EP1080106A1 - Secreted proteins and polynucleotides encoding them - Google Patents

Secreted proteins and polynucleotides encoding them

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Publication number
EP1080106A1
EP1080106A1 EP99925654A EP99925654A EP1080106A1 EP 1080106 A1 EP1080106 A1 EP 1080106A1 EP 99925654 A EP99925654 A EP 99925654A EP 99925654 A EP99925654 A EP 99925654A EP 1080106 A1 EP1080106 A1 EP 1080106A1
Authority
EP
European Patent Office
Prior art keywords
protein
polynucleotide
seq
amino acid
nucleotide
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Withdrawn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
EP99925654A
Other languages
German (de)
French (fr)
Inventor
Kenneth Jacobs
John M. Mccoy
Edward R. Lavallie
Lisa A. Collins-Racie
Cheryl Evans
David Merberg
Sha Mi
Maurice Treacy
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.)
Genetics Institute LLC
Original Assignee
Genetics Institute LLC
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Filing date
Publication date
Priority claimed from US09/175,928 external-priority patent/US6312921B1/en
Application filed by Genetics Institute LLC filed Critical Genetics Institute LLC
Publication of EP1080106A1 publication Critical patent/EP1080106A1/en
Withdrawn legal-status Critical Current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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:l from nucleotide 44 to nucleotide 1204; the nucleotide sequence of SEQ ID NO:l from nucleotide 1 to nucleotide 403; the nucleotide sequence of the full-length protein coding sequence of clone AJ26_3 deposited under accession number ATCC 98115; or the nucleotide sequence of the mature protein coding sequence of clone AJ26_3 deposited under accession number ATCC 98115.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
  • polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:9 from nucleotide 6 to nucleotide 2408; the nucleotide sequence of SEQ ED NO:9 from nucleotide 1295 to nucleotide 1705; the nucleotide sequence of the full-length protein coding sequence of clone BL89_13 deposited under accession number ATCC 98153; or the nucleotide sequence of the mature protein coding sequence of clone BL89_13 deposited under accession number ATCC 98153.
  • the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12 from amino acid 1 to amino acid 68.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ED NO:33;
  • protein comprises the amino acid sequence of SEQ ED NO:33 or the amino acid sequence of SEQ ED NO:33 from amino acid 24 to amino acid 96.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • Processes are also provided for producing a protein, which comprise:
  • Figure 5 demonstrates that AJ172_2 mediates actual cell fusion and does not operate through a mechanism of arrested cell division.
  • a first cell line was transfected with AJ172_2, luciferase and ERK.
  • a second cell line was transfected with AJ172_2 and MEK. When the cells were mixed, fusion occurred resulting in production of luciferase activity.
  • FIG. 7 demonstrates that the mechanism of AJ 172_2 induced cell fusion does not require homophilic or heterophilic protein-protein interactions.
  • COS cells transfected with AJ172_2 were mixed with liposomes containing a green fluorescent protein (GFP) expression plasmid. As shown in Figure 7, the COS cells fused with the liposomes, took up the expression plasmid, and began expressing GFP.
  • Figures 8-10 present data which demonstrate that AJ172_2 is expressed in the formation of cytotrophoblasts associated with choriocarcinoma (see Example 2).
  • the nucleotide sequence disclosed herein for AJ26_3 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. AJ26_3 demonstrated at least some similarity with sequences identified as U46493 (Cloning vector pFlp recombinase gene, complete eds). The predicted amino acid sequence disclosed herein for AJ26_3 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted AJ26_3 protein demonstrated at least some similarity to sequences identified as J01917 (DNA polymerase [Human adenovirus type 2]), J01969 (DNA polymerase [Human adenovirus type 5]), L24893 (HUMAAC02_1 myelin protein zero [Homo sapiens]), U43330 (CTX [Xenopus laevis]), and U43394 (CTX [Xenopus laevis]). Based upon sequence similarity, AJ26_3 proteins and each similar protein or peptide may share at least some activity.
  • the nucleotide sequence disclosed herein for BL89_13 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. No hits were found in the database.
  • the TopPredll computer program predicts a potential transmembrane domain within the BL89_13 protein sequence centered around amino acid 625 of SEQ ID NO:10.
  • the EcoRI/Notl restriction fragment obtainable from the deposit containing clone CC25_17 should be approximately 300 bp.
  • the EcoRI/Notl restriction fragment obtainable from the deposit containing clone D305_2 should be approximately 2400 bp.
  • D305_2 demonstrated at least some similarity with sequences identified as AA055703 (zl75d04.rl Stratagene colon (#937204) Homo sapiens cDNA clone 510439 5'), N49593 (yy58d05.sl Homo sapiens cDNA clone 277737 3'), R66646 (yi35b08.rl Homo sapiens cDNA clone 141207 5" similar to SP P24A_YEAST P32802 P24A PROTEIN), U81006 (Human p76 mRNA, complete eds), and Z48758 (S.cerevisiae chromosome EV cosmid 9727).
  • the EcoRI/Notl restriction fragment obtainable from the deposit containing clone G55_l should be approximately 2000 bp.
  • G55_l demonstrated at least some similarity with sequences identified as R83586 (ypl6a07.rl Homo sapiens cDNA clone 1875725'). Based upon sequence similarity, G55_l proteins and each similar protein or peptide may share at least some activity.
  • the EcoRI/Notl restriction fragment obtainable from the deposit containing clone K39_7 should be approximately 1675 bp.
  • K39_7 The predicted amino acid sequence disclosed herein for K39_7 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted K39_7 protein demonstrated at least some similarity to sequences identified as R86811 (Saccharomyces cerevisiae mutant LAGl protein) and U42438 (similar to S. cerevisiae longevity-assurance protein 1 (SP P38703) [Caenorhabditis elegans]). Based upon sequence similarity, K39_7 proteins and each similar protein or peptide may share at least some activity.
  • the nucleotide sequence of the 5' portion of K363_3 as presently determined is reported in SEQ ID NO:32. What appUcants presently beUeve is the proper reading frame for the coding region is indicated in SEQ ID NO:33.
  • the predicted amino acid sequence of the K363_3 protein conesponding to the foregoing nucleotide sequence is reported in SEQ ED NO:33. Additional nucleotide sequence from the 3' portion of K363_3, including the polyA tail, is reported in SEQ ID NO:34.
  • the EcoRI/Notl restriction fragment obtainable from the deposit containing clone K363_3 should be approximately 2690 bp.
  • K363_3 The nucleotide sequence disclosed herein for K363_3 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. K363_3 demonstrated at least some similarity with sequences identified as AA437876 (vd20h06.sl Knowles Solter mouse 2 cell Mus musculus cDNA clone 793115 5'), D21554 (Mouse embryonal carcinoma F9 cell cDNA, 67F09), and Y08460 (Mus musculus mRNA for Mdes transmembrane protein). The predicted amino acid sequence disclosed herein for K363_3 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted K363_3 protein demonstrated at least some similarity to sequences identified as Y08460 (Mdes protein [Mus musculus]). Based upon sequence similarity, K363_3 proteins and each similar protein or peptide may share
  • K464_4 A polynucleotide of the present invention has been identified as clone "K464_4".
  • K464_4 was refened to as K464_3 in previous appUcations.
  • K464_4 was isolated from a murine adult bone manow (stromal cell line FCM-4) cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein.
  • K464_4 is a full-length clone, including the entire coding sequence of a secreted protein (also refened to herein as "K464_4 protein").
  • K464_4 The predicted amino acid sequence disclosed herein for K464_4 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol.
  • the predicted K464_4 protein demonstrated at least some similarity to sequences identified as L33768 (JAK3 [Mus musculus]) and X16213 (MHC T7 class I antigen (64 AA) (119 is 2nd base in codon)
  • K464_4 proteins and each similar protein or peptide may share at least some activity.
  • Amino acids 184 to 196 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 197, or are a transmembrane domain.
  • the EcoRI/Notl restriction fragment obtainable from the deposit containing clone K483_l should be approximately 1500 bp.
  • L69_3 A polynucleotide of the present invention has been identified as clone "L69_3".
  • L69_3 was refened to as L69_2 in previous appUcations.
  • L69_3 was isolated from a murine adult thymus cDNA Ubrary using methods which are selective for cDNAs encoding secreted proteins (see U.S.
  • Amino acids 7 to 19 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 20, or are a transmembrane domain. Additional nucleotide sequence from the 3' portion of L69_3, including the polyA tail, is reported in SEQ ID NO:45.
  • Clones AJ26_3, AJ172_2, AP224_2, BL89_10, BL341_4, BV239_2, CC25_16, CC397_11, D305_2, G55_l, K39_7, K330_3, K363_3, K446_3, K464_4, K483_l, and L69_3 were deposited on July 25, 1996 with the American Type Culture Collection as an original deposit under the Budapest Treaty and were given the accession number ATCC 98115, from which each clone comprising a particular polynucleotide is obtainable.
  • the cDNA insert can still be isolated by digestion with EcoRl and Notl. However, Notl will then produce the 5' site and EcoRl wiU produce the 3' site for placement of the cDNA in proper orientation for expression in a suitable vector.
  • the cDNA may also be expressed from the vectors in which they were deposited.
  • the invention also includes polynucleotides with sequences complementary to those of the polynucleotides disclosed herein.
  • 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.
  • yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins.
  • yeast strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
  • the protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein.
  • the resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography.
  • the purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.
  • the protein of the invention may also be expressed in a form which will faciUtate purification.
  • it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively.
  • MBP maltose binding protein
  • GST glutathione-S-transferase
  • TRX thioredoxin
  • Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively.
  • the protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope.
  • One such epitope (“Flag") is commercially available from
  • the protein may also be produced by known conventional chemical synthesis. Methods for constructing the proteins of the present invention by synthetic means are known to those skilled in the art.
  • the synthetically-constructed protein sequences by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.
  • the proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deUberately engineered.
  • modifications in the peptide or DNA sequences can be made by those skiUed in the art using known techniques.
  • Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence.
  • one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule.
  • Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Patent No. 4,518,584).
  • such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.
  • polynucleotides and proteins of the present invention are expected to 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 conesponding 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 oUgomers for attachment to a "gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immun
  • 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 eUcit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively dete ⁇ nine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the conesponding 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 conelative receptors or Ugands.
  • the protein binds or potentially binds to another protein (such as, for example, in a receptor-Ugand 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.
  • 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 soUd 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 ceU 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 ceU 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 proUferation assays for cell Unes including, without Umitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DAI, 123, Tl 165, 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 proliferation and differentiation of hematopoietic and lymphopoietic cells include, without Umitation, 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. CoUgan 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 Umitation, those described in: Cunent Protocols in Immunology, Ed by J. E. CoUgan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene PubUshing 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.
  • 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, GuiUain-Bane syndrome, autoimmune thyroiditis, insuUn dependent diabetes melUtis, 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.
  • Down regulating or preventing one or more antigen functions (including without Umitation B lymphocyte antigen functions (such as , for example, B7)), e.g., preventing high level lymphokine synthesis by activated T ceUs, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD).
  • 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.
  • murine models of GVHD 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 eUminate disease symptoms.
  • Examples include murine experimental autoimmune encephaUtis, systemic lupus erythmatosis in MRUlpr/lpr mice or NZB hybrid mice, murine autoimmune coUagen arthritis, diabetes melUtus 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
  • Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response.
  • enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection.
  • systemic viral diseases such as influenza, the common cold, and encephaUtis 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 ceUs 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 ceUs express aU 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.
  • 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-l-like activity and/or B7-3-like activity.
  • the transfected tumor ceUs 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 activity of a protein of the invention may, among other means, be measured by the following methods:
  • Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without Umitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559,
  • erythroid progenitor ceUs alone or in combination with other cytokines, thereby indicating utiUty, 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 proUferation 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 proUferation 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 compUmentary to platelet transfusions; and or in supporting the growth and proUferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoi
  • Such agents may provide an environment to attract bone-forming ceUs, 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.
  • compositions of the present invention may provide an environment to attract tendon- or Ugament-forming cells, stimulate growth of tendon- or Ugament-forming cells, induce differentiation of progenitors of tendon- or Ugament-forming cells, or induce growth of tendon Ugament 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.
  • a protein of the present invention may also exhibit activin- or inhibin-related activities.
  • the protein of the invention may be useful as a fertility inducing therapeutic, based upon the abiUty 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 fertiUty in sexually immature mammals, so as to increase the Ufetime reproductive performance of domestic animals such as cows, sheep and pigs.
  • a protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian ceUs, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast ceUs, eosinophils, epithelial and/or endotheUal 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 locaUzed 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 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 abiUty of a protein to induce the migration of cells across a membrane as weU as the abiUty of a protein to induce the adhesion of one cell population to another ceU population.
  • Suitable assays for movement and adhesion include, without Umitation, those described in: Current Protocols in Immunology, Ed by J.E. CoUgan, A.M. Kruisbeek, D.H. MarguUes, E.M. Shevach, W.Strober, Pub.
  • 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 Umitation, those described in: Linet et al., J. CUn. 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.
  • Ugands including without Umitation, cellular adhesion molecules (such as selectins, integrins and their Ugands) and receptor/Ugand pairs involved in antigen presentation, antigen recognition and development of ceUular and humoral immune responses).
  • Receptors and Ugands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/Ugand interaction.
  • a protein of the present invention including, without Umitation, fragments of receptors and Ugands may themselves be useful as inhibitors of receptor/Ugand interactions.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Cadherin malfunction is also impUcated in other human diseases, such as pemphigus vulgaris and pemphigus fohaceus (auto-immune bUstering skin diseases), Crohn's disease, and some developmental abnormaUties.
  • the cadherin superfamily includes well over forty members, each with a distinct pattern of expression. All members of the superfamily have in common conserved extracellular repeats
  • E-cadherin one member of the cadherin superfamily, is expressed in epitheUal cell types. Pathologically, if E-cadherin expression is lost in a tumor, the maUgnant cells become invasive and the cancer metastasizes. Transf ection of cancer cell lines with polynucleotides expressing E- cadherin has reversed cancer-associated changes by returning altered cell shapes to normal, restoring cells' adhesiveness to each other and to their substrate, decreasing the cell growth rate, and drastically reducing anchorage-independent ceU growth. Thus, reintroducing E-cadherin expression reverts carcinomas to a less advanced stage. It is likely that other cadherins have the same invasion suppressor role in carcinomas derived from other tissue types.
  • proteins of the present invention with cadherin activity can be used to treat cancer.
  • Introducing such proteins or polynucleotides into cancer cells can reduce or eUminate the cancerous changes observed in these cells by providing normal cadherin expression.
  • Cancer cells have also been shown to express cadherins of a different tissue type than their origin, thus aUowing these cells to invade and metastasize in a different tissue in the body.
  • Proteins of the present invention with cadherin activity can be substituted in these cells for the inappropriately expressed cadherins, restoring normal cell adhesive properties and reducing or eliminating the tendency of the cells to metastasize.
  • proteins of the present invention with cadherin activity can be used to generate antibodies recognizing and binding to cadherins.
  • Such antibodies can be used to block the adhesion of inappropriately expressed tumor-ceU cadherins, preventing the cells from forming a tumor elsewhere.
  • Such an anti- cadherin antibody can also be used as a marker for the grade, pathological type, and prognosis of a cancer, i.e. the more progressed the cancer, the less cadherin expression there will be, and this decrease in cadherin expression can be detected by the use of a cadherin-binding antibody.
  • Fragments of proteins of the present invention with cadherin activity preferably a polypeptide comprising a decapeptide of the cadherin recognition site, and poly-nucleotides of the present invention encoding such protein fragments, can also be used to block cadherin function by binding to cadherins and preventing them from binding in ways that produce undesirable effects. Additionally, fragments of proteins of the present invention with cadherin activity, preferably truncated soluble cadherin fragments which have been found to be stable in the circulation of cancer patients, and polynucleotides encoding such protein fragments, can be used to disturb proper cell- cell adhesion.
  • Assays for cadherin adhesive and invasive suppressor activity include, without limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-18817, 1995; Miyaki et al. Oncogene 11: 2547-2552, 1995; Ozawa et al. Cell 63: 1033-1038, 1990.
  • 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.
  • 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 kilUng, infectious agents, including, without Umitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without Umitation, 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 caricadic cycles or rhythms; effecting the fertiUty of male or female subjects; effecting the metaboUsm, cataboUsm, anaboUsm, processing, utiUzation, storage or elimination of dietary fat, Upid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component(s); effecting behavioral characteristics, including, without Umitation, appetite, Ubido, stress, cognition (including cognitive disorders),
  • a protein of the present invention may be used in a pharmaceutical composition when combined with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabiUzers, solubilizers, and other materials well known in the art.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration.
