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WO2006074370A2 - Compositions et methodes destinees a traiter une maladie immunologique - Google Patents

Compositions et methodes destinees a traiter une maladie immunologique Download PDF

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Publication number
WO2006074370A2
WO2006074370A2 PCT/US2006/000465 US2006000465W WO2006074370A2 WO 2006074370 A2 WO2006074370 A2 WO 2006074370A2 US 2006000465 W US2006000465 W US 2006000465W WO 2006074370 A2 WO2006074370 A2 WO 2006074370A2
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tnfrii
cd120b
cells
tnf
protein
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WO2006074370A3 (fr
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F. Xavier Valencia
Peter. E. Lipsky
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US Department of Health and Human Services
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates

Definitions

  • This invention generally relates to compositions and methods for treating a immune-related disease in a mammal, a method for determining the presence of or predisposition to a immune-related disease in a mammal, and a pharmaceutical composition for treating a immune-related disease in a mammal.
  • Tumor necrosis factor is a pleiotropic cytokine critical for cell trafficking, inflammation, maintenance of secondary lymphoid organ structure and host defense against various pathogens. Pasparakis et ⁇ /., J Exp Med 184:1397-1411, 1996. Because of this panoply of effects, TNF plays a critical role in bridging innate and adaptive immunity. However, its role in regulating the function of Tregs or their impact on effector cells is presently unknown. More specifically neutralizing anti-TNF antibodies increased CD4+CD25+ T regulatory cell number and ameliorated the disease in a NOD diabetes model. Wu et al., Proc. Natl. Acad. Sd.
  • SLE Systemic lupus erythematosus
  • T- and B-cell autoimmune responses result in the generation of autoantibodies and immune complexes, along with autoreactive T cells, which together cause pathology in several target organs, including skin, blood vessels, lung and kidney.
  • autoreactive T cells which together cause pathology in several target organs, including skin, blood vessels, lung and kidney.
  • Methods for regulating immune function in primates using the FoxP3 protein are discussed.
  • U.S. patent publication 2003/0170648 Rheumatoid arthritis (RA) is one of the most common human autoimmune diseases, with a prevalence of nearly 1%.
  • the present invention generally relates to a method for preventing or treating immune-related disease in a mammal comprising administering a modulator of TNFRII/CD120b in a therapeutic amount to the mammal in need thereof, wherein the immune-related disease is prevented or treated.
  • TNF tumor necrosis factor
  • T R T regulatory
  • the present invention demonstrates that human Treg express TNFRII and that the percentage of Treg that express TNFRII can be enhanced by exposure to TNF.
  • the addition of TNF or agonistic antibody to TNFRII reversed their suppressive activity by downmodulating the expression of FoxP3.
  • the beneficial effect of TNF blockade in autoimmune/inflammatory diseases could involve the restoration of immune homeostasis by permitting the full expression of CD4+CD25+ hl Treg function.
  • a method for preventing or treating autoimmune disease in a mammal comprising administering an antagonist of TNFRII/CD120b in a therapeutic amount to the mammal in need thereof, wherein the autoimmune disease is prevented or treated.
  • a method for treating neoplastic disease in a mammal comprising administering an agonist of TNFRII/CD120b in a therapeutic amount to the mammal in need thereof, wherein neoplastic disease is treated.
  • a method for treating allogeneic tissue rejection in a mammal comprising administering an agonist of TNFRII/CD120b in a therapeutic amount to the mammal in need thereof, wherein allogeneic tissue rejection is treated, hi a further aspect, a method for inhibiting graft versus host disease in a mammal is provided wherein the method comprises administering to the mammal an agonist of TNFR ⁇ /CD120b, wherein inhibiting graft versus host disease in the mammal is inhibited.
  • the present invention further provides a method for inhibiting an autoimmune response in a mammal comprising: isolating T cells from the mammal; transducing the T cells with an antagonist of TNFRII/CD120b; expanding the transduced T cells; and reintroducing the transduced T cells into the mammal, wherein an autoimmune response in the mammal is inhibited, hi another aspect, a method for enhancing an immune response to a disease in a mammal is provided comprising: isolating T cells from the mammal; transfecting the T cells with an antagonist of TNFRII/CD120b; expanding the transfected T cells; and reintroducing the transfected T cells into the mammal, wherein an immune response to a disease in the mammal is enhanced.
  • a method for preventing or treating autoimmune disease in a mammal comprise administering an antagonist of TNFRII/CD120b in a therapeutic amount to the mammal in need thereof, wherein the autoimmune disease is prevented or treated.
  • the antagonist prevents binding of tumor necrosis factor- ⁇ to TNFRII/CD120b.
  • the autoimmune disease is systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, diabetes, inflammatory bowel disease, psoriasis, or asthma.
  • a method for treating neoplastic disease in a mammal comprises administering an agonist of TNFRII/CD120b in a therapeutic amount to the mammal in need thereof, wherein neoplastic disease is treated.
  • the agonist prevents binding of tumor necrosis factor- ⁇ to TNFRII/CD120b.
  • the neoplastic disease is cancer, solid tumor, sarcoma, melanoma, carcinoma, leukemia, or lymphoma.
  • the method comprises providing an immunotherapeutic agent to the mammal, and treating the mammal with the immunotherapeutic agent and the antagonist.
  • a method for treating allogeneic tissue rejection in a mammal comprises administering an agonist of TNFRII/CD120b in a therapeutic amount to the mammal in need thereof, wherein allogeneic tissue rejection is treated.
  • the agonist prevents binding of tumor necrosis factor- ⁇ to TNFRII/CD120b.
  • a method for inhibiting an autoimmune response in a mammal comprises isolating T cells from the mammal; transducing the T cells with an antagonist of TNFRII/CD120b; expanding the transduced T cells; and reintroducing the transduced T cells into the mammal, wherein an autoimmune response in the mammal is inhibited.
  • the T cells are CD4 + CD25+ hl regulatory T cells.
  • the autoimmune disease is systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, diabetes, inflammatory bowel disease, psoriasis, or asthma
  • the antagonist is interfering RNA, short hairpin RNA, ribozyme, antisense oligonucleotide, or protein inhibitor.
  • a method for enhancing an immune response to a disease in a mammal comprises isolating T cells from the mammal; transfecting the T cells with an antagonist of TNFRII/CD120b; expanding the transfected T cells; and reintroducing the transfected T cells into the mammal, wherein an immune response to a disease in the mammal is enhanced, hi one aspect, the antagonist is interfering RNA, short hairpin RNA, ribozyme, antisense oligonucleotide, or protein inhibitor, hi a further aspect, the disease is infectious disease, HIV infection, or AJDS-related disease.
  • a method for inhibiting graft versus host disease in a mammal comprises administering to the mammal an agonist of TNFRII/CD120b, wherein inhibiting graft versus host disease in the mammal is inhibited.
  • a method for identifying a compound which modulates signaling in cells via a TNFRII/CD120b pathway comprises contacting a test compound with a cell- based assay system comprising a cell expressing TNFRII/CD120b and capable of signaling ' responsiveness to tumor necrosis factor- ⁇ , providing tumor necrosis factor- ⁇ to the assay system in an amount selected to be effective to modulate TNFRII/CD120b signaling, and detecting an effect of the test compound on TNFRII/CD120b signaling in the assay system, effectiveness of the test compound in the assay being indicative of the modulation, hi one aspect, a compound is identified according to the methods of the present invention.
  • the compound is an agonist of TNFRII/CD120b and activates TNFRII/CD120b pathway signaling.
  • the compound is an antagonist of TNFRII/CD120b and inhibits TNFRII/CD120b pathway signaling
  • the compound is an inhibitor ofFoxP3 expression, or the compound is an activator oiFoxP3 expression
  • the compound is an inhibitor of TRAF2 expression, or the compound is an activator of TRAF2 expression.
  • the test compound is a monoclonal antibody, a polyclonal antibody, a peptide, or a small molecule.
  • the test compound is an organic molecule, a natural product, a peptide, an oligosaccharide, a nucleic acid, a lipid, an antibody, or binding fragment thereof, hi a further detailed aspect, the test compound is from a library of compounds, hi a further aspect, the library is a random peptide library, a combinatorial library, an oligosaccharide library or a phage display library.
  • a method for identifying a compound which induces transplantation tolerance in a tissue comprises contacting a test compound with a cell-based assay system comprising a cell expressing TNFRIFCD 120b and capable of signaling responsiveness to tumor necrosis factor- ⁇ , providing tumor necrosis factor- ⁇ to the assay system in an amount selected to be effective to activate TNFRII/CD120b signaling; and detecting an effect of the test compound on TNFRH/CD120b signaling in the assay system, effectiveness of the test compound in the assay being indicative of inducing transplantation tolerance in the tissue, hi one aspect, the compound is an agonist of TNFRlI/CD120b. In a further aspect, the compound is an inhibitor of FoxP3. In a further aspect, the compound is an inhibitor of TRAF2. In one aspect, a compound is identified according to the methods of the present invention.
  • a method for identifying a compound which reduces immune tolerance in a cancer cell comprises contacting a test compound with a cell-based assay system comprising a cell expressing TNFRII/CD120b and capable of signaling responsiveness to tumor necrosis factor- ⁇ , providing tumor necrosis factor- ⁇ to the assay system in an amount selected to be effective to suppress TNFRII/CD120b signaling; and detecting an effect of the test compound on TNFRII/CD120b signaling in the assay system, effectiveness of the test compound in the assay being indicative of reducing tolerance in cancer immunotherapy in the tissue.
  • the compound is an agonist of TNFRII/CD120b.
  • the compound is an inhibitor of FoxP3, or the compound is an inhibitor of TRAF2.
  • a method for identifying a compound which reduces self recognition in a cell comprises contacting a test compound with a cell-based assay system comprising a cell expressing TNFRII/CD120b and capable of signaling responsiveness to a ligand, providing TNF and the ligand to the assay system in an amount selected to be effective to suppress TNFRII/CD120b signaling; and detecting an effect of the test compound on TNFRII/CD120b signaling in the assay system, effectiveness of the test compound in the assay being indicative of reducing self recognition in a cell.
  • the compound is an antagonist of TNFRII/CD120b.
  • the compound is an activator o ⁇ FoxP3, or the compound is an activator of TRAF2.
  • a compound is identified according to the methods of the present invention.
  • Figure 1 shows phenotypes of CD4+CD25+ hl T R cells from normal donors and CD4+CD25+ hl T R cells from donors with active systemic lupus erythematosus (SLE).
  • SLE systemic lupus erythematosus
  • Figure 2 shows phenotypes of CD4+CD25+ 1 " T R cells from normal donors and CD4+CD25+ hl T R cells from donors with active systemic lupus erythematosus (SLE) following overnight incubation of CD4+CD25+ hi T R cells on TNF ⁇ .
  • Figure 3 shows neither IL-10 nor TGF ⁇ l mediate the suppressive effect of human CD4+CD25+ hi T R cells.
  • FIG. 4 shows CD4+CD25+ hi T R cells from active SLE lack in vitro suppressive function.
  • Figure 5 shows CD4+CD25+ hi T R cells from normal donors can suppress CD4+ effectors from active SLE patients.
  • Figure 6 shows TNF ⁇ reverses the CD4+CD25+ hi T R cells suppression on CD4+ effectors.
  • Figure 7 shows TNFRII crosslinking reverses the CD4+CD25+ hi TR cell suppression on CD4+ effector cells.
  • Figure 8 shows TNF ⁇ preincubation reverses the CD4+CD25+ hi T R cell suppression on CD4+ effectors.
  • Figure 9 shows CD4+CD25+ hi T R cells and CD4+CD25- effectors from SLE patients were in vitro stimulated on plate bound CD3 and IL-2.
  • FIG. 10 shows TNFRII crosslinking abrogates the suppression of CD4+CD25+ hi T R cells in a CFSE dilution assay.
  • FIG. 11 shows signaling through TNFRII abolishes the suppression of IFN ⁇ secretion.
  • FIG 12 shows TNF ⁇ down modulates FoxP3 expression in freshly isolated T R cells from donors with clinically active SLE and normal donors.
  • Figure 13 shows levels of FoxP3 messenger RNA in CD4+CD25+ hi T cells relative to CD4+CD25- T cells.
  • Figure 14 shows TNFRII crosslinking reverses TGF ⁇ l induced suppressive function.
  • Figure 15 shows kinetics of TNFRII expression on CD4+CD25- T cells at the indicated time points after stimulation in vitro with anti-CD3 in the presence of T-depleted splenocytes.
  • Figure 16A shows concentration dependent loss of suppressive function of CD4+CD25 hi Treg by TNF.
  • Figure 16B shows TNFRII crosslinking reverses the CD4+CD25 hi Treg mediated suppression of the proliferation of CD4+CD25- effectors.
  • Figure 17 shows a phenotype of CD4+CD25 hi Treg from RA patients.
  • Figures 18A, 18B show CD4+CD25 hi T cells from active RA patients fail to suppress proliferation.
  • Figures 19 A, 19B, and 19C show CD4+CD25 hi Treg from active RA patients are defective suppressors.
  • Figures 2OA and 2OB show CD4+CD25 hi Treg from RA patients recover their suppressive function after Infliximab therapy.
  • the present invention generally relates to a method for preventing or treating immune-related disease in a mammal comprising administering a modulator of TNFRII/CD120b in a therapeutic amount to the mammal in need thereof, wherein the immune-related disease is prevented or treated.
  • T regulatory (T R ) cells A small population of CD4+ T cells, identified by the co-expression of CD25, e.g., CD4+CD25+ 1 " T regulatory (T R ) cells, have the ability to regulate immune responses. These T- regulatory (T R ) cells have been found and characterized in humans and rodents. Furthermore, studies in both human and mice have demonstrated that a defective regulatory T cell function contributes to autoimmune diseases in animal models. Viglietta et al., J. Exp. Med. 199: 971-979, 2004; Shevach, Nature Review Immunol 2:389, 2002.
  • the present invention examined whether a similar defect in CD4+CD25+ hl T R cell occurs in an autoimmune disease, for example, systemic lupus erythematosus (SLE).
  • SLE systemic lupus erythematosus
  • Studies in humans have identified the regulatory effector function in the brightest 2% of CD25+ which is referred to as CD4+CD25+ hi T R cell, and this population was selected to study.
  • the present invention provides evidence for a functional defect in the CD4+CD25+ 1 " T R cells in SLE.
  • a novel function has been demonstrated for tumor necrosis factor (TNF) as an important factor down modulating T regulatory cell function.
  • TNF tumor necrosis factor
  • the present invention provides a method for preventing or treating autoimmune disease in a mammal comprising administering an antagonist of TNFRII/CD120b in a therapeutic amount to the mammal in need thereof, wherein the autoimmune disease is prevented or treated.
  • the present invention also suggests the mechanism of TNF effect on TNFRII/CD120b as a modulator of T regulatory cell function.
  • the mechanism of the TNF effect is a reduction in FoxP3 expression in CD4+CD25+ hl T R cells.
  • the present invention provides methods for treatment of immune-related disease utilizing strategies designed to enhance the limited and/or inadequate numbers of these regulatory T cells in the periphery or diminish the numbers of regulatory T cells, as needed, for therapeutic intervention in diseases including, but not limited to, autoimmune diseases, cancer, allogeneic graft rejection, and HIV infection.
  • Tumor necrosis factor TNF or TNF- ⁇
  • lymphotoxin LT
  • TNF tumor necrosis factor
  • LT lymphotoxin
  • Type I 55-60 kDa, 426 amino acids
  • Type II 75-80 kDa, 439 amino acids
  • TNF Receptors e.g., TNFRI/CD120a and TNFRII/CD120b, respectively.
  • Monomeric forms of TNF and LTa are not biologically active.
  • the TNFRs are type II transmembrane glycoproteins whose extracellular regions are comprised of four, well-conserved cysteine-rich domains, but whose intracellular domains are distinctly different.
  • TNFRI is constitutively expressed by most cell types.
  • TNFRII is expressed primarily by endothelial cells and cells of hematopoietic origin.
  • Transmembrane TNF effects cell signaling (e.g., cell death or costimulation) by direct cell-to-cell contact.
  • the transmembrane form of TNF may bind preferentially to the TNFRII.
  • Most proinflammatory activities of TNF appear to be mediated through the TNFRI. There is evidence that TNFRII can mediate cytotoxicity and costimulate thymocyte and T cell proliferation.
  • Inhibitors include inhibitors and activators.
  • Inhibitors are agents that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of TNFRII/CD120b signaling, e.g., antagonists.
  • Activators are agents that, e.g., bind to, stimulate, increase, open, activate, facilitate, enhance activation, sensitize or up regulate the activity of TNFRII/CD120b signaling, e.g., agonists.
  • Modulators include agents that, e.g., alter the interaction of TNFRII/CD120b with: proteins that bind activators or inhibitors, receptors, including proteins, peptides, lipids, carbohydrates, polysaccharides, or combinations of the above, e.g., lipoproteins, glycoproteins, and the like.
  • Modulators include genetically modified versions of naturally-occurring TNFRII/CD120b ligands, e.g., TNF- ⁇ , with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like.
  • Such assays for inhibitors and activators include, e.g., applying putative modulator compounds to a cell expressing TNF- ⁇ and then determining the functional effects on TNFRII/CD120b signaling, as described herein. Samples or assays comprising TNF- ⁇ and TNFRII/CD120b that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition.
  • Control samples can be assigned a relative TNFRII/CD120b activity value of 100%.
