently discovered that IL-2 plays a critical role in the differentiation and maintenance of FoxP3+ regulatory T cells (see, for example, Turka et al., Front Biosci 13, 1440-1446 (2008)) and it also expands and activates human immunoregulatory CD56^bπght NK cells (see, for example, Bielekova et al., PNAS 103, 5941-5946 (2006)).

CD25 blocking antibodies (Ab) were developed as potential immunosuppressive strategies in humans (Waldmann et al., Blood 72, 1805-1816 (1988)). They proved their therapeutic efficacy in solid-organ transplantation (see, for example, Waldmann et al., Curr Opin Immunol 10, 507-512 (1998)) and in immune-mediated diseases such as inflammatory uveitis (Nussenblatt et al., Ophthalmology 112, 764-770 (2005)) and multiple sclerosis (MS) (see, for example, Bielekova et al., Proc Natl Acad Sci U SA 101, 8705-8708 (2004)). Furthermore, CD25 deficiency in humans has more severe consequences for the immune system than in mice (see, for example, Roifman et al., Pediatr Res 48, 6-11 (2000)). Human loss-of-function mutation of CD25 is characterized by repeated infectious complications such as CMV pneumonia, candidiasis and adenoviral gastroenteritis, in addition to the development of lymphadenopathy and tissue infiltration by T cells (see, for example, Sharfe et al., Proc Natl Acad Sci U SA 94, 3168-3171 (1997)). Collectively these data demonstrate that in addition to its role in terminating T cell responses, in humans, IL-2 also plays an essential role in the activation and development of effector functions of T cells.

A CD25 -blocking Ab (daclizumab) has only a mild inhibitory effect on human T cell proliferation or cytokine production after polyclonal (CD3/CD28 Ab) stimulation (Bielekova et al., PNAS 103, 5941-5946 (2006)). Previously, the main therapeutic effect of daclizumab in MS was attributed to profound expansion of CD56^bπght regulatory NK cells. However, an MS patient was identified who had only transient (i.e. several weeks) expansion of CD56^bπght NK cells but sustained (>10 months) inhibition of brain inflammatory activity while on daclizumab therapy. This observation prompted critical reconsideration of a possible second mechanism of action of daclizumab in MS.

Polyclonal T cell activation by CD3/CD28 Ab represents a non- physiological CD4/CD8 co-receptor independent type of T cell receptor (TCR) signaling. Because co-receptor/MHC-dependent TCR signaling is more restrictive than co-receptor independent signaling, the effect of daclizumab on a more physiological type of T cell activation by antigen (Ag)-loaded mature dendritic cells (mDCs) was tested. A profound (>80%) inhibitory role of daclizumab was observed on T cell activation in this setting. A series of mechanistic experiments is presented herein that provide an explanation for the two divergent outcomes of IL-2 signaling in human T cells: The experiments demonstrated that IL-2 plays a crucial role in T cell activation and development of effector functions when it is presented to primed T cells in-trans, by CD25 on mDCs; on the other hand it primes activated T cells for later cell death. These experiments also demonstrated that IL-2 provides an AICD signal, especially when it is received outside of the immune synapse, by CD25 expressed on T cells. These observations led to the design of agents that selectively inhibit CD25 on either T cell or dendritic cells.

Example 1 Materials and Methods Isolation of peripheral blood mononuclear cells and generation of DCs:

Studies were performed in accordance with the institutional guidelines and all subjects signed informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated from apheresis samples by lymphocyte separation medium (Lonza, Walkers ville, MD USA). CD 14+ monocytes were isolated from fresh PBMC by positive selection using magnetic sorting (IMag, CD14 Magnetic Particles, BD Biosciences, San Jose, CA, USA). Isolated cell purity was routinely evaluated above 92% (Fig. 12). Isolated cells were plated at IxIO⁶ cell/ml in IMDM medium containing 50ng/ml rhGM-CSF and lOOng/ml rhIL-4 (both PeproTech, Rocky Hill, NJ USA) and 5% human serum for 6 days. Medium was replenished every 3 days. Immature DCs (iDCs) were harvested, extensively washed and re-plated at 1x10 cell/ml and loaded with either (0.5μg/ml) influenza hemagglutinin vaccine (FIuHA; Fluzone, Sanofi Pasteur, Doylestown, PA, USA) or (lOμg/ml) Brain Protein Medley (BPM; Clontech, Takara Bio USA, Madison, WI) and maturation cocktail, which consisted of 50ng/ml TNF-α, lOng/ml IL- lβ, lOng/ml IL-6 (all Peprotech), lμM PGE2 (Sigma, St Louis, MS USA) for 2 days. The phenotype of DCs at different maturation stages was characterized by staining with antibodies against the following surface markers; CDl Ic, CD25, CD80, CD83, CD122, CD132 and MHC- II (all BD Biosciences).

T cell proliferation assays: Autologous T cells were purified from cryo- preserved PBMC by negative selection with IMag T Lymphocyte Enrichment Set (BD Biosciences) according to the manufacturer's protocol. T cells purity was checked periodically and exceeded 95%. T cells were labeled for 8 min with 1 μM CFSE (carboxyfluorescein diacetate succinimidyl diester; Molecular Probes, Invitrogen, Carlsbad, CA USA) as described (Bielekova et al., PNAS 103, 5941- 5946 (2006)). CFSE stained T cells were cultured with autologous antigen (Ag)- loaded mDC (10:1 T cell:DC ratio) in the presence of lOμg/ml daclizumab (Roche, Nutley, NJ) or equal concentration of MA-251 antibody (BD Biosciences) as a control. MA-251 is an antibody that binds CD25 outside of the tac epitope and therefore does not inhibit IL-2 signaling. The dose of daclizumab selected is the peak dose achievable in humans with lmg/kg administered intravenously every 4 weeks (Bielekova et al, supra). Alternatively, mDCs were pre-treated with daclizumab or MA-251 (for 30min at 37°C and extensively washed) before co- culture with T cells. After 7-10 d, T cells cultures were collected, washed and were stained with anti-CD4, anti-CD8 and anti CD3 (BD Biosciences) and were analyzed on a BD LSR-II flow cytometer (BD Biosciences). Data were analyzed with BD FACSDiva v6.1 (BD Biosciences) software and FCS Express v3 software (De Novo Software, Los Angeles, CA, USA).

