WO2004097009A2 - Procédé d'identification d'agents d'activation ou d'inhibition de ikki - Google Patents

Procédé d'identification d'agents d'activation ou d'inhibition de ikki Download PDF

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WO2004097009A2
WO2004097009A2 PCT/EP2004/004604 EP2004004604W WO2004097009A2 WO 2004097009 A2 WO2004097009 A2 WO 2004097009A2 EP 2004004604 W EP2004004604 W EP 2004004604W WO 2004097009 A2 WO2004097009 A2 WO 2004097009A2
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Prior art keywords
lkki
cell
ikki
expression
activity
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PCT/EP2004/004604
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WO2004097009A3 (fr
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Vladimir Kravchenko
Frank Mercurio
Richard J. Ulevitch
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Novartis Ag
Novartis Pharma Gmbh
The Scripps Research Institute
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Publication of WO2004097009A2 publication Critical patent/WO2004097009A2/fr
Publication of WO2004097009A3 publication Critical patent/WO2004097009A3/fr

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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/80—Immunoglobulins specific features remaining in the (producing) cell, i.e. intracellular antibodies or intrabodies
    • 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
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • the invention relates to the field of gene regulation. More specifically, the invention relates to the modulation of immunological and inflammatory responses.
  • NF-kB Nuclear factor kappa beta
  • IkB inhibitory molecules
  • NF-kB is activated by a variety of signals that include cytokines, such as tumor necrosis factor (TNF) and IL-1, bacterial products such as lipopolysaccharide (LPS), oxidative stress, viruses, and DNA-damaging agents (Baldwin, Annu. Rev, hrmunol., 14:649 (1996)).
  • TNF tumor necrosis factor
  • LPS lipopolysaccharide
  • oxidative stress viruses
  • viruses DNA-damaging agents
  • IKK IB kinase
  • IKK ⁇ and IKK ⁇ are catalytic subunits, whereas NEMO serves a non-enzymatic, regulatory function. Both kinases directly phosphorylate IkB.
  • the activity of IKK ⁇ and IKK ⁇ is stimulated by TNF and IL-1 treatment.
  • IKKi is a recently identified kinase that is related to IKK ⁇ and IKK ⁇ (Shimada et al., Int. Immunol. 11:1357-1362 (1999)). Although IKKi has homology with IKK ⁇ and IKK ⁇ , the amino acid identity between IKKi and IKK ⁇ is only 24% in the kinase domain. Over- expression of IKKi activates NF-kB. IKKi is expressed preferentially in immune cells, and is induced in response to LPS or inflammatory cytokines. The kinase activity can be regulated by IKKi expression levels (Shimada et al., Int. Immunol.. 11: 1357-1362 (1999)). LKKi phosphorylates the IkB proteins of the complex that inhibits NF-kB activity. Phosphorylation of these IkB proteins causes them to be degraded, which allows NF-kB to become active.
  • C/EBPs The CCAAT/enhancer-binding proteins (C/EBPs) encompass a family of transcription factors with structural as well as functional homologies. Six C/EBPs have been identified that interact with each other and transcription factors in other protein families to regulate mRNA transcription. These proteins vary in tissue specificity and transactivating ability. The pleiotropic effects of C EBPs are observed in part because of tissue-specific and stage-specific expression, leaky ribosomal reading, post-transcriptional modifications, and variable DNA binding specificities. These mechanisms result in variable amounts of the C/EBP isoforms available to cognate sites in different tissues. These factors influence normal tissue development and cellular function, cellular proliferation, and functional differentiation.
  • C/EBP and NF-kB function to regulate overlapping physiological processes, such as inflammatory responses and liver regeneration, in part, by the coordinated transcriptional regulation of a common subset of genes
  • Methods to control or modulate gene expression that is affected by the NF-kB and C/EBP proteins offer great promise for treating many diseases, such as diseases that are related to cellular proliferation, inflammation, autoimmune diseases and immune responses. Accordingly, methods to identify and use agents that for modulating these responses are needed.
  • IKKi function plays an essential role in numerous cellular functions, for example, in hematopoietic cells and non-hematopoietic cells, and in embryonic cells as well as differentiated cells. Modulation of IKKi function may have implications for the treatment of inflammation, tissue regeneration, tissue rejection, cancer, apoptosis, neurodegeneration, osteoporosis and cachexia.
  • the invention provides a method to reduce inappropriate immune responses, a method to identify agents that modulate IKKi, a method to promote apoptosis of a cell, a method to inhibit apoptosis of a cell, a method to reduce lipopolysaccharide induced septic shock, a method to promote tissue proliferation, a method to inhibit proliferation of a cell, a method to treat neurodegeneration, a nucleic acid segment that encodes an IKKi polypeptide that lacks kinase activity, an expression cassette that includes a nucleic acid segment that encodes an IKKi polypeptide that lacks kinase activity, a cell that includes a nucleic acid segment that encodes an IKKi polypeptide that lacks kinase activity, a cell that includes an expression cassette that includes a nucleic acid segment that encodes an LKKi polypeptide that lacks kinase activity, a non-human embryo and animal that includes a nucleic acid segment that encodes
  • the invention provides a method to modulate an immune response in a mammal that involves administering to the mammal with an agent that can modulate LKKi activity or expression.
  • the agent is an IKKi inhibitor, for example, a small interfering RNA (siRNA), ribozyme, antisense nucleic acid, kinase inhibitor, anti-TKKi antibody, small molecule, peptide inhibitor, mutant IKKi polypeptide and the like.
  • Agents that increase IKKi expression or activity include interferons (e.g. interferon-gamma, IFN ⁇ ), tumor necrosis factor (TNF), liposaccharides (e.g.
  • bacterial lipopolysaccharides e.g., dexamethasone, methylisobutylxanthine and insulin
  • agents that promote differentiation e.g., dexamethasone, methylisobutylxanthine and insulin
  • IKKi polypeptides IKKi nucleic acids
  • anti-IKKi antibodies small molecules
  • small molecules peptides, and the like.
  • the invention provides a method to promote apoptosis of a cell that involves contacting the cell with an agent that is an IKKi inhibitor.
  • the method includes contacting the cell with an inhibitor of NF-kB.
  • the cell can be a eukaryotic cell.
  • the eukaryotic cell is a human cell. More preferably, the eukaryotic cell is a human cancer cell.
  • the invention provides a method to inhibit apoptosis of a cell that involves contacting the cell with an agent that is an IKKi activator.
  • the cell can be a eukaryotic cell.
  • the eukaryotic cell is a human cell.
  • the invention provides a method to inhibit inflammation in a mammal that involves administering an effective amount of an agent that is an IKKi inhibitor to the mammal.
  • the mammal is a human.
  • the IKKi inhibitor is administered to the mammal as a pharmaceutical composition.
  • the invention provides a method to reduce lipopolysaccharide induced septic shock in a mammal that involves administering an effective amount of an agent that is an IKKi inhibitor to the mammal.
  • an agent that is an IKKi inhibitor to the mammal.
  • gabexate mesilate is administered to the mammal in addition to the IKKi inhibitor.
  • the mammal is a human.
  • the IKKi inhibitor is administered to the mammal as a pharmaceutical composition.
  • the invention provides a method for promoting regeneration of a mammalian tissue by administering an effective amount of an agent that promotes LKKi expression or IKKi activity to the tissue.
  • the invention provides a method to stimulate IKKi expression in a cell that involves contacting the cell with an agent that is a CCAAT enhancer binding protein (C/EBP) activator.
  • C/EBP CCAAT enhancer binding protein
  • the agent activates C/EBP beta or C/EBP delta. More preferably, the agent activates C/EBP beta and C EBP delta.
  • the cell can be a eukaryotic cell.
  • the eukaryotic cell is a mammalian cell. More preferably the eukaryotic cell is a human cell.
  • the eukaryotic cell can, for example, be a human embryonic cell or a human embryonic kidney cell.
  • the invention provides a nucleic acid segment that encodes a kinase inactive IKKi polypeptide
  • the nucleic acid segment can encode a kinase inactive polypeptide having an amino acid sequence corresponding to SEQ ID NO: 3.
  • the nucleic acid segment can also encode a kinase active polypeptide having an amino acid sequence corresponding to SEQ ID NO:2.
  • the nucleic acid segment can be codon optimized for prokaryotic cells. However, preferably, the nucleic acid segment is codon optimized for eukaryotic cells. Moor preferably, the nucleic acid segment is codon optimized for human cells.
  • the nucleic acid segment can be included within an expression cassette.
  • the expression cassette can be contained within an expression vector.
  • the expression cassette or expression vector can be contained within a cell.
  • the invention provides a polypeptide that is a kinase inactive LKKi polypeptide.
  • the kinase inactive polypeptide can have an amino acid sequence corresponding to SEQ ID NO: 3.
  • Agents that modulate IKKi can also be identified according to the method of the invention.
  • the method involves determining if a candidate agent increases or decreases IKKi expression or enzymatic activity.
  • the method can involve contacting a test cell with a candidate agent and determining if the candidate agent increases or decreases expression of an IKKi regulated gene in the test cell when compared to expression of an IKKi regulated gene in a control cell that was not contacted with the candidate agent.
  • the test cell can optionally be contacted with an IKKi inducer.
  • the control cell can optionally be contacted with an IKKi inducer.
  • the test cell and the control cell are contacted with an IKKi inducer.
  • the IKKi inducer is interleukin-1, interleukin-6, or interferon-gamma. More preferably, the LKKi inducer is phorbol myristate acetate. Even more preferably, the LKKi inducer is lipopolysaccharide. Most preferably, the IKKi inducer is tumor necrosis factor.
  • the test cell can optionally be contacted with epidermal growth factor.
  • the control cell can optionally be contacted with epidermal growth factor.
  • the test cell and the control cell are contacted with epidermal growth factor.
  • the IKKi regulated gene can be interleukin-1, interleukin-6, or interleukin-8.
  • the LKKi regulated gene is IP- 10 or COX-2. More preferably, the IKKi regulated gene is RANTES. Most preferably, the IKKi regulated gene is A20.
  • the test cell and the control cell can be a eukaryotic cell.
  • the eukaryotic cell is a mammalian cell. More preferably the eukaryotic cell is a human cell. Even more preferably, the eukaryotic cell is a human embryonic cell.
  • the eukaryotic cell is a human embryonic kidney cell.
  • the test cell can include an expression cassette that encodes IKKi.
  • the control cell can include an expression cassette that encodes IKKi.
  • the test cell and the control cell include an expression cassette that encodes IKKi.
  • the test cell can include an expression cassette that encodes a kinase inactive IKKi.
  • the control cell can include an expression cassette that encodes a kinase inactive IKKi.
  • the test cell and the control cell can include an expression cassette that encodes a kinase inactive IKKi.
  • the kinase inactive JKKi is IKKi (KM) (K38M).
  • the immune response is a complement response. More preferably, the immune response is a C3 response. Even more preferably, the immune response is a lipopolysaccharide response. Most preferably, the immune response is an inflammatory response. An agent that decreases IKKi expression or enzymatic activity can hinder a proliferative response. Preferably the proliferative response is cancer. An agent that increases IKKi expression or enzymatic activity can activate an immune response.
  • the immune response is a complement response. More preferably, the immune response is a C3 response.
  • An agent that increases IKKi expression or enzymatic activity can activate a proliferative response.
  • the proliferative response is tissue regeneration. More preferably, the proliferative response is liver regeneration.
  • the method also provides an agent identified according to the method.
  • a method to identify an agent that is an inhibitor of IKKi can involve contacting a test cell with tumor necrosis factor, epidermal growth factor, and a candidate agent; and determining if the candidate agent decreases survival of the test cell when compared to survival of a control cell that was contacted with tumor necrosis factor and epidermal growth factor.
  • the test cell and the control cell can be a eukaryotic cell.
  • the eukaryotic cell is a mammalian cell. More preferably the eukaryotic cell is a human cell. Even more preferably, the eukaryotic cell is a human embryonic cell. Most preferably, the eukaryotic cell is a human embryonic kidney cell.
  • the test cell can include an expression cassette that encodes IKKi.
  • the control cell can include an expression cassette that encodes LKKi.
  • the test cell and the control cell include an expression cassette that encodes IKKi.
  • a method to identify an agent that modulates TKKi kinase activity can involve incubating a test reaction mixture that includes IKKi kinase, a nucleotide having a gamma-label, a kinase substrate, and a candidate agent under conditions where LKKi can transfer the gamma-label into the kinase substrate to form a labeled product; and determining if the presence of the candidate agent increases or decreases an amount of the labeled product formed when compared to the amount of labeled product formed in a control reaction mixture lacking the candidate agent.
  • the kinase substrate is a fusion protein that includes a portion of IkB-alpha. More preferably, the kinase substrate is IkB- alpha.
  • the gamma label is a radioactive label. More preferably the gamma label is
  • FIG. 1 shows the development and characterization of cell lines that regulate the expression of an IKKi transgene that is either a wild type (IKKi) or kinase inactive version
  • IKKiKM in a manner that mimics that of the endogenous IKKi gene.
  • FIG. 1A shows the endogenous kinase activity of the IKK signalsome and IKKi which were evaluated from extracts of Jurkat or HEK 293 cells treated with TNF for 15 min or with PM A for 30 min.
  • the endogenous IKK signalsome and IKKi protein was immunoprecipitated with anti-NEMO or anti-IKKi antibodies, respectively, and subsequently subjected to a kinase assay (KA) using GST-IkB ⁇ 1-54 as a substrate (P-IkB).
  • KA kinase assay
  • the samples were also analyzed by Western blot (WB) to determine the level of U K2 and
  • FIG. IB shows a northern blot analysis to establish the level of LKKi mRNA expressed in Jurkat and HEK 293 cells. Hybridizatiori of the northern blot for GAPDH mRNA was used as a loading control.
  • FIG. 1C shows western blot analysis of Flag-IKKiKM and Flag-IKKi proteins isolated from extracts of TNF- or PMA-treated HEK (negative control), HEK-IKKiKM and HEK-TKKi cells. Flag-IKKi or Flag-TKKiKM protein was first immunoprecipitated from the lysate with anti-Flag monoclonal antibodies and the immune complex was then subjected to western blot analysis using the anti-IKKi antibodies.
  • FIG. ID shows an electrophoretic mobility shift assay (EMS A) of nuclear extracts from HEK, HEK-IKKiKM and HEK-IKKi cells treated with TNF or PMA for indicated times in minutes.
  • the DNA binding activity of the NF- ⁇ B transcription factor is shown (NF- kB).
  • the amount of non-specific DNA binding activity provides an internal loading control.
  • FIG. 2 shows that TKKi functions to protect cells from TNF-induced cytotoxicity.
  • FIG. 2A shows agarose gel analysis of DNA fragmentation in samples from HEK, HEK-LKKiKM and HEK-IKKi cells treated with TNF in the presence or absence of 10% FBS.
  • the RNase untreated samples were subjected to Northern blot (NB) analysis for GAPDH mRNA.
  • FIG. 2B shows agarose gel analysis of DNA fragmentation in samples from HEK and HEK-IKKiKM cells treated with TNF, EGF or TNF+EGF in the absence of 10% FBS.
  • the RNase untreated samples were subjected to Northern blot (NB) analysis for GAPDH mRNA.
  • FIG. 2C shows northern blot (NB) analysis of Egr- 1 , c-jun, IkB ⁇ and GAPDH mRNA expression, and Western blot (WB) analysis of phospho-Akt/PKB, phospho-GSK-3 ⁇ and actin proteins, in samples from EGF-treated HEK (P) and HEK-TKKiKM (M) cells.
  • FIG. 2D shows Northern blot analysis of A20, Egr-1 and GAPDH mRNA expression in samples from TNF+EGF, TNF or EGF treated HEK (P), HEK-IKKiKM (M) and HEK- IKKi (W) cells.
  • FIG. 2E shows LKKi or IKK2 kinase assays (KA) from extracts of TNF, TNF+EGF or EGF treated HEK (P), HEK-IKKiKM (M) and HEK-TKKi (W) cells.
  • Total cell extracts were immunoprecipitated (IP) with anti-IKKi or anti-NEMO antibodies, and kinase activity was assessed using a GST-IkB (amino acids 1-54) substrate.
  • the immunoprecipitated samples were also subjected to Western blot (WB) analysis for the presence of TKKi or IKK2. Data on all panels represent one of six experiments (three independent clones were used) with similar results.
