WO2004058805A2 - T cell activating gene - Google Patents

T cell activating gene Download PDF

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Publication number
WO2004058805A2
WO2004058805A2 PCT/JP2003/016715 JP0316715W WO2004058805A2 WO 2004058805 A2 WO2004058805 A2 WO 2004058805A2 JP 0316715 W JP0316715 W JP 0316715W WO 2004058805 A2 WO2004058805 A2 WO 2004058805A2
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Prior art keywords
protein
activation
cell activation
nucleotide sequence
cells
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PCT/JP2003/016715
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French (fr)
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WO2004058805A3 (en
Inventor
Akio Matsuda
Shiho Yoneta
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Asahi Kasei Pharma Corporation
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Priority to AU2003290432A priority Critical patent/AU2003290432A1/en
Publication of WO2004058805A2 publication Critical patent/WO2004058805A2/en
Publication of WO2004058805A3 publication Critical patent/WO2004058805A3/en

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    • 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

Definitions

  • the present invention relates to a protein which is involved in T cell activation, 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 disorder of T cell.
  • the present invention also relates to a method for screening a substance capable of inhibiting or promoting T cell activation by using the protein, DNA, recombinant vector and transformant.
  • T cells recognize non-self molecules and an abnormality of self cells and exclude them, and thus play an important role in biological defence.
  • the dysfunction of T cells is a cause of various immune diseases. For example, if T cells attack a self antigen as a foreign substance, autoimmune diseases such as diabetes Type I, rheumatoid arthritis or multiple sclerosis are induced. Also, a response against a foreign antigen becomes excessive, allergy diseases such as asthma or atopic dermatitis occur. Therefore, elucidation of molecular mechanism of T cell activation which is important for these diseases is expected to contribute to elucidation of pathological conditions and development of therapeutic means for various immune diseases.
  • T cell activation starts by the following process; T cells recognize a complex of an antigen on antigen presenting cells and major histocompatibility antigen (MHC) via T cell receptor (TCR) and then crossl nking occurs between several TCRs, and further costimulation via CD28 is provided.
  • MHC major histocompatibility antigen
  • TCR T cell receptor
  • Lck a Src type tyrosine kinase, which associates with CD4 or CD8 phosphorylates two Tyr of an activation motif (immunoreceptor tyrosine-based activation motif ; ITAM) which is common in an intracellular region of CD3.
  • ITAM activation motif
  • ZAP70 a Syk type tyrosine kinase, is associated to the ITAM motifs and activated by Lck.
  • the signal is transmitted to serme/threonine protein phosphatases (for example, Calcineurin;Cn), serme/threonine protein kinase (for example, PKC, MAPK super family) and the like via various adaptor proteins (for example, LAT, SLP-76), and finally transcription factors such as NF K B, NFAT or AP-1 are activated.
  • serme/threonine protein phosphatases for example, Calcineurin;Cn
  • serme/threonine protein kinase for example, PKC, MAPK super family
  • various adaptor proteins for example, LAT, SLP-76
  • transcription factors such as NF K B, NFAT or AP-1 are activated.
  • IL-2 is a most important cytokine for regulating the proliferation and function of various immunocompetent cells including T cell itself.
  • the NFAT activation pathway is already an action target of an immunosuppressant.
  • Cyclosporin A (CsA) and FK506 inhibit an activity of Cn, an enzyme which dephosphorylates NFAT which is normally present in cytoplasm in phosphorylated state, and prevent the transmittance of NFAT into nucleus, thereby repressing the production of IL-2 (Liu .et al.,Cell 66,807-815(1991) ; and Flanagan,W.M.et al., Nature 352,803-807(1991)).
  • NF K B and AP-1 are also considered to play an important role in IL-2 transcription activation in view of the results of the experiment using IL-2 promoter having a mutation in its binding sequence (Rao A.,Immunol.Today 15,274-281(1994) ; Marian F.et al.,Oncogene 20,2476-2489(2001) ; Serfling E.et al.,Biochimica Biophysica Acta 1263,181-200(1995) ; and Kane L.P.et al.,Trends in Immunol.23,413-420(2002)).
  • the object of the present invention is to identify a new gene and protein which is involved in T cell activation which is useful as mentioned above, 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 which is involved in T cell activation, 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 T cell activation by using the protein, DNA, recombinant vector and transformant, a kit for the screening, a substance capable of inhibiting or promoting T cell activation which is obtained 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 T cell activation, etc.
  • the present inventors have intensively studied to solve the above objects. 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 TAg Jurkat cells (Jurkat (human T cell line) which expresses SV40 large antigen; gifted from Dr Crabtree GR. Stanford University); and isolating a new DNA (cDNA) encoding a protein having a function of NF ⁇ B or NFAT activation in T cell by using the assay system. It was confirmed that these new DNAs could activate NF B or NFAT by its expression in TAg Jurkat cells. This result shows that these new DNAs are signal transduction molecules involved in the NF K B or NFAT activation in T cell, namely the T cell activation pathway. Thus, the present invention has been completed.
  • the present invention provides the folio wings: ( 1 ) A purified protein of the following (a) or (b) :
  • a protein that consists of an 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, 190, 192, 19
  • a protein that consists of an 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, 190, 192, 194, 196
  • nucleotide sequence which encodes a protein that is involved in T cell activation, and which hybridizes with a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of (a) under stringent conditions;
  • nucleotide sequence which encodes a protein that is involved in T cell activation, and which consists of a nucleotide sequence having at least one nucleotide deletion, substitution or addition in a nucleotide sequence of any of SEQ JD 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,
  • nucleotide sequence which encodes a protein that is involved in T cell activation, and which hybridizes with a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of (a) under stringent conditions;
  • nucleotide sequence which encodes a protein that is involved in T cell activation, and which consists of a nucleotide sequence having at least one nucleotide deletion, substitution or addition in a coding region of a nucleotide sequence of any of SEQ JD 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,
  • the coding region in the present invention refers to a region which encodes a protein that is involved in T cell activation.
  • An isolated polynucleotide comprising a nucleotide sequence which encodes a protein that is involved in T cell activation and has at least 95% identity to the polynucleotide according to (3) over the entire length thereof.
  • An isolated polynucleotide comprising a nucleotide sequence which encodes a protein that is involved in T cell activation and has at least 95% identity to the polynucleotide 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 agent for gene therapy which comprises the recombinant vector according to (9) as an active ingredient.
  • (12) A membrane of the transformant according to (11) which has the protein according to (1) or (2), which is a membrane protein.
  • a process for diagnosing a disease or susceptibiHty 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 which inhibit or promote T cell activation which comprises the steps of:
  • a method for screening compounds which inhibit or promote T cell activation which comprises the steps of:
  • a compound which inhibits or promotes T cell 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 which inhibits or promotes an activation of NF c B or NFAT in T cells which comprises:
  • a transformant comprising a gene encoding a protein which promotes an activation of NF K B or NFAT in T cells according to (1), (2) or (8) and a gene encoding a signal which can detect promotion of activity of NF K B or NFAT; 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).
  • a ribozyme or deoxyribozyme capable of inhibiting activation of NF /c B or NFAT in T cells 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 which is involved in a pathway leading to activation of NF K B or NFAT in T cells.
  • a method for treating a disease associated with T cell 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 ohgonucleotide according to (24), and/or a ribozyme or deoxyribozyme according to (25), and/or a double strand RNA according to (26) in an effective amount to treat and/or prevent a disease selected from the group consisting of autoimmune diseases, allergic, diseases, infectious diseases and AJDS or to treat acute or chronic rejection at organ transplant or bone-marrow transplant.
  • a method of treating autoimmune diseases, allergic diseases, infectious diseases or AIDS, or acute or chronic rejection at organ transplant or bone-marrow transplant which comprises administering a pharmaceutical composition produced by the process according to (18) or (19) to a patient suffering from a disease associated with T cell activation.
  • (31) 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 ohgonucleotide according to (24) as an active ingredient.
  • a pharmaceutical composition which comprises a ribozyme or deoxyribozyme according to (25) as an active ingredient.
  • a pharmaceutical composition or a gene therapy agent which comprises a double strand RNA according to (26) or a vector capable of expressing said double strand RNA, an active ingredient.
  • a disease which is selected from the group consisting of autoimmune diseases, allergic diseases, infectious diseases or AJDS, or for the treatment of acute or chronic rejection at organ transplant or bone-marrow transplant.
  • a computer-readable medium on which a sequence data set has been stored said sequence data set comprising at least one of nucleotide sequence or that of coding region which is selected from the group consisting of SEQ JD 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,
  • a method for calculating identity to other nucleotide sequences and/or amino acid sequences which comprises comparing data on a medium according to (36) 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 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,
  • a full-length cDNA was produced from mRNA prepared from human peripheral blood monocyte (PBMC), 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 pNF K B-Luc reporter plasmid or the pNFAT-Luc reporter plasmid both from STRATAGENE, each of which contains DNA encoding luciferase, and the above full-length cDNA plasmid were cotransfected into TAg Jurkat cells.
  • the phrase "involved in T cell activation” means that NF K B or NFAT is activated (alternatively, activation of NF K B or NFAT is induced), which is a transcription factor which is necessary for the expression of IL-2 gene, when a gene is introduced into a suitable cell and the protein encoded by the gene is excessively expressed.
  • Activation of NF c B or NFAT can be measured, for example, by an assay using NF K B dependant reporter gene or NFAT dependant reporter gene.
  • the phrase " having a function of NF K B or NFAT activation” means that it has a function of increasing the reporter activity compared to control experiment (host 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.
  • 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 a suitable 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 ⁇ -galactosidase.
  • Suitable host cells include cells which induce an activation signal transduction leading to IL-2 production by co-stimulation with an antibody against TCR/CD3 and CD28 or co-stimulation with phorbol ester and calcium ionophore. Examples thereof include Jurkat cells.
  • a clone which expresses the activation function by synergistic effect with the stimulation can be obtained.
  • Cell culture and introduction of genes into cells (transfection) can be performed and optimized by a person skilled in the art by known techniques.
  • TAg Jurkat cells human T cell line, Jurkat, which expresses
  • FBS Fetal Bovine Serum
  • RPMU640 containing lOmM HEPES and 50 ⁇ M 2-ME
  • STRATAGENE pNF ⁇ B-Luc or pNFAT-Luc reporter plasmid
  • QJAGEN SuperFect
  • an expression vector for example, Ras(Val-12) expression vector
  • DNA encoding an active type protein of a signal transduction molecule which is known to belong to AP-1 activation pathway is co-transfected, or alternatively an agent which is known to activate AP-1, such as PMA, is added in the medium.
  • culturing is carried out for 24 hours at 37°C, and the luciferase activity is measured by using a long term luciferase assay system, Picagene LT2.0 (Toyo Ink Mfg) to measure the activation of NF K B or NFAT.
  • luciferase activity can be measured using PerkinElmer's WaUac ARVOTMST 1420 MULTILABEL COUNTER.
  • the method for gene introduction by SuperFect and measurement of luciferase activity by Picagene LT2.0 can be performed respectively according to the attached protocols.
  • the amount of SuperFect per 1 well is suitably 0.3 to 0.8 ⁇ 1, preferably 0.5 ⁇ 1;
  • the amount of pNF K B-LUC or pNFAT-Luc reporter plasmid is suitably 50 to lOOng, preferably lOOng; and the amount of expression vector is suitably 50 to lOOng, preferably lOOng.
  • the concentration of PMA to be added in the NFAT assay system is suitably 5 to 25 ng/ml.
  • NF K B or NFAT An ability of activating NF K B or NFAT can be confirmed by regarding the ability of increasing the reporter activity (luciferase activity) relative to the control experiment (for cells into which only a null vector was introduced) as an index.
  • Increase in reporter activity as an index 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.
  • proteins that is involved in T cell activation consist of an amino acid sequence having 1 or several (preferably a few) amino acid deletion, substitution or addition in the above amino acid sequences:
  • Identity used herein means a relationship between two or more protein sequence or two or more nucleotide sequences, as determined by comparing the sequences, as known in the art. In the art, “identity” also means the degree of sequence relatedness between protein or nucleotide sequences, as dete ⁇ nined by the match between protein or nucleotide 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, for example, 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, Miller W, Lipman DJ,. Nucleic Acids Res. 25:p3389-3402 81997)), however methods of determining identity are not limited to this. 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, BLOSUM62, is used.
  • the theory of this amino acids substitution matrix is shown in Altschul S.F., J. Mol. Biol. 219: 555-565 (1991), and its applicability to DNA sequence comparison is shown in States D. J., Gish W., Altschul S.F., Methods, 3: 66-70 (1991).
  • optimal gap cost is determined empirically and in the case of BLOSUM62, preferably parameters, 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 sequence identity to the amino acid sequence of SEQ ID NO: 2 may have an amino acid sequence that includes up to 5 amino acid changes per 100 amino acids of the amino acid sequence of SEQ JD 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 teiminus or the carboxy terminus of the subject sequence, or may form one or more groups of changes.
  • polynucleotides comprising a nucleotide sequence, which has at least 95% identity, preferably 97-99% identity, to any of the above sequences, and which encodes a protein which is involved in T cell activation;
  • a polynucleotide which has a nucleotide sequence that encodes a protein, wherein the protein has an amino acid sequence having at least 95% identity, preferably at least 97-99% identity, to the amino acid sequence 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
  • Polynucleotides which are identical or substantially identical to a nucleotide sequence contained in the above nucleotide sequence may be used as hybridization probes to isolate full-length cDNA or genomic clones encoding proteins of the present invention or cDNA and genomic clones of other genes that have a high sequence similarity to the above sequences, or as primers for a 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 that is involved in T cell activation in the same way as in the protein having an amino acid sequence represented by SEQ JD 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,
  • 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 involved in T cell activation and which has at least one amino acid deletion, substitution or addition compared to the protein having an amino acid sequence represented 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
  • 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, glutarnine), (serine, threonine), (lysine, arginine) and (phenylalanine, tyrosine).
  • nucleotide sequences e.g., a polynucleotide of SEQ JD 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,
  • the present invention also includes a protein that is involved in T cell activation and comprises an amino acid sequence having a high identity to the amino acid sequence of 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
  • High identity refers to an amino acid sequence having an identity of at least 90%, preferably at least 97-99% over the entire length of an amino acid sequence represented by above SEQ JD 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,
  • 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 protein encoded by the gene of the present invention includes secretory proteins (growth factors, cytokines, hormones, etc.), protein modifying enzymes (protein phosphorylases, protein phosphatases, proteases, etc), signal transduction molecules (interaction molecules between proteins, etc.), intranuclear proteins (intranuclear receptors, transcription factors) and membrane proteins.
