JP2002510488A - Secreted proteins and polynucleotides encoding them - Google Patents

Abstract

  (57) [Summary] Disclosed are novel polynucleotides and the proteins encoded by them.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, as well as therapeutic, diagnostic and research utilities for these polynucleotides and proteins.

BACKGROUND proteinaceous factors invention (e.g., lymphokines, interferons, including cytokine such as CSFs and interleukins) has methods for the purpose of finding this 10
Matured rapidly over the years. Currently, conventional hybridization and expression cloning methods rely on information directly related to the discovered protein (ie,
In the case of hybridization cloning, depending on the partial DNA / amino acid sequence of the protein; in the case of expression cloning, the activity of the protein).
The new polypeptide is cloned "directly". Signal sequence cloning (
DNA sequences are isolated based on the presence of the currently well-recognized secretory leader sequence motifs), as well as more recent "indirect" cloning methods, such as various PCR or low stringency hybridization cloning methods. A large number of DNA / amino acid sequences for proteins known to have biological activity due to their secreted nature in the case of cloning of the leader sequence, or due to the cell or tissue source in the case of the PCR method. Has made progress by enabling. The present invention is directed to these proteins and the polynucleotides that encode them.

[0003] In an embodiment of the outline 1 of the invention, the present invention is isolated to provide a composition comprising a polynucleotide selected from the group consisting of: comprising one of the nucleotide sequences: (a) SEQ ID NO: (B) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 from nucleotide 45 to nucleotide 590; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 from nucleotide 126 to nucleotide 590; (d) A polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone ya91 deposited under accession number ATCC 98724; (e) c of clone ya91 deposited under accession number ATCC 98724
A polynucleotide encoding the full length protein encoded by the DNA insert; (f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone ya91 deposited under accession number ATCC98724; (g) a deposition number accession number ATCC98724 C of cloned ya91
A polynucleotide encoding a mature protein encoded by the DNA insert; (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2; (i) a SEQ ID NO: 2 having biological activity A polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 2
(J) a polynucleotide that is an allelic variant of the polynucleotide of (a) to (g) above; (k) a species homolog of the protein of (h) or (i) above. A coding polynucleotide; (l) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i); and (m) under stringent conditions (a) A polynucleotide that hybridizes to any one of the polynucleotides set forth in (i) and has a length of at least 25% of the length of SEQ ID NO: 1. Preferably, such a polynucleotide is a nucleotide sequence from nucleotide 45 to nucleotide 590 of SEQ ID NO: 1; nucleotide 1 of SEQ ID NO: 1.
Nucleotide sequence from 26 to nucleotide 590; accession number ATCC 987
Nucleotide sequence of the full-length protein coding sequence of clone ya91 deposited as accession number 24; or clone ya deposited as accession number ATCC 98724
9 contains the nucleotide sequence of the mature protein coding sequence. In another preferred embodiment, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone ya91 deposited under accession number ATCC 98724. In a further preferred embodiment, the present invention relates to a polynucleotide encoding a protein comprising a fragment of the amino acid sequence SEQ ID NO: 2 having biological activity, said fragment comprising SEQ ID NO: 2, preferably 8, A polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 2, which has more preferably 20 and most preferably 30 contiguous amino acids) or a fragment having the biological activity (SEQ ID NO: 2 amino acids 8
From 6 to 95 amino acids). Another embodiment provides a gene corresponding to the cDNA sequence of SEQ ID NO: 1. A further embodiment of the invention provides a method for producing an isolated polynucleotide, wherein the method is selected from the group consisting of: (a) a method comprising the steps of: (i) 65 in 6X SSC. Preparing one or more polynucleotide probes that hybridize at <RTIgt; 0 C </ RTI> to a nucleotide sequence selected from the group consisting of: (aa) SEQ ID NO: 1 (at the 3 'end of SEQ ID NO: 1). (Above the poly (A) tail); and (ab) the cDNA of clone ya91 deposited as ATCC 98724.
Nucleotide sequence of NA insert (ii) Human genomic DN under conditions of stringency of at least 4 × SSC, 50 ° C.
Hybridizing the probe (s) to A; then (iii) isolating the DNA polynucleotide detected with the probe (s); and (b) a method comprising the steps of: i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 ° C. to a nucleotide sequence selected from the group consisting of: (ba) SEQ ID NO: 1 (SEQ ID NO: (Except for the poly (A) tail at the 3 'end of 1: 1); and (bb) the cD of clone ya91 deposited as ATCC 98724.
Nucleotide sequence of NA insert (ii) Human genomic DN under conditions of stringency of at least 4 × SSC, 50 ° C.
A. hybridizing the primer (s) to A; (iii) amplifying the human DNA sequence; and (iv) isolating the polynucleotide product of step (b) (iii). Preferably, the nucleotide sequence of the polynucleotide isolated by this method corresponds to the cDNA sequence of SEQ ID NO: 1, from the nucleotide sequence corresponding to the 5 'end of SEQ ID NO: 1 to 3' of SEQ ID NO: 1. The sequence is continued up to the nucleotide sequence corresponding to the terminus, except for the poly (A) tail at the 3 ′ terminus of SEQ ID NO: 1. Also preferably, the nucleotide sequence of the polynucleotide isolated by the above method corresponds to nucleotide 45 to nucleotide 590 of the cDNA sequence of SEQ ID NO: 1, and corresponds to nucleotide 45 to nucleotide 590 of SEQ ID NO: 1. From the nucleotide sequence corresponding to the 5 'end of the sequence,
It is a continuous sequence from nucleotide 45 to nucleotide 590 of SEQ ID NO: 1 up to the nucleotide sequence corresponding to the 3 ′ end of the sequence. Also preferably, the nucleotide sequence of the polynucleotide isolated by the above method corresponds to nucleotides 126 to 590 of the cDNA sequence of SEQ ID NO: 1, and corresponds to nucleotides 126 to 590 of SEQ ID NO: 1. From the nucleotide sequence corresponding to the 5 'end of the sequence, nucleotide 1 of SEQ ID NO: 1
It is a continuation from 26 to nucleotide 590 up to the nucleotide sequence corresponding to the 3 'end of the sequence. In another embodiment, the present invention provides a composition comprising a protein, comprising: (a) an amino acid sequence of SEQ ID NO: 2; (b) a fragment of the amino acid sequence of SEQ ID NO: 2, wherein the fragment is (Comprising eight consecutive amino acids of SEQ ID NO: 2); and (c) c of clone ya91 deposited under accession number ATCC 98724.
A composition comprising an amino acid sequence selected from the group consisting of an amino acid sequence encoded by a DNA insert and substantially free from other mammalian proteins. Preferably, such a protein comprises the amino acid sequence of SEQ ID NO: 2. In a further preferred embodiment, the present invention provides
A protein comprising a fragment of the amino acid sequence of SEQ ID NO: 2 having biological activity (
The fragment comprises SEQ ID NO: 2, preferably comprising 8, more preferably 20, and most preferably 30 contiguous amino acids), or a fragment of the amino acid sequence SEQ ID NO: 2 having biological activity. A protein comprising the amino acid sequence from amino acid 86 to amino acid 95 of SEQ ID NO: 2.

[0004] In one embodiment, the invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3; (B) a polynucleotide comprising the nucleotide sequence from nucleotide 194 to nucleotide 466 of SEQ ID NO: 3; (c) a polynucleotide comprising the nucleotide sequence from nucleotide 338 to nucleotide 466 of SEQ ID NO: 3; (d) accession number ATCC 98724 A polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone ya111 deposited under accession number; (e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone ya111 deposited under accession number ATCC 98724; (F) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone ya111 deposited under accession number ATCC98724; (g) a mature protein encoded by the cDNA insert of clone ya111 deposited under accession number ATCC98724; (H) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 4; (i) a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 4 having biological activity (The fragment is SEQ ID NO: 4 8)
(J) a polynucleotide that is an allelic variant of the polynucleotide of (a) to (g) above; (k) a species homolog of the protein of (h) or (i) above. A coding polynucleotide; (l) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i); and (m) under stringent conditions (a) A polynucleotide that hybridizes to any one of the polynucleotides set forth in (i) and has a length of at least 25% of the length of SEQ ID NO: 3. Preferably, such a polynucleotide is nucleotide 194 of SEQ ID NO: 3.
Nucleotide sequence from nucleotide 338 to nucleotide 466 of SEQ ID NO: 3; accession number ATCC 98
724, containing the nucleotide sequence of the full length protein coding sequence of clone ya11 1 deposited as 724; or the mature protein coding sequence of clone ya11 1 deposited as accession number ATCC 98724. In another preferred embodiment, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone ya11 deposited under accession number ATCC 98724. In a further preferred embodiment, the present invention relates to a polynucleotide encoding a protein comprising a fragment of the amino acid sequence SEQ ID NO: 4 having biological activity, said fragment comprising SEQ ID NO: 4, preferably 8 More preferably, the polynucleotide encodes a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 4 that has biological activity, including 20 and most preferably 30 contiguous amino acids. 4 amino acids 40 to 49). Another embodiment provides a gene corresponding to the cDNA sequence of SEQ ID NO: 3. A further embodiment of the invention provides a method for producing an isolated polynucleotide, wherein the method is selected from the group consisting of: (a) a method comprising the steps of: (i) 65 in 6X SSC. Preparing one or more polynucleotide probes that hybridize at 0 ° C. to a nucleotide sequence selected from the group consisting of: (aa) SEQ ID NO: 3 (at the 3 ′ end of SEQ ID NO: 3) (Except poly (A) tail); and (ab) c of clone ya11_11 deposited as ATCC 98724
Nucleotide sequence of DNA insert (ii) Human genomic DN under conditions of stringency of at least 4 × SSC, 50 ° C.
Hybridizing the probe (s) to A; then (iii) isolating the DNA polynucleotide detected with the probe (s); and (b) a method comprising the steps of: i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 ° C. to a nucleotide sequence selected from the group consisting of: (ba) SEQ ID NO: 3 (SEQ ID NO: 3) (Excluding the poly (A) tail at the 3 ′ end of 3: 3); and (bb) c of clone ya11_11 deposited as ATCC 98724.
Nucleotide sequence of DNA insert (ii) Human genomic DN under conditions of stringency of at least 4 × SSC, 50 ° C.
A. hybridizing the primer (s) to A; (iii) amplifying the human DNA sequence; and (iv) isolating the polynucleotide product of step (b) (iii). Preferably, the nucleotide sequence of the polynucleotide isolated by this method corresponds to the cDNA sequence of SEQ ID NO: 3, from the nucleotide sequence corresponding to the 5 'end of SEQ ID NO: 3 to 3' of SEQ ID NO: 3 It is a continuous sequence up to the nucleotide sequence corresponding to the terminus, except for the poly (A) tail at the 3 ′ terminus of SEQ ID NO: 3. Also preferably, the nucleotide sequence of the polynucleotide isolated by the above method corresponds to nucleotide 194 to nucleotide 466 of the cDNA sequence of SEQ ID NO: 3, and nucleotide 194 of SEQ ID NO: 3
To the nucleotide sequence corresponding to the 5 ′ end of the sequence from SEQ ID NO: 3 to nucleotide 466, to the nucleotide sequence corresponding to the 3 ′ end of the sequence from nucleotide 194 to nucleotide 466 of SEQ ID NO: 3. Also preferably, the nucleotide sequence of the polynucleotide isolated by the above method corresponds to nucleotides 338 to 466 of the cDNA sequence of SEQ ID NO: 3, and corresponds to nucleotides 338 to 466 of SEQ ID NO: 3. It is a continuous sequence from the nucleotide sequence corresponding to the 5 'end of the sequence to the nucleotide sequence corresponding to the 3' end of the sequence from nucleotide 338 to nucleotide 466 of SEQ ID NO: 3. In another embodiment, the present invention provides a composition comprising a protein, comprising: (a) an amino acid sequence of SEQ ID NO: 4; (b) a fragment of the amino acid sequence of SEQ ID NO: 4 And (c) an amino acid sequence selected from the group consisting of the amino acid sequence encoded by the cDNA insert of clone ya111 deposited under accession number ATCC 98724. Wherein the composition is substantially free of other mammalian proteins. Preferably, such a protein comprises the amino acid sequence of SEQ ID NO: 4. In a further preferred embodiment, the present invention provides
A protein comprising a fragment of the amino acid sequence of SEQ ID NO: 4 having biological activity (
The fragment comprises SEQ ID NO: 4, preferably containing 8, more preferably 20, most preferably 30 contiguous amino acids), or a fragment of the amino acid sequence SEQ ID NO: 4 having biological activity. The amino acid sequence of SEQ ID NO: 4 from amino acid 40 to amino acid 49.

