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EP1040188A2 - Menschliche proteine mit sekretionssignalsequenz und dafür kodierende cdnas - Google Patents

Menschliche proteine mit sekretionssignalsequenz und dafür kodierende cdnas

Info

Publication number
EP1040188A2
EP1040188A2 EP98945604A EP98945604A EP1040188A2 EP 1040188 A2 EP1040188 A2 EP 1040188A2 EP 98945604 A EP98945604 A EP 98945604A EP 98945604 A EP98945604 A EP 98945604A EP 1040188 A2 EP1040188 A2 EP 1040188A2
Authority
EP
European Patent Office
Prior art keywords
protein
proteins
cells
present
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98945604A
Other languages
English (en)
French (fr)
Inventor
Seishi Kato
Tomoko Kimura
Shingo Sekine
Midori Kobayashi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sagami Chemical Research Institute
Protegene Inc
Original Assignee
Sagami Chemical Research Institute
Protegene Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sagami Chemical Research Institute, Protegene Inc filed Critical Sagami Chemical Research Institute
Publication of EP1040188A2 publication Critical patent/EP1040188A2/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention relates to human proteins having secretory signal sequences and DNAs coding for these proteins as well as eucaryotic cells forming said proteins by secretory expression.
  • the proteins of the present invention can be employed as pharmaceuticals or as antigens for preparing antibodies against said proteins.
  • the human cDNAs of the present invention can be utilized as probes for the gene diagnosis and gene sources for the gene therapy.
  • the cDNAs can be utilized as gene sources for large-scale production of the proteins encoded by said cDNAs .
  • Animal cells wherein expression vectors of said cDNAs are introduced can be utilized for secretory production of the proteins encoded by said cDNA.
  • secretory proteins have been obtained by a method comprising isolation and purification of the target protein from a large amount of the blood or a cell culture supernatant by using a biological activity as an indicator, determination of its primary structure followed by cloning of the corresponding cDNA on the basis of the information on the thus-obtained ammo acid sequence, and production of a recombinant protein using said cDNA.
  • the contents of the secretory proteins are generally so low that the isolation and purification are difficult m many cases .
  • secretory proteins and type-1 membrane proteins possess hydrophobic sequences, defined as secretory signal sequences, consisting of about 20 ammo acid residues at the ammo acid termini (the N-termini) . Therefore, the cloning of genes coding for the secretory proteins or type-1 membrane proteins is expected to be carried out by using the presence or absence of these secretory signal sequences as indicators .
  • the object of the present invention is to provide proteins having secretory signal sequences and DNAs coding for these proteins as well as eucaryotic cells forming said proteins by secretory expression.
  • the present inventors have been successful m selective cloning of cDNAs having secretory signal sequences from a human full-length cDNA bank, thereby completing the present invention.
  • the present invention provides human proteins having secretory signal sequences, namely proteins containing any of the ammo acid sequences represented by Sequence No. 1 to Sequence 4.
  • the present invention provides DNAs coding for the above-mentioned proteins, exemplified by cDNAs containing any of the base sequences represented by Sequence Nos. 5 to 9, 11, 13, and 15, as well as transformation eucaryotic cells that are capable of expressing said cDNAs .
  • Figure 1 A figure depicting the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP01738.
  • Figure 2 A figure depicting the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP01766.
  • Figure 3 A figure depicting the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP01842.
  • Figure A figure depicting the hydrophobicity/hydrophilicity profile of the protein encoded by clone HP10484.
  • the proteins of the present invention can be obtained, for example, by a method for isolation from human organs, cell lines, etc., a method for preparation of peptides by the chemical synthesis, or a method for production with the recombinant DNA technology using the DNAs coding for the human secretory proteins of the present invention, wherein the method for obtance by the recombinant DNA technology is employed preferably.
  • vitro expression can be achieved by preparation of an RNA by m vitro transcription from a vector having one of cDNAs of the present invention, followed by vitro translation using this RNA as a template.
  • recombination of the translation region into a suitable expression vector by the method known m the art leads to expression of a large amount of the encoded protein by using prokaryotic cells such as Escheri chia coli , Bacill us suhtilis, etc., and eucaryotic cells such as yeasts, insect cells, mammalian cells, etc.