  • Secretory proteins growth factors, cytokines, hormones, etc.
  • protein modifying enzymes protein phosphorylases, protein phosphatases, proteases, etc
  • signal transduction molecules interaction molecules between proteins, etc.
  • intranuclear proteins intranuclear receptors, transcription factors
  • membrane proteins include receptors, cellular adhesion molecules, ion channels, transporters, etc.
  • the present invention also includes a polynucleotide encoding the above protein of the present invention.
  • nucleotide sequences encoding a protein consisting of an amino acid sequence 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
  • the DNA includes cDNA, genomic DNA, and chemicaUy synthesized DNA.
  • the DNA of the present invention includes a DNA which encodes a protein that is involved in T ceU activation and hybridizes under stringent conditions with the DNA of the above nucleotide sequence of SEQ JD 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,
  • 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, lOO ⁇ g/ml denatured, sheared salmon sperm DNA) 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 1 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 42 °C 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 having a function of activating NF K B or NFAT in T cells.
  • the present invention also includes a polynucleotide comprising a nucleotide sequence which encodes a protein that is involved in T cell activation and has a high sequence similarity to the nucleotide sequence of the polynucleotide according to above item (3), (4) or (5).
  • these nucleotide sequences are 95% identical, preferably 97% 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 protein according to the above item (1), (2) or (8) using recombinant DNA techniques.
  • 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;
  • 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 application, PCR amplification using a synthetic DNA having a portion of the nucleotide sequence of the present invention (e.g., SEQ JD 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,
  • 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 normaUy 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 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
  • the present invention also provides a recombinant vector, which comprises the above DNA.
  • the expression vector capable of expressing 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.
  • the vectors should be selected to be compatible with hosts. Suitable combinations of host-expression vector systems are selected depending on the desired expression product.
  • Plasmid vectors compatible with these bacteria 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 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 repUcable expression 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 of genes of 3-phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, alcohol dehydrogenase and the like.
  • promoters and te ⁇ ninators for use in expression vectors to express the DNA of the present invention in fungal cells include promoters and terminators derived from genes for ph ⁇ sphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPD), actin and the like.
  • 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 vector suitable for insect ceUs include baculovirus.
  • Recombinant vectors used to express the DNA of the present invention in animal cells normally contain functional sequences to regulate genes, a promoter to be placed upstream of the DNA of the present invention, a polyadenylation site and a transcription te ⁇ nination sequence.
  • Such functional sequences which can be used to express the DNA of the present invention in cells, can be obtained from viruses and viral substances.
  • Examples of such functional sequences include an SR ⁇ 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 pSV2-dhfr (ATCC 37146), pBPV- 1(9-1) (ATCC 37111), pcDNA3.1 (INVJTROGEN) and pME18S-FL3.
  • the present invention also provides a transformant which comprises the above recombinant vector.
  • Microorganisms or ceUs transformed with the recombinant vector of the present invention can be selected from remaining untransformed parent ceUs based on at least one phenotype conferred by the recombinant vector.
  • 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 ceUs 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. coli JM109 and E. coU 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 include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris.
  • fungi include Aspergillus nidulans, and Acremonium chrysogenum (ATCC 11550).
  • insect cells for example, Spodoptera frugiperda (Sf cells), High Five TM ceUs derived from eggs of Trichoplusiani, etc.
  • insect cells for example, Spodoptera frugiperda (Sf cells), High Five TM ceUs derived from eggs of Trichoplusiani, etc.
  • animal cells include HEK 293 cells, COS-1 cells, COS-7 ceUs, Hela cells, and Chinese hamster ovary (CHO) cells. Among them, CHO ceUs and HEK 293 cells are preferred.
  • combinations of expression vectors and hosts 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 ceUs by known methods such as DEAE-dextran method, calcium-phosphate-mediated transfection, electropofation, lipofection, etc.
  • ceUs which stably express 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.
  • transformants can be selected using the above selectable marker as an indication of successful transformation.
  • the animal ceUs 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 transformant 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 transformant 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 (namely, cell itself or culture medium).
  • the above transformant can be cultured by methods known to those skUled in the art (see e.g., "Bio Manual Series 4", YODOSHA CO., LTD.).
  • animal cells can be cultured by various known animal ceU culture methods including attachment culture such as Petri dish culture, multitray type culture and module culture, attachment culture in which cells are attached to ceU culture carriers (microcarriers), or 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 examples of medium for use in the culture include medium 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 based on solubUity 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 Uquid chromatography
  • 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.
  • high performance liquid chromatography is employed for purification.
  • WeU known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during intraceUular 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-transf erase (GST) and a hexa-histidine sequence (6 x His).
  • GST glutathione S-transf erase
  • 6 x His hexa-histidine sequence
  • the fusion protein of the protein of the present invention with another protein can be advantageously purified by affinity chromatography using a substance with ah affinity for the fusion partner protein.
  • fusion proteins with GST can be purified by affinity chromatography using glutathione as a Ugand.
  • membrane protein When the protein of the present invention is a membrane protein, a transformant 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 transformants 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 ceU can be prepared by a method known to those skiUed in the art. For example, cells are collected from the culture where transformants 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 provides 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 specificaUy react with the protein of the present invention or a fragment thereof, and to production of such an antibody.
  • specificaUy 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 specificaUy 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) or epitope-bearing fragments as an antigen 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.
  • 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 ceUs by using a reagent such as polyethylene glycol. From the resulting fused cells (i.e. hybridoma), the clone producing the antibody directed against the protein of the present invention can be selected. Then, the resulting clonal hybridoma can be implanted intraperitoneaUy 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.
  • humanized antibody or human antibody When the resulting antibody is used to administer to humans, it is preferable to use a humanized antibody or human antibody in order to reduce its immunogenicity.
  • humanized antibodies or human antibodies 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 complement ty-dete ⁇ nining region (CDR) from a mouse monoclonal antibody.
  • CDR complement ty-dete ⁇ nining 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 transformant producing the protein of the present invention.
  • These proteins can also be used to construct ELISA, RIA (radioimmunoassay) and western blotting systems.
  • ELISA electrospray
  • 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.
  • these antibodies can be used for diagnosis of a disease characterized by undesirable activation of T cells resulting from (expression) abnormality of the protein of the present invention, such as autoimmune diseases, allergic diseases, infectious diseases or AIDS, or acute or chronic rejection at organ transplant or bone-marrow transplant.
  • a standard value In order to provide a basis for diagnosis of a disease, a standard value must be established regarding the expression of the protein of the present invention.
  • this is a well-known technique to those skiUed in the art.
  • 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 function of activation of NF ⁇ B or NFAT in T ceUs which is possessed by the protein of present invention that is, neutralizing antibody).
  • the antibodies of the present invention can be purified and then administered to patients with a disease characterized by undesirable activation of T cells resulting from (expression) abnormality of the protein of the present invention, such as autoimmune diseases, allergic diseases, infectious diseases or AJDS, or acute or chronic rejection at organ transplant or bone-marrow transplant.
  • a disease characterized by undesirable activation of T cells resulting from (expression) abnormality of the protein of the present invention, such as autoimmune diseases, allergic diseases, infectious diseases or AJDS, or acute or chronic rejection at organ transplant or bone-marrow transplant.
  • the present invention provides 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 therapeuticaUy or preventively active ingredients or inactive ingredients (e.g., conventional pharmaceutically acceptable carriers or dUuents such as immunogenic adjuvants) and physiologically non-toxic stabilizers and excipients.
  • the resulting combinations can be sterilized by filtration,
  • Administration to a patient can be intra-arterial administration, 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 skiUed in the art.
  • the antibody of the present invention exhibits therapeutic activity by inhibiting the promotion of T cell activation mediated by the protein of the present invention. SpecificaUy, the antibody of the present invention can be usefulf as a medicament for treatment or prevention of diseased assciated with abnormal activity of T cells, such as autoimmune diseases, allergic diseases, infectious diseases or AIDS, or acute or chronic rejection at organ transplant or bone-marrow transplant.
  • 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, from cDNA library or genomic DNA library.
  • prey a protein which can associate with the protein of the present invention, from cDNA library or genomic DNA library.
  • it can be determined whether the protein of the present invention can associate with other cellular proteins e.g., ZAP70 or PKC ⁇ which are known be essential for T cell activation).
  • 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 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 susceptibiUty to the diseases through detection of mutation in a gene for the protein of the present invention which is involved in T cell activation.
  • diseases may be diagnosised by analyzing expression level of the gene in a sample derived from a subject at protein or mRNA level, and detecting an abnormaUy decreased or increased level of the expression.
  • Determination of the presence or absence of a mutation in the gene encoding the protein of the present invention which is involved in T ceU activation may involve RT-PCR using a part of the nucleotide sequences of genes as a primer, followed by conventional DNA sequencing to detect the presence or absence of the mutation.
  • PCR-SSCP Genetics 5:874-879 (1989); "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.
  • 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.
  • Decreased or increased expression of a gene in a sample can be measured at the RNA level using any of the methods weU known in the art for the quantitation of polynucleotides, for example, nucleic acid ampUfication 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 determine 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 abnormaUty of expression level of gene in a sample is not particularly limited.
  • 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 diagnosed 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 diagnosed 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.
  • the DNA of the present invention is useful for gene diagnosis regarding damage, mutations, and reduced, increased or over- expression of the DNA or mRNA.
  • the mutation may cause a disease associated with the expression and/or activity of NF K B or NFAT in T cells.
  • the abnormal expression of the novel protein of the present invention which is involved in T cell activation may be responsible for diseases associated with the expression and/or activity of NF K B or NFAT in T cells.
  • the present invention also relates to a method for screening compounds which inhibit or promote T cell activation mediated by the protein of the present invention.
  • the above screening method is for screening in respect of activity as an inhibiting agent or activating agent of T cells, and the above compound is a compound having activity as an inhibiting agent or activating agent of T cell function.
  • the above screening method comprises the following steps:
  • a compound that increases the detectable signal 2-fold or higher than normal is preferably isolated or identified as an activator compound, and a compound that decreases the detectable signal 50% or less than normal is preferably isolated or identified as an inhibitor compound.
  • genes encoding a signal which can detect involvement in T ceU activation include reporter genes. Reporter genes are used instead of directly detecting the activation of transcription factors of interest to be tested. 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.). A gene encoding any peptide or protein can be used as a reporter gene, so long as those skUled in the art can measure the activity or amount of the expression product (including the amount of the produced mRNA) of the reporter genes.
  • 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.
  • Other examples include NF K B dependent reporter plasmid described in Tanaka S.e al J.Vet.Med.Sci.Vol.59(7), Rothe M.e aLScience Vol.269 ⁇ l424-1427(1995).
  • Any reporter plasmids can be used to evaluate NFAT activation so long as the reporter plasmids have an NFAT recognition sequence inserted upstream of the reporter gene, and examples thereof include pNFAT-Luc(STRATAGENE). '
  • Any host can be used so long as it induces an activation signal transduction leading to IL-2 production by co-stimulation with an antibody against TCR/CD3 and CD28 or co- stimulation with phorbol ester and calcium ionophore.
  • Jurkat cells are preferably used. Transformation and culture of the cells can be carried out as described above.
  • the method for screening a compound which inhibits or promotes T cell activation comprises culturing the transformant 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 for the screening include, but not limited to, low molecular weight compounds, high molecular weight 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 Ubrary which was synthesized by combinatorial chemistry, a naruraUy occurring product containing ceUs, plants, animals or a part thereof, or an extracted product of such naruraUy occurring product.
  • the test substance which shows an activity of inhibiting or promoting NF K B or NFAT activation in T cells can be further screened to isolate a single substance having the activity. Isolation 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:
  • examples of the method of measuring an activation of NF ⁇ B or NFAT in T cells include a method of analysing the binding to NF / B or NFAT binding sequence using cell extraction solution by gel shift (for example, Hayashi T et al. 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 for example, IL-2
  • 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. CommerciaUy available antibodies(from, e.g., Wako Pure Chemical Industries, Ltd.) can also be used.
  • the present invention further provides a method of producing a pharmaceutical composition, which comprises the foUowing steps (a) to (e): (a) preparing a transformant by introducing a gene encoding a protein that is involved in T cell activation according to the present invention and a gene encoding a signal which can detect an involvement in T cell activation into a host cell;
  • a pharmaceutical composition may also be produced by the foUowing steps (a) to (e):
  • 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 50% 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) deteirmining 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;
  • the present invention also provides a compound which is selected by the above screening method.
  • This compound has an activity of inhibiting or promoting T cell activation. More specifically, this compound has an activity of inhibiting or promoting NF / B or NFAT activation in T cells which is promoted by the protein of the present invention.
  • the compounds obtained by the above screening methods have an activity of inhibiting or promoting T cell activation, they are useful as therapeutic or preventive pharmaceuticals for the diseases resulting from unfavorable activation or inactivation of T cells.
