WO2001002563A2 - Proteines humaines ayant des domaines hydrophobes et adn codant pour ces proteines - Google Patents

Proteines humaines ayant des domaines hydrophobes et adn codant pour ces proteines Download PDF

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WO2001002563A2
WO2001002563A2 PCT/JP2000/003943 JP0003943W WO0102563A2 WO 2001002563 A2 WO2001002563 A2 WO 2001002563A2 JP 0003943 W JP0003943 W JP 0003943W WO 0102563 A2 WO0102563 A2 WO 0102563A2
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protein
proteins
present
cells
cell
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PCT/JP2000/003943
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WO2001002563A3 (fr
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Seishi Kato
Tomoko Kimura
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Sagami Chemical Research Center
Protegene Inc.
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Priority to EP00937278A priority Critical patent/EP1196561A2/fr
Priority to JP2001508335A priority patent/JP2003504021A/ja
Priority to AU52498/00A priority patent/AU5249800A/en
Publication of WO2001002563A2 publication Critical patent/WO2001002563A2/fr
Publication of WO2001002563A3 publication Critical patent/WO2001002563A3/fr

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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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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 human proteins having hydrophobic domains, DNAs encoding these proteins, expression vectors for these DNAs, eukaryotic cells expressing these DNAs and antibodies directed to these proteins.
  • the proteins of the present invention can be employed as pharmaceuticals or as antigens for preparing antibodies directed to these proteins.
  • the human cDNAs of the present invention can be utilized as probes for genetic diagnosis and gene sources for gene therapy.
  • the cDNAs can be utilized as gene sources for producing the proteins encoded by these cDNAs in large quantities.
  • Cells into which these genes are introduced to express secretory proteins or membrane proteins in large quantities can be utilized for detection of the corresponding receptors or ligands, screening of novel small molecule pharmaceuticals and the like.
  • the antibodies of the present invention can be utilized for the detection, quantification, purification and the like of the proteins of the present invention.
  • membrane proteins play important roles, as signal receptors, ion channels, transporters and the like in the material transport and the signal transduction through the cell membrane.
  • Examples thereof include receptors for various cytokines, ion channels for the sodium ion, the potassium ion, the chloride ion and the like, transporters for saccharides and amino acids and the like.
  • the genes for many of them have already been cloned. It has been clarified that abnormalities in these membrane proteins are involved in a number of previously cryptogenic diseases. Therefore, discovery of a new membrane protein is expected to lead to elucidation of the causes of many diseases, so that isolation of new genes encoding the membrane proteins has been desired.
  • a general method is the so-called expression cloning method, in which a cDNA library is introduced into eukaryotic cells to express cDNAs, and the cells secreting, or expressing on the surface of membrane, the protein having the activity of interest are then screened.
  • a cDNA library is introduced into eukaryotic cells to express cDNAs, and the cells secreting, or expressing on the surface of membrane, the protein having the activity of interest are then screened.
  • genes for proteins with known functions can be cloned by using this method.
  • a secretory protein or a membrane protein possesses at least one hydrophobic domain within the protein. After synthesis on ribosomes, such domain works as a secretory signal or remains in the phospholipid membrane to be entrapped in the membrane. Accordingly, if the existence of a highly hydrophobic domain is observed in the amino acid sequence of a protein encoded by a cDNA when the whole base sequence of the full-length cDNA is determined, it is considered that the cDNA encodes a secretory protein or a membrane protein.
  • the main object of the present invention is to provide novel human proteins having hydrophobic domains,
  • Fig. 1 illustrates the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP03372.
  • Fig. 2 illustrates the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP03375.
  • Fig. 3 illustrates the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP03376.
  • Fig. 4 illustrates the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP03377.
  • Fig. 5 illustrates the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP03378.
  • Fig. 6 illustrates the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP03379.
  • Fig. 7 illustrates the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP03380.
  • Fig. 8 illustrates the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP03396.
  • Fig. 9 illustrates the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP10678.
  • Fig. 10 illustrates the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP10688.
  • the present invention provides a human protein having hydrophobic domain (s), namely a protein comprising any one of an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 10.
  • the present invention provides a DNA encoding said protein, exemplified by a cDNA comprising any one of a base sequence selected from the group consisting of SEQ ID NOS: 11 to 30, an expression vector that is capable of expressing said DNA by in vitro translation or in eukaryotic cells, a transformed eukaryotic cell that is capable of expressing said DNA and of producing said protein and an antibody directed to said protein.
  • the proteins of the present invention can be obtained, for example, by a method for isolating proteins from human organs, cell lines or the like, a method for preparing peptides by the chemical synthesis based on the amino acid sequence of the present invention, or a method for producing proteins by the recombinant DNA technology using the DNAs encoding the hydrophobic domains of the present invention.
  • the method for producing proteins by the recombinant DNA technology is preferably employed.
  • the proteins can be expressed in vitro by preparing an RNA by in vitro transcription from a vector having the cDNA of the present invention, and then carrying out in vitro translation using this RNA as a template.
  • incorporation of the translated region into a suitable expression vector by the method known in the art may lead to expression of a large amount of the encoded protein in prokaryotic cells such as Escherichia coli , Bacillus subtilis, etc., and eukaryotic cells such as yeasts, insect cells, mammalian cells, etc.
  • prokaryotic cells such as Escherichia coli , Bacillus subtilis, etc.
  • eukaryotic cells such as yeasts, insect cells, mammalian cells, etc.