  • Inhibition of TNFRII/CD120b is achieved when the TNFRII/CD120b activity value relative to the control is about 80%, optionally 50% or 25-0%.
  • Activation of TNFRII/CD120b is achieved when the TNFRII/CD120b activity value relative to the control is 110%, optionally 150%, optionally 200-500%, or 1000-3000% higher.
  • Antagonist is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a TNFRII/CD120b polypeptide or TNFRII/CD120b signaling.
  • agonist is used in the broadest sense and includes any molecule that mimics or enhances a biological activity of a TNFRII/CD120b polypeptide or TNFRII/CD120b signaling.
  • Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native TNF- ⁇ polypeptides, peptides, antisense oligonucleotides, small organic molecules, and the like.
  • Methods for identifying agonists or antagonists of a TNFRH/CD120b polypeptide can comprise contacting a TNFRII/CD120b polypeptide with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the TNFRII/CD120b polypeptide.
  • a ligand such as an endogenous or exogenous ligand, e.g., TNF- ⁇
  • receptors such as TNFRII/CD120b pathway signaling resulting in cell signaling to produce a response, for example, an autoimmune response or allogeneic tissue rejection.
  • Test compound refers to a nucleic acid, DNA, RNA, protein, polypeptide, or small chemical entity that is determined to effect an increase or decrease in a gene expression as a result of signaling through the TNFRTI/CD120b pathway.
  • the test compound can be an antisense RNA, ribozyme, polypeptide, or small molecular chemical entity.
  • the term "test compound” can be any small chemical compound, or a biological entity, such as a protein, sugar, nucleic acid or lipid. Typically, test compounds will be small chemical molecules and polypeptides.
  • a "test compound specific for signaling by TNFRII/CD120b” is determined to be a modulator of TNFRII/CD120b pathway signaling via TNF- ⁇ .
  • Cell-based assays include TNFRII/CD 120b binding assays, for example, radioligand or fluorescent ligand binding assays for TNFRII/CD 120b to cells, plasma membranes, detergent-solubilized plasma membrane proteins, immobilized collagen (Alberdi, J Biol Chem. 274:31605-12, lyyy; Meyer et al, 2002); TNFRII/CD120b -affinity column chromatography (Alberdi, J Biol Chem.
  • Exemplary TNFRII/CD120b binding activity assays of the present invention are: a TNF- ⁇ / TNFRII/CD120b ligand blot assay (Aymerich et al, Invest Ophthalmol Vis ScL 42:3287-93, 2001); a TNFRII/CD120b affinity column chromatography assay (Alberdi, J Biol Chem. 274:31605-12, 1999) and a TNF- ⁇ / TNFRII/CD120b ligand binding assay (Alberdi et al, J Biol Chem. 274:31605-12, 1999). Each incorporated by reference in their entirety.
  • TNF- ⁇ binding to TNFRII/CD 120b can be assayed by either immobilizing the ligand or the receptor.
  • the assay can include immobilizing TNFRII/CD 120b fused to a His tag onto Ni-activated NTA resin beads.
  • TNF- ⁇ can be added in an appropriate buffer and the beads incubated for a period of time at a given temperature. After washes to remove unbound material, the bound protein can be released with, for example, SDS, buffers with a high pH, and the like and analyzed.
  • Contacting refers to mixing a test compound in a soluble form into an assay system, for example, a cell-based assay system, such that an effect upon receptor-mediated signaling can be measured.
  • “Signaling responsiveness” or “effective to activate signaling” or “stimulating a cell-based assay system” refers to the ability of TNFRII/CD 120b pathway to signal via TNF- ⁇ to inhibit or enhance an immune response, or treat autoimmune disease, neoplastic disease, systemic lupus erthymatosus, or allogeneic tissue rejection
  • Detecting an effect refers to an effect measured in a cell-based assay system.
  • the effect detected can be TNFRII/CD 120b signaling in an assay system, for example, TNF cellular assay, TNFRII/CD 120b binding assay.
  • Assay being indicative of modulation refers to results of a cell-based assay system indicating that cell activation by TNFRII/CD 120b signaling via TNF- ⁇ induces a protective response in cells against inflammation.
  • Biological activity and “biologically active” with regard to a ligand of TNFRII/CD 120b of the present invention refer to the ability of the ligand molecule to specifically bind to and signal through a native or recombinant TNFRII/CD 120b, or to block the ability of a native or recombinant TNFRII/CD 120b to participate in signal transduction.
  • the (native and variant) ligands of TNFRII/CD 120b of the present invention include agonists and antagonists of a native or recombinant TNFRII/CD120b.
  • Preferred biological activities of the ligands of TNFRII/CD 120b of the present invention include the ability to induce or inhibit, for example, inhibiting or enhancing an immune response, or treating autoimmune disease, neoplastic disease, systemic lupus erthymatosus, or allogeneic tissue rejection. Accordingly, the administration of the compounds or agents of the present invention can prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with autoimmune disease, neoplastic disease, allogeneic tissue rejection, or other disorders.
  • High affinity for a ligand refers to an equilibrium association constant (Ka) of at least about 10 3 M '1 , at least about 10 4 M “1 , at least about 10 5 M “1 , at least about 10 6 M “1 , at least about 10 7 M “1 , at least about 10 8 M “1 , at least about 10 9 M “1 , at least about 10 10 M “1 , at least about 10 11 M “1 , or at least about 10 12 M “1 or greater, e.g., up to 10 13 M “1 or 10 14 M '1 or greater.
  • Ka equilibrium association constant
  • K a is intended to refer to the equilibrium association constant of a particular ligand-receptor interaction, e.g., antibody-antigen interaction. This constant has units of 1/M.
  • K d is intended to refer to the equilibrium dissociation constant of a particular ligand-receptor interaction. This constant has units of M.
  • k a is intended to refer to the kinetic association constant of a particular ligand-receptor interaction. This constant has units of I/Ms.
  • k d is intended to refer to the kinetic dissociation constant of a particular ligand-receptor interaction. This constant has units of 1/s.
  • Particular ligand-receptor interactions refers to the experimental conditions under which the equilibrium and kinetic constants are measured.
  • Isotype refers to the antibody class that is encoded by heavy chain constant region genes. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Additional structural variations characterize distinct subtypes of IgG (e.g., IgG 1 , IgG 2 , IgG 3 and IgG 4 ) and IgA (e.g., IgA 1 and IgA 2 )
  • the ability of a molecule to bind to TNFRII/CD 120b can be determined, for example, by the ability of the putative ligand to bind to TNFRII/CD 120b immunoadhesin coated on an assay plate. Specificity of binding can be determined by comparing binding to TNFRII/CD120b.
  • Control sequences or "regulatory sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • Tne control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, a ribosome binding site, and possibly, other as yet poorly understood sequences.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • Vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"), hi general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno- associated viruses), which serve equivalent functions.
  • a "label” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available (e.g., the polypeptides of the invention can be made detectable, e.g., by incorporating a radiolabel into the peptide, and used to detect antibodies specifically reactive with the peptide).
  • Sorting in the context of cells as used herein to refers to both physical sorting of the cells, as can be accomplished using, e.g., a fluorescence activated cell sorter, as well as to analysis of cells based on expression of cell surface markers, e.g., FACS analysis in the absence of sorting.
  • Cell Cell
  • cell line cell culture
  • all such designations include progeny.
  • progeny include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny cannot be precisely identical in DNA content, due to ' deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.
  • Antibody is used in the broadest sense and specifically covers monoclonal antibodies, antibody compositions with polyepitopic specificity, bispecific antibodies, diabodies, and single-chain molecules, as well as antibody fragments (e.g., Fab, F(ab') 2 , and Fv), so long as they exhibit the desired biological activity.
  • Antibodies can be labeled/conjugated to toxic or non-toxic moieties. Toxic moieties include, for example, bacterial toxins, viral toxins, radioisotopes, and the like.
  • Antibodies can be labeled for use in biological assays (e.g., radioisotope labels, fluorescent labels) to aid in detection of the antibody.
  • Antibodies can also be labeled/conjugated for diagnostic or therapeutic purposes, e.g., with radioactive isotopes that deliver radiation directly to a desired site for applications such as radioimmunotherapy (Garmestani. et al, Nucl Med Biol, 28:409, 2001), imaging techniques and radioimrnunoguided surgery or labels that allow for Un vivo imaging or detection of specific antibody/antigen complexes.
  • Antibodies can also be conjugated with toxins to provide an immunotoxin (see, Kreitman, RJ Adv Drug Del Rev, 31:53, 1998).
  • Monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention can be made by the hybridoma method first described by Kohler et al, Nature, 256: 495, 1975, or can be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567, Cabilly et al.).
  • the "monoclonal antibodies” can also be isolated from phage antibody libraries using the techniques described in Clackson et al, 624-628, 1991, and Marks et al, J. MoI Biol. 222:581-597, 1991, for example.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
  • chimeric antibodies immunoglobulins in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • CDR complementary-determining region
  • humanized antibodies can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • the humanized antibody includes a PrimatizedTM antibody wherein the antigen-binding region of the antibody is derived from an antibody produced by immunizing macaque monkeys with the antigen of interest.
  • Amino acids from the variable regions of the mature heavy and light chains of immunoglobulins are designated Hx and Lx respectively, where x is a number designating the position of an amino acids according to the scheme of Kabat et al, 1987 and 1991, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD).
  • Kabat et al. list many amino acid sequences for antibodies for each subclass, and list the most commonly occurring ammo acid tor eacn residue position in that subclass.
  • Kabat et al. use a method for assigning a residue number to each amino acid in a listed sequence, and this method for assigning residue numbers has become standard in the field. Kabat et al.
  • Non-imrnunogenic in a human means that upon contacting the polypeptide of interest in a physiologically acceptable carrier and in a therapeutically effective amount with the appropriate tissue of a human, no state of sensitivity or resistance to the polypeptide of interest is demonstrable upon the second administration of the polypeptide of interest after an appropriate latent period (e.g., 8 to 14 days).
  • Neutralizing antibody refers to an antibody which is able to block or significantly reduce an effector function of wild type or mutant TNF- ⁇ or TNFRII/CD120b.
  • a neutralizing antibody can inhibit or reduce TNFRII/CD120b activation by an agonist antibody, as determined, for example, in a TNF- ⁇ / TNFRII/CD120b binding assay, or other assays taught herein or known in the art.
  • Receptor denotes a cell-associated protein, for example, TNFRII/CD120b, that binds to a bioactive molecule termed a "ligand.” This interaction mediates the effect of the ligand on the cell.
  • Receptors can be membrane bound, cytosolic or nuclear; monomelic (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., TNF receptor I, TNF receptor II, PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).
  • Membrane-bound receptors for example TNFRII/CD120b, are characterized by a multi-domain structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction.
  • the extracellular ligand-binding domain and the intracellular effector domain are located in separate polypeptides that comprise the complete functional receptor.
  • the binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell, which in turn leads to an alteration in the metabolism of the cell.
  • Metabolic events that are often linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids.
  • Treating refers to any indicia of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology, or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a subject's physical or mental well-being.
  • the treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination.
  • the term “treating” includes the administration of the compounds or agents of the present invention to inhibit or enhance an immune response, or treat autoimmune disease, neoplastic disease, systemic lupus erthymatosus, or allogeneic tissue rejection. It also includes the administration of the compounds of the present invention to enhance an immune response in a subject toward infection with a pathogen. Accordingly, the term “treating” includes the administration of the compounds or agents of the present invention to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated autoimmune disease, neoplastic disease, systemic lupus erthymatosus, or allogeneic tissue rejection, or other disorders.
  • therapeutic effect refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.
  • Concomitant administration of a known drug with a compound of the present invention means administration of the drug and the compound at such time that both the known drug and the compound will have a therapeutic effect or diagnostic effect. Such concomitant administration can involve concurrent ⁇ i.e. at the same time), prior, or subsequent administration of the drug with respect to the administration of a compound of the present invention.
  • a person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compounds of the present invention.
  • Inflammation refers to an innate immune response that occurs when tissues are injured by bacteria, trauma, toxins, heat, or any other cause. The damaged tissue releases compounds including histamine, bradykinin, and serotonin. Inflammation refers to both acute responses (i.e., responses in which the inflammatory processes are active) and chronic responses ⁇ i.e., responses marked by slow progression and formation of new connective tissue). Acute and chronic inflammation can be distinguished by the cell types involved. Acute inflammation often involves polymorphonuclear neutrophils; whereas chronic inflammation is normally characterized by a lymphohistiocytic and/or granulomatous response. Inflammation includes reactions of both the specific and non-specific defense systems.
  • a specific defense system reaction is a specific immune system reaction response to an antigen (possibly including an autoantigen).
  • a non-specific defense system reaction is an inflammatory response mediated by leukocytes incapable of immunological memory. Such cells include granulocytes, macrophages, neutrophils and eosinophils. Examples of specific types of inflammation are diffuse inflammation, focal inflammation, croupous inflammation, interstitial inflammation, obliterative inflammation, parenchymatous inflammation, reactive inflammation, specific inflammation, toxic inflammation and traumatic inflammation.
  • Subject or “patient” refers to any mammalian patient or subject to which the compositions of the invention can be administered.
  • mammals human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals.
  • accepted screening methods are employed to determine risk factors associated with a targeted or suspected disease or condition or to determine the status of an existing disease or condition in a subject. These screening methods include, for example, conventional work-ups to determine risk factors that can be associated with the targeted or suspected disease or condition. These and other routine methods allow the clinician to select patients in need of therapy using the methods and formulations of the invention.
  • solid phase is meant a non-aqueous matrix to which a reagent of interest (e.g., TNFRII/CD120b or an antibody thereto) can adhere.
  • a reagent of interest e.g., TNFRII/CD120b or an antibody thereto
  • solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones.
  • the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g. ,an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149.
  • Specifically (or selectively) binds to an antibody refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies.
  • the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample.
  • bind(s) or "bind(s) specifically” when referring to a peptide refers to a peptide molecule which has intermediate or high binding affinity, exclusively or predominately, to a target molecule 1 .
  • the phrase "specifically binds to” refers to a binding reaction which is determinative of the presence of a target protein in the presence of a heterogeneous population of proteins and other biologies.
  • the specified binding moieties bind preferentially to a particular target protein and do not bind in a significant amount to other components present in a test sample. Specific binding to a target protein under such conditions can require a binding moiety that is selected for its specificity for a particular target antigen.
  • a variety of assay formats can be used to select ligands that are specifically reactive with a particular protein. For example, solid-phase ELISA immunoassays, immunoprecipitation, Biacore and Western blot are used to identify peptides that specifically react with TNFRII/CD120b domain-containing proteins. Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 times background.
  • Specific binding between a monovalent peptide and TNFRIFCD 120b means a binding affinity of at least 10 3 M '1 , and preferably 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or 10 10 M "1 .
  • the binding affinity of TNF- ⁇ to TNFR ⁇ /CD120b is between about 10 6 M "1 to about 10 10 M "1 .
  • the present invention is based on the discovery that the TNFRII/CD 120b is a specific sensor of endogenous and exogenous ligands which are necessary for signaling via the TNFRII/CD 120b pathway.
  • TNF- ⁇ is a protein that binds TNFRII/CD 120b with a high affinity.
  • the experiments described herein demonstrate that TNFRII/CD 120b is a receptor which can play a role in mediating responses to TNF- ⁇ .
  • the receptor, TNFRII/CD 120b, and TNF- ⁇ has been found to be present in a variety of tissue and cell populations, for example, CD4+CD25+ hl T R cells, and are necessary for inhibiting or enhancing an immune response, or treating autoimmune disease, neoplastic disease, systemic lupus erthymatosus, or allogeneic tissue rejection.
  • This invention relies on routine techniques in the field of recombinant genetics.
  • Basic texts disclosing the general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., 1989; Kriegler, Gene Transfer and Expression: A Laboratory Manual, 1990; and Ausubel et al., eds., , Current Protocols in Molecular Biology, 1994.
  • TNFRII/CD 120b nucleic acids, polymorphic variants, orthologs, and alleles that are substantially identical to sequences provided herein can be isolated using TNFRII/CD 120b nucleic acid probes and oligonucleotides under stringent hybridization conditions, by screening libraries.
  • expression libraries can be used to clone TNFRII/CD 120b protein, polymorphic variants, orthologs, and alleles by detecting expressed homologs immunologically with antisera or purified antibodies made against human TNFRII/CD 120b or portions thereof.
  • Identifying bioactive agents that modulate TNFRII/CD 120b pathway signaling via TNF- ⁇ the information is used in a wide variety of ways.
  • one of several cellular assays e.g., TNF assay, TNFRII/CD 120b binding assay, can be used in conjunction with high throughput screening techniques, to allow monitoring for antagonists or agonists of TNFRII/CD120b pathway signaling after treatment with a candidate agent, Zlokarnik, et al., Science 279:84-8, 1998; and Heid et al., Genome Res. 6:986, 1996; each incorporated herein by reference in their entirety.
  • the candidate agents are added to cells.
  • Candidadidate bioactive agent or “drug candidate” or grammatical equivalents as used herein describes any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, to be tested for bioactive agents that are capable of directly or indirectly altering the activity of TNFRII/CD 120b pathway signaling.
  • the bioactive agents modulate TNFRII/CD 120b pathway signaling via TNF- ⁇ .
  • the candidate agents induce an antagonist or agonist effect in a TNF assay, TNFRII/CD 120b binding assay, as further described below.
  • a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.
  • Candidate agents encompass numerous chemical classes, though typically they are organic molecules, e.g., small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons.