  • prokaryotic cells such as Escheri chia coli , Bacill us suhtilis, etc.
  • eucaryotic cells such as yeasts, insect cells, mammalian cells, etc.
  • a recombinant expression vector bearing the translation region m the cDNA of the present invention is constructed m an expression vector having an origin, a promoter, a ribosome-bmdmg site, a cDNA-cloning site, a terminator etc., which can be replicated in the prokaryotic cells, and, after transformation of the host cells with said expression vector, the thus-obtained transformant is incubated, whereby the protein encoded by said cDNA can be produced on a large scale in the prokaryotic cells.
  • a maturation protein can be obtained by carrying out the expression with inserting an initiation codon in the translation region wherein the secretory signal sequence is removed.
  • a fusion protein with another protein can be expressed. Only a protein portion coding for said cDNA can be obtained by cleavage of said fusion protein with a suitable protease.
  • the protein of the present invention can be produced by extracellular secretion, when the translation region of said cDNA is subjected to recombination to an expression vector for eucaryotic cells that has a promoter, a splicing region, a poly (A) addition site, etc., followed by introduction into the eucaryotic cells.
  • the expression vector is exemplified by pKAl , pED6dpc2, pCDM8, pSVK3, pMSG, pSVL, pBK- CMV, pBK-RSV, EBV vector, pRS, pYES2, and so on.
  • eucaryotic cells examples include mammalian culture cells such as simian kidney cells COS7, Chinese hamster ovary cells CHO, etc., budding yeasts, fission yeasts, silkworm cells, Xenopus laevis egg cells, and so on, but any eucaryotic cells may be used, provided that they are capable of forming the present proteins by secretory expression.
  • the expression vector can be introduced in the eucaryotic cells by methods known in the art such as the electroporation method, the potassium phosphate method, the liposome method, the DEAE-dextran method, and so on.
  • the objective protein can be isolated from the culture and purified by a combination of separation procedures known in the art.
  • separation procedures include treatment with a denaturing agent such as urea or a surface-active agent, sonication, enzymatic digestion, saltmg-out or solvent precipitation, dialysis, centrifugation, ultraflltration, gel filtration, SDS-PAGE, isoelectric focusing, ion-exchange chromatograpny, hydrophobic chromatography, affinity chromatography, reverse phase chromatography, and so on.
  • the proteins of the present invention include peptide fragments (more than 5 ammo acid residues) containing any partial ammo acid sequence m the ammo acid sequences represented by Sequence No. 1 to Sequence No. 4. These peptide fragments can be utilized as antigens for preparation of antibodies.
  • the proteins of the present invention are secreted m the form of maturation proteins outside the cells, after the signal sequences are removed. Therefore, these maturation proteins shall come with the scope of the present invention.
  • the N-termmal ammo acid sequences of the maturation proteins can be easily identified by using the method for the cleavage-site determination m a signal sequence [Japanese Patent Kokai Publication No. 1996-187100].
  • the DNAs of the present invention include all DNAs coding for the above-mentioned proteins. Said DNAs can be obtained by using a method by chemical synthesis, a method by cDNA cloning, and so on.
  • the cDNAs of the present invention can be cloned, for example, from cDNA libraries of the human cell origin. These cDNA are synthesized by using as templates poly (A) " RNAs extracted from human cells.
  • the human cells may be cells delivered from the human body, for example, by the operation or may be the culture cells.
  • the cDNAs can be synthesized by using any method selected from the Okayama-Berg method [Okayama, H. and Berg, P., Mol. Cell. Biol.
  • the primary selection of one of the cDNAs coding for the human proteins having secretory signal sequences is carried out by sequencing of a partial base sequence of a cDNA clone selected at random from cDNA libraries, sequencing of the amino acid sequence encoded by the base sequence, and recognition of the presence or absence of a hydrophobic site in the resulting N- terminal amino acid sequence region.
  • the secondary selection is carried out by determination of the whole sequence by the sequencing and the protein expression by in vitro translation.
  • Ascertainment of cDNAs of the present invention for encoding the proteins having secretory signal sequences is carried out by using the signal sequence detection method [Yokoyama-Kobayashi, M. et al . , Gene 163: 193-196 (1995)].