  • 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 dissolving or suspending the compound into a suitable solvent by conventional methods 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 foUowing.
  • the above compounds or their pharmaceuticaUy acceptable salts in an amount effective as an active ingredient, and pharmaceuticaUy 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 soUd form such as tablet, granule, capsule, pUl and powder, and solution form such as solution, syrup, elixir and dispersion.
  • a form useful for parenteral administration includes sterile solution, 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 polyaUcylene 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 provides a kit for screening a compound which inhibits or promotes T ceU activation.
  • the kit comprises: (a) a transformant comprising a gene encoding a protein that is involved in T ceU activation according to the present invention and a gene encoding a signal which can detect a promotion of NF / B or NFAT activation; and (b) reagents for measuring the signal.
  • the kit comprises reagents necessary for screening compounds which inhibits or promotes T ceU activation.
  • the present invention relates to a diagnostic kit which comprises: (a) a polynucleotide of the present invention having a nucleotide sequence represented by any one of SEQ JD 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
  • a protein of the present invention having an amino acid sequence represented by any one 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, 190, 192, 19
  • a diagnostic kit comprising at least any one of (a) to (d) is useful for diagnosing a disease or susceptibUity to a disease such as autoimmune diseases, allergic diseases, infectious diseases, AIDS, or acute or chronic rejection at organ transplant or bone-marrow transplant.
  • a disease or susceptibUity to a disease such as autoimmune diseases, allergic diseases, infectious diseases, AIDS, or acute or chronic rejection at organ transplant or bone-marrow transplant.
  • T cell is involved in a wide variety of pathological conditions such as autoimmune diseases, allergic diseases, infectious diseases, AJDS, or acute or chronic rejection at organ transplant or bone-marrow transplant, it is an attractive target for drug design and therapeutic intervention.
  • the finding of the new protein described herein that is involved in T cell activation has provided a new medicament and therapeutic method for controlling an abnormal T cell function.
  • the present invention also relates to use of a compound which inhibits the function of the protein that is involved in T cell activation described above, for inhibiting T ceU activation.
  • the present invention also relates to use of a compound which activates the function of the protein that is involved in T cell activation described above, for promoting T cell activation.
  • the compound obtained by the above screening method, which inhibits or promotes T cell activation is useful as a medicament to treat or prevent diseases characterized by undesirable activation of T cells, such as autoimmune diseases, aUergic diseases, infectious diseases, AIDS, or acute or chronic rejection at organ transplant or bone-marrow transplant.
  • autoimmune diseases rheumatoid arthritis, multiple sclerosis, insulin-dependent diabetes, thyroiditis, psoriasis, myasthenia gravis, systematic lupus erythematosus, lupus, and the like
  • allergic diseases asthma, atopic dermatitis, contact hypersensitivity, allergic rhinitis and the like
  • virus and bacterial infectious diseases hepatitis, tuberculosis, leprosy, and the like
  • AJDS acquired immunodeficiency syndrome
  • the gene encoding the protein of the present invention is useful for gene therapy to treat various diseases such as autoimmune diseases, allergic diseases, infectious diseases, AJDS, or acute or chronic rejection at organ transplant or bone-marrow transplant.
  • Gene therapy refers to administering into the human body a gene or a ceU into which a gene has been introduced.
  • 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.
  • RNAi RNA interference
  • the present invention provides a vector for gene therapy which expresses double strand RNA having a gene sequence 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 auxUiary 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 appUed 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 10jU 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 limited.
  • 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 the administration route may vary with the weight and age of the patient, but those skilled in the art can appropriately select the administration route, and can appropriately select suitable dosage which is suitable for the administration route.
  • 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 which inhibits or promotes T ceU activation as an active ingredient.
  • the above compound is useful as a medicament to treat or prevent diseases characterized by abnormal activity of T cells, such as autoimmune diseases, allergic diseases, infectious diseases, AJDS, or acute or chronic rejection at organ transplant or bone-marrow transplant.
  • diseases characterized by abnormal activity of T cells such as autoimmune diseases, allergic diseases, infectious diseases, AJDS, or acute or chronic rejection at organ transplant or bone-marrow transplant.
  • the present invention provides a pharmaceutical composition for the treatment or prevention of autoimmune diseases, aUergic diseases, infectious diseases, AJDS, or acute or chronic rejection at organ transplant or bone-marrow transplant, and the like, which comprises the compound which inhibits or promotes T ceU activation.
  • the pharmaceutical composition is useful as a medicament for the treatment or prevention of rheumatoid arthritis, multiple sclerosis, msulin-dependent diabetes, thyroiditis, psoriasis, myasthenia gravis, systematic lupus erythematosus, lupus, asthma, atopic dermatitis, contact hypersensitivity, allergic rhinitis, hepatitis, tuberculosis, leprosy, and AIDS.
  • the present invention also relates to the use of the above-mentioned compound for manufacturing a medicament for the treatment and/or prevention of autoimmune diseases, allergic diseases, infectious diseases, or AJDS, or for the treatment of acute or chronic rejection at organ transplant or bone-marrow transplant.
  • 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 overaU 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 ohgonucleotide 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 ohgonucleotide of the present invention may be a naturaUy 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 ACJD AND ENZYME) Vol.40, No.lO, 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 ceUular 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 oligonucleotide 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. All 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 antisense oUgonucleotide of the present invention can be produced by the well-known soUd phase synthesis technique. Devices for such synthesis are commerciaUy available from some manufactures including Applied Biosystems. Other oligonucleotides 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 oligonucleotides 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.lO 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 initiation 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 oUgonucleotide 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 oUgonucleotide 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, lipofection, electroporation, 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 retrovirus vector, then the expression vector can be introduced into the cells containing the target nucleic acid sequence by contacting the vector with the ceUs 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 T cell activation mediated by the protein of the present invention.
  • the antisense oligonucleotide 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 T ceUs, such as autoimmune diseases, aUergic diseases, infectious diseases, AJDS, or acute or chronic rejection at organ transplant or bone-marrow transplant.
  • the present invention also provides a pharmaceutical composition which comprises the above antisense oligonucleotide as an active ingredient.
  • the antisense oligonucleotide of the present invention can also be used to detect such diseases using northern hybridization or PCR.
  • the present invention also provides a ribozyme or deoxyribozyme which inhibits T cell 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 or deoxyribozyme has a complementary sequence to the mRNA for the protein of the present invention, complementarity 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 weU studied regarding their primary and secondary structure necessary for their cleavage activity, and those skUled in the art can easUy design the ribozymes nucleotided solely on the nucleotide sequence information for the DNA encoding the protein of the present invention [see e.g., 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)].
  • the hammerhead ribozymes or deoxyribozymes have a structure consisting of two recognition sites (recognition site I and recognition site II fo ⁇ ning 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 severakup to 10 to 20 complementary base pairs around that sequence.
  • the suitabiUty 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 DNA encoding a ribozyme into a suitable expression vector and introducing the vector into ceU strains, ceUs 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 have an activity of inhibiting the T ceU activation mediated by the protein of the present invention.
  • a double-stranded RNA is provided, which inhibits T cell activation.
  • RNA interference RNA interference
  • siRNA small interfering RNA
  • RNAi RNA sequence complementary to the sense RNA
  • siRNA Expression Vector available from Ambion can be used (Morita Takashi et al., Protein, Nucleic Acid and Enzyme, Vol.4 No.14 p. 1939-p. 1945 (2001); Sugimoto Asako, Kagaku to Seibutu (Chemistry and Biology), Vol.40 No.ll pp.713-718).
  • 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 tiansfo ⁇ ning a suitable cell with the reporter plasmid, culturing the transformant 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 ceU 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 or that of coding region which is selected from the group consisting of SEQ JD 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, 16
  • 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 dete ⁇ nining their secondary and tertiary structure, e.g., information for identifying other sequence having a simUar 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 program package. 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 flexible disks, tapes, chips, hard disk, compact disks and video disks.
  • the data on the medium aUows a method for calculating a homology by comparing the data with other nucleotide sequence data. This method comprises the steps of providing a first nucleotide sequence containing the nucleotide sequence of the present invention for the computer-readable medium, and then comparing the first nucleotide 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 polynucleotide comprising all or part of the nucleotide sequences selected from the group consisting of SEQ JD 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,
  • a plurality of the various polynucleotides which are DNA probes are fixed on a specificaUy processed solid substrates such as slide glass to form a DNA microarray 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 determined.
  • the present invention further relates to an insoluble substrate to which polypeptides comprising all or part of the amino acid sequences 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,
  • Example 1 Construction of a fuU-length cDNA Ubrary using the oligo-capping method (1) Preparation of RNA from human peripheral blood monocyte (PBMC)
  • PBMC peripheral blood monocyte
  • PBMC peripheral blood monocytes
  • RNA extraction source (2) One portion was cultured for further 24 hours, and the cells were collected as mentioned above to obtain the RNA extraction source (2).
  • the other portion was cultured for 24 hours in the medium containing PMA (Phorbol-12-myristate-13-acetate) at a final concentration of lOnM and A23187 at a final concentration of 10 M, and the ceUs were collected to obtain the RNA extraction source (3).
  • total RNAs were respectively obtained from these (1) to (3) (3 types of the recovered cells) by using the RNA extraction reagent ISOGEN (purchased from NIPPON GENE) according to the manufacture's protocol.
  • poly A + RNA was obtained from the total RNA by using an oUgo-dT cellulose column according to Maniatis et al., supra.
  • a full-length cDNA library was constructed from the above poly A + RNA of human PBMC 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, Y. & Sugano, S. "Shin Idenshi Kougaku Handbook
  • the full-length cDNA library constructed as above was transfected into E. coli strain TOP 10 by electroporation, then spread on LB agar medium containing lO ⁇ glvol ampicUlin, 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 E.coli colonies grown on ampicillin-containing LB agar medium.
  • Example 2 Cloning of DNA having a function of activating NF- K B (1) Screening of the cDNA encoding the protein having a function of activating NF- K B
  • TAg Jurkat cells (gifted from Dr Crabtree GR. Stanford University) were seeded at 4 x 10 4 ceUs/80 ⁇ 1/weU in a 96 well plate for cell culture in RPMI1640 medium containing 10% FBS. Then, lOOng of pNF K B-LUC (purchased from STRATAGENE) and 2 ⁇ 1 of the full-length cDNA prepared in above Example 1 were cotransfected into the ceUs in a well using SuperFect (purchased from QIAGEN) according to the manufacturer's protocol.
  • SuperFect purchased from QIAGEN
  • the luciferase activity was measured using long-term luciferase assay system, Picagene LT2.0 (TOYO INK) according to the attached manufacturer's instructions.
  • the luciferase activity was measured using Wallac ARVOTMST 1420 MULTILABEL COUNTER (Perkin Elmer).
  • sequencing was carried out using the reagent Thermo Sequenase II Dye Te ⁇ ninator Cycle Sequencing Kit (Amersham Pharmacia Biotech) or BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (AppUed Biosystems) and ABI PRISM 3100 sequencer according to the manufacturer's instructions.
  • amino acid sequences of the protein coding regions were deduced (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, 84, 86, 88, 90, 92, 94, 96, 98, 100, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, and 688
  • NF- K B reporter activity The results of measurement of NF- K B reporter activity (luciferase activity) of 49 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 empty pME18S-FL3 vector was introduced in place of full length cDNA).
  • the value of activity was shown as a relative value when the value obtained in the control experiment (luciferase activity of a cell into which empty pME18S-FL3 vector was introduced) was regarded to be 1.
  • Table 1 Results of measurement of NF K B reporter activity
  • Example 3 Cloning of DNA having a function of activating NFAT (1) Screening of the cDNA encoding the protein having a function of activating NFAT
  • TAg Jurkat cells were seeded at 4 x 10 4 cells/80 ⁇ 1/well in a 96 well plate for cell culture in RPMI1640 medium containing 10% FBS. Then, lOOng of pNFAT-Luc (purchased from STRATAGENE) and 2 ⁇ 1 of the fuU-length cDNA prepared in above Example 1 were cotransfected into the ceUs in a well using SuperFect (purchased from QIAGEN) according to the manufacturer's protocol. After 18 hours of culture at 37°C in the presence of 5% CO 2 , PMA which is a compound for activating AP-1 through activation of PKC was added at a final concentration of 20ng/ml.
  • PMA which is a compound for activating AP-1 through activation of PKC was added at a final concentration of 20ng/ml.
  • the luciferase activity was measured using long-term luciferase assay system, Picagene LT2.0 (TOYO INK) according to the attached manufacturer's instructions.
  • the luciferase activity was measured using Wallac ARVOTMST 1420 MULTILABEL COUNTER (Perkin Elmer).
  • amino acid sequences of the protein coding regions were deduced (SEQ JD NOS: 78, 80, 82, 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, 190, 192, 194, 196, 198, 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, 26
  • the results of measurement of NFAT reporter activity (luciferase activity) of 95 clones among the above obtained clones are shown in Table 2 below.
  • the value of activity shown in Table 2 is a relative value to the value of control experiment (luciferase activity of a cell into which empty pME18S-FL3 vector was introduced in place of full length cDNA).
  • the value of activity was shown as a relative value when the value obtained in the control experiment (luciferase activity of a cell into which empty pME18S-FL3 vector was introduced) was regarded to be 1.
  • Reporter assay was carried out using 293EBNA ceUs which are non-lymphocyte ceUs, and it was confirmed that the NF ⁇ B or NFAT activating function of the clones of the present invention was specific for T cells.
  • B-Luc or pNFAT-Luc purchased from STRATAGENE
  • lOOng of the clone obtained in above Examples 2 and 3 were cotransfected into the cells in a weU using FuGENE6
  • luciferase activity was measured using long-term luciferase assay system, Picagene LT2.0 (TOYO INK) according to the attached manufacturer's instructions.
  • the luciferase activity was measured using WaUac ARVOTMST 1420
  • NF K B or NFAT activating function or showed only NF /c B or NFAT activating function which is clearly weaker as compared with the case of the assay using Jurkat cells.
  • the present invention provides industrially highly useful proteins which have a function of activating NF rc B or NFAT specifically in T cells and are involved in T cell activation, and genes encoding the proteins.
  • the proteins of the present invention and the genes encoding the proteins aUow not only screening for compounds useful for treating and preventing diseases associated with the disorder of T cells, 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.