  • the pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, D -3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, E -10, IL-11, EL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNF1, TNF2, G- CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin.
  • the pharmaceutical composition may further contain other agents which either enhance the activity of the protein or compUment its activity or use in treatment.
  • Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein of the invention, or to minimize side effects.
  • protein of the present invention may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.
  • a protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins.
  • pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.
  • the term "therapeutically effective amount” means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, heaUng, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • a meaningful patient benefit i.e., treatment, heaUng, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions.
  • the term refers to that ingredient alone.
  • the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • a therapeutically effective amount of protein of the present invention is administered to a mammal having a condition to be treated.
  • Protein of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors.
  • protein of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factors), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.
  • Administration of protein of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical appUcation or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is prefened.
  • the liquid form of the pharmaceutical composition may further contain physiological satine solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
  • the pharmaceutical composition When administered in liquid form, contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention.
  • protein of the present invention When a therapeuticaUy effective amount of protein of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
  • a prefened pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
  • the pharmaceutical composition of the present invention may also contain stabiUzers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
  • the amount of protein of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of protein of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein of the present invention and observe the patient's response. Larger doses of protein of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.
  • the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 ⁇ g to about 100 mg (preferably about 0.1 ng to about 10 mg, more preferably about 0.1 ⁇ g to about 1 mg) of protein of the present invention per kg body weight.
  • the duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the protein of the present invention will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.
  • Protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specificaUy react with the protein. Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen.
  • the peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole Umpet hemocyanin (KLH).
  • KLH keyhole Umpet hemocyanin
  • Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein.
  • NeutraUzing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved.
  • neutraUzing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein.
  • the therapeutic method includes administering the composition topically, systematicaUy, or locaUy as an implant or device.
  • the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form.
  • the composition may desirably be encapsulated or injected in a viscous form for dehvery to the site of bone, cartilage or tissue damage.
  • Topical administration may be suitable for wound heaUng and tissue repair.
  • Therapeutically useful agents other than a protein of the invention which may also optionaUy be included in the composition as described above, may altematively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention.
  • the composition would include a matrix capable of deUvering the protein-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body.
  • a matrix capable of deUvering the protein-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body.
  • Such matrices may be formed of materials presently in use for other implanted medical applications.
  • the choice of matrix material is based on biocompatibility, biodegradabiUty, mechanical properties, cosmetic appearance and interface properties.
  • the particular appUcation of the compositions wiU define the appropriate formulation.
  • Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycohc acid and polyanhydrides.
  • Other potential materials are biodegradable and biologicaUy weU-defined, such as bone or dermal coUagen.
  • Further matrices are comprised of pure proteins or extracellular matrix components.
  • Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or other ceramics.
  • Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate.
  • the bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradabiUty.
  • CMC carboxymethylcellulose
  • Other prefened sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly( vinyl alcohol).
  • the amount of sequestering agent useful herein is 0.5-20 wt%, preferably 1-10 wt% based on total formulation weight, which represents the amount necessary to prevent desorbtion of the protein from the polymer matrix and to provide appropriate handhng of the composition, yet not so much that the progenitor ceUs are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.
  • proteins of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question.
  • agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF- ⁇ and TGF- ⁇ ), and insuUn-Uke growth factor (IGF).
  • EGF epidermal growth factor
  • PDGF platelet derived growth factor
  • TGF- ⁇ and TGF- ⁇ transforming growth factors
  • IGF insuUn-Uke growth factor
  • the therapeutic compositions are also presently valuable for veterinary appUcations. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins of the present invention.
  • the dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors.
  • the dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition.
  • polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into ceUs for expression in a mammahan subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA). Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proUferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic pu ⁇ oses.
  • IGF I insuUn like growth factor I
  • AJ172_2 a novel human cDNA isolated using a yeast signal sequence trap, encodes a protein that exhibits significant homology to a baboon endogenous retrovirus envelope protein. Genomic DNA sequences flanking the AJ172_2 gene reveal that it is part of a previously uncharacterized defective provirus, indicating that the sequence may be an example of a retroviral gene that has been "captured" by the human host. Southern blot analyses show that AJ172_2- hybridizing sequences are restricted to humans and monkeys; being absent from cows, dogs, rats, mice, rabbits, chickens and yeast.
  • AJ172_2 expression in COS cells reproducibly causes the formation of giant multinucleated COS-ceU syncytia which closely resemble these fused placental syncytiotrophoblasts, suggesting that AJ172_2 plays a role in mediating cell fusion events in human placenta and fusion of other types of cells expressing AJ172_2.
  • a number of independent binary reporter gene approaches indicate that AJ172_2-induced COS syncytia are truly derived from ceU-ceU fusions and do not result from incomplete ceU divisions.
  • AJ172_2 can mediate fusion of many different cell types, including human, monkey, rodent and insect lines.
  • AJ172_2 cDNA encodes a typical signal sequence, an extraceUular domain, a transmembrane domain and a short cytoplasmic tail.
  • the recent appearance in pubhc databases of many very closely related EST sequences derived from many independent cDNA libraries provides additional supporting evidence for the expression of AJ172_2 in vivo.
  • This provirus exhibits a typical mo ⁇ hology; 5' and 3'- long terminal repeat sequences, putative gag and pol genes, and a third open reading frame encoding a putative viral envelope protein.
  • the gag and pol genes are punctuated by multiple chain termination codons and are defective, whereas the third, envelope ORF is intact.
  • This third ORF is identical to AJ172_2, which we conclude to be the envelope gene of an ancient retrovirus.
  • AJ172_2 may play a critical role in the normal placental biology of humans and primates, mediating ceU fusion events which may be important in processes such as blastocyst implantation, the control of uterine wall infiltration by fetal trophoblasts, and in optimizing the efficiency of placental transporter and secretory function.
  • AJ172_2 may thus be the first described example of a captured viral gene performing an important biological role in a mammaUan host organism.
  • Figure 4 demonstrates that AJ172_2 expression in transfected COS cells can cause the formation of giant multinucleated syncytia by a fusigenic mechanism.
  • the cells expressing AJ172_2 can be seen to have formed multinucleate syncytia, while the non-transfected cells remained mononucleate.
  • Figure 5 demonstrates that AJ172_2 mediates actual cell fusion and does not operate through a mechanism of anested cell division.
  • a first cell Une was transfected with AJ172_2, luciferase and ERK.
  • a second cell line was transfected with AJ172_2 and MEK. When the cells were mixed, fusion occuned resulting in production of luciferase activity.
  • FIG. 6 demonstrates that AJ 172_2 can mediate fusion between cells of differing types and between a cell expressing AJ172_2 and a cell not expressing AJ172_2.
  • HELA cells were transfected with a cDNA encoding a P-selectin glycoprotein Ugand-1/Fc fusion protein (PSGL-Fc).
  • COS cells were transfected with AJ172_2.
  • Another batch of COS cells was transfected with AJ172_2 in reverse orientation.
  • the transfected HELA cells were mixed with each type of COS cells. As shown in Figure 6, mixture with the AJ172_ transfected COS cells caused fusion with the HELA cells, resulting in multinucleate fusions.
  • Mixture with the COS cells transfected with A 172_2 in reverse orientation resulted in no fusion (mononucleate cells remained).
  • syncytiotrophoblasts found in the placental vilU form a continuous boundary layer between maternal and fetal tissue. This vitally important structure is responsible for the efficient transfer to the fetus of nutrients, growth factors and antibodies, and for the removal of waste products. It is possible that cytotrophoblast ceU fusion is an adaptation to increase the efficiency of these transport and secretory processes.
  • Harris proposed that the invasiveness of the fetal trophoblast was a trait acquired by a mammaUan antecedent following an ancient retroviral infection. He went on to suggest that this event may even have been seminal in the evolution of aU modern placental mammals.
  • AJ172_2 may be the first example of a viral gene co-opted to serve a vital role in a mammalian host.
  • Trophoblast syncytia may make a number of important contributions to overall placental efficiency. One of these may simply be to provide an extended surface area for maternal fetal exchange. In this regard the theoretical geometry of syncytia formation predicts that the ratio of overall cell surface area to cytoplasmic volume will decrease progressively as cells fuse together.
  • Antagonists to AJ172_2 may be useful as birth control agents, either by preventing initial implantation of embryos into the uterine wall or by interrupting normal placental development and leading to abortion of the conceptus.
  • AJ172_2 Since the regulation of AJ172_2 expression is very tight, small molecules designed to positively or negatively modulate the control of AJ172_2 gene expression may be useful as birth control agents. They may also be useful in treatment of placental pathologies such as pre-eclampsia or choriocarcinoma, where aberrant cytotrophoblast fusion events have been observed.
  • AJ172_2 as a cell fusion agent may have applications as a means of increasing DNA transfection efficiencies in vitro (research applications, moving genes into cultured cell Unes or primary cell Unes with greater efficiency) or in vivo (gene therapy applications, moving genes into cells in the intact organism with greater efficiency). AJ172_2 may also be impUcated in osteoclast fusion. Therefore, AJ172_2 antagonists or small molecules directed to control of AJ172_2 gene expression may be useful in the treatment of bone disorders such as osteoporosis or osteopetrosis.
  • BeWo is a human trophoblastic choriocarcinoma line which can be induced by forskolin to form syncytiotrophblasts.
  • BeWo-derived syncytiotrophblasts are morpologically very similar to AJ172_2-fused COS cells.
  • Figure 8 shows that AJ172_2 transcription in BeWo cells increases at least five fold in response to forskolin treatment, correlating well with cell fusion ( Figure 9).
  • pre-eclampsia is disregulated in pre-eclampsia. This would indicate that pre-eclamsia and other diseases of placental morphogenesis may be caused by temporal or spatial disregulation of AJ172 expression, quantitative disregulation in AJ172 expression levels, or by mutations in the AJ172 gene.
  • antibodies to AJ172 are useful in detecting serum levels of AJ172, and thus are useful as a dignostic tools to monitor for pre-eclampsia, for other placental pathologies or for cancerous conditions.
  • Agents modulating AJ172 expression or function have therapeutic potential in treatment of neoplastic diseases in addition to choriocarcinoma.
  • Patent and Uterature references cited herein are inco ⁇ orated by reference as if fully set forth.

Abstract

Novel polynucleotides and the proteins encoded thereby are disclosed.

Description

SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
This application is a continuation-in-part of application Ser. No. 09/080,478, filed May 18,
1998, which was a continuation-in-part of application Ser. No. 08/976,110, filed November 21, 1997, which was a continuation-in-part of the following applications: Ser. No. 08/686,878, filed July 26, 1996 and issued as U.S. Patent No. 5,708,157 on January 13, 1998; Ser. No. 08/702,081, filed August 23, 1996, which is a continuation-in-part of Ser. No. 08/686,878; Ser. No. 08/721,489, filed September 27, 1996 and issued as U.S. Patent No. 5,786,465 on July 28, 1998, which was a continuation-in-part of Ser. No. 08/686,878; and 08/721,924, filed September 27, 1996, which is a continuation-in-part of Ser. No. 08/686,878. All of such applications are incorporated by reference herein.
FIELD OF THE INVENTION
The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.
BACKGROUND OF THE INVENTION
Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, CSFs and interleukins) has matured rapidly over the past decade. The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides "directly" in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning). More recent "indirect" cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity by virtue of their secreted nature in the case of leader sequence cloning, or by virtue of the cell or tissue source in the case of PCR-based techniques. It is to these proteins and the polynucleotides encoding them that the present invention is directed. SUMMARY OF THE INVENTION In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 ;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 44 to nucleotide 1204;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 1 to nucleotide 403; (d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone AJ26_3 deposited under accession number ATCC 98115;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone AJ26_3 deposited under accession number ATCC 98115;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AJ26_3 deposited under accession number ATCC 98115;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AJ26_3 deposited under accession number ATCC 98115;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2; (i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 2 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:l from nucleotide 44 to nucleotide 1204; the nucleotide sequence of SEQ ID NO:l from nucleotide 1 to nucleotide 403; the nucleotide sequence of the full-length protein coding sequence of clone AJ26_3 deposited under accession number ATCC 98115; or the nucleotide sequence of the mature protein coding sequence of clone AJ26_3 deposited under accession number ATCC 98115. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone AJ26_3 deposited under accession number ATCC 98115. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 120.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:l. In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:2;
(b) the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 120; (c) fragments of the amino acid sequence of SEQ ID NO:2; and
(d) the amino acid sequence encoded by the cDNA insert of clone AJ26_3 deposited under accession number ATCC 98115; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:2 or the amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid 120.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 928 to nucleotide 2541 ;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3 from nucleotide 988 to nucleotide 2541;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 684 to nucleotide 1128; (e) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone AJ172_2 deposited under accession number ATCC 98115;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone AJ172_2 deposited under accession number ATCC 98115; (g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AJ172_2 deposited under accession number ATCC 98115;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AJ172_2 deposited under accession number ATCC 98115;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4; (j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above; (1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:3 from nucleotide 928 to nucleotide 2541 ; the nucleotide sequence of SEQ ID NO:3 from nucleotide 988 to nucleotide 2541; the nucleotide sequence of SEQ ID NO:3 from nucleotide 684 to nucleotide 1128; the nucleotide sequence of the full-length protein coding sequence of clone AJ172_2 deposited under accession number ATCC 98115; or the nucleotide sequence of the mature protein coding sequence of clone AJ172_2 deposited under accession number ATCC 98115. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone AJ172_2 deposited under accession number ATCC 98115. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4 from amino acid 1 to amino acid 67.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:3.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:4;
(b) the amino acid sequence of SEQ ID NO:4 from amino acid 1 to amino acid 67;
(c) fragments of the amino acid sequence of SEQ ID NO:4; and
(d) the amino acid sequence encoded by the cDNA insert of clone AJ172_2 deposited under accession number ATCC 98115; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:4 or the amino acid sequence of SEQ ID NO:4 from amino acid 1 to amino acid 67.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:6; (b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:6 from nucleotide 185 to nucleotide 385;
(c) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone AP224_2 deposited under accession number ATCC 98115;
(d) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone AP224_2 deposited under accession number ATCC 98115;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AP224_2 deposited under accession number ATCC 98115; (f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AP224_2 deposited under accession number ATCC 98115;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:7;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:7 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(f) above;
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above ; and (k) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:6 from nucleotide 185 to nucleotide 385; the nucleotide sequence of the full-length protein coding sequence of clone AP224_2 deposited under accession number ATCC 98115; or the nucleotide sequence of the mature protein coding sequence of clone AP224_2 deposited under accession number ATCC 98115. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone AP224_2 deposited under accession number ATCC 98115. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:7 from amino acid 1 to amino acid 28.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:6, SEQ ID NO:5 or SEQ ID NO:8 .
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:7; (b) the amino acid sequence of SEQ ED NO:7 from amino acid 1 to amino acid 28;
(c) fragments of the amino acid sequence of SEQ ID NO:7; and
(d) the amino acid sequence encoded by the cDNA insert of clone AP224_2 deposited under accession number ATCC 98115; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: 7 from amino acid 1 to amino acid 28.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 6 to nucleotide 2408;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 1295 to nucleotide 1705;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone BL89_13 deposited under accession number ATCC 98153;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone BL89_13 deposited under accession number ATCC 98153;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone BL89_13 deposited under accession number ATCC 98153;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone BL89_13 deposited under accession number ATCC 98153; (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 10;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 10 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i). Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:9 from nucleotide 6 to nucleotide 2408; the nucleotide sequence of SEQ ED NO:9 from nucleotide 1295 to nucleotide 1705; the nucleotide sequence of the full-length protein coding sequence of clone BL89_13 deposited under accession number ATCC 98153; or the nucleotide sequence of the mature protein coding sequence of clone BL89_13 deposited under accession number ATCC 98153. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone BL89_13 deposited under accession number ATCC 98153. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 10 from amino acid 431 to amino acid 567. Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID
NO:9.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ED NO: 10; (b) the amino acid sequence of SEQ ID NO: 10 from amino acid 431 to amino acid 567;
(c) fragments of the amino acid sequence of SEQ ID NO: 10; and
(d) the amino acid sequence encoded by the cDNA insert of clone BL89_13 deposited under accession number ATCC 98153; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 10 from amino acid 431 to amino acid 567.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 11 ;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ED NO: 11 from nucleotide 2113 to nucleotide 2337;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 11 from nucleotide 2036 to nucleotide 2316; (d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone BL341_4 deposited under accession number ATCC 98115;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone BL341_4 deposited under accession number ATCC 98115; (f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone BL341_4 deposited under accession number ATCC 98115;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone BL341_4 deposited under accession number ATCC 98115; (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 12 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i). Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ED NO: 11 from nucleotide 2113 to nucleotide 2337; the nucleotide sequence of SEQ ID NO: 11 from nucleotide
2036 to nucleotide 2316; the nucleotide sequence of the full-length protein coding sequence of clone BL341_4 deposited under accession number ATCC 98115; or the nucleotide sequence of the mature protein coding sequence of clone BL341_4 deposited under accession number ATCC 98115. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone BL341_4 deposited under accession number ATCC 98115.
