WO1997039122A2 - PHOSPHOPROTEINE DE 78 kDa ISOLEE ET CLONEE DE MASTOCYTES (AGENT INHIBITEUR DE LA DEGRANULATION DES MASTOCYTES) ET SON UTILISATION - Google Patents

PHOSPHOPROTEINE DE 78 kDa ISOLEE ET CLONEE DE MASTOCYTES (AGENT INHIBITEUR DE LA DEGRANULATION DES MASTOCYTES) ET SON UTILISATION Download PDF

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Publication number
WO1997039122A2
WO1997039122A2 PCT/US1997/006042 US9706042W WO9739122A2 WO 1997039122 A2 WO1997039122 A2 WO 1997039122A2 US 9706042 W US9706042 W US 9706042W WO 9739122 A2 WO9739122 A2 WO 9739122A2
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Prior art keywords
polynucleotide
amino acid
protein
seq
acid sequence
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PCT/US1997/006042
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English (en)
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WO1997039122A3 (fr
WO1997039122A9 (fr
Inventor
Theoharis C. Theoharides
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Muro Pharmaceutical, Inc.
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Priority to AU24563/97A priority Critical patent/AU2456397A/en
Publication of WO1997039122A2 publication Critical patent/WO1997039122A2/fr
Publication of WO1997039122A3 publication Critical patent/WO1997039122A3/fr
Publication of WO1997039122A9 publication Critical patent/WO1997039122A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—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
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention provides novel proteins, along with therapeutic, diagnostic and research utilities for these proteins.
  • the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO: l from nucleotide 70 to nucleotide 505; the nucleotide sequence of the full length protein coding sequence of clone AP162 deposited under accession number ATCC 98026 ; or the nucleotide sequence of the mature protein coding sequence of clone API 62 deposited under accession number ATCC 98026 .
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone API 62 deposited under accession number ATCC 98026 .
  • such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO:2 from amino acid 42 to amino acid 61.
  • 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 protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:4 from nucleotide 230 to nucleotide 791 ; the nucleotide sequence of SEQ ID NO:4 from nucleotide 311 to nucleotide 791 ; the nucleotide sequence of the full length protein coding sequence of clone AM931 deposited under accession number ATCC 98026 ; or the nucleotide sequence of the mature protein coding sequence of clone AM931 deposited under accession number ATCC 98026 .
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AM931 deposited under accession number ATCC 98026 . In yet other preferred embodiments, such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO:5 from amino acid 32 to amino acid 51.
  • 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:5;
  • protein comprises the amino acid sequence of SEQ ID NO: 5 or the amino acid sequence of SEQ ID NO:5 from amino acid 32 to amino acid 51.
  • the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:6 from nucleotide 14 to nucleotide 491 ; the nucleotide sequence of SEQ ID NO:6 from nucleotide 83 to nucleotide 491 ; the nucleotide sequence of the full length protein coding sequence of clone AM610 deposited under accession number ATCC 98026 ; or the nucleotide sequence of the mature protein coding sequence of clone AM610 deposited under accession number ATCC 98026 .
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AM610 deposited under accession number ATCC 98026 . In yet other preferred embodiments, such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO:7 from amino acid 31 to amino acid 50.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: 7 from amino acid 31 to amino acid 50.
  • the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9;
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:9 from nucleotide 1 to nucleotide 483; the nucleotide sequence of the full length protein coding sequence of clone AM340 deposited under accession number ATCC 98026 ; or the nucleotide sequence of the mature protein coding sequence of clone AM 340 deposited under accession number ATCC 98026 .
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AM340 deposited under accession number ATCC 98026 .
  • such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO:9 from nucleotide 1 to nucleotide 483; the nucleotide sequence of the full length protein coding sequence of clone AM340 deposited under accession number ATCC 98026 ; or the nucleotide sequence of the mature protein coding sequence of clon
  • 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: 10;
  • the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 11 from nucleotide 15 to nucleotide 462; the nucleotide sequence of SEQ ID NO: 11 from nucleotide 87 to nucleotide 462; the nucleotide sequence of the full length protein coding sequence of clone AM282 deposited under accession number ATCC 98026 ; or the nucleotide sequence of the mature protein coding sequence of clone AM282 deposited under accession number ATCC 98026 .
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AM282 deposited under accession number ATCC 98026 . In yet other preferred embodiments, such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO: 12 from amino acid 28 to amino acid 47.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • AM282 deposited under accession number ATCC 98026; the protein being substantially free from other mammalian proteins.
  • the protein comprises the amino acid sequence of SEQ ID NO: 12 or the amino acid sequence of SEQ
  • the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 14 from nucleotide 185 to nucleotide 519; the nucleotide sequence of SEQ ID NO: 14 from nucleotide 260 to nucleotide 519; the nucleotide sequence of the full length protein coding sequence of clone AK647 deposited under accession number ATCC 98026 ; or the nucleotide sequence of the mature protein coding sequence of clone AK647 deposited under accession number ATCC 98026 .
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AK647 deposited under accession number ATCC 98026 . In yet other preferred embodiments, such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO: 15 from amino acid 27 to amino acid 46.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO: 15 or the amino acid sequence of SEQ ID NO: 15 from amino acid 27 to amino acid 46.
  • the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 17 from nucleotide 257 to nucleotide 536; the nucleotide sequence of SEQ ID NO: 17 from nucleotide 329 to nucleotide 536; the nucleotide sequence of the full length protein coding sequence of clone AK583 deposited under accession number ATCC 98026 ; or the nucleotide sequence of the mature protein coding sequence of clone AK583 deposited under accession number ATCC 98026 .
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AK583 deposited under accession number ATCC 98026 . In yet other preferred embodiments, such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO: 18 from amino acid 14 to amino acid 33.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO: 18 or the amino acid sequence of SEQ ID NO: 18 from amino acid 14 to amino acid 33.
  • the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
  • a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone AK533 deposited under accession number ATCC 98026 ;
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:20 from nucleotide 179 to nucleotide 476; the nucleotide sequence of the full length protein coding sequence of clone AK533 deposited under accession number ATCC 98026 ; or the nucleotide sequence of the mature protein coding sequence of clone AK533 deposited under accession number ATCC 98026 .
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AK533 deposited under accession number ATCC 98026 .
  • such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO:21 from amino acid 35 to amino acid 57.
  • 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 protein encoded by a polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:23;
  • a polynucleotide comprising the nucleotide sequence of the mamre protein coding sequence of clone AK296 deposited under accession number ATCC 98026 ;
  • polynucleotide which encodes a species homologue of the protein of (h) or (i) above.
  • polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
  • polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AK296 deposited under accession number ATCC 98026 .
  • polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO:23
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO:24 or the amino acid sequence of SEQ ID NO:24 from amino acid 81 to amino acid 90.
