WO2006042101A1 - Methode de traitement de l'uveite active - Google Patents

Methode de traitement de l'uveite active Download PDF

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Publication number
WO2006042101A1
WO2006042101A1 PCT/US2005/036125 US2005036125W WO2006042101A1 WO 2006042101 A1 WO2006042101 A1 WO 2006042101A1 US 2005036125 W US2005036125 W US 2005036125W WO 2006042101 A1 WO2006042101 A1 WO 2006042101A1
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dose
uveitis
daclizumab
pulsatile
subject
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PCT/US2005/036125
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English (en)
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Robert B. Nussenblatt
Thomas Waldmann
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The Government Of The United States As Represented By The Secretary Of Health And Human Services
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • This relates to the field of uveitis, specifically to the use of an antibody that binds the EL-2 receptor for the treatment of active uveitis.
  • Intraocular inflammatory diseases grouped under the term “uveitis” are a major cause of visual loss in industrialized nations.
  • "Uveitis” refers to an intraocular inflammation of the uveal tract, namely, the iris, choroids, and ciliary body. Uveitis is responsible for about 10% of legal blindness in the United States (National Institutes of Health, Interim Report of the Advisory Eye Council Support or Visual Research, U.S. Department of Health Education and Welfare, Washington, DC, 1976, pp. 20-22). Complications associated with uveitis include posterior synechia, cataract, glaucoma and retinal edema (Smith et al., Immunol. Cell Biol 76:497-512, 1998).
  • Treatment of uveitis often focuses on the control of the inflammatory symptoms. In such cases, corticosteroids are often used to suppress inflammation in the eye. Anterior uveitis often responds to local steroid treatment with eye drops. However, drops do not usually provide therapeutic levels of steroids in the posterior part of the eye for the treatment of intermediate uveitis, posterior uveitis or panuveitis. Periocular injections are then indicated. These injections can be given sub-conjunctivally or beneath Tenon's capsule. Steroids can also be used intraocularly.
  • systemic treatments with corticosteroids are often used when local injections fail. It is believed that approximately 100,000 American citizens currently require the use of some systemic corticosteroids or other immunosuppressive agents as treatment for ocular inflammation. However, many of the most severe cases of uveitis do not respond to high dose systemic corticosteroid therapy or the patient cannot continue with adequate therapeutic doses because of adverse events.
  • the side effects of such systemic therapies can include hypertension, hyperglycemia, peptic ulceration, Cushingoid features, osteoporosis, growth limitation, myopathy, psychosis and increased susceptibility to infection can be devastating.
  • Newer immunosuppressive agents are being investigated for use in uveitis treatment, such as Tacrolimus, Sirolimus and mycophelonate mofetil, which also have serious side effects (Anglade and Whitcup, Drugs 49:213-223, 1995).
  • SUMMARY Methods are described for treating a subject with active uveitis. These methods include administering to the subject a high pulsatile dose of an interleukin- 2 (IL-2) receptor antagonist, hi one example, the IL-2 receptor antagonist is an antibody that binds the IL-2 receptor, such as daclizumab. Methods are also provided for the treatment of corneal transplant rejection, limbal stem cell rejection following transplantation, optic neuritis and/or dry eye, that include administering to the subject a high pulsatile dose of an IL-2 receptor antagonist.
  • IL-2 receptor antagonist is an antibody that binds the IL-2 receptor, such as daclizumab.
  • FIG. 1 is a schematic diagram of an exemplary treatment protocol.
  • FIG. 2 is a set of plots of data obtained from a fluorescence activated cell sorting (FACS) analysis showing the number of NK cells of different subpopulations obtained from 5 high dose HAT (daclizumab) patients before and after >6 weeks of HAT treatment. NK subpopulations were measured by contacting the cells with antibodies to CD122, CXCR3 and CD56, and detecting expression using three fluorescent markers.
  • FACS fluorescence activated cell sorting
  • FIG. 3 is a set of plots of data obtained from a fluorescence activated cell sorting (FACS) analysis showing the number of NK cells of different subpopulations obtained from 5 high dose HAT (daclizumab) patients before and after >6 weeks of HAT treatment. NK subpopulations were measured by contacting the cells with antibodies to CD122, CXCR3 and CD56, and detecting expression using three fluorescent markers. The expression of high CD122 (X-axis) and high CD56 (Y-axis) is shown.
  • FACS fluorescence activated cell sorting
  • FIGS. 4A-4C are a set of plots and graphs showing that in vivo blockade of human IL-2R induced expansion of the CD56 b ⁇ ght NK cell subset.
  • FIG. 4A is a set of plots showing a comparison of the CD56 b ⁇ ght NK subset before and after Daclizumab infusion. Whole blood samples from patients were collected and stained with indicated antibodies and percentages of CD56 bright CX3CRl ' NK cells in total lymphocytes were analyzed. The gated cells in dot plots represent the CD56 b ⁇ ght NK subset.
  • FIG. 4B is a graph showing a time course analysis of the expansion of the CD56 bright NK subset.
  • FIG. 4C is a bar graph of the results from uveitis patients with active disease had significantly lower CD56 b ⁇ ght NK cells.
  • FIG. 5A-5C are bar graphs, plots and a digital image showing the phenotypic and functional analysis of the CD56 b ⁇ ght NK cells from patients receiving Daclizumab therapy.
  • FIG. 5 A is a bar graph showing a comparison of cytokine profiles between the induced CD56 bright NK subpopulation and CD56 dim NK subpopulation. Sorted CD56 bright NK and CD56 dim NK cells were stimulated with IL- 12 and IL- 15 for 72 hours and culture supernatants were analyzed for multiple cytokine levels as described in the Examples section. Data was representative of two independent sorting and cytokine array analysis experiments.
  • FIG. 5 A is a bar graph showing a comparison of cytokine profiles between the induced CD56 bright NK subpopulation and CD56 dim NK subpopulation. Sorted CD56 bright NK and CD56 dim NK cells were stimulated with IL- 12 and IL- 15 for 72 hours and culture supernatants were analyzed for multiple cytokine
  • FIG. 5B is a plot showing that in vivo expanded CD56 b ⁇ ght NK cells after daclizumab therapy appeared to have much lower expression of CD 161 as compared to those from normal donors. Representative data from 1 out of 5 patients and 1 out of 5 normal donors. The gated cells are the CD56 bright NK subset.
  • FIG. 5C is a digital image showing that daclizumab does not trigger downstream signaling events. Purified PBMCs from normal human donors were treated with either daclizumab (100 ⁇ g/ml) or recombinant human IL-2 (20 ng/ml). Cells were lysed over time by RIPA buffer and phosphorylated STAT5 was examined by Western blot analysis using a phospho-STAT5 specific antibody. Arrow indicates band that corresponding to phosphorylated STAT5.
  • CME crystoid macular edema
  • ETDRS early treatment diabetic retinopathy study
  • FACS fluorescence activated cells sorting
  • GM-CSF granulocyte macrophage colony stimulating factor HAHA: human anti-human antibodies
  • IFN interferon
  • Ig immunoglobulin IL: interleuckin
  • IL-2 interleukin 2
  • IL-2R interleukin 2 receptor
  • IV intravenous kg: kilogram mg: milligram mm: millimeter
  • NK natural killer
  • PBMC peripheral blood mononuclear cells
  • SC subcutaneous
  • TNF tumor necrosis factor
  • VKH Vogt-Koganagi-Harada syndrome
  • VH variable heavy
  • VL variable light
  • 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.
  • Antagonist of an IL-2 Receptor An agent that specifically binds to the IL-2R, or a component thereof, and inhibits a biological function of the IL-2 receptor or the component.
  • Exemplary 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, and proliferation and/or activation of lymphocytes such as T cells in response to IL-2.
  • IL-2R antagonists of use in the methods disclosed herein inhibit at least one of these functions.
  • IL-2R antagonist of use in the methods disclosed herein can inhibit more than one or all of these functions.
  • an IL-2 receptor antagonist is an antibody that specifically binds Tac ( ⁇ 55), such as Zenapax® (also known as daclizumab, 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.,
  • an exemplary human anti-p55 antibody of use in the methods of the invention is HuMax-TAC, being developed by Genmab.
