U.S. patent application number 12/016817 was filed with the patent office on 2008-06-05 for mouse cytokine receptor zcytor16.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Zhi Chen, Wayne Kindsvogel, Scott R. Presnell, Wenfeng Xu.
Application Number | 20080131931 12/016817 |
Document ID | / |
Family ID | 26955895 |
Filed Date | 2008-06-05 |
United States Patent
Application |
20080131931 |
Kind Code |
A1 |
Presnell; Scott R. ; et
al. |
June 5, 2008 |
MOUSE CYTOKINE RECEPTOR ZCYTOR16
Abstract
Cytokines and their receptors have proven usefulness in both
basic research, animal models, and as therapeutics. The present
invention provides polynucleotides encoding a new cytokine receptor
designated as "mouse Zcytor16", which can bind and antagonize the
IL-TIF cytokine. The present invention also provides corresponding
vectors, cells, and methods of production.
Inventors: |
Presnell; Scott R.; (Tacoma,
WA) ; Xu; Wenfeng; (Mukilteo, WA) ;
Kindsvogel; Wayne; (Seattle, WA) ; Chen; Zhi;
(Bellevue, WA) |
Correspondence
Address: |
ZYMOGENETICS, INC.;INTELLECTUAL PROPERTY DEPARTMENT
1201 EASTLAKE AVENUE EAST
SEATTLE
WA
98102-3702
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
26955895 |
Appl. No.: |
12/016817 |
Filed: |
January 18, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11045944 |
Jan 28, 2005 |
7351555 |
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12016817 |
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10090365 |
Mar 4, 2002 |
6875845 |
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11045944 |
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60273035 |
Mar 2, 2001 |
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60279232 |
Mar 27, 2001 |
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Current U.S.
Class: |
435/69.1 ;
435/252.3; 435/254.11; 435/255.1; 435/320.1; 435/325; 435/348;
435/419; 536/23.4; 536/23.5 |
Current CPC
Class: |
C07K 14/7155 20130101;
A61P 29/00 20180101; C07K 2319/00 20130101; Y10S 530/81 20130101;
A01K 2217/075 20130101; A61P 43/00 20180101; A01K 2217/05
20130101 |
Class at
Publication: |
435/69.1 ;
536/23.5; 435/320.1; 435/252.3; 435/255.1; 435/254.11; 435/348;
435/325; 435/419; 536/23.4 |
International
Class: |
C12P 21/04 20060101
C12P021/04; C07H 21/04 20060101 C07H021/04; C12N 15/00 20060101
C12N015/00; C12N 1/20 20060101 C12N001/20; C12N 1/00 20060101
C12N001/00; C12N 5/06 20060101 C12N005/06; C12N 5/00 20060101
C12N005/00; C12N 5/10 20060101 C12N005/10 |
Claims
1. An isolated polynucleotide comprising the nucleic acid sequence
from the group consisting of: (a) the polynucleotide sequence from
8 to 697 of SEQ ID NO:47; (b) the polynucleotide sequence from 77
to 697 of SEQ ID NO:47; (c) the polynucleotide sequence from 86 to
697 of SEQ ID NO:47; and (d) polynucleotide sequences complementary
to the polynucleotides encoding the polypeptides consisting of a
sequence of amino acid residues as shown in (a), (b), and (c).
2. An isolated polynucleotide of claim 1, wherein the
polynucleotide consists of the nucleic acid sequence from the group
consisting of: (a) the polynucleotide sequence from 8 to 697 of SEQ
ID NO:47; (b) the polynucleotide sequence from 77 to 697 of SEQ ID
NO:47; (c) the polynucleotide sequence from 86 to 697 of SEQ ID
NO:47; and (d) polynucleotide sequences complementary to the
polynucleotides encoding the polypeptides consisting of a sequence
of amino acid residues as shown in (a), (b), and (c).
3. An isolated polynucleotide encoding a polypeptide comprising the
sequence of amino acid residues selected from the group consisting
of: (a) amino acid residues 27 to 230 of SEQ ID NO:48; (b) amino
acid residues 27 to 126 of SEQ ID NO:48; and (c) amino acid
residues 131 to 230 of SEQ ID NO:48.
4. An isolated polynucleotide of claim 3, wherein the
polynucleotide encodes the polypeptide consisting of a sequence of
amino acid residues selected from the group consisting of: (a)
amino acid residues 27 to 230 of SEQ ID NO:48; (b) amino acid
residues 27 to 126 of SEQ ID NO:48; and (c) amino acid residues 131
to 230 of SEQ ID NO:48.
5. An isolated polynucleotide encoding a soluble cytokine receptor
polypeptide comprising a sequence of amino acid residues as shown
from amino acid 27 to 230 of SEQ ID NO:48.
6. A vector, comprising the isolated polynucleotide of claim 3.
7. An expression vector, comprising the isolated polynucleotide of
claim 3, a transcription promoter, and a transcription terminator,
wherein the promoter is operably linked with the polynucleotide,
and wherein the polynucleotide is operably linked with the
transcription terminator.
8. A recombinant host cell comprising the expression vector of
claim 7, wherein the host cell is selected from the group
consisting of bacterium, yeast cell, fungal cell, insect cell,
mammalian cell, and plant cell.
9. A method of producing a polypeptide, the method comprising
culturing recombinant host cells that comprise the expression
vector of claim 7, and that produce the polypeptide.
10. The method of claim 9, further comprising isolating the
polypeptide from the cultured recombinant host cells.
11. An isolated polynucleotide that encodes the soluble cytokine
receptor polypeptide of claim 5, wherein the soluble cytokine
receptor polypeptide encoded by the polynucleotide sequence binds
IL-10-Related T Cell-Derived Inducible Factor (IL-TIF) or
antagonizes IL-TIF activity.
12. The isolated polynucleotide according to claim 11, wherein the
soluble cytokine receptor polypeptide encoded by the polynucleotide
comprises a homodimeric, heterodimeric or multimeric receptor
complex.
13. The isolated polynucleotide according to claim 12, wherein the
soluble cytokine receptor polypeptide encoded by the polynucleotide
comprises a heterodimeric or multimeric receptor complex further
comprising a soluble Class I or Class II cytokine receptor.
14. The isolated polynucleotide according to claim 12, wherein the
soluble cytokine receptor polypeptide encoded by the polynucleotide
comprises a heterodimeric or multimeric receptor complex further
comprising a soluble Cytokine Receptor Family 2-4 (CRF2-4) receptor
polypeptide (SEQ ID NO:35), a soluble IL-10 receptor polypeptide
(SEQ ID NO:36), or soluble zcytor11 receptor polypeptide (SEQ ID
NO:34).
15. An isolated polynucleotide that encodes the soluble cytokine
receptor polypeptide of claim 5, wherein the soluble cytokine
receptor polypeptide encoded by the polynucleotide comprises a
homodimeric, heterodimeric or multimeric receptor complex.
16. An isolated polynucleotide according to claim 15, wherein the
soluble cytokine receptor polypeptide encoded by the polynucleotide
further comprises a soluble Class I or Class II cytokine
receptor.
17. An isolated polynucleotide according to claim 15, wherein the
soluble cytokine receptor polypeptide encoded by the polynucleotide
comprises a heterodimeric or multimeric receptor complex further
comprising a soluble Cytokine Receptor Family 2-4 (CRF2-4) receptor
polypeptide (SEQ ID NO:35), a soluble IL-10 receptor polypeptide
(SEQ ID NO:36), or soluble zcytor11 receptor polypeptide (SEQ ID
NO:34).
18. An isolated polynucleotide according to claim 15, wherein the
soluble cytokine receptor polypeptide further encodes an
intracellular domain.
19. An isolated polynucleotide according to claim 15, wherein the
soluble cytokine receptor polypeptide further comprises an affinity
tag.
20. An expression vector comprising the following operably linked
elements: (a) a transcription promoter; a first DNA segment
encoding the soluble cytokine receptor polypeptide of claim 5; and
a transcription terminator; and (b) a second transcription
promoter; a second DNA segment encoding a soluble Class I or Class
II cytokine receptor polypeptide; and a transcription terminator;
and wherein the first and second DNA segments are contained within
a single expression vector or are contained within independent
expression vectors.
21. An expression vector according to claim 20, further comprising
a secretory signal sequence operably linked to the first and second
DNA segments.
22. An expression vector according to claim 20, wherein the second
DNA segment encodes a polypeptide comprising a soluble Cytokine
Receptor Family 2-4 (CRF2-4) receptor polypeptide (SEQ ID NO:35), a
soluble IL-10 receptor polypeptide (SEQ ID NO:36), or soluble
zcytor11 receptor polypeptide (SEQ ID NO:34).
23. A cultured cell comprising an expression vector according to
claim 20, wherein the cell expresses the polypeptides encoded by
the DNA segments.
24. A cultured cell comprising an expression vector according to
claim 20, wherein the first and second DNA segments are located on
independent expression vectors and are co-transfected into the
cell, and cell expresses the polypeptides encoded by the DNA
segments.
25. A cultured cell into which has been introduced an expression
vector according to claim 20, wherein the cell expresses a
heterodimeric or multimeric soluble receptor polypeptide encoded by
the DNA segments.
26. A cell according to claim 23, wherein the cell secretes a
soluble cytokine receptor polypeptide heterodimer or multimeric
complex.
27. A cell according to claim 23, wherein the cell secretes a
soluble cytokine receptor polypeptide heterodimer or multimeric
complex that binds IL-10-Related T Cell-Derived Inducible Factor
(IL-TIF) or antagonizes IL-TIF activity.
28. A DNA construct encoding a fusion protein comprising: a first
DNA segment encoding the soluble cytokine receptor polypeptide of
claim 5; and at least one other DNA segment encoding a soluble
Class I or Class II cytokine receptor polypeptide, wherein the
first and other DNA segments are connected in-frame; and wherein
the first and other DNA segments encode the fusion protein.
29. A DNA construct encoding a fusion protein according to claim
28, wherein at least one other DNA segment encodes a polypeptide
comprising a soluble Cytokine Receptor Family 2-4 (CRF2-4) receptor
polypeptide (SEQ ID NO:35), a soluble IL-10 receptor polypeptide
(SEQ ID NO:36), or soluble zcytor11 receptor polypeptide (SEQ ID
NO:34).
30. An expression vector comprising the following operably linked
elements: a transcription promoter; a DNA construct encoding a
fusion protein according to claim 28; and a transcription
terminator, wherein the promoter is operably linked to the DNA
construct, and the DNA construct is operably linked to the
transcription terminator.
31. A cultured cell comprising an expression vector according to
claim 30, wherein the cell expresses a polypeptide encoded by the
DNA construct.
32. A method of producing a fusion protein comprising: culturing a
cell according to claim 31; and isolating the polypeptide produced
by the cell.
33. A method of producing a soluble cytokine receptor polypeptide
that comprises a heterodimeric or multimeric complex comprising:
culturing a cell according to claim 23; and isolating the soluble
receptor polypeptides produced by the cell.
34. The isolated polynucleotide according to claim 15, wherein the
soluble cytokine receptor polypeptide further comprises an affinity
tag, chemical moiety, toxin, label, biotin/avidin label,
radionuclide, enzyme, substrate, cofactor, inhibitor, fluorescent
marker, chemiluminescent marker, cytotoxic molecule or an
immunoglobulin Fc domain.
35. The isolated polynucleotide according to claim 3, wherein the
polypeptide by the polynucleotide further comprises an affinity
tag, chemical moiety, toxin, label, biotin/avidin label,
radionuclide, enzyme, substrate, cofactor, inhibitor, fluorescent
marker, chemiluminescent marker, cytotoxic molecule or an
immunoglobulin Fc domain.
36. The isolated polynucleotide of claim 3, wherein the polypeptide
encoded by the polynucleotide binds IL-10-Related T Cell-Derived
Inducible Factor (IL-TIF) or antagonizes IL-TIF activity.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/045,944, filed Jan. 28, 2005, which is a divisional of U.S.
application Ser. No. 10/090,365, filed Mar. 4, 2002, which claims
benefit of Provisional Application 60/273,035, filed on Mar. 2,
2001, and Provisional Application 60/279,232, filed on Mar. 27,
2001, all of which are incorporated herein by reference. Under 35
U.S.C. .sctn. 119(e)(1), this application claims benefit of said
Provisional Applications.
TECHNICAL FIELD
[0002] The present invention relates generally to a new protein
expressed by mouse cells that is a murine ortholog to human
Zcytor16 (U.S. patent application Ser. No. 09/728,911). In
particular, the present invention relates to a novel gene that
encodes a receptor, designated as "mouse Zcytor16," and to nucleic
acid molecules encoding mouse Zcytor16 polypeptides, and antibodies
to the polypeptide.
BACKGROUND OF THE INVENTION
[0003] Cytokines are soluble, small proteins that mediate a variety
of biological effects, including the regulation of the growth and
differentiation of many cell types (see, for example, Arai et al.,
Ann. Rev. Biochem. 59:783 (1990); Mosmann, Curr. Opin. Immunol.
3:311 (1991); Paul and Seder, Cell 76:241 (1994)). Proteins that
constitute the cytokine group include interleukins, interferons,
colony stimulating factors, tumor necrosis factors, and other
regulatory molecules. For example, human interleukin-17 is a
cytokine which stimulates the expression of interleukin-6,
intracellular adhesion molecule 1, interleukin-8, granulocyte
macrophage colony-stimulating factor, and prostaglandin E2
expression, and plays a role in the preferential maturation of
CD34+ hematopoietic precursors into neutrophils (Yao et al., J.
Immunol. 155:5483 (1995); Fossiez et al., J. Exp. Med. 183:2593
(1996)).
[0004] Receptors that bind cytokines are typically composed of one
or more integral membrane proteins that bind the cytokine with high
affinity and transduce this binding event to the cell through the
cytoplasmic portions of the certain receptor subunits. Cytokine
receptors have been grouped into several classes on the basis of
similarities in their extracellular ligand binding domains. For
example, the receptor chains responsible for binding and/or
transducing the effect of interferons are members of the class II
cytokine receptor family, based upon a characteristic about 200
residue extracellular domain.
[0005] The demonstrated in vivo activities of cytokines and their
receptors illustrate the clinical potential of, and need for, other
cytokines, cytokine receptors, cytokine agonists, and cytokine
antagonists.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1 is an alignment of human Zcytor16 (hZcytor16) (SEQ ID
NO:2), and mouse Zcytor16 (mZcytor16) (SEQ ID NO:38). The ":" in
the FIGURE indicates amino acids that are identical between the
mouse and human sequences, and the "." in the FIGURE indicates
amino acids that are conserved substitutions. There is a 66.2%
identity between the human and mouse sequences over the entire
sequence (231 amino acid overlap).
BRIEF SUMMARY OF THE INVENTION
[0007] The present invention provides a novel receptor, designated
"mouse Zcytor16." The present invention also provides mouse
Zcytor16 polypeptides and mouse Zcytor16 fusion proteins, as well
as nucleic acid molecules encoding such polypeptides and proteins,
and methods for using these nucleic acid molecules and amino acid
sequences.
DETAILED DESCRIPTION OF THE INVENTION
1. Overview
[0008] An illustrative nucleotide sequence that encodes mouse
Zcytor16 is provided by SEQ ID NO:37. The encoded polypeptide has
the following amino acid sequence: MMPKHCLLGL LIILLSSATE IQPARVSLTL
QKVRFQSRNF HNILHWQAGS SLPSNNSIYF VQYKMYGQSQ WEDKVDCWGT TALFCDLTNE
TLDPYELYYG RV MTACAGRH SAWTRTPRFT PWWETKLDPP VVTITRVNAS LRVLLRPPEL
PNRNQSGKNA SMETYYGLVY RVFTINNSLE KEQKAYEGTQ RAVEIEGLIP HSSYCVVAEM
YQPMFDRRSP RSKERCVQIP (SEQ ID NO:38). The 230 amino acid
polypeptide represents the extracellular domain, also called a
cytokine-binding domain, of a new class II cytokine receptor.
Features of the Zcytor16 polypeptide include putative signal
sequences at amino acid residues 1 to 23 of SEQ ID NO:38, and a
mature soluble receptor polypeptide from residues 24 to 230 of SEQ
ID NO:38. The receptor has two fibronectin III domains, also called
immunoglobulin superfamily (Ig) domains, characteristic of the
class II cytokine receptor family that comprise amino acid residues
31 to 122 (fibronectin III domain I), and 131 to 229 (fibronectin
III domain II) of SEQ ID NO:38, and a linker that resides between
the Ig domains (i.e., at amino acid residues 127-130 of SEQ ID
NO:38). Thus molecules of the present invention include
polypeptides that include a cytokine binding domain comprising
amino acids 31 to 229 of SEQ ID NO:38. Moreover, additional
variants of the zcytor16 polypeptide include polypeptides that
comprise amino acid residues 27 to 122, 24 to 122, 31 to 126, 27 to
126, or 24 to 126, (fibronectin III domain I), and 131 to 229 or
230 (fibronectin III domain II) of SEQ ID NO:38, and a linker that
resides between the Ig domains (i.e., at amino acid residues
123-130, or 127-130 of SEQ ID NO:38). Thus molecules of the present
invention include polypeptides that include a cytokine binding
domain comprising amino acids 24, or 27, to 229 or 230 of SEQ ID
NO:38. In addition, zcytor16 contains conserved motifs and residues
characteristic of class II cytokines: an SXWS (SEQ ID NO:46)-like
motif from residue 219-222 of SEQ ID NO:38; conserved Tryptophan
residues at residues 46, 71, and 113 of SEQ ID NO:38; and conserved
Cysteine residues at residues 77, 85, 205, and 226 of SEQ ID NO:38.
The mouse Zcytor16 gene is specifically expressed in Lung,
Pancreas, Placenta, Salivary Gland, Skeletal Muscle, Skin, Small
Intestine, Smooth Muscle, Spleen, Stomach, and Testis cDNAs and is
expected to be expressed in specific mononuclear cells, such as
activated T-cell and B-cell subsets. Alignment of the polypeptide
sequences shows that mouse Zcytor16 (SEQ ID NO:38) is an ortholog
of the human Zcytor16 sequence (SEQ ID NO:2) (U.S. patent
application Ser. No. 09/728,911) as shown in FIG. 1.
[0009] Another illustrative nucleotide sequence that encodes a
preferred mouse Zcytor16 is provided by SEQ ID NO:47. The encoded
polypeptide has the following amino acid sequence: MMPKHCLLGL
LIILLSSATE IQPARVSLTPQ KVRFQSRNFH
NILHWQAGSSLPSNNSIYFVQYKMYGQSQWEDKVDCWGTTALFCDLTNETLDPYELYYGRV
MTACAGRHSAWTRTPRFTPWWETKLDPPVVTITRVNASLRVLLRPPELPNRNQSGKNASME
TYYGLVYRVFTINNSLEKEQKAYEGTQRAVEIEGLIPHSSYCVVAEMYQPMFDRRSPRSKER
CVHIP (SEQ ID NO:48). The 230 amino acid polypeptide represents the
extracellular domain, also called a cytokine-binding domain, of a
new class II cytokine receptor. The SEQ ID NO:48 sequence differs
from SEQ ID NO:38 by two amino acids: amino acid 30 (Leu) and 228
(Gln) shown in SEQ ID NO:38 are amino acids 30 (Pro) and 228 (His)
shown in SEQ ID NO:48. Features of the Zcytor16 polypeptide include
putative signal sequences at amino acid residues 1 to 23 of SEQ ID
NO:48, and a mature soluble receptor polypeptide from residues 24
to 230 of SEQ ID NO:48. The receptor has two fibronectin III
domains, also called immunoglobulin superfamily (Ig) domains,
characteristic of the class II cytokine receptor family that
comprise amino acid residues 31 to 122 (fibronectin III domain I),
and 131 to 229 (fibronectin III domain II) of SEQ ID NO:48, and a
linker that resides between the Ig domains (i.e., at amino acid
residues 127-130 of SEQ ID NO:48). Thus molecules of the present
invention include polypeptides that include a cytokine binding
domain comprising amino acids 31 to 229 of SEQ ID NO:48. Moreover,
additional variants of the zcytor16 polypeptide include
polypeptides that comprise amino acid residues 27 to 122, 24 to
122, 31 to 126, 27 to 126, or 24 to 126, (fibronectin III domain
I), and 131 to 229 or 230 (fibronectin III domain II) of SEQ ID
NO:48, and a linker that resides between the Ig domains (i.e., at
amino acid residues 123-130, or 127-130 of SEQ ID NO:48). Thus
molecules of the present invention include polypeptides that
include a cytokine binding domain comprising amino acids 24, or 27,
to 229 or 230 of SEQ ID NO:48. In addition, zcytor16 contains
conserved motifs and residues characteristic of class II cytokines:
an SXWS (SEQ ID NO:46)-like motif from residue 219-222 of SEQ ID
NO:48; conserved Tryptophan residues at residues 46, 71, and 113 of
SEQ ID NO:48; and conserved Cysteine residues at residues 77, 85,
205, and 226 of SEQ ID NO:48.
[0010] An illustrative nucleotide sequence that encodes human
Zcytor16 is provided by SEQ ID NO:1. The encoded polypeptide has
the following amino acid sequence: MMPKHCFLGF LISFFLTGVA GTQSTHESLK
PQRVQFQSRN FHNILQWQPG RALTGNSSVY FVQYKIYGQR QWKNKEDCWG TQELSCDLTS
ETSDIQEPYY GRVRAASAGS YSEWSMTPRF TPWWETKIDP PVMNITQVNG SLLVILHAPN
LPYRYQKEKN VSIEDYYELL YRVFIINNSL EKEQKVYEGA HRAVEIEALT PHSSYCVVAE
IYQPMLDRRS QRSEERCVEI P (SEQ ID NO:2). The 231 amino acid
polypeptide represents the extracellular domain, also called a
cytokine-binding domain, of a new class II cytokine receptor.
Features of the Zcytor16 polypeptide include putative signal
sequences at amino acid residues 1 to 21, or 1 to 22 of SEQ ID
NO:2, and a mature soluble receptor polypeptide from residues 22 to
231 or 23 to 231 of SEQ ID NO:2. The receptor has two fibronectin
III domains, also called immunoglobulin superfamily (Ig) domains,
characteristic of the class II cytokine receptor family that
comprise amino acid residues 32 to 123 (fibronectin III domain I),
and 132 to 230 (fibronectin III domain II) of SEQ ID NO:2, and a
linker that resides between the Ig domains (i.e., at amino acid
residues 128-131 of SEQ ID NO:2). Thus molecules of the present
invention include polypeptides that include a cytokine binding
domain comprising amino acids 32 to 230 of SEQ ID NO:2. Moreover,
additional variants of the zcytor16 polypeptide include
polypeptides that comprise amino acid residues 28 to 123, 23 to
123, 22 to 123, or 32 to 127, 28 to 127, 23 to 127, 22 to 127,
(fibronectin III domain I), and 132 to 230 or 231 (fibronectin III
domain II) of SEQ ID NO:2, and a linker that resides between the Ig
domains (i.e., at amino acid residues 124-131, or 128-131 of SEQ ID
NO:2). Thus molecules of the present invention include polypeptides
that include a cytokine binding domain comprising amino acids 22,
23, or 28 to 132 to 230 or 231 of SEQ ID NO:2. In addition,
zcytor16 contains conserved motifs and residues characteristic of
class II cytokines: an SXWS (SEQ ID NO:46) motif from residue
220-223 of SEQ ID NO:2; conserved Tryptophan residues at residues
47, 72, and 114 of SEQ ID NO:2; and conserved Cysteine residues at
residues 78, 86, 206, and 227 of SEQ ID NO:2. The Zcytor16 gene is
expressed in monocytes, lymphoid, placenta, spleen, tonsil and
other tissues, and resides in human chromosome 6q23-q24.
[0011] The corresponding polynucleotides encoding the zcytor16
polypeptide regions, domains, motifs, residues and sequences
described above are as shown in SEQ ID NO:1 (human zcytor16) and
SEQ ID NO:37, and SEQ ID NO:47 (mouse zcytor16).
[0012] As described below, the present invention provides isolated
polypeptides comprising an amino acid sequence that is at least
70%, at least 80%, or at least 90% identical to a reference amino
acid sequence of SEQ ID NO:38 or SEQ ID NO:48 selected from the
group consisting of: (a) amino acid residues amino acid residues 24
to 230, or 27 to 230, (b) amino acid residues 27 to 126, (c) amino
acid residues 131 to 230 and (d) amino acid residues amino acid
residues 1 to 230. The present invention also provides isolated
polypeptides as disclosed above that specifically bind with an
antibody that specifically binds with a polypeptide consisting of
the amino acid sequence of SEQ ID NO:38 or SEQ ID NO:48.
Illustrative polypeptides include polypeptides comprising either
amino acid residues 24 to 230, or 27 to 230 of SEQ ID NO:38 or SEQ
ID NO:48 or a fragment thereof described herein. Moreover, the
present invention also provides isolated polypeptides as disclosed
above that bind IL-TIF (e.g., human IL-TIF polypeptide sequence as
shown in SEQ ID NO:15 or mouse IL-TIF polypeptide sequence as shown
in SEQ ID NO:41). The human IL-TIF polynucleotide sequence is shown
in SEQ ID NO:14. The mouse IL-TIF polynucleotide sequence is shown
in SEQ ID NO:40.
[0013] The present invention also provides isolated polypeptides
comprising at least 15 contiguous amino acid residues of an amino
acid sequence of SEQ ID NO:38 or SEQ ID NO:48 selected from the
group consisting of: (a) amino acid residues amino acid residues 24
to 230, or 27 to 230, (b) amino acid residues 27 to 126, (c) amino
acid residues 131 to 230 and (d) amino acid residues amino acid
residues 1 to 230. Illustrative polypeptides include polypeptides
that either comprise, or consist of, amino acid residues (a) to
(d). Moreover, the present invention also provides isolated
polypeptides as disclosed above that bind IL-TIF.
[0014] The present invention also includes variant mouse Zcytor16
polypeptides, wherein the amino acid sequence of the variant
polypeptide shares an identity with amino acid residues 24 to 230,
or 27 to 230 of SEQ ID NO:38 or SEQ ID NO:48 selected from the
group consisting of at least 70% identity, at least 80% identity,
at least 90% identity, at least 95% identity, or greater than 95%
identity, and wherein any difference between the amino acid
sequence of the variant polypeptide and the corresponding amino
acid sequence of SEQ ID NO:38 or SEQ ID NO:48 is due to one or more
conservative amino acid substitutions. Moreover, the present
invention also provides isolated polypeptides as disclosed above
that bind IL-TIF.
[0015] The present invention further provides antibodies and
antibody fragments that specifically bind with such polypeptides.
Exemplary antibodies include polyclonal antibodies, murine
monoclonal antibodies, humanized antibodies derived from murine
monoclonal antibodies, and human monoclonal antibodies.
Illustrative antibody fragments include F(ab').sub.2, F(ab).sub.2,
Fab', Fab, Fv, scFv, and minimal recognition units. The present
invention further includes compositions comprising a carrier and a
peptide, polypeptide, or antibody described herein.
[0016] The present invention also provides isolated nucleic acid
molecules that encode a mouse Zcytor16 polypeptide, wherein the
nucleic acid molecule is selected from the group consisting of: (a)
a nucleic acid molecule comprising the nucleotide sequence of SEQ
ID NO:39 or SEQ ID NO:49, (b) a nucleic acid molecule encoding an
amino acid sequence that comprises either amino acid residues 24 to
230, or 27 to 230 of SEQ ID NO:38 or SEQ ID NO:48 and (c) a nucleic
acid molecule that remains hybridized following stringent wash
conditions to a nucleic acid molecule comprising the nucleotide
sequence of nucleotides 77 or 86 to 697 of SEQ ID NO:37 or SEQ ID
NO:47, or the complement of the nucleotide sequence of nucleotides
77 or 86 to 697 of SEQ ID NO:37 or SEQ ID NO:47. Illustrative
nucleic acid molecules include those in which any difference
between the amino acid sequence encoded by nucleic acid molecule
(c) and the corresponding amino acid sequence of SEQ ID NO:38 or
SEQ ID NO:48 is due to a conservative amino acid substitution. The
present invention further contemplates isolated nucleic acid
molecules that comprise nucleotides 8 to 697 of SEQ ID NO:37 or SEQ
ID NO:47. Moreover, the present invention also provides isolated
polynucleotides that encode polypeptides as disclosed above that
bind IL-TIF.
[0017] The present invention also includes vectors and expression
vectors comprising such nucleic acid molecules. Such expression
vectors may comprise a transcription promoter, and a transcription
terminator, wherein the promoter is operably linked with the
nucleic acid molecule, and wherein the nucleic acid molecule is
operably linked with the transcription terminator. The present
invention further includes recombinant host cells and recombinant
viruses comprising these vectors and expression vectors.
Illustrative host cells include bacterial, yeast, fungal, insect,
mammalian, and plant cells. Recombinant host cells comprising such
expression vectors can be used to produce mouse Zcytor16
polypeptides by culturing such recombinant host cells that comprise
the expression vector and that produce the mouse Zcytor16 protein,
and, optionally, isolating the mouse Zcytor16 protein from the
cultured recombinant host cells.
[0018] In addition, the present invention provides pharmaceutical
compositions comprising a pharmaceutically acceptable carrier and
at least one of such an expression vector or recombinant virus
comprising such expression vectors. The present invention further
includes pharmaceutical compositions, comprising a pharmaceutically
acceptable carrier and a polypeptide described herein.
[0019] The present invention also contemplates methods for
detecting the presence of mouse Zcytor16 RNA in a biological
sample, comprising the steps of (a) contacting a mouse Zcytor16
nucleic acid probe under hybridizing conditions with either (i)
test RNA molecules isolated from the biological sample, or (ii)
nucleic acid molecules synthesized from the isolated RNA molecules,
wherein the probe has a nucleotide sequence comprising a portion of
the nucleotide sequence of SEQ ID NO:37 or SEQ ID NO:47, or its
complement, and (b) detecting the formation of hybrids of the
nucleic acid probe and either the test RNA molecules or the
synthesized nucleic acid molecules, wherein the presence of the
hybrids indicates the presence of mouse Zcytor16 RNA in the
biological sample. For example, suitable probes consist of the
following nucleotide sequences of SEQ ID NO:37 or SEQ ID NO:47:
nucleotides 77, 86, or 100 to 373; nucleotides 77, 86, or 100 to
694 or 697; nucleotides 401 to 694 or 697; and nucleotides 8 to 694
or 697. Other suitable probes consist of the complement of these
nucleotide sequences, or a portion of the nucleotide sequences or
their complements.
[0020] The present invention further provides methods for detecting
the presence of mouse Zcytor16 polypeptide in a biological sample,
comprising the steps of: (a) contacting the biological sample with
an antibody or an antibody fragment that specifically binds with a
polypeptide consisting of the amino acid sequence of SEQ ID NO:38
or SEQ ID NO:48, wherein the contacting is performed under
conditions that allow the binding of the antibody or antibody
fragment to the biological sample, and (b) detecting any of the
bound antibody or bound antibody fragment. Such an antibody or
antibody fragment may further comprise a detectable label selected
from the group consisting of radioisotope, fluorescent label,
chemiluminescent label, enzyme label, bioluminescent label, and
colloidal gold.
[0021] The present invention also provides kits for performing
these detection methods. For example, a kit for detection of
Zcytor16 gene expression may comprise a container that comprises a
nucleic acid molecule, wherein the nucleic acid molecule is
selected from the group consisting of (a) a nucleic acid molecule
comprising the nucleotide sequence of nucleotides 77, 86, or 100 to
694 or 697, or 8 to 694 or 697 of SEQ ID NO:37 or SEQ ID NO:47, (b)
a nucleic acid molecule that is a fragment of (a) consisting of at
least eight nucleotides. Such a kit may also comprise a second
container that comprises one or more reagents capable of indicating
the presence of the nucleic acid molecule. On the other hand, a kit
for detection of mouse Zcytor16 protein may comprise a container
that comprises an antibody, or an antibody fragment, that
specifically binds with a polypeptide consisting of the amino acid
sequence of SEQ ID NO:38 or SEQ ID NO:48.
[0022] The present invention also contemplates anti-idiotype
antibodies, or anti-idiotype antibody fragments, that specifically
bind an antibody or antibody fragment that specifically binds a
polypeptide consisting of the amino acid sequence of SEQ ID NO:38
or SEQ ID NO:48. An exemplary anti-idiotype antibody binds with an
antibody that specifically binds a polypeptide consisting of amino
acid residues 24 to 230, or 27 to 230 of SEQ ID NO:38 or SEQ ID
NO:48.
[0023] The present invention also provides isolated nucleic acid
molecules comprising a nucleotide sequence that encodes a mouse
Zcytor16 secretion signal sequence and a nucleotide sequence that
encodes a biologically active polypeptide, wherein the mouse
Zcytor16 secretion signal sequence comprises an amino acid sequence
of residues 1 to 23 of SEQ ID NO:38 or SEQ ID NO:48. Illustrative
biologically active polypeptides include Factor VIIa, proinsulin,
insulin, follicle stimulating hormone, tissue type plasminogen
activator, tumor necrosis factor, interleukin, colony stimulating
factor, interferon, erythropoietin, and thrombopoietin. Moreover,
the present invention provides fusion proteins comprising a mouse
Zcytor16 secretion signal sequence and a polypeptide, wherein the
mouse Zcytor16 secretion signal sequence comprises an amino acid
sequence of residues 1 to 23, of SEQ ID NO:38 or SEQ ID NO:48.
[0024] The present invention also provides fusion proteins,
comprising a mouse Zcytor16 polypeptide and an immunoglobulin
moiety. In such fusion proteins, the immunoglobulin moiety may be
an immunoglobulin heavy chain constant region, such as a human
F.sub.c fragment. The present invention further includes isolated
nucleic acid molecules that encode such fusion proteins.
[0025] The present invention also provides monomeric, homodimeric,
heterodimeric and multimeric receptors comprising a mouse Zcytor16
extracellular domain. Such receptors are soluble or membrane bound,
and act as antagonists of the Zcytor16 ligand, IL-TIF (e.g., the
human IL-TIF as shown in SEQ ID NO:15, and mouse IL-TIF shown in
SEQ ID NO:41). In a preferred embodiment, such receptors are
soluble receptors comprising at least one mouse Zcytor16
extracellular domain polypeptide comprising amino acids 24 to 230,
or 27 to 230 of SEQ ID NO:38 or SEQ ID NO:48. The present invention
further includes isolated nucleic acid molecules that encode such
receptor polypeptides.
[0026] The present invention also provides polyclonal and
monoclonal antibodies to monomeric, homodimeric, heterodimeric and
multimeric receptors comprising a mouse Zcytor16 extracellular
domain such as those described above. Moreover, such antibodies can
be used antagonize the binding to the Zcytor16 ligand, IL-TIF (SEQ
ID NO:15, and SEQ ID NO:41), to the Zcytor16 receptor.
[0027] The present invention also provides a method for detecting a
cancer in a patient, comprising: obtaining a tissue or biological
sample from a patient; incubating the tissue or biological sample
with an antibody as described above under conditions wherein the
antibody binds to its complementary polypeptide in the tissue or
biological sample; visualizing the antibody bound in the tissue or
biological sample; and comparing levels of antibody bound in the
tissue or biological sample from the patient to a normal control
tissue or biological sample, wherein an increase in the level of
antibody bound to the patient tissue or biological sample relative
to the normal control tissue or biological sample is indicative of
a cancer in the patient.
[0028] The present invention also provides a method for detecting a
cancer in a patient, comprising: obtaining a tissue or biological
sample from a patient; labeling a polynucleotide comprising at
least 14 contiguous nucleotides of SEQ ID NO:37 or SEQ ID NO:47 or
the complement of SEQ ID NO:37 or SEQ ID NO:47; incubating the
tissue or biological sample with under conditions wherein the
polynucleotide will hybridize to complementary polynucleotide
sequence; visualizing the labeled polynucleotide in the tissue or
biological sample; and comparing the level of labeled
polynucleotide hybridization in the tissue or biological sample
from the patient to a normal control tissue or biological sample,
wherein an increase in the labeled polynucleotide hybridization to
the patient tissue or biological sample relative to the normal
control tissue or biological sample is indicative of a cancer in
the patient.
[0029] These and other aspects of the invention will become evident
upon reference to the following detailed description. In addition,
various references are identified below and are incorporated by
reference in their entirety.
2. Definitions
[0030] In the description that follows, a number of terms are used
extensively. The following definitions are provided to facilitate
understanding of the invention.
[0031] As used herein, "nucleic acid" or "nucleic acid molecule"
refers to polynucleotides, such as deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA), oligonucleotides, fragments generated by
the polymerase chain reaction (PCR), and fragments generated by any
of ligation, scission, endonuclease action, and exonuclease action.
Nucleic acid molecules can be composed of monomers that are
naturally-occurring nucleotides (such as DNA and RNA), or analogs
of naturally-occurring nucleotides (e.g., .alpha.-enantiomeric
forms of naturally-occurring nucleotides), or a combination of
both. Modified nucleotides can have alterations in sugar moieties
and/or in pyrimidine or purine base moieties. Sugar modifications
include, for example, replacement of one or more hydroxyl groups
with halogens, alkyl groups, amines, and azido groups, or sugars
can be functionalized as ethers or esters. Moreover, the entire
sugar moiety can be replaced with sterically and electronically
similar structures, such as aza-sugars and carbocyclic sugar
analogs. Examples of modifications in a base moiety include
alkylated purines and pyrimidines, acylated purines or pyrimidines,
or other well-known heterocyclic substitutes. Nucleic acid monomers
can be linked by phosphodiester bonds or analogs of such linkages.
Analogs of phosphodiester linkages include phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the
like. The term "nucleic acid molecule" also includes so-called
"peptide nucleic acids," which comprise naturally-occurring or
modified nucleic acid bases attached to a polyamide backbone.
Nucleic acids can be either single stranded or double stranded.
[0032] The term "complement of a nucleic acid molecule" refers to a
nucleic acid molecule having a complementary nucleotide sequence
and reverse orientation as compared to a reference nucleotide
sequence. For example, the sequence 5' ATGCACGGG 3' is
complementary to 5' CCCGTGCAT 3'.
[0033] The term "contig" denotes a nucleic acid molecule that has a
contiguous stretch of identical or complementary sequence to
another nucleic acid molecule. Contiguous sequences are said to
"overlap" a given stretch of a nucleic acid molecule either in
their entirety or along a partial stretch of the nucleic acid
molecule.
[0034] The term "degenerate nucleotide sequence" denotes a sequence
of nucleotides that includes one or more degenerate codons as
compared to a reference nucleic acid molecule that encodes a
polypeptide. Degenerate codons contain different triplets of
nucleotides, but encode the same amino acid residue (i.e., GAU and
GAC triplets each encode Asp).
[0035] The term "structural gene" refers to a nucleic acid molecule
that is transcribed into messenger RNA (mRNA), which is then
translated into a sequence of amino acids characteristic of a
specific polypeptide.
[0036] An "isolated nucleic acid molecule" is a nucleic acid
molecule that is not integrated in the genomic DNA of an organism.
For example, a DNA molecule that encodes a growth factor that has
been separated from the genomic DNA of a cell is an isolated DNA
molecule. Another example of an isolated nucleic acid molecule is a
chemically-synthesized nucleic acid molecule that is not integrated
in the genome of an organism. A nucleic acid molecule that has been
isolated from a particular species is smaller than the complete DNA
molecule of a chromosome from that species.
[0037] A "nucleic acid molecule construct" is a nucleic acid
molecule, either single- or double-stranded, that has been modified
through human intervention to contain segments of nucleic acid
combined and juxtaposed in an arrangement not existing in
nature.
[0038] "Linear DNA" denotes non-circular DNA molecules having free
5' and 3' ends. Linear DNA can be prepared from closed circular DNA
molecules, such as plasmids, by enzymatic digestion or physical
disruption.
[0039] "Complementary DNA (cDNA)" is a single-stranded DNA molecule
that is formed from an mRNA template by the enzyme reverse
transcriptase. Typically, a primer complementary to portions of
mRNA is employed for the initiation of reverse transcription. Those
skilled in the art also use the term "cDNA" to refer to a
double-stranded DNA molecule consisting of such a single-stranded
DNA molecule and its complementary DNA strand. The term "cDNA" also
refers to a clone of a cDNA molecule synthesized from an RNA
template.
[0040] "Probes and/or primers" as used herein can be RNA or DNA.
DNA can be either cDNA or genomic DNA. Polynucleotide probes and
primers are single or double-stranded DNA or RNA, generally
synthetic oligonucleotides, but may be generated from cloned cDNA
or genomic sequences or its complements. Analytical probes will
generally be at least 20 nucleotides in length, although somewhat
shorter probes (14-17 nucleotides) can be used. PCR primers are at
least 5 nucleotides in length, preferably 15 or more nt, more
preferably 20-30 nt. Short polynucleotides can be used when a small
region of the gene is targeted for analysis. For gross analysis of
genes, a polynucleotide probe may comprise an entire exon or more.
Probes can be labeled to provide a detectable signal, such as with
an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer,
paramagnetic particle and the like, which are commercially
available from many sources, such as Molecular Probes, Inc.,
Eugene, Oreg., and Amersham Corp., Arlington Heights, Ill., using
techniques that are well known in the art.
[0041] A "promoter" is a nucleotide sequence that directs the
transcription of a structural gene. Typically, a promoter is
located in the 5' non-coding region of a gene, proximal to the
transcriptional start site of a structural gene. Sequence elements
within promoters that function in the initiation of transcription
are often characterized by consensus nucleotide sequences. These
promoter elements include RNA polymerase binding sites, TATA
sequences, CAAT sequences, differentiation-specific elements (DSEs;
McGehee et al., Mol. Endocrinol. 7:551 (1993)), cyclic AMP response
elements (CREs), serum response elements (SREs; Treisman, Seminars
in Cancer Biol. 1:47 (1990)), glucocorticoid response elements
(GREs), and binding sites for other transcription factors, such as
CRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye
et al., J. Biol. Chem. 269:25728 (1994)), SP1, cAMP response
element binding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and
octamer factors (see, in general, Watson et al., eds., Molecular
Biology of the Gene, 4th ed. (The Benjamin/Cummings Publishing
Company, Inc. 1987), and Lemaigre and Rousseau, Biochem. J. 303:1
(1994)). If a promoter is an inducible promoter, then the rate of
transcription increases in response to an inducing agent. In
contrast, the rate of transcription is not regulated by an inducing
agent if the promoter is a constitutive promoter. Repressible
promoters are also known.
[0042] A "core promoter" contains essential nucleotide sequences
for promoter function, including the TATA box and start of
transcription. By this definition, a core promoter may or may not
have detectable activity in the absence of specific sequences that
may enhance the activity or confer tissue specific activity.
[0043] A "regulatory element" is a nucleotide sequence that
modulates the activity of a core promoter. For example, a
regulatory element may contain a nucleotide sequence that binds
with cellular factors enabling transcription exclusively or
preferentially in particular cells, tissues, or organelles. These
types of regulatory elements are normally associated with genes
that are expressed in a "cell-specific," "tissue-specific," or
"organelle-specific" manner.
[0044] An "enhancer" is a type of regulatory element that can
increase the efficiency of transcription, regardless of the
distance or orientation of the enhancer relative to the start site
of transcription.
[0045] "Heterologous DNA" refers to a DNA molecule, or a population
of DNA molecules, that does not exist naturally within a given host
cell. DNA molecules heterologous to a particular host cell may
contain DNA derived from the host cell species (i.e., endogenous
DNA) so long as that host DNA is combined with non-host DNA (i.e.,
exogenous DNA). For example, a DNA molecule containing a non-host
DNA segment encoding a polypeptide operably linked to a host DNA
segment comprising a transcription promoter is considered to be a
heterologous DNA molecule. Conversely, a heterologous DNA molecule
can comprise an endogenous gene operably linked with an exogenous
promoter. As another illustration, a DNA molecule comprising a gene
derived from a wild-type cell is considered to be heterologous DNA
if that DNA molecule is introduced into a mutant cell that lacks
the wild-type gene.
[0046] A "polypeptide" is a polymer of amino acid residues joined
by peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 10 amino acid residues are commonly
referred to as "peptides."
[0047] A "protein" is a macromolecule comprising one or more
polypeptide chains. A protein may also comprise non-peptidic
components, such as carbohydrate groups. Carbohydrates and other
non-peptidic substituents may be added to a protein by the cell in
which the protein is produced, and will vary with the type of cell.
Proteins are defined herein in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are generally
not specified, but may be present nonetheless.
[0048] A peptide or polypeptide encoded by a non-host DNA molecule
is a "heterologous" peptide or polypeptide.
[0049] An "integrated genetic element" is a segment of DNA that has
been incorporated into a chromosome of a host cell after that
element is introduced into the cell through human manipulation.
Within the present invention, integrated genetic elements are most
commonly derived from linearized plasmids that are introduced into
the cells by electroporation or other techniques. Integrated
genetic elements are passed from the original host cell to its
progeny.
[0050] A "cloning vector" is a nucleic acid molecule, such as a
plasmid, cosmid, or bacteriophage, that has the capability of
replicating autonomously in a host cell. Cloning vectors typically
contain one or a small number of restriction endonuclease
recognition sites that allow insertion of a nucleic acid molecule
in a determinable fashion without loss of an essential biological
function of the vector, as well as nucleotide sequences encoding a
marker gene that is suitable for use in the identification and
selection of cells transformed with the cloning vector. Marker
genes typically include genes that provide antibiotic resistance,
e.g., tetracycline resistance or ampicillin resistance.
[0051] An "expression vector" is a nucleic acid molecule encoding a
gene that is expressed in a host cell. Typically, an expression
vector comprises a transcription promoter, a gene, and a
transcription terminator. Gene expression is usually placed under
the control of a promoter, and such a gene is said to be "operably
linked to" the promoter. Similarly, a regulatory element and a core
promoter are operably linked if the regulatory element modulates
the activity of the core promoter.
[0052] A "recombinant host" is a cell that contains a heterologous
nucleic acid molecule, such as a cloning vector or expression
vector. In the present context, an example of a recombinant host is
a cell that produces mouse Zcytor16 from an expression vector. In
contrast, mouse Zcytor16 can be produced by a cell that is a
"natural source" of mouse Zcytor16, and that lacks an expression
vector.
[0053] "Integrative transformants" are recombinant host cells, in
which heterologous DNA has become integrated into the genomic DNA
of the cells.
[0054] A "fusion protein" is a hybrid protein expressed by a
nucleic acid molecule comprising nucleotide sequences of at least
two polynucleotides, genes, or cDNAs. For example, a fusion protein
can comprise at least part of a mouse Zcytor16 polypeptide fused
with a polypeptide that binds an affinity matrix. Such a fusion
protein provides a means to isolate large quantities of mouse
Zcytor16 using affinity chromatography.