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, for example, at least two of the functional chemical groups.
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • candidate agents are peptides.
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents can be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs.
  • the candidate bioactive agents are proteins.
  • protein herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.
  • the protein can be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures.
  • amino acid or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the methods herein.
  • Amino acid also includes imino acid residues such as proline and hydroxyproline.
  • the side chains can be in either the (R) or the (S) configuration. In further embodiments, the amino acids are in the (S) or (L)-configuration. If non-naturally occurring side chains are used, non-amino acid substituents can be used, for example to prevent or retard in vivo degradations.
  • the candidate bioactive agents are naturally occurring proteins or fragments of naturally occurring proteins.
  • cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts can be used.
  • libraries of procaryotic and eucaryotic proteins can be made for screening using the methods herein.
  • the libraries can be bacterial, fungal, viral, and mammalian proteins, and human proteins.
  • the candidate bioactive agents are peptides of from about 5 to about 30 amino acids, typically from about 5 to about 20 amino acids, and typically from about 7 to about 15 being.
  • the peptides can be digests of naturally occurring proteins as is outlined above, random peptides, or "biased” random peptides.
  • randomized or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they can incorporate any nucleotide or amino acid at any position.
  • the synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.
  • the library can be fully randomized, with no sequence preferences or constants at any position.
  • the library can be biased. Some positions within the sequence are either held constant, or are selected from a limited number of possibilities.
  • the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of nucleic acid binding domains, the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, or to purines.
  • the candidate bioactive agents are nucleic acids, as defined above.
  • nucleic acid candidate bioactive agents can be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids.
  • digests of procaryotic or eucaryotic genomes can be used as is outlined above for proteins.
  • the candidate bioactive agents are organic chemical moieties.
  • Several different drug screening methods can be accomplished to identify drugs or bioactive agents that modulate TNFRII/CD120b pathway signaling via TNF-oc.
  • One such method is the screening of candidate agents that can act as an antagonist or agonist of TNFRII/CD120b pathway signaling, thus generating the associated phenotype.
  • candidate agents that can act as an antagonist to TNFRII/CD120b pathway signaling are expected to result in the immunosuppressive phenotype.
  • candidate agents that can act as an agonist to TNFRII/CD120b pathway signaling are expected to result in the immunostimulant phenotype, upon challenge with a pathogen.
  • candidate agents can be determined that mimic or alter TNFRII/CD120b pathway signaling.
  • screening can be done to alter the biological function of the TNFRII/CD120b pathway signaling. Again, having identified the importance of a TNF- ⁇ in TNFRII/CD120b pathway signaling, screening for agents that bind and/or modulate the biological activity of the TNFRII/CD120b can be performed as outlined below.
  • a candidate agent can be administered in any one of several cellular assays, e.g., TNF assay, TNFRII/CD120b binding assay.
  • administration or “contacting” herein is meant that the candidate agent is added to the cells in such a manner as to allow the agent to act upon the cell, whether by uptake and intracellular action, or by action at the cell surface.
  • nucleic acid encoding a proteinaceous candidate agent ⁇ i.e., a peptide can be put into a viral construct such as a retroviral construct and added to the cell, such that expression of the peptide agent is accomplished; see PCT US97/01019, incorporated herein by reference in its entirety.
  • the cells can be washed if desired and are allowed to incubate under physiological conditions for some period of time. The cells are then harvested and a new gene expression profile is generated, as outlined herein.
  • TNFRII/CD120b pathway signaling via TNF- ⁇ can be screened for agents that produce an immunosuppressive or immune-stimulating phenotype.
  • a change in a binding assay or cellular assay indicates that the agent has an effect on TNFRII/CD120b pathway signaling activity.
  • an immunosuppressive or immune-stimulating profile is induced or maintained, before, during, and/or after stimulation with ligand.
  • the agent acts as an agonist or antagonist in one of several cellular or binding assays, e.g., TNF assay or TNF- ⁇ / TNFRII/CD120b binding assay.
  • screens can be done on individual genes and gene products. After having identified a cellular or binding assay as indicative of inhibition or enhancement of an immune response, or treatment of autoimmune disease, neoplastic disease, systemic lupus erthymatosus, or allogeneic tissue rejection, screening of modulators of cellular or binding assay can be completed.
  • screening for modulators of cellular or binding assay can be completed. This will be done as outlined above, but in general a few cellular or binding assay are evaluated. In some methods, screens are designed to first find candidate agents that can affect a cellular activity or binding assay, and then these agents can be used in other assays that evaluate the ability of the candidate agent to modulate TNFRII/CD120b pathway signaling.
  • [0106] hi general, purified or isolated gene product can be used for binding assays; that is, the gene products of TNF- ⁇ and TNFRII/CD120b are made.
  • the expression vectors can be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome.
  • these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding a TNFRII/CD120b or TNF- ⁇ protein.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase.
  • transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express TNF- ⁇ or TNFRH7CD120b protein; for example, transcriptional and translational regulatory nucleic acid sequences from Bacillus are used to express the protein in Bacillus. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.
  • the transcriptional and translational regulatory sequences can include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
  • the regulatory sequences include a promoter and transcriptional start and stop sequences.
  • Promoter sequences encode either constitutive or inducible promoters.
  • the promoters can be either naturally occurring promoters or hybrid promoters.
  • Hybrid promoters which combine elements of more than one promoter, are also known in the art, and are useful in the methods herein.
  • the expression vector can comprise additional elements.
  • the expression vector can have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a procaryotic host for cloning and amplification.
  • the expression vector contains at least one sequence homologous to the host cell genome, and typically two homologous sequences which flank the expression construct.
  • the integrating vector can be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art. Methods to effect homologous recombination are described in PCT US93/03868 and PCT US98/05223, each incorporated herein by reference in their entirety.
  • the expression vector contains a selectable marker gene to allow the selection of transformed host cells.
  • Selection genes are well known in the art and will vary with the host cell used.
  • One expression vector system is a retroviral vector system such as is generally described in PCT/US97/01019 and PCT/US97/01048, each incorporated herein by reference in their entirety.
  • the TNF- ⁇ or TNFRII/CD 120b proteins of the present methods and compositions are produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding TNF- ⁇ or TNFRII/CD 120b, under the appropriate conditions to induce or cause expression of the protein.
  • the conditions appropriate for TNF- ⁇ or TNFRII/CD 120b expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation.
  • the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction.
  • the timing of the harvest is important.
  • the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield.
  • Appropriate host cells include yeast, bacteria, archebacteria, fungi, and insect and animal cells, including mammalian cells. Of particular interest are Drosophila melanogaster cells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis, SF9 cells, C 129 cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa cells.
  • CD4+CD25+ hi T R cells are host cells as provided herein, which for example, include non-recombinant cell lines, such as primary cell lines, hi addition, purified primary CD4+CD25+ hl T R cells for TNF assay derived from either transgenic or non-transgenic strains can also be used.
  • the host cell can alternatively be an cell type known to have immunodeficiency disorder.
  • the TNF- ⁇ or TNFRII/CD120b proteins are expressed in mammalian cells.
  • Mammalian expression systems can include retroviral systems.
  • a mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence for TNF- ⁇ or TNFRH/CD120b protein into mRNA.
  • a promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and a TATA box, using a located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site.
  • a mammalian promoter will also contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box.
  • An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation.
  • mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.
  • transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence.
  • the 3' terminus of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation.
  • transcription terminator and polyadenlytion signals include those derived form SV40.
  • nucleic acid into mammalian hosts, as well as other hosts, is well known in the art, and will vary with the host cell used. Techniques include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
  • TNF- ⁇ or TNFRII/CD 120b proteins are expressed in bacterial systems which are well known in the art.
  • TNF- ⁇ or TNFRII/CD120b proteins can be produced in insect cells.
  • Expression vectors for the transformation of insect cells, and in particular, baculovirus- based expression vectors, are well known in the art.
  • TNF- ⁇ or TNFRII/CD 120b proteins are produced in yeast cells.
  • Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica.
  • a TNF- ⁇ or TNFRII/CD120b protein can also be made as a fusion protein, using techniques well known in the art. For example, for the creation of monoclonal antibodies, if the desired epitope is small, the protein can be fused to a carrier protein to form an immunogen. Alternatively, TNF- ⁇ or TNFRII/CD120b protein can be made as a fusion protein to increase expression. For example, when a protein is a shorter peptide, the nucleic acid encoding the peptide can be linked to other nucleic acid for expression purposes. Similarly, TNF- ⁇ or TNFRII/CD120b proteins of the methods and compositions herein can be linked to protein labels, such as green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP).
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • YFP yellow fluorescent protein
  • BFP blue fluorescent protein
  • the proteins are recombinant.
  • a "recombinant protein” is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid as depicted above.
  • a recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics.
  • the protein can be isolated or purified away from some or all of the proteins and compounds with which it is normally associated in its wild type host, and thus can be substantially pure.
  • an isolated protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, typically constituting at least about 0.5%, typically at least about 5% by weight of the total protein in a given sample.
  • a substantially pure protein comprises at least about 75% by weight of the total protein, at least about 80%, and typically at least about 90%.
  • the definition includes the production of TNF- ⁇ or TNFRII/CD120b protein from one organism in a different organism or host cell.
  • the protein can be made at a significantly higher concentration than is normally seen, through the use of a inducible promoter or high expression promoter, such that the protein is made at increased concentration levels.
  • the protein can be in a form not normally found in nature, as in the addition of an epitope tag or amino acid substitutions, insertions and deletions, as discussed below.
  • the protein when the TNF- ⁇ or TNFRII/CD120b protein is to be used to generate antibodies, the protein must share at least one epitope or determinant with the full length transcription product of the nucleic acids.
  • epitope or “determinant” herein is meant a portion of a protein which will bind an antibody.
  • the epitope is unique; that is, antibodies generated to a unique epitope show little or no cross-reactivity.
  • the antibodies provided herein can be capable of reducing or eliminating the biological function of a TNF- ⁇ or TNFRII/CD120b protein, as is described below.
  • the addition of antibodies (either polyclonal or monoclonal) to the protein (or cells containing the protein) can reduce or eliminate the protein's activity. Generally, at least a 25% decrease in activity is observed, with typically at least about 50% and typically about a 95-100% decrease being observed.
  • the proteins can be variant proteins, comprising one more amino acid substitutions, insertions and deletions.
  • a TNF- ⁇ or TNFRII/CD120b protein is purified or isolated after expression.
  • Proteins can be isolated or purified in a variety of ways. Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromato focusing.
  • a TNF- ⁇ or TNFRII/CD120b protein can be purified using a standard anti-TNF- ⁇ or anti-TNFRII/CD120b protein antibody column. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful.
  • binding assays can be done. These methods comprise combining a TNF- ⁇ or TNFRII/CD120b protein and a candidate bioactive agent, and determining the binding of the candidate agent to the TNF- ⁇ or TNFRII/CD120b protein. Methods utilize a human TNF- ⁇ or TNFRII/CD120b protein, although other mammalian proteins can also be used, including rodents (mice, rats, hamsters, guinea pigs), farm animals (cows, sheep, pigs, horses) and primates. These latter methods can be used for the development of animal models of human disease. In some methods, variant or derivative TNF- ⁇ or TNFRII/CD120b proteins can be used, including deletion TNF- ⁇ or TNFRII/CD120b proteins as outlined above.
  • the assays herein utilize TNF- ⁇ or TNFRII/CD120b proteins as defined herein. In some assays, portions of proteins can be utilized, hi other assays, portions having different activities can be used. In addition, the assays described herein can utilize either isolated TNF- ⁇ or TNFRII/CD120b proteins or cells comprising the TNF- ⁇ or TNFRII/CD120b proteins. In some methods, the protein or the candidate agent is non-diffusably bound to an insoluble support having isolated sample receiving areas ⁇ e.g., a microtiter plate or an array). The insoluble supports can be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening.
  • the surface of such supports can be solid or porous and of any convenient shape.
  • suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic ⁇ e.g., polystyrene), polysaccharides, nylon or nitrocellulose, and teflonTM. Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. In some cases magnetic beads and the like are included.
  • the particular manner of binding of the composition is not crucial so long as it is compatible with the reagents and overall methods described herein, maintains the activity of the composition and is nondiffusable.
  • Methods of binding include the use of antibodies (which do not sterically block either the ligand binding site or activation sequence when the protein is bound to the support), direct binding to ionic supports, chemical crosslinking, or by the synthesis of the protein or agent on the surface. Following binding of the protein or agent, excess unbound material is removed by washing. The sample receiving areas can then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety. Also included in the methods and compositions herein are screening assays wherein solid supports are not used.
  • BSA bovine serum albumin
  • the TNF- ⁇ or TNFRII/CD120b protein is bound to the support, and a candidate bioactive agent is added to the assay.
  • the candidate agent is bound to the support and the protein is added.
  • Novel binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, and peptide analogs. Of particular interest are screening assays for agents that have a low toxicity for human cells. A wide variety of assays can be used for this purpose, including labeled in vitro protein- protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (such as phosphorylation assays) and the like.
  • the determination of the binding of the candidate bioactive agent to a TNF- ⁇ or TNFRII/CD120b protein can be done in a number of ways.
  • the candidate bioactive agent is labeled, and binding determined directly. For example, this can be done by attaching all or a portion of a TNF- ⁇ or TNFRII/CD120b protein to a solid support, adding a labeled candidate agent (for example a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support.
  • a labeled candidate agent for example a fluorescent label
  • washing off excess reagent for example a fluorescent label
  • labeled herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e.g., radioisotope, fluorescers, enzyme, antibodies, particles such as magnetic particles, chemiluminescers, or specific binding molecules.
  • Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin.
  • the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above.
  • the label can directly or indirectly provide a detectable signal. [U132J Jn some methods, only one of the components is labeled.
  • the proteins can be labeled at tyrosine positions using 125 I, or with fluorophores.
  • more than one component can be labeled with different labels; using 125 I for the proteins, for example, and a fluorophor for the candidate agents.
  • the binding of the candidate bioactive agent is determined through the use of competitive binding assays.
  • the competitor is a binding moiety known to bind to the target molecule such as an antibody, peptide, binding partner, or ligand.
  • the target molecule such as an antibody, peptide, binding partner, or ligand.
  • This assay can be used to determine candidate agents which interfere with binding between proteins and the competitor.
  • the candidate bioactive agent is labeled.
  • Either the candidate bioactive agent, or the competitor, or both, is added first to the protein for a time sufficient to allow binding, if present.
  • Incubations can be performed at any temperature which facilitates optimal activity, typically between about 4 0 C and 40 0 C. Incubation periods are selected for optimum activity, but can also be optimized to facilitate rapid high through put screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.
  • the competitor is added first, followed by the candidate bioactive agent.
  • Displacement of the competitor is an indication that the candidate bioactive agent is binding to the TNF- ⁇ or TNFRII/CD120b protein and thus is capable of binding to, and potentially modulating, the activity of the protein.
  • either component can be labeled.
  • the presence of label in the wash solution indicates displacement by the agent.
  • the candidate bioactive agent is labeled, the presence of the label on the support indicates displacement.
  • the candidate bioactive agent is added first, with incubation and washing, followed by the competitor.
  • the absence of binding by the competitor can indicate that the bioactive agent is bound to the TNF- ⁇ or TNFRII/CD120b protein with a higher affinity.
  • the candidate bioactive agent is labeled, the presence of the label on the support, coupled with a lack of competitor binding, can indicate that the candidate agent is capable of binding to the protein.
  • ком ⁇ онент binding methods can also be run as differential screens. These methods can comprise a TNF- ⁇ or TNFRII/CD120b protein and a competitor in a first sample.
  • a second sample comprises a candidate bioactive agent, a TNF- ⁇ or TNPRII/CD120b protein and a competitor.
  • the binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the TNF- ⁇ or TNFRII/CD120b protein and potentially modulating its activity. If the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the protein.
  • TNFRII/CD120b proteins that bind to the native TNF- ⁇ or TNFRII/CD120b protein, but cannot bind to modified proteins.
  • the structure of the protein can be modeled, and used in rational drug design to synthesize agents that interact with that site.
  • Drug candidates that affect TNFRII/CD120b bioactivity are also identified by screening drugs for the ability to either enhance or reduce the activity of the protein.
  • screening for agents that modulate the activity of proteins are performed. In general, this will be done on the basis of the known biological activity of the TNF- ⁇ or TNFRII/CD120b protein.
  • a candidate bioactive agent is added to a sample of the protein, as above, and an alteration in the biological activity of the protein is determined.
  • Moduleating the activity includes an increase in activity, a decrease in activity, or a change in the type or kind of activity present.
  • the candidate agent should both bind to TNF- ⁇ or TNFRII/CD120b (although this may not be necessary), and alter its biological or biochemical activity as defined herein.
  • the methods include both in vitro screening methods, as are generally outlined above, and in vivo screening of cells for alterations in the presence, distribution, activity or amount of the protein.
  • Some methods comprise combining a TNF- ⁇ or TNFRII/CD120b sample and a candidate bioactive agent, then evaluating the effect on TNFRII/CD120b activity to inhibit or enhance an immune response.
  • TNFRII/CD120b activity or grammatical equivalents herein is meant one of TNFRII/CD120b biological activities, including, but not limited to, its ability to affect immune activation or inhibition.
  • One activity herein is the capability to bind to a target gene, or modulate TNFRII/CD120b pathway signaling. TNFRII/CD120b pathway signaling is induced or maintained.