  • an IL-2 receptor antagonist is an antibody that specifically binds the p75 or ⁇ 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.5,152,980; 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- ⁇ l antibody is an antagonist that specifically binds the beta chain of human IL-2R.
  • 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-2R ⁇ ) are also known.
  • peptide antagonists for p55 and p75 are disclosed in U.S. Patent No. 5,635,597. These peptides are also of use in the methods disclosed herein.
  • 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
  • Fab 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
  • Fab' 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
  • (FaV) 2 the fragment of the antibody obtained by treating whole antibody with the enzyme pepsin without subsequent reduction or equivalently by genetic engineering
  • F(Ab ') 2 a dimer of two FAb' fragments held together by disulfide bonds
  • Fv a genetically engineered fragment containing the variable region of the light
  • CDR Complementarity-determining region
  • CDRs are involved in antigen-antibody binding, and the CDR3 comprises a unique region specific for antigen-antibody binding.
  • An antigen-binding site 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.
  • Corneal transplant The placement of a cornea, or a piece of a cornea, from one subject into another. When the clear part of the eye, the cornea, becomes diseased, it can become opaque and medication will not help. Often the only way to deal with the problem is to transplant a corneal "button" obtained from a cadaver. Since it is foreign tissue (non-self), immunosuppressive agents are often used to prevent rejection. For example, corticosteroid drops can be used. Those recipients who do reject a cornea and require a supplemental transplant, and those recipients who have high risk characteristics, often require more aggressive immunosuppression. Steroid use to prevent rejection of the transplant can lead to glaucoma and reactivation of herpes in the eye.
  • Cytokine A generic name for a diverse group of soluble proteins and peptides which act as humoral regulators at nano- to picomolar concentrations and which, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues. These proteins also mediate interactions between cells directly and regulate processes taking place in the extracellular environment. Many growth factors and cytokines act as cellular survival factors by preventing programmed cell death. Cytokines and interleukins include both naturally occurring peptides and variants that retain full or partial biological activity. Although specific cytokines/interleukins are described in the specification, they are not limited to the specifically disclosed peptides.
  • Cytokines include the interleukins, such as, but not limited to, interleukin (IL)-IO, IL-2, and IL-4. Cytokines also include interferon (IFN)- ⁇ , tumor necrosis factor (TNF)- ⁇ , granulocyte macrophage stimulating factor.
  • IL interleukin
  • IL-2 interleukin-2
  • IL-4 interferon
  • TNF tumor necrosis factor
  • granulocyte macrophage stimulating factor granulocyte macrophage stimulating factor
  • Dry eye The result of lack of lubrication (tears) in the eye. There are many causes of dry eye, including immune-mediated causes. For example, dry eye can be the result of Sjogren's syndrome.
  • 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.
  • EAU Experimental Autoimmune Uveoretinitis
  • EAU autoimmune uveoretinitis
  • S-Ag retinal S-antigen
  • S-Ag binds phosphorylated cytopigments and blocks the interaction of transducin with the photoexcited light receptor of the visual cascade.
  • S-Ag is the only retinal autoantigen to which a substantial number of human patients with endogenous intermediate and posterior uveitis consistently demonstrate in vitro proliferative responses (Nussenblatt et al., Am. J. Ophthalmol. 89:173, 1980; Nussenblatt et al., Am. J. Ophthalmol. 94:147, 1982).
  • Framework region 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.
  • CDRs complementarity-determining regions
  • 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.
  • 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.
  • 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 1 , IgG 2 , IgG 3 , IgG 4 ), 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 acids 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. Sd.
  • An immunoglobulin light or heavy chain variable region includes a framework region interrupted by three hypervariable regions, also called complementarity determining regions (CDRs) (see, Sequences of Proteins of Immunological Interest, E. Kabat et al., U.S. Department of Health and Human
  • 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.
  • 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.
  • 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.
  • 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.”
  • 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.
  • 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
  • Human antibodies can also be prepared by using transgenic animals carrying a human immunoglobulin gene (e.g., see Lonberg et al., PCT PublicationNo. WO93/12227; and Kucherlapati, PCT Publication No. WO91/10741, which are herein incorporated by reference).
  • Inflammation A series of local tissue reactions that take place at a site of injury and have an immunological component.
  • the injury may be due to trauma, lack of blood supply, hemorrhage, autoimmune attack, transplanted exogenous tissue or infection.
  • This generalized response by the body includes the release of many components of the immune system (such as cytokines), attraction of cells to the site of the damage, swelling of tissue due to the release of fluid and other processes.
  • Inflammation can be of an infectious or a non-infectious etiology. In the eye, inflammation produces vascular dilation, fluid leakage into extra- vascular spaces, migration of leukocytes and other cells.
  • Infectious agent An agent that can infect a subject, including, but not limited to, viruses, bacteria, and fungi.
  • Interleukin 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.
  • 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).
  • IL-2 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.
  • IL-2 receptor A cellular receptor that binds IL-2 and mediates its biological effects.
  • 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).
  • High affinity interleukin 2 receptor (IL-2R) complexes are expressed on activated lymphoid cells, including T cells and natural killer (NK) cells. They consist of 3 subunits (alpha, beta, and gamma), each capable of binding
  • 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
  • p55 has a length of 251 amino acids with an extracellular domain of 219 amino acids and 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 .
  • the gene encoding the gamma subunit of theTL2 receptor maps to human chromosome Xql3, spans approximately 4.2 kb and contains eight exons.
  • the 55 kDa IL-2 ⁇ (p55, Tac, or CD-25) subunit is expressed by most T, B and NK cells only after they have been activated by interaction with an antigen or with IL-2.
  • Tac subunit associated with the IL-2R beta-gamma subunits forms the high affinity IL-2R complex.
  • expression of the Tac subunit marks the activation of all T cells that are major contributors to both allograft destruction as well as autoimmune disorders such as uveitis.
  • Limbal stem cell transplant A transplant of limbal cells used to treat diseases of the front of the eye. There are some cases of corneal dysfunction which are due to a lack of limbal stem cells. Before performing a corneal transplant, limbal stem cells are transplanted, often from a foreign (non-self) donor. Subjects who receive a limbal stem cell transplant need to be immunosuppressed aggressively to prevent rejection.
  • 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.
  • NK cells A type of lymphocyte that shares a common progenitor with T cells. Generally, natural killer cells and have been described as large, granular, bone-marrow derived lymphocytes. These cells weaken the target cell's plasma membrane.
  • Human NK cells are generally characterized immunohistochemically by the presence of CD56 and the absence of CD3 on the cell membrane. In one specific, non-limiting example, NK cells can be characterized as being of CD56 bright cells. In one example, CD56 bright NK cells are capable of cytokine production.
  • CD56 b ⁇ ght NK cells can express one or more of CXCR3, CD122 and CD62L, CD94, and/or NKGD2D.
  • CD56 bright NK cells can also express no or low levels of CD 161.
  • Murine NK cells also express NKl .1.
  • 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 are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins.
  • polypeptide is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced.
  • fragment refers to a portion of a polypeptide that is at least 8, 10, 15, 20 or 25 amino acids in length.
  • 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.
  • 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.
  • compositions and formulations suitable for pharmaceutical delivery of the IL-2 receptor antagonists herein disclosed 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 IL-2 receptor antagonists herein disclosed.
  • 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.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • 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.
  • compositions to be administered can contain non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, salts, amino acids, and pH buffering agents and the like, for example sodium or potassium chloride or phosphate, Tween, sodium acetate or sorbitan monolaurate.
  • Pulsatile Dose A dose administered as a bolus.
  • a pulsatile dose can be administered to a subject as a single administration, such as by direct injection or by an intravenous infusion during a specified time period.
  • the pulsatile dose can be a "push" or rapid dose, but need not be, as it can be administered over a defined time period, such as in an infusion.
  • Repeated pulsatile doses can be administered to a subject, such as a bolus administered repeatedly, such as about every one, two, or three months, or about every one, two, three or four weeks or about every one, two or three days.