  • the protein of the present invention can be produced in vitro by incorporating the translated region of this cDNA into a vector having an RNA polymerase promoter, and then adding the vector to an in vitro translation system such as a rabbit reticulocyte lysate or a wheat germ extract, which contains an RNA polymerase corresponding to the promoter.
  • RNA polymerase promoters are exemplified by T7, T3, SP ⁇ and the like.
  • the vectors containing promoters for these RNA polymerases are exemplified by pKAl, pCDM8, pT3/T7 18, pT7/3 19, pBluescript II and the like.
  • the protein of the present invention can be expressed in the secreted form or the form incorporated in the microsome membrane when a canine pancreas microsome or the like is added to the reaction system.
  • the protein of the present invention is produced by expressing the DNA in a microorganism such as Escherichia coli etc.
  • a recombinant expression vector in which the translated region of the cDNA of the present invention is incorporated into an expression vector having an origin which is capable of replicating in the microorganism, a promoter, a ribosome-binding site, a cDNA-cloning site, a terminator and the like is constructed. After transformation of the host cells with this expression vector, the resulting transformant is grown, whereby the protein encoded by the cDNA can be produced in large quantities in the microorganism.
  • a protein fragment containing any translated region can be obtained by adding an initiation codon and a termination codon in front of and behind the selected translated region to express the protein.
  • the protein can be expressed as a fusion protein with another protein. Only the portion of the protein encoded by the cDNA can be obtained by cleaving this fusion protein with a suitable protease.
  • the expression vectors for Escherichia coli are exemplified by the pUC series, pBluescript II, the pET expression system, the pGEX expression system and the like.
  • the protein of the present invention is produced by expressing the DNA in eukaryotic cells
  • the protein of the present invention can be produced as a secretory protein, or as a membrane protein on the surface of cell membrane, by incorporating the translated region of the cDNA into an expression vector for eukaryotic cells that has a promoter, a splicing region, a poly (A) addition site and the like, and then introducing the vector into the eukaryotic cells.
  • the expression vectors are exemplified by pKAl, pED6dpc2, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vectors, pRS, pYES2 and the like.
  • eukaryotic cells to be used in general include mammalian cultured cells such as monkey kidney COS7 cells, Chinese hamster ovary CHO cells and the like, budding yeasts, fission yeasts, silkworm cells, Xenopus oocytes and the like. Any eukaryotic cells may be used as long as they are capable of expressing the proteins of the present invention.
  • the expression vector can be introduced into the eukaryotic cells by using a method known in the art such as the electroporation method, the calcium phosphate method, the liposome method, the DEAE-dextran method and the like.
  • the protein of interest can be isolated and purified from the culture by a combination of separation procedures known in the art.
  • separation procedures include treatment with a denaturing agent such as urea or a detergent, sonication, enzymatic digestion, salting-out or solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, ion-exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography and the like.
  • the proteins of the present invention also include peptide fragments (of 5 amino acid residues or more) containing any partial amino acid sequences in the amino acid sequences represented by SEQ ID NOS: 1 to 10. These peptide fragments can be utilized as antigens for preparation of antibodies.
  • proteins of the present invention those having the signal sequences are secreted in the form of mature proteins after the signal sequences are removed. Therefore, these mature proteins shall come within the scope of the protein of the present invention.
  • the N- terminal amino acid sequences of the mature proteins can be easily determined by using the method for the determination of cleavage site of a signal sequence [JP-A 8-187100] .
  • some membrane proteins undergo the processing on the cell surface to be converted to the secreted forms.
  • proteins or peptides in the secreted forms shall also come within the scope of the protein of the present invention.
  • expression of the proteins in appropriate eukaryotic cells affords the proteins to which sugar chains are added. Accordingly, such proteins or peptides to which sugar chains are added shall also come within the scope of the protein of the present invention.
  • the DNAs of the present invention include all the
  • DNAs encoding the above-mentioned proteins can be obtained by using a method for chemical synthesis, a method for cDNA cloning and the like.
  • the cDNAs of the present invention can be cloned, for example, from cDNA libraries derived from the human cells.
  • the cDNAs are synthesized by using poly (A) + RNAs extracted from human cells as templates.
  • the human cells may be cells delivered from the human body, for example, by the operation or may be the cultured cells.
  • the cDNAs can be synthesized by using any method such as the Okayama-Berg method [Okayama, H. and Berg, P., Mol. Cell. Biol. 2: 161- 170 (1982)], the Gubler-Hoffman method [Gubler, U. and Hoffman, J. , Gene 25: 263-269 (1983)] and the like.
  • cDNAs of the present invention can be cloned from the cDNA libraries by synthesizing an oligonucleotide on the basis of base sequences of any portion in the cDNA of the present invention and screening the cDNA libraries using this oligonucleotide as a probe for colony or plaque hybridization according to a method known in the art.
  • the cDNA fragments of the present invention can be prepared from an mRNA isolated from human cells by the RT-PCR method in which oligonucleotides which hybridize with both termini of the cDNA fragment of interest are synthesized, which are then used as the primers.
  • the cDNAs of the present invention are characterized in that they comprise any one of the base sequences represented by SEQ ID NOS: 11 to 20 or the base sequences represented by SEQ ID NOS: 21 to 30.
  • Table 1 summarizes the clone number (HP number) , the cells from which the cDNA clone was obtained, the total number of bases of the cDNA, and the number of the amino acid residues of the encoded protein, for each of the cDNAs.