Maturation of DCs in the presence of IL-2 and daclizumab for surface and ICCS: Monocyte-derived iDCs were generated as described above. iDCs were harvested, loaded with Ag and matured by addition of maturation cocktail in the presence or absence of lOμg/ml daclizumab, 100IU of IL-2 or a combination of both for 2 days. 48h later, a portion of mDCs cultures were surface stained using anti- CD25, anti-CD80 and anti-CD83 (all BD Biosciences), while the remaining mDCs were incubated with lμl/ml brefeldin A (BFA, BD Biosciences) for 5 h. mDCs were fixed and permeabilized using BD Cytofix/Cytoperm kit (BD Biosciences) according to manufacturer's protocol. DCs were then stained using antibodies against IL-2, IL-6 and IL-10. For all stainings, proper isotype controls were used in parallel for setting up gates and subtracting non-specific staining.

Flow cytometry based signaling assay: mDCs and autologous T cells were harvested and extensively washed and incubated for 1 hour in RPMI without serum to avoid any possible cytokine signal that may be initiated by human serum. Cells were incubated with 100 IU/ml IL-2 or 20ng/ml IL-4 for 10-20 minutes at 37⁰C, 5%CO₂- For trans-presentation experiments, T cells were pre-incubated with daclizumab (lOμg/ml), washed extensively and then co-cultured with mDCs in the presence of exogenous IL-2 for 10-50 minutes. In order to stop signaling after selected time-points, cells were immediately fixed with 10% paraformaldehyde for 10 minutes (37⁰C, 5%CO₂), permeabilized with PhosFlow Perm Buffer II (BD Biosciences) and then washed and stained for pStat5 and pStatό (BD Biosciences) and were analyzed on a BD LSR-II.

Fluorescent microscopy: mDCs and autologous T cells were plated onto glass bottom poly-d-lysine coated dishes (MatTek, Ashland, MA, USA) and incubated at 37⁰C, 5%CC>₂ overnight. Adherent cells were carefully washed with FACS buffer (IXPBS, 5% FBS, and 0.1% sodium azide) and incubated for 1 hour at RT with primary un-conjugated anti-CD25 and anti-CD3 (BD Biosciences). Plates were carefully washed with FACS buffer and incubated with appropriate florescent labeled secondary antibodies Alexa Fluor 488-conjugated goat anti-mouse IgGlκ and Alexa Flour 594-conjugated goat anti-mouse IgG2a (Invitrogen). Washed cells were fixed with 4% paraformaldehyde and stained with DAPI (4,6-diamidino-2- phenylindole dihydrochloride) (Invitrogen). Images were obtained on a Zeiss Axiovert 200M Fluorescence cell Imaging Microscope (Carl Zeiss Microimaging GmbH, Gottingen, Germany), using Volocity software, (Improvision Inc, Waltham, MA, USA). Post-processing combination of images was performed using Adobe Photoshop (Adobe Systems, Inc. San Jose, CA, USA).

T cell siRNA experiments: T cells were isolated by negative selection from cryopreserved PBMC samples using the Pan T cell Isolation Kit II (Miltenyi Biotech, Auburn, CA, USA) according to the manufacturer's protocol. Purity of separation was checked periodically by flow cytometry and was routinely >95%.

Isolated T cells were then CFSE-stained. In preparation for nucleofection, cells were rested overnight in media containing 10% human serum without antibiotics. Nucleofection was performed with control or CD25 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted in RNase-free water at 3μg/5xlO⁶ cells using the Amaxa Nucleofection system and T cell Nucleofection kit (Amaxa, Gaithersburg, MD, USA). After nucleofection, cells were immediately rested (37°C, 5% CO₂) in media without antibiotics for 6 hours before further culture. A GFP siRNA control transfected in CFSE-unstained cells was 71.9% ±0.82 effective as analyzed by flow cytometry. T cells were then cocultured in a 10:1 ratio (T:DC) with autologous, Ag-loaded mDCs or with CD3/CD28 Dynabeads (0.3:1 bead:T cell ratio) (Invitrogen, Carlsbad, CA, USA). T cells stimulated by CD3/CD28 beads were stained for surface expression of CD25 between 12 and 48 hours following stimulation and analyzed by flow cytometry. Within the first 24 hours, CD25 expression was inhibited by 33.4-72.3% in CD25 siRNA nucleofected T cells as compared to control siRNA. In half of the coculture conditions mDCs were pre- incubated with daclizumab or MA251 anti-CD25Ab as control for 30 minutes and were thoroughly washed (3X) prior to culture with T cells. IL-7 (5ng/ml) was added to cultures as indicated. Seven days later, T cells were harvested, stained for intracellular cytokines IL- 17, IL-2 and IFN-γ as described (Bielekova et al, supra) and analyzed by flow cytometry for proliferation by CFSE dilution and cytokine production. Flow cytometry analyses were run in duplicates and gating was set on isotype controls.

Statistical analysis: Differences between different treatment groups were analyzed using non-parametric Mann-Whitney rank-sum test (SigmaStat, San Jose, CA, USA). P values smaller than 0.05 were considered significant. Antibodies: The following antibodies were used in FACS staining; CD3-

APC (clone UCHTl), CD3-AmCyan (clone Sk7), CD4-Cy5.5 (clone RPA-T4), CD4-Pacific Blue (clone RPA-T4), CD8-PE (clone HIT8a), CD8-APC-Cy7 (clone SKl), CD8-AmCyan (clone SKl), CDl Ic-PE (clone B-ly6), CD14-PE (clone M5E2) CD25-APC (clone MA-251), CD80-FITC (clone BBl), CD83-APC (clone HB15e), CD86-FITC (clone 2331/FUN-l), IL-2-APC (clone MQ1-17H12), IL-6- AF488 (clone JES3-9D7), pStat5-PE (clone 47), pStat6-AF488 (clone 18) (all from BD Biosciences), PE-IL-17 (clone eBio64DEC17), and IFN-γ-PerCP-Cy5.5 (clone 4S.B3) (both from eBiosciences) and anti CD25-FITC (clone Bl.49.9; Immunotech, Westbrook, ME USA) for analysis of the efficacy of CD25 Tac epitope blocking by daclizumab. Maturation of DCs in the presence ofIL-2 and daclizumab: iDCs were harvested, loaded with Ag and matured by addition of maturation cocktail in the presence or absence of lOμg/ml daclizumab, 100 IU/ml of IL-2 or a combination of both for 2 days. 48h later, a portion of mDC cultures were surface stained using anti-CD25, anti-CD80 and anti-CD83 (all BD Biosciences), while the remaining mDCs were incubated with lμl/ml brefeldin A (BFA, BD Biosciences) for 5 hours. mDCs were fixed and permeabilized using BD Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer's protocol. DCs were then stained using antibodies against IL-2, IL-6. For all stainings, proper isotype controls were used in parallel for setting up gates and subtracting non-specific staining.