[0055] The term "receptor" denotes a cell-associated protein that
binds to a bioactive molecule termed a "ligand." This interaction
mediates the effect of the ligand on the cell. Receptors can be
membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid
stimulating hormone receptor, beta-adrenergic receptor) or
multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3
receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor
and IL-6 receptor). Membrane-bound receptors are characterized by a
multi-domain structure comprising an extracellular ligand-binding
domain and an intracellular effector domain that is typically
involved in signal transduction. In certain membrane-bound
receptors, the extracellular ligand-binding domain and the
intracellular effector domain are located in separate polypeptides
that comprise the complete functional receptor.
[0056] In general, the binding of ligand to receptor results in a
conformational change in the receptor that causes an interaction
between the effector domain and other molecule(s) in the cell,
which in turn leads to an alteration in the metabolism of the cell.
Metabolic events that are often linked to receptor-ligand
interactions include gene transcription, phosphorylation,
dephosphorylation, increases in cyclic AMP production, mobilization
of cellular calcium, mobilization of membrane lipids, cell
adhesion, hydrolysis of inositol lipids and hydrolysis of
phospholipids.
[0057] A "soluble receptor" is a receptor polypeptide that is not
bound to a cell membrane. Soluble receptors are most commonly
ligand-binding receptor polypeptides that lack transmembrane and
cytoplasmic domains, and other linkage to the cell membrane such as
via glycophosphoinositol (gpi). Soluble receptors can comprise
additional amino acid residues, such as affinity tags that provide
for purification of the polypeptide or provide sites for attachment
of the polypeptide to a substrate, or immunoglobulin constant
region sequences. Many cell-surface receptors have naturally
occurring, soluble counterparts that are produced by proteolysis or
translated from alternatively spliced mRNAs. Soluble receptors can
be monomeric, homodimeric, heterodimeric, or multimeric, with
multimeric receptors generally not comprising more than 9 subunits,
preferably not comprising more than 6 subunits, and most preferably
not comprising more than 3 subunits. Receptor polypeptides are said
to be substantially free of transmembrane and intracellular
polypeptide segments when they lack sufficient portions of these
segments to provide membrane anchoring or signal transduction,
respectively. Soluble receptors of class I and class II cytokine
receptors generally comprise the extracellular cytokine binding
domain free of a transmembrane domain and intracellular domain. For
example, representative soluble receptors include a soluble
receptor for CRF2-4 (Genbank Accession No. Z17227) as shown in SEQ
ID NO:35; a soluble receptor for IL-10R (Genbank Accession No.s
U00672 and NM-001558) as shown in SEQ ID NO:36; and a soluble
receptor for zcytor11 (U.S. Pat. No. 5,965,704) as shown in SEQ ID
NO:34. It is well within the level of one of skill in the art to
delineate what sequences of a known class I or class II cytokine
sequence comprise the extracellular cytokine binding domain free of
a transmembrane domain and intracellular domain. Moreover, one of
skill in the art using the genetic code can readily determine
polynucleotides that encode such soluble receptor polypeptides.
[0058] The term "secretory signal sequence" denotes a DNA sequence
that encodes a peptide (a "secretory peptide") that, as a component
of a larger polypeptide, directs the larger polypeptide through a
secretory pathway of a cell in which it is synthesized. The larger
polypeptide is commonly cleaved to remove the secretory peptide
during transit through the secretory pathway.
[0059] An "isolated polypeptide" is a polypeptide that is
essentially free from contaminating cellular components, such as
carbohydrate, lipid, or other proteinaceous impurities associated
with the polypeptide in nature. Typically, a preparation of
isolated polypeptide contains the polypeptide in a highly purified
form, i.e., at least about 80% pure, at least about 90% pure, at
least about 95% pure, greater than 95% pure, or greater than 99%
pure. One way to show that a particular protein preparation
contains an isolated polypeptide is by the appearance of a single
band following sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis of the protein preparation and Coomassie Brilliant
Blue staining of the gel. However, the term "isolated" does not
exclude the presence of the same polypeptide in alternative
physical forms, such as dimers or alternatively glycosylated or
derivatized forms.
[0060] The terms "amino-terminal" and "carboxyl-terminal" are used
herein to denote positions within polypeptides. Where the context
allows, these terms are used with reference to a particular
sequence or portion of a polypeptide to denote proximity or
relative position. For example, a certain sequence positioned
carboxyl-terminal to a reference sequence within a polypeptide is
located proximal to the carboxyl terminus of the reference
sequence, but is not necessarily at the carboxyl terminus of the
complete polypeptide.
[0061] The term "expression" refers to the biosynthesis of a gene
product. For example, in the case of a structural gene, expression
involves transcription of the structural gene into mRNA and the
translation of mRNA into one or more polypeptides.
[0062] The term "splice variant" is used herein to denote
alternative forms of RNA transcribed from a gene. Splice variation
arises naturally through use of alternative splicing sites within a
transcribed RNA molecule, or less commonly between separately
transcribed RNA molecules, and may result in several mRNAs
transcribed from the same gene. Splice variants may encode
polypeptides having altered amino acid sequence. The term splice
variant is also used herein to denote a polypeptide encoded by a
splice variant of an mRNA transcribed from a gene.
[0063] As used herein, the term "immunomodulator" includes
cytokines, stem cell growth factors, lymphotoxins, co-stimulatory
molecules, hematopoietic factors, and synthetic analogs of these
molecules.
[0064] The term "complement/anti-complement pair" denotes
non-identical moieties that form a non-covalently associated,
stable pair under appropriate conditions. For instance, biotin and
avidin (or streptavidin) are prototypical members of a
complement/anti-complement pair. Other exemplary
complement/anti-complement pairs include receptor/ligand pairs,
antibody/antigen (or hapten or epitope) pairs, sense/antisense
polynucleotide pairs, and the like. Where subsequent dissociation
of the complement/anti-complement pair is desirable, the
complement/anti-complement pair preferably has a binding affinity
of less than 10.sup.9 M.sup.-1.
[0065] An "anti-idiotype antibody" is an antibody that binds with
the variable region domain of an immunoglobulin. In the present
context, an anti-idiotype antibody binds with the variable region
of an anti-mouse Zcytor16 antibody, and thus, an anti-idiotype
antibody mimics an epitope of mouse Zcytor16.
[0066] An "antibody fragment" is a portion of an antibody such as
F(ab').sub.2, F(ab).sub.2, Fab', Fab, and the like. Regardless of
structure, an antibody fragment binds with the same antigen that is
recognized by the intact antibody. For example, an anti-mouse
Zcytor16 monoclonal antibody fragment binds with an epitope of
mouse Zcytor16.
[0067] The term "antibody fragment" also includes a synthetic or a
genetically engineered polypeptide that binds to a specific
antigen, such as polypeptides consisting of the light chain
variable region, "Fv" fragments consisting of the variable regions
of the heavy and light chains, recombinant single chain polypeptide
molecules in which light and heavy variable regions are connected
by a peptide linker ("scFv proteins"), and minimal recognition
units consisting of the amino acid residues that mimic the
hypervariable region.
[0068] A "chimeric antibody" is a recombinant protein that contains
the variable domains and complementary determining regions derived
from a rodent antibody, while the remainder of the antibody
molecule is derived from a human antibody.
[0069] "Humanized antibodies" are recombinant proteins in which
murine complementarity determining regions of a monoclonal antibody
have been transferred from heavy and light variable chains of the
murine immunoglobulin into a human variable domain.
[0070] As used herein, a "therapeutic agent" is a molecule or atom
which is conjugated to an antibody moiety to produce a conjugate
which is useful for therapy. Examples of therapeutic agents include
drugs, toxins, immunomodulators, chelators, boron compounds,
photoactive agents or dyes, and radioisotopes.
[0071] A "detectable label" is a molecule or atom which can be
conjugated to an antibody moiety to produce a molecule useful for
diagnosis. Examples of detectable labels include chelators,
photoactive agents, radioisotopes, fluorescent agents, paramagnetic
ions, or other marker moieties.
[0072] The term "affinity tag" is used herein to denote a
polypeptide segment that can be attached to a second polypeptide to
provide for purification or detection of the second polypeptide or
provide sites for attachment of the second polypeptide to a
substrate. In principal, any peptide or protein for which an
antibody or other specific binding agent is available can be used
as an affinity tag. Affinity tags include a poly-histidine tract,
protein A (Nilsson et al., EMBO J. 4:1075 (1985); Nilsson et al.,
Methods Enzymol. 198:3 (1991)), glutathione S transferase (Smith
and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer
et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)), substance P,
FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),
streptavidin binding peptide, or other antigenic epitope or binding
domain. See, in general, Ford et al., Protein Expression and
Purification 2:95 (1991). DNA molecules encoding affinity tags are
available from commercial suppliers (e.g., Pharmacia Biotech,
Piscataway, N.J.).
[0073] A "naked antibody" is an entire antibody, as opposed to an
antibody fragment, which is not conjugated with a therapeutic
agent. Naked antibodies include both polyclonal and monoclonal
antibodies, as well as certain recombinant antibodies, such as
chimeric and humanized antibodies.
[0074] As used herein, the term "antibody component" includes both
an entire antibody and an antibody fragment.
[0075] An "immunoconjugate" is a conjugate of an antibody component
with a therapeutic agent or a detectable label.
[0076] As used herein, the term "antibody fusion protein" refers to
a recombinant molecule that comprises an antibody component and a
mouse Zcytor16 polypeptide component. Examples of an antibody
fusion protein include a protein that comprises a mouse Zcytor16
extracellular domain, and either an Fc domain or an antigen-biding
region.
[0077] A "target polypeptide" or a "target peptide" is an amino
acid sequence that comprises at least one epitope, and that is
expressed on a target cell, such as a tumor cell, or a cell that
carries an infectious agent antigen. T cells recognize peptide
epitopes presented by a major histocompatibility complex molecule
to a target polypeptide or target peptide and typically lyse the
target cell or recruit other immune cells to the site of the target
cell, thereby killing the target cell.
[0078] An "antigenic peptide" is a peptide which will bind a major
histocompatibility complex molecule to form an MHC-peptide complex
which is recognized by a T cell, thereby inducing a cytotoxic
lymphocyte response upon presentation to the T cell. Thus,
antigenic peptides are capable of binding to an appropriate major
histocompatibility complex molecule and inducing a cytotoxic T
cells response, such as cell lysis or specific cytokine release
against the target cell which binds or expresses the antigen. The
antigenic peptide can be bound in the context of a class I or class
II major histocompatibility complex molecule, on an antigen
presenting cell or on a target cell.
[0079] In eukaryotes, RNA polymerase II catalyzes the transcription
of a structural gene to produce mRNA. A nucleic acid molecule can
be designed to contain an RNA polymerase II template in which the
RNA transcript has a sequence that is complementary to that of a
specific mRNA. The RNA transcript is termed an "anti-sense RNA" and
a nucleic acid molecule that encodes the anti-sense RNA is termed
an "anti-sense gene." Anti-sense RNA molecules are capable of
binding to mRNA molecules, resulting in an inhibition of mRNA
translation.
[0080] An "anti-sense oligonucleotide specific for mouse Zcytor16"
or a "mouse Zcytor16 anti-sense oligonucleotide" is an
oligonucleotide having a sequence (a) capable of forming a stable
triplex with a portion of the mouse Zcytor16 gene, or (b) capable
of forming a stable duplex with a portion of an mRNA transcript of
the mouse Zcytor16 gene.
[0081] A "ribozyme" is a nucleic acid molecule that contains a
catalytic center. The term includes RNA enzymes, self-splicing
RNAs, self-cleaving RNAs, and nucleic acid molecules that perform
these catalytic functions. A nucleic acid molecule that encodes a
ribozyme is termed a "ribozyme gene."
[0082] An "external guide sequence" is a nucleic acid molecule that
directs the endogenous ribozyme, RNase P, to a particular species
of intracellular mRNA, resulting in the cleavage of the mRNA by
RNase P. A nucleic acid molecule that encodes an external guide
sequence is termed an "external guide sequence gene."
[0083] The term "variant mouse Zcytor16 gene" refers to nucleic
acid molecules that encode a polypeptide having an amino acid
sequence that is a modification of SEQ ID NO:38 or SEQ ID NO:48.
Such variants include naturally-occurring polymorphisms of mouse
Zcytor16 genes, as well as synthetic genes that contain
conservative amino acid substitutions of the amino acid sequence of
SEQ ID NO:38 or SEQ ID NO:48. Additional variant forms of mouse
Zcytor16 genes are nucleic acid molecules that contain insertions
or deletions of the nucleotide sequences described herein. A
variant mouse Zcytor16 gene can be identified, for example, by
determining whether the gene hybridizes with a nucleic acid
molecule having the nucleotide sequence of SEQ ID NO:37 or SEQ ID
NO:47, or its complement, under stringent conditions.
[0084] Alternatively, variant mouse Zcytor16 genes can be
identified by sequence comparison. Two amino acid sequences have
"100% amino acid sequence identity" if the amino acid residues of
the two amino acid sequences are the same when aligned for maximal
correspondence. Similarly, two nucleotide sequences have "100%
nucleotide sequence identity" if the nucleotide residues of the two
nucleotide sequences are the same when aligned for maximal
correspondence. Sequence comparisons can be performed using
standard software programs such as those included in the LASERGENE
bioinformatics computing suite, which is produced by DNASTAR
(Madison, Wis.). Other methods for comparing two nucleotide or
amino acid sequences by determining optimal alignment are
well-known to those of skill in the art (see, for example, Peruski
and Peruski, The Internet and the New Biology: Tools for Genomic
and Molecular Research (ASM Press, Inc. 1997), Wu et al. (eds.),
"Information Superhighway and Computer Databases of Nucleic Acids
and Proteins," in Methods in Gene Biotechnology, pages 123-151 (CRC
Press, Inc. 1997), and Bishop (ed.), Guide to Human Genome
Computing, 2nd Edition (Academic Press, Inc. 1998)). Particular
methods for determining sequence identity are described below.
[0085] Regardless of the particular method used to identify a
variant mouse Zcytor16 gene or variant mouse Zcytor16 polypeptide,
a variant gene or polypeptide encoded by a variant gene may be
functionally characterized the ability to bind specifically to an
anti-mouse Zcytor16 antibody. A variant mouse Zcytor16 gene or
variant mouse Zcytor16 polypeptide may also be functionally
characterized the ability to bind to its ligand, IL-TIF, using a
biological or biochemical assay described herein.
[0086] The term "allelic variant" is used herein to denote any of
two or more alternative forms of a gene occupying the same
chromosomal locus. Allelic variation arises naturally through
mutation, and may result in phenotypic polymorphism within
populations. Gene mutations can be silent (no change in the encoded
polypeptide) or may encode polypeptides having altered amino acid
sequence. The term allelic variant is also used herein to denote a
protein encoded by an allelic variant of a gene.
[0087] The term "ortholog" denotes a polypeptide or protein
obtained from one species that is the functional counterpart of a
polypeptide or protein from a different species. Sequence
differences among orthologs are the result of speculation.
[0088] "Paralogs" are distinct but structurally related proteins
made by an organism. Paralogs are believed to arise through gene
duplication. For example, .alpha.-globin, .beta.-globin, and
myoglobin are paralogs of each other.
[0089] The present invention includes functional fragments of mouse
Zcytor16 genes. Within the context of this invention, a "functional
fragment" of a mouse Zcytor16 gene refers to a nucleic acid
molecule that encodes a portion of a mouse Zcytor16 polypeptide
which is a domain described herein or at least specifically binds
with an anti-mouse Zcytor16 antibody.
[0090] Due to the imprecision of standard analytical methods,
molecular weights and lengths of polymers are understood to be
approximate values. When such a value is expressed as "about" X or
"approximately" X, the stated value of X will be understood to be
accurate to 110%.
3. Production of Mouse Zcytor16 Polynucleotides or Genes
[0091] Nucleic acid molecules encoding a mouse Zcytor16 gene can be
obtained by screening a mouse cDNA or genomic library using
polynucleotide probes based upon SEQ ID NO:37 or SEQ ID NO:47.
These techniques are standard and well-established.
[0092] As an illustration, a nucleic acid molecule that encodes a
mouse Zcytor16 gene can be isolated from a cDNA library. In this
case, the first step would be to prepare the cDNA library by
isolating RNA from a tissue, such as a tissue wherein mouse
Zcytor16 is specifically expressed, using methods well-known to
those of skill in the art. In general, RNA isolation techniques
must provide a method for breaking cells, a means of inhibiting
RNase-directed degradation of RNA, and a method of separating RNA
from DNA, protein, and polysaccharide contaminants. For example,
total RNA can be isolated by freezing tissue in liquid nitrogen,
grinding the frozen tissue with a mortar and pestle to lyse the
cells, extracting the ground tissue with a solution of
phenol/chloroform to remove proteins, and separating RNA from the
remaining impurities by selective precipitation with lithium
chloride (see, for example, Ausubel et al. (eds.), Short Protocols
in Molecular Biology, 3.sup.rd Edition, pages 4-1 to 4-6 (John
Wiley & Sons 1995) ["Ausubel (1995)"]; Wu et al., Methods in
Gene Biotechnology, pages 33-41 (CRC Press, Inc. 1997) ["Wu
(1997)"]).
[0093] Alternatively, total RNA can be isolated by extracting
ground tissue with guanidinium isothiocyanate, extracting with
organic solvents, and separating RNA from contaminants using
differential centrifugation (see, for example, Chirgwin et al.,
Biochemistry 18:52 (1979); Ausubel (1995) at pages 4-1 to 4-6; Wu
(1997) at pages 33-41).
[0094] In order to construct a cDNA library, poly(A).sup.+ RNA must
be isolated from a total RNA preparation. Poly(A).sup.+ RNA can be
isolated from total RNA using the standard technique of
oligo(dT)-cellulose chromatography (see, for example, Aviv and
Leder, Proc. Nat'l Acad. Sci. USA 69:1408 (1972); Ausubel (1995) at
pages 4-11 to 4-12).
[0095] Double-stranded cDNA molecules are synthesized from
poly(A).sup.+ RNA using techniques well-known to those in the art.
(see, for example, Wu (1997) at pages 41-46). Moreover,
commercially available kits can be used to synthesize
double-stranded cDNA molecules. For example, such kits are
available from Life Technologies, Inc. (Gaithersburg, Md.),
CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Promega
Corporation (Madison, Wis.) and STRATAGENE (La Jolla, Calif.).
[0096] Various cloning vectors are appropriate for the construction
of a cDNA library. For example, a cDNA library can be prepared in a
vector derived from bacteriophage, such as a .lamda.gt10 vector.
See, for example, Huynh et al., "Constructing and Screening cDNA
Libraries in .lamda.gt10 and .lamda.gt11," in DNA Cloning: A
Practical Approach Vol. 1, Glover (ed.), page 49 (IRL Press, 1985);
Wu (1997) at pages 47-52.
[0097] Alternatively, double-stranded cDNA molecules can be
inserted into a plasmid vector, such as a pBLUESCRIPT vector
(STRATAGENE; La Jolla, Calif.), a LAMDAGEM-4 (Promega Corp.) or
other commercially available vectors. Suitable cloning vectors also
can be obtained from the American Type Culture Collection
(Manassas, Va.).
[0098] To amplify the cloned cDNA molecules, the cDNA library is
inserted into a prokaryotic host, using standard techniques. For
example, a cDNA library can be introduced into competent E. coli
DH5 or DH10B cells, which can be obtained, for example, from Life
Technologies, Inc. or GIBCO BRL (Gaithersburg, Md.).
[0099] A human genomic library can be prepared by means well known
in the art (see, for example, Ausubel (1995) at pages 5-1 to 5-6;
Wu (1997) at pages 307-327). Genomic DNA can be isolated by lysing
tissue with the detergent Sarkosyl, digesting the lysate with
proteinase K, clearing insoluble debris from the lysate by
centrifugation, precipitating nucleic acid from the lysate using
isopropanol, and purifying resuspended DNA on a cesium chloride
density gradient.
[0100] DNA fragments that are suitable for the production of a
genomic library can be obtained by the random shearing of genomic
DNA or by the partial digestion of genomic DNA with restriction
endonucleases. Genomic DNA fragments can be inserted into a vector,
such as a bacteriophage or cosmid vector, in accordance with
conventional techniques, such as the use of restriction enzyme
digestion to provide appropriate termini, the use of alkaline
phosphatase treatment to avoid undesirable joining of DNA
molecules, and ligation with appropriate ligases. Techniques for
such manipulation are well known in the art (see, for example,
Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) at pages
307-327).
[0101] Alternatively, human genomic libraries can be obtained from
commercial sources such as Research Genetics (Huntsville, Ala.) and
the American Type Culture Collection (Manassas, Va.).
[0102] A library containing cDNA or genomic clones can be screened
with one or more polynucleotide probes based upon SEQ ID NO:37 or
SEQ ID NO:47, using standard methods (see, for example, Ausubel
(1995) at pages 6-1 to 6-11).
[0103] Nucleic acid molecules that encode a mouse Zcytor16 gene can
also be obtained using the polymerase chain reaction (PCR) with
oligonucleotide primers having nucleotide sequences that are based
upon the nucleotide sequences of the mouse Zcytor16 gene, as
described herein. General methods for screening libraries with PCR
are provided by, for example, Yu et al., "Use of the Polymerase
Chain Reaction to Screen Phage Libraries," in Methods in Molecular
Biology, Vol. 15: PCR Protocols: Current Methods and Applications,
White (ed.), pages 211-215 (Humana Press, Inc. 1993). Moreover,
techniques for using PCR to isolate related genes are described by,
for example, Preston, "Use of Degenerate Oligonucleotide Primers
and the Polymerase Chain Reaction to Clone Gene Family Members," in
Methods in Molecular Biology, Vol. 15: PCR Protocols: Current
Methods and Applications, White (ed.), pages 317-337 (Humana Press,
Inc. 1993).
[0104] Anti-mouse Zcytor16 antibodies, produced as described below,
can also be used to isolate DNA sequences that encode mouse
Zcytor16 genes from cDNA libraries. For example, the antibodies can
be used to screen .lamda.gt11 expression libraries, or the
antibodies can be used for immunoscreening following hybrid
selection and translation (see, for example, Ausubel (1995) at
pages 6-12 to 6-16; Margolis et al., "Screening X expression
libraries with antibody and protein probes," in DNA Cloning 2:
Expression Systems, 2nd Edition, Glover et al. (eds.), pages 1-14
(Oxford University Press 1995)).
[0105] As an alternative, a mouse Zcytor16 gene can be obtained by
synthesizing nucleic acid molecules using mutually priming long
oligonucleotides and the nucleotide sequences described herein
(see, for example, Ausubel (1995) at pages 8-8 to 8-9). Established
techniques using the polymerase chain reaction provide the ability
to synthesize DNA molecules at least two kilobases in length (Adang
et al., Plant Molec. Biol. 21:1131 (1993), Bambot et al., PCR
Methods and Applications 2:266 (1993), Dillon et al., "Use of the
Polymerase Chain Reaction for the Rapid Construction of Synthetic
Genes," in Methods in Molecular Biology, Vol. 15: PCR Protocols:
Current Methods and Applications, White (ed.), pages 263-268,
(Humana Press, Inc. 1993), and Holowachuk et al., PCR Methods Appl.
4:299 (1995)).
[0106] The nucleic acid molecules of the present invention can also
be synthesized with "gene machines" using protocols such as the
phosphoramidite method. If chemically-synthesized double stranded
DNA is required for an application such as the synthesis of a gene
or a gene fragment, then each complementary strand is made
separately. The production of short genes (60 to 80 base pairs) is
technically straightforward and can be accomplished by synthesizing
the complementary strands and then annealing them. For the
production of longer genes (>300 base pairs), however, special
strategies may be required, because the coupling efficiency of each
cycle during chemical DNA synthesis is seldom 100%. To overcome
this problem, synthetic genes (double-stranded) are assembled in
modular form from single-stranded fragments that are from 20 to 100
nucleotides in length. For reviews on polynucleotide synthesis,
see, for example, Glick and Pasternak, Molecular Biotechnology,
Principles and Applications of Recombinant DNA (ASM Press 1994),
Itakura et al., Ann. Rev. Biochem. 53:323 (1984), and Climie et
al., Proc. Nat'l Acad. Sci. USA 87:633 (1990).
[0107] The sequence of a mouse Zcytor16 cDNA or mouse Zcytor16
genomic fragment can be determined using standard methods. Mouse
Zcytor16 polynucleotide sequences disclosed herein can also be used
as probes or primers to clone 5' non-coding regions of a mouse
Zcytor16 gene. Because mouse Zcytor16 is expressed in a limited
number of specific tissues, promoter elements from a mouse Zcytor16
gene can be used to direct the expression of heterologous genes in,
for example, specific tissues of transgenic animals or patients
treated with gene therapy. The identification of genomic fragments
containing a mouse Zcytor16 promoter or regulatory element can be
achieved using well-established techniques, such as deletion
analysis (see, generally, Ausubel (1995)).
[0108] Cloning of 5' flanking sequences also facilitates production
of mouse Zcytor16 proteins by "gene activation," as disclosed in
U.S. Pat. No. 5,641,670. Briefly, expression of an endogenous mouse
Zcytor16 gene in a cell is altered by introducing into the mouse
Zcytor16 locus a DNA construct comprising at least a targeting
sequence, a regulatory sequence, an exon, and an unpaired splice
donor site. The targeting sequence is a mouse Zcytor16 5'
non-coding sequence that permits homologous recombination of the
construct with the endogenous mouse Zcytor16 locus, whereby the
sequences within the construct become operably linked with the
endogenous mouse Zcytor16 coding sequence. In this way, an
endogenous mouse Zcytor16 promoter can be replaced or supplemented
with other regulatory sequences to provide enhanced,
tissue-specific, or otherwise regulated expression.
4. Production of Mouse Zcytor16 Gene Variants
[0109] The present invention provides a variety of nucleic acid
molecules, including DNA and RNA molecules, that encode the mouse
Zcytor16 polypeptides disclosed herein. Those skilled in the art
will readily recognize that, in view of the degeneracy of the
genetic code, considerable sequence variation is possible among
these polynucleotide molecules. SEQ ID NO:3 is a degenerate
nucleotide sequence that encompasses all nucleic acid molecules
that encode the human Zcytor16 polypeptide of SEQ ID NO:2.
Similarly, SEQ ID NO:39 is a degenerate nucleotide sequence that
encompasses all nucleic acid molecules that encode the mouse
Zcytor16 polypeptide of SEQ ID NO:38; and SEQ ID NO:49 is a
degenerate nucleotide sequence that encompasses all nucleic acid
molecules that encode the mouse Zcytor16 polypeptide of SEQ ID
NO:48. Those skilled in the art will recognize that the degenerate
sequence of SEQ ID NO:39 and SEQ ID NO:49 also provides all RNA
sequences encoding SEQ ID NO:38 and SEQ ID NO:48 respectively, by
substituting U for T. Moreover, the present invention also provides
isolated soluble monomeric, homodimeric, heterodimeric and
multimeric receptor polypeptides that comprise at least one mouse
Zcytor16 receptor subunit that is substantially homologous to the
receptor polypeptide of SEQ ID NO:38 or SEQ ID NO:48. Thus, the
present invention contemplates mouse Zcytor16 polypeptide-encoding
nucleic acid molecules comprising nucleotide 1 to nucleotide 693 of
SEQ ID NO:39, and nucleic acid molecules comprising nucleotide 1 to
nucleotide 693 of SEQ ID NO:49, and their RNA equivalents.
Moreover, mouse zcytor16 fragments described herein in SEQ ID
NO:37, SEQ ID NO:38, SEQ ID NO:47 and SEQ ID NO:48 with reference
to SEQ ID NO:39 and SEQ ID NO:49, are also contemplated.
[0110] Table 1 sets forth the one-letter codes used within SEQ ID
NO:39 or SEQ ID NO:49 to denote degenerate nucleotide positions.
"Resolutions" are the nucleotides denoted by a code letter.
"Complement" indicates the code for the complementary
nucleotide(s). For example, the code Y denotes either C or T, and
its complement R denotes A or G, A being complementary to T, and G
being complementary to C.
TABLE-US-00001 TABLE 1 Nucleotide Resolution Complement Resolution
A A T T C C G G G G C C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T
K G|T M A|C S C|G S C|G W A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G
V A|C|G B C|G|T D A|G|T H A|C|T N A|C|G|T N A|C|G|T
[0111] The degenerate codons used in SEQ ID NO:39 or SEQ ID NO:49,
encompassing all possible codons for a given amino acid, are set
forth in Table 2.
TABLE-US-00002 TABLE 2 One Amino Letter Degenerate Acid Code Codons
Codon Cys C TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA
ACC ACG ACT ACN Pro P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN
Gly G GGA GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E
GAA GAG GAR Gln Q CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA
CGC CGG CGT MGN Lys K AAA AAG AAR Met M ATG ATG Ile I ATA ATC ATT
ATH Leu L CTA CTC CTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe
F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGG TGG Ter .cndot. TAA TAG
TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN
[0112] One of ordinary skill in the art will appreciate that some
ambiguity is introduced in determining a degenerate codon,
representative of all possible codons encoding an amino acid. For
example, the degenerate codon for serine (WSN) can, in some
circumstances, encode arginine (AGR), and the degenerate codon for
arginine (MGN) can, in some circumstances, encode serine (AGY). A
similar relationship exists between codons encoding phenylalanine
and leucine. Thus, some polynucleotides encompassed by the
degenerate sequence may encode variant amino acid sequences, but
one of ordinary skill in the art can easily identify such variant
sequences by reference to the amino acid sequences of SEQ ID NO:38
or SEQ ID NO:48. Variant sequences can be readily tested for
functionality as described herein.
[0113] Different species can exhibit "preferential codon usage." In
general, see, Grantham et al., Nucl. Acids Res. 8:1893 (1980), Haas
et al. Curr. Biol. 6:315 (1996), Wain-Hobson et al., Gene 13:355
(1981), Grosjean and Fiers, Gene 18:199 (1982), Holm, Nuc. Acids
Res. 14:3075 (1986), Ikemura, J. Mol. Biol. 158:573 (1982), Sharp
and Matassi, Curr. Opin. Genet. Dev. 4:851 (1994), Kane, Curr.
Opin. Biotechnol. 6:494 (1995), and Makrides, Microbiol. Rev.
60:512 (1996). As used herein, the term "preferential codon usage"
or "preferential codons" is a term of art referring to protein
translation codons that are most frequently used in cells of a
certain species, thus favoring one or a few representatives of the
possible codons encoding each amino acid (See Table 2). For
example, the amino acid threonine (Thr) may be encoded by ACA, ACC,
ACG, or ACT, but in mammalian cells ACC is the most commonly used
codon; in other species, for example, insect cells, yeast, viruses
or bacteria, different Thr codons may be preferential. Preferential
codons for a particular species can be introduced into the
polynucleotides of the present invention by a variety of methods
known in the art. Introduction of preferential codon sequences into
recombinant DNA can, for example, enhance production of the protein
by making protein translation more efficient within a particular
cell type or species. Therefore, the degenerate codon sequences
disclosed herein serve as a template for optimizing expression of
polynucleotides in various cell types and species commonly used in
the art and disclosed herein. Sequences containing preferential
codons can be tested and optimized for expression in various
species, and tested for functionality as disclosed herein.
[0114] The present invention further provides variant polypeptides
and nucleic acid molecules that represent counterparts from other
species (orthologs). These species include, but are not limited to
mammalian, avian, amphibian, reptile, fish, insect and other
vertebrate and invertebrate species. Of particular interest are
Zcytor16 polypeptides from other mammalian species, including
human, rat, porcine, ovine, bovine, canine, feline, equine, and
other primate polypeptides. Orthologs of mouse Zcytor16 can be
cloned using information and compositions provided by the present
invention in combination with conventional cloning techniques. For
example, a mouse Zcytor16 cDNA can be cloned using mRNA obtained
from a tissue or cell type that expresses mouse Zcytor16 as
disclosed herein. Suitable sources of mRNA can be identified by
probing northern blots with probes designed from the sequences
disclosed herein. A library is then prepared from mRNA of a
positive tissue or cell line.
[0115] A Zcytor16-encoding cDNA can be isolated by a variety of
methods, such as by probing with a complete or partial cDNA or with
one or more sets of degenerate probes based on the disclosed
sequences. A cDNA can also be cloned using the polymerase chain
reaction with primers designed from the representative mouse
Zcytor16 sequences disclosed herein. In addition, a cDNA library
can be used to transform or transfect host cells, and expression of
the cDNA of interest can be detected with an antibody to mouse
Zcytor16 polypeptide.
[0116] Those skilled in the art will recognize that the sequence
disclosed in SEQ ID NO:37 or SEQ ID NO:47 represents a single
allele of mouse Zcytor16, and that allelic variation and
alternative splicing are expected to occur. Allelic variants of
this sequence can be cloned by probing cDNA or genomic libraries
from different individuals according to standard procedures.
Allelic variants of the nucleotide sequences disclosed herein,
including those containing silent mutations and those in which
mutations result in amino acid sequence changes, are within the
scope of the present invention, as are proteins which are allelic
variants of the amino acid sequences disclosed herein. cDNA
molecules generated from alternatively spliced mRNAs, which retain
the properties of the mouse Zcytor16 polypeptide are included
within the scope of the present invention, as are polypeptides
encoded by such cDNAs and mRNAs. Allelic variants and splice
variants of these sequences can be cloned by probing cDNA or
genomic libraries from different individuals or tissues according
to standard procedures known in the art.
[0117] Using the methods discussed above, one of ordinary skill in
the art can prepare a variety of polypeptides that comprise a
soluble receptor subunit that is substantially homologous to SEQ ID
NO:37 or SEQ ID NO:47, or SEQ ID NO:38 or SEQ ID NO:48, amino acids
24 to 230, or 27 to 230 of SEQ ID NO:38 or SEQ ID NO:48, or allelic
variants thereof and retain the ligand-binding properties of the
wild-type mouse Zcytor16 receptor. Such polypeptides may also
include additional polypeptide segments as generally disclosed
herein.
[0118] Within certain embodiments of the invention, the isolated
nucleic acid molecules can hybridize under stringent conditions to
nucleic acid molecules comprising nucleotide sequences disclosed
herein. For example, such nucleic acid molecules can hybridize
under stringent conditions to nucleic acid molecules comprising the
nucleotide sequence of SEQ ID NO:37 or SEQ ID NO:47, to nucleic
acid molecules consisting of the nucleotide sequence of nucleotides
8 to 694 or 697; or 77, 86, or 100 to 694 or 697 of SEQ ID NO:37 or
SEQ ID NO:47, or to nucleic acid molecules comprising a nucleotide
sequence complementary to SEQ ID NO:37 or SEQ ID NO:47 or to
nucleotides 8 to 694 or 697; or 77, 86, or 100 to 694 or 697 of SEQ
ID NO:37 or SEQ ID NO:47, or fragments thereof. In general,
stringent conditions are selected to be about 5.degree. C. lower
than the thermal melting point (T.sub.m) for the specific sequence
at a defined ionic strength and pH. The T.sub.m is the temperature
(under defined ionic strength and pH) at which 50% of the target
sequence hybridizes to a perfectly matched probe.
[0119] A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA
and DNA-RNA, can hybridize if the nucleotide sequences have some
degree of complementarity. Hybrids can tolerate mismatched base
pairs in the double helix, but the stability of the hybrid is
influenced by the degree of mismatch. The T.sub.m of the mismatched
hybrid decreases by 1.degree. C. for every 1-1.5% base pair
mismatch. Varying the stringency of the hybridization conditions
allows control over the degree of mismatch that will be present in
the hybrid. The degree of stringency increases as the hybridization
temperature increases and the ionic strength of the hybridization
buffer decreases. Stringent hybridization conditions encompass
temperatures of about 5-25.degree. C. below the T.sub.m of the
hybrid and a hybridization buffer having up to 1 M Na.sup.+. Higher
degrees of stringency at lower temperatures can be achieved with
the addition of formamide which reduces the T.sub.m of the hybrid
about 1.degree. C. for each 1% formamide in the buffer solution.
Generally, such stringent conditions include temperatures of
20-70.degree. C. and a hybridization buffer containing up to
6.times.SSC and 0-50% formamide. A higher degree of stringency can
be achieved at temperatures of from 40-70.degree. C. with a
hybridization buffer having up to 4.times.SSC and from 0-50%
formamide. Highly stringent conditions typically encompass
temperatures of 42-70.degree. C. with a hybridization buffer having
up to 1.times.SSC and 0-50% formamide. Different degrees of
stringency can be used during hybridization and washing to achieve
maximum specific binding to the target sequence. Typically, the
washes following hybridization are performed at increasing degrees
of stringency to remove non-hybridized polynucleotide probes from
hybridized complexes.
[0120] The above conditions are meant to serve as a guide and it is
well within the abilities of one skilled in the art to adapt these
conditions for use with a particular polynucleotide hybrid. The
T.sub.m for a specific target sequence is the temperature (under
defined conditions) at which 50% of the target sequence will
hybridize to a perfectly matched probe sequence. Those conditions
which influence the T.sub.m include, the size and base pair content
of the polynucleotide probe, the ionic strength of the
hybridization solution, and the presence of destabilizing agents in
the hybridization solution. Numerous equations for calculating
T.sub.m are known in the art, and are specific for DNA, RNA and
DNA-RNA hybrids and polynucleotide probe sequences of varying
length (see, for example, Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Edition (Cold Spring Harbor Press 1989);
Ausubel et al., (eds.), Current Protocols in Molecular Biology
(John Wiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide
to Molecular Cloning Techniques, (Academic Press, Inc. 1987); and
Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227 (1990)). Sequence
analysis software such as OLIGO 6.0 (LSR; Long Lake, Minn.) and
Primer Premier 4.0 (Premier Biosoft International; Palo Alto,
Calif.), as well as sites on the Internet, are available tools for
analyzing a given sequence and calculating T.sub.m based on user
defined criteria. Such programs can also analyze a given sequence
under defined conditions and identify suitable probe sequences.
Typically, hybridization of longer polynucleotide sequences, >50
base pairs, is performed at temperatures of about 20-25.degree. C.
below the calculated T.sub.m. For smaller probes, <50 base
pairs, hybridization is typically carried out at the T.sub.m or
5-10.degree. C. below. This allows for the maximum rate of
hybridization for DNA-DNA and DNA-RNA hybrids.
[0121] The length of the polynucleotide sequence influences the
rate and stability of hybrid formation. Smaller probe sequences,
<50 base pairs, reach equilibrium with complementary sequences
rapidly, but may form less stable hybrids. Incubation times of
anywhere from minutes to hours can be used to achieve hybrid
formation. Longer probe sequences come to equilibrium more slowly,
but form more stable complexes even at lower temperatures.
Incubations are allowed to proceed overnight or longer. Generally,
incubations are carried out for a period equal to three times the
calculated Cot time. Cot time, the time it takes for the
polynucleotide sequences to reassociate, can be calculated for a
particular sequence by methods known in the art.
[0122] The base pair composition of polynucleotide sequence will
effect the thermal stability of the hybrid complex, thereby
influencing the choice of hybridization temperature and the ionic
strength of the hybridization buffer. A-T pairs are less stable
than G-C pairs in aqueous solutions containing sodium chloride.
Therefore, the higher the G-C content, the more stable the hybrid.
Even distribution of G and C residues within the sequence also
contribute positively to hybrid stability. In addition, the base
pair composition can be manipulated to alter the T.sub.m of a given
sequence. For example, 5-methyldeoxycytidine can be substituted for
deoxycytidine and 5-bromodeoxuridine can be substituted for
thymidine to increase the T.sub.m, whereas
7-deazz-2'-deoxyguanosine can be substituted for guanosine to
reduce dependence on T.sub.m.
[0123] The ionic concentration of the hybridization buffer also
affects the stability of the hybrid. Hybridization buffers
generally contain blocking agents such as Denhardt's solution
(Sigma Chemical Co., St. Louis, Mo.), denatured salmon sperm DNA,
tRNA, milk powders (BLOTTO), heparin or SDS, and a Na.sup.+ source,
such as SSC (1.times.SSC: 0.15 M sodium chloride, 15 mM sodium
citrate) or SSPE (1.times.SSPE: 1.8 M NaCl, 10 mM
NaH.sub.2PO.sub.4, 1 mM EDTA, pH 7.7). Typically, hybridization
buffers contain from between 10 mM-1 M Na.sup.+. The addition of
destabilizing or denaturing agents such as formamide,
tetralkylammonium salts, guanidinium cations or thiocyanate cations
to the hybridization solution will alter the T.sub.m of a hybrid.
Typically, formamide is used at a concentration of up to 50% to
allow incubations to be carried out at more convenient and lower
temperatures. Formamide also acts to reduce non-specific background
when using RNA probes.
[0124] As an illustration, a nucleic acid molecule encoding a
variant mouse Zcytor16 polypeptide can be hybridized with a nucleic
acid molecule having the nucleotide sequence of SEQ ID NO:37 or SEQ
ID NO:47 (or its complement) at 42.degree. C. overnight in a
solution comprising 50% formamide, 5.times.SSC, 50 mM sodium
phosphate (pH 7.6), 5.times.Denhardt's solution
(100.times.Denhardt's solution: 2% (w/v) Ficoll 400, 2% (w/v)
polyvinylpyrrolidone, and 2% (w/v) bovine serum albumin), 10%
dextran sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm
DNA. One of skill in the art can devise variations of these
hybridization conditions. For example, the hybridization mixture
can be incubated at a higher temperature, such as about 65.degree.
C., in a solution that does not contain formamide. Moreover,
premixed hybridization solutions are available (e.g., EXPRESSHYB
Hybridization Solution from CLONTECH Laboratories, Inc.), and
hybridization can be performed according to the manufacturer's
instructions.
[0125] Following hybridization, the nucleic acid molecules can be
washed to remove non-hybridized nucleic acid molecules under
stringent conditions, or under highly stringent conditions. Typical
stringent washing conditions include washing in a solution of
0.5.times.-2.times.SSC with 0.1% sodium dodecyl sulfate (SDS) at
55-65.degree. C. As an illustration, nucleic acid molecules
encoding a variant mouse Zcytor16 polypeptide remain hybridized
with a nucleic acid molecule having the nucleotide sequence of SEQ
ID NO:37 or SEQ ID NO:47 (or its complement) under stringent
washing conditions, in which the wash stringency is equivalent to
0.5.times.-2.times.SSC with 0.1% SDS at 55-65.degree. C., including
0.5.times.SSC with 0.1% SDS at 55.degree. C., or 2.times.SSC with
0.1% SDS at 65.degree. C. One of skill in the art can readily
devise equivalent conditions, for example, by substituting SSPE for
SSC in the wash solution.
[0126] Typical highly stringent washing conditions include washing
in a solution of 0.1.times.-0.2.times.SSC with 0.1% sodium dodecyl
sulfate (SDS) at 50-65.degree. C. For example, nucleic acid
molecules encoding a variant mouse Zcytor16 polypeptide remain
hybridized with a nucleic acid molecule having the nucleotide
sequence of SEQ ID NO:37 or SEQ ID NO:47 (or its complement) under
highly stringent washing conditions, in which the wash stringency
is equivalent to 0.1.times.-0.2.times.SSC with 0.1% SDS at
50-65.degree. C., including 0.1.times.SSC with 0.1% SDS at
50.degree. C., or 0.2.times.SSC with 0.1% SDS at 65.degree. C.
[0127] The present invention also provides isolated mouse Zcytor16
polypeptides that have a substantially similar sequence identity to
the polypeptides of SEQ ID NO:38 or SEQ ID NO:48, or their
orthologs. The term "substantially similar sequence identity" is
used herein to denote polypeptides having at least 70%, at least
80%, at least 90%, at least 95% or greater than 95% sequence
identity to the sequences shown in SEQ ID NO:38 or SEQ ID NO:48, or
their orthologs.
[0128] The present invention also contemplates mouse Zcytor16
variant nucleic acid molecules that can be identified using two
criteria: a determination of the similarity between the encoded
polypeptide with the amino acid sequence of SEQ ID NO:38 or SEQ ID
NO:48, and a hybridization assay, as described above. Such mouse
Zcytor16 variants include nucleic acid molecules (1) that remain
hybridized with a nucleic acid molecule having the nucleotide
sequence of SEQ ID NO:37 or SEQ ID NO:47 (or its complement) under
stringent washing conditions, in which the wash stringency is
equivalent to 0.5.times.-2.times.SSC with 0.1% SDS at 55-65.degree.
C., and (2) that encode a polypeptide having at least 70%, at least
80%, at least 90%, at least 95% or greater than 95% sequence
identity to the amino acid sequence of SEQ ID NO:38 or SEQ ID
NO:48. Alternatively, mouse Zcytor16 variants can be characterized
as nucleic acid molecules (1) that remain hybridized with a nucleic
acid molecule having the nucleotide sequence of SEQ ID NO:37 or SEQ
ID NO:47 (or its complement) under highly stringent washing
conditions, in which the wash stringency is equivalent to
0.1.times.-0.2.times.SSC with 0.1% SDS at 50-65.degree. C., and (2)
that encode a polypeptide having at least 70%, at least 80%, at
least 90%, at least 95% or greater than 95% sequence identity to
the amino acid sequence of SEQ ID NO:38 or SEQ ID NO:48.
[0129] Percent sequence identity is determined by conventional
methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603
(1986), and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA
89:10915 (1992). Briefly, two amino acid sequences are aligned to
optimize the alignment scores using a gap opening penalty of 10, a
gap extension penalty of 1, and the "BLOSUM62" scoring matrix of
Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are
indicated by the standard one-letter codes). The percent identity
is then calculated as: ([Total number of identical matches]/[length
of the longer sequence plus the number of gaps introduced into the
longer sequence in order to align the two sequences])(100).
TABLE-US-00003 TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4
R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0
2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3
-3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3
1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3
-3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1
-2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1
-1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2
-3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2
-2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4
[0130] Those skilled in the art appreciate that there are many
established algorithms available to align two amino acid sequences.
The "FASTA" similarity search algorithm of Pearson and Lipman is a
suitable protein alignment method for examining the level of
identity shared by an amino acid sequence disclosed herein and the
amino acid sequence of a putative mouse Zcytor16 variant. The FASTA
algorithm is described by Pearson and Lipman, Proc. Nat'l Acad.
Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63
(1990). Briefly, FASTA first characterizes sequence similarity by
identifying regions shared by the query sequence (e.g., SEQ ID
NO:38 or SEQ ID NO:48) and a test sequence that have either the
highest density of identities (if the ktup variable is 1) or pairs
of identities (if ktup=2), without considering conservative amino
acid substitutions, insertions, or deletions. The ten regions with
the highest density of identities are then rescored by comparing
the similarity of all paired amino acids using an amino acid
substitution matrix, and the ends of the regions are "trimmed" to
include only those residues that contribute to the highest score.
If there are several regions with scores greater than the "cutoff"
value (calculated by a predetermined formula based upon the length
of the sequence and the ktup value), then the trimmed initial
regions are examined to determine whether the regions can be joined
to form an approximate alignment with gaps. Finally, the highest
scoring regions of the two amino acid sequences are aligned using a
modification of the Needleman-Wunsch-Sellers algorithm (Needleman
and Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, SIAM J. Appl.
