WO2003048202A2 - Nf-kappab activating genes - Google Patents

Nf-kappab activating genes Download PDF

Info

Publication number
WO2003048202A2
WO2003048202A2 PCT/JP2002/012644 JP0212644W WO03048202A2 WO 2003048202 A2 WO2003048202 A2 WO 2003048202A2 JP 0212644 W JP0212644 W JP 0212644W WO 03048202 A2 WO03048202 A2 WO 03048202A2
Authority
WO
WIPO (PCT)
Prior art keywords
protein
activation
seq
gene
polynucleotide
Prior art date
Application number
PCT/JP2002/012644
Other languages
French (fr)
Other versions
WO2003048202A3 (en
Inventor
Akio Matsuda
Shuji Muramatsu
Original Assignee
Asahi Kasei Pharma Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from JP2001368692A external-priority patent/JP2006166701A/en
Priority claimed from JP2002291302A external-priority patent/JP2006166705A/en
Application filed by Asahi Kasei Pharma Corporation filed Critical Asahi Kasei Pharma Corporation
Priority to AU2002349784A priority Critical patent/AU2002349784A1/en
Publication of WO2003048202A2 publication Critical patent/WO2003048202A2/en
Publication of WO2003048202A3 publication Critical patent/WO2003048202A3/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a protein capable of activating NF- ⁇ B, a DNA sequence encoding the protein, a method for obtaining the DNA, a recombinant vector containing the DNA, a transformant containing the recombinant vector, and an antibody which specifically reacts with the protein.
  • the present invention also relates to use of the protein, DNA or antibody of the invention in the diagnosis, treatment or prevention of diseases associated with the excessive activation or inhibition of NF- K B.
  • the present invention also relates to a method for screening a substance capable of inhibiting or promoting NF- K B activation by using the protein, DNA, recombinant vector and transformant.
  • NF- K B (nuclear factor kappa B) plays an important role in the transcriptional regulation of various genes involved in inflammation and immunological reactions.
  • NF- K B is a homo- or heterodimer protein which belongs to the Rel family. In unstimulated conditions, NF- K B normally resides in the cytoplasm as an inactive form by forming a complex with an I K B (inhibitory protein of NF- K B) to mask the nuclear transport signal of NF- K B.
  • I K B When cytokines such as interleukin (IL)-1 and tumor necrosis factor (TNF)- stimulate cells, I K B is phosphorylated by IKK (I B kinase) and degraded by the 26S proteasome through ubiquitination. The released NF- K B moves to the nucleus, where it binds to the DNA sequence called the NF- K B binding sequence and induces the transcription of the gene under control of NF- K B.
  • NF- K B is believed to regulate the expression of genes such as those for immunoglobulins, inflammatory cytokines (e.g., IL-1 and TNF- ), interferons and cell adhesion factors.
  • NF- K B is involved in inflammation and immune responses through the expression induction of these genes.
  • NF- K B The inhibition of the function or activation of NF- K B may inhibit the expression of many factors (proteins) involved in inflammatory or immunological diseases or other diseases such as tumor proliferation.
  • NF- / B is a promising target for medicaments against diseases caused or characterized by autoimmunity or inflammation [see e.g., Clinical Chemistry 45, 7-17 (1999); J Clin. Pharmacol. 38, 981-993 (1998); Gut 43, 856-860 (1998); The New England Journal of Medicine 366, 1066-1071 (1997); TiPS 46-50 (1997); The FASEB Journal 9, 899-909 (1995); Nature 395, 225-226 (1998); Science 278, 818-819 (1997); Cell 91, 299-302 (1997)].
  • Extracellular information is converted into a certain signal, which passes through the cell membrane and goes through the cytoplasm to the nucleus, where it regulates the expression of the target gene and causes cell responses. Therefore the elucidation of the mechanism of intracellular signal transduction from extracellular stimuli to NF- K B activation is of very important significance, because it provides very important means of developing new medicaments or therapies against autoimmune diseases and diseases exhibiting inflammatory symptoms.
  • NF- K B activation includes many steps mediated by various transmitters such as protein kinases. Therefore it is desirable for more efficient drug discovery to identify the transmitters which play a key role in the pathway, and to focus research on the transmitters to establish a new drug-screening method.
  • Some signaling molecules involved in NF- K B activation have been identified [e.g., IKK, ubiquitination enzymes and the 26S proteasome described above, as well as TNF receptor associated factor 2 (TRAF2) and NF- K B inducing kinase (NIK)].
  • TNF receptor associated factor 2 TNF receptor associated factor 2
  • NIK NF- K B inducing kinase
  • the object of the present invention is to identify a new gene and protein capable of directly, or indirectly, activating NF- K B, and to provide a method of use of them in medicaments, diagnostics and therapy. That is, an object of the present invention is to provide a new protein capable of activating NF- K B, a DNA sequence encoding the protein, a recombinant vector containing the DNA, a transformant containing the recombinant vector, a process for producing the protein, an antibody directed against the protein or a peptide fragment thereof, and a process for producing the antibody.
  • Another object of the present invention is to provide a method for screening a substance capable of inhibiting or promoting NF- K B activation using the protein, the DNA, the recombinant vector or the transformant, a kit for the screening, a substance capable of inhibiting or promoting NF- K B activation obtainable by the screening method or the screening kit, a process for producing the substance, a pharmaceutical composition containing a substance capable of inhibiting or promoting NF- K B activation, etc.
  • the present inventors have intensively studied to solve the above problems. As a result, the present inventors have succeeded in constructing a full-length cDNA library by using the oligo-capping method; establishing a gene function assay system by expression cloning using 293-EBNA cells; and isolating a new DNA (cDNA) encoding a protein having a function of activating NF- K B by using the assay system.
  • This new DNA molecule induced NF- K B activation by its expression in 293-EBNA cells.
  • This result shows that this new DNA is a signal transduction molecule involved in NF- K B activation.
  • the present invention has been completed.
  • a purified protein selected from the group consisting of: (a) a protein which comprises an amino acid sequence represented by any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,
  • a protein that activates NF- K B (Nuclear factor kappa B) and comprises an amino acid sequence having at least one amino acid deletion, substitution or addition in an amino acid sequence represented by any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170,
  • a protein which comprises an amino acid sequence represented by any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206,
  • a protein that activates NF- K B comprises an amino acid sequence having at least one amino acid deletion, substitution or addition in an amino acid sequence represented by any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,
  • a polynucleotide sequence which encodes a protein that activates NF- K B and consists of a polynucleotide sequence having at least one nucleotide deletion, substitution or addition in a polynucleotide sequence represented by any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151,
  • a polynucleotide sequence which encodes a protein that activates NF- K B and consists of a polynucleotide sequence having at least one nucleotide deletion, substitution or addition in a polynucleotide sequence represented by a coding region of any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,
  • An isolated polynucleotide comprising a nucleotide sequence which encodes a protein that activates NF- K B and has at least 95% identity to the polynucleotide sequence according to (3) over the entire length thereof.
  • An isolated polynucleotide comprising a nucleotide sequence which encodes a protein that activates NF- K B and has at least 95% identity to the polynucleotide sequence according to (4) or (5) over the entire length thereof.
  • a recombinant vector which comprises a polynucleotide according to any one of (3) to (7).
  • a process for diagnosing a disease or susceptibility to a disease related to expression or activity of the protein of (1), (2) or (8) in a subject comprising the steps of:
  • a method for screening compounds capable of inhibiting or promoting NF- K B activation which comprises the steps of:
  • a method for screening compounds capable of inhibiting or promoting NF- K. B activation which comprises the steps of:
  • a compound capable of inhibiting or promoting NF- K B activation which is selected by the method for screening according to (15) or (16).
  • a process for producing a pharmaceutical composition which comprises the steps of:
  • a process for producing a pharmaceutical composition which comprises the steps of:
  • a kit for screening a compound capable of inhibiting or promoting NF- K B activation which comprises:
  • (21) A monoclonal or polyclonal antibody or a fragment thereof, which recognizes the protein according to (1), (2) or (8).
  • a ribozyme or deoxyribozyme capable of inhibiting NF- K B activation which has an action of cleavage of RNA that encodes the protein according to (1), (2) or (8) or an action of cleavage of RNA that encodes a protein involved in a pathway leading to NF- K B activation.
  • a method for treating a disease associated with NF- K B activation which comprises administering to a subject a compound screened by the process according to (15) or (16), and/or a monoclonal or polyclonal antibody or a fragment thereof according to (21) or (22), and/or an antisense oligonucleotide according to (24), and/or a ribozyme or deoxyribozyme according to (25) in an effective amount to treat a disease selected from the group consisting of inflammation, autoimmune diseases, cancers, infectious disease, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • the pharmaceutical composition according to (27) for the treatment of inflammation, autoimmune diseases, cancers, infectious disease, bone diseases, AIDS, neurodegenerative diseases and/or ischemic disorders is provided.
  • a method of treating inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases or ischemic disorders which comprises administering a pharmaceutical composition produced by the process according to (18) or (19) to a patient suffering from a disease associated with NF- K B activation.
  • a pharmaceutical composition which comprises a monoclonal or polyclonal antibody or a fragment thereof according to (21) or (22) as an active ingredient.
  • a pharmaceutical composition which comprises an antisense oligonucleotide according to (24) as an active ingredient.
  • a pharmaceutical composition which comprises a ribozyme or deoxyribozyme according to (25) as an active ingredient.
  • composition according to any one of (30) to (32) for the treatment and/or prevention of a disease which is selected from the group consisting of inflammation, autoimmune diseases, infectious diseases, cancers, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • a computer-readable medium on which a sequence data set has been stored comprising at least one of nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175,
  • a method for calculating identity to other nucleotide sequences and/or amino acid sequences which comprises comparing data on a medium according to (34) with data of said other nucleotide sequences and/or amino acid sequences.
  • polypeptides comprising all or a part of the amino acid sequences selected from the group consisting of SEQ D NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,
  • Fig. 1 is a graph showing NF- K B reporter activity inhibition by the proteasome inhibitor MG-132 in Example 3.
  • the axis of abscissa is MG-132 concentration and the transversal axis is relative luciferase activity.
  • a full-length cDNA was produced from mRNA prepared from normal human lung fibroblasts (purchased from Sanko Junyaku Co., Ltd.) and the like, and a full-length cDNA library was constructed in which the cDNA was inserted into the vector pME18S-FL3 (GenBank Accession AB009864).
  • the cDNA library was introduced into E. coli cells, and plasmid preparation was carried out per clone.
  • the pNK K B-LUC reporter plasmid (STRATAGENE) containing a DNA encoding luciferase and the above full-length cDNA plasmid were cotransfected into 293-EBNA cells (Invitrogen).
  • luciferase activity was measured, and the plasmid with significantly increased luciferase activity compared to that of a control experiment (vector pME18S-FL3 is introduced into a cell in place of a full-length cDNA) was selected (the selected plasmid showed a 5-fold or more increase in luciferase activity compared to that of the control experiment), and the entire nucleotide sequence of the cDNA cloned into the plasmid was determined.
  • the protein encoded by the cDNA thus obtained shows that this protein is a signal transduction molecule involved in NF- K B activation.
  • activation of NF- K B refers to activation of NF- K B (including induction of NF- K B activation) when a gene is introduced into a suitable cell and the protein encoded by the gene is excessively expressed.
  • Activation of NF- K B can be measured, for example, by an assay using an NF- K B dependant reporter gene.
  • Activation of NF- / B is reflected by increasing the reporter activity compared to control cells (cells into which the vector only was introduced).
  • Increase in reporter activity is preferably by a factor of 1.5 or more, more preferably by a factor of 2 or more, and still more preferably by a factor of 5 or more.
  • Reporter activity can be measured by cloning a polynucleotide (e.g. cDNA) encoding the protein to be expressed into a suitable expression vector, co-transfecting the expression vector and an NF- K B dependant reporter plasmid into a suitable cell, and after culturing for a certain period, then measuring reporter activity.
  • a polynucleotide e.g. cDNA
  • Suitable expression vectors are well known to those skilled in the art, examples of which include pME18S-FL3, pcDNA3.1 (Invitrogen).
  • the reporter gene can be one which enables a person skilled in the art to easily detect the expression thereof, and examples include a gene encoding luciferase, chloramphenicol acetyl transferase, or ⁇ -galactosidase.
  • Use of a gene encoding luciferase is most preferable, and examples of an NF- K B dependent reporter plasmid include pNF- K B-LUC (STRATAGENE).
  • Suitable cells include cells which exhibit an NF- K B activation response to stimulation by IL-1, TNF- and the like. Examples include 293-EBNA cells. Cell culture and introduction of genes into cells (transfection) can be performed and optimized by a person skilled in the art by known techniques.
  • 293-EBNA cells are inoculated on DMEM medium (Dulbecco's Modified Eagle Medium) containing 5% FBS (Fetal Bovine Serum) in a 96-well cell culture plate to a final cell density of 1 x IO 4 cells/well, and cultured for 24 hours at 37°C in the presence of 5% CO 2 .
  • reporter plasmid pNF- K B-Luc (STRATAGENE) and the expression vector are cotransfected into the cells in a well using FuGENE 6 (Roche).
  • NF- rc B activation is then measured by measuring luciferase activity using a long term luciferase assay system, Picagene LT2.0 (Toyo Ink Mfg).
  • Picagene LT2.0 Toyo Ink Mfg
  • luciferase activity can be measured using PerkinElmer's Wallac ARVOTMST 1420 MUL ⁇ LABEL COUNTER.
  • the method for gene introduction by FuGENE ⁇ , and measurement of luciferase activity by Picagene LT2.0 can be performed respectively according to the attached protocols.
  • the amount of FuGENE ⁇ per 1 well is suitably 0.3 to 0.5 ⁇ 1, preferably 0.3 ⁇ 1; the amount of pNF- K B-Luc plasmid is suitably 50 to lOOng, preferably 50ng; the amount of expression vector is suitably 50-100ng, and preferably lOOng.
  • An ability to activate NF- rc B can be confirmed by an ability to increase the reporter activity (luciferase activity) relative to the control experiment (cells into which only a null vector was introduced).
  • Increase in reporter activity is preferably by a factor of 1.5 or more, more preferably by a factor of 2 or more, and still more preferably by a factor of 5 or more.
  • a protein which comprises an amino acid sequence, which has at least 95% identity, preferably at least 97-99% identity, to one of the above amino acid sequences over the entire length thereof.
  • identity used herein is a relationship between two or more protein sequence or two or more polynucleotide sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between protein or polynucleotide sequences, as determined by the match between protein or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
  • Identity and similarity can be readily calculated by known methods. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs.
  • BLAST Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. Biol., 215: p403-410 (1990), Altschul SF, Madden TL, Schaffer AA, Zhang Z, MiUer W, Lipman DJ,. Nucleic Acids Res. 25: p3389-3402 (1997)).
  • BLAST Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. Biol., 215: p403-410 (1990)
  • Altschul SF Madden TL
  • Schaffer AA Zhang Z, MiUer W, Lipman DJ,. Nucleic Acids Res. 25: p3389-3402 (1997).
  • the main initial conditions generally used in a BLAST search are as follows, but are not limited to these.
  • An amino acid substitution matrix is a matrix numerically representing the degree of analogy of each pairing of each of the 20 types of amino acid, and normally the default matrix of BLOSUM62 is used.
  • the theory of this amino acid substitution matrix is shown in Altschul S.F., J. Mol. Biol. 219: 555-565 (1991), and apphcability to DNA sequence comparison is shown on States D. J., Gish W., Altschul S.F., Methods, 3: 66-70 (1991).
  • optimal gap cost is determined by experience and in the case of BLOSUM62 preferably parameters of Existence 11, Extension 1 are used.
  • the expected value (EXPECT) is the threshold value concerning statistical significance for a match with a database sequence, and the default value is 10.
  • a protein having, for example, 95% or more identity to the amino acid sequence of SEQ ID NO: 2 may contain in the amino acid sequence up to 5 amino acid changes per 100 amino acids of the amino acid sequence of SEQ ID NO: 2.
  • a protein having 95% or more amino acid sequence identity to a subject amino acid sequence may have amino acids up to 5% of the total number of amino acids within the subject sequence, deleted or substituted by other amino acids, or amino acids up to 5% of the total number of amino acids within the subject sequence may be inserted within the subject sequence.
  • These changes within the subject sequence may exist at the amino terminus or the carboxy te ⁇ ninus of the subject sequence, or may exist at any position between these termini, or may form one or more groups of changes.
  • the Examples described below demonstrate that the protein consisting of an amino acid sequence of any one of the above SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110.
  • a polynucleotide which encodes a protein that activates NF- r B, and comprises a nucleotide sequence which has at least 95% identity, preferably at least 97-99% identity to any one of the above sequences;
  • Polynucleotides which are identical or almost identical to nucleotide sequences contained in the above nucleotide sequences may be used as hybridization probes to isolate full-length cDNA and genomic clones encoding the protein of the present invention, or cDNA or genomic clones of other genes that have a high sequence similarity to the above sequences, or genomic clones, or may be used as primers for nucleic acid amplification reactions.
  • these nucleotide sequences are 70% identical, preferably 80% identical, more preferably 90% identical, most preferably 95% identical to the above sequences.
  • the probes or primers will generally comprises at least 15 nucleotides, preferably 30 nucleotides and may have 50 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides. Particularly preferred primers have between 20 and 25 nucleotides.
  • the polynucleotide of the present invention may be either in the form of a DNA such as cDNA ,a genomic DNA obtained by cloning or synthetically produced, or may be in the form of RNA such as mRNA.
  • the polynucleotide may be single-stranded or double-stranded.
  • the double-stranded polynucleotides may be double-stranded DNA, double- stranded RNA or DNA:RNA hybrid.
  • the single-stranded polynucleotide may be sense strand also known as coding strand or antisense strand also known as non-coding strand.
  • a protein having the same activity that activates NF- K B as the protein having an amino acid sequence of any one of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168 170, 172, 174, 176, 178, 180, 182, 184, 186
  • One such method involves using conventional mutagenesis procedures for the DNA encoding the protein.
  • Another method is, for example, site-directed mutagenesis (e.g., Mutan-Super Express Km Kit from Takara Shuzo Co., Ltd.). Mutations of amino acids in proteins may also occur in nature.
  • the present invention also includes a mutated protein which is capable of activating NF- K B and which has at least one amino acid deletion, substitution or addition relative to the protein of any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176
  • substitutions of amino acids are preferably conservative substitutions, specific examples of which are substitutions within the following groups: (glycine, alanine), (valine, isoleucine, leucine), (aspartic acid, glutamic acid), (asparagine, glutamine), (serine, threonine), (lysine, arginine) and (phenylalanine, tyrosine).
  • the present invention also includes a protein that activates NF- K B and comprises an amino acid sequence having a high identity to the amino acid sequence of any one of the above SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180
  • High identity refers to an amino acid sequence having an identity of at least 90%, preferably at least 97 to 99% over the entire length of an amino acid sequence expressed by any one of the above SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,
  • the proteins of the present invention may be natural proteins derived from any human or animal cells or tissues, chemically synthesized proteins, or proteins obtained by genetic recombination techniques.
  • the protein may or may not be subjected to post-translational modifications such as sugar chain addition or phosphorylation.
  • proteins which are encoded by the genes of the present invention include secretion proteins (growth factors, cytokines, hormones, and the like), protein modification enzymes (protein kinase, protein phosphatase, protease, and the like), signal transduction molecules (protein-protein interaction moleculaes and the like), nuclear proteins (nuclear receptor, transcription factors and the like), and membrane proteins .
  • the membrane proteins include receptors, cell adhesion molecules, ion channels, and transporters.
  • the present invention also includes a polynucleotide encoding the above protein of the present invention.
  • the DNA includes cDNA, genomic DNA, and chemically synthesized DNA.
  • the DNA of the present invention includes a DNA which encodes a protein capable of activating NF- K B and hybridizes under stringent conditions with the DNA sequence of the above nucleotide sequence of any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167
  • stringent conditions refer to overnight incubation at 37 °C in a hybridization solution containing 30% formamide, 5 x SSC (0.75 M NaCl, 75mM trisodium citrate), 5 x Denhardt's solution, 0.5% SDS, 100 ⁇ g/ml denatured, sheared salmon sperm DNA) followed by washing (three times) in 2 x SSC, 0.1% SDS for 10 rninutes at room temperature, then followed by washing (two times) in 0.2 x SSC, 0.1% SDS for 10 minutes at 37 °C (low stringency).
  • Preferred stringent conditions are overnight incubation at 42°C in a hybridization solution containing 40% formamide, followed by washing (three times) in 2 x SSC, 0.1% SDS for 10 minutes at room temperature, then followed by washing (two times) in 0.2 x SSC, 0.1% SDS for 10 minutes at 42 °C (moderate stringency). More preferred stringent conditions are overnight incubation at 42T ⁇ in a hybridization solution containing 50% formamide, followed by washing (three times) in 2 x SSC, 0.1% SDS for 10 minutes at room temperature, followed by washing (two times) in 0.2 x SSC, 0.1% SDS for 10 minutes at 50°C (high stringency).
  • the DNA sequence thus obtained must encode a protein capable of activating NF- K B.
  • the present invention also includes a polynucleotide comprising a nucleotide sequence which encodes a protein capable of activating NF- K B and has a high sequence similarity to the nucleotide sequence of the polynucleotide according to above item (3), (4) or (5).
  • these nucleotide sequence are 95% identical, preferably 97% identical, more preferably 98-99% identical, most preferably at least 99% identical to the nucleotide sequence of the polynucleotide according to above item (3), (4) or (5) over the entire length thereof.
  • the above DNA of the present invention can be used to produce the above protein using recombinant DNA techniques.
  • the DNA and peptide of the present invention can be obtained by:
  • Techniques for cloning the DNA encoding the protein of the present invention in the above step (A) include, in addition to the methods described in the specification of the present appHcation, PCR amplification using a synthetic DNA having a part of the nucleotide sequence of the present invention (e.g., any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,
  • a DNA inserted into a suitable vector for example, a commercially available library (e.g., from CLONTECH and STRATAGENE) can be used. Techniques for hybridization are normally used in the art, and can be easily carried out in accordance with various laboratory manuals such as T Maniatis et al., supra. Depending on the intended purpose, the cloned DNA encoding the protein of the present invention can be used as such or if desired after digestion with a restriction enzyme or addition of a linker.
  • the DNA thus obtained may have a nucleotide sequence of any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191,
  • the present invention also provides a recombinant vector, which comprises the above DNA sequence.
  • the expression vector for the protein of the present invention can be produced, for example, by excising the desired DNA fragment from the DNA encoding the protein of the present invention, and ligating the DNA fragment downstream of a promoter in a suitable expression vector.
  • Expression vectors for use in the present invention may be any vectors derived from prokaryotes (e.g., E. coli), yeast, fungi, insect viruses and vertebrate viruses so long as such vectors are replicable.
  • the vectors should be selected to be compatible with microorganisms or cells used as hosts. Suitable combinations of host cell - expression vector systems are selected depending on the desired expression product.
  • Plasmid vectors compatible with these microorganisms are generally used as replicable expression vectors for recombinant DNA molecules.
  • the plasmids pBR322 and pBR327 can be used to transform E. coli.
  • Plasmid vectors normally contain an origin of replication, a promoter, and a marker gene conferring upon a recombinant DNA a phenotype useful for selecting the cells transformed with the recombinant DNA.
  • promoters include a j3 -lactamase promoter, lactose promoter and tryptophan promoter.
  • marker genes include an ampicillin resistance gene, and a tetracycline resistance gene.
  • suitable expression vectors include the plasmids pUC18 and pUC19 in addition to ⁇ BR322, pBR327.
  • YEp24 can be used as a replicable vector.
  • the plasmid YEp24 contains the URA3 gene, which can be employed as a marker gene.
  • promoters in expression vectors for yeast cells include promoters derived from genes for 3- ⁇ hosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase.
  • promoters and terminators for use in expression vectors to express the DNA of the present invention in fungal cells include promoters and terminators derived from genes for phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPD) and actin.
  • suitable expression vectors include the plasmids pPGACY2 and pBSFAHY83.
  • promoters for use in expression vectors to express the DNA of the present invention in insect cells include a polyhedrin promoter and P10 promoter.
  • expression vectors which are suitable for insect cells include baculo virus vector.
  • Recombinant vectors used to express the DNA of the present invention in animal cells normally contain functional sequences to regulate genes, such as an origin of replication, a promoter to be placed upstream of the DNA of the present invention, a ribosome-binding site, a polyadenylation site and a transcription termination sequence.