Example 2 Daclizumab has a prominent inhibitory effect on T cell priming by mDCs

In order to investigate the effect of daclizumab on T cell activation under physiological, co-receptor-dependent conditions, CFSE-stained autologous T cells were incubated with Ag-loaded mDCs in the presence of daclizumab or control anti- CD25 Ab MA-251, which does not block the IL-2-binding Tac epitope. 7-10 days later we evaluated T cell proliferation by CFSE-dilution as measured by flow cytometry (Fig. 1). T cell responses were tested to influenza- HA (FIu-HA), as protypic foreign antigen, and to a commercially available pool of proteins derived from human brain. (Brain protein medley, PBM) as a protypic self antigen.

While MA-251 Ab had no inhibitory effect on T cell proliferation, the addition of daclizumab to mDC:T cell co-cultures in a representative subject decreased FIu-HA- specific CD4⁺ T cell proliferation from 60.3% to 13.6% (Fig. Ia). A similar level of inhibition of proliferation (from 43.7% to 5.5%) was noted for BPM-specific CD4⁺ T cells. Overall, daclizumab inhibited Ag-specific T cell proliferation by 78-88%, irrespective of the Ag studied (Fig. Ib; P<0.001 Paired T- test). As FIu-HA- specific T cells produced significantly higher levels of cytokines than BPM-specific T cells FIu-HA ws used for all subsequent experiments. Because the inhibition of T cell proliferation in mDC:T cell co-cultures was significantly higher than the mild inhibition (0- 20%; representative experiment in Fig. Ic) was demonstrated previously in CD3/CD28 stimulated CD4⁺ T cells (Bielekova et al., supra) attention was focused on the effect of daclizumab on DCs.

Example 3 DCs do not express a complete IL-2 receptor and they do not phosphorylate

Stat5 in response to IL-2

Myeloid DCs were isolated from freshly isolated CD14⁺ monocytes from apheresis samples of healthy donors (see methods). First, the phenotype of immature DCs (iDCs) and mDCs were confirmed based on their surface expression of DC lineage and maturation markers (Fig. 2a). Next, to establish whether mDCs have a functional signaling IL-2R, the expression of IL-2R chains on the surface of DCs was examined by flow cytometry (Fig. 2b). It was observed that iDCs and mDCs both express CD132 but lack expression of CD122. CD122 was readily detectable on activated T cells using identical antibodies and staining procedures (Fig. 9). Only mDCs, but not iDCs, express CD25.

To determine whether mDCs are capable of signaling through the IL-2R, mDCs were incubated with IL-2 and Stat5 phosphorylation was examined by intracellular flow cytometry (Fig 2c). Resting human lymphocytes used as a positive control phosphorylated Stat5 in the presence of IL-2 (Fig 2c, upper panels). However, mDCs failed to phosphorylate Stat5 in the presence of IL-2 (Fig. 2c, lower panels). As a signaling control, IL-4 stimulation was used after the pilot experiments to demonstrate that both DC and lymphocytes readily phosphorylate Stat6 in response to IL-4 (Fig 2d).

Example 4

Neither exogenous IL-2 nor daclizumab affect DC maturation or function

Even though there was no Stat5 phosphorylation on mDCs in response to IL- 2, the possibility was considered that IL-2 provides a maturation signal to DCs using pathways alternative to Stat5 phosphorylation. Therefore, it was examined if CD25 expression on mDCs has any role on DC maturation or function. DCs were cultured in the presence or absence of IL-2, daclizumab, or a combination of both for 48 hours and their maturation markers and cytokine production were examined by flow cytometry. mDCs matured in the presence or absence of daclizumab or IL-2 express equivalent levels of maturation surface markers CD83, CD25 and CD80 and produce equal levels of the cytokines IL-2, and IL-6 (Fig. 10).

Example 5 In T cell/mDC co-cultures, blocking CD25 on mDCs, but not on T cells inhibits

T cell proliferation

Significant inhibition of T cell proliferation is seen when T cells were primed by mDCs in the presence of daclizumab, but in subsequent experiments it was demonstrated that DCs do not receive an IL-2 signal. Therefore, it was investigated if it was the CD25 blockade on the side of mDCs or T cells that is responsible for this observed inhibitory effect. Before the assembly of the proliferation assay mDCs or autologous T cells were co-incubated with daclizumab for 1 hour and extensively washed it away afterwards. A co-culture of T cells and mDCs was assembled (Fig. 3). Pre-treatment of mDCs with daclizumab was sufficient to significantly inhibit T cell proliferation when compared to untreated mDCs (Fig. 3a, 3b, compare left and middle panels; p>0.001; Mann-Whitney Rank Sum Test). Pre-treatment of T cells with daclizumab had no significant inhibitory effect on T cell activation (Fig. 3a, 3b right panels).

There are two possible explanations for this observation: the first suggests that CD25 expression on mDCs is crucially important for T cell activation. The alternative explanation is that mDCs might trans-present Fc receptor (FcR)-bound daclizumab to activated T cells and thus inhibit T cell proliferation. The latter possibility was investigated by conducting the following experiments; first, it was observed that as DCs mature, they down-modulate expression of all FcR, so that CD25⁺ mDCs do not express CD16, CD32 or CD64 (Fig. Ha). Additionally, the assays were repeated using the original murine anti-Tac Ab (IgG2a isotype) instead of daclizumab. It was demonstrated that this Ab binds only minimally to human FcR and that this nonspecific binding is completely abrogated by pre-treatment of DCs with a pool of human immunoglobulins used for intravenous administration (IVIg) (Fig. lib). Using a combination of IVIg followed by anti-Tac Ab pre- treatment of mDCs, identical inhibition of mDC-mediated trans -presentation of IL-2 and T cell proliferation (Fig. lie, lid) was observed. Therefore, it was concluded that it is CD25 expression on mDCs that is critical for effective T cell activation.

