EP1198241A1

EP1198241A1 - Novel proteins

Info

Publication number: EP1198241A1
Application number: EP00930284A
Authority: EP
Inventors: Kenneth Jacobs; John M. Mccoy; Edward R. Lavallie; Lisa A. Collins-Racie; Cheryl Evans; David Merberg
Original assignee: Genetics Institute LLC
Current assignee: Genetics Institute LLC
Priority date: 1999-04-29
Filing date: 2000-04-29
Publication date: 2002-04-24
Also published as: WO2000066148A9; WO2000066148A1; AU4813600A; JP2002542799A

Abstract

Novel proteins are disclosed.

Description

NOVEL PROTEINS

RELATED APPLICATION This application claims the benefit of priority under 35 U.S.C. §119(e) to copending U.S. Provisional Application No. 60/131,596, filed on April 29, 1999, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION The present invention provides novel proteins , along with therapeutic, diagnostic and research utilities for these proteins.

BACKGROUND OF THE INVENTION Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, CSFs and interleukins) has matured rapidly over the past decade. The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides "directly" in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning). More recent "indirect" cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity by virtue of their secreted nature in the case of leader sequence cloning, or by virtue of the cell or tissue source in the case of PCR-based techniques. It is to these proteins that the present invention is directed.

SUMMARY OF THE INVENTION In one embodiment, 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:l;

(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 156 to nucleotide 251; (c) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone AW179_li deposited under accession number ATCC 207186;

(d) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone AW179_li deposited under accession number ATCC 207186;

(e) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone AW179_li deposited under accession number ATCC 207186;

(f) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone AW179_li deposited under accession number ATCC 207186;

(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2;

(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 2 having biological activity, the fragment comprising eight contiguous amino acids of SEQ ID NO:2;

(i) a 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. Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:l from nucleotide 156 to nucleotide 251; the nucleotide sequence of the full-length protein coding sequence of clone AW179_li deposited under accession number ATCC 207186; or the nucleotide sequence of a mature protein coding sequence of clone AW179_li deposited under accession number ATCC 207186. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone AW179_li deposited under accession number ATCC 207186.

In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:2;

(b) a fragment of the amino acid sequence of SEQ ID NO:2, the fragment comprising eight contiguous amino acids of SEQ ID NO:2; and

(c) the amino acid sequence encoded by the cDNA insert of clone AW179_li deposited under accession number ATCC 207186; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:2. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino acids of SEQ ID NO:2, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2, the fragment comprising the amino acid sequence from amino acid 11 to amino acid 20 of SEQ ID NO:2.

In one embodiment, 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:3;

(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 1039 to nucleotide 1239; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID

NO:3 from nucleotide 1090 to nucleotide 1239;

(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone BG221_li deposited under accession number ATCC 207186; (e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone BG221_li deposited under accession number ATCC 207186; (f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone BG221_li deposited under accession number ATCC 207186; (g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone BG221_li deposited under accession number ATCC 207186;

(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4;

(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity, the fragment comprising eight contiguous amino acids of SEQ ID NO:4;

(j) a polynucleotide which is an allelic variant of a polynucleotide of

(a)-(g) above; and (k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above.

Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:3 from nucleotide 1039 to nucleotide 1239; the nucleotide sequence of SEQ ID NO:3 from nucleotide 1090 to nucleotide 1239; the nucleotide sequence of the full-length protein coding sequence of clone BG221_li deposited under accession number ATCC 207186; or the nucleotide sequence of a mature protein coding sequence of clone BG221_li deposited under accession number ATCC 207186. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone BG221_li deposited under accession number ATCC 207186.

In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO:4; (b) a fragment of the amino acid sequence of SEQ ID NO:4, the fragment comprising eight contiguous amino acids of SEQ ID NO:4; and

(c) the amino acid sequence encoded by the cDNA insert of clone

BG221_li deposited under accession number ATCC 207186; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:4. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino acids of SEQ ID NO:4, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4, the fragment comprising the amino acid sequence from amino acid 28 to amino acid

37 of SEQ ID NO:4.

(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5;

(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 49 to nucleotide 972; (c) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone K139_li deposited under accession number ATCC 207186;

(d) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone K139_li deposited under accession number ATCC 207186;

(e) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone K139_li deposited under accession number ATCC 207186;

(f) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone K139_li deposited under accession number ATCC 207186;

(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6;

(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 having biological activity, the fragment comprising eight contiguous amino acids of SEQ ID NO:6;

(i) a 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. Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:5 from nucleotide 49 to nucleotide 972; the nucleotide sequence of the full-length protein coding sequence of clone K139_li deposited under accession number ATCC 207186; or the nucleotide sequence of a mature protein coding sequence of clone K139_li deposited under accession number ATCC 207186. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone K139_li deposited under accession number ATCC 207186.

In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:6;

(b) a fragment of the amino acid sequence of SEQ ID NO:6, the fragment comprising eight contiguous amino acids of SEQ ID NO: 6; and

(c) the amino acid sequence encoded by the cDNA insert of clone K139_li deposited under accession number ATCC 207186; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:6. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous a ino acids of SEQ ID NO:6, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6, the fragment comprising the amino acid sequence from amino acid 149 to amino acid 158 of SEQ ID NO:6.

(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7;

(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 7 from nucleotide 90 to nucleotide 569; (c) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone K511_li deposited tinder accession number ATCC 207186;

(d) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone K511_li deposited under accession number ATCC 207186; (e) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone K511_li deposited under accession number ATCC 207186;

(f) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone K511_li deposited under accession number ATCC 207186; (g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8;

(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 having biological activity, the fragment comprising eight contiguous amino acids of SEQ ID NO:8; (i) a 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. Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:7 from nucleotide 90 to nucleotide 569; the nucleotide sequence of the full-length protein coding sequence of clone K511_li deposited under accession number ATCC 207186; or the nucleotide sequence of a mature protein coding sequence of clone K511_li deposited under accession number ATCC 207186. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone K511_li deposited under accession number ATCC 207186.

(a) the amino acid sequence of SEQ ID NO:8;

(b) a fragment of the amino acid sequence of SEQ ID NO:8, the fragment comprising eight contiguous amino acids of SEQ ID NO:8; and

(c) the amino acid sequence encoded by the cDNA insert of clone K511_li deposited under accession number ATCC 207186; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:8. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino acids of SEQ ID NO:8, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8, the fragment comprising the amino acid sequence from amino acid 75 to amino acid 84 of SEQ ID NO:8.

(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9;

(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 125 to nucleotide 505; (c) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone N154_li deposited under accession number ATCC 207186;

(d) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone N154_li deposited under accession number ATCC 207186; (e) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone N154_li deposited under accession number ATCC 207186;

(f) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone N154_li deposited under accession number ATCC 207186;

(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10;

(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity, the fragment comprising eight contiguous amino acids of SEQ ID NO: 10;

(i) a 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. Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID NO:9 from nucleotide 125 to nucleotide 505; the nucleotide sequence of the full-length protein coding sequence of clone N154_li deposited under accession number ATCC 207186; or the nucleotide sequence of a mature protein coding sequence of clone N154_li deposited under accession number ATCC 207186. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone N154_li deposited under accession number ATCC 207186.

In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:10;

(b) a fragment of the amino acid sequence of SEQ ID NO:10, the fragment comprising eight contiguous amino acids of SEQ ID NO:10; and

(c) the amino acid sequence encoded by the cDNA insert of clone N154_li deposited under accession number ATCC 207186; the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:10. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino acids of SEQ ID NO: 10, or a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10, the fragment comprising the amino acid sequence from amino acid 58 to amino acid 67 of SEQ ID NO:10.