  • FIG. 3 shows that TKKi promotes expression of pro-inflammatory cytokines in response to TNF and the rumor-promoter PMA.
  • FIG. 3A illustrates the results of RNase protection analysis for Rantes, IP-10, MCP-1 and L32 mRNA expression in samples from HEK (P), HEK-IKKiKM and HEK-LKKi cells stimulated with TNF for the indicated times.
  • FIG. 3B shows Northern blot analysis of I ⁇ B ⁇ , IL-8, Egr-1 and GAPDH mRNA expression in samples from HEK (P), HEK-IKKiKM (M) and HEK-TKKi (W) cells treated with PMA for indicated times.
  • FIG. 3C shows Northern blot analysis of I ⁇ B ⁇ , IL-8 and GAPDH mRNA expression in samples from HEK (P), HEK-IKKiKM (M) and HEK-IKKi (W) cells treated with TNF for indicated times. Data on all panels represent one of three independent experiments with similar results.
  • FIG. 4 shows that IKKi is required for activation of transcription factor C/EBP in response to treatment of cells with PMA or TNF+EGF.
  • FIG.4A shows electrophoretic mobility gel shift assays (EMS A) for C EBP DNA binding activity (EMSA for C/EBP) or Oct-1 DNA binding activity (EMSA for Oct-1) in nuclear extracts from HEK-IKKiKM (KM) and HEK-TKKi (WT) cells treated with TNF+PMA, TNF or PMA for 4 hours. The nuclear extracts were also incubated in the presence of antibodies specific to C/EBP ⁇ ( ⁇ ) or C/EBP ⁇ ( ⁇ ) as indicated on top of each lane. Arrows indicate the position of the DNA:protein complexes that appeared in the presence of antibodies against C/EBP ⁇ and C/EBP ⁇ .
  • FIG. 4B shows EMSA of a polypeptide composition of the C/EBP-specific complexes in HEK-IKKiKM (KM) and HEK-IKKi (WT) cells exposed to TNF+EGF or PMA (as a positive control) for 4 hours.
  • the nuclear extracts were incubated in the presence of specific antibodies to C/EBP ⁇ or C/EBP ⁇ as indicated on the top of each lane.
  • FIG. 4C1 shows an analysis of P incorporation into proteins prepared from HEK- TKKi (KM) and HEK-IKKi (WT) cells before and after treatment with PMA for 4 hours. After pre-incubation with 32 P-orthophosphate for 1 hour and PMA treatment, the nuclear extracts were prepared, immunoprecipitated (iP) by anti-C/EBP ⁇ or anti-p65 antibodies and subsequently subjected to SDS-PAGE and autoradiography. The phosphorylated products ( 32 P-C/EBP ⁇ and 32 P-p65) are shown on the right.
  • FIG. 1 shows an analysis of P incorporation into proteins prepared from HEK- TKKi (KM) and HEK-IKKi (WT) cells before and after treatment with PMA for 4 hours.
  • the nuclear extracts were prepared, immunoprecipitated (iP) by anti-C/EBP ⁇ or anti-p65 antibodies and subsequently subjected to SDS-PAGE and autoradiography.
  • 4C2 shows a western blot analysis of C/EBP ⁇ and actin (as a loading control) protein expression in total extracts from HEK-IKKiKM (KM) and HEK-TKKi (WT) cells before and after a 4 hour treatment with PMA (50 ng/ml).
  • FIG. 5 shows that JKK " ⁇ MEFs confirm a key role for IKKi in the expression of pro- inflammatory cytokines and the regulation of the transcription factors C EBP ⁇ and C/EBP ⁇ .
  • FIG. 5A shows northern blot analysis of IKKi, I ⁇ B ⁇ , JE/MCP-1, TP-10 and Egr-1 mRNA expression in samples from TKKi "7" and IKKi + + mouse embryonic fibroblasts (MEFs) treated with TNF or PMA for the indicated times (hours).
  • FIG. 5B shows northern blot analysis of IkB ⁇ , IL-6, IP-10 and Rantes mRNA expression in samples from JKKi "/” and IKKi +/+ MEFs treated with TNF or LL- 1 for the indicated times (hours) .
  • FIG. 5C shows an electrophoretic mobility gel shift assay (EMSA) for C/EBP (on the left) and NF-kB (on the right) DNA binding activity in nuclear extracts from TKKi " " and LKKi +/+ MEFs treated with TNF for the indicated time (hours).
  • ESA electrophoretic mobility gel shift assay
  • FIG. 5D shows EMSA of a polypeptide composition of the C/EBP-specific complexes in KKi "7" and IKKi + + MEFs exposed to TNF for 4 hours.
  • the nuclear extracts were incubated in the presence of normal rabbit IgG (NR) or specific antibodies to C/EBP ⁇ , C/EBP ⁇ or C/EBP ⁇ as indicated on the top of each lane.
  • NR normal rabbit IgG
  • FIG. 6 is a schematic representation depicting a role for the transcriptional up regulation of LKKi in promoting integration of the NF-kB and C/EBP pathways in response to a variety of cellular insults .
  • FIG. 7 shows that TNF-mediated induction of IKKi mRNA is TKK2-dependent.
  • One set of total RNAs were prepared from human umbilical endothelial cells (HUVEC) infected with adenovirus expressing vector (Ad) for wild type LKK2 (TKK2WT) or a Green Fluorescent Protein (GFP) as a control.
  • a second set of total RNAs were prepared from IJXrV ⁇ C infected with Ad for a kinase inactive mutant of TKK2 (IKK2KM) or GFP (control) and then treated with TNF.
  • the results of cDNA micro array analysis of IKKi mRNA are shown. Light-gray indicates increased expression of IKKi mRNA, whereas dark- grey indicates reduced expression of IKKi mRNA under these conditions.
  • FIG. 8 shows the characterization of basal levels of IKKiKM and IKKi mRNA expressed in randomly selected populations of HEK-IKKiKM (FKM7 and FKM13) and HEK-LKKi (FW1 and FW6) cell lines.
  • Northern blot analysis was used for genotyping and detection of IKKiKM and TKKi transcripts.
  • a sample of total RNAs from Jurkat cells was used as positive and negative controls.
  • Hybridization of the northern blot for GAPDH mRNA was used as a loading control.
  • FIG. 9 illustrates LPS responsiveness of cells derived from LKK2 '7" and IKK2 + + mouse embryos.
  • FIG. 9A shows northern blot analysis of steady-state mRNA levels in LKK2 "7" and
  • IKK2 + + MEFs treated with LPS 100 ng/ml for the indicated times (hours).
  • FIG. 9B shows electrophoretic mobility shift assay (EMSA) of nuclear extracts from LKK2 "7" and IKK2 + + MEFs before and after a 1 hour treatment with LPS.
  • ESA electrophoretic mobility shift assay
  • FIG. 9C shows northern blot analysis of steady-state mRNA levels in
  • FIG. 9D shows northern blot of steady-state mRNA levels in p65 " " and p65 +7+ imMEFs treated with LPS (100 ng/ml) for the indicated times (hours).
  • FIG. 10 shows that LKKi is required for C3 gene induction by LPS in MEFs and 3T3 cells.
  • FIG. 10A shows Northern blot analysis of steady-state mRNA levels in IKK2 "7” , IKK2 +7+ , LKKi “7” and LKKi +7+ MEFs treated with LPS (100 ng/ml) for the indicated times (hours).
  • FIG. 10B shows northern blot analysis of steady-state C3, I ⁇ B ⁇ or GAPDH mRNA levels in LKKi "7" and IKKi +7+ imMEFs treated with LPS (100 ng/ml) for the indicated times (hours).
  • FIG. 10C shows Northern blot analysis of steady-state Egr-1, C3, LKKi or GAPDH mRNA levels in Egr-1 "7" and Egr-l +7+ MEFs treated with LPS (100 ng/ml).
  • FIG. 11 shows that LPS induces expression of LKKi protein in MEFs.
  • FLG. 11A shows western blot analysis for IKKi protein expression in LKK2 "7" , LKK2 + + , LKKi "7” and LKKi +7+ MEFs before and after a 5 hour treatment with LPS (100 ng/ml).
  • An anti-LKKi immunoprecipitation was performed followed by Western blot analysis with an anti-LKKi.
  • the arrows indicate the position of LKKi and IgG (heavy chain).
  • FLG. 1 IB shows western blot analysis for LKK2, LKKI or NEMO protein expression in LKK2 "7" , IKK2 +7+ , LKKi "7 - and LKKi +/+ MEFs before and after a 5 hour treatment with LPS (100 ng/ml).
  • FIG. 11C shows LKK or IKKi kinase assay (KA) from extracts of LKKi "7" or LKKi + + MEFs treated with LPS for the indicated times (minutes).
  • An anti-NEMO or anti-LKKi immunoprecipitation (LP) was performed followed by kinase assay (KA) with GST-LkB ⁇ (l- 44) as a substrate.
  • the samples were also subjected to Western blot (WB) analysis for the relevant proteins as indicated.
  • FIG. 12 shows that the absence of LKKi affects LPS-mediated induction of genes for immune and inflammatory modulators in MEFs.
  • FIG. 12A shows Northern blot analysis of steady-state mRNA levels in LKKi " " and LKKi +7+ MEFs treated with LPS (100 ng/ml) for the indicated times (hours).
  • FIG. 12B shows western blot analysis of TNF in culture medium from LPS-treated LKKi * " and LKK + + MEFs for the indicated time (hours).
  • FIG. 12C1 shows IL-6 cytokine production by LKK "7" and LKK + + MEFs in response to
  • FIG. 12C1 shows IL-1 cytokine production by LKK "7" and LKK +7+ MEFs in response to LPS. Cytokine production was measured by ELISA after a 6 hour treatment with LPS (l ⁇ g/ml).
  • FIG. 12D shows electrophoretic mobility shift assay (EMSA) of nuclear extracts from LKKi + + and LKKi "7" MEFs treated with LPS (100 ng/ml) for the indicated times (hours).
  • ESA electrophoretic mobility shift assay
  • FIG. 12E shows EMSA of a polypeptide composition of the B-specif ⁇ c complexes in LKKi "7" and IKK2 +7+ MEFs exposed to 100 ng/ml LPS for lhour.
  • the nuclear extracts were incubated in the presence of specific antibody (Ab) to members of the NF- B/Rel family (c- Rel, p50 and p65) of proteins indicated on top of each lane or with normal rabbit IgG (NR).
  • FIG. 12F shows EMSA of nuclear extract from LKKi "7" and LKKi + + MEFs untreated or treated with LPS for 2 hours (the nuclear extracts were the same as in FIG. 12D). Analysis of the ISRE binding activity is shown.
  • FIG. 13 shows that LKKi is required for post-transcriptional regulation of C/EBP ⁇ .
  • FIG. 13 A shows electrophoretic mobility shift assay (EMSA) of nuclear extracts from
  • FIG. 13B shows western blot analysis for C/EBP ⁇ and actin (as a loading control) protein expression in LKKi + + and LKKi "7" MEFs before and after a 4 hour treatment with LPS (100 ng/ml).
  • 13C shows analysis of 32 P-labeled C/EBP ⁇ and p65 (as a control) proteins in nuclear extracts from LKKi +/+ and LKKi "7" MEFs before and after a 3 hour treatment with LPS.
  • the nuclear extracts were prepared, immunoprecipitated (LP) by anti-C/EBP ⁇ or anti-p65 antibodies and consequently subjected to SDS-PAGE and autoradiography.
  • the phosphorylated products (P-C/EBP ⁇ and P-p65) are shown on the left.
  • the positions of size markers are shown on the right.
  • FIG. 13D shows northern blot analysis of steady-state IKKi, IL-6, I ⁇ B ⁇ or GAPDH mRNA levels in untreated or treated with LPS HUVEC cells. The cells were pre-transfected with siRNA as indicated.
  • FIG. 13E illustrates IL-8, Egr-1 and GAPDH mRNA levels in IKKi "7" cells, LKKi(KM), and IKKi wild-type cells that were not treated with TNF + PMA, or treated with TNF + PMA.
  • FIG. 13F illustrates the effects of small interfering RNAs (siRNAs) on LKKi, LL-6, and LicB ⁇ induction by lipopolysaccharide.
  • the cells were treated with LPS as indicated.
  • the cells were transfected with a non-specific (ns) siRNA, or a siRNA specific for LKKi.
  • FIG. 14 shows that LKKi is a key molecule coupling NF-kB and C/EBP pathways.
  • FIG. 14A shows northern blot analysis of IKKi, C/EBP ⁇ , C/EBP ⁇ and GAPDH steady-state mRNA levels in p65 "7" and p65 + + imMEFs treated with LPS (100 ng/ml) for the indicated times (hours).
  • FIG. 14B shows northern blot analysis of C EBP ⁇ , C/EBP ⁇ and GAPDH steady-state mRNA levels in LKK2 "7" and LKK2 +/+ imMEFs treated with LPS (100 ng/ml) for the indicated times (hours).
  • FIG. 14C illustrates the 36-nucleotide sequence (SEQ ID NO:25) of mouse or human (shown in bold) chromosome 1 identified 480 base pairs (bp) upstream of the translation initiation site of the LKKi gene. The sequence of a C/EBP-like binding site is underlined.
  • FIG. 14D shows EMSA of nuclear extract from normal imMEFs treated with LPS for 2 hours.
  • the nuclear extracts were incubated with a 32 P-labeled 36 bp DNA fragment (see the sequence of the upper strand in FLG. 14C) in the absence or in the presence of competitive (Comp) unlabeled oligonucleotide (2 p ol) containing the wild type 36 bp fragment (W), the C/EBP consensus sequence (C), or the wild type 36 bp fragment containing the mutation of a C EBP binding motif (M).
  • W wild type 36 bp fragment
  • C C/EBP consensus sequence
  • M wild type 36 bp fragment containing the mutation of a C EBP binding motif
  • some samples were incubated in the presence of normal rabbit IgG (N) or in the presence of specific antibodies (2 ⁇ g per reaction) against C/EBP ⁇ , C/EBP ⁇ , or C/EBP ⁇ as indicated.
  • FIG. 14E shows chromatin immunoprecipitation (ChLP) assays that were carried out on chromatin samples from LKK2 " " (negative control), LKKi +7+ and LKKi “7” (positive control) imMEFs untreated or treated with LPS or TNF for 2 hours.
  • the chromatin was immunoprecipitated with antibodies to C/EBP ( ⁇ or ⁇ ) or p65 as an additional control. Shown is an IKKi or an I ⁇ B ⁇ (positive control) promoter fragment amplified by PCR from the ChLP samples.
  • FIGs. 15A-D illustrate that LKKi is required for cytokine gene induction by LFN ⁇ and TNF in MEF.
  • FIG. 15A provides a Northern blot analysis of steady-state mRNA levels in LKKi "7" and LKKi + + MEF treated with IFN ⁇ (10 ng/ml) for the indicated times.
  • FIG. 15B provides a Northern blot analysis of steady-state mRNA levels in IKKi “7” and IKKi +7+ MEF treated with TNF (40 ng/ml) for the indicated times.
  • FIG. 15C provides a Northern blot analysis of steady-state LKKi and GAPDH mRNA levels in LKKi "7” and LKKi + + MEF. LKKi "7" MEF and LKKi +7+ MEF were transfected with LKKi expression vector pkBLKKi using the amounts indicated.
  • FLG. 15D provides a Northern blot analysis of steady-state mRNA levels in untreated or treated with TNF (40 ng/ml, 2 h) or LFN ⁇ (lOng/ml, 3 h) LKKi "7" MEF.
  • MEF were pre- transfected with IKKi expression vectors pkBLKKi as indicated and LacZ expression vector to mark transfected cells.
  • FIGs. 16A-D illustrate that absence of LKKi affects cytokine responsiveness of MEF on the level of induction of C/EBP pathway.
  • FLG. 16A provides a Western blot for l ⁇ B ⁇ , phosphor-p65 (P-p65), phosphor-STATl (P-STATl), phosphor-p38 (P-p38) and actin proteins in MEF treated with IFN ⁇ (10 ng/ml), TNF (40 ng/ml) or LPS (100 ng/ml) for the indicated times.
  • FIG. 16B provides a Western blot for ⁇ hosphor-p65 (P- ⁇ 65), phosphor-STATl (P- STAT1), phosphor-p38 (P-p38), I B ⁇ , STAT1 and actin proteins in LKKi "7" and LKKi +7+ MEF treated with LFN ⁇ (10 ng/ml), TNF (40 ng/ml) for the indicated times.