  • Such functional sequences which can be used to express the DNA of the present invention in eukaryotic cells, can be obtained from viruses and viral substances.
  • Examples of such functional sequences include an SR ct promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter and HSV-TK promoter. Among them, a CMV promoter and SR ⁇ promoter can be preferably used.
  • any promoters can be used so long as they are suitable for use in the above host- vector systems.
  • origins of replication include foreign origins of replication, for example, those derived from viruses such as adenovirus, polyoma virus and SV40 virus. When vectors capable of integration into host chromosomes are used as expression vectors, origins of replication of the host chromosomes may be employed.
  • Suitable expression vectors include the plasmids pSV-dhfr (ATCC 37146), pBPV-l(9-l) (ATCC 37111), pcDNA3.1 (INVITROGEN) and pME18S-FL3.
  • the present invention also provides a transformed cell, which comprises the above recombinant vector.
  • Microorganisms or cells transformed with the replicable recombinant vector of the present invention can be selected from remaining untransformed parent cells based on at least one phenotype conferred by the recombinant vector as memtioned above. Phenotypes can be conferred by inserting at least one marker gene into the recombinant vector. Marker genes naturally contained in replicable vectors can be employed. Examples of marker genes include drug resistance genes such as neomycin resistance genes, and genes encoding dihydrofolate reductase.
  • any of prokaryotes e.g., E. coli
  • microorganisms e.g., yeast and fungi
  • insect and animal cells can be used so long as such hosts are compatible with the expression vectors used.
  • microorganisms include Escherichia coli strains such as E. coli K12 strain 294 (ATCC 31446), E. coli X1776 (ATCC 31537), E. coli C600, E. coh JM109 and E. coli B strain; bacterial strains belonging to the genus Bacillus such as Bacillus subtilis; intestinal bacteria other than E.
  • yeast such as Salmonella typhimurium or Serratia marcescens
  • various strains belonging to the genus Pseudomonas include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris.
  • yeast examples include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris.
  • fungi examples include Aspergillus nidulans, and Acremonium chrysogenum (ATCC 11550).
  • insect cells for example, Spodoptera frugiperda (Sf cells), High FiveTM cells derived from eggs of Trichoplusiani, etc.
  • insect cells for example, Spodoptera frugiperda (Sf cells), High FiveTM cells derived from eggs of Trichoplusiani, etc.
  • animal cells include HEK 293 cells, COS-1 cells, COS-7 cells, Hela cells, and Chinese hamster ovary (CHO) cells.
  • CHO cells and HEK 293 cells are preferred.
  • combinations of expression vectors and host cells to be used vary with experimental objects. According to such combinations, two types of expression (i.e. transient expression and constitutive expression) can be included.
  • Transformation of microorganisms and cells in the above step (C) refers to introducing DNA into microorganisms or cells by forcible methods or phagocytosis of cells and then transiently or constitutively expressing the trait of the DNA in a plasmid or an intra-chromosome integrated form.
  • Those skilled in the art can carry out transformation by known methods [see e.g., "Idenshi Kougaku Handbook (Genetic Engineering Handbook)", an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.].
  • DNA can be introduced into cells by known methods such as DEAE-dextran method, calcium-phosphate-mediated transfection, electroporation, hpofection, etc.
  • selection can be carried out by clonal selection of the animal cells containing the chromosomes into which the introduced expression vectors have been integrated.
  • transformants can be selected using the above selectable marker as an indication of successful transformation, hi addition, the animal cells thus obtained using the selectable marker can be subjected to repeated clonal selection to obtain stable animal cell strains highly capable of expressing the protein of the present invention.
  • DHFR dihydrofolate reductase
  • MTX methotrexate
  • the above transformed cells can be cultured under conditions which permit the expression of the DNA encoding the protein of the present invention to produce and accumulate the protein of the present invention. In this manner, the protein of the present invention can be produced.
  • the present invention also provides a process for producing a protein, which comprises culturing a transformed cell comprising the isolated polynucleotide according to above item (3) to (7) under conditions providing expression of the encoded protein, and recovering the protein from the culture (that is, cells or culture medium).
  • animal cells can be cultured by methods known to those skilled in the art (see e.g., "Bio Manual Series 4", YODOSHA CO., LTD.).
  • animal cells can be cultured by various known animal cell culture methods including attachment culture such as Petri dish culture, multitray type culture and module culture, attachment culture in which cells are attached to cell culture carriers (microcarriers), suspension culture in which productive cells themselves are suspended.
  • attachment culture such as Petri dish culture, multitray type culture and module culture
  • suspension culture in which productive cells themselves are suspended.
  • media for use in the culture include media commonly used for animal cell culture, such as D-MEM and RPMI 1640.
  • suitable combinations of per se known separation and purification methods can be used.
  • methods include methods based on solubility, such as salting-out and solvent precipitation; methods based on the difference in charges, such as ion-exchange chromatography; methods mainly based on the difference in molecular weights, such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis; methods based on specific affinity, such as affinity chromatography; methods based on the difference in hydrophobicity, such as reverse phase high performance liquid chromatography; and methods based on the difference in isoelectric points, such as isoelectric focusing.
  • a protein of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during intracellular synthesis, isolation or purification.
  • the protein of the present invention can also be produced as a fusion protein with another protein. These fusion proteins are also included within the present invention.
  • any vectors can be used so long as the DNA encoding the protein can be inserted into the vectors and the vectors can express the fusion protein.
  • proteins to which a polypeptide of the present invention can be fused include glutathione S-transferase (GST) and a hexa-histidine sequence (6 x His).
  • GST glutathione S-transferase
  • 6 x His hexa-histidine sequence
  • membrane protein When the protein of the present invention is a membrane protein, a transformed cell into which DNA encoding the protein of the present invention has been introduced can express the protein on its membrane.
  • the membrane which is prepared from such transformed cells and contains the protein of the present invention is also included within the present invention.
  • membrane of a cell includes cell membrane, and membrane of cell organelle.
  • the membrane of a cell can be prepared by a method known to those skilled in the art. For example, cells are collected from the culture where transformed cells are cultured, and suspended in a suitable buffer. Then, the cells are lysed by a homogenizer or by vortex after addition of glassbeads.
  • the obtained solution is centrifuged to remove uncrushed cells and the like, and the supernatant is ultracentrifuged under a sutable condition, and the obtained precipitate is suspended in a buffer to prepare a membrabe fraction.
  • the condition for ultracentrifugation can be suitably selected depending on the type of membrane and the like.
  • the present invention also includes a protein capable of inhibiting the activity of the protein of the present invention.
  • proteins include antibodies, or other proteins that bind to active sites of the protein of the present invention, thereby inhibiting the expression of their activity.
  • the present invention also relates to an antibody that reacts with the protein of the present invention or a fragment thereof, and to production of such an antibody. More preferably, the present invention relates to an antibody that specifically react with the protein of the present invention or a fragment thereof, and to production of such an antibody.
  • “specifically” means that closs-reactivity is low, more preferably closs-reactivity is not present.
  • the antibody of the present invention is not specifically limited so long as it can recognize the protein of the present invention.
  • examples of such antibodies include polyclonal antibodies, monoclonal antibodies and their fragments, single chain antibodies and humanized antibodies.
  • Antibody fragments can be produced by known techniques. Examples of such antibody fragments include, but not limited to, F(ab') 2 fragments, Fab' fragments, Fab fragments and Fv fragments.
  • a monoclonal or polyclonal antibody can be produced by administering the protein according to above item (1) or (2) as an antigen or epitope-bearing fragments to a non-human animal.
  • the antibody against the protein of the present invention can be produced by using the protein of the present invention or a peptide thereof as an immunogen according to per se known process for producing antibodies or antisera.
  • Such methods are described, for example, in "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", the third edition, an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.
  • polyclonal antibodies for example, the protein of the present invention or a peptide thereof can be injected to animals such as rabbits to produce antibodies directed against the protein or peptide, and then their blood can be collected.
  • the polyclonal antibodies can be purified from the blood, for example, by ammonium sulfate precipitation or ion-exchange chromatography, or by using the affinity column on which the protein has been immobilized.
  • mice In the case of monoclonal antibodies, for example, animals such as mice are immunized with the protein of the present invention, their spleen is removed and homogenized to obtain spleen cells, which are then fused with mouse myeloma cells by using a reagent such as polyethylene glycol. From the resulting hybrid cells (i.e. hybridoma cells), the clone producing the antibody directed against the protein of the present invention can be selected. Then, the resulting clonal hybridoma cells can be implanted intraperitoneally into mice, the ascitic fluid recovered from the mice. The resulting monoclonal antibody can be purified, for example, by ammonium sulfate precipitation or ion-exchange chromatography, or by using the affinity column on which the protein has been immobilized.
  • a reagent such as polyethylene glycol.
  • the resulting antibody When the resulting antibody is used to administer it to humans, it is preferably used as a humanized antibody or human antibody in order to reduce its immunogenicity.
  • the humanized antibody can be produced using transgenic mice or other mammals.
  • Humanized chimeric antibodies can be produced by linking a V region of a mouse antibody to a C region of a human antibody. Humanized antibodies can be produced by substituting a sequence derived from a human antibody for a region other than a complementarity-determining region (CDR) from a mouse monoclonal antibody.
  • CDR complementarity-determining region
  • human antibodies can be directly produced in the same manner as the production of conventional monoclonal antibodies by immunizing the mice whose immune systems have been replaced with human immune systems. These antibodies can be used to isolate or to identify clones expressing the protein.
  • these antibodies can be used to purify the protein of the present invention from a cell extract or transformed cells producing the protein of the present invention.
  • These proteins can also be used to construct ELISA, RIA (radioimmunoassay) and western blotting systems.
  • ELISA radioimmunoassay
  • RIA radioimmunoassay
  • western blotting systems can be used for diagnostic purposes for detecting an amount of the protein of the present invention present in a body sample in a tissue or a fluid in the blood of an animal, preferably human.
  • they can be used for diagnosis of a disease characterized by undesirable activation of HF- K B resulting from (expression) abnormality of the protein of the present invention, such as inflammation, autoimmune diseases, infectious diseases, cancers, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • a standard value (that is, a normal value for the expression of the protein of the present invention) must be established.
  • a method of calculating the standard value comprises binding a body fluid or a cell extract of normal individual of a human or an animal to an antibody against the protein of the present invention under a suitable condition for the complex formation, detecting the amount of the antibody-protein complex by chemical or physical means and then calculating the standard value for the normal sample using a standard curve prepared from a standard solution containing a known amount of an antigen (the protein of the present invention).
  • the presence of a disease can be confirmed by deviation from the standard value obtained by comparison of the standard value with the value obtained from a sample of an individual latently suffering from a disease associated with the protein of the present invention.
  • These antibodies can also be used as reagents for studying functions of the protein of the present invention.
  • the antibody of the present invention can be used as a medicament as mentioned below.
  • an antibody capable of inhibiting the activity of activating NF- K B of the protein of the present invention that is, neutralizing antibody.
  • the present invention is a pharmaceutical composition which comprises the above antibody as an active ingredient, and a method for therapy and/or prevention using the antibody of the present invention.
  • the active ingredient may be combined with other therapeutically or preventively active ingredients or inactive ingredients (e.g., conventional pharmaceutically acceptable carriers or diluents such as immunogenic adjuvants) and physiologically non-toxic stabilizers and excipients.
  • the resulting combinations can be sterilized by filtration, and formulated into vials after lyophilization or into various dosage forms in stabilized and preservable aqueous preparations.
  • Administration to a patient can be intra-arterial aclministration, intravenous administration and subcutaneous administration, which are well known to those skilled in the art.
  • the dosage range depends upon the weight and age of the patient, route of administration and the like. Suitable dosages can be determined by those skilled in the art.
  • These antibodies exhibit therapeutic activity by inhibiting the NF- K B activation mediated by the protein of the present invention. More specifically, the antibody of the present invention is useful as a medicament for treating or preventing a disease associated with abnormality of NF- K B activity such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • the DNA of the present invention can also be used to isolate, identify and clone other proteins involved in intracellular signal transduction processes.
  • the DNA sequence encoding the protein of the present invention can be used as a "bait" in yeast two-hybrid systems (see e.g., Nature 340:245-246 (1989)) to isolate and clone the sequence encoding a protein ("prey") which can associate with the protein of the present invention.
  • prey a protein which can associate with the protein of the present invention.
  • proteins which can associate with the protein of the present invention can be isolated from cell extracts by immunoprecipitation [see e.g., "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.] using antibodies directed against the protein of the present invention.
  • the protein of the present invention can be expressed as a fusion protein with another protein as described above, and immunoprecipitated with an antibody directed against the fusion protein in order to isolate a protein which can associate with the protein of the present invention.
  • the present invention provides a process for diagnosing a disease or susceptibility to a disease related to expression or activity of the protein of present invention in a subject comprising the steps of:
  • the diagnostic assays offer a process for diagnosing diseases or determining a susceptibility to the diseases through detection of mutation in a gene for the protein of the present invention which has a function of activating NF- K B, by the methods described.
  • diseases may be diagnosised by methods comprising determining from a sample derived from a subject an abonormally decreased or increased level of protein or mRNA.
  • the determination of the presence or absence of a mutation in the nucleotide seqeunce of a the gene encoding the protein of the present invention which has a function of activating NF- K B may involve RT-PCR using a part of the nucleotide sequences of genes encoding these proteins as a primer, followed by conventional DNA sequencing to detect the presence or absence of the mutation.
  • PCR-SSCP Geneomics 5:874-879 (1989); "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.] can also be used to determine the presence or absence of the mutation.
  • Decreased or increased expression of a gene in a sample can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, for example, nucleic acid amplification methods such as RT-PCR, and methods such as RNase protection assay, Northern blotting and other hybridization methods.
  • Assay techniques that can be used to dete ⁇ nine levels of a protein in a sample derived from a host are well-known to those skilled in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western blot analysis and ELISA assays.
  • the antibody of the present invention mentioned above can be used.
  • the degree of abnormality of expression level of gene in a sample is not particularly Umited.
  • the level of the expressed protein is 2 or more times, or 1/2 or less, as compared with normal case, the subject may be disgnosed to be a disease.
  • the level of the expressed protein is 3 or more times, or 1/3 or less, as compared with normal case, the subject may be disgnosed to be a disease.
  • the DNA of the present invention can be used to detect abnormality in the DNA or mRNA encoding the protein of the present invention or a peptide fragment thereof. Therefore, the DNA is useful for gene diagnosis such as detection of damage, mutation, decreased, increased or excessively increased expression of said DNA or mRNA.
  • the mutation may cause a disease associated with the expression and/or activity of NF- /c B.
  • the abnormal expression of the novel protein of the present invention which acts to activate NF- K B may be responsible for diseases associated with the expression and/or activity of NF- K B.
  • the present invention also relates to a method for screening compounds which inhibit or promote NF- /c B activation using the proteins of the invention.
  • a compound which inhibits NF- K B activation has an activity as an inhibitor of NF- K B in vivo or in vitro as a result of this function, while a compound which promotes NF- K B activation has an activity as an activator of NF- K B in vivo or in vitro as a result of this function. Therefore, an activity as an inhibitor or activator of NF- K B is screened in the method for screening of the present invention.
  • the above compounds have an activity as an inhibitor or activator of NF- K B.
  • the method for screening comprises the steps of: (a) preparing a transformed cell by introducing a gene encoding a protein that promotes activation of NF- K B according to the present invention and a gene encoding a signal which can detect activation of NF- K B into a cell;
  • an activator compound a compound that increases said detectable signal 2-fold or higher than normal
  • an inhibitor compound a compound that decreases said detectable signal half or less than normal
  • genes encoding a signal which can detect activation of NF- K B include reporter genes. Reporter genes are used instead of directly detecting the activation of transcription factors of interest. The transcriptional activity of a promoter of a gene is analyzed by linking the promoter to a reporter gene and measuring the activity of the product of the reporter gene ("Bio Manual Series 4" (1994), YODOSHA CO., LTD.).
  • Any peptide or protein can be used so long as those skilled in the art can measure the activity or amount of the expression product (including the amount of the produced mRNA) of the reporter genes. For example, enzymatic activity of chloramphenicol acetyltransferase, ⁇ -galactosidase, luciferase, etc., can be measured.
  • Any reporter plasmids can be used to evaluate NF- K B activation so long as the reporter plasmids have an NF- K B recognition sequence inserted upstream of the reporter gene.
  • pNF- r B-Luc STRATAGEGE
  • Other examples include NF- K B dependent reporter plasmids described in Tanaka S. et al., J. Vet. Med. Sci. Vol.59 (7); Rothe M. et al., Science Vol.269, p.1424-1427 (1995).
  • Any host cells may be used so long as NF- K B activation can be detected in the host cells.
  • Preferred host cells are mammalian cells such as 293-EBNA cells. Transformation and culture of the cells can be carried out as described above.
  • the method for screening a compound which inhibits or promotes NF- K B activation comprises culturing the transformed cell for a certain period of time, adding a certain amount of a test compound, measuring the reporter activity expressed by the cell after a certain period of time, and comparing the activity with that of a cell to which the test compound has not been added.
  • the reporter activity can be measured by methods known in the art (see e.g., "Bio Manual Series 4" (1994), YODOSHA CO., LTD.).
  • test compounds include, but not limited to, low molecular weight compounds, haigh molecular compounds, and peptides.
  • Test compounds may be artificially synthesized compounds or naturally occurring compounds.
  • Test compounds may be a single compound or mixtures. Usable examples includes a library of low molecular weight compounds, a compound library which was synthesized by combinatorial chemistry, a narurally occurring product containing cells, plants, animals or a part thereof, or an extracred product of such narurally occurring product.
  • the test substance which shows an activity of inhibiting or promoting NF- K B activation can be further screened to isolate a single substance having the activity. Isolate and purification of a desired compound from a mixture can be carried out by using any knonw method such as filteration, extraction, washing, drying, concentration, crystallization or various chromatography in combination.
  • the method for screening according to the present invention can be carried out by the following steps:
  • Methods for measuring the activation of NF- K B in the above method include a method of analyzing the binding of NF- /c B to its binding sequence using cell extraction solution by gel shift (for example, Hayashi T. etal. J.Biol.Chem.268, p.26790-26795 (1993), Nauman M. et al. EMBO J. 13, p4597-4607 (1994)).
  • the amount of mRNA or proteins for genes whose expression is known to be induced by NF- K B activation e.g., genes for IL-1 and TNF- a
  • the amount of mRNA can be measured, for example, by northern hybridization, RT-PCR, etc.
  • the amount of proteins can be measured, for example, by using antibodies.
  • the antibodies may be produced by known methods. Commercially available antibodies(from, e.g., Wako Pure Chemical Industries, Ltd.) can also be used.
  • the present invention further provided a method of producing a pharmaceutical composition, which comprises the following steps (a) to (f):
  • a pharmaceutical composition can be produced by the following steps: (a) preparing a transformed cell by introducing a gene encoding a protein that promotes activation of NF- K B according to the present invention into a cell;
  • step (d) of the method of producing a pharmaceutical composition it is preferable to isolate or identify as an activator compound, a compound that increases said detectable signal 2-fold or higher than normal, and to isolate or identify as an inhibitor compound, a compound that decreases said detectable signal half or less than normal.
  • the protein of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the protein, by:
  • the present invention also provides a compound which is selected by the above screening method.
  • This compound has a function of inhibiting or promoting NF- /c B activation. More specifically, this compound has a function of inhibiting or promoting NF- K B activation by the protein of the present invention.
  • the compounds obtained by the above screening methods have a function of inhibiting or promoting NF- K B activation, they are useful as therapeutic or preventive pharmaceuticals for the treament of diseases resulting from unfavorable activation or inactivation of NF- K B.
  • a compound which is obtained in the form of a salt can be purified as it is.
  • a compound which is obtained in the free form can be converted into a salt by isolating and purifying a salt obtained by dispersing or dissolving the compound into a suitable solvent and then adding a desired acid or base.
  • Examples of a step to optimize the compounds or salts thereof obtained by the method of the present invention as a pharmceutical composition include methods of formulating according to ordinary processes such as the following.
  • the above compounds or their pharmaceutically acceptable salts in an amount effective as an active ingredient, and pharmaceutically acceptable carriers can be mixed.
  • a form of formulation suitable for the mode of administration is selected.
  • a composition suitable for oral administration includes a solid form such as tablet, granule, capsule, pill and powder, and solution form such as solution, syrup, elixir and dispersion.
  • a form useful for parenteral administration includes sterile solution, dispersion, emulsion and suspension.
  • the above carriers include, for example, sugars such as gelatin, lactose and glucose, starches such as corn, wheat, rice and maize, fatty acids such as stearic acid, salts of fatty acids such as calcium stearate, magnesium stearate, talc, vegetable oil,alcohol such as stearyl alcohol and benzyl alcohol, gum, and polyalkylene glycol.
  • examples of such liquid carriers include generally water, saline, sugar solution of dextrose and the like, glycols such as ethylene glycol, propylene glycol and polyethylene glycol.
  • the present invention also provides a kit for screening compounds which inhibit or promote NF- K B activation.
  • the kit comprises reagents and the like necessary for screening compounds which inhibit or promote NF- K B activation, including:
  • the present invention relates to a diagnostic kit which comprises:
  • a polynucleotide of the present invention having a nucleotide sequence expressed by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,
  • a protein of the present invention having an amino acid seqeunce expressed by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,
  • a kit comprising at least one of (a) to (d) is useful for diagnosing a disease or susceptibility to a disease such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • a disease or susceptibility to a disease such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • NF- K B is involved in a wide variety of pathological conditions such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • NF- K B activity may have significant physiological effects [see e.g., Ann. Rheum. Ds. 57:738-741 (1998); American Journal of Pathology 152:793-803 (1998); ARTHRITIS & RHEUMA ⁇ SM 40:226-236 (1997); Am. J. Respir. Crit. Care Med. 158:1585-1592 (1998); J. Exp. Med. 188:1739-1750 (1998); Gut 42:477-484 (1998); The Journal of Immunology 161:4572-4582 (1998); Nature Medicine 3:894-899 (1997)].
  • the present invention also relates to a method of use of a compound which inhibits the function of the protein capable of activating NF- /c B described above, for inhibiting NF- K B activation. Further, the present invention relates to a method of using a compound which activates the function of the protein capable of activating NF- K B described above, for promoting NF- K B activation.
  • the compound obtained by the above screening method which inhibits NF- K B activation, is useful as a medicament to treat or prevent diseases characterized by undesirable activation of NF- K B, such as inflammation, autoimmune diseases (such as rheumatoid arthritis, systemic lupus erythematosus, asthma, etc), infectious diseases, bone diseases, and graft rejection.
