WO2008080195A1 - Compositions and methods for treating or preventing unwanted immune responses - Google Patents

Compositions and methods for treating or preventing unwanted immune responses Download PDF

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Publication number
WO2008080195A1
WO2008080195A1 PCT/AU2008/000004 AU2008000004W WO2008080195A1 WO 2008080195 A1 WO2008080195 A1 WO 2008080195A1 AU 2008000004 W AU2008000004 W AU 2008000004W WO 2008080195 A1 WO2008080195 A1 WO 2008080195A1
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antigen
cells
presenting cells
reib
immune response
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PCT/AU2008/000004
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French (fr)
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Ranjeny Thomas
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The University Of Queensland
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4621—Cellular immunotherapy characterized by the effect or the function of the cells immunosuppressive or immunotolerising
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46433—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule

Definitions

  • This invention relates generally to methods and compositions for modulating immune responses. More particularly, the present invention relates to increasing the number of antigen-presenting cells in which both the canonical and non- canonical NF- ⁇ B pathways are activated for the treatment or prophylaxis of undesirable immune responses associated with defective NF- ⁇ B activation, including autoimmune diseases, organ-specific diseases and allergies.
  • the treatment or prophylaxis of the undesirable immune responses is achieved by increasing the number of antigen-presenting cells in which ReIB is activated or activatable.
  • NF- ⁇ B transmits signals from the cell surface to the nucleus. Signaling through cell surface receptors to activate NF- ⁇ B and MAP kinases through adaptor molecules is of critical importance to survival and activation of all cells in the body, including those regulating innate and adaptive immunity, including antigen- presenting cells such as dendritic cells (DC). As such, NF- ⁇ B is a key signaling component in autoimmunity and an attractive target for autoimmune disease therapy. NF-KB FUNCTION
  • NF- ⁇ B proteins p50, p52, c-Rel, p65/RelA and ReIB are present in mammals. All share a rel homology domain (RHD) that mediates DNA-binding, dimerisation and nuclear translocation. p50 and p52 homodimers are transcriptionally inactive, but have the capacity to bind DNA. In contrast, c-rel or ReIA are able to bind DNA and p50 or p52 are able to bind DNA and mediate transcriptional activation. As a result, the availability of subunits and affinity determines the NF- ⁇ B composition of the cell (Hoffmann A, et al, Immunol. Rev. (2006) 210:171-186).
  • NF- ⁇ B is present as an inactive form in the cytoplasm bound to inhibitory proteins or IKBS, including I ⁇ B ⁇ , I ⁇ B ⁇ , I ⁇ B ⁇ , I ⁇ B ⁇ , IKBNS, Bcl-3, plOO and plO5 (Ghosh S, Cell (2002) 109 Suppl:S81-96).
  • IKBS inhibitory proteins
  • I ⁇ B ⁇ , I ⁇ B ⁇ , I ⁇ B ⁇ , I ⁇ B ⁇ , IKBNS, Bcl-3, plOO and plO5 Ghosh S, Cell (2002) 109 Suppl:S81-96.
  • These proteins contain ankyrin repeats consisting of two tightly packed helices followed by a loop and a tight hairpin turn, which facilitate binding to NF- ⁇ B dimers.
  • the NLS region of NF- KB enables dimer nuclear import. I ⁇ B ⁇ masks the NLS, preventing nuclear import of dimers.
  • I ⁇ B ⁇ only masks the NLS of p65 and not p50. Nuclear retention is normally prevented by the presence of a nuclear export sequence in I ⁇ B ⁇ . If this NF- ⁇ B export sequence is blocked, RelA/p50 complexes are retained in the nucleus (Huang T, et al, Proc. Natl. Acad. Sci. U. S. A. (2000) 97(3): 1014-1019).
  • a variety of receptor-ligand pairs activates NF -KB including TLR/pathogen signals, inflammatory receptors (TNFR/TNF and IL-lR/IL-1), T cell (CD40/CD40L, TCR/MHC peptide) and B cell signals (BAFFR/BAFF, BCR/Ag) and differentiation signals such as lymphotoxin/LT ⁇ and RANK/RANKL. Signaling these pathways leads to activation of serine/threonine kinase IKB kinase (IKK) (Yamamoto Y, et al. , Trends Biochem. Sci. (2004) 29(2):72-79).
  • IKK serine/threonine kinase IKB kinase
  • IKK phosphorylates IKB which is recognized by a specific ubiquitin ligase complex, b-TrCP-SCF. Ubiquitinated IKB is degraded by the 26S proteasome, leading to release of NF- ⁇ B, nuclear import and transcriptional activation.
  • the IKK complex consists of 3 subunits including IKK ⁇
  • IKKl IKK
  • IKK ⁇ IKK2
  • NEMO essential modulator
  • IKK may be activated through phosphorylation by mitogen activated protein kinase kinase kinase (MAPKKK) or NF- ⁇ B inducing kinase (NIK), leading to subsequent autophosphorylation of the IKK complex and full activity.
  • MAPKKK mitogen activated protein kinase kinase kinase kinase
  • NIK NF- ⁇ B inducing kinase
  • IKK ⁇ and NEMO deficient mice have impaired NF- ⁇ B activation in response to cytokine and TLR activation, particularly activation of RelA/p50.
  • IKK ⁇ has a particular role in activation of RelB/p52 complexes and histone phosphorylation to enhance NF- ⁇ B DNA binding.
  • the non-canonical pathway is activated by signals associated with cell differentiation, including LT ⁇ , CD40L and BAFF.
  • RelB/p52 heterodimers are the predominant NF- ⁇ B proteins induced, regulated by pi 00, the precursor to p52, which contains an IKB domain target site for phosphorylation by IKK ⁇ .
  • Signal-specific activation of IKK ⁇ results in processing of pi 00 to p52 and activation of RelB/p52.
  • This pathway is characterized by sustained IKK ⁇ and long lasting activation of NF- ⁇ B.
  • the non- canonical pathway appears to be an adaptation of the canonical NF -KB pathway for cellular differentiation processes and is important in B cell and DC differentiation and lymphoid organogenesis.
  • NIK appears to be an upstream kinase that activates IKK ⁇ .
  • NIK, IKK ⁇ and ReIB knockout mice share similar defects in lymphoid organogenesis.
  • LT ⁇ signals both pathways and resulting target genes are activated
  • LPS a typical classical pathway activator, also leads to activation of the non-canonical pathway (Mordmuller B, et al, EMBO Rep. (2003) 4(l):82-87).
  • Activation of the non- canonical pathway ensures that although newly synthesized I ⁇ B ⁇ inhibits RelA/p50, newly synthesized ReIB and processing of pi 00 to p52 leads to dimer replacement or exchange with RelB/p52 and sustained DC differentiation (Saccani S, et al., MoI. Cell (2003) 11(6): 1563-1574).
  • ReIB and p52 are activated slowly after an activation event with late translocation to the nucleus, due to nuclear exchange of non-canonical pathway NF- ⁇ B dimers with those derived from the classical pathway.
  • ReIB not only heterodimerizes with p52 in the regulation of the non-canonical pathway, but it also has a role in the regulation of the canonical pathway through its capacity to heterodimerize with p50.
  • NF- ⁇ B target genes are involved in inflammation, cellular organization and differentiation and proliferation. Tissue macrophages are the major source of NF- ⁇ B-induced pro-inflammatory cytokines.
  • NF-KB induced cytokines such as TNF ⁇ , IL-I and IL-6 activate innate responses leading to the release of c-reactive protein (CRP) and complement, and up-regulation of adhesion molecules by local endothelial cells.
  • NF- ⁇ B-induced chemokines including IL-8, MIP- l ⁇ , MCP-I, RANTES and eotaxin, and growth factors such as GM-CSF mobilize and redirect myeloid cells to local tissue.
  • RI rheumatoid arthritis
  • IBD inflammatory bowel disease
  • NF- ⁇ B has a role in lymphoid organogenesis through the induction of the chemokines CXC12, CXCL13, CCL21 and CCL19.
  • NF- ⁇ B has a role in many stages of B and T cell differentiation (Claudio E, et al, (2006) 13(5):697-701) including a role for the non-canonical pathway in NKT cell development and for the canonical and non-canonical pathways in regulatory T cell (Treg) development (Schmidt-Supprian M, et al, Proc. Natl. Acad. Sci. U. S. A.
  • c-Rel is also required for efficient IL-2 production by naive T cells (Banerjee D, et al, Immunity (2005) 23(4):445-458) and T reg are critically dependent on IL-2 for post thymic survival (D'Cruz LM, et al, Nat. Immunol. (2005) 6(11):1152- 1159; Fontenot JD, et al, Nat. Immunol. (2005) 6(11):1142-1151).
  • NF- ⁇ B plays an important role in proliferation of lymphocytes as well as non-hematopoetic cells such as synoviocytes, that hyperproliferate in RA.
  • Relevant NF- ⁇ B target genes include c-myc, cyclin Dl and anti-apoptotic genes including c-IAP and Bcl-2.
  • those T cells reactive to self-antigen presented by medullary antigen-presenting cells which include medullary epithelial cells (mTEC) and medullary dendritic cells (DC) are deleted by negative selection above a threshold of affinity for self antigens presented by those APC (Kappler JW, et al, Cell (1987) 49:273-280). Since an affinity threshold applies for central deletion of self-reactive T cells, circulation of low-affinity self- reactive T cells in the periphery is therefore inevitable. Low-level thymic expression and presentation of self-antigens normally expressed by peripheral somatic cells is common.
  • APC medullary antigen-presenting cells
  • mTEC medullary epithelial cells
  • DC medullary dendritic cells
  • DC are the critical decision making cells in the immune system (Fazekas de St Groth B. Immunol Today. 1998;19:448-54).
  • DC are likely to play essential roles in both the initiation and perpetuation of autoimmunity and autoimmune diseases.
  • the understanding of the means by which DC contribute to peripheral tolerance has opened the exciting possibility of harnessing them for antigen-specific immunotherapy of autoimmune diseases and transplantation.
  • DC are now recognized as essential regulators of both innate and acquired arms of the immune system (Banchereau J, et al, Nature. 1998 Mar
  • DC are responsible for the stimulation of naive T lymphocytes, a property that distinguishes them from all other antigen presenting cells (APC).
  • APC antigen presenting cells
  • DC are also essential accessory cells in the generation of primary antibody responses (Inaba K, et al, Proc Natl Acad Sci U S A. 1983 Oct;80(19):6041-5) and are powerful enhancers of NK cell cytotoxicity (Kitamura H, et al, J Exp Med. 1999 Apr 5;189(7):1121-8).
  • DC are crucial for the initiation of primary immune responses of both helper and cytotoxic T lymphocytes, and thus act as "nature's adjuvant" (Schuler G, et al, J Exp Med.
  • DC are also involved in the maintenance of tolerance to antigens.
  • DC contribute to thymic central tolerance and shaping of the T cell repertoire by presenting antigens to T cells and deleting those T cells that exhibit strong autoreactivity (Brocker T. J Exp Med. 1997 Oct 20; 186(8): 1223-32).
  • DC also play a role in peripheral tolerance.
  • DC contribute by deletion of autoreactive lymphocytes and expansion of the population of regulatory T cells (Treg). Accordingly, DC offer potential utility in protective and therapeutic strategies for tolerance restoration in autoimmune diseases.
  • DC precursors from the bone marrow migrate via the bloodstream to peripheral tissues where they reside as immature DC.
  • Immature DC efficiently capture invading pathogens and other particulate and soluble antigens (Ag).
  • Ag uptake DC rapidly cross the endothelium of lymphatic vessels and migrate to the draining secondary lymphoid organs.
  • DC undergo a process of maturation, which is characterized by downregulation of the capacity to capture Ag and upregulation of Ag processing and presentation, expression of co-stimulatory molecules and altered dendritic morphology (Steinman RM. Annu Rev Immunol.
  • DC are important directors of immune responsiveness, through the interactions with responding lymphocytes and other accessory cells. Broadly, evidence suggests that under steady state conditions, recruitment of DC precursors into tissues and migration/maturation into secondary lymphoid organs occurs at low rates and may favour tolerance induction. On the other hand, stimulation of immature DC leading to DC maturation and activation may induce a productive immune response (Sallusto F, et al., J Exp Med. 1999 Feb 15;189(4):611- 4).
  • the process of DC maturation can be stimulated by various mechanisms, including pathogen-derived molecules (LPS, DNA, RNA), proinflammatory cytokines (TNF ⁇ , IL-I, IL-6), tissue factors such as hyaluronan fragments, migration of DC across endothelial barriers between inflamed tissues and lymphatics, and T cell-derived signals (CD 154) (Sparigan T, et al., Eur J Immunol. 1998 Jun;28(6):2045-54; Cella M, et al., J Exp Med. 1999 Mar l;189(5):821-9; De Smedt T, et al. , J Exp Med. 1996 Oct 1 ; 184(4): 1413-24).
  • LPS pathogen-derived molecules
  • TNF ⁇ proinflammatory cytokines
  • IL-6 proinflammatory cytokines
  • tissue factors such as hyaluronan fragments
  • CD 154 T cell-derived signals
  • DC represent an attractive therapeutic target, either to enhance or to attenuate immunity for modulation of disease.
  • ex vivo modulation of DC and exposure to antigen before transfer into an animal or human recipient has been the major approach to achieve protective and therapeutic immunity. This relates in part to complexity of the DC system in the context of a whole person with an immune system disorder, and in part to the difficulty of delivery of specific Ags and immunomodulators Xo OC in vivo.
  • NF- ⁇ B activity leads to transcription of a number of genes involved in the immune response.
  • ReIB activity is required for myeloid DC differentiation and the development of CD4 + CD8 " splenic DC (Burkly L, et al, Nature. 1995 Feb 9;373(6514):531-6; Weih F, et al, Cell. 1995;80(2):331-40; Wu L, et al,
  • ReIB regulates DC and B cell APC function through regulation of CD40 and MHC molecule expression
  • ReIB regulates DC and B cell APC function through regulation of CD40 and MHC molecule expression
  • the inventors also determined that DC in certain undesirable immune responses including autoimmune diseases ⁇ e.g., type 1 diabetes), organ-specific diseases and allergies have impaired NF- ⁇ B activation and that effective restoration of NF- ⁇ B function in these DC, in order to restore Treg suppressor function and reverse inflammatory disease, requires activation of both the canonical and non- canonical NF- ⁇ B pathways, including the restoration of ReIB function.
  • autoimmune diseases e.g., type 1 diabetes
  • organ-specific diseases and allergies have impaired NF- ⁇ B activation and that effective restoration of NF- ⁇ B function in these DC, in order to restore Treg suppressor function and reverse inflammatory disease, requires activation of both the canonical and non- canonical NF- ⁇ B pathways, including the restoration of ReIB function.
  • the present invention provides methods for modulating an immune response, especially an undesirable or deleterious immune response associated with defective NF- ⁇ B activation in a subject.
  • These methods generally comprise increasing the number of antigen-presenting cells in the subject, in which both the canonical and non-canonical NF- ⁇ B pathways are activated or activatable, suitably in response to a pro-inflammatory signal (e.g., tumor necrosis factor, C5a, interleukin-1, CDl 54 and lipolysaccharide).
  • a pro-inflammatory signal e.g., tumor necrosis factor, C5a, interleukin-1, CDl 54 and lipolysaccharide.
  • the methods comprise increasing the number of antigen-presenting cells in which ReIB is activated or activatable.
  • the present invention provides methods for modulating an undesirable or deleterious immune response associated with defective NF- ⁇ B activation in a subject, wherein the methods comprise increasing in the subject the number of antigen-presenting cells in which ReIB is activated or activatable, e.g., in response to a pro-inflammatory signal.
  • Antigen-presenting cells in which both the canonical and non-canonical NF- ⁇ B pathways are activated or activatable, or in which ReIB is activated or activatable (e.g., in response to a pro-inflammatory signal) are also referred to herein as "operable NF- ⁇ B antigen-presenting cells" or "antigen- presenting cells with operable NF- ⁇ B function.”
  • the methods comprise administering to the subject an effective amount of an immune modulator that increases the number of operable NF- ⁇ B antigen-presenting cells.
  • the immune modulator may comprise at least one agent that activates the canonical and non-canonical NF- ⁇ B pathways (e.g., an agent that increases the level or functional activity of ReIB) and is suitably administered in an amount sufficient to restore NF- ⁇ B function, including ReIB function, suitably to a basal state, in antigen-presenting cells of the subject.
  • the agent or agents may be administered in soluble or in particulate form.
  • Non-limiting antigen presenting cells include dendritic cells, macrophages and Langerhans cells.
  • the operable NF- ⁇ B antigen-presenting cells test positive for NF- ⁇ B restoration when they stimulate the production of regulatory T lymphocytes that suppress or otherwise reduce the undesirable or deleterious immune response.
  • the regulatory T lymphocyte expresses markers of constitutive regulatory or suppressor T lymphocytes, including CD4, CD25, CD62L, GITR, CTLA4 and the transcription factor FoxP3.
  • Illustrative regulatory T lymphocytes include, but are not limited to, CD4 + CD25 + regulatory T lymphocytes, TrI lymphocytes, Th3 lymphocytes, Th2 lymphocytes, CD8 + CD28 " regulatory T lymphocytes, natural killer (NK) T lymphocytes and ⁇ T lymphocytes.
  • the regulatory T lymphocyte is CD4 + CD25 + .
  • the immune modulator comprises operable NF-
  • the operable NF- ⁇ B antigen-presenting cells are derived from a donor, who is suitably histocompatible with the subject.
  • the operable NF- ⁇ B antigen-presenting cells are derived by harvesting antigen-presenting cells from the subject and treating them ex vivo with at least one agent that activates the canonical and non-canonical NF- ⁇ B pathways (e.g., an agent that increases the level or functional activity of ReIB) in an amount sufficient to restore NF- ⁇ B function, including ReIB function, suitably to a basal state, in those cells.
  • the immune modulator may be administered by injection, by topical application or by the nasal or oral route including sustained-release modes of administration, over a period of time and in amounts which are suitably effective to ameliorate the symptoms of the undesirable or deleterious immune response.
  • the immune modulator is administered systemically.
  • the undesirable or deleterious immune response is typically selected from autoimmune diseases, organ specific diseases and allergies.
  • the undesirable or deleterious immune response is associated with the presence of or predisposition to an autoimmune disease or related condition such as but not limited to type 1 diabetes mellitus (also referred to herein as "TlDM"), systemic lupus erythematosus, Churg Strauss disease, scleroderma, Wegener granulomatosus, Wiskott Aldrich syndrome.
  • TlDM type 1 diabetes mellitus
  • the undesirable or deleterious immune response is associated with the presence of or predisposition to an allergy, illustrative examples of which include allergic eczema and allergic asthma.
  • the undesirable or deleterious immune response is associated with the presence of or predisposition to an organ-specific disease representative examples of which include TlDM, thyroiditis, adrenal insufficiency, alopecia, atrophic gastritis, vitiligo, premature ovarian failure, autoimmune polyendocrine syndromes (APS), parathyroiditis, hypoparathyroidism, autoimmune adrenal insufficiency (Addison's disease), autoimmune hepatitis, Sjogren's syndrome, celiac disease, exocrine pancreatitis, keratitis and mucocutaneous candidiasis.
  • organ-specific disease representative examples of which include TlDM, thyroiditis, adrenal insufficiency, alopecia, atrophic gastritis, vitiligo, premature ovarian failure, autoimmune polyendocrine syndromes (APS), parathyroiditis, hypoparathyroidism, autoimmune adrenal insufficiency (Addison's disease), autoimmune hepati
  • the agent(s) increase(s) the level or functional activity of a canonical pathway kinase selected from IKK and IKK ⁇ /NEMO and the level of a non-canonical pathway kinase selected from NIK and IKK ⁇ .
  • the at least one agent decreases the level or functional activity of SHP-I .
  • the at least one agent stimulates or otherwise increases the level or functional activity of ReIB.
  • the defective NF- ⁇ B activation which associates with the undesirable or deleterious immune response, is typically associated with aberrant signaling through the NF- ⁇ B pathway, suitably in response to a pro-inflammatory signal.
  • Representative members of the NF- ⁇ B pathway include, but are not limited to, BTK, LYN, BCR Ig ⁇ , BCR Ig ⁇ , Syk, Blnk, PLC ⁇ 2, PKC ⁇ , DAG, CARMAl, BCLlO, MALTl, PDK, PIP3, AKT, COT, IKK ⁇ , IKK ⁇ , IKK ⁇ , NIK, RelA/p65, P105/p50, c-Rel, ReIB, p52, NIK, Leul3, CD81, CD19, CD21 and its ligands in the complement and coagulation cascade, ubiquitin mediated proteolysis, TRAF6, ubiquitin ligase, Tab2, TAKl, NEMO, NOD2, RIP2, Lck,
  • the defective NF- ⁇ B activation associates with a decrease in the level or functional activity of an expression product of at least one gene selected from genes that encode RelA/p65, P105/p50, c-Rel, ReIB or p52. In other embodiments, the defective NF- ⁇ B activation associates with an increase in the level or functional activity of an expression product of the SHP-I gene.
  • Aberrant signaling is suitably detected by detecting aberrant expression of a gene belonging to the canonical or non-canonical NF- ⁇ B pathway, e.g., aberrant expression of ReIB, as described in more detail below.
  • the methods further comprise administering to the subject an effective amount of antigen that corresponds to at least a portion of a target antigen associated (e.g. , an allergen, autoantigen or alloantigen) with the undesirable or deleterious immune response.
  • a target antigen associated e.g. , an allergen, autoantigen or alloantigen
  • the antigen is administered concurrently with an immune modulator as broadly described above.
  • the antigen may be selected from proteinaceous antigens, lipid antigens, glycolipid antigens and carbohydrate antigens.
  • the antigen is in the form of a nucleic acid construct from which it is expressible.
  • the invention thus provides compositions for modulating an immune response, especially an undesirable or deleterious immune response associated with defective NF- ⁇ B activation in a subject, wherein the compositions generally comprise an immune modulator that increases the number of operable NF- ⁇ B antigen-presenting cells as defined herein and an antigen that corresponds to at least a portion of a target antigen associated with the undesirable or deleterious immune response.
  • the composition may further comprise a pharmaceutically acceptable carrier or diluent.
  • the methods of the present invention are useful for inducing a tolerogenic response including the induction of an anergic response, or the suppression of a future or existing immune response, to a specified antigen or group of antigens.
  • the immune response includes, but is not limited to, a response mediated by immunoglobulin molecules (e.g., IgE) and/or T lymphocytes (e.g., cytotoxic T lymphocytes (CTLs) and T helper lymphocytes).
  • immunoglobulin molecules e.g., IgE
  • T lymphocytes e.g., cytotoxic T lymphocytes (CTLs) and T helper lymphocytes.
  • the immune response is typically but not exclusively directed to an antigen selected from a protein antigen, a particulate antigen, an alloantigen, an autoantigen, an allergen, a bacterial antigen, a viral antigen, a parasitic antigen or an immune complex.
  • an antigen selected from a protein antigen, a particulate antigen, an alloantigen, an autoantigen, an allergen, a bacterial antigen, a viral antigen, a parasitic antigen or an immune complex.
  • the invention extends to the use of an immune modulator as broadly described above and optionally an antigen that corresponds to at least a portion of a target antigen that associates with the presence or risk of an undesirable or deleterious immune response and with defective NF- ⁇ B function in antigen-presenting cells, in the manufacture of a medicament for suppressing the immune response.
  • the present invention provides methods for treating a condition associated with an undesirable or deleterious immune response and with defective NF- ⁇ B activation in a subject.
  • These methods generally comprise administering to the subject an effective amount of an immune modulator that increases the number of antigen-presenting cells in the subject, in which both the canonical and non-canonical NF- ⁇ B pathways are activated or activatable, suitably in response to a pro-inflammatory signal.
  • the methods comprise administering to the subject an effective amount of an immune modulator that increases the number of antigen-presenting cells in the subject, in which ReIB is activated or activatable, suitably in response to a pro-inflammatory signal.
  • the methods further comprise contacting antigen-presenting cells with an antigen that corresponds to at least a portion of a target antigen that associates with the presence or risk of the undesirable or deleterious immune response, in an amount that is effective for the antigen-presenting cells to present the antigen or a processed form thereof on their surface.
  • an antigen that corresponds to at least a portion of a target antigen that associates with the presence or risk of the undesirable or deleterious immune response, in an amount that is effective for the antigen-presenting cells to present the antigen or a processed form thereof on their surface.
  • the antigen-presenting cells which may be autologous or allogeneic, can be contacted with the antigen ex vivo.
  • the antigen-presenting cells which may be autologous or allogeneic, can be contacted with the antigen in vivo and in illustrative examples of this type, the antigen is administered to the subject in particulate form.
  • the condition is from autoimmune diseases (e.g., type 1 diabetes), organ specific diseases and allergies.
  • the methods further comprise, prior to increasing the number of the operable NF- ⁇ B antigen-presenting cells in the subject, the step of diagnosing the presence, stage or degree or risk of development of the undesirable or deleterious immune response by detecting in the subject aberrant (e.g., reduced or abrogated) signaling through the NF- ⁇ B pathway, suitably in response to a pro-inflammatory signal, as explained in more detail below.
  • the step of diagnosing the presence, stage or degree or risk of development of the undesirable or deleterious immune response by detecting in the subject aberrant (e.g., reduced or abrogated) signaling through the NF- ⁇ B pathway, suitably in response to a pro-inflammatory signal, as explained in more detail below.
  • the invention also encompasses the use of an immune modulator as broadly described above and optionally an antigen that corresponds to at least a portion of a target antigen that associates with the presence or risk of an undesirable or deleterious immune response and with defective NF- ⁇ B function in antigen-presenting cells, in the study and modulation of the immune response.
  • the present invention provides methods for diagnosing the presence or risk of development of an undesirable or deleterious immune response associated with defective NF- ⁇ B activation, including defective ReIB activation, in a subject. These methods generally comprise detecting in the subject aberrant (e.g., reduced or abrogated) signaling through the NF- ⁇ B pathway, suitably in response to a pro-inflammatory signal.
  • the undesirable or deleterious immune response is associated with a condition selected from autoimmune diseases, organ specific diseases and allergies.
  • the condition is other than TlDM.
  • Aberrant signaling is suitably detected by directly or indirectly detecting aberrant expression of a gene belonging to the canonical or non-canonical NF- KB pathway (such genes are also referred to herein as "NF- ⁇ B pathway genes").
  • the aberrant expression is detected by: (1) measuring in a biological sample obtained from the subject the level or functional activity of an expression product of at least one NF- ⁇ B pathway gene and (2) comparing the measured level or functional activity of each expression product to the level or functional activity of a corresponding expression product in a reference sample obtained from one or more normal subjects or from one or more subjects lacking the undesirable or deleterious immune response, wherein a difference in the level or functional activity of the expression product in the biological sample as compared to the level or functional activity of the corresponding expression product in the reference sample is indicative of the presence or risk of development of the undesirable or deleterious immune response in the subject.
  • the methods further comprise diagnosing the presence, stage or degree or risk of development of the undesirable or deleterious immune response in the subject when the measured level or functional activity of the or each expression product is different than the measured level or functional activity of the or each corresponding expression product.
  • the difference typically represents an at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, or even an at least about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% increase, or an at least about 10%, 20%, 30% 40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 97%, 98% or 99%, or even an at least about 99.5%, 99.9%, 99.95%, 99.99%, 99.995% or 99.999% decrease in the level or functional activity of an individual expression product as compared to the level or functional activity of an individual corresponding expression product.
  • the presence or risk of development of the unwanted or deleterious immune response is determined by detecting a decrease in the level or functional activity of an expression product of at least one NF- ⁇ B pathway gene, which is suitably selected from genes that encode BTK, LYN, BCR Ig ⁇ , BCR Ig ⁇ , Syk, Blnk, PLC ⁇ 2, PKC ⁇ , DAG, CARMAl, BCLlO, MALTl, PI3K, PIP3, AKT, COT, IKK ⁇ , IKK ⁇ , IKK ⁇ , NIK, RelA/p65, P105/p50, c-Rel, ReIB, P 52, NIK, Leul3, CD81, CD19, CD21 and its ligands in the complement and coagulation cascade, TRAF6, ubiquitin ligase, Tab2, TAKl, NEMO, NOD2, RIP2, Lck, fyn, Zap70, LAT, GRB2, SOS, CD3
  • NF- ⁇ B pathway gene which
  • the presence or risk of development of the unwanted or deleterious immune response is determined by detecting a decrease in the level or functional activity of an expression product of at least one gene selected from genes that encode RelA/p65, P105/p50, c-Rel, ReIB or p52.
  • the presence or risk of development of the unwanted or deleterious immune response is determined by detecting an increase in the level or functional activity of an expression product of at least one NF- ⁇ B pathway gene, which is suitably selected from genes that encode SHP- 1, SHIP, PIR-B, CD22, CD72, FcgRIIB, IKB, PlOO, CTLA4, CDIa, TGF- ⁇ , PD-I, CbI, KIR3DL1, KIR3DL2, KIR2DL and Csk.
  • NF- ⁇ B pathway gene which is suitably selected from genes that encode SHP- 1, SHIP, PIR-B, CD22, CD72, FcgRIIB, IKB, PlOO, CTLA4, CDIa, TGF- ⁇ , PD-I, CbI, KIR3DL1, KIR3DL2, KIR2DL and Csk.
  • the presence or risk of development of the unwanted or deleterious immune response is determined by detecting an increase in the level or functional activity of an expression product of a gene that is common to both the canonical and non-canonical NF- ⁇ B pathways.
  • the gene is the SHP-I gene.
  • the methods further comprise diagnosing the absence of the unwanted or deleterious immune response or a low risk of developing that immune response when the measured level or functional activity of the or each expression product is the same as or similar to the measured level or functional activity of the or each corresponding expression product.
  • the measured level or functional activity of an individual expression product varies from the measured level or functional activity of an individual corresponding expression product by no more than about 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.1%.
  • NF- ⁇ B pathway polynucleotides polypeptide expression products of NF- ⁇ B pathway genes are referred to herein as "NF- ⁇ B pathway polypeptides.”
  • the methods comprise measuring the level or functional activity of individual expression products of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or even 30, 40 or 50 NF- ⁇ B pathway genes.
  • the methods may comprise measuring the level or functional activity of a
  • the methods may comprise measuring the level or functional activity of a NF- ⁇ B pathway polypeptide either alone or in combination with as much as 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 other NF- ⁇ B pathway polypeptides(s).
  • the biological sample comprises blood, especially peripheral blood, which suitably includes leukocytes.
  • the expression product is selected from a RNA molecule or a polypeptide.
  • the expression product is the same as the corresponding expression product.
  • the expression product is a variant (e.g., an allelic variant) of the corresponding expression product.
  • the biological sample comprises cells, which have been exposed to a pro-inflammatory signal.
  • the reference sample also comprises cells, which have been exposed to the pro-inflammatory signal.
  • the expression product or corresponding expression product is a target RNA (e.g., mRNA) or a DNA copy of the target RNA whose level is measured using at least one nucleic acid probe that hybridizes under at least low, medium, or high stringency conditions to the target RNA or to the DNA copy, wherein the nucleic acid probe hybridizes to at least 15 contiguous nucleotides of a NF- KB pathway polynucleotide.
  • the measured level or abundance of the target RNA or its DNA copy is normalized to the level or abundance of a reference RNA or a DNA copy of the reference RNA that is present in the same sample.
  • the nucleic acid probe is immobilized on a solid or semi-solid support.
  • the nucleic acid probe forms part of a spatial array of nucleic acid probes.
  • the level of nucleic acid probe that is bound to the target RNA or to the DNA copy is measured by hybridization (e.g. , using a nucleic acid array).
  • the level of nucleic acid probe that is bound to the target RNA or to the DNA copy is measured by nucleic acid amplification (e.g. , using a polymerase chain reaction (PCR)).
  • PCR polymerase chain reaction
  • the level of nucleic acid probe that is bound to the target RNA or to the DNA copy is measured by nuclease protection assay.
  • the expression product or corresponding expression product is a NF- ⁇ B pathway polypeptide whose level is measured using at least one antigen-binding molecule that is immuno-interactive with the NF- ⁇ B pathway polypeptide.
  • the measured level of the NF- ⁇ B pathway polypeptide is normalized to the level of a reference polypeptide that is present in the same sample.
  • the antigen-binding molecule is immobilized on a solid or semisolid support.
  • the antigen-binding molecule forms part of a spatial array of antigen-binding molecule.
  • the level of antigen-binding molecule that is bound to the NF- ⁇ B pathway polypeptide is measured by immunoassay (e.g., using an ELISA).
  • the expression product or corresponding expression product is a NF- ⁇ B pathway polypeptide whose level is measured using at least one substrate for that polypeptide with which it reacts to produce a reaction product.
  • the measured functional activity of the NF- ⁇ B pathway polypeptide is normalized to the functional activity of a reference polypeptide that is present in the same sample.
  • the expression product or corresponding expression product is a NF- ⁇ B pathway polypeptide whose level is measured using at least one oligonucleotide that binds to a nucleic acid binding site of the marker polypeptide (e.g., ReIB, p65, p50 etc).
  • the oligonucleotide comprises a detectable label and the level of oligonucleotide that is bound to the NF- ⁇ B pathway polypeptide is measured by quantifying the amount of detectable label that associates with the polypeptide (e.g., by autoradiography, fiuorimetry, luminometry, or phosphoimage analysis).
  • aberrant signaling of the NF- ⁇ B pathway is detected by detecting aberrant phosphorylation of a polypeptide involved in or belonging to the NF- ⁇ B signaling pathway.
  • aberrant phosphorylation is detected by: (1) determining in a biological sample obtained from the subject the phosphorylation state of at least one phosphorylatable polypeptide selected from IRAK, TAK 1 , TAB 1 , TAB2, PKR, Akt, Cot, IKK ⁇ , IKK ⁇ and IKK ⁇ ; and (2) comparing each determined phosphorylation state to the phosphorylation state of a corresponding phosphorylatable polypeptide in a reference sample obtained from one or more normal subjects or from one or more subjects lacking disease, wherein a difference in the phosphorylation state of the phosphorylatable polypeptide in the biological sample as compared to the phosphorylation state of the corresponding phosphorylatable polypeptide in the reference sample is indicative of the presence or risk of development of the unwanted
  • the present invention provides methods for treating, preventing or inhibiting the development of the unwanted or deleterious immune response in a subject.
  • These methods generally comprise detecting in the subject aberrant signaling through the NF- ⁇ B pathway, suitably in response to an inflammatory signal, as broadly described above and administering to the subject an effective amount of an agent that treats or ameliorates the symptoms or reverses or inhibits the development of the unwanted or deleterious immune response in the subject.
  • Representative examples of such treatments or agents include, but are not limited to, anti-CD3 therapy (e.g., monoclonal antibody hOKT3gammal (Ala- AIa)) and antigen- specific tolerogenic therapies.
  • the present invention provides probes for interrogating nucleic acid for the presence of a NF- ⁇ B pathway polynucleotide as broadly described above. These probes generally comprise a nucleotide sequence that hybridizes under at least low stringency conditions to a NF- ⁇ B pathway polynucleotide as broadly described above. [0045] In yet another aspect, the present invention provides oligonucleotides for interrogating protein samples for the presence of a NF- ⁇ B pathway polypeptide that comprises a nucleic acid binding site.
  • oligonucleotides generally comprise a nucleotide sequence that binds to the nucleic acid binding site of the NF- ⁇ B pathway polypeptide (e.g., ReIB, p65, p50 etc).
  • the invention provides a solid or semi-solid support comprising at least one nucleic acid probe as broadly described above or at least one polypeptide-binding oligonucleotide as broadly described above immobilized to the support.
  • the solid or semi-solid support comprises a spatial array of nucleic acids immobilized thereon.
  • Still a further aspect of the present invention provides an antigen- binding molecule that is immuno-interactive with a NF- ⁇ B pathway polypeptide as broadly described above.
  • the invention provides a solid or semi-solid support comprising at least one antigen-binding molecule as broadly described above immobilized thereon.
  • the solid or semi-solid support comprises a spatial array of antigen-binding molecules immobilized thereon.
  • Still another aspect of the invention provides the use of one or more
  • NF- ⁇ B polynucleotides as broadly described above, or the use of one or more probes as broadly described above, or the use of one or more NF- ⁇ B pathway polypeptides as broadly described above, or the use of one or more antigen-binding molecules as broadly described above, or the use of one or more polypeptide-binding oligonucleotides as broadly described above, in the manufacture of a kit for diagnosing the presence of or predisposition to an unwanted or deleterious immune response associated with defective NF- ⁇ B function, including defective ReIB function, in a subject.
  • Yet further aspects of the invention are directed to the use of the diagnostic methods as broadly described above, or one or more NF- ⁇ B pathway polynucleotides as broadly described above, or the use of one or more probes as broadly described above, or the use of one or more NF- ⁇ B pathway polypeptides as broadly described above, or the use of one or more antigen-binding molecules as broadly described above, or the use of one or more polypeptide-binding oligonucleotides as broadly described above, for diagnosing the presence or risk of development of an unwanted or deleterious immune response associated with defective NF- ⁇ B function, including defective ReIB function, in animals, especially vertebrates animals including mammals.
  • FIG. 1 is a graphical representation showing reduced LPS-induced NF- ⁇ B activity in TlDM DC.
  • Cytoplasmic or nuclear extracts were prepared from DC cultured for 72h from TlDM patients or healthy control (HC) subjects without (-) or with (+) addition of 100 ng/ml LPS for the last 24 h of culture, or from a positive control lymphoblastoid cell line (LCL). After electrophoresis, membranes were immunoblotted with NF- ⁇ B subunit antibodies, or with Ponceau S protein stain Representative of 5 experiments analyzing 5 individual pairs of donors.
  • FIG. 2 is a graphical representation showing that SHP-I expression is increased in PBMC of TlDM patients.
  • A PBMC from 13 healthy control (HC) subjects, 18 Tl DM and 6 T2DM patients were cultured for 48h. DC from healthy controls or TlDM patients were harvested at 72 h. All cells were then permeabilised and stained for SHP-I . Data are expressed as ⁇ MFI, in gated monocytes or DC. ** p ⁇ 0.01, * p ⁇ 0.05.
  • FIG. 3 is a graphical representation showing that SHP-I inhibits NF- kB activity in TlDM.
  • A PBMC from 7 healthy control (HC) subjects and 4 TlDM patients were cultured for 30 min without (UT) or with 100 ng/ml LPS, then permeabilised and stained for phospho-I- ⁇ B ⁇ . Data are expressed as ⁇ MFI, in gated monocytes. ** p ⁇ 0.01.
  • B TlDM monocytes were cultured with GM-CSF and IL-4 with or without 10 ⁇ g/ml sodium stibogluconate for 48 hours, then cells were analyzed for phospho-I ⁇ B ⁇ by flow cytometry.
  • TlDM monocytes were cultured with GM-CSF and IL-4 with or without 10 ⁇ g/ml sodium stibogluconate for 48 hours, with LPS added for the last 24 hours.
  • CD40 expression was compared, with relative values shown for 2 individuals.
  • TlDM PBMC cultured with or without SHP-I inhibitor and LPS for 24 hours were analyzed by ELISA for nuclear ReIB DNA binding, with relative values shown for 2 individuals.
  • FIG. 4 is a graphical representation showing that adoptive transfer of wt DC reverses inflammatory organ pathology: (A) 12.5 x 10 6 bead-purified or 2 x 10 6 sorted DC from progenipoietin-1 -treated RelB +/" mice were injected i.v into ReIB 7" mice. Recipients were monitored serially for weight gain and well being. Untreated ReIB + " mice and ReIB " " mice served as controls. The % cumulative weight gain above individual baseline weight, and final weights at the end of the monitoring period are shown. *** p ⁇ 0.001, ** p ⁇ 0.01 comparing ReIB " ' " with and without DC treatment.
  • FIG. 5 is a graphical representation showing defective ReIB activation in Sjogren's patients.
  • DCs derived from healthy controls or from patients with Sjogren's syndrome were cultured for 72 h ex vivo. For the final 24 h of cell culture, DCs were incubated with or without 100 ng/ml LPS. Nuclear extracts from the DCs were bound to wells of a NF- ⁇ B oligonucleotide-coated ELISA plate (10 ⁇ g per well), and revealed with antibodies against either ReIB, ReIA or p50. Light output was measured in photon units after reading for 5 seconds.
  • DNA binding of NF- ⁇ B following LPS exposure was compared with DNA binding without LPS (nil) in cells derived from representative healthy control or Sjogren's syndrome subjects, and expressed in photon units (Figure 5A) or as fold change in DNA binding by NF- ⁇ B after LPS treatment in cells derived from healthy control and Sjogren's syndrome subjects ( Figure 5B).
  • Figure 6A is a graphical representation showing defective ReIB, ReIA, p50 and c-Rel activation in a representative patient with type 1 diabetes mellitus (TlDM).
  • TlDM type 1 diabetes mellitus
  • Figure 6B is a graphical representation showing defective LPS- induced ReIA, ReIB, c-Rel and p50 activation over time in representative patients with TlDM.
  • Nuclear extracts from DCs prepared from two healthy controls and two TlDM patients were incubated with LPS for between 0 and 24 h.
  • DNA binding by ReIA, p50, c-Rel or ReIB was assessed at intervals and expressed in photon units. Representative of data from nine individuals from each group in 6A and 6B.
  • Figure 7A is a graphical representation showing defective ReIB, ReIA, and p50 activation in TlDM monocytes.
  • Peripheral blood monocytes from healthy control subjects or TlDM patients were cultured with or without LPS for 24h. Nuclear extracts were prepared and tested for NF- ⁇ B binding to DNA as described in Figure 6.
  • Data are expressed as fold change in NF- ⁇ B binding to DNA after LPS treatment.
  • Figure 7B is a graphical representation showing increased ReIB and ReIA DNA binding at baseline (without LPS) in TlDM monocytes. DNA binding of ReIB, ReIA, c-Rel and p50 for 8 untreated healthy control subjects and 16 TlDM subjects' PB monocytes is shown with median, p ⁇ 0.05 comparing ReIB and ReIA medians of healthy and Tl DM subjects by Mann Whitney test.
  • Figure 8 is a graphical representation showing defective LPS-induced ReIB activation of DCs of siblings of a TlDM proband (child), but not their father.
  • Nuclear extracts from DCs prepared from first degree relatives of a child with TlDM were incubated with or without LPS for 24 h as described in Figure 6.
  • DNA binding by ReIB was assessed at 24h and expressed in photon units.
  • NF- ⁇ B pathway gene product e.g., transcript or polypeptide
  • a specific NF- ⁇ B pathway gene is aberrantly expressed if the level of expression of such a gene product is at least about 10% ( X 0 ), 20% ( X ), 30% ( X 0 ), 40% ( % ), 50% ( y 2 ), 60% ( % ), 70% ( X 0 ), 80% ( % ) or 90% ( X 0 ), or even at least about 100% (1-fold), 200% (2-fold), 300% (3-fold), 400% (4-fold), 500% (5-fold), 600% (6-fold), 700% (7-fold), 800% (8-fold), 900% (9-fold) or 1000% (10-fold) higher than the level of expression of a corresponding NF- ⁇ B pathway gene product in a tissue sample or body fluid obtained from a healthy subject or from a subject without the unwanted or deleterious immune response.
  • a specific NF- ⁇ B pathway gene is aberrantly expressed if the level of expression of such a gene product is about Xo 5 A i Xo 5 A ⁇ > Yi ⁇ > A ⁇ > Xo J /5 ' Xo ' or ev ⁇ about ⁇ 00 , j4oo » /300 ⁇ > /400 > Xoo » Xoo > /700 > Xoo > Xoo > Xooo or l ess of the level of expression of a corresponding NF- ⁇ B pathway gene product in a tissue sample or body fluid obtained from a healthy subject or from a subject without an unwanted or deleterious immune response as defined herein.
  • cytokines e.g., IL-I, IL-6, IL-8 and TNF
  • chemokines e.g., IL-I, IL-6, IL-8 and TNF
  • adhesion molecules e.g., IL-6, IL-8 and TNF
  • MMPs matrix metalloproteinases
  • COX2 cyclooxygenase 2
  • iNOS inducible nitric oxide synthase
  • “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • administration concurrently or “administering concurrently” or “co-administering” and the like refer to the administration of a single composition containing two or more actives, or the administration of each active as separate compositions and/or delivered by separate routes either contemporaneously or simultaneously or sequentially within a short enough period of time that the effective result is equivalent to that obtained when all such actives are administered as a single composition.
  • simultaneous is meant that the active agents are administered at substantially the same time, and desirably together in the same formulation.
  • the active agents are administered closely in time, e.g., one agent is administered within from about one minute to within about one day before or after another. Any contemporaneous time is useful. However, it will often be the case that when not administered simultaneously, the agents will be administered within about one minute to within about eight hours and preferably within less than about one to about four hours. When administered contemporaneously, the agents are suitably administered at the same site on the subject.
  • the term "same site” includes the exact location, but can be within about 0.5 to about 15 centimeters, preferably from within about 0.5 to about 5 centimeters.
  • the term “separately” as used herein means that the agents are administered at an interval, for example at an interval of about a day to several weeks or months.
  • the active agents may be administered in either order.
  • the term “sequentially” as used herein means that the agents are administered in sequence, for example at an interval or intervals of minutes, hours, days or weeks. If appropriate the active agents may be administered in a regular repeating cycle.
  • the term "agent” refers to an organic or inorganic chemical and in some cases refers to a biochemical substance that includes, but is not limited to, proteins, peptides or amino acids; nucleic acids such as DNA, such as full- length genes or fragments thereof derived from genomic, cDNA or artificial coding sequences, gene regulatory elements, RNA, including mRNA, tRNA, ribosomal RNA, ribozymes and antisense RNA, oligonucleotides and oligoribonucleotides, deoxyribonucleotides and ribonucleotides; carbohydrates; lipids; proteoglycans; such agents may be administered as isolated (purified) compounds or in crude mixtures, such as in a tissue, cell or cell lysate.
  • nucleic acids such as DNA, such as full- length genes or fragments thereof derived from genomic, cDNA or artificial coding sequences, gene regulatory elements, RNA, including mRNA, tRNA, ribo
  • amplicon refers to a target sequence for amplification, and/or the amplification products of a target sequence for amplification. In certain other embodiments an “amplicon” may include the sequence of probes or primers used in amplification.
  • amplicon refers to a suppressed response, or a state of non-responsiveness, to a specified antigen or group of antigens by an immune system. For example, T lymphocytes and B lymphocytes are anergic when they cannot respond to their specific antigen under optimal conditions of stimulation.
  • antigen is meant all, or part of, a protein, peptide, or other molecule or macromolecule capable of eliciting an immune response in a vertebrate animal, especially a mammal. Such antigens are also reactive with antibodies from animals immunized with that protein, peptide, or other molecule or macromolecule.
  • antigen-binding molecule a molecule that has binding affinity for a target antigen. It will be understood that this term extends to immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity.
  • autologous is meant something (e.g., cells, tissues etc) derived from the same organism.
  • allogeneic refers to cells, tissues, organisms etc that are of different genetic constitution.
  • alloantigen is meant an antigen found only in some members of a species, such as blood group antigens.
  • a “xenoantigen” refers to an antigen that is present in members of one species but not members of another.
  • an “allograft” is a graft between members of the same species and a “xenograft” is a graft between members of a different species.
  • the term “basal state” as applied to NF- ⁇ B or ReIB function refers to the level of activity of the canonical and/or non-canonical NF- ⁇ B pathways, or to the level of activity of ReIB, in antigen-presenting cells of normal individuals or of individuals that lack the undesirable or deleterious immune response, as defined herein. Accordingly, the term “basal state” includes and encompasses a "normal level" of activity of the canonical and/or non-canonical NF- ⁇ B pathways, or of ReIB.
  • biological sample refers to a sample that may be extracted, untreated, treated, diluted or concentrated from an animal.
  • the biological sample may include a biological fluid such as whole blood, serum, plasma, saliva, urine, sweat, ascitic fluid, peritoneal fluid, synovial fluid, amniotic fluid, cerebrospinal fluid, tissue biopsy, and the like.
  • the biological sample is blood, especially peripheral blood.
  • an antigen which encodes an amino acid sequence that displays substantial similarity to an amino acid sequence in a target antigen.
  • the antigen will display at least about 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % similarity to at least a portion of the target antigen.