  • FLG. 16C provides an electrophoretic mobility shift assay (EMSA) of nuclear extracts from LKKi "7” and LKKi + + MEF treated with LFN ⁇ (10 ng/ml) or TNF (40 ng/ml) for the indicated times.
  • ESA electrophoretic mobility shift assay
  • 16D provides a Northern blot analysis of steady-state C/EBP ⁇ or C/EBP ⁇ mRNA levels in IKKi "7" and IKKi +/+ MEF treated with LFN ⁇ (10 ng/ml) or TOF (40 ng/ml) for the indicated times.
  • FIG. 17 illustrates the responsiveness of IKKi "7" and LKKi +/+ MEF to LFN ⁇ plus TNF.
  • a Northern blot analysis is shown of steady-state Nos2, TP-10, RANTES, IRF-1, L ⁇ B ⁇ 5 C/EBP ⁇ or C/EBP ⁇ mRNA levels in LKKi "7” and LKKi +7+ MEF treated with LFN ⁇ (10 ng/ml) plus TNF (40 ng/ml) for the indicated times.
  • FIGs. 18 A-G illustrate that IKKi is required for activation of genes encoding immune and inflammatory modulators regulated by C/EBP and NF- ⁇ B pathways in MEF.
  • FIG. 18 A provides a Northern blot analysis of steady-state mRNA levels in p65 "7" .
  • FIG. 18B provides a Northern blot analysis of steady-state mRNA levels in p65 "7" , LKKi “7” and LKKi + + MEF treated with LPS (100 ng/ml) plus LFN ⁇ (10 ng/ml) for the indicated times.
  • FIG. 18C shows nitrite production from wild type (WT), p65 “7” and LKKi “7” MEF treated by LFN ⁇ alone (control) or in combination with TNF or LPS for 48 h.
  • FIG. 18D shows IL-6 production from wild type (WT), ⁇ 65 “7” and IKKi “7” MEF treated by IFN ⁇ alone (control) or in combination with TNF or LPS for 20 h.
  • FIG. 18E provides an electrophoretic mobility shift assay (EMSA) of nuclear extracts from p65 '7" , LKKi “7” and LKKi +/+ MEF before and after 6 h treatment with IFN ⁇ (10 ng/ml), TNF (40 ng/ml) and or LPS (100 ng/ml), as indicated.
  • ESA electrophoretic mobility shift assay
  • FIG. 18F provides an electrophoretic mobility shift assay (EMSA) showing the polypeptide composition of the C/EBP-specific complexes in nuclear extracts from IKKi "7" and LKKi +7+ MEF treated with LFN ⁇ (10 ng/ml) plus TNF (T) or LPS (L) for 6 h.
  • the nuclear extracts were incubated in the presence of specific antibody (Ab) to C/EBP ⁇ ( ⁇ ) or C/EBP ⁇ ( ⁇ ) as indicated.
  • the DNA binding activity of C EBP or Octl (as a control) transcription factor is shown.
  • FLG. 18G provides a Western blot analysis of LKKi protein expression in P65 "7" , LKKi "
  • FIGs. 19A-F illustrate that LKKi is a key molecule coupling the inflammatory responses to TNF and LFN ⁇ with the adipocyte differentiation program through the C/EBP pathway.
  • FIG. 19A provides a Western blot analysis of IKKi protein expression in p65 “7" , LKKi +/+ or TKKi " " MEF occurring during an adipocyte differentiation program induced by standard differentiation induction media (DM). The same membrane was striped and re- probed for actin (as a loading control).
  • FIG. 19B provides a Northern blot analysis of steady-state mRNA levels in p65 "7" , IKKi “7” and LKKi + + MEF before and after treatment with differentiation induction media (DM) or with DM in the presence of LFN ⁇ (10 ng/ml) and TNF (40 ng/ml) as indicated.
  • DM differentiation induction media
  • FIG. 19C provides an electrophoretic mobility shift assay (EMSA) of nuclear extracts from p65 “7” , LKKi “7” and IKKi +/+ MEF before and after treatment with differentiation induction media (DM) or with DM in the presence of LFN ⁇ (10 ng/ml) and TNF (40 ng/ml) as indicated.
  • DM differentiation induction media
  • LFN ⁇ 10 ng/ml
  • TNF 40 ng/ml
  • FIG. 19D provides an electrophoretic mobility shift assay (EMSA) showing the polypeptide composition of the C/EBP-specific complexes in nuclear extracts from LKKi +7+ MEF before and after treatment with differentiation induction media (DM) or with DM in the presence of IFN ⁇ (10 ng/ml) and TNF (40 ng) as indicated.
  • DM differentiation induction media
  • IFN ⁇ 10 ng/ml
  • TNF 40 ng
  • the nuclear extracts were incubated in the presence of specific antibody (Ab) to C/EBP ⁇ or C/EBP ⁇ as indicated.
  • FIG. 19E provides a Northern blot analysis of aP2 or C/EBP ⁇ steady-state mRNA levels in IKKi +7+ and LKKi "7" MEF before and after treatment with differentiation induction media (DM) as indicated.
  • FIG. 19F provides a Northern blot analysis of aP2, C/EBP ⁇ or C/EBP ⁇ mRNA levels in LKKi + + and LKKi "7" MEF before and after incubation for 2 days in differentiation induction media (DM) followed by treatment with 10%o FBS containing 5 ⁇ g/ml insulin (Ins) as indicated.
  • DM differentiation induction media
  • FIGs. 20A-D illustrate that IKKi is required for survival of growth-arrested cells.
  • FIG. 20A graphically illustrates that TNF+LFN ⁇ is cytotoxic to LKKi +7+ , LKKi “7” and p65 “7” MEFs, but that wild type (LKKi + + ) cells are more robust than LKKi "7” and p65 “7” MEFs.
  • MEFs were treated with TNF (10 ng/ml) plus IFN ⁇ (5 ng/ml) for 24, 48 or 72 hours in growth media containing 10% FBS. Viable cells remaining after the treatment are shown as a percentage of viable untreated cells.
  • FIG. 20B graphically illustrates the viability of wild type (WT, both LKKi +7+ and p65 +7+ were tested), p65 "7” and LKKi “7” MEFs after 48 hours of incubation in 10% or 0.5% FBS. Viable cells remaining after the treatment are shown as a percentage of viable untreated cells.
  • FIG. 20C graphically illustrates that TNF, LFN ⁇ or TNF+LFN ⁇ is cytotoxic to LKKi + + ,
  • MEFs LKKi "7" and ⁇ 65 “7” MEFs.
  • MEFs were treated with TNF (10 ng/ml) plus LFN ⁇ (5 ng/ml) for 48 hours in media containing 0.5% FBS. Viable cells remaining after the treatment are shown as a percentage of viable untreated cells.
  • FIG. 20D graphically illustrates that TNF+LFN ⁇ (T/T) is cytotoxic to IKKi “7” and p65 “7” MEFs.
  • Wild type both LKKi “7” and p65 +7+ were tested
  • p65 "7” or LKKi “7” MEFs were untreated (ctl) or treated for 1 (Id) or 2 (2d) days with adipocyte differentiation media (DM) containing TNF (10 ng/ml) and LFN ⁇ (5 ng/ml) as indicated. Viable cells remaining after the treatment are shown as a percentage of viable untreated cells.
  • DM adipocyte differentiation media
  • FIGs. 21 A-D illustrate that IKKi +7+ MEF are more responsive to amyloid beta peptide (A ⁇ ) and MDP (a bacterial component) than are IKKi "7" MEFs.
  • FIG. 21A provides a Northern blot analysis of steady-state MCP-1, IL-6, LRF-1 (as a control for LFN ⁇ ) and GAPDH (as a loading control) mRNA levels in LKKi "7" and LKKi +/+ MEF treated with LPS (as a positive control) or A ⁇ ( 10 ⁇ M) plus LFN ⁇ (10 ng/ml) as indicated.
  • FIG. 21 A provides a Northern blot analysis of steady-state Nos2, IL-6, RANTES and
  • GAPDH (as a loading control) mRNA levels in IKKi "7" and LKKi +7+ MEF treated with LPS (as a positive control) or MDP ( 10 ⁇ M) plus LFN ⁇ (10 ng/ml) as indicated.
  • FIG. 21 C graphically illustrates nitrite production from LKKi +/+ and LKKi "7" MEF that were untreated (ctl), IFN ⁇ treated, IFN ⁇ + A ⁇ (Abeta) treated, or LFN ⁇ + MDP treated for 40 h.
  • FIG. 21D graphically illustrates IL-6 production from LKKi + + and LKKi "7" MEF treated by IFN ⁇ alone (ctl), by LFN ⁇ in combination with A ⁇ or by LFN ⁇ in combination with MDP for 20 h.
  • the inducible kinase known as LKKi/LKK ⁇ is a key player in immunological and mflammatory responses.
  • LKKi is required for expression of a group of genes induced by pro-inflammatory stimuli such as bacterial lipopolysaccharide (LPS).
  • LKKi modulates the expression of genes that are coordinately regulated by nuclear factor kappa beta (NF- ⁇ B) and CAAT / enhancer binding protein (C/EBP).
  • NF- ⁇ B nuclear factor kappa beta
  • C/EBP CAAT / enhancer binding protein
  • LKKi plays an important role in cellular functions associated with inflammation (including chronic inflammation), cancer development, apoptosis, tissue regeneration and neurodegenerative diseases (e.g., Alzheimer's disease).
  • modulation of IKKi activity or expression can be used to modulate these cellular functions and thereby treat inflammatory disorders, cancer, apoptosis, tissue injuries and neurodegenerative diseases.
  • loss of LKKi makes cells less susceptible to gene activation by lipopolysaccharides and Amyloid- ⁇ peptide.
  • IKKi provides a link between the NF- ⁇ B and C/EBP pathways. This link includes NF- ⁇ B-dependent regulation of C/EBP ⁇ and C/EBP ⁇ gene transcription, and IKKi-mediated activation of C/EBP. Disruption of the NF- ⁇ B pathway results in the blockade of the induction of C/EBP ⁇ , C/EBP ⁇ and IKKi genes.
  • cells lacking IKKi are normal in activation of the canonical NF- ⁇ B pathway, but fail to induce C/EBP ⁇ activity and transcription of C/EBP and C/EBP-NF- ⁇ B target genes in response to LPS. Additionally, in response to LPS or tumor necrosis factor ⁇ (TNF- ⁇ ), both ⁇ and ⁇ subunits of C/EBP interact with the IKKi promoter and act through a feedback mechanism to regulate IKKi-dependent cellular processes.
  • TNF- ⁇ tumor necrosis factor ⁇
  • LKKi has been found to play an unexpected and novel role in coordinating the cross-talk among key pathways that effect cell survival and inflammation, and therefore provides a selective target for anti-inflammatory, anti-cancer, tissue transplantation and neurodegenerative therapies.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • FLAG hydrophilic 8-amino acid peptide DYKDDDDK
  • LKKi inducer is an agent that causes an increase in LKKi mRNA production or LKKi protein production through expression of the IKKi mRNA.
  • IKKi mRNA production is induced by a wide variety of stimuli. Examples of these stimuli include, lipopolysaccharide (LPS), tumor necrosis factor (TNF), phorbol myristate acetate (PMA), interleukin-1, and interleukin-6.
  • LPS lipopolysaccharide
  • TNF tumor necrosis factor
  • PMA phorbol myristate acetate
  • interleukin-1 interleukin-6.
  • modulate refers to an increase or decrease in LKKi expression or activity.
  • modulation of IKKi expression can refer to an increase or decrease in the production of mRNA that encodes IKKi.
  • Modulation can also refer to an increase or decrease in translation of the mRNA that encodes LKKi which results in an increase or decrease production of the LKKi protein.
  • Modulation can also refer to an increase or decrease in LKKi enzymatic activity.
  • LKKi activators and LKKi inhibitors modulate LKKi expression and or LKKi activity.
  • IKKi inducers modulate IKKi gene transcription and or expression.
  • LKKi activity is the effect of the LKKi protein in biological systems.
  • LKKi plays a key role in modulating the immune response.
  • the invention therefore provides methods for modulating an immune response by modulating the expression or activity of IKKi.
  • the invention also provides methods for modulating inflammation by modulating the expression or activity of LKKi.
  • the invention provides a method to inhibit LPS induced septic shock in a mammal.
  • the invention provides methods for modulating amyloid- ⁇ peptide-mediated transcription by modulating the expression or activity of TKKi.
  • LKKi-deficiency resulted in a marked reduction in amyloid- ⁇ -mediated and MDP-mediated mRNA expression of MCP-1, LL-6, LRF-1, Nos2 and Rantes genes. Accordingly, inappropriate immune responses, inflammation, septic shock and neurodegenerative diseases such as Alzheimer's disease may be treated by administering an agent that inhibits LKKi expression or activity to a mammal in need thereof.
  • the methods of the invention can be used for, but not limited to, the treatment of inflammation in a mammal, and for treatment of other inflammation-associated disorders, such as, as an analgesic in the treatment of pain and headaches, or as an antipyretic for the treatment of fever.
  • Inflammation is defined as the reaction of vascularized living tissue to injury. As such, inflammation is a fundamental, stereotyped complex of cytologic and chemical reactions of affected blood vessels and adjacent tissues in response to an injury or abnormal stimulation caused by a physical, chemical or biological agent. Inflammation usually leads to the accumulation of fluid and blood cells at the site of injury, and can be a healing process. However, inflammation sometimes causes harm, usually through a dysfunction of the normal progress of inflammation.
  • Inflammatory diseases are those pertaining to, characterized by, causing, resulting from, or becoming affected by inflammation.
  • inflammatory diseases or disorders include, without limitation, asthma, bronchitis, lung inflammation, osteoarthritis, juvenile arthritis, rheumatoid arthritis, spondylo arthopathies, gouty arthritis, chronic granulomatous diseases such as tuberculosis, leprosy, sarcoidosis, and silicosis, nephritis, amyloidosis, ankylosing spondylitis, chronic bronchitis, scleroderma, systemic lupus erythematosus, polymyositis, appendicitis, inflammatory bowel disease, Crohn's disease, gastritis, irritable bowel syndrome, ulcerative colitis and for the prevention of colorectal cancer, Sjorgen's syndrome, Reiter's syndrome, psoriasis, pelvic inflammatory disease, orbit
  • the methods of the invention are also useful for treating inflammation in vascular diseases, migraine headaches, periarteritis nodosa, thyroiditis, aplastic anemia, Hodgkin's disease, sclerodoma, rheumatic fever, type I diabetes, myasthenia gravis, sarcoidosis, nephrotic syndrome, Behcet's syndrome, polymyositis, gingivitis, hypersensitivity, conjunctivitis, swelling occurring after injury, myocardial ischemia, and the like.
  • IKKi function plays an essential role in several cellular functions involving tissue regeneration, including suppressing inflammation, protecting against apoptosis, promoting survival of growth arrested cells, promoting cellular differentiation and related functions.
  • modulation of IKKi function can have many positive effects on the treatment of damaged or diseased tissues.
  • the invention therefore provides a method for promoting regeneration of a mammalian tissue by administering an effective amount of an LKKi polypeptide or an IKKi nucleic acid to the tissue.
  • LKKi is important for cellular functioning in cells of hematopoietic origin as well as non- hematopoietic origin. As illustrated herein, LKKi has far-reaching effects on mouse embryonic fibroblasts. Mouse embryonic fibroblasts are pluripotent mesenchymal stem cells that give rise to numbers of non-hematopoietic cell types, including myocytes, chondrocytes, osteoblasts, and adipocytes. Also, as illustrated herein siRNA directed against LKKi can affect cellular functioning in human umbilical vein endothelial cells (HUVEC). Hence, LKKi can be also used to influence endothelial cell functioning and other cells of non- hematopoietic origin.
  • UAVEC umbilical vein endothelial cells
  • LKKi polypeptides and/or nucleic acids are useful for tissue regeneration of liver, heart, vascular tissues, kidney, bones and muscles as well as for treatment of diseases such as osteoporosis, cachexia and chronic inflammatory diseases.
  • the invention provides a method to promote apoptosis of a cell.
  • the method involves contacting a cell with an agent that is an inhibitor of IKKi such that the cell undergoes TNF induced apoptosis.
  • a cell may be contacted with an agent that inhibits IKKi and TNF.
  • the cell may be contacted with an LKKi mutant polypeptide that does not have LKKi activity.