  • the ascertainment for a coding portion of an inserted cDNA fragment to function as a signal sequence is provided by fusing a cDNA fragment coding for the N-terminus of the target protein with a cDNA coding for the protease domain of urokinase and then expressing the resulting cDNA in C0S7 cells to detect the urokinase activity in the cell culture medium.
  • the same clones as the cDNAs of the present invention can be easily obtained by screening of the cDNA libraries constructed from the human cell lines and human tissues utilized m the present invention by the use of an ol gonucleotide probe synthesized on the basis of the cDNA base sequence described m any of Sequence Nos. 9, 11 and 13.
  • any protein that is formed by these modifications comprising insertion or deletion of one or plural ammo acids and/or substitution with other ammo acids shall come withm the scope of the present invention, as far as the protein possesses the activity of any protein having the ammo acid sequences represented by Sequence No. 1 to Sequence No. 4.
  • the cDNAs of the present invention include cDNA fragments (more than 10 bp) containing any partial base sequence m the base sequences represented by Sequence No. 5 to Sequence No. 8 or in the base sequences represented by Sequence Nos. 9, 11, 13 and 15.
  • the portion coding for the secretory signal sequence can be utilized as means to secrete an optionally selected protein outside the cells by fusing with a cDNA encoding another protein.
  • DNA fragments consisting of a sense chain and an anti-sense chain shall come with this scope. These DNA fragments can be utilized as the probes for the gene diagnosis.
  • 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) identifledbelow.
  • 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 researcn community for various purposes.
  • the polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues m which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or m 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 m 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 m 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
  • the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, m a receptor-ligand interaction)
  • the polynucleotide can also be used m interaction trap assays (such as, for example, that described m 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 m assay to determine biological activity, including m 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) m assays designed to quantitatively determine levels of the protein (or its receptor) m biological fluids; as markers for tissues m which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or m 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, m 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 m these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
  • Polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements . 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. Cytokine and Cell Pro! i feration/Differentiation Activity
  • 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 m 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 m certain cell populations.
  • Many protein factors discovered to date, including all known cytokines have exhibited activity m 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, 39/11, BaF3, MC9/G, M+ (preBM+), 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 m: Current Protocols m 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 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 m: 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
  • 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 m: 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 m Humans) ; Weinberger et al., Proc. Natl. Acad. Sci. USA 77 : 6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11:405-411, 1981; Takai et al . , J. Immunol.
  • 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 m 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 orfungal 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, Guillam-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
  • T cell responses may be inhibited by suppressing T cell responses or by inducing specific tolerance m 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 m that it is generally antigen-specific and persists after exposure to the tolerizmg agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen the absence of the tolerizmg 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 lymphokme 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 m reduced tissue destruction m 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 m 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 m 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 m 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 mMRL/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 may also be useful 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 m cases of viral infection. In addition, systemic viral diseases such as influenza, the commoncold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically.
  • anti-viral immune responses may be enhanced m an infected patient by removing T cells from the patient, costimulatmg 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 remtroducmg the m 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 remtroduce the transfected cells into the patient.
  • the infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells m 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
  • transfected with a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance m 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-l ⁇ ke activity alone, or m conjunction with a peptide having B7-l-like activity and/or B7-3-l ⁇ ke 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.
  • 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 Hoc chain protein and an MHC class H ⁇ 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 Hoc chain protein and an MHC class H ⁇ 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 m: Current Protocols m Immunology, Edby 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 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.
  • T-cell-dependent immunoglobulin responses and isotype switching include, without limitation, those described m: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J.J.
  • MLR Mixed lymphocyte reaction
  • Dendritic cell-depenoent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Gueryetal., 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 m: 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.
  • 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 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 complimentary 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 (such as those usually treated with
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for proliferation and differentiation of various hematopoietic lines are cited above.
  • 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.
  • 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 (coUagenase 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.
  • De novo tendon/ligament-like tissue formation induced by a composition 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.
  • 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 tissue
  • 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 m: International Patent Publication
  • Assays for wound healing activity include, without limitation, those described m: Winter, Epidermal Wound Healing, pps . 71-112 (Maibach, HI and Rovee, DT, eds.), Year Book Medical
  • Aprotem of the present invention may also exhibit activin- or mhibm-related activities.