Math. 26:787 (1974)), which allows for amino acid insertions and
deletions. Illustrative parameters for FASTA analysis are: ktup=1,
gap opening penalty=10, gap extension penalty=1, and substitution
matrix=BLOSUM62. These parameters can be introduced into a FASTA
program by modifying the scoring matrix file ("SMATRIX"), as
explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63
(1990).
[0131] FASTA can also be used to determine the sequence identity of
nucleic acid molecules using a ratio as disclosed above. For
nucleotide sequence comparisons, the ktup value can range between
one to six, preferably from three to six, most preferably three,
with other parameters set as described above.
[0132] The present invention includes nucleic acid molecules that
encode a polypeptide having a conservative amino acid change,
compared with an amino acid sequence disclosed herein. For example,
variants can be obtained that contain one or more amino acid
substitutions of SEQ ID NO:38 or SEQ ID NO:48, in which an alkyl
amino acid is substituted for an alkyl amino acid in a mouse
Zcytor16 amino acid sequence, an aromatic amino acid is substituted
for an aromatic amino acid in a mouse Zcytor16 amino acid sequence,
a sulfur-containing amino acid is substituted for a
sulfur-containing amino acid in a mouse Zcytor16 amino acid
sequence, a hydroxy-containing amino acid is substituted for a
hydroxy-containing amino acid in a mouse Zcytor16 amino acid
sequence, an acidic amino acid is substituted for an acidic amino
acid in a mouse Zcytor16 amino acid sequence, a basic amino acid is
substituted for a basic amino acid in a mouse Zcytor16 amino acid
sequence, or a dibasic monocarboxylic amino acid is substituted for
a dibasic monocarboxylic amino acid in a mouse Zcytor16 amino acid
sequence. Among the common amino acids, for example, a
"conservative amino acid substitution" is illustrated by a
substitution among amino acids within each of the following groups:
(1) glycine, alanine, valine, leucine, and isoleucine, (2)
phenylalanine, tyrosine, and tryptophan, (3) serine and threonine,
(4) aspartate and glutamate, (5) glutamine and asparagine, and (6)
lysine, arginine and histidine. The BLOSUM62 table is an amino acid
substitution matrix derived from about 2,000 local multiple
alignments of protein sequence segments, representing highly
conserved regions of more than 500 groups of related proteins
(Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 89:10915
(1992)). Accordingly, the BLOSUM62 substitution frequencies can be
used to define conservative amino acid substitutions that may be
introduced into the amino acid sequences of the present invention.
Although it is possible to design amino acid substitutions based
solely upon chemical properties (as discussed above), the language
"conservative amino acid substitution" preferably refers to a
substitution represented by a BLOSUM62 value of greater than -1.
For example, an amino acid substitution is conservative if the
substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
According to this system, preferred conservative amino acid
substitutions are characterized by a BLOSUM62 value of at least 1
(e.g., 1, 2 or 3), while more preferred conservative amino acid
substitutions are characterized by a BLOSUM62 value of at least 2
(e.g., 2 or 3).
[0133] Particular variants of mouse Zcytor16 are characterized by
having at least 70%, at least 80%, at least 90%, at least 95% or
greater than 95% sequence identity to the corresponding amino acid
sequence (e.g., SEQ ID NO:38 or SEQ ID NO:48), wherein the
variation in amino acid sequence is due to one or more conservative
amino acid substitutions.
[0134] Conservative amino acid changes in a mouse Zcytor16 gene can
be introduced, for example, by substituting nucleotides for the
nucleotides recited in SEQ ID NO:37 or SEQ ID NO:47. Such
"conservative amino acid" variants can be obtained by
oligonucleotide-directed mutagenesis, linker-scanning mutagenesis,
mutagenesis using the polymerase chain reaction, and the like (see
Ausubel (1995) at pages 8-10 to 8-22; and McPherson (ed.), Directed
Mutagenesis: A Practical Approach (IRL Press 1991)). A variant
mouse Zcytor16 polypeptide can be identified by the ability to
specifically bind anti-mouse Zcytor16 antibodies.
[0135] The proteins of the present invention can also comprise
non-naturally occurring amino acid residues. Non-naturally
occurring amino acids include, without limitation,
trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,
trans-4-hydroxyproline, N-methylglycine, allo-threonine,
methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine,
nitroglutamine, homoglutamine, pipecolic acid, thiazolidine
carboxylic acid, dehydroproline, 3- and 4-methylproline,
3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.
Several methods are known in the art for incorporating
non-naturally occurring amino acid residues into proteins. For
example, an in vitro system can be employed wherein nonsense
mutations are suppressed using chemically aminoacylated suppressor
tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA
are known in the art. Transcription and translation of plasmids
containing nonsense mutations is typically carried out in a
cell-free system comprising an E. coli S30 extract and commercially
available enzymes and other reagents. Proteins are purified by
chromatography. See, for example, Robertson et al., J. Am. Chem.
Soc. 113:2722 (1991), Ellman et al., Methods Enzymol. 202:301
(1991), Chung et al., Science 259:806 (1993), and Chung et al.,
Proc. Nat'l Acad. Sci. USA 90:10145 (1993).
[0136] In a second method, translation is carried out in Xenopus
oocytes by microinjection of mutated mRNA and chemically
aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.
271:19991 (1996)). Within a third method, E. coli cells are
cultured in the absence of a natural amino acid that is to be
replaced (e.g., phenylalanine) and in the presence of the desired
non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
The non-naturally occurring amino acid is incorporated into the
protein in place of its natural counterpart. See, Koide et al.,
Biochem. 33:7470 (1994). Naturally occurring amino acid residues
can be converted to non-naturally occurring species by in vitro
chemical modification. Chemical modification can be combined with
site-directed mutagenesis to further expand the range of
substitutions (Wynn and Richards, Protein Sci. 2:395 (1993)).
[0137] A limited number of non-conservative amino acids, amino
acids that are not encoded by the genetic code, non-naturally
occurring amino acids, and unnatural amino acids may be substituted
for mouse Zcytor16 amino acid residues.
[0138] Essential amino acids in the polypeptides of the present
invention can be identified according to procedures known in the
art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081 (1989), Bass et
al., Proc. Nat'l Acad. Sci. USA 88:4498 (1991), Coombs and Corey,
"Site-Directed Mutagenesis and Protein Engineering," in Proteins:
Analysis and Design, Angeletti (ed.), pages 259-311 (Academic
Press, Inc. 1998)). In the latter technique, single alanine
mutations are introduced at every residue in the molecule, and the
resultant mutant molecules are tested for biological activity to
identify amino acid residues that are critical to the activity of
the molecule. See also, Hilton et al., J. Biol. Chem. 271:4699
(1996).
[0139] Although sequence analysis can be used to further define the
Zcytor16 ligand binding region, amino acids that play a role in
mouse Zcytor16 binding activity (such as binding of mouse Zcytor16
to ligand IL-TIF, or to an anti-mouse Zcytor16 antibody) can also
be determined by physical analysis of structure, as determined by
such techniques as nuclear magnetic resonance, crystallography,
electron diffraction or photoaffinity labeling, in conjunction with
mutation of putative contact site amino acids. See, for example, de
Vos et al., Science 255:306 (1992), Smith et al., J. Mol. Biol.
224:899 (1992), and Wlodaver et al., FEBS Lett. 309:59 (1992).
[0140] Multiple amino acid substitutions can be made and tested
using known methods of mutagenesis and screening, such as those
disclosed by Reidhaar-Olson and Sauer (Science 241:53 (1988)) or
Bowie and Sauer (Proc. Nat'l Acad. Sci. USA 86:2152 (1989)).
Briefly, these authors disclose methods for simultaneously
randomizing two or more positions in a polypeptide, selecting for
functional polypeptide, and then sequencing the mutagenized
polypeptides to determine the spectrum of allowable substitutions
at each position. Other methods that can be used include phage
display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner et
al., U.S. Pat. No. 5,223,409, Huse, international publication No.
WO 92/06204, and region-directed mutagenesis (Derbyshire et al.,
Gene 46:145 (1986), and Ner et al., DNA 7:127, (1988)). Moreover,
mouse Zcytor16 labeled with biotin or FITC can be used for
expression cloning of Zcytor16 ligands.
[0141] Variants of the disclosed mouse Zcytor16 nucleotide and
polypeptide sequences can also be generated through DNA shuffling
as disclosed by Stemmer, Nature 370:389 (1994), Stemmer, Proc.
Nat'l Acad. Sci. USA 91:10747 (1994), and international publication
No. WO 97/20078. Briefly, variant DNA molecules are generated by in
vitro homologous recombination by random fragmentation of a parent
DNA followed by reassembly using PCR, resulting in randomly
introduced point mutations. This technique can be modified by using
a family of parent DNA molecules, such as allelic variants or DNA
molecules from different species, to introduce additional
variability into the process. Selection or screening for the
desired activity, followed by additional iterations of mutagenesis
and assay provides for rapid "evolution" of sequences by selecting
for desirable mutations while simultaneously selecting against
detrimental changes.
[0142] Mutagenesis methods as disclosed herein can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides in host cells. Mutagenized DNA
molecules that encode biologically active polypeptides, or
polypeptides that bind with anti-mouse Zcytor16 antibodies, can be
recovered from the host cells and rapidly sequenced using modern
equipment. These methods allow the rapid determination of the
importance of individual amino acid residues in a polypeptide of
interest, and can be applied to polypeptides of unknown
structure.
[0143] The present invention also includes "functional fragments"
of mouse Zcytor16 polypeptides and nucleic acid molecules encoding
such functional fragments. Routine deletion analyses of nucleic
acid molecules can be performed to obtain functional fragments of a
nucleic acid molecule that encodes a mouse Zcytor16 polypeptide. As
an illustration, DNA molecules having the nucleotide sequence of
SEQ ID NO:37 or SEQ ID NO:47 can be digested with Bal31 nuclease to
obtain a series of nested deletions. The fragments are then
inserted into expression vectors in proper reading frame, and the
expressed polypeptides are isolated and tested for the ability to
bind anti-mouse Zcytor16 antibodies. One alternative to exonuclease
digestion is to use oligonucleotide-directed mutagenesis to
introduce deletions or stop codons to specify production of a
desired fragment. Alternatively, particular fragments of a mouse
Zcytor16 gene can be synthesized using the polymerase chain
reaction.
[0144] This general approach is exemplified by studies on the
truncation at either or both termini of interferons have been
summarized by Horisberger and Di Marco, Pharmac. Ther. 66:507
(1995). Moreover, standard techniques for functional analysis of
proteins are described by, for example, Treuter et al., Molec. Gen.
Genet. 240:113 (1993), Content et al., "Expression and preliminary
deletion analysis of the 42 kDa 2-5A synthetase induced by human
interferon," in Biological Interferon Systems, Proceedings of
ISIR-TNO Meeting on Interferon Systems, Cantell (ed.), pages 65-72
(Nijhoff 1987), Herschman, "The EGF Receptor," in Control of Animal
Cell Proliferation, Vol. 1, Boynton et al., (eds.) pages 169-199
(Academic Press 1985), Coumailleau et al., J. Biol. Chem. 270:29270
(1995); Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi
et al., Biochem. Pharmacol. 50:1295 (1995), and Meisel et al.,
Plant Molec. Biol. 30:1 (1996).
[0145] Analysis of the particular sequences disclosed herein
provide a set of illustrative functional fragments presented in
Table 4, wherein "F.sub.m Domain" is used to denote fibronectin III
domains. The nucleotides encoding additional mouse zcytor16
functional variant domains described herein, not show in Table 4,
can be determined with reference to SEQ ID NO:37 or SEQ ID NO:47.
Such functional fragments include for example, the following
nucleotide sequences of SEQ ID NO:37 or SEQ ID NO:47: nucleotides
77, 86, or 100 to 373; nucleotides 77, 86, or 100 to 694 or 697;
nucleotides 401 to 694 or 697; and nucleotides 8 to 694 or 697, and
amino acid sequences encoded thereby, e.g., such as those shown in
SEQ ID NO:38 or SEQ ID NO:48 respectively.
TABLE-US-00004 TABLE 4 Amino acid residues Nucleotides Mouse
Zcytor16 (SEQ ID NO: 38 or (SEQ ID NO: 37 or Feature SEQ ID NO: 48)
SEQ ID NO: 47) First F.sub.III Domain 31-122 100-373 Second
F.sub.III Domain 131-229 401-694 Both F.sub.III Domains 31-229
100-694
[0146] The present invention also contemplates functional fragments
of a mouse Zcytor16 gene that have amino acid changes, compared
with an amino acid sequence disclosed herein. A variant mouse
Zcytor16 gene can be identified on the basis of structure by
determining the level of identity with disclosed nucleotide and
amino acid sequences, as discussed above. An alternative approach
to identifying a variant gene on the basis of structure is to
determine whether a nucleic acid molecule encoding a potential
variant mouse Zcytor16 gene can hybridize to a nucleic acid
molecule comprising a nucleotide sequence, such as SEQ ID NO:37 or
SEQ ID NO:47.
[0147] For any mouse Zcytor16 polypeptide, including variants and
fusion proteins, one of ordinary skill in the art can readily
generate a fully degenerate polynucleotide sequence encoding that
variant using the information set forth in Tables 1 and 2 above.
Moreover, those of skill in the art can use standard software to
devise mouse Zcytor16 variants based upon the nucleotide and amino
acid sequences described herein. Accordingly, the present invention
includes a computer-readable medium encoded with a data structure
that provides at least one of the following sequences: SEQ ID NO:37
or SEQ ID NO:47, SEQ ID NO:38 or SEQ ID NO:48, and SEQ ID NO:39 or
SEQ ID NO:49. Suitable forms of computer-readable media include
magnetic media and optically-readable media. Examples of magnetic
media include a hard or fixed drive, a random access memory (RAM)
chip, a floppy disk, digital linear tape (DLT), a disk cache, and a
ZIP disk. Optically readable media are exemplified by compact discs
(e.g., CD-read only memory (ROM), CD-rewritable (RW), and
CD-recordable), and digital versatile/video discs (DVD) (e.g.,
DVD-ROM, DVD-RAM, and DVD+RW).
[0148] The present invention also provides polypeptide fragments or
peptides comprising an epitope-bearing portion of a mouse Zcytor16
polypeptide described herein. Such fragments or peptides may
comprise an "immunogenic epitope," which is a part of a protein
that elicits an antibody response when the entire protein is used
as an immunogen. Immunogenic epitope-bearing peptides can be
identified using standard methods (see, for example, Geysen et al.,
Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).
[0149] In contrast, polypeptide fragments or peptides may comprise
an "antigenic epitope," which is a region of a protein molecule to
which an antibody can specifically bind. Certain epitopes consist
of a linear or contiguous stretch of amino acids, and the
antigenicity of such an epitope is not disrupted by denaturing
agents. It is known in the art that relatively short synthetic
peptides that can mimic epitopes of a protein can be used to
stimulate the production of antibodies against the protein (see,
for example, Sutcliffe et al., Science 219:660 (1983)).
Accordingly, antigenic epitope-bearing peptides and polypeptides of
the present invention are useful to raise antibodies that bind with
the polypeptides described herein.
[0150] Antigenic epitope-bearing peptides and polypeptides can
contain at least four to ten amino acids, at least ten to fifteen
amino acids, or about 15 to about 30 amino acids of an amino acid
sequence disclosed herein. Such epitope-bearing peptides and
polypeptides can be produced by fragmenting a mouse Zcytor16
polypeptide, or by chemical peptide synthesis, as described herein.
Moreover, epitopes can be selected by phage display of random
peptide libraries (see, for example, Lane and Stephen, Curr. Opin.
Immol. 5:268 (1993), and Cortese et al., Curr. Opin. Biotechnol.
7:616 (1996)). Standard methods for identifying epitopes and
producing antibodies from small peptides that comprise an epitope
are described, for example, by Mole, "Epitope Mapping," in Methods
in Molecular Biology, Vol. 10, Manson (ed.), pages 105-116 (The
Humana Press, Inc. 1992), Price, "Production and Characterization
of Synthetic Peptide-Derived Antibodies," in Monoclonal Antibodies:
Production, Engineering, and Clinical Application, Ritter and
Ladyman (eds.), pages 60-84 (Cambridge University Press 1995), and
Coligan et al. (eds.), Current Protocols in Immunology, pages
9.3.1-9.3.5 and pages 9.4.1-9.4.11 (John Wiley & Sons
1997).
5. Production of Mouse Zcytor16 Polypeptides
[0151] The polypeptides of the present invention, including
full-length polypeptides; soluble monomeric, homodimeric,
heterodimeric and multimeric receptors; full-length receptors;
receptor fragments (e.g. ligand-binding fragments), functional
fragments, and fusion proteins, can be produced in recombinant host
cells following conventional techniques. To express a mouse
Zcytor16 gene, a nucleic acid molecule encoding the polypeptide
must be operably linked to regulatory sequences that control
transcriptional expression in an expression vector and then,
introduced into a host cell. In addition to transcriptional
regulatory sequences, such as promoters and enhancers, expression
vectors can include translational regulatory sequences and a marker
gene which is suitable for selection of cells that carry the
expression vector.
[0152] Expression vectors that are suitable for production of a
foreign protein in eukaryotic cells typically contain (1)
prokaryotic DNA elements coding for a bacterial replication origin
and an antibiotic resistance marker to provide for the growth and
selection of the expression vector in a bacterial host; (2)
eukaryotic DNA elements that control initiation of transcription,
such as a promoter; and (3) DNA elements that control the
processing of transcripts, such as a transcription
termination/polyadenylation sequence. As discussed above,
expression vectors can also include nucleotide sequences encoding a
secretory sequence that directs the heterologous polypeptide into
the secretory pathway of a host cell. For example, a mouse Zcytor16
expression vector may comprise a mouse Zcytor16 gene and a
secretory sequence derived from any secreted gene.
[0153] Mouse Zcytor16 proteins of the present invention may be
expressed in mammalian cells. Examples of suitable mammalian host
cells include African green monkey kidney cells (Vero; ATCC CRL
1587), human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby
hamster kidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL
10314), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster
ovary cells (CHO-K1; ATCC CCL61; CHO DG44 (Chasin et al., Som.
Cell. Molec. Genet. 12:555, 1986)), rat pituitary cells (GH1; ATCC
CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E;
ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1; ATCC
CRL 1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658).
[0154] For a mammalian host, the transcriptional and translational
regulatory signals may be derived from viral sources, such as
adenovirus, bovine papilloma virus, simian virus, or the like, in
which the regulatory signals are associated with a particular gene
which has a high level of expression. Suitable transcriptional and
translational regulatory sequences also can be obtained from
mammalian genes, such as actin, collagen, myosin, and
metallothionein genes.
[0155] Transcriptional regulatory sequences include a promoter
region sufficient to direct the initiation of RNA synthesis.
Suitable eukaryotic promoters include the promoter of the mouse
metallothionein I gene (Hamer et al., J. Molec. Appl. Genet. 1:273
(1982)), the TK promoter of Herpes virus (McKnight, Cell 31:355
(1982)), the SV40 early promoter (Benoist et al., Nature 290:304
(1981)), the Rous sarcoma virus promoter (Gorman et al., Proc.
Natl. Acad. Sci. USA 79:6777 (1982)), the cytomegalovirus promoter
(Foecking et al., Gene 45:101 (1980)), and the mouse mammary tumor
virus promoter (see, generally, Etcheverry, "Expression of
Engineered Proteins in Mammalian Cell Culture," in Protein
Engineering: Principles and Practice, Cleland et al. (eds.), pages
163-181 (John Wiley & Sons, Inc. 1996)).
[0156] Alternatively, a prokaryotic promoter, such as the
bacteriophage T3 RNA polymerase promoter, can be used to control
mouse Zcytor16 gene expression in mammalian cells if the
prokaryotic promoter is regulated by a eukaryotic promoter (Zhou et
al., Mol. Cell. Biol. 10:4529 (1990), and Kaufman et al., Nucl.
Acids Res. 19:4485 (1991)).
[0157] In certain embodiments, a DNA sequence encoding a mouse
Zcytor16 monomeric or homodimeric soluble receptor polypeptide, or
a DNA sequence encoding an additional subunit of a heterodimeric or
multimeric mouse Zcytor16 soluble receptor, e.g., CRF2-4 or IL10R,
polypeptide is operably linked to other genetic elements required
for its expression, generally including a transcription promoter
and terminator, within an expression vector. The vector will also
commonly contain one or more selectable markers and one or more
origins of replication, although those skilled in the art will
recognize that within certain systems selectable markers may be
provided on separate vectors, and replication of the exogenous DNA
may be provided by integration into the host cell genome. Selection
of promoters, terminators, selectable markers, vectors and other
elements is a matter of routine design within the level of ordinary
skill in the art. Many such elements are described in the
literature and are available through commercial suppliers. Multiple
components of a soluble receptor complex can be co-transfected on
individual expression vectors or be contained in a single
expression vector. Such techniques of expressing multiple
components of protein complexes are well known in the art.
[0158] An expression vector can be introduced into host cells using
a variety of standard techniques including calcium phosphate
transfection, liposome-mediated transfection,
microprojectile-mediated delivery, electroporation, and the like.
The transfected cells can be selected and propagated to provide
recombinant host cells that comprise the expression vector stably
integrated in the host cell genome. Techniques for introducing
vectors into eukaryotic cells and techniques for selecting such
stable transformants using a dominant selectable marker are
described, for example, by Ausubel (1995) and by Murray (ed.), Gene
Transfer and Expression Protocols (Humana Press 1991).
[0159] For example, one suitable selectable marker is a gene that
provides resistance to the antibiotic neomycin. In this case,
selection is carried out in the presence of a neomycin-type drug,
such as G-418 or the like. Selection systems can also be used to
increase the expression level of the gene of interest, a process
referred to as "amplification." Amplification is carried out by
culturing transfectants in the presence of a low level of the
selective agent and then increasing the amount of selective agent
to select for cells that produce high levels of the products of the
introduced genes. A suitable amplifiable selectable marker is
dihydrofolate reductase, which confers resistance to methotrexate.
Other drug resistance genes (e.g., hygromycin resistance,
multi-drug resistance, puromycin acetyltransferase) can also be
used. Alternatively, markers that introduce an altered phenotype,
such as green fluorescent protein, or cell surface proteins such as
CD4, CD8, Class I MHC, placental alkaline phosphatase may be used
to sort transfected cells from untransfected cells by such means as
FACS sorting or magnetic bead separation technology.
[0160] Mouse Zcytor16 polypeptides can also be produced by cultured
mammalian cells using a viral delivery system. Exemplary viruses
for this purpose include adenovirus, retroviruses, herpesvirus,
vaccinia virus and adeno-associated virus (AAV). Adenovirus, a
double-stranded DNA virus, is currently the best studied gene
transfer vector for delivery of heterologous nucleic acid (for a
review, see Becker et al., Meth. Cell Biol. 43:161 (1994), and
Douglas and Curiel, Science & Medicine 4:44 (1997)). Advantages
of the adenovirus system include the accommodation of relatively
large DNA inserts, the ability to grow to high-titer, the ability
to infect a broad range of mammalian cell types, and flexibility
that allows use with a large number of available vectors containing
different promoters.
[0161] By deleting portions of the adenovirus genome, larger
inserts (up to 7 kb) of heterologous DNA can be accommodated. These
inserts can be incorporated into the viral DNA by direct ligation
or by homologous recombination with a co-transfected plasmid. An
option is to delete the essential E1 gene from the viral vector,
which results in the inability to replicate unless the E1 gene is
provided by the host cell. Adenovirus vector-infected human 293
cells (ATCC Nos. CRL-1573, 45504, 45505), for example, can be grown
as adherent cells or in suspension culture at relatively high cell
density to produce significant amounts of protein (see Garnier et
al., Cytotechnol. 15:145 (1994)).
[0162] Mouse Zcytor16 can also be expressed in other higher
eukaryotic cells, such as avian, fungal, insect, yeast, or plant
cells. The baculovirus system provides an efficient means to
introduce cloned mouse Zcytor16 genes into insect cells. Suitable
expression vectors are based upon the Autographa californica
multiple nuclear polyhedrosis virus (AcMNPV), and contain
well-known promoters such as Drosophila heat shock protein (hsp) 70
promoter, Autographa californica nuclear polyhedrosis virus
immediate-early gene promoter (ie-1) and the delayed early 39K
promoter, baculovirus p10 promoter, and the Drosophila
metallothionein promoter. A second method of making recombinant
baculovirus utilizes a transposon-based system described by Luckow
(Luckow, et al., J. Virol. 67:4566 (1993)). This system, which
utilizes transfer vectors, is sold in the BAC-to-BAC kit (Life
Technologies, Rockville, Md.). This system utilizes a transfer
vector, PFASTBAC (Life Technologies) containing a Tn7 transposon to
move the DNA encoding the mouse Zcytor16 polypeptide into a
baculovirus genome maintained in E. coli as a large plasmid called
a "bacmid." See, Hill-Perkins and Possee, J. Gen. Virol. 71:971
(1990), Bonning, et al., J. Gen. Virol. 75:1551 (1994), and
Chazenbalk, and Rapoport, J. Biol. Chem. 270:1543 (1995). In
addition, transfer vectors can include an in-frame fusion with DNA
encoding an epitope tag at the C- or N-terminus of the expressed
mouse Zcytor16 polypeptide, for example, a Glu-Glu epitope tag
(Grussenmeyer et al., Proc. Nat'l Acad. Sci. 82:7952 (1985)). Using
a technique known in the art, a transfer vector containing a mouse
Zcytor16 gene is transformed into E. coli, and screened for bacmids
which contain an interrupted lacZ gene indicative of recombinant
baculovirus. The bacmid DNA containing the recombinant baculovirus
genome is then isolated using common techniques.
[0163] The illustrative PFASTBAC vector can be modified to a
considerable degree. For example, the polyhedrin promoter can be
removed and substituted with the baculovirus basic protein promoter
(also known as Pcor, p6.9 or MP promoter) which is expressed
earlier in the baculovirus infection, and has been shown to be
advantageous for expressing secreted proteins (see, for example,
Hill-Perkins and Possee, J. Gen. Virol. 71:971 (1990), Bonning, et
al., J. Gen. Virol. 75:1551 (1994), and Chazenbalk and Rapoport, J.
Biol. Chem. 270:1543 (1995). In such transfer vector constructs, a
short or long version of the basic protein promoter can be used.
Moreover, transfer vectors can be constructed which replace the
native mouse Zcytor16 secretory signal sequences with secretory
signal sequences derived from insect proteins. For example, a
secretory signal sequence from Ecdysteroid Glucosyltransferase
(EGT), honey bee Melittin (Invitrogen Corporation; Carlsbad,
Calif.), or baculovirus gp67 (PharMingen: San Diego, Calif.) can be
used in constructs to replace the native mouse Zcytor16 secretory
signal sequence.
[0164] The recombinant virus or bacmid is used to transfect host
cells. Suitable insect host cells include cell lines derived from
IPLB-Sf-21, a Spodoptera frugiperda pupal ovarian cell line, such
as Sf9 (ATCC CRL 1711), Sf21AE, and Sf21 (Invitrogen Corporation;
San Diego, Calif.), as well as Drosophila Schneider-2 cells, and
the HIGH FIVEO cell line (Invitrogen) derived from Trichoplusia ni
(U.S. Pat. No. 5,300,435). Commercially available serum-free media
can be used to grow and to maintain the cells. Suitable media are
Sf900 II.TM. (Life Technologies) or ESF 921.TM. (Expression
Systems) for the Sf9 cells; and Ex-cellO405.TM. (JRH Biosciences,
Lenexa, Kans.) or Express FiveO.TM. (Life Technologies) for the T.
ni cells. When recombinant virus is used, the cells are typically
grown up from an inoculation density of approximately
2-5.times.10.sup.5 cells to a density of 1-2.times.10.sup.6 cells
at which time a recombinant viral stock is added at a multiplicity
of infection (MOI) of 0.1 to 10, more typically near 3.
[0165] Established techniques for producing recombinant proteins in
baculovirus systems are provided by Bailey et al., "Manipulation of
Baculovirus Vectors," in Methods in Molecular Biology, Volume 7:
Gene Transfer and Expression Protocols, Murray (ed.), pages 147-168
(The Humana Press, Inc. 1991), by Patel et al., "The baculovirus
expression system," in DNA Cloning 2: Expression Systems, 2nd
Edition, Glover et al. (eds.), pages 205-244 (Oxford University
Press 1995), by Ausubel (1995) at pages 16-37 to 16-57, by
Richardson (ed.), Baculovirus Expression Protocols (The Humana
Press, Inc. 1995), and by Lucknow, "Insect Cell Expression
Technology," in Protein Engineering: Principles and Practice,
Cleland et al. (eds.), pages 183-218 (John Wiley & Sons, Inc.
1996).
[0166] Fungal cells, including yeast cells, can also be used to
express the genes described herein. Yeast species of particular
interest in this regard include Saccharomyces cerevisiae, Pichia
pastoris, and Pichia methanolica. Suitable promoters for expression
in yeast include promoters from GAL1 (galactose), PGK
(phosphoglycerate kinase), ADH (alcohol dehydrogenase), AOX1
(alcohol oxidase), HIS4 (histidinol dehydrogenase), and the like.
Many yeast cloning vectors have been designed and are readily
available. These vectors include YIp-based vectors, such as YIp5,
YRp vectors, such as YRp17, YEp vectors such as YEp13 and YCp
vectors, such as YCp19. Methods for transforming S. cerevisiae
cells with exogenous DNA and producing recombinant polypeptides
therefrom are disclosed by, for example, Kawasaki, U.S. Pat. No.
4,599,311, Kawasaki et al., U.S. Pat. No. 4,931,373, Brake, U.S.
Pat. No. 4,870,008, Welch et al., U.S. Pat. No. 5,037,743, and
Murray et al., U.S. Pat. No. 4,845,075. Transformed cells are
selected by phenotype determined by the selectable marker, commonly
drug resistance or the ability to grow in the absence of a
particular nutrient (e.g., leucine). A suitable vector system for
use in Saccharomyces cerevisiae is the POT1 vector system disclosed
by Kawasaki et al. (U.S. Pat. No. 4,931,373), which allows
transformed cells to be selected by growth in glucose-containing
media. Additional suitable promoters and terminators for use in
yeast include those from glycolytic enzyme genes (see, e.g.,
Kawasaki, U.S. Pat. No. 4,599,311, Kingsman et al., U.S. Pat. No.
4,615,974, and Bitter, U.S. Pat. No. 4,977,092) and alcohol
dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446, 5,063,154,
5,139,936, and 4,661,454.
[0167] Transformation systems for other yeasts, including Hansenula
polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis,
Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia
methanolica, Pichia guillermondii and Candida maltosa are known in
the art. See, for example, Gleeson et al., J. Gen. Microbiol.
132:3459 (1986), and Cregg, U.S. Pat. No. 4,882,279. Aspergillus
cells may be utilized according to the methods of McKnight et al.,
U.S. Pat. No. 4,935,349. Methods for transforming Acremonium
chrysogenum are disclosed by Sumino et al., U.S. Pat. No.
5,162,228. Methods for transforming Neurospora are disclosed by
Lambowitz, U.S. Pat. No. 4,486,533.
[0168] For example, the use of Pichia methanolica as host for the
production of recombinant proteins is disclosed by Raymond, U.S.
Pat. No. 5,716,808, Raymond, U.S. Pat. No. 5,736,383, Raymond et
al., Yeast 14:11-23 (1998), and in international publication Nos.
WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA
molecules for use in transforming P. methanolica will commonly be
prepared as double-stranded, circular plasmids, which are
preferably linearized prior to transformation. For polypeptide
production in P. methanolica, the promoter and terminator in the
plasmid can be that of a P. methanolica gene, such as a P.
methanolica alcohol utilization gene (AUG1 or AUG2). Other useful
promoters include those of the dihydroxyacetone synthase (DHAS),
formate dehydrogenase (FMD), and catalase (CAT) genes. To
facilitate integration of the DNA into the host chromosome, it is
preferred to have the entire expression segment of the plasmid
flanked at both ends by host DNA sequences. A suitable selectable
marker for use in Pichia methanolica is a P. methanolica ADE2 gene,
which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC
4.1.1.21), and which allows ade2 host cells to grow in the absence
of adenine. For large-scale, industrial processes where it is
desirable to minimize the use of methanol, host cells can be used
in which both methanol utilization genes (AUG1 and AUG2) are
deleted. For production of secreted proteins, host cells can be
deficient in vacuolar protease genes (PEP4 and PRB1).
Electroporation is used to facilitate the introduction of a plasmid
containing DNA encoding a polypeptide of interest into P.
methanolica cells. P. methanolica cells can be transformed by
electroporation using an exponentially decaying, pulsed electric
field having a field strength of from 2.5 to 4.5 kV/cm, preferably
about 3.75 kV/cm, and a time constant (t) of from 1 to 40
milliseconds, most preferably about 20 milliseconds.
[0169] Expression vectors can also be introduced into plant
protoplasts, intact plant tissues, or isolated plant cells. Methods
for introducing expression vectors into plant tissue include the
direct infection or co-cultivation of plant tissue with
Agrobacterium tumefaciens, microprojectile-mediated delivery, DNA
injection, electroporation, and the like. See, for example, Horsch
et al., Science 227:1229 (1985), Klein et al., Biotechnology 10:268
(1992), and Miki et al., "Procedures for Introducing Foreign DNA
into Plants," in Methods in Plant Molecular Biology and
Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press,
1993).
[0170] Alternatively, mouse Zcytor16 genes can be expressed in
prokaryotic host cells. Suitable promoters that can be used to
express mouse Zcytor16 polypeptides in a prokaryotic host are
well-known to those of skill in the art and include promoters
capable of recognizing the T4, T3, Sp6 and T7 polymerases, the
P.sub.R and P.sub.L promoters of bacteriophage lambda, the trp,
recA, heat shock, lacUV5, tac, Ipp-lacSpr, phoA, and lacZ promoters
of E. coli, promoters of B. subtilis, the promoters of the
bacteriophages of Bacillus, Streptomyces promoters, the int
promoter of bacteriophage lambda, the bla promoter of pBR322, and
the CAT promoter of the chloramphenicol acetyl transferase gene.
Prokaryotic promoters have been reviewed by Glick, J. Ind.
Microbiol. 1:277 (1987), Watson et al., Molecular Biology of the
Gene, 4th Ed. (Benjamin Cummins 1987), and by Ausubel et al.
(1995).
[0171] Suitable prokaryotic hosts include E. coli and Bacillus
subtilus. Suitable strains of E. coli include BL21(DE3),
BL21(DE3)pLysS, BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF',
DH5IMCR, DH10B, DH10B/p3, DH11S, C600, HB101, JM101, JM105, JM109,
JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, and ER1647 (see, for
example, Brown (ed.), Molecular Biology Labfax (Academic Press
1991)). Suitable strains of Bacillus subtilus include BR151, YB886,
MI119, MI120, and B170 (see, for example, Hardy, "Bacillus Cloning
Methods," in DNA Cloning: A Practical Approach, Glover (ed.) (IRL
Press 1985)).
[0172] When expressing a mouse Zcytor16 polypeptide in bacteria
such as E. coli, the polypeptide may be retained in the cytoplasm,
typically as insoluble granules, or may be directed to the
periplasmic space by a bacterial secretion sequence. In the former
case, the cells are lysed, and the granules are recovered and
denatured using, for example, guanidine isothiocyanate or urea. The
denatured polypeptide can then be refolded and dimerized by
diluting the denaturant, such as by dialysis against a solution of
urea and a combination of reduced and oxidized glutathione,
followed by dialysis against a buffered saline solution. In the
latter case, the polypeptide can be recovered from the periplasmic
space in a soluble and functional form by disrupting the cells (by,
for example, sonication or osmotic shock) to release the contents
of the periplasmic space and recovering the protein, thereby
obviating the need for denaturation and refolding.
[0173] Methods for expressing proteins in prokaryotic hosts are
well-known to those of skill in the art (see, for example, Williams
et al., "Expression of foreign proteins in E. coli using plasmid
vectors and purification of specific polyclonal antibodies," in DNA
Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),
page 15 (Oxford University Press 1995), Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, page 137 (Wiley-Liss, Inc.
1995), and Georgiou, "Expression of Proteins in Bacteria," in
Protein Engineering: Principles and Practice, Cleland et al.
(eds.), page 101 (John Wiley & Sons, Inc. 1996)).
[0174] Standard methods for introducing expression vectors into
bacterial, yeast, insect, and plant cells are provided, for
example, by Ausubel (1995).
[0175] General methods for expressing and recovering foreign
protein produced by a mammalian cell system are provided by, for
example, Etcheverry, "Expression of Engineered Proteins in
Mammalian Cell Culture," in Protein Engineering: Principles and
Practice, Cleland et al. (eds.), pages 163 (Wiley-Liss, Inc. 1996).
Standard techniques for recovering protein produced by a bacterial
system is provided by, for example, Grisshammer et al.,
"Purification of over-produced proteins from E. coli cells," in DNA
Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),
pages 59-92 (Oxford University Press 1995). Established methods for
isolating recombinant proteins from a baculovirus system are
described by Richardson (ed.), Baculovirus Expression Protocols
(The Humana Press, Inc. 1995).
[0176] As an alternative, polypeptides of the present invention can
be synthesized by exclusive solid phase synthesis, partial solid
phase methods, fragment condensation or classical solution
synthesis. These synthesis methods are well-known to those of skill
in the art (see, for example, Merrifield, J. Am. Chem. Soc. 85:2149
(1963), Stewart et al., "Solid Phase Peptide Synthesis" (2nd
Edition), (Pierce Chemical Co. 1984), Bayer and Rapp, Chem. Pept.
Prot. 3:3 (1986), Atherton et al., Solid Phase Peptide Synthesis: A
Practical Approach (IRL Press 1989), Fields and Colowick,
"Solid-Phase Peptide Synthesis," Methods in Enzymology Volume 289
(Academic Press 1997), and Lloyd-Williams et al., Chemical
Approaches to the Synthesis of Peptides and Proteins (CRC Press,
Inc. 1997)). Variations in total chemical synthesis strategies,
such as "native chemical ligation" and "expressed protein ligation"
are also standard (see, for example, Dawson et al., Science 266:776
(1994), Hackeng et al., Proc. Nat'l Acad. Sci. USA 94:7845 (1997),
Dawson, Methods Enzymol. 287: 34 (1997), Muir et al., Proc. Nat'l
Acad. Sci. USA 95:6705 (1998), and Severinov and Muir, J. Biol.
Chem. 273:16205 (1998)).
[0177] Peptides and polypeptides of the present invention comprise
at least six, at least nine, or at least 15 contiguous amino acid
residues of SEQ ID NO:38 or SEQ ID NO:48. As an illustration,
polypeptides can comprise at least six, at least nine, or at least
15 contiguous amino acid residues of any of the following amino
acid sequences of SEQ ID NO:38 or SEQ ID NO:48: amino acid residues
amino acid residues 1 to 230, amino acid residues 24 to 230, or 27
to 230, and amino acid residues 27 to 126, amino acid residues 131
to 230, or other fragments disclosed herein. Within certain
embodiments of the invention, the polypeptides comprise 20, 30, 40,
50, 100, or more contiguous residues of these amino acid sequences.
Nucleic acid molecules encoding such peptides and polypeptides are
useful as polymerase chain reaction primers and probes.
[0178] Moreover, mouse Zcytor16 polypeptides can be expressed as
monomers, homodimers, heterodimers, or multimers within higher
eukaryotic cells. Such cells can be used to produce mouse Zcytor16
monomeric, homodimeric, heterodimeric and multimeric receptor
polypeptides that comprise at least one mouse Zcytor16 polypeptide
("mouse Zcytor16-comprising receptors" or "mouse
Zcytor16-comprising receptor polypeptides"), or can be used as
assay cells in screening systems. Within one aspect of the present
invention, a polypeptide of the present invention comprising the
mouse Zcytor16 extracellular domain is produced by a cultured cell,
and the cell is used to screen for ligands for the receptor,
including the natural ligand, IL-TIF, as well as agonists and
antagonists of the natural ligand. To summarize this approach, a
cDNA or gene encoding the receptor is combined with other genetic
elements required for its expression (e.g., a transcription
promoter), and the resulting expression vector is inserted into a
host cell. Cells that express the DNA and produce functional
receptor are selected and used within a variety of screening
systems. Each component of the monomeric, homodimeric,
heterodimeric and multimeric receptor complex can be expressed in
the same cell. Moreover, the components of the monomeric,
homodimeric, heterodimeric and multimeric receptor complex can also
be fused to a transmembrane domain or other membrane fusion moiety
to allow complex assembly and screening of transfectants as
described above.
[0179] Mammalian cells suitable for use in expressing mouse
Zcytor16 receptors and transducing a receptor-mediated signal
include cells that express other receptor subunits that may form a
functional complex with mouse Zcytor16. These subunits may include
those of the interferon receptor family or of other class II or
class I cytokine receptors, e.g., CRF2-4 (Genbank Accession No.
Z17227), IL-1 OR (Genbank Accession No.s U00672 and
NM.sub.--001558), zcytor11 (U.S. Pat. No. 5,965,704), zcytor7 (U.S.
Pat. No. 5,945,511), and IL-9R. It is also preferred to use a cell
from the same species as the receptor to be expressed. Within a
preferred embodiment, the cell is dependent upon an exogenously
supplied hematopoietic growth factor for its proliferation.
Preferred cell lines of this type are the human TF-1 cell line
(ATCC number CRL-2003) and the AML-193 cell line (ATCC number
CRL-9589), which are GM-CSF-dependent human leukemic cell lines and
BaF3 (Palacios and Steinmetz, Cell 41: 727-734, (1985)) which is an
IL-3 dependent murine pre-B cell line. Other cell lines include
BHK, COS-1 and CHO cells.
[0180] Suitable host cells can be engineered to produce the
necessary receptor subunits or other cellular component needed for
the desired cellular response. This approach is advantageous
because cell lines can be engineered to express receptor subunits
from any species, thereby overcoming potential limitations arising
from species specificity. Species orthologs of the mouse receptor
cDNA can be cloned and used within cell lines from the same
species, such as a mouse cDNA in the BaF3 cell line. Cell lines
that are dependent upon one hematopoietic growth factor, such as
GM-CSF or IL-3, can thus be engineered to become dependent upon
another cytokine that acts through the mouse Zcytor16 receptor,
such as IL-TIF.
[0181] Cells expressing functional receptor are used within
screening assays. A variety of suitable assays are known in the
art. These assays are based on the detection of a biological
response in a target cell. One such assay is a cell proliferation
assay. Cells are cultured in the presence or absence of a test
compound, and cell proliferation is detected by, for example,
measuring incorporation of tritiated thymidine or by colorimetric
assay based on the metabolic breakdown of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
(Mosman, J. Immunol. Meth. 65: 55-63, (1983)). An alternative assay
format uses cells that are further engineered to express a reporter
gene. The reporter gene is linked to a promoter element that is
responsive to the receptor-linked pathway, and the assay detects
activation of transcription of the reporter gene. A preferred
promoter element in this regard is a serum response element, or
SRE. See, e.g., Shaw et al., Cell 56:563-572, (1989). A preferred
such reporter gene is a luciferase gene (de Wet et al., Mol. Cell.
Biol. 7:725, (1987)). Expression of the luciferase gene is detected
by luminescence using methods known in the art (e.g., Baumgartner
et al., J. Biol. Chem. 269:29094-29101, (1994); Schenborn and
Goiffin, Promega.sub.--Notes 41:11, 1993). Luciferase activity
assay kits are commercially available from, for example, Promega
Corp., Madison, Wis. Target cell lines of this type can be used to
screen libraries of chemicals, cell-conditioned culture media,
fungal broths, soil samples, water samples, and the like. For
example, a bank of cell-conditioned media samples can be assayed on
a target cell to identify cells that produce ligand. Positive cells
are then used to produce a cDNA library in a mammalian expression
vector, which is divided into pools, transfected into host cells,
and expressed. Media samples from the transfected cells are then
assayed, with subsequent division of pools, re-transfection,
subculturing, and re-assay of positive cells to isolate a cloned
cDNA encoding the ligand.
[0182] A natural ligand for the mouse Zcytor16 receptor can also be
identified by mutagenizing a cell line expressing the full-length
receptor or receptor fusion (e.g., comprising the mouse Zcytor16
extracellular domain fused to the transmembrane and signaling
domain of another cytokine receptor) and culturing it under
conditions that select for autocrine growth. See WIPO publication
WO 95/21930. Within a typical procedure, IL-3 dependent BaF3 cells
expressing mouse Zcytor16 and the necessary additional subunits are
mutagenized, such as with 2-ethylmethanesulfonate (EMS). The cells
are then allowed to recover in the presence of IL-3, then
transferred to a culture medium lacking IL-3 and IL-4. Surviving
cells are screened for the production of a Zcytor16 ligand (e.g.,
IL-TIF), such as by adding soluble receptor to the culture medium
or by assaying conditioned media on wild-type BaF3 cells and BaF3
cells expressing the receptor. Using this method, cells and tissues
expressing IL-TIF can be identified. Such methods can be employed
to detect IL-TIF from different species, e.g., human, and as such
cells, cancers, and tissues expressing human IL-TIF can be
identified using the mouse Zcytor16 polypeptides of the present
invention.
[0183] Moreover several IL-TIF responsive cell lines are known
(Dumontier et al., J. Immunol. 164:1814-1819, 2000; Dumoutier, L.
et al., Proc. Nat'l. Acad. Sci. 97:10144-10149, 2000; Xie M H et
al., J. Biol. Chem. 275: 31335-31339, 2000; Kotenko S V et al., JBC
in press), as well as those that express the IL-TIF receptor
subunit zcytor11. For example, the following cells are responsive
to IL-TIF: TK-10 (Xie M H et al., supra.) (human renal carcinoma);
SW480 (ATCC No. CCL-228) (human colon adenocarcinoma); HepG2 (ATCC
No. HB-8065) (human hepatoma); PC12 (ATCC No. CRL-1721) (murine
neuronal cell model; rat pheochromocytoma); and MES13 (ATCC No.
CRL-1927) (murine kidney mesangial cell line). In addition, some
cell lines express zcytor11 (IL-TIF receptor) are also candidates
for responsive cell lines to IL-TIF: A549 (ATCC No. CCL-185) (human
lung carcinoma); G-361 (ATCC No. CRL-1424) (human melanoma); and
Caki-1 (ATCC No. HTB-46) (human renal carcinoma). These cells can
be used in assays to assess the functionality of mouse Zcytor16 as
an IL-TIF antagonist or anti-inflammatory factor, even in
heterologous systems, such as human.