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.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and IB are schematic representations of the pED6 and pNOTs vectors, respectively, used for deposit of clones disclosed herein.

DETAILED DESCRIPTION ISOLATED PROTEINS

Nucleotide and amino acid sequences, as presently determined, are reported below for each clone and protein disclosed in the present application. The nucleotide sequence of each clone can readily be determined by sequencing of the deposited clone in accordance with known methods. The predicted amino acid sequence (both full-length and mature forms) 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. As used herein a "secreted" protein is one which, when expressed in a suitable host cell, is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence. "Secreted" proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g. , receptors) from the cell in which they are expressed. "Secreted" proteins also include without limitation proteins which are transported across the membrane of the endoplasmic reticulum.

Protein "AW179 li"

One protein of the present invention has been identified as protein "AW179_li". A partial cDNA clone encoding AW179_li was first isolated from a human adult ovary (PA-1 teratocarcrnoma, pool of retinoic-acid-treated, activin-treated, and untreated tissue) cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank and GeneSeq databases using BLASTN/BLASTX and FASTA search protocols. The search revealed at least some sequence similarity to sequences identified as AA861423 (ak34c06.sl Soares testis NHT Homo sapiens cDNA clone IMAGE:1407850 3¹ similar to TR 008608 008608 SEIZURE RELATED PROTEIN 15; mRNA sequence), AF057297 (Homo sapiens ornithine decarboxylase antizyme 2 (OAZ2) mRNA, complete cds), H93950 (yvl3bl0.sl Homo sapiens cDNA clone IMAGEJ42587 3'), R97297 (yq74h08.rl Homo sapiens cDNA clone IMAGEJ01567 5'), T81992 (yd36a06.rl Homo sapiens cDNA clone IMAGE:1102905'), W38584 (zbl9cl2.rl Soares fetal lung NbHL19W Homo sapiens cDNA clone IMAGE:302518 5'), and W76088 (zd59a04.rl Soares fetal heart NbHH19W Homo sapiens cDNA clone IMAGE:344910 5'). The predicted amino acid sequence disclosed herein for AW179_li was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted AW179_li protein demonstrated at least some similarity to sequences identified as AF057297 (ornithine decarboxylase antizyme 2 [Homo sapiens]). Ornithine decarboxylase antizyme binds to, and destabilizes, ornithine decarboxylase which is then degraded. Ornithine decarboxylase antizyme is expressed through the mechanism of translational frameshifting: an autoregulatory mechanism enables modulation of frameshifting according to the cellular concentration of poly amines. A 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 5¹ end and 3' UTR , including a poly(A) tail. This full-length clone is also referred to herein as "AW179_li".

Applicants' methods identified clone AW179_li as encoding a secreted protein. The nucleotide sequence of AW179_li as presently determined is reported in SEQ

ID NO:l, and includes the poly(A) tail. The predicted reading frame and amino acid sequence of the AW179_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:2. However, through the mechanism of translational frameshifting as described above, a naturally occuring frameshift at nucleotide 251 of SEQ ID NO:l from frame 1 to frame 2, skipping nucleotide 252, produces the amino acid sequence shown in SEQ ID NO:15, encoded by nucleotides 156 to 251 of SEQ ID NO:l followed by nucleotides 253 to 726 of SEQ ID NO:l.

The EcoRI/Notl restriction fragment obtainable from the deposit containing clone AW179_li should be approximately 990 bp.

Protein "BG221 li"

One protein of the present invention has been identified as protein "BG221_li". A partial cDNA clone encoding BG221_li was first isolated from a human adult brain cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank and GeneSeq databases using BLASTN/BLASTX and FASTA search protocols. The search revealed at least some sequence similarity to sequences identified as AI075660 (oy27g04.sl Soares_senescent_fibroblasts_NbHSF Homo sapiens cDNA clone IMAGEJ667094 3', mRNA sequence), AI341673 (qq95g04.xl Soares total fetus Nb2HF8_9w Homo sapiens cDNA clone IMAGEJ939158 3', mRNA sequence), R43552 (ygl9g07.sl Homo sapiens cDNA clone IMAGEJ2793 3'), R63117 (yi01c07.rl Homo sapiens cDNA clone IMAGEJ379645'), and T24072 (Human gene signature HUMGS06057). 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 poly(A) tail. This full-length clone is also referred to herein as "BG221_li".

Applicants' methods identified clone BG221_li as encoding a secreted protein. The nucleotide sequence of BG221_li as presently determined is reported in SEQ ID NO:3, and includes the poly(A) tail. The predicted reading frame and amino acid sequence of the BG221_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:4. Amino acids 5 to 17 of SEQ ID NO:4 are a predicted leader/ signal sequence, with the predicted mature amino acid sequence beginning at amino acid 18. Due to the hydrophobic nature of the predicted leader/ signal sequence, it is likely to act as a transmembrane domain should the predicted leader/ signal sequence not be separated from the remainder of the BG221_li protein. The EcoRI/Notl restriction fragment obtainable from the deposit containing clone BG221_li should be approximately 1400 bp.

Protein "K139 li" One protein of the present invention has been identified as protein "K139_li". A partial cDNA clone encoding K139_li was first isolated from a mouse adult bone marrow (stromal cell line FCM-4) cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank and GeneSeq databases using BLASTN/BLASTX and FASTA search protocols. The search revealed at least some sequence similarity to an EST identified as AA058874 (zl96al2.rl Stratagene corneal stroma (#937222) Homo sapiens cDNA clone IMAGE:512446 5' similar to contains element MSR1 repetitive element; mRNA sequence), AA064738 (zml3hl2.rl Stratagene pancreas (#937208) Homo sapiens cDNA clone IMAGE:525575 5' similar to WP K10D3.5 CE06174), T20987 (Human gene signature HUMGS02264), V59121 (Nucleotide sequence of BCOM3, a kinase), and W79308 (zdβOcOl.rl Soares fetal heart NbHH19W Homo sapiens cDNA clone IMAGEJ46944 5'). The predicted amino acid sequence disclosed herein for K139_li was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted K139_li protein demonstrated at least some similarity to sequences identified as W77299 (Amino acid sequence of BCOM3, a kinase), Z46636 (protein kinase PkpA [Phycomyces blakesleeanus]), and Z75545 (K10D3.5; weak similarity with many protein kinases [Caenorhabditis elegans]). 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 poly(A) tail. This human full-length clone is also referred to herein as "K139_li". Applicants' methods identified clone K139_li as encoding a secreted protein.

The nucleotide sequence of K139_li as presently determined is reported in SEQ ID NO:5, and includes the poly(A) tail. The predicted reading frame and amino acid sequence of the K139_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:6. The EcoRI/Notl restriction fragment obtainable from the deposit containing clone K139_li should be approximately 1818 bp.

Protein "K511 li" One protein of the present invention has been identified as protein "K511_li". A partial cDNA clone encoding K511_li was first isolated from a mouse adult bone marrow

(stromal line FCM-4) cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. The nucleotide sequence of such partial cDNA was determined and searched against the GenBank and GeneSeq databases using BLASTN/BLASTX and

FASTA search protocols. The search revealed at least some sequence similarity to an EST identified as AA008157 (mg70e07.rl Soares mouse embryo NbME13.5 14.5 Mus musculus cDNA clone IMAGE:4383725'), AI147016 (qa82a09.xl Soares fetal heart NbHH19W Homo sapiens cDNA clone IMAGE:1693240 3', mRNA sequence), R36551 (yg33h04.r9 Homo sapiens cDNA clone IMAGEJ4549 5'), R42054 (yg05h04.sl Homo sapiens cDNA clone

31562 3'), and T21796 (Human gene signature HUMGS03261). A 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 poly(A) tail. This full-length human clone is also referred to herein as

"K511_li".

Applicants' methods identified clone K511_li as encoding a secreted protein. The nucleotide sequence of K511_li as presently determined is reported in SEQ ID NO:7, and includes the poly(A) tail. The predicted reading frame and amino acid sequence of the K511_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NO:8. The TopPredll computer program predicts a potential transmembrane domain within the K511_li protein sequence, centered around amino acid 96 of SEQ ID NO:8. A predicted reading frame within the K511_li clone extends from nucleotide 696 to nucleotide 857 of SEQ ID NO:7, and encodes the amino acid sequence reported in SEQ ID NO:16.

The EcoRI/Notl restriction fragment obtainable from the deposit containing clone K511_li should be approximately 1869 bp. Protein "N154 li"

One protein of the present invention has been identified as protein "N154_li". A partial cDNA clone encoding N154_li was first isolated from a rat fetal pancreas cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S.

Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. The nucleotide sequence of such partial cDNA was determined and searched against the

GenBank and GeneSeq databases using BLASTN/BLASTX and FAST A search protocols. The search revealed at least some sequence similarity to an EST identified as AA081656

(zn22bl0.rl Stratagene neuroepithelium NT2RAMI 937234 Homo sapiens cDNA clone

IMAGE:548155 5', mRNA sequence), H10911 (ym06g04.rl Homo sapiens cDNA clone

IMAGE:47242 5'), H97815 (yw02h01.sl Homo sapiens cDNA clone IMAGEJ51089 3'),

T26563 (Human gene signature HUMGS08809), T34128 (EST63020 Homo sapiens cDNA 5' end similar to None), and W08671 (mb48el2.rl Soares mouse p3NMF19.5 Mus musculus cDNA clone IMAGEJ326865'). A 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 poly(A) tail. This full-length human clone is also referred to herein as "N154_li".

Applicants' methods identified clone N154_li as encoding a secreted protein. The nucleotide sequence of N154_li as presently determined is reported in SEQ ID NO:9, and includes the poly(A) tail. The predicted reading frame and amino acid sequence of the N154_li protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID NOJO.

The EcoRI/Notl restriction fragment obtainable from the deposit containing clone N154_li should be approximately 1470 bp.

Deposit of Clones Clones AW179_li, BG221_li, K139_li, K511_li, and N154_li were deposited on

April 2, 1999 with the American Type Culture Collection (10801 University Boulevard, Manassas, Virginia 20110-2209 U.S.A.) as an original deposit under the Budapest Treaty and were given the accession number ATCC 207186, from which each clone comprising a particular polynucleotide is obtainable. All restrictions on the availability to the public of the deposited material will be irrevocably removed upon the granting of the patent, except for the requirements specified in 37 C.F.R. § 1.808(b), and the term of the deposit will comply with 37 C.F.R. § 1.806.

Each clone has been transfected into separate bacterial cells (E. coli) in this composite deposit. Each clone can be removed from the vector in which it was deposited by performing an EcoRI/ Notl digestion (5' site, EcoRI; 3' site, Notl) to produce the appropriate fragment for such clone. Each clone was deposited in either the pED6 or pNOTs vector depicted in Figures 1A and IB, respectively. The pED6dpc2 vector ("pED6") was derived from pED6dpcl by insertion of a new polylinker to facilitate cDNA cloning (Kaufman et al, 1991, Nucleic Acids Res. 19: 4485-4490); the pNOTs vector was derived from pMT2 (Kaufman et al, 1989, Mol Cell Biol. 9: 946-958) by deletion of the DHFR sequences, insertion of a new polylinker, and insertion of the Ml 3 origin of replication in the Clal site. In some instances, the deposited clone can become "flipped" (i.e., in the reverse orientation) in the deposited isolate. In such instances, the cDNA insert can still be isolated by digestion with EcoRI and Notl. However, Notl will then produce the 5' site and EcoRI will produce the 3' site for placement of the cDNA in proper orientation for expression in a suitable vector. The cDNA may also be expressed from the vectors in which they were deposited.

Bacterial cells containing a particular clone can be obtained from the composite deposit as follows:

An 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 sequence of an oligonucleotide probe that was used to isolate or to sequence each full-length clone is identified below, and should be most reliable in isolating the clone of interest.

Clone Probe Sequence¹

BG221_li SEQ ID NO:ll

K139_li SEQ ID NO:12 K511_li SEQ ID NOJ3

N154_li SEQ ID NO:14

References are to sequences in the Sequence Listing attached hereto. The following SEQ ID NO:s in the attached Sequence Listing correspond to the indicated clone: AW179_li: SEQ ID NO:l ; BG221_li: SEQ ID NO:3 ; K139_li: SEQ ID NO:5 ; K511_li: SEQ ID NO:7 ; and N154_li: SEQ ID NO:9.

In the sequences listed above which include an N at position 2, that position is occupied in preferred probes/ rimers by a biotinylated phosphoaramidite residue rather than a nucleotide (such as, for example, that produced by use of biotin phosphoramidite (l-dimemoxytiityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-0-(2-cyanoethyl)-(N,N- diisopropyl)-phosphoramadite) (Glen Research, cat. no. 10-1953)).

The design of the oligonucleotide probe should preferably follow these parameters: (a) It should be designed to an area of the sequence which has the fewest ambiguous bases ("N's"), if any; (b) It should be designed to have a T_m of approx. 80 ° C (assuming 2° for each

A or T and 4 degrees for each G or C).

The oligonucleotide should preferably be labeled with γ-³²P ATP (specific activity 6000 Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for labeling oligonucleotides. Other labeling techniques can also be used. Unincorporated label should preferably be removed by gel filtration chromatography or other established methods. The amount of radioactivity incorporated into the probe should be quantitated by measurement in a scintillation counter. Preferably, specific activity of the resulting probe should be approximately 4e+6 dpm/pmole.

The bacterial culture containing the pool of full-length clones should preferably be thawed and 100 μl of the stock used to inoculate a sterile culture flask containing 25 ml of sterile L-broth containing ampicillin at 100 μg/ml. The culture should preferably be grown to saturation at 37°C, and the saturated culture should preferably be diluted in fresh L- broth. 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 μg/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at 37°C. Other known methods of obtaining distinct, well-separated colonies can also be employed. Standard colony hybridization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them.

The filter is then preferably incubated at 65°C for 1 hour with gentle agitation in 6X SSC (20X stock is 175J g NaCl/liter, 88J g Na citrate/ liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100 μg/ml of yeast RNA, and 10 mM EDTA (approximately 10 mL per 150 mm filter). Preferably, the probe is then added to the hybridization mix at a concentration greater than or equal to le+6 dpm/mL. The filter is then preferably incubated at 65°C with gentle agitation overnight. The filter is then preferably washed in 500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably followed by 500 mL of 2X SSC/0J% SDS at room temperature with gentle shaking for 15 minutes. A third wash with 0.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is optional. The filter is then preferably dried and subjected to autoradiography for sufficient time to 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. Clones in the deposit are of human. The clones can then be verified by restriction analysis, hybridization analysis, or DNA sequencing.

Once a particular clone has been isolated from the composite deposit, the cDNA can be removed from the vector in which it was deposited by performing an EcoRI/ Notl digestion (5' site, EcoRI; 3' site, Notl). The resultant EcoRI/ Notl fragments should have the following approximate sizes:

Clone Size

AW179_li 990 bp BG221_li 1400 bp

K139_li 1818 bp

K511_li 1869 bp

N154_li 1470 bp

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, etal, Bio/ Technology 10, 773-778 (1992) and in R.S. McDowell, etal, J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by reference. Such fragments may be fused to carrier molecules such as irrtmunoglobulrns for many purposes, including increasing the valency of protein binding sites. For example, fragments of the protein may be fused through "linker" sequences to the Fc portion of an immunoglobulin. For a bivalent form of the protein, such a fusion could be to the Fc portion of an IgG molecule. Other irnrnxinoglobulin isotypes may also be used to generate such fusions. For example, a protein - IgM fusion would generate a decavalent form of the protein of the invention.

The present invention also provides both full-length and mature forms of the disclosed proteins. The full-length form of the such proteins is identified in the sequence listing by translation of the nucleotide sequence of each disclosed clone. The mature form(s) 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(s) of the mature form(s) of the protein may also be determinable from the amino acid sequence of the full-length form. Where the protein of the present invention is membrane-bound (e.g., is a receptor), the present invention also provides for soluble forms of such protein. In such forms, part or all of the intracellular and transmembrane domains of the protein are deleted such that the protein is fully secreted from the cell in which it is expressed. The intracellular and transmembrane domains of proteins of the invention can be identified in accordance with known techniques for determination of such domains from sequence information. For example, the TopPredll computer program can be used to predict the location of transmembrane domains in an amino acid sequence, domains which are described by the location of the center of the transmsmbrane domain, with at least ten transmembrane amino acids on each side of the reported central residue (s). Proteins and protein fragments of the present invention include proteins with amino acid sequence lengths that are at least 25% (more preferably at least 50%, and most preferably at least 75%) of the length of a disclosed protein and have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with that disclosed protein, where sequence identity is determined by comparing the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while n inimizing sequence gaps. Also included in the present invention are proteins and protein fragments that contain a segment preferably comprising 8 or more (more preferably 20 or more, most preferably 30 or more) contiguous amino acids that shares at least 75% sequence identity (more preferably, at least 85% identity; most preferably at least 95% identity) with any such segment of any of the disclosed proteins. In particular, sequence identity may be determined using WU-BLAST (Washington University BLAST) version 2.0 software, which builds upon WU-BLAST version 1.4, which in turn is based on the public domain NCBI-BLAST version 1.4 (Altschul and Gish, 1996, Local alignment statistics, Doolittle ed., Methods in Enzymology 266: 460-480; Altschul et al, 1990, Basic local alignment search tool, Journal of Molecular Biology 215: 403-410; Gish and States, 1993, Identification of protein coding regions by database similarity search, Nature Genetics 3: 266-272; Karlin and Altschul, 1993, Applications and statistics for multiple high-scoring segments in molecular sequences, Proc. Natl. Acad. Sci. USA 90: 5873-5877; all of which are incorporated by reference herein). WU-BLAST version 2.0 executable programs for several UNIX platforms can be downloaded from ftp://blast.wustl.edu/blast/executables. The complete suite of search programs (BLASTP, BLASTN, BLASTX, TBLASTN, and TBLASTX) is provided at that site, in addition to several support programs. WU-BLAST 2.0 is copyrighted and may not be sold or redistributed in any form or manner without the express written consent of the author; but the posted executables may otherwise be freely used for commercial, nonprofit, or academic purposes. In all search programs in the suite - BLASTP, BLASTN, BLASTX, TBLASTN and TBLASTX — the gapped alignment routines are integral to the database search itself, and thus yield much better sensitivity and selectivity while producing the more easily inteφreted output. Gapping can optionally be turned off in all of these programs, if desired. The default penalty (Q) for a gap of length one is Q=9 for proteins and BLASTP, and Q=10 for BLASTN, but may be changed to any integer value including zero, one through eight, nine, ten, eleven, twelve through twenty, twenty-one through fifty, fifty-one through one hundred, etc. The default per-residue penalty for extending a gap (R) is R=2 for proteins and BLASTP, and R=10 for BLASTN, but may be changed to any integer value including zero, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve through twenty, twenty-one through fifty, fifty-one through one hundred, etc. Any combination of values for Q and R can be used in order to align sequences so as to maximize overlap and identity while minimizing sequence gaps. The default amino acid comparison matrix is BLOSUM62, but other amino acid comparison matrices such as PAM can be utilized.

A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences and determination of percent homology between two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incoφorated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison puφoses, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Research 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) Comput Appl Biosci. 4:11-17. Such an algorithm is incoφorated into the ALIGN program available, for example, at the GENESTREAM network server, IGH Montpellier, FRANCE (http://vega.igh.cnrs.fr) or at the ISREC server (http : //www. ch. embnet . org) . When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. In another preferred embodiment, the percent homology between two amino acid sequences can be accomplished using the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4. In yet another preferred embodiment, the percent homology between two nucleic acid sequences can be accomplished using the GAP program in the GCG software package (available at http://www.gcg.com), using a gap weight of 50 and a length weight of 3.

Species homologues of the disclosed proteins are also provided by the present invention. As used herein, a "species homologue" is a protein or polynucleotide with a different species of origin from that of a given protein or polynucleotide, but with significant sequence similarity to the given protein or polynucleotide. Preferably, polynucleotide species homologues have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% identity) with the given polynucleotide, and protein species homologues have at least 30% sequence identity (more preferably, at least 45% identity; most preferably at least 60% identity) with the given protein, where sequence identity is determined by comparing the nucleotide sequences of the polynucleotides or the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while ininimizing sequence gaps. Species homologues 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. Preferably, species homologues are those isolated from mammalian species. Most preferably, species homologues are those isolated from certain mammalian species such as, for example, Pan troglodytes, Gorilla gorilla, Pongo pygmaeus, Hylobates concolor, Macaca mulatta, Papio papio, Papio hamadryas, Cercopithecus aethiops, Cebus capucinus, Aotus trivirgatus, Sanguinus oedipus, Microcebus urinus, Mus musculus, Rattus norvegicus, Cricetulus griseus, Felis catus, Mustela vison, Cam^'s familiaris, Oryctolagus cuniculus, Bos taurus, Ovis aries, Sus scrofa, and Equus caballus, for which genetic maps have been created allowing the identification of syntenic relationships between the genomic organization of genes in one species and the genomic organization of the related genes in another species (O'Brien and Seuanez, 1988, Ann. Rev. Genet. 22: 323-351; O'Brien etal, 1993, Nature Genetics 3:103-112; Johansson etal, 1995, Genomics 25: 682-690; Lyons etal, 1997, Nature Genetics 15: 47-56; O'Brien et al, 1997, Trends in Genetics 13(10): 393-399; Carver and Stubbs, 1997, Genome Research 7 112?>-11?>7; all of which are incorporated by reference herein).

The invention also encompasses allelic variants of the disclosed proteins; that is, naturally-occurring alternative forms of the isolated proteins which are identical or have significantly similar sequences to those encoded by the disclosed polynucleotides. Preferably, allelic variants have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90% identity) with the given polynucleotide, where sequence identity is determined by comparing the nucleotide sequences of the polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps. Allelic variants may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from individuals of the appropriate species.

The invention also includes polynucleotides with sequences complementary to those of the polynucleotides disclosed herein.

The isolated polynucleotide encoding 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 recombinantly. 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 185, 537-566 (1990). As defined herein "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. A number of types of cells may act as suitable host cells for expression of the protein. 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.

Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escheήchia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.

The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/ insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, 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), incorporated herein by reference. As used herein, an insect cell capable of expressing a polynucleotide of the present invention is "transformed."

The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein. The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GA

Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or inrmunoaffinity chromatography. Alternatively, the protein of the invention may also be expressed in a form which will facilitate purification. For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin

(TRX). Kits for expression and purification of such fusion proteins are commercially available from New England BioLabs (Beverly, MA), Pharmacia (Piscataway, NJ) and Invitrogen Corporation (Carlsbad, CA), respectively. The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope.

One such epitope ("Flag") is commercially available from the Eastman Kodak Company

(New Haven, CT).

Finally, one or more reverse-phase high performance liquid chromatography (RP- HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein. 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-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and/ or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.

The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or 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. For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Patent No. 4,518,584). Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein. Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and may thus be useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein. Such modifications are believed to be encompassed by the present invention.

USES AND BIOLOGICAL ACTIVITY

The proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified below. Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).

Research Uses and Utilities The proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands. Where 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. Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without limitation "Molecular Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Berger, S.L. and A.R. Kimmel eds., 1987.

Nutritional Uses

Proteins of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate. In such cases the protein 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. In the case of microorganisms, the protein of the invention can be added to the medium in or on which the microorganism is cultured.

Cytokine and Cell Proliferation /Differentiation Activity

A protein of the present invention may exhibit cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor-dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DAI, 123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK. The activity of a protein of the invention may, among other means, be measured by the following methods:

Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley- Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., J. Immunol. 149:3778-3783, 1992; Bowman et al., J. Immunol. 152: 1756-1761, 1994.

Assays for cytokine production and/ or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and

Measurement of mouse and human Interferon γ, Schreiber, R.D. In Current Protocols in

Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.

Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Current

Protocols in Immunology. J.E.e.a. CoUgan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons,

Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature

336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983;

Measurement of mouse and human interleukin 6 - Nordan, R. In Current Protocols in Immunology. J.E.e.a. CoUgan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991;

Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of human

Interleukin 11 - Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. J. In Current Protocols in Immunology. J.E.e.a. CoUgan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991;

Measurement of mouse and human Interleukin 9 - Ciarletta, A., Giannotti, J., Clark, S.C. and Turner, K.J. In Current Protocols in Immunology. J.E.e.a. CoUgan eds. Vol 1 pp. 6.13.1,

John Wiley and Sons, Toronto. 1991.

Assays for T-ceU clone responses to antigens (which will identify, among others, proteins that affect APC-T ceU interactions as weU as direct T-ceU effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. CoUgan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their ceUular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Lmmun. 11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988.

Immune Stimulating or Suppressing Activity A protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without 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 (SOD)), e.g., in regulating (up or down) growth and proliferation of T and/ or B lymphocytes, as weU as effecting the cytolytic activity of NK ceUs and other cell populations. These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as weU as bacterial or fungal infections, or may result from autoimmune disorders. More specificaUy, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis. Of course, in this regard, a protein of the present invention may also be useful where a boost to the immune system generaUy may be desirable, i.e., in the treatment of cancer.

Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, GuiUain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes meUitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease. Such a protein of the present invention may also to be useful in the treatment of aUergic reactions and conditions, such as asthma (particularly aUergic asthma) or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein of the present invention.

Using the proteins of the invention it may also be possible to regulate immune responses in a number of ways. Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response. The functions of activated T cells may be inhibited by suppressing T ceU responses or by inducing specific tolerance in T ceUs, or both. Immunosuppression of T ceU responses is generaUy an active, non-antigen-specific, process which requires continuous exposure of the T ceUs to the suppressive agent. Tolerance, which involves inducing non-responsiveness or anergy in T ceUs, is distinguishable from immunosuppression in that it is generaUy antigen-specific and persists after exposure to the tolerizing agent has ceased. OperationaUy, tolerance can be demonstrated by the lack of a T ceU 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 ceUs, wiU be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD). For example, blockage of T ceU function should result in reduced tissue destruction in tissue transplantation. TypicaUy, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T ceUs, foUowed by an immune reaction that destroys the transplant. The administration of a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune ceUs (such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7- 1, B7-3) or blocking antibody), prior to transplantation can lead to the binding of the molecule to the natural ligand(s) on the immune ceUs without transmitting the corresponding costimulatory signal. Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune ceUs, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may also be sufficient to anergize the T ceUs, thereby inducing tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.

The efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of appropriate systems which can be used include aUogeneic cardiac grafts in rats and xenogeneic pancreatic islet ceU grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow etal, Science 257:789-792 (1992) and Turka etal, Proc. Natl. Acad. Sci USA, ££11102-11105 (1992). In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease.

Blocking antigen function may also be therapeuticaUy useful for treating autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation of T ceUs 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 ceUs may reduce or eliminate disease symptoms. Administration of reagents which block costimulation of T ceUs by disrupting receptor:ligand interactions of B lymphocyte antigens can be used to inhibit T ceU activation and prevent production of autoantibodies or T ceU-derived cytokines which may be involved in the disease process. AdditionaUy, blocking reagents may induce antigen- specific tolerance of autoreactive T ceUs which could lead to long-term relief from the disease. The efficacy of blocking reagents in preventing or aUeviating autoimmune disorders can be determined using a number of weU-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 coUagen arthritis, diabetes meUitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856). Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the common cold, and encephaUtis might be aUeviated by the administration of stimulatory forms of B lymphocyte antigens systemicaUy.

Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T ceUs from the patient, costimulating the T ceUs in vitro with viral antigen- pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T ceUs into the patient. Another method of enhancing anti- viral immune responses would be to isolate infected ceUs from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the ceUs express aU or a portion of the protein on their surface, and reintroduce the transfected ceUs into the patient. The infected ceUs would now be capable of delivering a costimulatory signal to, and thereby activate, T ceUs in vivo.

In another application, up regulation or enhancement of antigen function (preferably B lymphocyte antigen function) may be useful in the induction of tumor immunity. Tumor ceUs (e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleic acid encoding at least one peptide of the present invention can be administered to a subject to overcome tumor-specific tolerance in the subject. If desired, the tumor ceU can be transfected to express a combination of peptides. For example, tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-l-like activity and/ or B7-3-like activity. The transfected tumor ceUs are returned to the patient to result in expression of the peptides on the surface of the transfected cell. Alternatively, gene therapy techniques can be used to target a tumor cell for transf ection in vivo.

The presence of the peptide of the present invention having the activity of a B lymphocyte antigen(s) on the surface of the tumor ceH provides the necessary costimulation signal to T ceUs to induce a T ceU mediated immune response against the transfected tumor ceUs. In addition, tumor ceUs which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding aU or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I α chain protein and β₂ microglobulin protein or an MHC class II α chain protein and an MHC class II β chain protein to thereby express MHC class I or MHC class II proteins on the ceU surface. Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T ceU mediated immune response against the transfected tumor ceU. OptionaUy, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity. Thus, the induction of a T ceU mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.

The activity of a protein of the invention may, among other means, be measured by the foUowing methods: Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. CoUgan, 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 5 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 10 135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet al., J. Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988; BertagnoUi et al., CeUular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092, 1994.

Assays for T-cell-dependent immunoglobulin responses and isotype switching (which wiU identify, among others, proteins that modulate T-ceU dependent antibody 15 responses and that affect Thl/Th2 profUes) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B ceU 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 WUey and Sons, Toronto. 1994.

Mixed lymphocyte reaction (MLR) assays (which wiU identify, among others, 20 proteins that generate predominantly Thl and CTL responses) 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; Takai et 25 al., J. Immunol. 140:508-512, 1988; BertagnoUi et al., J. Immunol. 149:3778-3783, 1992.

Dendritic ceU-dependent assays (which wiU identify, among others, proteins expressed by dendritic ceUs that activate naive T-ceUs) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 30 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990. Assays for lymphocyte survival/ apoptosis (which wiU identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et 5 al., Cancer Research 53:1945-1951, 1993; Itoh et al., CeU 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.

Assays for proteins that influence early steps of T-ceU commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 10 1994; Fine et al., CeUular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.

Hematopoiesis Regulating Activity

A protein of the present invention may be useful in regulation of hematopoiesis

15 and, consequently, in the treatment of myeloid or lymphoid ceU deficiencies. Even marginal biological activity in support of colony forming cells or of factor-dependent ceU lines indicates involvement in regulating hematopoiesis, e.g. in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction

20 with irradiation/ chemotherapy to stimulate the production of erythroid precursors and/ or erythroid ceUs; in supporting the growth and proliferation of myeloid ceUs such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo- suppression; in supporting the growth and proliferation of megakaryocytes and

25 consequently of platelets thereby aUowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generaUy for use in place of or complimentary to platelet transfusions; and/ or in supporting the growth and proliferation of hematopoietic stem ceUs which are capable of maturing to any and aU of the above- mentioned hematopoietic ceUs and therefore find therapeutic utility in various stem ceU

30 disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as weU as in repopulating the stem ceU compartment post irradiation/ chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with bone marrow transplantation or with peripheral progenitor ceU transplantation (homologous or heterologous)) as normal ceUs or genetically manipulated for gene therapy.

The activity of a protein of the invention may, among other means, be measured by the foUowing methods: Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.

Assays for embryonic stem cell differentiation (which wUl identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; KeUer et al., Molecular and CeUular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915,

1993.

Assays for stem ceU survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: MethylceUulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, WUey-Liss, Inc., New York, NY. 1994;

Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I.K. and BriddeU, R.A. In

Culture of Hematopoietic Cells. R.I. Freshney, etal. eds. Vol pp. 23-39, WUey-Liss, Inc.,

New York, NY. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming ceU assay, Ploemacher, R.E. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 1-21, WUey-Liss, Inc., New York, NY. 1994; Long term bone marrow cultures in the presence of stromal ceUs, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R.I. Freshney, et al eds. Vol pp. 163-179, WUey-Liss, Inc., New

York, NY. 1994; Long term culture initiating ceU assay, Sutherland, H.J. In Culture of Hematopoietic Cells. R.I. Freshney, etal eds. Vol pp. 139-162, WUey-Liss, Inc., New York,

NY. 1994.

Tissue Growth Activity

A protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and/ or nerve tissue growth or regeneration, as weU 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 normaUy 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 weU as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.

A protein of this invention may also be used in the treatment of periodontal disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming ceUs, stimulate growth of bone-forming ceUs or induce differentiation of progenitors of bone-forming ceUs. A protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and/ or cartilage repair or by blocking inflammation or processes of tissue destruction (coUagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.

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 normaUy formed, has appUcation in the healing of tendon or Ugament 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 weU as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue. De novo tendon/ ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or Ugament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments. The compositions of the present invention may provide an environment to attract tendon- or

Ugament-forming ceUs, stimulate growth of tendon- or ligament-forming ceUs, induce differentiation of progenitors of tendon- or ligament-forming ceUs, or induce growth of tendon/ Ugament ceUs or progenitors ex vivoior return in vivo to effect tissue repair. The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or Ugament defects. The compositions may also include an appropriate matrix and/ or sequestering agent as a carrier as is weU known in the art.

The protein of the present invention may also be useful for proliferation of neural ceUs and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as weU as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural ceUs 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. It is expected that a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endotheUum) tissue, or for promoting the growth of ceUs comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to aUow normal tissue to regenerate. A protein of the invention may also exhibit angiogenic activity.

A protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage. A protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or ceUs; 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 foUowing methods: Assays for tissue generation activity include, without limitation, those described in:

International Patent Publication No. WO95/16035 (bone, cartilage, tendon); International Patent PubUcation No. W095/ 05846 (nerve, neuronal); International Patent PubUcation No. W091/ 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 modtfied by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84 (1978).

Activin/Inhibin Activity

A protein of the present invention may also exhibit activin- or inhibin-related activities. Inhibins are characterized by their abUity to inhibit the release of foUicle stimulating hormone (FSH), whUe activins and are characterized by their abUity to stimulate the release of foUicle stimulating hormone (FSH). Thus, a protein of the present invention, alone or in heterodimers with a member of the inhibin α famUy, may be useful as a contraceptive based on the abUity 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. Alternatively, the protein of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin-β group, may be useful as a fertility inducing therapeutic, based upon the abUity of activin molecules in stimulating FSH release from ceUs 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 sexuaUy immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.

The activity of a protein of the invention may, among other means, be measured by the following methods:

Assays for activin/ inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986.

Chemotactic/Chemokinetic Activity

A protein of the present invention may have chemotactic or chemokinetic activity (e.g., act as a chemokine) for mammalian ceUs, including, for example, monocytes, fibroblasts, neutrophUs, T-ceUs, mast ceUs, eosinophils, epitheUal and/ or endothehal ceUs.

Chemotactic and chemokinetic proteins can be used to mobUize or attract a desired ceU population to a desired site of action. Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as weU as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophUs to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.

A protein or peptide has chemotactic activity for a particular ceU population if it can stimulate, directly or indirectly, the directed orientation or movement of such ceU population. Preferably, the protein or peptide has the ability to directly stimulate directed movement of ceUs. Whether a particular protein has chemotactic activity for a population of cells can be readUy 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 foUowing methods:

Assays for chemotactic activity (which wiU identify proteins that induce or prevent chemotaxis) consist of assays that measure the abUity of a protein to induce the migration of ceUs across a membrane as weU as the abUity of a protein to induce the adhesion of one cell population to another ceU population. Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and WUey-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995; Lmd et al. APMIS 103:140-146, 1995; MuUer et al Eur. J. Immunol. 25: 1744-1748; Gruber et al. J. of Immunol. 152:5860-5867, 1994; Johnston et al. J. of Immunol. 153: 1762-1768, 1994.

Hemostatic and Thrombolytic Activity

A protein of the invention may also exhibit hemostatic or thrombolytic activity. As a result, such a protein is expected to be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes. A protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).

The activity of a protein of the invention may, among other means, be measured by the foUowing 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.

Receptor /Ligand Activity A protein of the present invention may also demonstrate activity as receptors, receptor Ugands or inhibitors or agonists of receptor/Ugand interactions. Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in ceU-ceU interactions and their ligands (including without limitation, ceUular adhesion molecules (such as selectins, integrins and their ligands) and receptor/Ugand 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/Ugand interaction.

A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/Ugand interactions.

Suitable assays for receptor-ligand activity include without limitation those described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and

WUey-Interscience (Chapter 7.28, Measurement of CeUular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstern et al., J. Exp. Med. 169:149-160 1989;

Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., CeU 80:661-670, 1995.

Anti-Inflammatory Activity

Proteins of the present invention may also exhibit anti-inflammatory activity. The anti-iriflarnmatory activity may be achieved by providing a stimulus to ceUs involved in the inflammatory response, by inhibiting or promoting ceU-ceU interactions (such as, for example, ceU adhesion), by inhibiting or promoting chemotaxis of ceUs involved in the inflammatory process, inhibiting or promoting ceU 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.

Cadherin /Tumor Invasion Suppressor Activity

Cadherins are calcium-dependent adhesion molecules that appear to play major roles during development, particularly in defining specific cell types. Loss or alteration of normal cadherin expression can lead to changes in ceU adhesion properties linked to tumor growth and metastasis. Cadherin malfunction is also implicated in other human diseases, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune blistering skin diseases), Crohn's disease, and some developmental abnormalities. The cadherin superfamUy includes well over forty members, each with a distinct pattern of expression. AU members of the superfamUy have in common conserved extraceUular repeats (cadherin domains), but structural differences are found in other parts of the molecule. The cadherin domains bind calcium to form their tertiary structure and thus calcium is required to mediate their adhesion. Only a few amino acids in the first cadherin domain provide the basis for homophUic adhesion; modification of this recognition site can change the specificity of a cadherin so that instead of recognizing only itself, the mutant molecule can now also bind to a different cadherin. In addition, some cadherins engage in heterophUic adhesion with other cadherins.

E-cadherin, one member of the cadherin superfamUy, is expressed in epitheUal ceU types. PathologicaUy, if E-cadherin expression is lost in a tumor, the malignant ceUs become invasive and the cancer metastasizes. Transfection of cancer ceU lines with polynucleotides expressing E-cadherin has reversed cancer-associated changes by returning altered ceU shapes to normal, restoring ceUs' adhesiveness to each other and to their substrate, decreasing the ceU growth rate, and drasticaUy reducing anchorage- independent ceU growth. Thus, reintroducing E-cadherin expression reverts carcinomas to a less advanced stage. It is likely that other cadherins have the same invasion suppressor role in carcinomas derived from other tissue types. Therefore, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be used to treat cancer. Introducing such proteins or polynucleotides into cancer ceUs can reduce or eliminate the cancerous changes observed in these cells by providing normal cadherin expression.

Cancer ceUs have also been shown to express cadherins of a different tissue type than their origin, thus aUowing these cells to invade and metastasize in a different tissue in the body. Proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be substituted in these ceUs for the inappropriately expressed cadherins, restoring normal cell adhesive properties and reducing or eliminating the tendency of the ceUs to metastasize.

AdditionaUy, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can used to generate antibodies recognizing and binding to cadherins. Such antibodies can be used to block the adhesion of inappropriately expressed tumor-cell cadherins, preventing the ceUs from forming a tumor elsewhere. Such an anti-cadherin antibody can also be used as a marker for the grade, pathological type, and prognosis of a cancer, i.e. the more progressed the cancer, the less cadherin expression there wUl be, and this decrease in cadherin expression can be detected by the use of a cadherin-binding antibody.

Fragments of proteins of the present invention with cadherin activity, preferably a polypeptide comprising a decapeptide of the cadherin recognition site, and polynucleotides of the present invention encoding such protein fragments, can also be used to block cadherin function by binding to cadherins and preventing them from binding in ways that produce undesirable effects. Additionally, fragments of proteins of the present invention with cadherin activity, preferably truncated soluble cadherin fragments which have been found to be stable in the circulation of cancer patients, and polynucleotides encoding such protein fragments, can be used to disturb proper cell-ceU adhesion. Assays for cadherin adhesive and invasive suppressor activity include, without limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-18817, 1995; Miyaki et al. Oncogene 11: 2547-2552, 1995; Ozawa et al. CeU 63: 1033-1038, 990.

Tumor Inhibition Activity

In addition to the activities described above for immunological treatment or prevention of tumors, a protein of the invention may exhibit other anti-tumor activities. A protein may inhibit tumor growth directly or indirectly (such as, for example, via antibody-dependent ceU-mediated cytotoxicity (ADCC)). A protein may exhibit its tumor inhibitory activity by acting on tumor tissue or tumor precursor tissue, by inhibiting formation of tissues necessary to support tumor growth (such as, for example, by inhibiting angiogenesis), by causing production of other factors, agents or ceU types which inhibit tumor growth, or by suppressing, eliminating or inhibiting factors, agents or ceU types which promote tumor growth.

Other Activities

A protein of the invention may also exhibit one or more of the foUowing 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) bodUy 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 and growth of embryonic stem ceUs in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); imrnunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein. ADMINISTRATION AND DOSING

A protein of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources) may be used in a pharmaceutical composition when combined with a pharmaceuticaUy acceptable carrier. Such a composition may also contain (in addition to protein and a carrier) dUuents, fillers, salts, buffers, stabilizers, solubilizers, and other materials weU known in the art. The term "pharmaceuticaUy 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 wiU depend on the route of administration. The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, 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 ceU 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. Such additional factors and/ or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein of the invention, or to minimize side effects. Conversely, protein of the present invention may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti- thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti- inflammatory agent.

A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itseU or other proteins. As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.

The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) of present invention along with protein or peptide antigens. The protein and/ or peptide antigen wiU deliver a stimulatory signal to both B and T lymphocytes. B lymphocytes wiU respond to antigen through their surface immunoglobulin receptor. T lymphocytes wiU respond to antigen through the T ceU receptor (TCR) foUowing presentation of the antigen by MHC proteins. MHC and structuraUy related proteins including those encoded by class I and class II MHC genes on host ceUs wUl serve to present the peptide antigen(s) to T lymphocytes. The antigen components could also be suppUed as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T ceUs. Alternatively antibodies able to bind surface immunolgobulin and other molecules on B ceUs as weU as antibodies able to bind the TCR and other molecules on T ceUs can be combined with the pharmaceutical composition of the invention. The pharmaceutical composition of the invention may be in the form of a Uposome in which protein of the present invention is combined, in addition to other pharmaceuticaUy acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as miceUes, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, suUatides, lysolecithin, phospholipids, saponin, bUe acids, and the like. Preparation of such Uposomal 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, aU of which are incorporated herein by reference. As used herein, the term "therapeuticaUy 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. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, seriaUy or simultaneously. In practicing the method of treatment or use of the present invention, a therapeuticaUy effective amount of protein of the present invention is administered to a mammal having a condition to be treated. Protein of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, protein of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentiaUy. If administered sequentiaUy, the attending physician wiU 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.

When a therapeuticaUy 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 eUxir. When administered in tablet form, the pharmaceutical composition of the invention may additionaUy 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. When administered in Uquid form, a Uquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oU, mineral oU, soybean oU, or sesame oU, or synthetic oUs 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. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention. When a therapeuticaUy effective amount of protein of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein of the present invention wiU be in the form of a pyrogen-free, parenteraUy acceptable aqueous solution. The preparation of such parenteraUy acceptable protein solutions, having due regard to pH, isotonicity, stabUity, and the like, is within the skUl in the art. 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 stabUizers, 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 wiU depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician wUl decide the amount of protein of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein of the present invention and observe the patient's response. Larger doses of protein of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 μg to about 100 mg (preferably about OJng to about 10 mg, more preferably about 0J μg to about 1 mg) of protein of the present invention per kg body weight.

The duration of intravenous therapy using the pharmaceutical composition of the present invention wiU 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 wiU be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician wUl decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.

Protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specificaUy react with the protein. As used herein, the term "antibody" includes without limitation a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single-chain antibody, a CDR-grafted antibody, a humanized antibody, or fragments thereof which bind to the indicated protein. Such term also includes any other species derived from an antibody or antibody sequence which is capable of binding the indicated protein.

Antibodies to a particular protein can be produced by methods well known to those skilled in the art. For example, monoclonal antibodies can be produced by generation of antibody-producing hybridomas in accordance with known methods (see for example, Goding, 1983, Monoclonal antibodies: principles and practice, Academic Press

Inc., New York; and Yokoyama, 1992, "Production of Monoclonal Antibodies" in

Current Protocols in Immunology, Unit 2.5, Greene Publishing Assoc. and John Wiley

& Sons). Polyclonal sera and antibodies can be produced by inoculation of a mammalian subject with the relevant protein or fragments thereof in accordance with known methods.

Fragments of antibodies, receptors, or other reactive peptides can be produced from the corresponding antibodies by cleavage of and collection of the desired fragments in accordance with known methods (see for example, Goding, supra; and Andrew et al., 1992, "Fragmentation of Immunoglobulins" in Current Protocols in Immunology, Unit 2.8, Greene Publishing Assoc. and John Wiley & Sons). Chimeric antibodies and single chain antibodies can also be produced in accordance with known recombinant methods (see for example, 5,169,939, 5,194,594, and 5,576,184). Humanized antibodies can also

5 be made from corresponding murine antibodies in accordance with well known methods (see for example, U.S. Patent Nos. 5,530,101, 5,585,089, and 5,693,762). Additionally, human antibodies may be produced in non-human animals such as mice that have been genetically altered to express human antibody molecules (see for example Fishwild et al. , 1996, Nature Biotechnology 14: 845-851 ; Mendez et al, 1997, Nature Genetics 15: 146-

10 156 (erratum Nature Genetics 16: 410); and U.S. Patents 5,877,397 and 5,625,126). Such antibodies may be obtained using either the entire protein or fragments thereof as an im unogen. The peptide immunoge s additionaUy may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH). Methods for synthesizing such peptides are known in the art, for example, as in

15 R.P. Merrifield, J. Amer.Chem.Soc. 85, 2149-2154 (1963); J.L. Krstenansky, etal, FEBS Lett. 211, 10 (1987).

Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein. Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions

20 associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved. In the case of cancerous ceUs or leukemic ceUs, neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous ceUs, which may be mediated by the protein.

25 For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topicaUy, systematicaUy, or locaUy as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be

30 encapsulated or injected in a viscous form for deUvery to the site of bone, cartilage or tissue damage. Topical administration may be suitable for wound healing and tissue repair. TherapeuticaUy useful agents other than a protein of the invention which may also optionaUy be included in the composition as described above, may alternatively or additionaUy, be administered simultaneously or sequentiaUy with the composition in the methods of the invention. Preferably for bone and/ or cartilage formation, the composition would include a matrix capable of deUvering the protein-containing composition to the site of bone and/ or cartilage damage, providing a structure for the developing bone and cartilage and optimaUy 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 biocompatibUity, biodegradabUity, 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 chemicaUy defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. Other potential materials are biodegradable and biologicaUy well- defined, such as bone or dermal coUagen. Further matrices are comprised of pure proteins or extraceUular 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 coUagen and tricalciumphosphate. The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradabUity.

Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns. In some applications, it wUl be useful to utilize a sequestering agent, such as carboxymethyl ceUulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.

A preferred famUy of sequestering agents is ceUulosic materials such as aUcylceUuloses (including hydroxyalkylceUuloses), including methylcellulose, ethylceUulose, hydroxyethylceUulose, hydroxypropylceUulose, hydroxypropyl- methylceUulose, and carboxymethylceUulose, the most preferred being cationic salts of carboxymethylceUulose (CMC). 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 ceUs are prevented from infUtrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor ceUs.

In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the bone and/ or cartilage defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor

(EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-α and

TGF-β), and insulin-like growth factor (IGF).

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-containing pharmaceutical composition to be used in tissue regeneration wiU be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I

(insuUn like growth factor I), to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of tissue/ bone growth and/ or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.

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 ceU or organism (including, without limitation, in the form of viral vectors or naked DNA).

CeUs may also be cultured 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 ceUs. Treated ceUs can then be introduced in vivo for therapeutic purposes. Patent and Uterature references cited herein are incorporated by reference as if fully set forth.

Claims

What is claimed is:

1. An isolated protein encoded by a polynucleotide selected from the group consisting of:

(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l;

(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 156 to nucleotide 251;

(c) a polynucleotide comprising the nucleotide sequence of the fuU- length protein coding sequence of clone AW179_li deposited under accession number ATCC 207186;

(e) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2; and (f) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2, the fragment comprising eight contiguous amino acids of SEQ ID NO:2.

2. A composition comprising the protein of claim 1 and a pharmaceuticaUy acceptable carrier.

3. A protein comprising an amino acid sequence selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO:2; (b) a fragment of the amino acid sequence of SEQ ID NO:2, the fragment comprising eight contiguous amino acids of SEQ ID NO:2; and

(c) the amino acid sequence encoded by the cDNA insert of clone AW179_li deposited under accession number ATCC 207186; the protein being substantiaUy free from other mammalian proteins.

4. The protein of claim 3, wherein said protein comprises the amino acid sequence of SEQ ID NO:2.

5. The protein of claim 3, wherein said protein comprises a fragment of the amino acid sequence of SEQ ID NO:2, the fragment comprising eight contiguous amino acids of SEQ ID NOJ.

6. A composition comprising the protein of claim 3 and a pharmaceuticaUy acceptable carrier.

7. An isolated protein encoded by a polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID

NOJ;

(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NOJ from nucleotide 1039 to nucleotide 1239;

(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NOJ from nucleotide 1090 to nucleotide 1239;

(d) a polynucleotide comprising the nucleotide sequence of the full- length protein coding sequence of clone BG221_li deposited under accession number ATCC 207186;

(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone BG221_li deposited under accession number ATCC 207186;

(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone BG221_li deposited under accession number ATCC 207186;

(g) a polynucleotide encoding a mature protein encoded by the cDNA insert of clone BG221_li deposited under accession number ATCC 207186;

(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4; and

(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4, the fragment comprising eight contiguous amino acids of SEQ ID NO:4.

8. A protein comprising an amino acid sequence selected from the group consisting of:

(c) the amino acid sequence encoded by the cDNA insert of clone BG221_li deposited under accession number ATCC 207186; the protein being substantiaUy free from other mammalian proteins.

9. An isolated protein encoded by a polynucleotide selected from the group consisting of:

(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5;

(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 49 to nucleotide 972;

(c) a polynucleotide comprising the nucleotide sequence of the fuU- length protein coding sequence of clone K139_li deposited under accession number ATCC 207186;

(e) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6; and (f) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6, the fragment comprising eight contiguous amino acids of SEQ ID NO:6.

10. A protein comprising an amino acid sequence selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO:6;

(b) a fragment of the amino acid sequence of SEQ ID NO:6, the fragment comprising eight contiguous amino acids of SEQ ID NO:6; and

(c) the amino acid sequence encoded by the cDNA insert of clone K139_li deposited under accession number ATCC 207186; the protein being substantiaUy free from other mammalian proteins.

11. An isolated protein encoded by a polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NOJ;

(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NOJ from nucleotide 90 to nucleotide 569; (c) a polynucleotide comprising the nucleotide sequence of the fuU- length protein coding sequence of clone K511_li deposited under accession number ATCC 207186;

(d) a polynucleotide encoding the fuU-length protein encoded by the cDNA insert of clone K511_li deposited under accession number ATCC 207186; (e) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8; and

(f) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8, the fragment comprising eight contiguous amino acids of SEQ ID NO:8.

12. A protein comprising an amino acid sequence selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO:8;

(c) the amino acid sequence encoded by the cDNA insert of clone K511_li deposited under accession number ATCC 207186; the protein being substantiaUy free from other mammalian proteins.

13. 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;

(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 125 to nucleotide 505;

(c) a polynucleotide comprising the nucleotide sequence of the fuU- length protein coding sequence of clone N154_li deposited under accession number ATCC 207186; (d) a polynucleotide encoding the fuU-length protein encoded by the cDNA insert of clone N154_li deposited under accession number ATCC 207186;

(e) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10; and (f) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10, the fragment comprising eight contiguous ammo acids of SEQ ID NO:10.

14. A protein comprising an amino acid sequence selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO:10;

(c) the amino acid sequence encoded by the cDNA insert of clone N154_li deposited under accession number ATCC 207186; the protein being substantiaUy free from other mammalian proteins.