  • the method can be used to treat numerous conditions through elimination of undesirable cells.
  • the methods of the invention are also useful for treating cancer.
  • the methods of the invention can be used as proapoptotic, anti-apoptotic, anti-cell cycle progressive, anti-invasive, and anti-metastatic agents.
  • carcinoma such as bladder, breast, colon, kidney, liver, lung, including small cell lung cancer, esophagus, gall-bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, and skin, including squamous cell carcinoma
  • hematopoietic tumors of lymphoid lineage including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T- cell-lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkett's lymphoma
  • hematopoietic tumors of myeloid lineage including acute and chronic myclogenous leukemias, myelodysplastic syndrome and promyelocytic leukemia
  • tumors of mesenchymal origin including fibrosarcom
  • an IKKi inhibitor can be injected into or adjacent to a tumor alone, or in combination with TNF, to cause the tumor cells to undergo apoptosis. Accordingly, the method may be used to treat cancer. Due to the key role of the LKKi protein kinase in the regulation of cellular proliferation, these methods are also useful in the treatment of a variety of cell proliferative disorders such as, for instance, benign prostate hyperplasia, familial adenomatosis, polyposis, neuro-fibromatosis, psoriasis, vascular smooth cell proliferation associated with atherosclerosis, pulmonary fibrosis, arthritis glomerulonephritis and post-surgical stenosis and restenosis.
  • benign prostate hyperplasia familial adenomatosis, polyposis, neuro-fibromatosis, psoriasis, vascular smooth cell proliferation associated with atherosclerosis, pulmonary fibrosis, arthritis glomerulonephritis and post-surgical stenosis and reste
  • the invention also provides a method to inhibit apoptosis of a cell.
  • the method involves contacting a cell with an agent that activates LKKi such that the cell is protected from TNF-induced apoptosis.
  • an agent that activates LKKi such that the cell is protected from TNF-induced apoptosis.
  • expression of LKKi can protect cells from TNF-mediated cytotoxicity.
  • LKKi activity is required to promote the protective effects; hence an inactive IKKi mutant polypeptide should not be used ' .
  • a method that involves administering LKKi, agents that promote LKKi expression or agents that activate LKKi is useful to protect healthy tissue, for example, when the tissue is stressed or suffers from an undesirable disease or condition.
  • tissues involved in surgery or transplantation can be treated with agents that promote LKKi expression or activity.
  • Such tissues include, for example, liver tissue, heart tissue, vascular tissue or kidney tissue.
  • healthy tissues that are located next to a tumor can be contacted with an TKKi activator, while the adjoining tumor tissue is contacted with TNF and an LKKi inhibitor.
  • TKKi activator vascular tissue or kidney tissue.
  • TNF and an LKKi inhibitor Such a treatment scheme will provide protection to the healthy tissue, while allowing the tumor to be induced to undergo apoptosis.
  • the method can be used to promote the regeneration of tissues.
  • LKKi or an LKKi activator can be contacted with liver cells (or other transplantation tissues) following transplantation of a liver (or other tissues) into a recipient.
  • This treatment scheme will promote regeneration of the liver (or other tissues) in the recipient following transplantation.
  • Agents that Modulate LKKi Expression or Activity Any agent that inhibits or increases TKKi expression or activity can be used in the methods of the invention.
  • Agents that inhibit LKKi expression or activity include small interfering RNAs (siRNAs), ribozymes, antisense nucleic acids, kinase inhibitors, anti-LKKi antibodies, small molecules, peptides, mutant LKKi polypeptides and the like.
  • Agents that increase LKKi expression or activity include interferons (e.g. interferon-gamma, IFN ⁇ ), tumor necrosis factor (TNF), liposaccharides (e.g.
  • bacterial lipopolysaccharides e.g., bacterial lipopolysaccharides, agents that promote differentiation (e.g., dexamethasone, methylisobutylxanthine and insulin), LKKi polypeptides, LKKi nucleic acids, anti-LKKi antibodies, small molecules, peptides, and the like.
  • agents that promote differentiation e.g., dexamethasone, methylisobutylxanthine and insulin
  • LKKi polypeptides e.g., LKKi nucleic acids, anti-LKKi antibodies, small molecules, peptides, and the like.
  • siRNA small interfering RNAs
  • HAVECs human umbilical vein endothelial cells
  • siRNAs employed to reduce IKKi mRNA levels had the following sequences: 5'-GUGAAGGUCUUCAACACUACC-3' (SEQ LD NO: 6) and S'-UAGUGUUGAAGACCUUCACAG-S' (SEQ LD NO: 7).
  • inflammation or a disease involving an inappropriate immune response can be treated by administering to a mammal a nucleic acid that can inhibit the functioning of an LKKi RNA.
  • Nucleic acids that can inhibit the function of an LKKi RNA can be generated from coding and non-coding regions of the TKKi gene.
  • nucleic acids that can inhibit the function of an IKKi RNA are often selected to be complementary to sequences near the 5' end of the coding region.
  • the nucleic acid that can inhibit the functioning of an TKKi RNA can be complementary to sequences near the 5' end of SEQ ID NO:4 or 5.
  • nucleic acids that can inhibit the function of an LKKI RNA having SEQ LD NO:4 can be complementary to SEQ LD NO:4, SEQ LD NO:5 or to LKKi RNAs from other species (e.g., mouse, rat, cat, dog, goat, pig or a monkey IKKi RNA).
  • a nucleic acid that can inhibit the functioning of an LKKi RNA need not be 100% complementary to a selected region of SEQ ID NO:4 or 5. Instead, some variability the sequence of the nucleic acid that can inhibit the functioning of an LKKi RNA is permitted.
  • a nucleic acid that can inhibit the functioning of a human TKKi RNA can be complementary to a nucleic acid encoding a mouse or rat TKKi gene product.
  • Nucleic acids encoding mouse IKKi gene product for example, can be found in the NCBI database at GenBank Accession No. AB016589, NM 019777 and NT 0399180; a mouse TKKi polypeptide sequence has GenBank Accession No. NP 062751.
  • This mouse TKKi polypeptide is about 94% identical to the human IKKi polypeptide with GenBank Accession No. XP 375834.
  • the mouse cDNA clone (GenBank Accession No. AB016589) is about 84% identical to the human LKKi cDNA (GenBank Accession No. XM 375834).
  • the rat IKKi cDNA (GenBank Accession No. XM 344139) is also about 84% identical to the human LKKi cDNA (GenBank Accession No. XM 375834).
  • the LKKi gene and protein are conserved among different species.
  • nucleic acids that can hybridize under moderately or highly stringent hybridization conditions are sufficiently complementary to inhibit the functioning of an LKKi RNA and can be utilized in the compositions of the invention.
  • stringent hybridization conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • stringent conditions encompass temperatures in the range of about 1°C to about 20°C lower than the thermal pointing point of the selected sequence, depending upon the desired degree of stringency as otherwise qualified herein.
  • the nucleic acids that can inhibit the functioning of LKKi RNA can hybridize to an LKKi RNA under physiological conditions, for example, physiological temperatures and salt concentrations.
  • inhibitory nucleic acid molecules that comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides that are precisely complementary to an IKKi coding sequence, each separated by a stretch of contiguous nucleotides that are not complementary to adjacent LKKi coding sequences, can inhibit the function of LKKi mRNA.
  • each stretch of contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length.
  • Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length.
  • One skilled in the art can easily use the calculated melting point of a nucleic acid hybridized to a sense nucleic acid to estimate the degree of mismatching that will be tolerated between a particular nucleic acid for inhibiting expression of a particular IKKi RNA.
  • a nucleic acid that can inhibit the function of an endogenous LKKi RNA is an anti-sense oligonucleotide.
  • the anti-sense oligonucleotide is complementary to at least a portion of the coding sequence of a gene comprising SEQ ID NO:4 or 5.
  • Such anti-sense oligonucleotides are generally at least six nucleotides in length, but can be about 8, 12, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides long. Longer oligonucleotides can also be used.
  • LKKi anti-sense oligonucleotides can be provided in a DNA construct, or expression cassette and introduced into cells whose division is to be decreased, for example, into cells expressing LKKi, such as immune cells, neuronal cells or lymphocyte precursor cells.
  • ribozyme is an RNA molecule with catalytic activity. See, e.g., Cech, 1987, Science 236: 1532-1539; Cech, 1990, Ann. Rev. Biochem. 59:543-568; Cech, 1992, Curr. Opin. Struct. Biol. 2: 605-609; Couture and Stinchcomb, 1996, Trends Genet. 12: 510-515. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (see, e.g., Haseloff et al., U.S. Pat. No. 5,641,673).
  • LKKi nucleic acids complementary to SEQ LD NO:4 or 5 can be used to generate ribozymes that will specifically bind to mRNA transcribed from an IKKi gene.
  • Methods of designing and constructing ribozymes that can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. (1988), Nature 334:585-591).
  • the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al., EP 321,201).
  • the target sequence can be a segment of about 10, 12, 15, 20, or 50 contiguous nucleotides selected from a nucleotide sequence having SEQ ID NO:4 or 5. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target.
  • the hybridizing and cleavage regions of the ribozyme can be integrally related; thus, upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
  • RNA interference involves post-transcriptional gene silencing (PTGS) induced by the direct introduction of dsRNA.
  • PTGS post-transcriptional gene silencing
  • siRNAs small interfering RNAs
  • siRNAs are generally 21-23 nucleotide dsRNAs that mediate post-transcriptional gene silencing.
  • siRNAs can induce post-transcriptional gene silencing in mammalian cells.
  • siRNAs can also be produced in vivo by cleavage of dsRNA introduced directly or via a transgene or virus. Amplification by an RNA-dependent RNA polymerase may occur in some organisms.
  • siRNAs are incorporated into the RNA-induced silencing complex, guiding the complex to the homologous endogenous mRNA where the complex cleaves the transcript.
  • siRNAs are available. See, e.g., Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001). Duplexes of 21 -nucleotide RNAs mediate RNA interference in mammalian cell culture. Nature 411: 494-498; J. Harborth, S. M. Elbashir, K. Vandenburgh, H. Manninga, S. A. Scaringe, K. Weber and T. Tuschl (2003). Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing, Antisense Nucleic Acid Drug Dev. 13: 83-106.
  • an effective siRNA can be made by selecting target sites within SEQ D NO: 4 or 5 that begin with AA, that have 3' TJU overhangs for both the sense and antisense siRNA strands, and that have an approximate 50% G/C content.
  • a siRNA of the invention can hybridize to LKKi nucleic acids of the following sequences: AATTACCTGT GGCACACAGA TG (SEQ ID NO: 14)
  • a siRNA of the invention can thus be a double-stranded RNA having one of the following sequences:
  • AACAAGAAAU CCGGAGAGCU GUU SEQ ID NO : 21
  • AAAUCCGGAG AGCUGGUUGC UU SEQ ID NO : 22
  • AAGGUCUUCA ACACUACCAG CU SEQ ID NO : 23 )
  • This double stranded siRNA having SEQ ID NO: 19 would have the following structure.
  • Nucleic acids that can decrease LKKi expression or translation can hybridize to a nucleic acid comprising SEQ LD NO:4 or 5 under physiological conditions. In other embodiments, these nucleic acids can hybridize to a nucleic acid comprising SEQ LD NO:4 or 5 under stringent hybridization conditions. Examples of nucleic acids that can modulate the expression or translation of an TKKi polypeptide include a siRNA that consists essentially of a double-stranded RNA with any one of SEQ ID NO:6, 7, 19-24.
  • a method to identify an agent that modulates TKKi activity provides a method to identify an agent that modulates LKKi activity. This activity includes LKKi expression as well as LKKi enzymatic activity.
  • the method involves contacting a test cell with a candidate agent and determining if the agent causes LKKi enzymatic activity to increase or decrease within the test cell.
  • the method involves determining if a candidate agent increases or decreases LKKi enzymatic activity in vitro.
  • the method of the invention can be used to identify agents that increase or decrease LKKi activity.
  • An increase or decrease in LKKi activity within a cell can be determined by comparing the LKKi activity within a test cell that was contacted with a candidate agent, with the LKKi activity within a control cell that was not contacted with a candidate agent.
  • the IKKi activity in a control cell may be determined before, concurrently, or after the IKKi activity within the control cell is determined.
  • LKKi activity can be determined by detecting expression of an LKKi regulated gene.
  • LKKi regulated genes include A20, interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), LP-10, COX-2, RANTES, and the like.
  • An increase or decrease in transcription of an IKKi regulated gene can be determined through use of many methods.
  • the presence and quantity of messenger RNA (mRNA) encoded by an LKKi regulated gene in a cell or other sample can be determined through use of hybridization based procedures, such as northern blotting, gene chip technologies, or through production and hybridization of complimentary DNA (cDNA).
  • mRNA messenger RNA
  • Additional examples of methods that can be used to detect and quantify mRNA of LKKi regulated genes include nucleic acid amplification based methods, such as polymerase chain reaction, ligase chain reaction, and the like. Instrumental methods may be used to detect and quantify mRNA of TKKi regulated genes. For example, probes containing a detectable label may be hybridized to the mRNA. Such probes may be labeled with a fluorescent tag that allows for rapid detection of the mRNA, and therefore provides for high-throughput screening of candidate agents that modulate LKKi. Such methods can be automated according to procedures in common practice in the pharmaceutical industry. Numerous labeled probes may be constructed, and include those that use fluorescence resonance energy transfer (FRET) or fluorescence quenching for detection.
  • FRET fluorescence resonance energy transfer
  • Candidate agents can also be identified that cause an increase or decrease in the transcription or translation of the gene encoding IKKi. Accordingly, a test cell can be contacted with a candidate agent. Production of IKKi mRNA or IKKi protein within the cell can be determined and compared to production in a control cell to determine if a candidate agent increases of decreases production of LKKi mRNA or protein. Such methods have been described herein and are known in the art.
  • Antibodies have been described herein and can also be produced that bind to the LKKi protein. These antibodies can be used to determine if a candidate agent increases or decreases expression of the LKKi protein within a cell.
  • the antibodies can be utilized in immunosorbant assays, such as enzyme-linked immunosorbant (ELIZA) or radio- immunosorbant assays (RLA), to detect IKKi protein.
  • ELIZA enzyme-linked immunosorbant
  • RLA radio- immunosorbant assays
  • Test cells can also be constructed that express an IKKi protein that includes a tag.
  • a fusion protein can be constructed such that the tag is an epitope that can be bound by an antibody (Shimada et al., nternat. Immunol., 11: 1357-1362 (1999)).
  • An example of such a tag is the FLAG ® tag.
  • An increase or decrease in the production of the fusion protein can then be readily followed through use of immunological techniques as are known in the art and described herein (Harlow et al., Antibodies: A Laboratory Manual, page 319 (Cold Spring Harbor Pub. 1988)).
  • the enzymatic activity of LKKi can also be assessed to determine if a candidate agent increases or decreases LKKi activity.
  • the kinase activity of LKKi can be assessed by incubating a kinase substrate, a 32 P- ⁇ -labeled nucleotide, and IKKi under conditions in which LKKi can transfer 32 P from the nucleotide onto the kinase substrate.
  • the level of phosphorylation of the kinase substrate can then be assessed to determine if a candidate agent increases or decreases the activity of LKKi relative to a control that was not contacted with the candidate agent.
  • kinase activity of LKKi is known in the art and are disclosed herein (Shimada et al., Lnternat. Lmmunol. 11:1357-1362 (1999)).
  • the ability of a candidate agent to modulate the kinase activity of TKKi within a cell can also be assessed. This can be done by contacting a test cell with the candidate agent and then lysing the cell to produce a cellular lysate.
  • the TKKi kinase activity in the cellular lysate can be assessed with an in vitro kinase assay to determine if the candidate agent increased or decreased the kinase activity of LKKi within the cell.
  • Antibodies can be used to determine if a candidate agent modulates the activity of LKKi within a cell. This can be done by obtaining an antibody that recognizes an LKKi kinase substrate that is in phosphorylated form, and obtaining another antibody that recognizes the LKKi substrate in non- phosphorylated form.
  • An example, of an LKKi substrate that can be used according to the method is LkB- ⁇ .
  • cells are contacted with a candidate agent.
  • a lysate is prepared from the contacted cells. The lysate is then assayed with antibodies that recognize the LKKi subsfrate in phosphorylated and non-phosphorylated form.