  • an effective amount in the context of modulating an immune response or treating or preventing a disease or condition, is meant the administration of that amount of composition to an individual in need thereof, either in a single dose or as part of a series, that is effective for that modulation, treatment or prevention.
  • the effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • RNA message or translation of RNA message into proteins or polypeptides. Detection of either types of gene expression in use of any of the methods described herein are part of the invention.
  • the term "functional activity” generally refers to the ability of a molecule (e.g., a transcript or polypeptide) to perform its designated function including a biological, enzymatic, or therapeutic function.
  • the functional activity of a molecule corresponds to its specific activity as determined by any suitable assay known in the art.
  • gene is used in its broadest context to include both a genomic DNA region corresponding to the gene as well as a cDNA sequence corresponding to exons or a recombinant molecule engineered to encode a functional form of a product.
  • immuno-interactive includes reference to any interaction, reaction, or other form of association between molecules and in particular where one of the molecules is, or mimics, a component of the immune system.
  • a level or functional activity in the context of a gene expression product (e.g., a protein or a transcript) produced by a specified cell is to be taken in its broadest sense and includes a level or functional activity of the expression product that is produced in a single cell or in a plurality or population of cells. In the latter case, therefore, it will be understood that the phrase will encompass a mean level or functional activity of the protein produced by a plurality or population of cells.
  • normal activity refers to a value that is at least 25% of the activity of one or more of NF- ⁇ B and its subunits (e.g., p50, plO5, p52, p 100, ReIA, ReIB and cRel) observed in the basal state, as defined herein, suitably in the range of 30-90% and more suitably in the range of 95-100%, and all integer percentages therebetween.
  • a sample such as, for example, a cell extract or nucleic acid or polypeptide extract is isolated from, or derived from, a particular source.
  • the extract may be isolated directly from biological fluid or tissue of the subject.
  • patient refers to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired.
  • Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates, rodents ⁇ e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards etc), and fish.
  • a preferred subject is a human in need of treatment or prophylaxis for a condition or disease,
  • pharmaceutically-acceptable carrier is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in topical or systemic administration.
  • polynucleotide or “nucleic acid” as used herein designates mRNA, RNA, cRNA, cDNA or DNA.
  • the term typically refers to oligonucleotides greater than 30 nucleotides in length.
  • Polypeptide “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers.
  • primer an oligonucleotide which, when paired with a strand of DNA, is capable of initiating the synthesis of a primer extension product in the presence of a suitable polymerizing agent.
  • the primer is preferably single-stranded for maximum efficiency in amplification but can alternatively be double-stranded.
  • a primer must be sufficiently long to prime the synthesis of extension products in the presence of the polymerization agent. The length of the primer depends on many factors, including application, temperature to be employed, template reaction conditions, other reagents, and source of primers.
  • the primer may be at least about 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500, to one base shorter in length than the template sequence at the 3' end of the primer to allow extension of a nucleic acid chain, though the 5' end of the primer may extend in length beyond the 3' end of the template sequence.
  • primers can be large polynucleotides, such as from about 35 nucleotides to several kilobases or more.
  • Primers can be selected to be “substantially complementary” to the sequence on the template to which it is designed to hybridize and serve as a site for the initiation of synthesis.
  • substantially complementary it is meant that the primer is sufficiently complementary to hybridize with a target polynucleotide.
  • the primer contains no mismatches with the template to which it is designed to hybridize but this is not essential.
  • non-complementary nucleotide residues can be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the template.
  • non-complementary nucleotide residues or a stretch of non- complementary nucleotide residues can be interspersed into a primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize therewith and thereby form a template for synthesis of the extension product of the primer.
  • regulatory lymphocyte is meant a lymphocyte that is involved in regulating or suppressing responses and actions of other cells, especially of other immune cells such as B lymphocytes and T helper lymphocytes.
  • Probe refers to a molecule that binds to a specific sequence or subsequence or other moiety of another molecule. Unless otherwise indicated, the term '"probe” typically refers to a polynucleotide probe that binds to another polynucleotide, often called the " 'target polynucleotide", through complementary base pairing. Probes can bind target polynucleotides lacking complete sequence complementarity with the probe, depending on the stringency of the hybridization conditions. Probes can be labeled directly or indirectly and include primers within their scope.
  • the term "related" when used with respect to subjects indicates that the subjects are known to share a common line of descent: that is, the subjects have a known ancestor in common.
  • Illustrative examples of related subjects include siblings (brothers and sisters), parents, grandparents, children, grandchildren, aunts, uncles, cousins, second cousins and third cousins.
  • Subjects less closely related than third cousins are typically not sufficiently related to be useful as "related" subjects for the methods of this invention, even if they share a known ancestor, unless some related individuals that lie between the distantly related subjects are also included.
  • each subject shares a known ancestor within three generations or less with at least one other subject in the group, and desirably with all other subjects in the group or has at least that degree of consanguinity due to multiple known common ancestors.
  • subjects share a common ancestor within two generations or less, or otherwise have equivalent level of consanguinity.
  • unrelated when used in respect of subjects, refers to subjects who do not share a known ancestor within 3 generations or less, or otherwise have known relatedness at that degree.
  • suppression any attenuation or regulation of an immune response, including B-lymphocyte and T lymphocyte immune responses, to an antigen or group of antigens.
  • the attenuation is mediated at least in part by suppressor T lymphocytes (e.g., CD4 + CD25 + regulatory T lymphocytes).
  • surrogate marker refers to a biological or clinical parameter that is measured in place of the biologically definitive or clinically most meaningful parameter. In comparison to definitive markers, surrogate markers are generally either more convenient, less expensive, provide earlier information or provide pharmacological or physiological information not directly obtainable with definitive markers.
  • Non-limiting examples of surrogate biological parameters include testing cell NF- ⁇ B levels in subjects having or at risk of developing type 1 diabetes, whereas examples of definitive biological parameters include insulin independence, blood glucose and C peptide production. The measurement of a surrogate marker may be an endpoint in a clinical study or clinical trial, hence "surrogate endpoint".
  • template refers to a nucleic acid that is used in the creation of a complementary nucleic acid strand to the "template” strand.
  • the template may be either RNA and/or DNA, and the complementary strand may also be RNA and/or DNA.
  • the complementary strand may comprise all or part of the complementary sequence to the "template,” and/or may include mutations so that it is not an exact, complementary strand to the "template”. Strands that are not exactly complementary to the template strand may hybridize specifically to the template strand in detection assays described here, as well as other assays known in the art, and such complementary strands that can be used in detection assays are part of the invention.
  • treatment By “treatment, “treat,” “treated” and the like is meant to include both therapeutic and prophylactic treatment.
  • wild-type and "normal” are used interchangeably to refer to the phenotype that is characteristic of most of the members of the species occurring naturally and contrast for example with the phenotype of a mutant.
  • underscoring or italicizing the name of a gene shall indicate the gene, in contrast to its protein product, which is indicated by the name of the gene in the absence of any underscoring or italicizing.
  • "ReIB” shall mean the ReIB gene, whereas “ReIB” shall indicate the protein product or products generated from transcription and translation and alternative splicing of the "ReIB” gene.
  • the present invention arises in part from the determination that certain undesired or deleterious immune responses, including particular autoimmune diseases, organ-specific diseases, and allergic disorders either constitutively or as a result of interaction between pathogen or allergen with antigen presenting cells, are associated with defective NF- ⁇ B function through aberrant signaling of the canonical and/or non- canonical NF- ⁇ B pathways.
  • the invention provides methods for diagnosing the presence or risk of development in the subject of an undesirable or deleterious immune response that associates with defective NF- ⁇ B function ⁇ e.g., defective ReIB function), wherein these methods generally comprise detecting (e.g., directly or indirectly) a reduced or abrogated level or functional activity of NF- ⁇ B or one of its subunits (e.g., p50, plO5, p52, plOO, ReIA, ReIB and cRel), as compared to a reference level or functional activity corresponding to a basal state of NF- ⁇ B function or one of its subunits, which is indicative of the presence of the undesirable or deleterious immune response.
  • a reduced or abrogated level or functional activity of NF- ⁇ B or one of its subunits e.g., p50, plO5, p52, plOO, ReIA, ReIB and cRel
  • NF- ⁇ B pathway genes illustrative examples of which are listed above, by detecting for example the level or functional activity of an expression product of those genes, e.g., the level or functional activity of a transcript or the level or functional activity of a polypeptide.
  • the presence, degree, stage or risk of development of undesirable or deleterious immune response is detected when an NF- ⁇ B pathway gene is expressed at a detectably lower level in the biological sample as compared to the level at which that gene is expressed in a reference sample obtained from normal subjects or from subjects lacking the undesirable or deleterious immune response.
  • the gene is selected from genes encoding BTK, LYN, BCR Ig ⁇ , BCR Ig ⁇ , Syk, Blnk, PLC ⁇ 2, PKC ⁇ , DAG, CARMAl, BCLlO, MALTl, PI3K, PIP3, AKT, COT, IKK ⁇ , IKK ⁇ , IKK ⁇ , NIK, RelA/p65, P105/p50, c-Rel, ReIB, p52, NIK, Leul3, CD81, CD 19, CD21 and its ligands in the complement and coagulation cascade, TRAF6, and other ubiquitin ligases, Tab2, TAKl, NEMO, NOD2, RIP2, Lck, fyn, Zap70, LAT, GRB2, SOS, CD3 zeta, Slp-76, GADS, ITK, PLC ⁇ l, PKC ⁇ , ICOS, CD28, SHP-2, SAP, SLAM and 2
  • the presence, degree, stage or risk of development of the undesirable or deleterious immune response is detected when an NF- ⁇ B pathway gene is expressed at a detectably higher level in the biological sample as compared to the level at which that gene is expressed in a reference sample obtained from normal subjects or from subjects lacking the deleterious or unwanted immune response.
  • the gene is selected from genes encoding SHP-I, SHIP, PIR-B, CD22, CD72, FcgRIIB, IKB, PlOO, CTLA4, PD-I, CbI, KIR3DL1, KIR3DL2, KIR2DL and Csk.
  • the presence, degree, stage or risk of development of the undesirable or deleterious immune response is detected when the level or functional activity of an NF- ⁇ B pathway gene product in the biological sample varies from the level or functional activity of a corresponding NF- ⁇ B pathway gene product in the reference sample by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 97%, 98% or 99%, or even by at least about 99.5%, 99.9%, 99.95%, 99.99%, 99.995% or 99.999%, or even by at least about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000%.
  • the corresponding gene product is generally selected from the same gene product that is present in the biological sample, a gene product expressed from a variant gene (e.g., an homologous or orthologous gene) including an allelic variant, or a splice variant or protein product thereof.
  • the method comprises measuring the level or functional activity of individual expression products of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19 or 20 NF- ⁇ B pathway genes.
  • the method comprises measuring the level or functional activity of an expression product of the ReIB gene.
  • defective NF- ⁇ B function may be determined either through failure of NF- ⁇ B to direct the transcription of downstream genes, physical characteristics or DNA- or protein-binding activity in comparison to those of the basal state.
  • NF- ⁇ B activity may be assayed either in vivo or in vitro using an ELISA system of specific antibody to detect nuclear NF- ⁇ B subunit binding to a consensus oligonucleotide binding sequence.
  • activity can be detected using an NF- ⁇ B-dependent reporter gene expression construct and a substrate for enzymatic detection (such as chloramphenicol acetyl transferase or ⁇ -galactosidase, depending on the specificity of the enzyme encoded by the reporter gene), wherein comparative quantitation of the product of the diagnostic enzymatic reaction (or, in the absence of a reaction substrate, the level of the reporter mRNA or its encoded protein) in biological samples derived from a test subject and a normal control individual allow for the assessment of NF- ⁇ B functional loss.
  • a substrate for enzymatic detection such as chloramphenicol acetyl transferase or ⁇ -galactosidase, depending on the specificity of the enzyme encoded by the reporter gene
  • comparative quantitation of the product of the diagnostic enzymatic reaction or, in the absence of a reaction substrate, the level of the reporter mRNA or its encoded protein
  • the biological sample may contain blood, especially peripheral blood, or a fraction or extract thereof.
  • the biological sample comprises blood cells such as mature, immature and developing leukocytes, including lymphocytes, polymorphonuclear leukocytes, neutrophils, monocytes, reticulocytes, basophils, coelomocytes, haemocytes, eosinophils, megakaryocytes, macrophages, dendritic cells natural killer cells, or fraction of such cells (e.g., a nucleic acid or protein fraction).
  • the biological sample comprises leukocytes including peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • the diagnostic tests of the invention may be used to diagnose the presence, degree, stage or risk of development of an undesirable or deleterious immune response in any subject, which response is associated with defective NF- ⁇ B function, including defective ReIB function.
  • these tests are used with subjects related to a patient with clinical signs (one or more definitive markers) of the undesirable or deleterious immune response. These tests are desirably used at a stage and frequency to enable early the immune response, its progression, or to diagnose the risk of developing disease associated with that response.
  • the diagnostic tests of the invention may be used to diagnose the presence, degree, stage or risk of development of the unwanted or deleterious immune response in any subject.
  • these tests are used with subjects related to a patient with clinical signs (one or more definitive markers) of the unwanted or deleterious immune response.
  • These tests are desirably used at a stage and frequency to enable early detection of the unwanted or deleterious immune response or a condition related to that response, the progression of the immune response or related condition, or to diagnose the risk of developing that immune response or related condition.
  • Nucleic acids used in polynucleotide-based assays can be isolated from cells contained in the biological sample, according to standard methodologies (Sambrook, et al, 1989, supra; and Ausubel et al, 1994, supra).
  • the nucleic acid is typically fractionated (e.g., poly A+ RNA) or whole cell RNA. Where RNA is used as the subject of detection, it may be desired to convert the RNA to a complementary DNA.
  • the nucleic acid is amplified by a template-dependent nucleic acid amplification technique. A number of template dependent processes are available to amplify the NF- ⁇ B pathway gene sequences present in a given template sample.
  • PCR polymerase chain reaction
  • TlDM marker sequence is present in a sample
  • the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides.
  • the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated.
  • a reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al. , 1989, supra. Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641. Polymerase chain reaction methodologies are well known in the art.
  • the template-dependent amplification involves the quantification of transcripts in real-time.
  • RNA or DNA may be quantified using the Real-Time PCR technique (Higuchi, 1992, et al. , Biotechnology 10:413-417).
  • the concentration of the amplified products of the target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture. If the DNA mixtures are cDNAs synthesized from RNAs isolated from different tissues or cells, the relative abundance of the specific mRNA from which the target sequence was derived can be determined for the respective tissues or cells.
  • LCR ligase chain reaction
  • An isothermal amplification method in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5' ⁇ -thio-triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention, Walker et al, (1992, Proc. Natl. Acad. Sci. U.S.A 89:392-396).
  • SDA Strand Displacement Amplification
  • RCR Repair Chain Reaction
  • CPR cyclic probe reaction
  • Still another amplification method described in GB Application No. 2 202 328, and in PCT Application No. PCT/US89/01025, may be used.
  • modified primers are used in a PCR-like, template- and enzyme- dependent synthesis.
  • the primers may be modified by labeling with a capture moiety ⁇ e.g., biotin) and/or a detector moiety (e.g., enzyme).
  • a capture moiety e.g., biotin
  • a detector moiety e.g., enzyme
  • an excess of labeled probes are added to a sample.
  • the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.
  • nucleic acid amplification procedures include transcription- based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et a!., 1989, Proc. Natl. Acad. Sci. U.S.A., 86: 1173; Gingeras et al., PCT Application WO 88/10315).
  • TAS transcription- based amplification systems
  • NASBA nucleic acid sequence based amplification
  • 3SR Zaoh et a!., 1989, Proc. Natl. Acad. Sci. U.S.A., 86: 1173; Gingeras et al., PCT Application WO 88/10315.
  • NASBA the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of
  • DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again.
  • the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization.
  • the double-stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6.
  • the RNAs are reverse transcribed into single stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6.
  • T7 or SP6 an isothermal cyclic reaction
  • the resulting products whether truncated or complete, indicate target specific sequences.
  • ssRNA single-stranded RNA
  • dsDNA double-stranded DNA
  • the ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase).
  • RNA-dependent DNA polymerase reverse transcriptase
  • the RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA).
  • RNase H ribonuclease H
  • the resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template.
  • This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence.
  • This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification.
  • the starting sequence can be chosen to be in the form of either DNA or RNA.
  • Miller et al. in PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts.
  • Other amplification methods include "RACE” and "one-sided PCR” (Frohman, M.
  • the NF- ⁇ B pathway nucleic acid of interest is identified in the sample directly using a template-dependent amplification as described, for example, above, or with a second, known nucleic acid following amplification.
  • the identified product is detected.
  • the detection may be performed by visual means ⁇ e.g., ethidium bromide staining of a gel).
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax Technology; Bellus, 1994, J Macromol. Sci. Pure, Appl. Chem., A31(l):1355-1376).
  • amplification products or "amplicons" are visualized in order to confirm amplification of the NF- ⁇ B pathway gene sequences.
  • One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light.
  • the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.
  • visualization is achieved indirectly. Following separation of amplification products, a labeled nucleic acid probe is brought into contact with the amplified NF- ⁇ B pathway gene sequence.
  • the probe is suitably conjugated to a chromophore but may be radiolabeled.
  • the probe is conjugated to a binding partner, such as an antigen-binding molecule, or biotin, and the other member of the binding pair carries a detectable moiety or reporter molecule.
  • a binding partner such as an antigen-binding molecule, or biotin
  • the other member of the binding pair carries a detectable moiety or reporter molecule.
  • the techniques involved are well known to those of skill in the art and can be found in many standard texts on molecular protocols (e.g., see Sambrook et al., 1989, supra and Ausubel et al. 1994, supra).
  • chromophore or radiolabel probes or primers identify the target during or following amplification.
  • target nucleic acids are quantified using blotting techniques, which are well known to those of skill in the art.
  • Southern blotting involves the use of DNA as a target
  • Northern blotting involves the use of RNA as a target.
  • cDNA blotting is analogous, in many aspects, to blotting or RNA species.
  • a probe is used to target a DNA or RNA species that has been immobilized on a suitable matrix, often a filter of nitrocellulose. The different species should be spatially separated to facilitate analysis. This often is accomplished by gel electrophoresis of nucleic acid species followed by "blotting" on to the filter.
  • the blotted target is incubated with a probe (usually labeled) under conditions that promote denaturation and rehybridization. Because the probe is designed to base pair with the target, the probe will bind a portion of the target sequence under renaturing conditions. Unbound probe is then removed, and detection is accomplished as described above.
  • a probe usually labeled
  • genotyping methods and allelic discrimination methods and technologies such as those described by Kristensen et al (Biotechniques 30(2):318-322), including the use of single nucleotide polymorphism analysis, high performance liquid chromatography, TaqManTM, liquid chromatography, and mass spectrometry.
  • biochip-based technologies such as those described by Hacia et al. (1996, Nature Genetics 14:441-447) and Shoemaker et al. (1996, Nature Genetics 14:450-456). Briefly, these techniques involve quantitative methods for analyzing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ biochip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization. See also Pease et al. (1994, Proc. Natl. Acad. Sci. U.S.A.
  • nucleic acid probes to NF- ⁇ B pathway polynucleotides are made and attached to biochips to be used in screening and diagnostic methods, as outlined herein.
  • the nucleic acid probes attached to the biochip are designed to be substantially complementary to specific expressed NF- KB pathway nucleic acids, i.e., the target sequence (either the target sequence of the sample or to other probe sequences, for example in sandwich assays), such that hybridization of the target sequence and the probes of the present invention occurs.
  • This complementarity need not be perfect; there may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the nucleic acid probes of the present invention. However, if the number of mismatches is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence.
  • more than one probe per sequence is used, with either overlapping probes or probes to different sections of the target being used. That is, two, three, four or more probes, with three being desirable, are used to build in a redundancy for a particular target.
  • the probes can be overlapping (i.e. have some sequence in common), or separate.
  • nucleic acids can be attached to or immobilized on a solid support in a wide variety of ways.
  • immobilized and grammatical equivalents herein is meant the association or binding between the nucleic acid probe and the solid support is sufficient to be stable under the conditions of binding, washing, analysis, and removal as outlined below.
  • the binding can be covalent or non-covalent.
  • non-covalent binding and grammatical equivalents herein is meant one or more of either, electrostatic, hydrophilic, and hydrophobic interactions.
  • non-covalent binding is the covalent attachment of a molecule, such as, streptavidin to the support and the non-covalent binding of the biotinylated probe to the streptavidin.
  • covalent binding and grammatical equivalents herein is meant that the two moieties, the solid support and the probe, are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds.
  • Covalent bonds can be formed directly between the probe and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules. Immobilization may also involve a combination of covalent and non-covalent interactions.
  • the probes are attached to the biochip in a wide variety of ways, as will be appreciated by those in the art.
  • the nucleic acids can either be synthesized first, with subsequent attachment to the biochip, or can be directly synthesized on the biochip.
  • the biochip comprises a suitable solid or semi-solid substrate or solid support.
  • substrate or “solid support” is meant any material that can be modified to contain discrete individual sites appropriate for the attachment or association of the nucleic acid probes and is amenable to at least one detection method.
  • the number of possible substrates are very large, and include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonTM, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, etc.
  • the substrates allow optical detection and do not appreciably fluorescese.
  • the substrate is planar, although as will be appreciated by those of skill in the art, other configurations of substrates may be used as well.
  • the probes may be placed on the inside surface of a tube, for flow-through sample analysis to minimize sample volume.
  • the substrate may be flexible, such as a flexible foam, including closed cell foams made of particular plastics.
  • oligonucleotides probes are synthesized on the substrate, as is known in the art.
  • photoactivation techniques utilizing photopolymerization compounds and techniques can be used.
  • the nucleic acids are synthesized in situ, using well known photolithographic techniques, such as those described in WO 95/25116; WO 95/35505; U.S. Pat. Nos.
  • oligonucleotide probes on the biochip are exposed to or contacted with a nucleic acid sample suspected of containing one or more NF- ⁇ B pathway polynucleotides under conditions favoring specific hybridization.
  • Sample extracts of DNA or RNA may be prepared from fluid suspensions of biological materials, or by grinding biological materials, or following a cell lysis step which includes, but is not limited to, lysis effected by treatment with SDS (or other detergents), osmotic shock, guanidinium isothiocyanate and lysozyme.
  • Suitable DNA which may be used in the method of the invention, includes cDNA. Such DNA may be prepared by any one of a number of commonly used protocols as for example described in Ausubel, et al. , 1994, supra, and Sambrook, et al, 1989, supra.
  • RNA which may be used in the method of the invention, includes messenger RNA, complementary RNA transcribed from DNA (cRNA) or genomic or subgenomic RNA. Such RNA may be prepared using standard protocols as for example described in the relevant sections of Ausubel, et al. 1994, supra and Sambrook, et al. , 1989, supra).
  • cDNA may be fragmented, for example, by sonication or by treatment with restriction endonucleases.
  • cDNA is fragmented such that resultant DNA fragments are of a length greater than the length of the immobilized oligonucleotide probe(s) but small enough to allow rapid access thereto under suitable hybridization conditions.
  • fragments of cDNA may be selected and amplified using a suitable nucleotide amplification technique, as described for example above, involving appropriate random or specific primers.
  • the target NF- ⁇ B pathway polynucleotides are detectably labeled so that their hybridization to individual probes can be determined.
  • the target polynucleotides are typically detectably labeled with a reporter molecule illustrative examples of which include chromogens, catalysts, enzymes, fluorochromes, chemiluminescent molecules, bioluminescent molecules, lanthanide ions (e.g., Eu 34 ), a radioisotope and a direct visual label.
  • a reporter molecule illustrative examples of which include chromogens, catalysts, enzymes, fluorochromes, chemiluminescent molecules, bioluminescent molecules, lanthanide ions (e.g., Eu 34 ), a radioisotope and a direct visual label.
  • a direct visual label use may be made of a colloidal metallic or non-metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like.
  • Illustrative labels of this type include large colloids, for example, metal colloids such as those from gold, selenium, silver, tin and titanium oxide.
  • an enzyme is used as a direct visual label
  • biotinylated bases are incorporated into a target polynucleotide. Hybridization is detected by incubation with streptavidin-reporter molecules.
  • Suitable fluorochromes include, but are not limited to, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), R-Phycoerythrin (RPE), and Texas Red.
  • FITC fluorescein isothiocyanate
  • TRITC tetramethylrhodamine isothiocyanate
  • RPE R-Phycoerythrin
  • Texas Red Texas Red
  • Other exemplary fluorochromes include those discussed by Dower et al. (International Publication WO 93/06121). Reference also may be made to the fluorochromes described in U.S. Patents 5,573,909 (Singer et al), 5,326,692 (Brinkley et al). Alternatively, reference may be made to the fluorochromes described in U.S. Patent Nos.
  • fluorescent labels include, for example, fluorescein phosphoramidites such as FluoreprimeTM (Pharmacia), FluorediteTM (Millipore) and FAM (Applied Biosystems International)
  • Radioactive reporter molecules include, for example, 32 P, which can be detected by an X-ray or phosphoimager techniques.
  • the hybrid-forming step can be performed under suitable conditions for hybridizing oligonucleotide probes to test nucleic acid including DNA or RNA.
  • suitable conditions for hybridizing oligonucleotide probes to test nucleic acid including DNA or RNA.
  • whether hybridization takes place is influenced by the length of the oligonucleotide probe and the polynucleotide sequence under test, the pH, the temperature, the concentration of mono- and divalent cations, the proportion of G and C nucleotides in the hybrid-forming region, the viscosity of the medium and the possible presence of denaturants.
  • Such variables also influence the time required for hybridization.
  • the preferred conditions will therefore depend upon the particular application. Such empirical conditions, however, can be routinely determined without undue experimentation.
  • high discrimination hybridization conditions are used.
  • Wallace et al. (1979, Nucl. Acids Res. 6:3543) who describe conditions that differentiate the hybridization of 11 to 17 base long oligonucleotide probes that match perfectly and are completely homologous to a target sequence as compared to similar oligonucleotide probes that contain a single internal base pair mismatch.
  • Wood et al. (1985, Proc. Natl. Acid. Sci.
  • a hybridization reaction can be performed in the presence of a hybridization buffer that optionally includes a hybridization optimizing agent, such as an isostabilizing agent, a denaturing agent and/or a renaturation accelerant.
  • a hybridization optimizing agent such as an isostabilizing agent, a denaturing agent and/or a renaturation accelerant.
  • isostabilizing agents include, but are not restricted to, betaines and lower tetraalkyl ammonium salts.
  • Denaturing agents are compositions that lower the melting temperature of double stranded nucleic acid molecules by interfering with hydrogen bonding between bases in a double stranded nucleic acid or the hydration of nucleic acid molecules.
  • Denaturing agents include, but are not restricted to, formamide, formaldehyde, dimethylsulphoxide, tetraethyl acetate, urea, guanidium isothiocyanate, glycerol and chaotropic salts.
  • Hybridization accelerants include heterogeneous nuclear ribonucleoprotein (hnRP) Al and cationic detergents such as cetyltrimethylammonium bromide (CTAB) and dodecyl trimethylammonium bromide (DTAB), polylysine, spermine, spermidine, single stranded binding protein (SSB), phage T4 gene 32 protein and a mixture of ammonium acetate and ethanol.
  • CAB cetyltrimethylammonium bromide
  • DTAB dodecyl trimethylammonium bromide
  • polylysine polylysine
  • spermine spermine
  • spermidine single stranded binding protein
  • SSB single
  • Hybridization buffers may include target polynucleotides at a concentration between about 0.005 nM and about 50 nM, preferably between about 0.5 nM and 5 nM, more preferably between about 1 nM and 2 nM.
  • a hybridization mixture containing the target NF- ⁇ B pathway polynucleotides is placed in contact with the array of probes and incubated at a temperature and for a time appropriate to permit hybridization between the target sequences in the target polynucleotides and any complementary probes.
  • Contact can take place in any suitable container, for example, a dish or a cell designed to hold the solid support on which the probes are bound.
  • incubation will be at temperatures normally used for hybridization of nucleic acids, for example, between about 20° C and about 75° C, example, about 25° C, about 30° C, about 35° C, about 40° C, about 45° C, about 50° C, about 55° C, about 60° C, or about 65° C.
  • temperatures normally used for hybridization of nucleic acids for example, between about 20° C and about 75° C, example, about 25° C, about 30° C, about 35° C, about 40° C, about 45° C, about 50° C, about 55° C, about 60° C, or about 65° C.
  • 20° C to 50° C is desirable.
  • lower temperatures are preferred.
  • a sample of target polynucleotides is incubated with the probes for a time sufficient to allow the desired level of hybridization between the target sequences in the target polynucleotides and any complementary probes.
  • the hybridization may be carried out at about 45° C +/-10° C in formamide for 1-2 days.
  • the probes are washed to remove any unbound nucleic acid with a hybridization buffer, which can typically comprise a hybridization optimizing agent in the same range of concentrations as for the hybridization step. This washing step leaves only bound target polynucleotides.
  • the probes are then examined to identify which probes have hybridized to a target polynucleotide.
  • a signal may be instrumentally detected by irradiating a fluorescent label with light and detecting fluorescence in a fluorimeter; by providing for an enzyme system to produce a dye which could be detected using a spectrophotometer; or detection of a dye particle or a colored colloidal metallic or non metallic particle using a reflectometer; in the case of using a radioactive label or chemiluminescent molecule employing a radiation counter or autoradiography.
  • a detection means may be adapted to detect or scan light associated with the label which light may include fluorescent, luminescent, focussed beam or laser light.
  • a charge couple device (CCD) or a photocell can be used to scan for emission of light from a probe:target polynucleotide hybrid from each location in the micro-array and record the data directly in a digital computer.
  • electronic detection of the signal may not be necessary. For example, with enzymatically generated colour spots associated with nucleic acid array format, visual examination of the array will allow interpretation of the pattern on the array.
  • the detection means is suitably interfaced with pattern recognition software to convert the pattern of signals from the array into a plain language genetic profile.
  • oligonucleotide probes specific for different NF- ⁇ B pathway gene products are in the form of a nucleic acid array and detection of a signal generated from a reporter molecule on the array is performed using a 'chip reader'.
  • a detection system that can be used by a 'chip reader' is described for example by Pirrung et al (U.S. Patent No. 5,143,854).
  • the chip reader will typically also incorporate some signal processing to determine whether the signal at a particular array position or feature is a true positive or maybe a spurious signal.
  • Exemplary chip readers are described for example by Fodor et al (U.S. Patent No., 5,925,525).
  • the reaction may be detected using flow cytometry.
  • the presence of an aberrant concentration of a NF- ⁇ B pathway protein is indicative of the presence, degree, activity, stage or risk of development of an unwanted or deleterious immune response associated with defective NF- ⁇ B signaling, including defective ReIB signaling, or related condition.
  • NF- ⁇ B pathway protein levels in biological samples can be assayed using any suitable method known in the art.
  • a NF- ⁇ B pathway protein is an enzyme
  • the protein can be quantified based upon its catalytic activity or based upon the number of molecules of the protein contained in a sample.
  • Antibody-based techniques may be employed, such as, for example, immunohistological and immunohistochemical methods for measuring the level of a protein of interest in a tissue sample.
  • a primary antibody polyclonal or monoclonal
  • a secondary detection system is used to detect presence (or binding) of the primary antibody.
  • Detectable labels can be conjugated to the secondary antibody, such as a fluorescent label, a radiolabel, or an enzyme (e.g., alkaline phosphatase, horseradish peroxidase) which produces a quantifiable, e.g., colored, product.
  • the primary antibody itself can be detectably labeled.
  • immunohistological labeling of a tissue section is provided.
  • a protein extract is produced from a biological sample (e.g., tissue, cells) for analysis.
  • Such an extract e.g., a detergent extract
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • a protein-specific monoclonal antibody can be used both as an immunoadsorbent and as an enzyme-labeled probe to detect and quantify a NF- ⁇ B pathway protein of interest.
  • the amount of such protein present in a sample can be calculated by reference to the amount present in a standard preparation using a linear regression computer algorithm (see Lacobilli et ⁇ l. , 1988, Breast Cancer Research and Treatment 11 :19-30).
  • two different monoclonal antibodies to the protein of interest can be employed, one as the immunoadsorbent and the other as an enzyme-labeled probe.
  • recent developments in the field of protein capture arrays permit the simultaneous detection and/or quantification of a large number of proteins.
  • low-density protein arrays on filter membranes such as the universal protein array system (Ge, 2000 Nucleic Aeids Res. 28(2):e3) allow imaging of arrayed antigens using standard ELISA techniques and a scanning charge-coupled device (CCD) detector.
  • CCD scanning charge-coupled device
  • Protein capture arrays typically comprise a plurality of protein-capture agents each of which defines a spatially distinct feature of the array.
  • the protein-capture agent can be any molecule or complex of molecules which has the ability to bind a protein and immobilize it to the site of the protein-capture agent on the array.
  • the protein-capture agent may be a protein whose natural function in a cell is to specifically bind another protein, such as an antibody or a receptor.
  • the protein- capture agent may instead be a partially or wholly synthetic or recombinant protein which specifically binds a protein.
  • the protein-capture agent may be a protein which has been selected in vitro from a mutagenized, randomized, or completely random and synthetic library by its binding affinity to a specific protein or peptide target.
  • the selection method used may optionally have been a display method such as ribosome display or phage display, as known in the art.
  • the protein- capture agent obtained via in vitro selection may be a DNA or RNA aptamer which specifically binds a protein target (see, e.g., Potyrailo et al., 1998 Anal. Chem. 70:3419- 3425; Cohen et al, 1998, Proc. Natl. Acad. Sci.
  • aptamers are selected from libraries of oligonucleotides by the SelexTM process and their interaction with protein can be enhanced by covalent attachment, through incorporation of brominated deoxyuridine and UV-activated crosslinking (photoaptamers). Aptamers have the advantages of ease of production by automated oligonucleotide synthesis and the stability and robustness of DNA; universal fluorescent protein stains can be used to detect binding.
  • the in vitro selected protein-capture agent may be a polypeptide ⁇ e.g., an antigen) (see, e.g., Roberts and Szostak, 1997 Proc. Natl. Acad. Sci. USA, 94: 12297-12302).
  • An alternative to an array of capture molecules is one made through 'molecular imprinting' technology, in which peptides (e.g., from the C-terminal regions of proteins) are used as templates to generate structurally complementary, sequence- specific cavities in a polymerizable matrix; the cavities can then specifically capture (denatured) proteins which have the appropriate primary amino acid sequence (e.g. , available from ProteinPrintTM and Aspira Biosystems).
  • Exemplary protein capture arrays include arrays comprising spatially addressed antigen-binding molecules, commonly referred to as antibody arrays, which can facilitate extensive parallel analysis of numerous proteins defining a proteome or subproteome.
  • Antibody arrays have been shown to have the required properties of specificity and acceptable background, and some are available commercially (e.g., BD Biosciences, Clontech, BioRad and Sigma). Various methods for the preparation of antibody arrays have been reported (see, e.g., Lopez et al, 2003 J. Chromatogr. B 787: 19-27; Cahill, 2000 Trends in Biotechnology 7:47-51 ; U.S. Pat. App. Pub. 2002/0055186; U.S. Pat. App. Pub.
  • the antigen-binding molecules of such arrays may recognize at least a subset of proteins expressed by a cell or population of cells, illustrative examples of which include growth factor receptors, hormone receptors, neurotransmitter receptors, catecholamine receptors, amino acid derivative receptors, cytokine receptors, extracellular matrix receptors, antibodies, lectins, cytokines, serpins, proteases, kinases, phosphatases, ras- like GTPases, hydrolases, steroid hormone receptors, transcription factors, heat-shock transcription factors, DNA-binding proteins, zinc-finger proteins, leucine-zipper proteins, homeodomain proteins, intracellular signal transduction modulators and effectors, apoptosis-related factors, DNA synthesis factors, DNA repair factors, DNA recombination factors, cell-surface antigens, hepatitis C virus (HCV) proteases and HIV proteases.
  • HCV hepatitis C virus
  • Antigen-binding molecules for antibody arrays are made either by conventional immunization (e.g. , polyclonal sera and hybridomas), or as recombinant fragments, usually expressed in E. coli, after selection from phage display or ribosome display libraries (e.g., available from Cambridge Antibody Technology, Biolnvent, Affitech and Biosite).
  • phage display or ribosome display libraries e.g., available from Cambridge Antibody Technology, Biolnvent, Affitech and Biosite.
  • 'combibodies' comprising non-covalent associations of VH and VL domains, can be produced in a matrix format created from combinations of diabody-producing bacterial clones (e.g., available from Domantis).
  • antigen-binding molecules for use as protein-capture agents include monoclonal antibodies, polyclonal antibodies, Fv, Fab, Fab' and F(ab')2 immunoglobulin fragments, synthetic stabilised Fv fragments, e.g., single chain Fv fragments (scFv), disulphide stabilised Fv fragments (dsFv), single variable region domains (dAbs) minibodies, combibodies and multivalent antibodies such as diabodies and multi-scFv, single domains from camelids or engineered human equivalents.
  • synthetic stabilised Fv fragments e.g., single chain Fv fragments (scFv), disulphide stabilised Fv fragments (dsFv), single variable region domains (dAbs) minibodies, combibodies and multivalent antibodies such as diabodies and multi-scFv, single domains from camelids or engineered human equivalents.
  • Individual spatially distinct protein-capture agents are typically attached to a support surface, which is generally planar or contoured.
  • Common physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, and magnetic and other microbeads.
  • microdrops of protein delivered onto planar surfaces are widely used, related alternative architectures include CD centrifugation devices based on developments in microfluidics (e.g., available from Gyros) and specialised chip designs, such as engineered microchannels in a plate (e.g., The Living ChipTM, available from Biotrove) and tiny 3D posts on a silicon surface (e.g., available from Zyomyx).
  • microfluidics e.g., available from Gyros
  • specialised chip designs such as engineered microchannels in a plate (e.g., The Living ChipTM, available from Biotrove) and tiny 3D posts on a silicon surface (e.g., available from Zyomyx).
  • Particles in suspension can also be used as the basis of arrays, providing they are coded for identification; systems include colour coding for microbeads (e.g., available from Luminex, Bio-Rad and Nanomics Biosystems) and semiconductor nanocrystals (e.g., QDotsTM, available from Quantum Dots), and barcoding for beads (UltraPlexTM, available from Smartbeads) and multimetal microrods (NanobarcodesTM particles, available from Surromed). Beads can also be assembled into planar arrays on semiconductor chips (e.g., available from LEAPS technology and BioArray Solutions).
  • colour coding for microbeads e.g., available from Luminex, Bio-Rad and Nanomics Biosystems
  • semiconductor nanocrystals e.g., QDotsTM, available from Quantum Dots
  • barcoding for beads UltraPlexTM, available from Smartbeads
  • NanobarcodesTM particles available
  • individual protein- capture agents are typically attached to an individual particle to provide the spatial definition or separation of the array.
  • the particles may then be assayed separately, but in parallel, in a compartmentalised way, for example in the wells of a microtiter plate or in separate test tubes.
  • a protein sample which is optionally fragmented to form peptide fragments (see, e.g., U.S. Pat. App. Pub. 2002/0055186), is delivered to a protein-capture array under conditions suitable for protein or peptide binding, and the array is washed to remove unbound or non-specifically bound components of the sample from the array.
  • the presence or amount of protein or peptide bound to each feature of the array is detected using a suitable detection system.
  • the amount of protein bound to a feature of the array may be determined relative to the amount of a second protein bound to a second feature of the array. In certain embodiments, the amount of the second protein in the sample is already known or known to be invariant.
  • a protein sample of a first cell or population of cells is delivered to the array under conditions suitable for protein binding.
  • a protein sample of a second cell or population of cells to a second array is delivered to a second array which is identical to the first array. Both arrays are then washed to remove unbound or non-specifically bound components of the sample from the arrays.
  • the amounts of protein remaining bound to the features of the first array are compared to the amounts of protein remaining bound to the corresponding features of the second array.
  • the amount of protein bound to individual features of the first array is subtracted from the amount of protein bound to the corresponding features of the second array.
  • fluorescence labeling can be used for detecting protein bound to the array.
  • the same instrumentation as used for reading DNA microarrays is applicable to protein-capture arrays.
  • capture arrays e.g. antibody arrays
  • fluorescently labelled proteins from two different cell states, in which cell lysates are labeled with different fluorophores (e.g., Cy-3 and Cy-5) and mixed, such that the color acts as a readout for changes in target abundance.
  • Fluorescent readout sensitivity can be amplified 10-100 fold by tyramide signal amplification (TSA) (e.g., available from Perkin Elmer Lifesciences).
  • TSA tyramide signal amplification
  • Planar waveguide technology e.g., available from Zeptosens
  • High sensitivity can also be achieved with suspension beads and particles, using phycoerythrin as label (e.g., available from Luminex) or the properties of semiconductor nanocrystals (e.g., available from Quantum Dot).
  • Fluorescence resonance energy transfer has been adapted to detect binding of unlabelled ligands, which may be useful on arrays (e.g., available from Affibody).
  • nucleic acids can be used as a protein capture agents, in which the nucleic acids bind to or otherwise complex with a nucleic acid-binding domain of an NF- ⁇ B pathway transcription factor (e.g., ReIB, cRel, p65, p50 etc).
  • a transcription factor e.g., RNA or DNA
  • a nucleic acid e.g., RNA or DNA
  • cis sites are known to those of skill in the art and nucleic acids corresponding to these sites can be prepared using standard procedures.
  • nucleic acids can be used in any suitable nucleic acid- binding protein assay, non limiting examples of which include the assays described by Haukanes B I and Kvam C (Biotechnology, 1993 Jan 11 60-63), Alberts B et al.
  • aberrant signaling of the NF- ⁇ B pathway is detected by detecting aberrant phosphorylation of a phosphorylatable polypeptide (e.g.
  • the phosphorylation state of the phosphorylatable polypeptide is indicative of the activity of the NF- ⁇ B signaling pathway.
  • the phosphorylation state of the phosphorylatable polypeptide in the biological sample is higher or lower than the phosphorylation state of the same polypeptide in a reference sample, then this indicates that the phosphorylation state of phosphorylatable polypeptide in the biological sample is abnormal, indicative of aberrant signaling through the NF- ⁇ B pathway.
  • aberrant signaling through the NF- ⁇ B pathway is detected when the phosphorylation state of a NF- ⁇ B associated phosphorylatable polypeptide such as but not limited to IRAK, TAKl, TABl, TAB2, PKR, Akt, Cot, IKK ⁇ , IKK ⁇ and IKK ⁇ , is lower than the phosphorylation state of the corresponding polypeptide in the reference sample.
  • a NF- ⁇ B associated phosphorylatable polypeptide such as but not limited to IRAK, TAKl, TABl, TAB2, PKR, Akt, Cot, IKK ⁇ , IKK ⁇ and IKK ⁇ .
  • the techniques used for detection of NF- ⁇ B pathway gene expression products will include internal or external standards to permit quantitative or semi-quantitative determination of those products, to thereby enable a valid comparison of the level or functional activity of these expression products in a biological sample with the corresponding expression products in a reference sample or samples.
  • standards can be determined by the skilled practitioner using standard protocols.
  • absolute values for the level or functional activity of individual expression products are determined.
  • kits All the essential materials and reagents required for detecting and quantifying NF- ⁇ B pathway gene expression products may be assembled together in a kit.
  • the kit comprises: a) primers designed to produce double stranded DNA complementary to at least a portion of a NF- ⁇ B pathway gene; wherein at least one of the primers contains a sequence which hybridizes to RNA, cDNA or an EST corresponding to the marker gene to create an extension product and at least one other primer that hybridizes to the extension product; b) an enzyme with reverse transcriptase activity, and c) an enzyme with thermostable DNA polymerase activity; wherein the primers are used to detect the expression levels of the marker gene in a test subject.
  • the kit comprises at least one oligonucleotide which hybridizes to RNA, cDNA or an EST corresponding to a NF- ⁇ B pathway gene, wherein the oligonucleotide is used to detect the expression levels of the marker gene in a test sample.
  • the kit comprises at least one oligonucleotide that binds to or otherwise complexes with a nucleic acid-binding domain of a NF- ⁇ B pathway polypeptide (e.g., an NF- ⁇ B pathway transcription factor), wherein the oligonucleotide is used to detect the level of the transcription factor in a test sample.
  • a NF- ⁇ B pathway polypeptide e.g., an NF- ⁇ B pathway transcription factor
  • kits may optionally include appropriate reagents for detection of labels, positive and negative controls, washing solutions, blotting membranes, microtitre plates dilution buffers and the like.
  • a nucleic acid-based detection kit may include (i) a NF- ⁇ JB pathway polynucleotide (which may be used as a positive control), (ii) a primer or probe that specifically hybridizes to a NF- ⁇ B pathway polynucleotide.
  • enzymes suitable for amplifying nucleic acids including various polymerases (Reverse Transcriptase, Taq, SequenaseTM DNA ligase etc.
  • kits will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each primer or probe.
  • a protein-based detection kit may include (i) a NF- ⁇ B pathway polypeptide (which may be used as a positive control) and (ii) an antigen-binding molecule that is immuno-interactive with a NF- ⁇ B pathway polynucleotide, or (iii) an oligonucleotide that binds to or otherwise complexes with a nucleic acid-binding domain of the NF- ⁇ B pathway polypeptide (e.g., an NF- ⁇ B pathway transcription factor).
  • a NF- ⁇ B pathway polypeptide which may be used as a positive control
  • an antigen-binding molecule that is immuno-interactive with a NF- ⁇ B pathway polynucleotide
  • an oligonucleotide that binds to or otherwise complexes with a nucleic acid-binding domain of the NF- ⁇ B pathway polypeptide (e.g., an NF- ⁇ B pathway transcription factor).
  • the kit can also feature various devices and reagents for performing one of the assays described herein; and/or printed instructions for using the kit to quantify the expression of a NF- ⁇ B pathway polynucleotide or the level of a NF- KB pathway.
  • NF- KB function prevents antigen-presenting cells such as DC to license regulatory lymphocyte mediated suppression of T cell-associated inflammatory disease. They have also determined that the capacity of antigen-presenting cells to license this suppression is restorable by activating both the canonical and non-canonical NF- ⁇ B pathways (e.g., by activating ReIB or restoring its function or expressing it at basal levels).
  • the present invention provides methods for modulating an immune response, especially an undesirable or deleterious immune response associated with defective NF- ⁇ B activation, including defective ReIB activation, in a subject, wherein the methods generally comprise increasing the number of antigen-presenting cells in the subject, in which both the canonical and non-canonical NF- ⁇ B pathways are activated or activatable (e.g., in which ReIB is activated or activatable), suitably in response to a pro-inflammatory signal such as but not limited to tumor necrosis factor (e.g., TNF ⁇ ), C5a, interleukin- 1 (e.g., IL- l ⁇ ), CD 154 and lipolysaccharide (LPS).
  • TNF ⁇ tumor necrosis factor
  • C5a interleukin- 1
  • IL- l ⁇ interleukin- 1
  • CD 154 lipolysaccharide
  • NF- ⁇ B operable antigen-presenting cells are also referred to herein as NF- ⁇ B operable antigen-presenting cells.
  • the number of antigen- presenting cells with operable NF- ⁇ B function can be increased by use of (i) at least one agent (also referred to herein as an "NF- KB restoration agent") that activates the canonical and non-canonical NF- ⁇ B pathways, (ii) an antigen-presenting cell that has been contacted ex vivo with at least one NF- ⁇ B restoration agent, and (iii) an operable NF- ⁇ B antigen-presenting cell derived from a histocompatible donor.
  • one or more NF- ⁇ B restoration agents are used in vivo or ex vivo, to activate the canonical and non-canonical NF- ⁇ B pathways, in cells including antigen-presenting cells.
  • one method of achieving this is to use a first NF- ⁇ B restoration agent that activates the canonical pathway and a second NF- ⁇ B restoration agent that activates the non-canonical pathway.
  • NF- ⁇ B restoration agents that modulate both pathways may be employed.
  • NF- ⁇ B pathway include Toll-like receptor (TLR) agonists, Tumor Necrosis Factor- ⁇ (TNF- ⁇ ) agonists and agents that increase the level or functional activity of a canonical pathway kinase selected from IKK and IKK ⁇ /NEMO.