  • the same clones as the cDNAs of the present invention can be easily obtained by screening the cDNA libraries constructed from the human cell lines or human tissues utilized in the present invention using an oligonucleotide probe synthesized on the basis of the base sequence of the cDNA provided in any one of SEQ ID NOS: 11 to 30.
  • any cDNA in which one or plural nucleotides are added, deleted and/or substituted with other nucleotides in SEQ ID NOS: 11 to 30 shall come within the scope of the present invention.
  • any protein in which one or plural amino acids are added, deleted and/or substituted with other amino acids resulting from the above-mentioned changes shall come within the scope of the present invention, as long as the protein possesses the activity of the protein having any one of the amino acid sequences represented by SEQ ID NOS: 1 to 10.
  • the cDNAs of the present invention also include cDNA fragments (of 10 bp or more) containing any partial base sequence in the base sequences represented by SEQ ID NOS: 11 to 20 or in the base sequences represented by SEQ ID NOS: 21 to 30. Also, DNA fragments consisting of a sense strand and an anti-sense strand shall come within this scope. These DNA fragments can be utilized as the probes for the genetic diagnosis.
  • the antibody of the present invention can be obtained from a serum after immunizing an animal using the protein of the present invention as an antigen.
  • a peptide that is chemically synthesized based on the amino acid sequence of the present invention and a protein expressed in eukaryotic or prokaryotic cells can be used as an antigen.
  • an antibody can be prepared by introducing the above-mentioned expression vector for eukaryotic cells into the muscle or the skin of an animal by injection or by using a gene gun and then collecting a serum therefrom (JP-A 7-313187) .
  • Animals that can be used include a mouse, a rat, a rabbit, a goat, a chicken and the like.
  • a monoclonal antibody directed to the protein of the present invention can be produced by fusing B cells collected from the spleen of the immunized animal with myelomas to generate hybridomas.
  • polynucleotides and proteins of the present invention may exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified below.
  • Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA) .
  • the polynucleotides provided by the present invention can be used by the research community for various purposes.
  • the polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states) ; as molecular weight markers on Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to "subtract-out" known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a "gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization
  • the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
  • the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.
  • the proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high- throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state) ; and, of course, to isolate correlative receptors or ligands.
  • the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
  • the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
  • Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate.
  • the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules.
  • the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.
  • a protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.
  • cytokine cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.
  • Many protein factors discovered to date, including all known cytokines have exhibited activity in one or more factor dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity.
  • the activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+) , 2E8, RB5, DAI, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al . , J. Immunol.
  • Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human Interferon ⁇ , Schreiber, R.D. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
  • Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205- 1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A.
  • Assays for T-cell clone responses to antigens include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci.
  • a protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.
  • a protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID) ) , e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.
  • SCID severe combined immunodeficiency
  • These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders.
  • infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis.
  • a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.
  • Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.
  • a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems.
  • Other conditions, in which immune suppression is desired may also be treatable using a protein of the present invention.
  • T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.
  • Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent.
  • Tolerance which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen- specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.
  • Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as , for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD) .
  • B lymphocyte antigen functions such as , for example, B7
  • GVHD graft-versus-host disease
  • blockage of T cell function should result in reduced tissue destruction in tissue transplantation.
  • rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.
  • a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand (s) on immune cells can lead to the binding of the molecule to the natural ligand (s) on the immune cells without transmitting the corresponding costimulatory signal.
  • a B7 lymphocyte antigen e.g., B7-1, B7-3 or blocking antibody
  • Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant .
  • the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject.
  • Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents.
  • the efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans.
  • appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992).
  • murine models of GVHD see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease.
  • Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.
  • Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.
  • Administration of reagents which block costimulation of T cells by disrupting receptor : ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.
  • the efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856) .
  • Upregulation of an antigen function (preferably a B lymphocyte antigen function) , as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically.
  • anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.
  • Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient.
  • the infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.
  • up regulation or enhancement of antigen function may be useful in the induction of tumor immunity.
  • Tumor cells e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma
  • a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject. If desired, the tumor cell can be transfected to express a combination of peptides.
  • tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7- 1-like activity and/or B7-3-like activity.
  • the transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell.
  • gene therapy techniques can be used to target a tumor cell for transfection in vivo.
  • the presence of the peptide of the present invention having the activity of a B lymphocyte antigen (s) on the surface of the tumor cell provides the necessary costimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells.
  • tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I chain protein and ⁇ 2 microglobulin protein or an MHC class II ⁇ chain protein and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I chain protein and ⁇ 2 microglobulin protein or an MHC class II ⁇ chain protein and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity.
  • a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol.
  • T-cell-dependent immunoglobulin responses and isotype switching include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J.J. and Brunswick, M.
  • MLR Mixed lymphocyte reaction
  • Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al .
  • lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al .
  • Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111- 117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991. Hematopoiesis Regulating Activity
  • a protein of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g.
  • erythroid progenitor cells in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complementary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above- mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for embryonic stem cell differentiation include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.
  • Assays for stem cell survival and differentiation include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I.K. and Briddell, R.A.
  • a protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of burns, incisions and ulcers.
  • a protein of the present invention which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.
  • Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.
  • a protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone- forming cells.
  • a protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.
  • tissue regeneration activity that may be attributable to the protein of the present invention is tendon/ligament formation.
  • a protein of the present invention which induces tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals.
  • Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.