Abstract

Proteins which is involved in T cell activation, which are used for diagnosing, treating or preventing diseases associated with disorder of T cells, are provided. Using reporter plasmids pNFκB-Luc or pNFAT-Luc, cDNAs encoding proteins having a function of activating NFκB or NFAT in T cells were cloned from the cDNA library constructed from human peripheral blood monocyte (PBMC), and the DNA sequence and the deduced amino acid sequence are determined. The protein, the DNA encoding the protein, a recombinant vector containing the DNA, and a transformant containing the recombinant vector are used for screening a substance inhibiting or promoting T cell activation.

Description

DESCRIPTION T CELL ACTIVATING GENE
TECHNICAL FIELD
The present invention relates to a protein which is involved in T cell activation, 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 disorder of T cell.
The present invention also relates to a method for screening a substance capable of inhibiting or promoting T cell activation by using the protein, DNA, recombinant vector and transformant.
BACKGROUND ART
T cells recognize non-self molecules and an abnormality of self cells and exclude them, and thus play an important role in biological defence. The dysfunction of T cells is a cause of various immune diseases. For example, if T cells attack a self antigen as a foreign substance, autoimmune diseases such as diabetes Type I, rheumatoid arthritis or multiple sclerosis are induced. Also, a response against a foreign antigen becomes excessive, allergy diseases such as asthma or atopic dermatitis occur. Therefore, elucidation of molecular mechanism of T cell activation which is important for these diseases is expected to contribute to elucidation of pathological conditions and development of therapeutic means for various immune diseases.
Many signal transduction molecules involved in T cell activation have been identified by study on patients of immune disease, knock-out and transgenic mice or the like (Lin J. and Weiss A., J.Cell.Sci.ll4,243-244(2001); and Nel A.E., J.Allergy. Clin.Immunol 109,758-770(2002) ). T cell activation starts by the following process; T cells recognize a complex of an antigen on antigen presenting cells and major histocompatibility antigen (MHC) via T cell receptor (TCR) and then crossl nking occurs between several TCRs, and further costimulation via CD28 is provided. First, Lck, a Src type tyrosine kinase, which associates with CD4 or CD8 phosphorylates two Tyr of an activation motif (immunoreceptor tyrosine-based activation motif ; ITAM) which is common in an intracellular region of CD3. Then, ZAP70, a Syk type tyrosine kinase, is associated to the ITAM motifs and activated by Lck. Thereafter, the signal is transmitted to serme/threonine protein phosphatases (for example, Calcineurin;Cn), serme/threonine protein kinase (for example, PKC, MAPK super family) and the like via various adaptor proteins (for example, LAT, SLP-76), and finally transcription factors such as NF K B, NFAT or AP-1 are activated. These transcription factors are essential for the expression of IL-2 which is a most important cytokine for regulating the proliferation and function of various immunocompetent cells including T cell itself. The NFAT activation pathway is already an action target of an immunosuppressant. For example, Cyclosporin A (CsA) and FK506 inhibit an activity of Cn, an enzyme which dephosphorylates NFAT which is normally present in cytoplasm in phosphorylated state, and prevent the transmittance of NFAT into nucleus, thereby repressing the production of IL-2 (Liu .et al.,Cell 66,807-815(1991) ; and Flanagan,W.M.et al., Nature 352,803-807(1991)). NF K B and AP-1 are also considered to play an important role in IL-2 transcription activation in view of the results of the experiment using IL-2 promoter having a mutation in its binding sequence (Rao A.,Immunol.Today 15,274-281(1994) ; Marian F.et al.,Oncogene 20,2476-2489(2001) ; Serfling E.et al.,Biochimica Biophysica Acta 1263,181-200(1995) ; and Kane L.P.et al.,Trends in Immunol.23,413-420(2002)).
Recently, the analysis of mechanism of T cell activation has been rapidly progressed, but the immune system is a very ingenious and complex network, and thus the whole mechanism has not yet been revealed. The therapy and prevention of many diseases associated with T cells as mentioned above are still insufficient. It is desired to find out a novel substance which is involved in T cell activation, reveal the mechanism, and use them in the field of medicament, diagnosis and medical care.
DISCLOSURE OF THE INVENTION The object of the present invention is to identify a new gene and protein which is involved in T cell activation which is useful as mentioned above, 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 which is involved in T cell activation, 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 T cell activation by using the protein, DNA, recombinant vector and transformant, a kit for the screening, a substance capable of inhibiting or promoting T cell activation which is obtained 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 T cell activation, etc.
The present inventors have intensively studied to solve the above objects. 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 TAg Jurkat cells (Jurkat (human T cell line) which expresses SV40 large antigen; gifted from Dr Crabtree GR. Stanford University); and isolating a new DNA (cDNA) encoding a protein having a function of NF κ B or NFAT activation in T cell by using the assay system. It was confirmed that these new DNAs could activate NF B or NFAT by its expression in TAg Jurkat cells. This result shows that these new DNAs are signal transduction molecules involved in the NF K B or NFAT activation in T cell, namely the T cell activation pathway. Thus, the present invention has been completed.
That is, the present invention provides the folio wings: ( 1 ) A purified protein of the following (a) or (b) :
(a) a protein that consists of an 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 and 774; and (b) a protein that is involved in T cell activation and consists of an amino acid sequence having at least one amino acid deletion, substitution or addition in an 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 and 774.
(2) A purified protein that in involved in T cell activation 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 of the following (a) or (b):
(a) a protein that consists of an 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, 190, 192, 194, 196, 198, 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, 286 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 and 774; and (b) a protein that is involved in T cell activation and consists of an amino acid sequence having at least one amino acid deletion, substitution or addition in an amino acid sequence 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 and 774.
(4) An isolated polynucleotide comprising a nucleotide sequence of any of the following
(a) to (c):
(a) a nucleotide sequence represented by any one of SEQ JD 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, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771 and 773;
(b) a nucleotide sequence which encodes a protein that is involved in T cell activation, and which hybridizes with a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of (a) under stringent conditions; and
(c) a nucleotide sequence which encodes a protein that is involved in T cell activation, and which consists of a nucleotide sequence having at least one nucleotide deletion, substitution or addition in a nucleotide sequence of any of SEQ JD 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, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771 and 773.
(5) An isolated polynucleotide comprising a nucleotide sequence of any of the following (a) to (c):
(a) a nucleotide sequence represented by a coding region of any one of SEQ JD 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, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771 and 773;
(b) a nucleotide sequence which encodes a protein that is involved in T cell activation, and which hybridizes with a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of (a) under stringent conditions; and
(c) a nucleotide sequence which encodes a protein that is involved in T cell activation, and which consists of a nucleotide sequence having at least one nucleotide deletion, substitution or addition in a coding region of a nucleotide sequence of any of SEQ JD 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, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771 and 773.
The coding region in the present invention refers to a region which encodes a protein that is involved in T cell activation.
(6) An isolated polynucleotide comprising a nucleotide sequence which encodes a protein that is involved in T cell activation and has at least 95% identity to the polynucleotide according to (3) over the entire length thereof.
(7) An isolated polynucleotide comprising a nucleotide sequence which encodes a protein that is involved in T cell activation and has at least 95% identity to the polynucleotide 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 comprises the recombinant vector according to (9) as an active ingredient.
(11) A transformant which comprises the recombinant vector according to (9).
(12) A membrane of the transformant 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 transformant 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 susceptibiHty to a disease related to expression or activity of the protein of (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 gene in a sample derived from said subject.
(15) A method for screening compounds which inhibit or promote T cell activation, which comprises the steps of:
(a) preparing a transformant by introducing a gene encoding a protein that is involved in T cell activation according to (1), (2) or (8) and a gene encoding a signal which can detect an involvement in T cell activation into a host cell;
(b) culturing the transformant 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 an involvement in T cell activation; 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 which inhibit or promote T cell activation.
(16) A method for screening compounds which inhibit or promote T cell activation, which comprises the steps of:
(a) preparing a transformant by introducing a gene encoding a protein that is involved in T cell activation according to (1), (2) or (8) into a host cell;
(b) culturing the transformant under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring an activity of NF K B or NFAT in T cells; and
(d) selecting a candidate compound which can change the activity of NF / B or NFAT as compared with the case of the absence of candidate compounds, as a compound which inhibit or promote an activation of NF K B OΓ NFAT in T cells.
(17) A compound which inhibits or promotes T cell 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 transformant by introducing a gene encoding a protein that is involved in T cell activation according to (1), (2) or (8) and a gene encoding a signal which can detect an involvement in T cell activation into a host cell;
(b) culturing the transformant 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 an involvement in T cell activation;
(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 which inhibit or promote T cell 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 transformant by introducing a gene encoding a protein that is involved in T cell activation according to (1), (2) or (8) into a host cell;
(b) culturing the transformant under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring an activity of NF K B or NFAT in T cells;
(d) selecting a candidate compound which can change the activity of NF K B OΓ NFAT in T cells as compared with the case of the absence of candidate compounds, as a compound which inhibit or promote an activation of NF K B or NFAT in T cells; and
(e) producing a pharmaceutical composition which comprises a compound selected in the step of (d).
(20) A kit for screening a compound which inhibits or promotes an activation of NF c B or NFAT in T cells, which comprises:
(a) a transformant comprising a gene encoding a protein which promotes an activation of NF K B or NFAT in T cells according to (1), (2) or (8) and a gene encoding a signal which can detect promotion of activity of NF K B or NFAT; 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 or NFAT in T cells by the protein according to (1), (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 ohgonucleotide 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 activation of NF K B or NFAT in T cells.
(25) A ribozyme or deoxyribozyme capable of inhibiting activation of NF /c B or NFAT in T cells, 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 which is involved in a pathway leading to activation of NF K B or NFAT in T cells.
(26) A double strand RNA having a sequence corresponding to a part of the nucleotide sequence according to any one of (3) to (7), which inhibits expression of a protein that promotes activation of NF K B or NFAT in T cells.
(27) A method for treating a disease associated with T cell 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 ohgonucleotide according to (24), and/or a ribozyme or deoxyribozyme according to (25), and/or a double strand RNA according to (26) in an effective amount to treat and/or prevent a disease selected from the group consisting of autoimmune diseases, allergic, diseases, infectious diseases and AJDS or to treat acute or chronic rejection at organ transplant or bone-marrow transplant. (28) A pharmaceutical composition produced by the process according to (18) or (19) for inhibiting or promoting T cell activation.
(29) The pharmaceutical composition according to (28) for the treatment and/or prevention of autoimmune diseases, allergic diseases, infectious diseases and AJDS or for the treatment of acute or chronic rejection at organ transplant or bone-marrow transplant.
(30) A method of treating autoimmune diseases, allergic diseases, infectious diseases or AIDS, or acute or chronic rejection at organ transplant or bone-marrow transplant, which comprises administering a pharmaceutical composition produced by the process according to (18) or (19) to a patient suffering from a disease associated with T cell activation.
(31) A pharmaceutical composition which comprises a monoclonal or polyclonal antibody or a fragment thereof according to (21) or (22) as an active ingredient.
(32) A pharmaceutical composition which comprises an antisense ohgonucleotide according to (24) as an active ingredient.
(33) A pharmaceutical composition which comprises a ribozyme or deoxyribozyme according to (25) as an active ingredient.
(34) A pharmaceutical composition or a gene therapy agent, which comprises a double strand RNA according to (26) or a vector capable of expressing said double strand RNA, an active ingredient.
(35) The pharmaceutical composition according to any one of (31) to (33) for the treatment and/or prevention of a disease which is selected from the group consisting of autoimmune diseases, allergic diseases, infectious diseases or AJDS, or for the treatment of acute or chronic rejection at organ transplant or bone-marrow transplant.
(36) A computer-readable medium on which a sequence data set has been stored, said sequence data set comprising at least one of nucleotide sequence or that of coding region which is selected from the group consisting of SEQ JD 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, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771 and 773, 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302,- 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 and 774.
(37) A method for calculating identity to other nucleotide sequences and/or amino acid sequences, which comprises comparing data on a medium according to (36) with data of said other nucleotide sequences and/or amino acid sequences.
(38) An insoluble substrate to which polvnucleotides comprising all or part of the nucleotide sequences selected from the group consisting of SEQ JD 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, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771 and 773 are fixed.
(39) An insoluble substrate to which polypeptides comprising all 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724,' 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 and 774, are fixed.
The contents of the specifications and/or drawings of Japanese Patent Applications Nos.2002-376365, 2003-122113 and 2003-360559, U.S. Provisional AppUcations Nos.60/436,473, 60/465,792 and 60/512,846, which form the bases of priority of the instant application, are incorporated herein.
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 which is involved in T cell activation, 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 human peripheral blood monocyte (PBMC), 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 pNF K B-Luc reporter plasmid or the pNFAT-Luc reporter plasmid (both from STRATAGENE), each of which contains DNA encoding luciferase, and the above full-length cDNA plasmid were cotransfected into TAg Jurkat cells. In the detection system for NFAT activation, PMA was added after culturing for 18 hours. At 24 hours after gene transfection, 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 2-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 detemiined. The protein encoded by the cDNA thus obtained shows that this protein is a signal transduction molecule involved in activation of NF K B or NFAT in T cells.
The present invention is described in detail below.