  • the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:
  • polynucleotide which is an allelic variant of a polynucleotide of (a)-(f) above; and (j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:26 from nucleotide 58 to nucleotide 655; the nucleotide sequence of the full length protein coding sequence of clone H617 deposited under accession number ATCC 98026 ; or the nucleotide sequence of the mature protein coding sequence of clone H617 deposited under accession number ATCC 98026 .
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone H617 deposited under accession number ATCC 98026 .
  • such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO:27 from amino acid 65 to amino acid 84.
  • 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;
  • protein comprises the amino acid sequence of SEQ ID NO:27 or the amino acid sequence of SEQ ID NO:27 from amino acid 65 to amino acid 84.
  • the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:29 from nucleotide 14 to nucleotide 391; the nucleotide sequence of the full length protein coding sequence of clone BB9 deposited under accession number ATCC 98026 ; or the nucleotide sequence of the mature protein coding sequence of clone BB9 deposited under accession number ATCC 98026 .
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone BB9 deposited under accession number ATCC 98026 .
  • such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO:30 from amino acid 75 to amino acid 94.
  • 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 protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:32 from nucleotide 61 to nucleotide 514; the nucleotide sequence of SEQ ID NO:32 from nucleotide 115 to nucleotide 514; the nucleotide sequence of the full length protein coding sequence of clone AW191 deposited under accession number ATCC 98026 ; or the nucleotide sequence of the mature protein coding sequence of clone AW 191 deposited under accession number ATCC 98026 .
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AW191 deposited under accession number ATCC 98026 . In yet other preferred embodiments, such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO:33 from amino acid 24 to amino acid 43.
  • 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 protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:35 from nucleotide 180 to nucleotide 525; the nucleotide sequence of SEQ ID NO:35 from nucleotide 339 to nucleotide 525; the nucleotide sequence of the full length protein coding sequence of clone AT211 deposited under accession number ATCC 98026 ; or the nucleotide sequence of the mamre protein coding sequence of clone AT211 deposited under accession number ATCC 98026 .
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AT211 deposited under accession number ATCC 98026 . In yet other preferred embodiments, such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO:36 from amino acid 1 to amino acid 20.
  • 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:36;
  • protein comprises the amino acid sequence of SEQ ID NO: 36 or the amino acid sequence of SEQ ID NO:36 from amino acid 1 to amino acid 20.
  • the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:38 from nucleotide 225 to nucleotide 677; the nucleotide sequence of SEQ ID NO:38 from nucleotide 390 to nucleotide 677; the nucleotide sequence of the full length protein coding sequence of clone AT205 deposited under accession number ATCC 98026 ; or the nucleotide sequence of the mature protein coding sequence of clone AT205 deposited under accession number ATCC 98026 .
  • the polynucleotide encodes the full length or mamre protein encoded by the cDNA insert of clone AT205 deposited under accession number ATCC 98026 . In yet other preferred embodiments, such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO:39 from amino acid 6 to amino acid 25.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO: 39 or the amino acid sequence of SEQ ID NO:39 from amino acid 6 to amino acid 25.
  • the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
  • a polynucleotide comprising the nucleotide sequence of the mamre protein coding sequence of clone AS34 deposited under accession number ATCC 98026 ;
  • polynucleotide which encodes a species homologue of the protein of (h) or (i) above.
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:40 from nucleotide 128 to nucleotide 508; the nucleotide sequence of SEQ ID NO:40 from nucleotide 200 to nucleotide 508; the nucleotide sequence of the full length protein coding sequence of clone AS34 deposited under accession number ATCC 98026 ; or the nucleotide sequence of the mature protein coding sequence of clone AS34 deposited under accession number ATCC 98026 .
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AS34 deposited under accession number ATCC 98026 . In yet other preferred embodiments, such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO:41 from amino acid 27 to amino acid 46.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO:41 or the amino acid sequence of SEQ ID NO:41 from amino acid 27 to amino acid 46.
  • the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:43 from nucleotide 23 to nucleotide 676; the nucleotide sequence of the full length protein coding sequence of clone AS32 deposited under accession number ATCC 98026 ; or the nucleotide sequence of the mature protein coding sequence of clone AS32 deposited under accession number ATCC 98026 .
  • the polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone AS32 deposited under accession number ATCC 98026 .
  • such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO:44 from amino acid 78 to amino acid 97.
  • 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 protein encoded by a polynucleotide selected from the group consisting of
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO-46 from nucleotide 132 to nucleotide 479; the nucleotide sequence of SEQ ID NO:46 from nucleotide 201 to nucleotide 479; the nucleotide sequence of the full length protein coding sequence of clone AR260 deposited under accession number ATCC 98026 ; or the nucleotide sequence of the mamre protein coding sequence of clone AR260 deposited under accession number ATCC 98026 .
  • the polynucleotide encodes the full length or mamre protein encoded by the cDNA insert of clone AR260 deposited under accession number ATCC 98026 . In yet other preferred embodiments, such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO:47 from amino acid 40 to amino acid 59.
  • 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 protein encoded by a polynucleotide selected from the group consisting of:
  • polynucleotide which encodes a species homologue of the protein of (g) or (h) above.
  • polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1
  • polynucleotide encodes the full length or mature protein encoded by the cDNA insert of clone K640 deposited under accession number ATCC 98026 .
  • polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO:51 from amino acid 11 to amino acid 30.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO:51 or the amino acid sequence of SEQ ID NO:51 from amino acid 11 to amino acid 30.
  • the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO:54 from nucleotide 71 to nucleotide 377; the nucleotide sequence of the full length protein coding sequence of clone K39 deposited under accession number ATCC 98026 ; or the nucleotide sequence of the mamre protein coding sequence of clone K39 deposited under accession number ATCC 98026 .
  • the polynucleotide encodes the full length or mamre protein encoded by the cDNA insert of clone K39 deposited under accession number ATCC 98026 .
  • such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO:55 from amino acid 62 to amino acid 81.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • protein comprises the amino acid sequence of SEQ ID NO:55 or the amino acid sequence of SEQ ID NO:55 from amino acid 62 to amino acid 81.
  • the present invention provides a composition comprising an isolated protein encoded by a polynucleotide selected from the group consisting of:
  • such polynucleotide comprises the nucleotide sequence of SEQ ID NO: 57 from nucleotide 194 to nucleotide 423; the nucleotide sequence of the full length protein coding sequence of clone AT319 deposited under accession number ATCC 98026 ; or the nucleotide sequence of the mamre protein coding sequence of clone AT319 deposited under accession number ATCC 98026 .
  • the polynucleotide encodes the full length or mamre protein encoded by the cDNA insert of clone AT319 deposited under accession number ATCC 98026 .
  • such polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO:58 from amino acid 2 to amino acid 21.
  • the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
  • Such protein comprises the amino acid sequence of SEQ ID NO:58 or the amino acid sequence of SEQ ID NO:58 from amino acid 2 to amino acid 21.