  • the administered dose can be the same amount of an agent, or can be different amounts administered at several time points separated by periods wherein the agent is not administered to the subject, or wherein a decreased amount of the agent is administered to the subject.
  • purified does not require absolute purity or isolation; rather, it is intended as a relative term.
  • a purified or isolated protein preparation is one in which protein is more enriched than the protein is in its generative environment, for instance within a cell or in a biochemical reaction chamber.
  • a preparation of protein is purified such that the protein represents at least 50% of the total protein content of the preparation.
  • substantially purity of 90%, 95%, 98% or even 99% or higher of the active agent can be utilized.
  • Preventing or treating 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.
  • An example of a person with a known predisposition is someone with a history of a disease 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, such as uveitis, after it has begun to develop.
  • Sequence identity The similarity between two nucleic acid sequences, or two amino acid sequences, is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or orthologs of the IL-2R antibodies or antigen binding fragments, and the corresponding cDNA sequence, will possess a relatively high degree of sequence identity when aligned using standard methods. This homology will be more significant when the orthologous proteins or cDNAs are derived from species that are more closely related, compared to species more distantly related (e.g., human and murine sequences).
  • an agent that binds substantially only to a defined target an agent that binds substantially only to a defined target.
  • an IL-2 receptor-specific binding agent binds substantially only the IL- 2 receptor, or a component thereof.
  • 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.
  • Fabs, Fvs, and single-chain Fvs (SCF vs) that bind to an IL-2 receptor would be IL-2 receptor-specific binding agents.
  • Subject A human or non-human animal. In one embodiment, the subject has uveitis.
  • 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 an immune infiltrate in the uveal tract.
  • Therapeutically Effective Amount A dose sufficient to prevent advancement, or to cause regression of the disease, or which is capable of reducing symptoms caused by the disease, such as multiple sclerosis, hi one example, a therapeutically effective amount induces the expansion of CD56 b ⁇ ght NK cells.
  • 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.
  • Uveitis An intraocular inflammatory disease that includes crizos, rhinitis, rhinitis, rhinitis, rhinitis, rhinitis, aveitis, anterior uveitis.
  • Iritis is an inflammation of the iris.
  • Cyclitis is inflammation of the ciliary body.
  • Panuveitis refers to inflammation of the entire uveal (vascular) layer of the eye.
  • peripheral uveitis within which is the entity called peripheral uveitis, is centered in the area immediately behind the iris and lens in the region of the vitreous 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 ulceris. 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" or pan- 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
  • the inflammatory products 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 identifiable infectious agent.
  • infective agents can also cause uveitis.
  • an appropriate antimicrobial drug can be given to cure the disease.
  • the etiology of uveitis remains elusive in the majority of cases.
  • Active uveitis refers to intraocular inflammation wherein immune cells are present in the uveal tract, such as in the anterior chamber (active anterior uveitis) or in the vitreous (active posterior uveitis).
  • the number of immune cells in the anterior or vitreous can be quantified.
  • immune cells can be quantified using a system by Hogan and Kimura using a 1X1 mm slit lamp (American J. Opthal. Al: 155, 1959)
  • vitreous haze is a result of proteins in the vitreous (see Nussenblatt et al., Ophthalmology 92:467-471, 1985).
  • vitreous haze is ranked on a scale of 0-4, with 0 being no haze (no active inflammation) and 4 being inflammation so severe that the architecture of the back of the eye cannot be visualized using indirect ophthalmoscopy.
  • a score of 2 indicates the presence of active uveitis.
  • vitreous haze is visualized using an indirect ophthalmoscope.
  • 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 IgG 2a 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.
  • MLR mixed lymphocyte response
  • 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).
  • HAM/TSP human T cell lymphotrophic virus type 1 associated myelopathy/topical spastic paraparesis
  • Zenapax® is of use for the treatment of the eye. (see Nussenblatt et al., Proc. Natl. Acad. Set, 96:7462- 7466, 1999).
  • Methods are provided herein for the treatment of subjects that have active uveitis, hi one embodiment the subject has active posterior uveitis.
  • the methods disclosed herein can also be used for the treatment of subjects with other forms of active uveitis, such as active anterior uveitis or active diffuse uveitis.
  • the active uveitis can be the result of an infectious disease or have a non-infectious origin, hi one example, methods are provided for treating active posterior uveitis of infectious or non-infectious origin. Methods are also provided for the treatment of corneal transplant rejection, limbal stem cell rejection following transplantation, optic neuritis and/or dry eye.
  • the methods are of use in adult subjects and in children, hi one embodiment, the methods are of use for treating an adult subject with active uveitis, such as non-infectious uveitis, hi another embodiment, the methods are of use for treating children, such as children with juvenile rheumatoid arthritis, postviral neuroretinitis, pars planitis, sarcoidosis, idiopathic anterior uveitis or idiopathic intermediate uveitis or other non-infectious entities resulting in an intraocular inflammatory response.
  • an IL-2 receptor antagonist such as an antibody that binds a component of the IL-2 receptor (p55 or p75, such as an anti- Tac antibody), is administered to a subject.
  • the IL-2 receptor antagonist can induce CD56 b ⁇ ght NK cells.
  • the IL-2 receptor antagonist can be administered as a pulsatile high induction dose sufficient to treat the inflammation.
  • the IL-2 receptor antagonist can be administered as a single bolus.
  • the IL-2 receptor antagonist can be administered repeatedly, such as about every 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days or about every week, two weeks, three weeks, four, five or six weeks.
  • the IL-2 receptor antagonist can also be administered at irregular intervals, such as a first dose (induction bolus dose) followed by a second dose after about three to four weeks followed by a third dose after about two to three weeks.
  • the IL-2 receptor antagonist can also be administered about every one or about every two months.
  • at least two pulsatile doses are administered; these doses can be administered such that there is at least one to three weeks, such as about two weeks, between doses.
  • the doses can be administered about fourteen days apart, such as about two pulsatile doses administered about thirteen to about fifteen days apart. In one embodiment, no therapy is given between the pulsatile high doses.
  • a different therapeutic agent can be administered during the intervening period between the pulsatile high doses.
  • a low maintenance dose of the IL-2 receptor antagonist can be administered during the intervening period between the pulsatile high doses.
  • the pulsatile doses of the IL-2R antagonist can be administered parenterally, i.e., subcutaneously, intramuscularly or intravenously or by means of a needle-free injection device.
  • the pulsatile dose of the IL-2 receptor antagonist can also be administered intraocularly or periocularly.
  • the administration is intravenous (IV).
  • the compositions for parenteral administration will commonly include a solution of the IL-2R antagonist (such as an antibody) in a pharmaceutically acceptable carrier as described above.
  • the concentration of antibody in the formulations can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight or from 1 mg/niL to 100 mg/mL.
  • the concentration is selected primarily based on fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • IL-2 receptor antagonist is an antibody
  • the antibodies of use in the methods disclosed herein can be frozen or lyophilized for storage and reconstituted in a suitable carrier prior to use.
  • One of skill in the art can readily design appropriate lyophilization and reconstitution techniques.
  • the pulsatile high doses of an IL-2 receptor antagonist are high doses of an antibody, such as a monoclonal antibody, e.g., a chimeric, humanized or human monoclonal antibody.
  • a monoclonal antibody e.g., a chimeric, humanized or human monoclonal antibody.
  • daclizumab also known as ZenapaxTM
  • ZenapaxTM ZenapaxTM
  • 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 10 8 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.
  • the antibody can include two light chain/heavy chain dimers, and specifically binds to either p55 (such as the anti-Tac antibody) or p75.
  • IL-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.
  • 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.
  • 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 embodiments that antibody is an IgG, including but not limited to, IgG 1 , IgG 2 , IgG 3 and IgG 4 .