In the present invention, the phrase "involved in T cell activation" means that NF K B or NFAT is activated (alternatively, activation of NF K B or NFAT is induced), which is a transcription factor which is necessary for the expression of IL-2 gene, when a gene is introduced into a suitable cell and the protein encoded by the gene is excessively expressed. Activation of NF c B or NFAT can be measured, for example, by an assay using NF K B dependant reporter gene or NFAT dependant reporter gene. The phrase " having a function of NF K B or NFAT activation" means that it has a function of increasing the reporter activity compared to control experiment (host 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.
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 a suitable 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. Examples of an expression control sequences of the reporter gene include NF K B and NFAT binding sequences as mentioned herein, as well as AP-1 (activator protein- 1) binding sequence, or IL-2 promoter region. Suitable host cells include cells which induce an activation signal transduction leading to IL-2 production by co-stimulation with an antibody against TCR/CD3 and CD28 or co-stimulation with phorbol ester and calcium ionophore. Examples thereof include Jurkat cells. Depending on the purpose, by adding stimuli such as anti-CD3 antibody, anti-CD28 antibody, phorbol ester or calcium ionophore, a clone which expresses the activation function by synergistic effect with the stimulation can be obtained. 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, TAg Jurkat cells (human T cell line, Jurkat, which expresses
SV40 large antigen; gifted from Dr Crabtree GR. Stanford University) are seeded on 10%
FBS (Fetal Bovine Serum)-containing medium (RPMU640 containing lOmM HEPES and 50 μ M 2-ME) in a 96-well cell culture plate to a final cell density of 40,000cells/well, and pNF κ B-Luc or pNFAT-Luc reporter plasmid (STRATAGENE) and an expression vector are co-transfected using SuperFect (QJAGEN).
Since activation of AP-1 is essential for increase of transcription activity of pNFAT-Luc in NFAT assay, an expression vector (for example, Ras(Val-12) expression vector) containing DNA encoding an active type protein of a signal transduction molecule which is known to belong to AP-1 activation pathway is co-transfected, or alternatively an agent which is known to activate AP-1, such as PMA, is added in the medium. After gene transfection, culturing is carried out for 24 hours at 37°C, and the luciferase activity is measured by using a long term luciferase assay system, Picagene LT2.0 (Toyo Ink Mfg) to measure the activation of NF K B or NFAT. For example, luciferase activity can be measured using PerkinElmer's WaUac ARVOTMST 1420 MULTILABEL COUNTER. The method for gene introduction by SuperFect 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 SuperFect, the amount of SuperFect per 1 well is suitably 0.3 to 0.8 μ 1, preferably 0.5 μ 1; the amount of pNF K B-LUC or pNFAT-Luc reporter plasmid is suitably 50 to lOOng, preferably lOOng; and the amount of expression vector is suitably 50 to lOOng, preferably lOOng. The concentration of PMA to be added in the NFAT assay system is suitably 5 to 25 ng/ml. An ability of activating NF K B or NFAT can be confirmed by regarding the ability of increasing the reporter activity (luciferase activity) relative to the control experiment (for cells into which only a null vector was introduced) as an index. Increase in reporter activity as an index 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 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, 190, 192, 194, 196 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 and 774, the present invention provides the following proteins:
(a) proteins which comprise the above amino acid sequence;
(b) polypeptides having one of the above amino acid sequences;
(c) proteins that is involved in T cell activation and consist of an amino acid sequence having 1 or several (preferably a few) amino acid deletion, substitution or addition in the above amino acid sequences:
(d) proteins that s involved in T cell activation and comprises an amino acid sequence, which has at least 95% identity, preferably at least 97-99% identity, to the above amino acid sequences over the entire length thereof:
"Identity" used herein means a relationship between two or more protein sequence or two or more nucleotide sequences, as determined by comparing the sequences, as known in the art. In the art, "identity" also means the degree of sequence relatedness between protein or nucleotide sequences, as deteπnined by the match between protein or nucleotide 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, for example, 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, Miller W, Lipman DJ,. Nucleic Acids Res. 25:p3389-3402 81997)), however methods of determining identity are not limited to this. 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, BLOSUM62, is used. The theory of this amino acids substitution matrix is shown in Altschul S.F., J. Mol. Biol. 219: 555-565 (1991), and its applicability to DNA sequence comparison is shown in States D. J., Gish W., Altschul S.F., Methods, 3: 66-70 (1991). In this case, optimal gap cost is determined empirically and in the case of BLOSUM62, preferably parameters, 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 sequence identity to the amino acid sequence of SEQ ID NO: 2 may have an amino acid sequence that includes up to 5 amino acid changes per 100 amino acids of the amino acid sequence of SEQ JD 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 teiminus or the carboxy terminus of the subject sequence, or may form one or more groups of changes.
The Examples described below demonstrate that the protein consisting of an amino acid sequence 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 or 774 is involved in T cell activation.
Related to the nucleotide sequences of SEQ JD 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, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771 and 773, or the nucleotide sequences of the coding region of these sequences, the present invention further provides the following isolated polynucleotides:
(a) polynucleotides of any of the above sequences;
(b) polynucleotides comprising a nucleotide sequence, which has at least 95% identity, preferably 97-99% identity, to any of the above sequences, and which encodes a protein which is involved in T cell activation;
(c) a polynucleotide which has a nucleotide sequence that encodes a protein, wherein the protein has an amino acid sequence having at least 95% identity, preferably at least 97-99% identity, to the amino acid sequence 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 or 774, and is involved in T cell activation.
Polynucleotides which are identical or substantially identical to a nucleotide sequence contained in the above nucleotide sequence may be used as hybridization probes to isolate full-length cDNA or genomic clones encoding proteins of the present invention or cDNA and genomic clones of other genes that have a high sequence similarity to the above sequences, or as primers for a 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 that is involved in T cell activation in the same way as in the protein having an amino acid sequence represented by SEQ JD 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 or 774 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 involved in T cell activation and which has at least one amino acid deletion, substitution or addition compared to the protein having an amino acid sequence represented 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 or 774, as well as DNA encoding the protein. The number of mutation of amino acid 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, glutarnine), (serine, threonine), (lysine, arginine) and (phenylalanine, tyrosine). Based on the nucleotide sequences (e.g., a polynucleotide of SEQ JD 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, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771 and 773) encoding a protein consisting of an amino acid sequence of SEQ JD 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 or 774, or fragments 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 that is involved in T cell activation in the same way as in the protein having of an amino acid sequence of SEQ JD 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 or 774.
Thus, the present invention also includes a protein that is involved in T cell activation and comprises an amino acid sequence having a high identity to the amino acid sequence of 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 or 774. "High identity" refers to an amino acid sequence having an identity of at least 90%, preferably at least 97-99% over the entire length of an amino acid sequence represented by above SEQ JD 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 or 774.
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 protein encoded by the gene of the present invention includes secretory proteins (growth factors, cytokines, hormones, etc.), protein modifying enzymes (protein phosphorylases, protein phosphatases, proteases, etc), signal transduction molecules (interaction molecules between proteins, etc.), intranuclear proteins (intranuclear receptors, transcription factors) and membrane proteins. Membrane proteins include receptors, cellular adhesion molecules, ion channels, transporters, etc. Where the protein is a membrane protein, a compound selected by the below-described screening is more useful as a research tool of medical compound since it is expected to easily migrate into a cell or give signal transduction 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 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 or 774 include nucleotide sequences of SEQ JD 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, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733,- 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771 and 773. The DNA includes cDNA, genomic DNA, and chemicaUy 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 SEQ JD 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552,- 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 or 774, 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 that is involved in T ceU activation and hybridizes under stringent conditions with the DNA of the above nucleotide sequence of SEQ JD 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, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771 or 773 or complementary nucleotide 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, and 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, lOOμg/ml denatured, sheared salmon sperm DNA) 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 1 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 42 °C 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 having a function of activating NF K B or NFAT in T cells.
The present invention also includes a polynucleotide comprising a nucleotide sequence which encodes a protein that is involved in T cell activation and has a high sequence similarity to the nucleotide sequence of the polynucleotide according to above item (3), (4) or (5). Typically these nucleotide sequences are 95% identical, preferably 97% 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 protein according to the above item (1), (2) or (8) 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) transforming a host with the recombinant vector thus constructed; and
(D) culturing the obtained host to express the protein or its analogue, and then purifying it by column chromatography.
General procedures necessary to handle DNA and recombinant hosts (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 manufactures1 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 application, PCR amplification using a synthetic DNA having a portion of the nucleotide sequence of the present invention (e.g., SEQ JD 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, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771 or 773), as a primer, and selection of the DNA inserted into a suitable vector by hybridization with a labeled DNA fragment encoding a partial or fuU 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 normaUy 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 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, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771 or 773, or may be a polynucleotide of above items (3) to (7). The DNA to be inserted into an expression vector in the above step (B) may be a full-length cDNA encoding the above full-length protein or a DNA fragment thereof, or may be 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. The expression vector capable of expressing 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. The vectors should be selected to be compatible with hosts. Suitable combinations of host-expression vector systems are selected depending on the desired expression product.
When bacteria are used as hosts, plasmid vectors compatible with these bacteria are generally used as replicable expression vectors for recombinant DNA molecules. For example, the plasmids pBR322 and pBR327can 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 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 repUcable expression 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 of genes of 3-phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, alcohol dehydrogenase and the like.
Examples of promoters and teπninators for use in expression vectors to express the DNA of the present invention in fungal cells include promoters and terminators derived from genes for phόsphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPD), actin and the like. 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 vector suitable for insect ceUs include baculovirus.
Recombinant vectors used to express the DNA of the present invention in animal cells normally contain functional sequences to regulate genes, a promoter to be placed upstream of the DNA of the present invention, a polyadenylation site and a transcription teπnination sequence. Such functional sequences, which can be used to express the DNA of the present invention in cells, can be obtained from viruses and viral substances.
Examples of such functional sequences include an SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter and HSV-TK promoter. Among them, a CMV promoter and SRα 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 pSV2-dhfr (ATCC 37146), pBPV- 1(9-1) (ATCC 37111), pcDNA3.1 (INVJTROGEN) and pME18S-FL3.
The present invention also provides a transformant which comprises the above recombinant vector. Microorganisms or ceUs transformed with the recombinant vector of the present invention can be selected from remaining untransformed parent ceUs based on at least one phenotype conferred by the recombinant vector. 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 ceUs 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. coli JM109 and E. coU B strain; bacterial strains belonging to the genus Bacillus such as Bacillus subtilis; intestinal bacteria other than E. coli, such as Salmonella typhimurium or Serratia marcescens; and various strains belonging to the genus Pseudomonas. Examples of yeast include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris. Examples of fungi include Aspergillus nidulans, and Acremonium chrysogenum (ATCC 11550).
As insect cells, for example, Spodoptera frugiperda (Sf cells), High Five ™ ceUs derived from eggs of Trichoplusiani, etc., can be used when the virus is AcNPV. Examples of animal cells include HEK 293 cells, COS-1 cells, COS-7 ceUs, Hela cells, and Chinese hamster ovary (CHO) cells. Among them, CHO ceUs and HEK 293 cells are preferred. When animal cells are used as hosts, combinations of expression vectors and hosts 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 ceUs by known methods such as DEAE-dextran method, calcium-phosphate-mediated transfection, electropofation, lipofection, etc. For obtaining ceUs which stably express 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. In addition, the animal ceUs 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 whUe 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 to obtain animal ceU strains having higher levels of expression.
The above transformant 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 transformant 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 (namely, cell itself or culture medium).
The above transformant can be cultured by methods known to those skUled in the art (see e.g., "Bio Manual Series 4", YODOSHA CO., LTD.). For example, animal cells can be cultured by various known animal ceU culture methods including attachment culture such as Petri dish culture, multitray type culture and module culture, attachment culture in which cells are attached to ceU culture carriers (microcarriers), or suspension culture in which productive cells themselves are suspended. Examples of medium for use in the culture include medium 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 solubUity, 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 Uquid 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. Preferably, high performance liquid chromatography is employed for purification. WeU known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during intraceUular 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-transf erase (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 ah affinity for the fusion partner protein. For example, fusion proteins with GST can be purified by affinity chromatography using glutathione as a Ugand.