  • 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 mammalian subject a therapeutically effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier.
  • Fig. 1 is an autoradiograph evidencing the expression of clones API 62, AM931 , and AR260 in COS cells (expressed band(s) indicated by dot(s)).
  • Fig. 2 is an autoradiograph evidencing the expression of clone AM610 in COS cells
  • Fig. 3 is an autoradiograph evidencing the expression of clones AM340, AM282 and AK533 in COS cells (expressed band(s) indicated by dot(s)).
  • Fig. 4 is an autoradiograph evidencing the expression of clone AK647 in COS cells (expressed band(s) indicated by dot(s)).
  • Fig. 5 is an autoradiograph evidencing the expression of clones AH583, AK296, and AS32 in COS cells (expressed band(s) indicated by dot(s)).
  • Fig. 6 is an autoradiograph evidencing the expression of clones H617 and AT205 in COS cells (expressed band(s) indicated by dot(s)).
  • Fig. 7 is an autoradiograph evidencing the expression of clones BB9 and K39 in COS cells (expressed band(s) indicated by dot(s)).
  • Fig. 8 is an autoradiograph evidencing the expression of clones AW 191 and AS34 in COS cells (expressed band(s) indicated by dot(s)).
  • Fig. 9 is an autoradiograph evidencing the expression of clones AT211 and AT319 in COS cells (expressed band(s) indicated by dot(s)).
  • Fig. 10 is an autoradiograph evidencing the expression of clone K640 in COS cells (expressed band(s) indicated by dot(s)).
  • Nucleotide and amino acid sequences are reported below for each clone and protein disclosed in the present application. In some instances the sequences are preliminary and may include some incorrect or ambiguous bases or amino acids.
  • the actual 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 mamre) 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.
  • reported protein sequences include "Xaa” designators. These "Xaa” designators indicate either (1) a residue which cannot be identified because of nucleotide sequence ambiguity or (2) a stop codon in the determined nucleotide sequence where applicants believe one should not exist (if the nucleotide sequence were determined definitively).
  • 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 endoplpasmic reticulum.
  • a partial cDNA clone encoding API 62 was first isolated from a human adult placenta cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with an EST reported by the I. M.A. G.E. Consortium identified as "yu40d08.rl Homo sapiens cDNA clone 23671 5' " (GenBank accession number H62096). The search also found a hit at GenBank accession number H98192.
  • the human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc. , St. Louis, Mo, a distributor of the I. M.A. G.E. Consortium library.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "API 62".
  • Applicants' methods identified clone AP162 as encoding a secreted protein.
  • the nucleotide sequence of the 5' portion of API 62 as presently determined is reported in SEQ ID NO: 1. What applicants believe is the proper reading frame and the predicted N-terminal amino acid sequence of the full length API 62 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:2. Additional nucleotide sequence from the 3' portion of AP162, including the polyA tail, is reported in SEQ ID NO:3.
  • Protein "AM931" One protein of the present invention has been identified as protein "AM931 ".
  • a partial cDNA clone encoding AM931 was first isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with an EST reported by the I. M.A. G.E. Consortium identified as "yh63e02.rl Homo sapeins cDNA clone 134426 5' " (GenBank accession number R32076) . The search also found a hit at GenBank accession number N30331.
  • the human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I. M.A. G.E. Consortium library.
  • the clone received from the distributor was examined and dete ⁇ nined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AM931".
  • AM610 A partial cDNA clone encoding AM610 was first isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with an EST reported by the I. M.A. G.E. Consortium identified as "ymOlalO.rl Human EST 46249 5' " (GenBank accession number H09925). The search also found hits at GenBank accession numbers H09926 and R14298.
  • the human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I. M.A. G.E. Consortium library.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AM610".
  • Applicants' methods identified clone AM610 as encoding a secreted protein.
  • nucleotide sequence of the 5' portion of AM610 as presently determined is reported in SEQ ID NO:6. What applicants believe is the proper reading frame and the predicted N-terminal amino acid sequence of the full length AM610 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:7. Amino acids 1 to 23 are the predicted leader/signal sequence, with the predicted mamre amino acid sequence beginning at amino acid 24. Additional nucleotide sequence from the 3 ' portion of AM610, including the polyA tail, is reported in SEQ ID NO:8.
  • Protein "AM340” One protein of the present invention has been identified as protein "AM340".
  • a partial cDNA clone encoding AM340 was first isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with an EST reported by the I. M.A. G.E. Consortium identified as "yo68a05.rl Homo sapiens cDNA clone 183056 5' " (GenBank accession number H42936). The search also found a hit at GenBank accession number H42872.
  • the human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I. M.A. G.E. Consortium library.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AM340".
  • AM282 One protein of the present invention has been identified as protein "AM282".
  • a partial cDNA clone encoding AM282 was first isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the
  • GenBank database using BLASTA/BLASTX and FASTA search protocols.
  • the search revealed at least some identity with an EST reported by the I. M.A. G.E. Consortium identified as "yf95bl0.rl Human EST 30142 5' " (GenBank accession number R18560).
  • the search also found a thiat GenBank accession number T96696.
  • the human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc. , St.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AM282".
  • Applicants' methods identified clone AM282 as encoding a secreted protein.
  • the nucleotide sequence of the 5' portion of AM282 as presently determined is reported in SEQ ID NO: 11. What applicants believe is the proper reading frame and the predicted N-terminal amino acid sequence of the full length AM282 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO: 12.
  • Amino acids 1 to 24 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 25. Additional nucleotide sequence from the 3 ' portion of AM282, including the polyA tail, is reported in SEQ ID NO: 13.
  • AK647 A partial cDNA clone encoding AK647 was first isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with an EST reported by the I. M.A. G.E. Consortium identified as "ym40a05.rl Human EST 50483 5"' (GenBank accession number H17726). The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AK647" .
  • Applicants' methods identified clone AK647 as encoding a secreted protein.
  • nucleotide sequence of the 5' portion of AK647 as presently determined is reported in SEQ ID NO: 14. What applicants believe is the proper reading frame and the predicted N-terminal amino acid sequence of the full length AK647 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO: 15. Amino acids 1 to 25 are the predicted leader/signal sequence, with the predicted mamre amino acid sequence beginning at amino acid 26. Additional nucleotide sequence from the 3' portion of AK647, including the polyA tail, is reported in SEQ ID NO: 16.
  • AK583 A partial cDNA clone encoding AK583 was first isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with an EST reported by the I. M.A. G.E. Consortium identified as "yi90c06.rl Human EST 14656 5' " (GenBank accession number R77830). The search also found a hit at GenBank accession number H45398.
  • the human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I. M.A. G.E. Consortium library.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AK583".
  • Applicants' methods identified clone AK583 as encoding a secreted protein.