  • TLR Toll-like receptor
  • TNF- ⁇ Tumor Necrosis Factor- ⁇
  • TLR agonists include LPS, bacterial lipopeptides and lipoproteins, bacterial outer membrane proteins, lipoarabinomannan, glycosylphosphatidylinositol (GPI) anchors, taxol, zymosan, lipoteichoic acid, heat shock proteins (HSP) 60 and 70, fibrinogen, fibronectin, soluble heparin sulfate, hyaluronin, double stranded RNA, flagellin, pilin, single stranded viral, viral and bacterial unmethylated CpG DNA, chromatin-IgG complexes, endogenous mRNA, R837, R848, CPG 7909, loxoribine, uridine, poly-IC, and poly-AU, and combinations thereto.
  • LPS low-phosphatidylinositol
  • GPI glycosylphosphatidylinositol
  • HSP heat shock proteins
  • Illustrative TNF- ⁇ agonists include virulizin, a human or humanized monoclonal antibody TNF- ⁇ receptor agonist, Complete Freund's adjuvant (CFA), ISS-ODN, microbial cell wall components with LPS-like activity, cholera particles, E. coli heat labile enterotoxin, E.
  • ISCOMS immune stimulating complexes
  • polyethylene glycol poly(N-2-(hydroxypropyl)methacrylamide)
  • synthetic oligonucleotides containing CpG or CpA motifs monophosphoryl lipid A, Bacillus Calmette-Guerin, ⁇ -interferon, Tissue Plasminogen Activator, LPS, Interleukin-1,
  • Interleukin-2 Interleukin-2, UV light, a lymphotoxin, cachectin, a TNFR-2 agonist, an intracellular mediator of the TNF- ⁇ signaling pathway, IRF-I , STATl , a lymphokine, or the combination of TNF- ⁇ and an anti-TNFR-1 antibody.
  • Non-limiting NF- ⁇ B restoration agents for activating the non- canonical NF- ⁇ B pathway include agents that increase the level or functional activity of ReIB or a non-canonical pathway kinase selected from NIK and IKK ⁇ .
  • agents that increase the level or functional activity of ReIB include PP ARy antagonists or inhibitors, Vitamin D antagonists and estrogen antagonists or depletion agents.
  • Non-limiting examples of PPAR ⁇ antagonists or inhibitors include: 1- (p-chlorobenzyl)-5-chloro-3-thiophenylindole-2-carboxylic acid, as described for example in U.S. Patent Appl. Pub. No.
  • Vitamin D antagonists can be selected from: 2 ⁇ - methyl 19-nor vitamin D analog ester compounds illustrative examples of which include compounds of formula II ((22E)-(20S,24R)-25-carbobutoxy-2 ⁇ -methyl-26,27-cyclo-22- dehydro-1- ⁇ , 24-dihydroxy-19-norvitamin D 3 ), formula III ((22E)-(24R)-25- carbobutoxy-2 ⁇ -methyl-26,27-cyclo-22-dehydro- 1 - ⁇ ,24-dihydroxy- 19-norvitamin D 3 ), formula IV ((22E)-(20S)-25-carbopentoxy-2 ⁇ -methyl-24-oxo-22-dehydro-l ⁇ - hydroxy- 19-norvitamin D 3 ), formula V ((22E)-25-carbopentoxy-2 ⁇ -methyl-24-oxo-22- dehydro-1 ⁇ -hydroxy- 19-norvitamin D 3 ) and the like
  • Illustrative estrogen antagonists or depletion agents include tamoxifen, fulvestrant, toremifene and raloxifene, or a pharmaceutically acceptable salt thereof.
  • Illustrative estrogen-depletion agents are selected from: aromatase inhibitor such as but not limited to formestane, exemestane, anastrozole, vorozole, letrozole and aminoglutethimide, or a pharmaceutically acceptable salt thereof.
  • the level or functional activity of a NF- ⁇ B pathway polypeptide is increased, for example, by introducing into antigen-presenting cells a nucleic acid construct that expresses a nucleotide sequence encoding the NF- ⁇ B pathway polypeptide in the antigen-presenting cells.
  • nucleic acid constructs are typically in the form of vectors that are capable of being expressed in the desired subject host cell. Promoter, enhancer, stress or chemically-regulated promoters, antibiotic-sensitive or nutrient-sensitive regions may be included as required.
  • Non-limiting SHP-I inhibitors include pentavalent antimonial compounds (e.g., antimony dextran glucoside, antimony mannan, ethyl stibanime, ureastibamine, sodium stibogluconate, and glucantime) as described for example in U.S. Patent Application Publication No.
  • pentavalent antimonial compounds e.g., antimony dextran glucoside, antimony mannan, ethyl stibanime, ureastibamine, sodium stibogluconate, and glucantime
  • vanadate salts and complexes e.g., sodium ortho vanadate
  • dephosphatin dnacin Al, dnacin A2, STI-571
  • suramin gallium nitrate, meglumine antimonate
  • 2-(2-mercaptoethanol)-3-methyl-l,4- naphthoquinone 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime
  • DB289 Immtech
  • 2,5-bis(4-amidinophenyl)furan DB75, Immtech
  • SHP-I antisense compounds suitable for this use are known in the art (see, e.g., U.S. Patent Publication No. 2003/0232749).
  • Other antisense compounds that SHP-I activity can be identified using standard techniques.
  • accessible regions of the target SHP-I mRNA can be predicted using an RNA secondary structure folding program such as MFOLD (M. Zuker, D. H. Mathews & D. H. Turner, "Algorithms and Thermodynamics for RNA Secondary Structure Prediction: A Practical Guide. In: RNA Biochemistry and Biotechnology," J. Barciszewski & B. F. C. Clark, eds., NATO ASI Series, Kluwer Academic Publishers, (1999)).
  • Sub-optimal folds with a free energy value within 5% of the predicted most stable fold of the mRNA are predicted using a window of 200 bases within which a residue can find a complimentary base to form a base pair bond. Open regions that do not form a base pair are summed together with each suboptimal fold and areas that are predicted as open are considered more accessible to the binding to antisense nucleobase oligomers.
  • Other methods for antisense design are described, for example, in U.S. Pat. No. 6,472,521 ; Antisense Nucleic Acid Drug Dev. 7:439-444, 1997; Nucleic Acids Res. 28:2597-2604, 2000; and Nucleic Acids Res. 31 :4989-4994, 2003.
  • the biological activity of SHP-I can be reduced through the use of RNA interference (RNAi), employing, e.g., a double stranded RNA (dsRNA) or small interfering RNA (siRNA) directed to SHP-I (see, e.g., U.S. Patent Publication No. 2003/0232749).
  • RNA interference RNA interference
  • dsRNA double stranded RNA
  • siRNA small interfering RNA directed to SHP-I
  • Methods for designing such interfering RNAs are known in the art.
  • software for designing interfering RNA is available from Oligoengine (Seattle, Wash.).
  • the biological activity of SHP-I is reduced through the use of ribozymes.
  • Ribozymes which may be encoded in the genomes of the viruses or virus-like particles are described in Cech and Herschlag "Site-specific cleavage of single stranded DNA" U.S. Pat. No. 5,180,818; Altaian et al. "Cleavage of targeted RNA by RNAse P" U.S. Pat. No. 5,168,053, Cantin et al. "Ribozyme cleavage of HIV-I RNA” U.S. Pat. No. 5,149,796; Cech et al. "RNA ribozyme restriction endoribonucleases and methods", U.S. Pat. No. 5,1 16,742; Been et al.
  • RNA ribozyme polymerases, dephosphorylases, restriction endonucleases and methods U.S. Pat. No. 5,093,246; and Been et al. "RNA ribozyme polymerases, dephosphorylases, restriction endoribonucleases and methods; cleaves single-stranded RNA at specific site by transesterification", U.S. Pat. No. 4,987,071.
  • engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of target sequences.
  • J NF- KB restoration agents may be administered directly to the subject or contacted ex vivo with antigen-presenting cells before administering the treated cells to the subject, as described in more detail below.
  • an immune modulator selected from at least one NF- ⁇ B restoration agent as broadly described in Section 4.1 and an antigen- presenting cell with operable NF- ⁇ B function is administered concurrently with an antigen that corresponds to at least a portion of a target antigen that associates with the undesirable or deleterious immune response, for inducing a tolerogenic immune response to that target antigen.
  • compositions for modulating an immune response especially an undesirable or deleterious immune response associated with defective NF- ⁇ B activation in a subject
  • the compositions generally comprise an immune modulator that increases the number of operable NF- ⁇ B antigen-presenting cells as defined herein and an antigen that corresponds to at least a portion of a target antigen associated with the undesirable or deleterious immune response.
  • Illustrative target antigens include alloantigens and self antigens or peptide fragments thereof, which are presented in the context of MHC, as well as soluble proteins and fragments of insoluble complexes, particulate antigens, e.g., bacteria or parasites, and allergens.
  • exemplary antigens which are useful in the practice of the present invention include, but are not limited to, self antigens that are targets of autoimmune responses, allergens and transplantation antigens.
  • self antigens include, but are not restricted to, lupus autoantigen, Smith, Ro, La, Ul- RNP, fibrillin (scleroderma); proinsulin, insulin, IA2 and GAD65 in diabetes; collagen type II, HC gp39, dnaJpl , citrullinated proteins and peptides e.g.
  • citrullinated type II collagen, vimentin or fibrinogen in rheumatoid arthritis citrullinated type II collagen, vimentin or fibrinogen in rheumatoid arthritis
  • myelin basic protein and MOG in multiple sclerosis
  • gliadin in celiac disease
  • histones, PLP collagen, gIucose-6- phosphate isomerase, thyroglobulin, various tRNA synthetases, acetylcholine receptor (AchR), proteinase-3, myeloperoxidase etc.
  • AchR acetylcholine receptor
  • allergens include, but are not limited to, FeI d 1 (i.e., the feline skin and salivary gland allergen of the domestic cat Felis domesticus, the amino acid sequence of which is disclosed International Publication WO 91/06571), Der p I, Der p II, Der fl or Der fll (i.e., the major protein allergens from the house dust mite dermatophagoides, the amino acid sequence of which is disclosed in International Publication WO 94/24281).
  • FeI d 1 i.e., the feline skin and salivary gland allergen of the domestic cat Felis domesticus, the amino acid sequence of which is disclosed International Publication WO 91/06571
  • Der p I, Der p II, Der fl or Der fll i.e., the major protein allergens from the house dust mite dermatophagoides, the amino acid sequence of which is disclosed in International Publication WO 94/24281).
  • allergens may be derived, for example from the following: grass, tree and weed (including ragweed) pollens; fungi and moulds; foods such as fish, shellfish, crab, lobster, peanuts, nuts, wheat gluten, eggs and milk; stinging insects such as bee, wasp, and hornet and the chirnomidae (non-biting midges); other insects such as the housefly, fruit fly, sheep blow fly, screw worm fly, grain weevil, silkworm, honeybee, non-biting midge larvae, bee moth larvae, mealworm, cockroach and larvae of Tenibrio molitor beetle; spiders and mites, including the house dust mite; allergens found in the dander, urine, saliva, blood or other bodily fluid of mammals such as cat, dog, cow, pig, sheep, horse, rabbit, rat, guinea pig, mouse and gerbil; airborne particulates in general; latex; and protein detergent additives.
  • the antigen(s) may be isolated from a natural source or may be prepared by recombinant techniques as is known in the art.
  • peptide antigens can be eluted from the MHC and other presenting molecules of antigen- presenting cells obtained from a cell population or tissue for which a modified immune response is desired, e.g., an allogeneic tissue or cell population in transplantation medicine.
  • the eluted peptides can be purified using standard protein purification techniques known in the art (Rawson et al., 2000, Cancer Res 60(16), 4493-4498). If desired, the purified peptides can be sequenced and synthetic versions of the peptides produced using standard protein synthesis techniques as for example described below.
  • crude antigen preparations can be produced by isolating a sample of a cell population or tissue for which a modified immune response is desired, and either lysing the sample or subjecting the sample to conditions that will lead to the formation of apoptotic cells ⁇ e.g., irradiation with ultra violet or with gamma rays, viral infection, cytokines or by depriving cells of nutrients in the cell culture medium, incubation with hydrogen peroxide, or with drugs such as dexamethasone, ceramide chemotherapeutics and anti-hormonal agents such as Lupron or Tamoxifen).
  • the lysate or the apoptotic cells can then be used as a source of crude antigen for contact with the antigen- presenting cells.
  • the antigen When the antigen is known, it may be conveniently prepared in recombinant form using standard protocols as for example described in: Sambrook, et al, MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbor Press, 1989), in particular Sections 16 and 17; Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons, Inc. 1994-1998), in particular Chapters 10 and 16; and Coligan et al, CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5 and 6.
  • an antigen may be prepared by a procedure including the steps of (a) providing an expression vector from which the target antigen or analogue or mimetic thereof is expressible; (b) introducing the vector into a suitable host cell; (c) culturing the host cell to express recombinant polypeptide from the vector; and (d) isolating the recombinant polypeptide.
  • the antigen can be synthesized using solution synthesis or solid phase synthesis as described, for example, by Atherton and Sheppard (Solid Phase Peptide Synthesis: A Practical Approach, IRL Press at Oxford University Press, Oxford, England, 1989) or by Roberge et al. (1995, Science 269: 202).
  • the antigen is in the form of one or more peptides.
  • such peptides are at least 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 20, 25, 30 amino acid residues in length and suitably no more than about 500, 200, 100, 80, 60, 50, 40 amino acid residues in length.
  • the peptides can be in the form of a plurality of contiguous overlapping peptides whose sequences span at least a portion of a target antigen.
  • the peptide sequences are derived from at least about 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of the sequence corresponding to the target antigen.
  • each peptide of the plurality of contiguous overlapping peptide fragments can be 30-90 amino acids in length, e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 73, 75, 80, 81, 85, 86 and 90 amino acids in length.
  • the amino acid sequences of contiguous overlapping peptide fragments in the plurality overlap by about 10 to about 15 amino acids, e.g., 10, 11, 12, 13, 14 and 15 amino acids.
  • Exemplary methods for producing such peptide antigens are described, for example, by Astori et al. (2000 J. Immunol. 165, 3497-3505; and references cited therein) and in U.S. Pat. Appl. Pub. No. 2004/0241 178.
  • the antigen may be suitably modified, for example, by lipid modification to modify its physico-chemical properties.
  • At least one NF- ⁇ B restoration agent according to Section 4.1 and optionally at least one antigen according to Section 4.2 are provided in particulate form.
  • both an NF- ⁇ B restoration agent and an antigen are employed, they may be contained in or otherwise associated with the same particle or different particles.
  • a variety of particles may be used in the invention, including but not limited to, liposomes, micelles, lipidic particles, ceramic/inorganic particles and polymeric particles, and are typically selected from nanoparticles and microparticles.
  • the particles are suitably sized for phagocytosis or endocytosis by antigen-presenting cells.
  • Antigen-presenting cells include both professional and facultative types of antigen-presenting cells.
  • Professional antigen-presenting cells include, but are not limited to, macrophages, monocytes, B lymphocytes, cells of myeloid lineage, including monocytic-granulocytic-DC precursors, marginal zone Kupffer cells, microglia, T cells, Langerhans cells and dendritic cells including interdigitating dendritic cells and follicular dendritic cells.
  • facultative antigen-presenting cells include but are not limited to activated T cells, astrocytes, follicular cells, endothelium and fibroblasts.
  • the antigen-presenting cell is selected from monocytes, macrophages, B-lymphocytes, cells of myeloid lineage, dendritic cells or Langerhans cells.
  • the antigen-presenting cell expresses CDl Ic and includes a dendritic cell.
  • the particles have a dimension of less than about 100 ⁇ m, more suitably in the range of less than or equal to about 500 nm, although the particles may be as large as about 10 ⁇ m, and as small as a few nm.
  • Liposomes consist basically of a phospholipid bilayer forming a shell around an aqueous core.
  • Advantages include the lipophilicity of the outer layers which "mimic" the outer membrane layers of cells and that they are taken up relatively easily by a variety of cells.
  • Polymeric vehicles typically consist of micro/nanospheres and micro/nanocapsules formed of biocompatible polymers, which are either biodegradable (for example, polylactic acid) or non-biodegradable (for example, ethylenevinyl acetate).
  • biocompatible polymers which are either biodegradable (for example, polylactic acid) or non-biodegradable (for example, ethylenevinyl acetate).
  • the particles comprise an antigen-binding molecule on their surface, which is immuno-interactive with a marker that is expressed at higher levels on antigen-presenting cells (e.g., dendritic cells) than on non-antigen- presenting cells.
  • antigen-presenting cells e.g., dendritic cells
  • markers of this type include MGL, DCL-I, DEC-205, macrophage mannose R, DC-SIGN or other DC or myeloid specific (lectin) receptors, as for example disclosed by Hawiger et al. (2001, J Exp Med 194, 769), Kato et al. 2003, J Biol Chem 278, 34035), Benito et al.
  • the particles can be prepared from a combination of the bioactive agent(s), and a surfactant, excipient or polymeric material.
  • the particles are biodegradable and biocompatible, and optionally are capable of biodegrading at a controlled rate for delivery of a therapeutic or diagnostic agent.
  • the particles can be made of a variety of materials. Both inorganic and organic materials can be used. Polymeric and non-polymeric materials, such as fatty acids, may be used. Other suitable materials include, but are not limited to, gelatin, polyethylene glycol, trehalulose, dextran and chitosan. Particles with degradation and release times ranging from seconds to months can be designed and fabricated, based on factors such as the particle material.
  • Polymeric particles may be formed from any biocompatible and desirably biodegradable polymer, copolymer, or blend.
  • the polymers may be tailored to optimize different characteristics of the particle including: i) interactions between the bioactive agents to be delivered and the polymer to provide stabilization of the bioactive agents and retention of activity upon delivery; ii) rate of polymer degradation and, thereby, rate of agent release profiles; iii) surface characteristics and targeting capabilities via chemical modification; and iv) particle porosity.
  • polyanhydrides such as poly[(p-carboxyphenoxy)- hexane anhydride] (PCPH) may be used.
  • PCPH poly[(p-carboxyphenoxy)- hexane anhydride]
  • Biodegradable polyanhydrides are described in U.S. Pat. No. 4,857,311.
  • bulk eroding polymers such as those based on polyesters including poly(hydroxy acids) or poly(esters) can be used.
  • polyglycolic acid (PGA), polylactic acid (PLA), or copolymers thereof may be used to form the particles.
  • the polyester may also have a charged or functionalizable group, such as an amino acid.
  • particles with controlled release properties can be formed of poly(D,L-lactic acid) and/or poly(D,L-lactic-co-glycolic acid) ("PLGA”) which incorporate a surfactant such as DPPC.
  • polymers include poly(alkylcyanoacrylates), polyamides, polycarbonates, polyalkylenes such as polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly vinyl compounds such as polyvinyl alcohols, polyvinyl ethers, and polyvinyl esters, polymers of acrylic and methacrylic acids, celluloses and other polysaccharides, and peptides or proteins, or copolymers or blends thereof. Polymers may be selected with or modified to have the appropriate stability and degradation rates in vivo for different controlled drug delivery applications.
  • particles are formed from functionalized polyester graft copolymers, as described in Hrkach et al. (1995, Macromolecules, 28:4736-4739; and "Poly(L-Lactic acid-co-amino acid) Graft Copolymers: A Class of Functional Biodegradable Biomaterials" in Hydrogels and Biodegradable Polymers for Bioapplications, ACS Symposium Series No. 627, Raphael M. Ottenbrite et al., Eds., American Chemical Society, Chapter 8, pp. 93-101, 1996.)
  • Materials other than biodegradable polymers may be used to form the particles. Suitable materials include various non-biodegradable polymers and various excipients. The particles also may be formed of the bioactive agent(s) and surfactant alone.
  • Polymeric particles may be prepared using single and double emulsion solvent evaporation, spray drying, solvent extraction, solvent evaporation, phase separation, simple and complex coacervation, interfacial polymerization, and other methods well known to those of ordinary skill in the art. Particles may be made using methods for making microspheres or microcapsules known in the art, provided that the conditions are optimized for forming particles with the desired diameter.
  • the polymer is dissolved in a volatile organic solvent, such as methylene chloride.
  • a volatile organic solvent such as methylene chloride.
  • the bioactive agent(s) either in soluble form or dispersed as fine particles, is (are) added to the polymer solution, and the mixture is suspended in an aqueous phase that contains a surface-active agent such as polyvinyl alcohol).
  • the aqueous phase may be, for example, a concentration of 1% poly(vinyl alcohol) w/v in distilled water.
  • microspheres with different sizes (between 1 and 1000 ⁇ m) and morphologies can be obtained by this method.
  • Solvent removal was primarily designed for use with less stable polymers, such as the polyanhydrides.
  • the agent is dispersed or dissolved in a solution of a selected polymer in a volatile organic solvent like methylene chloride.
  • the mixture is then suspended in oil, such as silicon oil, by stirring, to form an emulsion.
  • oil such as silicon oil
  • the solvent diffuses into the oil phase and the emulsion droplets harden into solid polymer microspheres.
  • this method can be used to make microspheres from polymers with high melting points and a wide range of molecular weights. Microspheres having a diameter for example between one and 300 microns can be obtained with this procedure.
  • polymeric particles prepared using a single or double emulsion technique vary in size depending on the size of the droplets. If droplets in water-in-oil emulsions are not of a suitably small size to form particles with the desired size range, smaller droplets can be prepared, for example, by sonication or homogenation of the emulsion, or by the addition of surfactants. [0190] If the particles prepared by any of the above methods have a size range outside of the desired range, particles can be sized, for example, using a sieve, and further separated according to density using techniques known to those of skill in the art.
  • the polymeric particles can be prepared by spray drying.
  • Methods of spray drying such as that disclosed in PCT WO 96/09814 by Sutton and Johnson, disclose the preparation of smooth, spherical microparticles of a water-soluble material with at least 90% of the particles possessing a mean size between 1 and 10 ⁇ m.
  • Ceramic particles may also be used to deliver the bioactive agents of the invention. These particles are typically prepared using processes similar to the well known sol-gel process and usually require simple and room temperature conditions as described for example in Brinker et al. ("Sol-Gel Science: The Physics and Chemistry of Sol-Gel Processing;” Academic Press: San Diego, 1990, p-60), and Avnir et al. (1994, Chem. Mater. 6, 1605). Ceramic particles can be prepared with desired size, shape and porosity, and are extremely stable. These particles also effectively protect doped molecules (polypeptides, drugs etc.) against denaturation induced by extreme pH . and temperature (Jain et al, 1998, J. Am. Chem.
  • Publication WO 97/45367 discloses controllably dissolvable silica xerogels prepared via a sol-gel process, into which a biologically active agent is incorporated by impregnation into pre-sintered particles (1 to 500 ⁇ m) or disks.
  • International Publication WO 0050349 discloses controllably biodegradable silica fibres prepared via a sol-gel process, into which a biologically active agent is incorporated during synthesis of the fibre.
  • U.S. Pat. Appl. Pub. 20040180096 describes ceramic nanoparticles in which a bioactive substance is entrapped. The ceramic nanoparticles are made by formation of a micellar composition of the dye.
  • U.S. Pat. Appl. Pub. 2005012361 1 discloses controlled release ceramic particles comprising an active material substantially homogeneously dispersed throughout the particles. These particles are prepared by mixing a surfactant with an apolar solvent to prepare a reverse micelle solution; (b) dissolving a gel precursor, a catalyst, a condensing agent and a soluble active material in a polar solvent to prepare a precursor solution; (c) combining the reverse micelle solution and the precursor solution to provide an emulsion and (d) condensing the precursor in the emulsion.
  • 20060210634 discloses adsorbing bioactive substances onto ceramic particles comprising a metal oxide (e.g. , titanium oxide, zirconium oxide, scandium oxide, cerium oxide and yttrium oxide) by evaporation.
  • a metal oxide e.g. , titanium oxide, zirconium oxide, scandium oxide, cerium oxide and yttrium oxide
  • Kortesuo et al. disclose a spray drying method to produce spherical silica gel particles with a narrow particle size range for controlled delivery of drugs such as toremifene citrate and dexmedetomidine HCl.
  • Wang et al. 2006, Int J Pharm. 308(1- 2): 160- 167) describe the combination of adsorption by porous CaCO 3 microparticles and encapsulation by polyelectrolyte multilayer films for delivery of bioactive substances.
  • Liposomes can be produced by standard methods such as those reported by Kim et al. (1983, Biochim. Biophys. Acta 728, 339-348); Liu et al. (1992, Biochim. Biophys. Acta 1 104, 95-101); Lee et al. (1992, Biochim. Biophys. Acta, ⁇ 103, 185-197), Brey et al. (U.S. Pat. Appl. Pub. 20020041861), Hass et al. (U.S. Pat. Appl. Pub. 20050232984), Kisak et al. (U.S. Pat. Appl. Pub.
  • the lipids of choice (and any organic-soluble bioactive), dissolved in an organic solvent, are mixed and dried onto the bottom of a glass tube under vacuum.
  • the lipid film is rehydrated using an aqueous buffered solution containing any water-soluble bioactives to be encapsulated by gentle swirling.
  • the hydrated lipid vesicles can then be further processed by extrusion, submitted to a series of freeze-thawing cycles or dehydrated and then rehydrated to promote encapsulation of bioactives.
  • Liposomes can then be washed by centrifugation or loaded onto a size- exclusion column to remove unentrapped bioactive from the liposome formulation and stored at 4° C.
  • the basic method for liposome preparation is described in more detail in Thierry et al. (1992, Nuc. Acids Res. 20:5691-5698).
  • a particle carrying a payload of bioactive agent(s) can be made using the procedure as described in: Pautot et al. (2003, Proc. Natl. Acad. Sci. USA, 100(19): 10718-21).
  • Pautot et al. Using the Pautot et al. technique, streptavidin-coated lipids (DPPC, DSPC, and similar lipids) can be used to manufacture liposomes.
  • the drug encapsulation technique described by Needham et al (2001, Advanced Drug Delivery Reviews, 53(3): 285-305) can be used to load these vesicles with one or more active agents.
  • the liposomes can be prepared by exposing chloroformic solution of various lipid mixtures to high vacuum and subsequently hydrating the resulting lipid films (DSPC/CHOL) with pH 4 buffers, and extruding them through polycarbonated filters, after a freezing and thawing procedure. It is possible to use DPPC supplemented with DSPC or cholesterol to increase encapsulation efficiency or increase stability, etc.
  • a transmembrane pH gradient is created by adjusting the pH of the extravesicular medium to 7.5 by addition of an alkalinization agent.
  • a bioactive agent ⁇ e.g., small molecule NF- ⁇ B restoration agents, which are, for example, weak bases
  • a bioactive agent can be subsequently entrapped by addition of a solution of the bioactive agent in small aliquots to the vesicle solution, at an elevated temperature, to allow accumulation of the bioactive agent inside the liposomes.
  • lipid-based particles suitable for the delivery of the bioactive agents of the present invention such as niosomes are described by Copeland et al. (2005, Immunol. Cell Biol. 83: 95-105). 4.3.4 Ballistic particles
  • the bioactive agents of the present invention may be attached to ⁇ e.g., by coating or conjugation) or otherwise associated with particles suitable for use in needleless or "ballistic" (biolistic) delivery.
  • particles suitable for use in needleless or "ballistic" (biolistic) delivery are described, for example, in: International Publications WO 02/101412; WO 02/100380; WO 02/43774; WO 02/19989; WO 01/93829; WO 01/83528; WO 00/63385; WO 00/26385; WO 00/19982; WO 99/01 168; WO 98/10750; and WO 97/48485. It shall be understood, however, that such particles are not limited to their use with a ballistic delivery device and can otherwise be administered by any alternative technique ⁇ e.g., injection or microneedle delivery) through which particles are deliverable to immune cells.
  • the bioactive agents can be coated or chemically coupled to carrier particles ⁇ e.g., core carriers) using a variety of techniques known in the art.
  • Carrier particles are selected from materials which have a suitable density in the range of particle sizes typically used for intracellular delivery. The optimum carrier particle size will, of course, depend on the diameter of the target cells. Illustrative particles have a size ranging from about 0.01 to about 250 ⁇ m, from about 10 to about 150 ⁇ m, and from about 20 to about 60 ⁇ m; and a particle density ranging from about 0.1 to about 25 g/cm 3 , and a bulk density of about 0.5 to about 3.0 g/cm 3 , or greater.
  • Non-limiting particles of this type include metal particles such as, tungsten, gold, platinum and iridium carrier particles. Tungsten particles are readily available in average sizes of 0.5 to 2.0 ⁇ m in diameter. Gold particles or microcrystalline gold ⁇ e.g., gold powder Al 570, available from Engelhard Corp., East Newark, N.J.) may also be used. Gold particles provide uniformity in size (available from Alpha Chemicals in particle sizes of 1-3 ⁇ m, or available from Degussa, South Plainfield, N.J. in a range of particle sizes including 0.95 ⁇ m) and low toxicity. Microcrystalline gold provides a diverse particle size distribution, typically in the range of 0.1-5 ⁇ m.
  • microcrystalline gold provides for highly efficient coating with the active agents of the present invention.
  • bioactive molecules e.g., hydrophilic molecules such as proteins and nucleic acids
  • methods combine a predetermined amount of gold or tungsten with the bioactive molecules, CaCl 2 and spermidine.
  • ethanol is used to precipitate the bioactive molecules onto gold or tungsten particles (see, for example, Jumar et ai, 2004, Phys Med. Biol. 49:3603-3612).
  • the resulting solution is suitably vortexed continually during the coating procedure to ensure uniformity of the reaction mixture.
  • the particles can be transferred for example to suitable membranes and allowed to dry prior to use, coated onto surfaces of a sample module or cassette, or loaded into a delivery cassette for use in particular particle-mediated delivery instruments.
  • compositions may suitably be prepared as particles using standard techniques, such as by simple evaporation (air drying), vacuum drying, spray drying, freeze drying (lyophilization), spray-freeze drying, spray coating, precipitation, supercritical fluid particle formation, and the like. If desired, the resultant particles can be dandified using the techniques described in International Publication WO 97/48485.
  • Surfactants which can be incorporated into particles include phosphoglycerides.
  • Exemplary phosphoglycerides include phosphatidylcholines, such as the naturally occurring surfactant, L- ⁇ -phosphatidylcholine dipalmitoyl ("DPPC").
  • DPPC L- ⁇ -phosphatidylcholine dipalmitoyl
  • the surfactants advantageously improve surface properties by, for example, reducing particle-particle interactions, and can render the surface of the particles less adhesive.
  • the use of surfactants endogenous to the lung may avoid the need for the use of non- physiologic surfactants.
  • Providing a surfactant on the surfaces of the particles can reduce the tendency of the particles to agglomerate due to interactions such as electrostatic interactions, Van der Waals forces, and capillary action.
  • the presence of the surfactant on the particle surface can provide increased surface rugosity (roughness), thereby improving aerosolization by reducing the surface area available for intimate particle- particle interaction.
  • Surfactants known in the art can be used including any naturally occurring surfactant.
  • Other exemplary surfactants include diphosphatidyl glycerol (DPPG); hexadecanol; fatty alcohols such as polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surface active fatty acid, such as palmitic acid or oleic acid; sorbitan trioleate (Span 85); glycocholate; surfactin; a poloxamer; a sorbitan fatty acid ester such as sorbitan trioleate; tyloxapol and a phospholipid.
  • DPPG diphosphatidyl glycerol
  • hexadecanol fatty alcohols such as polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether
  • a surface active fatty acid such as palmitic acid or oleic acid
  • sorbitan trioleate Span 85
  • glycocholate surfactin
  • surfactin
  • the immune modulator that is used to increase the number of operable NF- ⁇ B antigen-presenting cells in the subject is an antigen- presenting cell or its precursor, which is obtained from the subject to be treated (i.e., an autologous antigen-presenting cell).
  • the antigen-presenting cell is contacted with at least one NF- ⁇ B restoration agent as described for example in Section 4.1, which is suitably in soluble form or in particulate form as described for example in Section 4.3, in an amount and for a time sufficient to restore NF- ⁇ B activity in the antigen-presenting cell to a basal state.
  • the immune modulator is an antigen-presenting cell that is derived from a donor that is MHC matched or mismatched with the subject (i.e., an allogeneic antigen-presenting cell).
  • the donor is histocompatible with the subject.
  • the antigen-presenting cell or its precursor is also contacted with an antigen according to Section 4.2, or with a polynucleotide from which the antigen is expressible, for a time and under conditions sufficient for the antigen or a processed form thereof to be presented by the antigen- presenting cell.
  • the antigen is in soluble form or in particulate form as described for example in Section 4.3.
  • Antigen-presenting cells or their precursors can be isolated by methods known to those of skill in the art. The source of such cells will differ depending upon the antigen-presenting cell required for modulating a specified immune response.
  • the antigen-presenting cell can be selected from dendritic cells, macrophages, monocytes and other cells of myeloid lineage.
  • precursors of antigen-presenting cells can be isolated from any tissue, but are most easily isolated from blood, cord blood or bone marrow (Sorg et al, 2001, Exp Hematol 29, 1289-1294; Zheng et al, 2000, J Hematother Stem Cell Res 9, 453-464). It is also possible to obtain suitable precursors from diseased tissues such as rheumatoid synovial tissue or fluid following biopsy or joint tap (Thomas et al, 1994a, J Immunol 153, 4016-4028; Thomas et al, 1994b, Arthritis Rheum 37(4)).
  • liver, spleen, heart, kidney, gut and tonsil Li et al, 1994, J Exp Med 179, 1823-1834; Mcllroy et al , 2001, Blood 97, 3470-3477; Vremec et al, 2000, J Immunol 159, 565-573; Hart and Fabre, 1981, J Exp Med 154(2), 347-361 ; Hart and McKenzie, 1988, J Exp Med 168(1), 157-170; Pavli et al, 1990, Immunology 70( 1 ), 40-47).
  • Leukocytes isolated directly from tissue provide a major source of antigen-presenting cell precursors. Typically, these precursors can only differentiate into antigen-presenting cells by culturing in the presence or absence of various growth factors. According to the practice of the present invention, the antigen-presenting cells may be so differentiated from crude mixtures or from partially or substantially purified preparations of precursors. Leukocytes can be conveniently purified from blood or bone marrow by density gradient centrifugation using, for example, Ficoll Hypaque which eliminates neutrophils and red cells (peripheral blood mononuclear cells or PBMCs), or by ammonium chloride lysis of red cells (leukocytes or white blood cells).
  • Ficoll Hypaque which eliminates neutrophils and red cells (peripheral blood mononuclear cells or PBMCs)
  • ammonium chloride lysis of red cells leukocytes or white blood cells.
  • antigen-presenting cells are present in peripheral blood as non- proliferating monocytes, which can be differentiated into specific antigen-presenting cells, including macrophages and dendritic cells, by culturing in the presence of specific cytokines.
  • Tissue-derived precursors such as precursors of tissue dendritic cells or of Langerhans cells are typically obtained by mincing tissue (e.g. , basal layer of epidermis) and digesting it with collagenase or dispase followed by density gradient separation, or selection of precursors based on their expression of cell surface markers.
  • tissue-derived precursors such as precursors of tissue dendritic cells or of Langerhans cells are typically obtained by mincing tissue (e.g. , basal layer of epidermis) and digesting it with collagenase or dispase followed by density gradient separation, or selection of precursors based on their expression of cell surface markers.
  • Langerhans cell precursors express CDl molecules as well as HLA-DR and can be purified on this basis.
  • the antigen-presenting cell precursor is a precursor of macrophages.
  • these precursors can be obtained from monocytes of any source and can be differentiated into macrophages by prolonged incubation in the presence of medium and macrophage colony stimulating factor (M-CSF) (Erickson- Miller et al, 1990, Int J Cell Cloning 8, 346-356; Metcalf and Burgess, 1982, J Cell Physiol, 111, 275-283).
  • M-CSF medium and macrophage colony stimulating factor
  • the antigen presenting cell precursor is a precursor of Langerhans cells.
  • Langerhans cells can be generated from human monocytes or CD34 + bone marrow precursors in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-4/TNF ⁇ and TGF ⁇ (Geissmann et al, 1998, J Exp Med, 187, 961-966; Strobl et al, 1997a, Blood 90, 1425- 1434; Strobl et al, 1997b, dv Exp Med Biol 417, 161-165; Strobl et al, 1996, J Immunol 157, 1499-1507).
  • GM-CSF granulocyte/macrophage colony-stimulating factor
  • IL-4/TNF ⁇ IL-4/TNF ⁇
  • TGF ⁇ granulocyte/macrophage colony-stimulating factor
  • the antigen-presenting cell precursor is a precursor of dendritic cells.
  • dendritic cell precursors can be obtained from peripheral blood, cord blood or bone marrow. These include monocytes, CD34 + stem cells, granulocytes, CD33 + CD1 Ic + DC precursors, and committed myeloid progenitors - described below.
  • Monocytes CD34 + stem cells, granulocytes, CD33 + CD1 Ic + DC precursors, and committed myeloid progenitors - described below.
  • Monocytes can be purified by adherence to plastic for 1-2 h in the presence of tissue culture medium ⁇ e.g., RPMI) and serum ⁇ e.g., human or foetal calf serum), or in serum- free medium (Anton et al, 1998, Scand J Immunol 47, 116-121; Araki et al, 2001, Br J Haematol 1 14, 681-689; Mackensen et al, 2000, Int J Cancer 86, 385-392; Nestle et al, 1998, Nat Med 4, 328-332; Romani et al, 1996, J Immunol Meth 196, 137-151; Thurner et al, 1999, J Immunol Methods 223, 1-15).
  • tissue culture medium ⁇ e.g., RPMI
  • serum e.g., human or foetal calf serum
  • Monocytes can also be elutriated from peripheral blood (Garderet et al, 2001, J Hematother Stem Cell Res 10, 553-567). Monocytes can also be purified by immunoaffinity techniques, including immunomagnetic selection, flow cytometric sorting or panning (Araki et al , 2001 , supra; Battye and Shortman, 1991 , Curr. Opin. Immunol. 3, 238-241 ), with anti- CD 14 antibodies to obtain CD14hi cells.
  • immunoaffinity techniques including immunomagnetic selection, flow cytometric sorting or panning (Araki et al , 2001 , supra; Battye and Shortman, 1991 , Curr. Opin. Immunol. 3, 238-241 ), with anti- CD 14 antibodies to obtain CD14hi cells.
  • the numbers (and therefore yield) of circulating monocytes can be enhanced by the in vivo use of various cytokines including GM-CSF (Groopman et al, 1987, N Engl J Med 317, 593-598; Hill et al, 1995, J Leukoc Biol 58, 634-642).
  • Monocytes can be differentiated into dendritic cells by prolonged incubation in the presence of GM-CSF and IL-4 (Romani et al , 1994, J Exp Med 180, 83-93; Romani et al, 1996, supra).
  • a combination of GM-CSF and IL-4 at a concentration of each at between about 200 to about 2000 U/mL, more preferably between about 500 to about 1000 U/mL and even more preferably between about 800 U/mL (GM-CSF) and 1000 U/mL (IL-4) produces significant quantities of immature dendritic cells, i.e., antigen-capturing phagocytic dendritic cells.
  • Other cytokines which promote differentiation of monocytes into antigen-capturing phagocytic dendritic cells include, for example, IL- 13.
  • CD34+ stem cells include, for example, IL- 13.
  • Dendritic cells can also be generated from CD34 + bone marrow derived precursors in the presence of GM-CSF, TNF ⁇ ⁇ stem cell factor (SCF, c-kitL), or GM-CSF, IL-4 ⁇ flt3L (Bai et al, 2002, Int J Oncol 20, 247-53; Chen et al, 2001, Clin Immunol 98, 280-292; Loudovaris et al, 2001, J Hematother Stem Cell Res 10, 569-578).
  • SCF TNF ⁇ ⁇ stem cell factor
  • c-kitL TNF ⁇ ⁇ stem cell factor
  • IL-4 ⁇ flt3L IL-4 ⁇ flt3L
  • CD34 + cells can be derived from a bone marrow aspirate or from blood and can be enriched as for monocytes using, for example, immunomagnetic selection or immunocolumns (Davis et al, 1994, J Immunol Meth 175, 247-257).
  • the proportion of CD34 + cells in blood can be enhanced by the in vivo use of various cytokines including (most commonly) G-CSF, but also flt3L and progenipoietin (Fleming et al, 2001, Exp Hematol 29, 943-951 ; Pulendran et al, 2000, J Immunol 165, 566-572; Robinson et al, 2000, J Hematother Stem Cell Res 9, 71 1-720).
  • various cytokines including (most commonly) G-CSF, but also flt3L and progenipoietin (Fleming et al, 2001, Exp Hematol 29, 943-951 ; Pulendran et al, 2000, J Immunol 165, 566-572; Robinson et al, 2000, J Hematother Stem Cell Res 9, 71 1-720).
  • DC can be generated from committed early myeloid progenitors in a similar fashion to CD34+ stem cells, in the presence of GM-CSF and IL-4/TNF.
  • myeloid precursors infiltrate many tissues in inflammation, including rheumatoid arthritis synovial fluid (Santiago-Schwarz et al, 2001, J Immunol. 167, 1758-1768).
  • Expansion of total body myeloid cells including circulating dendritic cell precursors and monocytes can be achieved with certain cytokines, including flt-3 ligand, granulocyte colony-stimulating factor (G-CSF) or progenipoietin (pro-GP) (Fleming et al, 2001, supra; Pulendran et al, 2000, supra; Robinson et al, 2000, supra). Administration of such cytokines for several days to a human or other mammal would enable much larger numbers of precursors to be derived from peripheral blood or bone marrow for in vitro manipulation.
  • cytokines including flt-3 ligand, granulocyte colony-stimulating factor (G-CSF) or progenipoietin (pro-GP)
  • Dendritic cells can also be generated from peripheral blood neutrophil precursors in the presence of GM-CSF, IL-4 and TNF ⁇ (Kelly et al , 2001 , Cell MoI Biol (Noisy-le-grand) 47, 43-54; Oehlcr et al, 1998, J Exp Med. 187, 1019-1028). It should be noted that dendritic cells can also be generated, using similar methods, from acute myeloid leukaemia cells (Oehler et al, 2000, Ann Hematol. 79, 355-62).
  • Tissue DC precursors and other sources of APC precursors include thymic precursors in the presence of IL-3 +/- GM-CSF, and liver DC precursors in the presence of GM-CSF and a collagen matrix.
  • Transformed or immortalised dendritic cell lines may be produced using oncogenes such as v-myc as for example described by (Paglia et al, 1993) or by myb (Banyer and Hapel, 1999; Gonda et al, 1993).
  • a second subset which lacks CD 14, CD 19, CD56 and CD3, known as plasmacytoid dendritic cell precursors, does not express CDl Ic, but does express CD123 (IL-3R chain) and HLA-DR (Farkas et al, 2001, Am J Pathol 159, 237-243; Grouard et al, 1997, J Exp Med 185, 1101-1111; Rissoan et al, 1999, Science 283, 1183-1186). Most circulating CDl Ic + dendritic cell precursors are HLA-DR + , however some precursors may be HLA-DR-. The lack of MHC class II expression has been clearly demonstrated for peripheral blood dendritic cell precursors (del Hoyo et al , 2002, Nature 415, 1043-1047).
  • CD33 + CD14 /l0 or CDl Ic + HLA-DR + , lineage marker- negative dendritic cell precursors described above can be differentiated into more mature antigen-presenting cells by incubation for 18-36 h in culture medium or in monocyte conditioned medium (Thomas et al, 1993b, supra; Thomas and Lipsky, 1994, J Immunol 153, 4016-4028) (O'Doherty et al, 1993, supra).
  • peripheral blood dendritic cells are characterised by low density and so can be purified on density gradients, including metrizamide and Nycodenz (Freudenthal and Steinman, 1990, Proc Natl Acad Sci U S A 87, 7698-7702; Vremec and Shortman, 1997, J Immunol 159, 565- 573), or by specific monoclonal antibodies, such as but not limited to the CMRF-44 mAb (Fearnley et al, 1999, Blood 93, 728-736; Vuckovic et al, 1998, Exp Hematol 26, 1255-1264).
  • Plasmacytoid dendritic cells can be purified directly from peripheral blood on the basis of cell surface markers, and then incubated in the presence of IL-3 (Grouard et al, 1997, supra; Rissoan et al, 1999, supra).
  • plasmacytoid DC can be derived from density gradients or CMRF-44 selection of incubated peripheral blood cells as above.
  • cytokines such as TNF- ⁇ , IL-6, IFN- ⁇ , IL- 1 ⁇ , necrotic cells, re-adherence, whole bacteria, membrane components, RNA or polylC
  • immature dendritic cells will become activated (Clark, 2002, J Leukoc Biol, 71, 388-400;hacker et al., 2002, Immunology 105, 245-251; Kaisho and Akira, 2002, Biochim Biophys Acta 1589, 1-13; Koski et al, 2001, Crit Rev Immunol 21, 179- 189).
  • This process of dendritic cell activation is inhibited in the presence of NF- KB inhibitors (O'Sullivan and Thomas, 2002, J Immunol 168, 5491-5498).
  • NF- ⁇ B restoration agents can be delivered into antigen-presenting cells in various forms, including small molecules, nucleic acids and polypeptides.
  • the NF- ⁇ B restoration agent s may be soluble or particulate.
  • the NF- ⁇ B restoration agent is typically in the form of a nucleic acid construct from which a NF- ⁇ B restoration agent ⁇ e.g., a proteinaceous NF- ⁇ B restoration agent) is expressible.
  • the amount of soluble or particulate NF- ⁇ B restoration agent to be placed in contact with antigen-presenting cells can be determined empirically by routine methods known to persons of skill in the art.
  • antigen-presenting cells are incubated with NF- ⁇ B restoration agent for about 10 min to about 12 hr at 35° C - 38° C or for as much time as required to activate NF- ⁇ B or to restore NF- ⁇ B function to a basal state.
  • the amount of soluble or particulate antigen to be placed in contact with antigen-presenting cells can be determined empirically by routine methods known to persons of skill in the art.
  • antigen-presenting cells are incubated with antigen for about 1 to 6 hr at 37° C, although it is also possible to expose antigen- presenting cells to antigen for the duration of incubation with growth factors and NF- ⁇ B restoration agent.
  • 0.1-10 ⁇ g/mL is suitable for producing antigen-specific antigen-presenting cells.
  • Dendritic cells are exposed to apoptotic bodies in approximately 1 : 1 ratio, and bacteria (Albert et al. , 1998, International Publication WO 99/42564; Corinti et al, 1999, J Immunol 163(6), 3029-
  • the antigen should be exposed to the antigen-presenting cells for a period of time sufficient for those cells to internalize the antigen.
  • the time and dose of antigen necessary for the cells to internalize and present the processed antigen may be determined using pulse-chase protocols in which exposure to antigen is followed by a washout period and exposure to a read-out system e.g., antigen reactive T cells. Once the optimal time and dose necessary for cells to express processed antigen on their surface is determined, a protocol may be used to prepare cells and antigen for inducing tolerogenic responses.
  • an antigen-presenting cell may vary depending on the antigen or form of antigen employed, its dose, and the antigen- presenting cell employed, as well as the conditions under which antigen loading is undertaken. These parameters can be determined by the skilled artisan using routine procedures.
  • the delivery of exogenous antigen to an antigen-presenting cell can be enhanced by methods known to practitioners in the art. For example, several different strategies have been developed for delivery of exogenous antigen to the endogenous processing pathway of antigen-presenting cells, especially dendritic cells. These methods include insertion of antigen into pH-sensitive liposomes (Zhou and Huang, 1994, Immunomethods 4, 229-235), osmotic lysis of pinosomes after pinocytic uptake of soluble antigen (Moore et ai, 1988, Cell 54, 777-785), coupling of antigens to potent adjuvants (Aichele et ai, 1990, J. Exp.
  • Recombinant bacteria eg. E. coli
  • transfected host mammalian cells may be pulsed onto dendritic cells (as particulate antigen, or apoptotic bodies respectively) for antigen delivery.
  • dendritic cells as particulate antigen, or apoptotic bodies respectively
  • a delivery system might be logically combined with a substance for inhibiting NF- ⁇ B, such as a plasmid encoding dominant negative I ⁇ B ⁇ (Pai et ai, 2002, J Virol 76, 1914- 1921).