  • the activity of the TNF- ⁇ or TNFRII/CD 120b protein is increased; in other methods, the activity of the TNF- ⁇ or TNFRII/CD 120b protein is decreased.
  • bioactive agents that are antagonists are useful in some methods, and bioactive agents that are agonists are useful in other methods.
  • Methods for screening for bioactive agents capable of modulating the activity of a TNF- ⁇ or TNFRII/CD120b protein are provided. These methods comprise adding a candidate bioactive agent, as defined above, to a cell comprising proteins. Cell types include almost any cell. The cells contain a recombinant nucleic acid that encodes a TNF- ⁇ or TNFRII/CD120b protein. In one method, a library of candidate agents are tested on a plurality of cells. The effect of the candidate agent on TNFRII/CD120b pathway signaling activity is then evaluated.
  • Positive controls and negative controls can be used in the assays. All control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the agent to the protein. Following incubation, all samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples can be counted in a scintillation counter to determine the amount of bound compound.
  • a variety of other reagents can be included in the screening assays. These include reagents like salts, neutral proteins (e.g., albumin and detergents) which can be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Reagents that otherwise improve the efficiency of the assay, (such as protease inhibitors, nuclease inhibitors, anti-microbial agents) can also be used. The mixture of components can be added in any order that provides for the requisite binding.
  • kits can be based on the use of the protein and/or the nucleic acid encoding the TNF- ⁇ or TNFRII/CD120b proteins. Assays regarding the use of nucleic acids are further described below.
  • nucleic acids which encode TNF- ⁇ or TNFRII/CD120b proteins or their modified forms can also be used to generate either transgenic animals, including "knock- in” and “knock out” animals which, in turn, are useful in the development and screening of therapeutically useful reagents.
  • a non-human transgenic animal e.g., a mouse or rat
  • a transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops, and can include both the addition of all or part of a gene or the deletion of all or part of a gene.
  • cDNA encoding a TNF- ⁇ or TNFRII/CD120b protein can be used to clone genomic DNA encoding a TNF- ⁇ or TNFRII/CD120b protein in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which either express (or overexpress) or suppress the desired DNA.
  • Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Patent Nos.
  • Transgenic animals that include a copy of a transgene encoding a TNFRII/CD120b protein introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of the desired nucleic acid. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression.
  • non-human homologues of a TNF- ⁇ or TNFRII/CD120b protein can be used to construct a transgenic animal comprising a protein "knock out" animal which has a defective or altered gene encoding a TNF- ⁇ or TNFRII/CD120b protein as a result of homologous recombination between the endogenous gene encoding a TNF- ⁇ or TNFRII/CD120b protein and altered genomic DNA encoding the protein introduced into an embryonic cell of the animal.
  • cDNA encoding a TNF- ⁇ or TNFRII/CD120b protein can be used to clone genomic DNA encoding the protein in accordance with established techniques.
  • a portion of the genomic DNA encoding a TNF- ⁇ or TNFRII/CD120b protein can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration.
  • another gene such as a gene encoding a selectable marker which can be used to monitor integration.
  • several kilobases of unaltered flanking DNA are included in the vector ⁇ see, e.g., Thomas and Capecchi, Cell 51:503, 1987, incorporated herein by reference in its entirety, for a description of homologous recombination vectors).
  • the vector is introduced into an embryonic stem cell line ⁇ e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected ⁇ see, e.g., Li et al., Cell 69:915, 1992, incorporated herein by reference in its entirety).
  • the selected cells are then injected into a blastocyst of an animal ⁇ e.g., a mouse or rat) to form aggregation chimeras ⁇ see, e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152).
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal.
  • Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA.
  • Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of a TNF- ⁇ or TNFRII/CD120b protein polypeptide.
  • Animal models for TNFRII/CD120b pathway signaling related disorders, or having a particular state of TNFRII/CD120b pathway signaling activity can include, for example, genetic models.
  • animal models for autoimmune diseases can include the nonobese diabetic (NOD) mouse (see, e.g., McDuffie, Curr Opin Immunol. 10(6):704-9, 1998; Tochino, Crit Rev Immunol 8(1):49-81, 1987), and experimental autoimmune encephalomyelitis (EAE) (see, e.g., Wong, Immunol Rev 169:93-104, 1999).
  • NOD nonobese diabetic
  • EAE experimental autoimmune encephalomyelitis
  • Schwartz et al. Autoimmunity and Autoimmune Diseases, Ch. 31, in Fundamental Immunology, Paul (ed.), Raven Press 1989, each incorporated herein by reference in their entirety.
  • Other models can include studies involving transplant rejection.
  • Animal models exhibiting TNFRII/CD120b pathway signaling related disorder- like symptoms can be engineered by utilizing, for example, TNF- ⁇ or TNFRII/CD120b sequences in conjunction with techniques for producing transgenic animals that are well known to those of skill in the art.
  • gene sequences can be introduced into, and overexpressed in, the genome of the animal of interest, or, if endogenous target gene sequences are present, they can either be overexpressed or, alternatively, can be disrupted in order to underexpress or inactivate target gene expression.
  • the coding portion of the target gene sequence can be ligated to a regulatory sequence which is capable of driving gene expression in the animal and cell type of interest.
  • a regulatory sequence which is capable of driving gene expression in the animal and cell type of interest.
  • Such regulatory regions will be well known to those of skill in the art, and can be utilized in the absence of undue experimentation.
  • an endogenous target gene sequence such a sequence can be isolated and engineered such that when reintroduced into the genome of the animal of interest, the endogenous target gene alleles will be inactivated.
  • the engineered target gene sequence is introduced via gene targeting such that the endogenous target sequence is disrupted upon integration of the engineered target sequence into the animal's genome.
  • Animals of any species including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates, e.g., baboons, monkeys, and chimpanzees can be used to generate animal models of TNFRII/CD120b pathway signaling related disorders or being a perpetually desired state of the TNFRII/CD120b pathway signaling.
  • Nucleic acids encoding TNF- ⁇ or TNFRII/CD120b polypeptides, antagonists or agonists can also be used in gene therapy.
  • a gene therapy vector is an exogenous polynucleotide which produces a medically useful phenotypic effect upon the mammalian cell(s) into which it is transferred.
  • a vector can or can not have an origin of replication.
  • it is useful to include an origin of replication in a vector for propagation of the vector prior to administration to a patient.
  • the origin of replication can often be removed before administration if the vector is designed to integrate into host chromosomal DNA or bind to host mRNA or DNA.
  • Vectors used in gene therapy can be viral or nonviral.
  • Viral vectors are usually introduced into a patient as components of a virus.
  • Nonviral vectors typically dsDNA
  • Viral vectors such as retroviruses, adenoviruses, adenoassociated viruses and herpes viruses, are often made up of two components, a modified viral genome and a coat structure surrounding it (see generally Smith et ah, Ann. Rev. Microbiol. 49:807-838, 1995, incorporated herein by reference in its entirety), although sometimes viral vectors are introduced in naked form or coated with proteins other than viral proteins. Most current vectors have coat structures similar to a wildtype virus. This structure packages and protects the viral nucleic acid and provides the means to bind and enter target cells. However, the viral nucleic acid in a vector designed for gene therapy is changed in many ways.
  • vector nucleic acids generally comprise two components: essential cis-acting viral sequences for replication and packaging in a helper line and the transcription unit for the exogenous gene. Other viral functions are expressed in trans in a specific packaging or helper cell line.
  • Nonviral nucleic acid vectors used in gene therapy include plasmids, RNAs, antisense oligonucleotides (e.g., methylphosphonate or phosphorothiolate), polyamide nucleic acids, interfering RNA (RNAi), hairpin RNA, and yeast artificial chromosomes (YACs).
  • Such vectors typically include an expression cassette for expressing a protein or RNA.
  • the promoter in such an expression cassette can be constitutive, cell type-specific, stage-specific, and/or modulatable (e.g., by hormones such as glucocorticoids; MMTV promoter). Transcription can be increased by inserting an enhancer sequence into the vector.
  • Enhancers are cis-acting sequences of between 10 to 300bp that increase transcription by a promoter. Enhancers can effectively increase transcription when either 5' or 3' to the transcription unit. They are also effective if located within an intron or within the coding sequence itself. Typically, viral enhancers are used, including SV40 enhancers, cytomegalovirus enhancers, polyoma enhancers, and adenovirus enhancers. Enhancer sequences from mammalian systems are also commonly used, such as the mouse immunoglobulin heavy chain enhancer.
  • Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application.
  • vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.
  • TNFRII/CD120b Pathway Signaling the level of TNFRII/CD120b signaling will be modulated in a cell by administering to the cell, in vivo or in vitro, any of a large number of TNFRII/CD120b-modulating molecules, e.g., polypeptides, antibodies, amino acids, nucleotides, lipids, carbohydrates, or any organic or inorganic molecule.
  • TNFRII/CD120b-modulating molecules e.g., polypeptides, antibodies, amino acids, nucleotides, lipids, carbohydrates, or any organic or inorganic molecule.
  • TNFRII/CD120b signaling can be assessed using a variety of in vitro and in vivo assays to determine functional, chemical, and physical effects, e.g., measuring the binding of TNFRII/CD120b to other molecules (e.g., radioactive binding to TNF- ⁇ ), measuring protein and/or RNA levels of TNFRII/CD120b pathway signaling via TNF- ⁇ that provides an immunosuppressive or immune-stimulating response, or measuring other aspects of pathway signaling, e.g., phosphorylation levels, transcription levels, receptor activity, ligand binding and the like.
  • Such assays can be used to test for both activators and inhibitors of TNFRII/CD120b signaling. Modulators thus identified are useful for, e.g., many diagnostic and therapeutic applications.
  • the TNFRII/CD120b pathway signaling via TNF- ⁇ in the assay will typically be a recombinant or naturally occurring polypeptide or a conservatively modified variant thereof.
  • the TNFRII/CD120b pathway signaling via TNF- ⁇ in the assay will be derived from a eukaryote and include an amino acid subsequence having amino acid sequence identity to the naturally occurring TNFRII/CD120b pathway signaling.
  • the amino acid sequence identity will be at least 70%, optionally at least 75%, 85%, or 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or greater.
  • the polypeptide of the assays will comprise a domain of an TNF- ⁇ or TNFRII/CD120b.
  • a domain of TNFRII/CD120b or TNF- ⁇ protein is bound to a solid substrate and used, e.g., to isolate any molecules that can bind to and/or modulate their activity.
  • a domain of a TNF- ⁇ or TNFRII/CD120b polypeptide e.g., anN-terminal domain, a C-terminal domain, is fused to a heterologous polypeptide, thereby forming a chimeric polypeptide.
  • Such chimeric polypeptides are also useful, e.g., in assays to identify modulators of an TNFRII/CD120b pathway signaling via TNF- ⁇ .
  • nucleic acids or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same ⁇ i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region ⁇ e.g., nucleotide sequence encoding a collectin described herein or amino acid sequence of a collectin described herein), when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection ⁇ see, e.g., NCBI web site).
  • sequences are then said to be "substantially identical.”
  • This term also refers to, or can be applied to, the compliment of a test sequence.
  • the term also includes sequences that have deletions and/or additions, as well as those that have substitutions.
  • the preferred algorithms can account for gaps and the like.
  • identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well- known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981 by the homology alignment algorithm of Needleman & Wunsch, J MoI. Biol.
  • a preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, Nuc. Acids Res 25:3389-3402, 1977 and Altschul et al., J. MoI. Biol. 215:403-410, 1990 respectively.
  • BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov ⁇ .
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence.
  • T is referred to as the neighborhood word score threshold (Altschul et al., supra).
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • polypeptide polypeptide
  • peptide protein
  • protein protein
  • amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ - carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups ⁇ e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein.
  • the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
  • the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • the following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
  • Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et ah, Molecular Biology of the Cell (3rd ed., 1994) and Cantor and Schimmel, Biophysical Chemistry Part I: The Conformation of Biological Macromolecules (1980).
  • Primary structure refers to the amino acid sequence of a particular peptide.
  • “Secondary structure” refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains, e.g., enzymatic domains, extracellular domains, transmembrane domains, pore domains, and cytoplasmic tail domains.
  • Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 15 to 350 amino acids long. Exemplary domains include domains with enzymatic activity, e.g., a kinase domain. Typical domains are made up of sections of lesser organization such as stretches of ⁇ -sheet and ⁇ -helices. "Tertiary structure” refers to the complete three dimensional structure of a polypeptide monomer. “Quaternary structure” refers to the three dimensional structure formed by the noncovalent association of independent tertiary units. Anisotropic terms are also known as energy terms.
  • a particular nucleic acid sequence also implicitly encompasses "splice variants.”
  • a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant of that nucleic acid.
  • “Splice variants,” as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript can be spliced such that different (alternate) nucleic acid splice products encode different polypeptides.
  • Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternate polypeptides derived from the same nucleic acid by read- through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition.
  • stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-1O 0 C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength pH.
  • T m thermal melting point
  • the T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary stringent hybridization conditions can be as following: 50% formamide, 5 ⁇ S'S'CV'aftd ⁇ % r "SDS';'meubating at 42 0 C, or, 5x SSC, 1% SDS, incubating at 65 0 C, with wash in 0.2x SSC, and 0.1% SDS at 65 0 C.
  • nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
  • Exemplary "moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37 0 C, and a wash in IX SSC at 45 0 C. A positive hybridization is at least twice background.
  • Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous reference, e.g., Ausubel et al, supra.
  • a temperature of about 36°C is typical for low stringency amplification, although annealing temperatures can vary between about 32°C and 48 0 C depending on primer length.
  • a temperature of about 62°C is typical, although high stringency annealing temperatures can range from about 50°C to about 65°C, depending on the primer length and specificity.
  • Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90°C - 95 0 C for 30 sec - 2 min., an annealing phase lasting 30 sec. - 2 min., and an extension phase of about 72 0 C for 1 - 2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al. PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N. Y. (1990).
  • Samples or assays that are treated with a potential TNFRII/CD120b pathway signaling inhibitor or activator are compared to control samples without the test compound, to examine the extent of modulation.
  • Control samples untreated with activators or inhibitors
  • TNFRII/CD120b activity value 100.
  • Inhibition of TNFRII/CD120b pathway signaling is achieved when the TNFRII/CD120b pathway signaling activity value relative to the control is about 90%, optionally about 50%, optionally about 25-0%.
  • Activation of a TNFRII/CD120b pathway signaling is achieved when the TNFRII/CD120b pathway signaling activity value relative to the control is about 110%, optionally about 150%, 200-500%, or about 1000-2000%.
  • test compounds upon the function of the polypeptides can be measured by examining any of the parameters described above. Any suitable physiological change that affects TNFRII/CD120b pathway signaling activity can be used to assess the influence of a test compound on the polypeptides of this invention. When the functional consequences are determined using intact cells or animals, one can also measure a variety of effects such as changes in cell growth or changes in cell-cell interactions.
  • Modulators of TNFRII/CD 120b pathway signaling that act by modulating gene expression can also be identified.
  • a host cell containing a TNFRII/CD 120b or TNF- ⁇ protein of interest is contacted with a test compound for a sufficient time to effect any interactions, and then the level of gene expression is measured.
  • the amount of time to effect such interactions can be empirically determined, such as by running a time course and measuring the level of transcription as a function of time.
  • the amount of transcription can be measured using any method known to those of skill in the art to be suitable.
  • mRNA expression of the protein of interest can be detected using Northern blots or by detecting their polypeptide products using immunoassays.
  • assays will be performed to identify molecules that physically interact with TNFRII/CD 120b or TNF- ⁇ .
  • molecules can be any type of molecule, including polypeptides, polynucleotides, amino acids, nucleotides, carbohydrates, lipids, or any other organic or inorganic molecule.
  • Such molecules can represent molecules that normally interact with TNFRII/CD 120b or TNF- ⁇ or can be synthetic or other molecules that are capable of interacting with TNFRIFCD 120b or TNF- ⁇ and that can potentially be used as lead compounds to identify classes of molecules that can interact with and/or modulate TNFRII/CD 120b pathway signaling via TNF- ⁇ .
  • Such assays can represent physical binding assays, such as affinity chromatography, immunoprecipitation, two-hybrid screens, or other binding assays, or can represent genetic assays.
  • TNFRII/CD 120b pathway signaling via TNF- ⁇ can be used.
  • the TNFRII/CD 120b or TNF- ⁇ has at least about 85% identity to the amino acid sequence of the naturally occurring TNFRII/CD120b or TNF- ⁇ .
  • a fragment of a TNFRII/CD 120b or TNF- ⁇ is used.
  • Such fragments can be used alone, in combination with other TNFRII/CD120b or TNF- ⁇ protein fragments, or in combination with sequences from heterologous proteins, e.g., the fragments can be fused to a heterologous polypeptides, thereby forming a chimeric polypeptide.
  • Compounds that interact with TNFRII/CD120b pathway signaling via TNF- ⁇ can be isolated based on an ability to specifically bind to a TNFRII/CD120b or TNF-oc or fragment thereof.
  • the TNF- ⁇ or TNFRII/CD120b or protein fragment will be attached to a solid support.
  • affinity columns are made using the TNF- ⁇ or TNFRII/CD120b polypeptide, and physically-interacting molecules are identified. It will be apparent to one of skill that chromatographic techniques can be performed at any scale and using equipment from many different manufactures (e.g., Pharmacia Biotechnology).