  • the amount of antibody binding to the phosphorylated and non-phosphorylated form of the LKKi substrate is then compared to the amount of antibody binding to the phosphorylated and non- phosphorylated form of the LKKi substrate in a lysate prepared from a control cell that was not contacted with the candidate agent.
  • An increase in the ratio of phosphorylated to non- phosphorylated IKKi substrate in a treated cell relative to a control cell will indicate that the candidate agent activates LKKi kinase activity.
  • a decrease in the ratio of phosphorylated to non-phosphorylated IKKi substrate in a treated cell relative to a control cell will indicate that the candidate agent inhibits LKKi kinase activity.
  • the ability of a candidate agent to modulate the kinase activity of LKKi can also be assessed through use of an in vitro kinase assay.
  • a cell lysate can be prepared. A portion of the cell lysate can be contacted with a candidate agent to produce a contacted lysate. The kinase activity of IKKi in the contacted lysate can then be compared to the kinase activity of IKKi in the lysate that was not contacted with the candidate agent to determine if the candidate agent modulates LKKi activity.
  • Conditions under which in vitro kinase assays can be conducted with IKKi are described herein and are known in the art (Shimada et al., Lnternat. Immunol 11:1357-1362 (1999)).
  • a method of the invention can optionally include the step of contacting a test cell and a control cell with an IKKi inducer.
  • IKKi inducers can be used within the invention.
  • LKKi inducers include tumor necrosis factor (TNF), lipopolysaccharide (LPS), interleukin-1 (IL-1), interleukin-6 (IL-6), interferon-gamma, phorbol myristate, and the like.
  • test cells may be used within the method of the invention as test cells or as control cells.
  • control cells include cells that are LKKi + + and LKKi "7” , such as LKKi “ 7" and LKKi “1"7”1" human embryonic kidney cells.
  • Cells that are LKKi +7+ and IKKi " " may be used in any desired combination within the method of the invention.
  • a method to screen for modulators of LKKi may be conducted by (1) contacting an LKKi +7+ test cell and an LKKi "7" test cell with a candidate agent, and comparing the expression of LKKi or an LKKi regulated gene, or LKKi enzymatic activity to that in control cells that were not contacted with a candidate agent and which are LKKi +7+ and IKKi " " .
  • a person of skill in the art can determine if the candidate agent acts on LKKi, or on other factors that are upstream or downstream of LKKi.
  • Cells may also be used within a method of the invention when those cells express an LKKi protein that lacks kinase activity.
  • An example of such a mutant is the LKKi(KM) mutant that is described herein.
  • Other such kinase deficient mutants can be identified by creating mutations in the gene encoding LKKi, and determining if the LKKi mutants exhibit kinase activity. This activity may be assessed through use of methods known in the art and described herein.
  • a method to identify an agent that inhibits LKKi enzymatic activity provides a method to identify an agent that inhibits LKKi enzymatic activity.
  • the method utilizes the discovery that LKKi protects a cell from tumor necrosis factor (TNF) induced apoptosis in the presence of epidermal growth factor (EGF).
  • TNF tumor necrosis factor
  • EGF epidermal growth factor
  • a test cell that expresses IKKi is contacted with TNF, EGF, and a candidate agent.
  • a control cell that expresses IKKi is contacted with the candidate agent and EGF. Death of the test cell, and survival of the control cell, indicates that the candidate agent inhibits LKKi activity and causes the test cell to undergo apoptosis.
  • An agent identified according to the method can be further characterized through use of methods described herein, such as those described for determining modulation of LKKi expression, modulation of LKKi regulated genes, or determining LKKi kinase activity.
  • test cell or control cell may be used that naturally expresses IKKi.
  • a test cell or control cell may be transformed with an expression construct that provides expression of LKKi, as described herein.
  • test cells and control cells can be grown on multi-well plates.
  • the cells within the plates can be contacted with the components of the assay through use of robotic methods.
  • Apoptosis of the cells can be determined through many art recognized methods, as well as through use of commercially available materials and protocols.
  • Examples of methods that can be used to detect apoptosis include, use of enhanced color variants of Annexin V conjugates, detection of caspase activity associated with apoptosis, direct and quantitative gel-based DNA fragmentation assays, use of ligation-mediated PCR, use of In Situ end-labeling, use of monoclonal antibodies to single-stranded DNA, use of flow and laser scanning cytometry, and the like (Chun, Apoptosis Detection and Assay Methods, BioTechniques Press, One Research Drive, Westborough, MA).
  • Nucleic acid segments, expression cassettes, nucleic acid constructs, transformed cells, trans genie animals, and polypeptides The present invention provides isolated nucleic acid segments that are complementary to LKKi nucleic acids or that encode LKKi proteins.
  • nucleic acids that are complementary to LKKi nucleic acids include siRNAs, ribozymes, antisense nucleic acids and the like.
  • LKKi proteins that can be encoded by the nucleic acid include, for example, wild type LKKi proteins or LKKi peptides as well as IKKi proteins that lack kinase activity.
  • LKKi protein that lacks kinase activity is the mutant LKKi (KM) in which the lysine at amino acid position 38 of SEQ LD NO: 2 has been substituted with methionine (K38M). This generates a mutant LKKi polypeptide having SEQ LD NO:2.
  • Nucleic acids encoding IKKi polypeptides include, for example, a wild type LKKi nucleic having SEQ LD NO:4 and a mutant LKKi nucleic acid encoding the IKKi(KM) mutant polypeptide having SEQ LD NO:5.
  • the invention also includes additional nucleic acid segments that encode related LKKi proteins (e.g. those from other mammalian species) that lack kinase activity. These proteins can be readily prepared by mutating the lysine at amino acid position 38 to other amino acids and determining if the mutated protein is kinase inactive.
  • LKKi proteins can readily be identified by screening for their lack of ability to protect cells against TNF-induced apoptosis.
  • a plasmid encoding LKKi can be randomly mutagenized according to methods known in the art, such as chemical or PCR based mutagenesis methods. These plasmids can then be amplified and purified through art recognized methods.
  • the mutagenized plasmids can be introduced into bacteria and then amplified and purified.
  • a purified plasmid can be introduced into an LKKi "7" cell, such as the LKKi "7" MEFs described herein.
  • transformed cells can be contacted with TNF and EGF such that cells which receive a plasmid that encodes a functional IKKi will survive, and cells receiving a kinase inactive LKKi will undergo apoptosis.
  • This method will allow a person skilled in the art to select for plasmids that encode a kinase inactive LKKi.
  • the nucleic acid encoding the kinase inactive LKKi can then be sequenced to determine the specific mutation or mutations which inactivate the kinase activity of LKKi. Accordingly, the invention includes additional kinase inactive LKKi proteins.
  • a nucleic acid segment of the invention can also include mutations of the sequence listed in SEQ LD NO: 4 or 5 that encode the same amino acids due to the degeneracy of the genetic code.
  • the amino acid threonine is encoded by ACU, ACC, ACA and ACG. It is intended that the invention includes all variations of the nucleic acid segments of SEQ LD NO: 4 or 5 that encode the same amino acids.
  • Such mutations are known in the art (Watson et al, Molecular Biology of the Gene, Benjamin Cummings 1987). Mutations also include alteration of a nucleic acid segment to encode LKKi proteins having conservative amino acid changes.
  • amino acid changes are exemplified by the following five groups which contain amino acids that are conservative substitutions for one another: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur containing: Methionine (M), Cysteine (C); Basic: Arginine (R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q).
  • the genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms.
  • the invention also provides an expression cassette which contains a DNA sequence capable of directing expression of a particular nucleic acid segment of the invention either in vitro or in a host cell.
  • a nucleic acid segment is that having SEQ LD NO:4 or 5, or nucleic acid sequences encoding the same amino acid sequence due to the degeneracy of the genetic code, or conservative mutations thereof.
  • Another example is a siRNA, a ribozyme or an antisense nucleic acid that is complementary to an IKKi nucleic acid, particularly an LKKi mRNA.
  • the expression cassette is a nucleic acid that includes a nucleic acid segment with a sequence that is homologous or complementary to an LKKi nucleic acid (e.g. SEQ LD NO:4 or 5) and that is functional during in vivo or in vitro transcription.
  • LKKi nucleic acid e.g. SEQ LD NO:4 or 5
  • the expression cassettes of the invention can also be used to produce IKKi proteins, polypeptides and nucleic acids either in vitro or in vivo.
  • the expression cassette can also be an isolatable unit such that the expression cassette may be in linear form and functional during in vitro transcription and translation assays.
  • the materials and procedures to conduct these assays are commercially available in the art, for example, from Promega Corp. (Madison, Wisconsin).
  • an in vitro transcript may be produced by placing a nucleic acid sequence under the control of a T7 promoter and then using T7 RNA polymerase to produce an in vitro transcript. This transcript may then be translated in vitro through use of a rabbit reticulocyte lysate.
  • the expression cassette can be incorporated into a vector allowing for replication and amplification of the expression cassette within a host cell with in vitro transcription and translation of a nucleic acid sequence.
  • An expression cassette may contain one or a plurality of restriction sites allowing for placement of the nucleic acid segment under the regulation of a regulatory sequence.
  • the expression cassette can also contain a termination signal operably linked to the nucleic acid segment as well as regulatory sequences required for proper translation of the nucleic acid segment.
  • the expression cassette containing the nucleic acid segment may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the expression cassette may also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Expression of the nucleic acid segment in the expression cassette may be under the control of a constitutive promoter or an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus.
  • the expression cassette may include in the 5'-3' direction of transcription, a transcriptional and translational initiation region, a nucleic acid segment and a transcriptional and translational termination region functional in vivo and /or in vitro.
  • the termination region may be native with the transcriptional initiation region, may be native with the nucleic acid segment, or may be derived from another source.
  • the regulatory sequence can be a nucleic acid sequence located upstream (5' non- coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influences the transcription, RNA processing or stability, or translation of the associated coding sequence.
  • Regulatory sequences can include, but are not limited to, enhancers, promoters, repressor binding sites, translation leader sequences, infrons, and polyadenylation signal sequences. They may include natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences. While regulatory sequences are not limited to promoters, some useful regulatory sequences include constitutive promoters, inducible promoters, regulated promoters, tissue-specific promoters, viral promoters and synthetic promoters.
  • a promoter is a nucleotide sequence that controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription.
  • a promoter includes a minimal promoter, consisting only, of all basal elements needed for transcription initiation, such as a TATA-box and/or initiator that is a short DNA sequence comprised of a TATA- box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression.
  • a promoter may be derived entirely from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments.
  • a promoter may contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions.
  • a promoter may also include a minimal promoter plus a regulatory element or elements that are capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal elements, the latter elements are often referred to as enhancers.
  • the promoter may also be inducible.
  • An enhancer is a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal or flipped), and is capable of functioning even when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence- specific DNA-binding proteins that mediate their effects.
  • the expression cassette can contain a 5' non-coding sequence which is a nucleotide sequence located 5' (upstream) to the coding sequence. It is present in the fully processed mRNA upstream of the initiation codon and may affect processing of the primary transcript to mRNA, stability of the mRNA or translation efficiency.
  • the expression cassette may also contain a 3' non-coding sequence which is a nucleotide sequence located 3' (downstream) to a coding sequence and includes polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
  • the polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the RNA precursor.
  • a nucleic acid segment of the invention may be contained within a vector.
  • a vector may include, but is not limited to, any plasmid, phagemid, F-factor, virus, cosmid, or phage in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable.
  • the vector can also transform a prokaryotic or eukaryotic host either by integration into the cellular genome or exist extra chromosomally (e.g. autonomous replicating plasmid with an origin of replication).
  • the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory element for transcription in vitro or in a host cell such as a eukaryotic cell or microbe, e.g. bacteria.
  • the vector may be a bi-functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of a promoter or other regulatory sequences for expression in a host cell.
  • Shuttle vectors are included and are DNA vehicles capable, naturally or by design, of replication in two different host organisms.
  • the vector may also be a cloning vector which typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion. Such insertion can occur without loss of essential biological function of the cloning vector.
  • a cloning vector may also contain a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Examples of marker genes are tetracycline resistance or ampicillin resistance. Many cloning vectors are commercially available (Stratagene, New England Biolabs, Clonetech).
  • nucleic acid segments of the invention may also be inserted into an expression vector.
  • an expression vector contains (1) prokaryotic DNA elements coding for a bacterial replication origin and an antibiotic resistance gene to provide for the amplification and selection of the expression vector in a bacterial host; (2) regulatory elements that control initiation of transcription such as a promoter; and (3) DNA elements that control the processing of transcripts such as infrons, transcription termination/polyadenylation sequence.
  • a vector into which the nucleic acid segment is to be inserted is treated with one or more restriction enzymes (restriction endonuclease) to produce a linearized vector having a blunt end, a "sticky" end with a 5 ' or a 3' overhang, or any combination of the above.
  • restriction enzymes restriction endonuclease
  • the vector may also be treated with a restriction enzyme and subsequently treated with another modifying enzyme, such as a polymerase, an exonuclease, a phosphatase or a kinase, to create a linearized vector that has characteristics useful for ligation of a nucleic acid segment into the vector.
  • a restriction enzyme such as a polymerase, an exonuclease, a phosphatase or a kinase
  • the nucleic acid segment that is to be inserted into the vector is freated with one or more restriction enzymes to create a linearized segment having a blunt end, a "sticky" end with a 5' or a 3' overhang, or any combination of the above.
  • the nucleic acid segment may also be treated with a restriction enzyme and subsequently treated with another DNA modifying enzyme.
  • DNA modifying enzymes include, but are not limited to, polymerase, exonuclease, phosphatase or a kinase, to create a nucleic acid segment that has characteristics useful for ligation of a nucleic acid segment into the vector.
  • the freated vector and nucleic acid segment are then ligated together to form a construct containing a nucleic acid segment according to methods known in the art (Sambrook et al, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001)).
  • the treated nucleic acid fragment and the treated vector are combined in the presence of a suitable buffer and ligase.
  • the mixture is then incubated under appropriate conditions to allow the ligase to ligate the nucleic acid fragment into the vector.
  • the nucleic acid fragment and the vector each have complimentary "sticky" ends to increase ligation efficiency, as opposed to blunt-end ligation.
  • the vector and nucleic acid fragment are each treated with two different restriction enzymes to produce two different complimentary "sticky” ends. This allows for directional ligation of the nucleic acid fragment into the vector, increases ligation efficiency and avoids ligation of the ends of the vector to reform the vector without the inserted nucleic acid fragment.
  • the invention also provides a construct containing a vector and an expression cassette.
  • the vector may be selected from, but not limited to, any vector described herein.
  • an expression cassette containing the nucleic acid sequences of the invention through methods known in the art and previously described (Sambrook et al, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001)).
  • the regulatory sequences of the expression cassette may be derived from a source other than the vector into which the expression cassette is inserted.
  • a construct containing a vector and an expression cassette is formed upon insertion of a nucleic acid segment of the invention into a vector that itself contains regulatory sequences.
  • an expression cassette is formed upon insertion of the nucleic acid segment into the vector.
  • Vectors containing regulatory sequences are available commercially and methods for their use are known in the art (Clonetech, Promega, Stratagene).
  • the expression cassette, or a vector construct containing the expression cassette may be inserted into a cell.
  • the expression cassette or vector construct may be carried episomally or integrated into the genome of the cell.
  • a variety of techniques are available and known to those skilled in the art for introduction of constructs into a cellular host. Transformation of bacteria and many eukaryotic cells may be accomplished through use of polyethylene glycol, calcium chloride, viral infection, phage infection, elecfroporation and other methods known in the art.
  • the present invention also provides for the production of fransgenic non-human animal models.
  • Animal species suitable for use in the animal models of the present invention include, but are not limited to, rats, mice, hamsters, guinea pigs, rabbits, dogs, cats, goats, sheep, pigs, and nonhuman primates (e.g., Rhesus monkeys, chimpanzees).
  • fransgenic rodents e.g., mice
  • mice are preferred due to their relative ease of maintenance.
  • a nucleic acid segment can be inserted into a germ line or stem cell using standard techniques of retroviral infection, oocyte microinjection, transfection, or microinjection into embryonic stem cells.
  • oocyte injection one or more copies of the recombinant DNA constructs of the present invention may be inserted into the pronucleus of a just-fertilized oocyte. This oocyte is then reimplanted into a pseudo-pregnant foster mother.
  • the live-born animals are screened for integrants using DNA analysis (e.g., from the tail veins of offspring mice) for the presence of the inserted recombinant transgene sequences.