  • diseases characterized by undesirable activation of NF- K B such as inflammation, autoimmune diseases (such as rheumatoid arthritis, systemic lupus erythematosus, asthma, etc), infectious diseases, bone diseases, and graft rejection.
  • NF- K B activation controls apoptosis of cells.
  • the compound obtained by the above screening method which inhibits NF- /c B activation, may be capable of stimulating apoptosis.
  • Diseases which may be treated by the induction of apoptosis include tumors.
  • diseases related to abnormality in NF- K B activation include AIDS (acquired immunodeficiency syndrome), neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, amyoxrophic lateral sclerosis, etc.), ischemic disorders (i.e. those caused by cardiac infarction, reperfusion injury, etc), myelogenesis incompetency syndrome (aplastic anemia, etc), skin diseases (Toxic epidermal necrolysis, etc), proliferative nephritis (IgA nephritis, purpuric nephritis, lupus nephritis, etc) and fulminant hepatitis.
  • a compound obtained by the above screening method which inhibits or promotes NF- K B activation, is useful as a medicament to treat or prevent these diseases.
  • the gene encoding the protein of the present invention is useful for gene therapy to treat various diseases such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • Gene therapy refers to adniinistering into the human body a gene or a cell into which a gene has been introduced for the purpose of therapy of diseases.
  • the protein of the present invention and the DNA encoding the protein can also be used for diagnostic purposes.
  • the present invention provides a agent for gene therapy which comprises a gene encoding the protein of the present invention.
  • the form of the agent for gene therapy is not particularly limited, but includes a pharmaceutical composition which comprises a expression vector containing a gene of the present invention in a pharmaceutical carrier of physiological buffer.
  • the pharmaceutical carrier may contain suitable stabilizer (for example, nuclease inhibitor), chelate agent (for example, EDTA), and/or other auxiliary agent.
  • the agent for gene therapy of the present invention may be provided as a complex of an expression vector containing a gene of the present invention and a liposome.
  • the agent for gene therapy may be applied using a catheter.
  • the agent for gene therapy of the present invention can be directly injected into a blood vessel of patient and the like.
  • the dosage of the agent for gene therapy of the present invention should be selected depending on the conditions such as age, sex, body weight and symptom of patient, and administration route, and is generally about 1 ⁇ g/kg to about 1000 mg/kg, more preferably about 10 ⁇ g/kg to about 100 mg/kg, as an amount of DNA (which is an effective ingredient) per one administration for adult.
  • the number of administration is not particularly hmited.
  • the compound obtained by the screening method of the present invention or a salt thereof can be formulated into the above pharmaceutical compositions (e.g., tablets, capsules, elixirs, microcapsules, sterile solutions and suspensions) according to conventional procedures.
  • the formulations thus obtained are safe and of low toxicity, and can be administered, for example, to humans and mammals (e.g., rats, rabbits, sheep, pigs, cattle, cats, dogs and monkeys).
  • Administration to patients can be carried out by methods known in the art, such as intra-arterial injection, intravenous injection and subcutaneous injection.
  • the dosage and administration mode may vary with the weight and age of the patient, but those skilled in the art can appropriately select suitable administration mode and can appropriately select suitable dosage depending on the administration mode.
  • the DNA can be inserted into a vector for gene therapy, and gene therapy can be carried out.
  • the present invention relates to a medicament which comprises a compound capable of inhibiting or promoting NF- K B activation as an active ingredient.
  • the above compound is useful as a medicament to treat or prevent diseases characterized by abnormal NF- K B activity, such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • diseases characterized by abnormal NF- K B activity such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • the present invention also relates to a medicament to treat or prevent inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases, ischemic disorders and the like, which comprises a compound capable of inhibiting or promoting NF- K B activation.
  • the compound is useful as a therapeutic and/or prophylactic drug against, for example, rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, diabetes, sepsis, asthma, allergic rhinitis, ischemic heart diseases, inflammatory intestinal diseases, subarachnoid hemonhage, viral hepatitis, AIDS, atherosclerosis, atopic dermatitis, viral infections, Crohn's disease, gout, hepatitis, multiple sclerosis, cardiac infarction, nephritis, osteoporosis, Alzheimer's, Parkinson's disease, Huntington's chorea, psoriasis, amyo trophic lateral sclerosis, or aplastic anemia.
  • rheumatoid arthritis arthritis
  • osteoarthritis systemic lupus erythematosus
  • diabetes sepsis
  • asthma allergic rhinitis
  • ischemic heart diseases inflammatory intestinal diseases,
  • the present invention also relates to the use of the above compound for manufacturing a medicament for the therapy and/or prevention of inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases, ischemic disorders and the like.
  • the present invention also provides an antisense ohgonucleotide against the polynucleotide of any one of above items (3) to (7).
  • An antisense ohgonucleotide refers to an oligonucleotide complementary to the target gene sequence.
  • the antisense oligonucleotide can inhibit the expression of the target gene by inhibiting RNA functions such as translation to proteins, transport to the cytoplasm and other activity necessary for overall biological functions.
  • the antisense ohgonucleotide may be RNA or DNA.
  • the DNA sequence of the present invention can be used to produce an antisense oligonucleotide capable of hybridizing with the mRNA transcribed from the gene encoding the protein of the present invention.
  • an antisense oligonucleotide generally has an inhibitory effect on the expression of the corresponding gene (see e.g., Saibou Kougaku Vol.13, No.4 (1994)).
  • the oligonucleotide containing an antisense coding sequence against a gene encoding the protein of the present invention can be introduced into a cell by standard methods.
  • the oligonucleotide effectively blocks the translation of mRNA of the gene encoding the protein of the present invention, thereby blocking its expression and inhibiting undesirable activity.
  • the antisense oligonucleotide of the present invention may be a naturally occurring oligonucleotide or its modified form [see e.g., Murakami & Makino, Saibou Kougaku Vol.13, No.4, p.259-266 (1994); Akira Murakami, Tanpakushitsu Kakusan Kouso (PROTEIN, NUCLEIC ACID AND ENZYME) Vol.40, No.10, p.1364-1370 (1995),Tunenari Takeuchi et al., Jikken Igaku (Experimental Medicien) Vol. 14, No. 4 p85-95(1996)].
  • the oligonucleotide may have modified sugar moieties or inter-sugar moieties.
  • modified forms include phosphothioates and other sulfur-containing species used in the art.
  • at least one phosphodiester bond in the oligonucleotide is substituted with the structure which can enhance the ability of the composition to permeate cellular regions where RNA with the activity to be regulated is located.
  • Such substitution preferably involves a phosphorothioate bond, a phosphoramidate bond, methylphosphonate bond, or a short-chain alkyl or cycloalkyl structure.
  • the antisense ohgonucleotide may also contain at least some modified base forms. Thus, it may contain purine and pyrimidine derivatives other than naturaUy occurring purine and pyrimidine.
  • the furanosyl moieties of the nucleotide subunits can be modified so long as the essential purpose of the present invention is attained. Examples of such modifications include 2'-O-alkyl and 2'-halogen substituted nucleotides.
  • sugar moieties at their 2-position examples include OH, SH, SCH 3 , OCH 3 , OCN or O(CH 2 ) n CH 3 , wherein n is 1 to about 10, and other substituents having similar properties.
  • the analogues are included in the scope of the present invention so long as they can hybridize with the mRNA of the gene of the present invention to inhibit functions of the mRNA.
  • the antisense oligonucleotide of the present invention contains about 3 to about 50 nucleotides, preferably about 8 to about 30 nucleotides, more preferably about 12 to about 25 nucleotides.
  • the oligonucleotide of the present invention can be produced by the well-known sohd phase synthesis technique. Devices for such synthesis are commercially available from some manufactures including Applied Biosystems. Other ohgonucleotides such as phosphothioates can also be produced by methods known in the art.
  • the antisense oligonucleotide of the present invention is designed to hybridize with the mRNA transcribed from the gene of the present invention.
  • Those skilled in the art can easily design an antisense ohgonucleotides based on a given gene sequence (For example, Murakami and Makino: Saibou Kougaku Vol. 13 No.4 p259-266 (1994), Akira Murakami: Tanpakushitsu Kakusan Kouso (PROTEIN, NUCLEIC ACID AND ENZYME) Vol. 40 No.10 pl364-1370 (1995), Tunenari Takeuchi et al., Jikken Igaku (Experimental Medicine) Vol. 14 No. 4 p85-95 (1996)).
  • antisense oligonucleotides which are designed in a region containing 5' region of mRNA, preferably, the translation initaiation site, are most effective for the inhibition of the expression of a gene.
  • the length of the antisense oligonucleotides is preferably 15 to 30 nucleotides and more preferably 20 to 25 nucleotides. It is important to confirm no interaction with other mRNA and no formation of secondary structure in the oligonucleotide sequence by homology search.
  • the evaluation of whether the designed antisense oligonucleotide is functional or not can be determined by introducing the antisence oligonucleotide into a suitable cell and measuring the amount of the target mRNA, for example by northern blotting or RT-PCR, or the amount of the target protein, for example by western blotting or fluorescent antibody technique, to confirm the effect of expression inhibition.
  • Another method includes the triple helix technique.
  • This technique involves forming a triple helix on the targeted intra-nuclear DNA sequence, thereby regulating its gene expression, mainly at the transcription stage.
  • the oligonucleotide is designed mainly in the gene region involved in the transcription and inhibits the transcription and the production of the protein of the present invention.
  • Such RNA, DNA and oligonucleotide can be produced using known synthesizers.
  • the antisense oligonucleotide may be introduced into the ceUs containing the target nucleic acid sequence by any of DNA transfection methods such as calcium phosphate method, electroporation,lipofection, microinjection, or gene transfer methods including the use of gene transfer vectors such as viruses.
  • An antisense oligonucleotide expression vector can be prepared using a suitable rexrovirus vector, then the expression vector can be introduced into the cells containing the target nucleic acid sequence by contacting the vector with the cells in vivo or ex vivo.
  • the DNA of the present invention can be used in the antisense RNA/DNA technique or the triple helix technique to inhibit NF- K B activation mediated by the protein of the present invention.
  • the antisense ohgonucleotide against the gene encoding the protein of the present invention is useful as a medicament to treat or prevent diseases characterized by undesirable activation of NF- K B, such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • diseases characterized by undesirable activation of NF- K B such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
  • the present invention also provides a medicament which comprises the above antisense ohgonucleotide as an active ingredient.
  • the antisense oligonucleotide can also be used to detect such diseases using northern hybridization or PCR.
  • the present invention also provides a ribozyme or deoxyribozyme which inhibits NF- n B activation.
  • a ribozyme and deoxyribozyme is an RNA capable of recognizing a nucleotide sequence of a nucleic acid and cleaving the nucleic acid (see e.g., Hiroshi Yanagawa, "Jikken Igaku (Experimental Medicine) Bioscience 12: New Age of RNA).
  • the ribozyme or deoxyribozyme can be produced so that it cleaves the selected target RNA (e.g., mRNA encoding the protein of the present invention).
  • the ribozyme or deoxyribozyme specifically cleaving the mRNA of the protein of the present invention can be designed.
  • Such ribozyme has a complementary sequence to the mRNA for the protein of the present invention, complementarily associates with the mRNA and then cleaves the mRNA, which results in reduction or entire loss of the expression of the protein of the present invention.
  • the level of the reduction of the expression is dependent on the level of the ribozyme or deoxyribozyme expression in the target cells.
  • ribozyme or deoxyribozyme commonly used: a hammerhead ribozyme and a hairpin ribozyme.
  • hammerhead ribozymes or deoxyribozymes have been well studied regarding their primary and secondary structure necessary for their cleavage activity, and those skilled in the art can easily design the ribozymes nucleotided solely on the nucleotide sequence information for the DNA encoding the protein of the present invention [see e.g., Iida et al., Saibou Kougaku Vol.16, No.3, p.438-445 (1997); Ohkawa & Taira, Jikken Igaku (Experimental Medicine) Vol.12, No.12, p.83-88 (1994)].
  • the hammerhead ribozymes or deoxyribozymes have a structure consisting of two recognition sites (recognition site I and recognition site II forming a chain complementary to target RNA) and an active site, and cleave the target RNA at the 3 'end of its sequence NUX (wherein N is A or G or C or U, and X is A or C or U) after the formation of a complementary pair with the target RNA in the recognition sites.
  • the sequence GUC (or GUA) has been found to have the highest activity [see e.g., Koizumi, M. et al., Nucl. Acids Res.
  • a ribozyme is designed to form several,up to 10 to 20 complementary base pairs around that sequence.
  • the suitability of the designed ribozyme can be evaluated by checking whether the prepared ribozyme can cleave the target mRNA in vitro according to the method described for example in Ohkawa & Taira, Jikken Igaku (Experimental Medicine) Vol.12, No.12, p.83-88 (1994).
  • the ribozyme can be prepared by methods known in the art to synthesize RNA molecules.
  • the sequence of the ribozyme can be synthesized on a DNA synthesizer and inserted into various vectors containing a suitable RNA polymerase promoter (e.g., T7 or SP6) to enzymatically synthesize an RNA molecule in vitro.
  • a suitable RNA polymerase promoter e.g., T7 or SP6
  • Such ribozymes can be introduced into cells by gene transfer methods such as microinjection.
  • Another method involves inserting a ribozyme DNA into a suitable expression vector and introducing the vector into ceU strains, cells or tissues. Suitable vectors can be used to introduce the ribozyme into a selected cell.
  • vectors commonly used for such purpose include plasmid vectors and animal virus vectors (e.g., retrovirus, adenovirus, herpes or vaccinia virus vectors).
  • animal virus vectors e.g., retrovirus, adenovirus, herpes or vaccinia virus vectors.
  • ribozymes are capable of inhibiting the NF- K B activation mediated by the protein of the present invention.
  • the present invention further provides a process for obtaining a new gene having a function, which comprises using the oligo-capping method to construct a full-length cDNA library, and detecting the presence of a protein having the function by using a signal factor.
  • a signal factor is a reporter gene.
  • the cDNA libraries produced using the oligo-capping method contain full-length cDNA clones in a ratio of 50 to 80%, namely, a 5 to 10-fold increase in full-length cDNA clones compared to the cDNA libraries produced by prior art methods (Sumio Sugano, the monthly magazine BIO INDUSTRY Vol.16, No.ll, p.19-26).
  • Full-length cDNA clones are essential for protein expression in functional analyses of genes, and full-length cDNA clones themselves are very important materials for activity measurement. Thus, cloning of full-length cDNA is necessary for functional analyses of genes.
  • Sequencing of the cDNA not only provides important information for establishing the primary sequence of the protein encoded by the cDNA, but also reveals the entire exon sequence. Thus, the full-length cDNA provides valuable information for identifying a gene, such as information for determining the primary sequence of a protein, exon-intron structure, the transcription initiation site of mRNA, the location of a promoter, etc.
  • the construction of full-length cDNA libraries by the oligo-capping method can be carried out, for example, according to the method described in "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", the third edition (1999), an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.
  • the oligo-capping method used herein involves substituting a cap structure with a synthetic oligo sequence by using BAP, TAP and an RNA ligase, as described in Suzuki & Sugano, "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", the third edition (1999), an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.
  • the reporter gene which can be used as a signal factor which indicates the presence of a protein having a function contains one or more suitable expression regulation sequence portion to which a protein factor such as a transcriptional factor can bind, and a structural gene portion which allows the measurement of the activation of the proteins factor.
  • the structural gene portion may encode any peptide or protein so long as those skiUed in the art can measure the activity or amount of its expression product (including the amount of the mRNA produced). For example, chloramphenicol acetyltransferase, ⁇ -galactosidase, luciferase, etc., can be used and their enzymatic activity measured.
  • CREB cAMP responsive element binding protein
  • AP-1 activator protein- 1
  • a CREB-dependent reporter plasmid and an expression vector comprising full-length cDNA produced by the oligo-capping method can be cotransfected into cells, and an expression vector having increased reporter activity can be selected from the cells to attain the purpose.
  • a gene capable of inhibiting CREB is to be obtained, a CREB-dependent reporter plasmid and an expression vector comprising full-length cDNA produced by the oligo-capping method can be cotransfected into cells, and an expression vector having decreased reporter activity can be selected from the cells to attain the purpose.
  • the cDNA clone (expression vector) to be transfected into the cells may be a single clone or multiple clones which may be transfected simultaneously.
  • One embodiment of the process of the present invention is detaUed in Examples herein.
  • a screening system for obtaining a gene capable of inhibiting NF- K B activation can also be constructed by cotransfecting an expression vector comprising full-length cDNA and a reporter gene into cells, stimulating the cells with IL-1 or TNF- and the like, and selecting a clone having subnormally increased reporter activity.
  • a gene encoding a protein capable of activating various factors for example, MAP kinase, transcription factor
  • a gene encoding the protein capable of activating NF- K B can be obtained in addition to a gene encoding the protein capable of activating NF- K B.
  • the process of the present invention uses an in vitro system or a cell-based system, preferably a cell-based system.
  • a cell-based system examples include cells of prokaryotes such as E. coli, microorganisms such as yeast and fungi, as well as insects and animals.
  • Preferred examples include animal cells, in particular, 293-EBNA cells and NIH3T3 cells.
  • the cDNA of the present invention is fuU-length, its 5' end sequence is the transcription initiation site of the corresponding mRNA. Therefore the cDNA sequence can be used to identify the promoter region of the gene by comparing the cDNA with the genomic nucleotide sequence. Genomic nucleotide sequences are available from various databases when the sequences have been deposited in the databases. Alternatively, the cDNA can also be used to clone the desired sequence from a genomic library, for example, by hybridization, and determine its nucleotide sequence. Thus, by comparing the nucleotide sequence of the cDNA of the present invention with a genomic sequence, the promoter region of the gene located upstream the cDNA can be identified.
  • the promoter fragment thus identified can be used to construct a reporter plasmid for evaluating the expression of the gene.
  • the DNA fragment spanning 2kb (preferably lkb) upstream from the transcription initiation site can be inserted upstream of the reporter gene to produce the reporter plasmid.
  • the reporter plasmid can be used to screen for a compound which enhances or reduces the expression of the gene.
  • such screening can be carried out by transforming a suitable cell with the reporter plasmid, culturing the transfonned ceU for a certain period of time, adding a certain amount of a test compound, measuring the reporter activity expressed by the cell after a certain period of time, and comparing the activity with that of a cell to which the test compound has not been added.
  • the present invention also relates to a computer-readable medium on which a sequence data set has been stored, said sequence data set comprising at least one of nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169
  • the present invention relates to a method for calculating a homology, which comprises comparing data on the above medium with data of other nucleotide sequences.
  • the gene and amino acid sequence of the present invention provide valuable information for determining their secondary and tertiary structure, e.g., information for identifying other sequence having a similar function and high homology.
  • These sequences are stored on the computer-readable medium, then a database is searched using data stored in a known macromolecule structure program and a known search tool such as GCG In this manner, a sequence in a database having a certain homology can be easily found.
  • the computer-readable medium may be any composition of materials used to store information or data. Examples of such media include commercially available floppy disks, tapes, chips, hard drives, compact disks and video disks.
  • the data on the medium allows a method for calculating a homology by comparing the data with other nucleotide sequence data. This method comprises the steps of providing a first polynucleotide sequence containing the polynucleotide sequence of the present invention for the computer-readable medium, and then comparing the first polynucleotide sequence with at least one-second polynucleotide or polypeptide sequence to identify the homology.
  • the present invention also relates to an insoluble substrate to which polynucleotides comprising all or part of the nucleotide sequences selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 17
  • a plurality of the various polynucleotides which are DNA probes are fixed on a specifically processed solid substrate such as slide glass to form a DNA microanay and then a labeled target polynucleotide is hybridized with the fixed polynucleotides to detect a signal from each of the probes.
  • the data obtained is analyzed and the gene expression is dete ⁇ nined.
  • the present invention further relates to an insoluble substrate to which polypeptides comprising all or a part of the amino acid sequences selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 18
  • Example 1 Construction of a fuU-length cDNA library using the oligo-capping method
  • Human lung fibroblasts (Cryo NHLF: purchased from Sanko Junyaku Co., Ltd.) were cultured according to the manufacture's protocol. After repeating subculturing the cells to obtain fifty 10cm dishes containing the resulting culture, the cells were recovered with a cell scraper. Then, total RNA was obtained from the recovered cells by using the RNA extraction reagent ISOGEN (purchased from NIPPON GENE) according to the manufacturer's protocol. Then, poly A + RNA was obtained from the total RNA by using an oligo-dT cellulose column according to Maniatis et al., supra.
  • RNA extraction reagent ISOGEN purchased from NIPPON GENE
  • ATDC5 a cell strain cloned from mouse EC (embryonal carcinoma) (Atsumi, T. et al.: CeU Diff. Dev, 30: ⁇ l09-116)(1990) was repeatedly subcultured to obtain fifty 10cm dishes containing the resultant culture. Thereafter, poly A + RNA was obtained by a method similar to that of (1) above. Culture of ATDC5 cells was performed according to the method described in Atsumi, T. et al.: Cell Diff. Dev., 30: pl09-116 (1990).
  • a human T ceU strain purchased from DAINIPPON PHARMACEUTICAL CO.,LTD
  • poly A + RNA was obtained in the same way as in the above (1).
  • Jurkat ceUs were cultured in PRMI 1640 medium (GLBCO) containing 10%FBS (Fetal Bovine Serum: GIBCO) and lOmM HEPES (GIBCO) in the presence of 5% CO 2 at 37T .
  • RAW264.7 cells a mause macrophage-like ceU starain (ATCC Number TTB-71), were cultured to obtain poly A + RNA in the same way as in the above (1).
  • a fuU-length cDNA library was constructed from poly A + RNA of the above human lung fibroblasts, ATDC5 cells, Jurkat cells and RAW264.7 cells by the oligo-capping method according to the method of Sugano S. et al. [e.g., Maruyama, K. & Sugano, S., Gene, 138:171-174 (1994); Suzuki, Y. et al., Gene, 200:149-156 (1997); Suzuki, Y. & Sugano, S. "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", the third edition (1999), an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.].
  • Sugano S. et al. e.g., Maruyama, K. & Sugano, S., Gene, 138:171-174 (1994); Suzuki, Y. et al., Gene, 200:149-156 (1997); Suzuki,
  • the full-length cDNA library constructed as above was transformed into E. coli strain TOP 10 by electroporation, then spread on LB agar medium containing 100 ⁇ g/ml of ampicillin, and incubated overnight at 37°C. Then, using QIAwell 96 Ultra Plasmid Kit (QIAGEN) according to the manufacturer's protocol, the plasmids were recovered from the colonies grown on ampicillin-containing LB agar medium.
  • the reporter activity of NF- K B was measured using long-term luciferase assay system,PIKKA GENE LT2.0 (TOYO INK) according to the attached manufacturer's instructions.
  • the luciferase activity was measured using WaUac ARVOTMST 1420 MULTILABEL COUNTER (Perkin Elmer).
  • the sequencing was carried out using the reagent Thermo Sequenase II Dye Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech) or BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (Applied Biosystems) and the device ABI PRISM 377 sequencer or ABI PRISM 3100 sequencer according to the manufacturer's instructions.
  • the fuU-length DNA sequences for the 144 new clones which were obtained by the above screening were determined (SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,
  • amino acid sequences of the protein coding regions were deduced (SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206
  • NF- /c B reporter activity The results of measurement of NF- /c B reporter activity (luciferase activity) of 41 clones among the above obtained clones are shown in Table 1 below.
  • the value of activity shown in Table 1 is a relative value to the value of control experiment (luciferase activity of a cell into which pME183-FL3 vector containing no insert has been introduced in place of full length cDNA).
  • Example 3 Screening compounds inhibiting NF- K B activation
  • 293-EBNA cells were seeded on DMEM medium containing 5% FBS in a 96-well cell culture plate to a final cell density of 1 x IO 4 cells/100 ⁇ 1/well, and cultured for 24 hours at 37°C in the presence of 5% CO 2 .
  • 50ng of the plasmid comprising the gene encoding NF- K B activating protein of SEQ ID NO: 12 which was obtained in Example 2 above, and 50ng of the reporter plasmid pNF K B-Luc were cotransfected into the cells in a weU using FuGENE 6.
  • the proteasome inhibitor MG-132 purchased form CALBIOCHEM
  • the present invention provides industrially highly useful proteins capable of activating NF- K B and genes encoding the proteins.
  • the proteins of the present invention and the genes encoding the proteins allow not only screening for compounds useful for treating and preventing diseases associated with the excessive activation or inhibition of NF- /c B, but also production of diagnostics for such diseases.
  • the genes of the present invention are also useful as a gene source used for gene therapy. AU publications, patents and patent applications cited herein are incorporated herein in their entirety.