  • the amount of an IL-2 receptor antagonist administered in a high pulsatile doses can determined by assessing the saturation of IL-2 receptors on cells in the immune system that are not in the circulation. For example, a dose of a primary IL-2 receptor antagonist can be administered to the subject. Peripheral blood mononuclear cells are then isolated from the subject. The ability of a second antagonist of the IL-2 receptor that binds a different site on the IL-2 receptor, such as an antibody that binds a different epitope, to bind the peripheral blood mononuclear cells (PBMC) is assessed. The ability of the primary antagonist of interest to bind the PBMC is also assessed. If the receptors are saturated, the second antagonist will bind the PBMC, while the primary antagonist will not bind the PBMC. This assay can used to determine if the primary antagonist saturates the IL- 2 receptor on PBMC.
  • PBMC peripheral blood mononuclear cells
  • the binding of the second antagonist of the IL-2 receptor is also assessed on immune cells that are not circulating, but are in one or more lymphoid organs (for example, lymph nodes, spleen, or tonsils) cells.
  • lymphoid organs for example, lymph nodes, spleen, or tonsils
  • the ability of a second antagonist of the IL-2 receptor that binds a different site on the IL-2 receptor, such as an antibody that binds a different epitope, to bind the cells from a sample (such as a biopsy) in a lymphoid organ is assessed.
  • the ability of the primary antagonist of interested to bind the cells in the lymphoid organ is also assessed. If the receptors are saturated, the second antagonist will bind the cells in the lymphoid organ, while the primary antagonist will not bind the cells in the lymphoid organ. This assay can used to dete ⁇ nine if the primary antagonist saturates the IL-2 receptor in a lymphoid tissue.
  • both the primary and the secondary IL-2 receptor antagonists are antibodies.
  • the binding of the primary and the secondary IL-2 receptor antagonist can be assessed by immunohistochemistry or by fluorescence activated cells sorting (FACS), using directly labeled antibodies or using additional antibodies that are labeled that bind only the primary or only the secondary IL-2 receptor antibody.
  • FACS fluorescence activated cells sorting
  • a "high pulsatile dose” saturates the IL-2 receptors in both the lymphoid sample and PBMC.
  • a "low pulsatile dose” saturates the IL-2 receptors on circulating PBMC, but not in the lymphoid organs.
  • a "high pulsatile dose” is a dose that produces effective levels in the serum between pulsatile doses or that reaches sequestered spaces outside the circulatory system (such as the lymphoid organs).
  • a “low pulsatile dose” is a dose that does not maintain effective levels in the serum between pulsatile doses.
  • a "high pulsatile dose” maintains effective concentrations in the serum, such as a concentration sufficient to saturate the IL-2 receptors on PBMC, for three to four weeks (the half life of this dose in the serum).
  • a "low pulsatile dose” does not provide a concentration sufficient to saturate the IL-2 receptors on PBMC for three to four weeks (the half life of this dose in the serum); levels that are not therapeutic are reached during this time period.
  • a dose that saturates IL-2 receptors outside of the circulating immune cells (such as T and natural killer (NK) cells), maintains therapeutic levels in the serum, and is not toxic to the subject is of use with the methods disclosed herein.
  • the does induces expansion of CD56 b ⁇ ght natural killer (NK) cells.
  • the IL-2 receptor antagonist can induce expansion of CD56 b ⁇ ght NK cells that are capable of cytokine production.
  • the IL-2 receptor antagonist can induce CD56 b ⁇ ght NK cells that express one or more of CXCR3, CD122 and CD62L, CD94, and/or NKGD2D.
  • the IL-2 receptor antagonist can induce CD56 b ⁇ ght NK cells that express no or low levels of CDl ⁇ l.
  • high pulsatile doses of daclizumab are administered to a subject with active uveitis, such as posterior uveitis of infectious or non-infectious origin.
  • a "high dose” is about 4 to about 10 mg/kg, such as about 5 to about 9 mg/kg, about 6 to about 8 mg/kg, about 7 mg/kg, or about 8 mg/kg.
  • a "low dose” of an IL-2 receptor antagonist can be administered as a bolus.
  • a low dose does not saturate all of the IL-2 receptors in the immune tissues, but may saturate the IL-2 receptors on circulating immune cells (such as T or NK cells in the peripheral blood),
  • a "low dose” of daclizumab is about 0.1 mg to 3 mg/kg, such as about 0.5-2 mg/kg, such as about 1 mg/kg.
  • One of skill in the art will be able to construct an administration regimen of a "low” dose to adjust serum levels of the IL-2 receptor antagonist.
  • a first pulsatile high dose of daclizumab of about 5 to about 10 mg/kg is administered.
  • a second pulsatile dose high dose is administered after about 12-16 days.
  • the first pulsatile dose and the second pulsatile dose can be administered intravenously.
  • the first and second intravenous doses are both administered as continuous IV infusions over a defined period, such as over about 1 to 3 hours.
  • No therapy can be administered during the intervening period between the administration of the first pulsatile dose and the second pulsatile dose.
  • a maintenance dose of an IL-2 receptor antagonist such as daclizumab, is administered subcutaneously.
  • Exemplary maintenance doses of daclizumab are "low" dosed that include about 0.5 to about 2.5 mg/kg, such as about 1 to about 2 mg/kg of daclizumab administered subcutaneously or intravenously.
  • the first pulsatile dose can be administered at the same concentration as the additional pulsatile doses, or at a different concentration.
  • about first high pulsatile dose of daclizumab is administered at about 5 to about 10 mg/kg
  • the second pulsatile dose of daclizumab is administered at about 5 to 10 mg/kg.
  • a first high pulsatile dose of daclizumab is administered at about 5 to 10 mg/kg followed by at least one pulsatile dose of about 4 to about 5 mg/kg daclizumab.
  • These doses can be intravenous.
  • Subsequent pulsatile doses can also be administered. In one embodiment, no treatments can be administered between the pulsatile doses.
  • a different agent (that is not an IL-2 receptor antagonist) is administered between the pulsatile doses.
  • Suitable agents include, but are not limited to, other immunosuppressive agents.
  • an IL-2 receptor antagonist is administered between the pulsatile doses.
  • This IL-2 receptor antagonist can be the same IL-2 receptor antagonist administered during the high pulsatile doses, but at a low maintenance concentration.
  • a different IL-2 receptor antagonist can be administered between the pulsatile doses.
  • the other agent such as another IL-2 receptor antagonist, or the low maintenance dose of the same IL-2 receptor antagonist, can be administered by the same route of administration as the high pulsatile doses or by a different route of administration.
  • one or more maintenance doses are administered after the pulsatile doses.
  • a different agent that is not an IL-2 receptor antagonist
  • Suitable agents include, but are not limited to, other immunosuppressive agents.
  • An IL-2 receptor antagonist can be administered after the pulsatile doses.
  • This IL-2 receptor antagonist can be the same IL-2 receptor antagonist administered during the high pulsatile doses, but at a low maintenance concentration.
  • a different IL-2 receptor antagonist can be administered after the pulsatile doses.
  • the other agent such as another IL-2 receptor antagonist, or the low maintenance dose of the same IL-2 receptor antagonist, can be administered by the same route of administration as the high pulsatile doses or by a different route of administration.
  • One example of a protocol of use for treating active uveitis is the administration of an intravenous first pulsatile dose of daclizumab at a dose of about 5 to about 10 mg/kg to the subject, about 12 to about 14 days later intravenously administering a second pulsatile dose of daclizumab at a dose of about 3 to about 5 mg/kg to the subject, and beginning about 14 days after administering the second induction dose, administering an intravenous or subcutaneous maintenance dose of daclizumab to the subject once a month.
  • the maintenance dose can be, for example, about 1 to about 2 mg/kg of daclizumab.
  • the antibody is basilimab, marketed as Simulect® by Novartis Pharma AG.
  • Simulect® is a chimeric (murine/human) monoclonal antibody (IgG 1 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.
  • Doses of Simulect® are likely to differ slightly, for example 0.25 mg/kg to 1 mg/kg, e.g., 0.5 mg/kg, is considered to be a "low dose.”
  • "high" doses are considered to be a dose of more than about 3 to 10 mg/kg, such as about 3, about 4, about 5, about 6 or about 7 mg/kg, or unit doses of 10, 20, 40, 50 or 100 mg.