  • compositions of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments.
  • the compositions of the present invention may provide an environment to attract tendon or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.
  • the compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects.
  • the compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.
  • the protein of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention.
  • Proteins of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds and the like.
  • a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium) , muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues.
  • organs including, for example, pancreas, liver, intestine, kidney, skin, endothelium
  • muscle smooth, skeletal or cardiac
  • vascular including vascular endothelium
  • a protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage .
  • a protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. WO95/16035 (bone, cartilage, tendon); International Patent Publication No. WO95/05846 (nerve, neuronal); International Patent Publication No. WO91/07491 (skin, endothelium ) .
  • Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps . 71-112 (Maibach, HI and Rovee, DT, eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84 (1978).
  • a protein of the present invention may also exhibit activin- or inhibin-related activities. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH) , while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH) .
  • FSH follicle stimulating hormone
  • a protein of the present invention alone or in heterodimers with a member of the inhibin a family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals.
  • the protein of the invention may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, United States Patent 4,798,885.
  • a protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
  • the activity of a protein of the invention may, among other means, be measured by the following methods: Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al . , Nature 321:779- 782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986.
  • a protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells.
  • Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action.
  • Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.
  • a protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.
  • the protein or peptide has the ability to directly stimulate directed movement of cells.
  • Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for chemotactic activity consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population.
  • Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest.
  • a protein of the invention may also exhibit hemostatic or thrombolytic activity. As a result, such a protein is expected to be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes.
  • a protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke)).
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467- 474, 1988.
  • a protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/ligand interactions.
  • receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses) .
  • Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.
  • a protein of the present invention may themselves be useful as inhibitors of receptor/ligand interactions.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Suitable assays for receptor-ligand activity include without limitation those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al . , Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med. 168:1145- 1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995. Anti-Inflammatory Activity
  • Proteins of the present invention may also exhibit anti-inflammatory activity.
  • the anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell- cell interactions (such as, for example, cell adhesion) , by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response.
  • Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions) , including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS) ) , ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
  • a protein of the invention may exhibit other anti-tumor activities.
  • a protein may inhibit tumor growth directly or indirectly
  • a protein may exhibit its tumor inhibitory activity by acting on tumor tissue or tumor precursor tissue, by inhibiting formation of tissues necessary to support tumor growth (such as, for example, by inhibiting angiogenesis) , by causing production of other factors, agents or cell types which inhibit tumor growth, or by suppressing, eliminating or inhibiting factors, agents or cell types which promote tumor growth.
  • a protein of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape) ; effecting biorhythms or cardiac cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component (s) ; effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders) , depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects;
  • the cDNA library of fibrosarcoma cell line HT-1080 (WO 98/11217) was used as a cDNA library. Additionally, the cDNA libraries constructed from human thymus mRNA (Clontech) , human kidney mRNA (Clontech) and human umbilical cord blood mRNA (Clontech) were also used.
  • Full-length cDNA clones were selected from the respective libraries and the whole base sequences thereof were determined to construct a homo-protein cDNA bank consisting of the full-length cDNA clones.
  • the hydrophobicity/hydrophilicity profiles were determined for the proteins encoded by the full-length cDNA clones registered in the homo-protein cDNA bank by the Kyte- Doolittle method [Kyte, J. & Doolittle, R. F. , J. Mol. Biol. 157: 105-132 (1982)] to examine the presence or absence of a hydrophobic domain.
  • a clone that has a hydrophobic region being assumed as a secretory signal or a transmembrane domain in the amino acid sequence of the encoded protein was selected as a clone candidate.
  • the plasmid vector bearing the cDNA of the present invention was used for in vitro transcription/translation with a T N T rabbit reticulocyte lysate kit (Promega) .
  • T N T rabbit reticulocyte lysate kit Promega
  • [ 35 S]methionine was added to label the expression product with a radioisotope.
  • Each of the reactions was carried out according to the protocols attached to the kit.
  • Two micrograms of the plasmid was subjected to the reaction at 30°C for 90 minutes in the reaction solution of a total volume of 25 ⁇ l containing 12.5 ⁇ l ⁇ of T N T rabbit reticulocyte lysate, 0.5 ⁇ l of a buffer solution (attached to the kit) , 2 ⁇ l of an amino acid mixture (without methionine) , 2 ⁇ l of [ 35 S]methionine (Amersham) (0.37 MBq/ ⁇ l) , 0.5 ⁇ l of T7 RNA polymerase, and 20 U of RNasin.
  • Escherichia coli cells harboring the expression vector for the protein of the present invention were cultured at 37°C for 2 hours in 2 ml of the 2 x YT culture medium containing 100 ⁇ g/ml of ampicillin, the helper phage M13K07 (50 ⁇ 1) was added thereto, and the cells were then cultured at 37 °C overnight.
  • Single-stranded phage particles were obtained by polyethylene glycol precipitation from a supernatant separated by centrifugation. The particles were suspended in 100 ⁇ l of 1 mM Tris-0.1 mM EDTA, pH 8 (TE) .
  • the cultured cells derived from monkey kidney, COS7 were cultured at 37 °C in the presence of 5% C0 2 in the Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum.
  • DMEM Dulbecco's modified Eagle's medium
  • 1 x IO 5 COS7 cells were inoculated into a 6-well plate (Nunc, well diameter: 3 cm) and cultured at 37 °C for 22 hours in the presence of 5% C0 2 . After the medium was removed, the cell surface was washed with a phosphate buffer solution followed by DMEM containing 50 mM Tris- hydrochloride (pH 7.5) (TDMEM) .