Abstract

Provided are proteins capable of activating NF-κB, which are used for diagnosing, treating or preventing diseases associated with the excessive activation or inhibition of NF-κB. Using plasmid pNFκB-Luc, cDNA encoding a protein capable of activating NF-κB has been cloned from a cDNA library constructed from human lung fibroblasts and the like, and the DNA sequence and the deduced amino acid sequence determined. The protein, the DNA encoding the protein, a recombinant vector containing the DNA, and a transformant containing the recombinant vector are useful for screening a substance inhibiting or promoting NF-κB activation.

Description

DESCRIPTION
NF- K B ACTIVATING GENE
TECHNICAL FIELD
The present invention relates to a protein capable of activating NF- κ B, a DNA sequence encoding the protein, a method for obtaining the DNA, a recombinant vector containing the DNA, a transformant containing the recombinant vector, and an antibody which specifically reacts with the protein. The present invention also relates to use of the protein, DNA or antibody of the invention in the diagnosis, treatment or prevention of diseases associated with the excessive activation or inhibition of NF- K B.
The present invention also relates to a method for screening a substance capable of inhibiting or promoting NF- K B activation by using the protein, DNA, recombinant vector and transformant.
BACKGROUND ART
The transcription factor NF- K B (nuclear factor kappa B) plays an important role in the transcriptional regulation of various genes involved in inflammation and immunological reactions. NF- K B is a homo- or heterodimer protein which belongs to the Rel family. In unstimulated conditions, NF- K B normally resides in the cytoplasm as an inactive form by forming a complex with an I K B (inhibitory protein of NF- K B) to mask the nuclear transport signal of NF- K B.
When cytokines such as interleukin (IL)-1 and tumor necrosis factor (TNF)- stimulate cells, I K B is phosphorylated by IKK (I B kinase) and degraded by the 26S proteasome through ubiquitination. The released NF- K B moves to the nucleus, where it binds to the DNA sequence called the NF- K B binding sequence and induces the transcription of the gene under control of NF- K B. NF- K B is believed to regulate the expression of genes such as those for immunoglobulins, inflammatory cytokines (e.g., IL-1 and TNF- ), interferons and cell adhesion factors. NF- K B is involved in inflammation and immune responses through the expression induction of these genes.
The inhibition of the function or activation of NF- K B may inhibit the expression of many factors (proteins) involved in inflammatory or immunological diseases or other diseases such as tumor proliferation. Thus, NF- / B is a promising target for medicaments against diseases caused or characterized by autoimmunity or inflammation [see e.g., Clinical Chemistry 45, 7-17 (1999); J Clin. Pharmacol. 38, 981-993 (1998); Gut 43, 856-860 (1998); The New England Journal of Medicine 366, 1066-1071 (1997); TiPS 46-50 (1997); The FASEB Journal 9, 899-909 (1995); Nature 395, 225-226 (1998); Science 278, 818-819 (1997); Cell 91, 299-302 (1997)].
Extracellular information is converted into a certain signal, which passes through the cell membrane and goes through the cytoplasm to the nucleus, where it regulates the expression of the target gene and causes cell responses. Therefore the elucidation of the mechanism of intracellular signal transduction from extracellular stimuli to NF- K B activation is of very important significance, because it provides very important means of developing new medicaments or therapies against autoimmune diseases and diseases exhibiting inflammatory symptoms.
It is believed that the signal transduction pathway from certain cell stimulation to NF- K B activation includes many steps mediated by various transmitters such as protein kinases. Therefore it is desirable for more efficient drug discovery to identify the transmitters which play a key role in the pathway, and to focus research on the transmitters to establish a new drug-screening method. Some signaling molecules involved in NF- K B activation have been identified [e.g., IKK, ubiquitination enzymes and the 26S proteasome described above, as well as TNF receptor associated factor 2 (TRAF2) and NF- K B inducing kinase (NIK)]. However, most of the mechanism of NF- K B activation remains unknown, and it has been desired to identify new signaling molecules and further to elucidate NF- rc B activation mechanism. DISCLOSURE OF THE INVENTION
The object of the present invention is to identify a new gene and protein capable of directly, or indirectly, activating NF- K B, and to provide a method of use of them in medicaments, diagnostics and therapy. That is, an object of the present invention is to provide a new protein capable of activating NF- K B, a DNA sequence encoding the protein, a recombinant vector containing the DNA, a transformant containing the recombinant vector, a process for producing the protein, an antibody directed against the protein or a peptide fragment thereof, and a process for producing the antibody.
Another object of the present invention is to provide a method for screening a substance capable of inhibiting or promoting NF- K B activation using the protein, the DNA, the recombinant vector or the transformant, a kit for the screening, a substance capable of inhibiting or promoting NF- K B activation obtainable by the screening method or the screening kit, a process for producing the substance, a pharmaceutical composition containing a substance capable of inhibiting or promoting NF- K B activation, etc.
The present inventors have intensively studied to solve the above problems. As a result, the present inventors have succeeded in constructing a full-length cDNA library by using the oligo-capping method; establishing a gene function assay system by expression cloning using 293-EBNA cells; and isolating a new DNA (cDNA) encoding a protein having a function of activating NF- K B by using the assay system. This new DNA molecule induced NF- K B activation by its expression in 293-EBNA cells. This result shows that this new DNA is a signal transduction molecule involved in NF- K B activation. Thus, the present invention has been completed.
That is, the present invention provides the folio wings: (1) A purified protein selected from the group consisting of: (a) a protein which comprises an amino acid sequence represented by any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288; and
(b) a protein that activates NF- K B (Nuclear factor kappa B) and comprises an amino acid sequence having at least one amino acid deletion, substitution or addition in an amino acid sequence represented by any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288.
(2) A purified protein that activates NF- K B and comprises an amino acid sequence having at least 95% identity to the protein according to (1) over the entire length thereof.
(3) An isolated polynucleotide which comprises a nucleotide sequence encoding a protein selected from the group consisting of:
(a) a protein which comprises an amino acid sequence represented by any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288; and
(b) a protein that activates NF- K B and comprises an amino acid sequence having at least one amino acid deletion, substitution or addition in an amino acid sequence represented by any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,
84, 86, 88, 90; 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,
160, 162 164 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206, 208 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244 246 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280 282 284, 286 and 288.
(4) An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of:
(a) a polynucleotide sequence represented by any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287;
(b) a polynucleotide sequence encoding a protein that activates NF- K B and hybridizing under stringent conditions with a polynucleotide having a polynucleotide sequence complementary to the polynucleotide sequence of (a); and
(c) a polynucleotide sequence which encodes a protein that activates NF- K B and consists of a polynucleotide sequence having at least one nucleotide deletion, substitution or addition in a polynucleotide sequence represented by any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287.
(5) An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of:
(a) a polynucleotide sequence represented by a coding region of any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287;
(b) a polynucleotide sequence encoding a protein that activates NF- K B and hybridizing under stringent conditions with a polynucleotide having a polynucleotide sequence complementary to the polynucleotide sequence of (a); and
(c) a polynucleotide sequence which encodes a protein that activates NF- K B and consists of a polynucleotide sequence having at least one nucleotide deletion, substitution or addition in a polynucleotide sequence represented by a coding region of any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287.
(6) An isolated polynucleotide comprising a nucleotide sequence which encodes a protein that activates NF- K B and has at least 95% identity to the polynucleotide sequence according to (3) over the entire length thereof.
(7) An isolated polynucleotide comprising a nucleotide sequence which encodes a protein that activates NF- K B and has at least 95% identity to the polynucleotide sequence according to (4) or (5) over the entire length thereof.
(8) A purified protein encoded by the polynucleotide according to any one of (3) to (7).
(9) A recombinant vector which comprises a polynucleotide according to any one of (3) to (7).
(10) A agent for gene therapy which compiises the recombinant vector according to (9) as an active ingredient. (11) A transformed cell which comprises the recombinant vector according to (9).
(12) A membrane of the cell according to (11), which has the protein according to (1) or (2) which is a membrane protein.
(13) A process for producing a protein according to (1), (2) or (8) comprising the steps of;
(a) culturing a transformed cell according to (11) under conditions providing expression of the protein according to (1), (2) or (8); and
(b) recovering the protein from the culture product.
(14) A process for diagnosing a disease or susceptibility to a disease related to expression or activity of the protein of (1), (2) or (8) in a subject comprising the steps of:
(a) detennining the presence or absence of a mutation in the gene encoding said protein in the genome of said subject; and/or
(b) analyzing the amount of expression of said protein in a sample derived from said subject.
(15) A method for screening compounds capable of inhibiting or promoting NF- K B activation, which comprises the steps of:
(a) preparing a transformed cell by introducing a gene encoding a protein that promotes activation of NF- K B according to (1), (2) or (8) and a gene encoding a signal which can detect activation of NF- K B into a cell;
(b) culturing the transformed cell under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring the signal which can detect activation of NF- K B; and
(d) selecting a candidate compound which can change the signal amount as compared with the case of the absence of candidate compounds, as a compound capable of inhibiting or promoting NF- K B activation.
(16) A method for screening compounds capable of inhibiting or promoting NF- K. B activation, which comprises the steps of:
(a) preparing a transformed cell by introducing a gene encoding a protein that promotes activation of NF- K B according to (1), (2) or (8) into a cell;
(b) culturing the transformed cell under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring the activation of NF- K B; and
(d) selecting a candidate compound which can change the activation of NF- K B as compared with the case of the absence of candidate compounds, as a compound capable of inhibiting or promoting NF- K B activation.
(17) A compound capable of inhibiting or promoting NF- K B activation, which is selected by the method for screening according to (15) or (16).
(18) A process for producing a pharmaceutical composition, which comprises the steps of:
(a) preparing a transformed cell by introducing a gene encoding a protein that promotes activation of NF- re B according to (1), (2) or (8) and a gene encoding a signal which can detect activation of NF- K B into a cell;
(b) culturing the transformed cell under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring the signal which can detect activation of NF- K B;
(d) selecting a candidate compound which can change the signal amount as compared with the case of the absence of candidate compounds, as a compound capable of inhibiting or promoting NF- K B activation; and
(e) producing a pharmaceutical composition which comprises a compound selected in the step of (d).
(19) A process for producing a pharmaceutical composition, which comprises the steps of:
(a) preparing a transformed cell by introducing a gene encoding a protein that promotes activation of NF- /c B according to (1), (2) or (8) into a cell;
(b) culturing the transformed cell under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring the activation of NF- K B; and
(d) selecting a candidate compound which can change the activation of NF- K B as compared with the case of the absence of candidate compounds, as a compound capable of inhibiting or promoting NF- K B activation; and
(e) producing a pharmaceutical composition which comprises a compound selected in the step of (d).
(20) A kit for screening a compound capable of inhibiting or promoting NF- K B activation, which comprises:
(a) a transformed cell comprising a gene encoding a protein that promotes activation of NF- K B according to (1), (2) or (8) and a gene encoding a signal which can detect activation of NF- K B; and
(b) reagents for measuring the signal.
(21) A monoclonal or polyclonal antibody or a fragment thereof, which recognizes the protein according to (1), (2) or (8).
(22) The monoclonal or polyclonal antibody or a fragment thereof according to (21), which inhibits the activity of promoting activation of NF- K B by the protein according to (l), (2) or (8). (23) A process for producing a monoclonal or polyclonal antibody according to (21) or (22), which comprises administering the protein according to (1), (2) or (8) or epitope-bearing fragments thereof to a non-human animal as an antigen.
(24) An antisense oligonucleotide having a sequence complementary to a part of the polynucleotide according to any one of (3) to (7), which prevents the expression of a protein which promotes NF- K B activation.
(25) A ribozyme or deoxyribozyme capable of inhibiting NF- K B activation, which has an action of cleavage of RNA that encodes the protein according to (1), (2) or (8) or an action of cleavage of RNA that encodes a protein involved in a pathway leading to NF- K B activation.
(26) A method for treating a disease associated with NF- K B activation, which comprises administering to a subject a compound screened by the process according to (15) or (16), and/or a monoclonal or polyclonal antibody or a fragment thereof according to (21) or (22), and/or an antisense oligonucleotide according to (24), and/or a ribozyme or deoxyribozyme according to (25) in an effective amount to treat a disease selected from the group consisting of inflammation, autoimmune diseases, cancers, infectious disease, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
(27) A pharmaceutical composition produced by the process according to (18) or (19) for inhibiting or promoting NF- K B activation.
(28) The pharmaceutical composition according to (27) for the treatment of inflammation, autoimmune diseases, cancers, infectious disease, bone diseases, AIDS, neurodegenerative diseases and/or ischemic disorders. (29) A method of treating inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases or ischemic disorders, which comprises administering a pharmaceutical composition produced by the process according to (18) or (19) to a patient suffering from a disease associated with NF- K B activation.
(30) A pharmaceutical composition which comprises a monoclonal or polyclonal antibody or a fragment thereof according to (21) or (22) as an active ingredient.
(31) A pharmaceutical composition which comprises an antisense oligonucleotide according to (24) as an active ingredient.
(32) A pharmaceutical composition which comprises a ribozyme or deoxyribozyme according to (25) as an active ingredient.
(33) The pharmaceutical composition according to any one of (30) to (32) for the treatment and/or prevention of a disease which is selected from the group consisting of inflammation, autoimmune diseases, infectious diseases, cancers, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
(34) A computer-readable medium on which a sequence data set has been stored, said sequence data set comprising at least one of nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287 or a coding region thereof, and/or at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288.
(35) A method for calculating identity to other nucleotide sequences and/or amino acid sequences, which comprises comparing data on a medium according to (34) with data of said other nucleotide sequences and/or amino acid sequences.
(36) An insoluble substrate to which polynucleotides comprising all or part of the nucleotide sequences selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287 are fixed.
(37) An insoluble substrate to which polypeptides comprising all or a part of the amino acid sequences selected from the group consisting of SEQ D NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,
60, 62, 64, 66. 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264; 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288, are fixed.
The contents of the specficiations and/or drawings of Japanese Patent Applications Nos. 2001-368692 and 2002-291302 and U.S. Provisional Applications Nos.60/335829 and 60/415,769, which from the bases of priority of the instant application, are incorporated herein.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a graph showing NF- K B reporter activity inhibition by the proteasome inhibitor MG-132 in Example 3. The axis of abscissa is MG-132 concentration and the transversal axis is relative luciferase activity.
BEST MODE FOR CARRYING OUT THE INVENTION
At first, in order to further clarify the basic feature of the present invention, the present invention is explained by following how the present invention is completed. In order to obtain a new gene having a function of activating NF- K B, the following experiments were carried out as shown in the examples.
First, using the oligo-capping method, a full-length cDNA was produced from mRNA prepared from normal human lung fibroblasts (purchased from Sanko Junyaku Co., Ltd.) and the like, and a full-length cDNA library was constructed in which the cDNA was inserted into the vector pME18S-FL3 (GenBank Accession AB009864). Next, the cDNA library was introduced into E. coli cells, and plasmid preparation was carried out per clone. Then, the pNK K B-LUC reporter plasmid (STRATAGENE) containing a DNA encoding luciferase and the above full-length cDNA plasmid were cotransfected into 293-EBNA cells (Invitrogen). After 24 or 48 hours of culture, luciferase activity was measured, and the plasmid with significantly increased luciferase activity compared to that of a control experiment (vector pME18S-FL3 is introduced into a cell in place of a full-length cDNA) was selected (the selected plasmid showed a 5-fold or more increase in luciferase activity compared to that of the control experiment), and the entire nucleotide sequence of the cDNA cloned into the plasmid was determined. The protein encoded by the cDNA thus obtained shows that this protein is a signal transduction molecule involved in NF- K B activation.
The present invention is described in detail below.
In the present invention, activation of NF- K B refers to activation of NF- K B (including induction of NF- K B activation) when a gene is introduced into a suitable cell and the protein encoded by the gene is excessively expressed. Activation of NF- K B can be measured, for example, by an assay using an NF- K B dependant reporter gene. Activation of NF- / B is reflected by increasing the reporter activity compared to control cells (cells into which the vector only was introduced). Increase in reporter activity is preferably by a factor of 1.5 or more, more preferably by a factor of 2 or more, and still more preferably by a factor of 5 or more.
Reporter activity can be measured by cloning a polynucleotide (e.g. cDNA) encoding the protein to be expressed into a suitable expression vector, co-transfecting the expression vector and an NF- K B dependant reporter plasmid into a suitable cell, and after culturing for a certain period, then measuring reporter activity. Suitable expression vectors are well known to those skilled in the art, examples of which include pME18S-FL3, pcDNA3.1 (Invitrogen). The reporter gene can be one which enables a person skilled in the art to easily detect the expression thereof, and examples include a gene encoding luciferase, chloramphenicol acetyl transferase, or β -galactosidase. Use of a gene encoding luciferase is most preferable, and examples of an NF- K B dependent reporter plasmid include pNF- K B-LUC (STRATAGENE). Suitable cells include cells which exhibit an NF- K B activation response to stimulation by IL-1, TNF- and the like. Examples include 293-EBNA cells. Cell culture and introduction of genes into cells (transfection) can be performed and optimized by a person skilled in the art by known techniques.
As a preferable method, 293-EBNA cells are inoculated on DMEM medium (Dulbecco's Modified Eagle Medium) containing 5% FBS (Fetal Bovine Serum) in a 96-well cell culture plate to a final cell density of 1 x IO4 cells/well, and cultured for 24 hours at 37°C in the presence of 5% CO2. Then, reporter plasmid pNF- K B-Luc (STRATAGENE) and the expression vector are cotransfected into the cells in a well using FuGENE 6 (Roche). After 24 hours of culture at 37°C, NF- rc B activation is then measured by measuring luciferase activity using a long term luciferase assay system, Picagene LT2.0 (Toyo Ink Mfg). For example, luciferase activity can be measured using PerkinElmer's Wallac ARVOTMST 1420 MULΗLABEL COUNTER. The method for gene introduction by FuGENEό, and measurement of luciferase activity by Picagene LT2.0 can be performed respectively according to the attached protocols. In a method of gene introduction with a 96-well plate using FuGENE6, the amount of FuGENEό per 1 well is suitably 0.3 to 0.5 μ 1, preferably 0.3 μ 1; the amount of pNF- K B-Luc plasmid is suitably 50 to lOOng, preferably 50ng; the amount of expression vector is suitably 50-100ng, and preferably lOOng. An ability to activate NF- rc B can be confirmed by an ability to increase the reporter activity (luciferase activity) relative to the control experiment (cells into which only a null vector was introduced). Increase in reporter activity is preferably by a factor of 1.5 or more, more preferably by a factor of 2 or more, and still more preferably by a factor of 5 or more.
Related to the amino acid sequences of any one of SEQ ID NOS. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288, the present invention provides the following proteins:
(a) a protein which comprises one of the above amino acid sequences;
(b) a peptide having one of the above amino acid sequences;
(c) a protein which activates NF- K B and consists of an amino acid sequence having at least one amino acid deletion, substitution or addition in the above amino acid sequences;
(d) a protein which comprises an amino acid sequence, which has at least 95% identity, preferably at least 97-99% identity, to one of the above amino acid sequences over the entire length thereof.
As known in the art, "identity" used herein is a relationship between two or more protein sequence or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between protein or polynucleotide sequences, as determined by the match between protein or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. "Identity" can be determined by using the BLAST program (for example, Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. Biol., 215: p403-410 (1990), Altschul SF, Madden TL, Schaffer AA, Zhang Z, MiUer W, Lipman DJ,. Nucleic Acids Res. 25: p3389-3402 (1997)). Where software such as BLAST is used, it is preferable to use default values. The main initial conditions generally used in a BLAST search are as follows, but are not limited to these. An amino acid substitution matrix is a matrix numerically representing the degree of analogy of each pairing of each of the 20 types of amino acid, and normally the default matrix of BLOSUM62 is used. The theory of this amino acid substitution matrix is shown in Altschul S.F., J. Mol. Biol. 219: 555-565 (1991), and apphcability to DNA sequence comparison is shown on States D. J., Gish W., Altschul S.F., Methods, 3: 66-70 (1991). In this case, optimal gap cost is determined by experience and in the case of BLOSUM62 preferably parameters of Existence 11, Extension 1 are used. The expected value (EXPECT) is the threshold value concerning statistical significance for a match with a database sequence, and the default value is 10.
As one example, a protein having, for example, 95% or more identity to the amino acid sequence of SEQ ID NO: 2 may contain in the amino acid sequence up to 5 amino acid changes per 100 amino acids of the amino acid sequence of SEQ ID NO: 2. In other words, a protein having 95% or more amino acid sequence identity to a subject amino acid sequence, may have amino acids up to 5% of the total number of amino acids within the subject sequence, deleted or substituted by other amino acids, or amino acids up to 5% of the total number of amino acids within the subject sequence may be inserted within the subject sequence. These changes within the subject sequence, may exist at the amino terminus or the carboxy teπninus of the subject sequence, or may exist at any position between these termini, or may form one or more groups of changes.
The Examples described below demonstrate that the protein consisting of an amino acid sequence of any one of the above SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110. 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288, is capable of activating NF- K B.
Related to the polynucleotide sequence of any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283 285 and 287 or the polynucleotide of a coding region (CDS) of these sequences, the present invention further provides the following isolated polynucleotides:
(a) a polynucleotide of any one of the above sequences;
(b) a polynucleotide which encodes a protein that activates NF- r B, and comprises a nucleotide sequence which has at least 95% identity, preferably at least 97-99% identity to any one of the above sequences; and
(c) a polynucleotide having a nucleotide sequence which encodes a protein that activates NF- K B and has an amino acid sequence which has at least 95% identity, preferably, at least 97-99% identity, to the amino acid sequence of any one of SEQ ID NOS: 2, 4, 6, 8 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288.
Polynucleotides which are identical or almost identical to nucleotide sequences contained in the above nucleotide sequences may be used as hybridization probes to isolate full-length cDNA and genomic clones encoding the protein of the present invention, or cDNA or genomic clones of other genes that have a high sequence similarity to the above sequences, or genomic clones, or may be used as primers for nucleic acid amplification reactions. Typically, these nucleotide sequences are 70% identical, preferably 80% identical, more preferably 90% identical, most preferably 95% identical to the above sequences. The probes or primers will generally comprises at least 15 nucleotides, preferably 30 nucleotides and may have 50 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides. Particularly preferred primers have between 20 and 25 nucleotides.
The polynucleotide of the present invention may be either in the form of a DNA such as cDNA ,a genomic DNA obtained by cloning or synthetically produced, or may be in the form of RNA such as mRNA. The polynucleotide may be single-stranded or double-stranded. The double-stranded polynucleotides may be double-stranded DNA, double- stranded RNA or DNA:RNA hybrid. The single-stranded polynucleotide may be sense strand also known as coding strand or antisense strand also known as non-coding strand.
Those skilled in the art can prepare a protein having the same activity that activates NF- K B as the protein having an amino acid sequence of any one of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288, by means of appropriate substitution of an amino acid in the protein using known methods. One such method involves using conventional mutagenesis procedures for the DNA encoding the protein. Another method is, for example, site-directed mutagenesis (e.g., Mutan-Super Express Km Kit from Takara Shuzo Co., Ltd.). Mutations of amino acids in proteins may also occur in nature. Thus, the present invention also includes a mutated protein which is capable of activating NF- K B and which has at least one amino acid deletion, substitution or addition relative to the protein of any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288, and the DNA encoding the protein. The number of mutations is preferably 1 to 10, more preferably 1 to 5, most preferably 1 to 3.
The substitutions of amino acids are preferably conservative substitutions, specific examples of which are substitutions within the following groups: (glycine, alanine), (valine, isoleucine, leucine), (aspartic acid, glutamic acid), (asparagine, glutamine), (serine, threonine), (lysine, arginine) and (phenylalanine, tyrosine).
Based on DNA (e.g., SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287) encoding a protein consisting of an amino acid sequence of any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288 or a fragment thereof, those skilled in the art can routinely isolate a DNA with a high sequence similarity to these nucleotide sequences by using hybridization techniques and the like, and obtain proteins having the same activity that activates NF- K B as the protein having of an amino acid sequence of any one of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288. Thus, the present invention also includes a protein that activates NF- K B and comprises an amino acid sequence having a high identity to the amino acid sequence of any one of the above SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288. "High identity" refers to an amino acid sequence having an identity of at least 90%, preferably at least 97 to 99% over the entire length of an amino acid sequence expressed by any one of the above SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288.
The proteins of the present invention may be natural proteins derived from any human or animal cells or tissues, chemically synthesized proteins, or proteins obtained by genetic recombination techniques. The protein may or may not be subjected to post-translational modifications such as sugar chain addition or phosphorylation.
Examples of the proteins which are encoded by the genes of the present invention include secretion proteins (growth factors, cytokines, hormones, and the like), protein modification enzymes (protein kinase, protein phosphatase, protease, and the like), signal transduction molecules (protein-protein interaction moleculaes and the like), nuclear proteins (nuclear receptor, transcription factors and the like), and membrane proteins . The membrane proteins include receptors, cell adhesion molecules, ion channels, and transporters. When the protein is a membrane protein, a compound which is selected by the screening menthioned herein below is more useful as a research tool for a pharmaceutical compound since the compound is expected to easily move into a cell or transmit a signal into a cell.
The present invention also includes a polynucleotide encoding the above protein of the present invention. Examples of nucleotide sequences encoding a protein consisting of an amino acid sequence of any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14. 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142. 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178 180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288 include nucleotide sequences of any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287. The DNA includes cDNA, genomic DNA, and chemically synthesized DNA. In accordance with the degeneracy of the genetic code, at least one nucleotide in the nucleotide sequence encoding a protein consisting of an amino acid sequence of any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288 can be substituted with other nucleotides without altering the amino acid sequence of the protein produced from the gene. Therefore, the DNA sequences of the present invention also include nucleotide sequences altered by substitution based on the degeneracy of the genetic code. Such DNA sequences can be synthesized using known methods.
The DNA of the present invention includes a DNA which encodes a protein capable of activating NF- K B and hybridizes under stringent conditions with the DNA sequence of the above nucleotide sequence of any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287, or any complementary sequence thereof. Stringent conditions are apparent to those skilled in the art, and can be easily attained in accordance with various laboratory manuals such as T. Maniatis et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory 1982, 1989.
That is, "stringent conditions" refer to overnight incubation at 37 °C in a hybridization solution containing 30% formamide, 5 x SSC (0.75 M NaCl, 75mM trisodium citrate), 5 x Denhardt's solution, 0.5% SDS, 100 μ g/ml denatured, sheared salmon sperm DNA) followed by washing (three times) in 2 x SSC, 0.1% SDS for 10 rninutes at room temperature, then followed by washing (two times) in 0.2 x SSC, 0.1% SDS for 10 minutes at 37 °C (low stringency). Preferred stringent conditions are overnight incubation at 42°C in a hybridization solution containing 40% formamide, followed by washing (three times) in 2 x SSC, 0.1% SDS for 10 minutes at room temperature, then followed by washing (two times) in 0.2 x SSC, 0.1% SDS for 10 minutes at 42 °C (moderate stringency). More preferred stringent conditions are overnight incubation at 42T^ in a hybridization solution containing 50% formamide, followed by washing (three times) in 2 x SSC, 0.1% SDS for 10 minutes at room temperature, followed by washing (two times) in 0.2 x SSC, 0.1% SDS for 10 minutes at 50°C (high stringency). The DNA sequence thus obtained must encode a protein capable of activating NF- K B.
The present invention also includes a polynucleotide comprising a nucleotide sequence which encodes a protein capable of activating NF- K B and has a high sequence similarity to the nucleotide sequence of the polynucleotide according to above item (3), (4) or (5). Typically these nucleotide sequence are 95% identical, preferably 97% identical, more preferably 98-99% identical, most preferably at least 99% identical to the nucleotide sequence of the polynucleotide according to above item (3), (4) or (5) over the entire length thereof.
The above DNA of the present invention can be used to produce the above protein using recombinant DNA techniques. In general, the DNA and peptide of the present invention can be obtained by:
(A) cloning the DNA encoding the protein of the present invention;
(B) inserting the DNA encoding the entire coding region of the protein or a part thereof into an expression vector to construct a recombinant vector;
(C) ttansforming host cells with the recombinant vector thus constructed; and
(D) culturing the obtained cells to express the protein or its analogue, and then purifying it by column chromatography.