  • the general principle of keeping the IL-2R saturated could also be used to guide the choice of dose levels of other IL-2R antagonists such as other monoclonal antibodies, using methods such as those described above
  • the IL-2R antagonist can also be a molecule that binds to other subunits of the IL-2 receptor, such as Mik- ⁇ l or Mik- ⁇ 2 or their chimeric or humanized versions, which bind to the beta chain of human IL-2R, or another antibody that specifically binds p75 (see U.S. Patent No. 5,530,101, which is incorporated herein by reference).
  • the IL-2R antagonist may also be a fragment of an antibody (e.g., a chimeric, humanized or human antibody) such as a Fab, (Fab') 2 , Fv, or scFv. Further, the fragment may be pegylated to increase its half-life.
  • the IL-2R antagonist is a combination of anti-IL-2R agents.
  • Zenapax® diaclizumab
  • Simulect® are administered together as a cocktail, or the agents are alternated in the administration schedule.
  • the IL-2R antagonist such as a humanized antibody that specifically binds the IL-2R, can be used in combination with other antibodies, particularly human monoclonal antibodies reactive with other markers on cells responsible for a disease.
  • suitable T cell markers can include those grouped into the so-called “Clusters of Differentiation,” (CD antigens, see the First International Leukocyte
  • the other antibody binds and inhibits a lymphokine, such as IFN-gamma, or a lymphokine receptor.
  • a lymphokine such as IFN-gamma
  • the other antibody binds ⁇ 5 ⁇ l integrin (VLA-5), of such as the antibody is Antegren® (Elan Pharmaceuticals and Biogen, Inc.).
  • An IL-2 antagonist such as the antibody that binds the IL-2R can be formulated with additional therapeutic agents. These can be used in one or more high pulsatile dose(s) or in the low maintenance dose.
  • exemplary agents include cyclosporine, FK506, steroids such as hydrocortisone, antibodies (such as anti-CD4 or antibodies that specifically bind the IL-2 receptor), cytokines (such as beta- interferon), or non-steroidal anti-inflammatory agents.
  • Additional agents include antibacterial antibiotics, such as minoglycosides (for example, amikacin, apramycin, arbekacin, bambermycins, butirosin, dibekacin, dihydrostreptomycin, fortimicin(s), gentamicin, isepamicin, kanamycin, micronomicin, neomycin, neomycin undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin, trospectomycin), amphenicols (for example, azidamfenicol, chloramphenicol, florfenicol, thiamphenicol), ansamycins (for example, rifamide, rifampin, rifamycin sv, rifapentine, rifaximin), ⁇ -lactams (for example, carbacephems (e.g.,
  • Agents of use also include synthetic antibacterials, such as 2,4-Diaminopyrimidines (for example, brodimoprim, tetroxoprim, trimethoprim), nitrofurans (for example, furaltadone, furazolium chloride, nifuradene, nifuratel, nifurfoline, nifurpirinol, nifurprazine, nifurtoinol, nitrofurantoin), quinolones and analogs (for example, cinoxacin, ciprofloxacin, clinafloxacin, difloxacin, enoxacin, fleroxacin, flumequine, grepafloxacin, lomefloxacin, miloxacin, nadifloxacin, nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, pazufloxacin, pefloxacin, pipemidic acid,
  • antifungal antibiotics such as polyenes (for example, amphotericin B, candicidin, beostatin, filipin, fungichromin, hachimycin, hamycin, lucensomycin, mepartricin, natamycin, nystatin, pecilocin, perimycin), others (for example, azaserine, griseofulvin, oligomycins, neomycin undecylenate, pyrrolnitrin, siccanin, tubercidin, viridin)allylamines (for example, butenafine, naftifine, terbinafme), imidazoles (for example, bifonazole, butoconazole, chlordantoin, chlormiidazole, cloconazole, clotrimazole, econazole, enilconazole, fenticonazole, flutrimazole, isoconazole, ketoconazoles (for example
  • Antineoplastic agents can also be of use including (1) antibiotics and analogs (for example, aclacinomycins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carubicin, carzinophilin, chromomycins, dactinomycin, daunorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, idarubicin, menogaril, mitomycins, mycophenolic acid, nogalamycin, olivomycines, peplomycin, pirarubicin, plicamycin, porflromycin, puromycin, streptonigrin, streptozocin, tubercidin, zinostatin, zorubicin), (2) antimetabolites such as folic acid analogs (for example, denopterin, edatrexate, methotrexate, piritrexim,
  • Steroidal anti-inflammatory agents can also be included in the therapeutic protocol, such as 21-acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clobetasone, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, cyclosporine, deflazacort, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortolone, fluorometholone, fluperolone acetate, fluprednidene
  • non ⁇ steroidal anti-inflammatory agents can be used. These include aminoarylcarboxylic acid derivatives (for example, enfenamic acid, etofenamate, flufenamic acid, isonixin, meclofenamic acid, mefenamic acid, niflumic acid, talniflumate, terofenamate, tolfenamic acid), arylacetic acid derivatives (for example, aceclofenac, acemetacin, alclofenac, amfenac, amtolmetin guacil, bromfenac, bufexamac, cinmetacin, clopirac, diclofenac sodium, etodolac, felbinac, fenclozic acid, fentiazac, glucametacin, ibufenac, indomethacin, isofezolac, isoxepac, lonazolac, metiazinic acid, mo
  • Uveitis refers to intraocular inflammatory diseases that are an important cause of visual loss.
  • Standard systemic immunosuppressive medications used for uveitis can cause significant toxic side effects, especially with prolonged use. Consequently, an effective treatment with a safer side effect profile is highly desirable.
  • IL-2 receptor antagonists can be used to treat active uveitis. These antagonists are also of use for treating other immune mediated disorders, such as corneal transplant rejection, limbal stem cell rejection following transplantation, optic neuritis and dry eye.
  • Daclizumab is a humanized monoclonal antibody directed against the high affinity IL-2 receptor CD25 or Tac subunit.
  • the IL-2 receptor system plays a central role in mediating immune responses. Blocking this system impedes immune responses and can inhibit local inflammatory responses, including uveitis. Pilot studies using intravenous or subcutaneous daclizumab treatments suggest that daclizumab treatments at 1 mg/kg every 2-4 weeks for quiescent uveitis may effectively replace the other immunosuppressive medications in a majority of cases.
  • the primary focus of the study was a short or acute response trial to relatively high-dose daclizumab infusions to observe if the vitreous haze associate with active uveitis was reduced. Li order to qualify for enrollment, each participant met all of the inclusion criteria (see below) and not meet any of the exclusion criteria (see below).
  • This study enrolled participants who currently had active non ⁇ infectious autoimmune uveitis. Two induction treatments were completed within 14 days, with the primary evaluation at Day 21. An induction regimen of intravenous (TV) daclizumab at 8 mg/kg was given on Day 0 followed by another IV dose of 4 mg/kg at Day 14, provided the safety endpoint was not been met. It was determined that if the induction regimen appears was ineffective by Day 21 (i.e., no improvements in the primary or secondary measures are observed), the participant exited the trial. However, this was not necessary, as described below.
  • the primary outcome was the reduction of vitreous haze by at least two steps in (such as 2+ to Trace, or 1+ to 0) from baseline to Day 21 in both eyes. Participants were considered a failure at Day 21 if either eye has not shown a 2-step reduction in vitreous haze. Participants were considered a treatment failure if at any time during the study either eye loses >3 lines (>15 letters or >0.30 logMAR in best- corrected visual acuity (BCVA), early treatment diabetic retinopathy study (ETDRS) method compared to baseline.
  • BCVA visual acuity
  • EDRS early treatment diabetic retinopathy study
  • Secondary outcome measures included the amount of retino- vascular leakage measured by fluorescein angiogram, retinal thickening as measured by OCT, presence or extent of cystoid macular edema (CME), weighted grading score of immunosuppressive medications (the immunosuppression load), ocular inflammation grades for anterior chamber (AC) cells, vitreous cells, and vitreous haze (Appendix 3), distance BCVA, Quality of Life assessment via the SF-36 questionnaire, and visual functioning measured by the.VFQ-25 questionnaire. Secondary outcomes were assessed at Day 21, and for participants who continue into maintenance therapy, at 6 and 12 months.