When the protein of the present invention is a membrane protein, a transformant 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 transformants 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 ceU can be prepared by a method known to those skiUed in the art. For example, cells are collected from the culture where transformants 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 provides 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 specificaUy 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 specificaUy 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) or epitope-bearing fragments as an antigen 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 ceUs by using a reagent such as polyethylene glycol. From the resulting fused cells (i.e. hybridoma), the clone producing the antibody directed against the protein of the present invention can be selected. Then, the resulting clonal hybridoma can be implanted intraperitoneaUy 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 to humans, it is preferable to use a humanized antibody or human antibody in order to reduce its immunogenicity. These humanized antibodies or human antibodies can be produced using transgenic mice or other mammals. For a general review of humanized antibodies or 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 complement ty-deteπnining 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 transformant 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, these antibodies can be used for diagnosis of a disease characterized by undesirable activation of T cells resulting from (expression) abnormality of the protein of the present invention, such as autoimmune diseases, allergic diseases, infectious diseases or AIDS, or acute or chronic rejection at organ transplant or bone-marrow transplant.
In order to provide a basis for diagnosis of a disease, a standard value must be established regarding the expression of the protein of the present invention. However, this is a well-known technique to those skiUed 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 function of activation of NF κ B or NFAT in T ceUs which is possessed by the protein of present invention (that is, neutralizing antibody).
The antibodies of the present invention can be purified and then administered to patients with a disease characterized by undesirable activation of T cells resulting from (expression) abnormality of the protein of the present invention, such as autoimmune diseases, allergic diseases, infectious diseases or AJDS, or acute or chronic rejection at organ transplant or bone-marrow transplant. Thus in another aspect, the present invention provides 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 of the present invention, the active ingredient may be combined with other therapeuticaUy or preventively active ingredients or inactive ingredients (e.g., conventional pharmaceutically acceptable carriers or dUuents 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 administration, 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 skiUed in the art. The antibody of the present invention exhibits therapeutic activity by inhibiting the promotion of T cell activation mediated by the protein of the present invention. SpecificaUy, the antibody of the present invention can be usefulf as a medicament for treatment or prevention of diseased assciated with abnormal activity of T cells, such as autoimmune diseases, allergic diseases, infectious diseases or AIDS, or acute or chronic rejection at organ transplant or bone-marrow transplant.
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, from cDNA library or genomic DNA library. In a similar manner, it can be determined whether the protein of the present invention can associate with other cellular proteins (e.g., ZAP70 or PKC θ which are known be essential for T cell activation). 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 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 susceptibiUty to the diseases through detection of mutation in a gene for the protein of the present invention which is involved in T cell activation. In addition, such diseases may be diagnosised by analyzing expression level of the gene in a sample derived from a subject at protein or mRNA level, and detecting an abnormaUy decreased or increased level of the expression.
Determination of the presence or absence of a mutation in the gene encoding the protein of the present invention which is involved in T ceU activation, may involve RT-PCR using a part of the nucleotide sequences of genes 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 weU known in the art for the quantitation of polynucleotides, for example, nucleic acid ampUfication 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 determine 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 abnormaUty of expression level of gene in a sample is not particularly limited. 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 diagnosed 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 diagnosed 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. Thus, for example, the DNA of the present invention is useful for gene diagnosis regarding damage, mutations, and reduced, increased or over- expression of the 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 K B or NFAT in T cells.
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 is involved in T cell activation may be responsible for diseases associated with the expression and/or activity of NF K B or NFAT in T cells. The present invention also relates to a method for screening compounds which inhibit or promote T cell activation mediated by the protein of the present invention.
It should be noted that compounds that inhibit T cell activation, wUl, as a result of this action, have in vivo and in vitro activity as a inhibiting agent for T cell function. Also, compounds that promote T cell activation, wUl, as a result of this action, have in vivo and in vitro activity as an activating agent for T cells Consequently, the above screening method is for screening in respect of activity as an inhibiting agent or activating agent of T cells, and the above compound is a compound having activity as an inhibiting agent or activating agent of T cell function.
The above screening method comprises the following steps:
(a) preparing a transformant by introducing a gene encoding a protein that is involved in T cell activation according to the present invention and a gene encoding a signal which can detect an involvement in T cell activation into a host cell;
(b) culturing the transformant 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 an involvement in T cell activation; 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 which inhibit or promote T cell activation.
A compound that increases the detectable signal 2-fold or higher than normal is preferably isolated or identified as an activator compound, and a compound that decreases the detectable signal 50% or less than normal is preferably isolated or identified as an inhibitor compound.
Examples of genes encoding a signal which can detect involvement in T ceU activation include reporter genes. Reporter genes are used instead of directly detecting the activation of transcription factors of interest to be tested. 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.). A gene encoding any peptide or protein can be used as a reporter gene, so long as those skUled 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. Other examples include NF K B dependent reporter plasmid described in Tanaka S.e al J.Vet.Med.Sci.Vol.59(7), Rothe M.e aLScience Vol.269 ρl424-1427(1995). Any reporter plasmids can be used to evaluate NFAT activation so long as the reporter plasmids have an NFAT recognition sequence inserted upstream of the reporter gene, and examples thereof include pNFAT-Luc(STRATAGENE). '
Any host can be used so long as it induces an activation signal transduction leading to IL-2 production by co-stimulation with an antibody against TCR/CD3 and CD28 or co- stimulation with phorbol ester and calcium ionophore. For example, Jurkat cells are preferably used. 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 T cell activation comprises culturing the transformant 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 for the screening include, but not limited to, low molecular weight compounds, high molecular weight 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 Ubrary which was synthesized by combinatorial chemistry, a naruraUy occurring product containing ceUs, plants, animals or a part thereof, or an extracted product of such naruraUy 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 or NFAT activation in T cells can be further screened to isolate a single substance having the activity. Isolation 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 transformant by introducing a gene encoding a protein that is involved in T cell activation according to the present invention into a cell;
(b) culturing the transformant under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring an activity of NF κ B or NFAT in T cells; and
(d) selecting a candidate compound which can change the activity of NF K B OΓ NFAT as compared with the case of the absence of candidate compounds, as a compound which inhibit or promote an activation of NF κ B or NFAT in T cells.
In the above method, examples of the method of measuring an activation of NF κ B or NFAT in T cells include a method of analysing the binding to NF / B or NFAT binding sequence using cell extraction solution by gel shift (for example, Hayashi T et al. 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 (for example, IL-2) whose expression is known to be induced by T cell activation is 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. CommerciaUy available antibodies(from, e.g., Wako Pure Chemical Industries, Ltd.) can also be used.
The present invention further provides a method of producing a pharmaceutical composition, which comprises the foUowing steps (a) to (e): (a) preparing a transformant by introducing a gene encoding a protein that is involved in T cell activation according to the present invention and a gene encoding a signal which can detect an involvement in T cell activation into a host cell;
(b) culturing the transformant 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 an involvement in T ceU activation;
(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 which inhibit or promote T ceU activation; and
(e) producing a pharmaceutical composition which comprises a compound selected in the step of (d).
In the present invention, a pharmaceutical composition may also be produced by the foUowing steps (a) to (e):
(a) preparing a transformant by introducing a gene encoding a protein that is involved in T cell activation according to the present invention into a host ceU;
(b) culturing the transformant under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring an activity of NF K B or NFAT in T cells;
(d) selecting a candidate compound which can change the activity of NF K B or NFAT in T cells as compared with the case of the absence of candidate compounds, as a compound which inhibit or promote an activation of NF K B OΓ NFAT in T cells; 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 50% 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) deteirmining 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 an activity of inhibiting or promoting T cell activation. More specifically, this compound has an activity of inhibiting or promoting NF / B or NFAT activation in T cells which is promoted by the protein of the present invention.
Since the compounds obtained by the above screening methods have an activity of inhibiting or promoting T cell activation, they are useful as therapeutic or preventive pharmaceuticals for the diseases resulting from unfavorable activation or inactivation of T cells.
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 dissolving or suspending the compound into a suitable solvent by conventional methods 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 foUowing. The above compounds or their pharmaceuticaUy acceptable salts in an amount effective as an active ingredient, and pharmaceuticaUy 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 soUd form such as tablet, granule, capsule, pUl and powder, and solution form such as solution, syrup, elixir and dispersion. A form useful for parenteral administration includes sterile solution, 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 polyaUcylene 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 provides a kit for screening a compound which inhibits or promotes T ceU activation. The kit comprises: (a) a transformant comprising a gene encoding a protein that is involved in T ceU activation according to the present invention and a gene encoding a signal which can detect a promotion of NF / B or NFAT activation; and (b) reagents for measuring the signal. The kit comprises reagents necessary for screening compounds which inhibits or promotes T ceU activation.
In another aspect, the present invention relates to a diagnostic kit which comprises: (a) a polynucleotide of the present invention having a nucleotide sequence represented by any one of SEQ JD 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, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771 and 773;
(b) a polynucleotide having a nucleotide sequence complementary to that of (a);
(c) a protein of the present invention having an amino acid sequence represented by any one 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 and 774, or a fragment thereof; or
(d) an antibody to a protein of the present invention of (c).
A diagnostic kit comprising at least any one of (a) to (d) is useful for diagnosing a disease or susceptibUity to a disease such as autoimmune diseases, allergic diseases, infectious diseases, AIDS, or acute or chronic rejection at organ transplant or bone-marrow transplant. Because T cell is involved in a wide variety of pathological conditions such as autoimmune diseases, allergic diseases, infectious diseases, AJDS, or acute or chronic rejection at organ transplant or bone-marrow transplant, it is an attractive target for drug design and therapeutic intervention.
The finding of the new protein described herein that is involved in T cell activation has provided a new medicament and therapeutic method for controlling an abnormal T cell function. The present invention also relates to use of a compound which inhibits the function of the protein that is involved in T cell activation described above, for inhibiting T ceU activation. The present invention also relates to use of a compound which activates the function of the protein that is involved in T cell activation described above, for promoting T cell activation. The compound obtained by the above screening method, which inhibits or promotes T cell activation, is useful as a medicament to treat or prevent diseases characterized by undesirable activation of T cells, such as autoimmune diseases, aUergic diseases, infectious diseases, AIDS, or acute or chronic rejection at organ transplant or bone-marrow transplant.
Examples of the diseases associated with an abnormal T cell activation include autoimmune diseases (rheumatoid arthritis, multiple sclerosis, insulin-dependent diabetes, thyroiditis, psoriasis, myasthenia gravis, systematic lupus erythematosus, lupus, and the like), allergic diseases (asthma, atopic dermatitis, contact hypersensitivity, allergic rhinitis and the like), virus and bacterial infectious diseases (hepatitis, tuberculosis, leprosy, and the like), AJDS (acquired immunodeficiency syndrome). Thus, the compound obtained by the above screening method, which inhibits T cell 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 autoimmune diseases, allergic diseases, infectious diseases, AJDS, or acute or chronic rejection at organ transplant or bone-marrow transplant. "Gene therapy" refers to administering into the human body a gene or a ceU into which a gene has been introduced. 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. When a gene encoding the protein of the present invention is used for a agent for gene therapy, a technique of RNA interference (RNAi) mentioned below may be applied. Thus, the present invention provides a vector for gene therapy which expresses double strand RNA having a gene sequence 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 auxUiary 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 appUed 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 10jU 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 limited.
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 the administration route may vary with the weight and age of the patient, but those skilled in the art can appropriately select the administration route, and can appropriately select suitable dosage which is suitable for the administration route. 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 which inhibits or promotes T ceU activation as an active ingredient.
In addition, the above compound is useful as a medicament to treat or prevent diseases characterized by abnormal activity of T cells, such as autoimmune diseases, allergic diseases, infectious diseases, AJDS, or acute or chronic rejection at organ transplant or bone-marrow transplant. Thus, the present invention provides a pharmaceutical composition for the treatment or prevention of autoimmune diseases, aUergic diseases, infectious diseases, AJDS, or acute or chronic rejection at organ transplant or bone-marrow transplant, and the like, which comprises the compound which inhibits or promotes T ceU activation. Specifically, the pharmaceutical composition is useful as a medicament for the treatment or prevention of rheumatoid arthritis, multiple sclerosis, msulin-dependent diabetes, thyroiditis, psoriasis, myasthenia gravis, systematic lupus erythematosus, lupus, asthma, atopic dermatitis, contact hypersensitivity, allergic rhinitis, hepatitis, tuberculosis, leprosy, and AIDS.