General procedures necessary to handle DNA and recombinant host cells (e.g., E. coli) in the above steps are well known to those skilled in the art, and can be easily carried out in accordance with various laboratory manuals such as T. Maniatis et al., supra. All the enzymes, reagents, etc., used in these procedures are commercially available, and unless otherwise stated, such commercially available products can be used according to the use conditions specified by the manufacturer's instructions to attain completely its objects. The above steps (A) to (D) can be further illustrated in more details as follows.
Techniques for cloning the DNA encoding the protein of the present invention in the above step (A) include, in addition to the methods described in the specification of the present appHcation, PCR amplification using a synthetic DNA having a part of the nucleotide sequence of the present invention (e.g., any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287) as a primer, and selection of the DNA inserted into a suitable vector by hybridization with a labeled DNA fragment encoding a partial or full coding region of the protein of the present invention or a labeled synthetic DNA. Another technique involves direct amplification from total RNAs or mRNA fractions prepared from cells or tissues, using the reverse transcriptase polymerase chain reaction (RT-PCR method).
As a DNA inserted into a suitable vector, for example, a commercially available library (e.g., from CLONTECH and STRATAGENE) can be used. Techniques for hybridization are normally used in the art, and can be easily carried out in accordance with various laboratory manuals such as T Maniatis et al., supra. Depending on the intended purpose, the cloned DNA encoding the protein of the present invention can be used as such or if desired after digestion with a restriction enzyme or addition of a linker. The DNA thus obtained may have a nucleotide sequence of any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287, or a polynucleotide of above items (3) to (7). The DNA sequence to be inserted into an expression vector in the above step (B) may be a full-length cDNA or a DNA fragment encoding the above full-length protein, or a DNA fragment constructed so that it expresses a part thereof.
Thus, the present invention also provides a recombinant vector, which comprises the above DNA sequence. The expression vector for the protein of the present invention can be produced, for example, by excising the desired DNA fragment from the DNA encoding the protein of the present invention, and ligating the DNA fragment downstream of a promoter in a suitable expression vector.
Expression vectors for use in the present invention may be any vectors derived from prokaryotes (e.g., E. coli), yeast, fungi, insect viruses and vertebrate viruses so long as such vectors are replicable. However, the vectors should be selected to be compatible with microorganisms or cells used as hosts. Suitable combinations of host cell - expression vector systems are selected depending on the desired expression product.
When microorganisms are used as hosts, plasmid vectors compatible with these microorganisms are generally used as replicable expression vectors for recombinant DNA molecules. For example, the plasmids pBR322 and pBR327 can be used to transform E. coli. Plasmid vectors normally contain an origin of replication, a promoter, and a marker gene conferring upon a recombinant DNA a phenotype useful for selecting the cells transformed with the recombinant DNA. Example of such promoters include a j3 -lactamase promoter, lactose promoter and tryptophan promoter. Examples of such marker genes include an ampicillin resistance gene, and a tetracycline resistance gene. Examples of suitable expression vectors include the plasmids pUC18 and pUC19 in addition to ρBR322, pBR327.
In order to express the DNA of the present invention in yeast, for example, YEp24 can be used as a replicable vector. The plasmid YEp24 contains the URA3 gene, which can be employed as a marker gene. Examples of promoters in expression vectors for yeast cells include promoters derived from genes for 3-ρhosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase.
Examples of promoters and terminators for use in expression vectors to express the DNA of the present invention in fungal cells include promoters and terminators derived from genes for phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPD) and actin. Examples of suitable expression vectors include the plasmids pPGACY2 and pBSFAHY83.
Examples of promoters for use in expression vectors to express the DNA of the present invention in insect cells include a polyhedrin promoter and P10 promoter. Examples of expression vectors which are suitable for insect cells include baculo virus vector.
Recombinant vectors used to express the DNA of the present invention in animal cells normally contain functional sequences to regulate genes, such as an origin of replication, a promoter to be placed upstream of the DNA of the present invention, a ribosome-binding site, a polyadenylation site and a transcription termination sequence. Such functional sequences, which can be used to express the DNA of the present invention in eukaryotic cells, can be obtained from viruses and viral substances.
Examples of such functional sequences include an SR ct promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter and HSV-TK promoter. Among them, a CMV promoter and SR α promoter can be preferably used. As promoters to be placed inherently upstream of the gene encoding the protein of the present invention, any promoters can be used so long as they are suitable for use in the above host- vector systems. Examples of origins of replication include foreign origins of replication, for example, those derived from viruses such as adenovirus, polyoma virus and SV40 virus. When vectors capable of integration into host chromosomes are used as expression vectors, origins of replication of the host chromosomes may be employed. Examples of suitable expression vectors include the plasmids pSV-dhfr (ATCC 37146), pBPV-l(9-l) (ATCC 37111), pcDNA3.1 (INVITROGEN) and pME18S-FL3.
The present invention also provides a transformed cell, which comprises the above recombinant vector. Microorganisms or cells transformed with the replicable recombinant vector of the present invention can be selected from remaining untransformed parent cells based on at least one phenotype conferred by the recombinant vector as memtioned above. Phenotypes can be conferred by inserting at least one marker gene into the recombinant vector. Marker genes naturally contained in replicable vectors can be employed. Examples of marker genes include drug resistance genes such as neomycin resistance genes, and genes encoding dihydrofolate reductase.
As hosts for use in the above step (C), any of prokaryotes (e.g., E. coli), microorganisms (e.g., yeast and fungi) as well as insect and animal cells can be used so long as such hosts are compatible with the expression vectors used. Examples of such microorganisms include Escherichia coli strains such as E. coli K12 strain 294 (ATCC 31446), E. coli X1776 (ATCC 31537), E. coli C600, E. coh JM109 and E. coli B strain; bacterial strains belonging to the genus Bacillus such as Bacillus subtilis; intestinal bacteria other than E. coh, such as Salmonella typhimurium or Serratia marcescens; and various strains belonging to the genus Pseudomonas. Examples of such yeast include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris. Examples of such fungi include Aspergillus nidulans, and Acremonium chrysogenum (ATCC 11550).
As insect cells, for example, Spodoptera frugiperda (Sf cells), High Five™ cells derived from eggs of Trichoplusiani, etc., can be used when the virus is AcNPV. Examples of such animal cells include HEK 293 cells, COS-1 cells, COS-7 cells, Hela cells, and Chinese hamster ovary (CHO) cells. Among them, CHO cells and HEK 293 cells are preferred. When cells are used as hosts, combinations of expression vectors and host cells to be used vary with experimental objects. According to such combinations, two types of expression (i.e. transient expression and constitutive expression) can be included.
"Transformation" of microorganisms and cells in the above step (C) refers to introducing DNA into microorganisms or cells by forcible methods or phagocytosis of cells and then transiently or constitutively expressing the trait of the DNA in a plasmid or an intra-chromosome integrated form. Those skilled in the art can carry out transformation by known methods [see e.g., "Idenshi Kougaku Handbook (Genetic Engineering Handbook)", an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.]. For example, in the case of animal cells, DNA can be introduced into cells by known methods such as DEAE-dextran method, calcium-phosphate-mediated transfection, electroporation, hpofection, etc. For stable expression of the protein of the present invention using animal cells, there is a method in which selection can be carried out by clonal selection of the animal cells containing the chromosomes into which the introduced expression vectors have been integrated. For example, transformants can be selected using the above selectable marker as an indication of successful transformation, hi addition, the animal cells thus obtained using the selectable marker can be subjected to repeated clonal selection to obtain stable animal cell strains highly capable of expressing the protein of the present invention. When a dihydrofolate reductase (DHFR) gene is used as a selectable marker, one can culture animal cells while gradually increasing the concentration of methotrexate (MTX) and select the resistant strains, thereby amplifying the DNA encoding the protein of the present invention together with the DHFR gene in the cell to obtain animal cell strains having higher levels of expression.
The above transformed cells can be cultured under conditions which permit the expression of the DNA encoding the protein of the present invention to produce and accumulate the protein of the present invention. In this manner, the protein of the present invention can be produced. Thus, the present invention also provides a process for producing a protein, which comprises culturing a transformed cell comprising the isolated polynucleotide according to above item (3) to (7) under conditions providing expression of the encoded protein, and recovering the protein from the culture (that is, cells or culture medium).
The above transformed cells can be cultured by methods known to those skilled in the art (see e.g., "Bio Manual Series 4", YODOSHA CO., LTD.). For example, animal cells can be cultured by various known animal cell culture methods including attachment culture such as Petri dish culture, multitray type culture and module culture, attachment culture in which cells are attached to cell culture carriers (microcarriers), suspension culture in which productive cells themselves are suspended. Examples of media for use in the culture include media commonly used for animal cell culture, such as D-MEM and RPMI 1640.
In order to separate and purify the protein of the present invention from the above culture, suitable combinations of per se known separation and purification methods can be used. Examples such methods include methods based on solubility, such as salting-out and solvent precipitation; methods based on the difference in charges, such as ion-exchange chromatography; methods mainly based on the difference in molecular weights, such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis; methods based on specific affinity, such as affinity chromatography; methods based on the difference in hydrophobicity, such as reverse phase high performance liquid chromatography; and methods based on the difference in isoelectric points, such as isoelectric focusing. For example, a protein of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during intracellular synthesis, isolation or purification.
The protein of the present invention can also be produced as a fusion protein with another protein. These fusion proteins are also included within the present invention. For the expression of such fusion proteins, any vectors can be used so long as the DNA encoding the protein can be inserted into the vectors and the vectors can express the fusion protein. Examples of proteins to which a polypeptide of the present invention can be fused include glutathione S-transferase (GST) and a hexa-histidine sequence (6 x His). The fusion protein of the protein of the present invention with another protein can be advantageously purified by affinity chromatography using a substance with an affinity for the fusion partner protein. For example, fusion proteins with GST can be purified by affinity chromatography using glutathione as a ligand.
When the protein of the present invention is a membrane protein, a transformed cell into which DNA encoding the protein of the present invention has been introduced can express the protein on its membrane. The membrane which is prepared from such transformed cells and contains the protein of the present invention is also included within the present invention. As used herein, "membrane of a cell" includes cell membrane, and membrane of cell organelle. The membrane of a cell can be prepared by a method known to those skilled in the art. For example, cells are collected from the culture where transformed cells are cultured, and suspended in a suitable buffer. Then, the cells are lysed by a homogenizer or by vortex after addition of glassbeads. The obtained solution is centrifuged to remove uncrushed cells and the like, and the supernatant is ultracentrifuged under a sutable condition, and the obtained precipitate is suspended in a buffer to prepare a membrabe fraction. The condition for ultracentrifugation can be suitably selected depending on the type of membrane and the like.
The present invention also includes a protein capable of inhibiting the activity of the protein of the present invention. Examples of such proteins include antibodies, or other proteins that bind to active sites of the protein of the present invention, thereby inhibiting the expression of their activity.
The present invention also relates to an antibody that reacts with the protein of the present invention or a fragment thereof, and to production of such an antibody. More preferably, the present invention relates to an antibody that specifically react with the protein of the present invention or a fragment thereof, and to production of such an antibody. As used herein, "specifically" means that closs-reactivity is low, more preferably closs-reactivity is not present.
The antibody of the present invention is not specifically limited so long as it can recognize the protein of the present invention. Examples of such antibodies include polyclonal antibodies, monoclonal antibodies and their fragments, single chain antibodies and humanized antibodies. Antibody fragments can be produced by known techniques. Examples of such antibody fragments include, but not limited to, F(ab')2 fragments, Fab' fragments, Fab fragments and Fv fragments. For example, a monoclonal or polyclonal antibody can be produced by administering the protein according to above item (1) or (2) as an antigen or epitope-bearing fragments to a non-human animal. The antibody against the protein of the present invention can be produced by using the protein of the present invention or a peptide thereof as an immunogen according to per se known process for producing antibodies or antisera. Such methods are described, for example, in "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", the third edition, an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.
In the case of polyclonal antibodies, for example, the protein of the present invention or a peptide thereof can be injected to animals such as rabbits to produce antibodies directed against the protein or peptide, and then their blood can be collected. The polyclonal antibodies can be purified from the blood, for example, by ammonium sulfate precipitation or ion-exchange chromatography, or by using the affinity column on which the protein has been immobilized.
In the case of monoclonal antibodies, for example, animals such as mice are immunized with the protein of the present invention, their spleen is removed and homogenized to obtain spleen cells, which are then fused with mouse myeloma cells by using a reagent such as polyethylene glycol. From the resulting hybrid cells (i.e. hybridoma cells), the clone producing the antibody directed against the protein of the present invention can be selected. Then, the resulting clonal hybridoma cells can be implanted intraperitoneally into mice, the ascitic fluid recovered from the mice. The resulting monoclonal antibody can be purified, for example, by ammonium sulfate precipitation or ion-exchange chromatography, or by using the affinity column on which the protein has been immobilized.
When the resulting antibody is used to administer it to humans, it is preferably used as a humanized antibody or human antibody in order to reduce its immunogenicity. The humanized antibody can be produced using transgenic mice or other mammals. For a general review of these humanized antibodies and human antibodies, see, for example, Morrison, S.L. et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984); Jones, P.T. et al., Nature 321:522-525 (1986); Hiroshi Noguchi, Igaku no Ayumi (J. Clin. Exp. Med.) 167:457-462 (1993); Takashi Matsumoto, Kagaku to Seibutsu (Chemistry and Biology) 36:448-456 (1998). Humanized chimeric antibodies can be produced by linking a V region of a mouse antibody to a C region of a human antibody. Humanized antibodies can be produced by substituting a sequence derived from a human antibody for a region other than a complementarity-determining region (CDR) from a mouse monoclonal antibody.
In addition, human antibodies can be directly produced in the same manner as the production of conventional monoclonal antibodies by immunizing the mice whose immune systems have been replaced with human immune systems. These antibodies can be used to isolate or to identify clones expressing the protein.
Also, these antibodies can be used to purify the protein of the present invention from a cell extract or transformed cells producing the protein of the present invention. These proteins can also be used to construct ELISA, RIA (radioimmunoassay) and western blotting systems. These assay systems can be used for diagnostic purposes for detecting an amount of the protein of the present invention present in a body sample in a tissue or a fluid in the blood of an animal, preferably human. For example, they can be used for diagnosis of a disease characterized by undesirable activation of HF- K B resulting from (expression) abnormality of the protein of the present invention, such as inflammation, autoimmune diseases, infectious diseases, cancers, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
In order to provide a basis for diagnosis of a disease, a standard value (that is, a normal value for the expression of the protein of the present invention) must be established. However, this is a well-known technique to those skilled in the art. For example, a method of calculating the standard value comprises binding a body fluid or a cell extract of normal individual of a human or an animal to an antibody against the protein of the present invention under a suitable condition for the complex formation, detecting the amount of the antibody-protein complex by chemical or physical means and then calculating the standard value for the normal sample using a standard curve prepared from a standard solution containing a known amount of an antigen (the protein of the present invention). The presence of a disease can be confirmed by deviation from the standard value obtained by comparison of the standard value with the value obtained from a sample of an individual latently suffering from a disease associated with the protein of the present invention. These antibodies can also be used as reagents for studying functions of the protein of the present invention.
The antibody of the present invention can be used as a medicament as mentioned below. When the antibody of the present invention is used as a medicament, it is preferred to use an antibody capable of inhibiting the activity of activating NF- K B of the protein of the present invention (that is, neutralizing antibody).
The antibodies of the present invention can be purified and then administered to patients of a disease characterized by undesirable activation of NF- K B resulting from (expression) abnormality of the protein of the present invention, such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders. Thus in another aspect, the present invention is a pharmaceutical composition which comprises the above antibody as an active ingredient, and a method for therapy and/or prevention using the antibody of the present invention. In such pharmaceutical compositions, the active ingredient may be combined with other therapeutically or preventively active ingredients or inactive ingredients (e.g., conventional pharmaceutically acceptable carriers or diluents such as immunogenic adjuvants) and physiologically non-toxic stabilizers and excipients. The resulting combinations can be sterilized by filtration, and formulated into vials after lyophilization or into various dosage forms in stabilized and preservable aqueous preparations.
Administration to a patient can be intra-arterial aclministration, intravenous administration and subcutaneous administration, which are well known to those skilled in the art. The dosage range depends upon the weight and age of the patient, route of administration and the like. Suitable dosages can be determined by those skilled in the art. These antibodies exhibit therapeutic activity by inhibiting the NF- K B activation mediated by the protein of the present invention. More specifically, the antibody of the present invention is useful as a medicament for treating or preventing a disease associated with abnormality of NF- K B activity such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
The DNA of the present invention can also be used to isolate, identify and clone other proteins involved in intracellular signal transduction processes. For example, the DNA sequence encoding the protein of the present invention can be used as a "bait" in yeast two-hybrid systems (see e.g., Nature 340:245-246 (1989)) to isolate and clone the sequence encoding a protein ("prey") which can associate with the protein of the present invention. In a similar manner, it can be determined whether the protein of the present invention can associate with other cellular proteins (e.g., NIK and TRAF2). In another method, proteins which can associate with the protein of the present invention can be isolated from cell extracts by immunoprecipitation [see e.g., "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.] using antibodies directed against the protein of the present invention. In still another method, the protein of the present invention can be expressed as a fusion protein with another protein as described above, and immunoprecipitated with an antibody directed against the fusion protein in order to isolate a protein which can associate with the protein of the present invention.
The present invention provides a process for diagnosing a disease or susceptibility to a disease related to expression or activity of the protein of present invention in a subject comprising the steps of:
(a) determining the presence or absence of a mutation in the gene encoding said protein in the genome of said subject; and/or
(b) analyzing the amount of expression of said protein in a sample derived from said subject.
The diagnostic assays offer a process for diagnosing diseases or determining a susceptibility to the diseases through detection of mutation in a gene for the protein of the present invention which has a function of activating NF- K B, by the methods described. In addition, such diseases may be diagnosised by methods comprising determining from a sample derived from a subject an abonormally decreased or increased level of protein or mRNA.
The determination of the presence or absence of a mutation in the nucleotide seqeunce of a the gene encoding the protein of the present invention which has a function of activating NF- K B, may involve RT-PCR using a part of the nucleotide sequences of genes encoding these proteins as a primer, followed by conventional DNA sequencing to detect the presence or absence of the mutation. PCR-SSCP [Genomics 5:874-879 (1989); "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.] can also be used to determine the presence or absence of the mutation.
Decreased or increased expression of a gene in a sample can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, for example, nucleic acid amplification methods such as RT-PCR, and methods such as RNase protection assay, Northern blotting and other hybridization methods. Assay techniques that can be used to deteπnine levels of a protein in a sample derived from a host are well-known to those skilled in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western blot analysis and ELISA assays. When an expression level is determined at a protein level, the antibody of the present invention mentioned above can be used. The degree of abnormality of expression level of gene in a sample is not particularly Umited. For example, when the level of the expressed protein is 2 or more times, or 1/2 or less, as compared with normal case, the subject may be disgnosed to be a disease. In another example, when the level of the expressed protein is 3 or more times, or 1/3 or less, as compared with normal case, the subject may be disgnosed to be a disease.
The DNA of the present invention can be used to detect abnormality in the DNA or mRNA encoding the protein of the present invention or a peptide fragment thereof. Therefore, the DNA is useful for gene diagnosis such as detection of damage, mutation, decreased, increased or excessively increased expression of said DNA or mRNA.
When the nucleotide sequence encoding the protein of the present invention in a genome of an individual contains a mutation, the mutation may cause a disease associated with the expression and/or activity of NF- /c B.
When the amount of the expression of the protein in a sample from an individual is different from the normal value, the abnormal expression of the novel protein of the present invention which acts to activate NF- K B may be responsible for diseases associated with the expression and/or activity of NF- K B.
The present invention also relates to a method for screening compounds which inhibit or promote NF- /c B activation using the proteins of the invention.
A compound which inhibits NF- K B activation has an activity as an inhibitor of NF- K B in vivo or in vitro as a result of this function, while a compound which promotes NF- K B activation has an activity as an activator of NF- K B in vivo or in vitro as a result of this function. Therefore, an activity as an inhibitor or activator of NF- K B is screened in the method for screening of the present invention. The above compounds have an activity as an inhibitor or activator of NF- K B.
The method for screening comprises the steps of: (a) preparing a transformed cell by introducing a gene encoding a protein that promotes activation of NF- K B according to the present invention and a gene encoding a signal which can detect activation of NF- K B into a cell;
(b) culturing the transformed cell under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring the signal which can detect activation of NF- K B; and
(d) selecting a candidate compound which can change the signal amount as compared with the case of the absence of candidate compounds, as a compound capable of inhibiting or promoting NF- K B activation.
Further, it is preferable to isolate or identify as an activator compound, a compound that increases said detectable signal 2-fold or higher than normal, and to isolate or identify as an inhibitor compound, a compound that decreases said detectable signal half or less than normal.
Examples of genes encoding a signal which can detect activation of NF- K B include reporter genes. Reporter genes are used instead of directly detecting the activation of transcription factors of interest. The transcriptional activity of a promoter of a gene is analyzed by linking the promoter to a reporter gene and measuring the activity of the product of the reporter gene ("Bio Manual Series 4" (1994), YODOSHA CO., LTD.).
Any peptide or protein can be used so long as those skilled in the art can measure the activity or amount of the expression product (including the amount of the produced mRNA) of the reporter genes. For example, enzymatic activity of chloramphenicol acetyltransferase, β -galactosidase, luciferase, etc., can be measured. Any reporter plasmids can be used to evaluate NF- K B activation so long as the reporter plasmids have an NF- K B recognition sequence inserted upstream of the reporter gene. For example, pNF- r B-Luc (STRATAGEGE) can be used. Other examples include NF- K B dependent reporter plasmids described in Tanaka S. et al., J. Vet. Med. Sci. Vol.59 (7); Rothe M. et al., Science Vol.269, p.1424-1427 (1995).
Any host cells may be used so long as NF- K B activation can be detected in the host cells. Preferred host cells are mammalian cells such as 293-EBNA cells. Transformation and culture of the cells can be carried out as described above.
In a specific embodiment, the method for screening a compound which inhibits or promotes NF- K B activation comprises culturing the transformed cell for a certain period of time, adding a certain amount of a test compound, measuring the reporter activity expressed by the cell after a certain period of time, and comparing the activity with that of a cell to which the test compound has not been added. The reporter activity can be measured by methods known in the art (see e.g., "Bio Manual Series 4" (1994), YODOSHA CO., LTD.).
Examples of test compounds include, but not limited to, low molecular weight compounds, haigh molecular compounds, and peptides. Test compounds may be artificially synthesized compounds or naturally occurring compounds. Test compounds may be a single compound or mixtures. Usable examples includes a library of low molecular weight compounds, a compound library which was synthesized by combinatorial chemistry, a narurally occurring product containing cells, plants, animals or a part thereof, or an extracred product of such narurally occurring product. When a mixture containing several compounds is used as a test substance for screening, the test substance which shows an activity of inhibiting or promoting NF- K B activation can be further screened to isolate a single substance having the activity. Isolate and purification of a desired compound from a mixture can be carried out by using any knonw method such as filteration, extraction, washing, drying, concentration, crystallization or various chromatography in combination.
The method for screening according to the present invention can be carried out by the following steps:
(a) preparing a transformed cell by introducing a gene encoding a protein that promotes activation of NF- K B according to the present invention into a cell;
(b) culturing the transformed cell under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring the activation of NF- K B; and (d) selecting a candidate compound which can change the activation of NF- K B as compared with the case of the absence of candidate compounds, as a compound capable of inhibiting or promoting NF- K B activation.
Methods for measuring the activation of NF- K B in the above method include a method of analyzing the binding of NF- /c B to its binding sequence using cell extraction solution by gel shift (for example, Hayashi T. etal. J.Biol.Chem.268, p.26790-26795 (1993), Nauman M. et al. EMBO J. 13, p4597-4607 (1994)). Alternatively, the amount of mRNA or proteins for genes whose expression is known to be induced by NF- K B activation (e.g., genes for IL-1 and TNF- a ), can be measured. The amount of mRNA can be measured, for example, by northern hybridization, RT-PCR, etc. The amount of proteins can be measured, for example, by using antibodies. The antibodies may be produced by known methods. Commercially available antibodies(from, e.g., Wako Pure Chemical Industries, Ltd.) can also be used.
The present invention further provided a method of producing a pharmaceutical composition, which comprises the following steps (a) to (f):
(a) preparing a transformed cell by introducing a gene encoding a protein that promotes activation of NF- K B according to the present invention and a gene encoding a signal which can detect activation of NF- r B into a cell;
(b) culturing the transformed cell under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring the signal which can detect activation of NF- re B;
(d) selecting a candidate compound which can change the signal amount as compared with the case of the absence of candidate compounds, as a compound capable of inhibiting or promoting NF- K B activation; and
(e) producing a pharmaceutical composition which comprises a compound selected in the step of (d).
In the present invention, a pharmaceutical composition can be produced by the following steps: (a) preparing a transformed cell by introducing a gene encoding a protein that promotes activation of NF- K B according to the present invention into a cell;
(b) culturing the transformed cell under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring the activation of NF- K B; and
(d) selecting a candidate compound which can change the activation of NF- K B as compared with the case of the absence of candidate compounds, as a compound capable of inhibiting or promoting NF- c B activation; and
(e) producing a pharmaceutical composition which comprises a compound selected in the step of (d).
In the step (d) of the method of producing a pharmaceutical composition, it is preferable to isolate or identify as an activator compound, a compound that increases said detectable signal 2-fold or higher than normal, and to isolate or identify as an inhibitor compound, a compound that decreases said detectable signal half or less than normal.
The protein of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the protein, by:
(a) detennining in the first instance the three-dimensional structure of the protein;
(b) deducing the three-dimensional structure for the likely reactive or binding site(s) of an agonist, antagonist or inhibitor;
(c) synthesising candidate compounds that are predicted to bind to or react with the deduced binding or reactive site; and
(d) testing whether the candidate compounds are indeed agonists, antagonists or inhibitor.
The present invention also provides a compound which is selected by the above screening method. This compound has a function of inhibiting or promoting NF- /c B activation. More specifically, this compound has a function of inhibiting or promoting NF- K B activation by the protein of the present invention.
The compounds obtained by the above screening methods have a function of inhibiting or promoting NF- K B activation, they are useful as therapeutic or preventive pharmaceuticals for the treament of diseases resulting from unfavorable activation or inactivation of NF- K B.
When obtainment of a salt of the compounds is desired, a compound which is obtained in the form of a salt can be purified as it is. A compound which is obtained in the free form can be converted into a salt by isolating and purifying a salt obtained by dispersing or dissolving the compound into a suitable solvent and then adding a desired acid or base. Examples of a step to optimize the compounds or salts thereof obtained by the method of the present invention as a pharmceutical composition, include methods of formulating according to ordinary processes such as the following. The above compounds or their pharmaceutically acceptable salts in an amount effective as an active ingredient, and pharmaceutically acceptable carriers can be mixed. A form of formulation suitable for the mode of administration is selected. A composition suitable for oral administration includes a solid form such as tablet, granule, capsule, pill and powder, and solution form such as solution, syrup, elixir and dispersion. A form useful for parenteral administration includes sterile solution, dispersion, emulsion and suspension. The above carriers include, for example, sugars such as gelatin, lactose and glucose, starches such as corn, wheat, rice and maize, fatty acids such as stearic acid, salts of fatty acids such as calcium stearate, magnesium stearate, talc, vegetable oil,alcohol such as stearyl alcohol and benzyl alcohol, gum, and polyalkylene glycol. Examples of such liquid carriers include generally water, saline, sugar solution of dextrose and the like, glycols such as ethylene glycol, propylene glycol and polyethylene glycol.