  • the present invention provides LKKi proteins that lack kinase activity.
  • LKKi(KM) in which the lysine at amino acid position 38 of SEQ LD NO: 2 has been substituted with methionine (K38M).
  • the invention also includes additional IKKi polypeptides that lack kinase activity.
  • These polypeptides can he identified according to the methods described herein. These polypeptides can be expressed through use of the expression cassettes and constructs described herein. Methods to purify the polypeptides include, but are not limited to, liquid chromatography, gel permeation chromatography, salt precipitation, immunopurification methods, affinity purification, and the like. Such methods are known in the art. These polypeptides can be used within the methods described herein to identify LKKi modulators.
  • Antibodies raised against LKKI can also be used to modulate IKKI activity. Ln some embodiments, such antibodies inhibit LKKI activity. Ln other embodiments, anti-IKKI antibodies can be used to activate or mimic IKKI activity.
  • the invention also contemplates antibodies that can bind to an LKKI polypeptide of the invention.
  • a disease where IKKi gene expression is undesirably active or inactive can be treated by administering to a mammal an antibody that can bind to LKKI polypeptide.
  • the antibody can be directed against an IKKI polypeptide comprising any one of SEQ LD NO:2, SEQ LD NO:3, or a combination thereof.
  • immunoglobulins All antibody molecules belong to a family of plasma proteins called immunoglobulins, whose basic building block, the immunoglobulin fold or domain, is used in various forms in many molecules of the immune system and other biological recognition systems.
  • a typical immunoglobulin has four polypeptide chains, containing an antigen binding region known as a variable region and a non-varying region known as the constant region.
  • Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced infrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end.
  • VH variable domain
  • VL variable domain at one end
  • the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains (Clothia et al, J. Mol. Biol. 186, 651-66, 1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82, 4592-4596 (1985).
  • immunoglobulins can be assigned to different classes. There are at least five (5) major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2.
  • the heavy chains constant domains that correspond to the different classes of immunoglobulins are called alpha ( ⁇ ), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ) and mu ( ⁇ ), respectively.
  • the light chains of antibodies can be assigned to one of two clearly distinct types, called kappa (K) and lambda ( ⁇ ), based on the amino sequences of their constant domain.
  • K kappa
  • ⁇ lambda
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
  • variable in the context of variable domain of antibodies, refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies.
  • the variable domains are for binding and determine the specificity of each particular antibody for its particular antigen.
  • CDRs complementarity determining regions
  • the more highly conserved portions of variable domains are called the framework
  • variable domains of native heavy and light chains each comprise four FR regions, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies.
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector function, such as participation of the antibody in antibody-dependent cellular toxicity.
  • an antibody that is contemplated for use in the present invention thus can be in any of a variety of forms, including a whole immunoglobulin, an antibody fragment such as Fv, Fab, and similar fragments, a single chain antibody that includes the variable domain complementarity determining regions (CDR), and the like forms, all of which fall under the broad term "antibody,” as used herein.
  • the present invention contemplates the use of any specificity of an antibody, polyclonal or monoclonal, and is not limited to antibodies that recognize and immunoreact with a specific epitope. In some embodiments, however, the antibodies of the invention may react with selected epitopes within various domains of the LKKL protein.
  • antibody fragment refers to a portion of a full-length antibody, generally the antigen binding or variable region.
  • antibody fragments include Fab, Fab', F(ab') 2 and Fv fragments.
  • Papain digestion of antibodies produces two identical antigen binding fragments, called the Fab fragment, each with a single antigen binding site, and a residual "Fc" fragment, so-called for its ability to crystallize readily.
  • Pepsin treatment yields an F(ab') 2 fragment that has two antigen binding fragments, which are capable of cross- linking antigen, and a residual other fragment (which is termed pFc').
  • Additional fragments can include diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.
  • “functional fragment” with respect to antibodies refers to Fv, F(ab) and F(ab') 2 fragments.
  • Antibody fragments retain some ability to selectively bind with its antigen or receptor and are defined as follows:
  • Fab is the fragment that contains a monovalent antigen-binding fragment of an antibody molecule.
  • a Fab fragment can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain.
  • Fab' is the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain. Two Fab' fragments are obtained per antibody molecule.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.
  • (Fab') 2 is the fragment of an antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction.
  • F(ab') 2 is a dimer of two Fab' fragments held together by two disulfide bonds.
  • Fv is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (V H -V L dimer). It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the V H -V dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • Single chain antibody defined as a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
  • Such single chain antibodies are also referred to as "single-chain Fv” or “sFv” antibody fragments.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding.
  • diabodies refers to a small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL).
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VH-VL polypeptide chain
  • monoclonal antibodies likewise is conventional. See, for example, Kohler & Milstein, Nature, 256:495 (1975); Coligan, et al, sections 2.5.1-2.6.7; and Harlow, et al, in: Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor Pub. (1988)), which are hereby incorporated by reference. Methods of in vitro and in vivo manipulation of monoclonal antibodies are also available to those skilled in the art.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, Nature 256, 495 (1975), or they may be made by recombinant methods, for example, as described in U.S.
  • Patent No. 4,816,567 The monoclonal antibodies for use with the present invention may also be isolated from antibody libraries using the techniques described in Clackson et al. Nature 352: 624-628 (1991), as well as in Marks et al, J. Mol Biol. 222: 581-597 (1991).
  • Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion- exchange chromatography. See, e.g., Coligan, et al, sections 2.7.1-2.7.12 and sections 2.9.1- 2.9.3; Barnes, et al, Purification of Immunoglobulin G (IgG), in: Methods in Molecular Biology. Vol. 10, pages 79-104 (Humana Press (1992).
  • SLAM Selected Lymphocyte Antibody Method
  • the SLAM technology permits the generation, isolation and manipulation of monoclonal antibodies without the process of hybridoma generation.
  • the methodology principally involves the growth of antibody forming cells, the physical selection of specifically selected antibody forming cells, the isolation of the genes encoding the antibody and the subsequent cloning and expression of those genes.
  • an animal is immunized with a source of specific antigen.
  • the animal can be a rabbit, mouse, rat, or any other convenient animal.
  • This immunization may consist of purified protein, in either native or recombinant form, peptides, DNA encoding the protein of interest or cells expressing the protein of interest.
  • Lymphocytes are isolated from the blood and cultured under specific conditions to generate antibody-forming cells, with antibody being secreted into the culture medium. These cells are detected by any of several means (complement mediated lysis of antigen-bearing cells, fluorescence detection or other) and then isolated using micromanipulation technology. The individual antibody forming cells are then processed for eventual single cell PCR to obtain the expressed Heavy and Light chain genes that encode the specific antibody. Once obtained and sequenced, these genes are cloned into an appropriate expression vector and recombinant, monoclonal antibody produced in a heterologous cell system.
  • Another method involves humanizing a monoclonal antibody by recombinant means to generate antibodies containing human specific and recognizable sequences. See, for review, Holmes, et al, J. Immunol, 158:2192-2201 (1997) and Vaswani, et al, Annals Allergy, Asthma & Immunol., 81 : 105-115 (1998).
  • the term "monoclonal antibody” as used herein 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 may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site.
  • each monoclonal antibody is directed against a single determinant on the antigen.
  • the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier "monoclonal” indicates the antibody is 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 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 (U.S. Pat. No. 4,816,567); Morrison et al. Proc. Natl. Acad Sci. 81, 6851-6855 (1984).
  • 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
  • Antibody fragments of the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab monovalent fragments.
  • a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
  • Fv fragments comprise an association of V H and V chains. This association may be noncovalent or the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde.
  • the Fv fragments comprise V H and V L chains connected by a peptide linker.
  • sFv single-chain antigen binding proteins
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick, et al, Methods: a Companion to Methods in Enzymology. Vol. 2, page 106 (1991). The invention further contemplates human and humanized forms of non-human (e.g. murine) antibodies.
  • humanized antibodies can be chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen- binding subsequences of antibodies) that 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 nonhuman species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that 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.
  • humanized antibodies can 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 Fv regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • mutant antibody refers to an amino acid sequence variant of an antibody.
  • one or more of the amino acid residues in the mutant antibody is different from what is present in the reference antibody.
  • Such mutant antibodies necessarily have less than 100% sequence identity or similarity with the reference amino acid sequence.
  • mutant antibodies have at least 75% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of the reference antibody.
  • mutant antibodies have at least 80%), more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of the reference antibody.
  • the antibodies of the invention are isolated antibodies.
  • An isolated antibody is one that has been identified and separated and/or recovered from a component of the environment in which it was produced. Contaminant components of its production environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • isolated antibody also includes antibodies within recombinant cells because at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • the antibodies of the invention can be purified by any available procedure.
  • the antibodies can be affinity purified by binding an antibody preparation to a solid support to which the antigen used to raise the antibodies is bound. After washing off contaminants, the antibody can be eluted by known procedures.
  • Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (see for example, Coligan, et al, Unit 9, Current Protocols in Immunology, Wiley Lnterscience, 1991, incorporated by reference) .
  • the antibody will be purified as measurable by at least three different methods: 1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight; 2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or 3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain.
  • An IKKi polypeptide, LKKi peptide, IKKi nucleic acid or LKKi modulator can be formulated as a pharmaceutical composition.
  • a pharmaceutical composition of the invention includes an LKKi polypeptide, LKKi nucleic acid or LKKi modulator in combination with a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions of the invention may be prepared in many forms that include tablets, hard or soft gelatin capsules, aqueous solutions, suspensions, and liposomes and other slow-release formulations, such as shaped polymeric gels.
  • An oral dosage form may be formulated such that the LKKi polypeptide, LKKi nucleic acid or LKKi modulator is released into the intestine after passing through the stomach.
  • Such formulations are described in U.S. Patent No. 6,306,434 and in the references contained therein.
  • Oral liquid pharmaceutical compositions may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid pharmaceutical compositions may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservatives.
  • LKKi polypeptide, LKKi nucleic acid or LKKi modulator can be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and maybe presented in unit dosage form in ampoules, pre-filled syringes, small volume infusion containers or multi-dose containers with an added preservative.
  • the pharmaceutical compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the LKKi polypeptide, IKKi nucleic acid or activators or inhibitors of LKKi may be in powder form, obtained by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile saline, before use.
  • a suitable vehicle e.g., sterile saline
  • Pharmaceutical compositions suitable for rectal administration can be prepared as unit dose suppositories.
  • Suitable carriers include saline solution and other materials commonly used in the art.
  • an LKKi polypeptide, LKKi nucleic acid or LKKi modulator can be conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray.
  • Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • an LKKi polypeptide for adminisfration by inhalation or insufflation, an LKKi polypeptide,
  • LKKi nucleic acid or LKKi modulator may take the form of a dry powder composition, for example, a powder mix of a modulator and a suitable powder base such as lactose or starch.
  • the powder composition may be presented in unit dosage form in, for example, capsules or cartridges or, e.g., gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflator.
  • an LKKi polypeptide, LKKi nucleic acid or LKKi modulator may be administered via a liquid spray, such as via a plastic bottle atomizer.
  • compositions of the invention may also contain other ingredients such as flavorings, colorings, anti-microbial agents, anti-inflammatory agents or preservatives. It will be appreciated that the amount of an LKKi polypeptide, IKKi nucleic acid or LKKi modulator required for use in treatment will vary not only with the particular carrier selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient. Ultimately the attendant health care provider may determine proper dosage.
  • IKKi (KM) (K38M) kinase defective mutant MQSTANYLWHTDDLLGQGATASVYKARNKKSGELVAVMVFNTTSYLRPREVQVR EFEVLRKLNHQNFVKLFAVEETGGSRQKVLVMEYCSSGSLLSVLESPENAFGLPEDE FLWLRCWAGMNHLRENGLVHRDLKPGNIMRLVGEEGQSTYKLTDFGAARELDDD EKFVSVYGTEEYLHPDMYERAVLRKPQQKAFGVTVDLWSIGVTLYHAATGSLPFIPF GGPRRNKE ⁇ MYRITTEKPAGALAGAQRRENGPLEWSYTLPITCQLSLGLQSQLVPILA NILEVEQAKCWGFDQFFAETSDILQRVVVHVFSLSQAVLHHIYLHAHNTIALFQEAVH KQTSVAPRHQEYLFEGHLCVLEPSVSAQHIAHTTASSPLTLFSTALPKGLAFRDPALD VPKFVPKV
  • Example 1 Cells Human embryonic kidney 293 (HEK) cells were obtained from ATCC. LKKi- deficient murine embryonic fibroblasts (MEFs) were generated and maintained using standard protocols as described by Takeda et al, Science, 284:313 (1999). Human umbilical vein endothelial cells (HUVEC) were purchased from Clonetics Corporation and maintained in EGM media.
  • HEK Human embryonic kidney 293
  • RelA/p65-, IKK2-, LKKi- or Egrl -deficient MEFs and the corresponding immortalized MEFs (imMEFs) were generated and maintained as described (Beg et al, Nature, 376:167 (1995); Li et al, Science, 284:321 (1999); Takeda et al, Science, 284:313 (1999); Yan et al, Nat. Med., 6:1355 (2000)).
  • Antibodies The JKK2, IKKI, NEMO, p65, p50, c-rel, C/EBP ⁇ and C/EBP ⁇ antibodies were purchased from Santa Cruz. The anti-LKKi rabbit polyclonal antibodies were raised against a TrpE-LKKi fusion protein. Oligonucleotides: The NF- ⁇ B, Oct-1 and C/EBP gel shift oligonucleotides were from
  • RNA oligonucleotides were purchased from Dharmacon Research. Double-stranded siRNAs (IKKi: 5'-GUGAAGGUCUUCAACACUACC-3' (SEQ LD NO: 6) x 5'- UAGUGUUGAAGACCUUCACAG-3' (SEQ LD NO: 7); firefly luciferase as a non-specific, (ns: 5'-CGUACGCGGAAUACUUCGAAA-3' (SEQ LD NO: 8) x 5'- UCGAAGUAUUCCGCGUACGUG-3' (SEQ LD NO: 9)) were prepared and used for transfection of HUVEC (approximately 5 xlO 6 cells per fransfection) by using electroporation performed as described (Gitlin et al, Nature. 418:430 (2002)).
  • LPS Lipopolysaccharide
  • Example 2 Nucleic acid constructs and stably transfected cell lines LKKi expression constructs were generated according to the following methods.
  • a DNA fragment containing the coding region of LKKi was obtained by PCR and cloned into the EcoRI/Xbal sites of a pcDNA3-derived plasmid. This introduced the Flag tag sequence to the 5' end of LKKi to create the plasmid pFlag-IKKi.
  • the plasmid pFlag-LKKiKM contains the catalytic inactive version of LKKi having a substitution of Lys38 to Met (K38M).
  • the plasmids, p ⁇ B-Flag-IKKiKM or p ⁇ B-Flag-LKKi were created by substitution of the CMV promoter fragment, NdeI/HindLII(blunt), from pFlag-LKKiKM with the NdeL/EcoRI(blunt) fragment containing an NF- ⁇ B -dependent promoter.
  • the NF- ⁇ B dependent promoter was isolated from the pNF- ⁇ B reporter plasmid (Stratagene).
  • HEK-LKKiKM and HEK-LKKi stable cell lines were generated by transfection of
  • nucleic acids were prepared and analyzed using a standard protocol. Briefly, nucleic acids (DNA+RNA) were isolated from the cytoplasm fraction by phenol-chloroform extraction and concentrated by ethanol precipitation. Samples were treated with RNase (0.1 mg/ml) for 1 h at 37°C and analyzed by 1.5% agarose gel for DNA using ethidium bromide staining. Samples without RNase treatment were analyzed by northern blot with a GAPDH probe.
  • Example 4 Northern blot assay: Samples of total RNA (10 ⁇ g) were analyzed by northern blot as described (Shimada et al, Int. Immunol, 11; 1357-1362 (1999)). A specific anti-sense oligonucleotide labeled by T4 polynucleotide kinase using ⁇ -[ 32 P]-ATP was hybridized to the RNA blot.
  • Electrophoretic mobility shift assay Nuclear extracts were prepared and used for EMSA as described (Kravchenko et al, J. Biol. Chem.. 270:14928-14934 (1995)).
  • the kinase activity of endogenous LKK2 or LKKi was measured by immune-complex kinase assay with GST-I ⁇ B ⁇ (l-46) as substrate (Li et al, Science.284:321-325 (1999); (Shimada et al, Int. Lmmunol, IT: 1357-1362 (1999)).