[0184] An additional screening approach provided by the present
invention includes the use of hybrid receptor polypeptides. These
hybrid polypeptides fall into two general classes. Within the first
class, the intracellular domain of mouse Zcytor16, is joined to the
ligand-binding domain of a second receptor. It is preferred that
the second receptor be a hematopoietic cytokine receptor, such as
mpl receptor (Souyri et al., Cell 63: 1137-1147, (1990). The hybrid
receptor will further comprise a transmembrane domain, which may be
derived from either receptor. A DNA construct encoding the hybrid
receptor is then inserted into a host cell. Cells expressing the
hybrid receptor are cultured in the presence of a ligand for the
binding domain (e.g., TPO in the case the mpl receptor
extracellular domain is used) and assayed for a response. This
system provides a means for analyzing signal transduction mediated
by mouse Zcytor16 while using readily available ligands. This
system can also be used to determine if particular cell lines are
capable of responding to signals transduced by mouse Zcytor16
monomeric, homodimeric, heterodimeric and multimeric receptors of
the present invention.
[0185] A second class of hybrid receptor polypeptides comprise the
extracellular (ligand-binding) domain of mouse Zcytor16
(approximately residues 24 to 230, or 27 to 230 of SEQ ID NO:38 or
SEQ ID NO:48) with an intracellular domain of a second receptor,
preferably a hematopoietic cytokine receptor, and a transmembrane
domain. Hybrid zacytor11 monomers, homodimers, heterodimers and
multimers of the present invention receptors of this second class
are expressed in cells known to be capable of responding to signals
transduced by the second receptor. Together, these two classes of
hybrid receptors enable the identification of a responsive cell
type for the development of an assay for detecting IL-TIF.
Moreover, such cells can be used in the presence of IL-TIF to assay
the soluble receptor antagonists of the present invention in a
competition-type assay. In such assay, a decrease in the
proliferation or signal transduction activity of IL-TIF in the
presence of a soluble receptor of the present invention
demonstrates antagonistic activity. Moreover IL-TIF-soluble
receptor binding assays can also be used to assess whether a
soluble receptor antagonizes IL-TIF activity.
6. Production of Mouse Zcytor16 Fusion Proteins and Conjugates
[0186] One general class of mouse Zcytor16 analogs are variants
having an amino acid sequence that is a mutation of the amino acid
sequence disclosed herein. Another general class of mouse Zcytor16
analogs is provided by anti-idiotype antibodies, and fragments
thereof, as described below. Moreover, recombinant antibodies
comprising anti-idiotype variable domains can be used as analogs
(see, for example, Monfardini et al., Proc. Assoc. Am. Physicians
108:420 (1996)). Since the variable domains of anti-idiotype mouse
Zcytor16 antibodies mimic mouse Zcytor16, these domains can provide
mouse or human Zcytor16 binding activity, e.g., to IL-TIF. Methods
of producing anti-idiotypic catalytic antibodies are known to those
of skill in the art (see, for example, Joron et al., Ann. NY Acad.
Sci. 672:216 (1992), Friboulet et al., Appl. Biochem. Biotechnol.
47:229 (1994), and Avalle et al., Ann. NY Acad. Sci. 864:118
(1998)).
[0187] Another approach to identifying Zcytor16 analogs is provided
by the use of combinatorial libraries. Methods for constructing and
screening phage display and other combinatorial libraries are
provided, for example, by Kay et al., Phage Display of Peptides and
Proteins (Academic Press 1996), Verdine, U.S. Pat. No. 5,783,384,
Kay, et. al., U.S. Pat. No. 5,747,334, and Kauffman et al., U.S.
Pat. No. 5,723,323.
[0188] Mouse Zcytor16 polypeptides have both in vivo and in vitro
uses. As an illustration, a soluble form of mouse Zcytor16 can be
added to cell culture medium to inhibit the effects of the Zcytor16
ligand produced by the cultured cells.
[0189] Fusion proteins of mouse Zcytor16 can be used to express
mouse Zcytor16 in a recombinant host, and to isolate the produced
mouse Zcytor16. As described below, particular mouse Zcytor16
fusion proteins also have uses in diagnosis and therapy. One type
of fusion protein comprises a peptide that guides a mouse Zcytor16
polypeptide from a recombinant host cell. To direct a mouse
Zcytor16 polypeptide into the secretory pathway of a eukaryotic
host cell, a secretory signal sequence (also known as a signal
peptide, a leader sequence, prepro sequence or pre sequence) is
provided in the mouse Zcytor16 expression vector. While the
secretory signal sequence may be derived from mouse Zcytor16, a
suitable signal sequence may also be derived from another secreted
protein or synthesized de novo. The secretory signal sequence is
operably linked to a mouse Zcytor16-encoding sequence such that the
two sequences are joined in the correct reading frame and
positioned to direct the newly synthesized polypeptide into the
secretory pathway of the host cell. Secretory signal sequences are
commonly positioned 5' to the nucleotide sequence encoding the
polypeptide of interest, although certain secretory signal
sequences may be positioned elsewhere in the nucleotide sequence of
interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland
et al., U.S. Pat. No. 5,143,830).
[0190] Although the secretory signal sequence of mouse Zcytor16 or
another protein produced by mammalian cells (e.g., tissue-type
plasminogen activator signal sequence, as described, for example,
in U.S. Pat. No. 5,641,655) is useful for expression of mouse
Zcytor16 in recombinant mammalian hosts, a yeast signal sequence is
preferred for expression in yeast cells. Examples of suitable yeast
signal sequences are those derived from yeast mating phermone
.alpha.-factor (encoded by the MF.alpha.1 gene), invertase (encoded
by the SUC2 gene), or acid phosphatase (encoded by the PHO5 gene).
See, for example, Romanos et al., "Expression of Cloned Genes in
Yeast," in DNA Cloning 2: A Practical Approach, 2.sup.nd Edition,
Glover and Hames (eds.), pages 123-167 (Oxford University Press
1995).
[0191] Mouse Zcytor16 monomeric, homodimeric, heterodimeric and
multimeric receptor polypeptides can be prepared by expressing a
truncated DNA encoding the extracellular domain, for example, a
polypeptide which contains 24 to 230, or 27 to 230 of SEQ ID NO:38
or SEQ ID NO:48, or the corresponding region of a non-mouse
receptor. It is preferred that the extracellular domain
polypeptides be prepared in a form substantially free of
transmembrane and intracellular polypeptide segments. To direct the
export of the receptor domain from the host cell, the receptor DNA
is linked to a second DNA segment encoding a secretory peptide,
such as a t-PA secretory peptide. To facilitate purification of the
secreted receptor domain, a C-terminal extension, such as a
poly-histidine tag, substance P, Flag.TM. peptide (Hopp et al.,
Biotechnology 6:1204-1210, (1988); available from Eastman Kodak
Co., New Haven, Conn.) or another polypeptide or protein for which
an antibody or other specific binding agent is available, can be
fused to the receptor polypeptide. Moreover, heterodimeric and
multimeric non-mouse Zcytor16 subunit extracellular cytokine
binding domains are a also prepared as above.
[0192] In an alternative approach, a receptor extracellular domain
of mouse Zcytor16 or other class I or II cytokine receptor
component can be expressed as a fusion with immunoglobulin heavy
chain constant regions, typically an F.sub.c fragment, which
contains two constant region domains and a hinge region but lacks
the variable region (See, Sledziewski, A Z et al., U.S. Pat. Nos.
6,018,026 and 5,750,375). The soluble mouse Zcytor16, soluble mouse
Zcytor16/CRF2-4 heterodimers, and monomeric, homodimeric,
heterodimeric and multimeric polypeptides of the present invention
include such fusions. Such fusions are typically secreted as
multimeric molecules wherein the Fc portions are disulfide bonded
to each other and two receptor polypeptides are arrayed in closed
proximity to each other. Fusions of this type can be used to
affinity purify the cognate ligand from solution, as an in vitro
assay tool, to block signals in vitro by specifically titrating out
ligand, and as antagonists in vivo by administering them
parenterally to bind circulating ligand and clear it from the
circulation. To purify ligand, a mouse Zcytor16-Ig chimera is added
to a sample containing the ligand (e.g., cell-conditioned culture
media or tissue extracts) under conditions that facilitate
receptor-ligand binding (typically near-physiological temperature,
pH, and ionic strength). The chimera-ligand complex is then
separated by the mixture using protein A, which is immobilized on a
solid support (e.g., insoluble resin beads). The ligand is then
eluted using conventional chemical techniques, such as with a salt
or pH gradient. In the alternative, the chimera itself can be bound
to a solid support, with binding and elution carried out as above.
The chimeras may be used in vivo to regulate inflammatory responses
including acute phase responses such as serum amyloid A (SAA),
C-reactive protein (CRP), and the like. Chimeras with high binding
affinity are administered parenterally (e.g., by intramuscular,
subcutaneous or intravenous injection). Circulating molecules bind
ligand and are cleared from circulation by normal physiological
processes. For use in assays, the chimeras are bound to a support
via the F.sub.c region and used in an ELISA format. Moreover, such
methods can be applied using the polypeptides of the present
invention to isolate mouse IL-TIF, as well as an orthologous
ligand, e.g., human IL-TIF.
[0193] The present invention further provides a variety of other
polypeptide fusions and related multimeric proteins comprising one
or more polypeptide fusions. For example, a soluble mouse Zcytor16
receptor or soluble mouse Zcytor16 heterodimeric polypeptide, such
as soluble mouse Zcytor16/CRF2-4, can be prepared as a fusion to a
dimerizing protein as disclosed in U.S. Pat. Nos. 5,155,027 and
5,567,584. Preferred dimerizing proteins in this regard include
immunoglobulin constant region domains, e.g., IgG.gamma.1, and the
human .kappa. light chain. Immunoglobulin-soluble mouse Zcytor16
receptor or immunoglobulin-soluble mouse Zcytor16 heterodimeric or
multimeric polypeptide, such as immunoglobulin-soluble mouse
Zcytor16/CRF2-4 fusions can be expressed in genetically engineered
cells to produce a variety of multimeric mouse Zcytor16 receptor
analogs. Auxiliary domains can be fused to soluble mouse Zcytor16
receptor or soluble mouse Zcytor16 heterodimeric or multimeric
polypeptides, such as soluble mouse Zcytor16/CRF2-4 to target them
to specific cells, tissues, or macromolecules (e.g., collagen, or
cells expressing the Zcytor16 ligand, IL-TIF). A mouse Zcytor16
polypeptide can be fused to two or more moieties, such as an
affinity tag for purification and a targeting domain. Polypeptide
fusions can also comprise one or more cleavage sites, particularly
between domains. See, Tuan et al., Connective Tissue Research
34:1-9, 1996.
[0194] In bacterial cells, it is often desirable to express a
heterologous protein as a fusion protein to decrease toxicity,
increase stability, and to enhance recovery of the expressed
protein. For example, mouse Zcytor16 can be expressed as a fusion
protein comprising a glutathione S-transferase polypeptide.
Glutathione S-transferease fusion proteins are typically soluble,
and easily purifiable from E. coli lysates on immobilized
glutathione columns. In similar approaches, a mouse Zcytor16 fusion
protein comprising a maltose binding protein polypeptide can be
isolated with an amylose resin column, while a fusion protein
comprising the C-terminal end of a truncated Protein A gene can be
purified using IgG-Sepharose. Established techniques for expressing
a heterologous polypeptide as a fusion protein in a bacterial cell
are described, for example, by Williams et al., "Expression of
Foreign Proteins in E. coli Using Plasmid Vectors and Purification
of Specific Polyclonal Antibodies," in DNA Cloning 2: A Practical
Approach, 2.sup.nd Edition, Glover and Hames (Eds.), pages 15-58
(Oxford University Press 1995). In addition, commercially available
expression systems are available. For example, the PINPOINT Xa
protein purification system (Promega Corporation; Madison, Wis.)
provides a method for isolating a fusion protein comprising a
polypeptide that becomes biotinylated during expression with a
resin that comprises avidin.
[0195] Peptide tags that are useful for isolating heterologous
polypeptides expressed by either prokaryotic or eukaryotic cells
include polyHistidine tags (which have an affinity for
nickel-chelating resin), c-myc tags, calmodulin binding protein
(isolated with calmodulin affinity chromatography), substance P,
the RYIRS tag (which binds with anti-RYIRS antibodies), the Glu-Glu
tag, and the FLAG tag (which binds with anti-FLAG antibodies). See,
for example, Luo et al., Arch. Biochem. Biophys. 329:215 (1996),
Morganti et al., Biotechnol. Appl. Biochem. 23:67 (1996), and Zheng
et al., Gene 186:55 (1997). Nucleic acid molecules encoding such
peptide tags are available, for example, from Sigma-Aldrich
Corporation (St. Louis, Mo.).
[0196] The present invention also contemplates that the use of the
secretory signal sequence contained in the mouse Zcytor16
polypeptides of the present invention to direct other polypeptides
into the secretory pathway. A signal fusion polypeptide can be made
wherein a secretory signal sequence derived from amino acid
residues 1 to 23 of SEQ ID NO:38 or SEQ ID NO:48 is operably linked
to another polypeptide using methods known in the art and disclosed
herein. The secretory signal sequence contained in the fusion
polypeptides of the present invention is preferably fused
amino-terminally to an additional peptide to direct the additional
peptide into the secretory pathway. Such constructs have numerous
applications known in the art. For example, these novel secretory
signal sequence fusion constructs can direct the secretion of an
active component of a normally non-secreted protein, such as a
receptor. Such fusions may be used in a transgenic animal or in a
cultured recombinant host to direct peptides through the secretory
pathway. With regard to the latter, exemplary polypeptides include
pharmaceutically active molecules such as Factor VIIa, proinsulin,
insulin, follicle stimulating hormone, tissue type plasminogen
activator, tumor necrosis factor, interleukins (e.g., interleukin-1
(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, and IL-15), colony stimulating factors
(e.g., granulocyte-colony stimulating factor (G-CSF) and
granulocyte macrophage-colony stimulating factor (GM-CSF)),
interferons (e.g., interferons-.alpha., -.beta., -.gamma.,
-.omega., -.delta., and -.tau.), the stem cell growth factor
designated "S1 factor," erythropoietin, and thrombopoietin. The
mouse Zcytor16 secretory signal sequence contained in the fusion
polypeptides of the present invention is preferably fused
amino-terminally to an additional peptide to direct the additional
peptide into the secretory pathway. Fusion proteins comprising a
mouse Zcytor16 secretory signal sequence can be constructed using
standard techniques.
[0197] Another form of fusion protein comprises a mouse Zcytor16
polypeptide and an immunoglobulin heavy chain constant region,
typically an F.sub.c fragment, which contains two or three constant
region domains and a hinge region but lacks the variable region. As
an illustration, Chang et al., U.S. Pat. No. 5,723,125, describe a
fusion protein comprising a human interferon and a human
immunoglobulin Fc fragment. The C-terminal of the interferon is
linked to the N-terminal of the Fc fragment by a peptide linker
moiety. An example of a peptide linker is a peptide comprising
primarily a T cell inert sequence, which is immunologically inert.
An exemplary peptide linker has the amino acid sequence: GGSGG
SGGGG SGGGG S (SEQ ID NO:4). In this fusion protein, an
illustrative Fc moiety is a human .gamma.4 chain, which is stable
in solution and has little or no complement activating activity.
Accordingly, the present invention contemplates a mouse Zcytor16
fusion protein that comprises a mouse Zcytor16 moiety and a human
Fc fragment, wherein the C-terminus of the mouse Zcytor16 moiety is
attached to the N-terminus of the Fc fragment via a peptide linker,
such as a peptide consisting of the amino acid sequence of SEQ ID
NO:4. The mouse Zcytor16 moiety can be a mouse Zcytor16 molecule or
a fragment thereof. For example, a fusion protein can comprise
amino acid residues 24 to 230, or 27 to 230 of SEQ ID NO:38 or SEQ
ID NO:48 and an Fc fragment (e.g., a human Fc fragment).
[0198] In another variation, a mouse Zcytor16 fusion protein
comprises an IgG sequence, a mouse Zcytor16 moiety covalently
joined to the aminoterminal end of the IgG sequence, and a signal
peptide that is covalently joined to the aminoterminal of the mouse
Zcytor16 moiety, wherein the IgG sequence consists of the following
elements in the following order: a hinge region, a CH.sub.2 domain,
and a CH.sub.3 domain. Accordingly, the IgG sequence lacks a
CH.sub.1 domain. The mouse Zcytor16 moiety displays a mouse
Zcytor16 activity, as described herein, such as the ability to bind
with a Zcytor16 ligand, including mouse and human IL-TIF. This
general approach to producing fusion proteins that comprise both
antibody and nonantibody portions has been described by LaRochelle
et al., EP 742830 (WO 95/21258).
[0199] Fusion proteins comprising a mouse Zcytor16 moiety and an Fc
moiety can be used, for example, as an in vitro assay tool. For
example, the presence of a Zcytor16 ligand, including mouse or
human IL-TIF, in a biological sample can be detected using a mouse
Zcytor16-immunoglobulin fusion protein, in which the mouse Zcytor16
moiety is used to bind the ligand, and a macromolecule, such as
Protein A or anti-Fc antibody, is used to bind the fusion protein
to a solid support. Such systems can be used to identify agonists
and antagonists that interfere with the binding of a Zcytor16
ligand to its receptor.
[0200] Other examples of antibody fusion proteins include
polypeptides that comprise an antigen-binding domain and a mouse
Zcytor16 fragment that contains a mouse Zcytor16 extracellular
domain. Such molecules can be used to target particular tissues for
the benefit of Zcytor16 binding activity.
[0201] The present invention further provides a variety of other
polypeptide fusions. For example, part or all of a domain(s)
conferring a biological function can be swapped between mouse
Zcytor16 of the present invention with the functionally equivalent
domain(s) from another member of the cytokine receptor family.
Polypeptide fusions can be expressed in recombinant host cells to
produce a variety of mouse Zcytor16 fusion analogs. A mouse
Zcytor16 polypeptide can be fused to two or more moieties or
domains, such as an affinity tag for purification and a targeting
domain. Polypeptide fusions can also comprise one or more cleavage
sites, particularly between domains. See, for example, Tuan et al.,
Connective Tissue Research 34:1 (1996).
[0202] Fusion proteins can be prepared by methods known to those
skilled in the art by preparing each component of the fusion
protein and chemically conjugating them. Alternatively, a
polynucleotide encoding both components of the fusion protein in
the proper reading frame can be generated using known techniques
and expressed by the methods described herein. General methods for
enzymatic and chemical cleavage of fusion proteins are described,
for example, by Ausubel (1995) at pages 16-19 to 16-25.
[0203] Mouse Zcytor16 polypeptides can be used to identify and to
isolate Zcytor16 ligands, including mouse and human IL-TIF. For
example, proteins and peptides of the present invention can be
immobilized on a column and used to bind ligands from a biological
sample that is run over the column (Hermanson et al. (eds.),
Immobilized Affinity Ligand Techniques, pages 195-202 (Academic
Press 1992)). As such, mouse Zcytor16 polypeptides of the present
invention can be used to identify and isolate IL-TIF for either
diagnostic, or production purposes.
[0204] The activity of a mouse Zcytor16 polypeptide can also be
observed by a silicon-based biosensor microphysiometer, which
measures the extracellular acidification rate or proton excretion
associated with receptor binding and subsequent physiologic
cellular responses. An exemplary device is the CYTOSENSOR
Microphysiometer manufactured by Molecular Devices, Sunnyvale,
Calif. A variety of cellular responses, such as cell proliferation,
ion transport, energy production, inflammatory response, regulatory
and receptor activation, and the like, can be measured by this
method (see, for example, McConnell et al., Science 257:1906
(1992), Pitchford et al., Meth. Enzymol. 228:84 (1997), Arimilli et
al., J. Immunol. Meth. 212:49 (1998), Van Liefde et al., Eur. J.
Pharmacol. 346:87 (1998)). The microphysiometer can be used for
assaying eukaryotic, prokaryotic, adherent, or non-adherent cells.
By measuring extracellular acidification changes in cell media over
time, the microphysiometer directly measures cellular responses to
various stimuli, including agonists, ligands, or antagonists of
mouse Zcytor16.
[0205] For example, the microphysiometer is used to measure
responses of an mouse Zcytor16-expressing eukaryotic cell, compared
to a control eukaryotic cell that does not express mouse Zcytor16
polypeptide. Suitable cells responsive to mouse Zcytor16-modulating
stimuli include recombinant host cells comprising a mouse Zcytor16
expression vector, and cells that naturally express mouse Zcytor16.
Extracellular acidification provides one measure for a mouse
Zcytor16-modulated cellular response. In addition, this approach
can be used to identify ligands, agonists, and antagonists of
Zcytor16 ligand, IL-TIF. For example, a molecule can be identified
as an agonist of Zcytor16 ligand by providing cells that express a
mouse Zcytor16 polypeptide, culturing a first portion of the cells
in the absence of the test compound, culturing a second portion of
the cells in the presence of the test compound, and determining
whether the second portion exhibits a cellular response, in
comparison with the first portion.
[0206] Alternatively, a solid phase system can be used to identify
a Zcytor16 ligand, or an agonist or antagonist of a Zcytor16
ligand. For example, a mouse Zcytor16 polypeptide or mouse Zcytor16
fusion protein, or mouse Zcytor16 monomeric, homodimeric,
heterodimeric or multimeric soluble receptor can be immobilized
onto the surface of a receptor chip of a commercially available
biosensor instrument (BIACORE, Biacore AB; Uppsala, Sweden). The
use of this instrument is disclosed, for example, by Karlsson,
Immunol. Methods 145:229 (1991), and Cunningham and Wells, J. Mol.
Biol. 234:554 (1993).
[0207] In brief, a mouse Zcytor16 polypeptide or fusion protein is
covalently attached, using amine or sulfhydryl chemistry, to
dextran fibers that are attached to gold film within a flow cell. A
test sample is then passed through the cell. If a ligand is present
in the sample, it will bind to the immobilized polypeptide or
fusion protein, causing a change in the refractive index of the
medium, which is detected as a change in surface plasmon resonance
of the gold film. This system allows the determination of on- and
off-rates, from which binding affinity can be calculated, and
assessment of stoichiometry of binding. This system can also be
used to examine antibody-antigen interactions, and the interactions
of other complement/anti-complement pairs.
[0208] Mouse Zcytor16 binding domains can be further characterized
by physical analysis of structure, as determined by such techniques
as nuclear magnetic resonance, crystallography, electron
diffraction or photoaffinity labeling, in conjunction with mutation
of putative contact site amino acids of Zcytor16 ligand agonists.
See, for example, de Vos et al., Science 255:306 (1992), Smith et
al., J. Mol. Biol. 224:899 (1992), and Wlodaver et al., FEBS Lett.
309:59 (1992).
[0209] The present invention also contemplates chemically modified
mouse Zcytor16 compositions, in which a mouse Zcytor16 polypeptide
is linked with a polymer. Illustrative mouse Zcytor16 polypeptides
are soluble polypeptides that lack a functional transmembrane
domain, such as a polypeptide consisting of amino acid residues 24
to 230, or 27 to 230 of SEQ ID NO:38 or SEQ ID NO:48. Typically,
the polymer is water soluble so that the mouse Zcytor16 conjugate
does not precipitate in an aqueous environment, such as a
physiological environment. An example of a suitable polymer is one
that has been modified to have a single reactive group, such as an
active ester for acylation, or an aldehyde for alkylation, In this
way, the degree of polymerization can be controlled. An example of
a reactive aldehyde is polyethylene glycol propionaldehyde, or
mono-(C1-C10) alkoxy, or aryloxy derivatives thereof (see, for
example, Harris, et al., U.S. Pat. No. 5,252,714). The polymer may
be branched or unbranched. Moreover, a mixture of polymers can be
used to produce mouse Zcytor16 conjugates.
[0210] Mouse Zcytor16 conjugates used for therapy can comprise
pharmaceutically acceptable water-soluble polymer moieties.
Suitable water-soluble polymers include polyethylene glycol (PEG),
monomethoxy-PEG, mono-(C 1-C 10)alkoxy-PEG, aryloxy-PEG,
poly-(N-vinyl pyrrolidone) PEG, tresyl monomethoxy PEG, PEG
propionaldehyde, bis-succinimidyl carbonate PEG, propylene glycol
homopolymers, a polypropylene oxide/ethylene oxide co-polymer,
polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol,
dextran, cellulose, or other carbohydrate-based polymers. Suitable
PEG may have a molecular weight from about 600 to about 60,000,
including, for example, 5,000, 12,000, 20,000 and 25,000. A mouse
Zcytor16 conjugate can also comprise a mixture of such
water-soluble polymers.
[0211] One example of a mouse Zcytor16 conjugate comprises a mouse
Zcytor16 moiety and a polyalkyl oxide moiety attached to the
N-terminus of the mouse Zcytor16 moiety. PEG is one suitable
polyalkyl oxide. As an illustration, mouse Zcytor16 can be modified
with PEG, a process known as "PEGylation." PEGylation of mouse
Zcytor16 can be carried out by any of the PEGylation reactions
known in the art (see, for example, EP 0 154 316, Delgado et al.,
Critical Reviews in Therapeutic Drug Carrier Systems 9:249 (1992),
Duncan and Spreafico, Clin. Pharmacokinet. 27:290 (1994), and
Francis et al., Int J Hematol 68:1 (1998)). For example, PEGylation
can be performed by an acylation reaction or by an alkylation
reaction with a reactive polyethylene glycol molecule. In an
alternative approach, mouse Zcytor16 conjugates are formed by
condensing activated PEG, in which a terminal hydroxy or amino
group of PEG has been replaced by an activated linker (see, for
example, Karasiewicz et al., U.S. Pat. No. 5,382,657).
[0212] PEGylation by acylation typically requires reacting an
active ester derivative of PEG with a mouse Zcytor16 polypeptide.
An example of an activated PEG ester is PEG esterified to
N-hydroxysuccinimide. As used herein, the term "acylation" includes
the following types of linkages between mouse Zcytor16 and a water
soluble polymer: amide, carbamate, urethane, and the like. Methods
for preparing PEGylated mouse Zcytor16 by acylation will typically
comprise the steps of (a) reacting a mouse Zcytor16 polypeptide
with PEG (such as a reactive ester of an aldehyde derivative of
PEG) under conditions whereby one or more PEG groups attach to
mouse Zcytor16, and (b) obtaining the reaction product(s).
Generally, the optimal reaction conditions for acylation reactions
will be determined based upon known parameters and desired results.
For example, the larger the ratio of PEG:mouse Zcytor16, the
greater the percentage of polyPEGylated mouse Zcytor16 product.
[0213] The product of PEGylation by acylation is typically a
polyPEGylated mouse Zcytor16 product, wherein the lysine 8-amino
groups are PEGylated via an acyl linking group. An example of a
connecting linkage is an amide. Typically, the resulting mouse
Zcytor16 will be at least 95% mono-, di-, or tri-pegylated,
although some species with higher degrees of PEGylation may be
formed depending upon the reaction conditions. PEGylated species
can be separated from unconjugated mouse Zcytor16 polypeptides
using standard purification methods, such as dialysis,
ultrafiltration, ion exchange chromatography, affinity
chromatography, and the like.
[0214] PEGylation by alkylation generally involves reacting a
terminal aldehyde derivative of PEG with mouse Zcytor16 in the
presence of a reducing agent. PEG groups can be attached to the
polypeptide via a --CH.sub.2--NH group.
[0215] Derivatization via reductive alkylation to produce a
monoPEGylated product takes advantage of the differential
reactivity of different types of primary amino groups available for
derivatization. Typically, the reaction is performed at a pH that
allows one to take advantage of the pKa differences between the
8-amino groups of the lysine residues and the .alpha.-amino group
of the N-terminal residue of the protein. By such selective
derivatization, attachment of a water-soluble polymer that contains
a reactive group such as an aldehyde, to a protein is controlled.
The conjugation with the polymer occurs predominantly at the
N-terminus of the protein without significant modification of other
reactive groups such as the lysine side chain amino groups. The
present invention provides a substantially homogenous preparation
of mouse Zcytor16 monopolymer conjugates.
[0216] Reductive alkylation to produce a substantially homogenous
population of monopolymer mouse Zcytor16 conjugate molecule can
comprise the steps of: (a) reacting a mouse Zcytor16 polypeptide
with a reactive PEG under reductive alkylation conditions at a pH
suitable to permit selective modification of the .alpha.-amino
group at the amino terminus of the mouse Zcytor16, and (b)
obtaining the reaction product(s). The reducing agent used for
reductive alkylation should be stable in aqueous solution and able
to reduce only the Schiff base formed in the initial process of
reductive alkylation. Illustrative reducing agents include sodium
borohydride, sodium cyanoborohydride, dimethylamine borane,
trimethylamine borane, and pyridine borane.
[0217] For a substantially homogenous population of monopolymer
mouse Zcytor16 conjugates, the reductive alkylation reaction
conditions are those that permit the selective attachment of the
water-soluble polymer moiety to the N-terminus of mouse Zcytor16.
Such reaction conditions generally provide for pKa differences
between the lysine amino groups and the .alpha.-amino group at the
N-terminus. The pH also affects the ratio of polymer to protein to
be used. In general, if the pH is lower, a larger excess of polymer
to protein will be desired because the less reactive the N-terminal
.alpha.-group, the more polymer is needed to achieve optimal
conditions. If the pH is higher, the polymer:mouse Zcytor16 need
not be as large because more reactive groups are available.
Typically, the pH will fall within the range of 3 to 9, or 3 to 6.
This method can be employed for making mouse Zcytor16-comprising
homodimeric, heterodimeric or multimeric soluble receptor
conjugates.
[0218] Another factor to consider is the molecular weight of the
water-soluble polymer. Generally, the higher the molecular weight
of the polymer, the fewer number of polymer molecules which may be
attached to the protein. For PEGylation reactions, the typical
molecular weight is about 2 kDa to about 100 kDa, about 5 kDa to
about 50 kDa, or about 12 kDa to about 25 kDa. The molar ratio of
water-soluble polymer to mouse Zcytor16 will generally be in the
range of 1:1 to 100:1. Typically, the molar ratio of water-soluble
polymer to mouse Zcytor16 will be 1:1 to 20:1 for polyPEGylation,
and 1:1 to 5:1 for monoPEGylation.
[0219] General methods for producing conjugates comprising a
polypeptide and water-soluble polymer moieties are known in the
art. See, for example, Karasiewicz et al., U.S. Pat. No. 5,382,657,
Greenwald et al., U.S. Pat. No. 5,738,846, Nieforth et al., Clin.
Pharmacol. Ther. 59:636 (1996), Monkarsh et al., Anal. Biochem.
247:434 (1997)). This method can be employed for making mouse
Zcytor16-comprising homodimeric, heterodimeric or multimeric
soluble receptor conjugates.
[0220] The present invention contemplates compositions comprising a
peptide or polypeptide described herein. Such compositions can
further comprise a carrier. The carrier can be a conventional
organic or inorganic carrier. Examples of carriers include water,
buffer solution, alcohol, propylene glycol, macrogol, sesame oil,
corn oil, and the like.
7. Isolation of Mouse Zcytor16 Polypeptides
[0221] The polypeptides of the present invention can be purified to
at least about 80% purity, to at least about 90% purity, to at
least about 95% purity, or greater than 95% purity with respect to
contaminating macromolecules, particularly other proteins and
nucleic acids, and free of infectious and pyrogenic agents. The
polypeptides of the present invention may also be purified to a
pharmaceutically pure state, which is greater than 99.9% pure. In
certain preparations, purified polypeptide is substantially free of
other polypeptides, particularly other polypeptides of animal
origin.
[0222] Fractionation and/or conventional purification methods can
be used to obtain preparations of mouse Zcytor16 purified from
natural sources (e.g., tonsil tissue), synthetic mouse Zcytor16
polypeptides, and recombinant mouse Zcytor16 polypeptides and
fusion mouse Zcytor16 polypeptides purified from recombinant host
cells. In general, ammonium sulfate precipitation and acid or
chaotrope extraction may be used for fractionation of samples.
Exemplary purification steps may include hydroxyapatite, size
exclusion, FPLC and reverse-phase high performance liquid
chromatography. Suitable chromatographic media include derivatized
dextrans, agarose, cellulose, polyacrylamide, specialty silicas,
and the like. PEI, DEAE, QAE and Q derivatives are suitable.
Exemplary chromatographic media include those media derivatized
with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF
(Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.),
Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins,
such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid
supports include glass beads, silica-based resins, cellulosic
resins, agarose beads, cross-linked agarose beads, polystyrene
beads, cross-linked polyacrylamide resins and the like that are
insoluble under the conditions in which they are to be used. These
supports may be modified with reactive groups that allow attachment
of proteins by amino groups, carboxyl groups, sulfhydryl groups,
hydroxyl groups and/or carbohydrate moieties.
[0223] Examples of coupling chemistries include cyanogen bromide
activation, N-hydroxysuccinimide activation, epoxide activation,
sulfhydryl activation, hydrazide activation, and carboxyl and amino
derivatives for carbodiimide coupling chemistries. These and other
solid media are well known and widely used in the art, and are
available from commercial suppliers. Selection of a particular
method for polypeptide isolation and purification is a matter of
routine design and is determined in part by the properties of the
chosen support. See, for example, Affinity Chromatography:
Principles & Methods (Pharmacia LKB Biotechnology 1988), and
Doonan, Protein Purification Protocols (The Humana Press 1996).
[0224] Additional variations in mouse Zcytor16 isolation and
purification can be devised by those of skill in the art. For
example, anti-mouse Zcytor16 antibodies, obtained as described
below, can be used to isolate large quantities of protein by
immunoaffinity purification.
[0225] The polypeptides of the present invention can also be
isolated by exploitation of particular properties. For example,
immobilized metal ion adsorption (IMAC) chromatography can be used
to purify histidine-rich proteins, including those comprising
polyhistidine tags. Briefly, a gel is first charged with divalent
metal ions to form a chelate (Sulkowski, Trends in Biochem. 3:1
(1985)). Histidine-rich proteins will be adsorbed to this matrix
with differing affinities, depending upon the metal ion used, and
will be eluted by competitive elution, lowering the pH, or use of
strong chelating agents. Other methods of purification include
purification of glycosylated proteins by lectin affinity
chromatography and ion exchange chromatography (M. Deutscher,
(ed.), Meth. Enzymol. 182:529 (1990)). Within additional
embodiments of the invention, a fusion of the polypeptide of
interest and an affinity tag (e.g., maltose-binding protein, an
immunoglobulin domain) may be constructed to facilitate
purification. Moreover, the ligand-binding properties of mouse
Zcytor16 extracellular domain can be exploited for purification,
for example, of mouse Zcytor16-comprising soluble receptors; for
example, by using affinity chromatography wherein IL-TIF ligand is
bound to a column and the mouse Zcytor16-comprising receptor is
bound and subsequently eluted using standard chromatography
methods.
[0226] mouse Zcytor16 polypeptides or fragments thereof may also be
prepared through chemical synthesis, as described above. mouse
Zcytor16 polypeptides may be monomers or multimers; glycosylated or
non-glycosylated; PEGylated or non-PEGylated; and may or may not
include an initial methionine amino acid residue.
8. Production of Antibodies to Mouse Zcytor16 Proteins
[0227] Antibodies to mouse Zcytor16 can be obtained, for example,
using the product of a mouse Zcytor16 expression vector or mouse
Zcytor16 isolated from a natural source as an antigen. Particularly
useful anti-mouse Zcytor16 antibodies "bind specifically" with
mouse Zcytor16. Antibodies are considered to be specifically
binding if the antibodies exhibit at least one of the following two
properties: (1) antibodies bind to mouse Zcytor16 with a threshold
level of binding activity, and (2) antibodies do not significantly
cross-react with polypeptides related to mouse Zcytor16.
[0228] With regard to the first characteristic, antibodies
specifically bind if they bind to a mouse Zcytor16 polypeptide,
peptide or epitope with a binding affinity (K.sub.a) of 10.sup.6
M.sup.-1 or greater, preferably 10.sup.7 M.sup.-1 or greater, more
preferably 10.sup.8 M.sup.-1 or greater, and most preferably
10.sup.9 M.sup.-1 or greater. The binding affinity of an antibody
can be readily determined by one of ordinary skill in the art, for
example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci.
51:660 (1949)). With regard to the second characteristic,
antibodies do not significantly cross-react with related
polypeptide molecules, for example, if they detect mouse Zcytor16,
but not presently known polypeptides using a standard Western blot
analysis. Examples of known related polypeptides include known
cytokine receptors.
[0229] Anti-mouse Zcytor16 antibodies can be produced using
antigenic mouse Zcytor16 epitope-bearing peptides and polypeptides.
Antigenic epitope-bearing peptides and polypeptides of the present
invention contain a sequence of at least nine, or between 15 to
about 30 amino acids contained within SEQ ID NO:38 or SEQ ID NO:48
or another amino acid sequence disclosed herein. However, peptides
or polypeptides comprising a larger portion of an amino acid
sequence of the invention, containing from 30 to 50 amino acids, or
any length up to and including the entire amino acid sequence of a
polypeptide of the invention, also are useful for inducing
antibodies that bind with mouse Zcytor16. It is desirable that the
amino acid sequence of the epitope-bearing peptide is selected to
provide substantial solubility in aqueous solvents (i.e., the
sequence includes relatively hydrophilic residues, while
hydrophobic residues are typically avoided). Moreover, amino acid
sequences containing proline residues may be also be desirable for
antibody production.
[0230] As an illustration, potential antigenic sites in mouse
Zcytor16 were identified using the Jameson-Wolf method, Jameson and
Wolf, CABIOS 4:181, (1988), as implemented by the PROTEAN program
(version 3.14) of LASERGENE (DNASTAR; Madison, Wis.). Default
parameters were used in this analysis.
[0231] The Jameson-Wolf method predicts potential antigenic
determinants by combining six major subroutines for protein
structural prediction. Briefly, the Hopp-Woods method, Hopp et al.,
Proc. Nat'l Acad. Sci. USA 78:3824 (1981), was first used to
identify amino acid sequences representing areas of greatest local
hydrophilicity (parameter: seven residues averaged). In the second
step, Emini's method, Emini et al., J. Virology 55:836 (1985), was
used to calculate surface probabilities (parameter: surface
decision threshold (0.6)=1). Third, the Karplus-Schultz method,
Karplus and Schultz, Naturwissenschaften 72:212 (1985), was used to
predict backbone chain flexibility (parameter: flexibility
threshold (0.2)=1). In the fourth and fifth steps of the analysis,
secondary structure predictions were applied to the data using the
methods of Chou-Fasman, Chou, "Prediction of Protein Structural
Classes from Amino Acid Composition," in Prediction of Protein
Structure and the Principles of Protein Conformation, Fasman (ed.),
pages 549-586 (Plenum Press 1990), and Garnier-Robson, Garnier et
al., J. Mol. Biol. 120:97 (1978) (Chou-Fasman parameters:
conformation table=64 proteins; a region threshold=103; .beta.
region threshold=105; Garnier-Robson parameters: .alpha. and .beta.
decision constants=0). In the sixth subroutine, flexibility
parameters and hydropathy/solvent accessibility factors were
combined to determine a surface contour value, designated as the
"antigenic index." Finally, a peak broadening function was applied
to the antigenic index, which broadens major surface peaks by
adding 20, 40, 60, or 80% of the respective peak value to account
for additional free energy derived from the mobility of surface
regions relative to interior regions. This calculation was not
applied, however, to any major peak that resides in a helical
region, since helical regions tend to be less flexible.
[0232] The results of this analysis indicated that the following
amino acid sequences of SEQ ID NO:38 or SEQ ID NO:48 would provide
suitable antigenic peptides: amino acids 35 to 40 ("antigenic
peptide 1"), amino acids 67 to 77 ("antigenic peptide 2"), 88 to 94
("antigenic peptide 3"), amino acids 108 to 119 ("antigenic peptide
4"), amino acids 108 to 130 ("antigenic peptide 5"), amino acids
125 to 130 ("antigenic peptide 6"), amino acids 147 to 161
("antigenic peptide 7"), amino acids 177 to 190 ("antigenic peptide
8"), and amino acids 216 to 225 ("antigenic peptide 9"). The
present invention contemplates the use of any one of antigenic
peptides 1 to 9 to generate antibodies to mouse Zcytor16. The
present invention also contemplates polypeptides comprising at
least one of antigenic peptides 1 to 9. A Hopp/Woods hydrophilicity
profile of the Zcytor16 protein sequence as shown in SEQ ID NO:38
or SEQ ID NO:48 can be generated (Hopp et al., Proc. Natl. Acad.
Sci. 78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986 and
Triquier et al., Protein Engineering 11:153-169, 1998). The profile
is based on a sliding six-residue window. Buried G, S, and T
residues and exposed H, Y, and W residues were ignored. For
example, in Zcytor16, hydrophilic regions that serve as suitable
antigens, include in reference to SEQ ID NO:38 or SEQ ID NO:48: (1)
amino acid number 220 to 225; (2) amino acid number 216 to 221; (3)
amino acid number 180 to 185; (4) amino acid number 179 to 184; and
(5) amino acid number 71 to 76. Moreover other suitable antigens
comprise residues, 24, 27 or 31 to 122 or 126; 131 to 229 or 230; 1
to 229 or 230; and 24 to 229 or 230 of SEQ ID NO:38 or SEQ ID
NO:48.
[0233] Moreover, suitable antigens also include the mouse Zcytor16
polypeptides comprising a mouse Zcytor16 cytokine binding, or
extracellular domain disclosed above in combination with another
class I or II cytokine extracellular domain, such as those that
form soluble mouse Zcytor16 heterodimeric or multimeric
polypeptides, such as soluble mouse Zcytor16/CRF2-4, mouse
Zcytor16/zcytor11, mouse Zcytor16/zcytor7, and the like.
[0234] Polyclonal antibodies to recombinant mouse Zcytor16 protein
or to mouse Zcytor16 isolated from natural sources can be prepared
using methods well-known to those of skill in the art. See, for
example, Green et al., "Production of Polyclonal Antisera," in
Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press
1992), and Williams et al., "Expression of foreign proteins in E.
coli using plasmid vectors and purification of specific polyclonal
antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition,
Glover et al. (eds.), page 15 (Oxford University Press 1995). The
immunogenicity of a mouse Zcytor16 polypeptide can be increased
through the use of an adjuvant, such as alum (aluminum hydroxide)
or Freund's complete or incomplete adjuvant. Polypeptides useful
for immunization also include fusion polypeptides, such as fusions
of mouse Zcytor16 or a portion thereof with an immunoglobulin
polypeptide or with maltose binding protein. The polypeptide
immunogen may be a full-length molecule or a portion thereof. If
the polypeptide portion is "hapten-like," such portion may be
advantageously joined or linked to a macromolecular carrier (such
as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or
tetanus toxoid) for immunization.
[0235] Although polyclonal antibodies are typically raised in
animals such as horses, cows, dogs, chicken, rats, mice, rabbits,
guinea pigs, goats, or sheep, an anti-mouse Zcytor16 antibody of
the present invention may also be derived from a subhuman primate
antibody. General techniques for raising diagnostically and
therapeutically useful antibodies in baboons may be found, for
example, in Goldenberg et al., international patent publication No.
WO 91/11465, and in Losman et al., Int. J. Cancer 46:310
(1990).
[0236] Alternatively, monoclonal anti-mouse Zcytor16 antibodies can
be generated. Rodent monoclonal antibodies to specific antigens may
be obtained by methods known to those skilled in the art (see, for
example, Kohler et al., Nature 256:495 (1975), Coligan et al.
(eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7
(John Wiley & Sons 1991) ["Coligan"], Picksley et al.,
"Production of monoclonal antibodies against proteins expressed in
E. coli," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover
et al. (eds.), page 93 (Oxford University Press 1995)).
[0237] Briefly, monoclonal antibodies can be obtained by injecting
mice with a composition comprising a mouse Zcytor16 gene product,
verifying the presence of antibody production by removing a serum
sample, removing the spleen to obtain B-lymphocytes, fusing the
B-lymphocytes with myeloma cells to produce hybridomas, cloning the
hybridomas, selecting positive clones which produce antibodies to
the antigen, culturing the clones that produce antibodies to the
antigen, and isolating the antibodies from the hybridoma
cultures.
[0238] In addition, an anti-mouse Zcytor16 antibody of the present
invention may be derived from a human monoclonal antibody. Human
monoclonal antibodies are obtained from transgenic mice that have
been engineered to produce specific human antibodies in response to
antigenic challenge. In this technique, elements of the human heavy
and light chain locus are introduced into strains of mice derived
from embryonic stem cell lines that contain targeted disruptions of
the endogenous heavy chain and light chain loci. The transgenic
mice can synthesize human antibodies against specific antigens,
e.g., Zcytor16, and the mice can be used to produce human
antibody-secreting hybridomas. Methods for obtaining human
antibodies from transgenic mice are described, for example, by
Green et al., Nature Genet. 7:13 (1994), Lonberg et al., Nature
368:856 (1994), and Taylor et al., Int. Immun. 6:579 (1994).
[0239] Monoclonal antibodies can be isolated and purified from
hybridoma cultures by a variety of well-established techniques.
Such isolation techniques include affinity chromatography with
Protein-A Sepharose, size-exclusion chromatography, and
ion-exchange chromatography (see, for example, Coligan at pages
2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., "Purification of
Immunoglobulin G (IgG)," in Methods in Molecular Biology, Vol. 10,
pages 79-104 (The Humana Press, Inc. 1992)).
[0240] For particular uses, it may be desirable to prepare
fragments of anti-mouse Zcytor16 antibodies. Such antibody
fragments can be obtained, for example, by proteolytic hydrolysis
of the antibody. Antibody fragments can be obtained by pepsin or
papain digestion of whole antibodies by conventional methods. As an
illustration, antibody fragments can be produced by enzymatic
cleavage of antibodies with pepsin to provide a 5S fragment denoted
F(ab').sub.2. This fragment can be further cleaved using a thiol
reducing agent to produce 3.5S Fab' monovalent fragments.
Optionally, the cleavage reaction can be performed using a blocking
group for the sulfhydryl groups that result from cleavage of
disulfide linkages. As an alternative, an enzymatic cleavage using
pepsin produces two monovalent Fab fragments and an Fc fragment
directly. These methods are described, for example, by Goldenberg,
U.S. Pat. No. 4,331,647, Nisonoff et al., Arch Biochem. Biophys.
89:230 (1960), Porter, Biochem. J. 73:119 (1959), Edelman et al.,
in Methods in Enzymology Vol. 1, page 422 (Academic Press 1967),
and by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
[0241] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0242] For example, Fv fragments comprise an association of V.sub.H
and V.sub.L chains. This association can be noncovalent, as
described by Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659
(1972). Alternatively, the variable chains can be linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
glutaraldehyde (see, for example, Sandhu, Crit. Rev. Biotech.
12:437 (1992)).
[0243] The Fv fragments may comprise V.sub.H and V.sub.L chains
which are connected by a peptide linker. These single-chain antigen
binding proteins (scFv) are prepared by constructing a structural
gene comprising DNA sequences encoding the V.sub.H and V.sub.L
domains which are connected by an oligonucleotide. The structural
gene is inserted into an expression vector which is subsequently
introduced into a host cell, such as E. coli. The recombinant host
cells synthesize a single polypeptide chain with a linker peptide
bridging the two V domains. Methods for producing scFvs are
described, for example, by Whitlow et al., Methods: A Companion to
Methods in Enzymology 2:97 (1991) (also see, Bird et al., Science
242:423 (1988), Ladner et al., U.S. Pat. No. 4,946,778, Pack et
al., Bio/Technology 11:1271 (1993), and Sandhu, supra).