  • VLPs Recombinant chimeric virus-like particles
  • an antigen may be linked to, or otherwise associated with, a cytolysin to enhance the transfer of the antigen into the cytosol of an antigen-presenting cell of the invention for delivery to the MHC class I pathway.
  • cytolysins include saponin compounds such as saponin-containing Immune Stimulating Complexes (ISCOMs) (see e.g., Cox and Coulter, 1997, Vaccine 15(3), 248-256 and U.S. Patent No. 6,352,697), phospholipases (see, e.g., Camilli et al., 1991, J. Exp. Med.
  • pore-forming toxins e.g., an alpha-toxin
  • natural cytolysins of gram-positive bacteria such as listeriolysin O (LLO, e.g., Mengaud et al., 1988, Infect. Immun. 56, 766-772 and Portnoy et al., 1992, Infect. Immun. 60, 2710- 2717
  • LLO listeriolysin O
  • SLO streptolysin O
  • PFO perfringolysin O
  • cytolysins may be advantageously used.
  • listeriolysin exhibits greater pore-forming ability at mildly acidic pH (the pH conditions within the phagosome), thereby facilitating delivery of vacuole (including phagosome and endosome) contents to the cytoplasm (see, e.g., Portnoy et al., 1992, Infect. Immun. 60, 2710-2717).
  • the cytolysin may be provided together with a pre-selected antigen in the form of a single composition or may be provided as a separate composition, for contacting the antigen-presenting cells.
  • the cytolysin is fused or otherwise linked to the antigen, wherein the fusion or linkage permits the delivery of the antigen to the cytosol of the target cell.
  • the cytolysin and antigen are provided in the form of a delivery vehicle such as, but not limited to, a liposome or a microbial delivery vehicle selected from virus, bacterium, or yeast.
  • a delivery vehicle such as, but not limited to, a liposome or a microbial delivery vehicle selected from virus, bacterium, or yeast.
  • the delivery vehicle is non-virulent.
  • the delivery vehicle is a non- virulent bacterium, as for example described by Portnoy et al. in U.S. Patent No. 6,287,556, comprising a first polynucleotide encoding a non-secreted functional cytolysin operably linked to a regulatory polynucleotide which expresses the cytolysin in the bacterium, and a second polynucleotide encoding one or more pre-selected antigens.
  • Non-secreted cytolysins may be provided by various mechanisms, e.g., absence of a functional signal sequence, a secretion incompetent microbe, such as microbes having genetic lesions (e.g., a functional signal sequence mutation), or poisoned microbes, etc.
  • a secretion incompetent microbe such as microbes having genetic lesions (e.g., a functional signal sequence mutation), or poisoned microbes, etc.
  • a wide variety of nonvirulent, non-pathogenic bacteria may be used; preferred microbes are relatively well characterised strains, particularly laboratory strains of E. coli, such as MC4100, MC 1061 , DH5. alpha., etc.
  • the bacteria are attenuated to be non- replicative, non-integrative into the host cell genome, and/or non-motile inter- or intra- cellularly.
  • the delivery vehicles described above can be used to deliver one or more antigens to virtually any antigen-presenting cell capable of endocytosis of the subject vehicle, including phagocytic and non-phagocytic antigen-presenting cells.
  • the subject methods generally require microbial uptake by the target cell and subsequent lysis within the antigen- presenting cell vacuole (including phagosomes and endosomes).
  • a proteinaceous NF- ⁇ B restoration agent and optionally an antigen of interest can be produced inside an antigen-presenting cell by introduction of one or more expression constructs that encode the restoration agent and/or the antigen.
  • expression constructs that encode the restoration agent and/or the antigen.
  • the expression of natural or synthetic nucleic acids is typically achieved by operably linking a nucleic acid of interest to a promoter (which may be either constitutive or inducible), preferably incorporating the construct into an expression vector, and introducing the vector into a suitable host cell.
  • Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular nucleic acid.
  • the vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (e.g. , shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems.
  • Vectors may be suitable for replication and integration in prokaryotes, eukaryotes, or preferably both. See, Giliman and Smith (1979), Gene, 8: 81-97; Roberts et al. (1987), Nature, 328: 731-734; Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, volume 152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989), MOLECULAR CLONING - A
  • Expression vectors containing regulatory elements from eukaryotic viruses are typically used for expression of nucleic acid sequences in eukaryotic cells.
  • SV40 vectors include pSVT7 and pMT2.
  • Vectors derived from bovine papilloma virus include pBV- IMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5.
  • exemplary vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • viral expression vectors are useful for modifying eukaryotic cells because of the high efficiency with which the viral vectors transfect target cells and integrate into the target cell genome.
  • Illustrative expression vectors of this type can be derived from viral DNA sequences including, but not limited to, adenovirus, adeno-associated viruses, herpes- simplex viruses and retroviruses such as B, C, and D retroviruses as well as spumaviruses and modified lentiviruses.
  • Suitable expression vectors for transfection of animal cells are described, for example, by Wu and Ataai (2000, Curr. Opin.
  • Biotechnol. 1 1(2), 205-208 Vigna and Naldini (2000, J. Gene Med. 2(5), 308-316), Kay et al. (2001, Nat. Med. 7(1), 33-40), Athanasopoulos, et al. (2000, Int. J. MoI. Med. 6(4),363-375) and Walther and Stein (2000, Drugs 60(2), 249-271).
  • the antigen-encoding portion of the expression vector may comprise a naturally-occurring sequence or a variant thereof, which has been engineered using recombinant techniques.
  • the codon composition of an antigen-encoding polynucleotide is modified to permit enhanced expression of the antigen in a target cell or tissue of choice using methods as set forth in detail in International Publications WO 99/02694 and WO 00/42215. Briefly, these methods are based on the observation that translational efficiencies of different codons vary between different cells or tissues and that these differences can be exploited, together with codon composition of a gene, to regulate expression of a protein in a particular cell or tissue type.
  • the replacement step affects 5%, 10%, 15%, 20%, 25%, 30%, more preferably 35%, 40%, 50%, 60%, 70% or more of the existing codons of a parent polynucleotide.
  • the expression vector is compatible with the antigen-presenting cell in which it is introduced such that the antigen-encoding polynucleotide is expressible by the cell.
  • the expression vector is introduced into the antigen-presenting cell by any suitable means which will be dependent on the particular choice of expression vector and antigen-presenting cell employed. Such means of introduction are well-known to those skilled in the art.
  • introduction can be effected by use of contacting (e.g., in the case of viral vectors), electroporation, transformation, transduction, conjugation or triparental mating, transfection, infection membrane fusion with cationic lipids, high-velocity bombardment with DNA-coated microprojectiles, incubation with calcium phosphate-DNA precipitate, direct microinjection into single cells, and the like.
  • contacting e.g., in the case of viral vectors
  • electroporation transformation, transduction, conjugation or triparental mating
  • transfection infection membrane fusion with cationic lipids
  • high-velocity bombardment with DNA-coated microprojectiles incubation with calcium phosphate-DNA precipitate
  • direct microinjection into single cells and the like.
  • the vectors are introduced by means of cationic lipids, e.g., liposomes.
  • liposomes are commercially available (e.g., Lipofectin®, LipofectamineTM, and the like
  • At least one immune modulator that increases the number of antigen-presenting cells with operable NF- ⁇ B function is useful in compositions and methods for treating or preventing an undesirable or deleterious immune response in a subject, wherein the immune response is associated with defective NF- ⁇ B activation (i.e., reduced or abrogated NF- ⁇ B activation), including defective ReIB activation (i.e., reduced or abrogated NF- ⁇ B activation).
  • the immune modulator(s) is (are) suitably selected from: (i) NF- ⁇ B restoration agents as described for example in Section 4.1 ; and (ii) antigen-presenting cells with operable NF- ⁇ B function as described for example in Section 4.4, wherein the immune modulators are in soluble form or in particulate form as described for example in Section 4.3 and are optionally associated with one or more antigens as described for example in Section 4.2.
  • inventive compositions and methods are useful, therefore, for treating or preventing an undesirable or deleterious immune response associated with defective NF- ⁇ B activation including, for example, allergies, organ-specific diseases,, parasitic diseases, and some inflammatory and autoimmune diseases.
  • allergies include seasonal respiratory allergies; allergy to aeroallergens such as hay fever; allergy treatable by reducing serum IgE and eosinophilia; asthma; eczema; animal allergies, food allergies; chronic urticaria; latex allergies; allergic rhinitis; atopic dermatitis; or allergies treatable by allergic desensitization.
  • Autoimmune diseases and related conditions that can be treated or prevented by the present invention include, for example, psoriasis, systemic lupus erythematosus, myasthenia gravis, stiff-man syndrome, thyroiditis, Sydenham chorea, rheumatoid arthritis, ankylosing spondylitis, autoimmune aplastic anemia, autoimmune hemolytic anemia, Churg Strauss disease, scleroderma, Wegener granulomatosus, Wiskott Aldrich syndrome, type 1 diabetes mellitus (TlDM) and multiple sclerosis.
  • psoriasis systemic lupus erythematosus
  • myasthenia gravis stiff-man syndrome
  • thyroiditis Sydenham chorea
  • rheumatoid arthritis ankylosing spondylitis
  • autoimmune aplastic anemia autoimmune hemolytic anemia
  • Churg Strauss disease scleroderma
  • inflammatory disease examples include Crohn's disease, chronic inflammatory eye diseases, chronic inflammatory lung diseases and chronic inflammatory liver diseases, autoimmune hemolytic anemia, idiopathic leucopoenia, ulcerative colitis, dermatomyositis, scleroderma, mixed connective tissue disease, irritable bowel syndrome, systemic lupus erythromatosus
  • SLE multiple sclerosis, myasthenia gravis, Guillan-Barre syndrome (antiphospholipid syndrome), primary myxoedema, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastris, alopecia totalis, Addison's disease, insulin-dependent diabetes mellitus (IDDM), Goodpasture's syndrome, Behcet's syndrome, Sjogren's syndrome, rheumatoid arthritis, sympathetic ophthalmia, Hashimoto's disease/hypothyroiditis, celiac disease/dermatitis herpetiformis, adult-onset idiopathic hypoparathyroidism (AOIH), amyotrophic lateral sclerosis, and demyelinating disease primary biliary cirrhosis, mixed connective tissue disease, chronic active hepatitis, polyendocrine failure, vitiligo, Celiac disease, chronic active hepatitis, CREST syndrome, dermatomyo
  • the undesirable or deleterious immune response is an organ-specific disease, non-limiting examples of which include TlDM, thyroiditis, adrenal insufficiency, alopecia, atrophic gastritis, vitiligo, premature ovarian failure, autoimmune polyendocrine syndromes (APS), parathyroiditis, hypoparathyroidism, autoimmune adrenal insufficiency (Addison's disease), autoimmune hepatitis, Sjogren's syndrome, celiac disease, exocrine pancreatitis, keratitis and mucocutaneous candidiasis.
  • TlDM thyroiditis
  • adrenal insufficiency alopecia
  • atrophic gastritis vitiligo
  • premature ovarian failure autoimmune polyendocrine syndromes (APS)
  • parathyroiditis hypoparathyroidism
  • autoimmune adrenal insufficiency autoimmune hepatitis
  • Sjogren's syndrome celiac disease
  • mice deficient in ReIB display autoimmune multi-organ inflammatory disease, including pancreatitis, salivary gland inflammation (Sjogren's syndrome), pneumonitis, hepatitis and keratitis and that mice deficient in the NIK (aly/aly mutant) and NF- ⁇ B2 (p52) components of the non-canonical NF- ⁇ B pathway exhibit milder organ-specific autoimmune inflammation (e.g., salivary and lacrimal glands, pancreas and liver).
  • the inventive compositions and methods are useful for restoring the capacity of antigen-presenting cells to license regulatory lymphocyte mediated suppression of T cell-associated inflammatory disease.
  • the efficiency of inducing lymphocytes, especially T lymphocytes, to exhibit tolerance to a specified antigen can be determined by assaying immune responses to that antigen including, but not limited to, assaying T lymphocyte cytolytic activity in vitro using for example the antigen- specific antigen-presenting cells as targets of antigen-specific cytolytic T lymphocytes (CTL); assaying antigen-specific T lymphocyte proliferation (see, e.g., Vollenweider and Groscurth, 1992, J. Immunol. Meth.
  • CTL antigen-specific cytolytic T lymphocytes
  • compositions of the present invention are typically in the form of pharmaceutical compositions, which may comprise a pharmaceutically acceptable carrier or diluent. In some embodiments, the compositions are administered to individuals having the unwanted or deleterious immune response.
  • compositions are administered to at-risk individuals who are autoantibody positive and/or HLA haplotype identified at risk e.g., TlDM first degree relatives with at least one and desirably two or more autoantibodies positive (see, e.g., Scofield, R. H., 2004. Lancet 363, 1544; Berglin et al, 2004, Arthritis Res Ther 6, R30336; Harrison et al, 2004, Diabetes Care 27, 2348), or individuals at risk of rheumatoid arthritis, with one or two HLA susceptibility genes and positive anti-CCP antibodies (Klarskog et al 2006, Arthritis Rheum. 54: 38) (Rantapaa-Dahlqvist S et al 2003, Arthritis Rheum. 48:2741).
  • compositions suitable for use in the present invention include compositions wherein the bioactive agents are contained in an effective amount to achieve their intended purpose.
  • the dose of active compounds administered to a patient should be sufficient to achieve a beneficial response in the patient over time such as a reduction in at least one symptom associated with the unwanted or deleterious immune response, which is suitably associated with a condition selected from an allergy, an autoimmune disease and a transplant rejection.
  • the quantity or dose frequency of the pharmaceutically active compounds(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. In this regard, precise amounts of the active compound(s) for administration will depend on the judgment of the practitioner.
  • the practitioner may evaluate inflammation, pro-inflammatory cytokine levels, lymphocyte proliferation, cytolytic T lymphocyte activity and regulatory T lymphocyte function.
  • those of skill in the art may readily determine suitable dosages of the antagonist and antigen.
  • the bioactive agents are administered to a subject to be treated in a manner compatible with the dosage formulation, and in an amount that will be prophylactically and/or therapeutically effective.
  • the amount of the composition to be delivered generally in the range of from 0.01 ⁇ g/kg to 100 ⁇ g/kg of bioactive molecule (e.g., NF- ⁇ B restoration agent or antigen) per dose, depends on the subject to be treated.
  • the NF- ⁇ B restoration agent-containing compositions will generally contain about 0.1% to 90%, about 0.5% to 50%, or about 1% to about 25%, by weight of the agent, the remainder being suitable pharmaceutical carriers and/or diluents etc and optionally an antigen.
  • the dosage of the agent can depend on a variety of factors, such as mode of administration, the species of the affected subject, age and/or individual condition.
  • the antigen-containing compositions will generally contain about 0.1% to 90%, about 0.5% to 50%, or about 1% to about 25%, by weight of antigen, the remainder being suitable pharmaceutical carriers and/or diluents etc and NF- ⁇ B restoration agent.
  • the particles may be formulated and administered systemically, topically or locally.
  • Techniques for formulation and administration may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition. Suitable routes may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • the particles of the invention may be formulated in aqueous solutions, suitably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • compositions of the present invention may be formulated for administration in the form of liquids, containing acceptable diluents (such as saline and sterile water), or may be in the form of lotions, creams or gels containing acceptable diluents or carriers to impart the desired texture, consistency, viscosity and appearance.
  • acceptable diluents such as saline and sterile water
  • Acceptable diluents and carriers are familiar to those skilled in the art and include, but are not restricted to, ethoxylated and nonethoxylated surfactants, fatty alcohols, fatty acids, hydrocarbon oils (such as palm oil, coconut oil, and mineral oil), cocoa butter waxes, silicon oils, pH balancers, cellulose derivatives, emulsifying agents such as non- ionic organic and inorganic bases, preserving agents, wax esters, steroid alcohols, triglyceride esters, phospholipids such as lecithin and cephalin, polyhydric alcohol esters, fatty alcohol esters, hydrophilic lanolin derivatives, and hydrophilic beeswax derivatives.
  • ethoxylated and nonethoxylated surfactants include, but are not restricted to, ethoxylated and nonethoxylated surfactants, fatty alcohols, fatty acids, hydrocarbon oils (such as palm oil, coconut oil, and mineral oil), cocoa butter waxes,
  • the particles of the present invention can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration, which is also preferred for the practice of the present invention.
  • Such carriers enable the compounds of the invention to be formulated in dosage forms such as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • These carriers may be selected from sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.
  • compositions for parenteral administration include aqueous solutions of the particles in water-soluble form. Additionally, suspensions of the particles may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • compositions for oral use can be obtained by combining the particles with solid excipients and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as., for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • PVP polyvinylpyrrolidone
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more therapeutic agents as described above with the carrier which constitutes one or more necessary ingredients.
  • the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g. by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of particle doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added.
  • compositions of the invention may be administered over a period of hours, days, weeks, or months, depending on several factors, including the severity of the neuropathic condition being treated, whether a recurrence of the condition is considered likely, etc.
  • the administration may be constant, e.g., constant infusion over a period of hours, days, weeks, months, etc.
  • the administration may be intermittent, e.g., particles may be administered once a day over a period of days, once an hour over a period of hours, or any other such schedule as deemed suitable.
  • compositions may also be administered to the respiratory tract as a nasal or pulmonary inhalation aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose, or with other pharmaceutically acceptable excipients.
  • the particles of the formulation may advantageously have diameters of less than 50 ⁇ m, suitably less than 10 ⁇ m.
  • Bioactive agent-containing particles are administered for active uptake by cells, for example by phagocytosis, as described for example in U.S. Pat. No. 5,783,567 (Pangaea).
  • phagocytosis by these cells may be improved by maintaining a particle size typically below about 20 ⁇ m, and preferably below about 11 ⁇ m.
  • the particles are delivered directly into the bloodstream (i.e., by intravenous or intra-arterial injection or infusion) if uptake by the phagocytic cells of the reticuloendothelial system (RES) , including liver and spleen, is desired.
  • RES reticuloendothelial system
  • the particles can also be introduced intradermally (i.e., to the APCs of the skin, such as dendritic cells and Langerhans cells) for example using ballistic or microneedle delivery.
  • Illustrative particle-mediated delivery techniques include explosive, electric or gaseous discharge delivery to propel carrier particles toward target cells as described, for example, in U.S. Pat. Nos. 4,945,050, 5,120,657, 5,149,655 and 5,630,796.
  • Non-limiting examples of microneedle delivery are disclosed in International Publication Nos. WO 2005/069736 and WO 2005/072630 and U.S. Pat. Nos. 6,503,231 and 5,457,041.
  • Another useful route of delivery is via the gastrointestinal tract, e.g., orally.
  • the particles can be introduced into organs such as the lung (e.g., by inhalation of powdered microparticles or of a nebulized or aerosolized solution containing the microparticles), where the particles are picked up by the alveolar macrophages, or may be administered intranasally or buccally.
  • a phagocytic cell phagocytoses the particle the NF- ⁇ B restoration agent and optionally the antigen are released into the interior of the cell.
  • the cells can be introduced into a patient by any means (e.g., injection), which produces the desired modified immune response to an antigen or group of antigens.
  • the cells may be derived from the patient (i.e., autologous cells) or from an individual or individuals who are MHC-matched or -mismatched (i.e., allogeneic) with the patient.
  • autologous cells are injected back into the patient from whom the source cells were obtained.
  • the injection site may be subcutaneous, intraperitoneal, intramuscular, intradermal, or intravenous.
  • the cells may be administered to a patient already suffering from the unwanted immune response or who is predisposed to the unwanted immune response in sufficient number to prevent or at least partially arrest the development, or to reduce or eliminate the onset of, that response.
  • the number of cells injected into the patient in need of the treatment or prophylaxis may vary depending on inter alia, the antigen or antigens and size of the individual. This number may range for example between about 10 3 and 10 11 , and more preferably between about 10 3 and 10 7 cells (e.g., dendritic cells).
  • Single or multiple administrations of the cells can be carried out with cell numbers and pattern being selected by the treating physician.
  • the cells should be administered in a pharmaceutically acceptable carrier, which is non-toxic to the cells and the individual.
  • Such carrier may be the growth medium in which the cells were grown, or any suitable buffering medium such as phosphate buffered saline.
  • the cells may be administered alone or as an adjunct therapy in conjunction with other therapeutics known in the art for the treatment or prevention of unwanted immune responses for example but not limited to glucocorticoids, methotrexate, D-penicillamine, hydroxychloroquine, gold salts, sulfasalazine, TNF ⁇ or interleukin-1 inhibitors, and/or other forms of specific immunotherapy.
  • the antigen-presenting cells are pre-contacted with one or more antigens associated with the unwanted or deleterious immune response to provide antigen-specific tolerogenic antigen presenting cells or are administered concurrently to the subject with one or more such antigens.
  • NF- ⁇ B expression by monocyte-derived DC cultured from healthy donors for 48-72 h (O'Sullivan BJ, et al. , J Immunol 168:5491-5498, 2002). Further, such DC efficiently translocate NF- ⁇ B to the nucleus after activation with TNF- ⁇ , LPS or CD 154 ((O'Sullivan BJ, et al, J Immunol 168:5491-5498, 2002).
  • LPS-induced ReIB activity was more variable among patients from each group than among healthy controls.
  • the mean LPS-induced ReIB activity was increased 1.4 fold in T2DM, 1.5 fold in RA and 1.9 fold in healthy controls relative to TlDM patients ( Figure 1C).
  • SHP-I is expressed by hematopoietic cells in PB, including circulating monocyte precursors of DC.
  • SHP-I was quantitated by intracellular staining and flow cytometric analysis. Expression of SHP-I by resting PB monocytes from 18 TlDM patients was significantly higher than expression by 13 healthy controls ( Figure 2A).
  • SHP-I decreased with prolonged cell culture in the presence of cytokines, and expression was low in both control and TlDM monocyte-derived DC cultured for 48 h in GM-CSF and IL-4. From subject to subject, the expression of SHP-I by PB monocytes predicted SHP-I expression by lymphocytes, whether healthy control or diabetic, suggesting similar regulatory processes in each cell type (Figure 2B).
  • mice deficient in ReIB the transcriptional mediator of the non- canonical NF- ⁇ B signaling pathway, display autoimmune and allergic multi-organ inflammatory disease and excessive myelopoiesis.
  • ReIB " mice inter-crossed with T cell deficient or RAG 7" mice fail to develop inflammatory diseases, indicating that this phenotype induces a defect in the control of effector or regulatory T cell functions.
  • C57BL/6 (wt) mice (Animal Resource Centre, Perth, Australia) were maintained in specific pathogen-free (SPF) conditions. Mice homozygous for an insertional mutation in the ReIB gene (designated RelB ⁇ ), on a C57BL/6 background, were originally generated in D. Lo's Laboratory (L. Burkly et al, 1995, Nature 373, 531). They were bred in clean conventional conditions and used at 5-7 weeks of age. RelB " ⁇ radiation chimeric mice, generated by transferring BM from RelB " ⁇ into lethally irradiated C57B1/6 hosts, were used after at least 6 weeks reconstitution.
  • PB mononuclear cells were prepared as described (O'Sullivan BJ, et al, J Immunol 168:5491-5498, 2002).
  • monocytes were purified from PBMC using CD 14 microbeads (Miltenyi Biotec, GmbH, Germany), then cultured in 24 well plates in X-VIVO 20 (BioWhittaker Walkersville, MD) with 800 U/ml rhGM-CSF and 600 U/ml rhIL-4 (Schering-Plough, Sydney, Australia) for 72 hours.
  • X-VIVO 20 BioWhittaker Walkersville, MD
  • 800 U/ml rhGM-CSF 600 U/ml rhIL-4 (Schering-Plough, Sydney, Australia) for 72 hours.
  • 10 ⁇ g/ml sodium stibogluconate were added at the commencement of DC culture.
  • 100 ng/ml LPS were added to some wells for the last 24 hours of the culture period.
  • CDl Ic + DC were purified from me v /me v or wild type mouse spleens using CDl Ic microbeads (Miltenyi Biotec), then cultured overnight with or without 100 ng/ml LPS in RPMI+10% FCS.
  • mice were administered 20ug progenipoietin-1 (Pfizer, NY, USA) s.c. daily for 7 days. Expanded splenic CDl Ic + DC were purified by MACS or sorted. Either 12.5 x 10 6 MACS-purified DC or 2 x 10 6 sorted DC were injected i.v into each ReIB " " recipient. Mice were monitored every five days for weight gain, gait, agility, alopecia, progression of lacrimal disease/keratoconjunctivits, dermatitis, knee joint swelling and feeding habits. Before sacrifice, mice received i.p. Brdu.
  • Binding was revealed with HRP-conjugated anti-rabbit antibody using ECL chemiluminescence reagents (Amersham).
  • ELISA-based format BDTM Transfactor Kits (BD biosciences, Palo Alto, CA) measured NF- ⁇ B DNA binding as described (O'Sullivan BJ, et ai, J Immunol 168:5491-5498, 2002). Absorbance data were expressed as relative units.
  • PBMC peripheral blood mononuclear cells

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Abstract

This present invention discloses methods and compositions for modulating immune responses. More particularly, the present invention discloses increasing the number of antigen-presenting cells in which both the canonical and non-canonical NFϰB pathways are activated for the treatment or prophylaxis of undesirable immune responses associated with defective NF-ϰB activation, including autoimmune diseases, organ-specific diseases and allergies. In specific embodiments, the treatment or prophylaxis of the undesirable immune responses is achieved by increasing the number of antigen-presenting cells in which RelB is activated or activatable.

Description

TITLE OF THE INVENTION
"COMPOSITIONS AND METHODS FOR TREATING OR PREVENTING UNWANTED IMMUNE
RESPONSES"
FIELD OF THE INVENTION [0001] This invention relates generally to methods and compositions for modulating immune responses. More particularly, the present invention relates to increasing the number of antigen-presenting cells in which both the canonical and non- canonical NF-κB pathways are activated for the treatment or prophylaxis of undesirable immune responses associated with defective NF-κB activation, including autoimmune diseases, organ-specific diseases and allergies. In specific embodiments, the treatment or prophylaxis of the undesirable immune responses is achieved by increasing the number of antigen-presenting cells in which ReIB is activated or activatable.
BACKGROUND OF THE INVENTION
[0002] NF-κB transmits signals from the cell surface to the nucleus. Signaling through cell surface receptors to activate NF-κB and MAP kinases through adaptor molecules is of critical importance to survival and activation of all cells in the body, including those regulating innate and adaptive immunity, including antigen- presenting cells such as dendritic cells (DC). As such, NF-κB is a key signaling component in autoimmunity and an attractive target for autoimmune disease therapy. NF-KB FUNCTION
[0003] Five NF-κB proteins p50, p52, c-Rel, p65/RelA and ReIB are present in mammals. All share a rel homology domain (RHD) that mediates DNA-binding, dimerisation and nuclear translocation. p50 and p52 homodimers are transcriptionally inactive, but have the capacity to bind DNA. In contrast, c-rel or ReIA are able to bind DNA and p50 or p52 are able to bind DNA and mediate transcriptional activation. As a result, the availability of subunits and affinity determines the NF-κB composition of the cell (Hoffmann A, et al, Immunol. Rev. (2006) 210:171-186).
[0004] In unstimulated cells, NF-κB is present as an inactive form in the cytoplasm bound to inhibitory proteins or IKBS, including IκBα, IκBβ, IκBε, IκBγ, IKBNS, Bcl-3, plOO and plO5 (Ghosh S, Cell (2002) 109 Suppl:S81-96). These proteins contain ankyrin repeats consisting of two tightly packed helices followed by a loop and a tight hairpin turn, which facilitate binding to NF-κB dimers. The NLS region of NF- KB enables dimer nuclear import. IκBβ masks the NLS, preventing nuclear import of dimers. In contrast, IκBα only masks the NLS of p65 and not p50. Nuclear retention is normally prevented by the presence of a nuclear export sequence in IκBα. If this NF-κB export sequence is blocked, RelA/p50 complexes are retained in the nucleus (Huang T, et al, Proc. Natl. Acad. Sci. U. S. A. (2000) 97(3): 1014-1019).
[0005] A variety of receptor-ligand pairs activates NF -KB, including TLR/pathogen signals, inflammatory receptors (TNFR/TNF and IL-lR/IL-1), T cell (CD40/CD40L, TCR/MHC peptide) and B cell signals (BAFFR/BAFF, BCR/Ag) and differentiation signals such as lymphotoxin/LTβ and RANK/RANKL. Signaling these pathways leads to activation of serine/threonine kinase IKB kinase (IKK) (Yamamoto Y, et al. , Trends Biochem. Sci. (2004) 29(2):72-79). IKK phosphorylates IKB which is recognized by a specific ubiquitin ligase complex, b-TrCP-SCF. Ubiquitinated IKB is degraded by the 26S proteasome, leading to release of NF-κB, nuclear import and transcriptional activation. The IKK complex consists of 3 subunits including IKKα
(IKKl), IKKβ (IKK2) and the associated non-catalytic regulatory subunit IKKγ/NF-κB essential modulator (NEMO). IKK may be activated through phosphorylation by mitogen activated protein kinase kinase kinase (MAPKKK) or NF-κB inducing kinase (NIK), leading to subsequent autophosphorylation of the IKK complex and full activity. IKKβ and NEMO deficient mice have impaired NF-κB activation in response to cytokine and TLR activation, particularly activation of RelA/p50. In contrast, IKKα has a particular role in activation of RelB/p52 complexes and histone phosphorylation to enhance NF-κB DNA binding.
[0006] The differential role of IKKα and IKKβ/NEMO in activating distinct NF-κB subunits has led to the classification of the NF-κB pathway into the classical (also known as "canonical") and alternate (also known as "non-canonical") pathways (Xiao G, et al, Cytokine Growth Factor Rev. (2006) 17(4):281-293). The canonical pathway is activated by TLR and pro-inflammatory cytokines, leading to DCKβ and NEMO-dependent phosphorylation, degradation of IKB, and subsequent activation of RelA/p50 heterodimers. In the absence of continual signaling the pathway is rapidly shut down, as a result of reduced IKKβ activity and induction of IKB. In contrast, the non-canonical pathway is activated by signals associated with cell differentiation, including LTβ, CD40L and BAFF. RelB/p52 heterodimers are the predominant NF-κB proteins induced, regulated by pi 00, the precursor to p52, which contains an IKB domain target site for phosphorylation by IKKα. Signal-specific activation of IKKα results in processing of pi 00 to p52 and activation of RelB/p52. This pathway is characterized by sustained IKKα and long lasting activation of NF-κB. The non- canonical pathway appears to be an adaptation of the canonical NF -KB pathway for cellular differentiation processes and is important in B cell and DC differentiation and lymphoid organogenesis. NIK appears to be an upstream kinase that activates IKKα. NIK, IKKα and ReIB knockout mice share similar defects in lymphoid organogenesis. Importantly, there is some overlap in activation of the canonical and non-canonical pathway; for example, LTβ signals both pathways and resulting target genes are activated (Dejardin E, et al., Immunity (2002) 17(4):525-535). LPS, a typical classical pathway activator, also leads to activation of the non-canonical pathway (Mordmuller B, et al, EMBO Rep. (2003) 4(l):82-87). This may be essential for efficient differentiation of DC, which up-regulate both NF-κB pathways following antigen encounter and migration into the secondary lymphoid organs. Activation of the non- canonical pathway ensures that although newly synthesized IκBα inhibits RelA/p50, newly synthesized ReIB and processing of pi 00 to p52 leads to dimer replacement or exchange with RelB/p52 and sustained DC differentiation (Saccani S, et al., MoI. Cell (2003) 11(6): 1563-1574). In contrast to members of the classical pathway (p50, ReIA, cRel), ReIB and p52 are activated slowly after an activation event with late translocation to the nucleus, due to nuclear exchange of non-canonical pathway NF-κB dimers with those derived from the classical pathway. In this regard, ReIB not only heterodimerizes with p52 in the regulation of the non-canonical pathway, but it also has a role in the regulation of the canonical pathway through its capacity to heterodimerize with p50. [0007] In immune responses, NF-κB target genes are involved in inflammation, cellular organization and differentiation and proliferation. Tissue macrophages are the major source of NF-κB-induced pro-inflammatory cytokines. NF- KB induced cytokines such as TNFα, IL-I and IL-6 activate innate responses leading to the release of c-reactive protein (CRP) and complement, and up-regulation of adhesion molecules by local endothelial cells. NF-κB-induced chemokines, including IL-8, MIP- lα, MCP-I, RANTES and eotaxin, and growth factors such as GM-CSF mobilize and redirect myeloid cells to local tissue. The same set of responses as occurs to infection also occurs in inflammatory autoimmune diseases, such as rheumatoid arthritis (RA) and inflammatory bowel disease (IBD).
[0008] NF-κB has a role in lymphoid organogenesis through the induction of the chemokines CXC12, CXCL13, CCL21 and CCL19. NF-κB has a role in many stages of B and T cell differentiation (Claudio E, et al, (2006) 13(5):697-701) including a role for the non-canonical pathway in NKT cell development and for the canonical and non-canonical pathways in regulatory T cell (Treg) development (Schmidt-Supprian M, et al, Proc. Natl. Acad. Sci. U. S. A. (2004) 101(13):4566-4571; Schmidt-Supprian M, et al, Immunity (2003) 19(3):377-389; Zheng Y, et al, J. Exp. Med. (2003) 197(7):861-874). c-Rel is also required for efficient IL-2 production by naive T cells (Banerjee D, et al, Immunity (2005) 23(4):445-458) and T reg are critically dependent on IL-2 for post thymic survival (D'Cruz LM, et al, Nat. Immunol. (2005) 6(11):1152- 1159; Fontenot JD, et al, Nat. Immunol. (2005) 6(11):1142-1151). NF-κB plays an important role in proliferation of lymphocytes as well as non-hematopoetic cells such as synoviocytes, that hyperproliferate in RA. Relevant NF-κB target genes include c-myc, cyclin Dl and anti-apoptotic genes including c-IAP and Bcl-2.
NF-κB IN AUTOIMMUNE INFLAMMATION
[0009] Autoimmune diseases result from a process involving three distinct but related components - a break in self tolerance, development of chronic inflammation in one or several organs, and if ongoing, tissue destruction and its resultant detrimental effects. "Central" tolerance defects are important contributors to spontaneous autoimmune disease. In the fetal and neonatal period, central tolerance is actively maintained in the thymus (Ardavin C: Immunol. Today (1997) 18:350-361). During this process, a repertoire of T cells restricted to self-MHC displayed by the thymic cortical epithelium (cTEC) is selected in each individual. In addition, those T cells reactive to self-antigen presented by medullary antigen-presenting cells (APC), which include medullary epithelial cells (mTEC) and medullary dendritic cells (DC), are deleted by negative selection above a threshold of affinity for self antigens presented by those APC (Kappler JW, et al, Cell (1987) 49:273-280). Since an affinity threshold applies for central deletion of self-reactive T cells, circulation of low-affinity self- reactive T cells in the periphery is therefore inevitable. Low-level thymic expression and presentation of self-antigens normally expressed by peripheral somatic cells is common. Expression of these antigens is transcriptionally controlled by AIRE, whose expression is in turn controlled by the alternate NF-κB pathway (Anderson MS, et al. , Science (2002) 298(5597): 1395-1401 ; Zhu M, et al., J. Clin. Invest. (2006) 116(1 1):2964-2971). In spontaneous autoimmune models, a variety of defects in the interaction of APC and thymocytes interferes with the normal process of negative selection, thus permitting the release of autoreactive T cells into the periphery, where subsequent environmental events more readily trigger autoimmune disease (Yoshitomi H, et al, J. Exp. Med. (2005) 201(6):949-960). Commonly, viral or modified self-antigens, which have not been expressed in the thymus, are presented by peripheral DC to initiate autoimmunity. Several modified self-antigens have been described in human autoimmune diseases. DENDRITIC CELLS
[0010] It has been proposed that DC are the critical decision making cells in the immune system (Fazekas de St Groth B. Immunol Today. 1998;19:448-54). Through their role in the generation of central and peripheral tolerance as well as in priming immune responses and stimulation of memory and effector T cells, DC are likely to play essential roles in both the initiation and perpetuation of autoimmunity and autoimmune diseases. However, the understanding of the means by which DC contribute to peripheral tolerance has opened the exciting possibility of harnessing them for antigen-specific immunotherapy of autoimmune diseases and transplantation.
[0011] DC are now recognized as essential regulators of both innate and acquired arms of the immune system (Banchereau J, et al, Nature. 1998 Mar
19;392(6673):245-52). They are responsible for the stimulation of naive T lymphocytes, a property that distinguishes them from all other antigen presenting cells (APC). DC are also essential accessory cells in the generation of primary antibody responses (Inaba K, et al, Proc Natl Acad Sci U S A. 1983 Oct;80(19):6041-5) and are powerful enhancers of NK cell cytotoxicity (Kitamura H, et al, J Exp Med. 1999 Apr 5;189(7):1121-8). DC are crucial for the initiation of primary immune responses of both helper and cytotoxic T lymphocytes, and thus act as "nature's adjuvant" (Schuler G, et al, J Exp Med. 1997 Oct 20; 186(8): 1183-7). Conversely, DC are also involved in the maintenance of tolerance to antigens. DC contribute to thymic central tolerance and shaping of the T cell repertoire by presenting antigens to T cells and deleting those T cells that exhibit strong autoreactivity (Brocker T. J Exp Med. 1997 Oct 20; 186(8): 1223-32). However, DC also play a role in peripheral tolerance. Here, DC contribute by deletion of autoreactive lymphocytes and expansion of the population of regulatory T cells (Treg). Accordingly, DC offer potential utility in protective and therapeutic strategies for tolerance restoration in autoimmune diseases.
[0012] DC precursors from the bone marrow migrate via the bloodstream to peripheral tissues where they reside as immature DC. Immature DC efficiently capture invading pathogens and other particulate and soluble antigens (Ag). After Ag uptake, DC rapidly cross the endothelium of lymphatic vessels and migrate to the draining secondary lymphoid organs. Following the uptake of immunogenic Ag and lymphatic migration, DC undergo a process of maturation, which is characterized by downregulation of the capacity to capture Ag and upregulation of Ag processing and presentation, expression of co-stimulatory molecules and altered dendritic morphology (Steinman RM. Annu Rev Immunol. 1991 ;9:271-96; Cella M, et al., Curr Opin Immunol. 1997 Feb;9(l): 10-6; CeIIa M, et al, J Exp Med. 1996 Aug l;184(2):747-52). After presentation of Ag to naive T cells in the T cell area of secondary lymphoid organs, most DC disappear, probably by apoptosis. Thus, under optimal conditions, the same DC sequentially carries out distinct functions such as capture and processing of Ag, Ag presentation to rare, naive Ag-specific T cells and induction of Ag-specific T cell clonal expansion.
[0013] Considering the crucial role of DC in Ag processing and presentation and thus in the regulation of immune reactivity, DC are important directors of immune responsiveness, through the interactions with responding lymphocytes and other accessory cells. Broadly, evidence suggests that under steady state conditions, recruitment of DC precursors into tissues and migration/maturation into secondary lymphoid organs occurs at low rates and may favour tolerance induction. On the other hand, stimulation of immature DC leading to DC maturation and activation may induce a productive immune response (Sallusto F, et al., J Exp Med. 1999 Feb 15;189(4):611- 4).
[0014] The process of DC maturation can be stimulated by various mechanisms, including pathogen-derived molecules (LPS, DNA, RNA), proinflammatory cytokines (TNFα, IL-I, IL-6), tissue factors such as hyaluronan fragments, migration of DC across endothelial barriers between inflamed tissues and lymphatics, and T cell-derived signals (CD 154) (Sparwasser T, et al., Eur J Immunol. 1998 Jun;28(6):2045-54; Cella M, et al., J Exp Med. 1999 Mar l;189(5):821-9; De Smedt T, et al. , J Exp Med. 1996 Oct 1 ; 184(4): 1413-24). In contrast, anti-inflammatory signals, such as IL-IO, TGFβ, prostaglandins, and corticosteroids tend to inhibit maturation (De Smedt T, et al, Eur J Immunol. 1997 May;27(5): 1229-35; Geissmann F, et al, J Immunol. 1999 Apr 15;162(8):4567-75; de Jong EC, et al, J Leukoc Biol. 1999 Aug;66(2):201-4). Thus, DC represent an attractive therapeutic target, either to enhance or to attenuate immunity for modulation of disease. To date, ex vivo modulation of DC and exposure to antigen before transfer into an animal or human recipient has been the major approach to achieve protective and therapeutic immunity. This relates in part to complexity of the DC system in the context of a whole person with an immune system disorder, and in part to the difficulty of delivery of specific Ags and immunomodulators Xo OC in vivo.
ROLE OF NF-KB IN REGULATING DC FUNCTION
[0015] The ability of a myeloid DC to induce immunity or tolerance is linked to its maturation state and thus to NF-κB activity (Dhodapkar MV, et al. , J Exp Med. 2001 Jan 15;193(2):233-8; Jonuleit H et al, J Exp Med. 2000 Nov 6; 192(9): 1213-22; Lutz MB, et al , Eur J Immunol. 2000 Apr;30(4): 1048-52: Mehling A, et al. , J
Immunol. 2000 Sep 1;165(5):2374-81). Immature DC generated from murine BM induce T cell unresponsiveness in vitro and prolonged cardiac allograft survival (Lutz MB, et al, Eur J Immunol. 2000 Jul;30(7): 1813-22). Various drugs and cytokines, and inhibitors of NF-κB inhibit myeloid DC maturation (de Jong EC, et al, et al, J Leukoc Biol. 1999 Aug;66(2):201 -4; Griffin MD, et al , Proc Natl Acad Sci U S A. 2001 Jun 5;98(12):6800-5; Hackstein H, et al, J Immunol. 2001 Jun 15;166(12):7053-62; Lee JI, et al, Transplantation. 1999 Nov 15;68(9):1255-63; Steinbrink K, et al, J Immunol. 1997 Nov l5;159(10):4772-80; Yoshimura S, et al, lnX Immunol. 2001 May;13(5):675- 83), including corticosteroids, salicylates, mycophenolate mofetil, transforming growth factor (TGF)-β IL-10. DC generated in the presence of these agents alter T cell function in vitro and in vivo, including promotion of allograft survival (Giannoukakis N, et al , MoI Ther. 2000; 1(5 Pt l):430-7; Griffin MD, et al, Proc Natl Acad Sci U S A. 2001;98(12):6800-5; Rea D, et al, Blood. 2000 May 15;95(10):3162-7; Adorini L, et al, J Cell Biochem. 2003 Feb l;88(2):227-33). [0016] NF-κB activity leads to transcription of a number of genes involved in the immune response. In particular, ReIB activity is required for myeloid DC differentiation and the development of CD4+CD8" splenic DC (Burkly L, et al, Nature. 1995 Feb 9;373(6514):531-6; Weih F, et al, Cell. 1995;80(2):331-40; Wu L, et al,
- 1 - Immunity. 1998 Dec;9(6):839-47). ReIB regulates DC and B cell APC function through regulation of CD40 and MHC molecule expression (O'Sullivan BJ, et al, Proc Natl Acad Sci U S A. 2000 Oct 10;97(21):l 1421-6; O'Sullivan BJ, et al., 3 Immunol. 2002 Jun 1;168(1 1):5491-8; Martin E, et al, Immunity. 2003 Jan;18(l):155-67). The present inventors have shown that antigen-exposed DC in which ReIB function is inhibited lack cell surface CD40, prevent priming of immunity, and suppress previously primed immune responses. While immature DC, which maintain the potential for subsequent activation, were only moderately suppressive of primed immune responses, ReIB- deficient DC lacking this potential were much more suppressive (Martin E, et al, Immunity. 2003 Jan; 18( 1 ) : 155-67).
[0017] In work leading up to the present invention, the inventors tested the hypothesis that inflammation develops in the absence of ReIB due to a deficiency in CD4+CD25+ Treg. Surprisingly, however, it was found that CD4+CD25+ Treg expand in the periphery of ReIB"7" mice without influencing the development of T cell-mediated inflammation. Additionally, the inventors found that DC-encoded ReIB enables Treg to suppress T cell associated pro-inflammatory mechanisms in RelB"A mice as transfer of RelB-sufficient wild type DC to RelB"A mice restores Treg suppressor function, reversing inflammatory disease. The inventors also determined that DC in certain undesirable immune responses including autoimmune diseases {e.g., type 1 diabetes), organ-specific diseases and allergies have impaired NF-κB activation and that effective restoration of NF-κB function in these DC, in order to restore Treg suppressor function and reverse inflammatory disease, requires activation of both the canonical and non- canonical NF-κB pathways, including the restoration of ReIB function. These discoveries have been reduced to practice in the form of immunomodulating compositions and methods of treating or preventing undesirable or deleterious immune responses, as described hereafter.
SUMMARY OF THE INVENTION
[0018] Accordingly, in one aspect, the present invention provides methods for modulating an immune response, especially an undesirable or deleterious immune response associated with defective NF-κB activation in a subject. These methods generally comprise increasing the number of antigen-presenting cells in the subject, in which both the canonical and non-canonical NF-κB pathways are activated or activatable, suitably in response to a pro-inflammatory signal (e.g., tumor necrosis factor, C5a, interleukin-1, CDl 54 and lipolysaccharide). In some embodiments, the methods comprise increasing the number of antigen-presenting cells in which ReIB is activated or activatable. Accordingly, in a related aspect, the present invention provides methods for modulating an undesirable or deleterious immune response associated with defective NF-κB activation in a subject, wherein the methods comprise increasing in the subject the number of antigen-presenting cells in which ReIB is activated or activatable, e.g., in response to a pro-inflammatory signal. Antigen-presenting cells in which both the canonical and non-canonical NF-κB pathways are activated or activatable, or in which ReIB is activated or activatable (e.g., in response to a pro-inflammatory signal), are also referred to herein as "operable NF-κB antigen-presenting cells" or "antigen- presenting cells with operable NF-κB function."
[0019] In some embodiments, the methods comprise administering to the subject an effective amount of an immune modulator that increases the number of operable NF-κB antigen-presenting cells. For example, the immune modulator may comprise at least one agent that activates the canonical and non-canonical NF-κB pathways (e.g., an agent that increases the level or functional activity of ReIB) and is suitably administered in an amount sufficient to restore NF-κB function, including ReIB function, suitably to a basal state, in antigen-presenting cells of the subject. The agent or agents may be administered in soluble or in particulate form. Non-limiting antigen presenting cells include dendritic cells, macrophages and Langerhans cells. Suitably, the operable NF-κB antigen-presenting cells test positive for NF-κB restoration when they stimulate the production of regulatory T lymphocytes that suppress or otherwise reduce the undesirable or deleterious immune response. In some embodiments, the regulatory T lymphocyte expresses markers of constitutive regulatory or suppressor T lymphocytes, including CD4, CD25, CD62L, GITR, CTLA4 and the transcription factor FoxP3. Illustrative regulatory T lymphocytes include, but are not limited to, CD4+CD25+regulatory T lymphocytes, TrI lymphocytes, Th3 lymphocytes, Th2 lymphocytes, CD8+CD28" regulatory T lymphocytes, natural killer (NK) T lymphocytes and γδ T lymphocytes. In specific embodiments, the regulatory T lymphocyte is CD4+CD25+. [0020] In other embodiments, the immune modulator comprises operable NF-
KB antigen-presenting cells. In illustrative examples of this type, the operable NF-κB antigen-presenting cells are derived from a donor, who is suitably histocompatible with the subject. In other illustrative examples, the operable NF-κB antigen-presenting cells are derived by harvesting antigen-presenting cells from the subject and treating them ex vivo with at least one agent that activates the canonical and non-canonical NF-κB pathways (e.g., an agent that increases the level or functional activity of ReIB) in an amount sufficient to restore NF-κB function, including ReIB function, suitably to a basal state, in those cells. The immune modulator may be administered by injection, by topical application or by the nasal or oral route including sustained-release modes of administration, over a period of time and in amounts which are suitably effective to ameliorate the symptoms of the undesirable or deleterious immune response. In specific embodiments, the immune modulator is administered systemically.