  • molecules that interact with TNF- ⁇ or TNFRII/CD120b in vivo can be identified by co- immunoprecipitation or other methods, i.e., immunoprecipitating TNF- ⁇ or TNFRII/CD120b using anti- TNF- ⁇ or anti- TNFRII/CD120b antibodies from a cell or cell extract, and identifying compounds, e.g., proteins, that are precipitated along with the TNF- ⁇ or TNFRII/CD120b.
  • this invention provides methods of treating neoplastic disease, allogeneic tissue rejection, or graft vs. host disease by increasing TNF- ⁇ or TNFRII/CD120b signaling or protein levels in a cell.
  • such methods are used to increase a reduced level of TNF- ⁇ or TNFRII/CD120b protein, e.g., a reduced level in a CD4+CD25+ hl T R cell, and can be performed in any of a number of ways, e.g.
  • the level of protein activity is increased to a level typical of a normal, cell, but the level can be increased to any level that is sufficient to increase TNFRII/CD120b signaling in the CD4+CD25+ hi T R cell, including to levels above or below those typical of normal cells.
  • such methods involve the use of activators of TNF- ⁇ or TNFRII/CD120b protein, where an "activator of TNF- ⁇ or TNFRII/CD120b protein" is a molecule that acts to increase TNF- ⁇ or TNFRII/CD120b gene polynucleotide levels, polypeptide levels and/or protein activity.
  • activators can include, but are not limited to, small molecule activators of TNF- ⁇ or TNFRII/CD120b protein.
  • this invention provides methods of treating autoimmune disease or inflammatory disease by reducing TNFRII/CD120b signaling or protein levels in a cell.
  • these methods are used to reduce an elevated level of TNFRII/CD 120b or TNF- ⁇ protein, e.g., an elevated level in a CD4+CD25+ hl T R cells can be performed in any of a number of ways, e.g., lowering the copy number of TNFRII/CD120b or TNF- ⁇ protein genes or decreasing the level of mRNA, protein, or protein activity in a cell.
  • the level of TNFRII/CD120b or TNF- ⁇ protein activity is lowered to a level typical of a normal CD4+CD25+ 1 " T R cell, but the level can be reduced to any level that is sufficient to increase TNFRII/CD120b or TNF- ⁇ signaling of the cell, including to levels above or below those typical of normal cells.
  • such methods involve the use of inhibitors of TNFRII/CD120b or TNF- ⁇ protein, where an "inhibitor of TNFRII/CD120b or TNF- ⁇ protein" is a molecule that acts to reduce TNFRII/CD120b or TNF- ⁇ protein polynucleotide levels, polypeptide levels and/or protein activity.
  • Such inhibitor s include, but are not limited to, antisense polynucleotides, ribozymes, antibodies, dominant negative TNFPJI/CD120b or TNF- ⁇ protein forms, and small molecule inhibitors of TNFRII/CD120b or TNF- ⁇ protein.
  • TNFRII/CD 120b or TNF- ⁇ protein levels or signaling through TNFRII/CD 120b will be reduced so as to treat autoimmune disease or inflammatory disease as a result of activated TNFRII/CD 120b or TNF- ⁇ protein levels.
  • the proliferation of a cell refers to the rate at which the cell or population of cells divides, or to the extent to which the cell or population of cells divides or increases in number. Proliferation can reflect any of a number of factors, including the rate of cell growth and division and the rate of cell death.
  • TNFRII/CD 120b or TNF- ⁇ induced gene in CD4+CD25+ 1 T R cells to inhibit or enhance an immune response, or treat autoimmune disease, neoplastic disease, systemic lupus erthymatosus, or allogeneic tissue rejection signaling.
  • Inhibition or activation of immune activity via TNFRII/CD 120b or TNF- ⁇ protein can act to treat autoimmune disease, neoplastic disease, allogeneic tissue rejection, or inflammatory disease.
  • TNFRII/CD 120b or TNF- ⁇ protein activity can be determined based on any of a number of factors, including, but not limited to, a level of TNFRII/CD 120b or TNF- ⁇ polynucleotide, e.g., mRNA or gDNA, the level of TNFRII/CD120b or TNF- ⁇ polypeptide, the degree of binding of a compound to a TNFRII/CD120b or TNF-a polynucleotide or polypeptide, TNFRH/CD120b or TNF- ⁇ protein intracellular localization, or any functional properties of TNFRII/CD120b or TNF- ⁇ protein, such as the ability of TNFRII/CD120b or TNF- ⁇ protein activity to inhibit or enhance an immune response, or treat autoimmune disease, neoplastic disease, allogeneic tissue rejection, or inflammatory disease.
  • a level of TNFRII/CD 120b or TNF- ⁇ polynucleotide e.g.
  • TNFRII/CD 120b or TNF- ⁇ protein activity is downregulated, or entirely inhibited, by the use of antisense polynucleotide, i.e., a nucleic acid complementary to, and which can preferably hybridize specifically to, a coding mRNA nucleic acid sequence, e.g, TNFRII/CD120b or TNF- ⁇ induced mRNA, or a subsequence thereof. Binding of the antisense polynucleotide to the mRNA reduces the translation and/or stability of the TNFRII/CD 120b or TNF- ⁇ induced mRNA.
  • antisense polynucleotide i.e., a nucleic acid complementary to, and which can preferably hybridize specifically to, a coding mRNA nucleic acid sequence, e.g, TNFRII/CD120b or TNF- ⁇ induced mRNA, or a subsequence thereof. Binding of the antisense poly
  • antisense polynucleotides can comprise naturally-occurring nucleotides, or synthetic species formed from naturally-occurring subunits or their close homologs. Antisense polynucleotides can also have altered sugar moieties or inter- sugar linkages. Exemplary among these are the phosphorothioate and other sulfur containing species which are known for use in the art. All such analogs are comprehended by this invention so long as they function effectively to hybridize with TNFRII/CD 120b or TNF- ⁇ induced mRNA.
  • antisense polynucleotides can readily be synthesized using recombinant means, or can be synthesized in vitro. Equipment for such synthesis is sold by several vendors, including Applied Biosystems. The preparation of other oligonucleotides such as phosphorothioates and alkylated derivatives is also well known to those of skill in the art.
  • ribozymes can be used to target and inhibit transcription of TNFRII/CD 120b or TNF- ⁇ protein.
  • a ribozyme is an RNA molecule that catalytically cleaves other RNA molecules.
  • Different kinds of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, RNAse P, and axhead ribozymes (see, e.g., Castanotto et al, Adv. in Pharmacology 25: 289-317, 1994 for a general review of the properties of different ribozymes).
  • hairpin ribozymes are described, e.g., in Hampel et at., Nucl. Acids Res., 18: 299-304, 1990; Hampel et al, European Patent Publication No. 0 360 257, 1990; U.S. Patent No. 5,254,678.
  • Methods of preparing are well known to those of skill in the art (see, e.g., Wong-Staal et al, WO 94/26877; Ojwang et al, Proc. Natl. Acad. Sd.
  • TNFRII/CD120b or TNF- ⁇ protein activity can also be decreased by the addition of an inhibitor of the TNFRII/CD120b or TNF- ⁇ protein.
  • an inhibitor of the TNFRII/CD120b or TNF- ⁇ protein can be accomplished in any of a number of ways, including by providing a dominant negative TNFRII/CD120b or TNF- ⁇ polypeptide, e.g., a form of TNFRII/CD120b or TNF- ⁇ protein that itself has no activity and which, when present in the same cell as a functional TNFRII/CD120b or TNF- ⁇ protein, reduces or eliminates the TNFRII/CD120b or TNF- ⁇ protein activity of the functional TNFRII/CD120b or TNF- ⁇ protein.
  • a dominant negative TNFRII/CD120b or TNF- ⁇ polypeptide e.g., a form of TNFRII/CD120b or TNF- ⁇ protein that itself has no activity and which, when present in the
  • inactive polypeptide variants can be used, e.g., by screening for the ability to inhibit TNFRII/CD120b or TNF- ⁇ protein activity.
  • Methods of making muteins are well known to those of skill (see, e.g., U.S. Patent Nos. 5,486,463; 5,422,260; 5,116,943; 4,752,585; and 4,518,504).
  • any small molecule e.g., any peptide, amino acid, nucleotide, lipid, carbohydrate, or any other organic or inorganic molecule can be screened for the ability to bind to or inhibit TNFRII/CD120b or TNF- ⁇ protein activity, as described below.
  • the compounds tested as modulators of a TNFRII/CD120b or TNF- ⁇ protein can be any small chemical compound, or a biological entity, such as a protein, sugar, nucleic acid or lipid.
  • test compounds will be small chemical molecules and peptides.
  • any chemical compound can be used as a potential modulator or binding compound in the assays of the invention, although most often compounds can be dissolved in aqueous or organic (especially DMSO-based) solutions.
  • the assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel ⁇ e.g., in microtiter formats on microtiter plates in robotic assays).
  • high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (potential modulator or binding compounds). Such "combinatorial chemical libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks” such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length ⁇ i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent No. 5,010,175; Furka, Int. J. Pept. Prot. Res. 37:487-493, 1991; and Houghton et al, Nature 354: 84-88, 1991).
  • Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication No.
  • WO 93/20242 random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Patent No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al, Proc. Nat. Acad. Sd. USA 90:6909-6913, 1993), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568, 1992), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc.
  • the invention provides soluble assays using molecules such as an N-terminal or C-terminal domain either alone or covalently linked to a heterologous protein to create a chimeric molecule.
  • the invention provides solid phase based Un vitro assays in a high throughput format, where a domain, chimeric molecule, TNF- ⁇ or TNFRII/CD120b protein, or cell or tissue expressing a TNF- ⁇ or TNFRII/CD120b protein is attached to a solid phase substrate.
  • each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator.
  • a single standard microtiter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different compounds. It is possible to assay several different plates per day; assay screens for up to about 6,000-20,000 different compounds is possible using the integrated systems of the invention. More recently, microfluidic approaches to reagent manipulation have been developed.
  • the molecule of interest can be bound to the solid state component, directly or indirectly, via covalent or non covalent linkage, e.g., via a tag.
  • the tag can be any of a variety of components, hi general, a molecule which binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest is attached to the solid support by interaction of the tag and the tag binder.
  • tags and tag binders can be used, based upon known molecular interactions well described in the literature.
  • a tag has a natural binder, for example, biotin, protein A, or protein G
  • tag binders avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.
  • Antibodies to molecules with natural binders such as biotin are also widely available and appropriate tag binders; see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis MO).
  • any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag/tag binder pair.
  • Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature.
  • the tag is a first antibody and the tag binder is a second antibody which recognizes the first antibody.
  • Synthetic polymers such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.
  • Common linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly-gly sequences of between about 5 and 200 amino acids.
  • polypeptide sequences such as poly-gly sequences of between about 5 and 200 amino acids.
  • Such flexible linkers are known to persons of skill in the art.
  • poly(ethelyne glycol) linkers are available from Shearwater Polymers, Inc. Huntsville, Alabama. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.
  • Tag binders are fixed to solid substrates using any of a variety of methods currently available.
  • Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent which fixes a chemical group to the surface which is reactive with a portion of the tag binder.
  • groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups.
  • Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces. The construction of such solid phase biopolymer arrays is well described in the literature. See, e.g., Merrifield, J. Am. Chem. Soc.
  • Nonchemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UV radiation, and the like.
  • Yet another assay for compounds that modulate TNF- ⁇ or TNFRII/CD 120b protein activity involves computer assisted drug design, in which a computer system is used to generate a three-dimensional structure of a TNF- ⁇ or TNFRII/CD 120b protein based on the structural information encoded by its amino acid sequence.
  • the input amino acid sequence interacts directly and actively with a pre-established algorithm in a computer program to yield secondary, tertiary, and quaternary structural models of the protein.
  • the models of the protein structure are then examined to identify regions of the structure that have the ability to bind. These regions are then used to identify compounds that bind to the protein.
  • the three-dimensional structural model of the protein is generated by entering protein amino acid sequences of at least 10 amino acid residues or corresponding nucleic acid sequences encoding a TNF- ⁇ or TNFRII/CD120b polypeptide into the computer system.
  • the amino acid sequence represents the primary sequence or subsequence of the protein, which encodes the structural information of the protein.
  • At least 10 residues of the amino acid sequence are entered into the computer system from computer keyboards, computer readable substrates that include, but are not limited to, electronic storage media (e.g., magnetic diskettes, tapes, cartridges, and chips), optical media (e.g., CD ROM), information distributed by internet sites, and by RAM.
  • electronic storage media e.g., magnetic diskettes, tapes, cartridges, and chips
  • optical media e.g., CD ROM
  • the three-dimensional structural model of the protein is then generated by the interaction of the amino acid sequence and the computer system, using software known to those of skill in the art.
  • the amino acid sequence represents a primary structure that encodes the information necessary to form the secondary, tertiary and quaternary structure of the protein of interest.
  • the software looks at certain parameters encoded by the primary sequence to generate the structural model. These parameters are referred to as "energy terms,” and primarily include electrostatic potentials, hydrophobic potentials, solvent accessible surfaces, and hydrogen bonding. Secondary energy terms include van der Waals potentials. Biological molecules form the structures that minimize the energy terms in a cumulative fashion. The computer program is therefore using these terms encoded by the primary structure or amino acid sequence to create the secondary structural model.
  • the tertiary structure of the protein encoded by the secondary structure is then formed on the basis of the energy terms of the secondary structure.
  • the user at this point can enter additional variables such as whether the protein is membrane bound or soluble, its location in the body, and its cellular location, e.g., cytoplasmic, surface, or nuclear. These variables along with the energy terms of the secondary structure are used to form the model of the tertiary structure, hi modeling the tertiary structure, the computer program matches hydrophobic faces of secondary structure with like, and hydrophilic faces of secondary structure with like.
  • potential modulator binding regions are identified by the computer system.
  • Three-dimensional structures for potential modulators are generated by entering amino acid or nucleotide sequences or chemical formulas of compounds, as described above.
  • the three-dimensional structure of the potential modulator is then compared to that of the TNF- ⁇ or TNFRII/CD120b protein to identify compounds that bind to the protein. Binding affinity between the protein and compound is determined using energy terms to determine which compounds have an enhanced probability of binding to the protein.
  • Computer systems are also used to screen for mutations, polymorphic variants, alleles and interspecies homologs of TNF- ⁇ or TNFRII/CD120b induced genes. Such mutations can be associated with disease states or genetic traits. GeneChipTM and related technology can also be used to screen for mutations, polymorphic variants, alleles and interspecies homologs. Once the variants are identified, diagnostic assays can be used to identify patients having such mutated genes. Identification of the mutated TNF- ⁇ or TNFRII/CD120b induced genes involves receiving input of a first nucleic acid or amino acid sequence of the naturally occurring TNF- ⁇ or TNFRII/CD120b induced gene, respectively, and conservatively modified versions thereof.
  • sequence is entered into the computer system as described above.
  • the first nucleic acid or amino acid sequence is then compared to a second nucleic acid or amino acid sequence that has substantial identity to the first sequence.
  • the second sequence is entered into the computer system in the manner described above. Once the first and second sequences are compared, nucleotide or amino acid differences between the sequences are identified.
  • sequences can represent allelic differences in various TNF- ⁇ or TNFRII/CD120b induced genes, and mutations associated with disease states and genetic traits.
  • Methods of identifying the genotype of an individual comprising determining all or part of the sequence of at least one TNF- ⁇ or TNFRII/CD120b gene of the individual are also provided. This is generally done in at least one tissue of the individual, and can include the evaluation of a number of tissues or different samples of the same tissue.
  • the method can include comparing the sequence of the sequenced mutant TNFRII/CD120b gene to a known TNFRII/CD120b gene, i.e., a wild-type gene. [0210]
  • the sequence of all or part of the TNF- ⁇ or TNFRII/CD 120b gene can then be compared to the sequence of a known TNF- ⁇ or TNFRII/CD 120b gene to determine if any differences exist.
  • the presence of a difference in the sequence between the TNF- ⁇ or TNFRII/CD 120b gene of the patient and the known TNF- ⁇ or TNFRII/CD 120b gene is indicative of a disease state or a propensity for a disease state, as outlined herein.
  • CD4+CD25+ hl T R cell states can be done using the methods and compositions herein.
  • the CD4+CD25+ hl T R cell state can be determined. This is particularly useful to verify the action of a drug, for example an immunosuppressive drug.
  • Other methods comprise administering the drug to a patient and removing a cell sample, particularly of CD4+CD25+ 1 " T R cells, from the patient.
  • the gene expression profile of the cell is then evaluated, as outlined herein, for example by comparing it to the expression profile from an equivalent sample from a healthy individual, hi this manner, both the efficacy (i.e., whether the correct expression profile is being generated from the drug) and the dose (is the dosage correct to result in the correct expression profile) can be verified.
  • the present discovery relating to the role of TNF- ⁇ or TNFRII/CD120b in inhibiting or enhancing an immune response, or e.g., treating autoimmune disease, neoplastic disease, systemic lupus erthymatosus, or allogeneic tissue rejection thus provides methods for treating differing disease states, hi one method, the TNF- ⁇ or TNFRII/CD 120b proteins, and particularly TNF- ⁇ or TNFRII/CD 120b protein fragments, are useful in the study or treatment of conditions which are mediated by various disease states, i.e., to diagnose, treat or prevent immune-mediated disorders.
  • immunological disorders can include conditions involving, for example, inhibition or enhancement of an immune response, autoimmune disease, neoplastic disease, systemic lupus erthymatosus, or allogeneic tissue rejection.