The present invention also provides a kit for screening compounds which inhibit or promote NF- K B activation. The kit comprises reagents and the like necessary for screening compounds which inhibit or promote NF- K B activation, including:
(a) a transformed cell comprising a gene encoding a protein that promotes activation of NF- K B according to the present invention and a gene encoding a signal which can detect activation of NF- K B; and
(b) reagents for measuring the signal. In another aspect, the present invention relates to a diagnostic kit which comprises:
(a) a polynucleotide of the present invention having a nucleotide sequence expressed by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,
167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287;
(b) a polynucleotide having a nucleotide sequence complementary to that of (a);
(c) a protein of the present invention having an amino acid seqeunce expressed by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,
168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288, or a fragment thereof; or
(d) an antibody to the protein of the present invention of (c).
A kit comprising at least one of (a) to (d) is useful for diagnosing a disease or susceptibility to a disease such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
Because NF- K B is involved in a wide variety of pathological conditions such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders, it is an attractive target for drug design and therapeutic intervention. Many experiments show that NF- K B activity may have significant physiological effects [see e.g., Ann. Rheum. Ds. 57:738-741 (1998); American Journal of Pathology 152:793-803 (1998); ARTHRITIS & RHEUMAΗSM 40:226-236 (1997); Am. J. Respir. Crit. Care Med. 158:1585-1592 (1998); J. Exp. Med. 188:1739-1750 (1998); Gut 42:477-484 (1998); The Journal of Immunology 161:4572-4582 (1998); Nature Medicine 3:894-899 (1997)].
The finding of the new protein described herein capable of activating NF- K B has provided a new medicament and method for controlling an abnormal NF- K B function. Thus, the present invention also relates to a method of use of a compound which inhibits the function of the protein capable of activating NF- /c B described above, for inhibiting NF- K B activation. Further, the present invention relates to a method of using a compound which activates the function of the protein capable of activating NF- K B described above, for promoting NF- K B activation. The compound obtained by the above screening method, which inhibits NF- K B activation, is useful as a medicament to treat or prevent diseases characterized by undesirable activation of NF- K B, such as inflammation, autoimmune diseases (such as rheumatoid arthritis, systemic lupus erythematosus, asthma, etc), infectious diseases, bone diseases, and graft rejection. Recently, it has also become apparent that NF- K B activation controls apoptosis of cells. The compound obtained by the above screening method, which inhibits NF- /c B activation, may be capable of stimulating apoptosis. Diseases which may be treated by the induction of apoptosis include tumors.
Further, examples of diseases related to abnormality in NF- K B activation include AIDS (acquired immunodeficiency syndrome), neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, amyoxrophic lateral sclerosis, etc.), ischemic disorders (i.e. those caused by cardiac infarction, reperfusion injury, etc), myelogenesis incompetency syndrome (aplastic anemia, etc), skin diseases (Toxic epidermal necrolysis, etc), proliferative nephritis (IgA nephritis, purpuric nephritis, lupus nephritis, etc) and fulminant hepatitis. Thus, a compound obtained by the above screening method, which inhibits or promotes NF- K B activation, is useful as a medicament to treat or prevent these diseases.
In addition, the gene encoding the protein of the present invention is useful for gene therapy to treat various diseases such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders. "Gene therapy" refers to adniinistering into the human body a gene or a cell into which a gene has been introduced for the purpose of therapy of diseases. The protein of the present invention and the DNA encoding the protein can also be used for diagnostic purposes. Thus, the present invention provides a agent for gene therapy which comprises a gene encoding the protein of the present invention.
The form of the agent for gene therapy is not particularly limited, but includes a pharmaceutical composition which comprises a expression vector containing a gene of the present invention in a pharmaceutical carrier of physiological buffer. The pharmaceutical carrier may contain suitable stabilizer (for example, nuclease inhibitor), chelate agent (for example, EDTA), and/or other auxiliary agent. Alternatively, the agent for gene therapy of the present invention may be provided as a complex of an expression vector containing a gene of the present invention and a liposome. The agent for gene therapy may be applied using a catheter. For example, the agent for gene therapy of the present invention can be directly injected into a blood vessel of patient and the like.
The dosage of the agent for gene therapy of the present invention should be selected depending on the conditions such as age, sex, body weight and symptom of patient, and administration route, and is generally about 1 μ g/kg to about 1000 mg/kg, more preferably about 10 μ g/kg to about 100 mg/kg, as an amount of DNA (which is an effective ingredient) per one administration for adult. The number of administration is not particularly hmited.
The compound obtained by the screening method of the present invention or a salt thereof can be formulated into the above pharmaceutical compositions (e.g., tablets, capsules, elixirs, microcapsules, sterile solutions and suspensions) according to conventional procedures. The formulations thus obtained are safe and of low toxicity, and can be administered, for example, to humans and mammals (e.g., rats, rabbits, sheep, pigs, cattle, cats, dogs and monkeys). Administration to patients can be carried out by methods known in the art, such as intra-arterial injection, intravenous injection and subcutaneous injection. The dosage and administration mode may vary with the weight and age of the patient, but those skilled in the art can appropriately select suitable administration mode and can appropriately select suitable dosage depending on the administration mode. When the compound can be encoded by DNA, the DNA can be inserted into a vector for gene therapy, and gene therapy can be carried out. Thus, the present invention relates to a medicament which comprises a compound capable of inhibiting or promoting NF- K B activation as an active ingredient.
In addition, the above compound is useful as a medicament to treat or prevent diseases characterized by abnormal NF- K B activity, such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders. Thus, the present invention also relates to a medicament to treat or prevent inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases, ischemic disorders and the like, which comprises a compound capable of inhibiting or promoting NF- K B activation. Specifically, the compound is useful as a therapeutic and/or prophylactic drug against, for example, rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, diabetes, sepsis, asthma, allergic rhinitis, ischemic heart diseases, inflammatory intestinal diseases, subarachnoid hemonhage, viral hepatitis, AIDS, atherosclerosis, atopic dermatitis, viral infections, Crohn's disease, gout, hepatitis, multiple sclerosis, cardiac infarction, nephritis, osteoporosis, Alzheimer's, Parkinson's disease, Huntington's chorea, psoriasis, amyo trophic lateral sclerosis, or aplastic anemia.
The present invention also relates to the use of the above compound for manufacturing a medicament for the therapy and/or prevention of inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases, ischemic disorders and the like.
The present invention also provides an antisense ohgonucleotide against the polynucleotide of any one of above items (3) to (7). An antisense ohgonucleotide refers to an oligonucleotide complementary to the target gene sequence. The antisense oligonucleotide can inhibit the expression of the target gene by inhibiting RNA functions such as translation to proteins, transport to the cytoplasm and other activity necessary for overall biological functions. In this case, the antisense ohgonucleotide may be RNA or DNA. The DNA sequence of the present invention can be used to produce an antisense oligonucleotide capable of hybridizing with the mRNA transcribed from the gene encoding the protein of the present invention. It is known that an antisense oligonucleotide generally has an inhibitory effect on the expression of the corresponding gene (see e.g., Saibou Kougaku Vol.13, No.4 (1994)). The oligonucleotide containing an antisense coding sequence against a gene encoding the protein of the present invention can be introduced into a cell by standard methods. The oligonucleotide effectively blocks the translation of mRNA of the gene encoding the protein of the present invention, thereby blocking its expression and inhibiting undesirable activity.
The antisense oligonucleotide of the present invention may be a naturally occurring oligonucleotide or its modified form [see e.g., Murakami & Makino, Saibou Kougaku Vol.13, No.4, p.259-266 (1994); Akira Murakami, Tanpakushitsu Kakusan Kouso (PROTEIN, NUCLEIC ACID AND ENZYME) Vol.40, No.10, p.1364-1370 (1995),Tunenari Takeuchi et al., Jikken Igaku (Experimental Medicien) Vol. 14, No. 4 p85-95(1996)]. Thus, the oligonucleotide may have modified sugar moieties or inter-sugar moieties. Examples of such modified forms include phosphothioates and other sulfur-containing species used in the art. According to several prefened embodiments of the present invention, at least one phosphodiester bond in the oligonucleotide is substituted with the structure which can enhance the ability of the composition to permeate cellular regions where RNA with the activity to be regulated is located.
Such substitution preferably involves a phosphorothioate bond, a phosphoramidate bond, methylphosphonate bond, or a short-chain alkyl or cycloalkyl structure. The antisense ohgonucleotide may also contain at least some modified base forms. Thus, it may contain purine and pyrimidine derivatives other than naturaUy occurring purine and pyrimidine. Similarly, the furanosyl moieties of the nucleotide subunits can be modified so long as the essential purpose of the present invention is attained. Examples of such modifications include 2'-O-alkyl and 2'-halogen substituted nucleotides. Examples of modifications in sugar moieties at their 2-position include OH, SH, SCH3, OCH3, OCN or O(CH2)nCH3, wherein n is 1 to about 10, and other substituents having similar properties. AU the analogues are included in the scope of the present invention so long as they can hybridize with the mRNA of the gene of the present invention to inhibit functions of the mRNA.
The antisense oligonucleotide of the present invention contains about 3 to about 50 nucleotides, preferably about 8 to about 30 nucleotides, more preferably about 12 to about 25 nucleotides. The oligonucleotide of the present invention can be produced by the well-known sohd phase synthesis technique. Devices for such synthesis are commercially available from some manufactures including Applied Biosystems. Other ohgonucleotides such as phosphothioates can also be produced by methods known in the art.
The antisense oligonucleotide of the present invention is designed to hybridize with the mRNA transcribed from the gene of the present invention. Those skilled in the art can easily design an antisense ohgonucleotides based on a given gene sequence (For example, Murakami and Makino: Saibou Kougaku Vol. 13 No.4 p259-266 (1994), Akira Murakami: Tanpakushitsu Kakusan Kouso (PROTEIN, NUCLEIC ACID AND ENZYME) Vol. 40 No.10 pl364-1370 (1995), Tunenari Takeuchi et al., Jikken Igaku (Experimental Medicine) Vol. 14 No. 4 p85-95 (1996)). Recent sutudy suggests that antisense oligonucleotides which are designed in a region containing 5' region of mRNA, preferably, the translation initaiation site, are most effective for the inhibition of the expression of a gene. The length of the antisense oligonucleotides is preferably 15 to 30 nucleotides and more preferably 20 to 25 nucleotides. It is important to confirm no interaction with other mRNA and no formation of secondary structure in the oligonucleotide sequence by homology search. The evaluation of whether the designed antisense oligonucleotide is functional or not can be determined by introducing the antisence oligonucleotide into a suitable cell and measuring the amount of the target mRNA, for example by northern blotting or RT-PCR, or the amount of the target protein, for example by western blotting or fluorescent antibody technique, to confirm the effect of expression inhibition.
Another method includes the triple helix technique. This technique involves forming a triple helix on the targeted intra-nuclear DNA sequence, thereby regulating its gene expression, mainly at the transcription stage. The oligonucleotide is designed mainly in the gene region involved in the transcription and inhibits the transcription and the production of the protein of the present invention. Such RNA, DNA and oligonucleotide can be produced using known synthesizers.
The antisense oligonucleotide may be introduced into the ceUs containing the target nucleic acid sequence by any of DNA transfection methods such as calcium phosphate method, electroporation,lipofection, microinjection, or gene transfer methods including the use of gene transfer vectors such as viruses. An antisense oligonucleotide expression vector can be prepared using a suitable rexrovirus vector, then the expression vector can be introduced into the cells containing the target nucleic acid sequence by contacting the vector with the cells in vivo or ex vivo.
The DNA of the present invention can be used in the antisense RNA/DNA technique or the triple helix technique to inhibit NF- K B activation mediated by the protein of the present invention.
The antisense ohgonucleotide against the gene encoding the protein of the present invention is useful as a medicament to treat or prevent diseases characterized by undesirable activation of NF- K B, such as inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders. Thus, the present invention also provides a medicament which comprises the above antisense ohgonucleotide as an active ingredient. The antisense oligonucleotide can also be used to detect such diseases using northern hybridization or PCR.
The present invention also provides a ribozyme or deoxyribozyme which inhibits NF- n B activation. A ribozyme and deoxyribozyme is an RNA capable of recognizing a nucleotide sequence of a nucleic acid and cleaving the nucleic acid (see e.g., Hiroshi Yanagawa, "Jikken Igaku (Experimental Medicine) Bioscience 12: New Age of RNA). The ribozyme or deoxyribozyme can be produced so that it cleaves the selected target RNA (e.g., mRNA encoding the protein of the present invention). Based on the nucleotide sequence of the DNA encoding the protein of the present invention, the ribozyme or deoxyribozyme specifically cleaving the mRNA of the protein of the present invention can be designed. Such ribozyme has a complementary sequence to the mRNA for the protein of the present invention, complementarily associates with the mRNA and then cleaves the mRNA, which results in reduction or entire loss of the expression of the protein of the present invention. The level of the reduction of the expression is dependent on the level of the ribozyme or deoxyribozyme expression in the target cells.
There are two types of ribozyme or deoxyribozyme commonly used: a hammerhead ribozyme and a hairpin ribozyme. In particular, hammerhead ribozymes or deoxyribozymes have been well studied regarding their primary and secondary structure necessary for their cleavage activity, and those skilled in the art can easily design the ribozymes nucleotided solely on the nucleotide sequence information for the DNA encoding the protein of the present invention [see e.g., Iida et al., Saibou Kougaku Vol.16, No.3, p.438-445 (1997); Ohkawa & Taira, Jikken Igaku (Experimental Medicine) Vol.12, No.12, p.83-88 (1994)]. It is known that the hammerhead ribozymes or deoxyribozymes have a structure consisting of two recognition sites (recognition site I and recognition site II forming a chain complementary to target RNA) and an active site, and cleave the target RNA at the 3 'end of its sequence NUX (wherein N is A or G or C or U, and X is A or C or U) after the formation of a complementary pair with the target RNA in the recognition sites. In particular, the sequence GUC (or GUA) has been found to have the highest activity [see e.g., Koizumi, M. et al., Nucl. Acids Res. 17:7059-7071 (1989); Uda et al., Saibou Kougaku Vol.16, No.3, p.438-445 (1997); Ohkawa & Taira, Jikken Igaku (Experimental Medicine) Vol.12, No.12, p.83-88 (1994); Kawasaki & Taira, Jikken Igaku (Experimental Medicine) Vol.18, No.3, p.381-386 (2000)].
Therefore the sequence GTC (or GTA) is searched out, and a ribozyme is designed to form several,up to 10 to 20 complementary base pairs around that sequence. The suitability of the designed ribozyme can be evaluated by checking whether the prepared ribozyme can cleave the target mRNA in vitro according to the method described for example in Ohkawa & Taira, Jikken Igaku (Experimental Medicine) Vol.12, No.12, p.83-88 (1994). The ribozyme can be prepared by methods known in the art to synthesize RNA molecules.
Alternatively, the sequence of the ribozyme can be synthesized on a DNA synthesizer and inserted into various vectors containing a suitable RNA polymerase promoter (e.g., T7 or SP6) to enzymatically synthesize an RNA molecule in vitro. Such ribozymes can be introduced into cells by gene transfer methods such as microinjection. Another method involves inserting a ribozyme DNA into a suitable expression vector and introducing the vector into ceU strains, cells or tissues. Suitable vectors can be used to introduce the ribozyme into a selected cell. Examples of vectors commonly used for such purpose include plasmid vectors and animal virus vectors (e.g., retrovirus, adenovirus, herpes or vaccinia virus vectors). Such ribozymes are capable of inhibiting the NF- K B activation mediated by the protein of the present invention.
The present invention further provides a process for obtaining a new gene having a function, which comprises using the oligo-capping method to construct a full-length cDNA library, and detecting the presence of a protein having the function by using a signal factor. An example of such signal factor is a reporter gene. Methods using a cDNA library containing a lot of non-full-length cDNAs are inefficient in obtaining many genes (cDNAs) having functions. Therefore libraries with a high ratio of the number of the full-length cDNA clones to the total number of the clones are necessary. "Full-length cDNA" refers to a complete DNA copy of mRNA from a gene. The cDNA libraries produced using the oligo-capping method contain full-length cDNA clones in a ratio of 50 to 80%, namely, a 5 to 10-fold increase in full-length cDNA clones compared to the cDNA libraries produced by prior art methods (Sumio Sugano, the monthly magazine BIO INDUSTRY Vol.16, No.ll, p.19-26). Full-length cDNA clones are essential for protein expression in functional analyses of genes, and full-length cDNA clones themselves are very important materials for activity measurement. Thus, cloning of full-length cDNA is necessary for functional analyses of genes. Sequencing of the cDNA not only provides important information for establishing the primary sequence of the protein encoded by the cDNA, but also reveals the entire exon sequence. Thus, the full-length cDNA provides valuable information for identifying a gene, such as information for determining the primary sequence of a protein, exon-intron structure, the transcription initiation site of mRNA, the location of a promoter, etc.
The construction of full-length cDNA libraries by the oligo-capping method can be carried out, for example, according to the method described in "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", the third edition (1999), an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD. The oligo-capping method used herein involves substituting a cap structure with a synthetic oligo sequence by using BAP, TAP and an RNA ligase, as described in Suzuki & Sugano, "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", the third edition (1999), an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.
The reporter gene which can be used as a signal factor which indicates the presence of a protein having a function contains one or more suitable expression regulation sequence portion to which a protein factor such as a transcriptional factor can bind, and a structural gene portion which allows the measurement of the activation of the proteins factor. The structural gene portion may encode any peptide or protein so long as those skiUed in the art can measure the activity or amount of its expression product (including the amount of the mRNA produced). For example, chloramphenicol acetyltransferase, β -galactosidase, luciferase, etc., can be used and their enzymatic activity measured.
Examples of reporter genes which indicate the presence of a protein having a function include reporter genes containing a CREB (cAMP responsive element binding protein) binding sequence or AP-1 (activator protein- 1) binding sequence at the expression regulation sequence region of the reporter genes, in addition to the NF- K B reporter genes described herein.
For example, if a gene capable of activating CREB is to be obtained, a CREB-dependent reporter plasmid and an expression vector comprising full-length cDNA produced by the oligo-capping method can be cotransfected into cells, and an expression vector having increased reporter activity can be selected from the cells to attain the purpose. If a gene capable of inhibiting CREB is to be obtained, a CREB-dependent reporter plasmid and an expression vector comprising full-length cDNA produced by the oligo-capping method can be cotransfected into cells, and an expression vector having decreased reporter activity can be selected from the cells to attain the purpose. These procedures may be carried out in the presence of a certain stimulus to the cells. The cDNA clone (expression vector) to be transfected into the cells may be a single clone or multiple clones which may be transfected simultaneously. One embodiment of the process of the present invention is detaUed in Examples herein. Alternatively, a screening system for obtaining a gene capable of inhibiting NF- K B activation can also be constructed by cotransfecting an expression vector comprising full-length cDNA and a reporter gene into cells, stimulating the cells with IL-1 or TNF- and the like, and selecting a clone having subnormally increased reporter activity. By preparing a reporter gene suitably, a gene encoding a protein capable of activating various factors (for example, MAP kinase, transcription factor) can be obtained in addition to a gene encoding the protein capable of activating NF- K B.
The process of the present invention uses an in vitro system or a cell-based system, preferably a cell-based system. Examples of such cells include cells of prokaryotes such as E. coli, microorganisms such as yeast and fungi, as well as insects and animals. Preferred examples include animal cells, in particular, 293-EBNA cells and NIH3T3 cells.
Because the cDNA of the present invention is fuU-length, its 5' end sequence is the transcription initiation site of the corresponding mRNA. Therefore the cDNA sequence can be used to identify the promoter region of the gene by comparing the cDNA with the genomic nucleotide sequence. Genomic nucleotide sequences are available from various databases when the sequences have been deposited in the databases. Alternatively, the cDNA can also be used to clone the desired sequence from a genomic library, for example, by hybridization, and determine its nucleotide sequence. Thus, by comparing the nucleotide sequence of the cDNA of the present invention with a genomic sequence, the promoter region of the gene located upstream the cDNA can be identified. In addition, the promoter fragment thus identified can be used to construct a reporter plasmid for evaluating the expression of the gene. In general, the DNA fragment spanning 2kb (preferably lkb) upstream from the transcription initiation site can be inserted upstream of the reporter gene to produce the reporter plasmid. The reporter plasmid can be used to screen for a compound which enhances or reduces the expression of the gene. For example, such screening can be carried out by transforming a suitable cell with the reporter plasmid, culturing the transfonned ceU for a certain period of time, adding a certain amount of a test compound, measuring the reporter activity expressed by the cell after a certain period of time, and comparing the activity with that of a cell to which the test compound has not been added. These methods are also included in the scope of the present invention.
The present invention also relates to a computer-readable medium on which a sequence data set has been stored, said sequence data set comprising at least one of nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287 or a coding region thereof, and/or at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288.
In another aspect, the present invention relates to a method for calculating a homology, which comprises comparing data on the above medium with data of other nucleotide sequences. Thus, the gene and amino acid sequence of the present invention provide valuable information for determining their secondary and tertiary structure, e.g., information for identifying other sequence having a similar function and high homology. These sequences are stored on the computer-readable medium, then a database is searched using data stored in a known macromolecule structure program and a known search tool such as GCG In this manner, a sequence in a database having a certain homology can be easily found.
The computer-readable medium may be any composition of materials used to store information or data. Examples of such media include commercially available floppy disks, tapes, chips, hard drives, compact disks and video disks. The data on the medium allows a method for calculating a homology by comparing the data with other nucleotide sequence data. This method comprises the steps of providing a first polynucleotide sequence containing the polynucleotide sequence of the present invention for the computer-readable medium, and then comparing the first polynucleotide sequence with at least one-second polynucleotide or polypeptide sequence to identify the homology.
The present invention also relates to an insoluble substrate to which polynucleotides comprising all or part of the nucleotide sequences selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287 are fixed. A plurality of the various polynucleotides which are DNA probes are fixed on a specifically processed solid substrate such as slide glass to form a DNA microanay and then a labeled target polynucleotide is hybridized with the fixed polynucleotides to detect a signal from each of the probes. The data obtained is analyzed and the gene expression is deteπnined.
The present invention further relates to an insoluble substrate to which polypeptides comprising all or a part of the amino acid sequences selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288, are fixed. By mixing organism-derived ceU extract with the insoluble substrate on which these proteins are fixed, it is possible to isolate or identify cell-derived components such as proteins captured on the insoluble substrate that can be expected to be useful in diagnosis or drug development.
EXAMPLES
The foUowing examples further illustrate, but do not Umit the present invention. Example 1 : Construction of a fuU-length cDNA library using the oligo-capping method
(1) Preparation of RNA from human lung fibroblasts (Cryo NHLF)
Human lung fibroblasts (Cryo NHLF: purchased from Sanko Junyaku Co., Ltd.) were cultured according to the manufacture's protocol. After repeating subculturing the cells to obtain fifty 10cm dishes containing the resulting culture, the cells were recovered with a cell scraper. Then, total RNA was obtained from the recovered cells by using the RNA extraction reagent ISOGEN (purchased from NIPPON GENE) according to the manufacturer's protocol. Then, poly A+ RNA was obtained from the total RNA by using an oligo-dT cellulose column according to Maniatis et al., supra.
(2) Preparation of RNA from mouse ATDC5 cells
ATDC5, a cell strain cloned from mouse EC (embryonal carcinoma) (Atsumi, T. et al.: CeU Diff. Dev, 30: ρl09-116)(1990) was repeatedly subcultured to obtain fifty 10cm dishes containing the resultant culture. Thereafter, poly A+ RNA was obtained by a method similar to that of (1) above. Culture of ATDC5 cells was performed according to the method described in Atsumi, T. et al.: Cell Diff. Dev., 30: pl09-116 (1990).
(3) Preparation of RNA from Jurkat cells
From Jurkat cells, a human T ceU strain (purchased from DAINIPPON PHARMACEUTICAL CO.,LTD), poly A+ RNA was obtained in the same way as in the above (1). Jurkat ceUs were cultured in PRMI 1640 medium (GLBCO) containing 10%FBS (Fetal Bovine Serum: GIBCO) and lOmM HEPES (GIBCO) in the presence of 5% CO2 at 37T .
(4) Preparation of RNA from RAW264.7 cells
RAW264.7 cells, a mause macrophage-like ceU starain (ATCC Number TTB-71), were cultured to obtain poly A+ RNA in the same way as in the above (1).
(5) Construction of a full-length cDNA library by the oligo-capping method
A fuU-length cDNA library was constructed from poly A+ RNA of the above human lung fibroblasts, ATDC5 cells, Jurkat cells and RAW264.7 cells by the oligo-capping method according to the method of Sugano S. et al. [e.g., Maruyama, K. & Sugano, S., Gene, 138:171-174 (1994); Suzuki, Y. et al., Gene, 200:149-156 (1997); Suzuki, Y. & Sugano, S. "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", the third edition (1999), an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.].
(6) Preparation of plasmid DNA
The full-length cDNA library constructed as above was transformed into E. coli strain TOP 10 by electroporation, then spread on LB agar medium containing 100 μ g/ml of ampicillin, and incubated overnight at 37°C. Then, using QIAwell 96 Ultra Plasmid Kit (QIAGEN) according to the manufacturer's protocol, the plasmids were recovered from the colonies grown on ampicillin-containing LB agar medium.
Example 2: Cloning of DNA capable of activating NF- K B
(1) Screening of the cDNA encoding the protein capable of activating NF- K B 293-EBNA cells (purchased from Invitrogen) were seeded on DMEM medium containing 5% FBS in a 96 weU ceU culture plate to a final ceU density of 1 x IO4 cells/100 μ 1/weU, and cultured for 24 hours at 37°C in the presence of 5% CO2. Then, 50ng of pNF K B-LUC (purchased from STRATAGENE) and 2 μ 1 of the full-length cDNA expression vector prepared in above Example 1.(6) were cotransfected into the cells in a well using FuGENE 6 (purchased from Roche) according to the manufacturer's protocol. After 24 hours of culture at 37°C, the reporter activity of NF- K B (luciferase activity) was measured using long-term luciferase assay system,PIKKA GENE LT2.0 (TOYO INK) according to the attached manufacturer's instructions. The luciferase activity was measured using WaUac ARVO™ST 1420 MULTILABEL COUNTER (Perkin Elmer).
(2) DNA sequencing
The above screening was carried out, and plasmids showing a 5-fold or more increase in luciferase activity compared to that of the control experiment (luciferase activity of the cell into which vacant vector pME18S-FL3 is introduced instead of full-length cDNA expression vector) were selected. One pass sequencing was carried out from the 5' end of the cloned cDNA (sequencing primer: 5'-CTTCTGCTCTAAAAGCTGCG-3' (SEQ ID NO: 289)) and from the 3* end (sequencing primer: 5'-CGACCTGCAGCTCGAGCACA-3' (SEQ ID NO: 290)) so that as long sequence as possible is determined. The sequencing was carried out using the reagent Thermo Sequenase II Dye Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech) or BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (Applied Biosystems) and the device ABI PRISM 377 sequencer or ABI PRISM 3100 sequencer according to the manufacturer's instructions.
(3) Full-length sequencing
The fuU-length DNA sequences for the 144 new clones which were obtained by the above screening, were determined (SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287). The amino acid sequences of the protein coding regions (open reading frames) were deduced (SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282 284, 286 and 288).
The results of measurement of NF- /c B reporter activity (luciferase activity) of 41 clones among the above obtained clones are shown in Table 1 below. The value of activity shown in Table 1 is a relative value to the value of control experiment (luciferase activity of a cell into which pME183-FL3 vector containing no insert has been introduced in place of full length cDNA).
Table 1
Clone activity (-fold)
SEQ TD NO:7 9
SEQ ID NO:9 12
SEQ TD NO:19 6
SEQ ID NO:23 5
SEQ T NO:29 92
SEQ JD NO:35 54
SEQ TD NO:37 33
SEQ JJD NO:39 22
SEQ ID NO:43 31
SEQ ID NO:47 63
SEQ JX> NO:51 12 SEQIDNO:75 9
SEQIDNO:77 13
SEQIDNO:85 13
SEQIDNO:91 7
SEQIDNO:103 7
SEQIDNO:lll 70
SEQIDNO:115 11
SEQIDNO:119 9
SEQ ID NO: 123 10
SEQ ID NO: 143 6
SEQfDNO:151 10
SEQIDNO:157 29
SEQIDNO:165 145
SEQ ID NO: 171 134
SEQ ID NO: 175 19
SEQIDNO:181 22
SEQ ID NO: 185 33
SEQ ID NO: 195 6
SEQIDNO:197 5
SEQ ID NO: 199 14
SEQIDNO:201 31
SEQIDNO:205 32
SEQIDNO:211 16
SEQIDNO:229 13
SEQIDNO:237 7
SEQIDNO:239 23
SEQIDNO:247 15
SEQIDNO:257 5 SEQ ID NO:261 17
SEQ ID NO:287 66
Example 3: Screening compounds inhibiting NF- K B activation
293-EBNA cells were seeded on DMEM medium containing 5% FBS in a 96-well cell culture plate to a final cell density of 1 x IO4 cells/100 μ 1/well, and cultured for 24 hours at 37°C in the presence of 5% CO2. Then, 50ng of the plasmid comprising the gene encoding NF- K B activating protein of SEQ ID NO: 12 which was obtained in Example 2 above, and 50ng of the reporter plasmid pNF K B-Luc were cotransfected into the cells in a weU using FuGENE 6. After 1 hour, the proteasome inhibitor MG-132 (purchased form CALBIOCHEM) (Uehara T et al., J. Biol. Chem. 274, p.15875-15882 (1999); Wang X. C. et al., Invest. Ophathalmol. Vis. Sci. 40, p.477-486) was added to the culture to final concentrations of 1.0 μ M. After 24 hours of culture at 37°C, the reporter activity was measured using PIKKA GENE LT2.0. The results showed that MG132 inhibited the expression of the reporter gene (Fig. 1).
INDUSTRIAL APPLICABILITY
As described above, the present invention provides industrially highly useful proteins capable of activating NF- K B and genes encoding the proteins. The proteins of the present invention and the genes encoding the proteins allow not only screening for compounds useful for treating and preventing diseases associated with the excessive activation or inhibition of NF- /c B, but also production of diagnostics for such diseases. The genes of the present invention are also useful as a gene source used for gene therapy. AU publications, patents and patent applications cited herein are incorporated herein in their entirety.