[0244] As an illustration, a scFV can be obtained by exposing
lymphocytes to mouse Zcytor16 polypeptide in vitro, and selecting
antibody display libraries in phage or similar vectors (for
instance, through use of immobilized or labeled mouse Zcytor16
protein or peptide). Genes encoding polypeptides having potential
mouse Zcytor16 polypeptide binding domains can be obtained by
screening random peptide libraries displayed on phage (phage
display) or on bacteria, such as E. coli. Nucleotide sequences
encoding the polypeptides can be obtained in a number of ways, such
as through random mutagenesis and random polynucleotide synthesis.
These random peptide display libraries can be used to screen for
peptides which interact with a known target which can be a protein
or polypeptide, such as a ligand or receptor, a biological or
synthetic macromolecule, or organic or inorganic substances.
Techniques for creating and screening such random peptide display
libraries are known in the art (Ladner et al., U.S. Pat. No.
5,223,409, Ladner et al., U.S. Pat. No. 4,946,778, Ladner et al.,
U.S. Pat. No. 5,403,484, Ladner et al., U.S. Pat. No. 5,571,698,
and Kay et al., Phage Display of Peptides and Proteins (Academic
Press, Inc. 1996)) and random peptide display libraries and kits
for screening such libraries are available commercially, for
instance from CLONTECH Laboratories, Inc. (Palo Alto, Calif.),
Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc.
(Beverly, Mass.), and Pharmacia LKB Biotechnology Inc. (Piscataway,
N.J.). Random peptide display libraries can be screened using the
mouse Zcytor16 sequences disclosed herein to identify proteins
which bind to mouse Zcytor16.
[0245] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing cells
(see, for example, Larrick et al., Methods: A Companion to Methods
in Enzymology 2:106 (1991), Courtenay-Luck, "Genetic Manipulation
of Monoclonal Antibodies," in Monoclonal Antibodies: Production,
Engineering and Clinical Application, Ritter et al. (eds.), page
166 (Cambridge University Press 1995), and Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, Birch et al., (eds.), page
137 (Wiley-Liss, Inc. 1995)).
[0246] Alternatively, an anti-mouse Zcytor16 antibody may be
derived from a "humanized" monoclonal antibody. Humanized
monoclonal antibodies are produced by transferring mouse
complementary determining regions from heavy and light variable
chains of the mouse immunoglobulin into a human variable domain.
Typical residues of human antibodies are then substituted in the
framework regions of the murine counterparts. The use of antibody
components derived from humanized monoclonal antibodies obviates
potential problems associated with the immunogenicity of murine
constant regions. General techniques for cloning murine
immunoglobulin variable domains are described, for example, by
Orlandi et al., Proc. Nat'l Acad. Sci. USA 86:3833 (1989).
Techniques for producing humanized monoclonal antibodies are
described, for example, by Jones et al., Nature 321:522 (1986),
Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285 (1992), Sandhu,
Crit. Rev. Biotech. 12:437 (1992), Singer et al., J. Immun.
150:2844 (1993), Sudhir (ed.), Antibody Engineering Protocols
(Humana Press, Inc. 1995), Kelley, "Engineering Therapeutic
Antibodies," in Protein Engineering Principles and Practice,
Cleland et al. (eds.), pages 399-434 (John Wiley & Sons, Inc.
1996), and by Queen et al., U.S. Pat. No. 5,693,762 (1997).
[0247] Polyclonal anti-idiotype antibodies can be prepared by
immunizing animals with anti-mouse Zcytor16 antibodies or antibody
fragments, using standard techniques. See, for example, Green et
al., "Production of Polyclonal Antisera," in Methods In Molecular
Biology: Immunochemical Protocols, Manson (ed.), pages 1-12 (Humana
Press 1992). Also, see Coligan at pages 2.4.1-2.4.7. Alternatively,
monoclonal anti-idiotype antibodies can be prepared using
anti-mouse Zcytor16 antibodies or antibody fragments as immunogens
with the techniques, described above. As another alternative,
humanized anti-idiotype antibodies or subhuman primate
anti-idiotype antibodies can be prepared using the above-described
techniques. Methods for producing anti-idiotype antibodies are
described, for example, by Irie, U.S. Pat. No. 5,208,146, Greene,
et. al., U.S. Pat. No. 5,637,677, and Varthakavi and Minocha, J.
Gen. Virol. 77:1875 (1996).
9. Use of Mouse Zcytor16 Nucleotide Sequences to Detect Gene
Expression and Gene Structure
[0248] Nucleic acid molecules can be used to detect the expression
of a mouse Zcytor16 gene in a biological sample. Suitable probe
molecules include double-stranded nucleic acid molecules comprising
the nucleotide sequence of SEQ ID NO:37 or SEQ ID NO:47, or a
portion thereof, as well as single-stranded nucleic acid molecules
having the complement of the nucleotide sequence of SEQ ID NO:37 or
SEQ ID NO:47, or a portion thereof. Probe molecules may be DNA,
RNA, oligonucleotides, and the like. As used herein, the term
"portion" refers to at least eight nucleotides to at least 20 or
more nucleotides. Illustrative probes bind with regions of the
mouse Zcytor16 gene that have a low sequence similarity to
comparable regions in other cytokine receptor genes.
[0249] In a basic assay, a single-stranded probe molecule is
incubated with RNA, isolated from a biological sample, under
conditions of temperature and ionic strength that promote base
pairing between the probe and target mouse Zcytor16 RNA species.
After separating unbound probe from hybridized molecules, the
amount of hybrids is detected.
[0250] In addition, as Zcytor16 expression in humans and mice is
tissue-specific, polynucleotide probes, anti-mouse Zcytor16
antibodies, and detection the presence of Zcytor16 polypeptides in
tissue can be used to assess whether a specific tissue is present,
for example, after surgery involving the excision of a diseased or
cancerous tissue. As such, the polynucleotides, polypeptides, and
antibodies of the present invention can be used as an aid to
determine whether all spleen tissue is excised after surgery, for
example, after surgery for spleen cancer. In such instances, it is
especially important to remove all potentially diseased tissue to
maximize recovery from the cancer, and to minimize recurrence.
Preferred embodiments include fluorescent, radiolabeled, or
calorimetrically labeled antibodies, that can be used in situ.
[0251] Moreover, anti-mouse Zcytor16 antibodies and binding
fragments can be used for tagging and sorting cells that
specifically-express mouse Zcytor16, such as mononuclear cells,
lymphoid cells, e.g, activated CD4+ T-cells and CD19+ B-cells, and
other described herein. Such methods of cell tagging and sorting
are well known in the art (see, e.g., "Molecular Biology of the
Cell", 3.sup.rd Ed., Albert, B. et al. (Garland Publishing, London
& New York, 1994). One of skill in the art would recognize the
importance of separating cell tissue types to study cells, and the
use of antibodies to separate specific cell tissue types.
Basically, antibodies that bind to the surface of a cell type are
coupled to various matrices such as collagen, polysaccharide beads,
or plastic to form an affinity surface to which only cells
recognized by the antibodies will adhere. The bound cells are then
recovered by conventional techniques. Other methods involve
separating cells by a fluorescence-activated cell sorter (FACS). In
this technique one labels cells with antibodies that are coupled to
a fluorescent dye. The labeled cells are then separated from
unlabeled cells in a FACS machine. In FACS sorting individual cells
traveling in single file pass through a laser beam and the
fluorescence of each cell is measured. Slightly further
down-stream, tiny droplets, most containing either one or no cells,
are formed by a vibrating nozzle. The droplets containing a single
cell are automatically give a positive or negative charge at the
moment of formation, depending on whether the cell they contain is
fluorescent, and then deflected by a strong electric field into an
appropriate container. Such machines can select 1 cell in 1000 and
sort about 5000 cells each second. This produces a uniform
population of cells for cell culture.
[0252] One of skill in the art would recognize that the antibodies
to the mouse Zcytor16 polypeptides of the present invention are
useful, because not all tissue types express the mouse Zcytor16
receptor and because it is important that biologists be able to
separate specific cell types for further study and/or therapeutic
re-implantation into the body. This is particularly relevant in
cells such as immune cells, wherein mouse Zcytor16 is
expressed.
[0253] Moreover, use of mouse Zcytor16 polynucleotide probes,
anti-mouse Zcytor16 antibodies, and detection the presence of mouse
Zcytor16 polypeptides in tissue can be used in the diagnosis and/or
prevention of spontaneous abortions, or to monitor placental health
and function. Since mouse Zcytor16 is expressed in the placenta, it
could play a role in the critical functions of placenta, such as
proliferation or survival of trophoblast cells, and the like. Thus,
mouse Zcytor16 could be essential for the function of the placenta,
thus maturation of embryos. Therefore, a supplement of mouse
Zcytor16 polypeptide, or anti-mouse Zcytor16 antibodies may be
beneficial in the prevention and treatment of certain types of
spontaneous abortions, or premature birth of babies caused by
abnormal expression of Zcytor16 in the placenta, or as a diagnostic
to assess the function of the placenta. For example, as mouse
Zcytor16 is normally expressed in placenta, the absence of mouse
Zcytor16 expression may be indicative of abnormal placenta
function. Similarly, use of mouse Zcytor16 polynucleotide probes,
anti-mouse Zcytor16 antibodies, can be used in animal husbandry,
for example in commercial mouse breeding colonies and transgenic
mouse embryo recipients in the prevention and treatment of certain
types of spontaneous abortions, or premature birth of pups caused
by abnormal expression of mouse Zcytor16 in the placenta, or as a
diagnostic to assess the function of the placenta.
[0254] Well-established hybridization methods of RNA detection
include northern analysis and dot/slot blot hybridization (see, for
example, Ausubel (1995) at pages 4-1 to 4-27, and Wu et al. (eds.),
"Analysis of Gene Expression at the RNA Level," in Methods in Gene
Biotechnology, pages 225-239 (CRC Press, Inc. 1997)). Nucleic acid
probes can be detectably labeled with radioisotopes such as
.sup.32P or .sup.35S. Alternatively, mouse Zcytor16 RNA can be
detected with a nonradioactive hybridization method (see, for
example, Isaac (ed.), Protocols for Nucleic Acid Analysis by
Nonradioactive Probes (Humana Press, Inc. 1993)). Typically,
nonradioactive detection is achieved by enzymatic conversion of
chromogenic or chemiluminescent substrates. Illustrative
nonradioactive moieties include biotin, fluorescein, and
digoxigenin.
[0255] Mouse Zcytor16 oligonucleotide probes are also useful for in
vivo diagnosis. As an illustration, .sup.18F-labeled
oligonucleotides can be administered to a subject and visualized by
positron emission tomography (Tavitian et al., Nature Medicine
4:467 (1998)).
[0256] Numerous diagnostic procedures take advantage of the
polymerase chain reaction (PCR) to increase sensitivity of
detection methods. Standard techniques for performing PCR are
well-known (see, generally, Mathew (ed.), Protocols in Human
Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCR
Protocols: Current Methods and Applications (Humana Press, Inc.
1993), Cotter (ed.), Molecular Diagnosis of Cancer (Humana Press,
Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols
(Humana Press, Inc. 1998), Lo (ed.), Clinical Applications of PCR
(Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis
(Humana Press, Inc. 1998)).
[0257] PCR primers can be designed to amplify a portion of the
mouse Zcytor16 gene that has a low sequence similarity to a
comparable region in other proteins, such as other cytokine
receptor proteins.
[0258] One variation of PCR for diagnostic assays is reverse
transcriptase-PCR (RT-PCR). In the RT-PCR technique, RNA is
isolated from a biological sample, reverse transcribed to cDNA, and
the cDNA is incubated with mouse Zcytor16 primers (see, for
example, Wu et al. (eds.), "Rapid Isolation of Specific cDNAs or
Genes by PCR," in Methods in Gene Biotechnology, pages 15-28 (CRC
Press, Inc. 1997)). PCR is then performed and the products are
analyzed using standard techniques.
[0259] As an illustration, RNA is isolated from biological sample
using, for example, the gunadinium-thiocyanate cell lysis procedure
described above. Alternatively, a solid-phase technique can be used
to isolate mRNA from a cell lysate. A reverse transcription
reaction can be primed with the isolated RNA using random
oligonucleotides, short homopolymers of dT, or mouse Zcytor16
anti-sense oligomers. Oligo-dT primers offer the advantage that
various mRNA nucleotide sequences are amplified that can provide
control target sequences. mouse Zcytor16 sequences are amplified by
the polymerase chain reaction using two flanking oligonucleotide
primers that are typically 20 bases in length.
[0260] PCR amplification products can be detected using a variety
of approaches. For example, PCR products can be fractionated by gel
electrophoresis, and visualized by ethidium bromide staining.
Alternatively, fractionated PCR products can be transferred to a
membrane, hybridized with a detectably-labeled mouse Zcytor16
probe, and examined by autoradiography. Additional alternative
approaches include the use of digoxigenin-labeled deoxyribonucleic
acid triphosphates to provide chemiluminescence detection, and the
C-TRAK colorimetric assay.
[0261] Another approach for detection of mouse Zcytor16 expression
is cycling probe technology (CPT), in which a single-stranded DNA
target binds with an excess of DNA-RNA-DNA chimeric probe to form a
complex, the RNA portion is cleaved with RNAase H, and the presence
of cleaved chimeric probe is detected (see, for example, Beggs et
al., J. Clin. Microbiol. 34:2985 (1996), Bekkaoui et al.,
Biotechniques 20:240 (1996)). Alternative methods for detection of
mouse Zcytor16 sequences can utilize approaches such as nucleic
acid sequence-based amplification (NASBA), cooperative
amplification of templates by cross-hybridization (CATCH), and the
ligase chain reaction (LCR) (see, for example, Marshall et al.,
U.S. Pat. No. 5,686,272 (1997), Dyer et al., J. Virol. Methods
60:161 (1996), Ehricht et al., Eur. J. Biochem. 243:358 (1997), and
Chadwick et al., J. Virol. Methods 70:59 (1998)). Other standard
methods are known to those of skill in the art.
[0262] Mouse Zcytor16 probes and primers can also be used to detect
and to localize Zcytor16 gene expression in tissue samples. Methods
for such in situ hybridization are well-known to those of skill in
the art (see, for example, Choo (ed.), In Situ Hybridization
Protocols (Humana Press, Inc. 1994), Wu et al. (eds.), "Analysis of
Cellular DNA or Abundance of mRNA by Radioactive In Situ
Hybridization (RISH)," in Methods in Gene Biotechnology, pages
259-278 (CRC Press, Inc. 1997), and Wu et al. (eds.), "Localization
of DNA or Abundance of mRNA by Fluorescence In Situ Hybridization
(RISH)," in Methods in Gene Biotechnology, pages 279-289 (CRC
Press, Inc. 1997)). Various additional diagnostic approaches are
well-known to those of skill in the art (see, for example, Mathew
(ed.), Protocols in Human Molecular Genetics (Humana Press, Inc.
1991), Coleman and Tsongalis, Molecular Diagnostics (Humana Press,
Inc. 1996), and Elles, Molecular Diagnosis of Genetic Diseases
(Humana Press, Inc., 1996)). Suitable test samples include blood,
urine, saliva, tissue biopsy, and autopsy material.
10. Use of Anti-Mouse Zcytor16 Antibodies to Detect Zcytor16 or
Antagonize Zcytor16 Binding to IL-TIF
[0263] The present invention contemplates the use of anti-mouse
Zcytor16 antibodies to screen biological samples in vitro for the
presence of Zcytor16. In one type of in vitro assay, anti-mouse
Zcytor16 antibodies are used in liquid phase. For example, the
presence of mouse Zcytor16 in a biological sample can be tested by
mixing the biological sample with a trace amount of labeled mouse
Zcytor16 and an anti-mouse Zcytor16 antibody under conditions that
promote binding between mouse Zcytor16 and its antibody. Complexes
of mouse Zcytor16 and anti-mouse Zcytor16 in the sample can be
separated from the reaction mixture by contacting the complex with
an immobilized protein which binds with the antibody, such as an Fc
antibody or Staphylococcus protein A. The concentration of mouse
Zcytor16 in the biological sample will be inversely proportional to
the amount of labeled mouse Zcytor16 bound to the antibody and
directly related to the amount of free labeled mouse Zcytor16.
Illustrative biological samples include blood, urine, saliva,
tissue biopsy, and autopsy material.
[0264] Alternatively, in vitro assays can be performed in which
anti-mouse Zcytor16 antibody is bound to a solid-phase carrier. For
example, antibody can be attached to a polymer, such as
aminodextran, in order to link the antibody to an insoluble support
such as a polymer-coated bead, a plate or a tube. Other suitable in
vitro assays will be readily apparent to those of skill in the
art.
[0265] In another approach, anti-mouse Zcytor16 antibodies can be
used to detect Zcytor16 in tissue sections prepared from a biopsy
specimen. Such immunochemical detection can be used to determine
the relative abundance of mouse Zcytor16 and to determine the
distribution of Zcytor16 in the examined tissue. General
immunochemistry techniques are well established (see, for example,
Ponder, "Cell Marking Techniques and Their Application," in
Mammalian Development: A Practical Approach, Monk (ed.), pages
115-38 (IRL Press 1987), Coligan at pages 5.8.1-5.8.8, Ausubel
(1995) at pages 14.6.1 to 14.6.13 (Wiley Interscience 1990), and
Manson (ed.), Methods In Molecular Biology, Vol. 10: Immunochemical
Protocols (The Humana Press, Inc. 1992)).
[0266] Immunochemical detection can be performed by contacting a
biological sample with an anti-mouse Zcytor16 antibody, and then
contacting the biological sample with a detectably labeled molecule
which binds to the antibody. For example, the detectably labeled
molecule can comprise an antibody moiety that binds to anti-mouse
Zcytor16 antibody. Alternatively, the anti-mouse Zcytor16 antibody
can be conjugated with avidin/streptavidin (or biotin) and the
detectably labeled molecule can comprise biotin (or
avidin/streptavidin). Numerous variations of this basic technique
are well-known to those of skill in the art.
[0267] Alternatively, an anti-mouse Zcytor16 antibody can be
conjugated with a detectable label to form an anti-mouse Zcytor16
immunoconjugate. Suitable detectable labels include, for example, a
radioisotope, a fluorescent label, a chemiluminescent label, an
enzyme label, a bioluminescent label or colloidal gold. Methods of
making and detecting such detectably-labeled immunoconjugates are
well-known to those of ordinary skill in the art, and are described
in more detail below.
[0268] The detectable label can be a radioisotope that is detected
by autoradiography. Isotopes that are particularly useful for the
purpose of the present invention are .sup.3H, .sup.125I, .sup.131I,
.sup.35S and .sup.14C.
[0269] Anti-mouse Zcytor16 immunoconjugates can also be labeled
with a fluorescent compound. The presence of a
fluorescently-labeled antibody is determined by exposing the
immunoconjugate to light of the proper wavelength and detecting the
resultant fluorescence. Fluorescent labeling compounds include
fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin,
allophycocyanin, o-phthaldehyde and fluorescamine.
[0270] Alternatively, anti-mouse Zcytor16 immunoconjugates can be
detectably labeled by coupling an antibody component to a
chemiluminescent compound. The presence of the
chemiluminescent-tagged immunoconjugate is determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of chemiluminescent labeling compounds
include luminol, isoluminol, an aromatic acridinium ester, an
imidazole, an acridinium salt and an oxalate ester.
[0271] Similarly, a bioluminescent compound can be used to label
anti-mouse Zcytor16 immunoconjugates of the present invention.
Bioluminescence is a type of chemiluminescence found in biological
systems in which a catalytic protein increases the efficiency of
the chemiluminescent reaction. The presence of a bioluminescent
protein is determined by detecting the presence of luminescence.
Bioluminescent compounds that are useful for labeling include
luciferin, luciferase and aequorin.
[0272] Alternatively, anti-mouse Zcytor16 immunoconjugates can be
detectably labeled by linking an anti-mouse Zcytor16 antibody
component to an enzyme. When the anti-mouse Zcytor16-enzyme
conjugate is incubated in the presence of the appropriate
substrate, the enzyme moiety reacts with the substrate to produce a
chemical moiety which can be detected, for example, by
spectrophotometric, fluorometric or visual means. Examples of
enzymes that can be used to detectably label polyspecific
immunoconjugates include .beta.-galactosidase, glucose oxidase,
peroxidase and alkaline phosphatase.
[0273] Those of skill in the art will know of other suitable labels
which can be employed in accordance with the present invention. The
binding of marker moieties to anti-mouse Zcytor16 antibodies can be
accomplished using standard techniques known to the art. Typical
methodology in this regard is described by Kennedy et al., Clin.
Chim. Acta 70:1 (1976), Schurs et al., Clin. Chim. Acta 81:1
(1977), Shih et al., Int'l J. Cancer 46:1101 (1990), Stein et al.,
Cancer Res. 50:1330 (1990), and Coligan, supra.
[0274] Moreover, the convenience and versatility of immunochemical
detection can be enhanced by using anti-mouse Zcytor16 antibodies
that have been conjugated with avidin, streptavidin, and biotin
(see, for example, Wilchek et al. (eds.), "Avidin-Biotin
Technology," Methods In Enzymology, Vol. 184 (Academic Press 1990),
and Bayer et al., "Immunochemical Applications of Avidin-Biotin
Technology," in Methods In Molecular Biology, Vol. 10, Manson
(ed.), pages 149-162 (The Humana Press, Inc. 1992).
[0275] Methods for performing immunoassays are well-established.
See, for example, Cook and Self, "Monoclonal Antibodies in
Diagnostic Immunoassays," in Monoclonal Antibodies: Production,
Engineering, and Clinical Application, Ritter and Ladyman (eds.),
pages 180-208, (Cambridge University Press, 1995), Perry, "The Role
of Monoclonal Antibodies in the Advancement of Immunoassay
Technology," in Monoclonal Antibodies: Principles and Applications,
Birch and Lennox (eds.), pages 107-120 (Wiley-Liss, Inc. 1995), and
Diamandis, Immunoassay (Academic Press, Inc. 1996).
[0276] The present invention also contemplates kits for performing
an immunological diagnostic assay for Zcytor16 gene expression.
Such kits comprise at least one container comprising an anti-mouse
Zcytor16 antibody, or antibody fragment. A kit may also comprise a
second container comprising one or more reagents capable of
indicating the presence of mouse Zcytor16 antibody or antibody
fragments. Examples of such indicator reagents include detectable
labels such as a radioactive label, a fluorescent label, a
chemiluminescent label, an enzyme label, a bioluminescent label,
colloidal gold, and the like. A kit may also comprise a means for
conveying to the user that mouse Zcytor16 antibodies or antibody
fragments are used to detect Zcytor16 protein. For example, written
instructions may state that the enclosed antibody or antibody
fragment can be used to detect mouse Zcytor16. The written material
can be applied directly to a container, or the written material can
be provided in the form of a packaging insert.
[0277] Alternative techniques for generating or selecting
antibodies useful herein include in vitro exposure of lymphocytes
to soluble mouse Zcytor16 monomeric receptor or soluble mouse
Zcytor16 homodimeric, heterodimeric or multimeric polypeptides, and
selection of antibody display libraries in phage or similar vectors
(for instance, through use of immobilized or labeled soluble mouse
Zcytor16 monomeric receptor or soluble mouse Zcytor16 homodimeric,
heterodimeric or multimeric polypeptides). Genes encoding
polypeptides having potential binding domains such as soluble mouse
Zcytor16 monomeric receptor or soluble mouse Zcytor16 homodimeric,
heterodimeric or multimeric polypeptide can be obtained by
screening random peptide libraries displayed on phage (phage
display) or on bacteria, such as E. coli. Nucleotide sequences
encoding the polypeptides can be obtained in a number of ways, such
as through random mutagenesis and random polynucleotide synthesis.
These random peptide display libraries can be used to screen for
peptides which interact with a known target which can be a protein
or polypeptide, such as a ligand or receptor, a biological or
synthetic macromolecule, or organic or inorganic substances.
Techniques for creating and screening such random peptide display
libraries are known in the art (Ladner et al., U.S. Pat. No.
5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al.,
U.S. Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698)
and random peptide display libraries and kits for screening such
libraries are available commercially, for instance from Clontech
(Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New
England Biolabs, Inc. (Beverly, Mass.) and Pharmacia LKB
Biotechnology Inc. (Piscataway, N.J.). Random peptide display
libraries can be screened using the soluble mouse Zcytor16
monomeric receptor or soluble mouse Zcytor16 homodimeric,
heterodimeric or multimeric polypeptide sequences disclosed herein
to identify proteins which bind to mouse Zcytor16-comprising
receptor polypeptides. These "binding polypeptides," which interact
with soluble mouse Zcytor16-comprising receptor polypeptides, can
be used for tagging cells, for example, those in which Zcytor16 is
expressed; for isolating homolog polypeptides by affinity
purification; they can be directly or indirectly conjugated to
drugs, toxins, radionuclides and the like. These binding
polypeptides can also be used in analytical methods such as for
screening expression libraries and neutralizing activity, e.g., for
blocking interaction between IL-TIF ligand and receptor, or viral
binding to a receptor. The binding polypeptides can also be used
for diagnostic assays for determining circulating levels of soluble
mouse Zcytor16-comprising receptor polypeptides; for detecting or
quantitating soluble or non-soluble Zcytor16-comprising receptors
as marker of underlying pathology or disease. These binding
polypeptides can also act as "antagonists" to block soluble or
membrane-bound mouse Zcytor16 monomeric receptor or mouse Zcytor16
homodimeric, heterodimeric or multimeric polypeptide binding (e.g.
to ligand) and signal transduction in vitro and in vivo. Again,
these binding polypeptides serve as anti-mouse Zcytor16 monomeric
receptor or anti-mouse Zcytor16 homodimeric, heterodimeric or
multimeric polypeptides and are useful for inhibiting IL-TIF
activity, as well as receptor activity or protein-binding.
Antibodies raised to the natural receptor complexes of the present
invention may be preferred embodiments, as they may act more
specifically against the IL-TIF, or more potently than antibodies
raised to only one subunit. Moreover, the antagonistic and binding
activity of the antibodies of the present invention can be assayed
in the IL-TIF proliferation, signal trap, luciferase or binding
assays in the presence of IL-TIF and Zcytor16-comprising soluble
receptors, and other biological or biochemical assays described
herein.
[0278] Antibodies to monomeric mouse Zcytor16 receptor or mouse
Zcytor16 homodimeric, heterodimeric or multimeric mouse
Zcytor16-containing receptors may be used for tagging cells that
express Zcytor16 receptors; for isolating soluble
Zcytor16-comprising receptor polypeptides by affinity purification;
for diagnostic assays for determining circulating levels of soluble
Zcytor16-comprising receptor polypeptides; for detecting or
quantitating soluble Zcytor16-comprising receptors as marker of
underlying pathology or disease; in analytical methods employing
FACS; for screening expression libraries; for generating
anti-idiotypic antibodies that can act as IL-TIF agonists; and as
neutralizing antibodies or as antagonists to block Zcytor16
receptor function, or to block IL-TIF activity in vitro and in
vivo. Suitable direct tags or labels include radionuclides,
enzymes, substrates, cofactors, biotin, inhibitors, fluorescent
markers, chemiluminescent markers, magnetic particles and the like;
indirect tags or labels may feature use of biotin-avidin or other
complement/anti-complement pairs as intermediates. Antibodies
herein may also be directly or indirectly conjugated to drugs,
toxins, radionuclides and the like, and these conjugates used for
in vivo diagnostic or therapeutic applications. Moreover,
antibodies to soluble mouse Zcytor16-comprising receptor
polypeptides, or fragments thereof may be used in vitro to detect
denatured or non-denatured mouse or orthologous Zcytor16-comprising
receptor polypeptides or fragments thereof in assays, for example,
Western Blots or other assays known in the art.
[0279] Antibodies to soluble mouse Zcytor16 receptor or soluble
mouse Zcytor16 homodimeric, heterodimeric or multimeric receptor
polypeptides are useful for tagging cells that express the
corresponding receptors and assaying their expression levels, for
affinity purification, within diagnostic assays for determining
circulating levels of receptor polypeptides, analytical methods
employing fluorescence-activated cell sorting. Moreover, divalent
antibodies, and anti-idiotypic antibodies may be used as agonists
to mimic the effect of the Zcytor16 ligand, IL-TIF.
[0280] Antibodies herein can also be directly or indirectly
conjugated to drugs, toxins, radionuclides and the like, and these
conjugates used for in vivo diagnostic or therapeutic applications.
For instance, antibodies or binding polypeptides which recognize
soluble mouse Zcytor16 receptor or soluble mouse Zcytor16
homodimeric, heterodimeric or multimeric receptor polypeptides of
the present invention can be used to identify or treat tissues or
organs that express a corresponding anti-complementary molecule
(i.e., a mouse Zcytor16-comprising soluble or membrane-bound
receptor). More specifically, antibodies to soluble mouse
Zcytor16-comprising receptor polypeptides, or bioactive fragments
or portions thereof, can be coupled to detectable or cytotoxic
molecules and delivered to a mammal having cells, tissues or organs
that express a Zcytor16-comprising receptor such as
Zcytor16-expressing cancers, or certain disease states.
[0281] Suitable detectable molecules may be directly or indirectly
attached to polypeptides that bind mouse Zcytor16-comprising
receptor polypeptides, such as "binding polypeptides," (including
binding peptides disclosed above), antibodies, or bioactive
fragments or portions thereof. Suitable detectable molecules
include radionuclides, enzymes, substrates, cofactors, inhibitors,
fluorescent markers, chemiluminescent markers, magnetic particles
and the like. Suitable cytotoxic molecules may be directly or
indirectly attached to the polypeptide or antibody, and include
bacterial or plant toxins (for instance, diphtheria toxin,
Pseudomonas exotoxin, ricin, abrin and the like), as well as
therapeutic radionuclides, such as iodine-131, rhenium-188 or
yttrium-90 (either directly attached to the polypeptide or
antibody, or indirectly attached through means of a chelating
moiety, for instance). Binding polypeptides or antibodies may also
be conjugated to cytotoxic drugs, such as adriamycin. For indirect
attachment of a detectable or cytotoxic molecule, the detectable or
cytotoxic molecule can be conjugated with a member of a
complementary/anticomplementary pair, where the other member is
bound to the binding polypeptide or antibody portion. For these
purposes, biotin/streptavidin is an exemplary
complementary/anticomplementary pair.
[0282] In another embodiment, binding polypeptide-toxin fusion
proteins or antibody-toxin fusion proteins can be used for targeted
cell or tissue inhibition or ablation (for instance, to treat
cancer cells or tissues). Alternatively, if the binding polypeptide
has multiple functional domains (i.e., an activation domain or a
ligand binding domain, plus a targeting domain), a fusion protein
including only the targeting domain may be suitable for directing a
detectable molecule, a cytotoxic molecule or a complementary
molecule to a cell or tissue type of interest. In instances where
the fusion protein including only a single domain includes a
complementary molecule, the anti-complementary molecule can be
conjugated to a detectable or cytotoxic molecule. Such
domain-complementary molecule fusion proteins thus represent a
generic targeting vehicle for cell/tissue-specific delivery of
generic anti-complementary-detectable/cytotoxic molecule
conjugates.
[0283] In another embodiment, mouse Zcytor16 binding
polypeptide-cytokine or antibody-cytokine fusion proteins can be
used for enhancing in vivo killing of target tissues (for example,
spleen, pancreatic, blood, lymphoid, colon, and bone marrow
cancers), if the binding polypeptide-cytokine or anti-mouse
Zcytor16 receptor antibody targets the hyperproliferative cell
(See, generally, Hornick et al., Blood 89:4437-47, 1997). The
described fusion proteins enable targeting of a cytokine to a
desired site of action, thereby providing an elevated local
concentration of cytokine. Suitable anti-mouse Zcytor16 monomer,
homodimer, heterodimer or multimer antibodies target an undesirable
cell or tissue (i.e., a tumor or a leukemia), and the fused
cytokine mediates improved target cell lysis by effector cells.
Suitable cytokines for this purpose include interleukin 2 and
granulocyte-macrophage colony-stimulating factor (GM-CSF), for
instance.
[0284] Alternatively, mouse Zcytor16 receptor binding polypeptides
or antibody fusion proteins described herein can be used for
enhancing in vivo killing of target tissues by directly stimulating
a Zcytor16 receptor-modulated apoptotic pathway, resulting in cell
death of hyperproliferative cells expressing Zcytor16-comprising
receptors.
11. Production of Transgenic Mice
[0285] Mice engineered to overexpress the mouse Zcytor16 gene,
referred to as "transgenic mice," and mice that exhibit a complete
absence of mouse Zcytor16 gene function, referred to as "knockout
mice," may also be generated (Snouwaert et al., Science 257:1083,
1992; Lowell et al., Nature 366:740-42, 1993; Capecchi, M. R.,
Science 244: 1288-1292, 1989; Palmiter, R. D. et al. Annu Rev
Genet. 20: 465-499, 1986). For example, transgenic mice that
over-express mouse Zcytor16, either ubiquitously or under a
tissue-specific or tissue-restricted promoter can be used to ask
whether over-expression causes a phenotype. For example,
over-expression of a wild-type Zcytor16 polypeptide, polypeptide
fragment or a mutant thereof may alter normal cellular processes,
or repress function of the Zcytor16 ligand IL-TIF, resulting in a
phenotype that identifies a tissue in which Zcytor16 expression is
functionally relevant and may indicate a therapeutic target for the
Zcytor16, its agonists or antagonists. For example, a preferred
transgenic mouse to engineer is one that over-expresses the full
length human Zcytor16 polypeptide (SEQ ID NO:2); or more preferably
the full length mouse Zcytor16 polypeptide (SEQ ID NO:38 or SEQ ID
NO:48), or mature mouse polypeptide (Amino acids 24 to 230, or 27
to 230 of SEQ ID NO:38 or SEQ ID NO:48). Preferred tissue-specific
or tissue-restricted promoters include monocyte and
lymphoid-restricted, epithelial-specific, lung-specific,
ovary-specific and skin-restricted promoters. Moreover, such
over-expression may result in a phenotype that shows similarity
with human diseases, or that counteracts the effects of increased
or administered IL-TIF (IL-TIF adenovirus in mice suggests that
IL-TIF over-expression increases the level of neutrophils and
platelets in vivo).
[0286] Similarly, knockout mouse Zcytor16 mice can be used to
determine where Zcytor16 is absolutely required in vivo. Such
knockout animals serve as models to understand the requirements of
Zcytor16 function in humans. A transgenic mouse that is a knockout
mouse would not expresses residue 1 to 230 or 24 to 230, or 27 to
230 of SEQ ID NO:38 or SEQ ID NO:48, because they would exhibit a
complete absence of endogenous Zcytor16 gene function. The
phenotype of knockout mice is predictive of the in vivo effects of
that a Zcytor16 antagonist, such as those described herein, may
have. The murine Zcytor16 mRNA, and cDNA is isolated (SEQ ID NO:37
or SEQ ID NO:47) and can be used to isolate mouse Zcytor16 genomic
DNA, which are subsequently used to generate knockout mice. These
transgenic and knockout mice may be employed to study the Zcytor16
gene and the protein encoded thereby in an in vivo system, and can
be used as in vivo models for corresponding human or animal
diseases (such as those in commercially viable animal populations).
The mouse models of the present invention are particularly relevant
as tumor models for the study of immune function, inflammatory
disease, cancer biology and progression. Such models are useful in
the development and efficacy of therapeutic molecules used in human
cancers, immune and inflammatory diseases. For example, because
increases in human Zcytor16 expression are associated with specific
human cancers, such as ovarian cancer, both transgenic mice and
knockout mice would serve as useful animal models for cancer.
Moreover, because increases in human Zcytor16 expression are
associated with specific monocyte cells, such CD4+ T-cells and
CD19+ B-cells, both mouse Zcytor16 transgenic mice and knockout
mice would serve as useful animal models for immune function,
inflammation, immune disorders, infection, anemia, hematopoietic
and other cancers. Moreover, because Zcytor16 is a receptor for
IL-TIF, use of mouse Zcytor16 transgenic and knockout micein a
mouse model employing IL-TIF (e.g., with and without IL-TIF) cans
serve as an additional animal model to study the development and
efficacy of therapeutic molecules used in human cancers, immune and
inflammatory diseases associated with IL-TIF, as well as assess the
efficacy and usefulness of IL-TIF antagonists in vivo, such as the
zcyr16 soluble receptors discussed herein. Moreover, in a preferred
embodiment, Zcytor16 transgenic mouse can serve as an animal model
for specific tumors, particularly ovarian cancer, stomach cancer,
uterine cancer, rectal cancer, lung cancer and esophageal cancer;
and as a model for IL-TIF-induced inflammation where Zcytor16
polypeptide is an antagonist. Such mouse zcytor16 transgenic and
knowckout mouse models can also be used to assess the therapeutic
aspects of IL-TIF, chemical therapeutics, anti-IL-TIF antibodies,
anti-Zcytor16 antibodies, or Zcytor16 soluble receptors therein.
Moreover, transgenic mice expression of mouse Zcytor16 antisense
polynucleotides or ribozymes directed against mouse Zcytor16,
described herein, can be used analogously to transgenic mice
described above.
[0287] Transgenic mice can be engineered to over-express the mouse
Zcytor16 gene in all tissues or under the control of a
tissue-specific or tissue-preferred regulatory element. These
over-producers of mouse Zcytor16 can be used to characterize the
phenotype that results from over-expression, and the transgenic
animals can serve as models for human disease caused by excess
mouse Zcytor16. Transgenic mice that over-express mouse Zcytor16
also provide model bioreactors for production of mouse Zcytor16,
such as soluble mouse Zcytor16, in the milk or blood of larger
animals. Methods for producing transgenic mice are well-known to
those of skill in the art (see, for example, Jacob, "Expression and
Knockout of Interferons in Transgenic Mice," in Overexpression and
Knockout of Cytokines in Transgenic Mice, Jacob (ed.), pages
111-124 (Academic Press, Ltd. 1994), Monastersky and Robl (eds.),
Strategies in Transgenic Animal Science (ASM Press 1995), and Abbud
and Nilson, "Recombinant Protein Expression in Transgenic Mice," in
Gene Expression Systems: Using Nature for the Art of Expression,
Fernandez and Hoeffler (eds.), pages 367-397 (Academic Press, Inc.
1999)).
[0288] For example, a method for producing a transgenic mouse that
expresses a mouse Zcytor16 gene can begin with adult, fertile males
(studs) (B6C3f1, 2-8 months of age (Taconic Farms, Germantown,
N.Y.)), vasectomized males (duds) (B6D2f1, 2-8 months, (Taconic
Farms)), prepubescent fertile females (donors) (B6C3f1, 4-5 weeks,
(Taconic Farms)) and adult fertile females (recipients) (B6D2f1,
2-4 months, (Taconic Farms)). The donors are acclimated for one
week and then injected with approximately 8 IU/mouse of Pregnant
Mare's Serum gonadotrophin (Sigma Chemical Company; St. Louis, Mo.)
I. P., and 46-47 hours later, 8 IU/mouse of human Chorionic
Gonadotropin (hCG (Sigma)) I. P. to induce superovulation. Donors
are mated with studs subsequent to hormone injections. Ovulation
generally occurs within 13 hours of hCG injection. Copulation is
confirmed by the presence of a vaginal plug the morning following
mating.
[0289] Fertilized eggs are collected under a surgical scope. The
oviducts are collected and eggs are released into urinanalysis
slides containing hyaluronidase (Sigma). Eggs are washed once in
hyaluronidase, and twice in Whitten's W640 medium (described, for
example, by Menino and O'Claray, Biol. Reprod. 77:159 (1986), and
Dienhart and Downs, Zygote 4:129 (1996)) that has been incubated
with 5% CO.sub.2, 5% O.sub.2, and 90% N.sub.2 at 37.degree. C. The
eggs are then stored in a 37.degree. C./5% CO.sub.2 incubator until
microinjection.
[0290] Ten to twenty micrograms of plasmid DNA containing a mouse
Zcytor16 encoding sequence is linearized, gel-purified, and
resuspended in 10 mM Tris-HCl (pH 7.4), 0.25 mM EDTA (pH 8.0), at a
final concentration of 5-10 nanograms per microliter for
microinjection. For example, the mouse Zcytor16 encoding sequences
can encode a polypeptide comprising the amino acid sequence of SEQ
ID NO:38 or SEQ ID NO:48.
[0291] Plasmid DNA is microinjected into harvested eggs contained
in a drop of W640 medium overlaid by warm, CO.sub.2-equilibrated
mineral oil. The DNA is drawn into an injection needle (pulled from
a 0.75 mm ID, 1 mm OD borosilicate glass capillary), and injected
into individual eggs. Each egg is penetrated with the injection
needle, into one or both of the haploid pronuclei.
[0292] Picoliters of DNA are injected into the pronuclei, and the
injection needle withdrawn without coming into contact with the
nucleoli. The procedure is repeated until all the eggs are
injected. Successfully microinjected eggs are transferred into an
organ tissue-culture dish with pre-gassed W640 medium for storage
overnight in a 37.degree. C./5% CO.sub.2 incubator.
[0293] The following day, two-cell embryos are transferred into
pseudopregnant recipients. The recipients are identified by the
presence of copulation plugs, after copulating with vasectomized
duds. Recipients are anesthetized and shaved on the dorsal left
side and transferred to a surgical microscope. A small incision is
made in the skin and through the muscle wall in the middle of the
abdominal area outlined by the ribcage, the saddle, and the hind
leg, midway between knee and spleen. The reproductive organs are
exteriorized onto a small surgical drape. The fat pad is stretched
out over the surgical drape, and a baby serrefine (Roboz,
Rockville, Md.) is attached to the fat pad and left hanging over
the back of the mouse, preventing the organs from sliding back
in.
[0294] With a fine transfer pipette containing mineral oil followed
by alternating W640 and air bubbles, 12-17 healthy two-cell embryos
from the previous day's injection are transferred into the
recipient. The swollen ampulla is located and holding the oviduct
between the ampulla and the bursa, a nick in the oviduct is made
with a 28 g needle close to the bursa, making sure not to tear the
ampulla or the bursa.
[0295] The pipette is transferred into the nick in the oviduct, and
the embryos are blown in, allowing the first air bubble to escape
the pipette. The fat pad is gently pushed into the peritoneum, and
the reproductive organs allowed to slide in. The peritoneal wall is
closed with one suture and the skin closed with a wound clip. The
mice recuperate on a 37.degree. C. slide warmer for a minimum of
four hours.
[0296] The recipients are returned to cages in pairs, and allowed
19-21 days gestation. After birth, 19-21 days postpartum is allowed
before weaning. The weanlings are sexed and placed into separate
sex cages, and a 0.5 cm biopsy (used for genotyping) is snipped off
the tail with clean scissors.
[0297] Genomic DNA is prepared from the tail snips using, for
example, a QIAGEN DNEASY kit following the manufacturer's
instructions. Genomic DNA is analyzed by PCR using primers designed
to amplify a mouse Zcytor16 gene or a selectable marker gene that
was introduced in the same plasmid. After animals are confirmed to
be transgenic, they are back-crossed into an inbred strain by
placing a transgenic female with a wild-type male, or a transgenic
male with one or two wild-type female(s). As pups are born and
weaned, the sexes are separated, and their tails snipped for
genotyping.
[0298] To check for expression of a transgene in a live animal, a
partial hepatectomy is performed. A surgical prep is made of the
upper abdomen directly below the zyphoid process. Using sterile
technique, a small 1.5-2 cm incision is made below the sternum and
the left lateral lobe of the liver exteriorized. Using 4-0 silk, a
tie is made around the lower lobe securing it outside the body
cavity. An atraumatic clamp is used to hold the tie while a second
loop of absorbable Dexon (American Cyanamid; Wayne, N.J.) is placed
proximal to the first tie. A distal cut is made from the Dexon tie
and approximately 100 mg of the excised liver tissue is placed in a
sterile petri dish. The excised liver section is transferred to a
14 ml polypropylene round bottom tube and snap frozen in liquid
nitrogen and then stored on dry ice. The surgical site is closed
with suture and wound clips, and the animal's cage placed on a
37.degree. C. heating pad for 24 hours post operatively. The animal
is checked daily post operatively and the wound clips removed 7-10
days after surgery. The expression level of mouse Zcytor16 mRNA is
examined for each transgenic mouse using an RNA solution
hybridization assay or polymerase chain reaction.
[0299] In addition to producing transgenic mice that over-express
mouse Zcytor16, it is useful to engineer transgenic mice with
either abnormally low or no expression of the gene. Such transgenic
mice provide useful models for diseases associated with a lack of
mouse Zcytor16. As discussed above, mouse Zcytor16 gene expression
can be inhibited using anti-sense genes, ribozyme genes, or
external guide sequence genes. To produce transgenic mice that
under-express the mouse Zcytor16 gene, such inhibitory sequences
are targeted to mouse Zcytor16 mRNA. Methods for producing
transgenic mice that have abnormally low expression of a particular
gene are known to those in the art (see, for example, Wu et al.,
"Gene Underexpression in Cultured Cells and Animals by Antisense
DNA and RNA Strategies," in Methods in Gene Biotechnology, pages
205-224 (CRC Press 1997)).
[0300] An alternative approach to producing transgenic mice that
have little or no mouse Zcytor16 gene expression is to generate
mice having at least one normal mouse Zcytor16 allele replaced by a
nonfunctional mouse Zcytor16 gene. One method of designing a
nonfunctional mouse Zcytor16 gene is to insert another gene, such
as a selectable marker gene, within a nucleic acid molecule that
encodes mouse Zcytor16. Standard methods for producing these
so-called "knockout mice" are known to those skilled in the art
(see, for example, Jacob, "Expression and Knockout of Interferons
in Transgenic Mice," in Overexpression and Knockout of Cytokines in
Transgenic Mice, Jacob (ed.), pages 111-124 (Academic Press, Ltd.
1994), and Wu et al., "New Strategies for Gene Knockout," in
Methods in Gene Biotechnology, pages 339-365 (CRC Press 1997)).
12. Therapeutic Uses of Polypeptides Having Zcytor16 Activity
[0301] Amino acid sequences having mouse Zcytor16 activity can be
used to modulate the immune system by binding Zcytor16 ligand, and
thus, preventing the binding of Zcytor16 ligand with endogenous
Zcytor16 receptor. Zcytor16 antagonists, such as anti-mouse
Zcytor16 antibodies, can also be used to modulate the immune system
by inhibiting the binding of Zcytor16 ligand with the endogenous
Zcytor16 receptor. Accordingly, the present invention includes the
use of proteins, polypeptides, and peptides having mouse Zcytor16
activity (such as mouse Zcytor16 polypeptides, mouse Zcytor16
analogs (e.g., anti-mouse Zcytor16 anti-idiotype antibodies), and
mouse Zcytor16 fusion proteins) to a subject which lacks an
adequate amount of this polypeptide, or which produces an excess of
Zcytor16 ligand. Mouse Zcytor16 antagonists (e.g., anti-mouse
Zcytor16 antibodies) can be also used to treat a subject that
produces an excess of either Zcytor16 ligand or Zcytor16 receptor.