  • nucleotide sequence of the 5' portion of AK583 as presently determined is reported in SEQ ID NO: 17. What applicants believe is the proper reading frame and the predicted N-terminal amino acid sequence of the full length AK583 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO: 18. Amino acids 1 to 24 are the predicted leader/signal sequence, with the predicted mamre amino acid sequence beginning at amino acid 25. Additional nucleotide sequence from the 3 ' portion of AK583, including the polyA tail, is reported in SEQ ID NO: 19.
  • AK533 A partial cDNA clone encoding AK533 was first isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with an EST reported by the I. M.A. G.E. Consortium identified as "yb82h07.rl Homo sapiens cDNA clone 77725 5' " (GenBank accession number T55939). The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc.
  • AK533 This full- length clone is also referred to herein as " AK533" .
  • Applicants' methods identified clone AK533 as encoding a secreted protein.
  • the nucleotide sequence of the 5' portion of AK533 as presently determined is reported in SEQ ID NO:20. What applicants believe is the proper reading frame and the predicted N-terminal amino acid sequence of the full length AK533 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:21. Additional nucleotide sequence from the 3' portion of AK533, including the polyA tail, is reported in SEQ ID NO:22.
  • AK296 protein of the present invention.
  • a partial cDNA clone encoding AK296 was first isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with an EST reported by the I. M.A. G.E. Consortium identified as "yc86gl2.rl Homo sapeins cDNA clone 22958 5' " (GenBank accession number T75226).
  • the human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I. M.A. G.E. Consortium library.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail).
  • This full- length clone is also referred to herein as "AK296" .
  • AK296 Applicants' methods identified clone AK296 as encoding a secreted protein.
  • nucleotide sequence of the 5' portion of AK296 as presently determined is reported in SEQ ID NO:23. What applicants believe is the proper reading frame and the predicted N-terminal amino acid sequence of the full length AK296 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:24. Amino acids 1 to 36 are the predicted leader/signal sequence, with the predicted mamre amino acid sequence beginning at amino acid 37. Additional nucleotide sequence from the 3 ' portion of AK296, including the polyA tail, is reported in SEQ ID NO: 25.
  • H617 A partial cDNA clone encoding H617 was first isolated from a human PBMC cDNA library using methods which are selective for cDNAs encoding secreted proteins.
  • the nucleotide sequence of such partial cDN A was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with an EST reported by the I. M.A. G.E. Consortium identified as "ysl lc!2.rl Homo sapeins cDNA clone 2144865'" (GenBank accession number H71514).
  • the search also found a hit at GenBank accession number R 10010.
  • the human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St.
  • H617 This full-length clone is also referred to herein as "H617”. Applicants' methods identified clone H617 as encoding a secreted protein.
  • nucleotide sequence of the 5' portion of H617 as presently determined is reported in SEQ ID NO:26. What applicants believe is the proper reading frame and the predicted N-terminal amino acid sequence of the full length H617 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:27. Additional nucleotide sequence from the 3' portion of H617, including the polyA tail, is reported in SEQ ID NO:28.
  • BB9 protein of the present invention
  • a partial cDNA clone encoding BB9 was first isolated from a human PBMC (TH1 or Th2) cDNA library using methods which are selective for cDNAs encoding secreted proteins.
  • the nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with an EST reported by the I. M.A. G.E. Consortium identified as "yd68g04.rl Human cDNA clone 113430 5' " (GenBank accession number T78562).
  • the search also found a thi at GenBank accession number R54388.
  • the human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I. M.A. G.E. Consortium library.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail).
  • This full-length clone is also referred to herein as "BB9".
  • Applicants' methods identified clone BB9 as encoding a secreted protein.
  • nucleotide sequence of the 5' portion of BB9 as presently determined is reported in SEQ ID NO:29. What applicants believe is the proper reading frame and the predicted N-terminal amino acid sequence of the full length BB9 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:30. Additional nucleotide sequence from the 3' portion of BB9, including the polyA tail, is reported in SEQ ID NO:31. Protein "AW191"
  • a partial cDNA clone encoding AW191 was first isolated from a human ovary (PA-1 ter ato carcinoma) cDNA library using methods which are selective for cDNAs encoding secreted proteins.
  • the nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with an EST reported by the I. M.A. G.E. Consortium identified as "ym03dl0.rl Homo sapiens cDNA clone 46942 5' " (GenBank accession number H 10314.
  • the search also found a hit at GenBank accession number H05460.
  • the human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I. M.A. G.E. Consortium library.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full- length clone is also referred to herein as "AW191 " .
  • Applicants' methods identified clone AW191 as encoding a secreted protein.
  • nucleotide sequence of the 5' portion of AW191 as presently determined is reported in SEQ ID NO: 32. What applicants believe is the proper reading frame and the predicted N-terminal amino acid sequence of the full length AW191 protein corresponding to the foregoing nucleotide sequence is repo ⁇ ed in SEQ ID NO:33. Amino acids 1 to 18 are the predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 19. Additional nucleotide sequence from the 3 ' portion of AW 191 , including the polyA tail, is reported in SEQ ID NO: 34.
  • AT211 A partial cDNA clone encoding AT211 was first isolated from a human lymphocyte and dendritic cell cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with an EST reported by the I. M.A. G.E. Consortium identified as "yq36f01.rl Homo sapiens cDNA clone 197881 5' " (GenBank accession number R96278). The search also found a hit at GenBank accession number R56077.
  • the human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I. M.A. G.E. Consortium library.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full- length clone is also referred to herein as "AT211 ".
  • Applicants' methods identified clone AT211 as encoding a secreted protein.
  • nucleotide sequence of the 5' portion of AT211 as presently determined is reported in SEQ ID NO:35. What applicants believe is the proper reading frame and the predicted N-terminal amino acid sequence of the full length AT211 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:36. Amino acids 1 to 53 are the predicted leader/signal sequence, with the predicted mamre amino acid sequence beginning at amino acid 54. Additional nucleotide sequence from the 3' portion of AT211, including the polyA tail, is reported in SEQ ID NO:37.
  • AT205 One protein of the present invention has been identified as protein "AT205" .
  • a partial cDNA clone encoding AT205 was first isolated from a human lymphocyte and dendritic cell cDNA library using methods which are selective for cDNAs encoding secreted proteins.
  • the nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with an EST repo ⁇ ed by the
  • I. M.A. G.E. Consortium identified as "yu83cl l .rl Homo sapiens cDNA clone 240404 5' " (GenBank accession number H78080). The search also found a hit at GenBank accession number H78081. The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I. M.A. G.E.
  • the clone received from the distributor was examined and dete ⁇ nined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full- length clone is also referred to herein as "AT205".
  • Applicants' methods identified clone AT205 as encoding a secreted protein.
  • nucleotide sequence of AT205 as presently determined is reported in SEQ ID NO:38. What applicants believe is the proper reading frame and the predicted amino acid sequence of the full length AT205 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:39. Amino acids 1 to 55 are the predicted leader/signal sequence, with the predicted mamre amino acid sequence beginning at amino acid 56.
  • AS34 A partial cDNA clone encoding AS34 was first isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with an EST reported by the I. M.A. G.E. Consortium identified as "yg71a01.rl Homo sapiens cDNA clone 38531 5' " (GenBank accession number R51118). The search also found a hit at GenBank accession number R 15801.
  • the human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I. M.A. G.E. Consortium library.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail).
  • This full-length clone is also referred to herein as "AS34".
  • AS34 Applicants' methods identified clone AS34 as encoding a secreted protein.
  • nucleotide sequence of the 5' portion of AS34 as presently determined is reported in SEQ ID NO:40. What applicants believe is the proper reading frame and the predicted N-terminal amino acid sequence of the full length AS34 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:41. Amino acids 1 to 24 are the predicted leader/signal sequence, with the predicted mamre amino acid sequence beginning at amino acid 25. Additional nucleotide sequence from the 3' portion of AS34, including the polyA tail, is reported in SEQ ID NO:42.
  • AS32 A partial cDNA clone encoding AS32 was first isolated from a human fetal brain cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with an EST reported by the I. M.A. G.E. Consortium identified as "yu75b08.rl Homo sapiens cDNA clone 239607 5' " (GenBank accession number H 80466). The search also found a hit at GenBank accession number H77627.
  • the human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I. M.A. G.E. Consortium library.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full-length clone is also referred to herein as "AS32".
  • Applicants' methods identified clone AS32 as encoding a secreted protein.
  • the nucleotide sequence of the 5' ponion of AS32 as presently determined is reported in SEQ ID NO:43. What applicants believe is the proper reading frame and the predicted N-terminal amino acid sequence of the full length AS32 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:44. Additional nucleotide sequence from the 3' portion of AS32, including the polyA tail, is reported in SEQ ID NO:45.
  • AR260 A partial cDNA clone encoding AR260 was first isolated from a human adult retina cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with an EST reported by the I. M.A. G.E. Consortium identified as "yg99gl2.rl Homo sapiens cDNA clone 41757 5'" (GenBank accession number R52804). The human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail). This full- length clone is also referred to herein as "AR260".
  • Applicants' methods identified clone AR260 as encoding a secreted protein.
  • the nucleotide sequence of the 5' portion of AR260 as presently determined is reported in SEQ ID NO:46. What applicants believe is the proper reading frame and the predicted N-terminal amino acid sequence of the full length AR260 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:47.
  • Amino acids 1 to 23 are the predicted leader/signal sequence, with the predicted mamre amino acid sequence beginning at amino acid 24. Additional nucleotide sequence from the 3' portion of AR260, including the polyA tail, is reported in SEQ ID NO:48.
  • K640 A partial cDNA clone encoding K640 was first isolated from a murine bone marrow (stromal cell line FCM-4) cDNA library using methods which are selective for cDNAs encoding secreted proteins.
  • the nucleotide sequence of such partial cDN A was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with an EST reported by the I.M.A.G.E. Consortium identified as "yf47a09.rl Homo sapiens cDNA clone 129976 5' " (GenBank accession number Rl 1595). The search also found a hit at GenBank accession number H09031.
  • the human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I.M.A.G.E. Consortium library.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail).
  • This full- length clone is also referred to herein as "K640" .
  • Applicants' methods identified clone K640 as encoding a secreted protein.
  • nucleotide sequence of the 5' portion of K640 as presently determined is reported in SEQ ID NO:49. What applicants believe is the proper reading frame and the predicted N-terminal amino acid sequence of the full length K640 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:50. Additional nucleotide sequence from the 3' portion of K640, including the polyA tail, is reported in SEQ ID NO:49.
  • K39 A partial cDNA clone encoding K39 was first isolated from a murine bone marrow (stromal cell line FCM-4) cDNA library using methods which are selective for cDNAs encoding secreted proteins.
  • the nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with an EST reported by the I.M.A.G.E. Consortium identified as "ym65b04.rl Homo sapiens cDNA clone 163759 5' " (GenBank accession number H14129). The search also found a hit at GenBank accession number H68304.
  • the human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis, Mo, a distributor of the I.M.A.G.E. Consortium library.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail).
  • This full-length clone is also referred to herein as "K39".
  • Applicants' methods identified clone K39 as encoding a secreted protein.
  • nucleotide sequence of the 5' portion of K39 as presently determined is reported in SEQ ID NO:52. What applicants believe is the proper reading frame and the predicted N-terminal amino acid sequence of the full length K39 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:53. Additional nucleotide sequence from the 3' portion of K39, including the polyA tail, is reported in SEQ ID NO:54.
  • AT319 A partial cDNA clone encoding AT319 was first isolated from a human lymphocyte and dendritic cell cDNA library using methods which are selective for cDNAs encoding secreted proteins. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank database using BLASTA/BLASTX and FASTA search protocols. The search revealed at least some identity with an EST reported by the I.M.A.G.E. Consortium identified as "yr21bl l.rl Homo sapiens cDNA clone 205917 5' " (GenBank accession number H57730). The search also found a hit at GenBank accession number H57731.
  • the human cDNA clone corresponding to the EST database entry was ordered from Genome Systems, Inc., St. Louis. Mo, a distributor of the I.M.A.G.E. Consortium library.
  • the clone received from the distributor was examined and determined to be a full length clone, including a 5' end and 3' UTR (including a polyA tail).
  • This full- length clone is also referred to herein as "AT319".
  • Applicants' methods identified clone AT319 as encoding a secreted protein.
  • nucleotide sequence of the 5' ponion of AT319 as presently determined is reponed in SEQ ID NO:55. What applicants believe is the proper reading frame and the predicted N-terminal amino acid sequence of the full length AT319 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:56. Additional nucleotide sequence from the 3' portion of AT319, including the polyA tail, is reported in SEQ ID NO:57.
  • Clones AP162, AM931, AM610, AM340, AM282, AK647, AK583, AK533, AK296, H617, BB9, AW 191 , AT211 , AT205, AS34, AS32, AR260, K640, K39 and AT319 were deposited on April 17, 1996 with the American Type Culture Collection under accession number ATCC 98026, from which each clone comprising a particular polynucleotide is obtainable. Each clone has been transfected into separate bacterial cells (E. coli) in this composite deposit. Bacterial cells containing a particular clone can be obtained from the composite deposit as follows:
  • oligonucleotide 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 design of the oligonucleotide probe should preferably follow these parameters:
  • the oligonucleotide should preferably be labeled with g- 32 P ATP (specific activity 6000
  • Ci/mmole Ci/mmole
  • T4 polynucleotide kinase using commonly employed techniques for labeling oligonucleotides. Other labeling techniques can also be used.
  • Uninco ⁇ orated label should preferably be removed by gel filtration chromatography or other established methods .
  • the amount of radioactivity inco ⁇ orated into the probe should be quantitated by measurement in a scintillation counter.
  • specific activity of the resulting probe should be approximately 4e + 6 dpm/pmole.
  • the bacterial culmre containing the pool of full-length clones should preferably be thawed and 100 ⁇ l of the stock used to inoculate a sterile culmre flask containing 25 ml of sterile L-broth containing ampicillin at 100 ⁇ g/ml.
  • the culmre should preferably be grown to saturation at 37 °C, and the samrated culmre should preferably be diluted in fresh L- broth. Aliquots of these dilutions should preferably be plated to determine the dilution and volume which will yield approximately 5000 distinct and well-separated colonies on solid bacteriological media containing L-broth containing ampicillin at 100 ⁇ g/ml and agar at
  • 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/liter, 88.2 g Na citrate/liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100 ⁇ g/ml of yeast RNA, and 10 mM EDTA (approximately 10 mL per 150 mm filter).
  • 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. IX 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 visualize 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 cyclized using known methods, for example, as described in H.U. Saragovi, et al , Bio/Technology iQ, 773-778 (1992) and in R.S. McDowell, et al , J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are inco ⁇ orated herein by reference.
  • Such fragments may be fused to carrier molecules such as immunoglobulins for many pu ⁇ oses, including increasing the valency of protein binding sites.
  • fragments of the protein may be fused through "linker" sequences to the Fc portion of an immunoglobulin.
  • a bivalent form of the protein such a fusion could be to the Fc portion of an IgG molecule.
  • Other immunoglobulin isotypes may also be used to generate such fusions.
  • 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 listing by translation of the nucleotide sequence of each disclosed clone.
  • the mature form of such protein may be obtained by expression of the disclosed full-length polynucleotide (preferably those deposited with ATCC) in a suitable mammalian cell or other host cell.
  • the sequence of the mamre form of the protein may also be determinable from the amino acid sequence of the full-length form.
  • the present invention also provides for soluble forms of such protein.
  • the intracellular and transmembrane domains of the protein are deleted such that the protein is fully secreted from the cell in which it is expressed.
  • the intracellular and transmembrane domains of proteins of the invention can be identified in accordance with known techniques for determination of such domains from sequence information.
  • Species homologs of the disclosed proteins are also provided by the present invention. Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.
  • the invention also encompasses allelic variants of the disclosed proteins; that is, naturally-occurring alternative forms of the isolated proteins which are identical, homologous or related to that encoded by the polynucleotides disclosed herein.
  • the isolated polynucleotide endcoing the protein of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al. , Nucleic Acids Res. 19_, 4485-4490(1991), in order to produce the protein recombinantiy.
  • an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al. , Nucleic Acids Res. 19_, 4485-4490(1991), in order to produce the protein recombinantiy.
  • Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology JL8_5_, 537-566 (1990).
  • operably linked 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 ligated polynucleotide/expression control sequence.
  • Mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, 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 cells.
  • monkey COS cells Chinese Hamster Ovary (CHO) cells
  • human kidney 293 cells human epidermal A431 cells
  • human Colo205 cells human Colo205 cells
  • CV-1 cells other transformed primate cell lines
  • 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 cells.
  • yeast eukaryotes
  • prokaryotes such as bacteria.
  • yeast strains include Saccharomyces cerevisiae, Schizosaccharomyc ⁇ 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.
  • 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, California, 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). inco ⁇ orated herein by reference.
  • 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 culmre conditions suitable to express the recombinant protein.
  • the resulting expressed protein may then be purified from such culture (i.e. , from culmre 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.
  • affinity resins as concanavalin A-agarose, heparin-toyopearl ® or Cibacrom blue 3GA Sepharose ®
  • hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether
  • immunoaffinity chromatography immunoaffinity chromatography
  • the protein of the invention may also be expressed in a form which will facilitate 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 Kod
  • RP- HPLC reverse-phase high performance liquid chromatography
  • hydrophobic RP-HPLC media e.g., silica gel having pendant methyl or other aliphatic groups
  • Some or all 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 mammalian 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-constructedprotein 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 deliberately engineered. For example, modifications in the peptide or DNA sequences can be made by those skilled 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.
  • 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 proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutive ly or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands.
  • the protein binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction)
  • the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction. Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.
  • 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 of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules.
  • the protein of the invention can be added to the medium in or on which the microorganism is culmred.
  • a protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations.
  • cytokine cytokine
  • cell proliferation either inducing or inhibiting
  • cell differentiation either inducing or inhibiting
  • the activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M-l- (preB M + ), 2E8, RB5, DAI, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies. E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al., J. Immunol.
  • Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp.
  • Assays for proliferation and differentiationof hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991 ; deVries et al., J. Exp. Med. 173: 1205-1211, 1991; Moreau et al., Namre 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A.
  • Assays for T-cell clone responses to antigens include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies,
  • a protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein.
  • a protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g. , in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.
  • SCID severe combined immunodeficiency
  • These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders.
  • infectious diseases causes by viral, bacterial , fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, he ⁇ esviruses, mycobacteria, Leishmania spp. , malaria spp. and various fungal infections such as candidiasis.
  • a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e. , in the treatment of cancer.
  • Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-hostdisease and autoimmune inflammatory eye disease.
  • a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems.
  • Other conditions, in which immune suppression is desired may also be treatable using a protein of the present invention.
  • T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.
  • Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent.
  • Tolerance which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.
  • Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as , for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft- versus-host disease (GVHD).
  • B lymphocyte antigen functions such as , for example, B7
  • GVHD graft- versus-host disease
  • blockage of T cell function should result in reduced tissue destruction in tissue transplantation.
  • rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant.
  • a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells 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
  • a B7 lymphocyte antigen 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
  • 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 costimulationmay 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.
  • appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al, Science 257:789-792 (1992) and Turka et al , Proc. Natl. Acad. Sci USA, 89: 1 1102-11105 (1992).
  • murine models of GVHD see Paul ed. , Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease.
  • Blocking antigen function may also be therapeutically useful for treating autoimmune diseases.
  • Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases.
  • Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.
  • Administration of reagents which block costimulation of T cells by disrupting receptor: ligand interactions of B lymphocyte antigens can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease.
  • the efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/ Ipr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed. , Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).
  • Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B lymphocyte antigens systemically.
  • anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen- pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient.
  • Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient.
  • the infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.
  • up regulation or enhancement of antigen function may be useful in the induction of mmor immunity .
  • Tumor cells e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma
  • a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject.
  • the mmor cell can be transfected to express a combination of peptides .
  • mmor 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 mmor cells are remrned 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 mmor cell for transfection in vivo.
  • tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated ponion) of an MHC class I a chain protein and ⁇ -, microglobuiin protein or an MHC class II ⁇ chain protein and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated ponion) of an MHC class I a chain protein and ⁇ -, microglobuiin protein or an MHC class II ⁇ chain protein and an MHC class II ⁇ chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
  • a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of mmor associated antigens and induce mmor specific immunity.
  • a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1- 3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol.
  • T-cell-dependent immunoglobulin responses and isotype switching include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
  • MLR Mixed lymphocyte reaction
  • Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol.
  • lymphocyte survival/apoptosis which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis
  • Assays for lymphocyte survival/apoptosis include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al.
  • 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 suppo ⁇ ing the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e.
  • the activity of a protein of the invention may, among other means, be measured by the following methods: Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.
  • Assays for embryonic stem cell differentiation include, without limitation, those described in: Johansson et al. Cellular Biology 15: 141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81 :2903-2915, 1993.
  • Assays for stem cell survival and differentiation include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama et al. , Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I.K. and Briddell, R.A.
  • a protein of the present invention also may have utility in compositions used for bone, canilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of burns, incisions and ulcers.
  • a protein of the present invention which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.
  • Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.
  • a protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells.
  • a protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and/or canilage 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/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals.
  • Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue.
  • compositions of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments.
  • the compositions of the present invention may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament- forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair.
  • the compositions of the invention may also be useful in the treatment of tendinitis, ca ⁇ al mnnel syndrome and other tendon or ligament defects.
  • compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.
  • the protein of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders , which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome.
  • Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke.
  • Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a protein of the invention. Proteins of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
  • a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues.
  • organs including, for example, pancreas, liver, intestine, kidney, skin, endothelium
  • muscle smooth, skeletal or cardiac
  • vascular including vascular endothelium
  • a protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.
  • a protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. W095/ 16035 (bone, cartilage, tendon); International Patent Publication No . WO95/05846 (nerve , neuronal); International Patent Publication No. WO91/07491 (skin, endothelium ).
  • Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT, eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71 :382-84 (1978).
  • a protein of the present invention may also exhibit activin- or inhibin-related activities. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH).
  • FSH follicle stimulating hormone
  • a protein of the present invention alone or in heterodimers with a member of the inhibin ⁇ family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals.
  • the protein of the invention may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, United States Patent 4,798,885.
  • a protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91 :562-572, 1972; Ling et al., Namre 321 :779-782, 1986; Vale et al. , Nature 321:776-779, 1986; Mason et al. , Namre 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986.
  • a protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells.
  • Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action.
  • Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to mmors or sites of infection may result in improved immune responses against the mmor or infecting agent.
  • a protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population.
  • the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
  • the activity of a protein of the invention may, among other means, be measured by the following methods: Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W. Strober, Pub.
  • a protein of the invention may also exhibit hemostatic or thrombolytic activity. As a result, such a protein is expected to be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes.
  • a protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin. Pharmacol. 26: 131-140, 1986; Burdick et al. , Thrombosis Res. 45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.
  • a protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/ligand interactions.
  • receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses).
  • Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction.
  • a protein of the present invention may themselves be useful as inhibitors of receptor/ligand interactions.
  • the activity of a protein of the invention may, among other means, be measured by the following methods:
  • Suitable assays for receptor-Iigand activity include without limitation those described in.Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H.
  • Proteins of the present invention may also exhibit anti-inflammatory activity.
  • the anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response.
  • Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia- reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine- induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Proteins of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material.
  • infection such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)
  • ischemia- reperfusion injury such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)
  • ischemia- reperfusion injury such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)
  • a protein of the invention may exhibit other ami-tumor activities.
  • a protein may inhibit mmor growth directly or indirectly (such as, for example, via ADCC).
  • a protein may exhibit its mmor inhibitory activity by acting on mmor tissue or tumor precursor tissue, by inhibiting formation of tissues necessary to support mmor growth (such as, for example, by inhibiting angiogenesis), by causing production of other factors, agents or cell types which inhibit mmor growth, or by suppressing, eliminating or inhibiting factors, agents or cell types which promote tumor growth.
  • a protein of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or caricadic cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation
  • 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, stabilizers, solubilizers, and other materials well known in the an.
  • 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 administratbn.
  • 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, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-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 compliment its activity or use in treatment.
  • 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-inflammaCry 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 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 will deliver 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.
  • TCR T cell receptor
  • MHC and strucmrally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes.
  • the antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells.
  • antibodies able to bind surface immunolgobulin and other molecules on B cells as well 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 liposome in which protein of the present invention is combined, in addition to other pharmaceutical ⁇ acceptable carriers, with amphipathc agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.
  • Suitable lipids for liposomal formulation include, without limitation, 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 inco ⁇ orated herein by reference.
  • 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, healing, 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, healing, 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 factor(s), 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 application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is preferred.
  • protein of the present invention When a therapeutically effective amount of protein of the present invention is administered orally, protein of the present invention will be in the form of a tablet, capsule, powder, solution or elixir.
  • the pharmaceutical composition of the invention may additionally contain a solid 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.
  • a liquid 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 saline 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 therapeutically 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 preferred 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 stabilizers, 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 namre and severity of the condition being treated, and on the namre 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 ⁇ g to about 100 mg (preferably about 0. l ⁇ g 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 specifically 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 limpet hemocyanin (KLH).
  • KLH keyhole limpet hemocyanin
  • Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein.
  • Neutralizing 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.
  • neutralizing 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, systematically, or locally 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 delivery to the site of bone, cartilage or tissue damage.
  • Topical administration may be suitable for wound healing and tissue repair.
  • Therapeutically useful agents other than a protein of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention.
  • the composition would include a matrix capable of delivering 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 delivering 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, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions will define the appropriate formulation.
  • Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and poiyanhydrides.
  • Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen.
  • 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, panicle size, particle shape, and biodegradability.
  • 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.
  • a sequestering agent such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.
  • a preferred family of sequestering agents is cellulosic materials such as alkylcelluloses(including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC).
  • CMC carboxymethylcellulose
  • Other preferred 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 handling of the composition, yet not so much that the progenitor cells 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
  • TGF- ⁇ and TGF- ⁇ transforming growth factors
  • IGF insulin-like growth factor
  • the therapeutic compositions are also presently valuable for veterinary applications. 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-containingpharmaceutical 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 'sage, 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 reconstimtionand with inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage.
  • IGF I insulin like growth factor I
  • Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian 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 culmred ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic pu ⁇ oses. Patent and literature references cited herein are inco ⁇ orated by reference as if fully set fo ⁇ h.