Example 6 mDCs use surface CD25 expression for trans-presentation of IL-2 to T cells via immune synapse Because the inhibitory effect of daclizumab on T cell proliferation resulted from the blockade of CD25 on mDCs, but because this did not affect mDC function, it was hypothesized that mDCs use CD25 for the trans-presentation of IL-2 to T cells. To address this question, the ability of mDCs to trans-present exogenous IL-2 to T cells was investigated by measuring Stat5 phosphorylation by flow cytometry (Fig. 4a). It was found that 22.9% of resting T cells were able to phosphorylate Stat5 after 20 min exposure to 50 IU/ml of IL-2 (Fig. 4a, 1^st panel). This early and late Stat5 phosphorylation was almost completely blocked by pre-incubating T cells with 10 μg/ml of daclizumab (Fig. 4a, 2^nd column). This is in agreement with the observation that this dose of daclizumab effectively blocks 100% of CD25 on the T cell surface (Fig. 12). The same daclizumab-treated T cells, unable to phosphorylate Stat5 in response to IL-2 by themselves, have fully restored and even enhanced (90- 173%, depending on the donor) Stat5 phosphorylation when co-incubated with mDCs that express CD25 (Fig. 4a, compare 1^st and 3^rd panels). Finally, unmodified T cells display significantly enhanced Stat5 phosphorylation when co-cultures with CD25-expressing mDCs are compared to cultures of unmodified T cells alone (Fig. 4a, 5^th panel). T cells with CD25 blocked by daclizumab do not phosphorylate Stat5 to exogenous IL-2 when co-incubated with iDCs that do not express CD25 (Fig. 4a, last panel). A saturating concentration of IL-15 blocking Ab was used in this assay to avoid Stat5 phosphorylation by IL-15. To confirm trans-presentation of IL-2 by mDCs to T cells the localization of

CD25 on mDCs during antigen presentation was studied by fluorescent microscopy. mDCs were cultured in the presence of autologous T cells and stained for the expression of CD25 and CD3 with fluorescently labeled antibodies. In the absence of mDC-T cell contact a homogenous distribution of CD25 on the surface of mDCs was observed. However, when a T cell comes in contact with a mDC there is consistent polarization of CD25 on the surface of the mDC toward the site of T cell contact (Fig. 5b). There is also polarization of CD3 on the T cell toward the interacting mDC indicating that the mDC and T cell have formed an immune synapse.

Example 7

CD25 on T cells is not necessary for T cell proliferation, but it limits the survival of effector T cells

Because T cells upregulate CD25 after activation via TCR, pre-incubation of T cells with daclizumab would block CD25 only on those T cells that express CD25 under resting conditions, but would not inhibit upregulation of CD25 on newly activated T cells. Therefore, the effect of CD25 on T cells was investigated further, using siRNA technology.

Resting, CFSE stained T cells were necleofected with control siRNA or CD25 siRNA and cocultured with mDCs that were pretreated with either daclizumab or the MA-251 control. The transfection efficacy, determined by GFP control 12-24 hours later, was on average 67%. Because in Ag-loaded mDC:T cell co-cultures the estimated precursor frequency of Ag-specific T cells is only a few per million, the inhibitory effect of CD25 siRNA on CD25 upregulation could not be investigated on activated T cells in this system. Instead, polyclonal T cell activation with CD3/CD28 Dynabeads was used to study CD25 expression on T cells nucleofected with control or CD25 siRNA in time-assay by flow cytometry. The maximal inhibition of CD25 expression by CD25 siRNA 12-48h post-stimulation was 95%, but the average efficacy from all experiments was 59%. The level of inhibition further decreased 48h post- stimulation (Fig. 5a) as cells started to divide. The expansion of Ag-specific T cells pre-treated with control or CD25 siRNA was evaluated 7-10 days post-stimulation by Ag-loaded mDCs. CD25 siRNA had a consistent inhibitory effect (2-73%, depending on the donor) on the percentage of CD4+ T cells that entered the proliferation cycle (Fig, 5b, %prolierating CD4), which we confirmed further by cell cycle analysis. This was in contrast to cultures with daclizumab, where T cell proliferation was almost completely abrogated (Fig. 5b, lower panels. Despite mild inhibition of T cell proliferation, the number of functional effector T cells was in fact increased in cultures with CD25 siRNA. The percentage of proliferating T cells that secreted IL- 2, IFN-γ or IL- 17 was consistently increased in CD25 siRNA conditions as compared to control siRNA (Fig. 5c and 13b). Unexpectedly, when using proportional enumeration of T cell numbers among different conditions, in some patients the enhanced survival of T cells pre-treated with CD25 siRNA was observed, leading to more than double the absolute numbers of Ag-specific CD4 effector T cells as compared to control siRNA (Fig. 5b; absolute number of proliferating CD4 and Fig. 5d). While this phenomenon was observed only in -50% of studied subjects 7-10 days post-stimulation (when the CFSE dilution assay was routinely performed),enhanced long-term (>14 days) survival of Ag-specific CD25 siRNA nucleofected T cells in the majority of subjects studied, a correlation of enhanced T cell survival with the efficacy of CD25 inhibition on activated T cells by CD25 siRNA was observed. Data from a representative subject who did not have enhanced survival of CD25 siRNA nucleofected T cells 7 days post-stimulation are provided in Fig. 13, in order to demonstrate comparative or increased numbers of functional effector T cells even in those subjects.

Example 8 T cells derived from a CD25 negative patient phosphorylate Stat5 and expand Ag-specific effector T cells when co-incubated with MHC-matched CD25 expressing mDCs

As shown in Fig 5a, even with a high transfection efficacy, CD25 siRNA was unable to completely silence CD25 expression on activated T cells. There are currently three children with a lack of CD25 expression described in the medical literature (see, for example, Caudy et al., J. Allergy Clin. Immunol. 119: 482-487, 2007). A single vial of 3x10 viable PBMC was obtained that were cryopreserved before one of these CD25 negative patients underwent bone marrow transplantation. All crucial experiments were repeated from this limited sample material (Fig. 6). Complete molecular MHC-I/II typing of the CD25 negative patient was obtained and used the NIH blood bank database to select the best MHC-matched healthy control subject, from which an apheresis was obtained, monocytes were isolated and the monocytes were matured into Ag-loaded, CD25 expressing mDCs.