[0005] 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: 5; (B) a polynucleotide comprising the nucleotide sequence from nucleotide 15 to nucleotide 233 of SEQ ID NO: 5; (c) a polynucleotide comprising the nucleotide sequence from nucleotide 174 to nucleotide 233 of SEQ ID NO: 5; (d) accession number ATCC 98724 A polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone ya281 deposited as (e) a polynucleic acid encoding the full-length protein encoded by the cDNA insert of clone ya281 deposited under accession number ATCC 98724 (F) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone ya281 deposited under accession number ATCC98724; (g) the mature protein encoded by the cDNA insert of clone ya281 deposited under accession number ATCC98724 (H) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 6; (i) a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 6 having biological activity (The fragment is SEQ ID NO: 6 8
(J) a polynucleotide that is an allelic variant of the polynucleotide of (a) to (g) above; (k) a species homolog of the protein of (h) or (i) above. A coding polynucleotide; (l) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i); and (m) under stringent conditions (a) A polynucleotide that hybridizes to any one of the polynucleotides set forth in (i) and has a length of at least 25% of the length of SEQ ID NO: 5. Preferably, such a polynucleotide is a nucleotide sequence from nucleotide 15 to nucleotide 233 of SEQ ID NO: 5; nucleotide 1 of SEQ ID NO: 5
Nucleotide sequence from 74 to nucleotide 233; accession number ATCC 987
Nucleotide sequence of the full-length protein coding sequence of clone ya281 deposited as accession number 24; or clone y deposited as accession number ATCC 98724
a281 contains the nucleotide sequence of the mature protein coding sequence. In another preferred embodiment, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone ya281 deposited under accession number ATCC 98724. In a further preferred embodiment, the present invention relates to a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 6 having biological activity, said fragment comprising SEQ ID NO: 6, preferably 8
, More preferably 20 and most preferably 30 contiguous amino acids)
Alternatively, a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 6 having biological activity (the fragment includes the amino acid sequence from amino acid 31 to amino acid 40 of SEQ ID NO: 6) is provided. Another embodiment provides a gene corresponding to the cDNA sequence of SEQ ID NO: 5. A further embodiment of the invention provides a method for producing an isolated polynucleotide, wherein the method is selected from the group consisting of: (a) a method comprising the steps of: (i) 65 in 6X SSC. Preparing one or more polynucleotide probes that hybridize at 0 ° C. to a nucleotide sequence selected from the group consisting of: (aa) SEQ ID NO: 5 (at the 3 ′ end of SEQ ID NO: 5) (Excluding poly (A) tail); and (ab) c of clone ya281 deposited as ATCC 98724
Nucleotide sequence of DNA insert (ii) Human genomic DN under conditions of stringency of at least 4 × SSC, 50 ° C.
Hybridizing the probe (s) to A; then (iii) isolating the DNA polynucleotide detected with the probe (s); and (b) a method comprising the steps of: i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 ° C. to a nucleotide sequence selected from the group consisting of: (ba) SEQ ID NO: 5 (SEQ ID NO: 5) : Bb except the poly (A) tail at the 3 'end of 5); and (bb) c of clone ya281 deposited as ATCC 98724.
Nucleotide sequence of DNA insert (ii) Human genomic DN under conditions of stringency of at least 4 × SSC, 50 ° C.
A. hybridizing the primer (s) to A; (iii) amplifying the human DNA sequence; and (iv) isolating the polynucleotide product of step (b) (iii). Preferably, the nucleotide sequence of the polynucleotide isolated by this method corresponds to the cDNA sequence of SEQ ID NO: 5, from the nucleotide sequence corresponding to the 5 'end of SEQ ID NO: 5 to 3' of SEQ ID NO: 5 It is a continuous sequence up to the nucleotide sequence corresponding to the terminus, except for the poly (A) tail at the 3 ′ terminus of SEQ ID NO: 5. Also preferably, the nucleotide sequence of the polynucleotide isolated by the above method corresponds to nucleotides 15 to 233 of the cDNA sequence of SEQ ID NO: 5, and preferably corresponds to nucleotides 15 to 233 of SEQ ID NO: 5. From the nucleotide sequence corresponding to the 5 'end of the sequence,
It is a continuous sequence from nucleotide 15 to nucleotide 233 of SEQ ID NO: 5 up to the nucleotide sequence corresponding to the 3 'end of the sequence. Also preferably, the nucleotide sequence of the polynucleotide isolated by the above method corresponds to nucleotides 174 to 233 of the cDNA sequence of SEQ ID NO: 5, and corresponds to nucleotides 174 to 233 of SEQ ID NO: 5. From the nucleotide sequence corresponding to the 5 'end of the sequence, nucleotide 1 of SEQ ID NO: 5
Contiguous from 74 to nucleotide 233 up to the nucleotide sequence corresponding to the 3 'end of the sequence. In another embodiment, the present invention relates to a composition comprising a protein, comprising: (a) an amino acid sequence of SEQ ID NO: 6; (b) a fragment of the amino acid sequence of SEQ ID NO: 6; And (c) an amino acid sequence selected from the group consisting of the amino acid sequence encoded by the cDNA insert of clone ya281 deposited under accession number ATCC 98724). And a composition that is substantially free of other mammalian proteins. Preferably, such a protein comprises the amino acid sequence of SEQ ID NO: 6. In a further preferred embodiment, the present invention provides
A protein comprising a fragment of the amino acid sequence of SEQ ID NO: 6 having biological activity (
The fragment comprises SEQ ID NO: 6, preferably comprising 8, more preferably 20, most preferably 30 contiguous amino acids), or a fragment of the amino acid sequence SEQ ID NO: 6 having biological activity. A protein comprising the amino acid sequence from amino acid 31 to amino acid 40 of SEQ ID NO: 6.

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: 7; (B) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 7 from nucleotide 102 to nucleotide 461; (c) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone yb811 deposited under accession number ATCC 98724; (d A) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone yb81_1 deposited under accession number ATCC 98724; (F) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone yb8111 deposited under accession number ATCC 98724; (g) a protein comprising the amino acid sequence of SEQ ID NO: 8 (H) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 8 having biological activity (the fragment is SEQ ID NO: 8;
(I) a polynucleotide that is an allelic variant of the polynucleotide of (a) to (f) above; and (j) a species homolog of the protein of (g) or (h) above. A polynucleotide that encodes; (k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(h); and (1) a (a) under stringent conditions A polynucleotide having a length of at least 25% of the length of SEQ ID NO: 7 which hybridizes to any one of the polynucleotides shown in (h). Preferably, such a polynucleotide is nucleotide 102 of SEQ ID NO: 7.
To nucleotide 461; accession number ATCC 98724
Nucleotide sequence of the full-length protein coding sequence of clone yb81_1 deposited as; or clone yb8 deposited as accession number ATCC 98724
11 includes the nucleotide sequence of the mature protein coding sequence. In another preferred embodiment, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone yb811 deposited under accession number ATCC 98724. In a further preferred embodiment, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 8 having biological activity, said fragment comprising SEQ ID NO: 8, preferably 8
More preferably, the polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 8 having biological activity, comprising 20 and most preferably 30 contiguous amino acids (the fragment is SEQ ID NO: 8 amino acid 55 to amino acid 64). Another embodiment provides a gene corresponding to the cDNA sequence of SEQ ID NO: 7. A further embodiment of the invention provides a method for producing an isolated polynucleotide, wherein the method is selected from the group consisting of: (a) a method comprising the steps of: (i) 65 in 6X SSC. Preparing one or more polynucleotide probes that hybridize at 0 ° C. to a nucleotide sequence selected from the group consisting of: (aa) SEQ ID NO: 7 (at the 3 ′ end of SEQ ID NO: 7) (Excluding the poly (A) tail); and (ab) c of clone yb81 1 deposited as ATCC 98724
Nucleotide sequence of DNA insert (ii) Human genomic DN under conditions of stringency of at least 4 × SSC, 50 ° C.
Hybridizing the probe (s) to A; then (iii) isolating the DNA polynucleotide detected with the probe (s); and (b) a method comprising the steps of: i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 ° C. to a nucleotide sequence selected from the group consisting of: (ba) SEQ ID NO: 7 (SEQ ID NO: : Except for the poly (A) tail at the 3 ′ end of 7); and (bb) c of clone yb811, deposited as ATCC 98724.
Nucleotide sequence of DNA insert (ii) Human genomic DN under conditions of stringency of at least 4 × SSC, 50 ° C.
A. hybridizing the primer (s) to A; (iii) amplifying the human DNA sequence; and (iv) isolating the polynucleotide product of step (b) (iii). Preferably, the nucleotide sequence of the polynucleotide isolated by this method corresponds to the cDNA sequence of SEQ ID NO: 7, from the nucleotide sequence corresponding to the 5 'end of SEQ ID NO: 7 to 3' of SEQ ID NO: 7 The sequence is continued up to the nucleotide sequence corresponding to the terminus, except for the poly (A) tail at the 3 ′ terminus of SEQ ID NO: 7. Also preferably, the nucleotide sequence of the polynucleotide isolated by the above method corresponds to nucleotide 102 to nucleotide 461 of the cDNA sequence of SEQ ID NO: 7, and the nucleotide sequence of nucleotide 102 of SEQ ID NO: 7
To the nucleotide sequence corresponding to the 5 'end of the sequence from to nucleotide 461 to the nucleotide sequence corresponding to the 3' end of the sequence from nucleotide 102 to nucleotide 461 of SEQ ID NO: 7. In another embodiment, the present invention provides a composition comprising a protein, comprising: (a) an amino acid sequence of SEQ ID NO: 8; (b) a fragment of the amino acid sequence of SEQ ID NO: 8 (wherein the fragment is And (c) an amino acid sequence selected from the group consisting of the amino acid sequence encoded by the cDNA insert of clone yb811 deposited under accession number ATCC 98724. And a composition that is substantially free of other mammalian proteins. Preferably, such a protein comprises the amino acid sequence of SEQ ID NO: 8. In a further preferred embodiment, the present invention provides
A protein comprising a fragment of the amino acid sequence of SEQ ID NO: 8 having biological activity (
The fragment comprises SEQ ID NO: 8, preferably comprising 8, more preferably 20, most preferably 30 contiguous amino acids), or a fragment of the amino acid sequence SEQ ID NO: 8 having biological activity. A protein comprising the amino acid sequence from amino acid 55 to amino acid 64 of SEQ ID NO: 8.

[0007] In one embodiment, the invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 9; (B) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 9 from nucleotide 170 to nucleotide 2968; (c) a polynucleotide comprising the nucleotide sequence of nucleotide 1370 to nucleotide 2968 of SEQ ID NO: 9; (d) accession number ATCC98724 A polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone yc141 deposited as (e) encoding the full-length protein encoded by the cDNA insert of clone yc141 deposited under accession number ATCC 98724 (F) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone yc141 deposited under accession number ATCC 98724; (g) the maturation encoded by the cDNA insert of clone yc141 deposited under accession number ATCC 98724; A polynucleotide encoding a protein; (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 10; (i) including a fragment of the amino acid sequence of SEQ ID NO: 10 having biological activity A polynucleotide encoding a protein (the fragment is SEQ ID NO: 10
(J) a polynucleotide that is an allelic variant of the polynucleotide of (a) to (g) above; (k) species homology of the protein of (h) or (i) above. (L) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i); and (m) under stringent conditions ( A polynucleotide that hybridizes to any one of the polynucleotides specified in a) to (i) and has a length of at least 25% of the length of SEQ ID NO: 9. Preferably, such a polynucleotide is nucleotide 170 of SEQ ID NO: 9.
To nucleotide 2968; nucleotide sequence from nucleotide 1370 to nucleotide 2968 of SEQ ID NO: 9; accession number ATC
The nucleotide sequence of the full length protein coding sequence of clone yc141, deposited as C98724; or the mature protein coding sequence of clone yc141, deposited as accession number ATCC 98724. In another preferred embodiment, the polynucleotide has accession number ATCC 98724.
And encodes the full-length or mature protein encoded by the cDNA insert of clone yc141. In a further preferred embodiment, 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, wherein said fragment comprises a fragment of SEQ ID NO: 10,
A polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 10 having preferably 8, more preferably 20, and most preferably 30 contiguous amino acids, or having biological activity. The fragment provides the amino acid sequence from amino acid 461 to amino acid 470 of SEQ ID NO: 10). Another embodiment provides a gene corresponding to the cDNA sequence of SEQ ID NO: 9. A further embodiment of the invention provides a method for producing an isolated polynucleotide, wherein the method is selected from the group consisting of: (a) a method comprising the steps of: (i) 65 in 6X SSC. Preparing one or more polynucleotide probes that hybridize at 0 ° C. to a nucleotide sequence selected from the group consisting of: (aa) SEQ ID NO: 9 (at the 3 ′ end of SEQ ID NO: 9) (Excluding the poly (A) tail); and (ab) c of clone yc141 deposited as ATCC 98724
Nucleotide sequence of DNA insert (ii) Human genomic DN under conditions of stringency of at least 4 × SSC, 50 ° C.
Hybridizing the probe (s) to A; then (iii) isolating the DNA polynucleotide detected with the probe (s); and (b) a method comprising the steps of: i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 ° C. to a nucleotide sequence selected from the group consisting of: (ba) SEQ ID NO: 9 (SEQ ID NO: : Bb except the poly (A) tail at the 3 'end of 9); and (bb) c of clone yc141 deposited as ATCC 98724.
Nucleotide sequence of DNA insert (ii) Human genomic DN under conditions of stringency of at least 4 × SSC, 50 ° C.
A. hybridizing the primer (s) to A; (iii) amplifying the human DNA sequence; and (iv) isolating the polynucleotide product of step (b) (iii). Preferably, the nucleotide sequence of the polynucleotide isolated by this method corresponds to the cDNA sequence of SEQ ID NO: 9, from the nucleotide sequence corresponding to the 5 'end of SEQ ID NO: 9 to 3' of SEQ ID NO: 9 The sequence is continued up to the nucleotide sequence corresponding to the terminus, except for the poly (A) tail at the 3 ′ terminus of SEQ ID NO: 9. Also preferably, the nucleotide sequence of the polynucleotide isolated by the above method corresponds to nucleotide 170 to nucleotide 2968 of the cDNA sequence of SEQ ID NO: 9, and nucleotide 17 to nucleotide 2968 of SEQ ID NO: 9.
Contiguous from a nucleotide sequence corresponding to the 5 'end of the sequence from 0 to nucleotide 2968 to a nucleotide sequence corresponding to the 3' end of the sequence from nucleotide 170 to nucleotide 2968 of SEQ ID NO: 9. Also preferably, the nucleotide sequence of the polynucleotide isolated by the above method corresponds to nucleotide 1370 to nucleotide 2968 of the cDNA sequence of SEQ ID NO: 9, and corresponds to nucleotide 1370 to nucleotide 2968 of SEQ ID NO: 9. It is a continuous sequence from the nucleotide sequence corresponding to the 5 'end of the sequence to the nucleotide sequence corresponding to the 3' end of the sequence from nucleotide 1370 to nucleotide 2968 of SEQ ID NO: 9. In another embodiment, the present invention relates to a composition comprising a protein, wherein the protein comprises: (a) an amino acid sequence of SEQ ID NO: 10; (b) a fragment of the amino acid sequence of SEQ ID NO: 10; (C) comprising an amino acid sequence selected from the group consisting of: the amino acid sequence encoded by the cDNA insert of clone yc141 deposited under accession number ATCC 98724; And compositions that are substantially free of other mammalian proteins. In a further preferred embodiment, the invention relates to a protein comprising a fragment of the amino acid sequence SEQ ID NO: 10 having biological activity, said fragment comprising SEQ ID NO: 10, preferably 8, more preferably 20, most preferably A protein comprising a fragment of the amino acid sequence of SEQ ID NO: 10 having biological activity (preferably containing 30 contiguous amino acids) (the fragment comprises the amino acid sequence from amino acid 461 to amino acid 470 of SEQ ID NO: 10) Including).

[0008] In certain preferred embodiments, the polynucleotide is operably linked to an expression control sequence. The present invention also provides host cells, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions. Also provided by the present invention are organisms having enhanced, reduced, or modified expression of a gene corresponding to a polynucleotide sequence disclosed herein. Further, a method for producing a protein, comprising: (a) growing a culture of a host cell transformed with such a polynucleotide composition in a suitable medium; and (b) purifying the protein from the culture. There is also provided a method comprising: The protein produced by such a method is also provided by the present invention. Preferred specific examples include those in which the protein produced by such a method is a mature form of the protein. The protein composition of the present invention may further contain a pharmaceutically acceptable carrier. Compositions comprising antibodies specifically reactive with such proteins are also provided by the present invention. Also provided is a method for preventing, treating or ameliorating a medical condition comprising administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the invention and a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION Isolated Proteins and Polynucleotides The nucleotide and amino acid sequences for each of the clones and proteins disclosed herein are reported below. By sequencing the deposited clones by known methods, the actual nucleotide sequence of each clone can be readily determined. The deduced amino acid sequence (both full length as well as mature) can then be determined from such nucleotide sequences. By expressing the clones in a suitable host cell, collecting the proteins, and then determining the sequence, the amino acid sequence of the protein encoded by each clone can also be determined. For each protein disclosed, Applicants have identified what has been determined to be the best identifiable reading frame using sequence information available at the time of filing. As used herein, the term "secreted" protein refers to that which is transported across a membrane when expressed in a suitable host cell, including transport as a result of a signal sequence in its amino acid sequence. “Secreted” proteins include, but are not limited to, proteins that are totally secreted (eg, soluble proteins) or partially secreted (eg, receptors) from the cell in which they are expressed. "Secreted" proteins also include, but are not limited to, those that are transported across the endoplasmic reticulum membrane.