  • Safety outcomes were tabulated by observing the nature, severity and frequency of systemic toxicities, adverse events, and infections throughout the study. Safety assessments were made routinely during the study, with a review of the previous visit interval performed at each scheduled visit. Each participant was encouraged to report any apparent adverse events between scheduled visits, and could return for additional evaluations and appropriate treatment between scheduled visits if needed.
  • the baseline (Week 0) evaluations must occur on Day 0 unless they are marked with an X . If marked X , the exams may occur after study consent is given and within 5 days prior to the initial daclizumab infusion. Medically-indicated evaluations may be repeated as needed.
  • Adverse events should be reported at any time. At each visit, a review by interview with the participant should be made with directed questions regarding adverse events for the interval since the last visit, including an assessment of the injection site(s). Medications are recorded at baseline and whenever a change occurs.
  • the baseline ETDRS visual acuity should be repeated and recorded as two independent evaluations, which may be performed on the same day, using two different sets of ETDRS letter charts.
  • Serum chemistry and LFTs include: sodium, potassium, chloride, CO 2 (total), creatinine, glucose, urea nitrogen, alkaline phosphatase, ALT/GPT, and AST/GOT. 6 Specimens were obtained from all participants. Serum samples must be collected immediately prior to a study treatment. Upon termination of study treatments, both PK and HAHA analysis samples should be taken at +4 and +8 weeks after the last study treatment. Inclusion Criteria
  • Participant had a diagnosis of active, non-infectious intermediate or posterior uveitis, which may include but is not restricted to the following conditions known to cause intermediate or posterior uveitis: panuveitis, intermediate uveitis of the pars planitis subtype, sarcoidosis, the Vogt-Koyanagi-Harada (VKH) syndrome, birdshot retinochoroidopathy, retinal vasculitis and sympathetic ophthalmia;
  • VKH Vogt-Koyanagi-Harada
  • Participant had active uveitis with > Grade 1 (1+) vitreous haze in at least one eye (see Appendix 3 for haze grading scale) including evidence of retinal vascular leakage using fluorescein angiography or the presence of cystoid macular edema (CME) at enrollment;
  • Participant did not plan to undergo elective ocular surgery (e.g., cataract extraction) during the study period;
  • Exclusion Criteria A volunteer was not permitted to enroll if they met any one of the following exclusion criteria:
  • Participant had received previous treatment with an IL-2 or IL-2R directed therapy within the past 90 days; 3. Participant had lens opacities or obscured anterior ocular media upon enrollment such that reliable evaluation and grading of posterior segment could not be performed (except that anterior chamber cells due to inflammation was not an exclusion);
  • Participant had a history of an active herpes zoster or varicella infection within 6 weeks before enrollment, or chicken pox exposure within 21 days before enrollment;
  • Participant was currently enrolled in another investigational or interventional therapeutic trial, or is using a therapy for a non-uveitis condition that would likely affect immune responses or interfere with trial logistics, or has received any investigational therapy within the 30 days prior to enrollment;
  • Participant had a history or diagnosis of Bechet's disease (since subsequent tapering or withdrawal of concomitant immunosuppressive medications is not a standard-of-care for Bechet's patients) or a primary diagnosis of anterior uveitis (e.g., HLA-B27 associated uveitis, or ocular conditions usually treated with local and not systemic medications);
  • Participant had a significant local or systemic infection requiring medical treatment at the time of enrollment;
  • Participant was currently pregnant or lactating; 10. Participant was a history of cancer (other than a non-melanoma skin cancer or in situ cervical cancer) diagnosed within the past 5 years;
  • Participant had a non-ocular, medically significant co-morbid condition that impaired normal activities, required immunosuppression, or had a condition with a prognosis that indicated a significant risk of disability or death if the condition were to continue or be exacerbated during the study period, or a medical condition that would likely have an impact on the participant's ability to comply with the visit schedule.
  • Such conditions included, for example, recent heart attack, significant COPD, brittle diabetes, kidney disease, severe emphysema, organ transplant (requiring corticosteroids or other immunosuppressive medications), hepatitis or other liver disease, or uncontrolled psychiatric illnesses.
  • IL-2 receptor antagonist such as daclizumab.
  • these exclusion criteria were set to limit variability between subjects, and thus to generate a controlled study that was amenable to statistical analysis.
  • the protocols disclosed herein can be used to treat any subject with uveitis, including for example those subjects infected with HIV, those who are pregnant or lactating, or those with a 5 history of cancer.
  • the immunosuppressive agents were assigned a relative weight, as disclosed below. Again, this was set for the purposes of statistical analyses; the assigned "weights" should not be construed to be limiting. Thus, a subject who is treated with an IL-2 receptor antagonist could also be treated with
  • the scale used to determine the grade is as follows:
  • Example 4 Daclizumab Formulation and Administration The IV daclizumab formulation contains 5 mg/mL of the anti-Tac monoclonal antibody and 0.2 mg/mL of Polysorbate-80 in 67 mM phosphate buffer (3.6 mg/mL of sodium phosphate monobasic monohydrate, 11 mg/mL sodium phosphate dibasic heptahydrate) and 4.6 mg/mL sodium chloride, with the pH adjusted to 6.9. No preservatives were added.
  • the formulation was diluted before use following the general instructions provided with the container or using the instructions provided for the Zenapax® package insert (the equivalent commercial preparation). The calculated dose was administered in a solution of at least 50 mL of 0.9% sodium chloride solution delivered over a period of 90 minutes.
  • the 800 mg maximum dose for a 100 kg person using the 5 mg/mL daclizumab IV formulation would be in a delivered volume of 210 mL.
  • An acceptable alternate for IV administration of daclizumab is to use the 100 mg/mL formulation described below, which must be diluted to the minimum volume of at least 50 mL of 0.9% sodium chloride solution before administration, and delivered over a period of 90 minutes.
  • the line was flushed with about 5 mL of 0.9% sodium chloride solution.
  • the start and end time of administration was recorded on the infusion sheet as wells as volume of fluids delivered. Vital signs were taken and recorded pre-infusion, immediately post infusion and 15 minutes after the infusion is completed. Additional vital signs were recorded if indicated.
  • the maximum amount of the study drug concentrate administered was 160 mL, which is the equivalent of 800 mg of daclizumab IV.
  • the subcutaneous (SC) daclizumab formulation contained 100 mg/niL of the anti-Tac monoclonal antibody in an isotonic aqueous buffer composed of 40 mM sodium succinate, 100 mM sodium chloride, and 0.03% Tween 80, with the pH adjusted to 6.0. This was supplied as 1.0 mL of a sterile solution in single-use, 2 mL glass vials for subcutaneous injection without dilution. No preservatives were added.
  • This SC formulation can also be used for IV administration provided it is diluted in at least 50 mL of 0.9% sodium chloride solution and administered in the same fashion as the IV formulation described above.
  • the SC daclizumab solution was prepared for SC injection by withdrawing the solution from the vial into a sterile, 1 mL plastic syringe using aseptic technique. No filtration was performed. After the SC daclizumab solution was drawn into a syringe for SC injection, it was stored at room temperature for up to 4 hours prior to use, after which it was administered.
  • aseptic techniques were used and the skin was prepared by swabbing with isopropyl alcohol (or other suitable disinfectant) and allowed to dry.
  • the skin was drawn taut or pinched into a fold to facilitate injection into the subcutaneous space using a needle such as a sterile 25-gauge x 5/8-inch needle. After injection, the participant was instructed to move their limb(s) about for a few moments to increase local circulation. Whenever the planned total injection volume for SC treatments exceeded 1 mL (100 mg), the treatment was administered at two or more injection sites, such that each injection site received ⁇ 1 mL. The time of administration of each injection was recorded. Vital signs were taken and recorded pre-injection and 30 minutes after the last injection. Additional vital signs were recorded if indicated. Daclizumab formulations were stored unopened under controlled, refrigerated conditions at 2° to 8° C.
  • the daclizumab induction treatments included an infusion of 8 mg/kg (up to a maximum of 800 mg total dose) administered IV at Day 0 followed by an FV infusion of 4 mg/kg (up to a maximum of 400 mg total dose) on Day 14 ( ⁇ 3 days).