First, it was demonstrated that polyclonally activated T cells from the CD25 negative patient lack surface (and intracellular) CD25 expression at all times during the proliferation cycle, while they express the intermediate affinity IL-2R (Fig. 6a, representative staining from 4 time-points on CD3/CD28 expanded T cells). Next, a T cell signaling experiment was performed using different doses of IL-2 and determined that the highest IL-2 dose that does not cause Stat5 phosphorylation of patient's T cells (i.e. does not signal via intermediate affinity receptor) is 50 IU/ml (Fig 6b; 2^nd panel). This concentration of IL-2 was used for mDC IL-2 trans- presentation experiments: when the patient's T cells were co-incubated with CD25 expressing mDCs in the absence of exogenous IL-2, only a minimal increase in Stat5 phosphorylation above the background was observed (Fig. 6b, 3^rd panel). However, when 50 IU/ml of IL-2 was added to mDC-T cell co-cultures, significant Stat5 phosphorylation of the patient's T cells (Fig 6b, 4^th panel) was now observed, which was diminished when donor mDCs were pre-incubated with daclizumab and extensively washed (Fig 6b, 5^th panel).

Simultaneously, a T cell proliferation assay was assermbled: CD25^" T cells were isolated from cryopreserved PBMCs by negative selection and were CFSE stained before they were co-cultured with donor, Ag-loaded, CD25 expressing mDCs, which were pre-incubated with control Ab (upper panels) or daclizumab (lower panels). Seven days later, cultures were briefly stimulated with PMA/ionomycin and assessed for the extent of CFSE dilution and cytokine production. Both CD4⁺ (purple) and CD8⁺ (blue) T cells from the CD25^" patient proliferated when they were co-incubated with CD25⁺ mDCs, but not when CD25 expression on mDCs was blocked by daclizumab (Fig 6c). Finally, these proliferating Ag-specific T cells differentiated to effector T cells, as demonstrated by their production of cytokines (Fig. 6d), at levels comparable to those effectors derived from healthy subjects (Fig. 5 and Fig. 13).

Finally, it was determined whether the lack of IL-2 trans-presented to primed T cells by mDCs is an absolute inhibitory signal for T cell expansion, or if their activation can be restored by other soluble factors. These studies focused on IL-7, as a T cell survival factor that is not produced by myeloid or lymphoid cells. It was observed that the addition of IL-7 to mDC:T cell co-cultures resulted in partial (60- 70%) restoration of T cell proliferation even in the presence of daclizumab (Fig. 7). Disclosed herein is the seminal observation that the inhibitory effect of CD25 blockade on T cell function is significantly greater when T cells are activated by Ag- loaded mDCs (78-88% inhibition) as compared to their polyclonal activation by CD3/CD28 Ab (-20% inhibition, Bielekova et al, supra). The ensuing experiments demonstrated that the inhibitory effect of daclizumab was dependent on the CD25 blockade on mDCs, rather than on T cells (Figs. 3-6). While it has been recently discovered that DCs produce IL-2 following microbial stimulation (Granuucci et al., Nat Immunol 2, 882-888 (2001)), and the expression of CD25 has been recognized as one of the activation markers of human mDCs (Mnasria et al., Int Immunopharmacol 8, 414-422 (2008); Grannuci et al., Nat Immunol 2, 882-888 (2001)) the relevance of this observation for T cell activation remained unclear. It was observed that both iDCs and mDCs lack expression of CD122 (Fig. 2b), which is essential for IL-2 signaling (Malek, Annu Rev Immunol 26, 453-479 (2008)). Consequently, IL-2 failed to induce Stat5 phosphorylation on DCs (Fig. 2c) no effects of IL-2 or daclizumab on DC maturation or function were observed. Therefore, it was concluded that CD25 expression on human myeloid mDCs must have a different role than facilitating IL-2 signaling on DCs.

The addition of high concentrations of exogenous IL-7 to in-vitro cultures truthfully may not reproduce the in-vivo situation, but this data provides proof-of- principle that other factors can, and in the case of CD25 negative patients do, partially compensate for the lack of IL-2 signal to primed T cells. This leads to their incomplete activation that is clearly ineffective for pathogen clearance, but causes persistent tissue infiltration due to the defective contraction of effector T cell responses.

Without being bound by theory, the source of IL-2 in mDC-mediated trans- presentation could be the mDC itself. It is known that DCs produce IL-2 and express CD25 only upon their activation. According to the model presented herein (Fig. 8) rather than indiscriminately releasing IL-2 to its environment, mDCs use CD25 to selectively trans-present IL-2 to primed Ag-specific T cells. This trans- presentation could require bilateral cross-talk between mDCs and T cells. Soon after activation (in the humans within 12h) the primed T cell can produce its own IL-2. However, the kinetics of CD25 expression on activated T cells are much slower: in polyclonally activated human T cells CD25 expression peaks 72 hours post- stimulation (Fig. 5a), at the time when IL-2 production by primed T cells has already declined. Therefore, in the critical time-period required for the formation of a stable immune synapse (i.e. first several hours) it is highly unlikely that T-cell derived CD25 contributes to the formation of the high-affinity IL-2R. This conclusion is consistent with the observation presented herein that blockade of CD25 on mDCs is sufficient for abrogation of T cell proliferation (Fig. 3). However, it remains unknown if early T cell secretion of IL-2 contributes to the pool of IL-2 that is being trans-presented by mDCs to T cells.

It is possible that CD25 expression on T cells does not contribute to the formation of the high affinity IL-2R during T cell priming. It is demonstrated herein that while CD25 expression on T cells is not crucial for T cell entry to the proliferation cycle, it clearly enhances proliferation and leads to greater expansion of effector T cells (Fig. 5 and Fig. 13). However, the resulting abundance of high- affinity IL-2 signaling has yet another effect: it limits long-term T cell survival. Greater long-term (>14d) in-vitro survival of expanded effector T cells from CD25 siRNA nucleofected T cells and also from the CD25 negative patient was consistently observed, in comparison to CD25 expressing T cells (Fig. 5). Without being bound by theory, activated T cells regulate the extent of final expansion and contraction of Ag-specific T cell pool by their paracrine IL-2 consumption (see, for example, Long et al., J. Immunol. 177: 4257-4261, 2006). This is dictated by T cell expression of CD25 that is linked in time to their TCR stimulation and by the availability of other freshly-activated T cells that provide a continuous source of IL- 2 (Fig. 8). This explains why humans with a CD25 deficiency are both immunodeficient and lymphopenic but have tissue infiltration with persistently- activated T cells (see, for example, Aoki et al. J. Autoimmun. 27: 50-53, 2006). However, in contrast to the in-vitro use of CD25⁺ matched mDCs for effective activation of CD25^" T cells (Fig. 6), as presented herein, CD25^" patients have to utilize a different mechanism of T cell activation in-vivo in order to develop lymphoproliferation. The studies presented herein are focused on IL-7, because the fact that daclizumab has such a profound inhibitory effect on T cell activation in mDC:T cell co-cultures made it unlikely that cytokines produced by either mDCs or T cells could provide sufficient alternative activation signals. Since IL-7 is not produced in high levels either by mDCs or T cells and because of its documented survival signal, it was tested if exogenous addition of IL-7 could substitute for the lack of DC-mediated IL-2 signal. It was observed that IL-7 indeed restores T cell proliferation, although not to the full extent seen without daclizumab (Fig. 7).