  • Inhibms are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH)
  • activms 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 m heterodimers with a member of the inhibin ⁇ family, may be useful as a contraceptive based on the ability of mhibms to decrease fertility m female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other mhibms can induce infertility m these mammals.
  • the protein of the invention may be useful as a fertility inducing therapeutic, based upon the ability of activm 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 m 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 activm/mhibm activity include, without limitation, those described m: 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. Chemotactic/Chemokineti c Activity
  • a protein of the present invention may have chemotactic or chemokmetic activity (e.g., act as a chemokme) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosmophils, epithelial and/or endothelial cells.
  • Chemotactic and chemokmetic proteins can be used to mobilize or attract a desired cell population to a desired site of action.
  • Chemotactic or chemokmetic proteins provide particular advantages m treatment of wounds and other trauma to tissues, as well as 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, Edby 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 m treatment of various coagulation disorders (mcludinghereditary 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 tnromboses 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 m: Linet et al . , J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al . , Thrombosis Res. 45:413-419, 1987; Humphrey et al . , Fibrmolysis 5:71-79 (1991); Schaub, Prostaglandms 35:467-474, 1988.
  • Receptor/Ligand Activity 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 m cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectms, mtegrms and their ligands) and receptor/ligand pairs involved m 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 m: Current Protocols m 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; Rosenste etal., 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-mflammatory activity.
  • the anti-mflammatory activity may be achieved by providing a stimulus to cells involved m the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved 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, endotoxm lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokme-mduced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of ytokines 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.
  • infection such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)
  • ischemia-reperfusion injury such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)
  • ischemia-reperfusion injury such as septic shock, sepsis or systemic inflammatory response syndrome (
  • a protein of the invention may exhibit other anti-tumor activities.
  • a protein may inhibit tumor growth directly or indirectly (such as, for example, via ADCC) .
  • 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 Other Activities
  • 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 m bone form or shape) ; effecting biorhythms or caricadic 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
  • the histiocyte lymphoma cell line U937 (ATCC CRL 1593) stimulated by phorbol ester and tissues of stomach cancer delivered by the operation were used for human cells to extract mRNAs.
  • the cell line was incubated by a conventional procedure.
  • RNA and 3 nmol of a chimeric DNA- RNA oligonucleotide 5' -dG-dG-dG-dG-dA-dA-dT-dT-dC-dG-dA-G-G- A-3' ) were dissolved in a solution containing 50 mM Tris- hydrochloride buffer solution (pH 7.5), 0.5 mM ATP, 5 mM MgCl 2 , 10 mM 2-mercaptoethanol, and 25% polyethylene glycol, whereto was added 50 units of T4RNA ligase and a total 30 ⁇ l volume of the resulting mixture was reacted at 20°C for 12 hours.
  • RNA was annealed with 1.2 ⁇ g of the vector primer, the resulting product was dissolved in a solution containing 50 mM Tris-hydrochloride buffer solution (pH 8.3), 75 mM KC1, 3 mM MgCl,, 10 mM dithiothreitol, and 1.25 mM dNTP (dATP + dCTP + dGTP + dTTP) , 200 units of a reverse transcriptase (GIBCO-BRL) were added, and the reaction in a total 20 ⁇ l volume was run at 42°C for one hour.
  • Tris-hydrochloride buffer solution pH 8.3
  • 75 mM KC1 75 mM KC1
  • 3 mM MgCl 3 mM MgCl
  • 10 mM dithiothreitol 1.25 mM dNTP (dATP + dCTP + dGTP + dTTP)
  • a reverse transcriptase
  • the reaction solution was subjected to phenol extraction, followed by ethanol precipitation, the resulting pellet was dissolved in a solution containing 50 mM Tris- hydrochloride buffer solution (pH 7.5), lOO mMNaCl, 10 mM MgCl,, and 1 mM dithiothreitol. Thereto were added 100 units of EcoRI and a total 20 ⁇ l volume of the resulting mixture was reacted at 37°C for one hour.
  • the resulting pellet was dissolved in a solution containing 20 mM Tris-hydrochloride buffer solution (pH7.5), lOOmMKCl, 4mMMgCl 2 , 10 mM (NH 4 ) 2 S0 4 , and 50 ⁇ g/ml of the bovine serum albumin. Thereto were added 60 units of an Escheri chia coli DNA ligase and the resulting mixture was reacted at 16°C for 16 hours .