  • the cell surface was washed with TDMEM, 2 ml per well of DMEM containing 10% fetal calf serum was added, and the cells were cultured at 37 °C for 2 days in the presence of 5% C0 2 .
  • the medium was exchanged for a medium containing [ 35 S] cysteine or [ 35 S]methionine, the cells were cultured for one hour. After the medium and the cells were separated each other by centrifugation, proteins in the medium fraction and the cell membrane fraction were subjected to SDS-PAGE.
  • a plasmid vector containing the cDNA of the present invention was dissolved in a phosphate buffer solution (PBS: 145 mM NaCl, 2.68 mM KC1, 8.09 mM Na 2 HP0 4 , 2 mM KH 2 P0 4 , pH 7.2) to a concentration of 2 ⁇ g/ ⁇ l. 25 ⁇ l each (a total of 50 ⁇ l) of the thus-prepared plasmid solution in PBS was injected into the right and left musculi quadriceps femoris of three mice (ICR line) using a 26 guage needle. After similar injections were repeated for one month at intervals of one week, blood was collected.
  • PBS phosphate buffer solution
  • the ORF encodes a protein consisting of
  • Figure 2 depicts the hydrophobicity/hydrophilicity profile, obtained by the Kyte- Doolittle method, of the present protein.
  • In vitro translation resulted in formation of a translation product of 30 kDa that was almost identical with the molecular weight of 29,598 predicted from the ORF.
  • the addition of a microsome led to the formation of a product of 29 kDa.
  • Application of the (-3,-1) rule a method for predicting the cleavage site of the secretory signal sequence, allows to expect that the mature protein starts from alanine at position 23.
  • the search of the GenBank using the base sequences of the present cDNA has revealed the registration of sequences that shared a homology of 90% or more (for example, Accession No. AA448958) among ESTs. However, since they are partial sequences, it can not be judged whether or not they encode the same protein as the protein of the present invention.
  • ⁇ HP03376> (SEQ ID NOS: 3, 13, and 23) Determination of the whole base sequence of the cDNA insert of clone HP03376 obtained from cDNA library of human fibrosarcoma cell line HT-1080 revealed the structure consisting of a 187-bp 5' -untranslated region, a 849-bp ORF, and a 1047-bp 3' -untranslated region.
  • the ORF encodes a protein consisting of 282 amino acid residues and there existed a putative secretory signal at the N-terminus and one putative transmembrane domain at the C-terminus.
  • Figure 3 depicts the hydrophobicity/hydrophilicity profile, obtained by the Kyte-Doolittle method, of the present protein.
  • the search of the protein data base using the amino acid sequence of the present protein revealed that the protein was similar to human glycoprotein gp55 (Accession No. CAA67711) .
  • Table 4 shows the comparison between amino acid sequences of the human protein of the present invention (HP) and human glycoprotein gp55 (GP) .
  • HP human protein of the present invention
  • GP human glycoprotein gp55
  • the marks of -, *, and . represent a gap, an amino acid residue identical with that of the protein of the present invention, and an amino acid residue similar to that of the protein of the present invention, respectively.
  • the both proteins shared a homology of 94.3% in the entire region.
  • the search of the GenBank using the base sequences of the present cDNA has revealed the registration of sequences that shared a homology of 90% or more (for example, Accession No. AA206770) among ESTs. However, since they are partial sequences, it can not be judged whether or not they encode the same protein as the protein of the present invention.
  • ⁇ HP03377> (SEQ ID NOS: 4, 14, and 24) Determination of the whole base sequence of the cDNA insert of clone HP03377 obtained from cDNA library of human fibrosarcoma cell line HT-1080 revealed the structure consisting of a 146-bp 5' -untranslated region, a 717-bp ORF, and a 397-bp 3' -untranslated region.
  • the ORF encodes a protein consisting of 238 amino acid residues and there existed three transmembrane domains.
  • Figure 4 depicts the hydrophobicity/hydrophilicity profile, obtained by the Kyte- Doolittle method, of the present protein.
  • the search of the GenBank using the base sequences of the present cDNA has revealed the registration of sequences that shared a homology of 90% or more (for example, Accession No. W25208) among ESTs. However, since they are partial sequences, it can not be judged whether or not they encode the same protein as the protein of the present invention.
  • ⁇ HP03378> (SEQ ID NOS: 5, 15, and 25) Determination of the whole base sequence of the cDNA insert of clone HP03378 obtained from cDNA library of human umbilical cord blood revealed the structure consisting of a 281-bp 5' -untranslated region, a 1119-bp ORF, and a 320-bp 3' -untranslated region.
  • the ORF encodes a protein consisting of 372 amino acid residues and there existed seven putative transmembrane domains.
  • Figure 5 depicts the hydrophobicity/hydrophilicity profile, obtained by the Kyte- Doolittle method, of the present protein. In vitro translation resulted in formation of a translation product of high molecular weight.
  • the search of the protein data base using the amino acid sequence of the present protein revealed that the protein was similar to human P2Y5-like receptor (Accession
  • the search of the GenBank using the base sequences of the present cDNA has revealed the registration of sequences that shared a homology of 90% or more (for example, Accession No. AA663042) among ESTs. However, since they are partial sequences, it can not be judged whether or not they encode the same protein as the protein of the present invention.
