EP1080106A1 - Proteines secretees et polynucleotides les codant - Google Patents

Proteines secretees et polynucleotides les codant

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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Withdrawn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)
English (en)
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
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from 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/fr
Withdrawn legal-status Critical Current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.

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Abstract

L'invention concerne de nouveaux polynucléotides et les protéines qui les codent.
EP99925654A 1998-05-18 1999-05-17 Proteines secretees et polynucleotides les codant Withdrawn EP1080106A1 (fr)

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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 (fr) 1998-05-18 1999-05-17 Proteines secretees et polynucleotides les codant

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WO2002022638A1 (fr) * 2000-09-12 2002-03-21 Human Genome Sciences, Inc. Proteines secretees humaines 22
FR2797889A1 (fr) * 1999-09-01 2001-03-02 Bio Merieux Procede de detection de l'expression d'une proteine d'enveloppe d'un retrovirus endogene humain et utilisations d'un gene codant pour cette proteine
AU2001229563A1 (en) * 2000-09-20 2002-04-02 Human Genome Sciences, Inc. 21 human secreted proteins
US7608704B2 (en) 2000-11-08 2009-10-27 Incyte Corporation Secreted proteins
EP1341812A2 (fr) * 2000-11-08 2003-09-10 Incyte Genomics, Inc. Proteines secretees
EP1565482B1 (fr) * 2002-10-23 2014-04-30 Ludwig Institute for Cancer Research Ltd. Adn et proteines 3 de type a34 et a33, anticorps associes et methodes de traitement associees
LT2769729T (lt) 2007-09-04 2019-05-10 Compugen Ltd. Polipeptidai ir polinukleotidai ir jų panaudojimas kaip vaistų taikinio vaistų ir biologinių preparatų gamybai
PT2190469E (pt) * 2007-09-04 2015-06-25 Compugen Ltd Polipéptidos e polinucleótidos, e utilizações dos mesmos como um alvo de fármacos para produzir fármacos e agentes biológicos
EP2726503B1 (fr) 2011-06-30 2019-09-04 Compugen Ltd. Polypeptides et leurs utilisations pour traiter les troubles auto-immuns et l'infection
JP2015512616A (ja) 2012-02-01 2015-04-30 コンピュゲン エルティーディー． Ｃ１ｏｒｆ３２抗体およびがんの治療のためのその使用

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US5712369A (en) * 1995-08-24 1998-01-27 Ludwig Institute For Cancer Research Isolated protein which binds to A33 antibody, and peptides corresponding to portions of the protein
US5708157A (en) * 1996-07-26 1998-01-13 Genetics Institute, Inc. Secreted proteins and polynucleotides encoding them
EP0973898A2 (fr) * 1997-04-10 2000-01-26 Genetics Institute, Inc. MARQUEURS SECRETES DE SEQUENCE EXPRIMEE (sEST)

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