  • the cDNA-synthesis reaction solution was used for transformation of Escheri chia coli DH12S (GIBCO-BRL) .
  • the transformation was carried out by the electroporation method. A portion of the transformant was sprayed on the 2xYT agar culture medium containing 100 ⁇ g/ml ampicillin and the mixture was incubated at 37°C overnight. A colony formed on the agar medium was picked up at random and inoculated on 2 ml of the 2xYT culture medium containing 100 ⁇ g/ml ampicillin. After incubation at 37°C overnight, the culture mixture was centrifuged to separate the mycelia, from which a plasmid DNA was prepared by the alkaline lysis method.
  • the plasmid DNA was subjected to double digestion with EcoRI and NotI, followed by 0.8% agarose gel electrophoresis, to determine the size of the cDNA insert. Furthermore, using the thus-obtained plasmid as a template, the sequence reaction was carried out by using an M13 universal primer labeled with a fluorescent dye and a Taq polymerase (a kit of Applied Biosystems) and then the product was examined with a fluorescent DNA sequencer (Applied Biosystems) to determine an about 400-bp base sequence at the 5' -terminus of the cDNA. The sequence data were filed as the homo/protein cDNA bank database.
  • the N-terminal hydrophobic region in the secretory protein clone candidate obtained in the above-mentioned steps functions as a secretory signal sequence.
  • the plasmid containing the target cDNA was cleaved at an appropriate restriction enzyme site existing at the downstream of the portion expected for encoding the secretory signal sequence. In the case in which this restriction site was a protruding 5' -terminus, the site was blunt-ended by the Klenow treatment.
  • Hindlll Digestion with Hindlll was further carried out and a DNA fragment containing the SV40 promoter and a cDNA encoding the secretory signal sequence at the downstream of the promoter was separated by agarose gel electrophoresis .
  • the resulting fragment was inserted between Hindlll in pSSD3 (DDBJ/EMBL/GenBank Registration No. AB007632) and a restriction enzyme site selected so as to match with the urokinase-coding frame, thereby constructing a vector expressing a fusion protein of the secretory signal sequence of the target cDNA and the urokinase protease domain.
  • Escheri chia coli (host: JM109) bearing the fusion-protein expression vector was incubated at 37°C for 2 hours in 2 ml of the 2xYT culture medium containing 100 ⁇ g/ml of ampicillin, the helper phage M13K07 (50 ⁇ l) was added and the incubation was continued at 37 °C overnight.
  • a supernatant separated by centrifugation underwent precipitation with polyethylene glycol to obtain single-stranded phage particles. These particles were suspended in 100 ⁇ l of 1 mM Tris-0.1 mM EDTA, pH 8 (TE) .
  • the culture cells originating from the simian kidney, C0S7 were incubated at 37°C in the presence of 5% CO, in the Dulbecco's modified Eagle's culture medium (DMEM) containing 10% fetal calf albumin.
  • DMEM Dulbecco's modified Eagle's culture medium
  • DMEM Dulbecco's modified Eagle's culture medium
  • Table 2 shows the restriction enzyme site used for cutting the cDNA fragment from each clone, the restriction enzyme site used for cleavage of pSSD3, and the presence or absence of a clear circle. Except for the case in which pSSD3 was used as the control, each of the samples formed a clear circle to identify that urokinase was secreted in the culture medium. In other words, it has been indicated that each of the cDNA fragments codes for the amino acid sequence that functions as the secretory signal sequence.
  • the plasmid vector bearing the cDNA of the present invention was used for m vitro transcription/translation with a T T rabbit reticulocyte lysate kit (Promega) .
  • T T rabbit reticulocyte lysate kit Promega
  • [ 5 S] methionme 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 reacted at 30°C for 90 minutes m a total 25 ⁇ l volume of the reaction solution containing 12.5 ⁇ l of T deliberatelyT rabbit reticulocyte lysate, 0.5 ⁇ l of a buffer solution (attached to kit), 2 ⁇ l of an ammo acid mixture (methionme-free) , 2 ⁇ l of [ j S] methionme (Amersham) (0.37 MBq/ ⁇ l), 0.5 ⁇ l of T7RNA polymerase, and 20 U of RNasin.