  • Table 7 shows the comparison between amino acid sequences of the human protein of the present invention (HP) and mouse putative sialyltransferase (MM) .
  • HP human protein of the present invention
  • MM mouse putative sialyltransferase
  • the marks of -, *, and . represent a gap, an amino acid residue identical with that of the protein of the present invention, and an amino acid residue similar to that of the protein of the present invention, respectively.
  • the both proteins shared a homology of 89.4% in the entire region.
  • the search of the GenBank using the base sequences of the present cDNA has revealed the registration of sequences that shared a homology of 90% or more (for example, Accession No. H50479) among ESTs. However, since they are partial sequences, it can not be judged whether or not they encode the same protein as the protein of the present invention.
  • ⁇ HP03396> (SEQ ID NOS: 8, 18, and 28) Determination of the whole base sequence of the cDNA insert of clone HP03396 obtained from cDNA library of human kidney revealed the structure consisting of a 245-bp 5' -untranslated region, a 585-bp ORF, and a 133-bp 3'- untranslated region.
  • the ORF encodes a protein consisting of 194 amino acid residues and there existed a putative secretory signal at the N-terminus.
  • Figure 8 depicts the hydrophobicity/hydrophilicity profile, obtained by the Kyte- Doolittle method, of the present protein.
  • the search of the GenBank using the base sequences of the present cDNA has revealed the registration of sequences that shared a homology of 90% or more (for example, Accession No. AA453324) among ESTs. However, since they are partial sequences, it can not be judged whether or not they encode the same protein as the protein of the present invention.
  • ⁇ HP10678> (SEQ ID NOS: 9, 19, and 29) Determination of the whole base sequence of the cDNA insert of clone HP10678 obtained from cDNA library of human fibrosarcoma cell line HT-1080 revealed the structure consisting of a 228-bp 5' -untranslated region, a 1629-bp ORF, and a 810-bp 3' -untranslated region.
  • the ORF encodes a protein consisting of 542 amino acid residues and there existed seven putative transmembrane domains.
  • Figure 9 depicts the hydrophobicity/hydrophilicity profile, obtained by the Kyte-Doolittle method, of the present protein. In vitro translation resulted in formation of a translation product of high molecular weight.
  • Table 9 shows the comparison between amino acid sequences of the human protein of the present invention (HP) and human hypothetical protein KIAA0758 (KI) .
  • HP human protein of the present invention
  • KI human hypothetical protein
  • the marks of -, *, and . represent a gap, an amino acid residue identical with that of the protein of the present invention, and an amino acid residue similar to that of the protein of the present invention, respectively.
  • the both proteins shared a homology of 37.9% in the entire region.
  • the search of the GenBank using the base sequences of the present cDNA has revealed the registration of sequences that shared a homology of 90% or more (for example, Accession No. AA035425) among ESTs. However, since they are partial sequences, it can not be judged whether or not they encode the same protein as the protein of the present invention.
  • ⁇ HP10688> (SEQ ID NOS: 10, 20, and 30) Determination of the whole base sequence of the cDNA insert of clone HP10688 obtained from cDNA library of human thymus revealed the structure consisting of a 173-bp 5' -untranslated region, a 831-bp ORF, and a 474-bp 3'- untranslated region.
  • the ORF encodes a protein consisting of 276 amino acid residues and there existed a putative secretory signal at the N-terminus and one transmembrane domain at the C-terminus.
  • Figure 10 depicts the hydrophobicity/hydrophilicity profile, obtained by the Kyte- Doolittle method, of the present protein.
  • the search of the GenBank using the base sequences of the present cDNA has revealed the registration of sequences that shared a homology of 90% or more (for example, Accession No. AA464826) among ESTs. However, since they are partial sequences, it can not be judged whether or not they encode the same protein as the protein of the present invention.
  • the present invention provides human proteins having hydrophobic domains, DNAs encoding these proteins, expression vectors for these DNAs and eukaryotic cells expressing these DNAs. Since all of the proteins of the present invention are secreted or exist in the cell membrane, they are considered to be proteins controlling the proliferation and/or the differentiation of the cells. Accordingly, the proteins of the present invention can be employed as pharmaceuticals such as carcinostatic agents which act to control the proliferation and/or the differentiation of the cells, or as antigens for preparing antibodies against these proteins.
  • the DNAs of the present invention can be utilized as probes for the genetic diagnosis and gene sources for the gene therapy. Furthermore, the DNAs can be utilized for expressing these proteins in large quantities.
  • Cells into which these genes are introduced to express these proteins can be utilized for detection of the corresponding receptors or ligands, screening of novel small molecule pharmaceuticals and the like.
  • the antibody of the present invention can be utilized for the detection, quantification, purification and the like of the protein of the present invention.
  • the present invention also provides genes corresponding to the polynucleotide sequences disclosed herein.
  • “Corresponding genes” are the regions of the genome that are transcribed to produce the mRNAs from which cDNA polynucleotide sequences are derived and may include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes may therefore include but are not limited to coding sequences, 5' and 3' untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements. The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein.
  • Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials.
  • An "isolated gene” is a gene that has been separated from the adjacent coding sequences, if any, present in the genome of the organism from which the gene was isolated.
  • Organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein are provided.
  • the desired change in gene expression can be achieved through the use of antisense polynucleotides or ribozymes that bind and/or cleave the mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol. Sci. 15(7): 250-254; Lavarosky et al., 1997, Biochem. Mol. Med. 62(1): 11-22; and Hampel, 1998, Prog. Nucleic Acid Res. Mol. Biol. 58: 1-39; all of which are incorporated by reference herein) .