  • the immune-complexes were also subjected to Western blot to estimate the amount of precipitated proteins.
  • ChLP Chromatin immunoprecipitation
  • Metabolic labeling Cells were plated on a 60 mm dish (1 x 10 6 cells/dish). On the second day, the cells were washed three times with phosphate-free DMEM containing 5% dialyzed FBS, and then incubated in the same medium containing 400 mCi/ml of [ 32 P]H 3 PO for 4 h. During the last 2 hours, some cells were incubated with 100 ng/ml LPS. The cells were then washed three times with cold PBS and used for preparation of nuclear extracts according to the EMSA protocol described above. The nuclear extracts were diluted by addition often volumes of standard RIPA buffer.
  • LKKi is a constitutively active kinase that does not regulate activation of the NF-kB pathway
  • the activities of endogenous LKK2 and LKKi were compared in exfracts from Jurkat and human embryonic kidney 293 (HEK) cells after stimulation with TNF or PMA. LKK2 activity was induced by both PMA and TNF (FIG. 1 A). Ln contrast, LKKi displayed significant activity when isolated from extracts of unstimulated cells.
  • LKKi activity was not induced endogenously in response to either PMA or TNF in these cells (Fig. 1 A). Furthermore, LKKi kinase activity correlated well with the protein level immunoprecipitated from Jurkat and HEK cell extracts. The level of IKKi mRNA determined by northern blot analysis paralleled that of IKKi protein levels (FIG. IB). TNF-induced expression of LKKi was blocked by expression of a kinase inactive version of LKK2 in primary human endothelial cells (FIG. 7). Accordingly, LKKi may represent a constitutively active kinase that is transcriptionally induced in response to inducers of the NF-kB pathway.
  • Example 7 Expression of LKKi or IKKiKM does not affect nuclear translocation of NF-kB in response to PMA or TNF Stable HEK cell lines that harbor an NF-kB-regulated expression vector encoding for either epitope-tagged LKKi (HEK-LKKi) or a kinase inactive version of LKKi (HEK- IKKiKM) were generated as described in Example 2 above. These cell lines regulate the expression of the IKKi and IKKiKM transgenes in a manner similar to that observed for the endogenous LKKi gene.
  • HEK-LKKi epitope-tagged LKKi
  • HEK- IKKiKM a kinase inactive version of LKKi
  • the HEK-LKKi and HEK-IKKiKM clones were selected to achieve a basal mRNA level of the respective LKKi transgene that was comparable with the basal expression of LKKi mRNA observed in Jurkat cells (FIG. 8).
  • the level of LKKi protein can function as a reporter for NF-kB activation because an NF-kB promoter drives expression of the LKKi transgene.
  • No difference in the level of stimulus-induced expression of LKKi as compared to IKKiKM was observed indicating that the expression of LKKi and IKKiKM did not affect NF-kB-dependent transcriptional activity.
  • Comparable levels of stimulus-induced NF-kB DNA binding activity was observed in the HEK, HEK-LKKi and HEK-LKKiKM cells (FIG. ID).
  • no change in the subunit composition of the NF-kB DNA-binding activity was observed as a function of LKKi or IKKiKM expression (data not shown).
  • cellular processes leading to nuclear translocation of NF-kB in response to PMA or TNF were not affected by the expression of either IKKi or IKKiKM.
  • TNF lymphothelial growth factor
  • TNF NF-kB
  • TNF plays a role in regulating cell survival pathways. TNF is thought to possess the capacity to promote opposing signals that impact cell survival. TNF can trigger apoptosis. However, TNF can also activate NF-kB that leads to induction of anti-apoptotic factors, which antagonize the pro-apoptotic effects of TNF (Beutler, Tumor Necrosis Factors. Raven Press, New York, 1991). Ln the end, the balance of these opposing signals dictates the ultimate outcome of a cell's fate.
  • DNA fragmentation was monitored to determine if LKKi plays a role in TNF- mediated cell survival signaling. DNA fragmentation is a biochemical parameter indicative of cell death.
  • HEK-IKKi cell lines subjected to TNF treatment were used. In the presence of serum, which provides a variety of factors that promote cell survival signals, TNF did not induce DNA fragmentation in either HEK or HEK-IKKi cells. However, under similar conditions, the HEK-LKKiKM cells displayed a slight increase in DNA fragmentation indicating a modest sensitivity to TNF-induced cell death (FIG. 2A). In contrast, the absence of serum sensitized HEK and HEK-LKKiKM cells, but not
  • HEK-IKKi cells to TNF-induced cell death. Therefore, expression of LKKi is sufficient to confer protection of HEK cells from TNF-mediated cytotoxicity.
  • EGF epidermal growth factor
  • Example 10 LKKi is independent from the LKK signalsome and NF-kB activation
  • the underlying mechanism by which LKKi confers its protective effect on cells against apoptosis was investigated.
  • High-density gene array analysis indicated that A20 mRNA was moderately elevated in HEK-LKKi cells relative to that in HEK-LKKiKM and HEK cells.
  • A20 has been shown to inhibit the pro-apoptotic effects induced by TNF (Dixit et al, J. Biol. Chem.. 265:2973 (1990); Opipari et al, J. Biol. Chem..
  • A20-def ⁇ cient mice are hypersensitive to TNF-induced apoptosis (Lee et al, Science, 289:2350 (2000)). Therefore, the level of A20 mRNA in the HEK, HEK-IKKi and HEK- IKKiKM cells that were cultured under conditions identical to that described for the cell survival studies was examined (FIG. 2 A and FIG. 2B). The cells were treated with TNF alone, or in combination with EGF, and subsequently monitored for A20 expression via northern blot analysis. A20 expression was strongly induced in the HEK-IKKi cells in response to co-stimulation with TNF and EGF.
  • A20 induction was completely absent in the HEK-LKKiKM cells (FIG. 2D).
  • the levels of Egr-1 , a well-documented EGF- regulated gene (Liu et al, Blood, 96:1772 (2000)), and GAPDH mRNA were not altered in these cells.
  • IKKi potentiates the induction of A20 gene expression in response to co- stimulation with TNF and EGF.
  • LKKi functions to promote cell survival in a manner that is independent of the NF-kB pathway.
  • the activities of endogenous LKKi and the LKK signalsome in all three HEK cell lines was measured using the same conditions in which LKKi-dependent changes in A20 gene expression were observed (FIG. 2D).
  • the activity of LKKi was not induced by either co- stimulation with EGF, TNF, or EGF and TNF.
  • stimulus-induced LKK signalsome activity was not affected by expression of either IKKi or IKKiKM (FIG. 2E).
  • Example 11 LKKi mediates changes in pro-inflammatory gene expression and C/EBP signaling
  • LKKi The role of LKKi in regulating immune and inflammatory responses was investigated. High-density gene array analysis performed on the HEK and HEK-LKKi cells suggested that LKKi affects TNF-induced expression of several chemokines, including Rantes, LP-10 and MCP-1. An RNase protection assay was used to confirm these findings. The results show that Rantes and LP-10 were markedly induced by TNF in the ITEK-LKKi cells, but not the HEK and HEK-IKKiKM cells (FIG. 3 A). Thus, LKKi is thought to play an important role in the regulation of TNF-mediated gene expression.
  • Example 12 The role of LKKi in PMA-mediated gene expression HEK, HEK-LKKi and HEK-LKKiKM cells were stimulated with PMA, and the expression of several diverse genes was monitored by northern blot analysis. Expression of the proinflammatory chemokine IL-8 was strongly induced in the HEK-LKKi cell line (FIG. 3B). There was only a modest induction of IL-8 by PMA in the HEK cells, which was inhibited in the HEK-LKKiKM cells. In addition, HEK-LKKi cells also displayed elevated expression of IkB ⁇ in response to PMA.
  • NF-kB was not activated to a greater extent in the HEK-LKKi cells relative to HEK or HEK-LKKiKM cells (FIG. ID). Therefore, activation of NF-kB is unlikely to be the mechanism by which IKKi functions to promote the expression of IkB ⁇ . In contrast to PMA, a significant effect on the expression of IL-8 or IkB ⁇ by TNF in the HEK-LKKi cells (FIG. 3C) was not observed. Thus, LKKi displays both stimulus and gene specific signaling in the LKKi HEK cell lines. In all cases, there were no changes in the expression of Ergl and GAPDH as a function of IKKi expression.
  • LKKi Since PMA is a mimetic of T cell receptor signaling and an activator of PKC, it seems reasonable that LKKi works in concert with physiologic stimuli that modulate these same cellular pathways.
  • IKKi effects on C/EBP DNA binding was analyzed in nuclear extracts from HEK-LKKi and HEK-LKKiKM cells stimulated with PMA, TNF, or TNF in combination with EGF to establish whether IKKi affects C/EBP signaling.
  • EMSA indicated that nuclear extracts from HEK-LKKi and HEK-LKKiKM cells display some differences in their pattern of C/EBP DNA-binding activity. These differences become more obvious in the super-shift studies using antibodies specific for either C/EBP ⁇ or C/EBP ⁇ (FIG. 4A).
  • Example 15 Phosphorylation status of C/EBP ⁇
  • the phosphorylation status of C/EBP ⁇ was analyzed by using metabolic labeling experiments carried out on HEK-TKKi and HEK-IKKiKM cells stimulated with PMA.
  • Nuclear extracts were prepared from 32 P-labeled cells and subjected to immunoprecipitation using antibodies against either C/EBP ⁇ or the p65/RelA subunit of NF-kB (as a control), and their phosphorylation status determined by autoradiography.
  • the net phosphorylation of C/EBP ⁇ , or an associated protein was markedly elevated in the HEK-LKKi cells, but not HEK-LKKiKM cells, in response to PMA (FIG. 4C, upper panels).
  • the total protein level of C/EBP ⁇ was measured via western blot analysis and found to be comparable in the HEK-IKKi and HEK-IKKiKM cells (FIG. 4C, lower panels).
  • the net phosphorylation of RelA was not effected by expression of either LKKi or IKKiKM.
  • LKKi effects two parameters of C/EBP biology, DNA-binding activity and phosphorylation.
  • LKKi-mediated effects on gene expression The capacity of LKKi to modulate the expression of genes involved in immune responses through use of HEK cell lines engineered to express LKKi trans genes in a NF-kB- dependent manner has been established herein. Modulation of gene expression by LKKi was further confirmed through use of mouse embryonic fibroblasts (MEFs) derived from LKKi- deficient (LKKi "7" ) and control animals (LKKi +7+ ).
  • MEFs mouse embryonic fibroblasts
  • LKKi-dependent effects on C/EBP activity was examined by determining whether IKKi is required for the appropriate regulation of IL-6 gene expression.
  • LKKi "7" and IKKi + + MEFs were stimulated with either IL-1 or TNF and subsequently monitored for expression of LL-6 by northern blot analysis.
  • LKKi-deficient MEFs were refractory to induction of IL-6 by IL-1 and TNF, whereas the induction of IkB ⁇ was not effected (FIG. 5B).
  • Example 18 Regulation of C/EBP DNA binding activity in LKKi-deficient MEFs EMSA studies revealed a marked reduction in TNF-induced C/EBP DNA-binding activity in the LKKi "7" MEFs as compared to the IKK +7+ MEFs (FIG. 5C). EMSA supershift studies showed that the absence of LKKi significantly affected activation of C/EBP ⁇ and C/EBP ⁇ in response to TNF (FIG. 5D). Ln contrast, the LKKi +7+ and LKKi "7" MEFs displayed nearly identical levels of NF-kB DNA-binding activity in response to TNF, which is consistent with results obtained in the HEK LKKi cell lines (compare FIG. ID and FIG. 5C, right panel). These results indicate that LKKi is not involved in the early events associated with NF-kB activation, but rather modulate a cellular process that acts to regulate C/EBP activity.
  • the promoter region of the C3 gene contains C/EBP sites (32), and C3 expression is regulated by C/EBP ⁇ (Juan et al, Proc. Natl Acad. Sci.. 90:2584 (1993)). While there are no identifiable NF-kB sites in the C3 promoter, transcription of the C3 gene is induced by NF- kB activators including LPS (Pahl, Oncogene, 1 . 8:6853 (1999); Rus et al, J. Immunol.
  • LKK2 "7" MEFs showed LPS-induced I B ⁇ mRNA and NF- kB DNA binding activity that was partially reduced when LKK2 '7" and IKK2 +7+ cells were compared (Li et al, Science, 284:321 (1999))(FIG. 9A and FIG. 9B). Similar results occurred with spontaneously immortalized fibroblasts (imMEFs) derived from LKK2 "7" and control cells (FIG. 9C). The absence of LKK2 revealed a deficiency in LPS-induced C3 induction that is not likely to result from loss of LPS responsiveness.
  • Example 20 p65 deficiency on LPS induction of C3 and IkB ⁇ mRNA
  • the effect of p65 deficiency on LPS induction of C3 and IkB ⁇ mRNA was investigated to determine the role of NF-kB pathway.
  • LPS-mediated induction of both I B ⁇ and C3 mRNAs was completely abolished in p65 '7" imMEFs (FIG. 9D).
  • Fs p65 '7" imMEFs
  • regulation of C3 gene expression is thought to require an intact NF-kB pathway.
  • the role of NF- kB is thought to involve indirect mechanisms, and requires additional gene expression under the control of NF-kB.
  • LPS-induced C3, I ⁇ B ⁇ and IKKi mRNA in IKKi "7" and control MEFs as well as in LKK2 "7” cells was measured (FIG. 10A).
  • LPS-mediated induction of I ⁇ B ⁇ mRNA was reduced in cells lacking IKK2, while LKKi-deficiency had no effect on LPS-induced expression of LkB ⁇ mRNA.
  • the induction of C3 mRNA was abolished in the LKKi "7” cells as well as in the LKK2 "7' cells. There was no observable induction of LKKi mRNA in the LKK2 "7” cells.
  • Extracts from LKK2 "7” , LKK2 + + , LKKi “7” (as negative control) or LKKi +7+ (as positive control) were immunoprecipitated to enrich the samples for LKKi.
  • the resultant immunoprecipitates were separated electrophoretically and then subjected to Western blot analysis (FIG. 11A).
  • Expression of IKKi protein was not detected in LKKi "7” cells.
  • IKKi +7+ or LKK2 + + cells showed detectable IKKi protein expression that was increased after LPS addition.
  • the basal level of TKKi protein was significantly reduced in IKK2-deficient cells. LPS addition failed to up-regulate LKKi expression in this cell type.
  • Example 23 IKKi regulation of LPS-induced C3 gene expression Whether the failure to induce C3 results from the absence of TKKi protein, or whether essential signaling including expression and/or activation of IKK2 is also affected by IKKi- deficiency was examined.
  • Western blot analysis was used to compare the levels of LKK2 protein in exfracts from IKKi "7” , IKKi +7+ , LKK2 "7” (as negative control) or lKK2 +7+ (as positive control) cells.
  • IKKi "7” , IKKi +7+ and LKK2 + + cells were determined to express nearly identical levels of LKK2 protein (FIG. 11B).
  • Example 24 Role of IKKi in innate immunity and inflammatory responses LPS-induced expression of TNF, IL-1, LL-6, LP-10, RANTES and COX-2 mRNA was measured to investigate whether LKKi deficiency altered the rate and extent of LPS-induced expression of a variety of genes associated with innate immune and inflammatory responses. LPS treatment increased mRNA expression for each of these genes in LKKi + + cells (FIG. 12A). TNF, LL-1 and LL-6 protein levels also increased in response to LPS treatment (FIG. 12B and FIG. 12C). LKKi deficiency resulted in a marked reduction of LPS-induced mRNA expression for each of this group of genes (FIG. 12A).
  • Example 25 Promoter analysis Comparative analysis of the promoters of the group of LKKi-modulated genes depicted in FIG. 12A identified the presence of binding sites for multiple transcription factors. These transcription factors included NF-kB, LRF-3 and C/EBP. Although NF-kB is known to be involved in the regulation all of these genes (Ghosh et al, Annu. Rev. Immunol, 16:225 (1998); Pahl, Oncogene. 18:6853 (1999)), the absence of LKKi is thought to have no effect on activation of the canonical NF-kB pathway. First, LPS-mediated activation of LKK complex activity (FIG.