In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12 from amino acid 1 to amino acid 68.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ED NO:l l.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 12;
(b) the amino acid sequence of SEQ ID NO: 12 from amino acid 1 to amino acid 68;
(c) fragments of the amino acid sequence of SEQ ED NO: 12; and
(d) the amino acid sequence encoded by the cDNA insert of clone BL341_4 deposited under accession number ATCC 98115; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ED NO: 12 or the amino acid sequence of SEQ ID NO: 12 from amino acid 1 to amino acid 68.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 13;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ED NO: 13 from nucleotide 1 to nucleotide 390;
(c) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone BV239_3 deposited under accession number ATCC
98153;
(d) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone BV239_3 deposited under accession number ATCC 98153;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone BV239_3 deposited under accession number ATCC 98153;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone BV239_3 deposited under accession number ATCC 98153;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 14; (h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 14 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(f) above;
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above ; and
(k) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 13 from nucleotide 1 to nucleotide 390; the nucleotide sequence of the full-length protein coding sequence of clone BV239_3 deposited under accession number ATCC 98153; or the nucleotide sequence of the mature protein coding sequence of clone BV239_3 deposited under accession number ATCC 98153. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone BV239_3 deposited under accession number ATCC 98153. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 14 from amino acid 50 to amino acid 130.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO.13. In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 14;
(b) the amino acid sequence of SEQ ED NO: 14 from amino acid 50 to amino acid 130; (c) fragments of the amino acid sequence of SEQ ID NO: 14; and
(d) the amino acid sequence encoded by the cDNA insert of clone BV239_3 deposited under accession number ATCC 98153; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO: 14 or the amino acid sequence of SEQ ID NO: 14 from amino acid 50 to amino acid 130.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 15 ;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 15 from nucleotide 144 to nucleotide 257;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ED NO: 15 from nucleotide 30 to nucleotide 271;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone CC25_17 deposited under accession number ATCC 98153;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone CC25_17 deposited under accession number ATCC 98153;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone CC25_17 deposited under accession number ATCC 98153; (g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone CC25_17 deposited under accession number ATCC 98153;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ED NO:16;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ED NO: 16 having biological activity; (j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 15 from nucleotide 144 to nucleotide 257; the nucleotide sequence of SEQ ID NO: 15 from nucleotide 30 to nucleotide 271; the nucleotide sequence of the full-length protein coding sequence of clone CC25_17 deposited under accession number ATCC 98153; or the nucleotide sequence of the mature protein coding sequence of clone CC25_17 deposited under accession number ATCC 98153. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone CC25_17 deposited under accession number ATCC 98153. Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ED
NO:15.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ED NO:16; (b) fragments of the amino acid sequence of SEQ ID NO: 16; and
(c) the amino acid sequence encoded by the cDNA insert of clone CC25_17 deposited under accession number ATCC 98153; the protein being substantially free from other mammahan proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO: 16. In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 17;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 17 from nucleotide 431 to nucleotide 520; (c) a polynucleotide comprising the nucleotide sequence of SEQ ED NO: 17 from nucleotide 266 to nucleotide 511;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone CC397_19 deposited under accession number ATCC 98153; (e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone CC397_19 deposited under accession number ATCC 98153;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone CC397_19 deposited under accession number ATCC 98153; (g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone CC397_19 deposited under accession number ATCC 98153;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:18;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 18 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 17 from nucleotide 431 to nucleotide 520; the nucleotide sequence of SEQ ED NO: 17 from nucleotide 266 to nucleotide 511; the nucleotide sequence of the full-length protein coding sequence of clone CC397_19 deposited under accession number ATCC 98153; or the nucleotide sequence of the mature protein coding sequence of clone CC397_19 deposited under accession number ATCC 98153. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone CC397_19 deposited under accession number ATCC 98153. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ED NO: 18 from amino acid 1 to amino acid 27.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:17.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ED NO: 18 ;
(b) the amino acid sequence of SEQ ID NO: 18 from amino acid 1 to amino acid 27;
(c) fragments of the amino acid sequence of SEQ ID NO: 18; and (d) the amino acid sequence encoded by the cDNA insert of clone CC397_19 deposited under accession number ATCC 98153; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ D NO: 18 or the amino acid sequence of SEQ ID NO: 18 from amino acid 1 to amino acid 27.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:20;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ED NO: 20 from nucleotide 253 to nucleotide 519;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ED NO:20 from nucleotide 298 to nucleotide 519;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone D305_2 deposited under accession number ATCC 98115; (e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone D305_2 deposited under accession number ATCC 98115;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone D305_2 deposited under accession number ATCC 98115;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone D305_2 deposited under accession number ATCC 98115 ;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:21;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ED NO:21 having biological activity; (j) ' polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ED NO:20 from nucleotide 253 to nucleotide 519; the nucleotide sequence of SEQ ID NO:20 from nucleotide 298 to nucleotide 519; the nucleotide sequence of the full-length protein coding sequence of clone
D305_2 deposited under accession number ATCC 98115 ; or the nucleotide sequence of the mature protein coding sequence of clone D305_2 deposited under accession number ATCC 98115. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone D305_2 deposited under accession number ATCC 98115.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ED NO:20, SEQ ED NO: 19 or SEQ DD NO:22 . In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:21 ;
(b) fragments of the amino acid sequence of SEQ ED NO:21 ; and
(c) the amino acid sequence encoded by the cDNA insert of clone D305_2 deposited under accession number ATCC 98115; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:21.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:23;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:23 from nucleotide 194 to nucleotide 622;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ED NO:23 from nucleotide 524 to nucleotide 622; (d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone G55_l deposited under accession number ATCC 98115 ;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone G55_l deposited under accession number ATCC 98115;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone G55_l deposited under accession number ATCC 98115;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone G55_l deposited under accession number ATCC 98115;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:24; (i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ED NO:24 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:23 from nucleotide 194 to nucleotide 622; the nucleotide sequence of SEQ ED NO:23 from nucleotide 524 to nucleotide 622; the nucleotide sequence of the full-length protein coding sequence of clone G55_l deposited under accession number ATCC 98115 ; or the nucleotide sequence of the mature protein coding sequence of clone G55_l deposited under accession number ATCC 98115. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone G55_l deposited under accession number ATCC 98115. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:24 from amino acid 1 to amino acid 32.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ED NO:23 or SEQ ED NO:25.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ED NO:24;
(b) the amino acid sequence of SEQ ID NO:24 from amino acid 1 to amino acid 32;
(c) fragments of the amino acid sequence of SEQ ID NO:24; and (d) the amino acid sequence encoded by the cDNA insert of clone G55_l deposited under accession number ATCC 98115; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:24 or the amino acid sequence of SEQ ID NO:24 from amino acid 1 to amino acid 32. In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:26;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:26 from nucleotide 402 to nucleotide 533; (c) a polynucleotide comprising the nucleotide sequence of SEQ ED NO:26 from nucleotide 447 to nucleotide 533;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone K39_7 deposited under accession number ATCC 98115;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone K39_7 deposited under accession number ATCC 98115 ; (f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone K39_7 deposited under accession number ATCC 98115;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone K39_7 deposited under accession number ATCC 98115; (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ED NO:27;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ED NO:27 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i). Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:26 from nucleotide 402 to nucleotide 533; the nucleotide sequence of SEQ ID NO:26 from nucleotide 447 to nucleotide 533; the nucleotide sequence of the full-length protein coding sequence of clone
K39_7 deposited under accession number ATCC 98115; or the nucleotide sequence of the mature protein coding sequence of clone K39_7 deposited under accession number ATCC 98115. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone K39_7 deposited under accession number ATCC 98115.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:26 or SEQ ID NO:28.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:27;
(b) fragments of the amino acid sequence of SEQ ID NO:27; and
(c) the amino acid sequence encoded by the cDNA insert of clone K39_7 deposited under accession number ATCC 98115; the protein being substantially free from other mammaUan proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:27.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ED NO:29; (b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:29 from nucleotide 241 to nucleotide 525;
(c) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone K330_3 deposited under accession number ATCC 98115; (d) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone K330_3 deposited under accession number ATCC 98115;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone K330_3 deposited under accession number ATCC 98115;
(f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone K330_3 deposited under accession number ATCC 98115;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ED NO:30;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ D NO:30 having biological activity; (i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(f) above;
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above ; and
(k) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ED NO:29 from nucleotide 241 to nucleotide 525; the nucleotide sequence of the full-length protein coding sequence of clone K330_3 deposited under accession number ATCC 98115; or the nucleotide sequence of the mature protein coding sequence of clone K330_3 deposited under accession number ATCC 98115. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone K330_3 deposited under accession number ATCC 98115. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ED NO:30 from amino acid 1 to amino acid 35. Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID
NO:29 or SEQ ID NO:31.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:30; (b) the amino acid sequence of SEQ ID NO:30 from amino acid 1 to amino acid 35;
(c) fragments of the amino acid sequence of SEQ ED NO:30; and
(d) the amino acid sequence encoded by the cDNA insert of clone K330_3 deposited under accession number ATCC 98115; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:30 or the amino acid sequence of SEQ ED NO:30 from amino acid 1 to amino acid 35.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ED NO:32;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ED NO:32 from nucleotide 158 to nucleotide 571;
(c) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone K363_3 deposited under accession number ATCC 98115;
(d) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone K363_3 deposited under accession number ATCC 98115;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone K363_3 deposited under accession number ATCC 98115; (f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone K363_3 deposited under accession number ATCC 98115;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:33;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ED NO:33 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(f) above;
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above ; and (k) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:32 from nucleotide 158 to nucleotide 571; the nucleotide sequence of the full-length protein coding sequence of clone K363_3 deposited under accession number ATCC 98115; or the nucleotide sequence of the mature protein coding sequence of clone K363_3 deposited under accession number ATCC 98115. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone K363_3 deposited under accession number ATCC 98115. In yet other prefened embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ED NO:33 from amino acid 24 to amino acid 96.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:32 or SEQ ID NO:34.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ED NO:33;
(b) the amino acid sequence of SEQ ED NO:33 from amino acid 24 to amino acid 96;
(c) fragments of the amino acid sequence of SEQ ED NO:33; and
(d) the amino acid sequence encoded by the cDNA insert of clone K363_3 deposited under accession number ATCC 98115; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ED NO:33 or the amino acid sequence of SEQ ED NO:33 from amino acid 24 to amino acid 96.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:35;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ED NO:35 from nucleotide 401 to nucleotide 526;
(c) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone K446_3 deposited under accession number ATCC 98115;
(d) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone K446_3 deposited under accession number ATCC 98115;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone K446_3 deposited under accession number ATCC 98115; (f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone K446_3 deposited under accession number ATCC 98115;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:36;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:36 having biological activity; (i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(f) above;
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above ; and (k) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:35 from nucleotide 401 to nucleotide 526; the nucleotide sequence of the full-length protein coding sequence of clone K446_3 deposited under accession number ATCC 98115; or the nucleotide sequence of the mature protein coding sequence of clone K446_3 deposited under accession number ATCC 98115. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone K446_3 deposited under accession number ATCC 98115.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ID NO:35 or SEQ ID NO:37.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ED NO:36;
(b) fragments of the amino acid sequence of SEQ ID NO:36; and (c) the amino acid sequence encoded by the cDNA insert of clone K446_3 deposited under accession number ATCC 98115; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ED NO:36.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:38;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:38 from nucleotide 380 to nucleotide 535;
(c) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone K464_4 deposited under accession number ATCC 98115;
(d) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone K464_4 deposited under accession number ATCC 98115;
(e) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone K464_4 deposited under accession number ATCC 98115; (f) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone K464_4 deposited under accession number ATCC 98115;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:39; (h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:39 having biological activity;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(f) above;
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above ; and
(k) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:38 from nucleotide 380 to nucleotide 535; the nucleotide sequence of the full-length protein coding sequence of clone K464_4 deposited under accession number ATCC 98115; or the nucleotide sequence of the mature protein coding sequence of clone K464_4 deposited under accession number ATCC 98115. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone K464_4 deposited under accession number ATCC 98115. Other embodiments provide the gene corcesponding to the cDNA sequence of SEQ ID
NO:38 or SEQ ID NO:40.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:39; (b) fragments of the amino acid sequence of SEQ ID NO:39; and
(c) the amino acid sequence encoded by the cDNA insert of clone K464_4 deposited under accession number ATCC 98115; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ED NO:39. In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:41 ;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ED NO:41 from nucleotide 218 to nucleotide 1159; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:41 from nucleotide 806 to nucleotide 1159;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ED NO:41 from nucleotide 217 to nucleotide 517; (e) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone K483_l deposited under accession number ATCC 98115;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone K483_l deposited under accession number ATCC 98115;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone K483_l deposited under accession number ATCC 98115;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone K483_l deposited under accession number ATCC 98115;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:42; (j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:42 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:41 from nucleotide 218 to nucleotide 1159; the nucleotide sequence of SEQ ID NO:41 from nucleotide 806 to nucleotide 1159; the nucleotide sequence of SEQ ED NO:41 from nucleotide 217 to nucleotide 517; the nucleotide sequence of the full-length protein coding sequence of clone K483_l deposited under accession number ATCC 98115; or the nucleotide sequence of the mature protein coding sequence of clone K483_l deposited under accession number ATCC 98115. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone K483_l deposited under accession number ATCC 98115. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ED NO:42 from amino acid 1 to amino acid 100.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ ED NO:41. In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:42;
(b) the amino acid sequence of SEQ ID NO:42 from amino acid 1 to amino acid 100;
(c) fragments of the amino acid sequence of SEQ ED NO:42; and
(d) the amino acid sequence encoded by the cDNA insert of clone K483_l deposited under accession number ATCC 98115; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:42 or the amino acid sequence of SEQ ID NO:42 from amino acid 1 to amino acid 100.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ED NO:43; (b) a polynucleotide comprising the nucleotide sequence of SEQ ED NO:43 from nucleotide 446 to nucleotide 835;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ED NO:43 from nucleotide 503 to nucleotide 835;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone L69_3 deposited under accession number ATCC 98115;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone L69_3 deposited under accession number ATCC 98115;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone L69_3 deposited under accession number ATCC 98115; (g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone L69_3 deposited under accession number ATCC 98115;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ D NO:44;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ED NO:44 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:43 from nucleotide 446 to nucleotide 835; the nucleotide sequence of SEQ ID NO:43 from nucleotide 503 to nucleotide 835; the nucleotide sequence of the full-length protein coding sequence of clone L69_3 deposited under accession number ATCC 98115; or the nucleotide sequence of the mature protein coding sequence of clone L69_3 deposited under accession number ATCC 98115. In other preferred embodiments, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone L69_3 deposited under accession number ATCC 98115. In yet other prefened embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ED NO:44 from amino acid 1 to amino acid 93.
Other embodiments provide the gene conesponding to the cDNA sequence of SEQ ID NO:43 or SEQ ID NO:45.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ED NO:44;
(b) the amino acid sequence of SEQ ED NO:44 from amino acid 1 to amino acid 93;
(c) fragments of the amino acid sequence of SEQ ED NO:44; and (d) the amino acid sequence encoded by the cDNA insert of clone L69_3 deposited under accession number ATCC 98115; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:44 or the amino acid sequence of SEQ ID NO:44 from amino acid 1 to amino acid 93. In certain prefened embodiments, the polynucleotide is operably linked to an expression control sequence. The invention also provides a host cell, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions.
Processes are also provided for producing a protein, which comprise:
(a) growing a culture of the host cell transformed with such polynucleotide compositions in a suitable culture medium; and
(b) purifying the protein from the culture.
The protein produced according to such methods is also provided by the present invention. Prefened embodiments include those in which the protein produced by such process is a mature form of the protein. Protein compositions of the present invention may further comprise a pharmaceutically acceptable carrier. Compositions comprising an antibody which specifically reacts with such protein are also provided by the present invention.
Methods are also provided for preventing, treating or ameliorating a medical condition which comprises administering to a mammahan subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable caπier.