Suitable subjects include mammals, such as humans. In addition,
human Zcytor16 and IL-TIF activity described herein emphasizes the
usefulness of using mouse Zcytor16 in animal models, such as a
mouse model described above for studying human inflammation,
inflammatory diseases, immune function and diseases and cancer, or
assessing therapeutic aspects of IL-TIF, chemical therapeutics,
anti-IL-TIF antibodies, anti-Zcytor16 antibodies, or Zcytor16
soluble receptors therein.
[0302] Moreover, we have shown that the human Zcytor16 receptor
binds a ligand called T-cell inducible Factor (IL-TIF) (SEQ ID
NO:15); Dumoutier, L. et al., Proc. Nat'l. Acad. Sci.
97:10144-10149, 2000; mouse IL-TIF sequence (SEQ ID NO:41) is shown
in Dumontier et al., J. Immunol. 164:1814-1819, 2000). Moreover,
commonly owned zcytor11 (U.S. Pat. No. 5,965,704) and CRF2-4
receptor also bind IL-TIF (See, WIPO publication WO 00/24758;
Dumontier et al., J. Immunol. 164:1814-1819, 2000; Spencer, S D et
al., J. Exp. Med. 187:571-578, 1998; Gibbs, V C and Pennica Gene
186:97-101, 1997 (CRF2-4 cDNA); Xie, M H et al., J. Biol. Chem.
275: 31335-31339, 2000; and Kotenko, S V et al., J. Biol. Chem.
manuscript in press M007837200). Moreover, IL-10.beta. receptor may
be involved as a receptor for IL-TIF, and it is believed to be
synonymous with CRF2-4 (Dumoutier, L. et al., Proc. Nat'l. Acad.
Sci. 97:10144-10149, 2000; Liu Y et al, J. Immunol. 152; 1821-1829,
1994 (IL-10R cDNA). Within preferred embodiments, the soluble
receptor form of mouse Zcytor16, residues 24-230 of SEQ ID NO:38 or
SEQ ID NO:48 is a monomer, homodimer, heterodimer, or multimer that
antagonizes the effects of IL-TIF in vivo. Antibodies and binding
polypeptides to such mouse Zcytor16 monomer, homodimer,
heterodimer, or multimers also serve as antagonists of Zcytor16
activity.
[0303] IL-TIF has been shown to be induced in the presence of IL-9,
and is suspected to be involved in promoting Th1-type immune
responses, and inflammation. IL-9 stimulates proliferation,
activation, differentiation and/or induction of immune function in
a variety of ways and is implicated in asthma, lung mastocytosis,
and other diseases, as well as activates STAT pathways. Antagonists
of IL-TIF or IL-9 function can have beneficial use against such
human diseases. The present invention provides such novel
antagonists of IL-TIF.
[0304] IL-TIF has been show to be involved in up-regulate the
production of acute phase reactants, such as serum amyloid A (SAA),
.alpha.1-antichymotrypsin, and haptoglobin, and that IL-TIF
expression is increased upon injection of lipopolysaccharide (LPS)
in vivo suggesting that IL-TIF is involved in inflammatory response
(Dumoutier, L. et al., Proc. Nat'l. Acad. Sci. 97:10144-10149,
2000). Production of acute phase proteins, such as SAA, is
considered s short-term survival mechanism where inflammation is
beneficial; however, maintenance of acute phase proteins for longer
periods contributes to chronic inflammation and can be harmful to
human health. For review, see Uhlar, C M and Whitehead, A S, Eur.
J. Biochem. 265:501-523, 1999, and Baumann H. and Gauldie, J.
Immunology Today 15:74-80, 1994. Moreover, the acute phase protein
SAA is implicated in the pathogenesis of several chronic
inflammatory diseases, is implicated in atherosclerosis and
rheumatoid arthritis, and is the precursor to the amyloid A protein
deposited in amyloidosis (Uhlar, C M and Whitehead, supra.). Thus,
as IL-TIF acts as a pro-inflammatory molecule and induces
production of SAA, antagonists would be useful in treating
inflammatory disease and other diseases associated with acute phase
response proteins induced by IL-TIF. Such antagonists are provided
by the present invention. For example, method of reducing
IL-TIF-induced or IL-9 induced inflammation comprises administering
to a mammal with inflammation an amount of a composition of soluble
mouse Zcytor16-comprising receptor sufficient to reduce
inflammation. Moreover, a method of suppressing an inflammatory
response in a mammal with inflammation can comprise: (1)
determining a level of serum amyloid A protein; (2) administering a
composition comprising a soluble mouse Zcytor16 cytokine receptor
polypeptide as described herein in an acceptable pharmaceutical
vehicle; (3) determining a post administration level of serum
amyloid A protein; (4) comparing the level of serum amyloid A
protein in step (1) to the level of serum amyloid A protein in step
(3), wherein a lack of increase or a decrease in serum amyloid A
protein level is indicative of suppressing an inflammatory
response.
[0305] The receptors of the present invention include at least one
mouse Zcytor16 receptor subunit. A second receptor polypeptide
included in the heterodimeric soluble receptor belongs to the
receptor subfamily that includes Interleukin-10 receptor, the
interferons (e.g., interferon-gamma alpha and beta chains and the
interferon-alpha/beta receptor alpha and beta chains), zcytor7,
zcytor11, and CRF2-4. A second soluble receptor polypeptide
included in a heterodimeric soluble receptor can also include a
zcytor11 soluble receptor subunit, disclosed in the commonly owned
U.S. Pat. No. 5,965,704; an IL-10R subunit, such as IL-10R.alpha.;
or a zcytor7 soluble receptor subunit, disclosed in the commonly
owned U.S. Pat. No. 5,945,511. The zcytor11 receptor in conjunction
with CRF2-4 and IL-10 Receptor was shown to signal JAK-STAT pathway
in response to IL-TIF (Xie et al., supra.; Kotenko et al., supra.).
According to the present invention, in addition to a monomeric or
homodimeric mouse Zcytor16 receptor polypeptide, a heterodimeric
soluble mouse Zcytor16 receptor, as exemplified by an embodiment
comprising a soluble mouse Zcytor16 receptor+soluble CRF2-4
receptor heterodimer (mouse Zcytor16/CRF2-4), can act as an
antagonist of the IL-TIF. Other embodiments include soluble
heterodimers comprising mouse Zcytor16/IL-10R, mouse
Zcytor16/IL-9R, mouse Zcytor16/zcytor11, mouse Zcytor16/zcytor7,
and other class II receptor subunits, as well as multimeric
receptors including but not limited to mouse
Zcytor16/CRF2-4/zcytor11 or mouse Zcytor16/CRF2-4/IL-10R.
[0306] Analysis of the tissue distribution of the mRNA
corresponding mouse Zcytor16 cDNA was similar to human Zcytor16
tissue distribution (U.S. patent application Ser. No. 09/728,911),
and showed that mRNA was expressed in placenta and spleen, and the
ligand to which Zcytor16 binds (IL-TIF) is implicated in inducing
inflammatory response including induction of the acute-phase
response (Dumoutier, L. et al., Proc. Nat'l. Acad. Sci.
97:10144-10149, 2000). Thus, particular embodiments of the present
invention are directed toward use of soluble mouse Zcytor16
heterodimers as antagonists in inflammatory and immune diseases or
conditions such as pancreatitis, type I diabetes (IDDM), pancreatic
cancer, pancreatitis, Graves Disease, inflammatory bowel disease
(IBD), Crohn's Disease, colon and intestinal cancer,
diverticulosis, autoimmune disease, sepsis, organ or bone marrow
transplant; inflammation due to trauma, surgery or infection;
amyloidosis; splenomegaly; graft versus host disease; and where
inhibition of inflammation, immune suppression, reduction of
proliferation of hematopoietic, immune, inflammatory or lymphoid
cells, macrophages, T-cells (including Th1 and Th2 cells),
suppression of immune response to a pathogen or antigen, or other
instances where inhibition of IL-TIF or IL-9 cytokine production is
desired.
[0307] Moreover, antibodies or binding polypeptides that bind mouse
Zcytor16 polypeptides, monomers, homodimers, heterodimers and
multimers described herein and/or mouse Zcytor16 polypeptides,
monomers, homodimers, heterodimers and multimers themselves are
useful to:
[0308] 1) Antagonize or block signaling via the IL-TIF receptors in
the treatment of acute inflammation, inflammation as a result of
trauma, tissue injury, surgery, sepsis or infection, and chronic
inflammatory diseases such as asthma, inflammatory bowel disease
(IBD), chronic colitis, splenomegaly, rheumatoid arthritis,
recurrent acute inflammatory episodes (e.g., tuberculosis), and
treatment of amyloidosis, and atherosclerosis, Castleman's Disease,
asthma, and other diseases associated with the induction of
acute-phase response.
[0309] 2) Antagonize or block signaling via the IL-TIF receptors in
the treatment of autoimmune diseases such as IDDM, multiple
sclerosis (MS), systemic Lupus erythematosus (SLE), myasthenia
gravis, rheumatoid arthritis, and IBD to prevent or inhibit
signaling in immune cells (e.g. lymphocytes, monocytes, leukocytes)
via mouse Zcytor16 (Hughes C et al., J. Immunol 153: 3319-3325,
1994). Alternatively antibodies, such as monoclonal antibodies
(MAb) to mouse Zcytor16-comprising receptors, can also be used as
an antagonist to deplete unwanted immune cells to treat autoimmune
disease. Asthma, allergy and other atopic disease may be treated
with an MAb against, for example, soluble mouse Zcytor16 soluble
receptors or mouse Zcytor16/CRF2-4 heterodimers, to inhibit the
immune response or to deplete offending cells. Blocking or
inhibiting signaling via Zcytor16, using the polypeptides and
antibodies of the present invention, may also benefit diseases of
the pancreas, kidney, pituitary and neuronal cells. IDDM, NIDDM,
pancreatitis, and pancreatic carcinoma may benefit. Mouse Zcytor16
may serve as a target for MAb therapy of cancer where an
antagonizing MAb inhibits cancer growth and targets immune-mediated
killing. (Holliger P, and Hoogenboom, H: Nature Biotech. 16:
1015-1016, 1998). Mabs to soluble mouse Zcytor16 monomers,
homodimers, heterodimers and multimers may also be useful to treat
nephropathies such as glomerulosclerosis, membranous neuropathy,
amyloidosis (which also affects the kidney among other tissues),
renal arteriosclerosis, glomerulonephritis of various origins,
fibroproliferative diseases of the kidney, as well as kidney
dysfunction associated with SLE, IDDM, type II diabetes (IDDM),
renal tumors and other diseases.
[0310] 3) Agonize or initiate signaling via the IL-TIF receptors in
the treatment of autoimmune diseases such as IDDM, MS, SLE,
myasthenia gravis, rheumatoid arthritis, and IBD. Anti-soluble
mouse Zcytor16, anti-soluble mouse Zcytor16/CRF2-4 heterodimers and
multimer monoclonal antibodies may signal lymphocytes or other
immune cells to differentiate, alter proliferation, or change
production of cytokines or cell surface proteins that ameliorate
autoimmunity. Specifically, modulation of a T-helper cell response
to an alternate pattern of cytokine secretion may deviate an
autoimmune response to ameliorate disease (Smith J A et al., J.
Immunol. 160:4841-4849, 1998). Similarly, agonistic Anti-soluble
mouse Zcytor16, anti-solublemouse Zcytor16/CRF2-4 heterodimers and
multimer monoclonal antibodies may be used to signal, deplete and
deviate immune cells involved in asthma, allergy and atopoic
disease. Signaling via mouse Zcytor16 may also benefit diseases of
the pancreas, kidney, pituitary and neuronal cells. IDDM, NIDDM,
pancreatitis, and pancreatic carcinoma may benefit. mouse Zcytor16
may serve as a target for MAb therapy of pancreatic cancer where a
signaling MAb inhibits cancer growth and targets immune-mediated
killing (Tutt, A L et al., J Immunol. 161: 3175-3185, 1998).
Similarly renal cell carcinoma may be treated with monoclonal
antibodies to mouse Zcytor16-comprising soluble receptors of the
present invention.
[0311] Soluble mouse Zcytor16 monomeric, homodimeric, heterodimeric
and multimeric polypeptides described herein can be used to
neutralize/block IL-TIF activity in the treatment of autoimmune
disease, atopic disease, NIDDM, pancreatitis and kidney dysfunction
as described above. A soluble form of mouse Zcytor16 may be used to
promote an antibody response mediated by Th cells and/or to promote
the production of IL-4 or other cytokines by lymphocytes or other
immune cells.
[0312] The soluble mouse Zcytor16-comprising receptors of the
present invention are useful as antagonists of the IL-TIF cytokine.
Such antagonistic effects can be achieved by direct neutralization
or binding of the IL-TIF. In addition to antagonistic uses, the
soluble receptors of the present invention can bind IL-TIF and act
as carrier proteins for the IL-TIF cytokine, in order to transport
the Ligand to different tissues, organs, and cells within the body.
As such, the soluble receptors of the present invention can be
fused or coupled to molecules, polypeptides or chemical moieties
that direct the soluble-receptor-Ligand complex to a specific site,
such as a tissue, specific immune cell, or tumor. For example, in
acute infection or some cancers, benefit may result from induction
of inflammation and local acute phase response proteins by the
action of IL-TIF. Thus, the soluble receptors of the present
invention can be used to specifically direct the action of the
IL-TIF. See, Cosman, D. Cytokine 5: 95-106, 1993; and
Femandez-Botran, R. Exp. Opin. Invest. Drugs 9:497-513, 2000.
[0313] Moreover, the soluble receptors of the present invention can
be used to stabilize the IL-TIF, to increase the bioavailability,
therapeutic longevity, and/or efficacy of the Ligand by stabilizing
the Ligand from degradation or clearance, or by targeting the
ligand to a site of action within the body. For example the
naturally occurring IL-6/soluble IL-6R complex stabilizes IL-6 and
can signal through the gp130 receptor. See, Cosman, D. supra., and
Femandez-Botran, R. supra. Moreover, mouse Zcytor16 may be combined
with a cognate ligand such as IL-TIF to comprise a ligand/soluble
receptor complex. Such complexes may be used to stimulate responses
from cells presenting a companion receptor subunit such as, for
example, zcytor11 or CRF2-4. The cell specificity of mouse
Zcytor16/ligand complexes may differ from that seen for the ligand
administered alone. Furthermore the complexes may have distinct
pharmacokinetic properties such as affecting half-life,
dose/response and organ or tissue specificity. ZcytoR16/IL-TIF
complexes thus may have agonist activity to enhance an immune
response or stimulate mesangial cells or to stimulate hepatic
cells. Alternatively only tissues expressing a signaling subunit
the heterodimerizes with the complex may be affected analogous to
the response to IL6/IL6R complexes (Hirota H. et al., Proc. Nat'l.
Acad. Sci. 92:4862-4866, 1995; Hirano, T. in Thomason, A. (Ed.)
"The Cytokine Handbook", 3.sup.rd Ed., p. 208-209). Soluble
receptor/cytokine complexes for IL12 and CNTF display similar
activities.
[0314] Mouse Zcytor16 homodimeric, heterodimeric and multimeric
receptor polypeptides may also be used within diagnostic systems
for the detection of circulating levels of IL-TIF ligand, and in
the detection of IL-TIF associated with acute phase inflammatory
response. Within a related embodiment, antibodies or other agents
that bind to mouse Zcytor16 soluble receptors of the present
invention can be used to detect circulating receptor polypeptides;
conversely, mouse Zcytor16 soluble receptors themselves can be used
to detect circulating or locally-acting IL-TIF polypeptides.
Elevated or depressed levels of ligand or receptor polypeptides may
be indicative of pathological conditions, including inflammation or
cancer. IL-TIF is known to induce associated acute phase
inflammatory response. Moreover, detection of acute phase proteins
or molecules such as IL-TIF can be indicative of a chronic
inflammatory condition in certain disease states (e.g., rheumatoid
arthritis). Detection of such conditions serves to aid in disease
diagnosis as well as help a physician in choosing proper
therapy.
[0315] Moreover, soluble mouse Zcytor16 receptor polypeptides of
the present invention can be used as a "ligand sink," i.e.,
antagonist, to bind ligand in vivo or in vitro in therapeutic or
other applications where the presence of the ligand is not desired.
For example, in chronic inflammatory conditions or cancers that are
expressing large amounts of bioactive IL-TIF, soluble mouse
Zcytor16 receptor or soluble mouse Zcytor16 heterodimeric and
multimeric receptor polypeptides, such as soluble mouse
Zcytor16/CRF2-4 can be used as a direct antagonist of the ligand in
vivo, and may aid in reducing progression and symptoms associated
with the disease, and can be used in conjunction with other
therapies (e.g., steroid or chemotherapy) to enhance the effect of
the therapy in reducing progression and symptoms, and preventing
relapse. Moreover, soluble mouse Zcytor16 receptor polypeptides can
be used to slow the progression of cancers that over-express
Zcytor16 receptors, by binding ligand in vivo that could otherwise
enhance proliferation of those cancers.
[0316] Moreover, soluble mouse Zcytor16 receptor polypeptides of
the present invention can be used in vivo or in diagnostic
applications to detect IL-TIF-expressing inflammation or cancers in
vivo or in tissue samples. For example, the soluble mouse Zcytor16
receptors of the present invention can be conjugated to a
radio-label or fluorescent label as described herein, and used to
detect the presence of the IL-TIF in a tissue sample using an in
vitro ligand-receptor type binding assay, or fluorescent imaging
assay. Moreover, radiolabeled soluble mouse Zcytor16 receptors of
the present invention could be administered in vivo to detect
Ligand-expressing solid tumors through a radio-imaging method known
in the art.
[0317] Generally, the dosage of administered mouse Zcytor16 (or
mouse Zcytor16 analog or fusion protein) will vary depending upon
such factors as the patient's age, weight, height, sex, general
medical condition and previous medical history. Typically, it is
desirable to provide the recipient with a dosage of mouse Zcytor16
polypeptide which is in the range of from about 1 pg/kg to 10 mg/kg
(amount of agent/body weight of patient), although a lower or
higher dosage also may be administered as circumstances
dictate.
[0318] Administration of a mouse Zcytor16 polypeptide to a subject
can be intravenous, intraarterial, intraperitoneal, intramuscular,
subcutaneous, intrapleural, intrathecal, by perfusion through a
regional catheter, or by direct intralesional injection. When
administering therapeutic proteins by injection, the administration
may be by continuous infusion or by single or multiple boluses.
[0319] Additional routes of administration include oral,
mucosal-membrane, pulmonary, and transcutaneous. Oral delivery is
suitable for polyester microspheres, zein microspheres, proteinoid
microspheres, polycyanoacrylate microspheres, and lipid-based
systems (see, for example, DiBase and Morrel, "Oral Delivery of
Microencapsulated Proteins," in Protein Delivery: Physical Systems,
Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)). The
feasibility of an intranasal delivery is exemplified by such a mode
of insulin administration (see, for example, Hinchcliffe and Illum,
Adv. Drug Deliv. Rev. 35:199 (1999)). Dry or liquid particles
comprising mouse Zcytor16 can be prepared and inhaled with the aid
of dry-powder dispersers, liquid aerosol generators, or nebulizers
(e.g., Pettit and Gombotz, TIBTECH 16:343 (1998); Patton et al.,
Adv. Drug Deliv. Rev. 35:235 (1999)). This approach is illustrated
by the AERX diabetes management system, which is a hand-held
electronic inhaler that delivers aerosolized insulin into the
lungs. Studies have shown that proteins as large as 48,000 kDa have
been delivered across skin at therapeutic concentrations with the
aid of low-frequency ultrasound, which illustrates the feasibility
of trascutaneous administration (Mitragotri et al., Science 269:850
(1995)). Transdermal delivery using electroporation provides
another means to administer a molecule having mouse Zcytor16
binding activity (Potts et al., Pharm. Biotechnol. 10:213
(1997)).
[0320] A pharmaceutical composition comprising a protein,
polypeptide, or peptide having mouse Zcytor16 binding activity can
be formulated according to known methods to prepare
pharmaceutically useful compositions, whereby the therapeutic
proteins are combined in a mixture with a pharmaceutically
acceptable carrier. A composition is said to be a "pharmaceutically
acceptable carrier" if its administration can be tolerated by a
recipient patient. Sterile phosphate-buffered saline is one example
of a pharmaceutically acceptable carrier. Other suitable carriers
are well-known to those in the art. See, for example, Gennaro
(ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack
Publishing Company 1995).
[0321] For purposes of therapy, molecules having mouse Zcytor16
binding activity and a pharmaceutically acceptable carrier are
administered to a patient in a therapeutically effective amount. A
combination of a protein, polypeptide, or peptide having mouse
Zcytor16 binding activity and a pharmaceutically acceptable carrier
is said to be administered in a "therapeutically effective amount"
if the amount administered is physiologically significant. An agent
is physiologically significant if its presence results in a
detectable change in the physiology of a recipient patient. For
example, an agent used to treat inflammation is physiologically
significant if its presence alleviates the inflammatory
response.
[0322] A pharmaceutical composition comprising mouse Zcytor16 (or
mouse Zcytor16 analog or fusion protein) can be furnished in liquid
form, in an aerosol, or in solid form. Liquid forms, are
illustrated by injectable solutions and oral suspensions. Exemplary
solid forms include capsules, tablets, and controlled-release
forms. The latter form is illustrated by miniosmotic pumps and
implants (Bremer et al., Pharm. Biotechnol. 10:239 (1997); Ranade,
"Implants in Drug Delivery," in Drug Delivery Systems, Ranade and
Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer et al.,
"Protein Delivery with Infusion Pumps," in Protein Delivery:
Physical Systems, Sanders and Hendren (eds.), pages 239-254 (Plenum
Press 1997); Yewey et al., "Delivery of Proteins from a Controlled
Release Injectable Implant," in Protein Delivery: Physical Systems,
Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)).
[0323] Liposomes provide one means to deliver therapeutic
polypeptides to a subject intravenously, intraperitoneally,
intrathecally, intramuscularly, subcutaneously, or via oral
administration, inhalation, or intranasal administration. Liposomes
are microscopic vesicles that consist of one or more lipid bilayers
surrounding aqueous compartments (see, generally, Bakker-Woudenberg
et al., Eur. J. Clin. Microbiol. Infect. Dis. 12 (Suppl. 1):S61
(1993), Kim, Drugs 46:618 (1993), and Ranade, "Site-Specific Drug
Delivery Using Liposomes as Carriers," in Drug Delivery Systems,
Ranade and Hollinger (eds.), pages 3-24 (CRC Press 1995)).
Liposomes are similar in composition to cellular membranes and as a
result, liposomes can be administered safely and are biodegradable.
Depending on the method of preparation, liposomes may be
unilamellar or multilamellar, and liposomes can vary in size with
diameters ranging from 0.02 .mu.m to greater than 10 .mu.m. A
variety of agents can be encapsulated in liposomes: hydrophobic
agents partition in the bilayers and hydrophilic agents partition
within the inner aqueous space(s) (see, for example, Machy et al.,
Liposomes In Cell Biology And Pharmacology (John Libbey 1987), and
Ostro et al., American J. Hosp. Pharm. 46:1576 (1989)). Moreover,
it is possible to control the therapeutic availability of the
encapsulated agent by varying liposome size, the number of
bilayers, lipid composition, as well as the charge and surface
characteristics of the liposomes.
[0324] Liposomes can adsorb to virtually any type of cell and then
slowly release the encapsulated agent. Alternatively, an absorbed
liposome may be endocytosed by cells that are phagocytic.
Endocytosis is followed by intralysosomal degradation of liposomal
lipids and release of the encapsulated agents (Scherphof et al.,
Ann. N.Y. Acad. Sci. 446:368 (1985)). After intravenous
administration, small liposomes (0.1 to 1.0 .mu.m) are typically
taken up by cells of the reticuloendothelial system, located
principally in the liver and spleen, whereas liposomes larger than
3.0 .mu.m are deposited in the lung. This preferential uptake of
smaller liposomes by the cells of the reticuloendothelial system
has been used to deliver chemotherapeutic agents to macrophages and
to tumors of the liver.
[0325] The reticuloendothelial system can be circumvented by
several methods including saturation with large doses of liposome
particles, or selective macrophage inactivation by pharmacological
means (Claassen et al., Biochim. Biophys. Acta 802:428 (1984)). In
addition, incorporation of glycolipid- or polyethelene
glycol-derivatized phospholipids into liposome membranes has been
shown to result in a significantly reduced uptake by the
reticuloendothelial system (Allen et al., Biochim. Biophys. Acta
1068:133 (1991); Allen et al., Biochim. Biophys. Acta 1150:9
(1993)).
[0326] Liposomes can also be prepared to target particular cells or
organs by varying phospholipid composition or by inserting
receptors or ligands into the liposomes. For example, liposomes,
prepared with a high content of a nonionic surfactant, have been
used to target the liver (Hayakawa et al., Japanese Patent
04-244,018; Kato et al., Biol. Pharm. Bull. 16:960 (1993)). These
formulations were prepared by mixing soybean phospatidylcholine,
.alpha.-tocopherol, and ethoxylated hydrogenated castor oil
(HCO-60) in methanol, concentrating the mixture under vacuum, and
then reconstituting the mixture with water. A liposomal formulation
of dipalmitoylphosphatidylcholine (DPPC) with a soybean-derived
sterylglucoside mixture (SG) and cholesterol (Ch) has also been
shown to target the liver (Shimizu et al., Biol. Pharm. Bull.
20:881 (1997)).
[0327] Alternatively, various targeting ligands can be bound to the
surface of the liposome, such as antibodies, antibody fragments,
carbohydrates, vitamins, and transport proteins. For example,
liposomes can be modified with branched type galactosyllipid
derivatives to target asialoglycoprotein (galactose) receptors,
which are exclusively expressed on the surface of liver cells (Kato
and Sugiyama, Crit. Rev. Ther. Drug Carrier Syst. 14:287 (1997);
Murahashi et al., Biol. Pharm. Bull. 20:259 (1997)). Similarly, Wu
et al., Hepatology 27:772 (1998), have shown that labeling
liposomes with asialofetuin led to a shortened liposome plasma
half-life and greatly enhanced uptake of asialofetuin-labeled
liposome by hepatocytes. On the other hand, hepatic accumulation of
liposomes comprising branched type galactosyllipid derivatives can
be inhibited by preinjection of asialofetuin (Murahashi et al.,
Biol. Pharm. Bull. 20:259 (1997)). Polyaconitylated human serum
albumin liposomes provide another approach for targeting liposomes
to liver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681
(1997)). Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe a
hepatocyte-directed liposome vesicle delivery system, which has
specificity for hepatobiliary receptors associated with the
specialized metabolic cells of the liver.
[0328] In a more general approach to tissue targeting, target cells
are prelabeled with biotinylated antibodies specific for a ligand
expressed by the target cell (Harasym et al., Adv. Drug Deliv. Rev.
32:99 (1998)). After plasma elimination of free antibody,
streptavidin-conjugated liposomes are administered. In another
approach, targeting antibodies are directly attached to liposomes
(Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).
[0329] Polypeptides having mouse Zcytor16 binding activity can be
encapsulated within liposomes using standard techniques of protein
microencapsulation (see, for example, Anderson et al., Infect.
Immun. 31:1099 (1981), Anderson et al., Cancer Res. 50:1853 (1990),
and Cohen et al., Biochim. Biophys. Acta 1063:95 (1991), Alving et
al. "Preparation and Use of Liposomes in Immunological Studies," in
Liposome Technology, 2nd Edition, Vol. III, Gregoriadis (ed.), page
317 (CRC Press 1993), Wassef et al., Meth. Enzymol. 149:124
(1987)). As noted above, therapeutically useful liposomes may
contain a variety of components. For example, liposomes may
comprise lipid derivatives of poly(ethylene glycol) (Allen et al.,
Biochim. Biophys. Acta 1150:9 (1993)).
[0330] Degradable polymer microspheres have been designed to
maintain high systemic levels of therapeutic proteins. Microspheres
are prepared from degradable polymers such as
poly(lactide-co-glycolide) (PLG), polyanhydrides, poly (ortho
esters), nonbiodegradable ethylvinyl acetate polymers, in which
proteins are entrapped in the polymer (Gombotz and Pettit,
Bioconjugate Chem. 6:332 (1995); Ranade, "Role of Polymers in Drug
Delivery," in Drug Delivery Systems, Ranade and Hollinger (eds.),
pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, "Degradable
Controlled Release Systems Useful for Protein Delivery," in Protein
Delivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92
(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney
and Burke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin.
Chem. Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated
nanospheres can also provide carriers for intravenous
administration of therapeutic proteins (see, for example, Gref et
al., Pharm. Biotechnol. 10:167 (1997)).
[0331] The present invention also contemplates chemically modified
polypeptides having binding mouse Zcytor16 activity such as mouse
Zcytor16 monomeric, homodimeric, heterodimeric or multimeric
soluble receptors, and mouse Zcytor16 antagonists, for example
anti-mouse Zcytor16 antibodies or binding polypeptides, which a
polypeptide is linked with a polymer, as discussed above.
[0332] Other dosage forms can be devised by those skilled in the
art, as shown, for example, by Ansel and Popovich, Pharmaceutical
Dosage Forms and Drug Delivery Systems, 5.sup.th Edition (Lea &
Febiger 1990), Gennaro (ed.), Remington's Pharmaceutical Sciences,
19.sup.th Edition (Mack Publishing Company 1995), and by Ranade and
Hollinger, Drug Delivery Systems (CRC Press 1996).
[0333] As an illustration, pharmaceutical compositions may be
supplied as a kit comprising a container that comprises a
polypeptide with a mouse Zcytor16 extracellular domain, e.g., mouse
Zcytor16 monomeric, homodimeric, heterodimeric or multimeric
soluble receptors, or a mouse Zcytor16 antagonist (e.g., an
antibody or antibody fragment that binds a mouse Zcytor16
polypeptide). Therapeutic polypeptides can be provided in the form
of an injectable solution for single or multiple doses, or as a
sterile powder that will be reconstituted before injection.
Alternatively, such a kit can include a dry-powder disperser,
liquid aerosol generator, or nebulizer for administration of a
therapeutic polypeptide. Such a kit may further comprise written
information on indications and usage of the pharmaceutical
composition. Moreover, such information may include a statement
that the mouse Zcytor16 composition is contraindicated in patients
with known hypersensitivity to mouse Zcytor16.
[0334] Polynucleotides and polypeptides of the present invention
will additionally find use as educational tools as a laboratory
practicum kits for courses related to genetics and molecular
biology, protein chemistry and antibody production and analysis.
Due to its unique polynucleotide and polypeptide sequence molecules
of mouse Zcytor16 can be used as standards or as "unknowns" for
testing purposes. For example, mouse Zcytor16 polynucleotides can
be used as an aid, such as, for example, to teach a student how to
prepare expression constructs for bacterial, viral, and/or
mammalian expression, including fusion constructs, wherein mouse
Zcytor16 is the gene to be expressed; for determining the
restriction endonuclease cleavage sites of the polynucleotides;
determining mRNA and DNA localization of mouse Zcytor16
polynucleotides in tissues (i.e., by Northern and Southern blotting
as well as polymerase chain reaction); and for identifying related
polynucleotides and polypeptides by nucleic acid hybridization.
[0335] Mouse Zcytor16 polypeptides can be used educationally as an
aid to teach preparation of antibodies; identifying proteins by
Western blotting; protein purification; determining the weight of
expressed mouse Zcytor16 polypeptides as a ratio to total protein
expressed; identifying peptide cleavage sites; coupling amino and
carboxyl terminal tags; amino acid sequence analysis, as well as,
but not limited to monitoring biological activities of both the
native and tagged protein (i.e., receptor binding, signal
transduction, proliferation, and differentiation) in vitro and in
vivo. mouse Zcytor16 polypeptides can also be used to teach
analytical skills such as mass spectrometry, circular dichroism to
determine conformation, especially of the four alpha helices, x-ray
crystallography to determine the three-dimensional structure in
atomic detail, nuclear magnetic resonance spectroscopy to reveal
the structure of proteins in solution. For example, a kit
containing the mouse Zcytor16 can be given to the student to
analyze. Since the amino acid sequence would be known by the
professor, the specific protein can be given to the student as a
test to determine the skills or develop the skills of the student,
the teacher would then know whether or not the student has
correctly analyzed the polypeptide. Since every polypeptide is
unique, the educational utility of mouse Zcytor16 would be unique
unto itself.
[0336] Moreover, since mouse Zcytor16 has a tissue-specific
expression and is a polypeptide with a class II cytokine receptor
structure and a distinct expression pattern, activity can be
measured using proliferation assays; luciferase and binding assays
described herein. Moreover, expression of mouse Zcytor16
polynucleotides and polypeptides in lymphoid and other tissues can
be analyzed in order to train students in the use of diagnostic and
tissue-specific identification and methods. Moreover mouse Zcytor16
polynucleotides can be used to train students on the use of
chromosomal detection and diagnostic methods. Such assays are well
known in the art, and can be used in an educational setting to
teach students about cytokine receptor proteins and examine
different properties, such as cellular effects on cells, enzyme
kinetics, varying antibody binding affinities, tissue specificity,
and the like, between mouse Zcytor16 and other cytokine receptor
polypeptides in the art.
[0337] The antibodies which bind specifically to mouse Zcytor16 can
be used as a teaching aid to instruct students how to prepare
affinity chromatography columns to purify mouse Zcytor16, cloning
and sequencing the polynucleotide that encodes an antibody and thus
as a practicum for teaching a student how to design humanized
antibodies. Moreover, antibodies which bind specifically to mouse
Zcytor16 can be used as a teaching aid for use in detection e.g.,
of activated CD91+ cells, cell sorting, or ovarian cancer tissue
using histological, and in situ methods amongst others known in the
art. The mouse Zcytor16 gene, polypeptide or antibody would then be
packaged by reagent companies and sold to universities and other
educational entities so that the students gain skill in art of
molecular biology. Because each gene and protein is unique, each
gene and protein creates unique challenges and learning experiences
for students in a lab practicum. Such educational kits containing
the mouse Zcytor16 gene, polypeptide or antibody are considered
within the scope of the present invention.
[0338] Within one aspect, the present invention provides an
isolated polypeptide, comprising at least 15 contiguous amino acid
residues of an amino acid sequence of SEQ ID NO:38 or SEQ ID NO:48
selected from the group consisting of: an amino acid sequence of
SEQ ID NO:38 or SEQ ID NO:48 selected from the group consisting of:
(a) amino acid residues 24 to 230; (b) amino acid residues 27 to
230; (c) amino acid residues 27 to 126; and (d) amino acid residues
131 to 230. In one embodiment, the isolated polypeptide disclosed
above comprises an amino acid sequence of SEQ ID NO:38 or SEQ ID
NO:48 selected from the group consisting of: (a) amino acid
residues 24 to 230; (b) amino acid residues 27 to 230; (c) amino
acid residues 27 to 126; and (d) amino acid residues 131 to 230. In
another embodiment, the isolated polypeptide disclosed above
consists of an amino acid sequence of SEQ ID NO:38 or SEQ ID NO:48
selected from the group consisting of: (a) amino acid residues 24
to 230; (b) amino acid residues 27 to 230; (c) amino acid residues
27 to 126; and (d) amino acid residues 131 to 230. In another
embodiment, the isolated polypeptide disclosed above comprises an
amino acid sequence that is at least 90% identical to a reference
amino acid sequence of SEQ ID NO:38 or SEQ ID NO:48 selected from
the group consisting of: (a) amino acid residues 24 to 230; (b)
amino acid residues 27 to 230; (c) amino acid residues 27 to 126;
and (d) amino acid residues 131 to 230. In another embodiment, the
isolated polypeptide disclosed above comprises an amino acid
sequence of SEQ ID NO:38 or SEQ ID NO:48 selected from the group
consisting of: (a) amino acid residues 24 to 230; (b) amino acid
residues 27 to 230; (c) amino acid residues 27 to 126; and (d)
amino acid residues 131 to 230.
[0339] Within a second aspect, the present invention provides an
isolated nucleic acid molecule, wherein the nucleic acid molecule
is either (a) a nucleic acid molecule comprising the nucleotide
sequence of SEQ ID NO:39 or SEQ ID NO:49, or (b) a nucleic acid
molecule that remains hybridized following stringent wash
conditions to a nucleic acid molecule consisting of the nucleotide
sequence of nucleotides 8 to 697 or 77 to 697 or 86 to 697 of SEQ
ID NO:37 or SEQ ID NO:47, or the complement of the nucleotide
sequence of nucleotides 8 to 697 or 77 to 697 or 86 to 697 of SEQ
ID NO:37 or SEQ ID NO:47. In one embodiment, the isolated nucleic
acid molecule is as disclosed above, wherein any difference between
the amino acid sequence encoded by the nucleic acid molecule and
the corresponding amino acid sequence of SEQ ID NO:38 or SEQ ID
NO:48 is due to a conservative amino acid substitution. In another
embodiment, the isolated nucleic acid molecule disclosed above
comprises the nucleotide sequence of nucleotides 8 to 697 or 77 to
697 or 86 to 697 of SEQ ID NO:37 or SEQ ID NO:47.
[0340] Within a third aspect, the present invention provides a
vector, comprising the isolated nucleic acid molecule as disclosed
above.
[0341] Within another aspect, the present invention provides an
expression vector, comprising the isolated nucleic acid molecule as
disclosed above, a transcription promoter, and a transcription
terminator, wherein the promoter is operably linked with the
nucleic acid molecule, and wherein the nucleic acid molecule is
operably linked with the transcription terminator.
[0342] Within another aspect, the present invention provides a
recombinant host cell comprising the expression vector as disclosed
above, wherein the host cell is selected from the group consisting
of bacterium, yeast cell, fungal cell, insect cell, mammalian cell,
and plant cell.
[0343] Within another aspect, the present invention provides a
method of producing mouse Zcytor16 protein, the method comprising
culturing recombinant host cells that comprise the expression
vector as disclosed above, and that produce the mouse Zcytor16
protein. In one embodiment, the method disclosed above further
comprises isolating the mouse Zcytor16 protein from the cultured
recombinant host cells.
[0344] Within another aspect, the present invention provides an
antibody or antibody fragment that binds with the polypeptide as
disclosed above. In one embodiment, the antibody disclosed above is
selected from the group consisting of: (a) polyclonal antibody, (b)
murine monoclonal antibody, (c) humanized antibody derived from
(b), and (d) human monoclonal antibody.
[0345] Within another aspect, the present invention provides an
anti-idiotype antibody that specifically binds with the antibody as
disclosed above.
[0346] Within another aspect, the present invention provides a
fusion protein, comprising the polypeptide as disclosed above. In
one embodiment, the fusion protein disclosed above further
comprises an immunoglobulin moiety.
[0347] Within another aspect, the present invention provides an
isolated polynucleotide that encodes a soluble cytokine receptor
polypeptide comprising a sequence of amino acid residues that is at
least 90% identical to the amino acid sequence as shown in SEQ ID
NO:38 or SEQ ID NO:48 from amino acid 24 to 230, or 27 to 230; and
wherein amino acid differences consist of conservative amino acid
substitutions; and wherein the soluble cytokine receptor
polypeptide encoded by the polynucleotide sequence binds IL-TIF or
antagonizes IL-TIF activity. In one embodiment, the isolated
polynucleotide is as disclosed above, wherein the soluble cytokine
receptor polypeptide encoded by the polynucleotide forms a
homodimeric, heterodimeric or multimeric receptor complex. In
another embodiment, the isolated polynucleotide is as disclosed
above, wherein the soluble cytokine receptor polypeptide encoded by
the polynucleotide forms a heterodimeric or multimeric receptor
complex further comprising a soluble Class I or Class II cytokine
receptor. In another embodiment, the isolated polynucleotide is as
disclosed above, wherein the soluble cytokine receptor polypeptide
encoded by the polynucleotide forms a heterodimeric or multimeric
receptor complex further comprising a soluble CRF2-4 receptor
polypeptide (SEQ ID NO:35), a soluble IL-10 receptor polypeptide
(SEQ ID NO:36), or soluble zcytor11 receptor polypeptide (SEQ ID
NO:34).
[0348] Within another aspect, the present invention provides an
isolated polynucleotide that encodes a soluble cytokine receptor
polypeptide comprising a sequence of amino acid residues as shown
in SEQ ID NO:38 or SEQ ID NO:48 from amino acid 24 to 230, or 27 to
230, wherein the soluble cytokine receptor polypeptide encoded by
the polynucleotide forms a homodimeric, heterodimeric or multimeric
receptor complex. In one embodiment, the isolated polynucleotide is
as disclosed above, wherein the soluble cytokine receptor
polypeptide encoded by the polynucleotide further comprises a
soluble Class I or Class II cytokine receptor. In another
embodiment, the isolated polynucleotide is as disclosed above,
wherein the soluble cytokine receptor polypeptide encoded by the
polynucleotide forms a heterodimeric or multimeric receptor complex
further comprising a soluble CRF2-4 receptor polypeptide (SEQ ID
NO:35), a soluble IL-10 receptor polypeptide (SEQ ID NO:36), or
soluble zcytor11 receptor polypeptide (SEQ ID NO:34). In another
embodiment, the isolated polynucleotide is as disclosed above,
wherein the soluble cytokine receptor polypeptide further encodes
an intracellular domain. In another embodiment, the isolated
polynucleotide is as disclosed above, wherein the soluble cytokine
receptor polypeptide further comprises an affinity tag.
[0349] Within another aspect, the present invention provides an
expression vector comprising the following operably linked
elements: (a) a transcription promoter; a first DNA segment
encoding a soluble cytokine receptor polypeptide having an amino
acid sequence as shown in SEQ ID NO:38 or SEQ ID NO:48 from amino
acid 24 to 230, or 27 to 230; and a transcription terminator; and
(b) a second transcription promoter; a second DNA segment encoding
a soluble Class I or Class II cytokine receptor polypeptide; and a
transcription terminator; and wherein the first and second DNA
segments are contained within a single expression vector or are
contained within independent expression vectors. In one embodiment,
the expression vector disclosed above further comprises a secretory
signal sequence operably linked to the first and second DNA
segments. In another embodiment, the expression vector is as
disclosed above, wherein the second DNA segment encodes a
polypeptide comprising a soluble CRF2-4 receptor polypeptide (SEQ
ID NO:35), a soluble IL-10 receptor polypeptide (SEQ ID NO:36), or
soluble zcytor11 receptor polypeptide (SEQ ID NO:34).
[0350] Within another aspect, the present invention provides a
cultured cell comprising an expression vector as disclosed above,
wherein the cell expresses the polypeptides encoded by the DNA
segments. In one embodiment, the cultured cell comprising an
expression vector is as disclosed above, wherein the first and
second DNA segments are located on independent expression vectors
and are co-transfected into the cell, and cell expresses the
polypeptides encoded by the DNA segments. In another embodiment,
the cultured cell comprising an expression vector is as disclosed
above, wherein the cell expresses a heterodimeric or multimeric
soluble receptor polypeptide encoded by the DNA segments. In
another embodiment, the cell disclosed above secretes a soluble
cytokine receptor polypeptide heterodimer or multimeric complex. In
another embodiment, the cell disclosed above secretes a soluble
cytokine receptor polypeptide heterodimer or multimeric complex
that binds IL-TIF or antagonizes IL-TIF activity.
[0351] Within another aspect, the present invention provides a DNA
construct encoding a fusion protein comprising: a first DNA segment
encoding a polypeptide having a sequence of amino acid residues as
shown in SEQ ID NO:38 or SEQ ID NO:48 from amino acid 24 to 230, or
27 to 230; and at least one other DNA segment encoding a soluble
Class I or Class II cytokine receptor polypeptide, wherein the
first and other DNA segments are connected in-frame; and wherein
the first and other DNA segments encode the fusion protein. In one
embodiment, the DNA construct encoding a fusion protein is as
disclosed above, wherein at least one other DNA segment encodes a
polypeptide comprising a soluble CRF2-4 receptor polypeptide (SEQ
ID NO:35), a soluble IL-10 receptor polypeptide (SEQ ID NO:36), or
soluble zcytor11 receptor polypeptide (SEQ ID NO:34).
[0352] Within another aspect, the present invention provides an
expression vector comprising the following operably linked
elements: a transcription promoter; a DNA construct encoding a
fusion protein as disclosed above; and a transcription terminator,
wherein the promoter is operably linked to the DNA construct, and
the DNA construct is operably linked to the transcription
terminator.
[0353] Within another aspect, the present invention provides a
cultured cell comprising an expression vector as disclosed above,
wherein the cell expresses a polypeptide encoded by the DNA
construct.
[0354] Within another aspect, the present invention provides a
method of producing a fusion protein comprising: culturing a cell
according as disclosed above; and isolating the polypeptide
produced by the cell.
[0355] Within another aspect, the present invention provides an
isolated soluble cytokine receptor polypeptide comprising a
sequence of amino acid residues that is at least 90% identical to
an amino acid sequence as shown in SEQ ID NO:38 or SEQ ID NO:48
from amino acid 24 to 230, or 27 to 230, and wherein the soluble
cytokine receptor polypeptide binds IL-TIF or antagonizes IL-TIF
activity. In one embodiment, the isolated polypeptide is as
disclosed above, wherein the soluble cytokine receptor polypeptide
forms a homodimeric, heterodimeric or multimeric receptor complex.
In another embodiment, the isolated polypeptide is as disclosed
above, wherein the soluble cytokine receptor polypeptide forms a
heterodimeric or multimeric receptor complex further comprising a
soluble Class I or Class II cytokine receptor. In another
embodiment, the isolated polypeptide is as disclosed above, wherein
the soluble cytokine receptor polypeptide forms a heterodimeric or
multimeric receptor complex further comprising a soluble CRF2-4
receptor polypeptide (SEQ ID NO:35), a soluble IL-10 receptor
polypeptide (SEQ ID NO:36), or soluble zcytor11 receptor
polypeptide (SEQ ID NO:34).
[0356] Within another aspect, the present invention provides an
isolated soluble cytokine receptor polypeptide comprising a
sequence of amino acid residues as shown in SEQ ID NO:38 or SEQ ID
NO:48 from amino acid 24 to 230, or 27 to 230, wherein the soluble
cytokine receptor polypeptide forms a homodimeric, heterodimeric or
multimeric receptor complex. In another embodiment, the isolated
polypeptide is as disclosed above, wherein the soluble cytokine
receptor polypeptide forms a heterodimeric or multimeric receptor
complex further comprising a soluble Class I or Class II cytokine
receptor. In another embodiment, the isolated polypeptide is as
disclosed above, wherein the soluble cytokine receptor polypeptide
forms a heterodimeric or multimeric receptor complex comprising a
soluble CRF2-4 receptor polypeptide (SEQ ID NO:35), a soluble IL-10
receptor polypeptide (SEQ ID NO:36), or soluble zcytor11 receptor
polypeptide (SEQ ID NO:34). In another embodiment, the isolated
polypeptide is as disclosed above, wherein the soluble cytokine
receptor polypeptide further comprises an affinity tag, chemical
moiety, toxin, or label.