  • I ⁇ B ⁇ gene a classical NF-kB target gene (Ghosh et al, Annu. Rev. Immunol. 16:225 (1998); Beg et al, Nature, 376:167 (1995)), is not affected by IKKi-deficiency (FIG. 10A and FIG. 10B).
  • Example 26 IKKi regulation of LRF-3 The effect of LPS on activation of interferon (LFN) regulatory factor-3 (LRF-3) in LKKi "7" MEFs was examined because the promoter region of LP-10 gene contains an LFN stimulus responsive element (LSRE), a binding site for transcription factor IRF-3 (Ohmori and Hamilton, J. Biol Chem., 268:6677 (1993); Kawai et al, J. Immunol.. 167:5887 (2001)). LPS treatment induced essentially the same levels of ISRE binding activity in IKKi "7” and LKKi +7+ MEFs (FLG. 12F). Thus, it is thought that LKKi is not involved in regulation of LRF- 3.
  • LFN interferon regulatory factor-3
  • C/EBP ⁇ is involved in IL-1 -induced regulation of the C3 gene (Juan et al, Proc. Natl. Acad. Sci.. 90:2584 (1993)). Moreover, C/EBP ⁇ appears to synergize with C/EBP ⁇ , another member of the C/EBP family, in transcriptional regulation of the IL-6 gene (Akira et al, EMBO J.. 9:1897 (1990); Kinoshita et al, Proc. Natl Acad. Sci.. 89:1473 (1992); Hu et al, J. Immunol, 160:2334 (1998)). LPS induction of C/EBP ⁇ and C/EBP ⁇ mRNA is reported herein, and is shown to occur to an identical extent in both LKKi "7" and LKKi +7+ MEFs (FIG. 12A).
  • results obtained from siRNA- transfected human umbilical vein endothelial cells demonstrated that specific reduction of LKKi transcripts leads to significant inhibition of LL-6 mRNA induced by LPS (FLG. 13D).
  • LPS induction of IkB ⁇ mRNA was unchanged by the reduction of IKKi expression (FIG. 13D).
  • Example 29 LKKi links NF-kB and C/EBP pathways It is thought that IKKi acts through C/EBP ⁇ to link the NF-kB and C/EBP pathways based on the results reported herein. Results obtained with LKK2 "7" MEFs indicate that activation of NF-kB is required for induction of IKKi and C3 mRNA. p65 “7” and IKK2 "7” cells were used to address this possibility.
  • LKKi regulation LKKi regulation through NF-kB, C/EBP, or through contribution from both pathways was investigated.
  • the 5 '-untranslated regions of human and mouse DNA in the vicinity of the IKKi gene was analyzed. The analysis did not reveal the presence of NF-kB sites, but rather indicated that both the murine and human genes have identical sequences containing a C EBP-like DNA binding site (FLG. 14C).
  • FLG. 14C C EBP-like DNA binding site
  • electrophoretic gel shift assay carried out on a nuclear extract prepared from LPS-freated cells, it was determined that this sequence has C/EBP-specific binding activity in vitro (FIG. 14D). Interaction of C EBP with the IKKi promoter was also demonstrated via chromatin immunoprecipitation (ChLP) assay.
  • ChLP chromatin immunoprecipitation
  • Example 31 LKKi is required for gene activation by TNF and IFN ⁇ through C/EBP pathway Biological responses to both IFN ⁇ and TNF are mediated mainly by the regulation of gene transcription. Because the transcription of genes encoding immuno-modulators such as LP-10, Rantes and IL-6 is induced in response to IFN ⁇ as well as to TNF, these immuno- modulators were used as a three-point marker for initial studies to investigate the possible role of LKKi in responses to LFN ⁇ and TNF. In this Example, mouse embryonic fibroblasts (MEFs) were isolated from mice bearing a targeted deletion of LKKi gene.
  • MEFs mouse embryonic fibroblasts
  • IKKi was directly provided to the LKKi "7" cells by transient transfection of IKKi "7" MEF. These MEFs were transfected with the pkB-LKKi expression plasmid, in which transcription of LKKi cDNA was controlled by NF- ⁇ B driven promoter to avoid non-specific effects of over-expression.
  • Tyrosine phosphorylation of STATl is a critical step in activation of the STAT pathway by LFN ⁇ ; phosphorylation-dependent degradation of an inhibitor of NF- ⁇ B (I ⁇ B ⁇ ) and phosphorylation of p65, a key subunit of NF- ⁇ B, are obligatory for activation of the NF- ⁇ B pathway by TNF.
  • I ⁇ B ⁇ inhibitor of NF- ⁇ B
  • p65 a key subunit of NF- ⁇ B
  • the promoter regions of LP-10, Rantes and IL-6 genes contain binding sites for multiple transcription factors, including NF- ⁇ B, IRFs, STAT and C/EBP, and different franscription factors normally collaborate to achieve optimal signal-induced franscription of these genes.
  • the transcription factors such as the p65 subunit of NF- ⁇ B and members of the C/EBP family, especially C/EBP ⁇ and C/EBP ⁇ , play a key role in transcriptional regulation of the IL-6 gene.
  • LFN ⁇ has no effect on activation of the canonical NF- ⁇ B pathway in MEFs, and TNF activation of the NF- KB pathway by means of phosphorylation of the p65 (Fig. 16B) and franscription of NF- ⁇ B were indistinguishable when LKKi + + and LKKi "7" cells were compared (Fig. 16C).
  • Example 32 LKKi is required for activation of genes encoding immune and inflammatory modulators regulated by C/EBP ⁇ and NF- ⁇ B pathways.
  • the Nos2 promoter contains a C/EBP binding motif, but the role of C/EBP in its regulation by LFN ⁇ and TNF is unclear (Goldring CEP Reveneau S, et al, 1996). TNF- and LFN ⁇ -related functions including innate and cellular immunity, and inactivation of bacterial infections, were significantly impaired by C/EBP ⁇ -deficiency, whereas a production of nitric oxide was comparable in C/EBP ⁇ "7" and C/EBP ⁇ + + proteose peptone-elicited macrophages treated with LFN ⁇ plus LPS (Tanaka, 1995).
  • C/EBP-NF- ⁇ B target genes such as IL-6 and JE/MCP-1 (Hu H-M et al, 1998).
  • C/EBP, STAT and NF- ⁇ B pathways are also considered to compensate for the absence of C/EBP ⁇ in transcriptional regulation of genes induced by LFN ⁇ plus LPS or TNF.
  • One compensatory mechanism may be the redundancy of ⁇ , ⁇ and ⁇ subunits of C/EBP in the LPS-induced franscription of C/EBP-NF- ⁇ B target genes such as IL-6 and JE/MCP-1 (Hu H-M et al, 1998).
  • Another possibility is the cooperation between C/EBP, STAT and NF- ⁇ B pathways according to sequential model of induction for acute phase genes regulated in response to activation of NF- ⁇ B and STAT by circulating cytokines such as TNF, LL-l ⁇ , LL- 6 and LFN ⁇ (Poli V, 1998).
  • LKKi appears to be essential for activation of the C EBP pathway by LPS as well as by TNF and LFN ⁇ (Fig.15 and 16). LKKi-deficient cells may represent a good system to dissect or address these issues.
  • LKKi '7" and p65 "7" animals revealed that the regulation of inducible ("an inducible” function of LKKi) but not the basal ("a basal" function of IKKi) expression of TKKi was impaired in cells lacking the p65 subunit of NF- ⁇ B.
  • the p65 subunit is an essential subunit for TNF-induced gene transcription regulated by NF- ⁇ B (Beg et al, 1995; Hoffmann et al, 2003).
  • Other workers have observed that TNF and LPS do not alter LKKi kinase activity but, rather, up- regulate its expression (Shimada et al, 1999; Peters et al, 2000; Kravchenko et al, 2003).
  • TNF+JJFN or LPS+LFN ⁇ (as a positive control) responses in p65 "7" , LKKi “7” and wild type MEFs were compared by northern blot analysis (Fig. 18A and B). Ln these experiments, the activation of several marker-genes that are known to be regulated by NF- ⁇ B, STATl or C/EBP were measured. Table 1 provides a listing of the genes whose expression was monitored, as well as a summary of the transcription factors shown by the inventors and others to operate on those genes. Table 1 : Distinct transcription factor requirements for inducible transcription of different genes.
  • LKKi "7" MEFs Fig. 18E and 18F.
  • p65-def ⁇ cient cells exhibited induction of C/EBP ⁇ expression in response to LPS+LFN ⁇ , but not to TNF+LFN ⁇ , however, C/EBP ⁇ - specific DNA binding activity was impaired in both cases (Fig. 18A, B, E and F).
  • the basal levels of expression of LKKi were not significantly impaired in p65 "7” cells, however, the inducible expression of LKKi mRNA and protein was observed only in wild type MEFs stimulated by TNF+LFN ⁇ (Fig. 18A and G).
  • the induction of LKKi gene expression depends on p65 subunit of NF- ⁇ B whereas "an inducible" function of LKKi is required for optimal activation of C/EBP ⁇
  • Example 33 LKKi is a key molecule coupling the inflammatory responses to TNF and LFN ⁇ with adipocyte differentiation through C/EBP pathway.
  • RNA was prepared from the same sets of cells and used for Northern blot analysis.
  • C/EBP ⁇ and C/EBP ⁇ mRNA expression was induced and DNA binding complexes containing C/EBP ⁇ and C/EBP ⁇ were detected (Fig. 19 B, C and D).
  • the reduction of C/EBP ⁇ -specific DNA binding activity on the second day of treatment was in an accord with the down-regulation of C/EBP ⁇ mRNA.
  • the reduction of C/EBP ⁇ -specific DNA binding activity in nuclear extracts from LKKi +/+ MEFs treated with DM for two days was not accompanied by a down-regulation of C/EBP ⁇ mRNA (Fig.
  • Example 34 LKKi is required for survival of growth-arrested cells.
  • LKKi may modulate cell survival, particularly under conditions involving suppression of cell growth.
  • differentiation inducers such as differentiation media (DM)
  • DM differentiation media
  • clonal expansion adipocyte specific genes
  • aP2 adipocyte specific genes
  • C EBP ⁇ expression levels are significantly up-regulated during clonal expansion phase of adipogenesis and then diminish upon growth arrest at confluence (Cao et al, Genes Gev., 5:1538 (1991; Hwang et al, 1997).
  • LKKi is required for gene activation by MDP and A ⁇ .
  • Long-term treatment of cells with cytokines such as TNF and LFN ⁇ mimics the events associated with chronic inflammatory diseases such as Crohn's and Alzheimer's diseases.
  • Crohn's disease results in loss of cellular responses to bacterial components such as MDP (muramyl dipeptide, a derivative of bacterial lipopolysaccharide).
  • Alzheimer's disease is believed to result in the effects of amyloid peptide beta (A ⁇ ) on gene expression.
  • LKKi "7" MEFs The inability of LKKi "7" MEFs to activate inflammatory genes prompted further experimentation to investigate whether LKKi-deficiency leads to alterations in A ⁇ - or MDP-mediated expression of a variety of genes for pro-inflammatory regulators such as IL-6, MCP- 1 , Nos2 and Rantes.
  • a ⁇ +LFN ⁇ or MDP+LFN ⁇ treatment induced mRNA expression of MCP- 1, IL-6, LRF- 1, Nos2 and Rantes genes in LKKi +7+ cells (Fig. 21 A and B).
  • LKKi-deficiency resulted in a marked reduction of A ⁇ - and MDP-mediated mRNA expression in these genes (Fig. 21 A and B).
  • Parallel changes in the amounts of nitric oxide and IL-6 produced were also observed in LKKi " ' 7' ' and LKKi "7” MEFs (Fig. 7C and D), confirming the results of the RNA analysis.
  • C/EBP CCAAT/enhancer binding
  • a reference to "a host cell” includes a plurality (for example, a culture or population) of such host cells, and so forth.
  • a host cell includes a plurality (for example, a culture or population) of such host cells, and so forth.
  • the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein.
  • the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.

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Abstract

La présente invention a trait à des procédés pour l'utilisation d'agents qui accroissent ou diminuent l'expression ou l'activité de IKKi pour la régulation de processus cellulaires, tels que l'inflammation, la différenciation cellulaire, la prolifération cellulaire, la régénération tissulaire, la neurodégénérescence et l'activation du complément. L'invention a également trait à un procédé d'identification d'agents qui accroissent ou diminuent l'expression ou l'activité de IKKi.
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WO2005034978A2 (fr) * 2003-10-02 2005-04-21 Xantos Biomedicine Ag Utilisation medicale de l'ikk$g(e) ou de ses inhibiteurs
WO2009120801A2 (fr) 2008-03-25 2009-10-01 The Regents Of The University Of Michigan Thérapies inhibitrices d’ikki et procédés de criblage, et diagnostics d’ikki apparentés
WO2015070027A1 (fr) * 2013-11-07 2015-05-14 University Of Southern California Utilisation d'inhibiteurs d'ikk epsilon pour activer nfat et la réponse des lymphocytes t
WO2017132538A1 (fr) 2016-01-29 2017-08-03 The Regents Of The University Of Michigan Analogues d'amlexanox
US9944652B2 (en) 2013-05-02 2018-04-17 The Regents Of The University Of Michigan Deuterated amlexanox
US9987242B2 (en) 2015-05-05 2018-06-05 Northwestern University Treatment of Levodopa-induced Dyskinesias
US10245255B2 (en) 2011-02-14 2019-04-02 The Regents Of The University Of Michigan Compositions and methods for the treatment of obesity and related disorders
US10435375B2 (en) 2015-05-05 2019-10-08 Northwestern University CXCR4 chemokine receptor modulators
US11246871B2 (en) 2016-07-15 2022-02-15 Northwestern University Targeting adenosine A2A receptors for the treatment of levodopa-induced dyskinesias
US11554103B2 (en) 2016-11-10 2023-01-17 Northwestern University Compositions and methods to reduce pharmaceutical-induced toxicity

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Cited By (16)

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WO2005034978A3 (fr) * 2003-10-02 2005-06-23 Xantos Biomedicine Ag Utilisation medicale de l'ikk$g(e) ou de ses inhibiteurs
WO2005034978A2 (fr) * 2003-10-02 2005-04-21 Xantos Biomedicine Ag Utilisation medicale de l'ikk$g(e) ou de ses inhibiteurs
WO2009120801A2 (fr) 2008-03-25 2009-10-01 The Regents Of The University Of Michigan Thérapies inhibitrices d’ikki et procédés de criblage, et diagnostics d’ikki apparentés
WO2009120801A3 (fr) * 2008-03-25 2010-02-18 The Regents Of The University Of Michigan Thérapies inhibitrices d’ikki et procédés de criblage, et diagnostics d’ikki apparentés
JP2011518126A (ja) * 2008-03-25 2011-06-23 ザ リージェンツ オブ ザ ユニバーシティ オブ ミシガン ＩＫＫｉ阻害剤の処置方法およびスクリーニング方法、ならびに関連するＩＫＫｉ診断方法
US10245255B2 (en) 2011-02-14 2019-04-02 The Regents Of The University Of Michigan Compositions and methods for the treatment of obesity and related disorders
US10590142B2 (en) 2013-05-02 2020-03-17 The Regents Of The University Of Michigan Deuterated amlexanox
US9944652B2 (en) 2013-05-02 2018-04-17 The Regents Of The University Of Michigan Deuterated amlexanox
WO2015070027A1 (fr) * 2013-11-07 2015-05-14 University Of Southern California Utilisation d'inhibiteurs d'ikk epsilon pour activer nfat et la réponse des lymphocytes t
US10370664B2 (en) 2013-11-07 2019-08-06 University Of Southern California Use of IKK epsilon inhibitors to activate NFAT and T cell response
US9987242B2 (en) 2015-05-05 2018-06-05 Northwestern University Treatment of Levodopa-induced Dyskinesias
US10435375B2 (en) 2015-05-05 2019-10-08 Northwestern University CXCR4 chemokine receptor modulators
US10214536B2 (en) 2016-01-29 2019-02-26 The Regents Of The University Of Michigan Amlexanox analogs
WO2017132538A1 (fr) 2016-01-29 2017-08-03 The Regents Of The University Of Michigan Analogues d'amlexanox
US11246871B2 (en) 2016-07-15 2022-02-15 Northwestern University Targeting adenosine A2A receptors for the treatment of levodopa-induced dyskinesias
US11554103B2 (en) 2016-11-10 2023-01-17 Northwestern University Compositions and methods to reduce pharmaceutical-induced toxicity

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