[0021] The undesirable or deleterious immune response is typically selected from autoimmune diseases, organ specific diseases and allergies. In some embodiments, therefore, the undesirable or deleterious immune response is associated with the presence of or predisposition to an autoimmune disease or related condition such as but not limited to type 1 diabetes mellitus (also referred to herein as "TlDM"), systemic lupus erythematosus, Churg Strauss disease, scleroderma, Wegener granulomatosus, Wiskott Aldrich syndrome. In other embodiments, the undesirable or deleterious immune response is associated with the presence of or predisposition to an allergy, illustrative examples of which include allergic eczema and allergic asthma. In still other embodiments, the undesirable or deleterious immune response is associated with the presence of or predisposition to an organ-specific disease representative examples of which include TlDM, thyroiditis, adrenal insufficiency, alopecia, atrophic gastritis, vitiligo, premature ovarian failure, autoimmune polyendocrine syndromes (APS), parathyroiditis, hypoparathyroidism, autoimmune adrenal insufficiency (Addison's disease), autoimmune hepatitis, Sjogren's syndrome, celiac disease, exocrine pancreatitis, keratitis and mucocutaneous candidiasis. [0022] In some embodiments in which at least one agent is used to activate the canonical and non-canonical NF-κB pathways, the agent(s) increase(s) the level or functional activity of a canonical pathway kinase selected from IKK and IKKβ/NEMO and the level of a non-canonical pathway kinase selected from NIK and IKKα. In other embodiments, the at least one agent decreases the level or functional activity of SHP-I . In still other embodiments, the at least one agent stimulates or otherwise increases the level or functional activity of ReIB.
[0023] The defective NF-κB activation, which associates with the undesirable or deleterious immune response, is typically associated with aberrant signaling through the NF-κB pathway, suitably in response to a pro-inflammatory signal. Representative members of the NF-κB pathway include, but are not limited to, BTK, LYN, BCR Igα, BCR Igβ, Syk, Blnk, PLCγ2, PKCβ, DAG, CARMAl, BCLlO, MALTl, PDK, PIP3, AKT, COT, IKKα, IKKβ, IKKγ, NIK, RelA/p65, P105/p50, c-Rel, ReIB, p52, NIK, Leul3, CD81, CD19, CD21 and its ligands in the complement and coagulation cascade, ubiquitin mediated proteolysis, TRAF6, ubiquitin ligase, Tab2, TAKl, NEMO, NOD2, RIP2, Lck, fyn, Zap70, LAT, GRB2, SOS, CD3 zeta, Slp-76, GADS, ITK, PLCγl, PKCΘ, ICOS, CD28, SHP-2, SAP, SLAM, 2B4, SHP-I, SHIP, PIR-B, CD22, CD72, FcgRIIB, IKB, PlOO, CTLA4, PD-I, CbI, KIR3DL1, KIR3DL2, KIR2DL and Csk. In some embodiments, the defective NF-κB activation associates with a decrease in the level or functional activity of an expression product of at least one gene selected from genes that encode RelA/p65, P105/p50, c-Rel, ReIB or p52. In other embodiments, the defective NF-κB activation associates with an increase in the level or functional activity of an expression product of the SHP-I gene.
[0024] Aberrant signaling is suitably detected by detecting aberrant expression of a gene belonging to the canonical or non-canonical NF-κB pathway, e.g., aberrant expression of ReIB, as described in more detail below.
[0025] In some embodiments, the methods further comprise administering to the subject an effective amount of antigen that corresponds to at least a portion of a target antigen associated (e.g. , an allergen, autoantigen or alloantigen) with the undesirable or deleterious immune response. In some embodiments, the antigen is administered concurrently with an immune modulator as broadly described above. The antigen may be selected from proteinaceous antigens, lipid antigens, glycolipid antigens and carbohydrate antigens. In some embodiments, the antigen is in the form of a nucleic acid construct from which it is expressible. In a related aspect, the invention thus provides compositions for modulating an immune response, especially an undesirable or deleterious immune response associated with defective NF-κB activation in a subject, wherein the compositions generally comprise an immune modulator that increases the number of operable NF-κB antigen-presenting cells as defined herein and an antigen that corresponds to at least a portion of a target antigen associated with the undesirable or deleterious immune response. In illustrative examples of this type, the composition may further comprise a pharmaceutically acceptable carrier or diluent.
[0026] The methods of the present invention are useful for inducing a tolerogenic response including the induction of an anergic response, or the suppression of a future or existing immune response, to a specified antigen or group of antigens. For example, the immune response includes, but is not limited to, a response mediated by immunoglobulin molecules (e.g., IgE) and/or T lymphocytes (e.g., cytotoxic T lymphocytes (CTLs) and T helper lymphocytes). The immune response is typically but not exclusively directed to an antigen selected from a protein antigen, a particulate antigen, an alloantigen, an autoantigen, an allergen, a bacterial antigen, a viral antigen, a parasitic antigen or an immune complex.
[0027] In a related aspect, the invention extends to the use of an immune modulator as broadly described above and optionally an antigen that corresponds to at least a portion of a target antigen that associates with the presence or risk of an undesirable or deleterious immune response and with defective NF-κB function in antigen-presenting cells, in the manufacture of a medicament for suppressing the immune response.
[0028] In yet another aspect, the present invention provides methods for treating a condition associated with an undesirable or deleterious immune response and with defective NF-κB activation in a subject. These methods generally comprise administering to the subject an effective amount of an immune modulator that increases the number of antigen-presenting cells in the subject, in which both the canonical and non-canonical NF-κB pathways are activated or activatable, suitably in response to a pro-inflammatory signal. In specific embodiments, the methods comprise administering to the subject an effective amount of an immune modulator that increases the number of antigen-presenting cells in the subject, in which ReIB is activated or activatable, suitably in response to a pro-inflammatory signal. In some embodiments, the methods further comprise contacting antigen-presenting cells with an antigen that corresponds to at least a portion of a target antigen that associates with the presence or risk of the undesirable or deleterious immune response, in an amount that is effective for the antigen-presenting cells to present the antigen or a processed form thereof on their surface. These embodiments are useful for producing antigen-specific antigen- presenting cells and for eliciting a focused or more directed tolerogenic immune response to the target antigen. The antigen-presenting cells, which may be autologous or allogeneic, can be contacted with the antigen ex vivo. Alternatively, the antigen- presenting cells, which may be autologous or allogeneic, can be contacted with the antigen in vivo and in illustrative examples of this type, the antigen is administered to the subject in particulate form. In some embodiments, the condition is from autoimmune diseases (e.g., type 1 diabetes), organ specific diseases and allergies.
[0029] In some embodiments, the methods further comprise, prior to increasing the number of the operable NF-κB antigen-presenting cells in the subject, the step of diagnosing the presence, stage or degree or risk of development of the undesirable or deleterious immune response by detecting in the subject aberrant (e.g., reduced or abrogated) signaling through the NF-κB pathway, suitably in response to a pro-inflammatory signal, as explained in more detail below.
[0030] The invention also encompasses the use of an immune modulator as broadly described above and optionally an antigen that corresponds to at least a portion of a target antigen that associates with the presence or risk of an undesirable or deleterious immune response and with defective NF-κB function in antigen-presenting cells, in the study and modulation of the immune response.
[003 IJ As noted above, the inventors have determined that antigen-presenting cells such as DC have impaired NF-κB activation, including defective ReIB activation, in certain undesirable or deleterious immune responses and have thus proposed that this defect is a surrogate marker for the presence of or predisposition to such immune responses. Accordingly, in another aspect, the present invention provides methods for diagnosing the presence or risk of development of an undesirable or deleterious immune response associated with defective NF-κB activation, including defective ReIB activation, in a subject. These methods generally comprise detecting in the subject aberrant (e.g., reduced or abrogated) signaling through the NF-κB pathway, suitably in response to a pro-inflammatory signal. In some embodiments, the undesirable or deleterious immune response is associated with a condition selected from autoimmune diseases, organ specific diseases and allergies. In specific embodiments, the condition is other than TlDM.
[0032] Aberrant signaling is suitably detected by directly or indirectly detecting aberrant expression of a gene belonging to the canonical or non-canonical NF- KB pathway (such genes are also referred to herein as "NF-κB pathway genes"). Typically, the aberrant expression is detected by: (1) measuring in a biological sample obtained from the subject the level or functional activity of an expression product of at least one NF-κB pathway gene and (2) comparing the measured level or functional activity of each expression product to the level or functional activity of a corresponding expression product in a reference sample obtained from one or more normal subjects or from one or more subjects lacking the undesirable or deleterious immune response, wherein a difference in the level or functional activity of the expression product in the biological sample as compared to the level or functional activity of the corresponding expression product in the reference sample is indicative of the presence or risk of development of the undesirable or deleterious immune response in the subject.
[0033] In some embodiments, the methods further comprise diagnosing the presence, stage or degree or risk of development of the undesirable or deleterious immune response in the subject when the measured level or functional activity of the or each expression product is different than the measured level or functional activity of the or each corresponding expression product. In these embodiments, the difference typically represents an at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, or even an at least about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% increase, or an at least about 10%, 20%, 30% 40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 97%, 98% or 99%, or even an at least about 99.5%, 99.9%, 99.95%, 99.99%, 99.995% or 99.999% decrease in the level or functional activity of an individual expression product as compared to the level or functional activity of an individual corresponding expression product. In illustrative examples of this type, the presence or risk of development of the unwanted or deleterious immune response is determined by detecting a decrease in the level or functional activity of an expression product of at least one NF-κB pathway gene, which is suitably selected from genes that encode BTK, LYN, BCR Igα, BCR Igβ, Syk, Blnk, PLCγ2, PKCβ, DAG, CARMAl, BCLlO, MALTl, PI3K, PIP3, AKT, COT, IKKα, IKKβ, IKKγ, NIK, RelA/p65, P105/p50, c-Rel, ReIB, P52, NIK, Leul3, CD81, CD19, CD21 and its ligands in the complement and coagulation cascade, TRAF6, ubiquitin ligase, Tab2, TAKl, NEMO, NOD2, RIP2, Lck, fyn, Zap70, LAT, GRB2, SOS, CD3 zeta, Slp-76, GADS, ITK, PLCγl, PKCΘ, ICOS, CD28, SHP-2, SAP, SLAM, PKR and 2B4. In specific embodiments, the presence or risk of development of the unwanted or deleterious immune response is determined by detecting a decrease in the level or functional activity of an expression product of at least one gene selected from genes that encode RelA/p65, P105/p50, c-Rel, ReIB or p52. In other illustrative examples, the presence or risk of development of the unwanted or deleterious immune response is determined by detecting an increase in the level or functional activity of an expression product of at least one NF-κB pathway gene, which is suitably selected from genes that encode SHP- 1, SHIP, PIR-B, CD22, CD72, FcgRIIB, IKB, PlOO, CTLA4, CDIa, TGF-β, PD-I, CbI, KIR3DL1, KIR3DL2, KIR2DL and Csk. In specific embodiments, the presence or risk of development of the unwanted or deleterious immune response is determined by detecting an increase in the level or functional activity of an expression product of a gene that is common to both the canonical and non-canonical NF-κB pathways. In illustrative examples of this type, the gene is the SHP-I gene.
[0034] In some embodiments, the methods further comprise diagnosing the absence of the unwanted or deleterious immune response or a low risk of developing that immune response when the measured level or functional activity of the or each expression product is the same as or similar to the measured level or functional activity of the or each corresponding expression product. In these embodiments, the measured level or functional activity of an individual expression product varies from the measured level or functional activity of an individual corresponding expression product by no more than about 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.1%.
[0035] As used herein, polynucleotide expression products of NF-κB pathway genes are referred to herein as "NF-κB pathway polynucleotides." Polypeptide expression products of NF-κB pathway genes are referred to herein as "NF-κB pathway polypeptides."
[0036] In some embodiments, the methods comprise measuring the level or functional activity of individual expression products of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or even 30, 40 or 50 NF-κB pathway genes. For example, the methods may comprise measuring the level or functional activity of a
NF-κB pathway polynucleotide either alone or in combination with as much as 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 other NF-κB pathway polynucleotide(s). In another example, the methods may comprise measuring the level or functional activity of a NF-κB pathway polypeptide either alone or in combination with as much as 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 other NF-κB pathway polypeptides(s).
[0037] Advantageously, the biological sample comprises blood, especially peripheral blood, which suitably includes leukocytes. In certain embodiments, the expression product is selected from a RNA molecule or a polypeptide. In some embodiments, the expression product is the same as the corresponding expression product. In other embodiments, the expression product is a variant (e.g., an allelic variant) of the corresponding expression product. In illustrative examples, the biological sample comprises cells, which have been exposed to a pro-inflammatory signal. In these examples, the reference sample also comprises cells, which have been exposed to the pro-inflammatory signal.
[0038] In certain embodiments, the expression product or corresponding expression product is a target RNA (e.g., mRNA) or a DNA copy of the target RNA whose level is measured using at least one nucleic acid probe that hybridizes under at least low, medium, or high stringency conditions to the target RNA or to the DNA copy, wherein the nucleic acid probe hybridizes to at least 15 contiguous nucleotides of a NF- KB pathway polynucleotide. In these embodiments, the measured level or abundance of the target RNA or its DNA copy is normalized to the level or abundance of a reference RNA or a DNA copy of the reference RNA that is present in the same sample. Suitably, the nucleic acid probe is immobilized on a solid or semi-solid support. In illustrative examples of this type, the nucleic acid probe forms part of a spatial array of nucleic acid probes. In some embodiments, the level of nucleic acid probe that is bound to the target RNA or to the DNA copy is measured by hybridization (e.g. , using a nucleic acid array). In other embodiments, the level of nucleic acid probe that is bound to the target RNA or to the DNA copy is measured by nucleic acid amplification (e.g. , using a polymerase chain reaction (PCR)). In still other embodiments, the level of nucleic acid probe that is bound to the target RNA or to the DNA copy is measured by nuclease protection assay. [0039] In other embodiments, the expression product or corresponding expression product is a NF-κB pathway polypeptide whose level is measured using at least one antigen-binding molecule that is immuno-interactive with the NF-κB pathway polypeptide. In these embodiments, the measured level of the NF-κB pathway polypeptide is normalized to the level of a reference polypeptide that is present in the same sample. Suitably, the antigen-binding molecule is immobilized on a solid or semisolid support. In illustrative examples of this type, the antigen-binding molecule forms part of a spatial array of antigen-binding molecule. In some embodiments, the level of antigen-binding molecule that is bound to the NF-κB pathway polypeptide is measured by immunoassay (e.g., using an ELISA).
[0040] In still other embodiments, the expression product or corresponding expression product is a NF-κB pathway polypeptide whose level is measured using at least one substrate for that polypeptide with which it reacts to produce a reaction product. In these embodiments, the measured functional activity of the NF-κB pathway polypeptide is normalized to the functional activity of a reference polypeptide that is present in the same sample.
[0041] In other embodiments, the expression product or corresponding expression product is a NF-κB pathway polypeptide whose level is measured using at least one oligonucleotide that binds to a nucleic acid binding site of the marker polypeptide (e.g., ReIB, p65, p50 etc). In illustrative examples, the oligonucleotide comprises a detectable label and the level of oligonucleotide that is bound to the NF-κB pathway polypeptide is measured by quantifying the amount of detectable label that associates with the polypeptide (e.g., by autoradiography, fiuorimetry, luminometry, or phosphoimage analysis). [0042] In other embodiments, aberrant signaling of the NF-κB pathway is detected by detecting aberrant phosphorylation of a polypeptide involved in or belonging to the NF-κB signaling pathway. In specific embodiments, aberrant phosphorylation is detected by: (1) determining in a biological sample obtained from the subject the phosphorylation state of at least one phosphorylatable polypeptide selected from IRAK, TAK 1 , TAB 1 , TAB2, PKR, Akt, Cot, IKKα, IKKβ and IKKγ; and (2) comparing each determined phosphorylation state to the phosphorylation state of a corresponding phosphorylatable polypeptide in a reference sample obtained from one or more normal subjects or from one or more subjects lacking disease, wherein a difference in the phosphorylation state of the phosphorylatable polypeptide in the biological sample as compared to the phosphorylation state of the corresponding phosphorylatable polypeptide in the reference sample is indicative of the presence or risk of development of the unwanted or deleterious immune response in the subject. [0043] In another aspect, the present invention provides methods for treating, preventing or inhibiting the development of the unwanted or deleterious immune response in a subject. These methods generally comprise detecting in the subject aberrant signaling through the NF-κB pathway, suitably in response to an inflammatory signal, as broadly described above and administering to the subject an effective amount of an agent that treats or ameliorates the symptoms or reverses or inhibits the development of the unwanted or deleterious immune response in the subject. Representative examples of such treatments or agents include, but are not limited to, anti-CD3 therapy (e.g., monoclonal antibody hOKT3gammal (Ala- AIa)) and antigen- specific tolerogenic therapies. [0044] In still another aspect, the present invention provides probes for interrogating nucleic acid for the presence of a NF-κB pathway polynucleotide as broadly described above. These probes generally comprise a nucleotide sequence that hybridizes under at least low stringency conditions to a NF-κB pathway polynucleotide as broadly described above. [0045] In yet another aspect, the present invention provides oligonucleotides for interrogating protein samples for the presence of a NF-κB pathway polypeptide that comprises a nucleic acid binding site. These oligonucleotides generally comprise a nucleotide sequence that binds to the nucleic acid binding site of the NF-κB pathway polypeptide (e.g., ReIB, p65, p50 etc). [0046] In related aspects, the invention provides a solid or semi-solid support comprising at least one nucleic acid probe as broadly described above or at least one polypeptide-binding oligonucleotide as broadly described above immobilized to the support. In some embodiments, the solid or semi-solid support comprises a spatial array of nucleic acids immobilized thereon. [0047] Still a further aspect of the present invention provides an antigen- binding molecule that is immuno-interactive with a NF-κB pathway polypeptide as broadly described above. [0048] In a related aspect, the invention provides a solid or semi-solid support comprising at least one antigen-binding molecule as broadly described above immobilized thereon. In some embodiments, the solid or semi-solid support comprises a spatial array of antigen-binding molecules immobilized thereon. [0049] Still another aspect of the invention provides the use of one or more
NF-κB polynucleotides as broadly described above, or the use of one or more probes as broadly described above, or the use of one or more NF-κB pathway polypeptides as broadly described above, or the use of one or more antigen-binding molecules as broadly described above, or the use of one or more polypeptide-binding oligonucleotides as broadly described above, in the manufacture of a kit for diagnosing the presence of or predisposition to an unwanted or deleterious immune response associated with defective NF-κB function, including defective ReIB function, in a subject.
[0050] Yet further aspects of the invention are directed to the use of the diagnostic methods as broadly described above, or one or more NF-κB pathway polynucleotides as broadly described above, or the use of one or more probes as broadly described above, or the use of one or more NF-κB pathway polypeptides as broadly described above, or the use of one or more antigen-binding molecules as broadly described above, or the use of one or more polypeptide-binding oligonucleotides as broadly described above, for diagnosing the presence or risk of development of an unwanted or deleterious immune response associated with defective NF-κB function, including defective ReIB function, in animals, especially vertebrates animals including mammals.
BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES
[0051] Figure 1 is a graphical representation showing reduced LPS-induced NF-κB activity in TlDM DC. (A) Cytoplasmic or nuclear extracts were prepared from DC cultured for 72h from TlDM patients or healthy control (HC) subjects without (-) or with (+) addition of 100 ng/ml LPS for the last 24 h of culture, or from a positive control lymphoblastoid cell line (LCL). After electrophoresis, membranes were immunoblotted with NF-κB subunit antibodies, or with Ponceau S protein stain Representative of 5 experiments analyzing 5 individual pairs of donors. (B) 10 μg nuclear extract from DC prepared from healthy controls (HC) or patients with TlDM with addition of 100 ng/ml LPS for varying periods of time were bound to wells of a NF-κB oligonucleotide-coated ELISA plate, and revealed with anti-RelB. Absorbance expressed in relative units. ReIB DNA binding was compared within groups at time 0 and 24h, and between groups at 24h. ** p < 0.01, *** p < 0.001. (C) nuclear extracts from DC prepared from HC or patients with TlDM, RA or T2D incubated with (LPS) or without (UT) LPS for 24 h. ReIB DNA binding assessed as in (b). Absorbance expressed in relative units. * p <0.05, ** p < 0.01, *** p < 0.001.
[0052] Figure 2 is a graphical representation showing that SHP-I expression is increased in PBMC of TlDM patients. (A) PBMC from 13 healthy control (HC) subjects, 18 Tl DM and 6 T2DM patients were cultured for 48h. DC from healthy controls or TlDM patients were harvested at 72 h. All cells were then permeabilised and stained for SHP-I . Data are expressed as Δ MFI, in gated monocytes or DC. ** p < 0.01, * p <0.05. (B) PBMC from 31 healthy control (HC) or TlDM patients were cultured for 48h, then permeabilised and stained for SHP-I. Δ MFI of SHP-I was determined for gated lymphocytes and monocytes in each sample, and then plotted. R =0.4573, p < 0.0001.
[0053] Figure 3 is a graphical representation showing that SHP-I inhibits NF- kB activity in TlDM. (A) PBMC from 7 healthy control (HC) subjects and 4 TlDM patients were cultured for 30 min without (UT) or with 100 ng/ml LPS, then permeabilised and stained for phospho-I-κBα. Data are expressed as Δ MFI, in gated monocytes. ** p < 0.01. (B) TlDM monocytes were cultured with GM-CSF and IL-4 with or without 10 μg/ml sodium stibogluconate for 48 hours, then cells were analyzed for phospho-IκBα by flow cytometry. Data are representative of three experiments, (c) TlDM monocytes were cultured with GM-CSF and IL-4 with or without 10 μg/ml sodium stibogluconate for 48 hours, with LPS added for the last 24 hours. CD40 expression was compared, with relative values shown for 2 individuals. TlDM PBMC cultured with or without SHP-I inhibitor and LPS for 24 hours were analyzed by ELISA for nuclear ReIB DNA binding, with relative values shown for 2 individuals.
[0054] Figure 4 is a graphical representation showing that adoptive transfer of wt DC reverses inflammatory organ pathology: (A) 12.5 x 106 bead-purified or 2 x 106 sorted DC from progenipoietin-1 -treated RelB+/" mice were injected i.v into ReIB7" mice. Recipients were monitored serially for weight gain and well being. Untreated ReIB+ " mice and ReIB" " mice served as controls. The % cumulative weight gain above individual baseline weight, and final weights at the end of the monitoring period are shown. *** p < 0.001, ** p < 0.01 comparing ReIB"'" with and without DC treatment. (B) Organs collected from RelB+/", ReIB7" and ReIB7" mice previously treated with either RelB+/" DC or anti-GITRL were fixed in formalin, sectioned and stained with either H&E, or anti-Brdu (revealed with diaminobenzidine) then counterstained with hematoxylin. Arrows indicate inflammatory infiltrate and arrow head (spleen) depicts megakaryocyte. The scale bar depicts 100 μm for all sections. Results are representative of two experiments containing two mice each per group.
[0055] Figure 5 is a graphical representation showing defective ReIB activation in Sjogren's patients. DCs derived from healthy controls or from patients with Sjogren's syndrome were cultured for 72 h ex vivo. For the final 24 h of cell culture, DCs were incubated with or without 100 ng/ml LPS. Nuclear extracts from the DCs were bound to wells of a NF-κB oligonucleotide-coated ELISA plate (10 μg per well), and revealed with antibodies against either ReIB, ReIA or p50. Light output was measured in photon units after reading for 5 seconds. DNA binding of NF-κB following LPS exposure (LPS) was compared with DNA binding without LPS (nil) in cells derived from representative healthy control or Sjogren's syndrome subjects, and expressed in photon units (Figure 5A) or as fold change in DNA binding by NF-κB after LPS treatment in cells derived from healthy control and Sjogren's syndrome subjects (Figure 5B).
[0056] Figure 6A is a graphical representation showing defective ReIB, ReIA, p50 and c-Rel activation in a representative patient with type 1 diabetes mellitus (TlDM). DCs derived from a healthy control subject or from a patients with TlDM were cultured for 72 h ex vivo. For the final 24 h of cell culture, DCs were incubated with or without
100 ng/ml LPS. Nuclear extracts from the DCs were bound to wells of a NF-κB oligonucleotide-coated ELISA plate (2-10 μg per well), and revealed with antibodies against either ReIB, ReIA, p50, c-Rel or p52. Light output was measured in photon units after reading for 5 seconds. DNA binding of NF-κB following LPS exposure was compared with DNA binding without LPS (untreated, UT) in cells derived from a representative healthy control and TlDM subject, and expressed in photon units.
[0057] Figure 6B is a graphical representation showing defective LPS- induced ReIA, ReIB, c-Rel and p50 activation over time in representative patients with TlDM. Nuclear extracts from DCs prepared from two healthy controls and two TlDM patients were incubated with LPS for between 0 and 24 h. DNA binding by ReIA, p50, c-Rel or ReIB was assessed at intervals and expressed in photon units. Representative of data from nine individuals from each group in 6A and 6B.
[0058] Figure 7A is a graphical representation showing defective ReIB, ReIA, and p50 activation in TlDM monocytes. Peripheral blood monocytes from healthy control subjects or TlDM patients were cultured with or without LPS for 24h. Nuclear extracts were prepared and tested for NF-κB binding to DNA as described in Figure 6.
Data are expressed as fold change in NF-κB binding to DNA after LPS treatment.
Values for cells derived from healthy controls and TlDM patients were compared by Mann Whitney test. *** p O.001, *p < 0.05.
[0059] Figure 7B is a graphical representation showing increased ReIB and ReIA DNA binding at baseline (without LPS) in TlDM monocytes. DNA binding of ReIB, ReIA, c-Rel and p50 for 8 untreated healthy control subjects and 16 TlDM subjects' PB monocytes is shown with median, p <0.05 comparing ReIB and ReIA medians of healthy and Tl DM subjects by Mann Whitney test.
[0060] Figure 8 is a graphical representation showing defective LPS-induced ReIB activation of DCs of siblings of a TlDM proband (child), but not their father. Nuclear extracts from DCs prepared from first degree relatives of a child with TlDM were incubated with or without LPS for 24 h as described in Figure 6. DNA binding by ReIB was assessed at 24h and expressed in photon units. DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0061] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.
[0062] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.
[0063] The term "aberrant expression" is used herein to describe the over- expression or under-expression of genes belonging to the NF-κB pathway, relative to the level of expression of the such genes in cells obtained from a healthy subject or from a subject lacking an unwanted or deleterious immune response as defined herein, and/or to a higher or lower level or functional activity of a NF-κB pathway gene product (e.g., transcript or polypeptide) in a tissue sample or body fluid obtained from a healthy subject or from a subject lacking the unwanted or deleterious immune response. In particular, a specific NF-κB pathway gene is aberrantly expressed if the level of expression of such a gene product is at least about 10% ( X0 ), 20% ( X ), 30% ( X0 ), 40% ( % ), 50% ( y2 ), 60% ( % ), 70% ( X0 ), 80% ( % ) or 90% ( X0 ), or even at least about 100% (1-fold), 200% (2-fold), 300% (3-fold), 400% (4-fold), 500% (5-fold), 600% (6-fold), 700% (7-fold), 800% (8-fold), 900% (9-fold) or 1000% (10-fold) higher than the level of expression of a corresponding NF-κB pathway gene product in a tissue sample or body fluid obtained from a healthy subject or from a subject without the unwanted or deleterious immune response. Alternatively, a specific NF-κB pathway gene is aberrantly expressed if the level of expression of such a gene product is about Xo 5 A i Xo 5 A ■> Yi ■> A ■> Xo J /5 ' Xo ' or evεπ about χ00 , j4oo » /300 ■> /400 > Xoo » Xoo > /700 > Xoo > Xoo > Xooo or less of the level of expression of a corresponding NF-κB pathway gene product in a tissue sample or body fluid obtained from a healthy subject or from a subject without an unwanted or deleterious immune response as defined herein. The term "aberrant expression" also encompasses aberrant expression of 'downstream' targets of NF-κB transcriptional regulation including pro-inflammatory cytokines (e.g., IL-I, IL-6, IL-8 and TNF), chemokines, adhesion molecules, matrix metalloproteinases (MMPs), cyclooxygenase 2 (COX2) and inducible nitric oxide synthase (iNOS).
[0064] By "about" is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
[0065] The terms "administration concurrently" or "administering concurrently" or "co-administering" and the like refer to the administration of a single composition containing two or more actives, or the administration of each active as separate compositions and/or delivered by separate routes either contemporaneously or simultaneously or sequentially within a short enough period of time that the effective result is equivalent to that obtained when all such actives are administered as a single composition. By "simultaneously" is meant that the active agents are administered at substantially the same time, and desirably together in the same formulation. By
"contemporaneously" it is meant that the active agents are administered closely in time, e.g., one agent is administered within from about one minute to within about one day before or after another. Any contemporaneous time is useful. However, it will often be the case that when not administered simultaneously, the agents will be administered within about one minute to within about eight hours and preferably within less than about one to about four hours. When administered contemporaneously, the agents are suitably administered at the same site on the subject. The term "same site" includes the exact location, but can be within about 0.5 to about 15 centimeters, preferably from within about 0.5 to about 5 centimeters. The term "separately" as used herein means that the agents are administered at an interval, for example at an interval of about a day to several weeks or months. The active agents may be administered in either order. The term "sequentially" as used herein means that the agents are administered in sequence, for example at an interval or intervals of minutes, hours, days or weeks. If appropriate the active agents may be administered in a regular repeating cycle. [0066] As used herein, the term "agent" refers to an organic or inorganic chemical and in some cases refers to a biochemical substance that includes, but is not limited to, proteins, peptides or amino acids; nucleic acids such as DNA, such as full- length genes or fragments thereof derived from genomic, cDNA or artificial coding sequences, gene regulatory elements, RNA, including mRNA, tRNA, ribosomal RNA, ribozymes and antisense RNA, oligonucleotides and oligoribonucleotides, deoxyribonucleotides and ribonucleotides; carbohydrates; lipids; proteoglycans; such agents may be administered as isolated (purified) compounds or in crude mixtures, such as in a tissue, cell or cell lysate.
[0067] The term "amplicon" refers to a target sequence for amplification, and/or the amplification products of a target sequence for amplification. In certain other embodiments an "amplicon" may include the sequence of probes or primers used in amplification. [0068] The term "anergy" as used herein refers to a suppressed response, or a state of non-responsiveness, to a specified antigen or group of antigens by an immune system. For example, T lymphocytes and B lymphocytes are anergic when they cannot respond to their specific antigen under optimal conditions of stimulation.
[0069] By "antigen" is meant all, or part of, a protein, peptide, or other molecule or macromolecule capable of eliciting an immune response in a vertebrate animal, especially a mammal. Such antigens are also reactive with antibodies from animals immunized with that protein, peptide, or other molecule or macromolecule.
[0070] By "antigen-binding molecule" is meant a molecule that has binding affinity for a target antigen. It will be understood that this term extends to immunoglobulins, immunoglobulin fragments and non-immunoglobulin derived protein frameworks that exhibit antigen-binding activity.
[0071] By "autologous" is meant something (e.g., cells, tissues etc) derived from the same organism.
[0072] The term "allogeneic" as used herein refers to cells, tissues, organisms etc that are of different genetic constitution.
[0073] By "alloantigen" is meant an antigen found only in some members of a species, such as blood group antigens. By contrast a "xenoantigen" refers to an antigen that is present in members of one species but not members of another. Correspondingly, an "allograft" is a graft between members of the same species and a "xenograft" is a graft between members of a different species.
[0074] As used herein, the term "basal state" as applied to NF-κB or ReIB function refers to the level of activity of the canonical and/or non-canonical NF-κB pathways, or to the level of activity of ReIB, in antigen-presenting cells of normal individuals or of individuals that lack the undesirable or deleterious immune response, as defined herein. Accordingly, the term "basal state" includes and encompasses a "normal level" of activity of the canonical and/or non-canonical NF-κB pathways, or of ReIB.
[0075] The term "biological sample" as used herein refers to a sample that may be extracted, untreated, treated, diluted or concentrated from an animal. The biological sample may include a biological fluid such as whole blood, serum, plasma, saliva, urine, sweat, ascitic fluid, peritoneal fluid, synovial fluid, amniotic fluid, cerebrospinal fluid, tissue biopsy, and the like. In certain embodiments, the biological sample is blood, especially peripheral blood.
[0076] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term
"comprising" and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of. Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.
[0077] By "corresponds to" or "corresponding to" is meant an antigen which encodes an amino acid sequence that displays substantial similarity to an amino acid sequence in a target antigen. In general the antigen will display at least about 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % similarity to at least a portion of the target antigen.
[0078] By "effective amount," in the context of modulating an immune response or treating or preventing a disease or condition, is meant the administration of that amount of composition to an individual in need thereof, either in a single dose or as part of a series, that is effective for that modulation, treatment or prevention. The effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
[0079] The terms "expression" or "gene expression" refer to either production of RNA message or translation of RNA message into proteins or polypeptides. Detection of either types of gene expression in use of any of the methods described herein are part of the invention.
[0080] As used herein, the term "functional activity" generally refers to the ability of a molecule (e.g., a transcript or polypeptide) to perform its designated function including a biological, enzymatic, or therapeutic function. In certain embodiments, the functional activity of a molecule corresponds to its specific activity as determined by any suitable assay known in the art.
[0081] The term "gene" is used in its broadest context to include both a genomic DNA region corresponding to the gene as well as a cDNA sequence corresponding to exons or a recombinant molecule engineered to encode a functional form of a product.
[0082] Reference herein to "immuno-interactive" includes reference to any interaction, reaction, or other form of association between molecules and in particular where one of the molecules is, or mimics, a component of the immune system.
[0083] Reference herein to "a level or functional activity" in the context of a gene expression product (e.g., a protein or a transcript) produced by a specified cell is to be taken in its broadest sense and includes a level or functional activity of the expression product that is produced in a single cell or in a plurality or population of cells. In the latter case, therefore, it will be understood that the phrase will encompass a mean level or functional activity of the protein produced by a plurality or population of cells.
[0084] By "modulating" is meant increasing or decreasing, either directly or indirectly, the immune response of an individual. [0085] As used herein, the term "normal activity" refers to a value that is at least 25% of the activity of one or more of NF-κB and its subunits (e.g., p50, plO5, p52, p 100, ReIA, ReIB and cRel) observed in the basal state, as defined herein, suitably in the range of 30-90% and more suitably in the range of 95-100%, and all integer percentages therebetween.
[0086] By "obtained from" is meant that a sample such as, for example, a cell extract or nucleic acid or polypeptide extract is isolated from, or derived from, a particular source. For instance, the extract may be isolated directly from biological fluid or tissue of the subject. [0087] The terms "patient," "subject," "host" or "individual" used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates, rodents {e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards etc), and fish. A preferred subject is a human in need of treatment or prophylaxis for a condition or disease, which is associated with the presence or aberrant expression of an antigen of interest. However, it will be understood that the aforementioned terms do not imply that symptoms are present.
[0088] By "pharmaceutically-acceptable carrier" is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in topical or systemic administration.
[0089] The term "polynucleotide" or "nucleic acid" as used herein designates mRNA, RNA, cRNA, cDNA or DNA. The term typically refers to oligonucleotides greater than 30 nucleotides in length.
[0090] "Polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers.
[0091] By "primer" is meant an oligonucleotide which, when paired with a strand of DNA, is capable of initiating the synthesis of a primer extension product in the presence of a suitable polymerizing agent. The primer is preferably single-stranded for maximum efficiency in amplification but can alternatively be double-stranded. A primer must be sufficiently long to prime the synthesis of extension products in the presence of the polymerization agent. The length of the primer depends on many factors, including application, temperature to be employed, template reaction conditions, other reagents, and source of primers. For example, depending on the complexity of the target sequence, the primer may be at least about 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500, to one base shorter in length than the template sequence at the 3' end of the primer to allow extension of a nucleic acid chain, though the 5' end of the primer may extend in length beyond the 3' end of the template sequence. In certain embodiments, primers can be large polynucleotides, such as from about 35 nucleotides to several kilobases or more. Primers can be selected to be "substantially complementary" to the sequence on the template to which it is designed to hybridize and serve as a site for the initiation of synthesis. By "substantially complementary", it is meant that the primer is sufficiently complementary to hybridize with a target polynucleotide. Desirably, the primer contains no mismatches with the template to which it is designed to hybridize but this is not essential. For example, non-complementary nucleotide residues can be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the template. Alternatively, non-complementary nucleotide residues or a stretch of non- complementary nucleotide residues can be interspersed into a primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize therewith and thereby form a template for synthesis of the extension product of the primer.
[0092] By "regulatory lymphocyte" is meant a lymphocyte that is involved in regulating or suppressing responses and actions of other cells, especially of other immune cells such as B lymphocytes and T helper lymphocytes.
[0093] "Probe" refers to a molecule that binds to a specific sequence or subsequence or other moiety of another molecule. Unless otherwise indicated, the term '"probe" typically refers to a polynucleotide probe that binds to another polynucleotide, often called the "'target polynucleotide", through complementary base pairing. Probes can bind target polynucleotides lacking complete sequence complementarity with the probe, depending on the stringency of the hybridization conditions. Probes can be labeled directly or indirectly and include primers within their scope.
[0094] As used herein, the term "related" when used with respect to subjects indicates that the subjects are known to share a common line of descent: that is, the subjects have a known ancestor in common. Illustrative examples of related subjects include siblings (brothers and sisters), parents, grandparents, children, grandchildren, aunts, uncles, cousins, second cousins and third cousins. Subjects less closely related than third cousins are typically not sufficiently related to be useful as "related" subjects for the methods of this invention, even if they share a known ancestor, unless some related individuals that lie between the distantly related subjects are also included. Thus, for a group of related individuals, each subject shares a known ancestor within three generations or less with at least one other subject in the group, and desirably with all other subjects in the group or has at least that degree of consanguinity due to multiple known common ancestors. In specific embodiments, subjects share a common ancestor within two generations or less, or otherwise have equivalent level of consanguinity. Conversely, as used herein the term "unrelated," when used in respect of subjects, refers to subjects who do not share a known ancestor within 3 generations or less, or otherwise have known relatedness at that degree.
[0095] By "suppression," "suppressing" and the like is meant any attenuation or regulation of an immune response, including B-lymphocyte and T lymphocyte immune responses, to an antigen or group of antigens. In some embodiments, the attenuation is mediated at least in part by suppressor T lymphocytes (e.g., CD4+CD25+ regulatory T lymphocytes).
[0096] As used herein the term "surrogate marker" refers to a biological or clinical parameter that is measured in place of the biologically definitive or clinically most meaningful parameter. In comparison to definitive markers, surrogate markers are generally either more convenient, less expensive, provide earlier information or provide pharmacological or physiological information not directly obtainable with definitive markers. Non-limiting examples of surrogate biological parameters include testing cell NF-κB levels in subjects having or at risk of developing type 1 diabetes, whereas examples of definitive biological parameters include insulin independence, blood glucose and C peptide production. The measurement of a surrogate marker may be an endpoint in a clinical study or clinical trial, hence "surrogate endpoint".
[0097] The term "template" as used herein refers to a nucleic acid that is used in the creation of a complementary nucleic acid strand to the "template" strand. The template may be either RNA and/or DNA, and the complementary strand may also be RNA and/or DNA. In certain embodiments, the complementary strand may comprise all or part of the complementary sequence to the "template," and/or may include mutations so that it is not an exact, complementary strand to the "template". Strands that are not exactly complementary to the template strand may hybridize specifically to the template strand in detection assays described here, as well as other assays known in the art, and such complementary strands that can be used in detection assays are part of the invention.
[0098] By "treatment, " "treat," "treated" and the like is meant to include both therapeutic and prophylactic treatment.
[0099] The terms "wild-type" and "normal" are used interchangeably to refer to the phenotype that is characteristic of most of the members of the species occurring naturally and contrast for example with the phenotype of a mutant.
[0100] As used herein, underscoring or italicizing the name of a gene shall indicate the gene, in contrast to its protein product, which is indicated by the name of the gene in the absence of any underscoring or italicizing. For example, "ReIB" shall mean the ReIB gene, whereas "ReIB" shall indicate the protein product or products generated from transcription and translation and alternative splicing of the "ReIB" gene.
2. Methods of detecting undesirable or deleterious immune responses [0101] The present invention arises in part from the determination that certain undesired or deleterious immune responses, including particular autoimmune diseases, organ-specific diseases, and allergic disorders either constitutively or as a result of interaction between pathogen or allergen with antigen presenting cells, are associated with defective NF-κB function through aberrant signaling of the canonical and/or non- canonical NF-κB pathways. Accordingly, the invention provides methods for diagnosing the presence or risk of development in the subject of an undesirable or deleterious immune response that associates with defective NF-κB function {e.g., defective ReIB function), wherein these methods generally comprise detecting (e.g., directly or indirectly) a reduced or abrogated level or functional activity of NF-κB or one of its subunits (e.g., p50, plO5, p52, plOO, ReIA, ReIB and cRel), as compared to a reference level or functional activity corresponding to a basal state of NF-κB function or one of its subunits, which is indicative of the presence of the undesirable or deleterious immune response.
[0102] To facilitate detection, it is generally desirable to qualitatively or quantitatively determine in a biological sample the level of expression of one or more NF-κB pathway genes, illustrative examples of which are listed above, by detecting for example the level or functional activity of an expression product of those genes, e.g., the level or functional activity of a transcript or the level or functional activity of a polypeptide. In some embodiments, the presence, degree, stage or risk of development of undesirable or deleterious immune response is detected when an NF-κB pathway gene is expressed at a detectably lower level in the biological sample as compared to the level at which that gene is expressed in a reference sample obtained from normal subjects or from subjects lacking the undesirable or deleterious immune response. In illustrative examples of this type, the gene is selected from genes encoding BTK, LYN, BCR Igα, BCR Igβ, Syk, Blnk, PLCγ2, PKCβ, DAG, CARMAl, BCLlO, MALTl, PI3K, PIP3, AKT, COT, IKKα, IKKβ, IKKγ, NIK, RelA/p65, P105/p50, c-Rel, ReIB, p52, NIK, Leul3, CD81, CD 19, CD21 and its ligands in the complement and coagulation cascade, TRAF6, and other ubiquitin ligases, Tab2, TAKl, NEMO, NOD2, RIP2, Lck, fyn, Zap70, LAT, GRB2, SOS, CD3 zeta, Slp-76, GADS, ITK, PLCγl, PKCΘ, ICOS, CD28, SHP-2, SAP, SLAM and 2B4. In other embodiments, the presence, degree, stage or risk of development of the undesirable or deleterious immune response is detected when an NF-κB pathway gene is expressed at a detectably higher level in the biological sample as compared to the level at which that gene is expressed in a reference sample obtained from normal subjects or from subjects lacking the deleterious or unwanted immune response. In illustrative examples of this type, the gene is selected from genes encoding SHP-I, SHIP, PIR-B, CD22, CD72, FcgRIIB, IKB, PlOO, CTLA4, PD-I, CbI, KIR3DL1, KIR3DL2, KIR2DL and Csk. Generally, the presence, degree, stage or risk of development of the undesirable or deleterious immune response is detected when the level or functional activity of an NF-κB pathway gene product in the biological sample varies from the level or functional activity of a corresponding NF-κB pathway gene product in the reference sample by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%, 96%, 97%, 98% or 99%, or even by at least about 99.5%, 99.9%, 99.95%, 99.99%, 99.995% or 99.999%, or even by at least about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000%. The corresponding gene product is generally selected from the same gene product that is present in the biological sample, a gene product expressed from a variant gene (e.g., an homologous or orthologous gene) including an allelic variant, or a splice variant or protein product thereof. In some embodiments, the method comprises measuring the level or functional activity of individual expression products of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19 or 20 NF-κB pathway genes. In specific embodiments, the method comprises measuring the level or functional activity of an expression product of the ReIB gene.
[0103] Alternatively, or in addition, defective NF-κB function may be determined either through failure of NF-κB to direct the transcription of downstream genes, physical characteristics or DNA- or protein-binding activity in comparison to those of the basal state. NF-κB activity may be assayed either in vivo or in vitro using an ELISA system of specific antibody to detect nuclear NF-κB subunit binding to a consensus oligonucleotide binding sequence. Alternatively activity can be detected using an NF-κB-dependent reporter gene expression construct and a substrate for enzymatic detection (such as chloramphenicol acetyl transferase or β-galactosidase, depending on the specificity of the enzyme encoded by the reporter gene), wherein comparative quantitation of the product of the diagnostic enzymatic reaction (or, in the absence of a reaction substrate, the level of the reporter mRNA or its encoded protein) in biological samples derived from a test subject and a normal control individual allow for the assessment of NF-κB functional loss. In other illustrative examples, immunological or other biochemical determination of whether or not IKB has been cleaved from NF-κB may be made.
[00100] The biological sample may contain blood, especially peripheral blood, or a fraction or extract thereof. Typically, the biological sample comprises blood cells such as mature, immature and developing leukocytes, including lymphocytes, polymorphonuclear leukocytes, neutrophils, monocytes, reticulocytes, basophils, coelomocytes, haemocytes, eosinophils, megakaryocytes, macrophages, dendritic cells natural killer cells, or fraction of such cells (e.g., a nucleic acid or protein fraction). In specific embodiments, the biological sample comprises leukocytes including peripheral blood mononuclear cells (PBMC).
[0104] The diagnostic tests of the invention may be used to diagnose the presence, degree, stage or risk of development of an undesirable or deleterious immune response in any subject, which response is associated with defective NF-κB function, including defective ReIB function. In specific embodiments, these tests are used with subjects related to a patient with clinical signs (one or more definitive markers) of the undesirable or deleterious immune response. These tests are desirably used at a stage and frequency to enable early the immune response, its progression, or to diagnose the risk of developing disease associated with that response.
[0105] The diagnostic tests of the invention may be used to diagnose the presence, degree, stage or risk of development of the unwanted or deleterious immune response in any subject. In specific embodiments, these tests are used with subjects related to a patient with clinical signs (one or more definitive markers) of the unwanted or deleterious immune response. These tests are desirably used at a stage and frequency to enable early detection of the unwanted or deleterious immune response or a condition related to that response, the progression of the immune response or related condition, or to diagnose the risk of developing that immune response or related condition.
2.1 Nucleic acid-based diagnostics [0106] Nucleic acids used in polynucleotide-based assays can be isolated from cells contained in the biological sample, according to standard methodologies (Sambrook, et al, 1989, supra; and Ausubel et al, 1994, supra). The nucleic acid is typically fractionated (e.g., poly A+ RNA) or whole cell RNA. Where RNA is used as the subject of detection, it may be desired to convert the RNA to a complementary DNA. In some embodiments, the nucleic acid is amplified by a template-dependent nucleic acid amplification technique. A number of template dependent processes are available to amplify the NF-κB pathway gene sequences present in a given template sample. An exemplary nucleic acid amplification technique is the polymerase chain reaction (referred to as PCR) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800, 159, Ausubel et al {supra), and in Innis et al. ("PCR Protocols", Academic Press, Inc., San Diego Calif., 1990). Briefly, in PCR, two primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence. An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase. If a cognate
TlDM marker sequence is present in a sample, the primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated. A reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified. Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al. , 1989, supra. Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641. Polymerase chain reaction methodologies are well known in the art.
[0107] In certain advantageous embodiments, the template-dependent amplification involves the quantification of transcripts in real-time. For example, RNA or DNA may be quantified using the Real-Time PCR technique (Higuchi, 1992, et al. , Biotechnology 10:413-417). By determining the concentration of the amplified products of the target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture. If the DNA mixtures are cDNAs synthesized from RNAs isolated from different tissues or cells, the relative abundance of the specific mRNA from which the target sequence was derived can be determined for the respective tissues or cells. This direct proportionality between the concentration of the PCR products and the relative mRNA abundance is only true in the linear range of the PCR reaction. The final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent of the original concentration of target DNA.
[0108] Another method for amplification is the ligase chain reaction ("LCR"), disclosed in EPO No. 320 308. In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCR, bound ligated units dissociate from the target and then serve as "target sequences" for ligation of excess probe pairs. U.S. Pat. No. 4,883,750 describes a method similar to
LCR for binding probe pairs to a target sequence. [0109] Qβ Replicase, described in PCT Application No. PCT/US87/00880, may also be used. In this method, a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence that can then be detected.
[0110] An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'α-thio-triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention, Walker et al, (1992, Proc. Natl. Acad. Sci. U.S.A 89:392-396).