  • the standard calendar schedule of all future treatments through the 52-week study period was fixed (see Table 2).
  • maintenance therapy began at 2 mg/kg (up to a maximum of 200 mg total dose) at four- week intervals (i.e., q 4 weeks or every 28 days ⁇ 7 days).
  • Scheduled treatments continued at q 4-week intervals through Week 52. Schedule creep was not permitted so if treatments could not be made during the ⁇ 7 day window as scheduled, they were skipped rather than adjusting the subsequent scheduled treatment dates
  • SC injections were made only in the upper limbs. If the participant had maintained visual acuity during that initial period, other locations could be used for subsequent injections. Subcutaneous injections of daclizumab were then given in any suitable location visible to the participant on the participant's limbs (e.g., upper arms or legs) or if desired, the trunk of the body. Needle placement sites were rotated between limbs and various limb or body locations to avoid using the same location for consecutive treatments, or injecting directly over a site that can be recognized from a previous treatment.
  • OCT Ocular computed tomography
  • Pregnancy test (urine or serum ⁇ -HCG) for females with childbearing potential (CBP);
  • Clinical laboratory tests including: CBC with differential, serum CHEM 20 panel (sodium, potassium, chloride, total CO 2 [bicarbonate], creatinine, glucose, urea nitrogen [BUN], albumin, calcium [total], magnesium [total], inorganic phosphorous, ALT/GPT, AST/GOT, total bilirubin, lactate dehydrogenase [LDH], total protein, total creatinine kinase, alkaline phosphatase, and uric acid), total cholesterol, LDL cholesterol, triglycerides, and urinalysis; 12. Serum levels of daclizumab for pharmacokinetic (PK) evaluation will be assessed in all participants at specified intervals.
  • PK pharmacokinetic
  • the default schedule for these PK evaluations will be at visits scheduled closest to Day 0*, 7, 14*, 21, 28*, 56*, 84*, 140*, 196*, and 364*. (The * means trough level taken immediately before the scheduled treatment for that day.)
  • HAHA Human-anti-daclizumab antibodies
  • serum samples taken immediately prior to study treatments from participants at the assessment scheduled closest to Day 0*, 7, 14*, 21, 28*, 56*, 196*, and 364*. Similar to PK analysis sampling, participants who terminate study treatments early during the trial were scheduled to provide two HAHA analysis samples during their washout period. These samples are scheduled at 4 and 8 weeks ( ⁇ 1 week) after the last study treatment. If a participant has terminated study treatments early, the washout HAHA specimen schedule superseded the existing default schedule. Specimens for serum daclizumab and the anti-daclizumab antibodies were assayed at a central laboratory. (Instructions for processing, temporary storage and shipping of PK and HAHA specimens were provided in the study MOP).
  • Clinical laboratory tests and immunology tests including: blood type and
  • HLA class I and II
  • ESR ESR
  • total protein hepatitis B screen
  • urine cytology all at baseline and study exit. If a participant tested negative for hepatitis B on enrollment, they were offered the possibility of immunization against hepatitis B during the trial.
  • FACS analysis or other research laboratory methods used on peripheral blood samples to determine the presence and functionality of IL-2 receptors on circulating T lymphocytes and other cell surface or internal markers. These determinations used, for example, CD25 antibody (anti- Tac receptor antibody), 7G7 antibody directed against another portion of the IL-2 receptor, or other related antibodies or reagents. Separate analyses were performed using functional suppressor cell assays [at baseline, Week 12, 28, and 52]. Blood specimens for suppressor assays were collected both two days prior to treatment and on the specified day of treatment. On those days, participants withheld taking their regular medications until after the specimens were drawn and optimally fast or have a minimal breakfast that day. Total combined blood volume for all these assays using peripheral blood did not exceed 100 mL for each of the four specified visit periods.
  • Example 6 Results Six participants enrolled and received the initial dose of 8 mg/kg intravenous daclizumab. Median follow-up duration of the five active participants was 12 weeks (range 8-24 weeks). Participant #002 chose to voluntarily withdraw and was terminated from the study immediately after enrollment, due to personal issues, and without any apparent adverse effects. This participant had signs of psychiatric instability before enrollment; behavior control became a significant issue when in the clinic. Patient demographic as shown below in Table 4:
  • Figs. 2-3 show the results obtained from five subjects treated with a high pulsatile dose daclizumab. The results were presented from before treatment and after >6 weeks of daclizumab treatment. As shown in Figs. 2-3, treatment of the subjects with the high pulsatile dose of daclizumab resulted in an increased in the population of NK cells expressing both CD56 and CXCR3 in all of the subjects.
  • Daclizumab Humanized anti- IL-2 receptor ⁇ chain (CD25) monoclonal antibody
  • CD25 humanized anti- IL-2 receptor ⁇ chain
  • the follow up of these patients ranged from 6-28 weeks.
  • Flow cytometry data were analyzed by Flow Jo (TreeStar Inc., Ashland, OR). Briefly, the lymphocyte population was gated based on forward scatter (FSC) and side scatter (SSC) characteristics. NK cells were gated based on CD56+CD3- staining. CD56 bright cells were gated based on CD56 bright CX3CRl-CD3- or
  • CD56 bright CXCR3+CD3- The results were represented by the percentage of the CD56 bright subset in either lymphocyte or the CD56+CD3- NK cell subpopulation. Student t-test was used for statistical analysis.
  • PBMCs were isolated from patients' whole blood using a gradient centrifugation procedure as previously described (21). Cells were then stained with FITC-CD3, PE-CX3CR1, PerCP-CD4 and APC-CD56 antibodies (BD biosciences, CA). CD56 bright CD3-CX3CRl-CD4- and CD56 dim CD3-CXCR3+CD4- cells were purified by a 4-way high speed sorting protocol using a FACSAria sorter (BD Bioscience, CA). The purified populations for downstream analysis were all of more than 95% purity based on flow cytometry analysis.
  • Cells were then plated in 96-well tissue culture plates in duplicates (1- 4xlO 5 /ml density) in RPMI 1640 medium with 10% FBS and stimulated with or without cytokine cocktails, IL- 12 (20 ng/ml plus IL- 15 100 ng/ml, PeproTech Inc., Rocky Hill, NJ). After 72 hours of stimulation, duplicates of culture supernatants were pooled and cytokine levels were measuerd by a multiplex cytokine array assay (Pierce Biotechology Inc., Boston, MA).
  • PBMCs from normal donors were used. About 5x10 /ml of cells were either treated or untreated with the IL-2R blocking antibody (100 ug/ml) or with 20 ng/ml of recombinant human IL-2 (PeproTech Inc.). Cells were immediately lysed with RIPA buffer starting from 10 minutes up to 120 minutes. Cell lysates were subjected to Western analysis using anti-phospho-STAT5 specific antibody (Cell Signaling Technology, Beverly, MA).
  • CD56 b ⁇ ght NK cell subpopulation has been defined either by its intensity of CD56 staining in CD56+CD3- population or by double staining as CD56 b ⁇ ght and CD16 low or negative, this criteria could result in ambiguous conclusion if the CD56 bright NK cell subpopulation is relatively small.
  • the CD56 bright NK cell subpopulation can be more readily and consistently defined by double staining of either CD56 bright CX3CRl- or CD56 bright CXCR3+ in the lymphocytes or in the CD56+CD3- NK cell population. Therefore, CD56 bright CD3-CX3CRl- was used as a primary criteria to define the CD56 regulatory NK cell subpopulation throughout the following study.
  • CD56 b ⁇ ght NK cells may have a beneficial effect on active uveitis due to the treatment with daclizumab
  • cytokine profiles between the induced CD56 b ⁇ ght NK cell subpopulation and the CD56 dlm NK cell subpopulation from the same patient were compared.
  • the induced CD56 bright and the CD56 dim NK cell populations were separated by cell sorting and 14 cytokines, including IL-2, 4, 5, 6, 10, 18, IFN ⁇ , IL-l ⁇ , TNF- ⁇ , IFN- ⁇ , GM-CSF, GCSF, RANTES, and lymphotoxin were analyzed by a multiplex cytokine array assay.