  • Methods of modulating immune-regulatory states in cells or organisms comprise administering to a cell an anti- TNF- ⁇ or anti- TNFRII/CD120b antibody or other agent identified herein or by the methods provided herein, that reduces or eliminates the biological activity of the endogeneous TNF- ⁇ or TNFRII/CD 120b protein.
  • the methods comprise administering to a cell or organism a recombinant nucleic acid encoding a TNF- ⁇ or TNFRII/CD 120b protein or modulator including anti-sense nucleic acids. As will be appreciated by those in the art, this can be accomplished in any number of ways.
  • the activity of TNFRII/CD120b receptor signaling is increased by increasing the amount or activity of TNF- ⁇ or TNFRII/CD120b in the cell, for example by overexpressing the endogeneous TNF- ⁇ or TNFRII/CD120b protein or by administering a TNF- ⁇ or TNFRII/CD120b gene, using known gene therapy techniques, for example.
  • the gene therapy techniques include the incorporation of the exogenous gene using enhanced homologous recombination (EHR), for example as described in PCT/US93/03868, hereby incorporated by reference in its entirety.
  • EHR enhanced homologous recombination
  • Methods for diagnosing a CD4+CD25+ 1 " T R cell activity related condition in an individual comprise measuring the activity of TNF- ⁇ or TNFRII/CD120b protein in a tissue from the individual or patient, which can include a measurement of the amount or specific activity of the protein. This activity is compared to the activity of TNF- ⁇ or TNFRII/CD120b protein from either an unaffected second individual or from an unaffected tissue from the first individual. When these activities are different, the first individual can be at risk for a CD4+CD25+ hl T R cell activity mediated disorder.
  • nucleotide sequences encoding a TNF- ⁇ or TNFRII/CD120b protein can also be used to construct hybridization probes for mapping the gene which encodes that TNF- ⁇ or TNFRII/CD120b protein and for the genetic analysis of individuals with genetic disorders.
  • the nucleotide sequences provided herein can be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization screening with libraries.
  • the TNF- ⁇ or TNFRII/CD 120b proteins can be used to generate polyclonal and monoclonal antibodies to TNF- ⁇ or TNFRII/CD 120b proteins, which are useful as described herein.
  • a number of immunogens are used to produce antibodies that specifically bind TNF- ⁇ or TNFRII/CD 120b polypeptides.
  • Full-length TNF- ⁇ or TNFRII/CD 120b polypeptides are suitable immunogens.
  • the immunogen of interest is a peptide of at least about 3 amino acids, more typically the peptide is at least 5 amino acids in length, the fragment is at least 10 amino acids in length and typically the fragment is at least 15 amino acids in length.
  • the peptides can be coupled to a carrier protein (e.g., as a fusion protein), or are recombinantly expressed in an immunization vector.
  • Antigenic determinants on peptides to which antibodies bind are typically 3 to 10 amino acids in length.
  • Naturally occurring polypeptides are also used either in pure or impure form.
  • Recombinant polypeptides are expressed in eukaryotic or prokaryotic cells and purified using standard techniques.
  • the 6 000465 polypeptide, or a synthetic version thereof, is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies can be generated for subsequent use in immunoassays to measure the presence and quantity of the polypeptide.
  • TNF- ⁇ or TNFRII/CD120b antibodies find use in a number of applications.
  • the TNF- ⁇ or TNFRII/CD120b antibodies can be coupled to standard affinity chromatography columns and used to purify TNF- ⁇ or TNFRII/CD120b proteins as further described below.
  • the antibodies can also be used as blocking polypeptides, as outlined above, since they will specifically bind to the TNF- ⁇ or TNFRII/CD120b protein.
  • the anti- TNF- ⁇ or anti- TNFRII/CD 120b protein antibodies can comprise polyclonal antibodies.
  • Methods for producing polyclonal antibodies are known to those of skill in the art.
  • an immunogen for example, a purified polypeptide, a polypeptide coupled to an appropriate carrier (e.g., GST and keyhole limpet hemocyanin), or a polypeptide incorporated into an immunization vector such as a recombinant vaccinia virus (see, U.S. Patent No. 4,722,848) is mixed with an adjuvant and animals are immunized with the mixture.
  • the animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the polypeptide of interest.
  • blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the polypeptide is performed where desired. See, e.g., Coligan, Current Protocols in Immunology, Wiley/Greene, NY, 1991; and Harlow and Lane, supra, each incorporated herein by reference in their entirety.
  • Antibodies including binding fragments and single chain recombinant versions thereof, against predetermined fragments of TNF- ⁇ or TNFRII/CD 120b proteins are raised by immunizing animals, e.g., with conjugates of the fragments with carrier proteins as described above.
  • the anti- TNF- ⁇ or anti- TNFRII/CD 120b protein antibodies can, alternatively, be monoclonal antibodies.
  • the monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies are screened for binding to normal or modified polypeptides, or screened for agonistic or antagonistic activity, e.g., activity mediated through the TNF- ⁇ or TNFRII/CD 120b proteins.
  • the immunizing agent will typically include the TNF- ⁇ or TNFRII/CD120b protein polypeptide or a fusion protein thereof.
  • PBLs peripheral blood lymphocytes
  • spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986, pp. 59-103, incorporated herein by reference in its entirety).
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells can be cultured in a suitable culture medium that contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT)
  • HGPRT or HPRT hypoxanthine guanine phosphoribosyl transferase
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • Immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • More immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the SaIk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Rockville, Maryland.
  • Human myeloma and mouse- human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol. 133:3001, 1984; Brön et ah, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, 1987, pp. 51-63, each incorporated herein by reference in their entirety).
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against TNF- ⁇ or TNFRII/CD120b protein.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art.
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et ah, Anal. Biochem. 107:220, 1980, incorporated herein by reference in its entirety.
  • the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the phrase "homotypic interaction” refers to the binding of a given protein to another molecule of the same protein ⁇ e.g., the binding of TNF- ⁇ to TNFRII/CD120b).
  • the phrase “heterotypic interaction” refers to the binding of a given protein to a different protein or other molecule (e.g., the binding of lipid to TNF- ⁇ protein or TNFRII/CD120b protein, or the binding of a transcription factor to DNA).
  • immune cell response refers to the response of immune system cells to external or internal stimuli ⁇ e.g., antigen, cytokines, chemokines, and other cells) producing biochemical changes in the immune cells that result in immune cell migration, killing of target cells, phagocytosis, production of antibodies, other soluble effectors of the immune response, and the like.
  • external or internal stimuli e.g., antigen, cytokines, chemokines, and other cells
  • Endogenous refers a protein, nucleic acid, lipid or other component produced within the body or within cells or organs of the body of a mammalian subject or originating within cells or organs of the body of a mammalian subject.
  • Exogenous refers a protein, nucleic acid, lipid, or other component originating outside the body of a mammalian subject.
  • Immuno response refers to the concerted action of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the human body of invading pathogens, cells or tissues infected with pathogens, cancerous cells, allogeneic tissue rejection, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
  • Signal transduction pathway or “signal transduction event” refers to at least one biochemical reaction, but more commonly a series of biochemical reactions, which result from interaction of a cell with a stimulatory compound or agent.
  • the interaction of a stimulatory compound with a cell generates a "signal” that is transmitted through the signal transduction patnway, ultimately resulting in a cellular response, e.g., an immune response described above.
  • recombinant immunoglobulins can be produced. See, U.S. Patent No. 4,816,567; and Queen et al, Proc. Natl Acad. Sd. 86:10029-10033, 1989, each incorporated herein by reference in their entirety.
  • nucleic acids encoding light and heavy chain variable regions are inserted into expression vectors.
  • the light and heavy chains can be cloned in the same or different expression vectors.
  • the DNA segments encoding antibody chains are operably linked to control sequences in the expression vector(s) that ensure the expression of antibody chains.
  • control sequences include a signal sequence, a promoter, an enhancer, and a transcription termination sequence.
  • Expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosome.
  • E. coli is one procaryotic host useful for expressing antibodies.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.
  • bacilli such as Bacillus subtilus
  • enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species.
  • prokaryotic hosts one can also make expression vectors, which typically contain expression control sequences compatible with the host cell (e.g., an origin of replication) and regulatory sequences such as a lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
  • trp tryptophan
  • Saccharomyces is one host, with suitable vectors having expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of replication, termination sequences and the like as desired.
  • Mammalian tissue cell culture can also be used to express and produce the antibodies (See Winnacker, From Genes to Clones, 1987, incorporated herein by reference in its entirety).
  • Eukaryotic cells are useful because a number of suitable host cell lines capable of secreting intact antibodies have been developed.
  • Suitable host cells for expressing nucleic acids encoding the immunoglobulins include: monkey kidney CVl line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293) (Graham et al, J. Gen. Virol. 36:59, 1977); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary-cells-DHFR (CHO, Urlaub and Chasin, Proc. Natl.
  • the vectors containing the polynucleotide sequences of interest can be transferred into the host cell.
  • Calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation can be used for other cellular hosts. See generally Sambrook et ah, Molecular Cloning: A Laboratory Manual, 2d ed., 1989, incorporated herein by reference in its entirety.
  • heavy and light chains are cloned on separate expression vectors, the vectors are co-transfected to obtain expression and assembly of intact immunoglobulins. After introduction of recombinant DNA, cell lines expressing immunoglobulin products are cell selected. Cell lines capable of stable expression are useful (i.e., undiminished levels of expression after fifty passages of the cell line).
  • the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like. See generally Scopes, Protein Purification, 1982, incorporated herein by reference in its entirety.
  • Substantially pure immunoglobulins are of at least about 90 to 95% homogeneity, and are typically 98 to 99% homogeneity or more.
  • the polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature.
  • an antibody used for detecting an analyte can be directly labeled with a detectable moiety, or can be indirectly labeled by, for example, binding to the antibody a secondary antibody that is, itself directly or indirectly labeled.
  • Antibodies are also used for affinity chromatography in isolating TNF- ⁇ or TNFRII/CD120b proteins.
  • Columns are prepared, e.g., with the antibodies linked to a solid support, e.g., particles, such as agarose, Sephadex, or the like, where a cell lysate is passed tnrough the column, washed, and treated with increasing concentrations of a mild denaturant, whereby purified TNF- ⁇ or TNFRII/CD120b polypeptides are released.
  • a further approach for isolating DNA sequences which encode a human monoclonal antibody or a binding fragment thereof is by screening a DNA library from human B cells according to the general protocol outlined by Huse et al, Science 246: 1275-1281, 1989, incorporated herein by reference in its entirety, and then cloning and amplifying the sequences which encode the antibody (or binding fragment) of the desired specificity.
  • B cells can be obtained from a human immunized with the desired antigen, fragments, longer polypeptides containing the antigen or fragments or anti-idiotypic antibodies.
  • B cells are obtained from an individual who has not been exposed to the antigen.
  • B cell can also be obtained from transgenic non-human animals expressing human immunoglobulin sequences.
  • the transgenic non-human animals can be immunized with an antigen or collection of antigens.
  • the animals can also be unimmunized.
  • B cell mRNA sequences encoding human antibodies are used to generate cDNA using reverse transcriptase.
  • the V region encoding segments of the cDNA sequences are then cloned into a DNA vector that directs expression of the antibody V regions.
  • the V region sequences are specifically amplified by PCR prior to cloning.
  • the V region sequences are cloned into a site within the DNA vector that is constructed so that the V region is expressed as a fusion protein.
  • fusion proteins include ml 3 coliphage gene 3 and gene 8 fusion proteins.
  • the collection of cloned V region sequences is then used to generate an expression library of antibody V regions.
  • the DNA vector comprising the cloned V region sequences is used to transform eukaryotic or prokaryotic host cells.
  • the vector can optionally encode all or part of a viral genome, and can comprise viral packaging sequences. In some cases the vector does not comprise an entire virus genome, and the vector is then used together with a helper virus or helper virus DNA sequences.
  • the expressed antibody V regions are found in, or on the surface of, transformed cells or virus particles from the transformed cells.
  • This expression library comprising the cells or virus particles, is then used to identify V region sequences that encode antibodies, or antibody fragments reactive with predetermined antigens.
  • the expression library is screened or selected for reactivity of the expressed V regions with the predetermined antigens.
  • the cells or virus particles comprising the cloned V region sequences, and having the expressed V regions are screened or selected by a method that identifies or enriches for cells or virus particles that have V regions reactive ⁇ e.g., binding association or catalytic activity) with a predetermined antigen.
  • radioactive or fluorescent labeled antigen that then binds to expressed V regions can be detected and used to identity or sort cells or virus particles.
  • Antigen bound to a solid matrix or bead can also be used to select cells or virus particles having reactive V regions on the surface.
  • the V region sequences thus identified from the expression library can then be used to direct expression, in a transformed host cell, of an antibody or fragment thereof, having reactivity with the predetermined antigen.
  • human antibodies having the binding specificity of a selected murine antibody can be produced. See, for example, WO 92/20791, incorporated herein by reference in its entirety.
  • this method either the heavy or light chain variable region of the selected murine antibody is used as a starting material. If, for example, a light chain variable region is selected as the starting material, a phage library is constructed in which members display the same light chain variable region (i.e., the murine starting material) and a different heavy chain variable region. The heavy chain variable regions are obtained from a library of rearranged human heavy chain variable regions.
  • a phage showing strong specific binding can then be selected.
  • the human heavy chain variable region from this phage then serves as a starting material for constructing a further phage library.
  • each phage displays the same heavy chain variable region (i.e., the region identified from the first display library) and a different light chain variable region.
  • the light chain variable regions are obtained from a library of rearranged human variable light chain regions.
  • phage showing strong specific binding for the selected are selected.
  • Artificial antibodies that are similar to human antibodies can be obtained from phage display libraries that incorporate random or synthetic sequences, for example, in CDR regions.
  • fragments of antibodies against TNF- ⁇ or TNFRTI/CD120b protein or protein analogs are provided. Typically, these fragments exhibit specific binding to the TNF- ⁇ or TNFRH/CD120b protein receptor similar to that of a complete immunoglobulin.
  • Antibody fragments include separate heavy chains, light chains F ab , F a b- F( ab ') 2 and F v . Fragments are produced by recombinant DNA techniques, or by enzymic or chemical separation of intact immunoglobulins.
  • the antibodies can be monovalent antibodies.
  • Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain.
  • the heavy chain is truncated generally at any point in the F c region so as to prevent heavy chain crosslinking.
  • the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.
  • An alternative approach is the generation of humanized immunoglobulins by linking the CDR regions of non-human antibodies to human constant regions by recombinant DNA techniques. See U.S. patent 5,585,089, incorporated herein by reference in its entirety.
  • Humanized forms of non-human (e.g., murine) antibodies are immunoglobulins, immunoglobulin chains or fragments thereof (such as F v , F a b, F ab' , F ab2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • donor antibody non-human species
  • F v framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies can also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an F c region, typically that of a human immunoglobulin. See Jones et ah, Nature 321: 522-525, 1986; Riechmann et al, Nature 332: 323-329, 1988; and Presta, Curr. Op. Struct. Biol, 2:593-596, 1992, each incorporated herein by reference in their entirety.
  • Chimeric and humanized antibodies have the same or similar binding specificity and affinity as a mouse or other nonhuman antibody that provides the starting material for construction of a chimeric or humanized antibody.
  • Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, typically by genetic engineering, from immunoglobulin gene segments belonging to different species. For example, the variable (V) segments of the genes from a mouse monoclonal antibody can be joined to human constant (C) segments, such as IgGi and IgG 4 . Human isotype IgGi is typically used.
  • a typical chimeric antibody is thus a hybrid protein consisting of the V or antigen-binding domain from a mouse antibody and the C or effector domain from a human antibody.
  • Humanized antibodies have variable region framework residues substantially from a human antibody (termed an acceptor antibody) and complementarity determining regions substantially from a mouse-antibody (referred to as the donor immunoglobulin). See, Queen et ah, Proc. Natl. Acad. Sd. U.S.A. 86:10029-10033, 1989; and WO 90/07861; U.S. 5,693,762; U.S. 5,693,761; U.S. 5,585,089; U.S. 5,530,101; and U.S. 5,225,539, each incorporated herein by reference in their entirety.
  • the constant region(s), if present, are also substantially or entirely from a human immunoglobulin.
  • the human variable domains are usually chosen from human antibodies whose framework sequences exhibit a high degree of sequence identity with the murine variable region domains from which the CDRs were derived.
  • the heavy and light chain variable region framework residues can be derived from the same or different human antibody sequences.
  • the human antibody sequences can be the sequences of naturally occurring human antibodies or can be consensus sequences of several human antibodies. See WO 92/22653, incorporated herein by reference in its entirety.
  • Certain amino acids from the human variable region framework residues are selected for substitution based on their possible influence on CDR conformation and/or binding to antigen. Investigation of such possible influences is by modeling, examination of the characteristics of the amino acids at particular locations, or empirical observation of the effects of substitution or mutagenesis of particular amino acids.
  • the human framework amino acid when an amino acid differs between a murine variable region framework residue and a selected human variable region framework residue, the human framework amino acid should usually be substituted by the equivalent framework amino acid from the mouse antibody when it is reasonably expected that the amino acid: (1) noncovalently binds antigen directly, (2) is adjacent to a CDR region, (3) otherwise interacts with a CDR region (e.g. is within about 6 A of a CDR region), or (4) participates in the VL-VH interface.
  • variable region frameworks of humanized immunoglobulins usually show at least 85% sequence identity to a human variable region framework sequence or consensus of such sequences.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries discussed above. Hoogenboom and Winter, J. MoI. Biol. 227:381, 1991; and Marks et al, J. MoI. Biol. 222:581, 1991. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies. Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985; and Boerner et al, J. Immunol. 147: 86-95, 1991.