Clone "ya91" The polynucleotide of the present invention has been identified as clone ya91. ya9
1 is a method selective for cDNA encoding a secretory protein (US Pat. No. 553).
No. 6637), or was identified as encoding a secreted or transmembrane protein based on computer analysis of the amino acid sequence of the encoded protein. ya91 is a full-length clone, containing the entire coding sequence of a secreted protein (also referred to herein as "ya91 protein"). The nucleotide sequence of ya91 determined this time is shown in SEQ ID NO: 1, which contains the poly (A) tail. What the applicant considers to be the correct reading frame and deduced amino acid sequence of the ya91 protein corresponding to the above nucleotide sequence is shown in SEQ ID NO: 2. Amino acids 15 to 27 of SEQ ID NO: 2 are a predicted leader / signal sequence, with the predicted mature amino acid sequence beginning at amino acid 28. Because the putative leader / signal sequence is hydrophobic, the putative leader /
If the signal sequence is not separated from the rest of the ya91 protein, it may act as a transmembrane domain. EcoRI / NotI obtainable from the deposit containing clone ya91
The restriction fragment should be about 950 bp in length. The nucleotide sequence of ya91 disclosed herein was searched against the GenBank and GenSeq nucleotide sequence databases using BLASTN / BLASTX and FASTA search protocols. ya91 is AA442366 (zv62c04.r1 Soares testis
NHT Homo sapiens cDNA clone 758214 5 ') and AA609166 (af12a08.s1 Soar
It showed at least some similarity to the sequence identified as es testis NHT Homo sapiens cDNA clone 1031414 3 '). Based on sequence similarity, ya9
One protein and each similar protein or peptide may share at least some activity. According to the TopPredII computer program, additional potential transmembrane domains centered around amino acid 62 of SEQ ID NO: 2 in the ya91 protein sequence are expected.

Clone "ya11 1" The polynucleotide of the present invention has been identified as clone ya11-1. ya
111 is a method selective for cDNA encoding a secretory protein (US Pat.
No. 5,366,637), and was identified as encoding a secreted or transmembrane protein based on computer analysis of the amino acid sequence of the encoded protein. ya11 1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as “ya11 1 protein”). The nucleotide sequence of ya11 1 determined this time is shown in SEQ ID NO: 3, which includes the poly (A) tail. The present applicant has determined that the y corresponding to the above nucleotide sequence
The correct reading frame and deduced amino acid sequence of the a11 1 protein are shown in SEQ ID NO: 4. Amino acids 36 to 48 of SEQ ID NO: 4 are a predicted leader / signal sequence, with the predicted mature amino acid sequence beginning at amino acid 49. Because the putative leader / signal sequence is hydrophobic, if it is not separated from the rest of the ya11 1 protein, it may act as a transmembrane domain. EcoRI / Not obtainable from the deposit containing clone ya111
The I restriction fragment should be approximately 500 bp in length. The nucleotide sequence of ya111 disclosed herein was searched against the GenBank and GeneSeq databases using BLASTA / BLASTX and FASTA search protocols. ya11 1 is Z68274 (Human DNA sequence from cosmid L129H
7, Huntington's Disease Region, chromosome 4p16.3 contains Pseudogene an
d CpG island) showed at least some similarity to the sequence identified. Based on sequence similarity, the ya11 1 protein and each similar protein or peptide may share at least some activity. According to the TopPredII computer program, the presence of a further potential transmembrane domain centering around amino acid 81 of SEQ ID NO: 4 in the ya11 1 protein sequence is expected.

Clone "ya281" The polynucleotide of the present invention has been identified as clone "ya281" . ya
281 describes a method selective for cDNA encoding a secretory protein (US Pat.
No. 5,366,637), and was identified as encoding a secreted or transmembrane protein based on computer analysis of the amino acid sequence of the encoded protein. ya281 is a full-length clone, which contains the entire coding sequence of a secreted protein (also referred to herein as "ya281 protein"). The nucleotide sequence of ya281 determined this time is shown in SEQ ID NO: 5, including the poly (A) tail. The present applicant has determined that the y corresponding to the above nucleotide sequence
SEQ ID NO: 6 shows the correct reading frame and deduced amino acid sequence of a281 protein. Amino acids 41-53 of SEQ ID NO: 6 are a predicted leader / signal sequence, with the predicted mature amino acid sequence beginning at amino acid 54. Because the putative leader / signal sequence is hydrophobic, if it is not separated from the rest of the ya281 protein, it may act as a transmembrane domain. EcoRI / Not obtainable from the deposit containing clone ya281
The I restriction fragment should be approximately 300 bp in length. The nucleotide sequence of ya281 disclosed herein was searched against the GenBank and GeneSeq databases using BLASTA / BLASTX and FASTA search protocols. ya28 1 is AA576255 (nm62b09.s1 NCI_CGAP_Br3 Homo sapie
ns cDNA clone IMAGE: 1072793) showed at least some similarity to the sequence identified. Based on sequence similarity, the ya281 protein and each similar protein or peptide may share at least some activity. The ya281 protein was expressed in a COS cell expression system, and an expressed protein band of about 23 kDa was detected in the membrane fraction using SDS-polyacrylamide gel electrophoresis.

Clone "yb81 1" The polynucleotide of the present invention has been identified as clone yb81-1. yb
811 is a method selective for cDNA encoding a secreted protein (US Pat.
No. 5,366,637) from a human fetal brain cDNA library;
Alternatively, it was identified as encoding a secreted protein or a transmembrane protein based on computer analysis of the amino acid sequence of the encoded protein. yb81 1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "yb81 1 protein"). The nucleotide sequence of yb81 1 determined this time is shown in SEQ ID NO: 7, which includes a poly (A) tail. SEQ ID NO: 8 shows what the applicant considers to be the correct reading frame and deduced amino acid sequence for yb81 1 protein corresponding to the above nucleotide sequence. EcoRI / Not obtainable from the deposit containing clone yb81_1
The I restriction fragment should be approximately 1200 bp in length. The nucleotide sequence of yb811 disclosed herein was searched against the GenBank and GeneSeq databases using BLASTA / BLASTX and FASTA search protocols. yb81 1 is T67164 (Human alpha-N-acetylglucosaminidase)
It showed at least some similarity to the sequence identified as (gene). Based on sequence similarity, the yb81 1 protein and each similar protein or peptide may share at least some activity. According to the TopPredII computer program, a potential transmembrane domain centering around amino acid 73 of SEQ ID NO: 8 in the yb811 protein sequence is expected.

Clone "yc141" The polynucleotide of the present invention has been identified as clone "yc141" . yc
141 is a method selective for cDNA encoding a secretory protein (US Pat.
No. 5,366,637) and isolated from a human fetal kidney (293 cell line) cDNA library or identified as encoding a secreted or transmembrane protein based on computer analysis of the amino acid sequence of the encoded protein. Was done. y
c141 is a full-length clone, and is a secretory protein (herein, "yc141 protein").
) Is also included. The nucleotide sequence of yc141 determined this time is shown in SEQ ID NO: 9, which includes the poly (A) tail. The present applicant has determined that the y corresponding to the above nucleotide sequence
SEQ ID NO: 10 shows the correct reading frame and deduced amino acid sequence of c141 protein. Amino acids 388-400 of SEQ ID NO: 10 are a predicted leader / signal sequence, and the predicted mature amino acid sequence is amino acid 401
Start with. Because the putative leader / signal sequence is hydrophobic, if it is not separated from the rest of the yc141 protein, it may act as a transmembrane domain. EcoRI / Not obtainable from the deposit containing clone yc141
The I restriction fragment should be approximately 3000 bp in length. The nucleotide sequence of yc141 disclosed herein was searched against the GenBank and GeneSeq databases using BLASTA / BLASTX and FASTA search protocols. yc141 is AA007392 (zh99a08.r1 Soares fetal liver sple
en 1NFLS S1 Homo sapiens cDNA clone 429398 5 '), AA573120 (nj41e10.s1 NCI
_CGAP_AA1 Homo sapiens cDNA clone IMAGE 995082 similar to TR G285999 G28
5999 ORF, COMPLETE CDS), and D13642 (Human mRNA for KIAA0017 gene, co
It showed at least some similarity to the sequence identified as mplete cds). The predicted amino acid sequence disclosed herein for the yc141 protein was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The putative yc141 protein is D13642 (KIAA0017 [Homo sapiens]) and
Z47816 (unknown [Saccharomyces cerevisiae] (S. cerevisiae chromosome XII
It showed at least some similarity to the sequence identified as I cosmid 9827, and translated products)). Based on sequence similarity, the yc141 protein and each similar protein or peptide may share at least some activity. The yc141 protein contains an immunoglobulin and a tumor histocompatibility complex protein signature at its N-terminal tip (starting at amino acid 3 of SEQ ID NO: 10). According to the TopPredII computer program, in the yc141 protein sequence, amino acids 50, 200, 210 and 830 of SEQ ID NO: 10
Four additional potential transmembrane domains, each centered around a neighborhood, are expected. The yc141 protein was expressed in the COS cell expression system, and an expressed protein band of about 92 kDa was detected in the membrane fraction using SDS-polyacrylamide gel electrophoresis.

Deposit of clones Clones ya91, ya111, ya281, yb811, and y
c141 was established on April 7, 1998 based on the Budapest Treaty by American Type
Culture Collection (10801 University Boulevard, Manassas, Virginia 20110
-2209 USA) as the original deposit and given accession number ATCC 98724 from which each clone containing an individual polynucleotide is available. All restrictions on public use of the deposited material are set forth in 37 CFR. F. R. § 1.808 (
Except for the provisions of b) above, it will be canceled by grant of patent, and the deposit period will be 37 C.E.
F. R. § 1.806. Each clone was transfected into separate bacterial cells (E. coli) as this complex deposit. EcoRI / NotI digestion (5 ′ site, EcoRI; 3)
Site, NotI) to obtain fragments of appropriate size for such clones so that each clone can be removed from the deposited vector. Each clone was deposited in either the pED6 or pNOTs vector shown in FIGS. 1A and 1B, respectively. The pED6dpc2 vector (pED6) was inserted into the pED6 vector by inserting a new polylinker to facilitate cDNA cloning.
Derived from ED6dpc1 (Kaufman et al. (1991). Nucleic Acids Res. 19
: 4485-4490). DHFR was deleted, a new polylinker was inserted, and the M13 replication start site was inserted into the ClaI site to convert pNOTs vector into pMT2 (
Kaufman et al. 1989. Mol. Cell. Biol. 9: 1741-1750). In some cases, the deposited clones "flip" (ie, flipped) in the deposited isolate. In such cases, the cDNA insert can still be isolated by EcoRI and NotI digestion. However, in that case, NotI is required to place the cDNA in the correct orientation for expression in the appropriate vector.
A 'site will be created and EcoRI will create a 3' site. The cDNA may be expressed from a vector in which the cDNA has been deposited. Bacterial cells containing individual clones can be obtained from the complex deposit as follows: The oligonucleotide probes or probes should be designed to be against the sequence known for the individual clone. This sequence can be derived from the sequences herein or a combination of those sequences. The oligonucleotide probe sequences used to isolate each full-length clone are shown below and should be the most reliable in isolating the clone of interest.

Clone probe sequence ya9 1 SEQ ID NO: 11 ya28 1 SEQ ID NO: 12 yc14 1 SEQ ID NO: 13 The sequence listed above contains an N at position 2, which is a nucleotide rather than a nucleotide in preferred probes / primers. Biotinylated phosphoramidite residues (for example, biotin phosphoramidite (1-dimethoxytrityloxy-2)
-(N-biotinyl-4-aminobutyl) -propyl-3-O- (2-cyanoethyl)-(N, N'-diisopropyl) -phosphoramidite (Glen Research catalog no. 10-1953). ). Preferably, the design of the oligonucleotide probe should follow these parameters: (a) Should be designed to be the region of the sequence where the number of undefined bases (N), if any, is minimal; (B) Tm of about 80 ° C. (2 ° C. for A or T, 4 ° C. for G or C)
(Assume ° C). Preferably, oligonucleotides should be labeled with [gamma]-< ³² > P-ATP (specific activity 6000 Ci / mmole) using T4 polynucleotide kinase, using methods widely used for oligonucleotide labeling. Other labeling methods can be used. Preferably, unincorporated label should be removed by gel filtration or other established methods. It should be quantified by measuring the amount of radioactivity incorporated into the probe with a scintillation counter. Preferably,
The specific activity of the resulting probe should be about 4e + 6 dpm / pmole. Preferably, the bacterial culture containing the pool of full-length clones should be thawed and 100 μl of the stock used to inoculate a sterile culture flask containing 25 ml of sterile L broth containing 100 μg / ml ampicillin. Preferably, the culture should be grown at 37 ° C. to reach saturation, and preferably the saturated culture should be fresh L
Should be diluted with broth. Preferably, a portion of these dilutions are plated out in a 150 mm Petri dish with 100 μg / ml ampicillin and 1.
The dilution and volume at which 5000 distinct, well-separated colonies result when grown overnight at 37 ° C. on solid bacterial culture medium containing L broth containing 5% agar should be determined. Other known methods for obtaining well-defined and well-separated colonies can also be used. The colonies should then be transferred to nitrocellulose filters using standard colony hybridization techniques and lysed, denatured, and baked. Then, preferably, 6 × SSC containing 0.5% SDS, 100 μg / ml yeast RNA, and 10 mM EDTA (205.3 × 175.3 g stock).
NaCl / liter, 88.2 g sodium citrate, pH 7.0 with NaOH
(About 10 ml per 150 mm filter) at 65 ° C. for 1 hour with gentle stirring. The probe is then preferably added to the hybridization mix so that the concentration is greater than or equal to 1e + 6 dpm / ml. The filter is then preferably washed with 500 ml of 2X SSC / 0.5% SDS without stirring at room temperature, preferably afterwards with gentle shaking at room temperature with 500 ml of 2X SSC / 0. Wash with 1% SDS. 65 ° C., 30 minutes to 1 hour, 0.1 × SSC / 0
. A third wash with 5% SDS is optimal. The filter is then preferably dried and subjected to autoradiography for a sufficient time to visualize positives on the X-ray film. Other known hybridization methods can be used. Positive colonies are picked, grown in medium, and the plasmid DNA is then isolated using standard procedures. The clone can then be verified by restriction analysis, hybridization analysis or DNA sequencing.

A fragment of the protein of the present invention that can exhibit a biological activity is also included in the present invention. Protein fragments may be linear or, for example, HUSaragovi
, et al., Bio / Technology 10, 773-778 (1992) and RSMcDowell, et al., J
Amer. Chem. Soc. 114, 9245-9253 (1992) (both references are deemed to be described herein by reference) may be cyclized using known methods. . For many purposes, including increasing the number of valencies at the protein binding site,
Such a fragment may be fused to a carrier molecule along with the immunoglobulin.
For example, a protein fragment may be fused to the Fc portion of an immunoglobulin via a "linker". For the bivalent form of the protein, such a fusion is made to the F molecule of the IgG molecule.
This can be done for part c. Such fusions may be obtained using other immunoglobulin isotypes. For example, a protein-IgM fusion will yield a ten-valent form of a protein of the invention. The invention also provides the full length and mature forms of the disclosed proteins. The full length form of such a protein has been identified in the sequence listing by translation of the nucleotide sequence of the disclosed clone. Mature forms of such proteins may be obtained by expressing the disclosed full-length polynucleotides (preferably those deposited with the ATCC) in appropriate mammalian cells or other host cells. The sequence of the mature form of the protein may be determined from the amino acid sequence of the full-length form.

[0018] The present invention also provides a gene corresponding to the cDNA sequence disclosed herein. "
A “corresponding gene” is a region of the genome that is transcribed to produce mRNA,
CDNA is derived from the RNA and the flanking regions of the genome (including coding sequences, 5 'and 3' untranslated regions) or spliced exons, introns, promoters, enhancers, and Also includes silencer or suppressor elements. Corresponding genes can be isolated using the sequence information disclosed herein. Corresponding genes can be isolated using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence information to identify and / or amplify genes in a suitable genomic library or other source of genomic material. An "isolated gene" is a gene that has been separated from flanking coding sequences, if present, present in the genome of the organism from which the gene was isolated. Chromosomal locations corresponding to the polynucleotide sequences disclosed herein may be determined, for example, by hybridizing an appropriately labeled polynucleotide of the invention to the chromosome in situ. By identifying significantly similar nucleotide sequences in public databases, such as expressed sequence tags (ESTs) that have already been mapped to specific chromosomal locations, the corresponding chromosomal location of the disclosed polynucleotides may be determined. For at least some of the polynucleotide sequences disclosed herein, public database sequences having at least some similarity to the polynucleotides of the present invention are listed by database accession number. A search using the GenBank accession number of these public database sequences to identify UniGene clusters of overlapping sequences was performed at the National Center for Biotechnology I
This can be done on the internet site provided by nformation, the address of which is http://www.ncbi.nlm.nih.gov/UniGene/. Many UniGene clusters so identified have already been mapped to specific chromosomal locations.

An organism is provided that has enhanced, reduced, or altered expression of a gene corresponding to a polynucleotide sequence disclosed herein. Desirable changes in gene expression are achieved by using antisense polynucleotides or by using ribozymes that bind and / or cleave mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol. Sci. 15 (7): 250-2
54; Lavarosky et al., 1997, Biochem.Mol. Med. 62 (1): 11-22; and Hampel,
1998, Prog. Nucleic Acid Res. Mol. Biol. 58: 1-39; all of which are herein incorporated by reference). A transgenic animal having a multicopy gene corresponding to a polynucleotide disclosed herein, preferably obtained by transforming cells with a genetic construct that is stably maintained in transformed cells and their progeny. A transgenic animal is provided. Also provided are transgenic animals having altered genetic control regions that increase or decrease gene expression levels or alter the temporal or spatial pattern of gene expression (see European Patent No. 0 649 464 B1; All of which are considered to be described herein by reference). In addition, an organism is provided wherein the gene corresponding to the polynucleotide sequence disclosed herein has been incompletely or completely inactivated, either by insertion of an external sequence or by deletion of all or part of the corresponding gene. By insertion, preferably by insertion after incorrect resection of the transposable element (Plasterk, 1992, Bioessays 14 (9): 629-633; Zwaal et al., 19).
93, Proc. Natl. Acad. Sci. USA 90 (16): 7431-7435; Clark et al., 1994, Pr.
USA 91 (2): 719-722; all of which are deemed to be described herein by reference), or by homologous recombination, preferably by positive / negative genetic selection. Homologous recombination (Mansour et a
l., 1988, Nature 336: 348-352; US 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 deemed herein to be incorporated by reference), incomplete or complete gene inactivation can be performed. These organisms with altered gene expression
Preferably they are eukaryotes, and more preferably mammals. Such organisms are useful for developing non-human models for studying diseases associated with the corresponding gene, and for developing assay systems for identifying molecules that interact with protein products from the corresponding gene. Where the protein of the invention is membrane-bound (eg, a receptor), the invention also provides soluble forms of such protein. In such a form, some or all of the intracellular and transmembrane domains of the protein are removed so that the protein is sufficiently secreted from the host in which it was expressed. The intracellular and transmembrane domains of the protein of the present invention can be identified by known methods for determining such domains from sequence information. For example, TopP
The position of the transmembrane domain in the amino acid sequence can be predicted using the redII computer program, and those domains are described by the central position of the transmembrane domain
With at least 10 transmembrane amino acids on either side of the described central residue. The proteins and protein fragments of the present invention comprise at least 25% (more preferably at least 50%, most preferably at least 75%) of the amino acid sequence of the disclosed protein.
And has at least 60% sequence identity (more preferably at least 75% identity, most preferably at least 90%) to the disclosed proteins.
Or 95% identity). Sequence identity is determined by comparing the amino acid sequences of the proteins when the sequences are aligned to maximize overlap and identity while minimizing sequence gaps. It has at least 75% sequence identity (more preferably at least 85% identity, most preferably at least 95% identity) to any of the disclosed protein segments, preferably 8 or more ( Proteins or protein fragments containing segments comprising more preferably 20 or more, most preferably 30 or more) contiguous amino acids are also encompassed by the present invention.

In detail, WU-BLAST (Washington University BLAST) version 2. 0 software may be used to determine sequence identity, which is a publicly available unrestricted WBI based on NCBI-BLAST version 1.4.
-Built based on BLAST (Altschul and Gish, 1996, Local ali
gnment statistics, Doolittle ed., Methods in Enzymology 266: 460-480; Al
tschul et al., 1990, Basic local alignment search tool, Journal of Molec
ular Biology 215: 403-410; Gish and States, 1993, Identification of prot
ein coding regions by database similarity search, Nature Genetics 3: 266
-272; Karlin and Altschul, 1993, Applications and statistics for multipl
e high-scoring segments in molecular sequences, Proc. Natl. Acad. Sci. U
SA 90: 5873-5877 (all of these references are hereby incorporated by reference)). WU-BLAST for some UNIX platforms
Ftp://blast.wustl.edu/blast/executab version 2.0 executable
You can download from les. In addition to several support programs, a complete set of search programs (BLASTP, BLASTN, BLASTX,
TBLASTN and TBLASTX) are provided at this site. WU
-BLAST 2.0 is copyrighted and must not be sold or redistributed in any form without the consent of the author, but may be used for non-business or research purposes. Can be used for Pair (BLASTP, BLAST
N, BLASTX, TBLASTN, and TBLASTX), the gap alignment routine (gapped alignment routine)
routines) are integrated into the database search itself, resulting in highly sensitive, highly selective and easily interpretable results. Optimally, gapping can be turned off in all these programs. The default penalty (Q) for the length gap is Q = 9 for BLASTP and Q = 10 for BLASTN, but 1 to 8, 9, 10, including zero. It may be changed to any integer value such as 11, 12 to 20, 21 to 50, and 51 to 100. Residue 1 to widen the gap
The default penalty (R) per unit is R = 2 for protein and BLASTP, and R = 10 for BLASTN, but 0, 1, 2, 3
, 4, 5, 6, 7, 8, 9, 10, 11, 12 to 20, 21 to 50, 5,
It may be changed to an integer value such as 1 to 100. Any combination of Q and R may be used to compare sequences side-by-side to maximize overlap and identity and minimize sequence gaps. The default amino acid comparison matrix is BLOS
Although UM62, other amino acid comparison matrices such as PAM can be used.

[0021] Species homologs of the disclosed polynucleotides and proteins are also provided by the invention. The term "species homolog" herein is a protein or polynucleotide that relates to a species different from the source of the protein or polynucleotide, but as determined by one of skill in the art,
Those having significant sequence similarity. Preferably, the polynucleotide species homolog has at least 60% (more preferably at least 75%, most preferably at least 90%) sequence identity to the particular polynucleotide, and the protein species homolog is At least 30% (more preferably at least 45%)
%, Most preferably at least 60%). Here, sequence identity is determined by comparing the nucleotide sequence of a polynucleotide or the amino acid sequence of a protein juxtaposed such that overlap and identity are maximized and sequence gaps are minimized. Species homologs may be isolated and identified by generating appropriate probes or primers from the sequences provided herein, and then screening for an appropriate nucleic acid source from the desired species. Preferably, the species homolog is isolated from a mammalian species. Most preferably, the species homolog is, for example, Pan troglodyte
s, Gorilla gorilla, Pongo pygmaeus, Hylobates concolor, Macaca mulatta,
Papio papio, Papio hamadryas, Cercopithecus aethiops, Cebus capucinus, A
otus trivirgatus, Sanguinus oedipus, Microcebus murinus, Mus musculus, R
attus norvegicus, Crisetulus griseus, Felis catus, Mustela vison, Canis
It has been isolated from specific mammals such as familiaris, Oryctolagus cuniculus, Bos taurus, Ovis aries, Sus scrofa and Equus caballus, and has been genetically mapped to identify the genes in one species. It is possible to identify the association of gene sequence order between genomic organization and genomic organization of 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., 199
5, 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 Stu
bbs, 1997, Genome Research 7: 1123-1137, all of which are deemed to be described herein by reference).

The present invention also relates to allelic variants of the disclosed polynucleotides, ie, the spontaneous identification of isolated polynucleotides encoding proteins identical, homologous or related to the protein encoded by the disclosed polynucleotides. Form. Preferably, the allelic variant is at least 60% (for a particular polynucleotide)
More preferably at least 75%, most preferably at least 90%), wherein the sequence identity is such that the polynucleotides are juxtaposed such that overlap and identity are maximized and sequence gaps are minimized. By comparing the nucleotide sequences of Allelic variants may be isolated and identified by generating appropriate probes or primers from the sequences described herein and screening for an appropriate source of nucleic acid from an individual of the appropriate species. The present invention also encompasses polynucleotides having sequences complementary to the sequences of the polynucleotides disclosed herein.

[0023] The present invention also relates to a polynucleotide that is capable of hybridizing to a polynucleotide described herein under reduced stringency conditions, more preferably under stringent conditions, and most preferably under very stringent conditions. Also encompasses nucleotides. The following table shows examples of strictness conditions. The very strict conditions are, for example, at least as strict as the conditions A to F, and the strict conditions are, for example, at least as strict as the conditions GL, and the reduced conditions are, for example, For example, it is at least as strict as the conditions M to R.

[Table 1] a): Hybrid length is the expected length for the hybridizing region (s) of the hybridizing polynucleotide. When hybridizing a polynucleotide to a target polynucleotide of unknown sequence, the hybrid length is assumed to be the length of the hybridizing polynucleotide. When polynucleotides of known sequence hybridize, the length of the hybrid can be determined by juxtaposing the polynucleotide sequences and identifying the region (s) of optimal sequence complementarity. b): Hybridization and SSPE (1X SSPE is 0.15 M NaCl in the wash buffer, 10mM NaH ₂ PO _4, and 1.25 mM EDTA, pH 7.4 at a) the SSC (IX SSC is 0.15 M NaCl and 15mM Sodium citrate). After completion of hybridization, washing is performed for 15 minutes. * T _B -T _R : The hybridization temperature for hybrids expected to be less than 50 base pairs in length should be 5-10 ° C below the melting temperature (T _m ) of the hybrid. T _m is determined by the following equation. For hybrids less than 18 base pairs in length, T _m (° C.) = 2 (number of A + T bases) +4 (number of G + C bases). For hybrids 18 to 49 base pairs in length, T _m (° C.) = 81.5 + 16.6 (log ₁₀ [Na ⁺ ]) + 0.41 (% G + C) − (600 / N), where N is the hybrid And the sodium ion concentration in the hybridization buffer ([Na ⁺ ] for 1 × SSC = 0.165 M). Further examples of stringency conditions for polynucleotide hybridization are described in Sambrook, J., EF Fritsch, and T. Maniatis, 1989, Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Ha
rbor, NY, chapters 9 and 11, and Current Protocols in Molecular Biology,
1995, FM Ausubel et al., Eds., John Wiley & Sons, Inc., sections 2.10
and 6.3-6.4 and are deemed to be incorporated herein by reference. Preferably, each such hybridizing polynucleotide is at least 25% of the polynucleotide of the invention to be hybridized.
(More preferably, at least 50%, most preferably at least 75%) and at least 60% sequence identity (more preferably at least 75%) to the polynucleotide of the invention to be hybridized. , Most preferably at least 90% or 95% identity). Sequence identity is determined by comparing the amino acid sequences of the proteins when the sequences are aligned to maximize overlap and identity while minimizing sequence gaps.

[0024] The isolated polynucleotide encoding the protein of the present invention is described in Kaufman et al., Nucl.
The protein may be recombinantly produced by operably linking it to an expression control sequence such as the pMT2 or pED expression vector disclosed in eic Acids Res. 19, 4485-4490 (1991). Many suitable expression control sequences are known in the art. Many suitable expression control sequences are known in the art. General methods for recombinant protein expression are also known and are described in R. Kaufman, Methods in Enzymology 185, 537-566.
(1990) is a typical example. "Operably linked" as defined herein means that the isolated polynucleotide of the present invention and the expression control sequence are placed in a vector or cell and transformed (transfected) with the ligated polynucleotide / expression control sequence. It is meant to be expressed by a host cell. Many types of cells can act as suitable host cells for expressing the proteins of the present invention.
Mammalian cells include, for example, monkey COS cells, Chinese hamster ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo2
05 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell lines obtained from in vitro culture of primary tissues, primary explants, HeLa cells, mouse cells, BHK HL-60, U937HaK or Ju
rkat cells. Alternatively, the protein can be produced in lower eukaryotic cells such as yeast or prokaryotic cells such as bacteria. A potentially suitable yeast strain is Saccharomyces ce
revisiae, Schizosaccharomyces pombe, Kluyveromyces strain, Candida, or yeast strains capable of expressing heterologous proteins. A potentially suitable bacterial strain is Escherichia
E. coli, Bacillus subtilis, Salmonella typhimurium, or bacterial strains capable of expressing heterologous proteins. If the protein is produced in yeast or bacteria, it may be necessary to modify the protein produced therein, for example, by phosphorylation or glycosylation at appropriate sites to obtain a functional protein. Such covalent linkages may be made using known chemical or enzymatic methods. The isolated polynucleotides of the present invention may be operably linked to appropriate control sequences in one or more insect expression vectors and the protein obtained by using an insect expression system. Materials and methods for baculovirus / insect cell expression systems are described, for example, in In
Commercially available in kit form from Vitrogen, San Diego, California, USA (MaxBac ^R kit), such methods are well known in the art and are described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987).
) (Assumed to be described herein by reference). As used herein, insect cells capable of expressing a polynucleotide of the present invention have been "transformed." The protein of the present invention may be produced by culturing the transformed host cell under culture conditions suitable for expressing the recombinant protein. The resulting expressed protein may then be purified from such culture (ie, medium or cell extract) using known purification processes, such as gel filtration and ion exchange chromatography. Purification of the protein may also be accomplished by affinity columns containing an agent that will bind to the protein; one or more column steps with an affinity column such as Concanavalin A-agarose, Heparin-Toyopearl ^R or Cibacrom blue 3GA Sepharose ^R ; One or more steps using hydrophobic interaction chromatography with a resin such as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography. Alternatively, the protein of the invention may be expressed in an easily purified form. For example, maltose binding protein (MBP), glutathione-S-tonsferase (GST)
Alternatively, it may be expressed as a fusion protein such as a fusion protein with thioredoxin (TRX). Kits for expression and purification of such fusion proteins are available from New En
Commercially available from glan BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen. The protein can also be tagged with an epitope and then purified by using a specific antibody directed against such epitope. One such epitope ("flag") is commercially available from Kodak (New Haeven, CT). Finally, the protein is further purified using one or more reverse phase high quality liquid chromatography (RP-PLC) steps using a hydrophobic RP-HPLC medium, such as silica gel with pendant methyl or other aliphatic groups. be able to. Some or all of the above purification steps may be used in various combinations to obtain a substantially homogeneous isolated recombinant protein. The protein thus purified is substantially free of other mammalian proteins and is defined in the present invention as "isolated protein". The protein of the present invention is expressed as a product of a transgenic animal, for example, as a component of milk of a transgenic cow, goat, pig, or sheep characterized by somatic cells or germ cells containing a nucleotide sequence encoding the protein. You may. The protein may be produced by known conventional chemical synthesis. Methods for constructing the protein of the present invention by synthetic means are known to those skilled in the art. Synthetically constructed protein sequences may have common biological properties, including protein activity, by sharing primary, secondary or tertiary structure and / or conformational properties. Thus, they may be used as biological activities or immunological substitutes for naturally occurring purified proteins in immunological processes for screening therapeutic compounds and generating antibodies.

The proteins described herein are characterized by an amino acid sequence similar to the amino acid sequence of the purified protein, but may be modified therein, either spontaneously or by derivatization. For example, modifications of the peptide or DNA sequence can be made by those skilled in the art using known methods. Modifications of interest in the protein sequence include changes, substitutions, exchanges, insertions or deletions of selected amino acid residues in the coding sequence. For example, one or more cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Methods for such changes, substitutions, exchanges, insertions or deletions are well known to those skilled in the art (eg, US Pat. No. 415).
No. 8584). Preferably, such changes, substitutions, exchanges, insertions or deletions retain the desired activity of the protein. Other fragments and derivatives of the protein sequence that are expected to retain protein activity in whole or in part, and thus may be useful in screening or other immunological methods, are also readily made by one of skill in the art from the disclosure herein. Can be done. Such modifications are believed to be encompassed by the present invention.

Uses and Biological Activities The polynucleotides and proteins of the present invention are believed to exhibit one or more of the following uses or biological activities, including those associated with the assays cited herein. Can be The uses or activities described with respect to the proteins of the present invention may be due to the administration or use of such proteins, or the administration or use of polynucleotides encoding such proteins (eg, such as a vector suitable for gene therapy or DNA transfer). Can be provided.

Research Uses and Utility The polynucleotides provided by the present invention can be used for various purposes by a research population. For analysis, characterization or therapeutic use, as a marker of tissue in which the corresponding protein is preferentially expressed (constitutively or at a particular stage of tissue differentiation or development or in a disease state), and To identify potential genetic diseases as compared to the patient's endogenous DNA, as chromosomal markers or tags (if labeled) for identification of chromosomes or mapping of relevant gene locations, as molecular weight markers for Southern gels The use of known sequences in the process of discovering other novel polynucleotides as a source for obtaining PCR primers for genetic fingerprinting to find new related DNA sequences to use as hybridization probes "Gene chips" or other probes as probes for subtraction To select and generate oligomers for binding to carriers, to test expression patterns, to generate anti-protein antibodies using DNA immunization techniques, and to generate anti-DNA antibodies or to generate additional immunizations. Polynucleotides can be used as antigens to induce a response. If encoding a protein that binds or potentially binds to another protein (e.g., as in a receptor-ligand interaction), identify the other protein that binds, and / or an inhibitor of the binding interaction Interaction trap assays to identify DNA (eg, Gyuris et al., Cell 75: 791-803 (1993) and Rossi et al., 19).
97, Proc. Natl. Acad. Sci. USA 94: 8405-8410). The proteins provided by the present invention may be used in assays to determine biological activity, including a group of multiple proteins for high-throughput screening; to generate antibodies or induce other immune responses; As a reagent (including a labeling reagent) in an assay designed to quantitatively determine the level of a protein (or its receptor) in a biological fluid; the corresponding protein is preferentially expressed (constitutively Or expressed at a particular stage of tissue differentiation or development, or in a disease state) as a marker for tissue; and, of course, may be used to isolate relevant receptors or ligands. Where a protein binds or potentially binds to another protein, the protein can be used to identify other proteins that bind, or to identify inhibitors of the binding interaction. Using proteins involved in these binding interactions, screening for peptides or small molecular inhibitors or agonists for the binding interaction can also be performed. Any or all of these research applications can be evolved into reagent grade or kit formats for commercialization as research products. Methods for performing the above uses are well known to those skilled in the art. A literature disclosing such a method can be found in "Molecular Cloning: A Laboratory Manual", 2nd ed., Cold Sp.
ring Harbor Laboratory Press, Sambrook, J., EF Fritsch and T. Maniatis
eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Tec
hniques ", Academic Press, Berger, SL and AR Kimmel eds., 1987.

Use as Nutrition The polynucleotides and proteins of the present invention can be used as nutrient sources or additives. Such uses include, but are not limited to, use as a protein or amino acid additive, use as a carbon source, use as a nitrogen source, and use as a carbohydrate source. In such cases, the proteins or polynucleotides of the present invention may be added as food for a particular organism, or may be administered as a separate solid liquid formulation such as in the form of a powder, pill, solution, suspension or capsule. . In the case of a microorganism, the protein or polynucleotide of the present invention can be added to a medium in which the microorganism is cultured.

Cytokines and Cell Proliferation / Differentiation Activity The protein of the present invention may exhibit cytokine activity, cell proliferation activity (induction or inhibition) or cell differentiation activity (induction or inhibition), or produce other cytokines in certain cell populations. Can be induced. Many proteinaceous factors (including all known cytokines) that have been found to date have demonstrated activity in one or more factor-dependent cell proliferation assays, thus making the assay a convenient method of confirming cytokine activity. Serve as. The activity of the protein of the present invention is determined in cell lines (32D, DA
2, DA1G, T10, B9, B9 / 11, BaF3, MC9 / G, M + (pr
eB M +), 2E8, RB5, DA1, 123, T1165, HT2, CTL L2, TF-1, Mo7e and CMK). Confirmed by either of The activity of the protein of the invention may be measured, inter alia, by the following method: Assays for T cell or thymocyte proliferation are described in Current Protocols in I.
mmunology, Ed by JE Coligan, AM Kruisbeek, DH Margulies, EM She
vach, W Strober, Pub.Green Publishing Associates and Wiley-Interscienc
e (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Ch
apter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137: 3
494-3500, 1986; Bertagnolli et al., J. Immunol. 145: 1706-1712, 1990; Ber
tagnolli et al., Cellular Immunology 133: 327-341, 1991; Bertagnolli, et
al., J. Immunol. 149: 3778-3783, 1992; Bowman et al., J. Immunol. 152: 17
56-1761, 1994, including, but not limited to. Assays for cytokine production and / or proliferation of spleen cells, lymph node cells or thymocytes are described in Polyclonal T cell stimulation, Kruisbeek, AM
d Shevach, EM In Current Protocols in Immunology. JEea Coligan eds
Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Meas
urement of mouse and human Interferon g, Schreiber, RD In Current Pro
tocols in Immunology. JEea Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John
Includes, but is not limited to, those described in Wiley and Sons, Toronto. 1994. Assays for proliferation and differentiation of hematopoietic and lymphogenic cells are described in the Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottoml
y, K., Davis, LS and Lipsky, PE In Current Protocols in Immunology.
JEea Coligan eds.Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toron
to. 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.
USA 80: 2931-2938, 1983; Measurement of mouse and human interleukin 6
-Nordan, R. In Current Protocols in Immunology. JEea Coligan eds. V
ol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto.1991; Smith et al.,
Proc. Natl. Acad. Sci. USA 83: 1857-1861, 1986; Measurement of human I
nterleukin 11-Bennett, F., Giannotti, J., Clark, SC and Turner, K. J
. In Current Protocols in Immunology. JEea Coligan eds. Vol 1 pp.
6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse and hum
an Interleukin 9-Ciarletta, A., Giannotti, J., Clark, SC and Turner,
KJ In Current Protocols in Immunology. JEea Coligan eds. Vol 1 p
p. 6.13.1, including but not limited to those described in John Wiley and Sons, Toronto. 1991. Assays for the response of T cell clones to antigens, specifically identifying APC-T cell interactions as well as direct T cell effects by measuring proliferation and cytokine production, are described in Current Protocols in Immunology, Ed by JE.
Coligan, AM Kruisbeek, DH Margulies, EM Shevach, W Strober, Pub.
Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro
assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their ce
llular receptors; Chapter 7, Immunologic studies in Humans); Weinberger
Natl. Acad. Sci. USA 77: 6091-6095, 1980; Weinberger et al.
, Eur. J. Immun. 11: 405-411, 1981; Takai et al., J. Immunol. 137: 3494-35.
00, 1986; Takai et al., J. Immunol. 140: 508-512, 1988, but are not limited thereto.

Immunostimulatory or Suppressive Activity The protein of the present invention may also exhibit immunostimulatory or immunosuppressive activity, including but not limited to the activity in the assays described herein. Protein is
It may be useful in a variety of deficiencies and disorders, including severe immunodeficiency complications (SCID), for example, in up-regulating or down-regulating T and / or B lymphocyte proliferation, and in N
It may be useful in activating the cytolytic activity of K cells and other cell populations. These immunodeficiencies can be genetic and can include viral (
For example, it may be caused by HIV) and bacterial or fungal infections, or may be caused by an autoimmune disease. More specifically, infectious diseases caused by viruses, bacteria, fungi or other infectious agents, for example, various fungal infections such as HIV, hepatitis virus, herpes virus, mycobacteria, Leishmania, malaria and Candida species, It can be treated using the protein of the present invention. Of course, in this regard, the proteins of the invention may be used where a boost to the immune system is needed, ie, in the treatment of cancer. Autoimmune diseases that may be treated using the protein of the present invention include, for example, multiple sclerosis,
Systemic deep elitomades, rheumatoid arthritis, autoimmune pneumonia, Guillain-Barre syndrome, autoimmune thyroiditis, insulin-dependent diabetes mellitus, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory Eye diseases. Such proteins of the invention may be used in the treatment of allergic reactions and conditions, such as asthma or other respiratory diseases. Other conditions in which immunosuppression is desired, including, for example, asthma (particularly allergic asthma) and related respiratory problems, may be treated with the proteins of the invention. Other conditions for which immunosuppression is desired (including, for example, organ transplantation)
May be treated using the protein of the present invention. The proteins of the present invention can also be used to enable an immune response in a number of ways. Down-regulation may be in the form of inhibiting or blocking an immune response already in progress, or may involve interfering with the induction of an immune response. The function of activated T cells may be inhibited by suppressing T cell responses, or by inducing specific resistance in T cells, or both. In general, immunosuppression of a T cell response is an active, non-antigen-specific process that requires continuous exposure of T cells to an inhibitor. Tolerance means non-responsiveness or anergy in T cells, and differs from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent is stopped.
Operationally, resistance may be indicated by a lack of a T cell response when exposed to a specific antigen in the absence of a tolerizing agent. Down-regulating or preventing one or more antigen functions, including but not limited to B-lymphocyte antigen functions (eg, such as B7), eg, high levels of lymphokines by activated T cells Interference with synthesis may be useful in tissue, skin and organ transplantation and graft versus host disease (GVHD). For example, blocking T cell function should reduce tissue destruction in tissue transplantation. Typically, in tissue transplants, rejection of the graft is initiated by the recognition of the tissue piece as foreign by T cells, followed by an immune response that destroys the graft. A monomeric form having the activity of a molecule that inhibits or blocks the interaction of a B7 lymphocyte antigen with its native ligand on immune cells (another B lymphocyte antigen (eg, B7-1, B7-3)) Mixed with peptides,
Alternatively, pre-transplant administration of a soluble, monomeric form of a peptide with B7-2 activity alone, induces binding of the molecule to its native ligand on immune cells without transmitting a responsive costimulatory signal. there is a possibility. Blocking B lymphocyte antigen function in this regard prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may be sufficient to cause a loss of T cell response. The induction of long-term tolerance by B lymphocyte antigen blocking reagents may obviate the need for repeated administration of these blocking reagents. To achieve sufficient immunosuppression or resistance in a subject, it may also be necessary to block the function of the B lymphocyte antigen combination. The efficacy of individual blocking reagents in preventing organ transplant rejection or GVHD can be evaluated using animal models that can predict efficacy in humans. Examples of suitable systems that can be used include allogeneic heart grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which are used to test the immunosuppressive effects of CTLA4Ig fusion proteins in vivo. (Lensch
ow et al., Science 257: 789-792 (1992) and Turka et al., Proc Natl. Acad.
Sci. USA, 89: 11102-11105 (1992)). In addition, a rat model of GVHD (Paul ed., Fundamental Immunology, Ravan Press, New York, 1
989, pp. 846-847) can be used to determine the blocking effect of B lymphocyte antigen function on the progression of the disease in vivo. Also, blocking antigen function can be therapeutically useful for treating autoimmune diseases. Many autoimmune diseases are the result of inappropriate activation of T cells that are reactive to self tissue and promote the production of cytokines and autoantibodies that are involved in the pathology of the disease. Interfering with the activation of autoreactive T cells may reduce or eliminate the symptoms of the disease. Receptors for B lymphocyte antigens: autoantibodies or T cell-derived that inhibit T cell activation using administration of reagents that block T cell costimulation by disrupting ligand interactions and may be involved in the disease process Can interfere with the production of cytokines. In addition, blocking reagents can induce antigen-specific resistance of autoreactive T cells that can lead to long-term remission of the disease. The effectiveness of a blocking reagent in preventing or ameliorating an autoimmune disease can be determined using a number of well-characterized animal models for human autoimmune diseases. Examples are murine experimental autoimmune encephalitis, MRLlpr / lpr mice or N
Systemic deep erythematosus, murine autoimmune collagen arthritis in ZB hybrid mice, diabetes in NOD mice and BB rats, and experimental myasthenia gravis in rats (Paul ed., Fundamental Immunology, Raven Press,
New York, 1989, pp. 840-856). Up-regulation of antigen function (preferably B-lymphocyte antigen function) as a means to up-regulate the immune response is also therapeutically useful.
Up-regulation of the immune response may be in the form of promoting an existing immune response or eliminating the initial immune response. For example, enhancing an immune response by stimulating B lymphocyte antigen function may be useful in the case of a viral infection.
In addition, systemic viral diseases such as influenza, common cold, and encephalitis may be ameliorated by systemic administration of stimulatory forms of B lymphocyte antigen. Alternatively, the T cells are taken from a patient and co-stimulated in vitro with the viral antigen to which the ACPs expressing the peptide of the invention have been added, or combined with a stimulatory form of the soluble peptide of the invention and then in vitro. By reintroducing the T cells activated in the above into the patient, the antiviral immune response in the infected patient may be promoted. Another method of promoting an antiviral immune response is
The infected cells are removed from the patient and transfected with them a nucleic acid encoding a protein of the invention as described herein so that the cells express all or part of the protein on their surface. Will be re-introduced. The infected cells would then be able to transmit the costimulatory signal to the T cells in vivo and thereby activate the T cells. In another application, up-regulation or promotion of antigen function (preferably B lymphocyte antigen function) may be useful in inducing tumor immunity. Administering a tumor cell (eg, sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleic acid encoding at least one peptide of the present invention to a tumor-specific tumor in the subject Can overcome resistance. If desired, tumor cells can be transfected to express the peptide combination. For example, an expression vector that directs the expression of tumor cells obtained from a patient in combination with a peptide having B7-2-like activity alone or with a peptide having B7-1-like activity and / or B7-3-like activity, It can be transfected ex vivo. The transfected tumor cells are returned to the patient and the peptide is expressed on the surface of the transfected cells. Alternatively, tumor cells can be targeted for transfection in vivo using gene therapy. The presence of a peptide of the invention having the activity of a B lymphocyte antigen on the surface of tumor cells provides the necessary costimulatory signals for T cells to induce an immune response to transfected tumor cells mediated by T cells I do. In addition, MHC
Tumor cells lacking class I or MHC class II molecules, or sufficient MHC
Class I or tumor cells can not be re-expressed MHC class II molecules, MHC class Iα chain protein and beta ₂ microglobulin protein or an MHC class IIα chain Contact and MHC class IIβ chain all or part of the protein (e.g., truncations protein The nucleic acid encoding the cytoplasmic domain can be transfected, thereby allowing the expression of class I or class II MHC proteins on the cell surface. Expression of appropriate Class I or Class II HMC in combination with a peptide having the activity of a B lymphocyte antigen (eg, B7-1, B7-2, B7-3) is transfected by T cells. Induces an immune response against tumor cells. If desired, a gene encoding an antisense construct that blocks expression of an MHC class II binding protein, such as an invariant chain, can be co-transfected with a DNA encoding a peptide having B lymphocyte antigen activity. It can also enhance the presentation of tumor-associated antigens and induce tumor-specific immunity. Thus, induction of an immune response mediated by T cells in a human subject may be sufficient to overcome tumor-specific resistance in the subject.

The activity of the protein of the invention may be measured, inter alia, by the following method: Assays suitable for thymocyte or spleen cell cytotoxicity are described in Current Protocols
in Immunology, Ed by JE Coligan, AM Kruisbeek, DH Margulies, EM
Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Intersc
ience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19
; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl
Acad. Sci. USA 78: 2488-2492, 1981; Herrmann et al., J. Immunol. 128:
1968-1974, 1982; Handa et al., J. Immunol. 135: 1564-1572, 1985; Takai e
t 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.
Herrmann et al., J. Immunol. 128: 1968-1974, 1982; Handa et al., 1981;
J. Immunol. 135: 1564-1572, 1985; Takai et al., J. Immunol. 137: 3494-3
500, 1986; Bowmanet al., J. Virology 61: 1992-1998; Takai et al., J. Imm
unol. 140: 508-512, 1988; Bertagnolli et al., Cellular Immunology 133: 3
27-341, 1991; including but not limited to the assays described in Brown et al., J. Immunol. 153: 3079-3092, 1994. Assays for T cell-dependent immunoglobulin responses and isotype switching, specifically to modulate T cell-dependent antibody responses and identify proteins that affect Th1 / Th2 profiles, are described in Maliazewski, J. Immunol. 144: 3028-3033, 1990, including but not limited to assays for B cell function as described in In vitro antibody production, Mond, JJ and Brunswick, M. In Current.
Protocols in Immunology JEColigan eds.Val 1 pp.3.8.1-3.8.16, John Wile
y and Sons, Tronto 1994. Mixed lymphocyte reaction (MLR) assays, which identify proteins that primarily produce Th1 and CTL responses, are described in Current Protocols in Immunology, Ed by
JE Coligan, AM Kruisbeek, DH Margulies, EM Shevach, W Strober,
Pub.Green Publishing Associates and Wiley-Interscience (Chapter 3, In
Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunol
ogic 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. Im.
munol. 149: 3778-3783, 1992, but is not limited thereto. Dendritic cell-dependent assays, specifically identifying proteins expressed by dendritic cells that activate intact T cells, are described by Guery et al., J. Immunol. 134: 536-544.
, 1995; Inaba et al., Journal of Experimental Medicine 173: 549-559, 199
1; Macatonia et al., Journal of Immunology 154: 5071-5079, 1995; Porgado
r et al., Journal of Experimental Medicine 182: 255-260, 1995; Nair et a
l., 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, including, but not limited to. Assays for lymphocyte survival / apoptosis, specifically identifying proteins that prevent apoptosis following superantibody induction, as well as proteins that regulate lymphocyte homeostasis, are described by Darzynkiewicz et al., Cytometry 13: 795-808, 1992; Gorczyca et al.
, Leukemia 7: 659-670, 1993; Gorczyca et al., Cancer Research 53: 1945-1
951, 1993; Itoh et al., Cell 66: 233-243, 1991; Zacharchuk, Journal of I
mmunology 145: 4037-4045, 1990; Zamai et al., Cytometry 14: 891-897, 199
3; including but not limited to the assays described in Gorczyca et al., International Journal of Oncology 1: 639-648, 1992. Early stage T cell commitment and development assays
Antica et al., Blood 84: 111-117, 1994; Fine et al., Cellular Immunology 15
5: 111-122, 1994; Galy et al., Blood 85: 2770-2778, 1995; Toki et al., Pr
USA 88: 7548-7551, 1991, including but not limited to oc. Natl. Acad. Sci.

Hematopoietic modulatory activity The proteins of the present invention are useful in the regulation of hematopoiesis and therefore in the treatment of bone marrow and lymphocyte deficiencies. Even minimal biological activity supporting colony forming cells or factor-dependent cell lines has been implicated in regulating hematopoiesis.
For example, supporting the proliferation of erythroid progenitor cells alone, or in combination with other cytokines, for example, showing usefulness in the treatment of various anemias, or in combination with radiation therapy / chemotherapy, erythroid progenitor cells And / or stimulating the production of erythroblasts; supporting the proliferation of bone marrow cells such as granulocytes and monocytes / macrophages (ie, traditional CSF activity), eg, in combination with chemotherapy, Useful in preventing myelosuppression that occurs; supporting proliferation of megakaryocytes and then platelets, and thereby preventing or treating various platelet disorders such as thrombocytopenia; It is also commonly used instead of or in complement to platelet infusion; and / or hematopoietic stem cells (mature Becomes all of hematopoietic cells, therefore, a variety of stem cell disease (
It is usually a disease that is treated by transplantation and is useful in supporting the growth of, for example, aplastic anemia and paroxysmal nocturnal hemoglobinuria), which is useful in vivo or ex vivo. It is also useful in repopulating the stem cell compartment as normal cells after radiation / chemotherapy with (ie, combined with bone marrow transplantation), or after being genetically engineered for gene therapy. In particular, the activity of the protein of the present invention may be measured by the following method. Assays suitable for the proliferation and differentiation of various hematopoietic cell lines have already been cited. Assays for embryonic stem cell differentiation, particularly those that identify proteins that affect embryonic hematopoiesis, are 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., B
lood 81: 2903-2915, 1993, including, but not limited to. Assays for stem cell survival and differentiation (particularly identifying proteins that regulate lympho-hematopoiesis) are described in Methylcellulose colony forming assays, Freshney, MG In Cultu
re of Hematopoietic Cells.RI 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 ce
lls with high proliferative potential, McNiece, IK and Briddell, RA
In Culture of Hematopoietic Cells.RI Freshney, et al. Eds.Vol pp. 2
3-39, Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental H
ematology 22: 353-359, 1994; Cobblestone area forming cell assay, Ploema
cher, RE In Culture of Hematopoietic Cells.RI Freshney, et al. eds.
Vol pp. 1-21, Wiley-Liss, Inc., New York, NY. 1994; Long term bone ma
rrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M.
and Allen, T. In Culture of Hematopoietic Cells.RI Freshney, et al.
eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, NY. 1994; Long term c
ulture initiating cell assay, Sutherland, HJ In Culture of Hematopoiet
ic Cells.RI Freshney, et al. eds.Vol pp. 139-162, Wiley-Liss, Inc.,
Includes, but is not limited to, the assays described in New York, NY. 1994.

Tissue Proliferation Activity The protein of the present invention is useful in compositions used for the growth or regeneration of bone, cartilage, key, ligament and / or nervous tissue, as well as for wound healing and tissue repair, and in the treatment of burns, lacerations and ulcers. Has utility. The protein of the present invention, which induces cartilage and / or bone growth in an environment where bone is not normally formed, finds use in healing fractures and cartilage damage or defects in humans and other animals. Such formulations using the protein of the present invention are useful for reducing closed and open fractures and for improving fixation of artificial joints. De novo osteogenesis induced by osteogenic agents contributes to the repair of congenital, traumatic or oncological resection-induced craniofacial defects and is also useful in cosmetic surgery. The protein of the present invention may be used in the treatment of periodontal disease and other tooth restoration processes. Such agents provide an environment for attracting osteogenic cells, stimulate the growth of osteogenic cells,
Alternatively, it can induce differentiation of osteogenic cell precursors. The protein of the present invention may be used to stimulate osteoporosis or osteoarthritis, for example, by stimulating bone and / or cartilage repair or by blocking the process of tissue destruction mediated by inflammation or inflammatory processes (collagenase activity, osteoclast activity, etc.). May also be useful in the treatment of Another category of tissue development activity that may be due to the proteins of the present invention is key / ligament formation. Proteins of the invention that induce the formation of key / ligament-like or other tissues in an environment in which such tissues are not normally formed can be used to prevent key or ligament laceration, deformation and other key or ligament defects in humans and other animals. Applied to healing, Such formulations using the key / ligament-like tissue inducing protein may be used to prevent damage to the key or ligament tissue as well as to improve the fixation of the key or ligament to bone or other tissue. The de novo key / ligament-like tissue formation induced by the compositions of the present invention contributes to the repair of congenital, traumatic, or oncological resection-induced craniofacial defects, and furthermore the attachment of keys or ligaments Or it is also useful in cosmetic surgery for repair. The compositions of the present invention provide an environment for attracting key- or ligament-forming cells, stimulate the growth of key- or ligament-forming cells, and induce the differentiation of key- or ligament-forming cell precursors. Or induce the growth of key / ligament cells or progenitors ex vivo so as to effect tissue repair when returned to vivo.
The compositions of the present invention are also useful for keratitis, carpal tunnel syndrome and other key or ligament defects. The compositions of the present invention may include a suitable matrix and / or sequestering agent, such as a carrier well known in the art. The protein of the present invention is useful for the proliferation of nerve cells and the regeneration of nerve and brain tissues, namely, central and peripheral nervous system diseases and neuropathies and mechanical diseases and traumatic diseases (including degeneration, death or trauma to nerve cells or nerve tissues). ) May also be useful. More particularly, diseases of the peripheral nervous system such as peripheral nerve injury, peripheral and localized neuropathy, and Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral spinal sclerosis and Shy-Drager
The protein may be used in the treatment of diseases of the central nervous system, such as the syndrome. Additional conditions that may be treated according to the present invention include mechanical and traumatic diseases such as spinal cord diseases, cerebrovascular diseases such as head trauma and stroke. Peripheral neuropathy caused by chemotherapy or other medical therapies can be treated with the proteins of the present invention. The protein of the present invention is also useful for promoting more preferable and prompt closure of non-healing wounds including (but not limited to) pressure ulcers, ulcers related to vascular dysfunction, surgical and trauma wounds and the like. It is possible. The protein of the present invention may also be used in other tissues such as organs (including pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth muscle, skeletal muscle or cardiac muscle), and vascular (including vascular endothelium) tissue. Or an activity for promoting the growth of cells constituting such a tissue. Part of the desired effect may be the regeneration of normal tissue by suppressing fibrotic scarring. The proteins of the present invention may also exhibit angiogenic activity. The proteins of the present invention may also be useful in protecting or regenerating intestines and treating conditions caused by lung or liver fibrosis, reperfusion injury of various tissues, and systemic cytokine damage. The protein of the present invention promotes or suppresses differentiation of the above-mentioned tissue from precursor tissue or cells;
Alternatively, it may be useful for suppressing the growth of the tissue. The activity of the protein of the invention may be measured, inter alia, by the following method: Tissue regeneration activity assay is described in WO 95/16035 (bone, cartilage, key).
International Publication WO95 / 05846 (nerves, neurons); International Publication WO91 / 05
7491 (skin, endothelium), but is not limited thereto. Wound healing activity assays are described in Winter, Epidermal Wound Healing, pps. 71-112.
(Maibach, HI and Rovee, DT, eds.), Year Book Medical Publishers, Inc.,
Including, but not limited to, those described in Chicago (modified by Eaglstein and Mertz, J. Invest. Dermatol 71: 382-384 (1978)).

Activin / Inhibin Activity The protein of the present invention may also exhibit activin- or inhibin-related activity. Inhibins are characterized by their ability to inhibit follicle stimulating hormone (FSH) release,
Activins are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, the protein of the present invention, alone or in the form of a heterodimer with a member of the inhibin α family, reduces the fertility of female mammals and reduces the spermatogenesis of male mammals. Can be useful as Administration of a sufficient amount of another inhibin can induce infertility in these mammals. Alternatively, the protein of the present invention, as a homodimer or as a heterodimer with subunits of other proteins of the inhibin-β group, can be used to stimulate fertility based on the ability of activin molecules to stimulate FSH release from anterior pituitary cells. May be useful as therapeutic agents that induce For example, US Pat.
See 98885. The proteins of the invention may also be useful for improving reproduction in sexually immature mammals, resulting in increased fertility during the life of livestock such as cattle, sheep and pigs. The activity of the protein of the invention may be measured, inter alia, by the following method: Activin / inhibin activity assays are described in Vale et al., Endocrinology 91:
562-572, 1972; Ling et al., Nature 321: 779-782, 1986; Vale et al., Nat
ure 321: 776-779, 1986; Mason et al., Nature 318: 659-663,1985; Forage e
Natl. Acad. Sci. USA 83: 3091-3095, 1986, including but not limited to the assays described in US Pat.

Chemotaxis / chemokinetic activity The protein of the present invention can be used for the chemotactic or chemokinetic activity of mammalian cells such as monocytes, neutrophils, T cells, mast cells, eosinophils and / or endothelial cells (for example, (Acting as a chemokine). Chemotactic and chemokinetic proteins can be used to recruit or attract a desired cell population to a desired site of action. Chemotaxis or chemokinesis proteins are particularly advantageous for treating injuries and other trauma to tissues and for treating local infections. For example, attracting lymphocytes, monocytes or neutrophils to a tumor or site of infection can improve the immune response to the tumor or infectious agent. Certain proteins or peptides have chemotactic activity on a particular cell population and, when stimulable, direct or indirectly direct the movement of such cell population. Preferably, the protein or peptide has the ability to directly stimulate the directed movement of the cell. Whether a particular protein has chemotactic activity on a cell population can be readily determined by using such a protein or peptide in a known assay for cell chemotaxis. The activity of the protein of the invention may be measured, inter alia, by the following method: Assays for chemotactic activity (assays to identify proteins that induce or interfere with chemotaxis) induce migration across cell membranes. Assays that measure the ability of a protein to induce adhesion of one cell population to another cell population as well as the ability of the protein to adhere to another cell population. Suitable assays for migration and adhesion are described in Current Protocols in Immunology, Ed by JE
Coligan, AM Kruisbeek, DH Margulies, EM Shevach, W. Strober, Pub.
reene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measur
ement 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; Mulle
r 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, including, but not limited to.

Hemostasis and thrombolytic activity The protein of the present invention may also exhibit hemostatic or thrombolytic activity. As a result, such proteins are useful in the treatment of various clotting disorders, including hereditary disorders such as hemophilia, or in the treatment of injuries caused by trauma, surgery, or other causes, as well as clotting and other hemostasis. Can be promoted. The protein of the present invention may be useful for the treatment and prevention of thrombolysis or inhibition of thrombus formation and conditions resulting therefrom (eg, myocardial infarction and infarction of central nervous system blood vessels (eg, stroke)). The activity of the protein of the invention may be measured, inter alia, by the following method: Assays for haemostatic and thrombolytic activities are described in Linet et al., J. Clin. Pharmacol.
26: 131-140,1986; Burdick et al., Thrombosis Res. 45: 413-419, 1987; Hum
phrey et al., Fibrinolysis 5: 71-79 (1991); Schaub, Prostaglandins 35: 4
67-474, 1988, including but not limited to the assays described.

Receptor / Ligand Activity The protein of the present invention may also exhibit activity as a receptor, receptor ligand or inhibitor or agonist of receptor / ligand interaction. Examples of such receptors and ligands include cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, cells-
Receptors involved in cell interactions and their ligands (such as cell adhesion molecules (selectins, integrins and their ligands)) and receptors / ligands involved in antigen presentation, antigen recognition, development of cellular and humoral immune responses But not limited thereto. Receptors and ligands are also useful for screening potential peptide or small molecule inhibitors for related receptor / ligand interactions. The proteins of the present invention, including but not limited to receptor and ligand fragments, may themselves be useful as inhibitors of receptor / ligand interaction. The activity of the protein of the invention may be measured, inter alia, by the following method: Suitable assays for receptor-ligand activity are described in Current Protocols in Immunolog.
y, Ed by JE Coligan, AM Kruisbeek, DH Margulies, EM Shevach,
W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (C
hapter 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, 1
987; 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. Metho.
ds 175: 59-68, 1994; Stitt et al., Cell 80: 661-670, 1995, including, but not limited to.

Anti-inflammatory activity The protein of the present invention can exhibit anti-inflammatory activity. Anti-inflammatory activity involves stimulating cells that are involved in the stimulus response, resulting in cell-cell interactions (eg, attachment).
By inhibiting or promoting, thereby inhibiting or promoting the chemotaxis of cells involved in the inflammatory process, thereby inhibiting or promoting cell extravasation,
Alternatively, it is exerted by stimulating or suppressing the production of other factors that more directly inhibit or promote the inflammatory response. Symptoms of inflammation (
Includes chronic or acute symptoms, infection-related inflammation (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, lethal endotoxin injury, arthritis, complement mediated Super-acute rejection,
Nephritis, lung injury induced by cytokines or chemokines, inflammatory bowel disease, Crohn's disease or diseases resulting from overproduction of cytokines such as TNF or IL-1). . The proteins of the invention may also be useful for treating anaphylaxis and hypersensitivity to antigenic substances or materials.

Cadherin / Tumor Inhibition Inhibitory Activity Cadherin is a calcium-dependent adhesion molecule and appears to play a major role during development, particularly in determining specific cell types. Loss or alteration of normal cadherin expression can induce changes to cell adhesion properties associated with tumor growth and metastasis. Cadherin dysfunction has also been implicated in other human diseases, such as pemphigus vulgaris and pemphigus foliaceus (autoimmune cutaneous bullous disease), Crohn's disease, and some developmental abnormalities. The cadherin superfamily contains over 40 members, each with a different expression pattern. All members of the superfamily share common conserved extracellular repeats (cadherin domains), but show structural differences in other parts of the molecule. Calcium is necessary for their adhesion, since cadherin domains bind to calcium to form their tertiary structure. Since the small number of amino acids in the first cadherin domain provides the basis for homophilic adhesion, modification of this recognition site can alter the specificity of cadherin, thereby recognizing only itself Instead, the mutant molecule will be able to bind to different cadherins. In addition, some cadherins are involved in heterophilic adhesion with other cadherins. E-cadherin, a member of the cadherin superfamily, is expressed in epithelial cell types. Pathologically, if E-cadherin expression is lost in the tumor, the malignant cells become invasive and the cancer metastasizes. Transfecting a polynucleotide expressing E-cadhedrin into a cancer cell line restores the altered cell morphology, restores adhesion between cells and to other substrates, reduces cell growth rates, By dramatically reducing the growth of dependent cells,
The cancer-related changes were reversed. Thus, reintroducing E-cadhedrin expression returns the carcinoma to a less advanced stage. Other cadherins may have the same inhibitory role in carcinomas from other tissue types. Therefore, cancer can be treated using the protein of the present invention having cadherin activity and the polynucleotide of the present invention encoding such a protein. Introducing such proteins or polynucleotides into cancer cells may reduce or eliminate the cancerous changes observed in these cells by providing normal cadherin expression. Cancer cells are also known to express cadherins of different tissue types than originally, so these cancer cells can invade different tissues and metastasize. The protein of the present invention having cadherin activity, and the polynucleotide of the present invention encoding such a protein replace cadherin inappropriately expressed in these cells, restore normal cell adhesion properties, and reduce the tendency of cells to metastasize. Or can be removed. Furthermore, an antibody that recognizes and binds to cadherin can be obtained using the protein of the present invention having cadherin activity and the polynucleotide of the present invention encoding such a protein. Such antibodies can also be used to block the adhesion of inappropriately expressed tumor cells cadherin, so that the cells do not form tumors elsewhere. Such anti-cadhedrin antibodies can be used as markers for cancer grade, pathological type, and prognosis. That is, as the cancer progresses, the expression of cadherin decreases, and by using a cadherin-binding antibody, the decrease of cadherin expression can be detected. Using a fragment of a protein of the present invention having cadherin activity, preferably a decapeptide at the cadherin recognition site, and a polynucleotide of the present invention encoding such a protein fragment, to bind to cadherin to prevent cadherin binding that produces undesirable effects. Can also block cadherin function. Further, using a fragment of the protein of the present invention having cadherin activity, preferably a truncated soluble cadherin fragment known to be stable in the circulatory system of cancer patients, and a polynucleotide encoding such a protein fragment, Correct cell-cell adhesion can also be disrupted. Assays for cadherin adhesion and entry inhibitory activity are described in Hortsch et al. J Bio
l Chem 270 (32): 18809-18817, 1995; Miyaki et al. Oncogene 11: 2547-2552
, 1995; Ozawa et al. Cell 63: 1033-1038, 1990.

Tumor Inhibitory Activity In addition to the activities described above for the immunological treatment or prevention of tumors, the proteins of the present invention may exhibit other antitumor activities. Certain proteins directly or indirectly affect tumor growth (eg,
(Via ADCC). Certain proteins act on tumor tissue or progenitor tissue to inhibit the formation of tissue necessary to support tumor growth (eg, by inhibiting angiogenesis) and to inhibit tumor growth. Tumor inhibitory activity may be exhibited by causing the production of a factor, agent or cell type, or by removing or inhibiting a factor, agent or cell type that promotes tumor growth.

Other Activities The protein of the present invention may exhibit one or more of the following additional activities or effects: infectious agents, including but not limited to bacteria, viruses, fungi, and other parasites. Killing; height, weight, hair color, eye color, skin or other tissue pigmentation, or the size or form of an organ or body part (eg, breast augmentation or vice versa)
Effects on body characteristics (suppression or promotion); effects on digestion of consumed fat, protein or carbohydrates; appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) Effects on behavioral characteristics, including but not limited to, violent behavior; providing analgesic or other pain-relieving effects; promoting differentiation and proliferation of embryonic stem cells in non-hematopoietic lineages; hormones or endocrine Activity; for enzymes,
Correcting enzyme deficiency and treating related disorders; treating hyperproliferative disorders (eg, psoriasis); immunoglobulin-like activity (eg, the ability to bind antibodies or complement);
And the ability to act as an antigen in a vaccine composition to generate an immune response to such proteins or other substances or entities that cross-react with such proteins.

Administration and Dose The protein of the present invention (which may be from any source, including but not limited to recombinant and non-recombinant methods) is mixed with a pharmaceutically acceptable carrier. May be used in pharmaceutical compositions. Such compositions may contain (in addition to proteins and carriers) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable"
A non-toxic substance that does not interfere with the effectiveness of the biological activity of the active ingredient. The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition of the present invention comprises M-CSF, GM-CSF, T
NF, 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, TNF0, TNF1, TNF2, G-CSF
, Meg-CSF, thrombopoietin, stem cell factor, and cytokines such as erythropoietin, lymphokines, or other hematopoietic factors. The pharmaceutical composition may further contain other agents that enhance protein activity or supplement its activity or utility in therapy. Such additional factors and / or agents may be included in a pharmaceutical composition to exert a synergistic effect with the protein of the invention or to minimize side effects. Conversely, specific cytokines, lymphokines, other hematopoietic factors, thrombolytic factors or antithrombotic factors, or by incorporating the protein of the present invention into the formulation of anti-inflammatory agents, cytokines, lymphokines, other hematopoietic factors, thrombolytic factors or Side effects of antithrombotic factors or anti-inflammatory agents may be minimized. A protein of the invention may be active in multimers (eg, heterodimers or homodimers) or in complexes with itself or other proteins. Consequently, the pharmaceutical composition of the present invention may contain such a multimeric or complexed form of the protein of the present invention. The pharmaceutical composition of the present invention may be in the form of a complex of the protein of the present invention and a protein or peptide antigen. Protein and / or peptide antigens can be
It will deliver a stimulus signal to the lymphocytes. B lymphocytes will respond to antigen through their surface immunoglobulin receptor. T lymphocytes will respond to antigen through the T cell receptor (TCR) after presentation of the antigen by MHC proteins.
MHC and structurally related proteins, including those encoded by the host cell's class I and class II MHC genes, will serve to present peptide antigens to T lymphocytes. The antigen component can be supplied as a purified MHC-peptide complex alone or with a costimulatory molecule that can directly signal T cells. Alternatively, surface immunoglobulins on B cells and T cells on T cells
Antibodies that can bind CR and other molecules can also be mixed with the pharmaceutical compositions of the present invention. The pharmaceutical composition of the present invention may be in the form of a liposome, in which the protein of the present invention further comprises another pharmaceutically acceptable carrier, an amphipathic agent such as a lipid (in the form of an aggregate such as micelles in an aqueous solution). , Present as an insoluble monolayer, liquid crystal or lamellar layer). Suitable lipids for liposome formulations include, but are not limited to, monoglycerides, diglycerides, sulfatide, lysolecithin, phospholipids, saponins, bile acids, and the like. The preparation of such liposome formulations is within the level of ordinary skill in the art and is described, for example, in US Pat.
And 4737323, all of which are herein incorporated by reference.

As used herein, the term “therapeutically effective amount” refers to each of the pharmaceutical compositions or methods sufficient to show significant patient benefit, eg, amelioration of symptoms of such symptoms, healing, increased rate of healing. Means the total amount of active ingredients. When used for an individual active ingredient administered alone, the term refers only to that ingredient. When used in a combination of active ingredients, it refers to the total amount of active ingredients that produces a therapeutic effect regardless of sequential or simultaneous administration. In practicing the therapeutic methods of the invention, a therapeutically effective amount of a protein of the invention is administered to a mammal having the condition to be treated. According to the method of the invention, the protein of the invention may be administered alone or with other therapeutic agents, such as cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, the proteins of the invention may be administered simultaneously or sequentially with cytokines, lymphokines, other hematopoietic factors, thrombolytic or antithrombotic factors. If administered sequentially, the attending physician will determine the appropriate order of administration for combinations of the proteins of the present invention with cytokines, lymphokines, other hematopoietic, thrombolytic or antithrombotic factors. The administration of the protein of the present invention in the pharmaceutical composition of the present invention or used in the practice of the present invention can be performed by various conventional methods, for example, oral feeding, inhalation, or intradermal, subcutaneous, or intravenous injection. Intravenous injection into a patient is preferred. When a therapeutically effective amount of a protein of the invention is administered orally, the protein of the 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 further contain a solid carrier such as gelatin or an adjuvant. Tablets, capsules, and powders contain about 5 to 95% of a protein of the present invention;
It preferably contains about 25-90% of the protein of the invention. When administered in liquid form, water, petroleum, oils of animal or vegetable origin, such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. Liquid form pharmaceutical compositions may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains about 0.5 to 90% by weight of the protein of the present invention, preferably about 1 to 50% of the protein of the present invention. When administering a therapeutically effective amount of a protein of the invention intravenously, intradermally or subcutaneously,
The protein of the present invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such a parenterally acceptable protein solution having appropriate pH, isotonicity, and stability is within the skill of the art. Preferred pharmaceutical compositions for intravenous, intradermal, or subcutaneous injection include, in addition to the protein of the present invention, sodium chloride for injection, Ringer's solution, dextrose for injection, dextrose and sodium chloride for injection, and Ringer's solution containing lactic acid for injection, Or it should contain other carriers known in the art. The pharmaceutical composition of the present invention may contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those skilled in the art. The amount of the protein of the invention in the pharmaceutical composition of the invention depends on the nature and severity of the condition to be treated,
As well as the nature of the treatment the patient has already received. Ultimately, your doctor
The amount of the protein of the invention to be treated for each patient will be determined. First, the attending physician administers a low dose of the protein of the invention and observes the patient's response. Higher doses of the protein of the invention may be administered until the optimal therapeutic effect is obtained, with no further increase in the dose at the time the optimal therapeutic effect is obtained. Various pharmaceutical compositions for carrying out the method of the present invention contain about 0.01 μg to about 100 mg of the protein of the present invention (preferably,
It should contain from about 0.1 μg to about 10 mg per kg body weight, more preferably from 0.1 μg to about 1 mg per kg body weight. The duration of treatment by intravenous administration using the compositions of the present invention will vary with the severity of the disease to be treated and the condition and response of each patient. It is believed that the duration of each application of the protein of the invention ranges from 12 to 24 hours for continuous intravenous administration. Ultimately, the attending physician will decide the duration of intravenous treatment using the pharmaceutical composition of the present invention.