  • the cytokines produced by the induced CD56 bright NK cells and the CD56 dim NK cells can be classified into 3 groups, those not secreted by both populations (IL- 4, IL-5, IL-l ⁇ , IFN ⁇ and GCSF); those secreted at low levels by both groups (IL-6 and IL- 18) and those secreted abundantly only by the induced CD56 bright NK cells.
  • the induced CD56 b ⁇ ght NK cells were capable of producing an array of cytokines (IL-10, IL-2, IFN ⁇ , TNF- ⁇ , GM-CSF and lymphotoxin) upon activation.
  • the CD56 dim NK cells produced minimum or undetectable cytokines. This data is consistent with previous studies in healthy normal donors that the CD56 b ⁇ ght NK cells were more prone to secreting multiple cytokines than CD56 dim NK cells (these cells are called "regulatory NK cells" (Cooper et al., Blood, Vol. 97, p. 3146, 2001).
  • Interleukin-10 has been demonstrated to be protective for uveitis and was shown to be required for the induction of oral tolerance to experimental uveitis (for example, see Rizzo et al., J. Immunol. 162:2613, 1999)
  • a recent study demonstrated that the CD56 b ⁇ ght NK cells were present in local lymph nodes participating in immune responses (Fehniger et al., Blood 101 :3052, 2003).
  • the data presented here suggest that the induced CD56 b ⁇ ght NK cells could play a beneficial role in the remission of active uveitis by secreting substantial amounts of the immunosuppressive cytokine, IL-10.
  • CD56 b ⁇ ght NK cells by IL- 2R blockade therapy shared the same phenotype with those from normal donors.
  • Data showed that those CD56 b ⁇ ght NK cells induced in the patients indeed shared most of the phenotypic markers observed in normal donors.
  • the cells were typically high in their expression of CXCR3, CD122, CD62L, CD94 and NKG2D, but they did not express CX3CR1 with low levels of CD16.
  • the CD56 bright NK cells induced in the patients after IL-2R blockade seemed to be CD161 low or CDl 61 " while naturally occurring CD56 bright NK cells from healthy donors were CD161 high (Fig. 2B).
  • CD161 is an activating NK molecule and may be involved in the regulation of NK maturation (Giorda et al., Science 249: 1298, 1990; Poggi et al., Res Immunol. 148: 179, 1997). It is also proposed as an early marker for NK cells during NK development (Colucci et al., Nat Rev Immunol. 3: 413, 2003). The data presented herein indicate that there might also be differences between the CD56 b ⁇ ght NK cells induced by IL-2R blockade therapy and naturally occurring CD56 bright NK cells in healthy individuals.
  • IL-2 receptor blockade therapy can similarly induce in vivo expansion of the same subpopulation.
  • Daclizumab is an antagonist for human IL-2 on T cells. It is demonstrated herein that an IL-2 antagonist can induce in vivo expansion of the CD56 b ⁇ ght NK subpopulation, similarly to as IL-2. However, it seems unlikely that IL-2 infusion and IL-2R blockade would share the same molecular mechanism in inducing in vivo expansion of the CD56 b ⁇ ght NK subpopulation.
  • the data presented herein may elucidate molecular mechanisms of IL-2R blockade therapy and suggest biological roles of the CD56 bright NK subpopulation.

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Abstract

Cette invention concerne des méthodes de traitement d'un sujet souffrant d'uvéite active. Ces méthodes consistent à administrer de fortes doses pulsatiles d'un antagoniste du récepteur de l'interleukine-2 (IL-2) tel qu'un anticorps qui se lie au récepteur de l'IL-2. Sont également décrites des méthodes de traitement en cas de rejet d'une greffe de la cornée, du rejet de cellules souches limbiques après greffe, de la névrite optique et de la kératoconjonctivite sèche, consistant à administrer au sujet une dose fortement pulsatile d'un antagoniste du récepteur d'IL-2.
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2026822A1 (fr) * 2006-06-12 2009-02-25 Therakine Limited Traitement topique de maladies associées à la surface oculaire
WO2010025321A2 (fr) * 2008-08-28 2010-03-04 Facet Biotech Corporation Procédé de traitement de patients souffrant de sclérose en plaques avec des anticorps anti-il2r
WO2011026117A1 (fr) * 2009-08-31 2011-03-03 Facet Biotech Corporation Utilisation d'une population de cellules nk immunorégulatrices pour surveiller l'efficacité d'anticorps anti-il-2r chez des patients atteints d'une sclérose en plaques
WO2011023696A1 (fr) 2009-08-26 2011-03-03 Nycomed Gmbh Méthylpyrrolopyrimidine-carboxamides
WO2012166932A2 (fr) * 2011-06-01 2012-12-06 The Regents Of The University Of California Traitement de troubles du film lacrymal par des cellules souches mésenchymateuses
WO2023283381A1 (fr) * 2021-07-07 2023-01-12 Singh Biotechnology, Llc Suppression d'uvéite par un anticorps à domaine unique

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GUEX-CROSIER Y ET AL: "HUMANIZED ANTI-IL-2 AND ANTI-IL-15 RECEPTOR ANTIBODIES IN THE TREATMENT OF UVEORETINITIS IN A MONKEY MODEL", INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, ASSOCIATION FOR RESEARCH IN VISION AND, US, vol. 37, no. 3, 15 February 1996 (1996-02-15), pages S896, XP008013716, ISSN: 0146-0404 *
HIGUCHI M ET AL: "COMBINED ANTI-INTERLEUKIN-2 RECEPTOR AND LOW-DOSE CYCLOSPORINE THERAPY IN EXPERIMENTAL AUTOIMMUNE UVEORETINITIS", JOURNAL OF AUTOIMMUNITY, vol. 4, no. 1, 1991, pages 113 - 124, XP002364335, ISSN: 0896-8411 *
NUSSENBLATT R B ET AL: "HUMANIZED ANTI-INTERLEUKIN-2 (IL-2) RECEPTOR ALPHA THERAPY: LONG-TERM RESULTS IN UVEITIS PATIENTS AND PRELIMINARY SAFETY AND ACTIVITY DATA FOR ESTABLISHING PARAMETERS FOR SUBCUTANEOUS ADMINISTRATION", JOURNAL OF AUTOIMMUNITY, LONDON, GB, vol. 21, no. 3, 2003, pages 283 - 293, XP001204919, ISSN: 0896-8411 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2026822A1 (fr) * 2006-06-12 2009-02-25 Therakine Limited Traitement topique de maladies associées à la surface oculaire
EP2026822A4 (fr) * 2006-06-12 2012-07-04 Therakine Ltd Traitement topique de maladies associées à la surface oculaire
WO2010025321A2 (fr) * 2008-08-28 2010-03-04 Facet Biotech Corporation Procédé de traitement de patients souffrant de sclérose en plaques avec des anticorps anti-il2r
WO2010025321A3 (fr) * 2008-08-28 2010-05-20 Facet Biotech Corporation Procédé de traitement de patients souffrant de sclérose en plaques avec des anticorps anti-il2r
WO2011023696A1 (fr) 2009-08-26 2011-03-03 Nycomed Gmbh Méthylpyrrolopyrimidine-carboxamides
WO2011026117A1 (fr) * 2009-08-31 2011-03-03 Facet Biotech Corporation Utilisation d'une population de cellules nk immunorégulatrices pour surveiller l'efficacité d'anticorps anti-il-2r chez des patients atteints d'une sclérose en plaques
WO2012166932A2 (fr) * 2011-06-01 2012-12-06 The Regents Of The University Of California Traitement de troubles du film lacrymal par des cellules souches mésenchymateuses
WO2012166932A3 (fr) * 2011-06-01 2013-01-31 The Regents Of The University Of California Traitement de troubles du film lacrymal par des cellules souches mésenchymateuses
WO2023283381A1 (fr) * 2021-07-07 2023-01-12 Singh Biotechnology, Llc Suppression d'uvéite par un anticorps à domaine unique

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