  • human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos.
  • Bispecific antibodies are monoclonal, typically human or humanized, antibodies that have binding specificities for at least two different antigens.
  • one of the binding specificities is for the TNF- ⁇ or TNFRII/CD120b protein, the other one is for any other antigen, and for a cell-surface protein or receptor or receptor subunit.
  • bispecific antibodies Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities. Milstein and Cuello, Nature 305:537-539, 1983. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture often different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBOJ. 10:3655-3659, 1991.
  • Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences.
  • the fusion is typically with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions.
  • the first heavy-chain constant region (CHl) contains the site necessary for light-chain binding present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
  • Heteroconjugate antibodies are also within the scope of the present methods and compositions.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360; WO 92/200373; EP 03089).
  • the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
  • immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond.
  • Suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980. Each citation incorporated herein by reference in their entirety.
  • anti- TNF- ⁇ or anti- TNFRII/CD120b protein antibodies have various utilities.
  • anti- TNF- ⁇ or anti- TNFRII/CD120b protein antibodies can be used in diagnostic assays for a TNF- ⁇ or TNFRII/CD120b protein, e.g., detecting its expression in specific cells, tissues, or serum.
  • diagnostic assay techniques can be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases. Zola, 1987, Monoclonal Antibodies: A Manual of Techniques 147-158.
  • the antibodies used in the diagnostic assays can be labeled with a detectable moiety.
  • the detectable moiety should be capable of producing, either directly or indirectly, a detectable signal.
  • the detectable moiety can be a radioisotope, such as 3H, 14 C, 32 P, 35 S, or 125 I, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta- galactosidase or horseradish peroxidase.
  • Anti- TNF- ⁇ or anti- TNFRII/CD 120b protein antibodies also are useful for the affinity purification of TNF- ⁇ or TNFRII/CD 120b protein from recombinant cell culture or natural sources.
  • the antibodies against TNF- ⁇ or TNFRII/CD 120b protein are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in the art.
  • the immobilized antibody then is contacted with a sample containing the protein to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the TNF- ⁇ or TNFRII/CD 120b protein, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the protein from the antibody.
  • the anti- TNF- ⁇ or anti- TNFRII/CD120b protein antibodies can also be used in treatment.
  • the genes encoding the antibodies are provided, such that the antibodies bind to and modulate the TNF- ⁇ or TNFRII/CD 120b protein within the cell.
  • a therapeutically effective amount of TNF- ⁇ or TNFRII/CD 120b protein, agonist or antagonist is administered to a patient.
  • a “therapeutically effective amount”, “pharmacologically acceptable dose”, “pharmacologically acceptable amount” means that a sufficient amount of an immunosuppressive agent or combination of agents is present to achieve a desired result, e.g., preventing, delaying, inhibiting or reversing a symptom of a disease or disorder or the progression of disease or disorder when administered in an appropriate regime.
  • compositions are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions (see, e.g., Alfonso R Gennaro (ed), 2003, Remington: The Science and Practice of Pharmacy, (Formerly Remington's Pharmaceutical Sciences) 20th ed., incorporated herein by reference in its entirety).
  • the pharmaceutical compositions generally comprise TNF- ⁇ or TNFRII/CD 120b protein, agonist or antagonist in a form suitable for administration to a patient.
  • the pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
  • GMP Good Manufacturing Practice
  • Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the packaged nucleic acid suspended in diluents, such as water, saline or FECi 4UU; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions.
  • liquid solutions such as an effective amount of the packaged nucleic acid suspended in diluents, such as water, saline or FECi 4UU
  • capsules, sachets or tablets each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin
  • suspensions in an appropriate liquid such as water, saline or FECi 4UU
  • Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers.
  • Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
  • a flavor usually sucrose and acacia or tragacanth
  • pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
  • the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
  • “Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like, particularly the ammonium, potassium, sodium, calcium, and magnesium salts.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
  • nucleic acids alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • Suitable formulations for rectal administration include, for example, suppositories, which consist of the packaged nucleic acid with a suppository base.
  • Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons.
  • gelatin rectal capsules which consist of a combination of the packaged nucleic acid with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • Compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally.
  • Formulations for injection can be presented in unit dosage form, e.g., in ampules or in multidose containers, with an added preservative.
  • Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • Cells transduced by the packaged nucleic acid as described above in the context of ex vivo therapy can also be administered intravenously or parenterally as described above.
  • the dose administered to a patient should be sufficient to effect a beneficial therapeutic response in the patient over time.
  • the dose will be determined by the efficacy of the particular vector employed and the condition of the patient, as well as the body weight or surface area of the patient to be treated.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular vector, or transduced cell type in a particular patient.
  • the physician evaluates circulating plasma levels of the vector, vector toxicities, progression of the disease, and the production of anti- vector antibodies.
  • the dose equivalent of a naked nucleic acid from a vector is from about 1 ⁇ g to 100 ⁇ g for a typical 70 kilogram patient, and doses of vectors which include a retroviral particle are calculated to yield an equivalent amount of therapeutic nucleic acid.
  • inhibitors and transduced cells can be administered at a rate determined by the LD 50 of the inhibitor, vector, or transduced cell type, and the side-effects of the inhibitor, vector or cell type at various concentrations, as applied to the mass and overall tiealth of the patient. Administration can be accomplished via single or divided doses.
  • U271J Transduced cells are prepared for reinfusion according to established methods. See Abrahamsen et al, J. CHn. Apheresis 6:48-53, 1991; Carter et al, J. Clin. Arpheresis 4:113- 117, 1998; Aebersold et al, J. Immunol. Meth. 112:1-7, 1998; Muul et al, J.
  • the cells should number between 1 x 10 8 and 1 x 10 12 .
  • the growth characteristics of cells vary from patient to patient and from cell type to cell type. About 72 hours prior to reinfusion of the transduced cells, an aliquot is taken for analysis of phenotype, and percentage of cells expressing the therapeutic agent.
  • the TNP- ⁇ or TNFRII7CD 120b protein, agonist or antagonist or their homologs are useful tools for examining expression and regulation of signaling in CD4+CD25+ hl T R cells via the TNF- ⁇ / TNFRII/CD 120b signaling pathway.
  • Reagents that specifically hybridize to nucleic acids encoding TNF- ⁇ or TNFRII/CD120b proteins (including probes and primers of the proteins), and reagents that specifically bind to the proteins, e.g., antibodies, are used to examine expression and regulation.
  • Nucleic acid assays for the presence of TNF- ⁇ or TNFRII/CD 120b proteins in a sample include numerous techniques are known to those skilled in the art, such as Southern analysis, northern analysis, dot blots, RNase protection, Sl analysis, amplification techniques such as PCR and LCR, high density oligonucleotide array analysis, and in situ hybridization.
  • in situ hybridization for example, the target nucleic acid is liberated from its cellular surroundings in such as to be available for hybridization within the cell while preserving the cellular morphology for subsequent interpretation and analysis.
  • TNF- ⁇ or TNFRII/CD 120b protein can be detected with the various immunoassay techniques described above.
  • the test sample is typically compared to both a positive control (e.g., a sample expressing recombinant TNF- ⁇ or TNFRII/CD 120b protein) and a negative control.
  • kits for screening CD4+CD25+ hi T R cell activity modulators can be prepared from readily available materials and reagents are provided.
  • such kits can comprise any one or more of the following materials: the TNF- ⁇ or TNFRII/CD 120b proteins, agonists, or antagonists, reaction tubes, and instructions for testing the activities of TNF- ⁇ or TNFRII/CD120b genes.
  • a wide variety of kits and components can be prepared depending upon the intended user of the kit and the particular needs of the user.
  • the kit can be tailored for in vitro or in vivo assays for measuring the activity of TNF- ⁇ or TNFRII/CD120b proteins or CD4+CD25+ hi TR cell activity modulators.
  • Kits comprising probe arrays as described above are provided.
  • additional components of the kit include, for example, other restriction enzymes, reverse- transcriptase or polymerase, the substrate nucleoside triphosphates, means used to label (for example, an avidin-enzyme conjugate and enzyme substrate and chromogen if the label is biotin), and the appropriate buffers for reverse transcription, PCR, or hybridization reactions.
  • kits also contain instructions for carrying out the methods.
  • Treatment with prednisone ⁇ 10mg/day, hydroxychloroquine and or nonsteroidal anti-inflammatory drugs did not disqualify a patient from the inactive disease group.
  • Active disease was defined as a SLEDAI score > 6, with or without immunosuppressive treatment.
  • Patients were excluded with a history of infection within 3 weeks and co-morbidities such as diabetes mellitus. The study was approved by the Institutional Review Board of the National Institute of Arthritis and Musculoskeletal and Skin Diseases/National Institute of Diabetes and Digestive and Kidney Diseases, NIH.
  • Cytokines All cytokines used in this study were recombinant human proteins. Final concentrations were: lOOng/ml GM-CSF and IL-4 (R&D systems). For final DC maturation, 500ng/ml TNF (R&D) was employed.
  • Cytokine Assays T cells were stimulated with allogeneic DCs or platebound anti-CD3 (lO ⁇ g/ml) in X-VrVO-20 plus 1% serum. Cytokine analysis was performed at different time points by analysis of supernatants with commercially available ELISA kits for human IL- 10, EFN ⁇ , IL-4 and TGF ⁇ l (BD- Pharmingen) according to the manufacturer's instructions or by the cytometric bead array kit (CBA; BD Biosciences). A direct comparison of capture ELISA and CBA demonstrated that the two methods were highly comparable in terms of the amount of cytokine detected.
  • Dendritic cells were generated from elutriated monocytes (obtained from the Department of Transfusion Medicine, Clinical Center) from healthy donors after informed consent was given. In brief, monocytes were cultured in RPMI 1640 supplemented with 10% FBS, IL-4 and GM-CSF. At day 6 TNF was added to fully mature the cells. At day 7 non-adherent cells were harvested and constituted mature DCs that were >90% double positive for costimulatory molecules (CD80, CD86) and CD83.
  • Non-CD4+ T cells were labeled from PBMC from healthy donors (buffy coats) or SLE (Leukapheresis) with a negative CD4+ T cell isolation kit (Miltenyi biotec) according to the manufacturer's instructions and depleted using depletion sensitive mode of the AutoMACS®.
  • CD4+CD25+ hi T cells were isolated from the negatively selected CD4+ T cells by ⁇ ouble staining with CD4-Cychrome and PE-conjugated anti-CD25 (15 ⁇ g/10 8 cells) in PBS/2% t ⁇ tor 20 mm at 4"C.
  • the cells were washed and resuspended in PBS/2% FBS for cell sorting with a MoFIo high speed cell sorter (Dako Cytomation, Carpinteria, CA).
  • the purity of the final CD4+CD25+ hi and CD4+ SP preparations were typically > 98%.
  • CD4+CD25- and CD4+CD25+ (10 6 /ml) were grown in X-VIVO 20/1% NHS, 10% FBS supplemented with, lOOUml IL-2 at 37 0 C for 72 hrs. in 24 well plates precoated with ImI of a solution of 5 ⁇ g/ml of anti-CD3 (clone 64.1). At the end of 48 hrs cells were transfer from anti-CD3 stimulation conditions and maintained in medium with IL- 2 and used in proliferation assays at day 4-7.
  • CD4+CD25+ hi T cells comprise ⁇ l-2% ofCD4+ T cells in human peripheral blood. Approximately one-third to one-half of the circulating human peripheral blood lymphocytes express CD4, and of these roughly 10% co-express the IL-2 receptor ⁇ -chain, CD25.
  • CD4+CD25+ M T cells exhibit distinct phenotypical differences to CD4+CD25- T cells.
  • CD4+ T cells were magnetically isolated (negative selection) from PBMC of randomly selected healthy volunteers or from lupus or rheumatoid arthritis patients.
  • CD4+CD25+ 1 " T or CD4+CD25- cells were then sorted routinely achieving a purity of >98% for both populations ( Figure 1).
  • CD25+ brightest subset contains most of the CD4+CD25+ hi T regulatory cells (T R ), the CD25+ brightest subset were studied further.
  • GITR glucocorticoid induced tumor factor receptor
  • FIG. 1 shows that freshly sorted CD4+CD25+ hi T regulatory cells and CD4+CD25- effector cells from both Normal donor and SLE patients were incubated overnight with TNF ⁇ at 5 ngml "1 and their surface phenotype characterized.
  • the figure shows and upregulation on the surface expression of TNFRII selectively on the CD4+CD25+ hl T R subset from 10 up to 30% in normal donors and from 25 up to 50% in SLE patients while maintaining the same level of expression of other markers.
  • CD4+CD25 hl Tregs uniformly expressed high levels of FoxP3 protein, whereas CD4+CD25- cells did not express this transcription factor that governs Treg function.
  • CD4+CD25 int cells expressed minimal levels.
  • CD4+CD25 hi Tregs were not larger or more complex that CD4+CD25- effector T cells.
  • Freshly isolated human CD4+CD25+ 1 " T cells are anergic and do not proliferate after allogeneic or polyclonal activation. A low proliferative potential is highly characteristic of T R cells both in the murine and human systems. The proliferative capacity of freshly isolated T R from healthy donors and autoimmune disease patients to different stimuli was tested. Cells from normal donor proliferated poorly to immobilized anti-CD3, but this anergic state was reversed upon the addition of IL-2 to the culture. In contrast freshly isolated CD4+CD25+ 1 " T R from lupus patients showed an increased proliferative response to cross-linked anti-CD3.
  • CD4+CD25+ 1 T R from healthy volunteers suppress the proliferative responses ofCD4+CD25- effectors to alloantigens.
  • the regulatory properties of CD4+CD25+ hi T cells were investigated by testing their ability to suppress the proliferative responses of na ⁇ ve CD4+CD25- to alloantigens.
  • CD4+CD25- T cells from healthy individuals and lupus patients were stimulated with allogeneic mDC as APC or polyclonally stimulated with plate bound anti- CD3.
  • At a ratio of 1:1 CD4+CD25+ hi T cells from healthy volunteers inhibited the proliferation of CD4+CD25- T cells by an average of 80% ⁇ 5 (n 10; Figure 4).
  • CD4+CD25+ hl T R cells from SLE & subjects by culturing them for 3 days with plate bound anti-CD3 and high doses of IL-2 restored their suppressive function as they were able to suppress the proliferation of autologous CD4+CD25- by as much as 85%.
  • in vitro activated CD4+CD25+ 1 TR from both normal donors and SLE suppressed the production of IFN ⁇ and IL-2 by CD4+CD25- activated with anti-CD3mAb or allogeneic APC.
  • Human CD4+CD25+'" T regulatory cells were analyzed for their capacity to produce cytokines. After stimulation with immobilized anti-CD3 mAb, with or without soluble anti-CD28 mAb no detectable amounts of IL-2, IL-10, IL-4, TGF ⁇ , IFN ⁇ or EL-6 were detected (by BD- cytokine array) in the supernatants from healthy volunteers where as CD4+CD25+ hl T regulatory cells from clinically active SLE patients had an increased secretion only of IL-10.
  • FoxP3 is T R specific. Recently, the role of Scurf ⁇ n the protein product of FoxP3 in the development of CD4+CD25+ 1 " TR cells was demonstrated. Khattri et al., Nat Immunol 4: 337, 2003. It was further determined whether FoxP3 is also specifically expressed in human CD4+CD25+ hi T R cells. RT-PCR was first performed for FoxP3 in CD4+CD25+ hi and CD4+CD25- from healthy individual and from patients with SLE. A specific FoxP3 product was detected exclusively on CD4+CD25+ hl cells from activated healthy donors and SLE patients but not on CD4+CD25- from either donor (data not shown).
  • TNFa and signaling through TNFRII block suppressive activity of CD4+CD25+ hl TR cells.
  • Human CD4+CD25+ Thymocytes are known to express TNFRH constitutively but the expression of TNFRII on peripheral CD4+CD25+ 1 " T R cells has not been reported. Annunziato et ah, J Exp Med 196: 379, 2002. Furthermore, it is presently unknown whether the expression of TNFRII has any consequences in the biology of CD4+CD25+ hl TR cells. It was first determined whether the constitutively expression of TNFRH on CD4+CD25+ hl thymocytes was also seen in peripheral blood CD4+CD25+ 1 " T R .
  • FIG. 7 shows that TNFRII crosslinking reverses the CD4+CD25+ hi T R suppression on CD4+ effectors.
  • Previously polyclonally activated T cells were used in in vitro regulatory assays as described in Materials and Methods except that in this case to specifically address the effect of signaling trough TNFRII purified anti-TNFRII was coated in a 96 well micro titer plate at O ⁇ gwell "1 .
  • Figure 7 shows that crosslinking TNFRII specifically on CD4+CD25+ hl TR does not affect their anergic phenotype and neither has a co-stimulatory effect of the CD4+ effectors.
  • FIG. 9 shows that CD4+CD25+ hi T R and CD4+CD25- effectors from SLE patients were in vitro stimulated on plate bound CD3 and IL-2 as described in methods.
  • the figure shows acquisition of suppressive activity of CD4+CD25+ hl T R from SLE after in vitro activation and its complete reversal in the presence on TNF ⁇ during the assay.
  • Figure 16 shows A: Concentration dependent loss of suppressive function of CD4+CD25 hi Treg by TNF.
  • CD4+CD25- T cells (5xlO 4 /well) or CD4+CD25 hi Treg (5xlO 4 /well) alone or mixed together at a ratio of 1 : 1 were stimulated with platebound anti-CD3 (l ⁇ g/well).