Claims

1. A purified protein selected from the group consisting of:
(a) a protein which comprises an amino acid sequence represented by any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288; and
(b) a protein that activates NF- K B (Nuclear factor kappa B) and comprises an amino acid sequence having at least one amino acid deletion, substitution or addition in an amino acid sequence represented by any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288.
2. A purified protein that activates NF- K B and comprises an amino acid sequence having at least 95% identity to the protein according to claim 1 over the entire length thereof.
3. An isolated polynucleotide which comprises a nucleotide sequence encoding a protein selected from the group consisting of:
(a) a protein which comprises an amino acid sequence represented by any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288; and
(b) a protein that activates NF- K B and comprises an amino acid sequence having at least one amino acid deletion, substitution or addition in an amino acid sequence represented by any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288.
4. An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of:
(a) a polynucleotide sequence represented by any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287;
(b) a polynucleotide sequence encoding a protein that activates NF- rc B and hybridizing under stringent conditions with a polynucleotide having a polynucleotide sequence complementary to the polynucleotide sequence of (a); and
(c) a polynucleotide sequence which encodes a protein that activates NF- K B and consists of a polynucleotide sequence having at least one nucleotide deletion, substitution or addition in a polynucleotide sequence represented by any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287.
5. An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of:
(a) a polynucleotide sequence represented by a coding region of any one of SEQ TD NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287;
(b) a polynucleotide sequence encoding a protein that activates NF- K B and hybridizing under stringent conditions with a polynucleotide having a polynucleotide sequence complementary to the polynucleotide sequence of (a); and
(c) a polynucleotide sequence which encodes a protein that activates NF- K B and consists of a polynucleotide sequence having at least one nucleotide deletion, substitution or addition in a polynucleotide sequence represented by a coding region of any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287.
6. An isolated polynucleotide comprising a nucleotide sequence which encodes a protein that activates NF- K B and has at least 95% identity to the polynucleotide sequence according to claim 3 over the entire length thereof.
7. An isolated polynucleotide comprising a nucleotide sequence which encodes a protein that activates NF- K. B and has at least 95% identity to the polynucleotide sequence according to claim 4 or 5 over the entire length thereof.
8. A purified protein encoded by the polynucleotide according to any one of claims 3 to 7.
9. A recombinant vector which comprises a polynucleotide according to any one of claims 3 to 7.
10. A agent for gene therapy which comprises the recombinant vector according to claim 9 as an active ingredient.
11. A transformed cell which comprises the recombinant vector according to claim 9.
12. A membrane of the ceU according to claim 11, which has the protein according to claim 1 or 2 which is a membrane protein.
13. A process for producing a protein according to claim 1, 2 or 8 comprising the steps of;
(a) culturing a transformed ceU according to claim 11 under conditions providing expression of the protein according to claim 1, 2 or 8; and
(b) recovering the protein from the culture product.
14. A process for diagnosing a disease or susceptibility to a disease related to expression or activity of the protein of claim 1, 2 or 8 in a subject comprising the steps of:
(a) determining the presence or absence of a mutation in the gene encoding said protein in the genome of said subject; and/or
(b) analyzing the amount of expression of said protein in a sample derived from said subject.
15. A method for screening compounds capable of inhibiting or promoting NF- K B activation, which comprises the steps of:
(a) preparing a transformed cell by introducing a gene encoding a protein that promotes activation of NF- K B according to claim 1, 2 or 8 and a gene encoding a signal which can detect activation of NF- rc B into a cell;
(b) culturing the transformed cell under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring the signal which can detect activation of NF- K B; and
(d) selecting a candidate compound which can change the signal amount as compared with the case of the absence of candidate compounds, as a compound capable of inhibiting or promoting NF- re B activation.
16. A method for screening compounds capable of inhibiting or promoting NF- re B activation, which comprises the steps of:
(a) preparing a transformed cell by introducing a gene encoding a protein that promotes activation of NF- re B according to claim 1, 2 or 8 into a cell;
(b) culturing the transformed cell under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring the activation of NF- K B; and
(d) selecting a candidate compound which can change the activation of NF- re B as compared with the case of the absence of candidate compounds, as a compound capable of inhibiting or promoting NF- re B activation.
17. A compound capable of inhibiting or promoting NF- re B activation, which is selected by the method for screening according to claim 15 or 16.
18. A process for producing a pharmaceutical composition, which comprises the steps of:
(a) preparing a transformed cell by introducing a gene encoding a protein that promotes activation of NF- K B according to claim 1, 2 or 8 and a gene encoding a signal which can detect activation of NF- re B into a cell; (b) culturing the transformed ceU under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring the signal which can detect activation of NF- K B;
(d) selecting a candidate compound which can change the signal amount as compared with the case of the absence of candidate compounds, as a compound capable of inhibiting or promoting NF- K B activation; and
(e) producing a pharmaceutical composition which comprises a compound selected in the step of (d).
19. A process for producing a pharmaceutical composition, which comprises the steps of:
(a) preparing a transformed cell by introducing a gene encoding a protein that promotes activation of NF- re B according to claim 1, 2 or 8 into a cell;
(b) culturing the transformed cell under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring the activation of NF- re B; and
(d) selecting a candidate compound which can change the activation of NF- K B as compared with the case of the absence of candidate compounds, as a compound capable of inhibiting or promoting NF- /e B activation; and
(e) producing a pharmaceutical composition which comprises a compound selected in the step of (d).
20. A kit for screening a compound capable of inhibiting or promoting NF- K B activation, which comprises:
(a) a transformed cell comprising a gene encoding a protein that promotes activation of NF- K B according to claim 1, 2 or 8 and a gene encoding a signal which can detect activation of NF- re B; and
(b) reagents for measuring the signal.
21. A monoclonal or polyclonal antibody or a fragment thereof, which recognizes the protein according to claim 1, 2 or 8.
22. The monoclonal or polyclonal antibody or a fragment thereof according to claim 21, which inhibits the activity of promoting activation of NF- K B by the protein according to claim 1, 2 or 8.
23. A process for producing a monoclonal or polyclonal antibody according to claim 21 or 22, which comprises administering the protein according to claim 1, 2 or 8 or epitope-bearing fragments thereof to a non-human animal as an antigen.
24. An antisense oligonucleotide having a sequence complementary to a part of the polynucleotide according to any one of claims 3 to 7, which prevents the expression of a protein which promotes NF- re B activation.
25. A ribozyme or deoxyribozyme capable of inhibiting NF- K B activation, which has an action of cleavage of RNA that encodes the protein according to claim 1, 2 or 8 or an action of cleavage of RNA that encodes a protein involved in a pathway leading to NF- K B activation.
26. A method for treating a disease associated with NF- re B activation, which comprises admmistering to a subject a compound screened by the process according to claim 15 or 16, and/or a monoclonal or polyclonal antibody or a fragment thereof according to claim 21 or 22, and/or an antisense ohgonucleotide according to claim 24, and/or a ribozyme or deoxyribozyme according to claim 25 in an effective amount to treat a disease selected from the group consisting of inflammation, autoimmune diseases, cancers, infectious disease, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
27. A pharmaceutical composition produced by the process according to claim 18 or 19 for inhibiting or promoting NF- re B activation.
28. The pharmaceutical composition according to claim 27 for the treatment of inflammation, autoimmune diseases, cancers, infectious disease, bone diseases, AIDS, neurodegenerative diseases and/or ischemic disorders.
29. A method of treating inflammation, autoimmune diseases, cancers, infectious diseases, bone diseases, AIDS, neurodegenerative diseases or ischemic disorders, which comprises administering a pharmaceutical composition produced by the process according to claim 18 or 19 to a patient suffering from a disease associated with NF- K B activation.
30. A pharmaceutical composition which comprises a monoclonal or polyclonal antibody or a fragment thereof according to claim 21 or 22 as an active ingredient.
31. A pharmaceutical composition which comprises an antisense oligonucleotide according to claim 24 as an active ingredient.
32. A pharmaceutical composition which comprises a ribozyme or deoxyribozyme according to claim 25 as an active ingredient.
33. The pharmaceutical composition according to any one of claims 30 to 32 for the treatment and/or prevention of a disease which is selected from the group consisting of inflammation, autoimmune diseases, infectious diseases, cancers, bone diseases, AIDS, neurodegenerative diseases and ischemic disorders.
34. A computer-readable medium on which a sequence data set has been stored, said sequence data set comprising at least one of nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287 or a coding region thereof, and/or at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288.
35. A method for calculating identity to other nucleotide sequences and/or amino acid sequences, which comprises comparing data on a medium according to claim 34 with data of said other nucleotide sequences and/or amino acid sequences.
36. An insoluble substrate to which polynucleotides comprising all or part of the nucleotide sequences selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,
179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285 and 287 are fixed.
37. An insoluble substrate to which polypeptides comprising aU or a part of the amino acid sequences selected from the group consisting of SEQ JD NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,
108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178,
180, 182, 184, 186, 188, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286 and 288, are fixed.
PCT/JP2002/012644 2001-12-03 2002-12-03 Nf-kappab activating genes WO2003048202A2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002349784A AU2002349784A1 (en) 2001-12-03 2002-12-03 Nf-kappab activating genes

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
JP2001-368692 2001-12-03
JP2001368692A JP2006166701A (en) 2001-12-03 2001-12-03 NF-kappaB ACTIVATING GENE
US33582901P 2001-12-05 2001-12-05
US60/335,829 2001-12-05
JP2002291302A JP2006166705A (en) 2002-10-03 2002-10-03 NF-kappaB ACTIVATING GENE
JP2002-291302 2002-10-03
US41576902P 2002-10-04 2002-10-04
US60/415,769 2002-10-04

Publications (2)

Publication Number Publication Date
WO2003048202A2 true WO2003048202A2 (en) 2003-06-12
WO2003048202A3 WO2003048202A3 (en) 2004-07-01

Family

ID=27482716

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2002/012644 WO2003048202A2 (en) 2001-12-03 2002-12-03 Nf-kappab activating genes

Country Status (2)

Country Link
AU (1) AU2002349784A1 (en)
WO (1) WO2003048202A2 (en)

Cited By (78)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004003191A1 (en) * 2002-06-26 2004-01-08 Bayer Healthcare Ag Regulation of human map kinase kinase kinase
WO2004038373A2 (en) * 2002-10-22 2004-05-06 Oregon Health And Science University Apoptotic response regulation through interaction of tripartite motif protein 32 with pias
WO2004058805A2 (en) * 2002-12-26 2004-07-15 Asahi Kasei Pharma Corporation T cell activating gene
WO2004106937A2 (en) * 2003-06-02 2004-12-09 Bayer Healthcare Ag Diagnostics and therapeutics for diseases associated with g protein-coupled p2y purinoreceptor 6 (p2y6)
WO2004111085A1 (en) * 2003-06-10 2004-12-23 Xantos Biomedicine Ag Angiogenic factor and its medical use
WO2007013391A1 (en) * 2005-07-26 2007-02-01 Japan Science And Technology Agency Interferon-inducible molecule ips-1
US7537896B2 (en) 2000-01-28 2009-05-26 Henry M. Jackson Foundation For The Advancement Of Military Medicine Androgen-regulated PMEPA1 gene and polypeptides
WO2010017248A3 (en) * 2008-08-04 2010-04-29 University Of Miami Sting (stimulator of interferon genes), a regulator of innate immune responses
WO2010053877A2 (en) * 2008-11-04 2010-05-14 The Regents Of The University Of Colorado, A Body Corporate Mhc ii associated protein and uses thereof
EP2260858A2 (en) 2003-11-06 2010-12-15 Seattle Genetics, Inc. Monomethylvaline compounds capable of conjugation to ligands
EP2286844A2 (en) 2004-06-01 2011-02-23 Genentech, Inc. Antibody-drug conjugates and methods
WO2011031870A1 (en) 2009-09-09 2011-03-17 Centrose, Llc Extracellular targeted drug conjugates
WO2011056983A1 (en) 2009-11-05 2011-05-12 Genentech, Inc. Zirconium-radiolabeled, cysteine engineered antibody conjugates
WO2011130598A1 (en) 2010-04-15 2011-10-20 Spirogen Limited Pyrrolobenzodiazepines and conjugates thereof
WO2011156328A1 (en) 2010-06-08 2011-12-15 Genentech, Inc. Cysteine engineered antibodies and conjugates
WO2012074757A1 (en) 2010-11-17 2012-06-07 Genentech, Inc. Alaninyl maytansinol antibody conjugates
WO2012155019A1 (en) 2011-05-12 2012-11-15 Genentech, Inc. Multiple reaction monitoring lc-ms/ms method to detect therapeutic antibodies in animal samples using framework signature pepides
CN103030688A (en) * 2011-09-30 2013-04-10 北京大学 Short chain polypeptide for inhibiting cancer cell growth as well as encoding gene and application of short chain polypeptide
WO2013130093A1 (en) 2012-03-02 2013-09-06 Genentech, Inc. Biomarkers for treatment with anti-tubulin chemotherapeutic compounds
WO2014057074A1 (en) 2012-10-12 2014-04-17 Spirogen Sàrl Pyrrolobenzodiazepines and conjugates thereof
WO2014140174A1 (en) 2013-03-13 2014-09-18 Spirogen Sàrl Pyrrolobenzodiazepines and conjugates thereof
WO2014140862A2 (en) 2013-03-13 2014-09-18 Spirogen Sarl Pyrrolobenzodiazepines and conjugates thereof
WO2014159981A2 (en) 2013-03-13 2014-10-02 Spirogen Sarl Pyrrolobenzodiazepines and conjugates thereof
WO2015023355A1 (en) 2013-08-12 2015-02-19 Genentech, Inc. 1-(chloromethyl)-2,3-dihydro-1h-benzo[e]indole dimer antibody-drug conjugate compounds, and methods of use and treatment
WO2015095212A1 (en) 2013-12-16 2015-06-25 Genentech, Inc. 1-(chloromethyl)-2,3-dihydro-1h-benzo[e]indole dimer antibody-drug conjugate compounds, and methods of use and treatment
WO2015095223A2 (en) 2013-12-16 2015-06-25 Genentech, Inc. Peptidomimetic compounds and antibody-drug conjugates thereof
WO2015095227A2 (en) 2013-12-16 2015-06-25 Genentech, Inc. Peptidomimetic compounds and antibody-drug conjugates thereof
WO2016040825A1 (en) 2014-09-12 2016-03-17 Genentech, Inc. Anthracycline disulfide intermediates, antibody-drug conjugates and methods
WO2016040856A2 (en) 2014-09-12 2016-03-17 Genentech, Inc. Cysteine engineered antibodies and conjugates
WO2016037644A1 (en) 2014-09-10 2016-03-17 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
WO2016090050A1 (en) 2014-12-03 2016-06-09 Genentech, Inc. Quaternary amine compounds and antibody-drug conjugates thereof
EP3088004A1 (en) 2004-09-23 2016-11-02 Genentech, Inc. Cysteine engineered antibodies and conjugates
WO2017059289A1 (en) 2015-10-02 2017-04-06 Genentech, Inc. Pyrrolobenzodiazepine antibody drug conjugates and methods of use
WO2017064675A1 (en) 2015-10-16 2017-04-20 Genentech, Inc. Hindered disulfide drug conjugates
WO2017068511A1 (en) 2015-10-20 2017-04-27 Genentech, Inc. Calicheamicin-antibody-drug conjugates and methods of use
WO2017165734A1 (en) 2016-03-25 2017-09-28 Genentech, Inc. Multiplexed total antibody and antibody-conjugated drug quantification assay
EP3235820A1 (en) 2014-09-17 2017-10-25 Genentech, Inc. Pyrrolobenzodiazepines and antibody disulfide conjugates thereof
WO2017201449A1 (en) 2016-05-20 2017-11-23 Genentech, Inc. Protac antibody conjugates and methods of use
WO2017205741A1 (en) 2016-05-27 2017-11-30 Genentech, Inc. Bioanalytical method for the characterization of site-specific antibody-drug conjugates
WO2017214024A1 (en) 2016-06-06 2017-12-14 Genentech, Inc. Silvestrol antibody-drug conjugates and methods of use
WO2018031662A1 (en) 2016-08-11 2018-02-15 Genentech, Inc. Pyrrolobenzodiazepine prodrugs and antibody conjugates thereof
US9919056B2 (en) 2012-10-12 2018-03-20 Adc Therapeutics S.A. Pyrrolobenzodiazepine-anti-CD22 antibody conjugates
US9931414B2 (en) 2012-10-12 2018-04-03 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US9931415B2 (en) 2012-10-12 2018-04-03 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
WO2018065501A1 (en) 2016-10-05 2018-04-12 F. Hoffmann-La Roche Ag Methods for preparing antibody drug conjugates
US9950078B2 (en) 2013-10-11 2018-04-24 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US9956299B2 (en) 2013-10-11 2018-05-01 Medimmune Limited Pyrrolobenzodiazepine—antibody conjugates
US10010624B2 (en) 2013-10-11 2018-07-03 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US10029018B2 (en) 2013-10-11 2018-07-24 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
WO2019060398A1 (en) 2017-09-20 2019-03-28 Ph Pharma Co., Ltd. Thailanstatin analogs
US10392393B2 (en) 2016-01-26 2019-08-27 Medimmune Limited Pyrrolobenzodiazepines
US10420777B2 (en) 2014-09-12 2019-09-24 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US10544223B2 (en) 2017-04-20 2020-01-28 Adc Therapeutics Sa Combination therapy with an anti-axl antibody-drug conjugate
US10543279B2 (en) 2016-04-29 2020-01-28 Medimmune Limited Pyrrolobenzodiazepine conjugates and their use for the treatment of cancer
WO2020049286A1 (en) 2018-09-03 2020-03-12 Femtogenix Limited Polycyclic amides as cytotoxic agents
WO2020086858A1 (en) 2018-10-24 2020-04-30 Genentech, Inc. Conjugated chemical inducers of degradation and methods of use
WO2020123275A1 (en) 2018-12-10 2020-06-18 Genentech, Inc. Photocrosslinking peptides for site specific conjugation to fc-containing proteins
US10695439B2 (en) 2016-02-10 2020-06-30 Medimmune Limited Pyrrolobenzodiazepine conjugates
US10695433B2 (en) 2012-10-12 2020-06-30 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
WO2020157491A1 (en) 2019-01-29 2020-08-06 Femtogenix Limited G-a crosslinking cytotoxic agents
US10736903B2 (en) 2012-10-12 2020-08-11 Medimmune Limited Pyrrolobenzodiazepine-anti-PSMA antibody conjugates
US10751346B2 (en) 2012-10-12 2020-08-25 Medimmune Limited Pyrrolobenzodiazepine—anti-PSMA antibody conjugates
US10780096B2 (en) 2014-11-25 2020-09-22 Adc Therapeutics Sa Pyrrolobenzodiazepine-antibody conjugates
US10799595B2 (en) 2016-10-14 2020-10-13 Medimmune Limited Pyrrolobenzodiazepine conjugates
US10881730B2 (en) 2017-02-01 2021-01-05 Modernatx, Inc. Immunomodulatory therapeutic MRNA compositions encoding activating oncogene mutation peptides
US11059893B2 (en) 2015-04-15 2021-07-13 Bergenbio Asa Humanized anti-AXL antibodies
US11135303B2 (en) 2011-10-14 2021-10-05 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US11160872B2 (en) 2017-02-08 2021-11-02 Adc Therapeutics Sa Pyrrolobenzodiazepine-antibody conjugates
WO2022023735A1 (en) 2020-07-28 2022-02-03 Femtogenix Limited Cytotoxic agents
US11318211B2 (en) 2017-06-14 2022-05-03 Adc Therapeutics Sa Dosage regimes for the administration of an anti-CD19 ADC
US11352324B2 (en) 2018-03-01 2022-06-07 Medimmune Limited Methods
US11370801B2 (en) 2017-04-18 2022-06-28 Medimmune Limited Pyrrolobenzodiazepine conjugates
US11517626B2 (en) 2016-02-10 2022-12-06 Medimmune Limited Pyrrolobenzodiazepine antibody conjugates
US11524969B2 (en) 2018-04-12 2022-12-13 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof as antitumour agents
US11612665B2 (en) 2017-02-08 2023-03-28 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US11649250B2 (en) 2017-08-18 2023-05-16 Medimmune Limited Pyrrolobenzodiazepine conjugates
US11702473B2 (en) 2015-04-15 2023-07-18 Medimmune Limited Site-specific antibody-drug conjugates
US11897888B1 (en) 2020-04-30 2024-02-13 Stinginn Llc Small molecular inhibitors of sting signaling compositions and methods of use