  • Escheri chia coli bearing the expression vector of the protein of the present invention was infected with helper phage M13K07 and single-stranded phage particles were obtained by the above-mentioned procedure.
  • the thus-obtained phage was used for introducing each expression vector in the culture cells originating from the simian kidney, COS7. After incubation at 37°C for 2 days in the presence of 5% CO, the incubation was continued for one hour in the culture medium containing [ 35 S]cystine or [ 35 S] methionme .
  • Table 3 shows the comparison of the amino acid sequence between the human protein of the present invention (HP) and the rat zymogen granule protein (RN) .
  • HP human protein of the present invention
  • RN rat zymogen granule protein
  • the marks of -, *, and . represent a gap, an amino acid residue identical with the protein of the present invention, and an amino acid residue analogous to the protein of the present invention, respectively.
  • the both proteins possessed a homology of 84.5%.
  • the present protein had a C-terminus longer by 18 amino acid residues.
  • Determination of the wnole base sequence of the cDNA insert of clone HP01766 obtained from cDNA libraries of human lymphoma cell line U937 revealed the structure consisting of a 96-bp 5' -nontranslation region, a 519-bp ORF, and a 799-bp 3' - nontranslation region .
  • the ORF codes for a protein consisting of 172 ammo acid residues and there existed a hydrophobic region of a putative secretory signal sequence at the N- terminal .
  • Figure 2 depicts the hydrophobicity/hydrophilicity profile, obtained by the Kyte-Doolittle method, of the present protein .
  • the search of the protein data base by using the ammo acid sequence of the present protein revealed that the protein was analogous to the Xenopus laevis putative secretory protein XAG (GenBank Accession No . U76752 ) .
  • Table 4 shows the comparison of the ammo acid sequence between the human protein of the present invention (HP) and the Xenopus laevis putative secretory protein XAG (XL) .
  • the marks of - , * , and . represent a gap, an ammo acid residue identical with the protein of the present invention, and an ammo acid residue analogous to the protein of the present invention, respectively .
  • the both proteins possessed a homology of 34 . 5% .
  • Determination of the whole base sequence of the cDNA insert of clone HP10484 obtained from cDNA libraries of human lymphoma cell line U937 revealed the structure consisting of a 52-bp 5' -nontranslation region, a 663-bp ORF, and a 519-bp 3'- nontranslation region.
  • the ORF codes for a protein consisting of 220 amino acid residues and there existed a hydrophobic region of a putative secretory signal sequence at the N-termmal.
  • Figure 4 depicts the hydrophobicity/hydrophilicity proflie, obtained by the Kyte-Doolittle method, of the present protein.
  • the search of tne protein data base using the ammo acid sequence of the present protein has not revealed the presence of any known protein having an analogy . Also, the search of the GenBank using the base sequences of the present cDNA has revealed the presence of sequences that possessed a homology of 90 & or more
  • the present invention provides human proteins having secretory signal sequences and DNAs coding for these proteins as well as eucaryotic cells forming said proteins by secretory expression. All of the proteins of the present invention are secreted outside the cells and exist m the extracellular liquid or on the cell membrane, so that they are considered to be proteins controlling the proliferation and the differentiation of the cells . Accordingly, the proteins of the present invention can be employed as pharmaceuticals or as antigens for preparing antibodies against said proteins. Furthermore, said DNAs can be utilized for expression of said proteins in large amounts. Eucaryotic cells wherein expression vectors of said cDNAs are introduced can be utilized for secretory production of the proteins encoded by said cDNAs .
  • 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, mtrons, promoters, enhancers, and silencer or suppressor elements. The corresponding genes can be isolated m 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 m 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.
  • 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.
  • the present invention also provides for soluble forms of such protein.
  • the intracellular and transmembrane domains of the protein are deleted such that the protein is fully secreted from the cell in which it is expressed.
  • the intracellular and transmembrane domains of proteins of the invention can be identified in accordance with known techniques for determination of such domains from sequence information.
  • 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 ammo 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 ammo 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 m 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 SSPE
  • IxSSC 0.15M NaCl and 15mM sodium citrate
  • T B - T R The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10°C less than the melting temperature (T m ) of the hybrid, where T m is determined according to the following equations.
  • T m (°C) 2(#of A + T bases) + 4(# of G + C bases).
  • 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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