[0357] Within another aspect, the present invention provides an
isolated heterodimeric or multimeric soluble receptor complex
comprising soluble receptor subunits, wherein at least one of the
soluble receptor subunits comprises a soluble cytokine receptor
polypeptide comprising a sequence of amino acid residues as shown
in SEQ ID NO:38 or SEQ ID NO:48 from amino acid 24 to 230, or 27 to
230. In one embodiment, the isolated heterodimeric or multimeric
soluble receptor complex disclosed above further comprises a
soluble Class I or Class II cytokine receptor polypeptide. In
another embodiment, the isolated heterodimeric or multimeric
soluble receptor complex disclosed above further comprises a
soluble CRF2-4 receptor polypeptide (SEQ ID NO:35), a soluble IL-10
receptor polypeptide (SEQ ID NO:36), or soluble zcytor11 receptor
polypeptide (SEQ ID NO:34).
[0358] Within another aspect, the present invention provides a
method of producing a soluble cytokine receptor polypeptide that
forms a heterodimeric or multimeric complex comprising: culturing a
cell as disclosed above; and isolating the soluble receptor
polypeptides produced by the cell.
[0359] Within another aspect, the present invention provides a
method of producing an antibody to soluble cytokine receptor
polypeptide comprising: inoculating an animal with a soluble
cytokine receptor polypeptide selected from the group consisting
of: (a) a polypeptide comprising a monomeric or homodimeric soluble
cytokine receptor comprising a polypeptide as shown in SEQ ID NO:38
or SEQ ID NO:48 from amino acid 24 to 230, or 27 to 230; (b) a
polypeptide of (a) further comprising a soluble cytokine receptor
heterodimeric or multimeric receptor complex comprising a soluble
Class I or Class II cytokine receptor polypeptide; (c) a
polypeptide of (a) further comprising a soluble cytokine receptor
heterodimeric or multimeric receptor complex comprising a soluble
CRF2-4 receptor polypeptide (SEQ ID NO:35); (d) a polypeptide of
(a) further comprising a soluble cytokine receptor heterodimeric or
multimeric receptor complex comprising a soluble IL-10 receptor
polypeptide (SEQ ID NO:36); and wherein the polypeptide elicits an
immune response in the animal to produce the antibody; and
isolating the antibody from the animal.
[0360] Within another aspect, the present invention provides an
antibody produced by the method as disclosed above, which
specifically binds to a homodimeric, heterodimeric or multimeric
receptor complex comprising a polypeptide as shown in SEQ ID NO:38
or SEQ ID NO:48 from amino acid 24 to 230, or 27 to 230. In one
embodiment, the antibody disclosed above is a monoclonal
antibody.
[0361] Within another aspect, the present invention provides an
antibody which specifically binds to a homodimeric, heterodimeric
or multimeric receptor complex as disclosed above.
[0362] Within another aspect, the present invention provides a
method for inhibiting IL-TIF-induced proliferation or
differentiation of hematopoietic cells and hematopoietic cell
progenitors comprising culturing bone marrow or peripheral blood
cells with a composition comprising an amount of soluble cytokine
receptor polypeptide as shown in SEQ ID NO:38 or SEQ ID NO:48 from
amino acid 24 to 230, or 27 to 230, sufficient to reduce
proliferation or differentiation of the hematopoietic cells in the
bone marrow or peripheral blood cells as compared to bone marrow or
peripheral blood cells cultured in the absence of soluble cytokine
receptor. In one embodiment, the method is as disclosed above,
wherein the hematopoietic cells and hematopoietic progenitor cells
are lymphoid cells. In another embodiment, the method is as
disclosed above, wherein the lymphoid cells are macrophages or T
cells.
[0363] Within another aspect, the present invention provides a
method of reducing IL-TIF-induced or IL-9 induced inflammation
comprising administering to a mammal with inflammation an amount of
a composition of a polypeptide as shown in SEQ ID NO:38 or SEQ ID
NO:48 from amino acid 24 to 230, or 27 to 230 sufficient to reduce
inflammation.
[0364] Within another aspect, the present invention provides a
method of suppressing an inflammatory response in a mammal with
inflammation comprising: (1) determining a level of serum amyloid A
protein; (2) administering a composition comprising a soluble mouse
Zcytor16 cytokine receptor polypeptide as disclosed above in an
acceptable pharmaceutical vehicle; (3) determining a post
administration level of serum amyloid A protein; (4) comparing the
level of serum amyloid A protein in step (1) to the level of serum
amyloid A protein in step (3), wherein a lack of increase or a
decrease in serum amyloid A protein level is indicative of
suppressing an inflammatory response.
[0365] Within another aspect, the present invention provides a
method for detecting a cancer in a patient, comprising: obtaining a
tissue or biological sample from a patient; incubating the tissue
or biological sample with an antibody as disclosed above under
conditions wherein the antibody binds to its complementary
polypeptide in the tissue or biological sample; visualizing the
antibody bound in the tissue or biological sample; and comparing
levels of antibody bound in the tissue or biological sample from
the patient to a normal control tissue or biological sample,
wherein an increase in the level of antibody bound to the patient
tissue or biological sample relative to the normal control tissue
or biological sample is indicative of a cancer in the patient.
[0366] Within another aspect, the present invention provides a
method for detecting a cancer in a patient, comprising: obtaining a
tissue or biological sample from a patient; labeling a
polynucleotide comprising at least 14 contiguous nucleotides of SEQ
ID NO:37 or SEQ ID NO:47 or the complement of SEQ ID NO:37 or SEQ
ID NO:47; incubating the tissue or biological sample with under
conditions wherein the polynucleotide will hybridize to
complementary polynucleotide sequence; visualizing the labeled
polynucleotide in the tissue or biological sample; and comparing
the level of labeled polynucleotide hybridization in the tissue or
biological sample from the patient to a normal control tissue or
biological sample, wherein an increase in the labeled
polynucleotide hybridization to the patient tissue or biological
sample relative to the normal control tissue or biological sample
is indicative of a cancer in the patient.
[0367] Within another aspect, the present invention provides a
transgenic mouse, wherein the mouse over-expresses residues 1 to
230 or 24 to 230, or 27 to 230 of SEQ ID NO:38 or SEQ ID NO:48. In
one embodiment, the transgenic mouse is as disclosed above, wherein
the expression of residues 1 to 230 or 24 to 230, or 27 to 230 of
SEQ ID NO:38 or SEQ ID NO:48 is expressed using a tissue-specific
or tissue-restricted promoter. In another embodiment, the
transgenic mouse is as disclosed above, wherein the expression of
residues 1 to 230 or 24 to 230, or 27 to 230 of SEQ ID NO:38 or SEQ
ID NO:48 is expressed using an epithelial-specific, colon-specific,
or ovary-specific promoter. In another embodiment, the transgenic
mouse is as disclosed above, wherein the mouse does not expresses
residues 1 to 230 or 24 to 230, or 27 to 230 of SEQ ID NO:38 or SEQ
ID NO:48, relative to a normal mouse.
[0368] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
Cloning of Mouse Zcytor16 and Construction of Mammalian Expression
Vectors that Express Human and Mouse Zcytor16 Soluble Receptors:
Zcytor16CEE, Zcytor16CFLG, Zcytor16CHIS and Zcytor16-Fc4
A. Cloning of Mouse Zcytor16 Extracellular Domain
[0369] A mouse ortholog of human Zcytor16 (U.S. patent application
Ser. No. 09/728,911) was identified and designated "mouse
Zcytor16." The polynucleotide sequence of the mouse Zcytor16 clone
is shown in SEQ ID NO:37 and SEQ ID NO:47 and polypeptide sequence
shown in SEQ ID NO:38 and SEQ ID NO:48 respectively.
B. Mammalian Expression Construction of Soluble Zcytor16 Receptor
Zcytor16-Fc4
[0370] Construction of Mammalian Expression Vectors that Express
Zcytor16 Soluble Receptor Zcytor16sR/Fc4
[0371] An expression vector was prepared to express the soluble
human Zcytor16 polypeptide (Zcytor16sR, i.e., from residue 22 (Thr)
to residue 231 (Pro) of SEQ ID NO:2; SEQ ID NO: 13) fused to a
C-terminal Fc4 tag (SEQ ID NO:5).
[0372] A PCR generated Zcytor16 DNA fragment of about 630 bp was
created using oligo ZC29,181 (SEQ ID NO:6) and oligo ZC29,182 (SEQ
ID NO:7) as PCR primers to add BamHI and Bgl2 restriction sites at
5' and 3' ends respectively, of the Zcytor16 DNA encoding the
soluble receptor. A plasmid containing the Zcytor16 cDNA (SEQ ID
NO:1) (Example 1A) was used as a template. PCR amplification of the
Zcytor16 fragment was performed as follows: One cycle at 94.degree.
C. for 1 minute; 25 cycles at 94.degree. C. for 30 seconds,
68.degree. C. for 90 seconds, followed by an additional 68.degree.
C. incubation for 4 minutes, and hold at 110.degree. C. The
reaction was purified by chloroform/phenol extraction and
isopropanol precipitation, and digested with BamHI and Bgl2
(Boehringer Mannheim, Indianapolis, Ind.). A band of approximately
630 bp, as visualized by 1% agarose gel electrophoresis, was
excised and the DNA was purified using a QiaexII.TM. purification
kit (Qiagen, Valencia, Calif.) according to the manufacturer's
instruction.
[0373] The Fc4/pzmp20 plasmid is a mammalian expression vector
containing an expression cassette having the CMV promoter, human
tPA leader peptide, multiple restriction sites for insertion of
coding sequences, a Fc4 tag, and a human growth hormone terminator.
The plasmid also has an E. coli origin of replication, a mammalian
selectable marker expression unit having an SV40 promoter, an
enhancer and an origin of replication, as well as a DHFR gene, and
SV40 terminator. The Zcytor16sR/Fc4/pzmp20 expression vector uses
the human tPA leader peptide (SEQ ID NO:8 and SEQ ID NO:9) and
attaches the Fc4 tag (SEQ ID NO:5) to the C-terminus of the
extracellular portion of the Zcytor16 polypeptide sequence. Fc4 is
the Fc region derived from human IgG, which contains a mutation so
that it no longer binds the Fc receptor
[0374] About 30 ng of the restriction digested Zcytor16sR insert
and about 10 ng of the digested vector (which had been cut with
Bgl2) were ligated at 11.degree. C. overnight. One microliter of
ligation reaction was electroporated into DH10B competent cells
(Gibco BRL, Rockville, Md.) according to manufacturer's direction
and plated onto LB plates containing 50 mg/ml ampicillin, and
incubated overnight. Colonies were screened by restriction analysis
of DNA, which was prepared from 2 ml liquid cultures of individual
colonies. The insert sequence of positive clones was verified by
sequence analysis. A large-scale plasmid preparation was done using
a Qiagen.RTM. Mega prep kit (Qiagen) according to manufacturer's
instruction.
[0375] Similar methods are used to prepare mouse Zcytor16-Fc4
fusions, and Fc4 fusions of non-Zcytor16 subunits of heterodimeric
and multimeric receptors, such as CRF2-4 and IL-10R tagged with
Fc4.
C. Construction of Zcytor16 Mammalian Expression Vector Containing
Zcytor16CEE, Zcytor16CFLG and Zcytor16CHIS
[0376] An expression vector is prepared for the expression of the
soluble, extracellular domain of the Zcytor16 polypeptide (e.g.,
amino acids 22-231 of SEQ ID NO:2; SEQ ID NO:13; or amino acids
24-230 or SEQ ID NO:38 or SEQ ID NO:48), pC4Zcytor16CEE, wherein
the construct is designed to express a Zcytor16 polypeptide
comprised of the predicted initiating methionine and truncated
adjacent to the predicted transmembrane domain, and with a
C-terminal Glu-Glu tag (SEQ ID NO:10).
[0377] A Zcytor16 DNA fragment comprising the Zcytor16
extracellular cytokine binding domain is created using PCR, and
purified. The excised DNA is subcloned into a plasmid expression
vector that has a signal peptide, e.g., the native Zcytor16 signal
peptide, tPA leader, and attaches a Glu-Glu tag (SEQ ID NO:10) to
the C-terminus of the Zcytor16 polypeptide-encoding polynucleotide
sequence. Such an expression vector mammalian expression vector
contains an expression cassette having a mammalian promoter,
multiple restriction sites for insertion of coding sequences, a
stop codon and a mammalian terminator. The plasmid can also have an
E. coli origin of replication, a mammalian selectable marker
expression unit having an SV40 promoter, enhancer and origin of
replication, a DHFR gene and the SV40 terminator.
[0378] Restriction digested Zcytor16 insert and previously digested
vector are ligated using standard molecular biological techniques,
and electroporated into competent cells such as DH10B competent
cells (GIBCO BRL, Gaithersburg, Md.) according to manufacturer's
direction and plated onto LB plates containing 50 mg/ml ampicillin,
and incubated overnight. Colonies are screened by restriction
analysis of DNA prepared from individual colonies. The insert
sequence of positive clones is verified by sequence analysis. A
large-scale plasmid preparation is done using a QIAGEN.RTM. Maxi
prep kit (Qiagen) according to manufacturer's instructions.
[0379] The same process is used to prepare mouse Zcytor16 soluble
homodimeric, heterodimeric or multimeric receptors (including
non-Zcytor16 soluble receptor subunits, such as, soluble CRF2-4 or
IL-10R), or soluble receptors with a C-terminal HIS tag, composed
of 6 His residues in a row (SEQ ID NO:12); and a C-terminal FLAG
(SEQ ID NO:11) tag, Zcytor16CFLAG. To construct these constructs,
the aforementioned vector has either the HIS or the FLAG.RTM. tag
in place of the glu-glu tag (SEQ ID NO:10).
Example 2
Transfection And Expression of Soluble Receptor Polypeptides
[0380] The day before the transfection, BHK 570 cells (ATCC No.
CRL-10314; ATCC, Manasas, Va.) were plated in a 10-cm plate with
50% confluence in normal BHK DMEM (Gibco/BRL High Glucose) media.
The day of the transfection, the cells were washed once with Serum
Free (SF) DMEM, followed by transfection with the
Zcytor16sR/Fc4/pzmp20 expression plasmids. Sixteen micrograms of
human Zcytor16sR-Fc4 DNA construct (Example 1B) were diluted into a
total final volume of 640 .mu.l SF DMEM. A diluted
LipofectAMINE.TM. (Gibco BRL, Gaithersburg, Md.) mixture (35 .mu.l
LipofectAMINE.TM. in 605 .mu.l SF media) was added to the DNA mix,
and incubated for 30 minutes at room temperature. Five milliliters
of SF media was added to the DNA/LipofectAMINE.TM. mixture, which
was then added to BHK cells. The cells were incubated at 37.degree.
C./5% CO.sub.2 for 5 hours, after which 6.4 ml of BHK media with
10% FBS was added. The cells were incubated overnight at 37.degree.
C./5% CO.sub.2.
[0381] Approximately 24 hours post-transfection, the BHK cells were
split into selection media with 1 .mu.M methotrexate (MTX). The
cells were repeatedly split in this manner until stable
Zcytor16sR-Fc4/BHK cell lines were identified. To detect the
expression level of the Zcytor16 soluble receptor fusion proteins,
the transfected BHK cells were washed with PBS and incubated in SF
media for 72 hours. The SF condition media was collected and 20
.mu.l of the sample was run on 10% SDS-PAGE gel under reduced
conditions. The protein bands were transferred to nitrocellulose
filter by Western blot, and the fusion proteins were detected using
goat-anti-human IgG/HRP conjugate (Jackson ImmunoResearch
Laboratories, Inc, West Grove, Pa.). An expression vector
containing a different soluble receptor fused to the Fc4 was used
as a control. The expression level of the stable Zcytor16sR-Fc4/BHK
cells was approximately 2 mg/L.
[0382] For protein purification, the transfected BHK cells were
transferred into T-162 flasks. Once the cells reached about 80%
confluence, they were washed with PBS and incubated in 100 ml SF
media for 72 hours, and then the condition media was collected for
protein purification (Example 11).
[0383] Using the above methods, mouse Zcytor16 soluble receptor
fusion proteins are similarly expressed.
Example 3
Expression of Zcytor16 Soluble Receptor in E. coli
[0384] A. Construction of Expression Vector pCZR225 that Expresses
huZcytor16/MBP-6H Fusion Polypeptide
[0385] An expression plasmid containing a polynucleotide encoding a
Zcytor16 soluble receptor fused C-terminally to maltose binding
protein (MBP) is constructed via homologous recombination. The
fusion polypeptide contains an N-terminal approximately 388 amino
acid MBP portion fused to the human Zcytor16 soluble receptor
(e.g., SEQ ID NO:13; or for the mouse Zcytor16, amino acids 24 to
230 of SEQ ID NO:38 or SEQ ID NO:48). A fragment of Zcytor16 cDNA
(SEQ ID NO:1; or SEQ ID NO:37 or SEQ ID NO:47) is isolated using
PCR as described herein. Two primers are used in the production of
the Zcytor16 fragment in a standard PCR reaction: (1) one
containing about 40 bp of the vector flanking sequence and about 25
bp corresponding to the amino terminus of the Zcytor16, and (2)
another containing about 40 bp of the 3' end corresponding to the
flanking vector sequence and about 25 bp corresponding to the
carboxyl terminus of the Zcytor16. Two .mu.l of the 100 .mu.l PCR
reaction is run on a 1.0% agarose gel with 1.times.TBE buffer for
analysis, and the expected approximately fragment is seen. The
remaining PCR reaction is combined with the second PCR tube and
precipitated with 400 .mu.l of absolute ethanol. The precipitated
DNA used for recombining into an appropriately restriction digested
recipient vector pTAP98 to produce the construct encoding the
MBP-Zcytor16 fusion, as described below.
[0386] Plasmid pTAP98 is derived from the plasmids pRS316 and
pMAL-c2. The plasmid pRS316 is a Saccharomyces cerevisiae shuttle
vector (Hieter P. and Sikorski, R., Genetics 122:19-27, 1989).
pMAL-C2 (EB) is an E. coli expression plasmid. It carries the tac
promoter driving MalE (gene encoding MBP) followed by a His tag, a
thrombin cleavage site, a cloning site, and the rrnB terminator.
The vector pTAP98 is constructed using yeast homologous
recombination. 100 ng of EcoR1 cut pMAL-c2 is recombined with 1
.mu.g Pvu1 cut pRS316, 1 .mu.g linker, and 1 .mu.g Sca1/EcoR1 cut
pRS316 are combined in a PCR reaction. PCR products are
concentrated via 100% ethanol precipitation.
[0387] Competent yeast cells (S. cerevisiae) are combined with
about 10 .mu.l of a mixture containing approximately 1 .mu.g of the
Zcytor16 receptor PCR product above, and 100 ng of digested pTAP98
vector, and electroporated using standard methods and plated onto
URA-D plates and incubated at 30.degree. C.
[0388] After about 48 hours, the Ura+ yeast transformants from a
single plate are picked, DNA isolated, and transformed into
electrocompetent E. Coli cells (e.g., MC1061, Casadaban et. al. J.
Mol. Biol. 138, 179-207), and plated on MM/CA+AMP 100 mg/L plates
(Pryor and Leiting, Protein Expression and Purification 10:309-319,
1997). using standard procedures. Cells are grown in MM/CA with 100
.mu.g/ml Ampicillin for two hours, shaking, at 37.degree. C. 1 ml
of the culture is induced with 1 mM IPTG. 2-4 hours later the 250
.mu.l of each culture is mixed with 250 .mu.l acid washed glass
beads and 250 .mu.l Thorner buffer with 5% .beta.ME and dye (8M
urea, 100 mM Tris pH7.0, 10% glycerol, 2 mM EDTA, 5% SDS). Samples
are vortexed for one minute and heated to 65.degree. C. for 10
minutes. 20 .mu.l are loaded per lane on a 4%-12% PAGE gel (NOVEX).
Gels are run in 1.times.MES buffer. The positive clones are
subjected to sequence analysis.
[0389] One microliter of sequencing DNA is used to transform strain
BL21. The cells are electropulsed at 2.0 kV, 25 .mu.F and 400 ohms.
Following electroporation, 0.6 ml MM/CA with 100 mg/L Ampicillin.
Cells are grown in MM/CA and induced with ITPG as described above.
The positive clones are used to grow up for protein purification of
the human or mouse Zcytor16/MBP-6H fusion protein using standard
techniques.
Example 4
Mouse Zcytor16 Soluble Receptor Polyclonal Antibodies
[0390] Polyclonal antibodies are prepared by immunizing female New
Zealand white rabbits with the purified mouse Zcytor16/MBP-6H
polypeptide (Example 3), or the purified recombinant mouse
Zcytor16CEE or mouse Zcytor16-Fc4 soluble receptor (Example 1;
Example 11). The rabbits are each given an initial intraperitoneal
(IP) injection of about 200 mg of purified protein in Complete
Freund's Adjuvant (Pierce, Rockford, Ill.) followed by booster IP
injections of 100 mg purified protein in Incomplete Freund's
Adjuvant every three weeks. Seven to ten days after the
administration of the third booster injection, the animals are bled
and the serum is collected. The rabbits are then boosted and bled
every three weeks.
[0391] The mouse Zcytor16-specific polyclonal antibodies are
affinity purified from the rabbit serum using an CNBr-SEPHAROSE 4B
protein column (Pharmacia LKB) that is prepared using about 10 mg
of the appropriate purified mouse Zcytor16 polypeptide per gram
CNBr-SEPHAROSE, followed by 20.times. dialysis in PBS overnight.
Zcytor16-specific antibodies are characterized by an ELISA titer
check using 1 mg/ml of the appropriate protein antigen as an
antibody target. The lower limit of detection (LLD) of the rabbit
anti-Zcytor16 affinity purified antibodies is determined using
standard methods.
Example 5
Mouse Zcytor16 Receptor Monoclonal Antibodies
[0392] Mouse Zcytor16 receptor Monoclonal antibodies are prepared
by immunizing male BalbC mice (Harlan Sprague Dawley, Indianapolis,
Ind.) with the purified recombinant mouse Zcytor16 proteins
described herein. The mice are each given an initial
intraperitoneal (IP) injection of 20 mg of purified protein in
Complete Freund's Adjuvant (Pierce, Rockford, Ill.) followed by
booster IP injections of 10 mg purified protein in Incomplete
Freund's Adjuvant every two weeks. Seven to ten days after the
administration of the third booster injection, the animals are bled
and the serum is collected, and antibody titer assessed.
[0393] Splenocytes are harvested from high-titer mice and fused to
murine SP2/0 myeloma cells using PEG 1500 (Boerhinger Mannheim, UK)
in two separate fusion procedures using a 4:1 fusion ratio of
splenocytes to myeloma cells (Antibodies: A Laboratory Manual, E.
Harlow and D. Lane, Cold Spring Harbor Press). Following 10 days
growth post-fusion, specific antibody-producing hybridomas are
identified by ELISA using purified recombinant mouse Zcytor16
soluble receptor protein (Example 6C) as an antibody target and by
FACS using Baf3 cells expressing the Zcytor16 sequence (Example 8)
as an antibody target. The resulting hybridomas positive by both
methods are cloned three times by limiting dilution.
Example 6
Assessing Mouse Zcytor16 Receptor Heterodimerization Using ORIGEN
Assay
[0394] Soluble mouse Zcytor16 receptor (Example 11), or gp130
(Hibi, M. et al., Cell 63:1149-1157, 1990) are biotinylated by
reaction with a five-fold molar excess of sulfo-NHS-LC-Biotin
(Pierce, Inc., Rockford, Ill.) according to the manufacturer's
protocol. Soluble mouse Zcytor16 receptor and another soluble
receptor subunit, for example, soluble IL-10R (sIL-10R) or CRF2-4
receptor (CRF2-4), soluble zcytor11 receptor (U.S. Pat. No.
5,965,704) or soluble zcytor7 receptor (U.S. Pat. No. 5,945,511)
are labeled with a five fold molar excess of Ru-BPY-NHS (Igen,
Inc., Gaithersburg, Md.) according to manufacturer's protocol. The
biotinylated and Ru-BPY-NHS-labeled forms of the soluble mouse
Zcytor16 receptor can be respectively designated Bio-Zcytor16
receptor and Ru-Zcytor16; the biotinylated and Ru-BPY-NHS-labeled
forms of the other soluble receptor subunit can be similarly
designated. Assays can be carried out using conditioned media from
cells expressing a ligand, such as IL-TIF, that binds mouse
Zcytor16 heterodimeric receptors, or using purified IL-TIF.
[0395] For initial receptor binding characterization a panel of
cytokines or conditioned medium are tested to determine whether
they can mediate homodimerization of mouse Zcytor16 receptor and if
they can mediate the heterodimerization of mouse Zcytor16 receptor
with the soluble receptor subunits described above. To do this, 50
.mu.l of conditioned media or TBS-B containing purified cytokine,
is combined with 50 .mu.l of TBS-B (20 mM Tris, 150 mM NaCl, 1
mg/ml BSA, pH 7.2) containing e.g., 400 ng/ml of Ru-mZcytor16
receptor and Bio-mZcytor16, or 400 ng/ml of Ru-Zcytor16 receptor
and e.g., Bio-gp130, or 400 ng/ml of e.g., Ru-CRF2-4 and
Bio-mZcytor16. Following incubation for one hour at room
temperature, 30 .mu.g of streptavidin coated, 2.8 mm magnetic beads
(Dynal, Inc., Oslo, Norway) are added and the reaction incubated an
additional hour at room temperature. 200 .mu.l ORIGEN assay buffer
(Igen, Inc., Gaithersburg, Md.) is then added and the extent of
receptor association measured using an M8 ORIGEN analyzer (Igen,
Inc.).
Example 7
Construct for Generating a mouse Zcytor16 Receptor Heterodimer
[0396] A vector expressing a secreted mouse Zcytor16 heterodimer is
constructed. In this construct, the extracellular cytokine-binding
domain of Zcytor16 (e.g., amino acids 24 to 230 of SEQ ID NO:38 or
SEQ ID NO:48) is fused to the heavy chain of IgG gamma 1
(IgG.gamma.1) while the extracellular portion of the heteromeric
cytokine receptor subunit (e.g., an CRF2-4, IL-9, IL-10, zcytor7,
zcytor11, IL-4 receptor component) is fused to a human kappa light
chain (human .kappa. light chain).
A. Construction of IgG Gamma 1 and Human .kappa. Light Chain Fusion
Vectors
[0397] The heavy chain of IgG.gamma.1 can be cloned into the
Zem229R mammalian expression vector (ATCC deposit No. 69447) such
that any desired cytokine receptor extracellular domain having a 5'
EcoRI and 3' NheI site can be cloned in resulting in an N-terminal
extracellular domain-C-terminal IgG.gamma.1 fusion. The IgG.gamma.1
fragment used in this construct is made by using PCR to isolate the
IgG.gamma.1 sequence from a Clontech hFetal Liver cDNA library as a
template. PCR products are purified using methods described herein
and digested with MluI and EcoRI (Boerhinger-Mannheim), ethanol
precipitated and ligated with oligos which comprise an desired
restriction site linker, into Zem229R previously digested with and
EcoRI using standard molecular biology techniques disclosed
herein.
[0398] The human .kappa. light chain can be cloned in the Zem228R
mammalian expression vector (ATCC deposit No. 69446) such that any
desired cytokine receptor extracellular domain having a 5' EcoRI
site and a 3' KpnI site can be cloned in resulting in a N-terminal
cytokine extracellular domain-C-terminal human .kappa. light chain
fusion. As a KpnI site is located within the human .kappa. light
chain sequence, a special primer is designed to clone the 3' end of
the desired extracellular domain of a cytokine receptor into this
KpnI site: The primer is designed so that the resulting PCR product
contains the desired cytokine receptor extracellular domain with a
segment of the human .kappa. light chain up to the KpnI site. This
primer preferably comprises a portion of at least 10 nucleotides of
the 3' end of the desired cytokine receptor extracellular domain
fused in frame 5' to fragment cleaved at the KpnI site. The human
.kappa. light chain fragment used in this construct is made by
using PCR to isolate the human .kappa. light chain sequence from
the same Clontech human Fetal Liver cDNA library used above. PCR
products are purified using methods described herein and digested
with MluI and EcoRI (Boerhinger-Mannheim), ethanol precipitated and
ligated with the MluI/EcoRI linker described above, into Zem228R
previously digested with and EcoRI using standard molecular biology
techniques disclosed herein.
B. Insertion of Zcytor16 Receptor or Heterodimeric Subunit
Extracellular Domains into Fusion Vector Constructs
[0399] Using the construction vectors above, a construct having
mouse Zcytor16 fused to IgG.gamma.1 is made. This construction is
done by PCRing the extracellular cytokine-binding domain of the
mouse Zcytor16 receptor (amino acids 24-230 of SEQ ID NO:38 or SEQ
ID NO:48) from a placenta or other cDNA library (Clontech) or
plasmid (Example 1A) using standard methods and oligos that provide
EcoRI and NheI restriction sites. The resulting PCR product is
digested with EcoRI and NheI, gel purified, as described herein,
and ligated into a previously EcoRI and NheI digested and
band-purified Zem229R/IgG.gamma.1 described above. The resulting
vector is sequenced to confirm that the Zcytor16/IgG gamma 1 fusion
is correct.
[0400] A separate construct having a heterodimeric cytokine
receptor subunit extracellular domain fused to K light is also
constructed as above. The CRF2-4/human .kappa. light chain
construction is performed as above by PCRing from, e.g., a
lymphocyte cDNA library (Clontech) using standard methods, and
oligos that provide EcoRI and KpnI restriction sites. The resulting
PCR product is digested with EcoRI and KpnI and then ligating this
product into a previously EcoRI and KpnI digested and band-purified
Zem228R/human .kappa. light chain vector described above. The
resulting vector is sequenced to confirm that the cytokine receptor
subunit/human .kappa. light chain fusion is correct.
[0401] D. Co-Expression of the Mouse Zcytor16 and Heterodimeric
Cytokine Receptor Subunit Extracellular Domain
[0402] Approximately 15 .mu.g of each of vectors above, are
co-transfected into mammalian cells, e.g., BHK-570 cells (ATCC No.
CRL-10314) using LipofectaminePlus.TM. reagent (Gibco/BRL), as per
manufacturer's instructions. The transfected cells are selected for
10 days in DMEM+5% FBS (Gibco/BRL) containing 1 .mu.M of
methotrexate (MTX) (Sigma, St. Louis, Mo.) and 0.5 mg/ml G418
(Gibco/BRL) for 10 days. The resulting pool of transfectants is
selected again in 10 .mu.m of MTX and 0.5 mg/ml G418 for about 10
days.
[0403] The resulting pool of doubly selected cells is used to
generate protein. Three Factories (Nunc, Denmark) of this pool are
used to generate 10 L of serum free conditioned medium. This
conditioned media is passed over a 1 ml protein-A column and eluted
in about 10, 750 microliter fractions. The fractions having the
highest protein concentration are pooled and dialyzed (10 kD MW
cutoff) against PBS. Finally the dialyzed material is submitted for
amino acid analysis (AAA) using routine methods.
Example 8
Determination of Receptor Subunits that Heterodimerize or
Multimerize with Mouse Zcytor16 Receptor Using a Proliferation
Assay
[0404] Using standard methods described herein, cells expressing a
BaF3/mZcytor16-MPL chimera (wherein the extracellular domain of the
mouse Zcytor16 (e.g., amino acids 24 to 230 of SEQ ID NO:38 or SEQ
ID NO:48) is fused in frame to the intracellular signaling domain
of the mpl receptor) are tested for proliferative response in the
presence of IL-TIF. Such cells serve as a bioassay cell line to
measure ligand binding of monomeric or homodimeric mouse Zcytor16
receptors. In addition, BaF3/mZcytor16-MPL chimera cells
transfected with an additional heterodimeric cytokine receptor
subunit can be assessed for proliferative response in the presence
of IL-TIF. In the presence of IL-TIF, if the BaF3/mZcytor16-MPL
cells signal, this would suggest that Zcytor16 receptor can
homodimerize to signal. Transfection of the BaF3/MPL-mZcytor16 cell
line with and additional MPL-class II cytokine receptor fusion that
signals in the presence of the IL-TIF ligand, such as CRF2-4,
determines which heterodimeric cytokine receptor subunits are
required for mouse Zcytor16 receptor signaling. Use of MPL-receptor
fusions for this purpose alleviates the requirement for the
presence of an intracellular signaling domain for the mouse
Zcytor16 receptor.
[0405] Each independent receptor complex cell line is then assayed
in the presence of IL-TIF and proliferation measured using routine
methods (e.g., Alamar Blue assay). The BaF3/MPL-mZcytor16 bioassay
cell line serves as a control for the monomeric or homodimeric
receptor activity, and is thus used as a baseline to compare
signaling by the various receptor complex combinations. The
untransfected bioassay cell line serves as a control for the
background activity, and is thus used as a baseline to compare
signaling by the various receptor complex combinations. A
BaF3/MPL-mZcytor16 without ligand (IL-TIF) is also used as a
control. The IL-TIF in the presence of the correct receptor
complex, is expected to increase proliferation of the
BaF3/mZcytor16-MPL receptor cell line approximately 5 fold over
background or greater in the presence of IL-TIF. Cells expressing
the components of Zcytor16 heterodimeric and multimeric receptors
should proliferate in the presence of IL-TIF.
Example 9
Reconstitution of Mouse Zcytor16 Receptor In Vitro
[0406] To identify components involved in the Zcytor16-signaling
complex, receptor reconstitution studies are performed as follows.
BHK 570 cells (ATCC No. CRL-10314) transfected, using standard
methods described herein, with a luciferase reporter mammalian
expression vector plasmid serve as a bioassay cell line to measure
signal transduction response from a transfected mouse Zcytor16
receptor complex to the luciferase reporter in the presence of
IL-TIF. BHK cells do not endogenously express the mouse Zcytor16
receptor. An exemplary luciferase reporter mammalian expression
vector is the KZ134 plasmid that was constructed with complementary
oligonucleotides that contain STAT transcription factor binding
elements from 4 genes. A modified c-fos S is inducible element
(m67SIE, or hSIE) (Sadowski, H. et al., Science 261:1739-1744,
1993), the p21 SIE1 from the p21 WAF1 gene (Chin, Y. et al.,
Science 272:719-722, 1996), the mammary gland response element of
the .beta.-casein gene (Schmitt-Ney, M. et al., Mol. Cell. Biol.
11:3745-3755, 1991), and a STAT inducible element of the Fcg R1
gene, (Seidel, H. et al., Proc. Natl. Acad. Sci. 92:3041-3045,
1995). These oligonucleotides contain Asp718-XhoI compatible ends
and are ligated, using standard methods, into a recipient firefly
luciferase reporter vector with a c-fos promoter (Poulsen, L. K. et
al., J. Biol. Chem. 273:6229-6232, 1998) digested with the same
enzymes and containing a neomycin selectable marker. The KZ134
plasmid is used to stably transfect BHK, or BaF3 cells, using
standard transfection and selection methods, to make a BHK/KZ134 or
BaF3/KZ134 cell line respectively.
[0407] The bioassay cell line is transfected with mzcytor16-mpl
fusion receptor alone, or co-transfected along with one of a
variety of other known receptor subunits. Receptor complexes
include but are not limited to mzcytor16-mpl receptor only, various
combinations of mzcytor16-mpl receptor with one or more of the
CRF2-4, IL-9, IL-10, zcytor11, zcytor7 class II cytokine receptor
subunits, or IL-4 receptor components, or the IL-2 receptor
components (IL-2R.alpha., IL-2R.beta., IL-2R.gamma.); Zcytor16-mpl
receptor with one or more of the IL-4/IL-13 receptor family
receptor components (IL-4R.alpha., IL-13R.alpha., IL-13R.alpha.'),
as well as other Interleukin receptors (e.g., IL-15 R.alpha.,
IL-7R.alpha., IL-9R.alpha., IL-21R (Zcytor16)). Each independent
receptor complex cell line is then assayed in the presence of
cytokine-conditioned media or purified cytokines and luciferase
activity measured using routine methods. The untransfected bioassay
cell line serves as a control for the background luciferase
activity, and is thus used as a baseline to compare signaling by
the various receptor complex combinations. The conditioned medium
or cytokine that binds the mouse zyctor16 receptor in the presence
of the correct receptor complex, is expected to give a luciferase
readout of approximately 5 fold over background or greater.
[0408] As an alternative, a similar assay can be performed wherein
BaB/mZcytor16-mpl cell lines are co-transfected as described above
and proliferation measured (Example 8).
Example 10
COS Cell Transfection and Secretion Trap
[0409] COS cell transfections were performed as follows: A mixture
of 0.5 .mu.g DNA and 5 .mu.l lipofectamine (Gibco BRL) in 92 ul
serum free DMEM media (55 mg sodium pyruvate, 146 mg L-glutamine, 5
mg transferrin, 2.5 mg insulin, 1 .mu.g selenium and 5 mg fetuin in
500 ml DMEM) was incubated at room temperature for 30 minutes and
then 400 .mu.l serum free DMEM media added. A 500 .mu.l mixture was
added onto COS cells plated on 12-well tissue culture plate at
1.5.times.10.sup.5 COS cells/well and previously incubated for 5
hours at 37.degree. C. An additional 500 .mu.l 20% FBS DMEM media
(100 ml FBS, 55 mg sodium pyruvate and 146 mg L-glutamine in 500 ml
DMEM) was added and the plates were incubated overnight.
[0410] The secretion trap was performed as follows: Media was
rinsed off cells with PBS and fixed for 15 minutes with 1.8%
Formaldehyde in PBS. Cells were then washed with TNT (0.1M
Tris-HCl, 0.15M NaCl, and 0.05% Tween-20 in H.sub.2O). Cells were
permeated with 0.1% Triton-X in PBS for 15 minutes and washed again
with TNT. C ells were blocked for 1 hour with TNB (0.1M Tris-HCl,
0.15M NaCl and 0.5% Blocking Reagent (NEN Renaissance TSA-Direct
Kit; NEN) in H.sub.2O Cells were again washed with TNT. Cells were
then incubated for 1 hour with 1-3 .mu.g/ml human Zcytor16 soluble
receptor Fc4 fusion protein (Zcytor16sR-Fc4) (Example 11) in TNB.
Cells were washed with TNT, and then incubated for another hour
with 1:200 diluted goat-anti-human Ig-HRP (Fc specific; Jackson
ImmunoResearch Laboratories, Inc.) in TNB. Cells were again washed
with TNT. Antibodies positively binding to the human Zcytor16sR-Fc4
were detected with fluorescein tyramide reagent diluted 1:50 in
dilution buffer (NEN kit) and incubated for 4-6 minutes. Cells were
again washed with TNT. Cells were preserved with Vectashield
Mounting Media (Vector Labs) diluted 1:5 in TNT. Cells were
visualized using FITC filter on fluorescent microscope.
[0411] Since Zcytor16 is a Class II cytokine receptor, the binding
of human Zcytor16sR/Fc4 fusion protein with known or orphan Class
II cytokines was tested. The pZP7 expression vectors containing
cDNAs of cytokines (including IL-TIF, interferon alpha, interferon
beta, interferon gamma, IL-10, amongst others were transfected into
COS cells, and the binding of Zcytor16sR/Fc4 to transfected COS
cells were carried out using the secretion trap assay described
above. Human IL-TIF showed positive binding. Based on these data,
human IL-TIF and human Zcytor16 is a potential ligand-receptor
pair. Similar methods are used to show that mouse Zcytor16 is an
orthologous class II receptor that binds IL-TIF.
Example 11
Purification of Zcytor16-Fc4 Polypeptide from Transfected BHK 570
Cells
[0412] Unless otherwise noted, all operations were carried out at
4.degree. C. The following procedure was used for purifying human
Zcytor16 polypeptide containing C-terminal fusion to human Fc4
(Zcytor16-Fc4; Example 1). About 16,500 ml of conditioned media
from BHK 570 cells transfected with human Zcytor16-Fc4 (Example 2)
was filtered through a 0.2 um sterilizing filter and then
supplemented with a solution of protease inhibitors, to final
concentrations of, 0.001 mM leupeptin (Boerhinger-Mannheim,
Indianapolis, Ind.), 0.001 mM pepstatin (Boerhinger-Mannheim) and
0.4 mM Pefabloc (Boerhinger-Mannheim). A Poros protein A50 column
(20 ml bed volume, Applied Biosystems) was packed and washed with
400 ml PBS (Gibco/BRL) The supplemented conditioned media was
passed over the column with a flow rate of 15 ml/minute, followed
by washing with 800 ml PBS (BRL/Gibco). Zcytor16-Fc4 was eluted
from the column with 0.1 M Glycine pH 3.0 and 5 ml fractions were
collected directly into 0.5 ml 2M Tris pH 7.8, to adjust the final
pH to 7.4 in the fractions.
[0413] Column performance was characterized through western
blotting of reducing SDS-PAGE gels of the starting media and column
pass through. Western blotting used anti-human IgG HRP (Amersham)
antibody, which showed an immunoreactive protein at 60,000 Da in
the starting media, with nothing in the pass through, suggesting
complete capture. The protein A50 eluted fractions were
characterized by reducing SDS PAGE gel. This gel showed an
intensely Coomassie stained band at 60,000 Da in fractions 3 to 11.
Fractions 3 to 11 were pooled.
[0414] Protein A 50 elution pool was concentrated from 44 ml to 4
ml using a 30,000 Da Ultrafree Biomax centrifugal concentrator (15
ml volume, Millipore). A Sephacryl S-300 gel filtration column (175
ml bed volume; Pharmacia) was washed with 350 ml PBS (BRL/Gibco).
The concentrated pool was injected over the column with a flow rate
of 1.5 ml/min, followed by washing with 225 ml PBS (BRL/Gibco).
Eluted peaks were collected into 2 ml fractions.
[0415] Eluted fractions were characterized by reducing and
non-reducing silver stained (Geno Technology) SDS PAGE gels.
Reducing silver stained SDS PAGE gels showed an intensely stained
band at 60,000 Da in fractions 14-31, while non-reducing silver
stained SDS PAGE gels showed an intensely stained band at 160,000
Da in fractions 14-31. Fractions 1-13 showed many bands of various
sizes. Fractions 14-31 were pooled, concentrated to 22 ml using
30,000 Da Ultrafree Biomax centrifugal concentrator (15 ml volume,
Millipore). This concentrate was filtered through a 0.2 .mu.m
Acrodisc sterilizing filter (Pall Corporation).
[0416] The protein concentration of the concentrated pooled
fractions was performed by BCA analysis (Pierce, Rockford, Ill.)
and the material was aliquoted, and stored at -80.degree. C.
according to our standard procedures. The concentration of the
pooled fractions was 1.50 mg/ml.
[0417] This method is also used to purify the mouse Zcytor16-Fc4
and heterologous class II receptor Fc4 fusions.
Example 12
Human and Mouse Zcytor16 Tissue Distribution in Tissue Panels Using
Northern Blot and PCR
[0418] A. Human Zcytor16 Tissue Distribution using Northern Blot
and Dot Blot
[0419] Northern blot analysis was performed using Human Multiple
Tissue Northern Blots I, II, III (Clontech) and an in house
generated U-937 northern blot. U-937 is a human monoblastic
promonocytic cell line. The cDNA probe was generated using oligos
ZC25,963 (SEQ ID NO:16) and ZC28,354 (SEQ ID NO:17). The PCR
conditions were as follows: 94.degree. for 1 minute; 30 cycles of
94.degree., 15 seconds; 60.degree., 30 seconds; 72.degree., 30
seconds and a final extension for 5 minutes at 72.degree.. The 364
bp product was gel purified by gel electrophoresis on a 1% TBE gel
and the band was excised with a razor blade. The cDNA was extracted
from the agarose using the QIAquick Gel Extraction Kit (Qiagen). 94
ng of this fragment was radioactively labeled with .sup.32P-dCTP
using Rediprime II (Amersham), a random prime labeling system,
according to the manufacturer's specifications. Unincorporated
radioactivity was removed using a Nuc-Trap column (Stratagene)
according to manufacturer's instructions. Blots were prehybridized
at 65.degree. for 3 hours in ExpressHyb (Clontech) solution. Blots
were hybridized overnight at 65.degree. in Expresshyb solution
containing 1.0.times.10.sup.6 cpm/ml of labeled probe, 0.1 mg/ml of
salmon sperm DNA and 0.5 .mu.g/ml of human cot-1 DNA. Blots were
washed in 2.times.SSC, 0.1% SDS at room temperature with several
solution changes then washed in 0.1.times.SSC. 0.1% SDS at
55.degree. for 30 minutes twice. Transcripts of approximately 1.6
kb and 3.0 kb size were detected in spleen and placenta, but not
other tissues examined. The same sized transcripts plus an
additional approximate 1.2 kb transcript was detected in U-937 cell
line.
B. Tissue Distribution of Human Zcytor16 in Tissue cDNA Panels
Using PCR
[0420] A panel of cDNAs from human tissues was screened for
Zcytor16 expression using PCR. The panel was made in-house and
contained 77 marathon cDNA and cDNA samples from various normal and
cancerous human tissues and cell lines are shown in Table 5, below.
The cDNAs came from in-house libraries or marathon cDNAs from
in-house RNA preps, Clontech RNA, or Invitrogen RNA. The marathon
cDNAs were made using the marathon-Ready.TM. kit (Clontech, Palo
Alto, Calif.) and QC tested with clathrin primers ZC21195 (SEQ ID
NO:18) and ZC21196 (SEQ ID NO:19) and then diluted based on the
intensity of the clathrin band. To assure quality of the panel
samples, three tests for quality control (QC) were run: (1) To
assess the RNA quality used for the libraries, the in-house cDNAs
were tested for average insert size by PCR with vector oligos that
were specific for the vector sequences for an individual cDNA
library; (2) Standardization of the concentration of the cDNA in
panel samples was achieved using standard PCR methods to amplify
full length alpha tubulin or G3PDH cDNA using a 5' vector oligo
ZC14,063 (SEQ ID NO:20) and 3' alpha tubulin specific oligo primer
ZC17,574 (SEQ ID NO:21) or 3' G3PDH specific oligo primer ZC17,600
(SEQ ID NO:22); and (3) a sample was sent to sequencing to check
for possible ribosomal or mitochondrial DNA contamination. The
panel was set up in a 96-well format that included a human genomic
DNA (Clontech, Palo Alto, Calif.) positive control sample. Each
well contained approximately 0.2-100 pg/.mu.l of cDNA.