[0111] Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation. A similar method, called Repair Chain Reaction (RCR), involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used in SDA. Target specific sequences can also be detected using a cyclic probe reaction (CPR). In CPR, a probe having 3' and 5' sequences of nonspecific DNA and a middle sequence of specific RNA is hybridized to DNA that is present in a sample. Upon hybridization, the reaction is treated with RNase H, and the products of the probe identified as distinctive products that are released after digestion. The original template is annealed to another cycling probe and the reaction is repeated.
[0112] Still another amplification method described in GB Application No. 2 202 328, and in PCT Application No. PCT/US89/01025, may be used. In the former application, "modified" primers are used in a PCR-like, template- and enzyme- dependent synthesis. The primers may be modified by labeling with a capture moiety {e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latter application, an excess of labeled probes are added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.
[0113] Other nucleic acid amplification procedures include transcription- based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et a!., 1989, Proc. Natl. Acad. Sci. U.S.A., 86: 1173; Gingeras et al., PCT Application WO 88/10315). In NASBA, the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a clinical sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA. These amplification techniques involve annealing a primer which has target specific sequences. Following polymerization, DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target specific primer, followed by polymerization. The double-stranded DNA molecules are then multiply transcribed by an RNA polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNAs are reverse transcribed into single stranded DNA, which is then converted to double stranded DNA, and then transcribed once again with an RNA polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate target specific sequences.
[0114] Davey et al, EPO No. 329 822 disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention. The ssRNA is a template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from the resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in duplex with either DNA or RNA). The resultant ssDNA is a template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5' to its homology to the template. This primer is then extended by DNA polymerase (exemplified by the large "Klenow" fragment of E. coli DNA polymerase I), resulting in a double-stranded DNA ("dsDNA") molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA. [0115] Miller et al. in PCT Application WO 89/06700 disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcription of many RNA copies of the sequence. This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts. Other amplification methods include "RACE" and "one-sided PCR" (Frohman, M. A., In: "PCR Protocols: A Guide to Methods and Applications", Academic Press, N. Y., 1990; Ohara et al, 1989, Proc. Natl Acad. Sci. U.S.A., 86:5673-567).
[0116] Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide, may also be used for amplifying target nucleic acid sequences. Wu et al, (1989, Genomics 4:560).
[0117] Depending on the format, the NF-κB pathway nucleic acid of interest is identified in the sample directly using a template-dependent amplification as described, for example, above, or with a second, known nucleic acid following amplification. Next, the identified product is detected. In certain applications, the detection may be performed by visual means {e.g., ethidium bromide staining of a gel). Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax Technology; Bellus, 1994, J Macromol. Sci. Pure, Appl. Chem., A31(l):1355-1376).
[0118] In some embodiments, amplification products or "amplicons" are visualized in order to confirm amplification of the NF-κB pathway gene sequences. One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light. Alternatively, if the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation. In some embodiments, visualization is achieved indirectly. Following separation of amplification products, a labeled nucleic acid probe is brought into contact with the amplified NF-κB pathway gene sequence. The probe is suitably conjugated to a chromophore but may be radiolabeled. Alternatively, the probe is conjugated to a binding partner, such as an antigen-binding molecule, or biotin, and the other member of the binding pair carries a detectable moiety or reporter molecule. The techniques involved are well known to those of skill in the art and can be found in many standard texts on molecular protocols (e.g., see Sambrook et al., 1989, supra and Ausubel et al. 1994, supra). For example, chromophore or radiolabel probes or primers identify the target during or following amplification. [0119] In certain embodiments, target nucleic acids are quantified using blotting techniques, which are well known to those of skill in the art. Southern blotting involves the use of DNA as a target, whereas Northern blotting involves the use of RNA as a target. Each provide different types of information, although cDNA blotting is analogous, in many aspects, to blotting or RNA species. Briefly, a probe is used to target a DNA or RNA species that has been immobilized on a suitable matrix, often a filter of nitrocellulose. The different species should be spatially separated to facilitate analysis. This often is accomplished by gel electrophoresis of nucleic acid species followed by "blotting" on to the filter. Subsequently, the blotted target is incubated with a probe (usually labeled) under conditions that promote denaturation and rehybridization. Because the probe is designed to base pair with the target, the probe will bind a portion of the target sequence under renaturing conditions. Unbound probe is then removed, and detection is accomplished as described above.
[0120] Following detection/quantification, one may compare the results seen in a given subject with a control reaction or a statistically significant reference group of normal subjects or of subjects lacking the unwanted or deleterious immune response. In this way, it is possible to correlate the amount of a NF-κB pathway gene nucleic acid detected with the progression or severity of the disease.
[0121] Also contemplated are genotyping methods and allelic discrimination methods and technologies such as those described by Kristensen et al (Biotechniques 30(2):318-322), including the use of single nucleotide polymorphism analysis, high performance liquid chromatography, TaqMan™, liquid chromatography, and mass spectrometry.
[0122] Also contemplated are biochip-based technologies such as those described by Hacia et al. (1996, Nature Genetics 14:441-447) and Shoemaker et al. (1996, Nature Genetics 14:450-456). Briefly, these techniques involve quantitative methods for analyzing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ biochip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization. See also Pease et al. (1994, Proc. Natl. Acad. Sci. U.S.A.
91 :5022-5026); Fodor et al. (1991, Science 251 :767-773). Briefly, nucleic acid probes to NF-κB pathway polynucleotides are made and attached to biochips to be used in screening and diagnostic methods, as outlined herein. The nucleic acid probes attached to the biochip are designed to be substantially complementary to specific expressed NF- KB pathway nucleic acids, i.e., the target sequence (either the target sequence of the sample or to other probe sequences, for example in sandwich assays), such that hybridization of the target sequence and the probes of the present invention occurs. This complementarity need not be perfect; there may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the nucleic acid probes of the present invention. However, if the number of mismatches is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence. In certain embodiments, more than one probe per sequence is used, with either overlapping probes or probes to different sections of the target being used. That is, two, three, four or more probes, with three being desirable, are used to build in a redundancy for a particular target. The probes can be overlapping (i.e. have some sequence in common), or separate.
[0123] As will be appreciated by those of ordinary skill in the art, nucleic acids can be attached to or immobilized on a solid support in a wide variety of ways. By "immobilized" and grammatical equivalents herein is meant the association or binding between the nucleic acid probe and the solid support is sufficient to be stable under the conditions of binding, washing, analysis, and removal as outlined below. The binding can be covalent or non-covalent. By "non-covalent binding" and grammatical equivalents herein is meant one or more of either, electrostatic, hydrophilic, and hydrophobic interactions. Included in non-covalent binding is the covalent attachment of a molecule, such as, streptavidin to the support and the non-covalent binding of the biotinylated probe to the streptavidin. By "covalent binding" and grammatical equivalents herein is meant that the two moieties, the solid support and the probe, are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds. Covalent bonds can be formed directly between the probe and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules. Immobilization may also involve a combination of covalent and non-covalent interactions. [0124] In general, the probes are attached to the biochip in a wide variety of ways, as will be appreciated by those in the art. As described herein, the nucleic acids can either be synthesized first, with subsequent attachment to the biochip, or can be directly synthesized on the biochip. [0125] The biochip comprises a suitable solid or semi-solid substrate or solid support. By "substrate" or "solid support" is meant any material that can be modified to contain discrete individual sites appropriate for the attachment or association of the nucleic acid probes and is amenable to at least one detection method. As will be appreciated by practitioners in the art, the number of possible substrates are very large, and include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, etc. In general, the substrates allow optical detection and do not appreciably fluorescese.
[0126] Generally the substrate is planar, although as will be appreciated by those of skill in the art, other configurations of substrates may be used as well. For example, the probes may be placed on the inside surface of a tube, for flow-through sample analysis to minimize sample volume. Similarly, the substrate may be flexible, such as a flexible foam, including closed cell foams made of particular plastics.
[0127] In certain embodiments, oligonucleotides probes are synthesized on the substrate, as is known in the art. For example, photoactivation techniques utilizing photopolymerization compounds and techniques can be used. In an illustrative example, the nucleic acids are synthesized in situ, using well known photolithographic techniques, such as those described in WO 95/25116; WO 95/35505; U.S. Pat. Nos.
5,700,637 and 5,445,934; and references cited within; these methods of attachment form the basis of the Affymetrix GeneChip™ technology.
[0128] In an illustrative biochip analysis, oligonucleotide probes on the biochip are exposed to or contacted with a nucleic acid sample suspected of containing one or more NF-κB pathway polynucleotides under conditions favoring specific hybridization. Sample extracts of DNA or RNA, either single or double-stranded, may be prepared from fluid suspensions of biological materials, or by grinding biological materials, or following a cell lysis step which includes, but is not limited to, lysis effected by treatment with SDS (or other detergents), osmotic shock, guanidinium isothiocyanate and lysozyme. Suitable DNA, which may be used in the method of the invention, includes cDNA. Such DNA may be prepared by any one of a number of commonly used protocols as for example described in Ausubel, et al. , 1994, supra, and Sambrook, et al, 1989, supra.
[0129] Suitable RNA, which may be used in the method of the invention, includes messenger RNA, complementary RNA transcribed from DNA (cRNA) or genomic or subgenomic RNA. Such RNA may be prepared using standard protocols as for example described in the relevant sections of Ausubel, et al. 1994, supra and Sambrook, et al. , 1989, supra).
[0130] cDNA may be fragmented, for example, by sonication or by treatment with restriction endonucleases. Suitably, cDNA is fragmented such that resultant DNA fragments are of a length greater than the length of the immobilized oligonucleotide probe(s) but small enough to allow rapid access thereto under suitable hybridization conditions. Alternatively, fragments of cDNA may be selected and amplified using a suitable nucleotide amplification technique, as described for example above, involving appropriate random or specific primers.
[0131] Usually the target NF-κB pathway polynucleotides are detectably labeled so that their hybridization to individual probes can be determined. The target polynucleotides are typically detectably labeled with a reporter molecule illustrative examples of which include chromogens, catalysts, enzymes, fluorochromes, chemiluminescent molecules, bioluminescent molecules, lanthanide ions (e.g., Eu34), a radioisotope and a direct visual label. In the case of a direct visual label, use may be made of a colloidal metallic or non-metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like. Illustrative labels of this type include large colloids, for example, metal colloids such as those from gold, selenium, silver, tin and titanium oxide. In some embodiments in which an enzyme is used as a direct visual label, biotinylated bases are incorporated into a target polynucleotide. Hybridization is detected by incubation with streptavidin-reporter molecules.
[0132] Suitable fluorochromes include, but are not limited to, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), R-Phycoerythrin (RPE), and Texas Red. Other exemplary fluorochromes include those discussed by Dower et al. (International Publication WO 93/06121). Reference also may be made to the fluorochromes described in U.S. Patents 5,573,909 (Singer et al), 5,326,692 (Brinkley et al). Alternatively, reference may be made to the fluorochromes described in U.S. Patent Nos. 5,227,487, 5,274,113, 5,405,975, 5,433,896, 5,442,045, 5,451,663, 5,453,517, 5,459,276, 5,516,864, 5,648,270 and 5,723,218. Commercially available fluorescent labels include, for example, fluorescein phosphoramidites such as Fluoreprime™ (Pharmacia), Fluoredite™ (Millipore) and FAM (Applied Biosystems International)
[0133] Radioactive reporter molecules include, for example, 32P, which can be detected by an X-ray or phosphoimager techniques.
[0134] The hybrid-forming step can be performed under suitable conditions for hybridizing oligonucleotide probes to test nucleic acid including DNA or RNA. In this regard, reference may be made, for example, to NUCLEIC ACID HYBRIDIZATION, A PRACTICAL APPROACH (Homes and Higgins, Eds.) (IRL press, Washington D. C, 1985). In general, whether hybridization takes place is influenced by the length of the oligonucleotide probe and the polynucleotide sequence under test, the pH, the temperature, the concentration of mono- and divalent cations, the proportion of G and C nucleotides in the hybrid-forming region, the viscosity of the medium and the possible presence of denaturants. Such variables also influence the time required for hybridization. The preferred conditions will therefore depend upon the particular application. Such empirical conditions, however, can be routinely determined without undue experimentation.
[0135] In certain advantageous embodiments, high discrimination hybridization conditions are used. For example, reference may be made to Wallace et al. (1979, Nucl. Acids Res. 6:3543) who describe conditions that differentiate the hybridization of 11 to 17 base long oligonucleotide probes that match perfectly and are completely homologous to a target sequence as compared to similar oligonucleotide probes that contain a single internal base pair mismatch. Reference also may be made to Wood et al. (1985, Proc. Natl. Acid. Sci. USA 82:1585) who describe conditions for hybridization of 11 to 20 base long oligonucleotides using 3M tetramethyl ammonium chloride wherein the melting point of the hybrid depends only on the length of the oligonucleotide probe, regardless of its GC content. In addition, Drmanac et al. {supra) describe hybridization conditions that allow stringent hybridization of 6-10 nucleotide long oligomers, and similar conditions may be obtained most readily by using nucleotide analogues such as 'locked nucleic acids (Christensen et al, 2001 Biochem J 354:481-4).
[0136] Generally, a hybridization reaction can be performed in the presence of a hybridization buffer that optionally includes a hybridization optimizing agent, such as an isostabilizing agent, a denaturing agent and/or a renaturation accelerant. Examples of isostabilizing agents include, but are not restricted to, betaines and lower tetraalkyl ammonium salts. Denaturing agents are compositions that lower the melting temperature of double stranded nucleic acid molecules by interfering with hydrogen bonding between bases in a double stranded nucleic acid or the hydration of nucleic acid molecules. Denaturing agents include, but are not restricted to, formamide, formaldehyde, dimethylsulphoxide, tetraethyl acetate, urea, guanidium isothiocyanate, glycerol and chaotropic salts. Hybridization accelerants include heterogeneous nuclear ribonucleoprotein (hnRP) Al and cationic detergents such as cetyltrimethylammonium bromide (CTAB) and dodecyl trimethylammonium bromide (DTAB), polylysine, spermine, spermidine, single stranded binding protein (SSB), phage T4 gene 32 protein and a mixture of ammonium acetate and ethanol. Hybridization buffers may include target polynucleotides at a concentration between about 0.005 nM and about 50 nM, preferably between about 0.5 nM and 5 nM, more preferably between about 1 nM and 2 nM.
[0137] In some embodiments, a hybridization mixture containing the target NF-κB pathway polynucleotides is placed in contact with the array of probes and incubated at a temperature and for a time appropriate to permit hybridization between the target sequences in the target polynucleotides and any complementary probes. Contact can take place in any suitable container, for example, a dish or a cell designed to hold the solid support on which the probes are bound. Generally, incubation will be at temperatures normally used for hybridization of nucleic acids, for example, between about 20° C and about 75° C, example, about 25° C, about 30° C, about 35° C, about 40° C, about 45° C, about 50° C, about 55° C, about 60° C, or about 65° C. For probes longer than 14 nucleotides, 20° C to 50° C is desirable. For shorter probes, lower temperatures are preferred. A sample of target polynucleotides is incubated with the probes for a time sufficient to allow the desired level of hybridization between the target sequences in the target polynucleotides and any complementary probes. For example, the hybridization may be carried out at about 45° C +/-10° C in formamide for 1-2 days. [0138] After the hybrid-forming step, the probes are washed to remove any unbound nucleic acid with a hybridization buffer, which can typically comprise a hybridization optimizing agent in the same range of concentrations as for the hybridization step. This washing step leaves only bound target polynucleotides. The probes are then examined to identify which probes have hybridized to a target polynucleotide.
[0139] The hybridization reactions are then detected to determine which of the probes has hybridized to a corresponding target sequence. Depending on the nature of the reporter molecule associated with a target polynucleotide, a signal may be instrumentally detected by irradiating a fluorescent label with light and detecting fluorescence in a fluorimeter; by providing for an enzyme system to produce a dye which could be detected using a spectrophotometer; or detection of a dye particle or a colored colloidal metallic or non metallic particle using a reflectometer; in the case of using a radioactive label or chemiluminescent molecule employing a radiation counter or autoradiography. Accordingly, a detection means may be adapted to detect or scan light associated with the label which light may include fluorescent, luminescent, focussed beam or laser light. In such a case, a charge couple device (CCD) or a photocell can be used to scan for emission of light from a probe:target polynucleotide hybrid from each location in the micro-array and record the data directly in a digital computer. In some cases, electronic detection of the signal may not be necessary. For example, with enzymatically generated colour spots associated with nucleic acid array format, visual examination of the array will allow interpretation of the pattern on the array. In the case of a nucleic acid array, the detection means is suitably interfaced with pattern recognition software to convert the pattern of signals from the array into a plain language genetic profile. In certain embodiments, oligonucleotide probes specific for different NF-κB pathway gene products are in the form of a nucleic acid array and detection of a signal generated from a reporter molecule on the array is performed using a 'chip reader'. A detection system that can be used by a 'chip reader' is described for example by Pirrung et al (U.S. Patent No. 5,143,854). The chip reader will typically also incorporate some signal processing to determine whether the signal at a particular array position or feature is a true positive or maybe a spurious signal. Exemplary chip readers are described for example by Fodor et al (U.S. Patent No., 5,925,525). Alternatively, when the array is made using a mixture of individually addressable kinds of labeled microbeads, the reaction may be detected using flow cytometry. 2.2 Protein-based diagnostics
[0140] Consistent with the present invention, the presence of an aberrant concentration of a NF-κB pathway protein is indicative of the presence, degree, activity, stage or risk of development of an unwanted or deleterious immune response associated with defective NF-κB signaling, including defective ReIB signaling, or related condition. NF-κB pathway protein levels in biological samples can be assayed using any suitable method known in the art. For example, when a NF-κB pathway protein is an enzyme, the protein can be quantified based upon its catalytic activity or based upon the number of molecules of the protein contained in a sample. Antibody-based techniques may be employed, such as, for example, immunohistological and immunohistochemical methods for measuring the level of a protein of interest in a tissue sample. For example, specific recognition is provided by a primary antibody (polyclonal or monoclonal) and a secondary detection system is used to detect presence (or binding) of the primary antibody. Detectable labels can be conjugated to the secondary antibody, such as a fluorescent label, a radiolabel, or an enzyme (e.g., alkaline phosphatase, horseradish peroxidase) which produces a quantifiable, e.g., colored, product. In another suitable method, the primary antibody itself can be detectably labeled. As a result, immunohistological labeling of a tissue section is provided. In some embodiments, a protein extract is produced from a biological sample (e.g., tissue, cells) for analysis. Such an extract (e.g., a detergent extract) can be subjected to western-blot or dot/slot assay of the level of the protein of interest, using routine immunoblotting methods (Jalkanen et o/., 1985, J. Cell. Biol. 101 :976-985; Jalkanen et αl. , 1987, J. Cell. Biol. 105:3087-3096).
[0141] Other useful antibody-based methods include immunoassays, such as the enzyme-linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). For example, a protein-specific monoclonal antibody, can be used both as an immunoadsorbent and as an enzyme-labeled probe to detect and quantify a NF-κB pathway protein of interest. The amount of such protein present in a sample can be calculated by reference to the amount present in a standard preparation using a linear regression computer algorithm (see Lacobilli et αl. , 1988, Breast Cancer Research and Treatment 11 :19-30). In other embodiments, two different monoclonal antibodies to the protein of interest can be employed, one as the immunoadsorbent and the other as an enzyme-labeled probe. [0142] Additionally, recent developments in the field of protein capture arrays permit the simultaneous detection and/or quantification of a large number of proteins. For example, low-density protein arrays on filter membranes, such as the universal protein array system (Ge, 2000 Nucleic Aeids Res. 28(2):e3) allow imaging of arrayed antigens using standard ELISA techniques and a scanning charge-coupled device (CCD) detector. Immuno-sensor arrays have also been developed that enable the simultaneous detection of clinical analytes. It is now possible using protein arrays, to profile protein expression in bodily fluids, such as in sera of healthy or diseased subjects, as well as in subjects pre- and post-drug treatment. [0143] Protein capture arrays typically comprise a plurality of protein-capture agents each of which defines a spatially distinct feature of the array. The protein-capture agent can be any molecule or complex of molecules which has the ability to bind a protein and immobilize it to the site of the protein-capture agent on the array. The protein-capture agent may be a protein whose natural function in a cell is to specifically bind another protein, such as an antibody or a receptor. Alternatively, the protein- capture agent may instead be a partially or wholly synthetic or recombinant protein which specifically binds a protein. Alternatively, the protein-capture agent may be a protein which has been selected in vitro from a mutagenized, randomized, or completely random and synthetic library by its binding affinity to a specific protein or peptide target. The selection method used may optionally have been a display method such as ribosome display or phage display, as known in the art. Alternatively, the protein- capture agent obtained via in vitro selection may be a DNA or RNA aptamer which specifically binds a protein target (see, e.g., Potyrailo et al., 1998 Anal. Chem. 70:3419- 3425; Cohen et al, 1998, Proc. Natl. Acad. Sci. USA 95:14272-14277; Fukuda, et al, 1997 Nucleic Acids Symp. Ser. 37:237-238; available from SomaLogic). For example, aptamers are selected from libraries of oligonucleotides by the Selex™ process and their interaction with protein can be enhanced by covalent attachment, through incorporation of brominated deoxyuridine and UV-activated crosslinking (photoaptamers). Aptamers have the advantages of ease of production by automated oligonucleotide synthesis and the stability and robustness of DNA; universal fluorescent protein stains can be used to detect binding. Alternatively, the in vitro selected protein-capture agent may be a polypeptide {e.g., an antigen) (see, e.g., Roberts and Szostak, 1997 Proc. Natl. Acad. Sci. USA, 94: 12297-12302). [0144] An alternative to an array of capture molecules is one made through 'molecular imprinting' technology, in which peptides (e.g., from the C-terminal regions of proteins) are used as templates to generate structurally complementary, sequence- specific cavities in a polymerizable matrix; the cavities can then specifically capture (denatured) proteins which have the appropriate primary amino acid sequence (e.g. , available from ProteinPrint™ and Aspira Biosystems).
[0145] Exemplary protein capture arrays include arrays comprising spatially addressed antigen-binding molecules, commonly referred to as antibody arrays, which can facilitate extensive parallel analysis of numerous proteins defining a proteome or subproteome. Antibody arrays have been shown to have the required properties of specificity and acceptable background, and some are available commercially (e.g., BD Biosciences, Clontech, BioRad and Sigma). Various methods for the preparation of antibody arrays have been reported (see, e.g., Lopez et al, 2003 J. Chromatogr. B 787: 19-27; Cahill, 2000 Trends in Biotechnology 7:47-51 ; U.S. Pat. App. Pub. 2002/0055186; U.S. Pat. App. Pub. 2003/0003599; PCT publication WO 03/062444; PCT publication WO 03/077851; PCT publication WO 02/59601 ; PCT publication WO 02/39120; PCT publication WO 01/79849; PCT publication WO 99/39210). The antigen-binding molecules of such arrays may recognize at least a subset of proteins expressed by a cell or population of cells, illustrative examples of which include growth factor receptors, hormone receptors, neurotransmitter receptors, catecholamine receptors, amino acid derivative receptors, cytokine receptors, extracellular matrix receptors, antibodies, lectins, cytokines, serpins, proteases, kinases, phosphatases, ras- like GTPases, hydrolases, steroid hormone receptors, transcription factors, heat-shock transcription factors, DNA-binding proteins, zinc-finger proteins, leucine-zipper proteins, homeodomain proteins, intracellular signal transduction modulators and effectors, apoptosis-related factors, DNA synthesis factors, DNA repair factors, DNA recombination factors, cell-surface antigens, hepatitis C virus (HCV) proteases and HIV proteases.
[0146] Antigen-binding molecules for antibody arrays are made either by conventional immunization (e.g. , polyclonal sera and hybridomas), or as recombinant fragments, usually expressed in E. coli, after selection from phage display or ribosome display libraries (e.g., available from Cambridge Antibody Technology, Biolnvent, Affitech and Biosite). Alternatively, 'combibodies' comprising non-covalent associations of VH and VL domains, can be produced in a matrix format created from combinations of diabody-producing bacterial clones (e.g., available from Domantis). Exemplary antigen-binding molecules for use as protein-capture agents include monoclonal antibodies, polyclonal antibodies, Fv, Fab, Fab' and F(ab')2 immunoglobulin fragments, synthetic stabilised Fv fragments, e.g., single chain Fv fragments (scFv), disulphide stabilised Fv fragments (dsFv), single variable region domains (dAbs) minibodies, combibodies and multivalent antibodies such as diabodies and multi-scFv, single domains from camelids or engineered human equivalents.
[0147] Individual spatially distinct protein-capture agents are typically attached to a support surface, which is generally planar or contoured. Common physical supports include glass slides, silicon, microwells, nitrocellulose or PVDF membranes, and magnetic and other microbeads.
[0148] While microdrops of protein delivered onto planar surfaces are widely used, related alternative architectures include CD centrifugation devices based on developments in microfluidics (e.g., available from Gyros) and specialised chip designs, such as engineered microchannels in a plate (e.g., The Living Chip™, available from Biotrove) and tiny 3D posts on a silicon surface (e.g., available from Zyomyx).
[0149] Particles in suspension can also be used as the basis of arrays, providing they are coded for identification; systems include colour coding for microbeads (e.g., available from Luminex, Bio-Rad and Nanomics Biosystems) and semiconductor nanocrystals (e.g., QDots™, available from Quantum Dots), and barcoding for beads (UltraPlex™, available from Smartbeads) and multimetal microrods (Nanobarcodes™ particles, available from Surromed). Beads can also be assembled into planar arrays on semiconductor chips (e.g., available from LEAPS technology and BioArray Solutions). Where particles are used, individual protein- capture agents are typically attached to an individual particle to provide the spatial definition or separation of the array. The particles may then be assayed separately, but in parallel, in a compartmentalised way, for example in the wells of a microtiter plate or in separate test tubes.
[0150] In operation, a protein sample, which is optionally fragmented to form peptide fragments (see, e.g., U.S. Pat. App. Pub. 2002/0055186), is delivered to a protein-capture array under conditions suitable for protein or peptide binding, and the array is washed to remove unbound or non-specifically bound components of the sample from the array. Next, the presence or amount of protein or peptide bound to each feature of the array is detected using a suitable detection system. The amount of protein bound to a feature of the array may be determined relative to the amount of a second protein bound to a second feature of the array. In certain embodiments, the amount of the second protein in the sample is already known or known to be invariant. [0151] For analyzing differential expression of proteins between two cells or cell populations, a protein sample of a first cell or population of cells is delivered to the array under conditions suitable for protein binding. In an analogous manner, a protein sample of a second cell or population of cells to a second array, is delivered to a second array which is identical to the first array. Both arrays are then washed to remove unbound or non-specifically bound components of the sample from the arrays. In a final step, the amounts of protein remaining bound to the features of the first array are compared to the amounts of protein remaining bound to the corresponding features of the second array. To determine the differential protein expression pattern of the two cells or populations of cells, the amount of protein bound to individual features of the first array is subtracted from the amount of protein bound to the corresponding features of the second array.
[0152] In an illustrative example, fluorescence labeling can be used for detecting protein bound to the array. The same instrumentation as used for reading DNA microarrays is applicable to protein-capture arrays. For differential display, capture arrays (e.g. antibody arrays) can be probed with fluorescently labelled proteins from two different cell states, in which cell lysates are labeled with different fluorophores (e.g., Cy-3 and Cy-5) and mixed, such that the color acts as a readout for changes in target abundance. Fluorescent readout sensitivity can be amplified 10-100 fold by tyramide signal amplification (TSA) (e.g., available from Perkin Elmer Lifesciences). Planar waveguide technology (e.g., available from Zeptosens) enables ultrasensitive fluorescence detection, with the additional advantage of no washing procedures. High sensitivity can also be achieved with suspension beads and particles, using phycoerythrin as label (e.g., available from Luminex) or the properties of semiconductor nanocrystals (e.g., available from Quantum Dot). Fluorescence resonance energy transfer has been adapted to detect binding of unlabelled ligands, which may be useful on arrays (e.g., available from Affibody). Several alternative readouts have been developed, including adaptations of surface plasmon resonance (e.g., available from HTS Biosystems and Intrinsic Bioprobes), rolling circle DNA amplification (e.g., available from Molecular Staging), mass spectrometry (e.g., available from Sense Proteomic, Ciphergen, Intrinsic and Bioprobes), resonance light scattering (e.g., available from Genicon Sciences) and atomic force microscopy (e.g., available from BioForce Laboratories). A microfluidics system for automated sample incubation with arrays on glass slides and washing has been co-developed by NextGen and Perkin Elmer Life Sciences.
[0153] In some embodiments, nucleic acids (typically oligonucleotides) can be used as a protein capture agents, in which the nucleic acids bind to or otherwise complex with a nucleic acid-binding domain of an NF-κB pathway transcription factor (e.g., ReIB, cRel, p65, p50 etc). The complexing of a transcription factor with a nucleic acid (e.g., RNA or DNA) generally requires a specific nucleic acid sequence that corresponds to a cis site to which the transcription factor binds. Such cis sites are known to those of skill in the art and nucleic acids corresponding to these sites can be prepared using standard procedures. The nucleic acids can be used in any suitable nucleic acid- binding protein assay, non limiting examples of which include the assays described by Haukanes B I and Kvam C (Biotechnology, 1993 Jan 11 60-63), Alberts B et al.
(Molecular Biology of the Cell, 1994, 3rd Edn., Garland Publications Inc), Kirigiti P and Machida C A (2000 Methods MoI Biol, 126, 431-51), Molecular Probes handbook and references therein (Molecular Probes, Inc., 4849 Pitchford Ave., Eugene, USA) and in Example 3 infra. [0154] In other embodiments, aberrant signaling of the NF-κB pathway is detected by detecting aberrant phosphorylation of a phosphorylatable polypeptide (e.g. , IRAK, TAKl, TABl, TAB2, PKR, Akt, Cot, IKKα, IKKβ and IKKγ) involved in or belonging to the NF-κB signaling pathway. The phosphorylation state of the phosphorylatable polypeptide is indicative of the activity of the NF-κB signaling pathway. Thus, if the phosphorylation state of the phosphorylatable polypeptide in the biological sample is higher or lower than the phosphorylation state of the same polypeptide in a reference sample, then this indicates that the phosphorylation state of phosphorylatable polypeptide in the biological sample is abnormal, indicative of aberrant signaling through the NF-κB pathway. In specific embodiments, aberrant signaling through the NF-κB pathway is detected when the phosphorylation state of a NF-κB associated phosphorylatable polypeptide such as but not limited to IRAK, TAKl, TABl, TAB2, PKR, Akt, Cot, IKKα, IKKβ and IKKγ, is lower than the phosphorylation state of the corresponding polypeptide in the reference sample. Methods of detecting the phosphorylation state of a protein are well-known in the art. However, it is desirable that antigen-binding molecules that specifically recognize phosphorylated or unphosphorylated forms of the phosphorylatable polypeptide be used. Non-limiting examples of such antigen-binding molecules are available from Cell Signaling Technology, Inc.(Danvers, Massachusetts).
[0155] In certain embodiments, the techniques used for detection of NF-κB pathway gene expression products will include internal or external standards to permit quantitative or semi-quantitative determination of those products, to thereby enable a valid comparison of the level or functional activity of these expression products in a biological sample with the corresponding expression products in a reference sample or samples. Such standards can be determined by the skilled practitioner using standard protocols. In specific examples, absolute values for the level or functional activity of individual expression products are determined.
3. Kits [0156] All the essential materials and reagents required for detecting and quantifying NF-κB pathway gene expression products may be assembled together in a kit. In some embodiments, the kit comprises: a) primers designed to produce double stranded DNA complementary to at least a portion of a NF-κB pathway gene; wherein at least one of the primers contains a sequence which hybridizes to RNA, cDNA or an EST corresponding to the marker gene to create an extension product and at least one other primer that hybridizes to the extension product; b) an enzyme with reverse transcriptase activity, and c) an enzyme with thermostable DNA polymerase activity; wherein the primers are used to detect the expression levels of the marker gene in a test subject. In other embodiments, the kit comprises at least one oligonucleotide which hybridizes to RNA, cDNA or an EST corresponding to a NF-κB pathway gene, wherein the oligonucleotide is used to detect the expression levels of the marker gene in a test sample. In still other embodiments, the kit comprises at least one oligonucleotide that binds to or otherwise complexes with a nucleic acid-binding domain of a NF-κB pathway polypeptide (e.g., an NF-κB pathway transcription factor), wherein the oligonucleotide is used to detect the level of the transcription factor in a test sample.
[0157] The kits may optionally include appropriate reagents for detection of labels, positive and negative controls, washing solutions, blotting membranes, microtitre plates dilution buffers and the like. For example, a nucleic acid-based detection kit may include (i) a NF-κJB pathway polynucleotide (which may be used as a positive control), (ii) a primer or probe that specifically hybridizes to a NF-κB pathway polynucleotide. Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (Reverse Transcriptase, Taq, Sequenase™ DNA ligase etc. depending on the nucleic acid amplification technique employed), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification. Such kits also generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each primer or probe. Alternatively, a protein-based detection kit may include (i) a NF-κB pathway polypeptide (which may be used as a positive control) and (ii) an antigen-binding molecule that is immuno-interactive with a NF-κB pathway polynucleotide, or (iii) an oligonucleotide that binds to or otherwise complexes with a nucleic acid-binding domain of the NF-κB pathway polypeptide (e.g., an NF-κB pathway transcription factor). The kit can also feature various devices and reagents for performing one of the assays described herein; and/or printed instructions for using the kit to quantify the expression of a NF-κB pathway polynucleotide or the level of a NF- KB pathway.
4. Methods of modulating undesirable or deleterious immune responses
[0158] The present inventors have also found that reduced or defective NF- KB function prevents antigen-presenting cells such as DC to license regulatory lymphocyte mediated suppression of T cell-associated inflammatory disease. They have also determined that the capacity of antigen-presenting cells to license this suppression is restorable by activating both the canonical and non-canonical NF-κB pathways (e.g., by activating ReIB or restoring its function or expressing it at basal levels). Accordingly, the present invention provides methods for modulating an immune response, especially an undesirable or deleterious immune response associated with defective NF-κB activation, including defective ReIB activation, in a subject, wherein the methods generally comprise increasing the number of antigen-presenting cells in the subject, in which both the canonical and non-canonical NF-κB pathways are activated or activatable (e.g., in which ReIB is activated or activatable), suitably in response to a pro-inflammatory signal such as but not limited to tumor necrosis factor (e.g., TNFα), C5a, interleukin- 1 (e.g., IL- lβ), CD 154 and lipolysaccharide (LPS). Such antigen- presenting cells are also referred to herein as NF-κB operable antigen-presenting cells. [0159] In accordance with the present invention, the number of antigen- presenting cells with operable NF-κB function (i.e., operable NF-κB antigen-presenting cells) can be increased by use of (i) at least one agent (also referred to herein as an "NF- KB restoration agent") that activates the canonical and non-canonical NF-κB pathways, (ii) an antigen-presenting cell that has been contacted ex vivo with at least one NF-κB restoration agent, and (iii) an operable NF-κB antigen-presenting cell derived from a histocompatible donor.
4.1 NF-κB restoration agents
[0160] In some embodiments, one or more NF-κB restoration agents are used in vivo or ex vivo, to activate the canonical and non-canonical NF-κB pathways, in cells including antigen-presenting cells. For example, one method of achieving this is to use a first NF-κB restoration agent that activates the canonical pathway and a second NF-κB restoration agent that activates the non-canonical pathway. Alternatively, NF-κB restoration agents that modulate both pathways may be employed. [0161] Non-limiting NF-κB restoration agents for activating the canonical
NF-κB pathway include Toll-like receptor (TLR) agonists, Tumor Necrosis Factor-α (TNF-α) agonists and agents that increase the level or functional activity of a canonical pathway kinase selected from IKK and IKKβ/NEMO. Representative examples of TLR agonists include LPS, bacterial lipopeptides and lipoproteins, bacterial outer membrane proteins, lipoarabinomannan, glycosylphosphatidylinositol (GPI) anchors, taxol, zymosan, lipoteichoic acid, heat shock proteins (HSP) 60 and 70, fibrinogen, fibronectin, soluble heparin sulfate, hyaluronin, double stranded RNA, flagellin, pilin, single stranded viral, viral and bacterial unmethylated CpG DNA, chromatin-IgG complexes, endogenous mRNA, R837, R848, CPG 7909, loxoribine, uridine, poly-IC, and poly-AU, and combinations thereto. Illustrative TNF-α agonists include virulizin, a human or humanized monoclonal antibody TNF-α receptor agonist, Complete Freund's adjuvant (CFA), ISS-ODN, microbial cell wall components with LPS-like activity, cholera particles, E. coli heat labile enterotoxin, E. coli heat labile enterotoxin complexed with lecithin vesicles, immune stimulating complexes (ISCOMS), polyethylene glycol, poly(N-2-(hydroxypropyl)methacrylamide), synthetic oligonucleotides containing CpG or CpA motifs, monophosphoryl lipid A, Bacillus Calmette-Guerin, γ-interferon, Tissue Plasminogen Activator, LPS, Interleukin-1,
Interleukin-2, UV light, a lymphotoxin, cachectin, a TNFR-2 agonist, an intracellular mediator of the TNF-α signaling pathway, IRF-I , STATl , a lymphokine, or the combination of TNF-α and an anti-TNFR-1 antibody.
[0162] Non-limiting NF-κB restoration agents for activating the non- canonical NF-κB pathway include agents that increase the level or functional activity of ReIB or a non-canonical pathway kinase selected from NIK and IKKα. Exemplary agents that increase the level or functional activity of ReIB include PP ARy antagonists or inhibitors, Vitamin D antagonists and estrogen antagonists or depletion agents.
[0163] Non-limiting examples of PPARγ antagonists or inhibitors include: 1- (p-chlorobenzyl)-5-chloro-3-thiophenylindole-2-carboxylic acid, as described for example in U.S. Patent Appl. Pub. No. 20030032581 ; 4-(4-(4-carboxyphenyl)butyl)2- heptyl4-oxo-5-thiazolidine N,N-dibenzylacetamide, (2S*,5S*)-4-(4-(4- carboxyphenyl)butyl)-2-heptyl-4-oxo-5-thiazolidin- e N,N-dibenzylacetamide, (2S,5S)- 4-(4-(4-carboxyphenyl)butyl)-2-heptyl-4-oxo-5thiazolidine N,N-dibenzylacetamide, (2S*,5S*)-4-(4-(4-carboxyphenyl)butyl)2-hexyl)-4-oxo-5-thiazolidine N,N- dibutylacetamide, (2S*,5S*)-4-(2-(4-carboxyphenyl)ethyl)-2-octyl-4-oxo-5-thiazolidine N,N-dibenzylacetamide, (2R*,5S*)-4-(2-(4-carboxyphenyl)ethyl)-2-octyl-4-oxo-5- thiazolidine N,N-dibenzylacetamide, (2S*,5S*)-4-(2-(4-carboxyphenyl)ethyl)-2-octyl- 4-oxo-5-thiazolidine N,N-di-(3-iodo)benzylacetamide, (2S*,5S*)-4-(3-(4- carboxyphenyl)propyl)-2-heptyl-4-oxo-5thiazolidin- e N,N-benzylacetamide, (2S*,5S*)-4-(4-(4-carboxyphenyl)butyl-2-heptyl-4-oxo-5-thiazolidine N,N-di-(3- benzylacetamide, (2S*,5S*)-4-(2-(4-carboxyphenyl)ethyl)-2-(6-phenylhexyl)-4-oxo-5- th- iazolidine N,N-dibenzylacetamide, (2S*,5S*)-4-(4-(4-carboxyphenyl)butyl)-2- (6phenylhexyl)-4-oxo-5-thi- azolidine N,N-dibenzylacetamide, 4-(4-(4- carboxyphenyl)butyl)-2-(4-phenylbutyl)-4-oxo-5-thiazolidine N,N-dibenzylacetamide, (2S*,5S*)-4-(2-(4-ureidophenyl)ethyl)-2-octyl-4-oxo-5-thiazolidine N,N- dibenzylacetamide, (2S*,5S*)-4-(2-(4-methylsulfonamidophenyl)ethyl)-2-octyl-4-oxo- 5-th- iazolidine N,N-dibenzylacetamide, (2S*,5S*)-4-(2-(4- aminosulfonylphenyl)ethyl)-2-octyl-4-oxo-5-thiazo- lidine N,N-dibenzylacetamide, (2S * ,5 S * )-4-(4-(4-carboxyphenyl)butyl)-2-hepyl-4-oxo-5-thiazolidine N-benzyl-N-(4- trifluorobenzyl)acetamide, (2R*,5S*)-4-(4(4-carboxyphenyl)butyl)-2-hepyl-4-oxo-5- thiazolidine N-benzyl-N-(4-trifluorobenzyl)acetamide, (2S*,5S*)-4-(2-(4-(3- hydantoino)phenyl)ethyl)-2-octyl-4-oxo-5-thiaz- olidine N,N-dibenzylacetamide, (2S*,5S*)-4-(2-(3,4-dioxomethylenephenyl)ethyl)-2-heptyl4-oxo-5-thi- azolidine N5N- dibenzylacetamide, (2S*,5S*)-4-(4-(4-carboxyphenyl)butyl)-2-octyl-4oxo-5- thiazolidine N-benzyl-N-(4-hydroxybuty l)acetamide, (2S * ,5 S * )-4-(2-(3 ,4- dioxomethylenephenyl)ethyl)-2-octyl-4-oxo-5-thi- azolidine N,N-dibenzylacetamide, (2S*,5S*)-4-(4-(4-carboxyphenyl)butyl)-2-heptyl-4-oxo-5-thiazolidin- e N-benzyl-N- (4-pyridyl)acetamide, (2S*,5S*)-4-(4carboxyphenyl)butyl)-2-heptyl-4-oxo-5- thiazolidine N-benzyl-N-(2-pyridyl)acetamide, (2S*,5S*)-4-(4-(4- carboxyphenyl)butyl)-2-heptyl-4-oxo-5-thiazolidin- e N-benzyl-N-(2- ethoxycarboxyethyl)acetamide, (2S*,5S*)-4-(4-(4-carboxyphenyl)butyl)-2-heptyl-4- oxo-5-thiazolidin- e N-benzyl-N-butylacetamide, (2S*,5S*)-4-(4-(4- carboxyphenyl)butyl)-2-heptyl4-oxo-thiazolidine N-benzyl-N-isopropylacetamide, (2S*,5S*)-4-(2-(4-hydroxyphenyl)ethyl)-2-heptyl-4-oxo-5-thiazolidin- e N,N- dibenzylacetamide, (2S*,5S*)-4-(4-(4-carboxyphenyl)butyl)-2-heptyl-4-oxo-5- thiazolidin- e N-enzyl-N-ethoxycarboxymethylacetamide, (2S*,5S*)-4-(4-(4- carboxyphenyl)butyl)-2-heptyl-4-oxo-5-thiazolidin- e N,N-di-(4- fluorobenzyl)acetamide, (2S*,5S*)-4-(2-(4-arboxymethoxyphenyl)ethyl)-2-heptyl-4- oxo-5-thiaz- olidine N,N-dibenzylacetamide, (2S*,5S*)-4-(2-(4- carboxyamidomethoxyphenyl)ethyl)-2-heptyl-4-oxo-5- -thiazolidine N5N- dibenzylacetamide, (2S*,5S*)-4-(2-(4-methoxyphenyl)ethyl)-2-heptyl-4-oxo-5- thiazolidin- e N,N-dibenzylacetamide, (2S*,5S*)-4-(4-(4arboxyphenyl)butyl)-2- heptyl4-oxo-thiazolidine N-benzyl-N-(2-thienylmethyl)acetamide, (2S*,5S*)-4-(4-(4- carboxyphenyl)butyl)-2-heptyl-4-oxo-5-thiazolidin- e N-benzyl-N-(2,3- dioxomethylenebenzyl)acetamide, (2S * , 5 S * )-4-(4-(4-carboxypheny l)butyl)-2-heptyl-4- oxo-5-thiazolidin- e N-benzyl-N-(2-thiazolemethyl)acetamide, (2S*,5S*)-4-(4-(4- carboxyphenyl)butyl)-2-heptyl-4-oxo-5-thiazolidin- e N-benzyl-N-(2- furfuryl)acetamide, and pharmaceutically acceptable salts and solvates thereof, as described fro example in U.S. Patent Appl. Pub. No. 20020151569; and GW9662 (2- chloro-5 -nitrobenzani 1 ide) .
[0164] Representative Vitamin D antagonists can be selected from: 2α- methyl 19-nor vitamin D analog ester compounds illustrative examples of which include compounds of formula II ((22E)-(20S,24R)-25-carbobutoxy-2α-methyl-26,27-cyclo-22- dehydro-1-α, 24-dihydroxy-19-norvitamin D3), formula III ((22E)-(24R)-25- carbobutoxy-2 α-methyl-26,27-cyclo-22-dehydro- 1 -α,24-dihydroxy- 19-norvitamin D3), formula IV ((22E)-(20S)-25-carbopentoxy-2 α-methyl-24-oxo-22-dehydro-l α - hydroxy- 19-norvitamin D3), formula V ((22E)-25-carbopentoxy-2 α-methyl-24-oxo-22- dehydro-1 α -hydroxy- 19-norvitamin D3) and the like; 2-methylene 19-nor vitamin D analog ester compounds such as, but are not limited to, compounds of formula VI ((22E)-(20S)-25-carbopentoxy-2-methylene-24-oxo-22-dehydro-lα -hydro- xy-19- norvitamin D3), formula VII ((22E)-25-carbopentoxy-2-methylene~ 24-oxo-22- dehydro-1 α-hydroxy- 19-norvitamin D3), formula VIII ((22E)-(20S,24R)-25- carbobutoxy-2-methylene-26,27-cyclo-22-dehydro- 1 α,24-dihydroxy- 19-norvitamin D3), formula IX ((22E)-(24R)-25-carbobutoxy-2-methylene-26,27-cyclo-22-dehydro- lα,24-dihydroxy- 19-norvitamin D3), and the like; 2α-methyl or 2-methylene 19-nor vitamin D analog ketone compounds, illustrative examples of which include compounds of formula X ((22E)-(20S)-25-heptanoyl-2α-methyl-24-oxo-2- 2-dehydro-lα-hydroxy- 19-norvitamin D3), formula XI ((22E)-25-heptanoyl-2.alpha.-methyl-24-oxo-22- dehydro- 1 α-hydroxy- 19- -norvitamin D3), formula XII ((22E)-(20S)-25-heptanoyl-2- methylene-24-oxo-22-dehydro-l α-hydroxy- 19-norvitamin D3), formula XIII ((22E)-25- heptanoyl-2-methylene-24-oxo-22-dehydro- 1 α-hydroxy- 19-nor- vitamin D3), and the like, as disclosed for example in U.S. Patent Appl. Pub. No. 20050182033; and Vitamin D3 lactone derivatives as disclosed for example in U.S. Patent Appl. Pub. No. 20060148768.
[0165] Illustrative estrogen antagonists or depletion agents include tamoxifen, fulvestrant, toremifene and raloxifene, or a pharmaceutically acceptable salt thereof.
Illustrative estrogen-depletion agents are selected from: aromatase inhibitor such as but not limited to formestane, exemestane, anastrozole, vorozole, letrozole and aminoglutethimide, or a pharmaceutically acceptable salt thereof.
[0166] In some embodiments, the level or functional activity of a NF-κB pathway polypeptide (e.g. , ReIB or a canonical or non-canonical pathway kinase) is increased, for example, by introducing into antigen-presenting cells a nucleic acid construct that expresses a nucleotide sequence encoding the NF-κB pathway polypeptide in the antigen-presenting cells. Such nucleic acid constructs are typically in the form of vectors that are capable of being expressed in the desired subject host cell. Promoter, enhancer, stress or chemically-regulated promoters, antibiotic-sensitive or nutrient-sensitive regions may be included as required.