  • Recombinant TNF was added at the beginning of the culture at increasing concentrations as indicated. After 72hrs, H thymidine incorporation was determined. Data are the mean ⁇ SEM of 3 independent experiments.
  • B: TNFRII crosslinking reverses the CD4+CD25 1 " Treg mediated suppression of the proliferation of CD4+CD25- effectors.
  • Previously activated T cells that had upregulated surface TNFRII expression were used for in vitro regulatory assays as described in Materials and Methods.
  • 96 well microtiter plates were coated with anti-TNFRTI mAb at increasing concentrations as indicated along with anti-CD3.
  • CD4+CD25- T cells (5xlO 4 /well) or CD4+CD25 hl Treg (5xlO 4 /well) alone or mixed together at a ratio of 1:1 were stimulated with platebound anti-CD3 (l ⁇ g/well), with or without anti-TNFRII. After 72hrs, 3 H thymidine incorporation was determined.
  • TNFRII Signaling through TNFRII regulates FoxPS expression.
  • T R activity correlates with FoxP3 expression (Khattri, Nat Immunol, 2003), although in this system the abundance ofFoxP3 transcript is not always directly proportional to suppressive activity as it is influenced by the nature of the stimulation present.
  • quantitative PCR was performed on freshly isolated and TNF ⁇ stimulated cell subsets. The results showed a 70% decrease in the FoxP3 expression on CD4+CD25+ hl T R both from normal donors and form in vitro activated CD4+CD25+ hi TR from SLE.
  • CD4+ T cells identified by the co-expression of CD25, (CD4+CD25+ hl T R cells) have the ability to regulate immune responses.
  • T cells known as T- regulatory cells, or TR cells have been found and characterized in humans and rodents.
  • TR cells have been found and characterized in humans and rodents.
  • studies in both human and mice have demonstrated that a defective regulatory T cell function contributes to autoimmune diseases in animal models. Viglietta et al., J. Exp. Med. 199: 971-979, 2004; Shevach, Nat Rev Immunol 2: 389, 2002.
  • Studies in humans have identified the regulatory effector function in the brightest 2% of CD25+ and this population was selected to study.
  • human CD4+CD25+ hi T R expressed considerably less GITR, only 10% of T R from healthy individuals and up to 20% in clinically active SLE patients, and cross-linking of GITR failed to reverse suppression in both healthy individual and SLE T R cells.
  • CD4+CD25+ hl T R cells were investigated by testing their ability to suppress the proliferative responses of na ⁇ ve CD4+CD25- to alloantigens.
  • CD4+CD25- effector T cells from healthy individuals and lupus patients were stimulated both with allogeneic mDC as APC or polyclonally stimulated with plate bound anti-CD3.
  • CD4+CD25+ hl T R cells suppressed the production of IFN ⁇ and IL-2 by CD4+CD25- effectors activated with anti-CD3mAb or allogeneic APC.
  • TNF impaired the ability of regulatory T cells to suppress disease in the NOD mouse model of diabetes. Wu et al, Proc. Natl. Acad. ScL USA 99: 12287-12292, 2002. It was suggested that endogenous levels of TNF might act centrally in the thymus to mediate these effects on regulatory T cells. Pertinent to this hypothesis is the recent observation that CD4 + CD25+ hl T cells derived from the thymus of healthy donors have an increased expression of TNFRII compared with CD4 + CD25 ⁇ T cells which might result in an increased susceptibility of regulatory T cells to the actions of TNF. Annunziato et ah, J Exp Med 196: 379, 2002. Also in the NOD mouse model a differential upregulation of TNFRII in T R cells in the inflammatory active site was demonstrated. Herman et ah, J Exp Med 199: 1479, 2004.
  • the present invention showed an increased constitutive expression of TNFRII in CD4+CD25+ hi T R from healthy volunteers (mean 10%) and clinically active SLE (mean 20%) compared to the CD4+CD25- effector subset from healthy donors (mean 2%) and SLE (4%). It was next determined whether signaling through TNFRII influenced the effector suppressive function of CD4+CD25+ hl T R as performed in a standard regulatory assay. Fresh or activated CD4+CD25+ hl TR from healthy and autoimune disease subject were able to suppress the proliferation of CD4+CD25- effector T cells. When TNFRII was crosslinked, they completely lost their regulatory activity (Figure 10).
  • Figure 14 shows TNFRII crosslinking reverses TGF ⁇ l induced suppressive function.
  • Figure 15 shows kinetics of TNFRII expression on CD4+CD25- T cells at the indicated time points after stimulation in vitro with anti-CD3 in the presence of T-depleted splenocytes.
  • TNF can reduce FoxP3 expression in CD4+CD25+ hl T R under conditions involving either anti-CD3 in the presence or absence of APC, suggesting the relevance of this control mechanism, since during the initiation of an adaptive immune response TNF will predominate in the site of immune activation precluding the CD4+CD25+ hl T R from suppressing the response thus allowing an effective adaptive immune response to take place.
  • the FoxP3 gene expression reduction is dependent on the levels of TNFRII cross-linking, suggesting a causal relationship.
  • CD4+CD25+ hi T R was isolated from TNF-, TNFRI- and TNFRII-deficient mice and wild type controls.
  • TNF reduces FoxP3 expression by up to a 60% reduction of FoxP 3 gene expression as measured by quantitative PCR in CD4+CD25+ 1
  • T R isolated from TNF-, TNFRI-deficient and wild type mice upon culturing them with soluble TNF but not from TNFRII confirming the specificity of the effect mediated by TNFRII.
  • the mechanisms of TNF mediated impairment of CD4+CD25+ hl TR becomes central in the pathogenesis of autoimmune diseases as TNF serum levels as well as soluble TNFRII are known to be increased in SLE and correlate with diseases activity (Smolen Lupus).
  • Treg function in these patients is compromised.
  • Ehrenstein et ah J Exp Med 200:277-285, 2004.
  • the phenotype of Tregs from patients with active RA was therefore examined.
  • CD4+CD25 1 " Tregs isolated from active RA were anergic, they did not suppress the proliferation of CD4+CD25- T cells ( Figure 18) and suppressed the production of IFN ⁇ from CD4+CD25- T cells less effectively than CD4+CD25 hi Treg from normals ( Figure 19A).
  • Figure 18 To determine whether the loss of regulatory function in active RA was explained by a decrease in the intrinsic function of CD4+CD25 1 " Treg or an increase in the resistance of CD4+CD25- effector T cells to inhibition, mixing experiments were carried out with cells from RA patients and normal controls.
  • Tregs from patients with active RA failed to suppress the proliferation and exhibited diminished regulation of IFN ⁇ production by autologous CD4+CD25- effector T cells, whereas CD4+CD25 hi Tregs from healthy controls readily suppressed the proliferative response of CD4+CD25- effectors from RA patients ( Figure 19B) as well as their IFN ⁇ secretion ( Figure 19A).
  • Figure 19B shows that the primary regulatory defect is in the function of CD4+CD25 hl Tregs isolated from the circulation of patients with active RA.
  • the levels of expression of the transcription factor FoxP3 in Tregs was modulated by TNF, the levels of FoxP3 expression on Tregs from normal patients and from patients with active RA were compared.
  • Figure 17 shows a phenotype of CD4+CD25 hi Treg from RA patients.
  • A Purified CD4+ T cells were stained with anti-CD4 Cychrome and anti-CD25 PE and the population was sorted into CD4+CD25- and CD4+CD25 hl subsets as indicated in the methods. The resultant purity of CD4+CD25 hi and CD4+CD25- T cells is shown. Purified populations were stained with anti-TNFRI-FITC, anti-TNFRII-APC, anti-GITR-FITC, anti-CD69-FITC and anti-CCR4-APC conjugated mAb as indicated. B.
  • CD4+CD25 hi T cells Freshly sorted CD4+CD25 hi T regulatory cells and CD4+CD25- effector cells were isolated from 15 RA patients treated with infliximab and their phenotype characterized. Numbers in each box indicate the percentages of positive cells and those in parentheses the mean fluorescence intensity of the stained cells. These changes in phenotype were compared to the phenotype of normal donors, shown in Figure 1.
  • FIG. 18 shows CD4+CD25 hi T cells from active RA patients fail to suppress proliferation.
  • CD4+CD25- responder (5xlO 4 /well) and CD4+CD25 hi Treg (5xlO 4 /well) were cultured with platebound anti-CD3 (l ⁇ g/well) either alone or at a 1:1 ratio. After 72hrs, 3 H thymidine incorporation was determined. Results are the mean ⁇ SEM of 15 separate experiments using patient samples compared to normal donors. Also shown is the percent inhibition of proliferation of these 15 experiments.
  • FIG. 19 shows CD4+CD25 hi Treg from active RA patients are defective suppressors.
  • A. CD4+CD25- responder (5xlO 4 /well) and CD4+CD25 hi Treg (5xlO 4 /well) were cultured with platebound anti-CD3 (l ⁇ g/well) either alone or at a 1:1 ratio. After 72hrs, 3 H thymidine incorporation was determined. CD4+CD25- effectors from active RA patients were also co-cultured with CD4+CD25 hl Treg from normal individuals. Results are the mean + SEM of 3 separate experiments.
  • Figure 20 shows CD4+CD25 1 " Treg from RA patients recover their suppressive function after Infliximab therapy.
  • CD4+CD25- and CD4+CD25 hl T cells were sorted as described from RA patients before (RA pre) and after (RA post) treatment with infliximab.
  • Real-time PCR was carried out in triplicate for FoxP3 mRNA and relative fold changes were normalized to GAPDH. Data represent the mean ⁇ SEM of 5 different experiments. The same RA patients were examined following 3 months of therapy with infliximab.
  • B. FoxP3 protein expression in CD4+CD25 hl Treg recovers after anti-TNF treatment of RA patients.
  • CD4+CD25- and CD4+CD25 hi T cells were sorted as described. Intracellular FoxP3 staining was carried out in CD4+CD25- and CD4+CD25 hi T cells sorted from an RA patient before and after infliximab therapy.
  • CD4+CD25- responder (5xlO 4 /well) and CD4+CD25 hl Treg (5x10 4 /well) were isolated from active RA patients before infliximab therapy and after 3 months of infliximab treatment.
  • Cells were cultured with platebound anti-CD3 (l ⁇ g/well) either alone or at a 1:1 ratio. After 72 hrs, 3 H thymidine incorporation was determined. Data represent the mean + SE of 15 RA patients before and after infliximab treatment.
  • TNF IS AN IMPORTANT FACTOR DOWN-MODULATING TREG FUNCTION
  • Tregs are governed by the effects of a transcriptional repressor, FoxP3.
  • m animals and humans lacking FoxP3 expression Treg function is absent, whereas overexpression of FoxP3 by in vitro transfection induces T cells to become both anergic and to exert a complete or partial suppressive activity.
  • expression of FoxP3 appears to play a necessary role in governing Treg action.
  • TNF by signaling through TNFRII downregulates FoxP3 expression suggests a mechanism by which this inflammatory cytokine can influence Treg function. It is notable that TNF altered the suppressive function of Tregs but not their proliferative unresponsiveness or inability to produce cytokines. These results suggest that different mechanisms may govern anergy and suppression in Tregs and that only the latter is importantly regulated by TNF.
  • the present invention provides that the suppressive function of Tregs can be modulated by TNF through its receptor TNFRII constitutively expressed on human Tregs.
  • TNFRII is largely confined to cells of the immune system, but this is the first report that TNFRII is constitutively expressed by Tregs and not by circulating CD4+CD25- effector cells. Carpentier et al, Curr Med Chem 11:2205-2212, 2004. TNFRI was expressed by neither Tregs nor effector cells.
  • the effect of TNF was mediated through TNFRII and the impact of TNF or the agonistic mAb to TNFRII was directed at the function of Tregs and not the effector T cells.
  • CD4+CD25 hl Treg prepared by flow cytometry were analyzed, whereas the previous report largely utilized CD4+CD25+ hl Treg isolated with magnetic beads that likely included both CD25 hl and CD25 int subpopulations.
  • CD4+CD25 1 " Treg from patients with active RA express high levels of TNFRII, markedly decreased levels of FoxP3 mRNA and protein, and a diminished capacity to suppress both proliferation and IFN ⁇ production by CD4+CD25- effector T cells.
  • TNF has previously been shown to upregulate TNFRII expression in human malignant epithelial cell lines, but no previous studies have examined this in Tregs.
  • the present invention provides evidence for a functional defect in the CD4+CD25+ hi T R cells in SLE.
  • a novel function has been demonstrated for TNF as an important factor down modulating T regulatory cell function.
  • the present invention also suggests a possible mechanism for this effect is a reduction in FoxPS expression in CD4+CD25+ 1 " T R cells.
  • the findings of the present invention provide evidence of defective T regulatory function in SLE and further provides a possible mechanism for this, that is, overproduction of TNF that causes a functional deficit in the T regulatory cell function.
  • the present invention provides a novel function of TNF as an important factor down-modulating Treg function.
  • the mechanism of this effect was uncovered.
  • a reduction in FoxP3 expression in CD4+CD25+ hl Treg that has been correlated with the suppressive function of CD4+CD25+ hl Tregs.
  • Treg function limits immune reactivity so as to avoid triggering immune responses to autologous tissue antigens revealed during inflammation, hi this way, TNF may play an important instructive role in controlling adaptive immunity.
  • Strategies designed to manipulate TNF signaling in Treg may result in novel therapeutic approaches to augment the limited and/or inadequate function of these regulatory T cells in inflammatory or autoimmune diseases.

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Abstract

La présente invention se rapporte à des compositions et à des méthodes destinées à traiter une maladie immunologique chez un mammifère. Un procédé selon l'invention permet de déterminer la présence d'une maladie immunologique chez un mammifère ou une prédisposition à une telle maladie de ce dernier. Une composition selon l'invention est destinée à traiter une maladie immunologique chez un mammifère.
PCT/US2006/000465 2005-01-07 2006-01-06 Compositions et methodes destinees a traiter une maladie immunologique Ceased WO2006074370A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008066784A3 (fr) * 2006-11-27 2008-10-16 Ludwig Inst Cancer Res Expression de foxp3 par des cellules cancéreuses
US20120094934A1 (en) * 2009-06-24 2012-04-19 Opko Curna, Llc Treatment of tumor necrosis factor receptor 2 (tnfr2) related diseases by inhibition of natural antisense transcript to tnfr2
WO2017070076A1 (fr) * 2015-10-20 2017-04-27 Albert Einstein College Of Medicine, Inc. Cibles de tnfr-1b thérapeutiques et leurs utilisations
US10988543B2 (en) 2015-11-11 2021-04-27 Opi Vi—Ip Holdco Llc Humanized anti-tumor necrosis factor alpha receptor 2 (anti-TNFR2) antibodies and methods of use thereof to elicit an immune response against a tumor
WO2023064315A1 (fr) * 2021-10-12 2023-04-20 Ampel Biosolutions, Llc Systèmes et procédés d'analyse de données de résultats rapportés par un patient

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US5606023A (en) * 1994-05-24 1997-02-25 Thomas Jefferson University Mutant tumor necrosis factor proteins
US6599710B1 (en) * 1999-03-10 2003-07-29 The General Hospital Corporation Treatment of autoimmune disease
AU7645700A (en) * 1999-10-07 2001-05-10 Maxygen Aps Single-chain antagonist polypeptides
GB0108689D0 (en) * 2001-04-05 2001-05-30 Medical Res Council Neuropathologies associated with expression of TNF-a
JP4660067B2 (ja) * 2001-04-24 2011-03-30 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツング 抗血管新生剤とＴＮＦαとを用いる組合せ療法

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008066784A3 (fr) * 2006-11-27 2008-10-16 Ludwig Inst Cancer Res Expression de foxp3 par des cellules cancéreuses
US8420791B2 (en) 2006-11-27 2013-04-16 Ludwig Institute For Cancer Research Ltd. Expression of FoxP3 by cancer cells
US20120094934A1 (en) * 2009-06-24 2012-04-19 Opko Curna, Llc Treatment of tumor necrosis factor receptor 2 (tnfr2) related diseases by inhibition of natural antisense transcript to tnfr2
EP2446036A4 (fr) * 2009-06-24 2013-09-18 Curna Inc Traitement de maladies associées au récepteur de facteur nécrosant des tumeurs 2 (tnfr2) par inhibition de la transcription antisens naturelle de tnfr2
US8859515B2 (en) * 2009-06-24 2014-10-14 Curna, Inc. Treatment of tumor necrosis factor receptor 2 (TNFR2) related diseases by inhibition of natural antisense transcript to TNFR2
WO2017070076A1 (fr) * 2015-10-20 2017-04-27 Albert Einstein College Of Medicine, Inc. Cibles de tnfr-1b thérapeutiques et leurs utilisations
US10717776B2 (en) 2015-10-20 2020-07-21 Albert Einstein College Of Medicine Tumor necrosis factor 1B (TNF-1B) mutants and methods of use thereof to screen for candidate therapeutic compounds
US10988543B2 (en) 2015-11-11 2021-04-27 Opi Vi—Ip Holdco Llc Humanized anti-tumor necrosis factor alpha receptor 2 (anti-TNFR2) antibodies and methods of use thereof to elicit an immune response against a tumor
WO2023064315A1 (fr) * 2021-10-12 2023-04-20 Ampel Biosolutions, Llc Systèmes et procédés d'analyse de données de résultats rapportés par un patient

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