Also disclosed are methods of promoting cell-cell fusion. Such a method comprises contacting a first cell and a second cell, wherein said first cell expresses an AJ172_2 protein. AJ172_2 expression in the first cell can occur naturally or be the result of transfection with a polynucleotide encoding an AJ172_2 protein. Preferably, the first cell is transfected with a polynucleotide or gene described above. The first cell and second cell can be of the same type or of different types. In other embodiments, at least one of said first cell and said second cell are transfected to express an additional protein other than the AJ172_2 protein.
In yet other embodiments, a method of inhibiting cell-cell fusion between a first cell which expresses an AJ172_2 protein and a second cell is disclosed, wherein the method comprises contacting said first cell with an AJ172_2 protein antagonist. Preferably, the antagonist is selected from the group consisting of an antibody or antibody fragment directed to an AJ172_2 protein, an antisense polynucleotide directed to a polynucleotide expressing an AJ172_2 protein, a nucleotide aptamer directed to an AJ172_2 protein, a peptide aptamer directed to an AJ172_2 protein and a small molecule which blocks the fusion-inducing activity of an AJ172_2 protein. In other preferred embodiments, the first cell is a placental cell (such as a cytotrophoblast) and the second cell is a cell from the maternal uterine lining.
Other embodiments provide for a method of inhibiting blastocyst implantation, wherein the method comprises contacting a cell within said blastocyst which expresses an AJ172_2 protein with an AJ 172_2 protein antagonist.
Yet other embodiments provide for a method of inhibiting trophoblast invasion, wherein the method comprises contacting a first cell which expresses an AJ172_2 protein with an AJ172_2 protein antagonist.
Further embodiments provide for a method of diagnosing or predicting the existence of a condition associated with disregulation of AH172_2 protein in a mammahan subject, such method comprising (a) determining a first level of expression of AJ172_2 protein in the subject, and (b) comparing such first level of expression to a second level of expression of AJ172_2 protein in one or more other mammalian subjects which do not have said condition. Preferably, the condition is selected from the group consisting of pre-eclampsia, placental pathology and cancer (including choriocarcinoma). In prefened embodiments, such first level of expression is determined in the serum of the subject, using an antibody or antibody fragment directed to AJ172_2 protein.
Other embodiments provide for a method of treating a neoplastic disease (including choriocarcinoma) in a mammahan subject, such method comprising administering to said subject a therapeutically effective amount of an agent which promotes the expression or function of AJ172_2.
Yet other embodiments provide for a method of inhibiting metastasis in a mammahan subject, such method comprising administering to the subject a therapeutically effective amount of an agent which inhibits the expression or function of AJ172_2.
BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and IB are schematic representations of the pED6 and pNOTs vectors, respectively, used for deposit of clones disclosed herein.
Figure 2 presents the results of Northern analyses of multiple human tissues which indicate that AJ172_2 is expressed highly in the placenta and weakly in the testes.
Figure 3 presents the results of in situ antisense-RNA hybridizations which localize AJ172_2 expression to placental syncytiotrophoblasts. Figure 4 demonstrates that AJ172_2 expression in transfected COS cells can cause the formation of giant multinucleated syncytia by a fusigenic mechanism. The cells expressing AJ172_2 can be seen to have formed multinucleate syncytia, while the non- transfected cells remained mononucleate.
Figure 5 demonstrates that AJ172_2 mediates actual cell fusion and does not operate through a mechanism of arrested cell division. A first cell line was transfected with AJ172_2, luciferase and ERK. A second cell line was transfected with AJ172_2 and MEK. When the cells were mixed, fusion occurred resulting in production of luciferase activity.
Figure 6 demonstrates that AJ172_2 can mediate fusion between cells of differing types and between a cell expressing AJ172_2 and a cell not expressing AJ172_2. HELA cells were transfected with a cDNA encoding a P-selectin glycoprotein ligand- 1/Fc fusion protein (PSGL-Fc). COS cells were transfected with AJ172_2. Another batch of COS cells was transfected with AJ172_2 in reverse orientation. The transfected HELA cells were mixed with each type of COS cells. As shown in Figure 6, mixture with the AJ172_2 transfected COS cells caused fusion with the HELA cells, resulting in multinucleate fusions. Mixture with the COS cells transfected with AJ172_2 in reverse orientation resulted in no fusion (mononucleate cells remained). Figure 7 demonstrates that the mechanism of AJ 172_2 induced cell fusion does not require homophilic or heterophilic protein-protein interactions. COS cells transfected with AJ172_2 were mixed with liposomes containing a green fluorescent protein (GFP) expression plasmid. As shown in Figure 7, the COS cells fused with the liposomes, took up the expression plasmid, and began expressing GFP. Figures 8-10 present data which demonstrate that AJ172_2 is expressed in the formation of cytotrophoblasts associated with choriocarcinoma (see Example 2).
Figures 11 and 12 present data which demonstrate that AJ172_2 is disregulated in pre- eclampsia (see Example 3).
Figures 13 and 14 present data which demonstrate activity of AJ172_2 in remodeling of extracellular matrices (see Example 4).
DETAILED DESCRIPTION ISOLATED PROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequences, as presently determined, are reported below for each clone and protein disclosed in the present application. The nucleotide sequence of each clone can readily be determined by sequencing of the deposited clone in accordance with known methods. The predicted amino acid sequence (both full-length and mature) can then be determined from such nucleotide sequence. The amino acid sequence of the protein encoded by a particular clone can also be determined by expression of the clone in a suitable host cell, collecting the protein and determining its sequence. For each disclosed protein applicants have identified what they have determined to be the reading frame best identifiable with sequence information available at the time of filing.
As used herein a "secreted" protein is one which, when expressed in a suitable host cell, is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence. "Secreted" proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g. , receptors) from the cell in which they are expressed. "Secreted" proteins also include without limitation proteins which are transported across the membrane of the endoplasmic reticulum. Clone "AJ26 3"
A polynucleotide of the present invention has been identified as clone "AJ26_3". AJ26_3 was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. AJ26_3 is a full-length clone, including the entire coding sequence of a secreted protein (also refened to herein as "AJ26_3 protein").
The nucleotide sequence of AJ26_3 as presently determined is reported in SEQ ED NO: 1. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the AJ26_3 protein conesponding to the foregoing nucleotide sequence is reported in SEQ ID NO:2.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone AJ26_3 should be approximately 2100 bp.
The nucleotide sequence disclosed herein for AJ26_3 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. AJ26_3 demonstrated at least some similarity with sequences identified as U46493 (Cloning vector pFlp recombinase gene, complete eds). The predicted amino acid sequence disclosed herein for AJ26_3 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted AJ26_3 protein demonstrated at least some similarity to sequences identified as J01917 (DNA polymerase [Human adenovirus type 2]), J01969 (DNA polymerase [Human adenovirus type 5]), L24893 (HUMAAC02_1 myelin protein zero [Homo sapiens]), U43330 (CTX [Xenopus laevis]), and U43394 (CTX [Xenopus laevis]). Based upon sequence similarity, AJ26_3 proteins and each similar protein or peptide may share at least some activity. The TopPredϋ computer program predicts four potential transmembrane domains within the AJ26_3 protein sequence, centered around amino acids 11, 41, 163, and 246 of SEQ ID NO:2. The AJ26_3 protein also has a possible signal sequence that could be cleaved to produce a mature protein starting at amino acid 17 of SEQ ID NO:2.
Clone "AJ172 2" A polynucleotide of the present invention has been identified as clone "AJ172_2".
AJ172_2 was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. AJ172_2 is a full-length clone, including the entire coding sequence of a secreted protein (also refened to herein as "AJ172_2 protein"). The nucleotide sequence of AJ172_2 as presently determined is reported in SEQ ED NO:3.
What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the AJ172_2 protein conesponding to the foregoing nucleotide sequence is reported in SEQ ED NO:4. Amino acids 8 to 20 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 21, or are a transmembrane domain.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone AJ172_2 should be approximately 3000 bp.
The nucleotide sequence disclosed herein for AJ172_2 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. AJ172_2 demonstrated at least some similarity with sequences identified as AA077794 (7H01C09 Chromosome 7 HeLa cDNA Library Homo sapiens cDNA clone 7H01C09), AC000064 (Human BAC clone RG083M05 from 7q21-7q22, complete sequence), D78692 (Human placenta cDNA 5'-end GEN-503H08), H12439 (yjllhlO.rl Homo sapiens cDNA clone 148483 5'), R27389 (yh46a09.sl Homo sapiens cDNA clone 132760 3'), and T09280 (Novel AMP/MCF virus clone 24 genome). The predicted amino acid sequence disclosed herein for AJ 172_2 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted AJ172_2 protein demonstrated at least some similarity to sequences identified as M26927 (pol polyprotein [Gibbon leukemia virus]), M93134 (pol protein [Friend murine leukemia virus]), and R75189 (Osteoinductive retrovirus RFB-14 pol gene product). AJ172_2 protein is similar to a number of viral env proteins, including those of baboon endogenous virus and many leukemia viruses, which associate with the membrane portion of the viral envelope. Based upon sequence similarity, AJ172_2 proteins and each similar protein or peptide may share at least some activity. The TopPredϋ computer program predicts five potential transmembrane domains within the AJ172_2 protein sequence, centered around amino acids 104, 267, 292, 328, and 457 of SEQ ID NO:4.
Clone "AP224 2"
A polynucleotide of the present invention has been identified as clone "AP224_2". AP224_2 was isolated from a human adult placenta cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. AP224_2 is a full-length clone, including the entire coding sequence of a secreted protein (also refened to herein as "AP224_2 protein").
The nucleotide sequence of the 5' portion of AP224_2 as presently determined is reported in SEQ ID NO:5. An additional internal nucleotide sequence from AP224_2 as presently determined is reported in SEQ ED NO:6. What applicants believe is the proper reading frame and the predicted amino acid sequence encoded by such internal sequence is reported in SEQ ED NO:7. Additional nucleotide sequence from the 3' portion of AP224_2, including the polyA tail, is reported in SEQ ID NO:8. The EcoRI/Notl restriction fragment obtainable from the deposit containing clone
AP224_2 should be approximately 2100 bp.
The nucleotide sequence disclosed herein for AP224_2 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. AP224_2 demonstrated at least some similarity with sequences identified as R37675 (yf61f08.sl Homo sapiens cDNA clone 26687 3'). Based upon sequence similarity, AP224_2 proteins and each similar protein or peptide may share at least some activity.
Clone "BL89 13"
A polynucleotide of the present invention has been identified as clone "BL89_13". BL89_13 was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. BL89_13 is a full-length clone, including the entire coding sequence of a secreted protein (also refened to herein as "BL89_13 protein"). The nucleotide sequence of BL89_13 as presently determined is reported in SEQ ED NO:9.
What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the BL89_13 protein conesponding to the foregoing nucleotide sequence is reported in SEQ ID NO: 10.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone BL89_13 should be approximately 3200 bp.
The nucleotide sequence disclosed herein for BL89_13 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. No hits were found in the database. The TopPredll computer program predicts a potential transmembrane domain within the BL89_13 protein sequence centered around amino acid 625 of SEQ ID NO:10.
Clone "BL341 4"
A polynucleotide of the present invention has been identified as clone "BL341_4". BL341_4 was isolated from a human adult testes cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. BL341_4 is a full-length clone, including the entire coding sequence of a secreted protein (also refened to herein as "BL341_4 protein"). The nucleotide sequence of BL341 _4 as presently determined is reported in SEQ ED NO : 11.
What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the BL341_4 protein conesponding to the foregoing nucleotide sequence is reported in SEQ ED NO: 12.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone BL341_4 should be approximately 2600 bp.
The nucleotide sequence disclosed herein for BL341_4 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. BL341_4 demonstrated at least some similarity with sequences identified as AA460103 (zx50al2.rl Soares testis NHT Homo sapiens cDNA clone) and Z63359 (H.sapiens CpG island DNA genomic Msel fragment, clone 81e7, reverse read cpg81e7.rtla). Based upon sequence similarity, BL341_4 proteins and each similar protein or peptide may share at least some activity.
Clone "BV239 3"
A polynucleotide of the present invention has been identified as clone "BV239_3". BV239_3 was isolated from a human adult brain cDNA hbrary using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. BV239_3 is a full-length clone, including the entire coding sequence of a secreted protein (also refened to herein as "BV239_3 protein"). The nucleotide sequence of BV239_3 as presently determined is reported in SEQ ED
NO:13. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the BV239_3 protein conesponding to the foregoing nucleotide sequence is reported in SEQ ID NO: 14.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone BV239_3 should be approximately 310 bp.
The nucleotide sequence disclosed herein for BV239_3 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. BV239_3 demonstrated at least some similarity with sequences identified as U46493
(Cloning vector pFlp recombinase gene, complete eds). Based upon sequence similarity, BV239_3 proteins and each similar protein or peptide may share at least some activity. Clone "CC25 17"
A polynucleotide of the present invention has been identified as clone "CC25_17". CC25_17 was isolated from a human adult brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. CC25_17 is a full-length clone, including the entire coding sequence of a secreted protein (also refened to herein as "CC25_17 protein").
The nucleotide sequence of CC25_17 as presently determined is reported in SEQ ID NO: 15. What appUcants presently beUeve to be the proper reading frame and the predicted amino acid sequence of the CC25_17 protein conesponding to the foregoing nucleotide sequence is reported in SEQ ID NO: 16.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone CC25_17 should be approximately 300 bp.
The nucleotide sequence disclosed herein for CC25_17 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. CC25_17 demonstrated at least some similarity with sequences identified as U46493
(Cloning vector pFlp recombinase gene, complete eds). Based upon sequence similarity, CC25_17 proteins and each similar protein or peptide may share at least some activity.
Clone "CC397 19"
A polynucleotide of the present invention has been identified as clone "CC397_19". CC397_19 was isolated from a human adult brain cDNA hbrary using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. CC397_19 is a full-length clone, including the entire coding sequence of a secreted protein (also refened to herein as "CC397_19 protein").
The nucleotide sequence of CC397_19 as presently determined is reported in SEQ ED NO: 17. What appUcants presently beUeve to be the proper reading frame and the predicted amino acid sequence of the CC397_19 protein conesponding to the foregoing nucleotide sequence is reported in SEQ ID NO: 18.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone CC397_19 should be approximately 1700 bp. The nucleotide sequence disclosed herein for CC397_19 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. CC397_19 demonstrated at least some similarity with sequences identified as AC002129
(Human DNA from chromsome 19 cosmid R33743, genomic sequence, complete sequence), D82019 (Mouse gene for basigin precursor, basigin signal precursor), G08688 (human STS
CHLC.GATA29D08.P14592 clone GATA29D08), M68516 (Human protein C inhibitor gene, complete eds), and Z68756 (Human DNA sequence from cosmid L191F1, Huntington's Disease
Region, chromosome 4pl6.3 contains Huntington Disease (HD) gene, CpG island ESTs and U7 small nuclear RNA). The predicted amino acid sequence disclosed herein for CC397_19 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted CC397_19 protein demonstrated at least some similarity to sequences identified as X52164 (Q300 protein (AA 1-77) [Mus musculus]). Based upon sequence similarity, CC397_19 proteins and each similar protein or peptide may share at least some activity.
The nucleotide sequence of CC397_19 indicates that it may contain an Alu repetitive element.
Clone "D305 2"
A polynucleotide of the present invention has been identified as clone "D305_2". D305_2 was isolated from a human adult blood (peripheral blood mononuclear cells treated with concanavahn A and phorbol myristate acetate) cDNA Ubrary using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. D305_2 is a full-length clone, including the entire coding sequence of a secreted protein (also refened to herein as "D305_2 protein").
The nucleotide sequence of the 5' portion of D305_2 as presently determined is reported in SEQ ID NO: 19. An additional internal nucleotide sequence from D305_2 as presently determined is reported in SEQ ID NO:20. What appUcants beUeve is the proper reading frame and the predicted amino acid sequence encoded by such internal sequence is reported in SEQ ED NO:21. Amino acids 3 to 15 of SEQ ID NO:21 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 16, or are a transmembrane domain. Additional nucleotide sequence from the 3' portion of D305_2, including the polyA tail, is reported in SEQ ID NO:22.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone D305_2 should be approximately 2400 bp.