An animal may be immunized using the protein of the present invention to obtain polyclonal and monoclonal antibodies that specifically react with the protein of the present invention. As used herein, the term “antibody” refers to a polyclonal, monoclonal, chimeric, single chain, CDR-grafted, humanized, or fragment thereof that binds to the indicated protein. But not limited to these. Such terms also include antibodies or other species derived from antibody sequences capable of binding the indicated proteins. Antibodies to a specific protein can be produced by methods well known in the art. For example, a monoclonal antibody can be produced by obtaining an antibody-producing hybridoma by a known method (for example, Goding, 1983, Monomer).
clonal antibodies: principles and practice, Academic Press Inc., New Yor
k, and Yokoyama, 1992, "Production of Monoclonal Antibodies" in Curren
t Protocols in Immunology, Unit 2.5, Greene Publishing Assoc. and John W
iley & Sons). Polyclonal sera and antibodies can be produced by inoculating a mammalian subject with the appropriate protein or fragment thereof by known methods. By cleaving and collecting the desired fragment by known methods, fragments of antibodies, receptors, or other reactive peptides can be obtained from the corresponding antibody (eg, Goding, supra, and Andrew et al., 1992, "Frag
mentation of Immunoglobulins "in Current Protocols in Immunology, Unit 2
.8, Greene Publishing Assoc. And John Wiley & Sons). In addition, known recombination methods (eg, US Pat.
76184), chimeric antibodies and single-chain antibodies can be produced. Well-known methods (eg, US Pat. No. 5,530,101, 5585089,
And 5,693,762) to generate humanized antibodies from the corresponding murine antibodies. In addition, humanized antibodies may be obtained in non-human animals, such as mice, which have been genetically altered to express human antibody molecules (eg,
Fishwild et al., 1996, Nature Biotechnology 14: 845-851, Mendez et al.,
1997, Nature, Genetics 15: 146-156 (error in Nature Genetics 16: 410) and US Pat. Nos. 5,877,397 and 5,625,126). Such antibodies may be obtained using whole proteins or fragments thereof as immunogens. The peptide immunogen may further include a cysteine residue at the carboxy terminus and binds to haptens such as keyhole limpet hemocyanin (KLH). Methods for synthesizing such peptides are known in the art, for example, RP Merrifield,
J. Amer. Chem. Soc. 85, 2149-2154 (1963); JL Krstenansky, et. Al., FE
BS Lett. 211, 10 (1987). A monoclonal antibody that binds to the protein of the present invention can be a useful diagnostic agent for immunodetection of the protein of the present invention. Neutralizing antibodies that bind to the protein of the present invention may be useful as therapeutics for conditions associated with the protein of the present invention, and may also be useful in the treatment of some forms of cancer in which abnormal expression of the protein of the present invention is involved. May be useful in the treatment of neoplasms. In the case of cancer cells or white blood cells, neutralizing monoclonal antibodies against the protein of the present invention may be useful in detecting and preventing metastatic spread of cancer cells mediated by the protein of the present invention. For compositions of the invention useful for regenerating bone, cartilage, tendons or ligaments, the method of treatment comprises:
It involves administering the composition locally, systemically, or locally, such as an implant or device. When administered, the therapeutic composition used in the present invention is, of course, in a pyrogen-free, physiologically acceptable form. In addition, if desired, the composition may be encapsulated or injected in a viscous form and delivered to the site of bone, cartilage or tissue damage. Topical administration is suitable for wound healing and tissue repair. Therapeutically useful agents other than the protein of the present invention, which may be contained in the above composition, may be alternatively or additionally administered simultaneously or sequentially with the composition in the method of the present invention. Preferably, for bone and / or cartilage formation, the composition delivers the protein-containing composition to the site of bone and / or cartilage damage, provides a structure for bone and cartilage development, and more optimally. Will contain a matrix that can be absorbed into the body. Such matrices may be made of materials currently used for other implanted medical devices. The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The appropriate formulation will be determined by the particular application of the composition. Possible matrices for the composition may be biodegradable and chemically known calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydride. Other usable materials are biodegradable and biologically well known, such as bone or skin collagen. Further, the matrix may contain pure proteins or extracellular matrix components.
Other possible matrices are not biodegradable, such as sintered hydroxyapatite, bioglass, aluminate, or other ceramics, but are chemically known. The matrix may comprise a combination of materials of the above type, for example polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. The bioceramics may be modified in the composition, for example in calcium-aluminate-phosphate, and processed to pore size, particle size,
The particle shape and biodegradability may be varied. A 50:50 (molar weight) copolymer of lactic acid and glycolic acid in the form of porous particles having a diameter in the range of 150 to 800 microns is presently preferred. In some applications, it may be useful to use a sequestering agent, such as carboxymethylcellulose or an autologous clot, to prevent dissociation of the protein composition from the matrix. A preferred family of sequestering agents is cellulosic materials such as alkylcellulose (including hydroxyalkylcellulose), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and carboxymethylcellulose, and carboxymethylcellulose (CMC). Most preferred are the cationic salts of
Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly (ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymers and poly (vinyl alcohol). The amount of sequestrant useful in the present invention is 0.5 to 20% by weight, preferably 1 to 10% by weight, based on the total formulation weight,
An amount that prevents detachment of the protein from the polymer matrix and allows for proper handling of the composition, but provides an opportunity for the progenitor cells to infiltrate the matrix, thereby promoting the osteogenic activity of the precursor cells. In order not to give too much.

In a further composition, the protein of the present invention may be mixed with other agents that are beneficial in treating bone and / or cartilage defects, wounds, or tissues. These agents include epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin-like growth factor (IGF). At present, the therapeutic compositions are also valuable for veterinary use. For details,
In addition to humans, livestock and thoroughbred horses are desirable patients for such treatments using the proteins of the present invention. The rules of administration of the protein-containing pharmaceutical composition used for tissue regeneration may include various factors that alter the action of the protein, such as the tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of the wound, The physician will decide on the type of tissue required (eg, bone), patient age, gender, and diet, severity of infection, duration of administration, and other clinical factors. Dosages may vary depending on the type of matrix used for reconstitution and the inclusion of other proteins in the pharmaceutical composition. For example, IGF
The addition of other known growth factors, such as I (insulin-like growth factor I), to the final composition can also affect the dose. Progress can be monitored by periodically assessing tissue / bone growth and / or repair, for example, by X-ray, histomorphological examination and tetracycline labeling. The polynucleotide of the present invention can also be used for gene therapy. Such polynucleotides can be introduced into cells in vivo or ex vivo and expressed in a mammalian subject. The polynucleotides of the present invention may be administered by other known methods for introducing nucleic acids into cells or organisms, including but not limited to viral vectors or in the form of naked DNA. ). The cells may be cultured and grown ex vivo in the presence of the protein of the present invention, or may have a desired effect on such cells, or may have a desired activity in such cells. The treated cells can then be introduced in vivo for therapeutic purposes. The patents and publications cited herein are considered to be entirely incorporated herein by reference.

[0046]

[Sequence list]

[Brief description of the drawings]

1A and 1B. PED used for depositing clones disclosed herein.
6 and pNOTs vectors are shown schematically.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 17/02 A61P 25/16 4H045 19/08 25/24 25/16 25/28 25/24 29/00 25/28 35/00 29/00 37/02 35/00 C07H 21/02 37/02 21/04 C07H 21/02 C07K 14/47 21/04 C12P 21/02 C C07K 14/47 C12N 15/00 ZNAA C12N 5/10 A61K 37/02 C12P 21/02 C12N 5/00 B (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT , LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM) , KE, LS, MW, SD, S , SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ , LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (72) Inventor Hillary F. Clark United States 02141 Cambridge, Mass., Webster Avenue Number 2, 146 (72 Inventor Kim Fechtel Ameri United States 02174 Marion Road, Arlington, Mass. 46 (72) Inventor Michael J. Agostino United States 18810 Waldorf Avenue, Andover, Mass., USA No. 26 F-term (reference) 4B024 AA01 AA05 AA11 AA12 BA21 BA80 CA04 DA02 EA04 GA11 HA01 HA12 HA15 HA17 4B064 AG01 AG02 CA10 CA19 CC24 CE07 CE11 CE12 DA01 DA05 DA13 DA14 4B065 AA90X AA91X AA93Y AB01 AC14 BA02 CA24 CA44 CA46 4C057 BB04 BB05 CC01 DD01 MM02 MM04 4C084 AA01 ZA23ZB12A22ZB12A22Z12B ZB352 ZC062 ZC212 ZC352 ZC552 4H045 AA11 AA20 AA30 BA10 CA40 CA45 DA01 DA45 DA65 DA86 EA24 EA28 FA74 GA22 GA26

Claims (19)

[Claims] 1. a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1; (b) a polynucleotide comprising the nucleotide sequence from nucleotide 45 to nucleotide 590 of SEQ ID NO: 1; A polynucleotide comprising the nucleotide sequence from nucleotide 126 to nucleotide 590; (d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone ya91 deposited under accession number ATCC 98724; (e) a polynucleotide comprising accession number ATCC 98724 Clone of clone ya91
A polynucleotide encoding the full length protein encoded by the DNA insert; (f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone ya91 deposited under accession number ATCC98724; (g) a deposition number accession number ATCC98724 C of cloned ya91
A polynucleotide encoding a mature protein encoded by the DNA insert; (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2; (i) a SEQ ID NO: 2 having biological activity A polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 2
(J) any one of the polynucleotides specified in (a)-(i) under conditions of at least 4X SSC, 65 ° C or 4X SSC, 42 ° C, 50% formamide stringency. And (k) at least one of the polynucleotides (a) to (i) under conditions of 4X SSC, 50 ° C or 6X SSC, 40 ° C, 50% formamide stringency. An isolated polynucleotide selected from the group consisting of a polynucleotide that hybridizes and has a length of at least 25% of the length of SEQ ID NO: 1. 2. The polynucleotide of claim 1, operably linked to at least one expression control sequence. 3. A host cell transformed with the polynucleotide of claim 2. 4. The host cell according to claim 3, which is a mammalian cell. 5. The polynucleotide encoded by the polynucleotide of claim 2, comprising: (a) growing the culture of the host cell of claim 3 in a suitable medium; and (b) purifying the protein from the culture. A method for producing a protein. 6. A protein produced by the method of claim 5. 7. An isolated polynucleotide encoding the protein of claim 6. 8. The polynucleotide of claim 7, comprising the cDNA insert of clone ya91 deposited as ATCC 98724. 9. (a) an amino acid sequence of SEQ ID NO: 2; (b) a fragment of the amino acid sequence of SEQ ID NO: 2 (the fragment comprises eight consecutive amino acids of SEQ ID NO: 2); and (c) ) C of clone ya91 deposited under accession number ATCC 98724
A protein comprising an amino acid sequence selected from the group consisting of an amino acid sequence encoded by a DNA insert, wherein the protein is substantially free of other mammalian proteins. 10. The protein of claim 9, wherein said protein comprises the amino acid sequence of SEQ ID NO: 2. 11. A composition comprising the protein of claim 9 and a pharmaceutically acceptable carrier. 12. (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3; (b) a polynucleotide comprising the nucleotide sequence from nucleotide 194 to nucleotide 466 of SEQ ID NO: 3; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3 A polynucleotide comprising the nucleotide sequence of from nucleotide 338 to nucleotide 466; (d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone ya111 deposited under accession number ATCC 98724; (e) a deposit comprising accession number ATCC 98724 A polynucleotide encoding the full-length protein encoded by the cDNA insert of clone ya111; (f) the coding sequence for the mature protein coding sequence of clone ya111 deposited under accession number ATCC 98724; (G) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone ya111 deposited under accession number ATCC 98724; (h) a protein comprising the amino acid sequence of SEQ ID NO: 4 (I) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 4 having biological activity (the fragment comprises 8 of SEQ ID NO: 4)
(J) any one of the polynucleotides specified in (a)-(i) under conditions of at least 4X SSC, 65 ° C or 4X SSC, 42 ° C, 50% formamide stringency. And (k) at least one of the polynucleotides (a) to (i) under conditions of 4X SSC, 50 ° C or 6X SSC, 40 ° C, 50% formamide stringency. An isolated polynucleotide selected from the group consisting of a polynucleotide that hybridizes and has a length of at least 25% of the length of SEQ ID NO: 3. 13. (a) an amino acid sequence of SEQ ID NO: 4; (b) a fragment of the amino acid sequence of SEQ ID NO: 4, said fragment comprising eight consecutive amino acids of SEQ ID NO: 4; and (c) A) a protein comprising an amino acid sequence selected from the group consisting of the amino acid sequence encoded by the cDNA insert of clone ya111 deposited under accession number ATCC 98724, and substantially free of other mammalian proteins; 14. (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 5; (b) a polynucleotide comprising the nucleotide sequence from nucleotide 15 to nucleotide 233 of SEQ ID NO: 5; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 5 A polynucleotide comprising the nucleotide sequence from nucleotide 174 to nucleotide 233; (d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone ya281 deposited under accession number ATCC 98724; (e) a polynucleotide comprising accession number ATCC 98724 A polynucleotide encoding the full-length protein encoded by the cDNA insert of clone ya281; (f) a sequence encoding the mature protein coding sequence of clone ya281 deposited under accession number ATCC 98724; (G) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone ya281 deposited under accession number ATCC 98724; (h) a protein comprising the amino acid sequence of SEQ ID NO: 6 (I) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 6 that has biological activity (the fragment comprises 8 of SEQ ID NO: 6)
(J) any one of the polynucleotides specified in (a) to (i) under conditions of at least 4X SSC, 65 ° C or 4X SSC, 42 ° C, 50% formamide stringency. And (k) at least one of the polynucleotides (a) to (i) under conditions of 4X SSC, 50 ° C or 6X SSC, 40 ° C, 50% formamide stringency. An isolated polynucleotide selected from the group consisting of a polynucleotide that hybridizes and has a length of at least 25% of the length of SEQ ID NO: 5. 15. (a) an amino acid sequence of SEQ ID NO: 6; (b) a fragment of the amino acid sequence of SEQ ID NO: 6, said fragment comprising eight consecutive amino acids of SEQ ID NO: 6; A) a protein comprising an amino acid sequence selected from the group consisting of the amino acid sequence encoded by the cDNA insert of clone ya281 deposited under accession number ATCC 98724, and substantially free of other mammalian proteins; 16. (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 7; (b) a polynucleotide comprising the nucleotide sequence from nucleotide 102 to nucleotide 461 of SEQ ID NO: 7; (c) a deposit comprising accession number ATCC 98724 A polynucleotide comprising the nucleotide sequence of the cloned full length protein coding sequence of clone yb81 1; (d) a polynucleotide encoding the full length protein encoded by the cDNA insert of clone yb81 1 deposited under accession number ATCC 98724; A) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 8; (f) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 8 having biological activity (the fragment SEQ ID NO: 8 of 8
(G) any one of the polynucleotides specified in (a)-(f) under conditions of stringency of at least 4X SSC, 65 ° C or 4X SSC, 42 ° C, 50% formamide. And (h) at least one of the polynucleotides (a) to (f) under conditions of 4X SSC, 50 ° C or 6X SSC, 40 ° C, 50% formamide stringency. An isolated polynucleotide selected from the group consisting of a polynucleotide that hybridizes and has a length of at least 25% of the length of SEQ ID NO: 7. 17. (a) an amino acid sequence of SEQ ID NO: 8; (b) a fragment of the amino acid sequence of SEQ ID NO: 8 (the fragment comprising eight consecutive amino acids of SEQ ID NO: 8); and (c) A) a protein comprising an amino acid sequence selected from the group consisting of the amino acid sequence encoded by the cDNA insert of clone yb811 deposited under accession number ATCC 98724, and substantially free of other mammalian proteins; 18. A polynucleotide comprising: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 9; (b) a polynucleotide comprising the nucleotide sequence from nucleotide 170 to nucleotide 2968 of SEQ ID NO: 9; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 9 A polynucleotide comprising the nucleotide sequence from nucleotide 1370 to nucleotide 2968; (d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone yc141 deposited under accession number ATCC98724; (e) a polynucleotide comprising accession number ATCC98724 A polynucleotide encoding the full-length protein encoded by the cDNA insert of clone yc141; (f) the mature protein coding of clone yc141 deposited under accession number ATCC 98724 (G) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone yc141 deposited under accession number ATCC 98724; (h) the amino acid sequence of SEQ ID NO: 10 (I) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 10 having biological activity (the fragment comprising SEQ ID NO: 10
(J) any of the polynucleotides specified in (a)-(i) under conditions of at least 4X SSC, 65 ° C or 4X SSC, 42 ° C, 50% formamide stringency. And (k) any of the polynucleotides specified in (a)-(i) under conditions of stringency of at least 4 × SSC, 50 ° C. or 6 × SSC, 40 ° C., 50% formamide. An isolated polynucleotide that hybridizes to one and is selected from the group consisting of polynucleotides having a length of at least 25% of the length of SEQ ID NO: 9. 19. (a) an amino acid sequence of SEQ ID NO: 10; (b) a fragment of the amino acid sequence of SEQ ID NO: 10 (the fragment comprises eight consecutive amino acids of SEQ ID NO: 10); and (c) A) a protein comprising an amino acid sequence selected from the group consisting of the amino acid sequence encoded by the cDNA insert of clone yc141 deposited under accession number ATCC 98724, and substantially free of other mammalian proteins;