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2935268B2 (en) 2012-12-21 2021-02-17 MedImmune Limited Pyrrolobenzodiazepines and conjugates thereof
US9567340B2 (en) 2012-12-21 2017-02-14 Medimmune Limited Unsymmetrical pyrrolobenzodiazepines-dimers for use in the treatment of proliferative and autoimmune diseases

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994023045A1 (en) * 1993-04-07 1994-10-13 Boehringer Ingelheim International Gmbh Method of inhibiting the transcription of genes
WO1996017927A2 (en) * 1994-12-05 1996-06-13 Karo Bio Ab Reporter cell line
US5932425A (en) * 1997-02-18 1999-08-03 Signal Pharmaceuticals, Inc. Compositions and methods for modulating cellular NF-κB activation
EP0955372A2 (en) * 1998-03-20 1999-11-10 Suntory Limited Method for screening substance inhibiting activation of NF-kappa B
WO2001072296A1 (en) * 2000-03-27 2001-10-04 Astacarotene Ab Method of inhibiting the expression of inflammatory cytokines and chemokines

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994023045A1 (en) * 1993-04-07 1994-10-13 Boehringer Ingelheim International Gmbh Method of inhibiting the transcription of genes
WO1996017927A2 (en) * 1994-12-05 1996-06-13 Karo Bio Ab Reporter cell line
US5932425A (en) * 1997-02-18 1999-08-03 Signal Pharmaceuticals, Inc. Compositions and methods for modulating cellular NF-κB activation
EP0955372A2 (en) * 1998-03-20 1999-11-10 Suntory Limited Method for screening substance inhibiting activation of NF-kappa B
WO2001072296A1 (en) * 2000-03-27 2001-10-04 Astacarotene Ab Method of inhibiting the expression of inflammatory cytokines and chemokines

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [Online] Homo spaiens, TTK protein kinase, retrieved from EBI Database accession no. BC000633 XP002233369 *
DOUVILLE E M J ET AL: "MULTIPLE CDNAS ENCODING THE ESK KINASE PREDICT TRANSMEMBRANE AND INTRACELLULAR ENZYME ISOFORMS" MOLECULAR AND CELLULAR BIOLOGY, vol. 12, no. 6, 1992, pages 2681-2689, XP009006945 ISSN: 0270-7306 & DATABASE EMBL [Online] 1 April 1992 (1992-04-01) retrieved from EBI Database accession no. M86377 *
KWON BYUNGSUK ET AL: "Identification of a novel activation-inducible protein of the tumor necrosis factor receptor superfamily and its ligand" JOURNAL OF BIOLOGICAL CHEMISTRY, THE AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, INC.,, US, vol. 274, no. 10, 5 March 1999 (1999-03-05), pages 6056-6061, XP002147323 ISSN: 0021-9258 *
MILLS G B ET AL: "EXPRESSION OF TTK A NOVEL HUMAN PROTEIN KINASE IS ASSOCIATED WITH CELL PROLIFERATION" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 267, no. 22, 1992, pages 16000-16006, XP002233366 ISSN: 0021-9258 & DATABASE EMBL [Online] 27 February 1992 (1992-02-27) retrieved from EBI Database accession no. M86699 *
WANG CUN-YU ET AL: "NK-kappaB antiapoptosis: Induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation." SCIENCE (WASHINGTON D C), vol. 281, no. 5383, pages 1680-1683, XP002233368 ISSN: 0036-8075 *

Cited By (111)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7537896B2 (en) 2000-01-28 2009-05-26 Henry M. Jackson Foundation For The Advancement Of Military Medicine Androgen-regulated PMEPA1 gene and polypeptides
WO2004003191A1 (en) * 2002-06-26 2004-01-08 Bayer Healthcare Ag Regulation of human map kinase kinase kinase
WO2004038373A2 (en) * 2002-10-22 2004-05-06 Oregon Health And Science University Apoptotic response regulation through interaction of tripartite motif protein 32 with pias
WO2004038373A3 (en) * 2002-10-22 2006-03-16 Univ Oregon Health & Science Apoptotic response regulation through interaction of tripartite motif protein 32 with pias
WO2004058805A2 (en) * 2002-12-26 2004-07-15 Asahi Kasei Pharma Corporation T cell activating gene
WO2004058805A3 (en) * 2002-12-26 2004-11-04 Asahi Kasei Pharma Corp T cell activating gene
WO2004106937A2 (en) * 2003-06-02 2004-12-09 Bayer Healthcare Ag Diagnostics and therapeutics for diseases associated with g protein-coupled p2y purinoreceptor 6 (p2y6)
WO2004106937A3 (en) * 2003-06-02 2005-05-26 Bayer Healthcare Ag Diagnostics and therapeutics for diseases associated with g protein-coupled p2y purinoreceptor 6 (p2y6)
WO2004111085A1 (en) * 2003-06-10 2004-12-23 Xantos Biomedicine Ag Angiogenic factor and its medical use
WO2004111088A2 (en) * 2003-06-10 2004-12-23 Xantos Biomedicine Ag Angiogenic factor and inhibitors thereof
WO2004111088A3 (en) * 2003-06-10 2005-02-10 Xantos Biomedicine Ag Angiogenic factor and inhibitors thereof
EP3120861A1 (en) 2003-11-06 2017-01-25 Seattle Genetics, Inc. Intermediate for conjugate preparation comprising auristatin derivatives and a linker
EP3858387A1 (en) 2003-11-06 2021-08-04 Seagen Inc. Monomethylvaline compounds capable of conjugation to ligands
EP3434275A1 (en) 2003-11-06 2019-01-30 Seattle Genetics, Inc. Assay for cancer cells based on the use of auristatin conjugates with antibodies
EP2489364A1 (en) 2003-11-06 2012-08-22 Seattle Genetics, Inc. Monomethylvaline compounds onjugated to antibodies
EP2260858A2 (en) 2003-11-06 2010-12-15 Seattle Genetics, Inc. Monomethylvaline compounds capable of conjugation to ligands
EP2486933A1 (en) 2003-11-06 2012-08-15 Seattle Genetics, Inc. Monomethylvaline compounds conjugated with antibodies
EP2478912A1 (en) 2003-11-06 2012-07-25 Seattle Genetics, Inc. Auristatin conjugates with anti-HER2 or anti-CD22 antibodies and their use in therapy
EP2286844A2 (en) 2004-06-01 2011-02-23 Genentech, Inc. Antibody-drug conjugates and methods
EP3088004A1 (en) 2004-09-23 2016-11-02 Genentech, Inc. Cysteine engineered antibodies and conjugates
WO2007013391A1 (en) * 2005-07-26 2007-02-01 Japan Science And Technology Agency Interferon-inducible molecule ips-1
EP2942357A1 (en) * 2008-08-04 2015-11-11 Glen N. Barber Sting (stimulator of inteferon genes), a regulator of innate immune responses
AU2015202068B2 (en) * 2008-08-04 2017-08-03 University Of Miami Sting (stimulator of interferon genes), a regulator of innate immune responses
WO2010017248A3 (en) * 2008-08-04 2010-04-29 University Of Miami Sting (stimulator of interferon genes), a regulator of innate immune responses
AU2009279682B2 (en) * 2008-08-04 2015-01-22 University Of Miami STING (stimulator of interferon genes), a regulator of innate immune responses
US20110212111A1 (en) * 2008-11-04 2011-09-01 Cambier John C MHC II Associated Protein and Uses Thereof
WO2010053877A3 (en) * 2008-11-04 2010-09-10 The Regents Of The University Of Colorado, A Body Corporate Mhc ii associated protein and uses thereof
WO2010053877A2 (en) * 2008-11-04 2010-05-14 The Regents Of The University Of Colorado, A Body Corporate Mhc ii associated protein and uses thereof
WO2011031870A1 (en) 2009-09-09 2011-03-17 Centrose, Llc Extracellular targeted drug conjugates
WO2011056983A1 (en) 2009-11-05 2011-05-12 Genentech, Inc. Zirconium-radiolabeled, cysteine engineered antibody conjugates
WO2011130598A1 (en) 2010-04-15 2011-10-20 Spirogen Limited Pyrrolobenzodiazepines and conjugates thereof
WO2011156328A1 (en) 2010-06-08 2011-12-15 Genentech, Inc. Cysteine engineered antibodies and conjugates
WO2012074757A1 (en) 2010-11-17 2012-06-07 Genentech, Inc. Alaninyl maytansinol antibody conjugates
WO2012155019A1 (en) 2011-05-12 2012-11-15 Genentech, Inc. Multiple reaction monitoring lc-ms/ms method to detect therapeutic antibodies in animal samples using framework signature pepides
CN103030688B (en) * 2011-09-30 2014-09-17 北京大学 Short chain polypeptide for inhibiting cancer cell growth as well as encoding gene and application of short chain polypeptide
CN103030688A (en) * 2011-09-30 2013-04-10 北京大学 Short chain polypeptide for inhibiting cancer cell growth as well as encoding gene and application of short chain polypeptide
US11135303B2 (en) 2011-10-14 2021-10-05 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
WO2013130093A1 (en) 2012-03-02 2013-09-06 Genentech, Inc. Biomarkers for treatment with anti-tubulin chemotherapeutic compounds
US10736903B2 (en) 2012-10-12 2020-08-11 Medimmune Limited Pyrrolobenzodiazepine-anti-PSMA antibody conjugates
US10751346B2 (en) 2012-10-12 2020-08-25 Medimmune Limited Pyrrolobenzodiazepine—anti-PSMA antibody conjugates
US11701430B2 (en) 2012-10-12 2023-07-18 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US11771775B2 (en) 2012-10-12 2023-10-03 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US10695433B2 (en) 2012-10-12 2020-06-30 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US11779650B2 (en) 2012-10-12 2023-10-10 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US10722594B2 (en) 2012-10-12 2020-07-28 Adc Therapeutics S.A. Pyrrolobenzodiazepine-anti-CD22 antibody conjugates
US10335497B2 (en) 2012-10-12 2019-07-02 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
EP2839860A1 (en) 2012-10-12 2015-02-25 Spirogen Sàrl Pyrrolobenzodiazepines and conjugates thereof
US11690918B2 (en) 2012-10-12 2023-07-04 Medimmune Limited Pyrrolobenzodiazepine-anti-CD22 antibody conjugates
WO2014057074A1 (en) 2012-10-12 2014-04-17 Spirogen Sàrl Pyrrolobenzodiazepines and conjugates thereof
US9931415B2 (en) 2012-10-12 2018-04-03 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US9931414B2 (en) 2012-10-12 2018-04-03 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US10994023B2 (en) 2012-10-12 2021-05-04 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US9919056B2 (en) 2012-10-12 2018-03-20 Adc Therapeutics S.A. Pyrrolobenzodiazepine-anti-CD22 antibody conjugates
US10799596B2 (en) 2012-10-12 2020-10-13 Adc Therapeutics S.A. Pyrrolobenzodiazepine-antibody conjugates
US10780181B2 (en) 2012-10-12 2020-09-22 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US10646584B2 (en) 2012-10-12 2020-05-12 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US9889207B2 (en) 2012-10-12 2018-02-13 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
WO2014140862A2 (en) 2013-03-13 2014-09-18 Spirogen Sarl Pyrrolobenzodiazepines and conjugates thereof
WO2014159981A2 (en) 2013-03-13 2014-10-02 Spirogen Sarl Pyrrolobenzodiazepines and conjugates thereof
WO2014140174A1 (en) 2013-03-13 2014-09-18 Spirogen Sàrl Pyrrolobenzodiazepines and conjugates thereof
WO2015023355A1 (en) 2013-08-12 2015-02-19 Genentech, Inc. 1-(chloromethyl)-2,3-dihydro-1h-benzo[e]indole dimer antibody-drug conjugate compounds, and methods of use and treatment
US10029018B2 (en) 2013-10-11 2018-07-24 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US9950078B2 (en) 2013-10-11 2018-04-24 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US9956299B2 (en) 2013-10-11 2018-05-01 Medimmune Limited Pyrrolobenzodiazepine—antibody conjugates
US10010624B2 (en) 2013-10-11 2018-07-03 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
WO2015095223A2 (en) 2013-12-16 2015-06-25 Genentech, Inc. Peptidomimetic compounds and antibody-drug conjugates thereof
WO2015095227A2 (en) 2013-12-16 2015-06-25 Genentech, Inc. Peptidomimetic compounds and antibody-drug conjugates thereof
WO2015095212A1 (en) 2013-12-16 2015-06-25 Genentech, Inc. 1-(chloromethyl)-2,3-dihydro-1h-benzo[e]indole dimer antibody-drug conjugate compounds, and methods of use and treatment
US10188746B2 (en) 2014-09-10 2019-01-29 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
WO2016037644A1 (en) 2014-09-10 2016-03-17 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
US10420777B2 (en) 2014-09-12 2019-09-24 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof
WO2016040856A2 (en) 2014-09-12 2016-03-17 Genentech, Inc. Cysteine engineered antibodies and conjugates
WO2016040825A1 (en) 2014-09-12 2016-03-17 Genentech, Inc. Anthracycline disulfide intermediates, antibody-drug conjugates and methods
EP3235820A1 (en) 2014-09-17 2017-10-25 Genentech, Inc. Pyrrolobenzodiazepines and antibody disulfide conjugates thereof
US10780096B2 (en) 2014-11-25 2020-09-22 Adc Therapeutics Sa Pyrrolobenzodiazepine-antibody conjugates
WO2016090050A1 (en) 2014-12-03 2016-06-09 Genentech, Inc. Quaternary amine compounds and antibody-drug conjugates thereof
US11702473B2 (en) 2015-04-15 2023-07-18 Medimmune Limited Site-specific antibody-drug conjugates
US11059893B2 (en) 2015-04-15 2021-07-13 Bergenbio Asa Humanized anti-AXL antibodies
WO2017059289A1 (en) 2015-10-02 2017-04-06 Genentech, Inc. Pyrrolobenzodiazepine antibody drug conjugates and methods of use
WO2017064675A1 (en) 2015-10-16 2017-04-20 Genentech, Inc. Hindered disulfide drug conjugates
WO2017068511A1 (en) 2015-10-20 2017-04-27 Genentech, Inc. Calicheamicin-antibody-drug conjugates and methods of use
US10392393B2 (en) 2016-01-26 2019-08-27 Medimmune Limited Pyrrolobenzodiazepines
US11517626B2 (en) 2016-02-10 2022-12-06 Medimmune Limited Pyrrolobenzodiazepine antibody conjugates
US10695439B2 (en) 2016-02-10 2020-06-30 Medimmune Limited Pyrrolobenzodiazepine conjugates
WO2017165734A1 (en) 2016-03-25 2017-09-28 Genentech, Inc. Multiplexed total antibody and antibody-conjugated drug quantification assay
EP4273551A2 (en) 2016-03-25 2023-11-08 F. Hoffmann-La Roche AG Multiplexed total antibody and antibody-conjugated drug quantification assay
US10543279B2 (en) 2016-04-29 2020-01-28 Medimmune Limited Pyrrolobenzodiazepine conjugates and their use for the treatment of cancer
WO2017201449A1 (en) 2016-05-20 2017-11-23 Genentech, Inc. Protac antibody conjugates and methods of use
WO2017205741A1 (en) 2016-05-27 2017-11-30 Genentech, Inc. Bioanalytical method for the characterization of site-specific antibody-drug conjugates
WO2017214024A1 (en) 2016-06-06 2017-12-14 Genentech, Inc. Silvestrol antibody-drug conjugates and methods of use
WO2018031662A1 (en) 2016-08-11 2018-02-15 Genentech, Inc. Pyrrolobenzodiazepine prodrugs and antibody conjugates thereof
WO2018065501A1 (en) 2016-10-05 2018-04-12 F. Hoffmann-La Roche Ag Methods for preparing antibody drug conjugates
US10799595B2 (en) 2016-10-14 2020-10-13 Medimmune Limited Pyrrolobenzodiazepine conjugates
US10881730B2 (en) 2017-02-01 2021-01-05 Modernatx, Inc. Immunomodulatory therapeutic MRNA compositions encoding activating oncogene mutation peptides
US11160872B2 (en) 2017-02-08 2021-11-02 Adc Therapeutics Sa Pyrrolobenzodiazepine-antibody conjugates
US11612665B2 (en) 2017-02-08 2023-03-28 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US11813335B2 (en) 2017-02-08 2023-11-14 Medimmune Limited Pyrrolobenzodiazepine-antibody conjugates
US11370801B2 (en) 2017-04-18 2022-06-28 Medimmune Limited Pyrrolobenzodiazepine conjugates
US10544223B2 (en) 2017-04-20 2020-01-28 Adc Therapeutics Sa Combination therapy with an anti-axl antibody-drug conjugate
US11938192B2 (en) 2017-06-14 2024-03-26 Medimmune Limited Dosage regimes for the administration of an anti-CD19 ADC
US11318211B2 (en) 2017-06-14 2022-05-03 Adc Therapeutics Sa Dosage regimes for the administration of an anti-CD19 ADC
US11649250B2 (en) 2017-08-18 2023-05-16 Medimmune Limited Pyrrolobenzodiazepine conjugates
WO2019060398A1 (en) 2017-09-20 2019-03-28 Ph Pharma Co., Ltd. Thailanstatin analogs
US11352324B2 (en) 2018-03-01 2022-06-07 Medimmune Limited Methods
US11524969B2 (en) 2018-04-12 2022-12-13 Medimmune Limited Pyrrolobenzodiazepines and conjugates thereof as antitumour agents
WO2020049286A1 (en) 2018-09-03 2020-03-12 Femtogenix Limited Polycyclic amides as cytotoxic agents
WO2020086858A1 (en) 2018-10-24 2020-04-30 Genentech, Inc. Conjugated chemical inducers of degradation and methods of use
WO2020123275A1 (en) 2018-12-10 2020-06-18 Genentech, Inc. Photocrosslinking peptides for site specific conjugation to fc-containing proteins
WO2020157491A1 (en) 2019-01-29 2020-08-06 Femtogenix Limited G-a crosslinking cytotoxic agents
US11897888B1 (en) 2020-04-30 2024-02-13 Stinginn Llc Small molecular inhibitors of sting signaling compositions and methods of use
WO2022023735A1 (en) 2020-07-28 2022-02-03 Femtogenix Limited Cytotoxic agents

Also Published As

Publication number Publication date
WO2003048202A3 (en) 2004-07-01
AU2002349784A8 (en) 2003-06-17
AU2002349784A1 (en) 2003-06-17

Similar Documents

Publication Publication Date Title
WO2003048202A2 (en) Nf-kappab activating genes
US7629453B2 (en) NF-κB activating gene
JP4344771B2 (en) NF-κB activating gene
US7129338B1 (en) Secretory protein or membrane protein
EP1514933A1 (en) Secretory protein or membrane protein
WO2004058805A2 (en) T cell activating gene
US20030092616A1 (en) STAT6 activation gene
US20040214167A9 (en) NF-kappa B activating gene
US20030092037A1 (en) Elk1 phosphorylation related gene
US7488808B2 (en) Janus family kinases and identification of immune modulators
US20030170719A1 (en) NF-kappa B activating gene
WO2004078112A2 (en) Apoptosis inducing gene
WO2002096943A1 (en) Stat6-activating genes
EP1474444A1 (en) A novel splice variant of myd88 and uses thereof
WO2003104277A2 (en) Stat6 activation gene
WO2004072277A2 (en) Elk1 phosphorylation related genes
US6500942B1 (en) Rin2, a novel inhibitor of Ras-mediated signaling
WO2003083117A2 (en) Vascularization controlling gene
JP2006166705A (en) NF-kappaB ACTIVATING GENE
JP2007527203A (en) Secreted protein family
JP2006166701A (en) NF-kappaB ACTIVATING GENE
US6346605B1 (en) Signal transducer for the TNF receptor super family, and uses thereof
WO2004013326A1 (en) Cartilage differentiation inhibiting gene
WO2003087375A1 (en) Cartilage differentiation regulating gene
JP2006166703A (en) Cartilage differentiation regulating gene

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LU MC NL PT SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase