SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
This application is a continuation-in-part of the following applications: (1) application Ser. No. 09/079,124, filed May 14, 1998, which is a continuation-in-part of provisional application Ser. No. 60/077,521, filed March 11, 1998; and (2) provisional application Ser. No. 60/077,522, filed March 11, 1998; all of which are incorporated by reference herein.
MELD OF THE INVENTION
The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.
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 iri the case of hybridization cloning; activity of the protein in the case of expression cloning). More recent "indirect" cloning techniques such as signal sequ~ce 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 3 0 techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity by virtue of their secreted nature in the case of leader sequence cloning, or by virtue of the cell or tissue source in the case of PCR-based techniques. It is to these proteins and the polynucleotides encoding them that the present invention is directed.
SL~1~ARY OF THE INVENTION
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1 from nucleotide 58 to nucleotide 1857;
(c) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone cb98 4 deposited under accession number ATCC 98689;
(d) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone cb98 4 deposited under accession number ATCC 98689;
(e) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone cb98 4 deposited under accession number ATCC
98689;
(f) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone cb98 4 deposited under accession number ATCC 98689;
(g) a polynucleotide encoding a protein comprising the amino acid 2 0 sequence of SEQ ID N0:2;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:2 having biological activity, the fragment comprising eight contiguous amino acids of SEQ ID N0:2;
(i) a polynucleotide which is an allelic variant of a polynucleotide of 2 5 (a)-{f) above; . , (j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above ;
{k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(h); and 3 0 (1) a polynudeotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a~(h) and that has a length that is at least 25% of the length of SEQ ID N0:1.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:1 from nucleotide 58 to nucleotide 1857; the nucleotide sequence of the full-length protein coding sequence of clone cb98 4 deposited under accession number ATCC
98689;
or the nucleotide sequence of a mature protein coding sequence of clone cb98 4 deposited under accession number ATCC 98689. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert of clone cb98_4 deposited under accession number ATCC 98689. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:2 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino acids of SEQ ID N0:2, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:2 having biological activity, the fragment comprising the amino acid sequence from amino acid 295 to amino acid 304 of SEQ ID N0:2.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:1.
Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
(a} a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group 2 0 consisting of:
(aa) SEQ ID N0:1, but excluding the poly(A) tail at the 3' end of SEQ ID NO:1; and (ab) the nucleotide sequence of the cDNA insert of clone cb98_4 deposited under accession number ATCC 98689;
2 5 (ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 50 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and 3 0 (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:1, but excluding the poly(A} tail at the 3' end of SEQ ID NO:1; and (bb) the nucleotide sequence of the cDNA insert of clone cb98_4 deposited under accession number ATCC 98689;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 4X SSC at 50 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:1, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
NO:1 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:1 , but excluding the poly(A} tail at the 3' end of SEQ ID NO:1. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:1 from nucleotide 58 to nucleotide 1857, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID NO:1 from nucleotide 58 to nucleotide 1857, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:1 from nucleotide 58 to nucleotide 1857.
2 0 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 N0:2;
(b) a fragment of the amino acid sequence of SEQ ID N0:2, the 2 5 fragment comprising eight contiguous amino acids of SEQ ID N0:2; and (c) the amino acid sequence encoded by the cDNA insert of clone cb98_4 deposited under accession number ATCC 98689;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:2. In further preferred 3 0 embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:2 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino acids of SEQ ID N0:2, or a protein comprising a fragment of the amino acid sequence of SEQ
WO 99!46287 PCT/US99/05243 ID N0:2 having biological activity, the fragment comprising the amino acid sequence from amino acid 295 to amino acid 304 of SEQ ID N0:2.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 170 to nucleotide 718;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 359 to nucleotide 718;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone du515_21 deposited under accession number ATCC 98689;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone du515 21 deposited under accession number ATCC 98689;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone du515_21 deposited under accession number ATCC 98689;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
2 0 insert of clone du515 21 deposited under accession number ATCC 98689;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID N0:4;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:4 having biological activity, the fragment 2 5 comprising eight contiguous amino acids of SEQ ID N0:4;, (j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ;
3 0 (1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i); and (m) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i) and that has a length that is at least 25% of the length of SEQ ID N0:3.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:3 from nucleotide 170 to nucleotide 718; the nucleotide sequence of SEQ ID
N0:3 from nucleotide 359 to nucleotide 718; the nucleotide sequence of the full-length protein coding sequence of clone du515 21 deposited under accession number ATCC 98689; or the nucleotide sequence of a mature protein coding sequence of clone du515 21 deposited under accession number ATCC 98689. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert of clone du515 21 deposited under accession number ATCC 98689. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:4 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino acids of SEQ ID N0:4, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:4 having biological activity, the fragment comprising the amino acid sequence from amino acid 86 to amino acid 95 of SEQ ID N0:4.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:3.
Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
2 0 (a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID N0:3, but excluding the poly(A) tail at the 2 5 3' end of SEQ ID N0:3; and (ab) the nucleotide sequence of the cDNA insert of clone du515_21 deposited under accession number ATCC 98689;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 50 degrees C; and 3 0 (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i} preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID N0:3, but excluding the poly(A) tail at the 3' end of SEQ ID N0:3; and (bb) the nucleotide sequence of the cDNA insert of clone du515 21 deposited under accession number ATCC 98689;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 4X SSC at 50 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:3, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
N0:3 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:3 , but excluding the poly(A) tail at the 3' end of SEQ ID N0:3. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:3 from nucleotide 170 to nucleotide 718, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of 2 0 SEQ ID N0:3 from nucleotide 170 to nucleotide 718, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:3 from nucleotide 170 to nucleotide 718. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
N0:3 from nucleotide 359 to nucleotide 718, and extending contiguously from a 2 5 nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:3 from nucleotide 359 to nucleotide 718, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:3 from nucleotide 359 to nucleotide 718.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group 3 0 consisting of:
(a) the amino acid sequence of SEQ ID N0:4;
(b) a fragment of the amino acid sequence of SEQ ID N0:4, the fragment comprising eight contiguous amino acids of SEQ ID N0:4; and (c) the amino acid sequence encoded by the cDNA insert of clone du515 21 deposited under accession number ATCC 98689;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:4. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:4 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino acids of SEQ ID N0:4, or a protein comprising a fragment of the amino acid sequence of SEQ
ID N0:4 having biological activity, the fragment comprising the amino acid sequence from amino acid 86 to amino acid 95 of SEQ ID N0:4.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 342 to nucleotide 638;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 411 to nucleotide 638;
(d) a polynucleotide comprising the nucleotide sequence of the full-2 0 length protein coding sequence of clone gn82_6 deposited under accession number ATCC 98689;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone gn82_6 deposited under accession number ATCC 98689;
(f) a polynucleotide comprising the nucleotide sequence of a mature 2 5 protein coding sequence of clone gn82_6 deposited under accession number ATCC
98689;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone gn82_6 deposited under accession number ATCC 98689;
(h) a poiynucleotide encoding a protein comprising the amino acid 3 0 sequence of SEQ ID N0:6;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:6 having biological activity, the fragment comprising eight contiguous amino acids of SEQ ID N0:6;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ;
(1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a}-(i); and (m) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i) and that has a length that is at least 25% of the length of SEQ ID N0:5.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:5 from nucleotide 342 to nucleotide 638; the nucleotide sequence of SEQ ID
N0:5 from nucleotide 411 to nucleotide 638; the nucleotide sequence of the full-length protein coding sequence of clone gn82_6 deposited under accession number ATCC 98689; or the nucleotide sequence of a mature protein coding sequence of clone gn82_6 deposited under accession number ATCC 98689. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone gn82_6 deposited under accession number ATCC 98689. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:6 having biological activity, the fragment 2 0 preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino acids of SEQ ID N0:6, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:6 having biological activity, the fragment comprising the amino acid sequence from amino acid 44 to amino acid 53 of SEQ
ID N0:6.
2 5 Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:5.
Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
(a) a process comprising the steps of:
3 0 (i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID N0:5, but excluding the poly(A) tail at the 3' end of SEQ ID N0:5; and (ab) the nucleotide sequence of the cDNA insert of clone gn82 6 deposited under accession number ATCC 98689;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 50 degrees C; and (iii) isolating the DNA polynucleotides detected with the probes};
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID N0:5, but excluding the poly(A) tail at the 3' end of SEQ ID N0:5; and (bb) the nucleotide sequence of the cDNA insert of clone gn82 6 deposited under accession number ATCC 98689;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 4X SSC at 50 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
2 0 Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:5, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
N0:5 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:5 , but excluding the poly(A) tail at the 3' end of SEQ ID N0:5. Also preferably the polynucleotide isolated 2 5 according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:5 from nucleotide 342 to nucleotide 638, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID N0:5 from nucleotide 342 to nucleotide 638, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:5 from nucleotide 342 to 3 0 nucleotide 638. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
N0:5 from nucleotide 411 to nucleotide 638, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:5 from nucleotide 411 to nucleotide 638, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:5 from nucleotide 411 to nucleotide 638.
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 N0:6;
(b) a fragment of the amino acid sequence of SEQ ID N0:6, the fragment comprising eight contiguous amino acids of SEQ ID N0:6; and (c) the amino acid sequence encoded by the cDNA insert of clone gn82_6 deposited under accession number ATCC 98689;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:6. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:6 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino acids of SEQ ID N0:6, or a protein comprising a fragment of the amino acid sequence of SEQ
ID N0:6 having biological activity, the fragment comprising the amino acid sequence from amino acid 44 to amino acid 53 of SEQ ID N0:6.
In one embodiment, the present invention provides a composition comprising an 2 0 isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 63 to nucleotide 959;
2 5 (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 252 to nucleotide 959;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 1 to nucleotide 385;
(e) a polynucleotide comprising the nucleotide sequence of the full-3 0 length protein coding sequence of clone ij1442_1 deposited under accession number ATCC 98755;
(f) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone ij1442_1 deposited under accession number ATCC 98755;
(g) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone ij1442_1 deposited under accession number ATCC 98755;
(h) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone ij1442_1 deposited under accession number ATCC 98755;
(i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID N0:8;
(j) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:8 having biological activity, the fragment comprising eight contiguous amino acids of SEQ ID N0:8;
(k) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein of (i) or (j) above ;
(m) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j); and (n) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(j} and that has a length that is at least 25% of the length of SEQ ID N0:7.
2 0 Preferably, such polynucleotide comprises the nucleotide sequence of SEQ
ID
N0:7 from nucleotide 63 to nucleotide 959; the nucleotide sequence of SEQ ID
N0:7 from nucleotide 252 to nucleotide 959; the nucleotide sequence of SEQ ID N0:7 from nucleotide 1 to nucleotide 385; the nucleotide sequence of the full-length protein coding sequence of clone ij1442_1 deposited under accession number ATCC 98755; or the 2 5 . nucleotide sequence of a mature protein coding sequence of clone ij1442 1 deposited under accession number ATCC 98755. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert of clone ijl4~_1 deposited under accession number ATCC 98755. In yet other preferred embodiments, the present invention provides a poiynucleotide encoding a protein 3 0 comprising the amino acid sequence of SEQ ID N0:8 from amino acid 1 to amino acid 268.
In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID
N0:8 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino acids of SEQ ID N0:8, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:8 having biological activity, the fragment comprising the amino acid sequence from amino acid 144 to amino acid 153 of SEQ ID N0:8.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:7.
Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID N0:7, but excluding the poly(A) tail at the 3' end of SEQ ID N0:7; and (ab) the nucleotide sequence of the cDNA insert of clone ij1442_1 deposited under accession number ATCC 98755;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 50 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
2 0 and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
2 5 (ba) SEQ ID N0:7, but excluding the pQly(A) tail at the 3' end of SEQ ID N0:7; and (bb) the nucleotide sequence of the cDNA insert of clone ij1442_1 deposited under accession number ATCC 98755;
(ii) hybridizing said primers) to human genomic DNA in 3 0 conditions at least as stringent as 4X SSC at 50 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:7, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
N0:7 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:7 , but excluding the poly{A) tail at the 3' end of SEQ ID N0:7. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:7 from nucleotide 63 to nucleotide 959, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID N0:7 from nucleotide 63 to nucleotide 959, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:7 from nucleotide 63 to nucleotide 959. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
N0:7 from nucleotide 252 to nucleotide 959, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:7 from nucleotide 252 to nucleotide 959, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:7 from nucleotide 252 to nucleotide 959. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:7 from nucleotide 1 to nucleotide 385, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID N0:7 from nucleotide 1 to nucleotide 385, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:7 from nucleotide 2 0 1 to nucleotide 385.
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 N0:8;
2 5 (b) the amino acid sequence of SEQ ID N0:8 from amino acid 1 to amino acid 268;
(c) a fragment of the amino acid sequence of SEQ ID N0:8, the fragment comprising eight contiguous amino acids of SEQ ID N0:8; and (d) the amino acid sequence encoded by the cDNA insert of clone 3 0 ij1442_1 deposited under accession number ATCC 98755;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:8 or the amino acid sequence of SEQ ID N0:8 from amino acid 1 to amino acid 268. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:8 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino acids of SEQ ID
N0:8, or a protein comprising a fragment of the amino acid sequence of SEQ ID
N0:8 having biological activity, the fragment comprising the amino acid sequence from amino acid 144 to amino acid 153 of SEQ ID N0:8.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9;
(b} a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 335 to nucleotide 895;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 806 to nucleotide 895;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone pe213_1 deposited under accession number ATCC 98689;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone pe213_1 deposited under accession number ATCC 98689;
(f) a polynucleotide comprising the nucleotide sequence of a mature 2 0 protein coding sequence of clone pe213_1 deposited under accession number ATCC 98689;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone pe213_1 deposited under accession number ATCC 98689;
(h) a polynucleotide encoding a protein comprising the amino acid 2 5 sequence of SEQ ID NO:10;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:10 having biological activity, the fragment comprising eight contiguous amino acids of SEQ ID N0:10;
(j) a polynucleotide which is an allelic variant of a polynucleotide of 3 0 (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ;
(1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a~(i); and (m) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i) and that has a length that is at least 25% of the length of SEQ ID N0:9.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:9 from nucleotide 335 to nucleotide 895; the nucleotide sequence of SEQ ID
N0:9 from nucleotide 806 to nucleotide 895; the nucleotide sequence of the full-length protein coding sequence of clone pe213_1 deposited under accession number ATCC 98689; or the nucleotide sequence of a mature protein coding sequence of clone pe213_1 deposited under accession number ATCC 98689. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert of clone pe213_1 deposited under accession number ATCC 98689. In further preferred embodiments, the present invention provides a polynucleotide encoding 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 polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity, the fragment comprising the amino acid sequence from amino acid 88 to amino acid 97 of SEQ ID N0:10.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
2 0 ID N0:9.
Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize 2 5 in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID N0:9, but excluding the poly(A) tail at the 3' end of SEQ ID N0:9; and (ab) the nucleotide sequence of the cDNA insert of clone 3 0 pe213_1 deposited under accession number ATCC 98689;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 50 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba} SEQ ID N0:9, but excluding the poly(A) tail at the 3' end of SEQ ID N0:9; and (bb} the nucleotide sequence of the cDNA insert of clone pe213_1 deposited under accession number ATCC 98689;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 4X SSC at 50 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:9, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
N0:9 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:9 , but excluding the poly(A) tail at the 3' end of SEQ ID N0:9. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the 2 0 cDNA sequence of SEQ ID N0:9 from nucleotide 335 to nucleotide 895, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID N0:9 from nucleotide 335 to nucleotide 895, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:9 from nucleotide 335 to nucleotide 895. Also preferably the polynucieotide isolated according to the above 2 5 process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
N0:9 from nucleotide 806 to nucleotide 895, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:9 from nucleotide 806 to nucleotide 895, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:9 from nucleotide 806 to nucleotide 895.
3 0 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 N0:10; and (c) the amino acid sequence encoded by the cDNA insert of clone pe213_1 deposited under accession number ATCC 98689;
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 having biological activity, the fragment comprising the amino acid sequence from amino acid 88 to amino acid 97 of SEQ ID N0:10.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:11;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:11 from nucleotide 305 to nucleotide 928;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 0 N0:11 from nucleotide 641 to nucleotide 928;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone pe318 4 deposited under accession number ATCC 98689;
(e) a polynucleotide encoding the full-length protein encoded by the 2 5 cDNA insert of clone pe318_4 deposited under accession number ATCC 98689;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone pe318_4 deposited under accession number ATCC 98689;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
3 0 insert of clone pe318 4 deposited under accession number ATCC 98689;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID N0:12;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:12 having biological activity, the fragment comprising eight contiguous amino acids of SEQ ID N0:12;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ;
(1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i); and (m) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i) and that has a length that is at least 25% of the length of SEQ ID NO:11.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:11 from nucleotide 305 to nucleotide 928; the nucleotide sequence of SEQ ID
NO:11 from nucleotide 641 to nucleotide 928; the nucleotide sequence of the full-length protein coding sequence of clone pe318_4 deposited under accession number ATCC 98689;
or the nucleotide sequence of a mature protein coding sequence of clone pe318 4 deposited under accession number ATCC 98689. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert 2 0 of clone pe318_4 deposited under accession number ATCC 98689. In further preferred embodiments, the present invention provides a poiynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:12 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino acids of SEQ ID N0:12, or a polynucleotide encoding .. ... - 2 5 a protein comprising a fragment of the amino acid sequence of SEQ
ID N0:12 having biological activity, the fragment comprising the amino acid sequence from amino acid 99 to amino acid 108 of SEQ ID N0:12.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:11.
3 0 Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
(a) a process comprising the steps of:
WO 99/46287 PC'f/US99/OS243 (i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID N0:11, but excluding the poly(A) tail at the 3' end of SEQ ID NO:11; and (ab) the nucleotide sequence of the cDNA insert of clone pe318 4 deposited under accession number ATCC 98689;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 50 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:11, but excluding the poly(A) tail at the 3' end of SEQ ID N0:11; and (bb) the nucleotide sequence of the cDNA insert of clone 2 0 pe318 4 deposited under accession number ATCC 98689;
(ii) hybridizing said primers) to human genamic DNA in conditions at least as stringent as 4X SSC at 50 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
2 5 Preferably the polynucleotide isolated according to the , above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:11, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ
ID N0:11 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:11 , but excluding the poly(A) tail at the 3' end of SEQ ID NO:11. Also preferably the 3 0 polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:11 from nucleotide 305 to nucleotide 928, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID N0:11 from nucleotide 305 to nucleotide 928, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:11 from nucleotide 305 to nucleotide 928. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
N0:11 from nucleotide 641 to nucleotide 928, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:11 from nucleotide 641 to nucleotide 928, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:11 from nucleotide 641 to nucleotide 928.
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 N0:12;
(b) a fragment of the amino acid sequence of SEQ ID N0:12, the fragment comprising eight contiguous amino acids of SEQ ID N0:12; and (c) the amino acid sequence encoded by the cDNA insert of clone pe318 4 deposited under accession number ATCC 98689;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:12. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:12 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino acids 2 0 of SEQ ID N0:12, or a protein comprising a fragment of the amino acid sequence of SEQ
ID N0:12 having biological activity, the fragment comprising the amino acid sequence from amino acid 99 to amino acid 108 of SEQ ID N0:12.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
2.5 . (a) a polynucleotide comprising. the nucleotide sequence of SEQ ID , .
N0:13;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13 from nucleotide 640 to nucleotide 792;
(c) a polynucleotide comprising the nucleotide sequence of the full-3 0 length protein coding sequence of clone pp85_1 deposited under accession number ATCC 98689;
(d) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone pp85_i deposited under accession number ATCC 98689;
(e) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone pp85_1 deposited under accession number ATCC 98689;
(f) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone pp85_1 deposited under accession number ATCC 98689;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID N0:14;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:14 having biological activity, the fragment comprising eight contiguous amino acids of SEQ ID N0:14;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(f) above;
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above ;
(k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(h); and (1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a}-(h) and that has a length that is at least 25% of the length of SEQ ID N0:13.
2 0 Preferably, such polynucleotide comprises the nucleotide sequence of SEQ
ID
N0:13 from nucleotide 640 to nucleotide 792; the nucleotide sequence of the full-length protein coding sequence of clone pp85_1 deposited under accession number ATCC
98689;
or the nucleotide sequence of a mature protein coding sequence of clone pp~ 1 deposited under accession number ATCC 98689. In other preferred embodiments, the 2 5 polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone pp85_1 deposited under accession number ATCC 98689. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:14 having biological activity, the fragment preferably comprising eight (more preferably twenty, most 3 0 preferably thirty) contiguous amino acids of SEQ ID N0:14, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:14 having biological activity, the fragment comprising the amino acid sequence from amino acid 20 to amino acid 29 of SEQ ID N0:14.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:13.
Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID N0:13, but excluding the poly(A) tail at the 3' end of SEQ ID N0:13; and (ab) the nucleotide sequence of the cDNA insert of clone pp85_1 deposited under accession number ATCC 98689;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 50 degrees C; and (iii} isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that 2 0 hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID N0:13, but excluding the poly(A) tail at the 3' end of SEQ ID N0:13; and (bb) the nucleotide sequence of the cDNA insert of clone 2 5 pp85_1 deposited under accession number ATCC 98689;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 4X SSC at 50 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
3 0 Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:13, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ
ID N0:13 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:13 , but excluding the poly(A) tail at the 3' end of SEQ ID N0:13. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:13 from nucleotide 640 to nucleotide 792, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID N0:13 from nucleotide 640 to nucleotide 792, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:13 from nucleotide 640 to nucleotide 792.
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 N0:14;
(b) a fragment of the amino acid sequence of SEQ ID N0:14, the fragment comprising eight contiguous amino acids of SEQ ID N0:14; and (c) the amino acid sequence encoded by the cDNA insert of clone pp85_1 deposited under accession number ATCC 98689;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:14. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:14 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino acids 2 0 of SEQ ID N0:14, or a protein comprising a fragment of the amino acid sequence of SEQ
ID N0:14 having biological activity, the fragment comprising the amino acid sequence from amino acid 20 to amino acid 29 of SEQ ID N0:14.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
2 5 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15 from nucleotide 641 to nucleotide 931;
(c) a polynucleotide comprising the nucleotide sequence of the,full-3 0 length protein coding sequence of clone pp325_9 deposited under accession number ATCC 98689;
(d) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone pp325_9 deposited under accession number ATCC 98689;
(e) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone pp325_9 deposited under accession number ATCC 98689;
(f) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone pp325 9 deposited under accession number ATCC 98689;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID N0:16;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:16 having biological activity, the fragment comprising eight contiguous amino acids of SEQ ID N0:16;
(i) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(f) above;
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above ;
(k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(h); and (1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a}-(h) and that has a length that is at least 25% of the length of SEQ ID N0:15.
2 0 Preferably, such polynucleotide comprises the nucleotide sequence of SEQ
ID
N0:15 from nucleotide 641 to nucleotide 931; the nucleotide sequence of the full-length protein coding sequence of clone pp325_9 deposited under accession number ATCC
98689; or the nucleotide sequence of a mature protein coding sequence of clone pp325_9 deposited under accession number ATCC 98689. In other preferred embodiments, the 2 5 polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone pp325_9 deposited under accession number ATCC 98689. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:16 having biological activity, the fragment preferably comprising eight (more preferably twenty, most 3 0 preferably thirty) contiguous amino ands of SEQ ID N0:16, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:16 having biological activity, the fragment comprising the amino acid sequence from amino acid 43 to amino acid 52 of SEQ ID N0:16.
WO 99/46287 PCT/US99/OS?A3 Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:15.
Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID N0:15, but excluding the poly(A} tail at the 3' end of SEQ ID N0:15; and (ab) the nucleotide sequence of the cDNA insert of clone pp325_9 deposited under accession number ATCC 98689;
(ii} hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 50 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that 2 0 hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID N0:15, but excluding the poly(A) tail at the 3' end of SEQ ID N0:15; and (bb) the nucleotide sequence of the cDNA insert of clone 2 5 pp325_9 deposited under accession number ATCC 98689;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 4X SSC at 50 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
3 0 Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:15, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ
ID N0:15 to a nucleotide sequence corresponding to the 3' end of SEQ ID Np:lS
, but excluding the poly(A) tail at the 3' end of SEQ ID N0:15. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:15 from nucleotide 641 to nucleotide 931, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID N0:15 from nucleotide 641 to nucleotide 931, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:15 from nucleotide 641 to nucleotide 931.
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 N0:16;
(b) a fragment of the amino acid sequence of SEQ ID N0:16, the fragment comprising eight contiguous amino acids of SEQ ID N0:16; and (c) the amino acid sequence encoded by the cDNA insert of clone pp325 9 deposited under accession number ATCC 98689;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:16. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:16 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino acids 2 0 of SEQ ID N0:16, or a protein comprising a fragment of the amino acid sequence of SEQ
ID N0:16 having biological activity, the fragment comprising the amino acid sequence from amino acid 43 to amino acid 52 of SEQ ID N0:16.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
2 5 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:17;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:17 from nucleotide 69 to nucleotide 443;
(c) a polynucleotide comprising the nucleotide sequence of the full-3 0 length protein coding sequence of clone qb401 6 deposited under accession number ATCC 98689;
(d) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone qb401 6 deposited under accession number ATCC 98689;
(e} a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone qb401 6 deposited under accession number ATCC 98689;
(f} a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone qb401 6 deposited under accession number ATCC 98689;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID N0:18;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:18 having biological activity, the fragment comprising eight contiguous amino acids of SEQ ID N0:18;
(i} a polynucleotide which is an allelic variant of a polynucleotide of (a)-(f) above;
(j) a polynucleotide which encodes a species homologue of the protein of (g) or (h) above ;
(k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(h); and (1) a polynucleotide that hybridizes under stringent conditions to any one of the polynudeotides specified in (ar(h) and that has a length that is at least 25% of the length of SEQ ID N0:17.
2 0 Preferably, such polynucleotide comprises the nucleotide sequence of SEQ
ID
N0:17 from nucleotide 69 to nucleotide 443; the nucleotide sequence of the full-length protein coding sequence of clone qb401 6 deposited under accession number ATCC
98689; or the nucleotide sequence of a mature protein coding sequence of clone qb401 6 deposited under accession number ATCC 98689. In other preferred embodiments, the 2 5 polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone qb401 6 deposited under accession number ATCC 98689. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:18 having biological activity, the fragment preferably comprising eight (more preferably twenty, most 3 0 preferably thirty) contiguous amino acids of SEQ ID N0:18, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:18 having biological activity, the fragment comprising the amino acid sequence from amino acid 57 to amino acid 66 of SEQ ID N0:18.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:17.
Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID N0:17, but excluding the poly(A) tail at the 3' end of SEQ ID N0:17; and (ab) the nucleotide sequence of the cDNA insert of clone qb401 6 deposited under accession number ATCC 98689;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 50 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that 2 0 hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID N0:17, but excluding the poly(A) tail at the 3' end of SEQ ID N0:17; and (bb) the nucleotide sequence of the cDNA insert of clone 2 5 qb401 6 deposited under accession number ATCC 98689;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 4X SSC at 50 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
3 0 Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:17, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ
ID N0:17 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:17 , but excluding the poly(A) tail at the 3' end of SEQ ID N0:17. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:17 from nucleotide 69 to nucleotide 443, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID N0:17 from nucleotide 69 to nucleotide 443, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:17 from nucleotide 69 to nucleotide 443.
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:18;
(b} a fragment of the amino acid sequence of SEQ ID N0:18, the fragment comprising eight contiguous amino acids of SEQ ID N0:18; and (c) the amino acid sequence encoded by the cDNA insert of clone qb401 6 deposited under accession number ATCC 98689;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:18. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:18 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino acids 2 0 of SEQ ID N0:18, or a protein comprising a fragment of the amino acid sequence of SEQ
ID N0:18 having biological activity, the fragment comprising the amino acid sequence from amino acid 57 to amino acid 66 of SEQ ID N0:18.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
2 5 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:19;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:19 from nucleotide 151 to nucleotide 381;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
3 0 N0:19 from nucleotide 226 to nucleotide 381;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone qc671 1 deposited under accession number ATCC 98689;
WO 99/46Z8~ PCTNS99/05243 (e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone qc671 1 deposited under accession number ATCC 98689;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone qc671 1 deposited under accession number ATCC 98689;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone qc671 1 deposited under accession number ATCC 98689;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID N0:20;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:20 having biological activity, the fragment comprising eight contiguous amino acids of SEQ ID N0:20;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ;
(I) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i); and (m) a polynucleotide that hybridizes under stringent conditions to any 2 0 one of the polynucleotides specified in (a)-(i) and that has a length that is at least 25% of the length of SEQ ID N0:19.
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:19 from nucleotide 151 to nucleotide 381; the nucleotide sequence of SEQ ID
N0:19 from nucleotide 226 to nucleotide 381; the nucleotide sequence of the full-length protein 2 5 coding sequence of clone qc671 1 deposited under accession number ATCC
98689; or the nucleotide sequence of a mature protein coding sequence of clone qc671 1 deposited under accession number ATCC 98689. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert of clone qc671 1 deposited under accession number ATCC 98689. In further preferred 3 0 embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:20 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino acids of SEQ ID N0:20, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:20 having biological activity, the fragment comprising the amino acid sequence from amino acid 33 to amino acid 42 of SEQ ID N0:20.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:19.
Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID N0:19, but excluding the poly(A) tail at the 3' end of SEQ ID N0:19; and (ab) the nucleotide sequence of the cDNA insert of clone qc671 1 deposited under accession number ATCC 98689;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 50 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and 2 0 (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID N0:19, but excluding the poly(A) tail at the 2 5 3' end of SEQ ID N0:19; and"
(bb) the nucleotide sequence of the cDNA insert of clone qc671 1 deposited under accession number ATCC 98689;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 4X SSC at 50 degrees C;
3 0 (iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:19, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ
ID N0:19 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:19 , but excluding the poly(A) tail at the 3' end of SEQ ID N0:19. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:19 from nucleotide 151 to nucleotide 381, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID N0:19 from nucleotide 151 to nucleotide 381, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:19 from nucleotide 151 to nucleotide 381. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
N0:19 from nucleotide 226 to nucleotide 381, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:19 from nucleotide 226 to nucleotide 381, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:19 from nucleotide 226 to nucleotide 381.
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 N0:20;
(b) a fragment of the amino acid sequence of SEQ ID N0:20, the fragment comprising eight contiguous amino acids of SEQ ID N0:20; and 2 0 (c) the amino acid sequence encoded by the cDNA insert of clone qc671 1 deposited under accession number ATCC 98689;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:20. In further preferred embodiments, the present invention provides a protein comprising a fragment of the 2.5 amino acid sequence of SEQ ID N0:20 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) contiguous amino acids of SEQ ID NO~O, or a protein comprising a fragment of the amino acid sequence of SEQ
ID N0:20 having biological activity, the fragment comprising the amino acid sequence from amino acid 33 to amino acid 42 of SEQ ID N0:20.
3 0 In certain preferred embodiments, the polynucleotide is operably linked to an expression control sequence. The invention also provides a host cell, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions.
Also provided by the present invention are organisms that have enhanced, reduced, or WO 99/46287 PC"T/US99/05243 modified expression of the genes) corresponding to the polynucleotide sequences disclosed herein.
Processes are also provided for producing a protein, which comprise:
(a) growing a culture of the host cell transformed with such polynucleotide compositions in a suitable culture medium; and (b) purifying the protein from the culture.
The protein produced according to such methods is also provided by the present invention.
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 lA and 1B are schematic representations of the pED6 and pNOTs vectors, respectively, used for deposit of clones disclosed herein.
DETAILED DESCRIPTION
ISOLATED PROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequences, as presently determined, are reported below for each clone and protein disclosed in the present application. The nucleotide 2 5 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 3 0 sequence. For each disclosed protein applicants have identified what they have determined to be the reading frame best identifiable with sequence information available at the time of filing.
As used herein a "secreted" protein is one which, when expressed in a suitable host cell, is transported across ar through a membrane, including transport as a result of signal sequences in its amino acid sequence. "Secreted" proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g. , receptors) from the cell in which they are expressed. "Secreted" proteins also include without limitation proteins which are transported across the membrane of the endoplasmic reticulum.
Clone "cb98 4"
A polynucleotide of the present invention has been identified as clone "cb98_4".
cb98 4 was isolated from a human fetal 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. cb98 4 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "cb98 4 protein").
The nucleotide sequence of cb98 4 as presently determined is reported in SEQ
ID
N0:1, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the cb98 4 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID N0:2.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone cb98 4 should be approximately 2200 bp.
2 0 The nucleotide sequence disclosed herein for cb98 4 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. cb98_4 demonstrated at least some similarity with sequences identified as AA178965 (zp39f09.s1 Stratagene muscle 937209 Homo sapiens cDNA
clone 611849 3'), AA290723 (zs44c07.r1 NCI_CGAP GCBl Homo sapiens cDNA clone 2 5 IMAGE:700332 5'), AA573499 (nk99fU1.s1 NCI_CGAP_Co3 Homo sapiens cDNA
clone IMAGE:1028953), 823530 (yg29g12.r1 Homo Sapiens cDNA clone 34025 5'), and (EST07267 Homo sapiens cDNA clone HIBBT41 5' end). The predicted amino acid sequence disclosed herein for cb98_4 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted cb98 4 3 0 protein demonstrated at least some similarity to sequences identified as (beta-myosin heavy chain [Homo sapiens]), U93871 (RaP2 interacting protein 8 [Homo sapiens]), and X78998 (endosomal protein [Homo Sapiens]). Based upon sequence similarity, cb98_4 proteins and each similar protein or peptide may share at least some activity. The TopPredII computer program predicts two potential transmembrane domains within the cb98 4 protein sequence, centered around amino acids 28 and 140 of SEQ ID N0:2; amino acids 15 to 27 are also a possible leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 28.
Clone "du515 21"
A polynucleotide of the present invention has been identified as clone "du515 21".
du515 21 was isolated from a human fetal 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. du515_21 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "du515_21 protein").
The nucleotide sequence of du515 21 as presently determined is reported in SEQ
ID N0:3, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the du515 21 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID N0:4.
Amino acids 51 to 63 of SEQ ID N0:4 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 64. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain 2 0 should the predicted leader/signal sequence not be separated from the remainder of the du515 21 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone du515 21 should be approximately 1300 bp.
The nucleotide sequence disclosed herein for du515 21 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. du515 21 demonstrated at least some similarity with sequences identified as AA124621 (mp72d01.r1 Soares 2NbMT Mus musculus cDNA clone 574753 5'), AA602397 (no30d06.s1 NCI_CGAP_Pr22 Homo sapiens cDNA clone IMAGE:1102187), M78422 (EST00570 Homo sapiens cDNA clone HFBCB84), Q59591 (Human brain 3 0 Expressed Sequence Tag EST00580), T04884 (EST02772 Homo sapiens cDNA clone HFBCB84), and W31206 (zb95a05.s1 Soares parathyroid tumor NbHPA Homo sapiens cDNA clone 320528 3'). The predicted amino acid sequence disclosed herein for du515 21 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted du515 21 protein demonstrated at least some similarity to sequences identified as L31611 (NMDA receptor subunit NR2D
[Rattus norvegicus]) and 215045 (glycine-rich_protein_(aal-291) [Brassica napus]).
Based upon sequence similarity, du515_21 proteins and each similar protein or peptide may share at least some activity.
Clone 'gn82 6"
A polynucleotide of the present invention has been identified as clone "gn82_6".
gn82_6 was isolated from a human adult blood (peripheral blood mononuclear cells treated with granulocyte-colony stimulating factor in vivo) 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. gn82_6 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "gn82_6 protein').
The nucleotide sequence of gn82_6 as presently determined is reported in SEQ
ID
N0:5, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the gn82_6 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID N0:6.
Amino acids 11 to 23 of SEQ ID N0:6 are a predicted leader/signal sequence, with the predicted 2 0 mature amino acid sequence beginning at amino acid 24. 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 gn82_6 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone 2 5 gn82_6 should be approximately 1000 bp.
The nucleotide sequence disclosed herein for gn82_6 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. gn82_6 demonstrated at least some similarity with sequences identified as AA102622 (zn43f05.s1 Stratagene HeLa cell s3 937216 Homo sapiens cDNA
3 0 clone 550209 3'), AA145264 (mr12h02.r1 Soares mouse 3NbMS Mus musculus cDNA clone 597267 5'), AA418655 (zv93g07.r1 Soares NhHMI'u Sl Homo Sapiens cDNA clone 5'), AA527345 (ng40a02.s1 NCI_CGAP_Co3 Homo sapiens cDNA clone IMAGE:937226), 898908 (yr31c08.r1 Homo Sapiens cDNA clone 206894 5'), and W72745 (zd71f04.s1 Soares fetal heart NbHHI9W Homo sapiens cDNA clone 346111 3' similar to WP:T26A5.7 CE00786 TRX). The predicted amino acid sequence disclosed herein for gn82_6 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted gn82 6 protein demonstrated at least some similarity to sequences identified as U00043 (similar to D. melanogaster trithorax protein (Caenorhabditis elegans)). Based upon sequence similarity, gn82 6 proteins and each similar protein or peptide may share at least some activity.
gn82_6 protein was expressed in a COS cell expression system, and an expressed protein band of approximately 18 kDa was detected in conditioned medium fractions using SDS polyacrylamide gel electrophoresis.
Clone "ij1442 1"
A polynucleotide of the present invention has been identified as clone "ij1442 1".
ij1442_1 was isolated from a human adult blood (peripheral blood mononuclear cells treated with granulocyte-colony stimulating factor in vivo) 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. ijl~ 1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "ij1442_1 protein").
2 0 The nucleotide sequence of ij1442_1 as presently determined is reported in SEQ
ID N0:7, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the ijl~ 1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID N0:8.
Amino acids 51 to 63 of SEQ ID N0:8 are a predicted Ieader/signal sequence, with the predicted 2 5 mature amino acid .sequence beginning at amino acid 64. 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 ijl~ 1 protein. The ATG sequence at positions 183-185 of SEQ ID N0:7 is another possible translation initiation codon.
3 0 The EcoRI/NotI restriction fragment obtainable from the deposit containing clone ij1442 1 should be approximately 1100 bp.
The nucleotide sequence disclosed herein for ij1442_1 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. ijl~ 1 demonstrated at least some similarity with sequences identified as AA150849 (z144bO1.r1 Soares pregnant uterus NbHPU Homo sapiens cDNA
clone 504745 5'), AF012536 (Homo Sapiens decoy receptor 1 (DcR 1 ) mRNA, complete cds), AF016267 (Homo sapiens TRAIL receptor 3 mRNA, complete cds). The predicted amino acid sequence disclosed herein for ij1442_1 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted ij1442_1 protein demonstrated at least some similarity to sequences identified as AF012536 (decoy receptor 1 [Homo sapiens)), AF012629 (antagonist decoy receptor for TRAILJApo-2L [Homo sapiens]), AF014794 (TNF related TRAIL receptor [Homo Sapiens]), AF016267 (TRAIL receptor 3 [Homo sapiens]), AF020502 (cytotoxic TRAIL
receptor-3 [Homo sapiens]), AF033854 (lymphocyte inhibitor of TRAIL [Homo sapiens]), and U90875 (cytotoxic ligand TRAIL receptor [Homo sapiens]). Based upon sequence similarity, ij 1442_1 proteins and each similar protein or peptide may share at least some activity. The TopPredII computer program predicts an additional potential trans-membrane domain within the ij 1442_1 protein sequence centered around amino acid 289 of SEQ ID
N0:8.
ij 1442_1 protein was expressed in a COS cell expression system, and an expressed protein band of approximately 23 kDa was detected in conditioned medium and membrane fractions using SDS polyacrylamide gel electrophoresis.
2 0 Clone "pe213 1"
A polynucleotide of the present invention has been identified as clone "pe213_1".
pe213_1 was isolated from a human adult blood (chronic myelogenous leukemia K562) 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 2 5 protein on the basis of computer analysis of the amino acid sequence of the encoded protein. pe213_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "pe213_1 protein").
The nucleotide sequence of pe213_1 as presently determined is reported in SEQ
ID N0:9, and includes a poly(A) tail. What applicants presently believe to be the proper 3 0 reading Name and the predicted amino acid sequence of the pe213_1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID
NO:10. Amino acids 145 to 157 of SEQ ID NO:20 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 158. 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 pe213_1 protein.
Another potential pe213_1 reading frame and predicted amino acid sequence is encoded by basepairs 645 to 947 of SEQ ID N0:9 and is reported in SEQ ID
N0:31.
Amino acids 38 to 50 of SEQ ID N0:31 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 51. Due to the hydrophobic nature of this predicted leader/signal sequence, it is likely to act as a transmembrane domain should it not be separated from the remainder of the protein of SEQ ID N0:31. The TopPredII computer program predicts an additional potential transmembrane domain within the SEQ ID N0:31 amino acid sequence centered around amino acid 11 of SEQ ID N0:31.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone pe213_1 should be approximately 1000 bp.
The nucleotide sequence disclosed herein for pe213_1 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. pe213_1 demonstrated at least some similarity with sequences identified as AA374638 (EST87065 HSC172 cells I Homo sapiens cDNA 5' end), (zv26a08.r1 Scares NhHMPu Sl Homo sapiens cDNA clone 754742 5'), AA424905 2 0 (zw03f01.s1 Scares NhHIvIPu S1 Homo sapiens cDNA clone 768217 3'), Q77233 (Human genome fragment), and 875637 (yi59d05.s1 Homo sapiens cDNA clone 143529 3').
Based upon sequence similarity, pe213_1 proteins and each similar protein or peptide may share at least some activity.
pe213_1 protein was expressed in a COS cell expression system, and an expressed 2 5 protein band of approximately 10 kDa was detected in membrane fractions using SDS
polyacrylamide gel electrophoresis.
Clone "pe318 4"
A polynucleotide of the present invention has been identified as clone "pe318 4".
3 0 pe318 4 was isolated from a human adult blood (chronic myelogenous leukemia K562) 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. pe318_4 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "pe318_4 protein").
The nucleotide sequence of pe318 4 as presently determined is reported in SEQ
ID N0:11, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the pe318 4 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:12. Amino acids 100 to 112 of SEQ ID N0:12 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 113. 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 pe318 4 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone pe318 4 should be approximately 1300 bp.
The nucleotide sequence disclosed herein for pe318_4 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. pe318_4 demonstrated at least some similarity with sequences identified as AA466632 (ve12h12.r1 Soares mouse NbMH Mus musculus cDNA clone 817991 5'}, AA583057 (nn80h04.s1 NCI_CGAP_Co9 Homo sapiens cDNA clone I1VIAGE:1090231), T25941 (Human gene signature HUMGS08174), and W02860 (za05c08.r1 2 0 Soares melanocyte 2NbHM Homo sapiens cDNA clone 291662 5'). The predicted amino acid sequence disclosed herein for pe318 4 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted pe318_4 protein demonstrated at least some similarity to sequences identified as 279597 (C33A11.2 [Caenorhabditis elegans]). Based upon sequence similarity, pe318 4 2 5 proteins and each similar protein or peptide may share at least some activity. The TopPredII computer program predicts four potential transmembrane domains within the pe318 4 protein sequence, centered around amino acids 40, 70,110, and 160 of SEQ ID
N0:12, respectively; amino acids 37 to 49 are also a possible leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 50.
,;lone "~,~85 1"
A polynucleotide of the present invention has been identified as clone "pp85_1".
pp85_1 was isolated from a human adult blood (lymphoblastic leukemia MOLT-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. pp85_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "pp85_1 protein").
The nucleotide sequence of pp85_1 as presently determined is reported in SEQ
ID
N0:13, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the pp85_1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:14.
Another potential pp85_1 reading frame and predicted amino acid sequence encoded by basepairs 121 to 384 of SEQ ID N0:13 is reported in SEQ ID N0:32.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone pp85_1 should be approximately 1200 bp.
The nucleotide sequence disclosed herein for pp85_1 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. No hits were found in the database. The TopPredII
computer program predicts a potential transmembrane domain within the pp85_1 protein sequence centered around amino acid 26 of SEQ ID N0:14; amino acids 13 to 25 of SEQ ID
N0:14 are also a possible leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 26. The nucleotide sequence of pp85_1 indicates that 2 0 it may contain an Alu repetitive element.
Clone "pp325 9"
A polynucleotide of the present invention has been identified as clone "pp325 9".
pp325_9 was isolated from a human adult blood (lymphoblastic leukemia MOLT-4) cDNA
2 5 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.
pp325 9 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "pp325_9 protein').
3 0 The nucleotide sequence of pp325_9 as presently determined is reported in SEQ
ID N0:15, and includes a poly(A} tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the pp325_9 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:16.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone pp325 9 should be approximately 2200 bp.
The nucleotide sequence disclosed herein for pp325 9 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. pp325_9 demonstrated at least some similarity with sequences identified as 834384 (yh85h08.s1 Homo sapiens cDNA clone 136575 3' similar to contains Alu repetitive element). Based upon sequence similarity, pp325_9 proteins and each similar protein or peptide may share at least some activity. The nucleotide sequence of pp325 9 indicates that it may contain an Alu repetitive element.
Clone "qb40I 6"
A polynucleotide of the present invention has been identified as clone "qb401 6".
qb401 6 was isolated from a human adult bladder (carcinoma 5637) 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. qb401 6 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "qb401 6 protein").
The nucleotide sequence of qb401 6 as presently determined is reported in SEQ
2 0 ID N0:17, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the qb401 6 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:18.
Another potential qb401_6 reading frame and predicted amino acid sequence encoded by basepairs 364 to 507 of SEQ ID N0:17 is reported in SEQ ID N0:33. Amino acids 6 to 18 . of SEQ ID N0:33 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 19. Due to the hydrophobic nature of this 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 protein of SEQ ID N0:33.
3 0 The EcoRI/NotI restriction fragment obtainable from the deposit containing clone qb401_6 should be approximately 2200 bp.
The nucleotide sequence disclosed herein for qb401 6 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. No hits were found in the database. The TopPredII
computer WO 99/46287 PC 1'/US99/05243 program predicts two potential cans-membrane domains within the qb401 6 protein sequence of SEQ ID N0:18.
Clone"qc671 1"
A polynucleotide of the present invention has been identified as clone "qc671 1".
qc671 1 was isolated from a human adult neural tissue (neuroepithelioma HTB-10 cell line) 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. qc671_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "qc671 1 protein').
The nucleotide sequence of qc671 1 as presently determined is reported in SEQ
ID N0:19, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the qc671 1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:20. Amino acids 13 to 25 of SEQ ID N0:20 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 26. 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 2 0 qc671 1 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone qc671 1 should be approximately 1100 bp.
The nucleotide sequence disclosed herein for qc671 1 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and 2 5 , . gA~A ~~~.protocols. qc671_1 demonstrated at least some similarity with sequences identified as AA699680 (zi78c08.s1 Soares fetal liver spleen 1NFLS Sl Homo sapiens cDNA clone 446894 3'). Based upon sequence similarity, qc671 1 proteins and each similar protein or peptide may share at least some activity.
qc671_1 protein was expressed in a COS cell expression system, and an expressed 3 0 protein band of approximately 11 kDa was detected in conditioned medium and membrane fractions using SDS polyacrylamide gel electrophoresis.
Deposit of Clones Clones cb98 4, du515_21, gn82_6, ij1442 3, pe213_l, pe318_4, pp85_l, pp325 9, qb401 6, and qc671_1 were deposited on March 11,1998 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
98689, from which each clone comprising a particular polynucleotide is obtainable. Clone ij1442_l, an isolate related to clone ij1442_3, was deposited on May 14, 1998 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 was given the accession number ATCC 98755, from which the ij1442_1 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. coIi) in these composite deposits. Each clone can be removed from the vector in which it was deposited by performing an EcoRI/NotI digestion (5' site, EcoRI; 3' site, NotI) to produce the appropriate fragment for such clone. Each clone was deposited in either the pED6 or pNOTs vector depicted in Figures lA and 1B, respectively. The pED6dpc2 vector ("pED6") was derived from pED6dpc1 by insertion of a new polylinker to facilitate 2 0 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 M13 origin of replication in the CIaI 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 . . . 2 5 ,. can till be isolated by digestion with EcoRL and Notl. However, NotI 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 3 0 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 Sequence cb98_4 SEQ ID N0:21 du515 21 SEQ ID N0:22 gn82_6 SEQ ID N0:23 ij1442_1 SEQ ID N0:24 pe213_1 SEQ ID N0:25 pe318_4 SEQ ID N0:26 pp85_1 SEQ ID N0:27 pp325_9 SEQ ID N0:28 qb401_6 SEQ ID N0:29 qc671 1 SEQ ID N0:30 In the sequences listed above which include an N at position 2, that position is occupied in preferred probes/primers by a biotinylated phosphoaramidite residue rather than a nucleotide (such as, for example, that produced by use of biotin phosphoramidite (1-dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-O-(2-cyanoethyl)-(N,N-2 0 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;
2 5 (b) , It should be designed to have a Tm of approx. 80 ° C
(assuming 2° for, each A or T and 4 degrees for each G or C).
The oligonucleotide should preferably be labeled with y 3zP 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 3 0 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 ul of the stock used to inoculate a sterile culture flask containing 25 ml of sterile L-broth containing ampicillin at 100 lzg/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 pg/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at 37°C. Other known methods of obtaining distinct, well-separated colonies can also be employed.
Standard colony hybridization procedures should then be used to transfer the colonies to nitrocellulose filters and lyse, denature and bake them.
The filter is then preferably incubated at 65°C for 1 hour with gentle agitation in 6X SSC (20X stock is 175.3 g NaCI/liter, 88.2 g Na citrate/liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS,100 pg/ml of yeast RNA, and 10 mM EDTA
(approximately 10 mL per 150 mm filfier). Preferably, the probe is then added to the hybridization mix at a concentration greater than or equal to 1e+6 dpm/mL. The filter is then preferably incubated at 65°C with gentle agitation overnight. The filter is then preferably washed in 500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably followed by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes.
2 0 A third wash with O.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 tune 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 2 5 using standard procedures. The clones can then be verified by restriction analysis, hybridization analysis, or DNA sequencing.
Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention. Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, 3 0 as described in H.U. Saragovi, et al., Bio/Technology ~Q, 773-778 (1992) and in R.S.
McDowell, et al., J. Amer. Chem. Soc. ~4 9245-9253 (1992), both of which are incorporated herein by reference. Such fragments may be fused to carrier molecules such as immunoglobulins 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 immunoglobulin 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 forms) 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 sequences) of the mature forms) of the protein may also be determinable from the amino acid sequence of the full-length form.
The present invention also provides genes corresponding to the polynucleotide sequences disclosed herein. "Corresponding genes" are the regions of the genome that are transcribed to produce the mRNAs from which cDNA polynucleotide sequences are derived and may include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes may therefore include but are not limited to coding sequences, 5' and 3' untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements. The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed 2 0 herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials. An "isolated gene" is a gene that has been separated from the adjacent coding sequences, if any, present in the genome of the organism from which the gene was isolated.
2 5 The chromosomal location corresponding to the polynucleotide sequences disclosed herein may also be determined, for example by hybridizing appropriately labeled polynucleotides of the present invention to chromosomes in situ. It may also be possible to determine the corresponding chromosomal location for a disclosed polynucleotide by identifying significantly similar nucleotide sequences in public 3 0 databases, such as expressed sequence tags (ESTs), that have akeady been mapped to particular chromosomal locations. For at least some of the polynucleotide sequences disclosed herein, public database sequences having at least some similarity to the polynucleotide of the present invention have been listed by database accession number.
Searches using the GenBank accession numbers of these public database sequences can then be performed at an Internet site provided by the National Center for Biotechnology Information having the address http://www.ncbi.nim.nih.gov/UruGene/, in order to identify "UruGene clusters" of overlapping sequences. Many of the "UniGene clusters"
so identified will already have been mapped to particular chromosomal sites.
Organisms that have enhanced, reduced, or modified expression of the genes) corresponding to the polynucleotide sequences disclosed herein are provided.
The desired change in gene expression can be achieved through the use of antisense polynucleotides or ribozymes that bind and/or cleave the mRNA transcribed from the gene (Albert and Morris,1994, Trends Pharmacol. Sci.15(7): 250-254; Lavarosky et al.,1997, Biochem. Mol. Med. 62(1):11-22; and Hampel,1998, Prog. Nucleic Acid Res. MoI.
Biol. 58:1-39; all of which are incorporated by reference herein). Transgenic animals that have multiple copies of the genes) corresponding to the polynucleotide sequences disclosed herein, preferably produced by transformation of cells with genetic constructs that are stably maintained within the transformed cells and their progeny, are provided.
Transgenic animals that have modified genetic control regions that increase or reduce gene expression levels, or that change temporal or spatial patterns of gene expression, are also provided (see European Patent No. 0 649 464 Bl, incorporated by reference herein).
In addition, organisms are provided in which the genes) corresponding to the polynucleotide sequences disclosed herein have been partially or completely inactivated, 2 0 through insertion of extraneous sequences into the corresponding genes) or through deletion of all or part of the corresponding gene(s). Partial or complete gene inactivation can be accomplished through insertion, preferably followed by imprecise excision, of transposable elements (Plasterk,1992, Bioessays 14(9): 629-633; Zwaal et al.,1993, Proc. Natl.
Acad. Sci. USA 90(16): 7431-7435; Clark et al.,1994, Proc. Natl. Acad. Sci.
LISA 91(2): 719-722;
2 5 all of which a;e incorporated by reference herein), or through homologous recombination, preferably detected by positive/negative genetic selection strategies (Mansour et al.,1988, Nature 336: 348-352; U.S. Patent Nos. 5,464,764; 5,487,992; 5,627,059;
5,631,153; 5;614, 396;
5,616,491; and 5,679,523; all of which are incorporated by reference herein).
These organisms with altered gene expression are preferably eukaryotes and more preferably 3 0 are mammals. Such organisms are useful for the development of non-human models for the study of disorders involving the corresponding gene(s), and for the development of assay systems for the identification of molecules that interact with the protein products) of the corresponding gene(s).
WO 99/46287 PC'T/US99/05243 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 TopPredII 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 minimizing sequence gaps. Also included in the present invention are proteins and protein fragments that contain a segment preferably comprising 8 or more (more preferably 20 or more, most preferably 30 or more) contiguous amino acids that 2 0 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 2 5 (Altschul andzGish; 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 3 0 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 interpreted 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.
Species homologues of the disclosed polynucleotides and proteins are also 2 0 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 (W ore preferably; at least 75% identity; most prefera131y at'least 90% identity) with 2 5 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 minimizing sequence gaps.
Species 3 0 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 murinus, Mus musculus, Rattus norvegicus, Cricetulus griseus, Felis catus, Mustela vison, Canis familiaris, Oryctolagus cuniculus, Bos taurus, Ovis aries, Sus scrofa, and Eguus 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 et al., 1993, Nature Genetics 3:103-112; Johansson et al.,1995, Genomics 25: 682-690; Lyons et al.,1997, Nature Genetics 15: 47-56; O'Brien et al.,1997, Trends in Genetics 13(10): 393-399;
Carver and Stubbs, 1997, Genome Research 7:1123-1137; all of which are incorporated by reference herein).
The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurnng alternative forms of the isolated polynucleotides which also encode 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 2 0 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.
2 5 ~ 'Fhe present irivent'ron also includes polynucleotides that hybridize under reduced stringency conditions, more preferably stringent conditions, and most preferably highly stringent conditions, to polynucleotides described herein. Examples of stringency conditions are shown in the table below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as 3 0 stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.
StringencyPolynucleotideHybridHybridization TemperatureWash ConditionHybrid Lengthand Temperature (bp); Buffer' and Buffer' A DNA:DNA 2 50 65C; lxSSC -or- 65C; 0.3xSSC
42C; lxSSC, 50% formamide B DNA:DNA <50 TH*; lxSSC TB*; lxSSC
C DNA:RNA 2 50 67C; lxSSC -or- 67C; 0.3xSSC
45C; lxSSC, 50% fonnamide D DNA:RNA <50 Tp*; lxSSC TD*; lxSSC
E RNA:RNA z 50 70C; lxSSC -or- 70C; 0.3xSSC
50C; lxSSC, 50% formamide F RNA:1Z1VA <50 TF*; lxSSC TF*; lxSSC
G DNA:DNA z 50 65C; 4xSSC -or- 65C; lxSSC
42C; 4xSSC, 50% formamide H DNA:DNA <50 TH*; 4xSSC T"*; 4xSSC
I DNA:RNA 2 50 67C; 4xSSC -or- 67C; lxSSC
45C; 4xSSC, 50% formamide j DNA:RNA <50 T~*; 4xSSC T~*; 4xSSC
K ItNA:RNA z 50 70C; 4xSSC -or- 67C; lxSSC
50C; 4xSSC, 50% formamide L RNA:RNA <50 T~*; 2xSSC T~*; 2xSSC
M DNA:DNA 2 50 50C; 4xSSC -or- 50C; 2xSSC
40C; 6xSSC, 50% formamide N DNA:DNA <50 TN*; 6xSSC TN*; 6xSSC
O DNA:RNA z 50 55C; 4xSSC -or- 55 C; 2xSSC
42C; 6xSSC, 50% formamide P DNA:RNA <50 TP*; 6xSSC TP*; 6xSSC
Q RNA:RNA Z 50 60C; 4xSSC -or- 60C; 2xSSC
45C; 6xSSC, 50% formamide ... 2 O . R ~A:RNA G50 TR*~ .. .. TR*: 4xSSC
t: The hybrid length is that anticipated for the hybridized regions) of the hybridizing polynudeotides. When hybridizing a polynucleotide to a target polynucleotide of unknown sequ~ce, the hybrid length is assumed to be that of the hybridizing polynucleotide. When polynucleotides of known sequence are hybridized, the 2 5 hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity.
': SSPE (lxSSPE is 0.15M NaCI, lOmM NaHZPO" and 1.25mM EDTA, pH 7.4) can be substituted for SSC
(lxSSC is 0.15M NaCI and lSmM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete.
3 0 "TB - TR: The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10°C less than the melting temperature (Tm) of the hybrid, where T," is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(°C) = 2(# of A + T bases) + 4(# of G +
C bases). For hybrids between 18 and 49 base pairs in length, T,"(°C} =
81.5 + 16.6(log~o[Na']) + 0.41 (%G+C) (600/N), where N is the number of bases in the hybrid, and [Na'] is the concentration of sodium ions in the 3 5 hybridization buffer ([Na'] for lxSSC = 0.165 M).
Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11, and Current Protocols in Molecular Biology,1995, F.M.
Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference.
Preferably, each such hybridizing polynucleotide has a length that is at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of the polynucleotide of the present invention to which it hybridizes, and has at least 60%
sequence identity (more preferably, at least 75% identity; most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to which it hybridizes, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps.
The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res. ~, 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 ~, 537-566 (1990). As defined herein "operably 2 0 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 Iigated polynucleotide/expression control sequence.
A number of types of cells may act as suitable host cells for expression of the 2 5 protein. Mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Co1o205 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.
3 0 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, KIuyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.
The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBacO 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 2 0 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 Sepharose(9; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.
2 5 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 (TItX). Kits for expression and purification of such fusion proteins are commercially available from New England BioLabs (Beverly, MA), Pharmacia (Piscataway, NJ) and 3 0 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 (1ZP-HPLC) steps employing hydrophobic 1ZP-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 Gammon 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.
2 0 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, 2 5 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, 3 0 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 polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified below. Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or by administration or use of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA).
Research Uses and Utilities The polynucleotides provided by the present invention can be used by the research community for various purposes. The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on Southern gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare 2 0 with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to "subtract-out"
known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a "gene chip" or other support, including for 2 5 examination of expression patterns; to raise anti-protein antibodies using DNA
immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response. Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, those 3 0 described in Gyuris et al.,1993, CeII 75: 791-803 and in Rossi et al.,1997, Proc. Natl. Acad.
Sci. LISA 94: 8405-8410, all of which are incorporated by reference herein) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.
The proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to 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 (imitation "Molecular Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E.F. Fritsch and T. Maruatis eds., 1989, and "Methods in Enzymology: Guide to 2 0 Molecular Cloning Techniques", Academic Press, Berger, S.L. and A.R.
ICimmel eds.,1987.
Nutritional Uses Polynucleotides and proteins of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein 2 5 or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate. In such cases the protein or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules. In the case of microorganisms, the protein or polynucleotide of the invention 3 0 can be added to the medium in or on which the microorganism is cultured.
~rtokine and Cell Proliferation/Differentiation Activi~
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/lI, BaF3, MC9/G, M+ (preB M+), 2E8, RBS, DA1,123, 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 Wfley-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 2 0 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 y, 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 2 5 cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Current Protocols in Immunology. j.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983;
3 0 Measurement of mouse and human interleukin 6 - Nordan, R. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.b.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. Coligan 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. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto.1991.
Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) 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; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans);
Weinberger et al., Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J.
Immun.
11:405-411,1981; Takai et al., J. Immunol. 137:3494-3500,1986; Takai et al., J. Immunol.
140:508-512, 1988.
Immune Stimulatin~or SupvrT ssing Activi~,r A protein of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein. A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., 2 0 in regulating (up or down) growth and proliferation of T and/or B
lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations.
These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders. More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a 2 5 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 generally may be desirable, i.e., in the treatment of cancer.
3 0 Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.
WO 99/462$7 PCT/US99/05243 Such a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein of the present invention.
Using the proteins of the invention it may also be possible to regulate immune responses in a number of ways. Down regulation may be in the form of inhibiting or blocking an immune response akeady in progress or may involve preventing the induction of an immune response. The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both.
Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent.
Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.
Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as , for example, B7)), e.g., preventing 2 0 high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD). For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation. Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys 2 5 the transplant. The administration of a molecule which inhibits or blocks interaction of a B7 lymphocyte antigen with its natural ligand(s) on immune cells (such as a soluble, monomeric form of a peptide having B7-2 activity alone or in conjunction with a monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7-1, B7-3) or blocking antibody), prior to transplantation can lead to the binding of the 3 0 molecule to the natural ligand(s) on the immune cells without transmitting the corresponding costimulatory signal. Blocking B lymphocyte antigen function in this matter prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.
The efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl. Acad.
Sci USA, 89:11102-11105 (1992). In addition, marine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of blocking B lymphocyte antigen function in vivo on the development of that disease.
Blocking antigen function may also be therapeutically useful for treating autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases.
Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms.
2 0 Administration of reagents which block costimulation of T cells by disrupting receptor:ligand interactions of B lymphocyte antigens can be used to inhibit T
cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from 5 the disease. The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include marine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/Ipr mice or NZB hybrid mice, marine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB
rats, and 3 0 marine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York,1989, pp. 840-856).
Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy.
Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B
lymphocyte antigens systemically.
Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient. Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient. The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.
In another application; up regulation or enhancement of antigen function (preferably B lymphocyte antigen function) may be useful in the induction of tumor immunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleic acid encoding at least one peptide of the present 2 0 invention can be administered to a subject to overcome tumor-specific tolerance in the subject. If desired, the tumor cell can be transfected to express a combination of peptides.
For example, tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-1-like activity and/or B7-3-like activity. The 2 5 transfected tumor cells are returned to the patient to result in expression of the peptides on the surface of the transfected cell. Alternatively, gene therapy techniques can be used to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a B
lymphocyte antigens) on the surface of the tumor cell provides the necessary 3 0 costimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells. In addition, tumor cells which lack MHC class I or MHC class II
molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC
class II
molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I a chain protein and biz microglobulin protein or an MHC class II a chain protein and an MHC class II
~i chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T
cell mediated immune response against the transfected tumor cell. Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity. Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E.
Coligan, A.M.
Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, immunologic studies in Humans); Herrmann et al., Proc. Natl.
Acad. Sci.
USA 78:2488-2492,1981; Herrmann et al., J. Immunol.128:1968-1974,1982; Handa et al., 2 0 J. immunol.135:1564-1572,1985; Takai et al., J. Immunol.137:3494-3500,1986; Takai et al., J. Immunol.140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.
Immunol.
135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet al., J.
Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988;
Bertagnolli et al., 2 5 Cellular 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 will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol.144:3028-3033,1990; and Assays for B cell function: In vitro 3 0 antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in Immunology.
].E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto.1994.
Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Th1 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 al., J. Immunol.140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783,1992.
Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without 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 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 will 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 al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991;
Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993;
2 0 Gorczyca et al., International Journal of Oncology 1:639-648,1992.
Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778,1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551,1991.
Hematopoiesis Re~ring_,Activ~
A protein of the present invention may be useful in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell deficiencies.
Even marginal biological activity in support of colony forming cells or of factor-dependent cell 3 0 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 with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF
activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytop~ia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stern cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous}) as normal cells or genetically manipulated for gene therapy.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.
Assays for embryonic stem cell differentiation (which will identify, among others, 2 0 proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151,1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915,1993.
Assays for stem cell survival and differentiation (which will identify, among 2 5 others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992;
Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, LK. and 3 0 Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et al.
eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, NY.1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. l-21, Wiley-Liss, l:nc.., New York, NY. 1994; Long fierm bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, NY.1994; Long term culture initiating cell assay, Sutherland, H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp.139-162, Wiley-Liss, Inc., New York, NY.1994.
Tissue Growth ActivitX
A protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of burns, incisions and ulcers.
A protein of the present invention, which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.
Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craruofacial defects, and also is useful in cosmetic plastic surgery.
A protein of this invention may also be used in the treatment of periodontal 2 0 disease, and in other tooth repair processes. Such agents may provide an environment to attract bone-forming cells, stimulate growth of bone-forming cells or induce differentiation of progenitors of bone-forming cells. A protein of the invention may also be useful in the treatment of osteoporosis or osteoarthritis, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue 2 5 destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes.
Another category of tissue regeneration activity that may be attributable to the protein of the present invention is tendon/ligament formation. A protein of the present invention, which induces tendon/ligament-like tissue or other tissue formation in 3 0 circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals. Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue. De novo tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments. The compositions of the present invention may provide an environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair. The compositions of the invention may also be useful in the treatment of tendirutis, carpal tunnel syndrome and other tendon or ligament defects. The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.
The protein of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a protein may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer s, 2 0 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 2 5 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 3 0 generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring to allow normal tissue to regenerate. A protein of the invention may also exhibit angiogenic activity.
A protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. W095/16035 (bone, cartilage, tendon);
International Patent Publication No. W095/05846 (nerve, neuronal);
International Patent Publication 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 modified by Eaglstein and Mertz, J.
Invest.
Dermatol 71:382-84 (1978).
Activin/Inh~in ActivitX
A protein of the present invention may also exhibit activin- or inhibin-related activities. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a protein of the present 2 5 invention, alone or in heterodimers with a member of the inhibin a family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals. Alternatively, the protein of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin-3 0 ji group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary.
See, for example, United States Patent 4,798,885. A protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for activin/inhibin activity include, without limitation, those described in:
Vale et al., Endocrinology 91:562-572,1972; Ling et al., 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 chemokineHc activity (e.g., act as a chemokine) for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells.
Chemotactic and chemokinetic proteins can be used to mobilize or attract a desired cell population to a desired site of action. Chemotactic or chemokinetic proteins provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.
A profiein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell 2 0 population. Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
The activity of a protein of the invention may, among other means, be measured 2 5 by the following methods:
Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for movement and adhesion 3 0 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 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376,1995; Lind et al.
APMIS 103:140-146,1995; Muller et al Eur. J. Immunol. 25:1744-1748; Gruber et al. J. of Immunol. 152:5860-5867,1994; Johnston et al. J. of Immunol.153: 1762-1768,1994.
Hemostatic and Thrombolytic ActivitX
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 hemophiliac) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes. A protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin. Pharmacol. 26:131-140,1986; Burdick et al., Thrombosis Res. 45:413-419,1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474,1988.
~tor/Ligand ActivitX
A protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agorusts of receptor/ligand 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, 2 5 receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selectins, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses). Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant 3 0 receptor/ligand interaction. A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.
The activity of a protein of the invention may, among other means, be measured by the following methods:
WO 99/46287 PC'f/US99/05243 Suitable assays for receptor-ligand activity include without limitation those described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M.
ICruisbeek, D.H.
Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987;
Bierer et al., J. Exp. Med.168:1145-1156, 1988; Rosenstein et al., J. Exp.
Med.169:149-160 1989; Stoltenborg et al., J. Immunol. Methods 175:59-68,1994; Stitt et al., Cell 80:661-670, 1995.
Anti-Inflammator;~ ActivitX
Proteins of the present invention may also exhibit anti-inflammatory activity.
The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response. Proteins exhibiting such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation inflammation associated with infection (such as septic shock, sepsis or systemic 2 0 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 Su~pressor Activity Cadherins are calcium-dependent adhesion molecules that appear to play major roles during development, particularly in defining specific cell types. Loss or alteration of normal cadherin expression can lead to changes in cell adhesion properties linked to 3 0 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 superfamily includes well over forty members, each with a distinct pattern of expression. All members of the superfamily have in common conserved WO 99/46287 PC"T/US99/05243 extracellular repeats (cadherin domains), but structural differences are found in other parts of the molecule. The cadherin domains bind calcium to form their tertiary structure and thus calcium is required to mediate their adhesion. Only a few amino acids in the first cadherin domain provide the basis for homophilic adhesion; modification of this recognition site can change the specificity of a cadherin so that instead of recognizing only itself, the mutant molecule can now also bind to a different cadherin. In addition, some cadherins engage in heterophilic adhesion with other cadherins.
E-cadherin, one member of the cadherin superfamily, is expressed in epithelial cell types. Pathologically, if E-cadherin expression is lost in a tumor, the malignant cells become invasive and the cancer metastasizes. Transfection of cancer cell lines with polynucleotides expressing E-cadherin has reversed cancer-associated changes by returning altered cell shapes to normal, restoring cells' adhesiveness to each other and to their substrate, decreasing the cell growth rate, and drastically reducing anchorage-independent cell growth. Thus, reintroducing E-cadherin expression reverts carcinomas to a less advanced stage. It is likely that other cadherins have the same invasion suppressor role in carcinomas derived from other tissue types. Therefore, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be used to treat cancer. Introducing such proteins or polynucleotides into cancer cells can reduce or eliminate the cancerous changes observed 2 0 in these cells by providing normal cadherin expression.
Cancer cells have also been shown to express cadherins of a different tissue type than their origin, thus allowing these cells to invade and metastasize in a different tissue in the body. Proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be substituted in these cells for the 2 5 inappropriately expressed cadherins, restoring normal cell adhesive properties and reducing or eliminating the tendency of the cells to metastasize.
Additionally, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can used to generate antibodies recognizing and binding to cadherins. Such antibodies can be used to block 3 0 the adhesion of inappropriately expressed tumor-cell cadherins, preventing the cells from foaming a tumor elsewhere. Such an anti-cadherin antibody can also be used as a marker for the grade, pathological type, and prognosis of a cancer, i.e. the more progressed the cancer, the less cadherin expression there will be, and this decrease in cadherin expression can be detected by the use of a cadherin-binding antibody.
Fragments of proteins of the present invention with cadherin activity, preferably a polypeptide comprising a decapeptide of the cadherin recognition site, and poly-nucleotides of the present invention encoding such protein fragments, can also be used to block cadherin function by binding to cadherins and preventing them from binding in ways that produce undesirable effects. Additionally, fragments of proteins of the present invention with cadherin activity, preferably truncated soluble cadherin fragments which have been found to be stable in the circulation of cancer patients, and polynucleotides encoding such protein fragments, can be used to disturb proper cell-cell adhesion.
Assays for cadherin adhesive and invasive suppressor activity include, without limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-18817, 1995;
Miyaki et al. Oncogene 11: 2547-2552,1995; Ozawa et al. Cell 63:1033-1038,1990.
Tumor Inhibition Activity In addition to the activities described above for immunological treatment or prevention of tumors, a protein of the invention may exhibit other anti-tumor activities.
A protein may inhibit tumor growth directly or indirectly (such as, for example, via antibody-dependent cell-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 2 0 inhibiting angiogenesis), by causing production of other factors, agents or cell types which inhibit tumor growth, or by suppressing, eliminating or inhibiting factors, agents or cell types which promote tumor growth.
Other Activities 2 5 A protein of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair rnlor, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ 3 0 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 cells 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); immunoglobulin-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 pharmaceutically acceptable Garner.
Such a composition may also contain (in addition to protein and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the 2 0 carrier will depend on the route of administration. The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL~-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. The pharmaceutical composition may further contain other 2 5 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, 3 0 thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor,'or anti-inflammatory agent.
A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins. As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.
The pharmaceutical composition of the invention may be in the form of a complex of the proteins) of present invention along with protein or peptide antigens.
The protein and /or peptide antigen will deliver a stimulatory signal to both B and T
lymphocytes. B
lymphocytes will respond to antigen through their surface immunoglobulin receptor. T
lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins. MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigens) to T lymphocytes. The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells. Alternatively antibodies able to bind surface immunolgobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention.
The pharmaceutical composition of the invention rnay be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers 2 0 in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the Level of skill in the art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent No.
4,501,728; U.S.
Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are incorporated herein 2 5 by reference.
As used herein, the term "therapeutically effective amount" means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, 3 0 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, serially or simultaneously.
In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein of the present invention is administered to a mammal having a condition to be treated. Protein of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, protein of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.
Administration of protein of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be earned 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 therapeutically effective amount of protein of the present invention is 2 0 administered orally, protein of the present invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95%
protein of the present invention, and preferably from about 25 to 90% protein of the present invention.
2 5 When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid 3 0 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 therapeutically effective amount of protein of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill 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 stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
The amount of protein of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone.
Ultimately, the attending physician will decide the amount of protein of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein of the present invention and observe the patient's response. Larger doses of protein of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 ug to about 100 2 0 mg (preferably about 0.lng to about 10 mg, more preferably about 0.1 pg to about 1 mg) of protein of the present invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient.
It is 2 5 contemplated that the duration of each application of the protein of the present invention will be in the range of 12 to 24 hours of continuous intravenous administration.
Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.
Protein of the invention may also be used to immunize animals to obtain 3 0 polyclonal and monoclonal antibodies which specifically react with the protein. Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen. The peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH). Methods for synthesizing such peptides are known in the art, for example, as in WO 99/46287 PC"TNS99/OSZ43 R.P. Merrifield, J. Amer.Chem.Soc. $5, 2149-2154 (1963); J.L. Krstenansky, et al., FEBS Lett.
211, 10 (198. Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein. Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved. In the case of cancerous cells or leukemic cells, neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein.
For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device.
When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage. Topical administration may be suitable for wound healing and tissue repair. Therapeutically useful agents other than a protein of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the 2 0 methods of the invention. Preferably for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body.
Such matrices may be formed of materials presently in use for other implanted medical 2 5 applications.
The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions will define the appropriate formulation.
Potential matrices for the compositions may be biodegradable and chemically defined calcium 3 0 sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix rnmponents. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. The bioceramics may be altered in composition, such as in calcium aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.
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 same applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.
A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate, polyethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and polyvinyl 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 2 0 provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.
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 2 5 question. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-a and TGR(i), 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 3 0 humans, are desired patients for such treatment with proteins of the present invention.
The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insulin 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 cell or organism (including, without limitation, in the form of viral vectors or naked DNA).
Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells.
Treated cells can then be introduced in vivo for therapeutic purposes.
Patent and literature references cited herein are incorporated by reference as if fully set forth.
SEQUENCE LISTING
<110> Jacobs, Kenneth McCoy, John M.
LaVallie, Edward R.
Collins-Racie, Lisa A.
Evans, Cheryl Merberg, David Treacy, Maurice Agostino, Michael J.
Steininger II, Robert J.
Genetics Institute, Inc.
<120> SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
<130> GI 6065A
<140> v E~ssMaiPmal~, Iabelnumber.~Ve~Sa yoa~s <141> ~ateofD~OSit arr.~ I(~ l 99 ' hereby certfly that this paper or fee is being <160>33 deposited with the Un~ed States Postal Ssrvioe .Express Mail Post Ofl'ke to Addrossee' service <170>Patentin Ver. 2 . 0 :~nde: 37 CFR 1 10 on the date indicabad above afld 1s ~dUassed t0 the A5EI8t8nt COn 1111111i0ner <210>1 ~r P tents, Washin n, .C. 20231 <211>2393 <212>DNA
<213>Homo sapiens <400> 1 gggctgggcg gcggggacag cggggacggc acggcgcgcg cagcttctaa gtgccagatg 60 atggaggagc gtgccaacct gatgcacatg atgaaactca gcatcaaggt gttgctccag 120 tcggctctga gcctgggccg cagcctggat gcggaccatg cccccttgca gcagttcttt 180 gtagtgatgg agcactgcct caaacatggg ctgaaagtta agaagagttt tattggccaa 240 aataaatcat tctttggtcc tttggagctg gtggagaaac tttgtccaga agcatcagat 300 atagcgacta gtgtcagaaa tcttccagaa ttaaagacag ctgtgggaag argccgagcg 360 tggctttatc ttgcactcat gcasaagaaa ctggcagatt atctgaaagt gcttatagac 420 aataaacatc tcttaagcga gttctatgag cctgaggctt taatgatgga ggaagaaggg 480 atggtgattg ttggtctgct ggtgggactc aatgttctcg atgccaatct ctgcttgaaa 540 ggagaagact tggattctca ggttggagta atagattttt ccctctacct taaggatgtg 600 caggatcttg atggtggcaa ggagcatgaa agaattactg atgtccttga tcaaaaaaat 660 tatgtggaag aacttaaccg gcacttgagc tgcacagttg gggatcttca aaccaagata 720 gatggcttgg aaaagactaa ctcaaagctt caagaagagc tttcagctgc aacagaccga 780 atttgctcac ttcaagaaga acagcagcag ttaagagaac aaaatgaatt aattcgagaa 840 agaagtgaaa~ aga~gtgtaga gataacaaaa caggatacca aagttgagct ggagacttac 900 aagcaaactc ggcaaggtct ggstgaaatg tacagtgatg tgtggaagca gctaaaagag 960 gagaagaaag tccggttgga actggaaaaa gaactggagt tacaaattgg aatgaaaacc 1020 gaaatggaaa ttgcaatgaa gttactggaa aaggacaccc acgagaagca ggacacacta 1080 gttgccctcc gccagcagct ggaagaagtc aaagcgatta atttacagat gtttcacsaa 1140 gctcagaatg cagagagcag tttgcagcag aagaatgaag ccatcacatc ctttgaagga 1200 aaaaccaacc aagttatgtc cagcatgaaa caaatggaag aaaggttgca gcactcggag 1260 cgggcgaggc agggggctga ggagcggagc cacaagctgc agcaggagct gggcgggagg 1320 atcggcgccc tgcagctgca gctctcccag ctgcacgagc aatgctcaag cctggagaaa 1380 gaattgaaat cagaaaaaga gcaaagacag gctcttcagc gcgaattaca gcacgagaaa 1440 gacacttcct ctctactcag gatggagctg caacaagtgg aaggactgaa aaaggagttg 1500 cgggagcttc aggacgagea ggcagagctg cagaagatct gcgaggagca ggaacaagcc 1560 ctccaggaaa tgggcctgca cctcagccag tccaagctga agatggaaga tataaaagaa 1620 gtgaaccagg cactgaaggg ccacgcctgg ctgaaagatg acgaagcgac acactgtagg 1680 cagtgtgaga aggagttctc catttcccgg agaaagcacc actgccggaa ctgtggccac 1740 atcttctgca acacctgctc cagcaacgag ctggccctgc cctcctaccc caagccggtg 1800 cgagtgtgcg acagctgcca caccctgctc ctgcagcgct gctcctccac ggcctcctga 1860 acgtccgtcc tcaggagcac agcctcacgg acagtgccaa accctgtggg tctccagggg 1920 cttgggaaat gtgttctttc ccaagagtat caaaggaaag aatcaaattt cttgcccggt 1980 cactggcact ccagaagaca gcgtgccgga accggcagct ctcacctttc tgtgacttgt 2040 tcggaattaa ctcctctgga tggaaacttc catcttactt ggttacatca cggctctggt 2100 tcagatacaa cttcatgatt ttgctactat catttttcac ttttcaaaga atttaaccta 2160 ttttacagca gttcagttyt gctagkgagt agttttcctc tcctaccttc cttctaaaaa 2220 cctgattcat gcacagcgtt tgacacacat ggagtctgcc agtgtgcctt ctctgcttca 2280 gacaagagat ytgccatttc atgcccttgt gactacctat cattggccct gcaataaaat 2340 catttatttt tcaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 2393 <210> 2 <211> 600 <212> PRT
<213> Homo Sapiens <220>
<221> UNSURE
<222> (99) <400> 2 Met Met Glu Glu Arg Ala Asn Leu Met His Met Met Lys Leu Ser Ile Lys Val Leu Leu Gln Ser Ala Leu Ser Leu Gly Arg Ser Leu Asp Ala Asp His Ala Pro Leu Gln Gln Phe Phe Val Val Met Glu His Cys Leu Lys His Gly Leu Lys Val Lys Lys Ser Phe Ile Gly Gln Asn Lys Ser Phe Phe Gly Pro Leu Glu Leu Val Glu Lys Leu Cys Pro Glu Ala Ser Asp Ile Ala Thr Ser Val Arg Asn Leu Pro Glu Leu Lys Thr Ala Val Gly Arg Xaa Arg Ala Trp Leu Tyr Leu Ala Leu Met Gln Lys Lys Leu Ala Asp Tyr Leu Lys Val Leu Ile Asp Asn Lys His Leu Leu Ser Glu ,, ,,.:._ , ~ ~...,,,,.;
Phe Tyr Glu Pro Glu Ala Leu Met Met Glu Glu Glu Gly Met Val Ile Val Gly Leu Leu Val Gly Leu Asn Val Leu Asp Ala Asn Leu Cys Leu Lys Gly Glu Asp Leu Asp Ser Gln Val Gly Val Ile Asp Phe Ser Leu Tyr Leu Lys Asp Val Gln Asp Leu Asp Gly Gly Lys Glu His Glu Arg Ile Thr Asp Val Leu Asp Gln Lys Asn Tyr Val Glu Glu Leu Asn Arg His Leu Ser Cys Thr Val Gly Asp Leu Gln Thr Lys Ile Asp Gly Leu Glu Lys Thr Asn Ser Lys Leu Gln Glu Glu Leu Ser Ala Ala Thr Asp Arg Ile Cys Ser Leu Gln Glu Glu Gln Gln Gln Leu Arg Glu Gln Asn Glu Leu Ile Arg Glu Arg Ser Glu Lys Ser Val Glu Ile Thr Lys Gln Asp Thr Lys Val Glu Leu Glu Thr Tyr Lys Gln Thr Arg Gln Gly Leu Asp Glu Met Tyr Ser Asp Val Trp Lys Gln Leu Lys Glu Glu Lys Lys Val Arg Leu Glu Leu Glu Lys Glu Leu Glu Leu Gln Ile Gly Met Lys Thr Glu Met Glu Ile Ala Met Lys Leu Leu Glu Lys Asp Thr His Glu Lys Gln Asp Thr Leu Val Ala Leu Arg Gln Gln Leu Glu Glu Val Lys Ala Ile Asn Leu Gln Met Phe His Lys Ala Gln Asn Ala Glu Ser Ser Leu Gln Gln Lys Asn Glu Ala Ile Thr Ser Phe Glu Gly Lys Thr Asn Gln Val Met Ser Ser Met Lys Gln Met Glu Glu Arg Leu Gln His Ser Glu Arg Ala Arg Gln Gly Ala Glu Glu Arg Ser His Lys Leu Gln Gln Glu Leu Gly Gly Arg Ile Gly Ala Leu Gln Leu Gln Leu Ser Gln Leu His Glu Gln Cys Ser Ser Leu Glu Lys Glu Leu Lys Ser Glu Lys Glu Gln Arg Gln Ala Leu Gln Arg Glu Leu Gln His Glu Lys Asp Thr Ser Ser Leu Leu Arg Met Glu Leu Gln Gln Val Glu Gly Leu Lys Lys Glu Leu Arg Glu Leu Gln Asp Glu Lys Ala Glu Leu Gln Lys Ile Cys Glu Glu Gln Glu Gln Ala Leu Gln Glu Met Gly Leu His Leu Ser Gln Ser Lys Leu Lys Met Glu Asp Ile Lys Glu Val Asn Gln Ala Leu Lys Gly His Ala Trp Leu Lys Asp Asp Glu Ala Thr His Cys Arg Gln Cys Glu Lys Glu Phe Ser Ile Ser Arg Arg Lys His His Cys Arg Asn Cys Gly His Ile Phe Cys Asn Thr Cys Ser Ser Asn Glu Leu Ala Leu Pro Ser Tyr Pro Lys Pro Val Arg Val Cys Asp Ser Cys His Thr Leu Leu Leu Gln Arg Cys Ser Ser Thr Ala Ser <210> 3 <211> 1284 <212> DNA
<2I3> Homo Sapiens <400> 3 ccctcatcca gccagcatca gggcttccct gggcggccga tgtggaatca gtttcagcct 60 cagtgaccca cggcagggaa acgtggacag tgctgcccga ctcataaggg cagagcccct 120 gccctagcct gaaacgggtc caggcctccc acggctctta gaatccaaca tgaacccctc 180 cctggccccc agaggctccg catggcccag ccgtctccct cccggcctct tccccctccc 240 gcttcactcc cactccagcc tccaagctgt tcctgttaca acaaatggct cctgcctcgg 300 cgcctttgca ctggtagccc tgctgctaag gacgctcctg ttccccagct ctcccccttg 360 gccggcgctc ttcgcaccta ggagcgtcac ttcttcaaga gccacccacc cgccctgtcc 420 aaggttcccc tgttggcctc acatcccccg cccagggcag caccgggacc tatcttgktc 480 ctggaggaga aggagaagcg gcgcctcgac cagctggaac gtaagaagga gacgcagcgc 540 ctactggagg aggaggactc caagctcaag ggcggcaagg cgccgcgggt ggccacgtcc 600 agcaaggtca cccgggccca gatcgaggac acgctgcgcc gagaccatca gctcagggag 660 gccccggaca cagccgagaa agccaagagc catctggagg tgccgctgga ggagaacgtg 720 aaccgccgcg tgctggagga gggcagcgtg gaggcgcgca ccatcgagga cgccattgca 780 gtgctcagcg tggcggagga ggcggccgac cggcacccag aaagacgcat gcgggcagcc 840 ttcacagcct ttgaggaagc ccagctgccg cggctcaaac aagagaaccc caacatgcgg 900 ctgtcgcagc tgaaacagct gctcaagaag gagtggctcc gctctcctga caaccccatg 960 aaccagcggg ccgtgccctt caatgccccc aagtgagccc agaacttggg gagccagttc 1020 acccacgggt ggtccaggtc acgactctgc acgcccttag gccaggtcag ctgcgagggt 1080 cacagagcgt tccgggggcc aaggcgccgg gccccggggc catgctctta tcaccagcca 1140 cccgtcctcc cgccagaggg tccctgcccc gagtgacacc ccatcccctc ccatcccccg 1200 gcgcgtgtgt gtagagcctc agggctgagt gcccaataaa ggtggcggca agccaaaaaa 1260 aaaaaaaaaa aaaaaaaaaa aaaa 1284 <210> 4 <211> 183 <212> PRT
<213> Homo Sapiens <220>
<221> UNSURE
<222> (103) <400> 4 Met Asn Pro Ser Leu Ala Pro Arg Gly Ser Ala Trp Pro Ser Arg Leu Pro Pro Gly Leu Phe Pro Leu Pro Leu His Ser His Ser Ser Leu Gln Ala Val Pro Val Thr Thr Asn Gly Ser Cys Leu Gly Ala Phe Ala Leu Val Ala Leu Leu Leu Arg Thr Leu Leu Phe Pro Ser Ser Pro Pro Trp Pro Ala Leu Phe Ala Pro Arg Ser Val Thr Ser Ser Arg Ala Thr His Pro Pro Cys Pro Arg Phe Pro Cys Trp Pro His Ile Pro Arg Pro Gly Gln His Arg Asp Leu Ser Xaa Ser Trp Arg Arg Arg Arg Ser Gly Ala Ser Thr Ser Trp Asn Val Arg Arg Arg Arg Ser Ala Tyr Trp Arg Arg Arg Thr Pro Ser Ser Arg Ala Ala Arg Arg Arg Gly Trp Pro Arg Pro Ala Arg Ser Pro Gly Pro Arg Ser Arg Thr Arg Cys Ala Glu Thr Ile Ser Ser Gly Arg Pro Arg Thr Gln Pro Arg Lys Pro Arg Ala Ile Trp Arg Cys Arg Trp Arg Arg Thr <210> 5 <211> 973 <212> DNA
<213> Homo sapiens <400> 5 gccaaaccaa actgcacgac atcgacggcg tacctcacct catcctcatc gcctcccgag 60 acatcgcggc tggggaggag ctcctgtatg actatgggga ccgcagcaag gcttccattg 120 aagcccaccc gtggctgaag cattaaccgg tgggccccgt gccctccccg ccccactttc 180 ccttcttcaa aggacaaagt gccctcaaag ggaattgaat ttttttttta cacacttaat 240 cttagcggat tacttcagat gtttttaaaa agtatattaa gatgcctttt cactgtagta 300 tttaaatatc tgttacaggt ttccaaggtg gacttgaaca gatggcctta tattaccaaa 360 acttttatat tctagtEgtt tttgtacttt ttttgcatac aagccgaacg tttgtgcttc 420 ccgtgcatgc agtcaaagac tcagcacagg ttttagagga aatagtcaaa catgaactag 480 gaagccaggt gagtctcctt tctccagtgg aagagccggg accttccccc tgcacccccg 540 acatccaggg acggggtgtg aggaagacgc tgcctcccaa tggcctggac gggatgtttc 600 caagctcttg ttcccctaac gtctcaacag gcgctcactg aagtgtatga atatttttta 660 aaaaggtttt tgcagtaagc tagtcttccc ctctgctttc tcgaaagctt actgagccct 720 gggccccaag cacgggccgg gcatagattt cctcttccac aagctgccgc ttttctgggc 780 accttgaagc atcagggcgt gaaatcaaac tagatgtggg cagggagagt gttgcttacc 840 tgccctgctg gggcagggtt tcctgaaact gggttaattc tttatagaaa tgtgaacact 900 gaatttattt taaaaaataa taataaaaat ttaaaaaaaa aaaaaaaaaa aaaaaaaaaa 960 aaaaaaaaaa aaa 973 <210> 6 <211> 99 <212> PRT
<213> Homo sapiens <400> 6 Met Ala Leu Tyr Tyr Gln Asn Phe Tyr Ile Leu Val Val Phe Val Leu Phe Leu His Thr Ser Arg Thr Phe Val Leu Pro Val His Ala Val Lys Asp Ser Ala Gln Val Leu Glu Glu Ile Val Lys His Glu Leu Gly Ser Gln Val Ser Leu Leu Ser Pro Val Glu Glu Pro Gly Pro Ser Pro Cys Thr Pro Asp Ile Gln Gly Arg Gly Val Arg Lys Thr Leu Pro Pro Asn Gly Leu Asp Gly Met Phe Pro Ser Ser Cys Ser Pro Asn Val Ser Thr Gly Ala His <210> 7 <211> 1121 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> (1035) <400> 7 cctctccacg cgcacgaact cagccaacga tttctgatag atttttggga gtttgaccag 60 agatgcaagg ggtgaaggag cgcttcctac cgttagggaa ctctggggac agagcgcccc 120 ggccgcctga tggccgaggc agggtgcgac ccaggaccca ggacggcgtc gggaaccata 180 ccatggcccg gatccccaag accctaaagt tcgtcgtcgt catcgtcgcg gtcctgctgc 240 cagtcctagc ttactctgcc accactgccc ggcaggagga agttccccag cagacagtgg 300 ccccacagca acagaggcac agcttcaagg gggaggagtg tccagcagga tctcatagat 360 cagaacatac tggagcctgt aacccatgca cagagggtgt ggattacacc aacgcttcca 420 acaatgaacc ttcttgcttc ccatgtacag tttgtaaatc agatcaaaaa cataaaagtt 480 cctgcaccat gaccagagac acagtgtgtc agtgtaaaga aggcaccttc cggaatgaas 540 actccccaga gatgtgccgg aagtgtagca ggtgccctag tggggaagtc caagtcagta 600 attgtacgtc ctgggatgat atccagtgtg ttgaagaatt tggtgccaat gccactgtgg 660 aaaccccagc tgctgaagag acaatgaaca ccagcccggg gactcctgcc ccagctgctg 720 aagagacaat gaacaccagc ccagggactc ctgccccagc tgctgaagag acaatgacca 780 ccagcccggg gactcctgcc ccagctgctg aagagacaat gaccaccagc ccggggactc 840 ctgccccagc tgctgaagag acaatgacca ccagcccggg gactcctgcc tcttctcatt 900 acctctcatg caccatcgta gggatcatag ttctaattgt gcttctgatt gtgtttgttt 960 gaaagacttc actgtggaag aaattccttc cttacctgaa aggttcaggt aggcgctggc 1020 tgagggcggg gggcnctaga cactctctgc cctgcctccc tctgctgtgt tcccacagac 1080 agaaacgcct gcccctgccc caaaaaaaaa aaaaaaaaaa a 1121 <210> 8 <211> 299 <212> PRT
<213> Homo sapiens <400> 8 Met Gln Gly Val Lys Glu Arg Phe Leu Pro Leu Gly Asn Ser Gly Asp Arg Ala Pro Arg Pro Pro Asp Gly Arg Gly Arg Val Arg Pro Arg Thr Gln Asp Gly Val Gly Asn His Thr Met Ala Arg Ile Pro Lys Thr Leu Lys Phe Val Val Val Ile Val Ala Val Leu Leu Pro Val Leu Ala Tyr Ser Ala Thr Thr Ala Arg Gln Glu Glu Val Pro Gln Gln Thr Val Ala Pro Gln Gln Gln Arg His Ser Phe Lys Gly Glu Glu Cys Pro Ala Gly Ser His Arg Ser Glu His Thr Gly Ala Cys Asn Pro Cys Thr Glu Gly Val Asp Tyr Thr Asn Ala Ser Asn Asn Glu Pro Ser Cys Phe Pro Cys Thr Val Cys Lys Ser Asp Gln Lys His Lys Ser Ser Cys Thr Met Thr Arg Asp Thr Val Cys Gln Cys Lys Glu Gly Thr Phe Arg Asn Glu Asn Ser Pro Glu Met Cys Arg Lys Cys Ser Arg Cys Pro Ser Gly Glu Val Gln Val Ser Asn Cys Thr Ser Trp Asp Asp Ile Gln Cys Val Glu Glu Phe Gly Ala Asn Ala Thr Val Glu Thr Pro Ala Ala Glu Glu Thr Met Asn Thr Ser Pro Gly Thr Pro Ala Pro Ala Ala Glu Glu Thr Met Asn Thr Ser Pro Gly Thr Pro Ala Pro Ala Ala Glu Glu Thr Met Thr Thr Ser Pro Gly Thr Pro Ala Pro Ala Ala Glu Glu Thr Met Thr Thr Ser Pro Gly Thr Pro Ala Pro Ala Ala Glu Glu Thr Met Thr Thr Ser Pro Gly Thr Pro Ala Ser Ser His Tyr Leu Ser Cys Thr Ile Val Gly Ile Ile Val Leu Ile Val Leu Leu Ile Val Phe Val <210> 9 <211> 1005 <212> DNA
<213> Homo Sapiens <400> 9 ggccgcactg gggagtgtgg gctgggccgc agatgtcatg tggcctgtgt tttggaccgt 60 ggttcgtacc tatgctcctt atgtcacatt ccctgttgcc ttcgtggtcg gggctgtggg 120 ttaccacctg gaatggttca tcaggggaaa ggacccccag cccgtggagg aggaaaagag 180 catctcagag cgccgggagg atcgcaagct ggatgagctt ctaggcaagg accacacgca 240 ggtggtgagc cttaaggaca agctagaatt tgccccgasa gctgtgctga acagaaaccg 300 cccagagaag aattaatgga ggacacaggg ccctatggtc ctactgtggg tggtgacttg 360 tcctgctacc atgttgacag agccccagaa cccacatcta attggctttg ttgcttattc 420 tggcccttcc cacaccacac agccacacaa atactggctg ctccttgatg gccaggcaga 480 cccagcagca gccgaggggc cagtgaagag gaaggccgca tctgttgtgt ggtggccaca 540 agcactcagg catctgagtt tactggtgca ctgctgggag gagagttatg agatgaacat 600 tggctgtcaa tctctgtggg caggcggttt ggcctctagt gggaatggct gggatttggg 660 cgttgccttt aggagggata cctgcatgtc tagttccagt ctgcactgga aagaattcaa 720 atatgcacct ggctcccttc actattttgc cctatccttt gtgctcattc ttactgaaat 780 ctgtcttgtc agctcaggaa tgggattccc ccaggaagga aagcactttt ctgttctggg 840 aagcccagac tgttcacttt ggggcaggga cgaacatgtg cctcgtgaat ttgcttgaaa 900 acagtcacca tcttctaccc ccatccctgt atagtgaaaa acctgattaa agtggtatct 960 gagaaccata aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 1005 <210> 10 <211> 187 <212> PRT
<213> Homo Sapiens <400> 10 Met Val Leu Leu Trp Val Val Thr Cys Pro Ala Thr Met Leu Thr Glu Pro Gln Asn Pro His Leu Ile Gly Phe Val Ala Tyr Ser Gly Pro Ser His Thr Thr Gln Pro His Lys Tyr Trp Leu Leu Leu Asp Gly Gln Ala Asp Pro Ala Ala Ala Glu Gly Pro Val Lys Arg Lys Ala Ala Ser Val Val Trp Trp Pro Gln Ala Leu Arg His Leu Ser Leu Leu Val His Cys Trp Glu Glu Ser Tyr Glu Met Asn Ile Gly Cys Gln Ser Leu Trp Ala Gly Gly Leu Ala Ser Ser Gly Asn Gly Trp Asp Leu Gly Val Ala Phe Arg Arg Asp Thr Cys Met Ser Ser Ser Ser Leu His Trp Lys Glu Phe Lys Tyr Ala Pro Gly Ser Leu His Tyr Phe Ala Leu Ser Phe Val Leu Ile Leu Thr Glu Ile Cys Leu Val Ser Ser Gly Met Gly Phe Pro Gln Glu Gly Lys His Phe Ser Val Leu Gly Ser Pro Asp Cys Ser Leu Trp g Gly Arg Asp Glu His Val Pro Arg Glu Phe Ala <210> 11 <211> 1198 <212> DNA
<213> Homo Sapiens <400> 11 aggtctagaa ttcaatcggg accagcggcc agcgctagtc ggtctggtaa ggatttacaa 60 aaggtgcagg tatgagcagg tctgaagact aacattttgt gaagttgtaa aacagaaaac 120 ctgttagaaa tgtggtggtt tcagcaaggc ctcagtttcc ttccttcagc ccttgtaatt 180 tggacatctg ctgctttcat attttcatac attactgcag taacactcca ccatatagac 240 ccggctttac cttatatcag tgacactggt acagtagctc cagaaaaatg cttattttgg 300 ggcaatgcta aatattgcgg cagttttatg cattgctacc atttatgttc gttataagca 360 agttcatgct ctgagtcctg aagagaacgt tatcatcaaa ttaaacaagg ctggccttgt 420 acttggaata ctgagttgtt taggactttc tattgtggca aacttccaga aaacaaccct 480 ttttgctgca catgtaagtg gagctgtgct tacctttggt atgggctcat tatatatgtt 540 tgttcagacc atcctttcct accaaatgca gcccaaaatc catggcaaac aagtcttctg 600 gatcagactg ttgttggtta tctggtgtgg agtaagtgca cttagcatgc tgacttgctc 660 atcagttttg cacagtggca attttgggac tgatttagaa cagaaactcc attggaaccc 720 cgaggacaaa ggttatgtgc ttcacatgat cactactgca gcagaatggt ctatgtcatt 780 ttccttcttt ggttttttcc tgacttacat tcgtgatttt cagaaaattt ctttacgggt 840 ggaagccaat ttacatggat taaccctcta tgacactgca ccttgcccta ttaacaatga 900 acgaacacgg ctactttcca gagatatttg atgaaaggat aaaatatttc tgtaatgatt 960 atgattctca gggattgggg aaaggttcac agaagttgct tattcttctc tgaaattttc 1020 aaccacttaa tcaaggctga cagtaacact gatgaatgct gataatcagg aaacatgaaa 1080 gaagccattt gatagattat tctaaaggat atcatcaaga agactattaa aaacacctat 1140 gcctatactt ttttatctca gaaaataaag tcaaaagact atgaaaaaaa aaaaaaaa 1198 <210> 12 <211> 208 <212> PRT
<213> Homo Sapiens <400> 12 Met Leu Asn Ile Ala Ala Val Leu Cys Ile Ala Thr Ile Tyr Val Arg Tyr Lys Gln Val His Ala Leu Ser Pro Glu Glu Asn Val Ile Ile Lys Leu Asn Lys Ala Gly Leu Val Leu Gly Ile Leu Ser Cys Leu Gly Leu Ser Ile Val Ala Asn Phe Gln Lys Thr Thr Leu Phe Ala Ala His Val Ser Gly Ala Val Leu Thr Phe Gly Met Gly Ser Leu Tyr Met Phe Val Gln Thr Ile Leu Ser Tyr Gln Met Gln Pro Lys Ile His Gly Lys Gln Val Phe Trp Ile Arg Leu Leu Leu Val Ile Trp Cys Gly Val Ser Ala Leu Ser Met Leu Thr Cys Ser Ser Val Leu His Ser Gly Asn Phe Gly Thr Asp Leu Glu Gln Lys Leu His Trp Asn Pro Glu Asp Lys Gly Tyr Val Leu His Met Ile Thr Thr Ala Ala Glu Trp Ser Met Ser Phe Ser Phe Phe Gly Phe Phe Leu Thr Tyr Ile Arg Asp Phe Gln Lys Ile Ser Leu Arg Val Glu Ala Asn Leu His Gly Leu Thr Leu Tyr Asp Thr Ala Pro Cys Pro Ile Asn Asn Glu Arg Thr Arg Leu Leu Ser Arg Asp Ile <210> 13 <211> 1164 <212> DNA
<213> Homo sapiens <400> 13 gtgggactct cttgaggcct ctctagtcca gccagggatg gtttttattt aaaaaataga 60 ttctgagatt tcatcttact aaggattcat gattcgcctc atttctgtaa ctgaggacta 120 atgtgctttc tgggtgacag caagactcta tctaaaaaca gagaaagaaa cccagacgag 180 cataagccca gtgtagacag aattgtgatg gagtgtattt catgtagcac cactggcctc 240 atgttcaaag gctttcatgt gccaagggcg ctggacccac tcccagggtg caccactgac 300 tcaccaccca gtgaaataat tgctgggttc aaaggcatag agttggaaaa gtgtcgattg 360 tatgtgtgcc tatatatgaa ggaatagtac ctgaaaaaag tgtcttcaga actggataga 420 ctgggtaaag gacaatggtc cacatccgtg ggtggcatga ctggagagct gtgggaagaa 480 taagaaagta agggagatta gaatgaatgg cgtctcaatt gaaaacagat tgatttttga 540 aagcctgaga aagcatttct gacatcctag gtctgatttg gtctctcttg cccaaggtca 600 caccatctgt cattgaataa gcatttactg tgtcaaacta tggtcaaggc atgcacctgt 660 ttcagattct tgaatatgac aagtttgttc ccagttttgt ggtatatcca tgccattccc 720 tctgcctgga atatttcccc tcacccccaa caccaggaaa agttgagagc taaatctcta 780 gttaggcaga gttgaatagg acatcttcaa tgtttgctct acttatttat tttttctttc 840 tttttttctt ccttagaaac agggtcttgg ccgggcgcac ggtggctcac acctgtaatc 900 ccaccacttt gggaggctga ggtgggcaga tcaccttagg tcaggggtcc aagaccagcc 960 tggccagcat ggtgaaaccc cgtctctact aaagatataa aattagctga gcatggtgac 1020 gtgcacttgt aatcccagct actcaggagg ctgaggcagg agaatctctt gaaaccagga 1080 ggtagaggtt gcagtgagcc aagatcacgc cattgcactc caccctgcac aagagtgtaa 1140 ctccatctca aaaaaaaaaa aaaa 1164 <210> 14 <211> 51 <212> PRT
<213> Homo sapiens <400> 14 Met Val Lys Ala Cys Thr Cys Phe Arg Phe Leu Asn Met Thr Ser Leu Phe Pro Val Leu Trp Tyr Ile His Ala Ile Pro Ser Ala Trp Asn Ile Ser Pro His Pro Gln His Gln Glu Lys Leu Arg Ala Lys Ser Leu Val 1~
Arg Gln Ser <210> 15 <211> 1938 <212> DNA
<213> Homo sapiens <400> 15 ggatagtcag aggtttgaac aaagtagaaa tgttttatgc ataagtgact gttttgagtt 60 tattcattga tgttttaaaa ataacgacat aaaacccaca gaagttttat cctttggcaa 120 acttttctcc catcttattt atttttctgt agatattttg attcattata aaacatctat 180 ttttaaaagt cagatttcag ctttgtaaaa tgaaaaatat accatcagta ttgaacaaaa 240 tattagttga atctcttctt ctggaatgtc ccaatagctt tttgtggtta ctcatcgtgg 300 aaggttgtaa tgccctagca ttacagattt tagggatgct aactgctggt gttgaagaat 360 aactgctgta ggaaaaaaaa ttagtgtaat tttaataaca ttgactatta gcatttaaca 420 cagaactatt ttcatcatca acaagtttat tcagctcatt ctaaatggtc ccttatataa 480 gggccaaaag tacttaactt ttaaaagtta gcaatataat ctcttcttgc ttataaggtc 540 aagtcttttg tgatagcctt actagcaata atagaaaatt gaaaaaaagc attttagttc 600 ccgtgtttaa aaatatttct tgtaagtgtt ggtattgcaa atgaattatt accaaatgtt 660 aataatctat tatgtcttgt tttttaaagt gaatgaattt ttagcttttg agggtcccat 720 cttgttggat atgagaatta aacatctaat caaaacaaat cagttaagtc aagcaactgc 780 tctagcaaag ctgtgttctg accatccaga gattggcata aaaggtagtt ttaagcaaac 840 ttaccttgtc tgtctttgta catcatcacc aaatggaaag ttaatcgaag aggtgagtat 900 gttttctttc attagtaatt atttttttag ttgagaaatt aatttttaat tagaatgtct 960 ttgatttcct tttctcttag atagcgaaga gatcattgtc aatcttacat ttttggtatg 1020 ttcttgagtg gtggcaactt gattctaagt agcttttaga attatatgta aactgattgc 1080 acctagtttc tgttttaaat aatatacatc ttttaaatgc tttggctaaa ttgtgggaaa 1140 gatagtggtg gcttaacagt ttagtaaaat atctatattt gtcagctttt ggccttaact 1200 tcaatcttgt ctgacttcaa gatacttgca catcttttaa actggctaat ttaagtaact 1260 ttatagaatt tttgtgactt atctctggaa atactttgtc atataaattc tatatagaat 1320 atatatatgc acatctatgc tatatatcat tttgagtacg tatttatttg cctatttaat 1380 gtaaaacctt attttctctc tgagacattg tcttttcact tctagtttgg tataaagcat 1440 agtattcaca tatttgtata gtttatgact acaaaattct gagaatggaa tcatgatcca 1500 aatgggaatt ttgcaatttt gcattttgag ggacttacaa tttcaaaagg aagttggaaa 1560 cagggtagga gaattagaag ttgttgatca agaaacaggg catgtatatt taaagaattt 1620 agataaaact atactagatt ggctgggtgc ctgtggctca cgcctgtaat cccagcactt 1680 tgggaggccg aggcgggcgg atcacgaggt caggagtttg agaccagcct ggccaacgta 1740 gtgaaactcc tctctctact aaaaatacca aaaattagct gggcatggtg gcgggcacct 1800 gtaatcccag ctacttggta ggctgaggca ggagaatcgt ttgaacctgg gaggtggggg 1860 ttgcagtgag ccaagatcat gccactgcac tccagcctgg gtgacagtgt gagactctgt 1920 cgcaaaaaaa aaaaaaaa 1938 <210> 16 <211> 97 <212> PRT
<213> Homo Sapiens <400> 16 Met Asn Tyr Tyr Gln Met Leu Ile Ile Tyr Tyr Val Leu Phe Phe Lys Val Asn Glu Phe Leu Ala Phe Glu Gly Pro Ile Leu Leu Asp Met Arg Ile Lys His Leu Ile Lys Thr Asn Gln Leu Ser Gln Ala Thr Ala Leu Ala Lys Leu Cys Ser Asp His Pro Glu Ile Gly Ile Lys Gly Ser Phe ~1 Lys Gln Thr Tyr Leu Val Cys Leu Cys Thr Ser Ser Pro Asn Gly Lys Leu Ile Glu Glu Val Ser Met Phe Ser Phe Ile Ser Asn Tyr Phe Phe Ser <210> 17 <211> 2185 <212> DNA
<213> Homo sapiens <400> 17 aaaaaaaaaa aaatccactg gctagaacca gtcgtgacgc ccagcctaat tgctaaggag 60 gcaggagtat gagacagagc agatggttat ttggtaagta ctctctcagc tacaggttac 120 ctcagttcca gggcgcttac agtaaggttc tgcctaactg tggcctacaa ggccctgcat 180 ggtctacctt tgcccggctc ttcaacctca cctctaccac tttccccctc agtcattccc 240 ctcagccact acggtcatcc ttcacttgcg gggatatgcc aagtgtttgc tgcctaagga 300 cctttgcaca cgtgttccct ctccttggaa tgctgtctct ccccagcttc cctacattct 360 tccatgctag tccttttcat cctctgggtg tctgcatatg tggccccttc tcatggcagc 420 ttttcctggc cagcctatgg aagtaggtcc atcaggcacc cctccctcga gtttgttttc 480 agcatagagc acctcccggt ttataattag atgtctttta tttaactagc ttaatgtcag 540 tattatggca gttttgataa aggattcccc attccctaac acaaaatgaa tgaatgaatg 600 aatgagaaag aatgagtcat ttcagcagca ggtggagtac aagttcagtg agctatggtt 660 gcaccagtgc actctagcct gggtcacaga gcaagactct gcctcaaaaa caaaaacaaa 720 aaatccagac tctgtctgag aactgccgtg tcaaccttct ggtgcctgtg gccacctaag 780 ctatagatta gctgttcctt ccgtcacctg cgtccccttc ctatccctgg gggtcccagc 840 agggagaacc ctgggctgcc cacctccagt gtgtgactga ggctggctgc caatggcgtg 900 ggcacaggcc cccacctccc ctgctgctgg gctcctatgt tactttgctc tcatggccac 960 tgtacctctc gggactgtgc accaagttgc tgcccaccta gaatcggttt ccattagagt 1020 gtttaaaatg ttaaaaatag ccttccaccc taaatgcttc cttcggaggc ccgagttggg 1080 aatcagaggt tcttttctat ctgcaccttt ctggcagaac tgaaggaggg tggctgtttg 1140 cacagtgttg tgggcccctg ccctgggcca gccacttcac atttgtcatc atggagccct 1200 gggacggccc tgggaggtgt ggggttatta ttctgattcc acaggagagg cgcatgaagc 1260 tcagagggtt aggaggtctg tgcctgggcg tgaagctgga atcgcagagc ttgggtctgg 1320 ctcaagccag agccgacgct tccccacgct tactcttgtg atcctagttc tactctttga 1380 tcacagtctg tgtttttgga tctcatgatc ctgcagaatc aactttatgt gtggaattct 1440 cttgagaatt taatcctggg aggaaagagt gctgtcttgg aagcctgagg ctgcccctgt 1500 ctatctgtga taagaagtct gtgagcagct cactcctgag ccttggcctt ggcagatagg 1560 aagctcagtg cgagagttaa agcttgtcac agccctttct tgtaggcatt tggcctcctt 1620 tgcctcatcc tgcctttggt gttctatttt tatttcacta gctctctggt gtgccagcct 1680 gaatgagtgg gcgttgcgtc tttctagcta gcagggagca ggtttggagg gatgaggaca 1740 gaggccattt tctccatctt catgtttgga cagtgggaca ggcatgcatg ggcctgcctg 1800 tccacccagc tcagaccatg ccggcagctc cagggtggag atgagggcgg ttcatgttta 1860 tacgcaccat ttattgcttg cattttaaag acatctaaaa actcaaagct tgagaagatg 1920 ggggtttcac aggaaaccag aacttcttgt tggggctgac atggggctct gttggcttac 1980 atccgtggct ggtgtgacct ggtgcctctt gtgtgggagg gtggatgtta gccccaagtg 2040 tgctgacagg tctggtcaca ggtagccctg cagacaccgc ccctccccat gtgttcctca 2100 ctcctgctca cttccaaatg ccctgtggtg ccagatgggg ccaccgcatg tcactgcaga 2160 aggaacaaga aaaaaaaaaa aaaaa 2185 <210> 18 <211> 125 <212> PRT
<213> Homo sapiens <400> 18 Met Arg Gln Ser Arg Trp Leu Phe Gly Lys Tyr Ser Leu Ser Tyr Arg Leu Pro Gln Phe Gln Gly Ala Tyr Ser Lys Val Leu Pro Asn Cys Gly Leu Gln Gly Pro Ala Trp Ser Thr Phe Ala Arg Leu Phe Asn Leu Thr Ser Thr Thr Phe Pro Leu Ser His Ser Pro Gln Pro Leu Arg Ser Ser Phe Thr Cys Gly Asp Met Pro Ser Val Cys Cys Leu Arg Thr Phe Ala His Val Phe Pro Leu Leu Gly Met Leu Ser Leu Pro Ser Phe Pro Thr g5 90 95 Phe Phe His Ala Ser Pro Phe His Pro Leu Gly Val Cys Ile Cys Gly Pro Phe Ser Trp Gln Leu Phe Leu Ala Ser Leu Trp Lys <210> 19 <211> 987 <212> DNA
<213> Homo sapiens <400> 19 ccacatccca gatgacattc ctcagaagct cgcagctgga gatgacatca ccttagacaa 60 catcacccta gcccaagaca tcgctattag agatacatca cctggacact aaagcctcca 120 acccagtgac actctcaagg tgctgacaaa atggacatgg acatttgttg cttttcttct 180 tttgaattag gaactctatt gtgtttcctg aatttactgt ctgcttggcc catgatcctg 240 gtatgttcct tgctctctgc caaaacatgc accgtcccct ccacacctaa aggaaaacct 300 ggtagaagct actactctca atgccattct aaaattgacg aagagtatta ttttccccaa 360 gaggcagctg ctgcaggcca gtgaatcctc tggaccctgg ggagagggtc atacactccc 420 tagtgacttc tactgtatgc tccagctttc caccaggacc tcctgccctg tccccaactg 480 taacgaccac aaccacccca tccccttggt tacacacasa tatcaccagt ctttacatgt 540 actttactga cttctcagat aataccctcc aacaagataa ctgcaggact tgccaccatg 600 acctgagaaa tacaccaaga tattattgtc caattttttt gagatatata tatatttctc 660 atacttttaa catactctgc aatccaccaa gatattggta gagaccctgg aaatttcaaa 720 tctatttcgt ctgctaatgc catgtgaatg ggaaacttag atacaggctg gcctacaagg 780 actccttata cttcaatttt tgttcctcct ttcaactctc ataaaaaata ttttctgtac 840 tatttagggc aaatgatggc cttctttcta agtctcatcc atcattgctg caaaggtgcc 900 ttagtttgat gttaaagtgt cacagggcca atgtattgac catgtttctg gcgtctataa 960 agtcgatgtt ttaaaaaaaa aaaaaaa 987 <210> 20 <211> 77 <212> PRT
<213> Homo sapiens <400> 20 Met Asp Met Asp Ile Cys Cys Phe Ser Ser Phe Glu Leu Gly Thr Leu Leu Cys Phe Leu Asn Leu Leu Ser Ala Trp Pro Met Ile Leu Val Cys Ser Leu Leu Ser Ala Lys Thr Cys Thr Val Pro Ser Thr Pro Lys Gly Lys Pro Gly Arg Ser Tyr Tyr Ser Gln Cys His Ser Lys Ile Asp Glu Glu Tyr Tyr Phe Pro Gln Glu Ala Ala Ala Ala Gly Gln <210> 21 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramidite residue <400> 21 tngctgcttc cacacatcac tgtacattt 29 <210> 22 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramidite residue <400> 22 cntgctctta tcaccagcca cccgtcctc 29 <210> 23 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramidite residue <400> 23 tnatataagg ccatctgttc aagtccacc 29 <210> 24 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramidite residue <400> 24 ancagaaatc gttggctgag ttcgtgcgc 29 <210> 25 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramidite residue <400> 25 cngtaagaat gagcacaaag gatagggca 29 <210> 26 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramidite residue <400> 26 cngcatttgg taggaaagga tggtctgaa 29 <210> 27 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) 1$
<223> biotinylated phosphoaramidite residue <400> 27 tncasgaatc tgaaacaggt gcatgcctt 29 <210> 28 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramidite residue <400> 28 tnaggctatc acaaaagact tgaccttat 29 <210> 29 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramidite residue <400> 29 tngcatggaa gaatgtaggg aagctgggg 29 <210> 30 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramidite residue <400> 30 antagcgatg tcttgggcta gggtgatgt 29 <210> 31 <211> 101 <212> PRT
<213> Homo sapiens <400> 31 Met A1a Gly Ile Trp Ala Leu Pro Leu Gly Gly Ile Pro Ala Cys Leu Val Pro Val Cys Thr Gly Lys Asn Ser Asn Met His Leu Ala Pro Phe Thr Ile Leu Pro Tyr Pro Leu Cys Ser Phe Leu Leu Lys Ser Val Leu Ser Ala Gln Glu Trp Asp Ser Pro Arg Lys Glu Ser Thr Phe Leu Phe Trp Glu Ala Gln Thr Val His Phe Gly Ala Gly Thr Asn Met Cys Leu Val Asn Leu Leu Glu Asn Ser His His Leu Leu Pro Pro Ser Leu Tyr Ser Glu Lys Pro Asp <210> 32 <211> 88 <212> PRT
<213> Homo sapiens <400> 32 Met Cys Phe Leu Gly Asp Ser Lys Thr Leu Ser Lys Asn Arg Glu Arg Asn Pro Asp Glu His Lys Pro Ser Val Asp Arg Ile Val Met Glu Cys Ile Ser Cys Ser Thr Thr Gly Leu Met Phe Lys Gly Phe His Val Pro Arg Ala Leu Asp Pro Leu Pro Gly Cys Thr Thr Asp Ser Pro Pro Ser Glu Ile Ile Ala Gly Phe Lys Gly Ile Glu Leu Glu Lys Cys Arg Leu Tyr Val Cys Leu Tyr Met Lys Glu <210> 33 <211> 48 <212> PRT
<213> Homo sapiens <400> 33 Met Leu Val Leu Phe Ile Leu Trp Val Ser Ala Tyr Val Ala Pro Ser His Gly Ser Phe Ser Trp Pro Ala Tyr Gly Ser Arg Ser Ile Arg His Pro Ser Leu Glu Phe Val Phe Ser Ile Glu His Leu Pro Val Tyr Asn