The nucleotide sequence disclosed herein for D305_2 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. D305_2 demonstrated at least some similarity with sequences identified as AA055703 (zl75d04.rl Stratagene colon (#937204) Homo sapiens cDNA clone 510439 5'), N49593 (yy58d05.sl Homo sapiens cDNA clone 277737 3'), R66646 (yi35b08.rl Homo sapiens cDNA clone 141207 5" similar to SP P24A_YEAST P32802 P24A PROTEIN), U81006 (Human p76 mRNA, complete eds), and Z48758 (S.cerevisiae chromosome EV cosmid 9727). The predicted amino acid sequence disclosed herein for D305_2 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted D305_2 protein demonstrated at least some similarity to sequences identified as U53880 (P24A protein (unknown function) (Swiss Prot. accession number P32802) [Saccharomyces cerevisiae]), U81006 (p76 [Homo sapiens]), X67316 (SCEMP70_1 p24a 70 kDa precursor [Saccharomyces cerevisiae]), and Z48758 (unknown [Saccharomyces cerevisiae]). Based upon sequence similarity, D305_2 proteins and each similar protein or peptide may share at least some activity.
Clone "G55 1"
A polynucleotide of the present invention has been identified as clone "G55_l ". G55_l was isolated from a human adult blood (peripheral blood mononuclear cells treated with concanavaUn A and phorbol myristate acetate) cDNA Ubrary using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. G55_l is a full-length clone, including the entire coding sequence of a secreted protein (also refened to herein as "G55_l protein").
The nucleotide sequence of the 5' portion of G55_l as presently determined is reported in SEQ ED NO:23. What appUcants presently beUeve is the proper reading frame for the coding region is indicated in SEQ ID NO:24. The predicted amino acid sequence of the G55_l protein conesponding to the foregoing nucleotide sequence is reported in SEQ ID NO:24. Amino acids 98 to 110 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 111, or are a transmembrane domain. Additional nucleotide sequence from the 3' portion of G55_l, including the polyA tail, is reported in SEQ ED NO:25.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone G55_l should be approximately 2000 bp.
The nucleotide sequence disclosed herein for G55_l was searched against the GenBank and
GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. G55_l demonstrated at least some similarity with sequences identified as R83586 (ypl6a07.rl Homo sapiens cDNA clone 1875725'). Based upon sequence similarity, G55_l proteins and each similar protein or peptide may share at least some activity.
Clone "K39 7" A polynucleotide of the present invention has been identified as clone "K39_7". K39_7 was refened to as K39_2 in previous appUcations. K39_7 was isolated from a murine adult bone marrow (stromal cell line FCM-4) cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. K39_7 is a full-length clone, including the entire coding sequence of a secreted protein (also refened to herein as "K39_7 protein").
The nucleotide sequence of the 5' portion of K39_7 as presently determined is reported in SEQ ID NO:26. What appUcants presently beUeve is the proper reading frame for the coding region is indicated in SEQ ID NO:27. The predicted amino acid sequence of the K39_7 protein conesponding to the foregoing nucleotide sequence is reported in SEQ ED NO:27. Amino acids 3 to 15 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 16, or are a transmembrane domain. Additional nucleotide sequence from the 3' portion of K39_7, including the polyA tail, is reported in SEQ ID NO:28.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone K39_7 should be approximately 1675 bp.
The nucleotide sequence disclosed herein for K39_7 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. K39_7 demonstrated at least some similarity with sequences identified as AA254326 (val5d06.rl Soares mouse lymph node NbMLN Mus musculus cDNA clone 722987 5' similar to WP:C09G4.1 CE03978), D18935 (Mouse 3'-directed cDNA, MUSGS01125, clone mc0564), H14129 (ym65b04.rl Homo sapiens cDNA clone 163759 5'), and R20230 (hUOG-1, DNA segment encoding a mammaUan GDF-1 protein). The predicted amino acid sequence disclosed herein for K39_7 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted K39_7 protein demonstrated at least some similarity to sequences identified as R86811 (Saccharomyces cerevisiae mutant LAGl protein) and U42438 (similar to S. cerevisiae longevity-assurance protein 1 (SP P38703) [Caenorhabditis elegans]). Based upon sequence similarity, K39_7 proteins and each similar protein or peptide may share at least some activity.
Clone "K330 3" A polynucleotide of the present invention has been identified as clone "K330_3". K330_3 was refened to as K330_2 in previous appUcations. K330_3 was isolated from a murine adult bone marrow (stromal cell line FCM-4) cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. K330_3 is a full-length clone, including the entire coding sequence of a secreted protein (also refened to herein as "K330_3 protein").
The nucleotide sequence of the 5' portion of K330_3 as presently determined is reported in SEQ ID NO:29. What applicants presently believe is the proper reading frame for the coding region is indicated in SEQ ID NO:30, The predicted amino acid sequence of the K330_3 protein conesponding to the foregoing nucleotide sequence is reported in SEQ ED NO:30. Additional nucleotide sequence from the 3' portion of K330_3, including the polyA tail, is reported in SEQ ID NO:31.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone K330_3 should be approximately 1300 bp.
The nucleotide sequence disclosed herein for K330_3 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. K330_3 demonstrated at least some similarity with sequences identified as A03900 (H.sapiens HuV(NP) gene), AA038010 (mi80al l.rl Soares mouse p3NMF19.5 Mus musculus cDNA clone 472892 5'), M30775 (Mouse thymidylate synthase pseudogene, 3' flank), R40824
(yf82c07.sl Homo sapiens cDNA clone 289393'), T23245 (Human gene signature HUMGS05046), and U23512 (Caenorhabditis elegans cosmid M01G4). Based upon sequence similarity, K330_3 proteins and each similar protein or peptide may share at least some activity.
Clone "K363 3"
A polynucleotide of the present invention has been identified as clone "K363_3". K363_3 was referred to as K363_2 in previous appUcations. K363_3 was isolated from a murine adult bone marrow (stromal cell Une FCM-4) cDNA Ubrary using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. K363_3 is a fuU-length clone, including the entire coding sequence of a secreted protein (also refened to herein as "K363_3 protein").
The nucleotide sequence of the 5' portion of K363_3 as presently determined is reported in SEQ ID NO:32. What appUcants presently beUeve is the proper reading frame for the coding region is indicated in SEQ ID NO:33. The predicted amino acid sequence of the K363_3 protein conesponding to the foregoing nucleotide sequence is reported in SEQ ED NO:33. Additional nucleotide sequence from the 3' portion of K363_3, including the polyA tail, is reported in SEQ ID NO:34.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone K363_3 should be approximately 2690 bp.
The nucleotide sequence disclosed herein for K363_3 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. K363_3 demonstrated at least some similarity with sequences identified as AA437876 (vd20h06.sl Knowles Solter mouse 2 cell Mus musculus cDNA clone 793115 5'), D21554 (Mouse embryonal carcinoma F9 cell cDNA, 67F09), and Y08460 (Mus musculus mRNA for Mdes transmembrane protein). The predicted amino acid sequence disclosed herein for K363_3 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted K363_3 protein demonstrated at least some similarity to sequences identified as Y08460 (Mdes protein [Mus musculus]). Based upon sequence similarity, K363_3 proteins and each similar protein or peptide may share at least some activity.
Clone "K446 3"
A polynucleotide of the present invention has been identified as clone "K446_3". K446_3 was refened to as K446_2 in previous appUcations. K446_3 was isolated from a murine adult bone manow (stromal cell line FCM-4) cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. K446_3 is a fuU-length clone, including the entire coding sequence of a secreted protein (also refened to herein as "K446_3 protein"). The nucleotide sequence of the 5' portion of K446_3 as presently determined is reported in SEQ ID NO:35. What appUcants presently beUeve is the proper reading frame for the coding region is indicated in SEQ ED NO:36. The predicted amino acid sequence of the K446_3 protein conesponding to the foregoing nucleotide sequence is reported in SEQ ID NO:36. Additional nucleotide sequence from the 3' portion of K446_3, including the polyA tail, is reported in SEQ ID NO:37.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone K446_3 should be approximately 2150 bp.
The nucleotide sequence disclosed herein for K446_3 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. No hits were found in the database. Clone "K464 4"
A polynucleotide of the present invention has been identified as clone "K464_4". K464_4 was refened to as K464_3 in previous appUcations. K464_4 was isolated from a murine adult bone manow (stromal cell line FCM-4) cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. K464_4 is a full-length clone, including the entire coding sequence of a secreted protein (also refened to herein as "K464_4 protein").
The nucleotide sequence of the 5' portion of K464_4 as presently determined is reported in SEQ ID NO:38. What applicants presently believe is the proper reading frame for the coding region is indicated in SEQ ID NO:39. The predicted amino acid sequence of the K464_4 protein conesponding to the foregoing nucleotide sequence is reported in SEQ ID NO:39. Additional nucleotide sequence from the 3' portion of K464_4, including the polyA tail, is reported in SEQ ID NO:40. The EcoRI Notl restriction fragment obtainable from the deposit containing clone K464_4 should be approximately 1250 bp.
The nucleotide sequence disclosed herein for K464_4 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. K464_4 demonstrated at least some similarity with sequences identified as AA260484 (va95a09.rl Soares mouse NML Mus musculus cDNA clone 747160 5'), AA419864 (vf49b08.rl Soares mouse NbM), L25338 (Mus musculus folate-binding protein gene, 5' end), M22527 (Mouse cytotoxic T lymphocyte-specific serine protease), T01176 (P815A antigen precursor gene P1A), T21224 (Human gene signature HUMGS02538), T41900 (Vector pAPEX-3p), U46493 (Cloning vector pFlp recombinase gene, complete eds), U89673 (Cloning vector pIRESlneo, complete plasmid sequence), W32699 (zc06bl l.sl Soares parathyroid tumor NbHPA Homo sapiens cDNA clone 321501 3'), and W36926 (mb82bl0.rl Soares mouse p3NMF19.5 Mus musculus cDNA clone 335899 5'). The predicted amino acid sequence disclosed herein for K464_4 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted K464_4 protein demonstrated at least some similarity to sequences identified as L33768 (JAK3 [Mus musculus]) and X16213 (MHC T7 class I antigen (64 AA) (119 is 2nd base in codon)
[Mus musculus]). Based upon sequence similarity, K464_4 proteins and each similar protein or peptide may share at least some activity.
Clone "K483 1 " A polynucleotide of the present invention has been identified as clone "K483_l ". K483_l was isolated from a murine adult bone manow (stromal cell line FCM-4) cDNA Ubrary using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. K483_l is a full-length clone, including the entire coding sequence of a secreted protein (also refened to herein as "K483_l protein").
The nucleotide sequence of K483_l as presently determined is reported in SEQ ID NO:41.
What appUcants presently believe to be the proper reading frame and the predicted amino acid sequence of the K483_l protein conesponding to the foregoing nucleotide sequence is reported in SEQ ID NO:42. Amino acids 184 to 196 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 197, or are a transmembrane domain.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone K483_l should be approximately 1500 bp.
The nucleotide sequence disclosed herein for K483_l was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. K483_l demonstrated at least some similarity with sequences identified as AA110914 (mm02c03.rl Stratagene mouse kidney (#937315) Mus musculus cDNA clone 520324 5'), AA318160 (EST20431 Retina π Homo sapiens cDNA 5' end), AA500150 (vi97c09.rl Barstead mouse pooled organs MPLRB4 Mus musculus cDNA clone 920176 5'), and N41895 (yw86b03.rl Homo sapiens cDNA clone 259085 5'). Based upon sequence similarity, K483_l proteins and each similar protein or peptide may share at least some activity. The TopPredEI computer program predicts three potential transmembrane domains within the K483_l protein sequence, centered around amino acids 18, 179, and 270 of SEQ ID NO:42. The K483_l protein also has a possible signal sequence that could be cleaved to produce a mature protein starting at amino acid 34 of SEQ ID N0.42.
Clone "L69 3"
A polynucleotide of the present invention has been identified as clone "L69_3". L69_3 was refened to as L69_2 in previous appUcations. L69_3 was isolated from a murine adult thymus cDNA Ubrary using methods which are selective for cDNAs encoding secreted proteins (see U.S.
Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. L69_3 is a full-length clone, including the entire coding sequence of a secreted protein (also refened to herein as "L69_3 protein"). The nucleotide sequence of the 5' portion of L69_3 as presently determined is reported in SEQ ED NO:43. What appUcants presently beUeve is the proper reading frame for the coding region is indicated in SEQ ID NO:44. The predicted amino acid sequence of the L69_3 protein conesponding to the foregoing nucleotide sequence is reported in SEQ ED NO:44. Amino acids 7 to 19 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 20, or are a transmembrane domain. Additional nucleotide sequence from the 3' portion of L69_3, including the polyA tail, is reported in SEQ ID NO:45.
The EcoRI/Notl restriction fragment obtainable from the deposit containing clone L69_3 should be approximately 1200 bp. The nucleotide sequence disclosed herein for L69_3 was searched against the GenBank and
GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. L69_3 demonstrated at least some similarity with sequences identified as H35162 (EST 108034 Rattus sp. cDNA similar to H.sapiens hypothetical protein (PIR:S25641)), U02442 (Cloning vector pADbeta, complete sequence), W74864 (md91bl0.rl Soares mouse embryo NbME13.5 14.5 Mus musculus cDNA), and X67698 (H.sapiens tissue specific mRNA). The predicted amino acid sequence disclosed herein for L69_3 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted L69_3 protein demonstrated at least some similarity to sequences identified as A 18921 (tissue-specific secretory protein [unidentified]). Based upon sequence similarity, L69_3 proteins and each similar protein or peptide may share at least some activity.
Deposit of Clones
Clones AJ26_3, AJ172_2, AP224_2, BL89_10, BL341_4, BV239_2, CC25_16, CC397_11, D305_2, G55_l, K39_7, K330_3, K363_3, K446_3, K464_4, K483_l, and L69_3 were deposited on July 25, 1996 with the American Type Culture Collection as an original deposit under the Budapest Treaty and were given the accession number ATCC 98115, from which each clone comprising a particular polynucleotide is obtainable. Clones K39_7, K330_3, K363_3, K446_3, K464_4, and L69_3 were referred to as K39_2, K330_2, K363_2, K446_2, K464_3, and L69_2, respectively, when the July 25, 1996 deposit was made. An additional isolate of each of clones BL89_10, BV239_2, CC25_16, and CC397_11 (namely isolates BL89_13, BV239_3, CC25_17, and CC397_19, respectively) were deposited with the American Type Culture Collection on August 23, 1996 under accession number 98153, from which each clone comprising a particular polynucleotide is obtainable. All restrictions on the availabiUty to the pubUc of the deposited material wiU be irrevocably removed upon the granting of the patent, except for the requirements specified in 37 C.F.R. § 1.808(b). Each clone has been transfected into separate bacterial cells (E. coli) in this composite deposit. Each clone can be removed from the vector in which it was deposited by performing an EcoRI/Notl digestion (5' site, EcoRl; 3' site, Notl) to produce the appropriate fragment for such clone. Each clone was deposited in either the pED6 or pNOTs vector depicted in Figure 1. The pED6dpc2 vector ("pED6") was derived from pEDόdpcl by insertion of a new polylinker to facilitate cDNA cloning (Kaufman et al, 1991, Nucleic Acids Res. 19: 4485-4490); the pNOTs vector was derived from pMT2 (Kaufman et al, 1989, Mol. Cell. Biol. 9: 946-958) by deletion of the DHFR sequences, insertion of a new polylinker, and insertion of the M 13 origin of replication in the Clal site. In some instances, the deposited clone can become "flipped" (i.e., in the reverse orientation) in the deposited isolate. In such instances, the cDNA insert can still be isolated by digestion with EcoRl and Notl. However, Notl will then produce the 5' site and EcoRl wiU produce the 3' site for placement of the cDNA in proper orientation for expression in a suitable vector. The cDNA may also be expressed from the vectors in which they were deposited.
Bacterial ceUs containing a particular clone can be obtained from the composite deposit as follows:
An oUgonucleotide probe or probes should be designed to the sequence that is known for that particular clone. This sequence can be derived from the sequences provided herein, or from a combination of those sequences. The sequence of the oligonucleotide probe that was used to isolate each full-length clone is identified below, and should be most reliable in isolating the clone of interest.
Clone Probe Sequence
AJ26_3 SEQ ID NO:46
AJ172_2 SEQ ED NO:47 AP224_2 SEQ ID NO:48
BL89_13 SEQ ID NO:49
BL341_4 SEQ ID NO:50
BV239_3 SEQ ED NO:51
CC25_17 SEQ ID NO:52 CC397_19 SEQ ID NO:53
D305_2 SEQ ID NO:54
G55_l SEQ ID NO:55
K39_7 SEQ ID NO:56
K330_3 SEQ ID NO:57 K363_3 SEQ ID NO:58 K446_3 SEQ ED NO:59
K464_4 SEQ ID NO:60
K483_l SEQ ID NO:61
L69_3 SEQ ID NO:62
In the sequences Usted above which include an N at position 2, that position is occupied in prefened probes/primers by a biotinylated phosphoaramidite residue rather than a nucleotide (such as , for example, that produced by use of biotin phosphoramidite (l-dimethoxytrityloxy-2-(N-biotinyl-4- aminobutyl)-propyl-3-0-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramadite) (Glen Research, cat. no. 10-1953)).
The design of the oUgonucleotide probe should preferably follow these parameters:
(a) It should be designed to an area of the sequence which has the fewest ambiguous bases ("N's"), if any;
(b) It should be designed to have a Tm of approx. 80 ° C (assuming 2° for each A or T and 4 degrees for each G or C).
The oUgonucleotide should preferably be labeled with g-32P ATP (specific activity 6000 Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for labeUng oUgonucleotides. Other labeUng techniques can also be used. Unincorporated label should preferably be removed by gel filtration chromatography or other estabhshed methods. The amount of radioactivity incoφorated into the probe should be quantitated by measurement in a scintillation counter. Preferably, specific activity of the resulting probe should be approximately 4e+6 dpm pmole.
The bacterial culture containing the pool of full-length clones should preferably be thawed and 100 μl of the stock used to inoculate a sterile culture flask containing 25 ml of sterile L-broth containing ampicillin at 100 μg ml. The culture should preferably be grown to saturation at 37°C, and the saturated culture should preferably be diluted in fresh L-broth. AUquots of these dilutions should preferably be plated to determine the dilution and volume which will yield approximately 5000 distinct and well-separated colonies on sohd bacteriological media containing L-broth containing ampicilUn at 100 μg/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at 37°C. Other known methods of obtaining distinct, well-separated colonies can also be employed. Standard colony hybridization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them.
The filter is then preferably incubated at 65°C for 1 hour with gentle agitation in 6X SSC
(20X stock is 175.3 g NaCl Uter, 88.2 g Na citrate/Uter, adjusted to pH 7.0 with NaOH) containing
0.5% SDS, 100 μg/ml of yeast RNA, and 10 mM EDTA (approximately 10 mL per 150 mm filter). Preferably, the probe is then added to the hybridization mix at a concentration greater than or equal to le+6 dpm/mL. The filter is then preferably incubated at 65°C with gentle agitation overnight. The filter is then preferably washed in 500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably followed by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes. A third wash with 0.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is optional. The filter is then preferably dried and subjected to autoradiography for sufficient time to visuaUze the positives on the X-ray film. Other known hybridization methods can also be employed.
The positive colonies are picked, grown in culture, and plasmid DNA isolated using standard procedures. The clones can then be verified by restriction analysis, hybridization analysis, or DNA sequencing.
Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention. Fragments of the protein may be in linear form or they may be cycUzed using known methods, for example, as described in H.U. Saragovi, etal, Bio/Technology 10, 773-778 (1992) and in R.S. McDowell, etal, J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incoφorated herein by reference. Such fragments may be fused to carrier molecules such as immunoglobuUns for many puφoses, including increasing the valency of protein binding sites. For example, fragments of the protein may be fused through "Unker" sequences to the Fc portion of an immunoglobuUn. For a bivalent form of the protein, such a fusion could be to the Fc portion of an IgG molecule. Other immunoglobuUn isotypes may also be used to generate such fusions. For example, a protein - IgM fusion would generate a decavalent form of the protein of the invention.
The present invention also provides both full-length and mature forms of the disclosed proteins. The full-length form of the such proteins is identified in the sequence Usting by translation of the nucleotide sequence of each disclosed clone. The mature form of such protein may be obtained by expression of the disclosed fiiU-length polynucleotide (preferably those deposited with
ATCC) in a suitable mammaUan cell or other host cell. The sequence of the mature form of the protein may also be determinable from the amino acid sequence of the full-length form.
The present invention also provides genes corresponding to the cDNA sequences disclosed herein. "Conesponding genes" are the regions of the genome that are transcribed to produce the mRNAs from which the cDNA sequences are derived and any contiguous regions of the genome necessary for the regulated expression of such genes, including but not Umited to coding sequences, 5' and 3' untranslated regions, alternatively spUced exons, introns, promoters, enhancers, and silencer or suppressor elements. The conesponding 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 ampUfication of genes in appropriate genomic libraries or other sources of genomic materials.
Where the protein of the present invention is membrane-bound (e.g., is a receptor), the present invention also provides for soluble forms of such protein. In such forms part or all of 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 amino acid sequences of the proteins when aUgned so as to maximize overlap and identity while minimizing sequence gaps. Also included in the present invention are 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. As used herein, 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 aUeUc 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. When hybridizing a polynucleotide to a target polynucleotide of unknown sequence, the hybrid length is assumed to be that of the hybridizing polynucleotide. When polynucleotides of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity.
*: SSPE (IxSSPE is 0.15M NaCl, lOmM NaH2P04, and 1.25mM EDTA, pH 7.4) can be substituted for SSC (IxSSC is 0.15M NaCl and 15mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete.
*TB - TR: 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_) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm( ° C) = 2(# of A + T bases) + 4(# of G + C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C) = 81.5 + 16.6(log10[Na+]) + 0.41 (%G+C) - (600/N), where N is the number of bases in the hybrid, and [Na*] is the concentration of sodium ions in the hybridization buffer ([Na+] for IxSSC = 0.165 M).
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. Ausubel et al., eds., John Wiley & Sons, Inc., sections
2.10 and 6.3-6.4, incorporated herein by reference.
Preferably, 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 aUgned so as to maximize overlap and identity while minimizing sequence gaps.
The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman etal., Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the protein recombinantly. Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exempUfied in R. Kaufman, Methods in Enzymology 185. 537-566
(1990). As defined herein "operably Unked" means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the Ugated polynucleotide/expression control sequence. A number of types of cells may act as suitable host cells for expression of the protein.
MammaUan host ceUs include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) ceUs, human kidney 293 ceUs, human epidermal A431 ceUs, human Colo205 ceUs, 3T3 ceUs, CV-1 cells, other transformed primate cell Unes, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat ceUs.
Altematively, it may be possible to produce the protein in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, CaUfornia, U.S.A. (the MaxBac® kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incoφorated herein by reference. As used herein, an insect cell capable of expressing a polynucleotide of the present invention is "transformed."
The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein. The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.
Alternatively, the protein of the invention may also be expressed in a form which will faciUtate purification. For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen, respectively. The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope. One such epitope ("Flag") is commercially available from Kodak (New Haven, CT).
Finally, one or more reverse-phase high performance Uquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., siUca gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein. Some or aU of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein. The protein thus purified is substantially free of other mammaUan proteins and is defined in accordance with the present invention as an "isolated protein." The protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.
The protein may also be produced by known conventional chemical synthesis. Methods for constructing the proteins of the present invention by synthetic means are known to those skilled in the art. The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.
The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deUberately engineered. For example, modifications in the peptide or DNA sequences can be made by those skiUed in the art using known techniques. Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence. For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Patent No. 4,518,584). Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein.
Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and may thus be useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein. Such modifications are beUeved to be encompassed by the present invention.
USES AND BIOLOGICAL ACTIVITY
The polynucleotides and proteins of the present invention are expected to 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).
Research Uses and Utilities
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 conesponding 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 oUgomers for attachment to a "gene chip" or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or eUcit another immune response. Where 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 eUcit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively deteπnine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the conesponding 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 conelative receptors or Ugands. Where the protein binds or potentially binds to another protein (such as, for example, in a receptor-Ugand 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.
Any or all of these research utiUties are capable of being developed into reagent grade or kit format for commerciaUzation as research products. Methods for performing the uses Usted above are well known to those skilled in the art.
References disclosing such methods include without limitation "Molecular Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor laboratory Press, Sambrook, J., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Berger, S.L. and A.R. Kimmel eds., 1987. Nutritional Uses
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. In such cases the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate soUd or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules. In the case of microorganisms, the protein or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.
Cytokine and Cell Proliferation/Differentiation Activity
A protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or ceU 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 proUferation assays for cell Unes including, without Umitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DAI, 123, Tl 165, 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-ceU or thymocyte proUferation include without Umitation those described in: Cunent Protocols in Immunology, Ed by J. E. CoUgan, A.M. Kruisbeek, D.H. MarguUes, E.M. Shevach, W Strober, Pub. Greene PubUshing 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; BertagnolU et al., J. Immunol. 145:1706-1712, 1990; BertagnolU et al., CeUular Immunology 133:327-341, 1991; BertagnolU, et al., J. Immunol. 149:3778-3783, 1992; Bowman et al., J. Immunol. 152: 1756-1761, 1994.
Assays for cytokine production and/or proUferation of spleen cells, lymph node cells or thymocytes include, without Umitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in Immunology. J.E.e.a. CoUgan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human Interferon γ, Schreiber, R.D. In Current Protocols in Immunology. J.E.e.a. CoUgan 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 Umitation, 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. CoUgan 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. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6 - Nordan, R. In Current Protocols in Immunology. J.E.e.a. CoUgan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991 ; Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11 - Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. J. In Current Protocols in Immunology. J.E.e.a. CoUgan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse and human Interleukin 9 - Ciarletta, A., Giannotti, J., Clark, S.C. and Turner, K.J. In Current Protocols in Immunology. J.E.e.a. CoUgan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991.
Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proUferation and cytokine production) include, without Umitation, those described in: Cunent Protocols in Immunology, Ed by J. E. CoUgan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene PubUshing 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. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988.
Immune Stimulating or Suppressing Activity A protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without Umitation 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 ceU populations. These immune deficiencies may be genetic or be caused by viral (e.g., HEV) as well as bacterial or fungal infections, or may result from autoimmune disorders. More specificaUy, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HEV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis. Of course, in this regard, 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, GuiUain-Bane syndrome, autoimmune thyroiditis, insuUn dependent diabetes melUtis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease. Such 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 (including, for example, organ transplantation), may also be treatable using a protein of the present invention.
Using the proteins of the invention it may also be possible to immune responses, in a number of ways. Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response. The functions of activated 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 ceUs to the suppressive agent. Tolerance, which involves inducing non-responsiveness or anergy in T ceUs, 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 Umitation B lymphocyte antigen functions (such as , for example, B7)), e.g., preventing high level lymphokine synthesis by activated T ceUs, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD). For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation. Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant. The administration of a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural Ugand(s) on immune ceUs (such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7-1, B7-3) or blocking antibody), prior to transplantation can lead to the binding of the molecule to the natural Ugand(s) on the immune cells without transmitting the conesponding costimulatory signal. Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, 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. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.
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. Examples of 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 I^enschow et al, Science 257:789-792 (1992) and Turka etα/., Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992). In addition, 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 eUminate disease symptoms. Administration of reagents which block costimulation of T cells by disrupting receptor:Ugand interactions of B lymphocyte antigens can be used to inhibit T ceU 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 ceUs which could lead to long-term reUef 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 encephaUtis, systemic lupus erythmatosis in MRUlpr/lpr mice or NZB hybrid mice, murine autoimmune coUagen arthritis, diabetes melUtus 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 encephaUtis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically.
Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T ceUs 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 ceUs express aU 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.
In another application, up regulation or enhancement of antigen function (preferably B lymphocyte 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 in the subject. If desired, the tumor cell can be transfected to express a combination of peptides. For example, 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-l-like activity and/or B7-3-like activity. The transfected tumor ceUs are returned to the patient to result in expression of the peptides on the surface of the transfected cell. Alternatively, 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. In addition, 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 aU or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I a chain protein and β2 microglobuUn protein or an MHC class π α chain protein and an MHC class II β chain protein to thereby express MHC class I or MHC class II proteins on the cell surface. Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell. Optionally, 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. Thus, the induction of a T ceU 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: Cunent Protocols in Immunology, Ed by J. E. CoUgan, A.M. Kruisbeek, D.H. MarguUes, 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. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; 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. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet al., J. Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988; BertagnolU et al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092, 1994.
Assays for T-cell-dependent immunoglobuUn responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl/Th2 profiles) include, without Umitation, those described in: MaUszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, JJ. and Brunswick, M. In Current Protocols in Immunology. J E.e.a. CoUgan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
Mixed lymphocyte reaction (MLR) assays (which wiU identify, among others, proteins that generate predominantly Thl and CTL responses) include, without Umitation, those described in:
Cunent Protocols in Immunology, Ed by J. E. CoUgan, A.M. Kruisbeek, D.H. MarguUes, E.M. Shevach, W Strober, Pub. Greene PubUshing 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; Takai et al., J. Immunol. 140:508-512, 1988; BertagnolU et al., J. Immunol. 149:3778-3783, 1992.
Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without Umitation, 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., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of CUnical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without Umitation, 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., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.
Assays for proteins that influence early steps of T-cell commitment and development include, without Umitation, 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. in supporting the growth and proliferation of erythroid progenitor ceUs alone or in combination with other cytokines, thereby indicating utiUty, 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 proUferation 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 proUferation 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 compUmentary to platelet transfusions; and or in supporting the growth and proUferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic ceUs and therefore find therapeutic utiUty in various stem cell disorders (such as those usually treated with transplantation, including, without Umitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with bone manow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy. The activity of a protein of the invention may, among other means, be measured by the following methods:
Suitable assays for proUferation and differentiation of various hematopoietic lines are cited above. Assays for embryonic stem ceU differentiation (which wiU identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without Umitation, 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 (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without Umitation, 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 BriddeU, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, NY. 1994; Long term bone manow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, NY. 1994; Long term culture initiating cell assay, Sutherland, H.J. In
Culture of Hematopoietic Cells. R.I. Freshney, etal. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, NY. 1994.
Tissue Growth Activity A protein of the present invention also may have utihty in compositions used for bone, cartilage, tendon, Ugament and/or nerve tissue growth or regeneration, as weU as for wound heaUng 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 appUcation in the heaUng 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 weU 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 ceUs, 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.
Another category of 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/Ugament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the heaUng of tendon or ligament tears, deformities and other tendon or Ugament defects in humans and other animals. Such a preparation employing a tendon/Ugament-Uke tissue inducing protein may have prophylactic use in preventing damage to tendon or Ugament 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 Ugament tissue. De novo tendon Ugament-Uke tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or Ugament 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 Ugament-forming cells, stimulate growth of tendon- or Ugament-forming cells, induce differentiation of progenitors of tendon- or Ugament-forming cells, or induce growth of tendon Ugament 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 proUferation 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 locahzed 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 Umitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
It is expected that 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, endotheiium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate. A protein of the invention may also exhibit angiogenic activity.
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 Umitation, those described in:
International Patent Publication No. WO95/16035 (bone, cartilage, tendon); International Patent PubUcation No. WO95/05846 (nerve, neuronal); International Patent PubUcation No. WO91/07491 (skin, endotheUum ).
Assays for wound heaUng 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).
Activin/Inhibin Activity 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 folUcle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of folUcle stimulating hormone (FSH). Thus, a protein of the present invention, alone or in heterodimers with a member of the inhibin α family, may be useful as a contraceptive based on the abiUty of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertiUty in these mammals. Altematively, the protein of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin-β group, may be useful as a fertility inducing therapeutic, based upon the abiUty 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 fertiUty in sexually immature mammals, so as to increase the Ufetime 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 Umitation, 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.
Chemotactic/Chemokinetic Activity
A protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian ceUs, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast ceUs, eosinophils, epithelial and/or endotheUal 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 locaUzed 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.
Preferably, the protein or peptide has the abiUty to directly stimulate directed movement of ceUs. 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 (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the abiUty of a protein to induce the migration of cells across a membrane as weU as the abiUty of a protein to induce the adhesion of one cell population to another ceU population. Suitable assays for movement and adhesion include, without Umitation, those described in: Current Protocols in Immunology, Ed by J.E. CoUgan, A.M. Kruisbeek, D.H. MarguUes, 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. 95:1370-1376, 1995; Lind et al. APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol. 25: 1744-1748; Gruber et al. J. of Immunol. 152:5860-5867, 1994; Johnston et al. J. of Immunol. 153: 1762-1768, 1994.
Hemostatic and Thrombolytic Activity
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 Umitation, those described in: Linet et al., J. CUn. 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.
Receptor/Ligand Activity
A protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/Ugand interactions. Examples of such receptors and Ugands include, without Umitation, cytokine receptors and their ligands, receptor kinases and their Ugands, receptor phosphatases and their Ugands, receptors involved in ceU-ceU interactions and their
Ugands (including without Umitation, cellular adhesion molecules (such as selectins, integrins and their Ugands) and receptor/Ugand pairs involved in antigen presentation, antigen recognition and development of ceUular and humoral immune responses). Receptors and Ugands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/Ugand interaction. A protein of the present invention (including, without Umitation, fragments of receptors and Ugands) may themselves be useful as inhibitors of receptor/Ugand interactions.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Suitable assays for receptor-Ugand activity include without Umitation those described in:Cunent Protocols in Immunology, Ed by J.E. CoUgan, A.M. Kruisbeek, D.H. MarguUes, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of CeUular 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 ceUs involved in the inflammatory response, by inhibiting or promoting ceU-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 (SE S)), ischemia-reperfusion injury, endotoxin lethaUty, 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 EL-1. Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
Cadherin/Tumor Invasion Suppressor Activity
Cadherins are calcium-dependent adhesion molecules that appear to play major roles during development, particularly in defining specific cell types. Loss or alteration of normal cadherin expression can lead to changes in cell adhesion properties Unked to tumor growth and metastasis.
Cadherin malfunction is also impUcated in other human diseases, such as pemphigus vulgaris and pemphigus fohaceus (auto-immune bUstering skin diseases), Crohn's disease, and some developmental abnormaUties.
The cadherin superfamily includes well over forty members, each with a distinct pattern of expression. All members of the superfamily have in common conserved extracellular repeats
(cadherin domains), but structural differences are found in other parts of the molecule. The cadherin domains bind calcium to form their tertiary structure and thus calcium is required to mediate their adhesion. Only a few amino acids in the first cadherin domain provide the basis for homophiUc adhesion; modification of this recognition site can change the specificity of a cadherin so that instead of recognizing only itself, the mutant molecule can now also bind to a different cadherin. In addition, some cadherins engage in heterophilic adhesion with other cadherins.
E-cadherin, one member of the cadherin superfamily, is expressed in epitheUal cell types. Pathologically, if E-cadherin expression is lost in a tumor, the maUgnant cells become invasive and the cancer metastasizes. Transf ection of cancer cell lines with polynucleotides expressing E- cadherin has reversed cancer-associated changes by returning altered cell shapes to normal, restoring cells' adhesiveness to each other and to their substrate, decreasing the cell growth rate, and drastically reducing anchorage-independent ceU growth. Thus, reintroducing E-cadherin expression reverts carcinomas to a less advanced stage. It is likely that other cadherins have the same invasion suppressor role in carcinomas derived from other tissue types. Therefore, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be used to treat cancer. Introducing such proteins or polynucleotides into cancer cells can reduce or eUminate the cancerous changes observed in these cells by providing normal cadherin expression. Cancer cells have also been shown to express cadherins of a different tissue type than their origin, thus aUowing these cells to invade and metastasize in a different tissue in the body. Proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be substituted in these cells for the inappropriately expressed cadherins, restoring normal cell adhesive properties and reducing or eliminating the tendency of the cells to metastasize.
Additionally, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can used to generate antibodies recognizing and binding to cadherins. Such antibodies can be used to block the adhesion of inappropriately expressed tumor-ceU cadherins, preventing the cells from forming a tumor elsewhere. Such an anti- cadherin antibody can also be used as a marker for the grade, pathological type, and prognosis of a cancer, i.e. the more progressed the cancer, the less cadherin expression there will be, and this decrease in cadherin expression can be detected by the use of a cadherin-binding antibody.
Fragments of proteins of the present invention with cadherin activity, preferably a polypeptide comprising a decapeptide of the cadherin recognition site, and poly-nucleotides of the present invention encoding such protein fragments, can also be used to block cadherin function by binding to cadherins and preventing them from binding in ways that produce undesirable effects. Additionally, fragments of proteins of the present invention with cadherin activity, preferably truncated soluble cadherin fragments which have been found to be stable in the circulation of cancer patients, and polynucleotides encoding such protein fragments, can be used to disturb proper cell- cell adhesion. Assays for cadherin adhesive and invasive suppressor activity include, without limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-18817, 1995; Miyaki et al. Oncogene 11: 2547-2552, 1995; Ozawa et al. Cell 63: 1033-1038, 1990.
Tumor Inhibition Activity
In addition to the activities described above for immunological treatment or prevention of tumors, 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 kilUng, infectious agents, including, without Umitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without Umitation, 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 caricadic cycles or rhythms; effecting the fertiUty of male or female subjects; effecting the metaboUsm, cataboUsm, anaboUsm, processing, utiUzation, storage or elimination of dietary fat, Upid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component(s); effecting behavioral characteristics, including, without Umitation, appetite, Ubido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic Uneages; hormonal or endocrine activity; in the case of enzymes, conecting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproUferative disorders (such as, for example, psoriasis); immunoglobuUn-Uke activity (such as, for example, the abiUty to bind antigens or complement); and the abiUty to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.
ADMINISTRATION AND DOSING A protein of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources) may be used in a pharmaceutical composition when combined with a pharmaceutically acceptable carrier. Such a composition may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabiUzers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, D -3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, E -10, IL-11, EL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNF1, TNF2, G- CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or compUment its activity or use in treatment. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein of the invention, or to minimize side effects. Conversely, protein of the present invention may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent. A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins. As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.
The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) of present invention along with protein or peptide antigens. The protein and/or peptide antigen wiU deUver a stimulatory signal to both B and T lymphocytes. B lymphocytes will respond to antigen through their surface immunoglobulin receptor. T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins. MHC and structuraUy related proteins including those encoded by class I and class π MHC genes on host cells wiU serve to present the peptide antigen(s) to T lymphocytes. The antigen components could also be suppUed as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells. Alternatively antibodies able to bind surface immunolgobuUn and other molecules on B ceUs as weU as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention.
The pharmaceutical composition of the invention may be in the form of a Uposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as Upids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without Umitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are incoφorated herein by reference.
As used herein, the term "therapeutically effective amount" means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, heaUng, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When appUed to an individual active ingredient, administered alone, the term refers to that ingredient alone. When appUed to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein of the present invention is administered to a mammal having a condition to be treated. Protein of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, protein of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factors), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.
Administration of protein of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical appUcation or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is prefened.
When a therapeutically effective amount of protein of the present invention is administered oraUy, protein of the present invention wiU be in the form of a tablet, capsule, powder, solution or eUxir. When administered in tablet form, the pharmaceutical composition of the invention may additionaUy contain a soUd carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and preferably from about 25 to 90% protein of the present invention. When administered in liquid form, a Uquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological satine solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention.
When a therapeuticaUy effective amount of protein of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stabiUty, and the like, is within the skill in the art. A prefened pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabiUzers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
The amount of protein of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of protein of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein of the present invention and observe the patient's response. Larger doses of protein of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 μg to about 100 mg (preferably about 0.1 ng to about 10 mg, more preferably about 0.1 μg to about 1 mg) of protein of the present invention per kg body weight. The duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the protein of the present invention will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.
Protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specificaUy react with the protein. Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen. The peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole Umpet hemocyanin (KLH). Methods for synthesizing such peptides are known in the art, for example, as in R.P. Merrifield, J. Amer.Chem.Soc. 85, 2149-2154 (1963); J.L. Krstenansky, et al, FEBS Lett. 211,, 10 (1987). Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein. NeutraUzing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved. In the case of cancerous cells or leukemic cells, neutraUzing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein.
For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematicaUy, or locaUy as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for dehvery to the site of bone, cartilage or tissue damage. Topical administration may be suitable for wound heaUng and tissue repair. Therapeutically useful agents other than a protein of the invention which may also optionaUy be included in the composition as described above, may altematively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention. Preferably for bone and/or cartilage formation, the composition would include a matrix capable of deUvering the protein-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body. Such matrices may be formed of materials presently in use for other implanted medical applications. The choice of matrix material is based on biocompatibility, biodegradabiUty, mechanical properties, cosmetic appearance and interface properties. The particular appUcation of the compositions wiU define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycohc acid and polyanhydrides. Other potential materials are biodegradable and biologicaUy weU-defined, such as bone or dermal coUagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradabiUty.
Presently prefened is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns. In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.
A prefened family of sequestering agents is cellulosic materials such as alkylcelluloses
(including hydroxyalkylceUuloses), including methylcellulose, ethylceUulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most prefened being cationic salts of carboxymethylcellulose (CMC). Other prefened sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly( vinyl alcohol). The amount of sequestering agent useful herein is 0.5-20 wt%, preferably 1-10 wt% based on total formulation weight, which represents the amount necessary to prevent desorbtion of the protein from the polymer matrix and to provide appropriate handhng of the composition, yet not so much that the progenitor ceUs are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.
In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insuUn-Uke growth factor (IGF).
The therapeutic compositions are also presently valuable for veterinary appUcations. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins of the present invention. The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insuUn like growth factor I), to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomoφhometric determinations and tetracychne labeUng. Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into ceUs for expression in a mammahan subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA). Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proUferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic puφoses.
Example 1 Characterization of AJ172 2 DNA and Protein
AJ172_2, a novel human cDNA isolated using a yeast signal sequence trap, encodes a protein that exhibits significant homology to a baboon endogenous retrovirus envelope protein. Genomic DNA sequences flanking the AJ172_2 gene reveal that it is part of a previously uncharacterized defective provirus, indicating that the sequence may be an example of a retroviral gene that has been "captured" by the human host. Southern blot analyses show that AJ172_2- hybridizing sequences are restricted to humans and monkeys; being absent from cows, dogs, rats, mice, rabbits, chickens and yeast. Northern blot analyses demonstrate that although the AJ172_2 gene is very highly transcribed in the human placenta and weakly transcribed in the testes, it is not expressed at aU in 21 additional human tissues. In situ antisense RNA hybridizations performed on full-term human placental sections revealed that AJ172_2 transcripts are specifically locaUzed to viUous syncytiotrophoblasts, a fused, multinucleated ceU type derived from fetal trophoblast tissue. We find that AJ172_2 expression in COS cells reproducibly causes the formation of giant multinucleated COS-ceU syncytia which closely resemble these fused placental syncytiotrophoblasts, suggesting that AJ172_2 plays a role in mediating cell fusion events in human placenta and fusion of other types of cells expressing AJ172_2. A number of independent binary reporter gene approaches indicate that AJ172_2-induced COS syncytia are truly derived from ceU-ceU fusions and do not result from incomplete ceU divisions. AJ172_2 can mediate fusion of many different cell types, including human, monkey, rodent and insect lines. Moreover even simple Uposomes can fuse readily to AJ172_2-transfected COS cells, suggesting that AJ172_2 requires neither homophiUc nor heterophiUc protein-protein interactions to promote membrane fusion events. We have also found that co-transfection of AJ172_2 with EL-11 or IL-12 into COS cells reproducibly leads to 2-5 fold increases in secreted cytokine yields. This phenomenon can be used in appUcations for AJ172_2 in enhancing mammalian cell recombinant gene expression.
The full-length AJ172_2 cDNA encodes a typical signal sequence, an extraceUular domain, a transmembrane domain and a short cytoplasmic tail. The recent appearance in pubhc databases of many very closely related EST sequences derived from many independent cDNA libraries provides additional supporting evidence for the expression of AJ172_2 in vivo. We also find that the DNA sequence of a segment of human chromosome 7 (genbank accession #AC000064) carries the entire AJ172_2 sequence. A closer examination of this chromosomal segment reveals the presence of a complete, previously uncharacterized, defective retrovirus. This provirus exhibits a typical moφhology; 5' and 3'- long terminal repeat sequences, putative gag and pol genes, and a third open reading frame encoding a putative viral envelope protein. The gag and pol genes are punctuated by multiple chain termination codons and are defective, whereas the third, envelope ORF is intact. This third ORF is identical to AJ172_2, which we conclude to be the envelope gene of an ancient retrovirus.
We have demonstrated that AJ172_2 is expressed highly and very specifically in human placental syncytiotrophoblasts. By means of in vitro transfection experiments we go on to show that AJ172_2, Uke many previously described viral envelope proteins, can mediate cell to cell fusion events leading to the formation of giant syncytia In a series of further experiments we demonstrate that neither homophiUc nor heterophiUc protein-protein interactions are required for AJ172_2 function, indeed the molecule can mediate efficient cell fusion to simple Uposomes. We suspect that AJ172_2 may play a critical role in the normal placental biology of humans and primates, mediating ceU fusion events which may be important in processes such as blastocyst implantation, the control of uterine wall infiltration by fetal trophoblasts, and in optimizing the efficiency of placental transporter and secretory function. AJ172_2 may thus be the first described example of a captured viral gene performing an important biological role in a mammaUan host organism.
Figure 2 presents the results of Northern analyses of multiple human tissues which indicate that AJ172_2 is expressed highly in the placenta and weakly in the testes.
As shown in Figure 3, in situ antisense-RNA hybridizations were used to specifically locaUze AJ172_2 expression to placental syncytiotrophoblasts.
Figure 4 demonstrates that AJ172_2 expression in transfected COS cells can cause the formation of giant multinucleated syncytia by a fusigenic mechanism. The cells expressing AJ172_2 can be seen to have formed multinucleate syncytia, while the non-transfected cells remained mononucleate. Figure 5 demonstrates that AJ172_2 mediates actual cell fusion and does not operate through a mechanism of anested cell division. A first cell Une was transfected with AJ172_2, luciferase and ERK. A second cell line was transfected with AJ172_2 and MEK. When the cells were mixed, fusion occuned resulting in production of luciferase activity. Figure 6 demonstrates that AJ 172_2 can mediate fusion between cells of differing types and between a cell expressing AJ172_2 and a cell not expressing AJ172_2. HELA cells were transfected with a cDNA encoding a P-selectin glycoprotein Ugand-1/Fc fusion protein (PSGL-Fc). COS cells were transfected with AJ172_2. Another batch of COS cells was transfected with AJ172_2 in reverse orientation. The transfected HELA cells were mixed with each type of COS cells. As shown in Figure 6, mixture with the AJ172_ transfected COS cells caused fusion with the HELA cells, resulting in multinucleate fusions. Mixture with the COS cells transfected with A 172_2 in reverse orientation resulted in no fusion (mononucleate cells remained).
Figure 7 demonstrates that the mechanism of AJ172_2 induced cell fusion does not require homophiUc or heterophiUc protein-protein interactions. COS cells transfected with AJ172_2 were mixed with hposomes containing a green fluorescent protein (GFP) expression plasmid. As shown in Figure 7, the COS cells fused with the liposomes, took up the expression plasmid, and began expressing GFP.
Cell fusion in the human placenta has been impUcated in a number of critical processes. Early in human placental development, embryonic trophoblastic cells are thought to fuse with epithelial cells during blastocyst implantation into the uterine wall. Subsequently, the carefully orchestrated invasion of fetal cytotrophoblast through the maternal decidua into the endometrium is perhaps controlled by cell fusion events, since invasive cytotrophoblasts become non-invasive when they fuse to form multinucleated syncytiotrophoblasts. Choriocarcinomas arise when cytotrophoblast fusion fails to occur and the invasive process continues unabated. Finally syncytiotrophoblasts found in the placental vilU form a continuous boundary layer between maternal and fetal tissue. This vitally important structure is responsible for the efficient transfer to the fetus of nutrients, growth factors and antibodies, and for the removal of waste products. It is possible that cytotrophoblast ceU fusion is an adaptation to increase the efficiency of these transport and secretory processes. In 1991 Harris proposed that the invasiveness of the fetal trophoblast was a trait acquired by a mammaUan antecedent following an ancient retroviral infection. He went on to suggest that this event may even have been seminal in the evolution of aU modern placental mammals. Harris based his hypothesis on circumstantial evidence, namely the frequent observation of retrovirus-like particles in placental preparations, and the presence in placenta of fused cells with a moφhology reminiscent of retrovirally-induced syncytia. The abiUty of AJ172_2 to mediate cell fusion events, its highly specific pattern of expression in placental syncytiotrophoblasts, and its presence in the genome as a part of a cryptic provirus all tend to support Harris's view. AJ172_2 may be the first example of a viral gene co-opted to serve a vital role in a mammalian host.
Although other mammalian placentas possess fused cell types, to date we have found AJ172_2-related sequences only in human and primate genomes. The simplest explanation for this would be that the protypical AJ172_2-like sequence has diverged extensively during the course of mammahan evolution. An alternative explanation would be that sequestration of a retrovirus to perform a role in placental development was a relatively common evolutionary event, and that the antecedents of different mammahan Uneages simply used different retroviruses to achieve the same goal. The fact that AJ172_2 needs no other receptor protein to function, the common abiUty of many known viruses to mediate cell-cell fusions, and the great diversity observed in placental moφhology even among closely related mammalian species would all be consistent with this explanation. Notwithstanding this, it is also possible that early primates acquired the AJ172_2 provirus for some unknown, primate-specific reason. Trophoblast syncytia may make a number of important contributions to overall placental efficiency. One of these may simply be to provide an extended surface area for maternal fetal exchange. In this regard the theoretical geometry of syncytia formation predicts that the ratio of overall cell surface area to cytoplasmic volume will decrease progressively as cells fuse together. If overall cytoplasmic volume and cell surface area remain constant during cell fusion, then the growing syncytium will progressively accumulate "excess" cell membrane over and above that which is required simply to enclose the cytoplasmic contents. This extra membrane should enable the syncytium to "spread" and cover a larger area of the substratum than would the equivalent number of unfused cells. Thus in the placenta the formation of syncytia would tend to increase placental efficiency by enlarging the area of the exchange surface. In support of this theoretical prediction we have observed in vitro that COS cells fused as a result of AJ172_2 expression adopt a "fried egg" appearance, with the nuclei gathered tightly together in one location and the cytoplasm covering an extended surface area.