There is a major difference between IL- 2Rα (CD25) and IL-15Rα affinity for their respective cytokines: in contrast to the very stable interaction of the IL- 15Rα for IL-15 (Dubbois et al., Immunity 17: 537-547, 2002), which is believed to be necessary for IL-15 trans-presentation, CD25 has only a low affinity for IL-2. The crystal structure of the quaternary IL-2 complex (Wang et al., Science 310: 1159-63, 2005) demonstrates that CD25 shares with IL-15Rα a long connecting peptidic arm between the globular head and the transmembrane region that is highly flexible and, in the case of IL-15, clearly capable of trans-presentation Dubbois et al., supra). Furthermore, CD25, like IL-15Rα, makes no contact with CD122 and CD 132 (Wang et al., supra). Yet, biophysical models predict that IL-2 binds first to CD25, before the CD25/IL-2 complex binds to CD122, which then recruits the common γ-chain (CD 132) (Wang et al., supra), leading to the formation of a high affinity IL-2R. So if the low affinity of CD25 for IL-2 truly hampered signaling, it could affect more α^'s-presentation as compared to Zrαns-presentation across the immune synapse. Clearly, the ability of CD25 to capture and concentrate IL-2 at the cell surface for presentation to intermediate affinity IL-2R (Wang et al., supra) would be only enhanced within the constraints of the immune synapse, where the diffusion of IL-2 is limited. Without being bound by theory, the low affinity of CD25 for IL-2 can be the reason why efficient IL-2 signaling under low IL-2 concentrations happens predominantly within the context of the immune synapse. On the other hand, high concentrations of IL-2, such as those present during an evolving immune response at the peak of effector T cell differentiation, are permissive for IL-2 cώ-presentation.

Example 9

Daclizumab or murine anti-Tac Ab bind to mDC via F_ab fragment Daclizumab shares with its original murine anti-Tac Ab only -10% structure, which mediates F_ab binding to CD25. The remaining 90% is human IgGl backbone, which binds avidly to F_c receptors (F_cRs). Therefore, it was asked if daclizumab binds to mDCs via F_c or F_ab fragments. First, flow cytometry revealed that while iDCs expressed high levels of all three human F_cRs, activation of iDCs with proinflammatory or PAMP stimuli resulted in their downmodulation, so that the expression of CD25 and F_cRs on DCs was mutually exclusive (Fig. 15a). Consequently, binding of anti-Tac to iDCs was inhibited by blocking F_cRs with polyclonal human immunoglobulin (IVIg) (Fig. 15b; upper panels), but the identical F_cRs blockade had no effect on anti-Tac binding to mDCs (Fig. 15b; lower panels). Murine anti-Tac was used in these experiments, because blockade of F_cRs with IVIg prevented us from selectively detecting daclizumab with secondary anti-human IgGl Ab. Therefore, as a control it was demonstrated that anti-Tac exerts identical inhibitory effects on Ag- specific T cell proliferation as daclizumab (Fig. 15c).

Example 10 mDCs do not express β-chain of IL-2R and therefore do not signal to IL-2 Previous experiments demonstrated that the inhibitory effect of daclizumab on Ag-specific proliferation of T cells resided in its ability to block the CD25 Tac epitope on mDCs. It was hypothesized that mDCs receive high-affinity IL-2 signal, necessary for effective T cell priming. The expression of IL- 2R chains in human myeloid DCs was investigated (Fig. 16a and 16b). Consistent with the mouse literature no CD 122 expression was observed either at the protein or mRNA level (Fig. 16c), despite the fact that varied population of HD and MS patients were studied, and that the experiments utilized multiple protocols for DC maturation, including the one reported by Mnasria et al (see Int Immunopharmacol 8, 414-422, 2008; Transplant Proc 41, 695-697, 2009; J Leukoc Biol 84, 460-467, 2008). Consequently, utilizing phospho-flow technology, which allows selective gating of target population, no IL-2 induced Stat5 phosphorylation was observed in mDCs

(Fig. 16d). In contrast, mDCs readily phosphorylated Stat6 in response to IL-4 (Fig. 16e) and lymphocytes phosphorylated both Stat5 and Stat6 to appropriate stimuli.

Even though no Stat5 phosphorylation was observed on DCs, the possibility exists that IL-2 provides a maturation signal to DCs using pathways besides Stat5 phosphorylation. Therefore, DCs were cultured in the presence or absence of IL-2, daclizumab, or a combination of both for 48h and examined their phenotype by flow cytometry. DCs matured in the presence or absence of daclizumab or IL-2 expressed equivalent levels of maturation markers CD83, CD25 and CD80 and produced equal levels of the cytokines IL-2, and IL-6.

Example 11 mDCs use their surface expression of CD25 to trans-present IL-2 to CD25- T cells, thus facilitating high affinity IL-2 signaling in primed T cells Because CD 122 expression could not be detected on mDCs and no direct effect of IL-2 or daclizumab was observed on DC phenotype, daclizumab must interferes with DC-mediated activation of T cells through a different route. It was tested whether mDC uses its own CD25 to complement the T cell expression of β- and γ_c-chains and facilitate assembly of high affinity IL-2R on T cells in-trans.

To test this hypothesis, the time course of CD25 and IL-2 expression was analyzed on polyclonally activated T cells. A small proportion of (likely memory) T cells produced high levels of IL-2 as early as 10 hours post-activation. (Fig. 17). However, no significant upregulation of CD25 was observed on T cells during the first 10 hours post-activation; in fact, the IL-2-producing T cells were conspicuously CD25 negative. All T cells upregulated CD25 at later time-points (>24h); but they also ceased to produce IL-2. This shut-down was IL-2-driven, as addition of IL-2 neutralizing Ab into T cell cultures resulted in retained IL-2 secretion by T cells as long as 24-48h post-activation (Fig. 17; lower panels). T cells activated in the presence of IL-2 resumed IL-2 production 48-72h post-stimulation, when they started to divide as demonstrated by CFSE-dilution. Paradoxically, this later surge of IL-2 production, and continuous increase in CD25 expression, were both dependent on early IL-2 signal, as they were significantly inhibited by IL-2 neutralizing Ab.