[0167] In other non-limiting examples, the canonical and non-canonical NF-
KB pathways can be activated using at least one agent that decreases the level or functional activity of SHP-I . Non-limiting SHP-I inhibitors include pentavalent antimonial compounds (e.g., antimony dextran glucoside, antimony mannan, ethyl stibanime, ureastibamine, sodium stibogluconate, and glucantime) as described for example in U.S. Patent Application Publication No. 20030092670, allopurinol, aminosidine, amphotericine/amphotericine B, miltefosine (an alkylphospholipid), paromomycin (aminosidine), pentamidine, levamisole, ketoconazole, bisperoxovanadium compounds (e.g., those described in Scrivens et o/.,.Mol. Cancer Ther. 2: 1053-1059, 2003; and U.S. Pat. No. 6,642,221), vanadate salts and complexes (e.g., sodium ortho vanadate), dephosphatin, dnacin Al, dnacin A2, STI-571, suramin, gallium nitrate, meglumine antimonate, 2-(2-mercaptoethanol)-3-methyl-l,4- naphthoquinone, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime, known as DB289 (Immtech), 2,5-bis(4-amidinophenyl)furan (DB75, Immtech), disclosed in U.S. Pat. No 5,843,980, and compounds described in Pestell et al, Oncogene 19:6607-6612, 2000; Lyon et al, Nat. Rev. Drug Discov. 1 :961-976, 2002, Ducruet et al, Bioorg. Med. Chem. 8:1451-1466, 2000; U.S. Patent Application Publication Nos.
2003/0114703; 2003/0144338; and 2003/0161893; and PCT Patent Publication Nos. WO99/46237; WO03/06788; and WO03/070158, linear or cyclic phosphopeptide ligands of SHP-I, as described for example by Imhof et al. (2005, J. Med. Chem. 48(5): 1528-1539). Still other analogs are those that fall within a formula provided in any of U.S. Pat. Nos. 5,428,051; 5,521,189; 5,602,172; 5,643,935; 5,723,495; 5,843,980; 6,008,247; 6,025,398; 6,172,104; 6,214,883; and 6,326,395; and U.S. Patent Application Publication Nos. 2001/0044468 and 2002/0019437; and the pentamidine analogs described in U.S. patent application Ser. No. 10/617,424 (see, e.g., Formula (II)). Alternatively, the biological activity of SHP-I can be reduced through the use of an antisense compound directed to RNA encoding the target protein. SHP-I antisense compounds suitable for this use are known in the art (see, e.g., U.S. Patent Publication No. 2003/0232749). Other antisense compounds that SHP-I activity can be identified using standard techniques. For example, accessible regions of the target SHP-I mRNA can be predicted using an RNA secondary structure folding program such as MFOLD (M. Zuker, D. H. Mathews & D. H. Turner, "Algorithms and Thermodynamics for RNA Secondary Structure Prediction: A Practical Guide. In: RNA Biochemistry and Biotechnology," J. Barciszewski & B. F. C. Clark, eds., NATO ASI Series, Kluwer Academic Publishers, (1999)). Sub-optimal folds with a free energy value within 5% of the predicted most stable fold of the mRNA are predicted using a window of 200 bases within which a residue can find a complimentary base to form a base pair bond. Open regions that do not form a base pair are summed together with each suboptimal fold and areas that are predicted as open are considered more accessible to the binding to antisense nucleobase oligomers. Other methods for antisense design are described, for example, in U.S. Pat. No. 6,472,521 ; Antisense Nucleic Acid Drug Dev. 7:439-444, 1997; Nucleic Acids Res. 28:2597-2604, 2000; and Nucleic Acids Res. 31 :4989-4994, 2003. In other illustrative examples, the biological activity of SHP-I can be reduced through the use of RNA interference (RNAi), employing, e.g., a double stranded RNA (dsRNA) or small interfering RNA (siRNA) directed to SHP-I (see, e.g., U.S. Patent Publication No. 2003/0232749). Methods for designing such interfering RNAs are known in the art. For example, software for designing interfering RNA is available from Oligoengine (Seattle, Wash.). In other embodiments, the biological activity of SHP-I is reduced through the use of ribozymes. Ribozymes which may be encoded in the genomes of the viruses or virus-like particles are described in Cech and Herschlag "Site-specific cleavage of single stranded DNA" U.S. Pat. No. 5,180,818; Altaian et al. "Cleavage of targeted RNA by RNAse P" U.S. Pat. No. 5,168,053, Cantin et al. "Ribozyme cleavage of HIV-I RNA" U.S. Pat. No. 5,149,796; Cech et al. "RNA ribozyme restriction endoribonucleases and methods", U.S. Pat. No. 5,1 16,742; Been et al. "RNA ribozyme polymerases, dephosphorylases, restriction endonucleases and methods", U.S. Pat. No. 5,093,246; and Been et al. "RNA ribozyme polymerases, dephosphorylases, restriction endoribonucleases and methods; cleaves single-stranded RNA at specific site by transesterification", U.S. Pat. No. 4,987,071. Within the scope of the present invention are engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of target sequences. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays. [0168 J NF- KB restoration agents according to the present invention may be administered directly to the subject or contacted ex vivo with antigen-presenting cells before administering the treated cells to the subject, as described in more detail below.
4.2 Antigen embodiments [0169] In some embodiments, an immune modulator selected from at least one NF-κB restoration agent as broadly described in Section 4.1 and an antigen- presenting cell with operable NF-κB function, is administered concurrently with an antigen that corresponds to at least a portion of a target antigen that associates with the undesirable or deleterious immune response, for inducing a tolerogenic immune response to that target antigen. The present invention thus provides compositions for modulating an immune response, especially an undesirable or deleterious immune response associated with defective NF-κB activation in a subject, wherein the compositions generally comprise an immune modulator that increases the number of operable NF-κB antigen-presenting cells as defined herein and an antigen that corresponds to at least a portion of a target antigen associated with the undesirable or deleterious immune response.
[0170] Illustrative target antigens include alloantigens and self antigens or peptide fragments thereof, which are presented in the context of MHC, as well as soluble proteins and fragments of insoluble complexes, particulate antigens, e.g., bacteria or parasites, and allergens. Thus, exemplary antigens which are useful in the practice of the present invention include, but are not limited to, self antigens that are targets of autoimmune responses, allergens and transplantation antigens. Examples of self antigens include, but are not restricted to, lupus autoantigen, Smith, Ro, La, Ul- RNP, fibrillin (scleroderma); proinsulin, insulin, IA2 and GAD65 in diabetes; collagen type II, HC gp39, dnaJpl , citrullinated proteins and peptides e.g. , citrullinated type II collagen, vimentin or fibrinogen in rheumatoid arthritis; myelin basic protein and MOG in multiple sclerosis; gliadin in celiac disease; histones, PLP, collagen, gIucose-6- phosphate isomerase, thyroglobulin, various tRNA synthetases, acetylcholine receptor (AchR), proteinase-3, myeloperoxidase etc. Examples of allergens include, but are not limited to, FeI d 1 (i.e., the feline skin and salivary gland allergen of the domestic cat Felis domesticus, the amino acid sequence of which is disclosed International Publication WO 91/06571), Der p I, Der p II, Der fl or Der fll (i.e., the major protein allergens from the house dust mite dermatophagoides, the amino acid sequence of which is disclosed in International Publication WO 94/24281). Other allergens may be derived, for example from the following: grass, tree and weed (including ragweed) pollens; fungi and moulds; foods such as fish, shellfish, crab, lobster, peanuts, nuts, wheat gluten, eggs and milk; stinging insects such as bee, wasp, and hornet and the chirnomidae (non-biting midges); other insects such as the housefly, fruit fly, sheep blow fly, screw worm fly, grain weevil, silkworm, honeybee, non-biting midge larvae, bee moth larvae, mealworm, cockroach and larvae of Tenibrio molitor beetle; spiders and mites, including the house dust mite; allergens found in the dander, urine, saliva, blood or other bodily fluid of mammals such as cat, dog, cow, pig, sheep, horse, rabbit, rat, guinea pig, mouse and gerbil; airborne particulates in general; latex; and protein detergent additives. Transplantation antigens can be derived from donor cells or tissues or from the donor antigen-presenting cells bearing MHC loaded with self antigen in the absence of exogenous antigen.
[0171] The antigen(s) may be isolated from a natural source or may be prepared by recombinant techniques as is known in the art. For example, peptide antigens can be eluted from the MHC and other presenting molecules of antigen- presenting cells obtained from a cell population or tissue for which a modified immune response is desired, e.g., an allogeneic tissue or cell population in transplantation medicine. The eluted peptides can be purified using standard protein purification techniques known in the art (Rawson et al., 2000, Cancer Res 60(16), 4493-4498). If desired, the purified peptides can be sequenced and synthetic versions of the peptides produced using standard protein synthesis techniques as for example described below. Alternatively, crude antigen preparations can be produced by isolating a sample of a cell population or tissue for which a modified immune response is desired, and either lysing the sample or subjecting the sample to conditions that will lead to the formation of apoptotic cells {e.g., irradiation with ultra violet or with gamma rays, viral infection, cytokines or by depriving cells of nutrients in the cell culture medium, incubation with hydrogen peroxide, or with drugs such as dexamethasone, ceramide chemotherapeutics and anti-hormonal agents such as Lupron or Tamoxifen). The lysate or the apoptotic cells can then be used as a source of crude antigen for contact with the antigen- presenting cells.
[0172] When the antigen is known, it may be conveniently prepared in recombinant form using standard protocols as for example described in: Sambrook, et al, MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbor Press, 1989), in particular Sections 16 and 17; Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley & Sons, Inc. 1994-1998), in particular Chapters 10 and 16; and Coligan et al, CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5 and 6. Typically, an antigen may be prepared by a procedure including the steps of (a) providing an expression vector from which the target antigen or analogue or mimetic thereof is expressible; (b) introducing the vector into a suitable host cell; (c) culturing the host cell to express recombinant polypeptide from the vector; and (d) isolating the recombinant polypeptide. [0173] Alternatively, the antigen can be synthesized using solution synthesis or solid phase synthesis as described, for example, by Atherton and Sheppard (Solid Phase Peptide Synthesis: A Practical Approach, IRL Press at Oxford University Press, Oxford, England, 1989) or by Roberge et al. (1995, Science 269: 202).
[0174] In some embodiments, the antigen is in the form of one or more peptides. Usually, such peptides are at least 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 20, 25, 30 amino acid residues in length and suitably no more than about 500, 200, 100, 80, 60, 50, 40 amino acid residues in length. In some embodiments in which two or more peptides are used, the peptides can be in the form of a plurality of contiguous overlapping peptides whose sequences span at least a portion of a target antigen. Suitably, the peptide sequences are derived from at least about 30, 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of the sequence corresponding to the target antigen. In some embodiments, each peptide of the plurality of contiguous overlapping peptide fragments can be 30-90 amino acids in length, e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 73, 75, 80, 81, 85, 86 and 90 amino acids in length. In various embodiments, the amino acid sequences of contiguous overlapping peptide fragments in the plurality overlap by about 10 to about 15 amino acids, e.g., 10, 11, 12, 13, 14 and 15 amino acids. Exemplary methods for producing such peptide antigens are described, for example, by Astori et al. (2000 J. Immunol. 165, 3497-3505; and references cited therein) and in U.S. Pat. Appl. Pub. No. 2004/0241 178. The antigen may be suitably modified, for example, by lipid modification to modify its physico-chemical properties.
4.3 Particle embodiments
[0175] In some embodiments, at least one NF-κB restoration agent according to Section 4.1 and optionally at least one antigen according to Section 4.2 (also collectively referred to herein as "the bioactive agents") are provided in particulate form. In embodiments in which both an NF-κB restoration agent and an antigen are employed, they may be contained in or otherwise associated with the same particle or different particles. A variety of particles may be used in the invention, including but not limited to, liposomes, micelles, lipidic particles, ceramic/inorganic particles and polymeric particles, and are typically selected from nanoparticles and microparticles. The particles are suitably sized for phagocytosis or endocytosis by antigen-presenting cells.
[0176] Antigen-presenting cells include both professional and facultative types of antigen-presenting cells. Professional antigen-presenting cells include, but are not limited to, macrophages, monocytes, B lymphocytes, cells of myeloid lineage, including monocytic-granulocytic-DC precursors, marginal zone Kupffer cells, microglia, T cells, Langerhans cells and dendritic cells including interdigitating dendritic cells and follicular dendritic cells. Examples of facultative antigen-presenting cells include but are not limited to activated T cells, astrocytes, follicular cells, endothelium and fibroblasts. In some embodiments, the antigen-presenting cell is selected from monocytes, macrophages, B-lymphocytes, cells of myeloid lineage, dendritic cells or Langerhans cells. In specific embodiments, the antigen-presenting cell expresses CDl Ic and includes a dendritic cell. In illustrative examples, the particles have a dimension of less than about 100 μm, more suitably in the range of less than or equal to about 500 nm, although the particles may be as large as about 10 μm, and as small as a few nm. Liposomes consist basically of a phospholipid bilayer forming a shell around an aqueous core. Advantages include the lipophilicity of the outer layers which "mimic" the outer membrane layers of cells and that they are taken up relatively easily by a variety of cells. Polymeric vehicles typically consist of micro/nanospheres and micro/nanocapsules formed of biocompatible polymers, which are either biodegradable (for example, polylactic acid) or non-biodegradable (for example, ethylenevinyl acetate). Some of the advantages of the polymeric devices are ease of manufacture and high loading capacity, range of size from nanometer to micron diameter, as well as controlled release and degradation profile.
[0177] In some embodiments, the particles comprise an antigen-binding molecule on their surface, which is immuno-interactive with a marker that is expressed at higher levels on antigen-presenting cells (e.g., dendritic cells) than on non-antigen- presenting cells. Illustrative markers of this type include MGL, DCL-I, DEC-205, macrophage mannose R, DC-SIGN or other DC or myeloid specific (lectin) receptors, as for example disclosed by Hawiger et al. (2001, J Exp Med 194, 769), Kato et al. 2003, J Biol Chem 278, 34035), Benito et al. (2004, J Am Chem Soc 126, 10355), Schjetne, et al. (2002, Int Immunol 14, 1423) and van Vliet et al, 2006, Nat Immunol Sep 24; [Epub ahead of print])(van Vliet et al, Immunobiology 2006, 21 1 :577-585).
[0178] The particles can be prepared from a combination of the bioactive agent(s), and a surfactant, excipient or polymeric material. In some embodiments, the particles are biodegradable and biocompatible, and optionally are capable of biodegrading at a controlled rate for delivery of a therapeutic or diagnostic agent. The particles can be made of a variety of materials. Both inorganic and organic materials can be used. Polymeric and non-polymeric materials, such as fatty acids, may be used. Other suitable materials include, but are not limited to, gelatin, polyethylene glycol, trehalulose, dextran and chitosan. Particles with degradation and release times ranging from seconds to months can be designed and fabricated, based on factors such as the particle material.
4.3.1 Polymeric Particles
[0179] Polymeric particles may be formed from any biocompatible and desirably biodegradable polymer, copolymer, or blend. The polymers may be tailored to optimize different characteristics of the particle including: i) interactions between the bioactive agents to be delivered and the polymer to provide stabilization of the bioactive agents and retention of activity upon delivery; ii) rate of polymer degradation and, thereby, rate of agent release profiles; iii) surface characteristics and targeting capabilities via chemical modification; and iv) particle porosity.
[0180] Surface eroding polymers such as polyanhydrides may be used to form the particles. For example, polyanhydrides such as poly[(p-carboxyphenoxy)- hexane anhydride] (PCPH) may be used. Biodegradable polyanhydrides are described in U.S. Pat. No. 4,857,311.
[0181] In other embodiments, bulk eroding polymers such as those based on polyesters including poly(hydroxy acids) or poly(esters) can be used. For example, polyglycolic acid (PGA), polylactic acid (PLA), or copolymers thereof may be used to form the particles. The polyester may also have a charged or functionalizable group, such as an amino acid. In illustrative examples, particles with controlled release properties can be formed of poly(D,L-lactic acid) and/or poly(D,L-lactic-co-glycolic acid) ("PLGA") which incorporate a surfactant such as DPPC.
[0182] Other polymers include poly(alkylcyanoacrylates), polyamides, polycarbonates, polyalkylenes such as polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly vinyl compounds such as polyvinyl alcohols, polyvinyl ethers, and polyvinyl esters, polymers of acrylic and methacrylic acids, celluloses and other polysaccharides, and peptides or proteins, or copolymers or blends thereof. Polymers may be selected with or modified to have the appropriate stability and degradation rates in vivo for different controlled drug delivery applications.
[0183] In some embodiments, particles are formed from functionalized polyester graft copolymers, as described in Hrkach et al. (1995, Macromolecules, 28:4736-4739; and "Poly(L-Lactic acid-co-amino acid) Graft Copolymers: A Class of Functional Biodegradable Biomaterials" in Hydrogels and Biodegradable Polymers for Bioapplications, ACS Symposium Series No. 627, Raphael M. Ottenbrite et al., Eds., American Chemical Society, Chapter 8, pp. 93-101, 1996.)
[0184] Materials other than biodegradable polymers may be used to form the particles. Suitable materials include various non-biodegradable polymers and various excipients. The particles also may be formed of the bioactive agent(s) and surfactant alone.
[0185] Polymeric particles may be prepared using single and double emulsion solvent evaporation, spray drying, solvent extraction, solvent evaporation, phase separation, simple and complex coacervation, interfacial polymerization, and other methods well known to those of ordinary skill in the art. Particles may be made using methods for making microspheres or microcapsules known in the art, provided that the conditions are optimized for forming particles with the desired diameter.
[0186] Methods developed for making microspheres for delivery of encapsulated agents are described in the literature, for example, as described in Doubrow, M., Ed., "Microcapsules and Nanoparticles in Medicine and Pharmacy," CRC Press, Boca Raton, 1992. Methods also are described in Mathiowitz and Langer (1987, J. Controlled Release 5, 13-22); Mathiowitz et al. (1987, Reactive Polymers 6, 275-283); and Mathiowitz et al. (1988, J. Appl. Polymer Sci. 35, 755-774) as well as in
U.S. Pat. No. 5,213,812, U.S. Pat. No. 5,417,986, U.S. Pat. No. 5,360,610, and U.S. Pat. No. 5,384,133. The selection of the method depends on the polymer selection, the size, external morphology, and crystallinity that is desired, as described, for example, by Mathiowitz et al. (1990, Scanning Microscopy 4: 329-340; 1992, J. Appl. Polymer Sci. 45, 125-134); and Benita et al. (1984, J. Pharm. Sci. 73, 1721-1724). [0187] In solvent evaporation, described for example, in Mathiowitz et al. ,
(1990), Benita; and U.S. Pat. No. 4,272,398 to Jaffe, the polymer is dissolved in a volatile organic solvent, such as methylene chloride. Several different polymer concentrations can be used, for example, between 0.05 and 2.0 g/mL. The bioactive agent(s), either in soluble form or dispersed as fine particles, is (are) added to the polymer solution, and the mixture is suspended in an aqueous phase that contains a surface-active agent such as polyvinyl alcohol). The aqueous phase may be, for example, a concentration of 1% poly(vinyl alcohol) w/v in distilled water. The resulting emulsion is stirred until most of the organic solvent evaporates, leaving solid microspheres, which may be washed with water and dried overnight in a lyophilizer. Microspheres with different sizes (between 1 and 1000 μm) and morphologies can be obtained by this method.
[0188] Solvent removal was primarily designed for use with less stable polymers, such as the polyanhydrides. In this method, the agent is dispersed or dissolved in a solution of a selected polymer in a volatile organic solvent like methylene chloride. The mixture is then suspended in oil, such as silicon oil, by stirring, to form an emulsion. Within 24 hours, the solvent diffuses into the oil phase and the emulsion droplets harden into solid polymer microspheres. Unlike the hot-melt microencapsulation method described for example in Mathiowitz et al. (1987, Reactive Polymers, 6:275), this method can be used to make microspheres from polymers with high melting points and a wide range of molecular weights. Microspheres having a diameter for example between one and 300 microns can be obtained with this procedure.
[0189] With some polymeric systems, polymeric particles prepared using a single or double emulsion technique, vary in size depending on the size of the droplets. If droplets in water-in-oil emulsions are not of a suitably small size to form particles with the desired size range, smaller droplets can be prepared, for example, by sonication or homogenation of the emulsion, or by the addition of surfactants. [0190] If the particles prepared by any of the above methods have a size range outside of the desired range, particles can be sized, for example, using a sieve, and further separated according to density using techniques known to those of skill in the art.
[0191] The polymeric particles can be prepared by spray drying. Methods of spray drying, such as that disclosed in PCT WO 96/09814 by Sutton and Johnson, disclose the preparation of smooth, spherical microparticles of a water-soluble material with at least 90% of the particles possessing a mean size between 1 and 10 μm.
4.3.2 Ceramic Particles [0192] Ceramic particles may also be used to deliver the bioactive agents of the invention. These particles are typically prepared using processes similar to the well known sol-gel process and usually require simple and room temperature conditions as described for example in Brinker et al. ("Sol-Gel Science: The Physics and Chemistry of Sol-Gel Processing;" Academic Press: San Diego, 1990, p-60), and Avnir et al. (1994, Chem. Mater. 6, 1605). Ceramic particles can be prepared with desired size, shape and porosity, and are extremely stable. These particles also effectively protect doped molecules (polypeptides, drugs etc.) against denaturation induced by extreme pH . and temperature (Jain et al, 1998, J. Am. Chem. Soc. 120, 11092-11095). In addition, their surfaces can be easily functionalized with different groups (LaI et al, 2000, Chem. Mater. 12, 2632-2639; Badley et al, 1990, Langmuir, 6, 792-801), and therefore they can be attached to a variety of monoclonal antibodies and other ligands in order to target them to desired sites in vivo.
[0193] Various ceramic particles have been described for delivery in vivo of active agent-containing payloads. For example, British Patent 1 590 574 discloses incorporation of biologically active components in a sol-gel matrix. International
Publication WO 97/45367 discloses controllably dissolvable silica xerogels prepared via a sol-gel process, into which a biologically active agent is incorporated by impregnation into pre-sintered particles (1 to 500 μm) or disks. International Publication WO 0050349 discloses controllably biodegradable silica fibres prepared via a sol-gel process, into which a biologically active agent is incorporated during synthesis of the fibre. U.S. Pat. Appl. Pub. 20040180096 describes ceramic nanoparticles in which a bioactive substance is entrapped. The ceramic nanoparticles are made by formation of a micellar composition of the dye. The ceramic material is added to the micellar composition and the ceramic nanoparticles are precipitated by alkaline hydrolysis. U.S. Pat. Appl. Pub. 2005012361 1 discloses controlled release ceramic particles comprising an active material substantially homogeneously dispersed throughout the particles. These particles are prepared by mixing a surfactant with an apolar solvent to prepare a reverse micelle solution; (b) dissolving a gel precursor, a catalyst, a condensing agent and a soluble active material in a polar solvent to prepare a precursor solution; (c) combining the reverse micelle solution and the precursor solution to provide an emulsion and (d) condensing the precursor in the emulsion. U.S. Pat. Appl. Pub. 20060210634 discloses adsorbing bioactive substances onto ceramic particles comprising a metal oxide (e.g. , titanium oxide, zirconium oxide, scandium oxide, cerium oxide and yttrium oxide) by evaporation. Kortesuo et al. (2000, Int J Pharm. May 10;200(2):223-229) disclose a spray drying method to produce spherical silica gel particles with a narrow particle size range for controlled delivery of drugs such as toremifene citrate and dexmedetomidine HCl. Wang et al. (2006, Int J Pharm. 308(1- 2): 160- 167) describe the combination of adsorption by porous CaCO3 microparticles and encapsulation by polyelectrolyte multilayer films for delivery of bioactive substances.
4.3.3 Liposomes [0194] Liposomes can be produced by standard methods such as those reported by Kim et al. (1983, Biochim. Biophys. Acta 728, 339-348); Liu et al. (1992, Biochim. Biophys. Acta 1 104, 95-101); Lee et al. (1992, Biochim. Biophys. Acta, ϊ 103, 185-197), Brey et al. (U.S. Pat. Appl. Pub. 20020041861), Hass et al. (U.S. Pat. Appl. Pub. 20050232984), Kisak et al. (U.S. Pat. Appl. Pub. 20050260260) and Smyth- Templeton et al. (U.S. Pat. Appl. Pub. 20060204566). Additionally, reference may be made to Copeland et al. (2005, Immunol. Cell Biol. 83: 95-105) who review lipid based particulate formulations for the delivery of antigen, and to Bramwell et al (2005, Crit Rev Ther Drug Carrier Syst. 22(2): 151-214; 2006, J Pharm Pharmacol. 58(6):717-728) who review particulate delivery systems for vaccines, including methods for the preparation of protein-loaded liposomes. Many liposome formulations using a variety of different lipid components have been used in various in vitro cell culture and animal experiments. Parameters have been identified that determine liposomal properties and are reported in the literature, for example, by Lee et al. (1992, Biochim. Biophys. Acta. 1 103, 185-197); Liu et al. (1992, Biochim. Biophys. Acta, 1 104, 95-101); and Wang et al. (1989, Biochem. 28, 9508-951).
[0195 j Briefly, the lipids of choice (and any organic-soluble bioactive), dissolved in an organic solvent, are mixed and dried onto the bottom of a glass tube under vacuum. The lipid film is rehydrated using an aqueous buffered solution containing any water-soluble bioactives to be encapsulated by gentle swirling. The hydrated lipid vesicles can then be further processed by extrusion, submitted to a series of freeze-thawing cycles or dehydrated and then rehydrated to promote encapsulation of bioactives. Liposomes can then be washed by centrifugation or loaded onto a size- exclusion column to remove unentrapped bioactive from the liposome formulation and stored at 4° C. The basic method for liposome preparation is described in more detail in Thierry et al. (1992, Nuc. Acids Res. 20:5691-5698).
[0196] A particle carrying a payload of bioactive agent(s) can be made using the procedure as described in: Pautot et al. (2003, Proc. Natl. Acad. Sci. USA, 100(19): 10718-21). Using the Pautot et al. technique, streptavidin-coated lipids (DPPC, DSPC, and similar lipids) can be used to manufacture liposomes. The drug encapsulation technique described by Needham et al (2001, Advanced Drug Delivery Reviews, 53(3): 285-305) can be used to load these vesicles with one or more active agents. [0197] The liposomes can be prepared by exposing chloroformic solution of various lipid mixtures to high vacuum and subsequently hydrating the resulting lipid films (DSPC/CHOL) with pH 4 buffers, and extruding them through polycarbonated filters, after a freezing and thawing procedure. It is possible to use DPPC supplemented with DSPC or cholesterol to increase encapsulation efficiency or increase stability, etc. A transmembrane pH gradient is created by adjusting the pH of the extravesicular medium to 7.5 by addition of an alkalinization agent. A bioactive agent {e.g., small molecule NF-κB restoration agents, which are, for example, weak bases) can be subsequently entrapped by addition of a solution of the bioactive agent in small aliquots to the vesicle solution, at an elevated temperature, to allow accumulation of the bioactive agent inside the liposomes.
[0198] Other lipid-based particles suitable for the delivery of the bioactive agents of the present invention such as niosomes are described by Copeland et al. (2005, Immunol. Cell Biol. 83: 95-105). 4.3.4 Ballistic particles
[0199] The bioactive agents of the present invention may be attached to {e.g., by coating or conjugation) or otherwise associated with particles suitable for use in needleless or "ballistic" (biolistic) delivery. Illustrative particles for ballistic delivery are described, for example, in: International Publications WO 02/101412; WO 02/100380; WO 02/43774; WO 02/19989; WO 01/93829; WO 01/83528; WO 00/63385; WO 00/26385; WO 00/19982; WO 99/01 168; WO 98/10750; and WO 97/48485. It shall be understood, however, that such particles are not limited to their use with a ballistic delivery device and can otherwise be administered by any alternative technique {e.g., injection or microneedle delivery) through which particles are deliverable to immune cells.
[0200] The bioactive agents can be coated or chemically coupled to carrier particles {e.g., core carriers) using a variety of techniques known in the art. Carrier particles are selected from materials which have a suitable density in the range of particle sizes typically used for intracellular delivery. The optimum carrier particle size will, of course, depend on the diameter of the target cells. Illustrative particles have a size ranging from about 0.01 to about 250 μm, from about 10 to about 150 μm, and from about 20 to about 60 μm; and a particle density ranging from about 0.1 to about 25 g/cm3, and a bulk density of about 0.5 to about 3.0 g/cm3, or greater. Non-limiting particles of this type include metal particles such as, tungsten, gold, platinum and iridium carrier particles. Tungsten particles are readily available in average sizes of 0.5 to 2.0 μm in diameter. Gold particles or microcrystalline gold {e.g., gold powder Al 570, available from Engelhard Corp., East Newark, N.J.) may also be used. Gold particles provide uniformity in size (available from Alpha Chemicals in particle sizes of 1-3 μm, or available from Degussa, South Plainfield, N.J. in a range of particle sizes including 0.95 μm) and low toxicity. Microcrystalline gold provides a diverse particle size distribution, typically in the range of 0.1-5 μm. The irregular surface area of microcrystalline gold provides for highly efficient coating with the active agents of the present invention. [0201] Many methods are known and have been described for adsorbing, coupling or otherwise attaching bioactive molecules {e.g., hydrophilic molecules such as proteins and nucleic acids) onto particles such as gold or tungsten particles. In illustrative examples, such methods combine a predetermined amount of gold or tungsten with the bioactive molecules, CaCl2 and spermidine. In other examples, ethanol is used to precipitate the bioactive molecules onto gold or tungsten particles (see, for example, Jumar et ai, 2004, Phys Med. Biol. 49:3603-3612). The resulting solution is suitably vortexed continually during the coating procedure to ensure uniformity of the reaction mixture. After attachment of the bioactive molecules, the particles can be transferred for example to suitable membranes and allowed to dry prior to use, coated onto surfaces of a sample module or cassette, or loaded into a delivery cassette for use in particular particle-mediated delivery instruments.
[0202] The formulated compositions may suitably be prepared as particles using standard techniques, such as by simple evaporation (air drying), vacuum drying, spray drying, freeze drying (lyophilization), spray-freeze drying, spray coating, precipitation, supercritical fluid particle formation, and the like. If desired, the resultant particles can be dandified using the techniques described in International Publication WO 97/48485.
4.3.5 Surfactants
[0203] Surfactants which can be incorporated into particles include phosphoglycerides. Exemplary phosphoglycerides include phosphatidylcholines, such as the naturally occurring surfactant, L-α-phosphatidylcholine dipalmitoyl ("DPPC"). The surfactants advantageously improve surface properties by, for example, reducing particle-particle interactions, and can render the surface of the particles less adhesive. The use of surfactants endogenous to the lung may avoid the need for the use of non- physiologic surfactants.
[0204] Providing a surfactant on the surfaces of the particles can reduce the tendency of the particles to agglomerate due to interactions such as electrostatic interactions, Van der Waals forces, and capillary action. The presence of the surfactant on the particle surface can provide increased surface rugosity (roughness), thereby improving aerosolization by reducing the surface area available for intimate particle- particle interaction.
[0205] Surfactants known in the art can be used including any naturally occurring surfactant. Other exemplary surfactants include diphosphatidyl glycerol (DPPG); hexadecanol; fatty alcohols such as polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surface active fatty acid, such as palmitic acid or oleic acid; sorbitan trioleate (Span 85); glycocholate; surfactin; a poloxamer; a sorbitan fatty acid ester such as sorbitan trioleate; tyloxapol and a phospholipid.
4.4 Antigen-presenting cell embodiments
[0206] In some embodiments, the immune modulator that is used to increase the number of operable NF-κB antigen-presenting cells in the subject is an antigen- presenting cell or its precursor, which is obtained from the subject to be treated (i.e., an autologous antigen-presenting cell). In these embodiments, the antigen-presenting cell is contacted with at least one NF-κB restoration agent as described for example in Section 4.1, which is suitably in soluble form or in particulate form as described for example in Section 4.3, in an amount and for a time sufficient to restore NF-κB activity in the antigen-presenting cell to a basal state. In other embodiments, the immune modulator is an antigen-presenting cell that is derived from a donor that is MHC matched or mismatched with the subject (i.e., an allogeneic antigen-presenting cell). Suitably, in these embodiments, the donor is histocompatible with the subject. [0207] In certain examples of the above embodiments, the antigen-presenting cell or its precursor is also contacted with an antigen according to Section 4.2, or with a polynucleotide from which the antigen is expressible, for a time and under conditions sufficient for the antigen or a processed form thereof to be presented by the antigen- presenting cell. Suitably, the antigen is in soluble form or in particulate form as described for example in Section 4.3.
4.4.1 Sources of antigen-presenting cells and their precursors
[0208] Antigen-presenting cells or their precursors can be isolated by methods known to those of skill in the art. The source of such cells will differ depending upon the antigen-presenting cell required for modulating a specified immune response. In this context, the antigen-presenting cell can be selected from dendritic cells, macrophages, monocytes and other cells of myeloid lineage.
[0209] Typically, precursors of antigen-presenting cells can be isolated from any tissue, but are most easily isolated from blood, cord blood or bone marrow (Sorg et al, 2001, Exp Hematol 29, 1289-1294; Zheng et al, 2000, J Hematother Stem Cell Res 9, 453-464). It is also possible to obtain suitable precursors from diseased tissues such as rheumatoid synovial tissue or fluid following biopsy or joint tap (Thomas et al, 1994a, J Immunol 153, 4016-4028; Thomas et al, 1994b, Arthritis Rheum 37(4)). Other examples include, but are not limited to liver, spleen, heart, kidney, gut and tonsil (Lu et al, 1994, J Exp Med 179, 1823-1834; Mcllroy et al , 2001, Blood 97, 3470-3477; Vremec et al, 2000, J Immunol 159, 565-573; Hart and Fabre, 1981, J Exp Med 154(2), 347-361 ; Hart and McKenzie, 1988, J Exp Med 168(1), 157-170; Pavli et al, 1990, Immunology 70( 1 ), 40-47).
[0210] Leukocytes isolated directly from tissue provide a major source of antigen-presenting cell precursors. Typically, these precursors can only differentiate into antigen-presenting cells by culturing in the presence or absence of various growth factors. According to the practice of the present invention, the antigen-presenting cells may be so differentiated from crude mixtures or from partially or substantially purified preparations of precursors. Leukocytes can be conveniently purified from blood or bone marrow by density gradient centrifugation using, for example, Ficoll Hypaque which eliminates neutrophils and red cells (peripheral blood mononuclear cells or PBMCs), or by ammonium chloride lysis of red cells (leukocytes or white blood cells). Many precursors of antigen-presenting cells are present in peripheral blood as non- proliferating monocytes, which can be differentiated into specific antigen-presenting cells, including macrophages and dendritic cells, by culturing in the presence of specific cytokines.
[0211] Tissue-derived precursors such as precursors of tissue dendritic cells or of Langerhans cells are typically obtained by mincing tissue (e.g. , basal layer of epidermis) and digesting it with collagenase or dispase followed by density gradient separation, or selection of precursors based on their expression of cell surface markers. For example, Langerhans cell precursors express CDl molecules as well as HLA-DR and can be purified on this basis. [0212] In some embodiments, the antigen-presenting cell precursor is a precursor of macrophages. Generally these precursors can be obtained from monocytes of any source and can be differentiated into macrophages by prolonged incubation in the presence of medium and macrophage colony stimulating factor (M-CSF) (Erickson- Miller et al, 1990, Int J Cell Cloning 8, 346-356; Metcalf and Burgess, 1982, J Cell Physiol, 111, 275-283).
[0213] In other embodiments, the antigen presenting cell precursor is a precursor of Langerhans cells. Usually, Langerhans cells can be generated from human monocytes or CD34+ bone marrow precursors in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-4/TNFα and TGFβ (Geissmann et al, 1998, J Exp Med, 187, 961-966; Strobl et al, 1997a, Blood 90, 1425- 1434; Strobl et al, 1997b, dv Exp Med Biol 417, 161-165; Strobl et al, 1996, J Immunol 157, 1499-1507). [0214] In still other embodiments, the antigen-presenting cell precursor is a precursor of dendritic cells. Several potential dendritic cell precursors can be obtained from peripheral blood, cord blood or bone marrow. These include monocytes, CD34+ stem cells, granulocytes, CD33+CD1 Ic+ DC precursors, and committed myeloid progenitors - described below. Monocytes:
[0215] Monocytes can be purified by adherence to plastic for 1-2 h in the presence of tissue culture medium {e.g., RPMI) and serum {e.g., human or foetal calf serum), or in serum- free medium (Anton et al, 1998, Scand J Immunol 47, 116-121; Araki et al, 2001, Br J Haematol 1 14, 681-689; Mackensen et al, 2000, Int J Cancer 86, 385-392; Nestle et al, 1998, Nat Med 4, 328-332; Romani et al, 1996, J Immunol Meth 196, 137-151; Thurner et al, 1999, J Immunol Methods 223, 1-15). Monocytes can also be elutriated from peripheral blood (Garderet et al, 2001, J Hematother Stem Cell Res 10, 553-567). Monocytes can also be purified by immunoaffinity techniques, including immunomagnetic selection, flow cytometric sorting or panning (Araki et al , 2001 , supra; Battye and Shortman, 1991 , Curr. Opin. Immunol. 3, 238-241 ), with anti- CD 14 antibodies to obtain CD14hi cells. The numbers (and therefore yield) of circulating monocytes can be enhanced by the in vivo use of various cytokines including GM-CSF (Groopman et al, 1987, N Engl J Med 317, 593-598; Hill et al, 1995, J Leukoc Biol 58, 634-642). Monocytes can be differentiated into dendritic cells by prolonged incubation in the presence of GM-CSF and IL-4 (Romani et al , 1994, J Exp Med 180, 83-93; Romani et al, 1996, supra). A combination of GM-CSF and IL-4 at a concentration of each at between about 200 to about 2000 U/mL, more preferably between about 500 to about 1000 U/mL and even more preferably between about 800 U/mL (GM-CSF) and 1000 U/mL (IL-4) produces significant quantities of immature dendritic cells, i.e., antigen-capturing phagocytic dendritic cells. Other cytokines which promote differentiation of monocytes into antigen-capturing phagocytic dendritic cells include, for example, IL- 13. CD34+ stem cells:
[0216] Dendritic cells can also be generated from CD34+ bone marrow derived precursors in the presence of GM-CSF, TNFα ± stem cell factor (SCF, c-kitL), or GM-CSF, IL-4 ± flt3L (Bai et al, 2002, Int J Oncol 20, 247-53; Chen et al, 2001, Clin Immunol 98, 280-292; Loudovaris et al, 2001, J Hematother Stem Cell Res 10, 569-578). CD34+ cells can be derived from a bone marrow aspirate or from blood and can be enriched as for monocytes using, for example, immunomagnetic selection or immunocolumns (Davis et al, 1994, J Immunol Meth 175, 247-257). The proportion of CD34+ cells in blood can be enhanced by the in vivo use of various cytokines including (most commonly) G-CSF, but also flt3L and progenipoietin (Fleming et al, 2001, Exp Hematol 29, 943-951 ; Pulendran et al, 2000, J Immunol 165, 566-572; Robinson et al, 2000, J Hematother Stem Cell Res 9, 71 1-720).
Other myeloid progenitors:
[0217] DC can be generated from committed early myeloid progenitors in a similar fashion to CD34+ stem cells, in the presence of GM-CSF and IL-4/TNF. Such myeloid precursors infiltrate many tissues in inflammation, including rheumatoid arthritis synovial fluid (Santiago-Schwarz et al, 2001, J Immunol. 167, 1758-1768). Expansion of total body myeloid cells including circulating dendritic cell precursors and monocytes, can be achieved with certain cytokines, including flt-3 ligand, granulocyte colony-stimulating factor (G-CSF) or progenipoietin (pro-GP) (Fleming et al, 2001, supra; Pulendran et al, 2000, supra; Robinson et al, 2000, supra). Administration of such cytokines for several days to a human or other mammal would enable much larger numbers of precursors to be derived from peripheral blood or bone marrow for in vitro manipulation. Dendritic cells can also be generated from peripheral blood neutrophil precursors in the presence of GM-CSF, IL-4 and TNFα (Kelly et al , 2001 , Cell MoI Biol (Noisy-le-grand) 47, 43-54; Oehlcr et al, 1998, J Exp Med. 187, 1019-1028). It should be noted that dendritic cells can also be generated, using similar methods, from acute myeloid leukaemia cells (Oehler et al, 2000, Ann Hematol. 79, 355-62).
Tissue DC precursors and other sources of APC precursors: [0218] Other methods for DC generation exist from, for example, thymic precursors in the presence of IL-3 +/- GM-CSF, and liver DC precursors in the presence of GM-CSF and a collagen matrix. Transformed or immortalised dendritic cell lines may be produced using oncogenes such as v-myc as for example described by (Paglia et al, 1993) or by myb (Banyer and Hapel, 1999; Gonda et al, 1993).
Circulating DC precursors:
[0219] These have been described in human and mouse peripheral blood. One can also take advantage of particular cell surface markers for identifying suitable dendritic cell precursors. Specifically, various populations of dendritic cell precursors can be identified in blood by the expression of CDl Ic and the absence or low expression of CD14, CD19, CD56 and CD3 (O'Doherty et al., 1994, Immunology 82, 487-493; O'Doherty et al, 1993, J Exp Med 178, 1067-1078). These cells can also be identified by the cell surface markers CD13 and CD33 (Thomas et al., 1993b, J
Immunol 151(12), 6840-6852). A second subset, which lacks CD 14, CD 19, CD56 and CD3, known as plasmacytoid dendritic cell precursors, does not express CDl Ic, but does express CD123 (IL-3R chain) and HLA-DR (Farkas et al, 2001, Am J Pathol 159, 237-243; Grouard et al, 1997, J Exp Med 185, 1101-1111; Rissoan et al, 1999, Science 283, 1183-1186). Most circulating CDl Ic+ dendritic cell precursors are HLA-DR+, however some precursors may be HLA-DR-. The lack of MHC class II expression has been clearly demonstrated for peripheral blood dendritic cell precursors (del Hoyo et al , 2002, Nature 415, 1043-1047).
[0220] Optionally, CD33+CD14 /l0 or CDl Ic+HLA-DR+, lineage marker- negative dendritic cell precursors described above can be differentiated into more mature antigen-presenting cells by incubation for 18-36 h in culture medium or in monocyte conditioned medium (Thomas et al, 1993b, supra; Thomas and Lipsky, 1994, J Immunol 153, 4016-4028) (O'Doherty et al, 1993, supra). Alternatively, following incubation of peripheral blood non-T cells or unpurified PBMC, the mature peripheral blood dendritic cells are characterised by low density and so can be purified on density gradients, including metrizamide and Nycodenz (Freudenthal and Steinman, 1990, Proc Natl Acad Sci U S A 87, 7698-7702; Vremec and Shortman, 1997, J Immunol 159, 565- 573), or by specific monoclonal antibodies, such as but not limited to the CMRF-44 mAb (Fearnley et al, 1999, Blood 93, 728-736; Vuckovic et al, 1998, Exp Hematol 26, 1255-1264). Plasmacytoid dendritic cells can be purified directly from peripheral blood on the basis of cell surface markers, and then incubated in the presence of IL-3 (Grouard et al, 1997, supra; Rissoan et al, 1999, supra). Alternatively, plasmacytoid DC can be derived from density gradients or CMRF-44 selection of incubated peripheral blood cells as above.
[0221] In general, for dendritic cells generated from any precursor, when incubated in the presence of activation factors such as monocyte-derived cytokines, lipopolysaccharide and DNA containing CpG repeats, cytokines such as TNF-α, IL-6, IFN-α, IL- 1 β, necrotic cells, re-adherence, whole bacteria, membrane components, RNA or polylC, immature dendritic cells will become activated (Clark, 2002, J Leukoc Biol, 71, 388-400; Hacker et al., 2002, Immunology 105, 245-251; Kaisho and Akira, 2002, Biochim Biophys Acta 1589, 1-13; Koski et al, 2001, Crit Rev Immunol 21, 179- 189). This process of dendritic cell activation is inhibited in the presence of NF- KB inhibitors (O'Sullivan and Thomas, 2002, J Immunol 168, 5491-5498).
4.4.2 Ex vivo delivery ofNF-κB restoration agents and antigen
[0222] NF-κB restoration agents can be delivered into antigen-presenting cells in various forms, including small molecules, nucleic acids and polypeptides. The NF-κB restoration agent s may be soluble or particulate. In nucleic acid embodiments, the NF-κB restoration agent is typically in the form of a nucleic acid construct from which a NF-κB restoration agent {e.g., a proteinaceous NF-κB restoration agent) is expressible. The amount of soluble or particulate NF-κB restoration agent to be placed in contact with antigen-presenting cells can be determined empirically by routine methods known to persons of skill in the art. Typically antigen-presenting cells are incubated with NF-κB restoration agent for about 10 min to about 12 hr at 35° C - 38° C or for as much time as required to activate NF-κB or to restore NF-κB function to a basal state.
[0223] The amount of soluble or particulate antigen to be placed in contact with antigen-presenting cells can be determined empirically by routine methods known to persons of skill in the art. Typically antigen-presenting cells are incubated with antigen for about 1 to 6 hr at 37° C, although it is also possible to expose antigen- presenting cells to antigen for the duration of incubation with growth factors and NF-κB restoration agent. Usually, for purified antigens and peptides, 0.1-10 μg/mL is suitable for producing antigen-specific antigen-presenting cells. Dendritic cells are exposed to apoptotic bodies in approximately 1 : 1 ratio, and bacteria (Albert et al. , 1998, International Publication WO 99/42564; Corinti et al, 1999, J Immunol 163(6), 3029-
3036). The antigen should be exposed to the antigen-presenting cells for a period of time sufficient for those cells to internalize the antigen. The time and dose of antigen necessary for the cells to internalize and present the processed antigen may be determined using pulse-chase protocols in which exposure to antigen is followed by a washout period and exposure to a read-out system e.g., antigen reactive T cells. Once the optimal time and dose necessary for cells to express processed antigen on their surface is determined, a protocol may be used to prepare cells and antigen for inducing tolerogenic responses. Those of skill in the art will recognise in this regard that the length of time necessary for an antigen-presenting cell to present an antigen may vary depending on the antigen or form of antigen employed, its dose, and the antigen- presenting cell employed, as well as the conditions under which antigen loading is undertaken. These parameters can be determined by the skilled artisan using routine procedures.
[0224] In some embodiments, the delivery of exogenous antigen to an antigen-presenting cell can be enhanced by methods known to practitioners in the art. For example, several different strategies have been developed for delivery of exogenous antigen to the endogenous processing pathway of antigen-presenting cells, especially dendritic cells. These methods include insertion of antigen into pH-sensitive liposomes (Zhou and Huang, 1994, Immunomethods 4, 229-235), osmotic lysis of pinosomes after pinocytic uptake of soluble antigen (Moore et ai, 1988, Cell 54, 777-785), coupling of antigens to potent adjuvants (Aichele et ai, 1990, J. Exp. Med., 171, 1815-1820; Gao et ai, 1991, J. Immunol., 147, 3268-3273; Schulz et ai, 1991, Proc. Natl. Acad. Sci. USA, 88, 991-993; Kuzu et ai, 1993, Euro. J. Immunol. 23, 1397-1400; and Jondal et ai, 1996, Immunity 5, 295-302), exosomes (Zitvogel et ai, 1998 Nat Med. 4, 594-600; 2002, Nat Rev Immunol. 2, 569-79), and apoptotic cell delivery of antigen (Albert et ai, 1998, Nature 392, 86-89; Albert et ai, 1998, Nature Med. 4, 1321-1324; and in
International Publications WO 99/42564 and WO 01/85207). Recombinant bacteria (eg. E. coli) or transfected host mammalian cells may be pulsed onto dendritic cells (as particulate antigen, or apoptotic bodies respectively) for antigen delivery. Such a delivery system might be logically combined with a substance for inhibiting NF-κB, such as a plasmid encoding dominant negative IκBα (Pai et ai, 2002, J Virol 76, 1914- 1921). Recombinant chimeric virus-like particles (VLPs) have also been used as vehicles for delivery of exogenous heterologous antigen to the MHC class I processing pathway of a dendritic cell line (Bachmann et ai, 1996, Eur. J. Immunol., 26(11), 2595-
2600). [0225] Alternatively, or in addition, an antigen may be linked to, or otherwise associated with, a cytolysin to enhance the transfer of the antigen into the cytosol of an antigen-presenting cell of the invention for delivery to the MHC class I pathway. Exemplary cytolysins include saponin compounds such as saponin-containing Immune Stimulating Complexes (ISCOMs) (see e.g., Cox and Coulter, 1997, Vaccine 15(3), 248-256 and U.S. Patent No. 6,352,697), phospholipases (see, e.g., Camilli et al., 1991, J. Exp. Med. 173, 751-754), pore-forming toxins (e.g., an alpha-toxin), natural cytolysins of gram-positive bacteria, such as listeriolysin O (LLO, e.g., Mengaud et al., 1988, Infect. Immun. 56, 766-772 and Portnoy et al., 1992, Infect. Immun. 60, 2710- 2717), streptolysin O (SLO, e.g., Palmer et al., 1998, Biochemistry 37(8), 2378-2383) and perfringolysin O (PFO, e.g., Rossjohn et al., Cell 89(5), 685-692). Where the antigen-presenting cell is phagosomal, acid activated cytolysins may be advantageously used. For example, listeriolysin exhibits greater pore-forming ability at mildly acidic pH (the pH conditions within the phagosome), thereby facilitating delivery of vacuole (including phagosome and endosome) contents to the cytoplasm (see, e.g., Portnoy et al., 1992, Infect. Immun. 60, 2710-2717).