  • Transgenic animals that have multiple copies of the gene(s) corresponding to the polynucleotide sequences disclosed herein, preferably produced by transformation of cells with genetic constructs that are stably maintained within the transformed cells and their progeny, are provided.
  • organisms are provided in which the gene(s) corresponding to the polynucleotide sequences disclosed herein have been partially or completely inactivated, through insertion of extraneous sequences into the corresponding gene(s) or through deletion of all or part of the corresponding gene(s) .
  • Partial or complete gene inactivation can be accomplished through insertion, preferably followed by imprecise excision, of transposable elements (Plasterk, 1992, Bioessays 14(9): 629-633; Zwaal et al., 1993, Proc. Natl. Acad. Sci. USA 90(16): 7431-7435; Clark et al., 1994, Proc. Natl. Acad. Sci. USA 91(2): 719-722; all of which are incorporated by reference herein) , or through homologous recombination, preferably detected by positive/negative genetic selection strategies (Mansour et al., 1988, Nature 336: 348-352; U.S. Patent Nos.
  • proteins and protein fragments of the present invention include proteins with amino acid sequence lengths that are at least 25% (more preferably at least 50%, and most preferably at least 75%) of the length of a disclosed protein and have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with that disclosed protein, where sequence identity is determined by comparing the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • proteins and protein fragments that contain a segment preferably comprising 8 or more (more preferably 20 or more, most preferably 30 or more) contiguous amino acids that shares at least 75% sequence identity (more preferably, at least 85% identity; most preferably at least 95% identity) with any such segment of any of the disclosed proteins.
  • Species homologs of the disclosed polynucleotides and proteins are also provided by the present invention.
  • a "species homologue" is a protein or polynucleotide with a different species of origin from that of a given protein or polynucleotide, but with significant sequence similarity to the given protein or polynucleotide, as determined by those of skill in the art.
  • Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.
  • the invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous, or related to that encoded by the polynucleotides .
  • the invention also includes polynucleotides with sequences complementary to those of the polynucleotides disclosed herein.
  • the present invention also includes polynucleotides capable of hybridizing under reduced stringency conditions, more preferably stringent conditions, and most preferably highly stringent conditions, to polynucleotides described herein. Examples of stringency conditions are shown in the table below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.
  • the hybrid length is that anticipated for the hybridized region (s) of the hybridizing polynucleotides.
  • the hybrid length is assumed to be that of the hybridizing polynucleotide.
  • the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity.
  • SSPE lxSSPE is 0.15M NaCl, lOmM NaH 2 P0 4 , and 1.25mM EDTA, pH7.4
  • IxSSC 0.15M NaCl and 15mM sodium citrate
  • T J melting temperature
  • Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F.M.
  • each such hybridizing polynucleotide has a length that is at least 25% (more preferably at least 50%, and most preferably at least 75%) of the length of the polynucleotide of the present invention to which it hybridizes, and has at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps.

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Abstract

L'invention concerne des protéines humaines ayant des domaines hydrophobes, des ADN codant pour ces protéines, des vecteurs d'expression pour ces ADN, des cellules eucaryotes transformées exprimant ces ADN et des anticorps dirigés contre ces protéines.
PCT/JP2000/003943 1999-07-02 2000-06-16 Proteines humaines ayant des domaines hydrophobes et adn codant pour ces proteines WO2001002563A2 (fr)

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EP00937278A EP1196561A2 (fr) 1999-07-02 2000-06-16 Proteines humaines ayant des domaines hydrophobes et adn codant pour ces proteines
JP2001508335A JP2003504021A (ja) 1999-07-02 2000-06-16 疎水性ドメインを有するヒトプロテイン並びにそれをコードするｄｎａ
AU52498/00A AU5249800A (en) 1999-07-02 2000-06-16 Human proteins having hydrophobic domains and dnas encoding these proteins

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WO2001068702A2 (fr) * 2000-03-16 2001-09-20 Bayer Aktiengesellschaft Regulation du recepteur couple a une proteine g humaine
WO2001087937A2 (fr) * 2000-05-18 2001-11-22 Incyte Genomics, Inc. Recepteurs couples aux proteines g
EP1157038A1 (fr) * 1999-02-26 2001-11-28 Smithkline Beecham Corporation Clonage du recepteur 7tm (axor 17) du type p2y
WO2002022665A1 (fr) * 2000-09-11 2002-03-21 Takeda Chemical Industries, Ltd. Proteines de recepteur couple a la proteine g et adn correspondants