The lack of CD25 expression on T cells within first 10 hours post-activation was supportive of the hypothesis that mDCs provide their CD25 to primed T cells for the formation of high affinity IL-2R. Thus, the ability of mDCs to complement formation of high-affinity IL-2R on CD25- T cells was tested. Whether T cells pre- treated with daclizumab, T cells from the CD25 negative patient, T cells pre-treated with murine anti-Tac were utilized, identical results were obtained: In each instance, CD25+ mDCs restored high-affinity IL-2 signaling on T cells, measured as Stat5 phosphorylation to IL-2 doses that were insufficient to trigger intermediate affinity IL-2R. Specifically, in a representative subject, 22.9% of resting T cells phosphorylated Stat5 10 minutes after exposure to 50IU of IL-2. Pre-treatment of T cells with daclizumab abrogated IL-2-driven Stat5 phosphorylation. High affinity IL-2 signaling on daclizumab-pretreated T cells was restored upon addition of CD25+ mDCs, but not when mDCs were pre-treated with daclizumab. iDCs, which do not express CD25 were unable to restore IL-2 signaling in daclizumab-treated T cells. A saturating concentration of IL- 15 blocking Ab was used in this assay to avoid Stat5 phosphorylation by IL- 15.

This experiment demonstrated that CD25 expression on mDCs complements high affinity IL- 2R formation on CD25 negative T cells in-trans, analogous to the IL-15 system. However, in contrast to IL-15R0C, which effectively captures IL-15 due to its high affinity, CD25 has very low affinity for IL-2. This would preclude CD25 from effectively capturing IL-2 under situations when IL-2 is produced in low concentrations, such as at the initiation of the immune response. It was hypothesized that rather than indiscriminately spilling IL-2 into the environment, mDCs (and possibly also memory T cells) release IL-2 directionally into the synaptic cleft, facilitating effective capture of IL-2 by mDC-derived CD25 and its trans-presentation to primed T cells. If this hypothesis was correct, the mDC CD25 should co-localize into the DC-T cell inter-phase upon formation of the immune synapse. Thus, CD25 expression on mDCs was studied in the resting state and upon contact with T cells by fluorescent microscopy. Homogeneous distribution of CD25 was observed on the surface of mDCs that were not in contact with T cells.

However, upon immune synapse formation clear co-localization of CD25 on the mDC with CD3 on the T cell was demonstrated.

The data presented herein provide an explanation for the diverse functions of IL-2 on human T cells and elucidate a mechanism which highlights how the expression of cytokine signaling chains on different cells of the immune system can divergently regulate T cell functions through a single cytokine.

Example 12 Clinical Trial

A clinical trial is conducted to determine the effects of administration of an agent that specifically binds dendritic cells covalently linked to a CD25 antagonist, in subjects having relapsing-remitting or secondary-progressive multiple sclerosis. In some studies, the agent that specifically binds dendritic cells is an antibody. In addition, the CD25 antagonist is an antibody that specifically bind the IL-2 receptor (for example p55), an siRNA, or a small molecule inhibitor.

Inclusion criteria: Subjects included in the trial are diagnosed with either relapsing-remitting or secondary-progressive multiple sclerosis. These subjects are between the ages of 16-65; scored between 2.5 and 6.5 on the EDSS. In one example, each subject must have had at least 3 gadolinium enhancing lesions in the first 3 pre-therapy MRI scans.

Exclusion criteria: Subjects are excluded from the trial if they are diagnosed with primary-progressive MS, pre-treatment blood tests were abnormal; they are diagnosed with a concurrent clinically significant major disease; contraindications to monoclonal antibody therapies are observed; they are determined to be positive for HIV; they are treated with glatiramer acetate or cyclophosphamide in the 26 weeks prior to the trial, or they are treated with intravenous immunoglobulin (IVIg), azathioprine (AZA), methotrexate (MTX), cyclosporin, cyclophosphamide (CTC), cladribine, or mitox in the 12 weeks prior to the trial, or if they are treated with corticosteroids or adrenocorticotrophic hormone (ACTH) in the 8 weeks prior to the trial, or if they are treated with any other investigational drug or procedure for MS; not practicing adequate contraception; or breastfeeding.

Course of Treatment: Once subjects are selected for inclusion in the trial, a baseline is determined. Four MRI scans are performed during the baseline period to determine a baseline number of contrast enhancing lesions, one at the beginning of the period and then at the end of each month of the baseline period with the fourth coinciding with the beginning of the combination therapy. Subjects are also evaluated on the EDSS, the Scripps Neurologic Rating Scale (NRS), and various ambulation and other motor skill tests.

Therapy begins after the 3 -month baseline is established. Therapy is administered for a six month period. For example, the therapy can be administered every other week or once a month. An exemplary dose is administered intravenously or subcutaneous administration of a dose of about 0.5 mg/kg 1 mg/kg of body weight, about 2 mg/kg, about 3 mg/kg, or about 4 mg/kg. MRI scans are performed during the treatment period to determine changes in the number of contrast enhancing lesions. For example, MRFs can be performed one every two weeks or monthly. A total of 6-8 MRI scans can be obtained. On the same schedule subjects are also evaluated on the EDSS, the Scripps NRS, and various ambulation and other motor skill tests.

Results: The combined administration of the therapeutic agent led to almost cessation of disease activity and clinical improvement in the subjects. The subject have either fewer or at least no increase in both new and total contrast enhancing lesions under the treatment as compared to the baseline period. Improvement on the EDSS following treatment is also observed (as compared to the baseline period). Improvement on the Scripps NRS is also observed following treatment (as compared to the baseline period). Improved ambulation on the ambulation index is also observed following treatment (as compared to the baseline period). Subjects either improved or had no change in a timed 20 minute walk. Subjects also demonstrated improved times with their dominant hand on the peg hole test and/or with their non- dominant hand on the peg hole test. The test subjects do not develop brain vaculitis, spinal cord vasculitis, or alopecia.

It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described invention. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

1. A composition comprising an agent that selectively inhibits CD25 on dendtric cells for use in treating an immune-mediated disorder, or that selectively inhibits CD25 on T cells for use in treating a tumor.