The present invention also relates to the use of the above-mentioned compound for manufacturing a medicament for the treatment and/or prevention of autoimmune diseases, allergic diseases, infectious diseases, or AJDS, or for the treatment of acute or chronic rejection at organ transplant or bone-marrow transplant.
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 overaU 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 ohgonucleotide 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 ohgonucleotide of the present invention may be a naturaUy 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 ACJD AND ENZYME) Vol.40, No.lO, 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 preferred 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 ceUular 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 oligonucleotide 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. All 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 antisense oUgonucleotide of the present invention can be produced by the well-known soUd phase synthesis technique. Devices for such synthesis are commerciaUy available from some manufactures including Applied Biosystems. Other oligonucleotides 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 oligonucleotides 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.lO 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 initiation 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 oUgonucleotide 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 oUgonucleotide 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, lipofection, electroporation, 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 retrovirus vector, then the expression vector can be introduced into the cells containing the target nucleic acid sequence by contacting the vector with the ceUs 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 T cell activation mediated by the protein of the present invention.
The antisense oligonucleotide 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 T ceUs, such as autoimmune diseases, aUergic diseases, infectious diseases, AJDS, or acute or chronic rejection at organ transplant or bone-marrow transplant. Thus, the present invention also provides a pharmaceutical composition which comprises the above antisense oligonucleotide as an active ingredient. The antisense oligonucleotide of the present invention can also be used to detect such diseases using northern hybridization or PCR.
The present invention also provides a ribozyme or deoxyribozyme which inhibits T cell 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 or deoxyribozyme has a complementary sequence to the mRNA for the protein of the present invention, complementarity 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 weU studied regarding their primary and secondary structure necessary for their cleavage activity, and those skUled in the art can easUy design the ribozymes nucleotided solely on the nucleotide sequence information for the DNA encoding the protein of the present invention [see e.g., 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)]. It is known that the hammerhead ribozymes or deoxyribozymes have a structure consisting of two recognition sites (recognition site I and recognition site II foπning 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); 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); Kawasaki & Taira, Jikken Igaku (Experimental Medicine) Vol.18, No.3, ρ.381-386 (2000)].
Therefore the sequence GTC (or GTA) is searched out, and a ribozyme is designed to form severakup to 10 to 20 complementary base pairs around that sequence. The suitabiUty 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 DNA encoding a ribozyme into a suitable expression vector and introducing the vector into ceU strains, ceUs 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 have an activity of inhibiting the T ceU activation mediated by the protein of the present invention. According to the present invention, a double-stranded RNA is provided, which inhibits T cell activation.
The introduction of the double-stranded RNA into a ceU enables the specific degradation of mRNA corresponding to the sequence of the RNA, and the degradation suppresses gene expression. Recently, this phenomenon, referred to as RNA interference (RNAi), has been revealed. One example of a method using RNAi is a method for introducing an artificially synthesized small interfering RNA (siRNA) into a cell. siRNA is a double-stranded RNA of 19 to 25 base pairs which is mentioned as an important trigger to induce RNAi phenomena. With regard to the 19-to-25-nucleotide sequence in a suitable region of the sequence of a gene encoding the protein of the present invention, a sense RNA (wherein DNA sequence is substituted by RNA sequence) and an antisense RNA (having a sequence complementary to the sense RNA) are synthesized to prepare siRNA, and the siRNA is introduced into a cell by lipofection using, for example, FuGENE6, thereby enabling the use of RNAi.
In addition to the introduction of synthesized siRNA, a method that is also effective has been recently and gradually unveiled, which comprises: incorporating the 19-to-25-nucleotide sequence in a suitable region of the sequence of a gene encoding the protein of the present invention and a sequence complementary thereto into a plasmid; and temporarily expressing siRNA in the cell. More specifically, for example, pSilencer siRNA Expression Vector available from Ambion can be used (Morita Takashi et al., Protein, Nucleic Acid and Enzyme, Vol.4 No.14 p. 1939-p. 1945 (2001); Sugimoto Asako, Kagaku to Seibutu (Chemistry and Biology), Vol.40 No.ll pp.713-718).
Further, 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 tiansfoπning a suitable cell with the reporter plasmid, culturing the transformant 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 ceU 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 or that of coding region which is selected from the group consisting of SEQ JD 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, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771 and 773, 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 and 774.
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 deteπnining their secondary and tertiary structure, e.g., information for identifying other sequence having a simUar 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 program package. 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 flexible disks, tapes, chips, hard disk, compact disks and video disks. The data on the medium aUows a method for calculating a homology by comparing the data with other nucleotide sequence data. This method comprises the steps of providing a first nucleotide sequence containing the nucleotide sequence of the present invention for the computer-readable medium, and then comparing the first nucleotide 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 polynucleotide comprising all or part of the nucleotide sequences selected from the group consisting of SEQ JD 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, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771 and 773 are fixed. A plurality of the various polynucleotides which are DNA probes are fixed on a specificaUy processed solid substrates such as slide glass to form a DNA microarray 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 determined.
The present invention further relates to an insoluble substrate to which polypeptides comprising all or part of the amino acid sequences 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 and 774 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 substances captured on the insoluble substrate that can be expected to be useful in diagnosis or drug development.
The foUowing examples further illustrate, but do not Umit the present invention.
EXAMPLES
Example 1 : Construction of a fuU-length cDNA Ubrary using the oligo-capping method (1) Preparation of RNA from human peripheral blood monocyte (PBMC)
About 2X 107 human peripheral blood monocytes (PBMC) were cultured in the presence of 5% CO2 at 37°C for 7 days in a RPMI1640 medium (purchased from GIBCO) containing 10% FBS (Fetal Bovine Serum; purchased from GIBCO) and PHA (Phytohemagglutinin) at a final concentration of 10 μ g/ml. The thus obtained culture solution was centrifuged and washed once with PBS, and about 1 X 108 ceUs were collected to obtain the RNA extraction source (1). The remaning cells (about 1 X 108 cells) were cultured for 24 hours in the aforementioned mediun lacking PHA, and were devided into 2 portions equally. One portion was cultured for further 24 hours, and the cells were collected as mentioned above to obtain the RNA extraction source (2). The other portion was cultured for 24 hours in the medium containing PMA (Phorbol-12-myristate-13-acetate) at a final concentration of lOnM and A23187 at a final concentration of 10 M, and the ceUs were collected to obtain the RNA extraction source (3). Then, total RNAs were respectively obtained from these (1) to (3) (3 types of the recovered cells) by using the RNA extraction reagent ISOGEN (purchased from NIPPON GENE) according to the manufacture's protocol. Then, poly A+ RNA was obtained from the total RNA by using an oUgo-dT cellulose column according to Maniatis et al., supra. (2) Construction of a full-length cDNA library by the oligo-capping method
A full-length cDNA library was constructed from the above poly A+ RNA of human PBMC 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.].
(3) Preparation of plasmid DNA
The full-length cDNA library constructed as above was transfected into E. coli strain TOP 10 by electroporation, then spread on LB agar medium containing lO μ glvol ampicUlin, 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 E.coli colonies grown on ampicillin-containing LB agar medium.
Example 2: Cloning of DNA having a function of activating NF- K B (1) Screening of the cDNA encoding the protein having a function of activating NF- K B
TAg Jurkat cells (gifted from Dr Crabtree GR. Stanford University) were seeded at 4 x 104 ceUs/80 μ 1/weU in a 96 well plate for cell culture in RPMI1640 medium containing 10% FBS. Then, lOOng of pNF K B-LUC (purchased from STRATAGENE) and 2 μ 1 of the full-length cDNA prepared in above Example 1 were cotransfected into the ceUs in a well using SuperFect (purchased from QIAGEN) according to the manufacturer's protocol. After 24 hours of culture at 37°C in the presence of 5% CO2, the luciferase activity was measured using long-term luciferase assay system, Picagene LT2.0 (TOYO INK) according to the attached manufacturer's instructions. The luciferase activity was measured using Wallac ARVO™ST 1420 MULTILABEL COUNTER (Perkin Elmer). (2) DNA sequencing
The above screening was carried out, and plasmids showing a 2-fold or more increase in luciferase activity compared to that of the control experiment (luciferase activity of the cell into which empty vector pME18S-FL3 is introduced instead of full-length cDNA) were selected. One pass sequencing was carried out from the 5' end of the cloned cDNA (sequencing primer: 5'-CTTCTGCTCTAAAAGCTGCG-3' (SEQ ID NO: 775)) and from the 3' end (sequencing primer: 5'-CGACCTGCAGCTCGAGCACA-3' (SEQ JD NO: 776)) so that as long sequence as possible is determined. The sequencing was carried out using the reagent Thermo Sequenase II Dye Teπninator Cycle Sequencing Kit (Amersham Pharmacia Biotech) or BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (AppUed Biosystems) and ABI PRISM 3100 sequencer according to the manufacturer's instructions. (3) Full-length sequencing
The full-length DNA sequences for the 97 new clones which were obtained by the above screening, were detennined (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, 83, 85, 87, 89, 91, 93, 95, 97, 99, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, and 687). The amino acid sequences of the protein coding regions (open reading frames) were deduced (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, 84, 86, 88, 90, 92, 94, 96, 98, 100, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, and 688).
The results of measurement of NF- K B reporter activity (luciferase activity) of 49 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 empty pME18S-FL3 vector was introduced in place of full length cDNA). In Table 1, the value of activity was shown as a relative value when the value obtained in the control experiment (luciferase activity of a cell into which empty pME18S-FL3 vector was introduced) was regarded to be 1. Table 1: Results of measurement of NF K B reporter activity
Figure imgf000072_0001
Figure imgf000073_0001
Example 3: Cloning of DNA having a function of activating NFAT (1) Screening of the cDNA encoding the protein having a function of activating NFAT
TAg Jurkat cells were seeded at 4 x 104 cells/80 μ 1/well in a 96 well plate for cell culture in RPMI1640 medium containing 10% FBS. Then, lOOng of pNFAT-Luc (purchased from STRATAGENE) and 2 μ 1 of the fuU-length cDNA prepared in above Example 1 were cotransfected into the ceUs in a well using SuperFect (purchased from QIAGEN) according to the manufacturer's protocol. After 18 hours of culture at 37°C in the presence of 5% CO2, PMA which is a compound for activating AP-1 through activation of PKC was added at a final concentration of 20ng/ml. After culturing for 6 hours, the luciferase activity was measured using long-term luciferase assay system, Picagene LT2.0 (TOYO INK) according to the attached manufacturer's instructions. The luciferase activity was measured using Wallac ARVO™ST 1420 MULTILABEL COUNTER (Perkin Elmer).
(2) DNA sequencing
Selection of the plasmids and one pass sequencing were carried out in the same way as in Example 2(2) above.
(3) Full-length sequencing
The fuU-length DNA sequences for the 290 new clones which were obtained by the above screening, were determined (SEQ ID NOS: 77, 79, 81, 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, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771 and 773). The amino acid sequences of the protein coding regions (open reading frames) were deduced (SEQ JD NOS: 78, 80, 82, 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 and 774).
The results of measurement of NFAT reporter activity (luciferase activity) of 95 clones among the above obtained clones are shown in Table 2 below. The value of activity shown in Table 2 is a relative value to the value of control experiment (luciferase activity of a cell into which empty pME18S-FL3 vector was introduced in place of full length cDNA). In Table 2, the value of activity was shown as a relative value when the value obtained in the control experiment (luciferase activity of a cell into which empty pME18S-FL3 vector was introduced) was regarded to be 1.
Table 2: Results of measurement of NFAT reporter activity
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Example 4: Confirmation of cell specificity
Reporter assay was carried out using 293EBNA ceUs which are non-lymphocyte ceUs, and it was confirmed that the NF κ B or NFAT activating function of the clones of the present invention was specific for T cells.
293EBNA ceUs (purchased from Invitrogen) were seeded at 1 x 104 ceUs/100 μ 1/weU in a 96 well plate for ceU culture in EvIDM medium (purchased from GIBCO) containing 10%
FBS, and cultured for 24 hours at 37°C in the presence of 5% CO2. Then, lOOng of pNF K
B-Luc or pNFAT-Luc (purchased from STRATAGENE) and lOOng of the clone obtained in above Examples 2 and 3 were cotransfected into the cells in a weU using FuGENE6
(purchased from Roche) according to the manufacturer's protocol. After 24 hours of culture at 37°C in a CO2 incubator, the luciferase activity was measured using long-term luciferase assay system, Picagene 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). The results are shown in Table 3 below. The value of activity shown in Table 3 is a relative value to the value of control experiment
(luciferase activity of a cell into which empty pME18S-FL3 vector was introduced in place of full length cDNA). In Table 3, the value of activity was shown as a relative value when the value obtained in the control experiment (luciferase activity of a cell into which empty pME18S-FL3 vector was introduced) was regarded to be 1.
In the assay using 293EBNA cells, the clones of the present invention did not show
NF K B or NFAT activating function, or showed only NF /c B or NFAT activating function which is clearly weaker as compared with the case of the assay using Jurkat cells.
Table 3: Cell specificity
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
INDUSTRIAL APPLICABILITY
As described above, the present invention provides industrially highly useful proteins which have a function of activating NF rc B or NFAT specifically in T cells and are involved in T cell activation, and genes encoding the proteins. The proteins of the present invention and the genes encoding the proteins aUow not only screening for compounds useful for treating and preventing diseases associated with the disorder of T cells, 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.
All publications, patents and patent appUcations cited herein are incorporated herein in their entirety.