  • AAATNTTAAA ATAATTCCAA GCTGAGTTTT CTAGATTGAG CAGAAATGGT GAAAGGAGTA 120
  • AAAAAAAAAA AAAAA 315 (2) INFORMATION FOR SEQ ID NO:4:
  • GGACATGCCA GGAATAAAAA GGATACTCAC TGTTACCATT CTGGCTCTCT GTCTTCCAAG 240
  • CCCTGGGAAT GCACAGGCAC AGTGCACGAA TGGCTTTGAC CTGGATCGCC AGTCAGGACA 300 GTGTTTAGAT ATTGATGAAT GCCGAACCAT CCCCGAGGCC TGCCGAGGAG ACATGATGTG 360
  • NGACACTTAC TGGTTAAACT TACGTTGCTA AAGATTTCTC TATAATAAGC CACACATTAT 120
  • GAGTGTTCTT CAGAGGATCC TCGCTGCCCA GGTTCCCTGC CAGAAGGACA GAATAATCCT 240
  • GAGACTCCWC AATTATTGAT CCAGGAACTG AGCAAGATCT TCCTTCCCCT GAAAATAGTT 120
  • MOLECULE TYPE cDNA
  • Xi SEQUENCE DESCRIPTION: SEQ ID NO:28.

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract

On a cloné une protéine de mastocyte de 78 kDa (moesine) depuis le rat et on a déterminé son ADNc et sa séquence d'acides aminés. Cette protéine est phosphorylée sur des résidus spécifiques de serine et de threonine sous l'effet exercé dans les mastocytes par un ou plusieurs isozymes de protéine kinase C, en particulier, l'isozyme κ, ce qui permet d'obtenir une phosphoprotéine qui inhibe la dégranulation des mastocytes (agent inhibiteur de la dégranulation des mastocytes). On peut stimuler la phosphorylation in vivo de cette protéine dans les mastocytes au moyen de médicaments, tels que cromolyne, nedocromil, de flavonoïdes, tels que quercetine et kaempherol, et de lodoxamide, On peut inhiber la dégranulation des mastocytes au moyen de l'administration d'inhibiteurs de phosphomoesine phosphatase. On peut identifier des tissus déficients en moesine de mastocytes au moyen d'un anticorps marqué anti-moesine ou anti-phosphomoesine et les traiter au moyen de la transfection de ces mastocytes par de l'ADNc de moesine dans un vecteur viral, tel que le vecteur du virus de la grippe.
PCT/US1997/006042 1996-04-12 1997-04-11 PHOSPHOPROTEINE DE 78 kDa ISOLEE ET CLONEE DE MASTOCYTES (AGENT INHIBITEUR DE LA DEGRANULATION DES MASTOCYTES) ET SON UTILISATION WO1997039122A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU24563/97A AU2456397A (en) 1996-04-12 1997-04-11 Isolated and cloned mast cell 78 kda phosphoprotein (mast cell degranulation inhibitory agent) and use thereof

Applications Claiming Priority (2)

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US63118496A 1996-04-12 1996-04-12
US08/631,184 1996-04-12

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999000410A2 (fr) * 1997-06-27 1999-01-07 Incyte Pharmaceuticals, Inc. Proteines de matricielles extracellulaires humaines
WO1999055864A1 (fr) * 1998-04-28 1999-11-04 Ono Pharmaceutical Co., Ltd. Nouveau polypeptide, adnc le codant et son utilisation
EP1037898A1 (fr) * 1997-06-27 2000-09-27 Genetics Institute, Inc. Proteines secretees
WO2000073458A1 (fr) * 1999-05-28 2000-12-07 Zymogenetics, Inc. Proteine-31 a helice alpha secretee
EP1557426A2 (fr) * 1998-02-09 2005-07-27 Human Genome Sciences, Inc. 45 Protéines humaines secrétées
US8410248B2 (en) 1999-03-12 2013-04-02 Human Genome Sciences Inc. HWBAO62 polypeptides
US9938341B2 (en) * 2002-06-14 2018-04-10 Stowers Institute For Medical Research Antibodies that specifcally bind Sost and Wise peptides

Citations (1)

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Publication number Priority date Publication date Assignee Title
EP0613945A2 (fr) * 1993-02-25 1994-09-07 The General Hospital Corporation Merlin, gène suppresseur de tumeurs, et ses utilisations

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
EP0613945A2 (fr) * 1993-02-25 1994-09-07 The General Hospital Corporation Merlin, gène suppresseur de tumeurs, et ses utilisations

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LANKES WT ET AL: "Moesin: a member of the protein 4.1-talin-ezrin family of proteins." PROC NATL ACAD SCI U S A, OCT 1 1991, 88 (19) P8297-301, UNITED STATES, XP002045604 *
NAKAMURA F ET AL: "Phosphorylation of threonine 558 in the carboxyl-terminal actin-binding domain of moesin by thrombin activation of human platelets." J BIOL CHEM, DEC 29 1995, 270 (52) P31377-85, UNITED STATES, XP002045605 *
SATO N ET AL: "A gene family consisting of ezrin, radixin and moesin. Its specific localization at actin filament/plasma membrane association sites." J CELL SCI, SEP 1992, 103 ( PT 1) P131-43, ENGLAND, XP002045603 *
SCHWARTZ-ALBIEZ R ET AL: "Differential expression of the microspike-associated protein moesin in human tissues." EUR J CELL BIOL, JUL 1995, 67 (3) P189-98, GERMANY, XP002045606 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6280739B1 (en) * 1996-04-18 2001-08-28 Genetics Institute, Inc. Method of inhibiting angiogenesis using secreted proteins
WO1999000410A2 (fr) * 1997-06-27 1999-01-07 Incyte Pharmaceuticals, Inc. Proteines de matricielles extracellulaires humaines
WO1999000410A3 (fr) * 1997-06-27 1999-03-18 Incyte Pharma Inc Proteines de matricielles extracellulaires humaines
EP1037898A1 (fr) * 1997-06-27 2000-09-27 Genetics Institute, Inc. Proteines secretees
US6303765B1 (en) 1997-06-27 2001-10-16 Incyte Genomics, Inc. Human extracellular matrix proteins
EP1037898A4 (fr) * 1997-06-27 2003-04-16 Inst Genetics Llc Proteines secretees
EP1557426A2 (fr) * 1998-02-09 2005-07-27 Human Genome Sciences, Inc. 45 Protéines humaines secrétées
EP1557426A3 (fr) * 1998-02-09 2005-08-03 Human Genome Sciences, Inc. 45 Protéines humaines secrétées
WO1999055864A1 (fr) * 1998-04-28 1999-11-04 Ono Pharmaceutical Co., Ltd. Nouveau polypeptide, adnc le codant et son utilisation
US8410248B2 (en) 1999-03-12 2013-04-02 Human Genome Sciences Inc. HWBAO62 polypeptides
WO2000073458A1 (fr) * 1999-05-28 2000-12-07 Zymogenetics, Inc. Proteine-31 a helice alpha secretee
US9938341B2 (en) * 2002-06-14 2018-04-10 Stowers Institute For Medical Research Antibodies that specifcally bind Sost and Wise peptides

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AU2456397A (en) 1997-11-07

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