WO2002029053A1 (fr) * 2000-10-05 2002-04-11 Takeda Chemical Industries, Ltd. Nouvelle proteine de recepteur couple a la proteine g et adn correspondant
WO2002079450A2 (fr) * 2001-03-30 2002-10-10 Pe Corporation (Ny) Recepteurs humains isoles couples a la proteine g, molecules d'acide nucleique codant pour les proteines humaines gpcr, et leurs utilisations
WO2002036753A3 (fr) * 2000-10-31 2002-10-10 Bayer Ag Régulation de la lipase triacylglycérol humaine
WO2002083856A2 (fr) * 2001-04-11 2002-10-24 Bristol-Myers Squibb Company Polynucleotides codant deux nouveaux recepteurs couples aux proteines g, hgprbmy28 et hgprbmy29, et leurs variants d'epissage
WO2002103005A1 (fr) * 2001-06-18 2002-12-27 National Institute Of Advanced Industrial Science And Technology Recepteurs couples aux proteines se fixant a la guanosine triphosphate
US7524638B1 (en) 2003-06-27 2009-04-28 Osi Pharmaceuticals, Inc. Methods for identification of modulators of OSGPR114 or OSGPR78 activity, and their use in the treatment of disease
US7947278B2 (en) 2006-07-17 2011-05-24 The Board Of Trustees Of The Leland Stanford Junior University Methods of modulating angiogenesis
US8404811B2 (en) 2009-05-08 2013-03-26 Genentech, Inc. Humanized anti-EGFL7 antibodies and methods using same
US8790646B2 (en) 2004-04-14 2014-07-29 Genentech Inc. Compositions and methods for modulating vascular development

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Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1157038A1 (fr) * 1999-02-26 2001-11-28 Smithkline Beecham Corporation Clonage du recepteur 7tm (axor 17) du type p2y
EP1157038A4 (fr) * 1999-02-26 2005-01-19 Smithkline Beecham Corp Clonage du recepteur 7tm (axor 17) du type p2y
WO2001068702A3 (fr) * 2000-03-16 2002-05-30 Bayer Ag Regulation du recepteur couple a une proteine g humaine
WO2001068702A2 (fr) * 2000-03-16 2001-09-20 Bayer Aktiengesellschaft Regulation du recepteur couple a une proteine g humaine
WO2001087937A2 (fr) * 2000-05-18 2001-11-22 Incyte Genomics, Inc. Recepteurs couples aux proteines g
WO2001087937A3 (fr) * 2000-05-18 2003-01-16 Incyte Genomics Inc Recepteurs couples aux proteines g
WO2002022665A1 (fr) * 2000-09-11 2002-03-21 Takeda Chemical Industries, Ltd. Proteines de recepteur couple a la proteine g et adn correspondants
WO2002029053A1 (fr) * 2000-10-05 2002-04-11 Takeda Chemical Industries, Ltd. Nouvelle proteine de recepteur couple a la proteine g et adn correspondant
WO2002036753A3 (fr) * 2000-10-31 2002-10-10 Bayer Ag Régulation de la lipase triacylglycérol humaine
WO2002079450A3 (fr) * 2001-03-30 2003-07-17 Pe Corp Ny Recepteurs humains isoles couples a la proteine g, molecules d'acide nucleique codant pour les proteines humaines gpcr, et leurs utilisations
WO2002079450A2 (fr) * 2001-03-30 2002-10-10 Pe Corporation (Ny) Recepteurs humains isoles couples a la proteine g, molecules d'acide nucleique codant pour les proteines humaines gpcr, et leurs utilisations
WO2002083856A3 (fr) * 2001-04-11 2004-03-11 Bristol Myers Squibb Co Polynucleotides codant deux nouveaux recepteurs couples aux proteines g, hgprbmy28 et hgprbmy29, et leurs variants d'epissage
US7635758B2 (en) 2001-04-11 2009-12-22 Bristol-Myers Squibb Company Antibodies directed to G-protein coupled receptor HGPRBMY29sv1
WO2002083856A2 (fr) * 2001-04-11 2002-10-24 Bristol-Myers Squibb Company Polynucleotides codant deux nouveaux recepteurs couples aux proteines g, hgprbmy28 et hgprbmy29, et leurs variants d'epissage
US7049096B2 (en) 2001-04-11 2006-05-23 Bristol-Meyers Squibb Company Polynucleotides encoding a novel human G-protein coupled receptor splice variant HGPRBMY29sv1
US7276354B2 (en) 2001-04-11 2007-10-02 Bristol-Myers Squibb Company Polynucleotides encoding a novel human G-protein coupled receptor splice variant, HGPRBMY29SV2
US7345148B2 (en) 2001-04-11 2008-03-18 Bristol-Myers Squibb Company Human G-protein coupled receptor, HGPRBMY29sv1 polypeptides
US8124729B2 (en) 2001-04-11 2012-02-28 Bristol-Myers Squibb Company Splice variants of human G-protein coupled receptor HGPRBMY29 (HGPRMBY29SV2)
WO2002103005A1 (fr) * 2001-06-18 2002-12-27 National Institute Of Advanced Industrial Science And Technology Recepteurs couples aux proteines se fixant a la guanosine triphosphate
US7879566B2 (en) 2003-06-27 2011-02-01 Osi Pharmaceuticals, Inc. Methods for the identification of modulators of OSGPR114 or OSGPR78 activity, and their use in the treatment of disease
US7524638B1 (en) 2003-06-27 2009-04-28 Osi Pharmaceuticals, Inc. Methods for identification of modulators of OSGPR114 or OSGPR78 activity, and their use in the treatment of disease
US8790646B2 (en) 2004-04-14 2014-07-29 Genentech Inc. Compositions and methods for modulating vascular development
US7947278B2 (en) 2006-07-17 2011-05-24 The Board Of Trustees Of The Leland Stanford Junior University Methods of modulating angiogenesis
US8404811B2 (en) 2009-05-08 2013-03-26 Genentech, Inc. Humanized anti-EGFL7 antibodies and methods using same
US8574576B2 (en) 2009-05-08 2013-11-05 Genentech, Inc. Humanized anti-EGFL7 antibodies and methods using same

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EP1196561A2 (fr) 2002-04-17

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