Claims

1. A purified protein of the following (a) or (b) :
(a) a protein that consists of an 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 and 774; and
(b) a protein that is involved in T ceU activation and consists of an amino acid sequence having at least one amino acid deletion, substitution or addition in an amino acid sequence 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 and 774.
2. A purified protein that in involved in T ceU activation 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 of the following (a) or (b):
(a) a protein that consists of an amino acid sequence 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 and 774; and (b) a protein that is involved in T ceU activation and consists of an amino acid sequence having at least one amino acid deletion, substitution or addition in an 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 and 774.
4. An isolated polynucleotide comprising a nucleotide sequence of any of the following
(a) to (c):
(a) a nucleotide sequence represented by any one of SEQ JD 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, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771 and 773;
(b) a nucleotide sequence which encodes a protein that is involved ih T cell activation, and which hybridizes with a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of (a) under stringent conditions; and
(c) a nucleotide sequence which encodes a protein that is involved in T cell activation, and which consists of a nucleotide sequence having at least one nucleotide deletion, substitution or addition in a nucleotide sequence of any 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, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771 and 773.
5. An isolated polynucleotide comprising a nucleotide sequence of any of the following (a) to (c):
(a) a nucleotide sequence represented by a coding region of any one of SEQ JD 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, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771 and 773;
(b) a nucleotide sequence which encodes a protein that is involved in T cell activation, and which hybridizes with a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of (a) under stringent conditions; and
(c) a nucleotide sequence which encodes a protein that is involved in T ceU activation, and which consists of a nucleotide sequence having at least one nucleotide deletion, substitution or addition in a coding region of a nucleotide sequence of any of SEQ JD 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, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, '489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771 and 773.
6. An isolated polynucleotide comprising a nucleotide sequence which encodes a protein that is involved in T cell activation and has at least 95% identity to the polynucleotide according to claim 3 over the entire length thereof.
7. An isolated polynucleotide comprising a nucleotide sequence which encodes a protein that is involved in T cell activation and has at least 95% identity to the polynucleotide 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 transformant which comprises the recombinant vector according to claim 9.
12. A membrane of the transformant 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 transformant 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 susceptibiUty 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) deteπnining 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 gene in a sample derived from said subject.
15. A method for screening compounds which inhibit or promote T cell activation, which comprises the steps of:
(a) preparing a transformant by introducing a gene encoding a protein that is involved in T cell activation according to claim 1, 2 or 8 and a gene encoding a signal which can detect an involvement in T cell activation into a host cell;
(b) culturing the transformant 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 an involvement in T cell activation; 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 which inhibit or promote T cell activation.
16. A method for screening compounds which inhibit or promote T cell activation, which comprises the steps of:
(a) preparing a transformant by introducing a gene encoding a protein that is involved in T cell activation according to claim 1, 2 or 8 into a host cell;
(b) culturing the transformant under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring an activity of NF K B or NFAT in T ceUs; and
(d) selecting a candidate compound which can change the activity of NF K B or NFAT as compared with the case of the absence of candidate compounds, as a compound which inhibit or promote an activation of NF κ B or NFAT in T cells.
17. A compound which inhibits or promotes T cell 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 transformant by introducing a gene encoding a protein that is involved in T cell activation according to claim 1, 2 or 8 and a gene encoding a signal which can detect an involvement in T cell activation into a host cell;
(b) culturing the transformant 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 an involvement in T cell activation;
(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 which inhibit or promote T cell 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 transformant by introducing a gene encoding a protein that is involved in T cell activation according to claim 1, 2 or 8 into a host cell;
(b) culturing the transformant under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring an activity of NF K B or NFAT in T cells;
(d) selecting a candidate compound which can change the activity of NF B or NFAT in T cells as compared with the case of the absence of candidate compounds, as a compound which inhibit or promote an activation of NF K B or NFAT in T cells; and
(e) producing a pharmaceutical composition which comprises a compound selected in the step of (d).
20. A kit for screening a compound which inhibits or promotes an activation of NF κ B or NFAT in T cells, which comprises:
(a) a transformant comprising a gene encoding a protein which promotes an activation of NF ( B or NFAT in T cells according to claim 1, 2 or 8 and a gene encoding a signal which can detect promotion of activity of NF K B or NFAT; 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 or NFAT in T cells 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 activation of NF K B or NFAT in T cells.
25. A ribozyme or deoxyribozyme capable of inhibiting activation of NF K B OΓ NFAT in T cells, 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 which is involved in a pathway leading to activation of NF K B or NFAT in T ceUs.
26. A double strand RNA having a sequence corresponding to a part of the nucleotide sequence according to any one of claims 3 to 7, which inhibits expression of a protein that promotes activation of NF K B or NFAT in T cells.
27. A method for treating a disease associated with T cell activation, which comprises administering 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 oligonucleotide according to claim 24, and/or a ribozyme or deoxyribozyme according to claim 25, and/or a double strand RNA according to claim 26 in an effective amount to treat and/or prevent a disease selected from the group consisting of autoimmune diseases, allergic diseases, infectious diseases and ADDS or to treat acute or chronic rejection at organ transplant or bone-marrow transplant.
28. A pharmaceutical composition produced by the process according to claim 18 or 19 for inhibiting or promoting T cell activation.
29. The pharmaceutical composition according to claim 28 for the treatment and/or prevention of autoimmune diseases, allergic diseases, infectious diseases and ADDS or for the treatment of acute or chronic rejection at organ transplant or bone-marrow transplant.
30. A method of treating autoimmune diseases, allergic diseases, infectious diseases or AIDS, or acute or chronic rejection at organ transplant or bone-marrow transplant, 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 T ceU activation.
31. A pharmaceutical composition which comprises a monoclonal or polyclonal antibody or a fragment thereof according to claim 21 or 22 as an active ingredient.
32. A pharmaceutical composition which comprises an antisense oligonucleotide according to claim 24 as an active ingredient.
33. A pharmaceutical composition which comprises a ribozyme or deoxyribozyme according to claim 25 as an active ingredient.
34. A pharmaceutical composition or a gene therapy agent, which comprises a double strand RNA according to claim 26 or a vector capable of expressing said double strand RNA, an active ingredient.
35. The pharmaceutical composition according to any one of claims 31 to 33 for the treatment and/or prevention of a disease which is selected from the group consisting of autoimmune diseases, allergic diseases, infectious diseases or AIDS, or for the treatment of acute or chronic rejection at organ transplant or bone-marrow transplant.
36. A computer-readable medium on which a sequence data set has been stored, said sequence data set comprising at least one of nucleotide sequence or that of coding region which is selected from the group consisting of SEQ JD 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, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771 and 773, and/or at least one amino acid sequence 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 and 774.
37. A method for calculating identity to other nucleotide sequences and/or amino acid sequences, which comprises comparing data on a medium according to claim 36 with data of said other nucleotide sequences and/or amino acid sequences.
38. An insoluble substrate to which polvnucleotides comprising all or part of the nucleotide sequences selected from the group consisting of SEQ JD 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, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 715, 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771 and 773 are fixed.
39. 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, 190, 192, 194, 196, 198, 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, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734, 736, 738, 740, 742, 744, 746, 748, 750, 752, 754, 756, 758, 760, 762, 764, 766, 768, 770, 772 and 774, are fixed.
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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1543018A1 (en) * 2002-08-14 2005-06-22 LG Life Sciences Ltd. Gene families associated with liver cancer
WO2005095447A2 (en) * 2004-03-29 2005-10-13 Isis Innovation Limited Low molecular weight forms of foxp1 proteins and splice variants thereof
WO2006040357A3 (en) * 2004-10-15 2006-11-02 Galapagos Nv Molecular targets and compounds, and methods to identify the same, useful in the treatment of joint degenerative and inflammatory diseases
EP1733743A4 (en) * 2004-04-09 2007-06-27 Takeda Pharmaceutical Preventives/remedies for cancer
JP2011523552A (en) * 2008-05-16 2011-08-18 ザ チルドレンズ ホスピタル オブ フィラデルフィア Chromosome 21q, 6q, and 15q gene mutations and methods for using them to diagnose and treat type 1 diabetes
US20120027801A1 (en) * 2006-06-29 2012-02-02 J. Craig Venter Institute, Inc. Polypeptides from neisseria meningitidis
US20120093807A1 (en) * 2005-03-29 2012-04-19 Sony Corporation Ror alpha promoting the induction of bmall
US8518638B2 (en) * 2003-08-19 2013-08-27 The Regents Of The University Of Michigan Compositions and methods for detecting and treating neurological conditions
WO2013158290A1 (en) * 2012-04-20 2013-10-24 The Wistar Institute Of Anatomy And Biology Methods and compositions for manipulating the immune system
US8673268B2 (en) 2004-10-15 2014-03-18 Galapagos N.V. Molecular targets and compounds, and methods to identify the same, useful in the treatment of joint degenerative and inflammatory diseases
WO2015200469A1 (en) * 2014-06-24 2015-12-30 University Of Massachusetts Manf as a regulator of immune system function
US9226936B2 (en) 2011-10-28 2016-01-05 The Wistar of Anatomy and Biology Methods and compositions for enhancing the therapeutic effect of anti-tumor T cells
WO2016134333A1 (en) * 2015-02-19 2016-08-25 Compugen Ltd. Anti-pvrig antibodies and methods of use
WO2016134335A3 (en) * 2015-02-19 2016-11-03 Compugen Ltd. Pvrig polypeptides and methods of treatment
US20170327536A1 (en) * 2011-12-29 2017-11-16 Tao Health Life Pharma Co., Ltd. Neurotoxic target for amylospheroid, method and material for reducing the neurotoxicity of amylospheroid, and use thereof
US10124061B2 (en) 2016-08-17 2018-11-13 Compugen Ltd. Anti-TIGIT antibodies, anti-PVRIG antibodies and combinations thereof
WO2018050852A3 (en) * 2016-09-16 2019-01-10 Genagon Therapeutics Ab Binding agents for use in therapy
EP3715848A1 (en) * 2019-03-25 2020-09-30 Université de Bretagne Occidentale (U.B.O.) Substance interacting with c terminal fragment of the stim1 fraction localized to the plasma membrane of the cells, for its use in the treatment of cancers, and in screening and diagnostic methods
US11214619B2 (en) 2018-07-20 2022-01-04 Surface Oncology, Inc. Anti-CD112R compositions and methods
US11225523B2 (en) 2017-06-01 2022-01-18 Compugen Ltd. Triple combination antibody therapies
US11406665B2 (en) 2017-01-12 2022-08-09 Genagon Therapeutics Ab Therapeutic peptides that target TGF-beta interaction

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002053737A1 (en) * 2000-12-28 2002-07-11 Asahi Kasei Kabushiki Kaisha Nf-kb activating gene
WO2003048202A2 (en) * 2001-12-03 2003-06-12 Asahi Kasei Pharma Corporation Nf-kappab activating genes

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1293569A3 (en) * 2001-09-14 2004-03-31 Research Association for Biotechnology Full-length cDNAs

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002053737A1 (en) * 2000-12-28 2002-07-11 Asahi Kasei Kabushiki Kaisha Nf-kb activating gene
WO2003048202A2 (en) * 2001-12-03 2003-06-12 Asahi Kasei Pharma Corporation Nf-kappab activating genes

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ALBRECHT BJ\RN ET AL: "Activation of nuclear factor of activated T cells by human T-lymphotropic virus type 1 accessory protein p12(I)." JOURNAL OF VIROLOGY. UNITED STATES APR 2002, vol. 76, no. 7, April 2002 (2002-04), pages 3493-3501, XP002277225 ISSN: 0022-538X *
DATABASE EMBL [Online] 13 December 2002 (2002-12-13), STRAUSBERG R.L., FEINGLOD E.A., GROUSE L.H. ET AL.: "Homo sapiens zinc finger CCCH type, antiviral 1, transcript variant 2, mRNA (cDNA clone MGC48898 IMAGE:5531412), complete cds." XP002277229 retrieved from EBI Database accession no. BC040956 *
DATABASE EMBL [Online] Sequence 2245 from Patent EP1293569 14 April 2003 (2003-04-14), ISOGAI T., SUGIYAMA T., OTSUKI T. ET AL.: "Full-length cDNAs" XP002277228 retrieved from EBI Database accession no. AX715561 -& EP 1 293 569 A (HELIX RESEARCH INSTITUTE) 19 March 2003 (2003-03-19) *
DATABASE EMBL [Online] Sequence 606 from Patent EP1293569 15 April 2003 (2003-04-15), ISOGAI T., SUGIYAMA T., OTSUKI T. ET AL.: "Full-length cDNAs" XP002277227 retrieved from EBI Database accession no. AX713922 -& EP 1 293 569 A (HELIX RESEARCH INSTITUTE) 19 March 2003 (2003-03-19) *
GRINGHUIS S I ET AL: "INTERLEUKIN-7 UPREGULATES THE INTERLEUKIN-2-GENE EXPRESSION IN ACTIVATED HUMAN T LYMPHOCYTES AT THE TRANSCRIPTIONAL LEVEL BY ENHANCING THE DNA BINDING ACTIVITIES OF BOTH NUCLEAR FACTOR OF ACTIVATED T CELLS AND ACTIVATOR PROTEIN-1" BLOOD, W.B. SAUNDERS, PHILADELPHIA, VA, US, vol. 90, no. 7, 1 October 1997 (1997-10-01), pages 2690-2700, XP001155562 ISSN: 0006-4971 *
ZHANG Z ET AL: "Dual regulation of T cell receptor-mediated signaling by oncogenic Cbl mutant 70Z." THE JOURNAL OF BIOLOGICAL CHEMISTRY. UNITED STATES 19 FEB 1999, vol. 274, no. 8, 19 February 1999 (1999-02-19), pages 4883-4889, XP002277226 ISSN: 0021-9258 *

Cited By (51)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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JP2008516593A (en) * 2004-10-15 2008-05-22 ガラパゴス・ナムローゼ・フェンノートシャップ Molecular targets and compounds useful for the treatment of joint degenerative and inflammatory diseases and methods for their identification
US8673268B2 (en) 2004-10-15 2014-03-18 Galapagos N.V. Molecular targets and compounds, and methods to identify the same, useful in the treatment of joint degenerative and inflammatory diseases
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US8491918B2 (en) * 2006-06-29 2013-07-23 J. Craig Venter Institute, Inc. Polypeptides from Neisseria meningitidis
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WO2015200469A1 (en) * 2014-06-24 2015-12-30 University Of Massachusetts Manf as a regulator of immune system function
US10550173B2 (en) 2015-02-19 2020-02-04 Compugen, Ltd. PVRIG polypeptides and methods of treatment
US10351625B2 (en) 2015-02-19 2019-07-16 Compugen Ltd. Anti-PVRIG antibodies and methods of use
US9714289B2 (en) 2015-02-19 2017-07-25 Compugen Ltd. Anti-PVRIG antibodies and methods of use
EP3295951A1 (en) * 2015-02-19 2018-03-21 Compugen Ltd. Anti-pvrig antibodies and methods of use
US11795209B2 (en) 2015-02-19 2023-10-24 Compugen Ltd. PVRIG polypeptides and methods of treatment
US11795220B2 (en) 2015-02-19 2023-10-24 Compugen Ltd. Anti-PVRIG antibodies and methods of use
US11623955B2 (en) 2015-02-19 2023-04-11 Compugen Ltd. Anti-PVRIG antibodies and methods of use
US10227408B2 (en) 2015-02-19 2019-03-12 Compugen Ltd. Anti-PVRIG antibodies and methods of use
WO2016134335A3 (en) * 2015-02-19 2016-11-03 Compugen Ltd. Pvrig polypeptides and methods of treatment
US11220542B2 (en) 2015-02-19 2022-01-11 Compugen Ltd. Anti-PVRIG antibodies and methods of use
EP3258951B1 (en) 2015-02-19 2020-01-29 Compugen Ltd. Anti-pvrig antibodies and methods of use
WO2016134333A1 (en) * 2015-02-19 2016-08-25 Compugen Ltd. Anti-pvrig antibodies and methods of use
EP3653221A1 (en) * 2015-02-19 2020-05-20 Compugen Ltd. Anti-pvrig antibodies and methods of use
CN107580500A (en) * 2015-02-19 2018-01-12 康姆普根有限公司 Anti- PVRIG antibody and application method
EP4129320A1 (en) * 2015-02-19 2023-02-08 Compugen Ltd. Anti-pvrig antibodies and methods of use
EP3653221B1 (en) 2015-02-19 2022-08-03 Compugen Ltd. Anti-pvrig antibodies and methods of use
EP3978929A1 (en) * 2015-02-19 2022-04-06 Compugen Ltd. Pvrig polypeptides and methods of treatment
US10751415B2 (en) 2016-08-17 2020-08-25 Compugen Ltd. Anti-TIGIT antibodies, anti-PVRIG antibodies and combinations thereof
US10213505B2 (en) 2016-08-17 2019-02-26 Compugen Ltd. Anti-TIGIT anibodies, anti-PVRIG antibodies and combinations thereof
US11701424B2 (en) 2016-08-17 2023-07-18 Compugen Ltd. Anti-TIGIT antibodies, anti-PVRIG antibodies and combinations thereof
US10124061B2 (en) 2016-08-17 2018-11-13 Compugen Ltd. Anti-TIGIT antibodies, anti-PVRIG antibodies and combinations thereof
WO2018050852A3 (en) * 2016-09-16 2019-01-10 Genagon Therapeutics Ab Binding agents for use in therapy
US11406665B2 (en) 2017-01-12 2022-08-09 Genagon Therapeutics Ab Therapeutic peptides that target TGF-beta interaction
US11225523B2 (en) 2017-06-01 2022-01-18 Compugen Ltd. Triple combination antibody therapies
US11279758B2 (en) 2018-07-20 2022-03-22 Surface Oncology, Inc. Anti-CD112R compositions and methods
US11214619B2 (en) 2018-07-20 2022-01-04 Surface Oncology, Inc. Anti-CD112R compositions and methods
WO2020193450A1 (en) * 2019-03-25 2020-10-01 Université De Bretagne Occidentale - Ubo Substance interacting with c terminal fragment of the stim1 fraction localized to the plasma membrane of the cells, for its use in the treatment of cancers, and in screening and diagnostic methods
EP3715848A1 (en) * 2019-03-25 2020-09-30 Université de Bretagne Occidentale (U.B.O.) Substance interacting with c terminal fragment of the stim1 fraction localized to the plasma membrane of the cells, for its use in the treatment of cancers, and in screening and diagnostic methods

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