Antagonists to AJ172_2 (either antibodies, antibody fragments, nucleotide aptamers, peptide aptamers, antisense nucleotides or small molecules) may be useful as birth control agents, either by preventing initial implantation of embryos into the uterine wall or by interrupting normal placental development and leading to abortion of the conceptus.
Since the regulation of AJ172_2 expression is very tight, small molecules designed to positively or negatively modulate the control of AJ172_2 gene expression may be useful as birth control agents. They may also be useful in treatment of placental pathologies such as pre-eclampsia or choriocarcinoma, where aberrant cytotrophoblast fusion events have been observed. AJ172_2 as a cell fusion agent may have applications as a means of increasing DNA transfection efficiencies in vitro (research applications, moving genes into cultured cell Unes or primary cell Unes with greater efficiency) or in vivo (gene therapy applications, moving genes into cells in the intact organism with greater efficiency). AJ172_2 may also be impUcated in osteoclast fusion. Therefore, AJ172_2 antagonists or small molecules directed to control of AJ172_2 gene expression may be useful in the treatment of bone disorders such as osteoporosis or osteopetrosis.
Example 2 Additional Evidence for AJ172 Expression in Choriocarcinoma Lines
Although the above experiments demonstrate that AJ172_2 can induce cell fusion, to establish that AJ172_2 expression could be correlated with cytotrophoblast fusion, we examined in vitro the fusion of BeWo cells, and monitored levels of AJ172_2 transcription in response to forskolin treatment. BeWo is a human trophoblastic choriocarcinoma line which can be induced by forskolin to form syncytiotrophblasts. (BeWo-derived syncytiotrophblasts are morpologically very similar to AJ172_2-fused COS cells). Figure 8 shows that AJ172_2 transcription in BeWo cells increases at least five fold in response to forskolin treatment, correlating well with cell fusion (Figure 9). In contrast a control choriocarcinoma line which fails to fuse in response to forskolin, JEG3, showed no expression of AJ172_2. Taken together these results establish that AJ172_2 can indeed mediate cell fusion in a trophoblastic cell type. Moreover DNA synthesis is arrested in BeWo cells which have been treated with forskolin (Figure 10), perhaps as a result of AJ172 expression and induction of cell fusion. Thus induction of AJ172 synthesis in vivo may be a therapy useful for controlling the growth of choricarcinomas.
Example 3 Additional Evidence for AJ172 Disregulation in Pre-eclampsia We performed in situ hybridizations on tissue sections prepared from the villous region of human pre-eclamptic placenta to examine if there were differences in the distribution of AJ172 expression compared to normal placenta. In the normal situation hybridization of a digoxygenin-labelled antisense AJ172_2 RNA probe is observed only to syncytiotrophoblasts comprising the layer of fused cells on the edges of the villi, bordering on the maternal blood space (Figure 11). In contrast for pre-eclamptic samples hybridization was observed in patches of fused cells throughout the placental villi (Figure 12). Thus it appears that AJ172 expression is disregulated in pre-eclampsia. This would indicate that pre-eclamsia and other diseases of placental morphogenesis may be caused by temporal or spatial disregulation of AJ172 expression, quantitative disregulation in AJ172 expression levels, or by mutations in the AJ172 gene.
In addition to uses and therapies discussed above, antibodies to AJ172 are useful in detecting serum levels of AJ172, and thus are useful as a dignostic tools to monitor for pre-eclampsia, for other placental pathologies or for cancerous conditions. Agents modulating AJ172 expression or function have therapeutic potential in treatment of neoplastic diseases in addition to choriocarcinoma.
Example 4 Additional Data Supporting a Role for AJ172 in Tissue Remodelling and Extracellular
Matrix Degradation We have observed that in BeWo choriocarcinoma cells, following treatments with forskolin and resulting in induction of both AJ172 and cell fusion, an increase in collagenase A expression at the RNA (Figure 13) and protein levels (Figure 14). These changes in expression may reflect the normal course of events in placenta, where it is known that a large amount of extracellular matrix remodelling occurs during placental morphogenesis. We see similar changes in COS cells transfected with AJ172, leading to the conclusion that changes in collagenase A expression levels may be a general cellular response to the cell fusion process. It is further possible that disregulated AJ172 expression, leading to increased local extracellular matrix breakdown, could be a contributing factor to metastatic processes in certain tumors. Thus, coπection of this disregulation using AJ172 DNA, protein and/or antibodies would inhibit such breakdown and the resulting metastases.
Patent and Uterature references cited herein are incoφorated by reference as if fully set forth.

Claims

What is claimed is:
1. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ED NO: 1 ;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ED NO:l from nucleotide 44 to nucleotide 1204;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ED NO: 1 from nucleotide 1 to nucleotide 403;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone AJ26_3 deposited under accession number ATCC 98115;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone AJ26_3 deposited under accession number ATCC 98115;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AJ26_3 deposited under accession number ATCC 98115;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AJ26_3 deposited under accession number ATCC 98115;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ED NO:2;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
2. A composition of claim 1 wherein said polynucleotide is operably linked to at least one expression control sequence.
3. A host cell transformed with a composition of claim 2.
4. The host cell of claim 3, wherein said cell is a mammahan cell.
5. A process for producing a protein encoded by a composition of claim 2, which process comprises:
(a) growing a culture of the host ceU of claim 3 in a suitable culture medium; and
(b) purifying said protein from the culture.
6. A protein produced according to the process of claim 5.
7. The protein of claim 6 comprising a mature protein.
8. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ED NO:2;
(b) the amino acid sequence of SEQ ED NO:2 from amino acid 1 to amino acid 120;
(c) fragments of the amino acid sequence of SEQ ED NO:2; and
(d) the amino acid sequence encoded by the cDNA insert of clone AJ26_3 deposited under accession number ATCC 98115; the protein being substantially free from other mammalian proteins.
9. The composition of claim 8, wherein said protein comprises the amino acid sequence of SEQ ED NO:2.
10. The composition of claim 8, wherein said protein comprises the amino acid sequence of SEQ ED NO:2 from amino acid 1 to amino acid 120.
11. The composition of claim 8, further comprising a pharmaceutically acceptable carrier.
12. A method for preventing, treating or ameliorating a medical condition which comprises administering to a mammaUan subject a therapeutically effective amount of a composition of claim 11.
13. An isolated gene conesponding to the cDNA sequence of SEQ ID NO: 1.
14. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ED NO:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 928 to nucleotide 2541;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ED NO:3 from nucleotide 988 to nucleotide 2541;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 684 to nucleotide 1128;
(e) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone AJ172_2 deposited under accession number ATCC 98115;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone AJ172_2 deposited under accession number ATCC 98115;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone AJ172_2 deposited under accession number ATCC 98115;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone AJ172_2 deposited under accession number ATCC 98115;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ED NO:4 having biological activity;
(k) a polynucleotide which is an allehc variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
15. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ED NO:4;
(b) the amino acid sequence of SEQ ED NO:4 from amino acid 1 to amino acid 67;
(c) fragments of the amino acid sequence of SEQ ID NO:4; and (d) the amino acid sequence encoded by the cDNA insert of clone AJ172_2 deposited under accession number ATCC 98115; the protein being substantially free from other mammalian proteins.
16. An isolated gene conesponding to the cDNA sequence of SEQ ED NO:3.
17. An isolated gene conesponding to the cDNA sequence of SEQ ED NO:6, SEQ ED NO:5 or SEQ ED NO:8 .
18. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ED NO:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ED NO:9 from nucleotide 6 to nucleotide 2408;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ED NO:9 from nucleotide 1295 to nucleotide 1705;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone BL89_13 deposited under accession number ATCC 98153;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone BL89_13 deposited under accession number ATCC 98153;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone BL89_13 deposited under accession number ATCC 98153;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone BL89_13 deposited under accession number ATCC 98153;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 10 having biological activity;
(j) a polynucleotide which is an allehc variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
19. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 10;
(b) the amino acid sequence of SEQ ID NO : 10 from amino acid 431 to amino acid 567;
(c) fragments of the amino acid sequence of SEQ ID NO: 10; and
(d) the amino acid sequence encoded by the cDNA insert of clone BL89_13 deposited under accession number ATCC 98153; the protein being substantially free from other mammalian proteins.
20. An isolated gene conesponding to the cDNA sequence of SEQ ED NO:9.
21. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ED NO: 11 ;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l 1 from nucleotide 2113 to nucleotide 2337;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ED NO:l 1 from nucleotide 2036 to nucleotide 2316;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone BL341_4 deposited under accession number ATCC 98115;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone BL341_4 deposited under accession number ATCC 98115;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone BL341_4 deposited under accession number ATCC 98115;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone BL341_4 deposited under accession number ATCC 98115;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ED NO:12;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ED NO: 12 having biological activity;
(j) a polynucleotide which is an allehc variant of a polynucleotide of (a)-(g) above; (k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
22. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ED NO: 12;
(b) the amino acid sequence of SEQ ID NO: 12 from amino acid 1 to amino acid 68;
(c) fragments of the amino acid sequence of SEQ ED NO: 12; and
(d) the amino acid sequence encoded by the cDNA insert of clone BL341_4 deposited under accession number ATCC 98115; the protein being substantially free from other mammalian proteins.
23. An isolated gene conesponding to the cDNA sequence of SEQ ED NO:l 1.
24. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ED NO: 15;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ED NO: 15 from nucleotide 144 to nucleotide 257;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 15 from nucleotide 30 to nucleotide 271 ;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone CC25_17 deposited under accession number ATCC 98153;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone CC25_17 deposited under accession number ATCC 98153;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone CC25_17 deposited under accession number ATCC 98153;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone CC25_17 deposited under accession number ATCC 98153;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:16; (i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ED NO: 16 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
25. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO: 16;
(b) fragments of the amino acid sequence of SEQ ID NO: 16; and
(c) the amino acid sequence encoded by the cDNA insert of clone CC25_17 deposited under accession number ATCC 98153; the protein being substantially free from other mammalian proteins.
26. An isolated gene conesponding to the cDNA sequence of SEQ ID NO: 15.
27. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ED NO: 17;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ED NO: 17 from nucleotide 431 to nucleotide 520;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ED NO: 17 from nucleotide 266 to nucleotide 511;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone CC397_19 deposited under accession number ATCC 98153;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone CC397_19 deposited under accession number ATCC 98153;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone CC397_19 deposited under accession number ATCC 98153;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone CC397_19 deposited under accession number ATCC 98153; (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ED NO: 18;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ED NO: 18 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).
28. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO : 18 ;
(b) the amino acid sequence of SEQ ED NO: 18 from amino acid 1 to amino acid 27;
(c) fragments of the amino acid sequence of SEQ ED NO: 18 ; and
(d) the amino acid sequence encoded by the cDNA insert of clone CC397_19 deposited under accession number ATCC 98153; the protein being substantially free from other mammalian proteins.
29. An isolated gene conesponding to the cDNA sequence of SEQ ID NO: 17.
30. A composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ED NO:41 ;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:41 from nucleotide 218 to nucleotide 1159;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:41 from nucleotide 806 to nucleotide 1159;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ED NO:41 from nucleotide 217 to nucleotide 517;
(e) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone K483_ l deposited under accession number ATCC 98115; (f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone K483_l deposited under accession number ATCC 98115;
(g) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone K483_l deposited under accession number ATCC 98115;
(h) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone K483_l deposited under accession number ATCC 98115;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ D NO:42;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:42 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j).
31. A composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:42;
(b) the amino acid sequence of SEQ ID NO:42 from amino acid 1 to amino acid 100;
(c) fragments of the amino acid sequence of SEQ ED NO:42; and
(d) the amino acid sequence encoded by the cDNA insert of clone K483_l deposited under accession number ATCC 98115; the protein being substantially free from other mammalian proteins.
32. An isolated gene corresponding to the cDNA sequence of SEQ ED NO:41.
33. A method of promoting ceU-ceU fusion, said method comprising contacting a first cell and a second cell, wherein said first cell expresses an AJ172_2 protein.
34. The method of claim 33, wherein said first cell naturally expresses said AJ172_2 protein.
35. The method of claim 34, wherein said first cell expresses said AJ172_2 protein as a result of transfection of said first cell.
36. The method of claim 35, wherein said first cell is transfected with a composition of claim 14.
37. The method of claim 35, wherein said first cell is transfected with a gene of claim 16.
38. The method of claim 33, wherein said AJ172_2 protein is a protein of claim 15.
39. The method of claim 33, wherein said first cell and said second ceU are ceUs of the same type.
40. The method of claim 33, wherein at least one of said first cell and said second cell are transfected to express an additional protein other than said AJ172_2 protein.
41. A method of inhibiting cell-cell fusion between a first cell which expresses an AJ172_2 protein and a second cell, said method comprising contacting said first cell with an AJ172_2 protein antagonist.
42. The method of claim 41, wherein said antagonist is selected from the group consisting of an antibody or antibody fragment directed to an AJ172_2 protein, an antisense polynucleotide directed to a polynucleotide expressing an AJ172_2 protein, a nucleotide aptamer directed to an AJ172_2 protein, a peptide aptamer directed to an AJ172_2 protein and a small molecule which blocks the fusion-inducing activity of an AJ172_2 protein.
43. The method of claim 41, wherein said first cell is a placental cell.
44. The method of claim 43 , wherein said second cell is a ceU from the maternal uterine Uning.
45. The method of claim 41, wherein said first cell is a cytotrophoblast.
46. A method of inhibiting blastocyst implantation, said method comprising contacting a cell within said blastocyst which expresses an AJ172_2 protein with an AJ172_2 protein antagonist.
47. A method of inhibiting trophoblast invasion, said method comprising contacting a first cell which expresses an AJ172_2 protein with an AJ172_2 protein antagonist.
48. The method of claim 42, wherein said antagonist is selected from the group consisting of an antibody or antibody fragment directed to an AJ172_2 protein, an antisense polynucleotide directed to a polynucleotide expressing an AJ172_2 protein, a nucleotide aptamer directed to an AJ172_2 protein, and a peptide aptamer directed to an AJ172_2 protein.
49. The method of claim 48, wherein said antagonist is selected from the group consisting of an antibody or antibody fragment directed to an AJ172_2 protein, and an antisense polynucleotide directed to a polynucleotide expressing an AJ172_2 protein.
50. A method of diagnosing or predicting the existence of a condition associated with disregulation of AH172_2 protein in a mammaUan subject, said method comprising (a) determining a first level of expression of AJ172_2 protein in said subject, and (b) comparing said first level of expression to a second level of expression of AJ172_2 protein in one or more other mammaUan subjects which do not have said condition.
51. The method of claim 50, wherein said condition is selected fromthe group consisting of pre-eclampsia, placental pathology and cancer.
52. The method of claim 50, wherein said first level of expression is determined in the serum of said subject.
53. The method of claim 50, wherein said first level of expression is determined using an antibody or antibody fragment directed to AJ172_2 protein.
54. The method of claim 51 , wherein said cancer is choriocarcinoma.
55. A method of treating a neoplastic disease in a mammaUan subject, said method comprising administering to said subject a therapeutically effective amount of an agent which modulates the expression or function of AJ172_2.
56. The method of claim 55, wherein said disease is choriocarcinoma.
57. A method of inhibiting metastasis in a mammaUna subject, said method comprising administering to said subject a therapeutically effective amount of an agent which modulates the expression or function of AJ172_2.
EP99925654A 1998-05-18 1999-05-17 Secreted proteins and polynucleotides encoding them Withdrawn EP1080106A1 (en)

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US8047898A 1998-05-18 1998-05-18
US80478 1998-05-18
US09/175,928 US6312921B1 (en) 1996-07-26 1998-10-20 Secreted proteins and polynucleotides encoding them
US175928 1998-10-20
PCT/US1999/010915 WO1999060020A1 (en) 1998-05-18 1999-05-17 Secreted proteins and polynucleotides encoding them

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