2. The composition of claim 1, wherein the agent selectively inhibits CD25 on dendritic cells, and wherein the immune-mediated disorder is multiple sclerosis.

3. The composition of claim 1, wherein the agent selectively inhibits CD25 on T cells, for use in treating a tumor.

4. A method of treating a subject with an immune mediated disease, comprising administering to the subject a therapeutically effective amount of an agent that selectively inhibits CD25 on dendritic cells, thereby treating the immune mediated disorder in the subject.

5. The method of claim 4, wherein the agent that selectively inhibits CD25 on dendritic cells is a composite molecule comprising an agent that selectively binds dendritic cells and a CD25 antagonist.

6. The method of claim 5, wherein the composite molecule comprises an antibody that specifically binds dendritic cells covalently linked to a CD25 antagonist.

7. The method of claim 6, wherein the antibody is DEC205, anti-BDCA-1, anti-BDCA-3 or anti-BDCA-2.

8. The method of any one of claims 4-7, wherein the CD25 antagonist is a small molecule inhibitor.

9. The method of claim 8, wherein the small molecule inhibitor is an acylphenylalanine compound.

10. The method of any one of claims 5-7, wherein the CD25 antagonist is a monoclonal antibody.

11. The method of claim 10, wherein the antibody is a humanized antibody or a fully human antibody.

12. The method of claim 10, wherein the antibody is daclizumab.

13. The method of any one of claims 5-7, wherein the CD25 antagonist is a CD25 siRNA or a CD25 antisense RNA.

14. The method of any one of claims 4-13, wherein the immune mediated disease is an autoimmune disorder.

15. The method of claim 14, wherein the autoimmune disorder is multiple sclerosis.

16. The method of claim 15, wherein the multiple sclerosis is relapsing- remitting multiple sclerosis.

17. The method of claim 15, wherein the multiple sclerosis is progressive multiple sclerosis.

18. The method of claim 15, wherein the subject does not develop brain vaculitis, spinal cord vasculitis, alopecia, or any combination thereof.

19. The method of any one of claims 4-14 wherein the immune-mediated disorder is uveitis or allograft rejection.

20. The method of claim 19, wherein the allograft is a heart transplant or a kidney transplant.

21. A composition comprising a composite molecule and a carrier, wherein the composite molecule comprises an agent that selectively binds dendritic cells or an agent that selectively binds T cells, wherein the agent is covalently bound to a CD25 antagonist.

22. The composition of claim 21, wherein the agent that selectively binds dendritic cells or selectively binds T cells is a monoclonal antibody.

23. The composition of claim 21, wherein the agent selectively binds dendritic cells, and wherein the agent is monoclonal antibody is DEC205, anti- BDCA-3 or anti-BDCA-2.

24. The composition of any one of claims 21-23, wherein the CD25 antagonist is a monoclonal antibody.

25. The composition of claim 24, wherein the monoclonal antibody is a fully human or a human monoclonal antibody.

26. The composition of claim 24, wherein the monoclonal antibody is daclizumab.

27. The composition of any one of claims 21-23, wherein the CD25 antagonist is a small molecule inhibitor.

28. The composition of claim 27, wherein the small molecule inhibitor is an acylphenylalanine compound.

29. A method of selecting a CD25 antagonist of use in treating an immune- mediated disorder, comprising contacting mature dendritic cell with the CD25 antagonist; priming T cells with the mature dendritic cells contacted with the CD25 antagonist; and determining the antagonist has an inhibitory effect on T cell priming by the mature dendritic cells as compared to a control; wherein an inhibitory effect of the antagonist on T cell priming by the mature dendritic cells indicates that agent is of use for treating the immune-mediated disorder.

30. The method of claim 29, wherein determining if the inhibitory effect of the antagonist on T cell priming comprises assessing T cell proliferation.

31. The method of any one of claims 29-30, further comprising determining if trans-presentation of IL-2 to T cells by dendritic cells is reduced in the presence of the antagonist, wherein a decrease in the trans- presentation of IL-2 to T cell by dendritic cells as compared to a control indicates the antagonist is of use for treating the immune-mediated disorder.

32. The method of claim 29, wherein the control is T cell priming by mature dendritic cells in the absence of the antagonist or a standard value.

33. The method of claim 31, wherein the control is trans-presentation of IL- 2 to T cells by dendritic cells in the absence of the antagonist or a standard value.

34. The method of any one of claims 29-33, wherein the immune-mediated disorder is an autoimmune disorder.

35. The method of claim 34, wherein the autoimmune disorder is multiple sclerosis.

36. The method of claim 37, wherein the immune mediated disorder is uveitis or allograft rejection.

37. A method of treating a subject having a tumor, comprising administering to the subject a therapeutically effective amount of an agent that selectively inhibits CD25 on T cells, thereby treating the tumor in the subject.

38. The method of claim 37, further comprising administering to the subject a therapeutically effective amount of an antigen expressed by cells in the tumor.

39. The method of any one of claims 37-38, wherein the agent that selectively inhibits CD25 on T cells is a composite molecule comprising an agent that selectively binds T cells and a CD25 antagonist.

40. The method of claim 39, wherein the composite molecule comprises an antibody that specifically binds T cells covalently linked to a CD25 antagonist.

41. The method of claim 40, wherein the antibody is a F(ab)₂ fragment.

42. The method of claim 40, wherein the CD25 antagonist is a small molecule inhibitor.

43. The method of claim 42, wherein the small molecule inhibitor is an acylphenylalanine compound.

44. The method of claim 40, wherein the CD25 antagonist is a monoclonal antibody.

45. The method of claim 44, wherein the antibody is a humanized antibody or a fully human antibody.

46. The method of claim 44, wherein the antibody is daclizumab.

47. The method of claim 40, wherein the CD25 antagonist is a CD25 siRNA or a CD25 antisense RNA.

48. The method any one of claims 38-47, wherein the tumor is a glioma.

49. Use of a therapeutically effective amount of an agent that selectively inhibits CD25 on dendritic cells for treating an immune mediated disorder in the subject.

50. The use of claim 49, wherein the agent that selectively inhibits CD25 on dendritic cells specifically binds dendritic cells but does not specifically bind T cells.

51. Use of a therapeutically effective amount of an agent that selectively inhibits CD25 on T cells for treating a tumor in a subject.

52. The use of claim 51, wherein the agent that selectively inhibits CD25 on T cells specifically binds T cells but does not specifically bind dendritic cells.