[0226] The cytolysin may be provided together with a pre-selected antigen in the form of a single composition or may be provided as a separate composition, for contacting the antigen-presenting cells. In one embodiment, the cytolysin is fused or otherwise linked to the antigen, wherein the fusion or linkage permits the delivery of the antigen to the cytosol of the target cell. In another embodiment, the cytolysin and antigen are provided in the form of a delivery vehicle such as, but not limited to, a liposome or a microbial delivery vehicle selected from virus, bacterium, or yeast. Suitably, when the delivery vehicle is a microbial delivery vehicle, the delivery vehicle is non-virulent. In a preferred embodiment of this type, the delivery vehicle is a non- virulent bacterium, as for example described by Portnoy et al. in U.S. Patent No. 6,287,556, comprising a first polynucleotide encoding a non-secreted functional cytolysin operably linked to a regulatory polynucleotide which expresses the cytolysin in the bacterium, and a second polynucleotide encoding one or more pre-selected antigens. Non-secreted cytolysins may be provided by various mechanisms, e.g., absence of a functional signal sequence, a secretion incompetent microbe, such as microbes having genetic lesions (e.g., a functional signal sequence mutation), or poisoned microbes, etc. A wide variety of nonvirulent, non-pathogenic bacteria may be used; preferred microbes are relatively well characterised strains, particularly laboratory strains of E. coli, such as MC4100, MC 1061 , DH5. alpha., etc. Other bacteria that can be engineered for the invention include well-characterised, nonvirulent, non-pathogenic strains of Listeria monocytogenes, Shigella flexneri, mycobacterium, Salmonella, Bacillus subtilis, etc. In a particular embodiment, the bacteria are attenuated to be non- replicative, non-integrative into the host cell genome, and/or non-motile inter- or intra- cellularly.
[0227] The delivery vehicles described above can be used to deliver one or more antigens to virtually any antigen-presenting cell capable of endocytosis of the subject vehicle, including phagocytic and non-phagocytic antigen-presenting cells. In embodiments when the delivery vehicle is a microbe, the subject methods generally require microbial uptake by the target cell and subsequent lysis within the antigen- presenting cell vacuole (including phagosomes and endosomes).
[0228] In other embodiments, a proteinaceous NF-κB restoration agent and optionally an antigen of interest can be produced inside an antigen-presenting cell by introduction of one or more expression constructs that encode the restoration agent and/or the antigen. As described, for example, in U.S. Pat. No. 5,976,567 (Inex), the expression of natural or synthetic nucleic acids is typically achieved by operably linking a nucleic acid of interest to a promoter (which may be either constitutive or inducible), preferably incorporating the construct into an expression vector, and introducing the vector into a suitable host cell. Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular nucleic acid. The vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (e.g. , shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems. Vectors may be suitable for replication and integration in prokaryotes, eukaryotes, or preferably both. See, Giliman and Smith (1979), Gene, 8: 81-97; Roberts et al. (1987), Nature, 328: 731-734; Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, volume 152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989), MOLECULAR CLONING - A
LABORATORY MANUAL (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, N. Y., (Sambrook); and F. M. Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel).
[0229] Expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are typically used for expression of nucleic acid sequences in eukaryotic cells. SV40 vectors include pSVT7 and pMT2. Vectors derived from bovine papilloma virus include pBV- IMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5. Other exemplary vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
[0230] While a variety of vectors may be used, it should be noted that viral expression vectors are useful for modifying eukaryotic cells because of the high efficiency with which the viral vectors transfect target cells and integrate into the target cell genome. Illustrative expression vectors of this type can be derived from viral DNA sequences including, but not limited to, adenovirus, adeno-associated viruses, herpes- simplex viruses and retroviruses such as B, C, and D retroviruses as well as spumaviruses and modified lentiviruses. Suitable expression vectors for transfection of animal cells are described, for example, by Wu and Ataai (2000, Curr. Opin.
Biotechnol. 1 1(2), 205-208), Vigna and Naldini (2000, J. Gene Med. 2(5), 308-316), Kay et al. (2001, Nat. Med. 7(1), 33-40), Athanasopoulos, et al. (2000, Int. J. MoI. Med. 6(4),363-375) and Walther and Stein (2000, Drugs 60(2), 249-271).
[0231] The antigen-encoding portion of the expression vector may comprise a naturally-occurring sequence or a variant thereof, which has been engineered using recombinant techniques. In one example of a variant, the codon composition of an antigen-encoding polynucleotide is modified to permit enhanced expression of the antigen in a target cell or tissue of choice using methods as set forth in detail in International Publications WO 99/02694 and WO 00/42215. Briefly, these methods are based on the observation that translational efficiencies of different codons vary between different cells or tissues and that these differences can be exploited, together with codon composition of a gene, to regulate expression of a protein in a particular cell or tissue type. Thus, for the construction of codon-optimised polynucleotides, at least one existing codon of a parent polynucleotide is replaced with a synonymous codon that has a higher translational efficiency in a target cell or tissue than the existing codon it replaces. Although it is preferable to replace all the existing codons of a parent nucleic acid molecule with synonymous codons which have that higher translational efficiency, this is not necessary because increased expression can be accomplished even with partial replacement. Suitably, the replacement step affects 5%, 10%, 15%, 20%, 25%, 30%, more preferably 35%, 40%, 50%, 60%, 70% or more of the existing codons of a parent polynucleotide.
[0232] The expression vector is compatible with the antigen-presenting cell in which it is introduced such that the antigen-encoding polynucleotide is expressible by the cell. The expression vector is introduced into the antigen-presenting cell by any suitable means which will be dependent on the particular choice of expression vector and antigen-presenting cell employed. Such means of introduction are well-known to those skilled in the art. For example, introduction can be effected by use of contacting (e.g., in the case of viral vectors), electroporation, transformation, transduction, conjugation or triparental mating, transfection, infection membrane fusion with cationic lipids, high-velocity bombardment with DNA-coated microprojectiles, incubation with calcium phosphate-DNA precipitate, direct microinjection into single cells, and the like. Other methods also are available and are known to those skilled in the art. Alternatively, the vectors are introduced by means of cationic lipids, e.g., liposomes. Such liposomes are commercially available (e.g., Lipofectin®, Lipofectamine™, and the like, supplied by Life Technologies, Gibco BRL, Gaithersburg, Md.).
5. Pharmaceutical formulations
[0233] In accordance with the present invention, at least one immune modulator that increases the number of antigen-presenting cells with operable NF-κB function is useful in compositions and methods for treating or preventing an undesirable or deleterious immune response in a subject, wherein the immune response is associated with defective NF-κB activation (i.e., reduced or abrogated NF-κB activation), including defective ReIB activation (i.e., reduced or abrogated NF-κB activation). The immune modulator(s) is (are) suitably selected from: (i) NF-κB restoration agents as described for example in Section 4.1 ; and (ii) antigen-presenting cells with operable NF-κB function as described for example in Section 4.4, wherein the immune modulators are in soluble form or in particulate form as described for example in Section 4.3 and are optionally associated with one or more antigens as described for example in Section 4.2.
[0234] The inventive compositions and methods are useful, therefore, for treating or preventing an undesirable or deleterious immune response associated with defective NF-κB activation including, for example, allergies, organ-specific diseases,, parasitic diseases, and some inflammatory and autoimmune diseases. Examples of allergies include seasonal respiratory allergies; allergy to aeroallergens such as hay fever; allergy treatable by reducing serum IgE and eosinophilia; asthma; eczema; animal allergies, food allergies; chronic urticaria; latex allergies; allergic rhinitis; atopic dermatitis; or allergies treatable by allergic desensitization. Autoimmune diseases and related conditions that can be treated or prevented by the present invention include, for example, psoriasis, systemic lupus erythematosus, myasthenia gravis, stiff-man syndrome, thyroiditis, Sydenham chorea, rheumatoid arthritis, ankylosing spondylitis, autoimmune aplastic anemia, autoimmune hemolytic anemia, Churg Strauss disease, scleroderma, Wegener granulomatosus, Wiskott Aldrich syndrome, type 1 diabetes mellitus (TlDM) and multiple sclerosis. Examples of inflammatory disease include Crohn's disease, chronic inflammatory eye diseases, chronic inflammatory lung diseases and chronic inflammatory liver diseases, autoimmune hemolytic anemia, idiopathic leucopoenia, ulcerative colitis, dermatomyositis, scleroderma, mixed connective tissue disease, irritable bowel syndrome, systemic lupus erythromatosus
(SLE), multiple sclerosis, myasthenia gravis, Guillan-Barre syndrome (antiphospholipid syndrome), primary myxoedema, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastris, alopecia totalis, Addison's disease, insulin-dependent diabetes mellitus (IDDM), Goodpasture's syndrome, Behcet's syndrome, Sjogren's syndrome, rheumatoid arthritis, sympathetic ophthalmia, Hashimoto's disease/hypothyroiditis, celiac disease/dermatitis herpetiformis, adult-onset idiopathic hypoparathyroidism (AOIH), amyotrophic lateral sclerosis, and demyelinating disease primary biliary cirrhosis, mixed connective tissue disease, chronic active hepatitis, polyendocrine failure, vitiligo, Celiac disease, chronic active hepatitis, CREST syndrome, dermatomyositis, dilated cardiomyopathy, eosinophilia-myalgia syndrome, epidermolisis bullosa acquisita (EBA), giant cell arteritis, Graves' disease/hyperthyroiditis, scleroderma, chronic idiopathic thrombocytopenic purpura, peripheral neuropathy, diabetic neuropathy , hemochromatosis, Henoch-Schonlein purpura, idiopathic IgA nephropathy, insulin-dependent diabetes mellitus (IDDM), Lambert-Eaton syndrome, linear IgA dermatosis, myocarditis, narcolepsy, necrotizing vasculitis, neonatal lupus syndrome (NLE), nephrotic syndrome, pemphigoid, pemphigus, polymyositis, primary sclerosing cholangitis, psoriasis, rapidly-progressive glomerulonephritis (RPGN), Reiter's syndrome, and septic shock. Other unwanted immune reactions that can also be treated or prevented by the present invention include antibodies to recombinant therapeutic agents such as anti-factor VIII antibodies in hemophilia or anti-insulin antibodies in diabetes.
[0235] In specific embodiments, the undesirable or deleterious immune response is an organ-specific disease, non-limiting examples of which include TlDM, thyroiditis, adrenal insufficiency, alopecia, atrophic gastritis, vitiligo, premature ovarian failure, autoimmune polyendocrine syndromes (APS), parathyroiditis, hypoparathyroidism, autoimmune adrenal insufficiency (Addison's disease), autoimmune hepatitis, Sjogren's syndrome, celiac disease, exocrine pancreatitis, keratitis and mucocutaneous candidiasis. Without wishing to be bound by any one particular theory or mode of operation, the inventors hypothesize that since activation of the NF- KB signaling pathway is dysfunctional in TlDM, it would also be dysfunctional in organ-specific diseases generally because they cluster within individuals and families that have or are predisposed to developing TlDM. This hypothesis is also consistent with the finding that mice deficient in ReIB (ReIB ^) display autoimmune multi-organ inflammatory disease, including pancreatitis, salivary gland inflammation (Sjogren's syndrome), pneumonitis, hepatitis and keratitis and that mice deficient in the NIK (aly/aly mutant) and NF- κB2 (p52) components of the non-canonical NF-κB pathway exhibit milder organ-specific autoimmune inflammation (e.g., salivary and lacrimal glands, pancreas and liver). [0236] The inventive compositions and methods are useful for restoring the capacity of antigen-presenting cells to license regulatory lymphocyte mediated suppression of T cell-associated inflammatory disease. The efficiency of inducing lymphocytes, especially T lymphocytes, to exhibit tolerance to a specified antigen can be determined by assaying immune responses to that antigen including, but not limited to, assaying T lymphocyte cytolytic activity in vitro using for example the antigen- specific antigen-presenting cells as targets of antigen-specific cytolytic T lymphocytes (CTL); assaying antigen-specific T lymphocyte proliferation (see, e.g., Vollenweider and Groscurth, 1992, J. Immunol. Meth. 149, 133-135), measuring B cell response to the antigen using, for example, Elispot assays, and Elisa assays; interrogating cytokine profiles; or measuring delayed-type hypersensitivity (DTH) responses by test of skin reactivity to a specified antigen (see, e.g., Chang et al 1993, Cancer Res. 53, 1043- 1050). [0237] The compositions of the present invention are typically in the form of pharmaceutical compositions, which may comprise a pharmaceutically acceptable carrier or diluent. In some embodiments, the compositions are administered to individuals having the unwanted or deleterious immune response. In other embodiments, the compositions are administered to at-risk individuals who are autoantibody positive and/or HLA haplotype identified at risk e.g., TlDM first degree relatives with at least one and desirably two or more autoantibodies positive (see, e.g., Scofield, R. H., 2004. Lancet 363, 1544; Berglin et al, 2004, Arthritis Res Ther 6, R30336; Harrison et al, 2004, Diabetes Care 27, 2348), or individuals at risk of rheumatoid arthritis, with one or two HLA susceptibility genes and positive anti-CCP antibodies (Klarskog et al 2006, Arthritis Rheum. 54: 38) (Rantapaa-Dahlqvist S et al 2003, Arthritis Rheum. 48:2741).
[0238] Pharmaceutical compositions suitable for use in the present invention include compositions wherein the bioactive agents are contained in an effective amount to achieve their intended purpose. The dose of active compounds administered to a patient should be sufficient to achieve a beneficial response in the patient over time such as a reduction in at least one symptom associated with the unwanted or deleterious immune response, which is suitably associated with a condition selected from an allergy, an autoimmune disease and a transplant rejection. The quantity or dose frequency of the pharmaceutically active compounds(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof. In this regard, precise amounts of the active compound(s) for administration will depend on the judgment of the practitioner. In determining the effective amount of the active compound(s) to be administered in the treatment or prophylaxis of the unwanted or deleterious immune response, the practitioner may evaluate inflammation, pro-inflammatory cytokine levels, lymphocyte proliferation, cytolytic T lymphocyte activity and regulatory T lymphocyte function. In any event, those of skill in the art may readily determine suitable dosages of the antagonist and antigen. [0239] Accordingly, the bioactive agents are administered to a subject to be treated in a manner compatible with the dosage formulation, and in an amount that will be prophylactically and/or therapeutically effective. The amount of the composition to be delivered, generally in the range of from 0.01 μg/kg to 100 μg/kg of bioactive molecule (e.g., NF-κB restoration agent or antigen) per dose, depends on the subject to be treated. In some embodiments, and dependent on the intended mode of administration, the NF-κB restoration agent-containing compositions will generally contain about 0.1% to 90%, about 0.5% to 50%, or about 1% to about 25%, by weight of the agent, the remainder being suitable pharmaceutical carriers and/or diluents etc and optionally an antigen. The dosage of the agent can depend on a variety of factors, such as mode of administration, the species of the affected subject, age and/or individual condition. In other embodiments, and dependent on the intended mode of administration, the antigen-containing compositions will generally contain about 0.1% to 90%, about 0.5% to 50%, or about 1% to about 25%, by weight of antigen, the remainder being suitable pharmaceutical carriers and/or diluents etc and NF-κB restoration agent.
[0240] Depending on the specific condition being treated, the particles may be formulated and administered systemically, topically or locally. Techniques for formulation and administration may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition. Suitable routes may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. For injection, the particles of the invention may be formulated in aqueous solutions, suitably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
[0241] The compositions of the present invention may be formulated for administration in the form of liquids, containing acceptable diluents (such as saline and sterile water), or may be in the form of lotions, creams or gels containing acceptable diluents or carriers to impart the desired texture, consistency, viscosity and appearance. Acceptable diluents and carriers are familiar to those skilled in the art and include, but are not restricted to, ethoxylated and nonethoxylated surfactants, fatty alcohols, fatty acids, hydrocarbon oils (such as palm oil, coconut oil, and mineral oil), cocoa butter waxes, silicon oils, pH balancers, cellulose derivatives, emulsifying agents such as non- ionic organic and inorganic bases, preserving agents, wax esters, steroid alcohols, triglyceride esters, phospholipids such as lecithin and cephalin, polyhydric alcohol esters, fatty alcohol esters, hydrophilic lanolin derivatives, and hydrophilic beeswax derivatives.
[0242] Alternatively, the particles of the present invention can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration, which is also preferred for the practice of the present invention. Such carriers enable the compounds of the invention to be formulated in dosage forms such as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. These carriers may be selected from sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.
[0243] Pharmaceutical formulations for parenteral administration include aqueous solutions of the particles in water-soluble form. Additionally, suspensions of the particles may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. [0244] Pharmaceutical preparations for oral use can be obtained by combining the particles with solid excipients and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as., for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more therapeutic agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g. by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
[0245] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of particle doses.
[0246] Pharmaceuticals which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.
[0247] The compositions of the invention may be administered over a period of hours, days, weeks, or months, depending on several factors, including the severity of the neuropathic condition being treated, whether a recurrence of the condition is considered likely, etc. The administration may be constant, e.g., constant infusion over a period of hours, days, weeks, months, etc. Alternatively, the administration may be intermittent, e.g., particles may be administered once a day over a period of days, once an hour over a period of hours, or any other such schedule as deemed suitable.
[0248] The compositions may also be administered to the respiratory tract as a nasal or pulmonary inhalation aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose, or with other pharmaceutically acceptable excipients. In such a case, the particles of the formulation may advantageously have diameters of less than 50 μm, suitably less than 10 μm. [0249] In some embodiments. Bioactive agent-containing particles are administered for active uptake by cells, for example by phagocytosis, as described for example in U.S. Pat. No. 5,783,567 (Pangaea). In some embodiments, phagocytosis by these cells may be improved by maintaining a particle size typically below about 20 μm, and preferably below about 11 μm. In specific embodiments, the particles are delivered directly into the bloodstream (i.e., by intravenous or intra-arterial injection or infusion) if uptake by the phagocytic cells of the reticuloendothelial system (RES) , including liver and spleen, is desired. Alternatively, one can target, via subcutaneous injection, take-up by the phagocytic cells of the draining lymph nodes. The particles can also be introduced intradermally (i.e., to the APCs of the skin, such as dendritic cells and Langerhans cells) for example using ballistic or microneedle delivery. Illustrative particle-mediated delivery techniques include explosive, electric or gaseous discharge delivery to propel carrier particles toward target cells as described, for example, in U.S. Pat. Nos. 4,945,050, 5,120,657, 5,149,655 and 5,630,796. Non-limiting examples of microneedle delivery are disclosed in International Publication Nos. WO 2005/069736 and WO 2005/072630 and U.S. Pat. Nos. 6,503,231 and 5,457,041.
[0250] Another useful route of delivery (particularly for DNAs encoding NF- KB restoration polypeptides or antigens) is via the gastrointestinal tract, e.g., orally. Alternatively, the particles can be introduced into organs such as the lung (e.g., by inhalation of powdered microparticles or of a nebulized or aerosolized solution containing the microparticles), where the particles are picked up by the alveolar macrophages, or may be administered intranasally or buccally. Once a phagocytic cell phagocytoses the particle, the NF-κB restoration agent and optionally the antigen are released into the interior of the cell. [0251] When antigen-presenting cells are employed as the immune modulator, the cells can be introduced into a patient by any means (e.g., injection), which produces the desired modified immune response to an antigen or group of antigens. The cells may be derived from the patient (i.e., autologous cells) or from an individual or individuals who are MHC-matched or -mismatched (i.e., allogeneic) with the patient. In specific embodiments, autologous cells are injected back into the patient from whom the source cells were obtained. The injection site may be subcutaneous, intraperitoneal, intramuscular, intradermal, or intravenous. The cells may be administered to a patient already suffering from the unwanted immune response or who is predisposed to the unwanted immune response in sufficient number to prevent or at least partially arrest the development, or to reduce or eliminate the onset of, that response. The number of cells injected into the patient in need of the treatment or prophylaxis may vary depending on inter alia, the antigen or antigens and size of the individual. This number may range for example between about 103 and 1011, and more preferably between about 103 and 107 cells (e.g., dendritic cells). Single or multiple administrations of the cells can be carried out with cell numbers and pattern being selected by the treating physician. The cells should be administered in a pharmaceutically acceptable carrier, which is non-toxic to the cells and the individual. Such carrier may be the growth medium in which the cells were grown, or any suitable buffering medium such as phosphate buffered saline. The cells may be administered alone or as an adjunct therapy in conjunction with other therapeutics known in the art for the treatment or prevention of unwanted immune responses for example but not limited to glucocorticoids, methotrexate, D-penicillamine, hydroxychloroquine, gold salts, sulfasalazine, TNFα or interleukin-1 inhibitors, and/or other forms of specific immunotherapy. In specific embodiments, the antigen-presenting cells are pre-contacted with one or more antigens associated with the unwanted or deleterious immune response to provide antigen-specific tolerogenic antigen presenting cells or are administered concurrently to the subject with one or more such antigens.
[0252] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.
EXAMPLES
EXAMPLE 1
LPS-INDUCED NF-KB ACTIVITY AND CELL SURFACE MARKER EXPRESSION IN TlDM
DC [0253] The present inventors have demonstrated constitutively low nuclear
NF-κB expression by monocyte-derived DC cultured from healthy donors for 48-72 h (O'Sullivan BJ, et al. , J Immunol 168:5491-5498, 2002). Further, such DC efficiently translocate NF-κB to the nucleus after activation with TNF-α, LPS or CD 154 ((O'Sullivan BJ, et al, J Immunol 168:5491-5498, 2002). Because DC from TlDM patients differentiate abnormally and NF-κB is required for the functional differentiation of DC, the present inventors examined NF-κB expression in DC generated from peripheral blood (PB) monocytes of TlDM patients and healthy controls in serum free medium, GM-CSF and IL-4 for 72 h with or without addition of LPS to induce differentiation for the final 24 h. While nuclear ReIB, ReIA, c-Rel, p52 and p50 proteins increased in response to LPS for DC cultured from healthy controls, this was not observed for TlDM DC (Figure IA). To determine the ability of NF-κB from TlDM and control subjects to bind its consensus DNA binding motif and therefore to exert transcriptional activity, binding was analyzed by ELISA using nuclear extracts from TlDM patients. In contrast to the similar ReIB DNA binding in resting DC from TlDM and healthy controls, binding of ReIB from healthy control DC was significantly higher than that from TlDM DC 24h after exposure to LPS (Figure IB). As reduced DC responsiveness to LPS might be a characteristic of cells derived from a hyperglycemic environment, or might occur generally in patients with autoimmune disease, the present inventors examined resting or LPS-exposed DC ReIB DNA binding for patients with type 2 diabetes (T2DM) or rheumatoid arthritis (RA). LPS-induced ReIB activity was more variable among patients from each group than among healthy controls. The mean LPS-induced ReIB activity was increased 1.4 fold in T2DM, 1.5 fold in RA and 1.9 fold in healthy controls relative to TlDM patients (Figure 1C).
[0254] Using an ELISA assay with a capacity for sensitive and quantitative detection of NF-κB DNA binding, due its readout in photon units, using the luminometer, greater sensitivity for diagnosis of TlDM subjects was obtained. Representative healthy control and TlDM patient DC NF-κB DNA binding with and without LPS is shown in Figures 6A and B. NF-κB binding of TlDM patients tends to increase early in response to LPS, then to repress at later time points (Figure 6B). PB monocytes displayed a similar lack of increase in NF-κB binding in response to LPS (depicted as fold increase in binding in response to LPS, in Figure 7A). Indeed DNA binding of ReIA and ReIB was higher in TlDM than healthy control monocytes, even in the absence of LPS activation (Figure 7B), suggesting that TlDM monocytes differentiate abnormally into DCs.
[0255] While LPS predominantly activates the classical NF-κB pathway in B cells, both classical and alternative pathways are activated by LPS in monocyte-derived DC (Zarnegar B, et al, Proc Natl Acad Sci USA 101 :8108-81 13, 2004; Mordmuller B, et al, EMBO Rep 4:82-87, 2003). TlD DC were similarly reduced in responsiveness to other upstream NF-kB activators, including TNFalpha and CD 154. The global reduction in NF-κB nuclear translocation in response to treatment with LPS, TNFalpha and CD 154, suggests a post-translational impairment in classical and alternative NF-κB pathway responsiveness to DC signaling.
EXAMPLE 2
INCREASED SHP l EXPRESSION IN TlDM PBMC
[0256] SHP-I is expressed by hematopoietic cells in PB, including circulating monocyte precursors of DC. To assess relative expression, SHP-I was quantitated by intracellular staining and flow cytometric analysis. Expression of SHP-I by resting PB monocytes from 18 TlDM patients was significantly higher than expression by 13 healthy controls (Figure 2A). In general, SHP-I decreased with prolonged cell culture in the presence of cytokines, and expression was low in both control and TlDM monocyte-derived DC cultured for 48 h in GM-CSF and IL-4. From subject to subject, the expression of SHP-I by PB monocytes predicted SHP-I expression by lymphocytes, whether healthy control or diabetic, suggesting similar regulatory processes in each cell type (Figure 2B). EXAMPLE 3
MONOCYTE SHP-I OVER-EXPRESSION INHIBITS NF-KB ACTIVITY AND DC
DIFFERENTIATION IN TlDM
[0257] To assess the functional effects of SHP-I on NF-kB in TlDM, PB monocytes were stimulated with LPS and then stained for phospho-I-κBα 30 minutes later. SHP-I expression in TlDM was negatively associated with induction of phospho- I-κBα expression by PB monocytes (Figure 3A). The inventors next addressed whether inhibition of SHP-I could rescue the NF-κB activation and DC differentiation in TlDM, using the SHP-I inhibitor sodium stibogluconate (Pathak MK, et al, J Immunol 167:3391-3397, 2001). Addition of sodium stibogluconate to cultures of TlDM monocyte-derived DC for 48 h promoted DC differentiation, associated with enhanced IκBα phosphorylation (Figure 3B). Moreover, when DC from these cultures were subsequently treated for 24 h with LPS, cell surface CD40 expression was augmented, relative to DC stimulated with LPS alone (Figure 3C). Finally, addition of sodium stibogluconate to TlDM PB mononuclear cells promoted ReIB DNA binding, and enhanced the ReIB response to LPS. These data indicate that SHP-I plays a functional role in TlDM to suppress NF-κB activity and inhibit DC differentiation from monocyte precursors.
EXAMPLE 4
ADOPTIVE TRANSFER OF WT DC RESTORES IMMUNOREGULATION AND REVERSES INFLAMMATORY ORGAN PATHOLOGY IN RELB DEFICIENT MICE
[0258] Mice deficient in ReIB, the transcriptional mediator of the non- canonical NF-κB signaling pathway, display autoimmune and allergic multi-organ inflammatory disease and excessive myelopoiesis. ReIB" " mice inter-crossed with T cell deficient or RAG7" mice fail to develop inflammatory diseases, indicating that this phenotype induces a defect in the control of effector or regulatory T cell functions. The inventors found in vitro, that T cell function was impaired in response to ReIB"'" DC, and in light of this finding, assessed the effect of DC in vivo by adoptive transfer of wt DC into ReIB"'" recipients. Either bead-purified or sorted CDl Ic+ RelB+/" splenic DC were transferred i.v. into ReIB"7" recipients, then these mice and untreated controls were monitored for 20 days. ReIB"'" mice receiving wt DC showed significant cumulative weight gain compared with untreated ReIB"'" mice, eventually reaching a similar weight to control ReIB+ " mice (Figure 4A). In addition hair regenerated and gait, tail and spine posture, agility, and eye lustre improved rapidly and remained healthy in treated mice. Untreated ReIB" mice showed signs of distress and disability after 10-15 days and required sacrifice after 20 days, whereas this early mortality was prevented by DC transfer to ReIB '''.
[0259] Compared with ReIB+7' pancreas by H&E staining, untreated ReIB'7" pancreas showed extensive inflammation with infiltration of lymphoid and myeloid cells into the exocrine pancreas and islets, disruption of acinar cell architecture, atrophy of pancreatic and islet cells, and numerous cells in the inflammatory infiltrate which incorporated Brdu. Pancreas from DC-treated ReIB"7" mice showed minimal inflammation, restoration of acinar cell architecture, normal pancreatic and islet cells, with rare parenchymal cells incorporating Brdu, suggestive of regeneration. In the liver, heavy peri-portal infiltration and Brdu incorporation by inflammatory cells observed in ReIB"7" mice was no longer observed after transfer of wt DC. In the spleen, untreated ReIB"7" animals showed extra-medullary hematopoiesis with increased numbers of erythroblasts and megakaryocytes, and inflammatory infiltrate into the red pulp. After wt DC transfer, this infiltrate decreased as did the number of erythroblasts and megakaryocytes, resulting in larger areas of red pulp (Figure 4B). Together the data indicate that adoptive transfer of wt DC restores immunoregulation to suppress organ inflammation in ReIB"7" mice.
EXAMPLE 5
DEFECTIVE RELB ACTIVATION IN PATIENTS WITH SJOGREN'S SYNDROME [0260] To examine whether patients with another organ-specific autoimmune disease also displayed evidence of abnormal NF-κB activation in response to LPS, patients with Sjogren's syndrome were examined, using similar methods to those described for TlDM patients. In contrast to the patients with rheumatoid arthritis (Figure 1), patients with Sjogren's syndrome also displayed a reduced ReIA, p50 and ReIB DNA binding capacity after 24h LPS incubation, compared with healthy control subjects using the same assay as for TlDM subjects (Figure 5) EXAMPLE 6
DEFECTIVE RELB ACTIVATION IN FIRST DEGREE RELATIVES OF CHILDREN WITH
TlDM.
[0261] To determine whether abnormal NF-κB activation might be associated with the risk of TlDM, first degree relatives in families in which at least one child had developed TlDM (proband) were examined for the NF-κB abnormality. Figure 8 shows one representative family in which father's DCs show normal NF-κB activation, but both siblings of the proband fail to increase DC NF-κB in response to LPS incubation.
EXPERIMENTAL
MATERIALS AND METHODS
MICE
[0262] C57BL/6 (wt) mice (Animal Resource Centre, Perth, Australia) were maintained in specific pathogen-free (SPF) conditions. Mice homozygous for an insertional mutation in the ReIB gene (designated RelB~Λ), on a C57BL/6 background, were originally generated in D. Lo's Laboratory (L. Burkly et al, 1995, Nature 373, 531). They were bred in clean conventional conditions and used at 5-7 weeks of age. RelB"Λ radiation chimeric mice, generated by transferring BM from RelB"Λ into lethally irradiated C57B1/6 hosts, were used after at least 6 weeks reconstitution.
CELL PREPARATION AND CULTURE FOR TlD STUDY [0263] Heparinized peripheral blood was collected from TlDM, type 2 diabetic (T2DM), rheumatoid arthritis (RA) patients or healthy controls (HC). The study was approved by the human ethics committee of the Princess Alexandra Hospital. Blood was collected from forty three otherwise healthy, insulin-treated TlDM patients (age range 7-72 years, mean age 33 years, 45% female). PB mononuclear cells (PBMC) were prepared as described (O'Sullivan BJ, et al, J Immunol 168:5491-5498, 2002). For DC, monocytes were purified from PBMC using CD 14 microbeads (Miltenyi Biotec, GmbH, Germany), then cultured in 24 well plates in X-VIVO 20 (BioWhittaker Walkersville, MD) with 800 U/ml rhGM-CSF and 600 U/ml rhIL-4 (Schering-Plough, Sydney, Australia) for 72 hours. In some experiments, 10 μg/ml sodium stibogluconate were added at the commencement of DC culture. 100 ng/ml LPS were added to some wells for the last 24 hours of the culture period. CDl Ic+ DC were purified from mev/mev or wild type mouse spleens using CDl Ic microbeads (Miltenyi Biotec), then cultured overnight with or without 100 ng/ml LPS in RPMI+10% FCS.
DC EXPANSION AND TRANSFER
[0264] RelB+/" mice were administered 20ug progenipoietin-1 (Pfizer, NY, USA) s.c. daily for 7 days. Expanded splenic CDl Ic+ DC were purified by MACS or sorted. Either 12.5 x 106 MACS-purified DC or 2 x 106 sorted DC were injected i.v into each ReIB" " recipient. Mice were monitored every five days for weight gain, gait, agility, alopecia, progression of lacrimal disease/keratoconjunctivits, dermatitis, knee joint swelling and feeding habits. Before sacrifice, mice received i.p. Brdu.
HISTOLOGY
[0265] Formalin-fixed liver, spleen, and pancreas from anti-GITR-L Ab- treated, DC-treated and control ReIB7- mice, as well as RelB+/" mice, were sectioned and stained with H&E at the histotechnology laboratory, Queensland Institute of Medical Research. Immunohistochemical analysis of Brdu incorporation in the tissue sections was detected using Brdu labeling and detection kit (Roche). Staining was revealed with HRP -conjugated anti-mouse Ig (Silenus, Victoria, Australia) and DAB (Biocare Medical, Concord, CA, USA). Haematoxylin counterstain was used on these sections to identify cell nuclei.
IMMUNOBLOTTING AND RELB DNA BINDING [0266] Cytoplasmic and nuclear extracts were prepared from 72 hour cultured
DC or PBMC cultured for 24 h with or without sodium stibogluconate, according to the iso-osmotic/nonidet P-40 method described (O'Sullivan BJ, et al, J Immunol 168:5491- 5498, 2002; Pettit AR, et al, J Immunol 159:3681-3691, 1997; Pathak MK, et al., J Immunol 167:3391-3397, 2001). Extracts were preserved at -70° C and protein was estimated using a Protein Assay kit (Bio-rad, Hercules, CA). For immunoblotting, 20 μg of protein were separated on 10% polyacrylamide gel and then transferred onto a nitrocellulose membrane (Amersham Biosciences, Suny, CA). The membrane was stained with 0.1% Ponceau S stain in 5% acetic acid to compare protein loading. After several washes, the membrane was blocked with skimmed milk and then incubated with rabbit NF-κB antibodies ReIA (sc-372), ReIB (sc-226), c-Rel (sc-70), p50 (sc-7178) from Santa Cruz Biotechnology (Santa Cruz, CA) and p52 (Upstate, Lake Placid, NY). Binding was revealed with HRP-conjugated anti-rabbit antibody using ECL chemiluminescence reagents (Amersham). ELISA-based format BD™ Transfactor Kits (BD biosciences, Palo Alto, CA) measured NF-κB DNA binding as described (O'Sullivan BJ, et ai, J Immunol 168:5491-5498, 2002). Absorbance data were expressed as relative units.
FLOW CYTOMETRY
[0267] For intracellular staining, PBMC were fixed with 2% PFA and permeabilized with 90% cold methanol for 30 min. Cells were incubated and washed with BD Perm/Wash™ buffer, then stained with either anti-phospho-p38 MAPK (T180/Y182):Alexa Fluor 647 (BD Pharmingen), anti-SHP-1 (Upstate) or anti- phospho-IκBα (Santa Cruz Biotechnology), then the latter two with biotinylated goat- anti-rabbit Ig, and finally with streptavidin-FITC. Isotype controls were included for every sample. Diabetic and control cells were included in every run and the same cytometry settings were maintained throughout. Some samples were run multiple times on different days to ensure reproducibility of the data over time. Delta mean fluorescence intensity (ΔMFI) was measured by subtracting MFI of isotype-matched controls from the specific marker MFI of the samples, using CellQuest Pro software.
INHIBITORS
[0268] Sodium stibogluconate (Calbiochem, Darmstadt, Germany) was dissolved at lmg/ml in water using the Mini-Beadbeater (Biospec Products Inc., Bartleville, OK). The p38 MAP kinase inhibitor SB230580 (Promega, Madison, WC) was used at 10 ug/ml.
STATISTICAL ANALYSIS [0269] Data were analyzed using paired or unpaired t tests.
[0270] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.
[0271] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application. [0272] Throughout the specification the aim has been to describe certain embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention. All such modifications and changes are intended to be included within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:
1. A method for modulating an undesirable or deleterious immune response associated with defective NF-KB activation in a subject, the method comprising increasing the number of operable NF-κB antigen-presenting cells in the subject, in which ReIB is activated or activatable in response to a pro-inflammatory signal.
2. A method according to claim 1 , comprising administering to the subject an effective amount of an immune modulator that increases the number of operable NF-κB antigen-presenting cells.
3. A method according to claim 2, wherein the immune modulator comprises at least one agent that activates ReIB and is provided in an amount sufficient to restore
ReIB function to a basal state in antigen-presenting cells of the subject.
4. A method according to claim 3, wherein an individual agent is administered in soluble form.
5. A method according to claim 3, wherein an individual agent is administered in particulate form.
6. A method according to claim 1 , wherein the antigen presenting cells are selected from dendritic cells, macrophages and Langerhans cell.
7. A method according to claim 1, wherein the operable NF-κB antigen- presenting cells stimulate the production of regulatory T lymphocytes that suppress or otherwise reduce the undesirable or deleterious immune response.
8. A method according to claim 6, wherein the regulatory T lymphocytes are CD4+CD25+regulatory T lymphocytes.
9. A method according to claim 2, wherein the immune modulator comprises operable NF-κB antigen-presenting cells.
10. A method according to claim 9, wherein the operable NF-κB antigen- presenting cells are derived from a donor, who is histocompatible with the subject.
11. A method according to claim 9, wherein the operable NF-κB antigen- presenting cells are derived by harvesting antigen-presenting cells from the subject and treating them ex vivo with at least one agent that activates ReIB in an amount sufficient to restore ReIB function to a basal state in those cells.
12. A method according to claim 3 or claim 11, wherein the agent(s) decrease(s) the level or functional activity of SHP-I .
13. A method according to claim 9, further comprising contacting the operable NF-κB antigen presenting cells with an antigen that corresponds to at least a portion of a target antigen that associates with the undesirable or deleterious immune response.
14. A method according to claim 1, wherein the undesirable or deleterious immune response associated with defective NF-κB activation is associated with the presence of or predisposition to an autoimmune disease.
15. A method according to claim 14, wherein the autoimmune disease is selected from TlDM, systemic lupus erythematosus, Churg Strauss disease, scleroderma, Wegener granulomatosus, Wiskott Aldrich syndrome
16. A method according to claim 14, wherein the autoimmune disease is TlDM.
17. A method according to claim 16, further comprising contacting the operable
NF-κB antigen presenting cells with an antigen that corresponds to at least a portion of a target antigen that associates with the undesirable or deleterious immune response, wherein the target antigen is selected from proinsulin, insulin, IA2 and GAD65.
18. A method according to claim 1, wherein the undesirable or deleterious immune response associated with defective NF-κB activation is associated with the presence of or predisposition to an allergy.
19. A method according to claim 18, wherein the allergy is selected from allergic asthma and allergic eczema.
20. A method according to claim 1 , wherein the undesirable or deleterious immune response associated with defective NF-κB activation is associated with the presence of or predisposition to an organ-specific disease.
21. A method according to claim 20, wherein the organ-specific disease is selected from TlDM, thyroiditis, adrenal insufficiency, alopecia, atrophic gastritis, vitiligo, premature ovarian failure, autoimmune polyendocrine syndromes (APS), parathyroiditis, hypoparathyroidism, autoimmune adrenal insufficiency (Addison's disease), autoimmune hepatitis, Sjogren's syndrome, celiac disease, exocrine pancreatitis, keratitis and mucocutaneous candidiasis.
22. A composition comprising an immune modulator that increases the number of operable NF-κB antigen-presenting cells in a subject, in which cells ReIB is activated or activatable in response to a pro-inflammatory signal, and an antigen that corresponds to at least a portion of a target antigen that associates with the presence or risk of an undesirable or deleterious immune response and with defective ReIB activation in antigen-presenting cells.
23. A composition according to claim 22, wherein the immune modulator comprises a SHP-I inhibitor.
24. A composition according to claim 22, wherein the immune modulator comprises a nucleic acid construct that expresses a nucleotide sequence encoding the NF-κB pathway polypeptide in antigen-presenting cells.
25. A composition according to any one of claims claim 22 to 23, wherein the target antigen is selected from proinsulin, insulin, IA2 and GAD65.
26. A composition comprising a SHP-I inhibitor and an antigen that corresponds to at least a portion of a target antigen that associates with the presence or risk of an undesirable or deleterious immune response and with defective ReIB activation in antigen-presenting cells.
27. Use of an immune modulator that increases the number of operable NF-κB antigen-presenting cells in the subject, in which cells ReIB is activated or activatable in response to a pro-inflammatory signal, and optionally an antigen that corresponds to at least a portion of a target antigen that associates with the presence or risk of an undesirable or deleterious immune response and with defective NF-κB activation in antigen-presenting cells in the manufacture of a medicament for suppressing the immune response.
28. A method for treating a condition associated with an undesirable or deleterious immune response and with defective NF-κB activation in a subject, the method comprising administering to the subject an effective amount of an immune modulator that increases the number of antigen-presenting cells in the subject, in which cells ReIB is activated or activatable, in response to a pro-inflammatory signal.
29. A method according to claim 27, further comprising contacting antigen- presenting cells with an antigen that corresponds to at least a portion of a target antigen that associates with the presence or risk of the undesirable or deleterious immune response, in an amount that is effective for the antigen-presenting cells to present the antigen or a processed form thereof on their surface.
30. A method according to claim 27, wherein the antigen-presenting cells are contacted with the antigen ex vivo.
31. A method according to claim 27, wherein the antigen-presenting cells are autologous relative to the subject.
32. A method according to claim 27 or claim 29, wherein the antigen-presenting cells are allogeneic relative to the subject.
33. A method according to claim 27. wherein the antigen-presenting cells are contacted with the antigen in vivo.
34. A method according to claim 27, wherein the antigen is administered to the subject in particulate form.
35. A method according to claim 27, wherein the condition is selected from an autoimmune disease, an allergy or an organ-specific disease.
36. A method according to claim 34, the autoimmune disease is selected from TlDM, systemic lupus erythematosus, Churg Strauss disease, scleroderma, Wegener granulomatosus, Wiskott Aldrich syndrome
37. A method according to claim 34, wherein the autoimmune disease is TlDM.
38. A method according to claim 34, wherein the allergy is selected from allergic asthma and allergic eczema.
39. A method according to claim 34, wherein the organ-specific disease is selected from TlDM, thyroiditis, adrenal insufficiency, alopecia, atrophic gastritis, vitiligo, premature ovarian failure, autoimmune polyendocrine syndromes (APS), parathyroiditis, hypoparathyroidism, autoimmune adrenal insufficiency (Addison's disease), autoimmune hepatitis, Sjogren's syndrome, celiac disease, exocrine pancreatitis, keratitis and mucocutaneous candidiasis.
40. A method for diagnosing the presence or risk of development of an undesirable or deleterious immune response associated with defective NF-κB activation in a subject, comprising detecting in the subject reduced or abrogated signaling through the NF-κB pathway in response to a pro-inflammatory signal.
41. A method according to claim 40, wherein the undesirable or deleterious immune response is associated with a condition selected from autoimmune diseases, organ specific diseases and allergies, wherein the condition is other than TlDM.
42. A method according to claim 40, wherein reduced or abrogated signaling is detected by detecting reduced or abrogated expression of ReIB.
43. A method according to claim 42, wherein the reduced or abrogated expression is detected by: (1) measuring in a biological sample obtained from the subject the level or functional activity of a ReIB expression product and (2) comparing the measured level or functional activity of the expression product to the level or functional activity of a corresponding expression product in a reference sample obtained from one or more normal subjects or from one or more subjects lacking the undesirable or deleterious immune response, wherein a change in the level or functional activity of the expression product in the biological sample as compared to the level or functional activity of the corresponding expression product in the reference sample is indicative of the presence or risk of development of the undesirable or deleterious immune response in the subject.
44. A method according to claim 43, wherein the presence or risk of development of the unwanted or deleterious immune response is determined by detecting an increase in the level or functional activity of an expression product of the SHP-I gene.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2247299A1 (en) * 2007-12-21 2010-11-10 University Of Rochester Molecular targets for treatment of inflammation
US8580520B2 (en) 2008-09-15 2013-11-12 Herlev Hospital YKL-40 as a marker for gastrointestinal cancers
US8697384B2 (en) 2008-01-23 2014-04-15 Herlev Hospital YKL-40 as a general marker for non-specific disease
RU2590696C2 (en) * 2014-09-22 2016-07-10 ФАНО России Федеральное государственное бюджетное учреждение науки Институт фундаментальных проблем биологии Российской академии наук (ИФПБ РАН) Agent for differentiation of monocyte-like cells

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001047543A2 (en) * 1999-12-24 2001-07-05 Synovis Limited Activation and inhibition of the immune system
EP1462111A1 (en) * 2003-03-28 2004-09-29 Universiteit Utrecht Holding B.V. Composition for inducing immunotolerance
JP2006112869A (en) * 2004-10-13 2006-04-27 Hamamatsu Photonics Kk Infrared detection system and its manufacturing method
WO2007134386A1 (en) * 2006-05-18 2007-11-29 The University Of Queensland Diagnostic agents and uses therefor

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001047543A2 (en) * 1999-12-24 2001-07-05 Synovis Limited Activation and inhibition of the immune system
EP1462111A1 (en) * 2003-03-28 2004-09-29 Universiteit Utrecht Holding B.V. Composition for inducing immunotolerance
EP1772152A2 (en) * 2003-03-28 2007-04-11 Universiteit Utrecht Holding B.V. Composition for inducing immunotolerance
EP1842550A2 (en) * 2003-03-28 2007-10-10 Universiteit Utrecht Holding B.V. Composition for inducing immunotolerance
JP2006112869A (en) * 2004-10-13 2006-04-27 Hamamatsu Photonics Kk Infrared detection system and its manufacturing method
WO2007134386A1 (en) * 2006-05-18 2007-11-29 The University Of Queensland Diagnostic agents and uses therefor

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FARIES M.B. ET AL.: "Calcium Signalling Interhibits Interleukin-12 Production and Activates CD83+ Dendritic Cells that Induce Th2 Cell Development", BLOOD, vol. 98, 2001, pages 2489 - 2497 *
VALERO R. ET AL.: "A Defective NF-kappaB/ReIB Pathway in Autoimmune-Prone New Zealand Black Mice is Associated with Inefficient Expansion of Thymocyte and Dendritic Cells", THE JOURNAL OF IMMUNOLOGY, vol. 169, 2002, pages 185 - 192, XP009158220, DOI: doi:10.4049/jimmunol.169.1.185 *
XING N. ET AL.: "Distinctive Dendritic Cell Modulation by Vitamin D3 and Glucocorticoid Pathway", BIOCHEMICAL AND BIOPHYSICS RESEARCH COMMUNICATION, vol. 297, 2002, pages 645 - 652 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2247299A1 (en) * 2007-12-21 2010-11-10 University Of Rochester Molecular targets for treatment of inflammation
EP2247299A4 (en) * 2007-12-21 2012-06-06 Univ Rochester Molecular targets for treatment of inflammation
US8697384B2 (en) 2008-01-23 2014-04-15 Herlev Hospital YKL-40 as a general marker for non-specific disease
US8580520B2 (en) 2008-09-15 2013-11-12 Herlev Hospital YKL-40 as a marker for gastrointestinal cancers
RU2590696C2 (en) * 2014-09-22 2016-07-10 ФАНО России Федеральное государственное бюджетное учреждение науки Институт фундаментальных проблем биологии Российской академии наук (ИФПБ РАН) Agent for differentiation of monocyte-like cells

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