[0421] The PCR reactions were set up using oligos ZC25,963 (SEQ ID
NO:30) and ZC25,964 (SEQ ID NO:31), Advantage 2 DNA Polymerase Mix
(Clontech) and Rediload dye (Research Genetics, Inc., Huntsville,
Ala.). The amplification was carried out as follow: 1 cycle at
94.degree. C. for 1 minute, 38 cycles of 94.degree. C. for 10
seconds, 60.degree. C. for 30 seconds and 72.degree. C. for 30
seconds, followed by 1 cycle at 72.degree. C. for 5 minutes. The
correct predicted DNA fragment size was observed in bone marrow,
fetal heart, fetal kidney, fetal muscle, fetal skin, heart, mammary
gland, placenta, salivary gland, skeletal muscle, small intestine,
spinal cord, spleen, kidney, fetal brain, esophageal tumor, uterine
tumor, stomach tumor, ovarian tumor, rectal tumor, lung tumor and
RPMI-1788 (a B-lymphocyte cell line). Zcytor16 expression was not
observed in the other tissues and cell lines tested in this panel.
The expression pattern of Zcytor16 shows expression in certain
tissue-specific tumors especially, e.g., ovarian cancer, stomach
cancer, uterine cancer, rectal cancer, lung cancer and esophageal
cancer, where zcytor16 is not expressed in normal tissue, but is
expressed in the tumor tissue. One of skill in the art would
recognize that the polynucleotides, polypeptides, antibodies, and
binding partners of the present invention can be used as a
diagnostic to detect cancer, or cancer tissue in a biopsy, tissue,
or histologic sample, particularly e.g., ovarian cancer, stomach
cancer, uterine cancer, rectal cancer, lung cancer and esophageal
cancer tissue. Such diagnostic uses for the molecules of the
present invention are known in the art and described herein.
[0422] In addition, because the expression pattern of human
Zcytor16, one of IL-TIF's receptors, shows expression in certain
specific tissues as well as tissue-specific tumors, binding
partners including the natural ligand, IL-TIF, can also be used as
a diagnostic to detect specific tissues (normal or abnormal),
cancer, or cancer tissue in a biopsy, tissue, or histologic sample,
where IL-TIF receptors are expressed, and particularly e.g.,
ovarian cancer, stomach cancer, uterine cancer, rectal cancer, lung
cancer and esophageal cancer tissue. IL-TIF can also be used to
target other tissues wherein its receptors, e.g., Zcytor16 and
zcytor11 are expressed. Moreover, such binding partners could be
conjugated to chemotherapeutic agents, toxic moieties and the like
to target therapy to the site of a tumor or diseased tissue. Such
diagnostic and targeted therapy uses are known in the art and
described herein.
[0423] A commercial 1st strand cDNA panel (Human Blood Fractions
MTC Panel, Clontech, Palo Alto, Calif.) was also assayed as above.
The panel contained the following samples: mononuclear cells,
activated mononuclear cells, resting CD4+ cells, activated CD4+
cells, resting CD8+ cells, activated CD8+ cells, resting CD14+
cells, resting CD19+ cells and activated CD19+ cells. Activated
CD4+ cells and activated CD19+ cells showed Zcytor16 expression,
whereas the other cells tested, including resting CD4+ cells and
resting CD19+ cells, did not.
TABLE-US-00005 TABLE 5 Tissue #samples adrenal gland 1 bone marrow
3 cervix 1 fetal brain 3 fetal kidney 1 fetal lung 1 heart 2 kidney
2 lung 1 mammary gland 1 ovary 1 pituitary 2 prostate 3 salivary
gland 2 small intestine 1 spleen 1 stomach 1 testis 5 thymus 1
thyroid 2 trachea 1 esophageal tumor 1 liver tumor 1 rectal tumor 1
uterine tumor 2 HaCAT library 1 HPVS library 1 K562 1 bladder 1
brain 2 colon 1 fetal heart 2 fetal liver 2 fetal skin 1 fetal
muscle 1 liver 1 lymph node 1 melanoma 1 pancreas 1 placenta 3
rectum 1 skeletal muscle 1 spinal cord 2 uterus 1 adipocyte library
1 islet 1 prostate SMC 1 RPMI 1788 1 WI38 1 lung tumor 1 ovarian
tumor 1 stomach tumor 1 CD3+ library 1 HPV library 1 MG63 library
1
C. Tissue Distribution of Human Zcytor16 in Human Tissue and Cell
Line RNA Panels Using RT-PCR
[0424] A panel of RNAs from human cell lines was screened for human
Zcytor16 expression using RT-PCR. The panels were made in house and
contained 84 RNAs from various normal and cancerous human tissues
and cell lines as shown in Tables 6-9 below. The RNAs were made
from in house or purchased tissues and cell lines using the RNAeasy
Midi or Mini Kit (Qiagen, Valencia, Calif.). The panel was set up
in a 96-well format with 100 ngs of RNA per sample. The RT-PCR
reactions were set up using oligos ZC25,963 (SEQ ID NO:30) and
ZC25,964 (SEQ ID NO:31), Rediload dye and SUPERSCRIPT One Step
RT-PCR System (Life Technologies, Gaithersburg, Md.). The
amplification was carried out as follows: one cycle at 55.degree.
for 30 minutes followed by 40 cycles of 94.degree., 15 seconds;
59.degree., 30 seconds; 72.degree., 30 seconds; then ended with a
final extension at 72.degree. for 5 minutes. 8 to 10 .mu.ls of the
PCR reaction product was subjected to standard Agarose gel
electrophoresis using a 4% agarose gel. The correct predicted cDNA
fragment size of 184 bps was observed in cell lines U-937, HL-60,
ARPE-19, HaCat#1, HaCat#2, HaCat#3, and HaCat#4; bladder, cancerous
breast, normal breast adjacent to a cancer, bronchus, colon,
ulcerative colitis colon, duodenum, endometrium, esophagus,
gastro-esophageal, heart left ventricle, heart ventricle, ileum,
kidney, lung, lymph node, lymphoma, mammary adenoma, mammary gland,
cancerous ovary, pancreas, parotid and skin, spleen lymphoma and
small bowel. Zcytor16 expression was not observed in the other
tissues and cell lines tested in this panel.
[0425] Zcytor16 is detectably expressed by PCR in normal tissues:
such as, the digestive system, e.g., esophagus, gastro-esophageal,
pancreas, duodenum, lleum, colon, small bowel; the female
reproductive system, e.g., mammary gland, endometrium, breast
(adjacent to cancerous tissues); and others systems, e.g., lymph
nodes, skin, parotid, bladder, bronchus, heart ventricles, and
kidney. Moreover, Zcytor16 is detectably expressed by PCR in
several human tumors: such as tumors associated with female
reproductive tissues e.g., mammary adenoma, ovary cancer, uterine
cancer, other breast cancers; and other tissues such as lymphoma,
stomach tumor, and lung tumor. The expression of Zcytor16 is found
in normal tissues of female reproductive organs, and in some tumors
associated with these organs. As such, Zcytor16 can serve as a
marker for these tumors wherein the Zcytor16 may be over-expressed.
Several cancers positive for Zcytor16 are associated with
ectodermal/epithelial origin (mammary adenoma, and other breast
cancers). Hence, Zcytor16 can serve as a marker for epithelial
tissue, such as epithelial tissues in the digestive system and
female reproductive organs (e.g., endometrial tissue, columnar
epithelium), as well as cancers involving epithelial tissues.
Moreover, in a preferred embodiment, Zcytor16 can serve as a marker
for certain tissue-specific tumors especially, e.g., ovarian
cancer, stomach cancer, uterine cancer, rectal cancer, lung cancer
and esophageal cancer, where zcytor 16 is not expressed in normal
tissue, but is expressed in the tumor tissue. Use of
polynucleotides, polypeptides, and antibodies of the present
invention for diagnostic purposes are known in the art, and
disclosed herein.
TABLE-US-00006 TABLE 6 Tissue #samples adrenal gland 6 bladder 3
brain 2 brain meningioma 1 breast 1 cancerous breast 4 normal
breast 5 adjacent to cancer bronchus 3 colon 15 cancerous colon 1
normal colon 1 adjacent to cancer ulcerative 1 colitis colon
duodenum 1 endometrium 5 cancerous endometrium 1 gastric cancer 1
esophagus 7 gastro-esophageal 1 heart aorta 1 heart left ventricle
4 heart right ventricle 2 heart ventricle 1 ileum 3 kidney 15
cancerous kidney 1
TABLE-US-00007 TABLE 7 Tissue/Cell Line #samples 293 1 C32 1
HaCat#1 1 HaCat#2 1 HaCat#3 1 HaCat#4 1 WI-38 1 WI-38 + 2 um
ionomycin #1 1 WI-38 + 2 um ionomycin #2 1 WI-38 + 5 um ionomycin#1
1 WI-38 + 5 um ionomycin#2 1 Caco-2, 1 Caco-2, differentiated 1
DLD-1 1 HRE 1 HRCE 1 MCF7 1 PC-3 1 TF-1 1 5637 1 143B 1 ME-180 1
prostate epithelia 1 U-2 OS 1 T-47D 1 Mg-63 1 Raji 1 U-373 MG 1
A-172 1 CRL-1964 1 CRL-1964 + butryic acid 1 HUVEC 1 SK-Hep-1 1
SK-Lu-1 1 Sk-MEL-2 1 K562 1 BeWo 1 FHS74.Int 1 HL-60 1 Malme 3M 1
FHC 1 HREC 1 HBL-100 1 Hs-294T 1 Molt4 1 RPMI 1 U-937 1 A-375 1
HCT-15 1 HT-29 1 MRC-5 1 RPT-1 1 RPT-2 1 WM-115 1 A-431 1 WERI-Rb-1
1 HEL-92.1.7 1 HuH-7 1 MV-4-11 1 U-138 1 CCRF-CEM 1 Y-79 1 A-549 1
EL-4 1 HeLa 229 1 HUT 78 1 NCI-H69 1 SaOS2 1 USMC 1 UASMC 2 AoSMC 1
UtSMC 1 HepG2 1 HepG2- IL6 1 NHEK#1 1 NHEK#2 1 NHEK#3 1 NHEK#4 1
ARPE-19 1 G-361 1 HISM 1 3AsubE 1 INT407 1
TABLE-US-00008 TABLE 8 Tissue #samples liver 10 lymph node 1
lymphoma 4 mammary adenoma 1 mammary gland 3 melinorioma 1
osteogenic sarcoma 2 pancreas 4 skin 5 sarcoma 2 lung 13 cancerous
lung 2 normal lung adjacent 1 to cancer muscle 3 neuroblastoma 1
omentum 2 ovary 6 cancerous ovary 2 parotid 7 salivary gland 4
TABLE-US-00009 TABLE 9 Tissue #samples small bowel 10 spleen 3
spleen lymphoma 1 stomach 13 stomach cancer 1 uterus 11 uterine
cancer 1 thyroid 9
D. Tissue Distribution of Mouse Zcytor16 in Tissue Panels Using
PCR
[0426] A panel of cDNAs from murine tissues was screened for mouse
Zcytor16 expression using PCR. The panel was made in-house and
contained 49 marathon cDNA and cDNA samples from various normal and
cancerous murine tissues and cell lines are shown in Table 10,
below. The cDNAs are either in-house cDNA libraries marathon cDNAs.
The RNA used to create these cDNA's came from either in-house RNA
preps, Clontech RNA, or Invitrogen RNA. The mouse marathon cDNAs
were made using the marathon-Ready.TM. kit (Clontech, Palo Alto,
Calif.) and QC tested with mouse transferrin receptor primers
ZC10,651 (SEQ ID NO:42) and ZC10,565 (SEQ ID NO:43) and then
diluted based on the intensity of the transferrin band. The
in-house libraries were diluted to provide 25 ng per well of cDNA.
To assure quality of the amplified library samples in the panel,
three tests for quality control (QC) were run: (1) To assess the
RNA quality used for the libraries, the in-house cDNAs were tested
for average insert size by PCR with vector oligos that were
specific for the vector sequences for an individual cDNA library;
(2) Standardization of the concentration of the cDNA in panel
samples was achieved using standard PCR methods to amplify full
length alpha tubulin or G3PDH cDNA using a 5' vector oligo:
ZC14,063 (SEQ ID NO:20) and 3' alpha tubulin specific oligo primer
ZC17,574 (SEQ ID NO:21) or 3' G3PDH specific oligo primer ZC17,600
(SEQ ID NO:22); and (3) a sample was sent to sequencing to check
for possible ribosomal or mitochondrial DNA contamination. The
panel was set up in a 96-well format that included a mouse genomic
DNA (Clontech, Palo Alto, Calif.) positive control sample. Each
well contained approximately 0.2-100 pg/.mu.l of cDNA. The PCR was
set up using oligos ZC38,001 (SEQ ID NO:44) and ZC38,022 (SEQ ID
NO:45), Advantage 2 Taq Polymerase (Clontech, Palo Alto, Calif.),
and Rediload dye (Research Genetics, Inc., Huntsville, Ala.). The
amplification was carried out as follows: 1 cycle at 94.degree. C.
for 2 minutes; 35 cycles of 94.degree. C. for 10 seconds,
60.degree. C. for 20 seconds and 68.degree. C. for 30 seconds,
followed by 1 cycle at 68.degree. C. for 7 minutes. About 12.5
.mu.l of the PCR reaction product was subjected to standard Agarose
gel electrophoresis using a 4% agarose gel.
[0427] The correct predicted DNA fragment size of 114 base pairs
was observed in Lung, Pancreas, Placenta, Salivary Gland, Skeletal
Muscle, Skin, Small Intestine, Smooth Muscle, Spleen, Stomach, and
Testis cDNAs. These initial expression results for the mouse
Zcytor16 corroborated the expression data seen for human Zcytor16
discussed herein, suggesting that the use of mouse Zcytor16 in an
animal model, such as mouse, would reasonably reflect the in vivo
expression and function of Zcytor16 seen in humans, and discussed
herein.
TABLE-US-00010 TABLE 10 Tissue/Cell line #samples 229 1 7F2 1
Adipocytes- 1 Amplified aTC1.9 1 Brain 4 CCC4 1 CD90+ Amplified 1
OC10B 1 Dentritic 1 Embyro 1 Heart 2 Kidney 3 Liver 2 Lung 2 MEWt#2
1 P388D1 1 Pancreas 1 Placenta 2 Jakotay-Prostate Cell Line 1
Nelix-Prostate Cell Line 1 Paris-Prostate Cell Line 1
Torres-Prostate Cell Line 1 Tuvak-Prostate Cell Line 1 Salivary
Gland 2 Skeletal Muscle 1 Skin 2 Small Intestine 1 Smooth Muscle 2
Spleen 2 Stomach 1 Testis 3 Thymus 1
Example 13
Construction of Expression Vector Expressing Full-Length
zcytor11
[0428] The entire zcytor11 receptor (commonly owned U.S. Pat. No.
5,965,704) was isolated by digestion with EcoRI and XhoI from
plasmid pZP7P, containing full-length zcytor11 receptor cDNA (SEQ
ID NO:24; amino acid sequence in SEQ ID NO:25) and a puromycin
resistance gene. The digest was run on a 1% low melting point
agarose (Boerhinger Mannheim) gel and the approximately 1.5 kb
zcytor11 cDNA was isolated using Qiaquick.TM. gel extraction kit
(Qiagen) as per manufacturer's instructions. The purified zcytor11
cDNA was inserted into an expression vector as described below.
[0429] Recipient expression vector pZP7Z was digested with EcoRI
(BRL) and XhoI (Boehringer Mannheim) as per manufacturer's
instructions, and gel purified as described above. This vector
fragment was combined with the EcoRI and XhoI cleaved zcytor11
fragment isolated above in a ligation reaction. The ligation was
run using T4 Ligase (BRL) at 12.degree. C. overnight. A sample of
the ligation was electroporated in to DH10B electroMAX.TM.
electrocompetent E. coli cells (25 .mu.F, 200.OMEGA., 1.8V).
Transformants were plated on LB+Ampicillin plates, and single
colonies were picked into 2 ml LB+Ampicillin and grown overnight.
Plasmid DNA was isolated using Wizard Minipreps (Promega), and each
was digested with EcoRI and XhoI to confirm the presence of insert.
The insert was approximately 1.5 kb, and was full-length. Digestion
with SpeI and PstI was used to confirm the identity of the
vector.
Example 14
Construction of BaF3 Cells Expressing the CRF2-4 Receptor
(BaF3/CRF2-4 Cells) and BaF3 Cells Expressing the CRF2-4 Receptor
with the zcytor11 Receptor (BaF3/CRF2-4/zcytor11 Cells)
[0430] BaF3 cells expressing the full-length CFR2-4 receptor were
constructed, using 30 .mu.g of a CFR2-4 expression vector,
described below. The BaF3 cells expressing the CFR2-4 receptor were
designated as BaF3/CFR2-4. These cells were used as a control, and
were further transfected with full-length zcytor11 receptor (U.S.
Pat. No. 5,965,704) and used to construct a screen for IL-TIF
activity as described below.
A. Construction of BaF3 Cells Expressing the CRF2-4 Receptor
[0431] The full-length cDNA sequence of CRF2-4 (Genbank Accession
No. Z17227) was isolated from a Daudi cell line cDNA library, and
then cloned into an expression vector pZP7P.
[0432] BaF3, an interleukin-3 (IL-3) dependent pre-lymphoid cell
line derived from murine bone marrow (Palacios and Steinmetz, Cell
41: 727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol. 6:
4133-4135, 1986), was maintained in complete media (RPMI medium
(JRH Bioscience Inc., Lenexa, Kans.) supplemented with 10%
heat-inactivated fetal calf serum, 2 ng/ml murine IL-3 (mIL-3) (R
& D, Minneapolis, Minn.), 2 mM L-glutaMax-1.TM. (Gibco BRL), 1
mM Sodium Pyruvate (Gibco BRL), and PSN antibiotics (GIBCO BRL)).
Prior to electroporation, CRF2-4/pZP7P was prepared and purified
using a Qiagen Maxi Prep kit (Qiagen) as per manufacturer's
instructions. For electroporation, BaF3 cells were washed once in
serum-free RPMI media and then resuspended in serum-free RPMI media
at a cell density of 10.sup.7 cells/ml. One ml of resuspended BaF3
cells was mixed with 30 .mu.g of the CRF2-4/pZP7P plasmid DNA and
transferred to separate disposable electroporation chambers (GIBCO
BRL). Following a 15-minute incubation at room temperature the
cells were given two serial shocks (800 lFad/300 V.; 1180 lFad/300
V.) delivered by an electroporation apparatus (CELL-PORATOR.TM.;
GIBCO BRL). After a 5-minute recovery time, the electroporated
cells were transferred to 50 ml of complete media and placed in an
incubator for 15-24 hours (37.degree. C., 5% CO.sub.2). The cells
were then spun down and resuspended in 50 ml of complete media
containing 2 .mu.g/ml puromycin in a T-162 flask to isolate the
puromycin-resistant pool. Pools of the transfected BaF3 cells,
hereinafter called BaF3/CRF2-4 cells, were assayed for signaling
capability as described below. Moreover these cells were further
transfected with zcytor11 receptor as described below.
B. Construction of BaF3 Cells Expressing CRF2-4 and zcytor11
Receptors
[0433] BaF3/CRF2-4 cells expressing the full-length zcytor11
receptor were constructed as per Example 5A above, using 30 .mu.g
of the zcytor11 expression vector, described in Example 6 above.
Following recovery, transfectants were selected using 200 .mu.g/ml
zeocin and 2 .mu.g/ml puromycin. The BaF3/CRF2-4 cells expressing
the zcytor11 receptor were designated as BaF3/CRF2-4/zcytor11
cells. These cells were used to screen for IL-TIF activity as well
as Zcytor16 antagonist activity described in Example 15.
Example 15
Screening for IL-TIF Antagonist Activity Using BaF3/CRF2-4/zcytor11
Cells Using an Alamar Blue Proliferation Assay
[0434] A. Screening for IL-TIF Activity Using BaF3/CRF2-4/zcytor11
Cells Using an Alamar Blue Proliferation Assay
[0435] Purified human IL-TIF-CEE (Example 19) was used to test for
the presence of proliferation activity as described below. Purified
human Zcytor16-Fc4 (Example 11) was used to antagonize the
proliferative response of the IL-TIF in this assay as described
below.
[0436] BaF3/CRF2-4/zcytor11 cells were spun down and washed in the
complete media, described in Example 7A above, but without mIL-3
(hereinafter referred to as "mIL-3 free media"). The cells were
spun and washed 3 times to ensure the removal of the mIL-3. Cells
were then counted in a hemacytometer. Cells were plated in a
96-well format at 5000 cells per well in a volume of 100 .mu.l per
well using the mIL-3 free media.
[0437] Proliferation of the BaF3/CRF2-4/zcytor11 cells was assessed
using IL-TIF-CEE protein diluted with mIL-3 free media to 50, 10,
2, 1, 0.5, 0.25, 0.13, 0.06 ng/ml concentrations. 100 .mu.l of the
diluted protein was added to the BaF3/CRF2-4/zcytor11 cells. The
total assay volume is 200 .mu.l. The assay plates were incubated at
37.degree. C., 5% CO.sub.2 for 3 days at which time Alamar Blue
(Accumed, Chicago, Ill.) was added at 20 .mu.l/well. Plates were
again incubated at 37.degree. C., 5% CO.sub.2 for 24 hours. Alamar
Blue gives a fluourometric readout based on number of live cells,
and is thus a direct measurement of cell proliferation in
comparison to a negative control. Plates were again incubated at
37.degree. C., 5% CO.sub.2 for 24 hours. Plates were read on the
Fmax.TM. plate reader (Molecular Devices Sunnyvale, Calif.) using
the SoftMax.TM. Pro program, at wavelengths 544 (Excitation) and
590 (Emmission). Results confirmed the dose-dependent proliferative
response of the BaF3/CRF2-4/zcytor11 cells to IL-TIF-CEE. The
response, as measured, was approximately 15-fold over background at
the high end of 50 ng/ml down to a 2-fold induction at the low end
of 0.06 ng/ml. The BaF3 wild type cells, and BaF3/CRF2-4 cells did
not proliferate in response to IL-TIF-CEE, showing that IL-TIF is
specific for the CRF2-4/zcytor11 heterodimeric receptor.
[0438] In order to determine if Zcytor16 is capable of antagonizing
IL-TIF activity, the assay described above was repeated using
purified soluble human Zcytor16/Fc4. When IL-TIF was combined with
Zcytor16 at 10 .mu.g/ml, the response to IL-TIF at all
concentrations was brought down to background. That the presence of
soluble human Zcytor16 ablated the proliferative effects of IL-TIF
demonstrates that it is a potent antagonist of the IL-TIF ligand.
This assay is also used to assess mouse Zcytor16 activity in
antagonizing IL-TIF. Similar results are expected.
Example 16
IL-TIF Activation of a Reporter Mini-Gene in MES13 Cells and
Inhibition of Activity by Human Zcytor16-Fc4
[0439] MES13 cells (ATCC No. CRL-1927) were plated at 10,000
cells/well in 96-well tissue culture clusters (Costar) in DMEM
growth medium (Life Technologies) supplemented with pyruvate and
10% serum (HyClone). Next day, the medium was switched to serum
free DMEM medium by substituting 0.1% BSA (Fraction V; Sigma) for
serum. This medium also contained the adenoviral construct KZ136
(below) that encodes a luciferase reporter mini-gene driven by SRE
and STAT elements, at a 1000:1 multiplicity of infection (m.o.i.),
i.e. 1000 adenoviral particles per cell. After allowing 24 h for
the incorporation of the adenoviral construct in the cells, the
media were changed and replaced with serum-free media. Human
recombinant IL-TIF with or without a recombinant Zcytor16-Fc4
fusion was added at the indicated final concentration in the well
(as described in Table 11, below). Dilutions of both the IL-TIF and
Zcytor16-Fc4 were performed in serum-free medium. 0.1% BSA was
added for a basal assay control. 4 h later, cells were lysed and
luciferase activity, denoting activation of the reporter gene, was
determined in the lysate using an Luciferase Assay System assay kit
(Promega) and a Labsystems Luminoskan luminometer (Labsystems,
Helsinki, Finland). Activity was expressed as luciferase units (LU)
in the lysate. Results are shown in Table 11, below.
TABLE-US-00011 TABLE 11 Level of IL-TIF (ng/ml) LU w/o zcytoR16 LU
w/10 .mu.g/ml zcytoR16 0 (basal BSA control) 103 .+-. 2 104 .+-. 2
0.03 105 .+-. 3 104 .+-. 4 0.3 108 .+-. 4 99 .+-. 6 3 134 .+-. 8 98
.+-. 15 30 188 .+-. 16 110 .+-. 3 300 258 .+-. 21 112 .+-. 30
[0440] These results demonstrate two things: First, that MES13
cells respond to human recombinant IL-TIF and therefore possess
endogenous functional receptors for the cytokine. Second, that the
human zcytoR16-Fc4 receptor fusion acts as an antagonist that
effectively blocks the response to IL-TIF, even at the highest dose
that this cytokine was used. Therefore, zcyto16 is an effective
antagonist of IL-TIF on cells (MES13) that are intrinsically
capable of responding to IL-TIF, i.e. cells that do not require
exogenous expression of additional receptor components to respond
to the cytokine. This assay is also used to assess mouse Zcytor16
activity in antagonizing IL-TIF. Similar results are expected.
[0441] The construction of the adenoviral KZ136 vector was as
follows. The original KZ136 vector is disclosed in Poulsen, L K et
al. J. Biol. Chem. 273:6228-6232, 1998. The CMV promoter/enhancer
and SV40 pA sequences were removed from pACCMV.pLpA (T. C. Becker
et al., Meth. Immunology 43:161-189, 1994.) and replaced with a
linker containing Asp718/KpnI and HindIII sites (oligos ZC13252
(SEQ ID NO:26) and ZC13453 (SEQ ID NO:27)). The STAT/SRE driven
luciferase reporter cassette was exised from vector KZ136 (Poulsen,
L K et al., supra.).) as a Asp718/KpnI-HindIII fragment and
inserted into the adapted pAC vector. Recombinant KZ136 Adenovirus
was produced by transfection with JM17 Adenovirus into 293 cells as
described in T. C. Becker et al. supra.). Plaque purified virus was
amplified and used to infect cultured cells at 5-50 pfu/cell 12-48
hours before assay. Luciferase reporter assays were performed as
described in 96 well microplates as per Poulsen, L K et al.,
supra.).
Example 17
Construct for Generating CEE-Tagged IL-TIF
[0442] Oligonucleotides were designed to generate a PCR fragment
containing the Kozak sequence and the coding region for human
IL-TIF, without its stop codon. These oligonucleotides were
designed with a KpnI site at the 5' end and a BamHI site at the 3'
end to facilitate cloning into pHZ200-CEE, our standard vector for
mammalian expression of C-terminal Glu-Glu tagged (SEQ ID NO:10)
proteins. The pHZ200 vector contains an MT-1 promoter.
[0443] PCR reactions were carried out using Turbo Pfu polymerase
(Stratagene) to amplify a IL-TIF cDNA fragment. About 20 ng human
IL-TIF polynucleotide template (SEQ ID NO:14), and oligonucleotides
ZC28590 (SEQ ID NO:28) and ZC28580 (SEQ ID NO:29) were used in the
PCR reaction. PCR reaction conditions were as follows: 95.degree.
C. for 5 minutes; 30 cycles of 95.degree. C. for 60 seconds,
55.degree. C. for 60 seconds, and 72.degree. C. for 60 seconds; and
72.degree. C. for 10 minutes; followed by a 4.degree. C. hold. PCR
products were separated by agarose gel electrophoresis and purified
using a QiaQuick.TM. (Qiagen) gel extraction kit. The isolated,
approximately 600 bp, DNA fragment was digested with KpnI and BamHI
(Boerhinger-Mannheim), gel purified as above and ligated into
pHZ200-CEE that was previously digested with KpnI and BamHI.
[0444] About one microliter of the ligation reaction was
electroporated into DH10B ElectroMax.TM. competent cells (GIBCO
BRL, Gaithersburg, Md.) according to manufacturer's direction and
plated onto LB plates containing 100 .mu.g/ml ampicillin, and
incubated overnight. Colonies were picked and screened by PCR using
oligonucleotides ZC28,590 (SEQ ID NO:28) and ZC28,580 (SEQ ID
NO:29), with PCR conditions as described above. Clones containing
inserts were then sequenced to confirm error-free IL-TIF inserts.
Maxipreps of the correct pHZ200-IL-TIF-CEE construct, as verified
by sequence analysis, were performed.
Example 18
Transfection and Expression of IL-TIF Soluble Receptor
Polypeptides
[0445] BHK 570 cells (ATCC No. CRL-10314), were plated at about
1.2.times.10.sup.6 cells/well (6-well plate) in 800 .mu.l of serum
free (SF) DMEM media (DMEM, Gibco/BRL High Glucose) (Gibco BRL,
Gaithersburg, Md.). The cells were transfected with an expression
plasmid containing IL-TIF-CEE described above (Example 17), using
Lipofectin.TM. (Gibco BRL), in serum free (SF) DMEM according to
manufacturer's instructions.
[0446] The cells were incubated at 37.degree. C. for approximately
five hours, then transferred to separate 150 mm MAXI plates in a
final volume of 30 ml DMEM/5% fetal bovine serum (FBS) (Hyclone,
Logan, Utah). The plates were incubated at 37.degree. C., 5%
CO.sub.2, overnight and the DNA: Lipofectin.TM. mixture was
replaced with selection media (5% FBS/DMEM with 1 .mu.M
methotrexate (MTX)) the next day.
[0447] Approximately 10-12 days post-transfection, colonies were
mechanically picked to 12-well plates in one ml of 5% FCS/DMEM with
5 .mu.M MTX, then grown to confluence. Positive expressing clonal
colonies Conditioned media samples were then tested for expression
levels via SDS-PAGE and Western analysis. A high-expressing clone
was picked and expanded for ample generation of conditioned media
for purification of the IL-TIF-CEE expressed by the cells (Example
19).
Example 19
Purification of IL-THF Soluble Receptors from BHK 570 Cells
[0448] Unless otherwise noted, all operations were carried out at
4.degree. C. The following procedure was used for purifying IL-TIF
polypeptide containing C-terminal GluGlu (EE) tags (SEQ ID NO:10).
Conditioned media from BHK cells expressing IL-TIF-CEE (Example 18)
was concentrated with an Amicon S10Y3 spiral cartridge on a ProFlux
A30. A Protease inhibitor solution was added to the concentrated
conditioned media to final concentrations of 2.5 mM
ethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St.
Louis, Mo.), 0.003 mM leupeptin (Boehringer-Mannheim, Indianapolis,
Ind.), 0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc
(Boehringer-Mannheim). Samples were removed for analysis and the
bulk volume was frozen at -80.degree. C. until the purification was
started. Total target protein concentrations of the concentrated
conditioned media were determined via SDS-PAGE and Western blot
analysis with the anti-EE HRP conjugated antibody.
[0449] About 100 ml column of anti-EE G-Sepharose (prepared as
described below) was poured in a Waters AP-5, 5 cm.times.10 cm
glass column. The column was flow packed and equilibrated on a
BioCad Sprint (PerSeptive BioSystems, Framingham, Mass.) with
phosphate buffered saline (PBS) pH 7.4. The concentrated
conditioned media was thawed, 0.2 micron sterile filtered, pH
adjusted to 7.4, then loaded on the column overnight with about 1
ml/minute flow rate. The column was washed with 10 column volumes
(CVs) of phosphate buffered saline (PBS, pH 7.4), then plug eluted
with 200 ml of PBS (pH 6.0) containing 0.5 mg/ml EE peptide
(Anaspec, San Jose, Calif.) at 5 ml/minute. The EE peptide used has
the sequence EYMPME (SEQ ID NO:10). The column was washed for 10
CVs with PBS, then eluted with 5 CVs of 0.2M glycine, pH 3.0. The
pH of the glycine-eluted column was adjusted to 7.0 with 2 CVs of
5.times.PBS, then equilibrated in PBS (pH 7.4). Five ml fractions
were collected over the entire elution chromatography and
absorbance at 280 and 215 nM were monitored; the pass through and
wash pools were also saved and analyzed. The EE-polypeptide elution
peak fractions were analyzed for the target protein via SDS-PAGE
Silver staining and Western Blotting with the anti-EE HRP
conjugated antibody. The polypeptide elution fractions of interest
were pooled and concentrated from 60 ml to 5.0 ml using a 10,000
Dalton molecular weight cutoff membrane spin concentrator
(Millipore, Bedford, Mass.) according to the manufacturer's
instructions.
[0450] To separate IL-TIF-CEE from other co-purifying proteins, the
concentrated polypeptide elution pooled fractions were subjected to
a POROS HQ-50 (strong anion exchange resin from PerSeptive
BioSystems, Framingham, Mass.) at pH 8.0. A 1.0.times.6.0 cm column
was poured and flow packed on a BioCad Sprint. The column was
counter ion charged then equilibrated in 20 mM TRIS pH 8.0 (Tris
(Hydroxymethyl Aminomethane)). The sample was diluted 1:13 (to
reduce the ionic strength of PBS) then loaded on the Poros HQ
column at 5 ml/minute. The column was washed for 10 CVs with 20 mM
Tris pH 8.0 then eluted with a 40 CV gradient of 20 mM Tris/1 M
sodium chloride (NaCl) at 10 ml/minute. 1.5 ml fractions were
collected over the entire chromatography and absorbance at 280 and
215 nM were monitored. The elution peak fractions were analyzed via
SDS-PAGE Silver staining. Fractions of interest were pooled and
concentrated to 1.5-2 ml using a 10,000 Dalton molecular weight
cutoff membrane spin concentrator (Millipore, Bedford, Mass.)
according to the manufacturer's instructions.
[0451] To separate IL-TIF-CEE polypeptide from free EE peptide and
any contaminating co-purifying proteins, the pooled concentrated
fractions were subjected to size exclusion chromatography on a
1.5.times.90 cm Sephadex S200 (Pharmacia, Piscataway, N.J.) column
equilibrated and loaded in PBS at a flow rate of 1.0 ml/min using a
BioCad Sprint. 1.5 ml fractions were collected across the entire
chromatography and the absorbance at 280 and 215 nM were monitored.
The peak fractions were characterized via SDS-PAGE Silver staining,
and only the most pure fractions were pooled. This material
represented purified IL-TIF-CEE polypeptide.
[0452] This purified material was finally subjected to a 4 ml
ActiClean Etox (Sterogene) column to remove any remaining
endotoxins. The sample was passed over the PBS equilibrated gravity
column four times then the column was washed with a single 3 ml
volume of PBS, which was pooled with the "cleaned" sample. The
material was then 0.2 micron sterile filtered and stored at
-80.degree. C. until it was aliquoted.
[0453] On Western blotted, Coomassie Blue and Silver stained
SDS-PAGE gels, the IL-TIF-CEE polypeptide was one major band. The
protein concentration of the purified material was performed by BCA
analysis (Pierce, Rockford, Ill.) and the protein was aliquoted,
and stored at -80.degree. C. according to standard procedures.
[0454] To prepare anti-EE Sepharose, a 100 ml bed volume of protein
G-Sepharose (Pharmacia, Piscataway, N.J.) was washed 3 times with
100 ml of PBS containing 0.02% sodium azide using a 500 ml Nalgene
0.45 micron filter unit. The gel was washed with 6.0 volumes of 200
mM triethanolamine, pH 8.2 (TEA, Sigma, St. Louis, Mo.), and an
equal volume of EE antibody solution containing 900 mg of antibody
was added. After an overnight incubation at 4.degree. C., unbound
antibody was removed by washing the resin with 5 volumes of 200 mM
TEA as described above. The resin was resuspended in 2 volumes of
TEA, transferred to a suitable container, and
dimethylpimilimidate-2HCl (Pierce, Rockford, Ill.) dissolved in
TEA, was added to a final concentration of 36 mg/ml of protein
G-Sepharose gel. The gel was rocked at room temperature for 45 min
and the liquid was removed using the filter unit as described
above. Nonspecific sites on the gel were then blocked by incubating
for 10 min. at room temperature with 5 volumes of 20 mM
ethanolamine in 200 mM TEA. The gel was then washed with 5 volumes
of PBS containing 0.02% sodium azide and stored in this solution at
4.degree. C.
Example 20
Human zcytor11 Tissue Distribution in Tissue Panels Using Northern
Blot and PCR
[0455] A. Human zcytor11 Tissue Distribution in Tissue Panels Using
PCR
[0456] A panel of cDNAs from human tissues was screened for
zcytor11 expression using PCR. The panel was made in-house and
contained 94 marathon cDNA and cDNA samples from various normal and
cancerous human tissues and cell lines are shown in Table 6 above.
Aside from the PCR reaction, the method used was as shown in
Example 12. The PCR reactions were set up using oligos ZC14,666
(SEQ ID NO: 32) and ZC14,742 (SEQ ID NO:33), Advantage 2 cDNA
polymerase mix (Clontech, Palo Alto, Calif.), and Rediload dye
(Research Genetics, Inc., Huntsville, Ala.). The amplification was
carried out as follows: 1 cycle at 94.degree. C. for 2 minutes, 40
cycles of 94.degree. C. for 15 seconds, 51.degree. C. for 30
seconds and 72.degree. C. for 30 seconds, followed by 1 cycle at
72.degree. C. for 7 minutes. The correct predicted DNA fragment
size was observed in bladder, brain, cervix, colon, fetal brain,
fetal heart, fetal kidney, fetal liver, fetal lung, fetal skin,
heart, kidney, liver, lung, melanoma, ovary, pancreas, placenta,
prostate, rectum, salivary gland, small intestine, testis, thymus,
trachea, spinal cord, thyroid, lung tumor, ovarian tumor, rectal
tumor, and stomach tumor. Zcytor11 expression was not observed in
the other tissues and cell lines tested in this panel.
[0457] A commercial 1st strand cDNA panel (Human Blood Fractions
MTC Panel, Clontech, Palo Alto, Calif.) was also assayed as above.
The panel contained the following samples: mononuclear cells,
activated mononuclear cells, resting CD4+ cells, activated CD4+
cells, resting CD8+ cells, activated CD8+ cells, resting CD14+
cells, resting CD19+ cells and activated CD19+ cells. All samples
except activated CD8+ and Activated CD19+ showed expression of
zcytor11.
[0458] B. Tissue Distribution of Zcytor11 in Human Cell Line and
Tissue Panels Using RT-PCR
[0459] A panel of RNAs from human cell lines was screened for
zcytor11 expression using RT-PCR. The panels were made in house and
contained 84 RNAs from various normal and cancerous human tissues
and cell lines as shown in Tables 7-10 above. The RNAs were made
from in house or purchased tissues and cell lines using the RNAeasy
Midi or Mini Kit (Qiagen, Valencia, Calif.). The panel was set up
in a 96-well format with 100 ngs of RNA per sample. The RT-PCR
reactions were set up using oligos ZC14,666 (SEQ ID NO:32) and
ZC14,742 (SEQ ID NO:33), Rediload dye and SUPERSCRIPT One Step
RT-PCR System (Life Technologies, Gaithersburg, Md.). The
amplification was carried out as follows: one cycle at 50.degree.
for 30 minutes followed by 45 cycles of 94.degree., 15 seconds;
52.degree., 30 seconds; 72.degree., 30 seconds; then ended with a
final extension at 72.degree. for 7 minutes. 8 to 10 uls of the PCR
reaction product was subjected to standard Agarose gel
electrophoresis using a 4% agarose gel. The correct predicted cDNA
fragment size was observed in adrenal gland, bladder, breast,
bronchus, normal colon, colon cancer, duodenum, endometrium,
esophagus, gastic cancer, gastro-esophageal cancer, heart
ventricle, ileum, normal kidney, kidney cancer, liver, lung, lymph
node, pancreas, parotid, skin, small bowel, stomach, thyroid, and
uterus. Cell lines showing expression of zcytor11 were A-431,
differentiated CaCO2, DLD-1, HBL-100, HCT-15, HepG2, HepG2+IL6,
HuH7, and NHEK #1-4. Zcytor11 expression was not observed in the
other tissues and cell lines tested in this panel.
[0460] In addition, because the expression pattern of zcytor11, one
of IL-TIF's receptors, shows expression in certain specific
tissues, binding partners including the natural ligand, IL-TIF, can
also be used as a diagnostic to detect specific tissues (normal or
abnormal), cancer, or cancer tissue in a biopsy, tissue, or
histologic sample, particularly in tissues where IL-TIF receptors
are expressed. IL-TIF can also be used to target other tissues
wherein its receptors, e.g., Zcytor16 and zcytor11 are expressed.
Moreover, such binding partners could be conjugated to
chemotherapeutic agents, toxic moieties and the like to target
therapy to the site of a tumor or diseased tissue. Such diagnostic
and targeted therapy uses are known in the art and described
herein.
[0461] The expression patterns of zcytor11 (above) and Zcytor16
(Example 12, and Example 21) indicated target tissues and cell
types for the action of IL-TIF, and hence IL-TIF antagonsists, such
as Zcytor16. The zcytor11 expression generally overlapped with
Zcytor16 expression in three physiologic systems: digestive system,
female reproductive system, and immune system. Moreover, the
expression pattern of the receptor (zcytor11) indicated that an
IL-TIF antagonist such as Zcytor16 would have therapeutic
application for human disease in two areas: inflammation (e.g.,
IBD, Chron's disease, pancreatitis) and cancer (e.g., ovary,
colon). That is, the polynucleotides, polypeptides and antibodies
of the present invention can be used to antagonize the
inflammatory, and other cytokine-induced effects of IL-TIF
interaction with the cells expressing the zcytor11 receptor.
[0462] Moreover, the expression of zcytor11 appeared to be
downregulated or absent in an ulcerative colitis tissue, HepG2
liver cell line induced by IL-6, activated CD8+ T-cells and CD19+
B-cells. However, Zcytor16 appeared to be upregulated in activated
CD19+ B-cells (Example 12), while zcytor11 is downregulated in
activated CD19+ cells, as compared to the resting CD19+ cells
(above). The expression of zcytor11 and Zcytor16 has a reciprocal
correlation in this case. These RT-PCR experiments demonstrate that
CD19+ peripheral blood cells, B lymphocytes, express receptors for
IL-TIF, namely zcytoR11 and zcytoR16. Furthermore B cells display
regulated expression of zcytoR11 and zcytoR16. B-lymphocytes
activated with mitogens decrease expression of zcytoR11 and
increase expression of zcytoR16. This represents a classical
feedback inhibition that would serve to dampen the activity of
IL-TIF on B cells and other cells as well. Soluble zcytoR16 would
act as an antagonist to neutralize the effects of IL-TIF on B
cells. This would be beneficial in diseases where B cells are the
key players: Autoimmune diseases including systemic lupus
erythmatosus (SLE), myasthenia gravis, immune complex disease, and
B-cell cancers that are exacerbated by IL-TIF. Also autoimmune
diseases where B cells contribute to the disease pathology would be
targets for zcytoR16 therapy: Multiple sclerosis, inflammatory
bowel disease (IBD) and rheumatoid arthritis are examples. ZcytoR16
therapy would be beneficial to dampen or inhibit B cells producing
IgE in atopic diseases including asthma, allergy and atopic
dermatitis where the production of IgE contributes to the
pathogenesis of disease.
[0463] B cell malignancies may exhibit a loss of the "feedback
inhibition" described above. Administration of zcytoR16 would
restore control of IL-TIF signaling and inhibit B cell tumor
growth. The administration of zcytoR16 following surgical resection
or chemotherapy may be useful to treat minimal residual disease in
patients with B cell malignancies. The loss of regulation may lead
to sustain or increased expression of zcytoR11. Thus creating a
target for therapeutic monoclonal antibodies targeting
zcytoR11.
Example 21
Identification of Cells Expressing Zcytor16 Using In Situ
Hybridization
[0464] Specific human tissues were isolated and screened for human
Zcytor16 expression by in situ hybridization. Various human tissues
prepared, sectioned and subjected to in situ hybridization included
cartilage, colon, appendix, intestine, fetal liver, lung, lymph
node, lymphoma, ovary, pancreas, placenta, prostate, skin, spleen,
and thymus. The tissues were fixed in 10% buffered formalin and
blocked in paraffin using standard techniques. Tissues were
sectioned at 4 to 8 microns. Tissues were prepared using a standard
protocol ("Development of non-isotopic in situ hybridization" at
The Laboratory of Experimental Pathology (LEP), NIEHS, Research
Triangle Park, N.C.; web address
http://dir.niehs.nih.gov/dirlep/ish.html). Briefly, tissue sections
were deparaffinized with HistoClear (National Diagnostics, Atlanta,
Ga.) and then dehydrated with ethanol. Next they were digested with
Proteinase K (50 .mu.g/ml) (Boehringer Diagnostics, Indianapolis,
Ind.) at 37.degree. C. for 2 to 7 minutes. This step was followed
by acetylation and re-hydration of the tissues.
[0465] One in situ probe was designed against the human Zcytor16
sequence (nucleotide 1-693 of SEQ ID NO:1), and isolated from a
plasmid containing SEQ ID NO:1 using standard methods. T3 RNA
polymerase was used to generate an antisense probe. The probe was
labeled with digoxigenin (Boehringer) using an In Vitro
transcription System (Promega, Madison, Wis.) as per manufacturer's
instruction.
[0466] In situ hybridization was performed with a
digoxigenin-labeled Zcytor16 probe (above). The probe was added to
the slides at a concentration of 1 to 5 .mu.mol/ml for 12 to 16
hours at 62.5.degree. C. Slides were subsequently washed in
2.times.SSC and 0.1.times.SSC at 55.degree. C. The signals were
amplified using tyramide signal amplification (TSA) (TSA, in situ
indirect kit; NEN) and visualized with Vector Red substrate kit
(Vector Lab) as per manufacturer's instructions. The slides were
then counter-stained with hematoxylin (Vector Laboratories,
Burlingame, Calif.).
[0467] Signals were observed in several tissues tested: The lymph
node, plasma cells and other mononuclear cells in peripheral
tissues were strongly positive. Most cells in the lymphatic nodule
were negative. In lymphoma samples, positive signals were seen in
the mitotic and multinuclear cells. In spleen, positive signals
were seen in scattered mononuclear cells at the periphery of
follicles were positive. In thymus, positive signals were seen in
scattered mononuclear cells in both cortex and medulla were
positive. In fetal liver, a strong signal was observed in a mixed
population of mononuclear cells in sinusoid spaces. A subset of
hepatocytes might also have been positive. In the inflamed
appendix, mononuclear cells in peyer's patch and infiltration sites
were positive. In intestine, some plasma cells and ganglia nerve
cells were positive. In normal lung, Zcytor16 was expressed in
alveolar epithelium and mononuclear cells in interstitial tissue
and circulation. In the lung carcinoma tissue, a strong signal was
observed in mostly plasma cells and some other mononuclear cells in
peripheral of lymphatic aggregates. In ovary carcinoma, epithelium
cells were strongly positive. Some interstitial cells, most likely
the mononuclear cells, were also positive. There was no signal
observed in the normal ovary. In both normal and pancreatitis
pancreas samples, acinar cells and some mononuclear cells in the
mesentery were positive. In the early term (8 weeks) placenta,
signal was observed in trophoblasts. In skin, some mononuclear
cells in the inflamed infiltrates in the superficial dermis were
positive. Keratinocytes were also weakly positive. In prostate
carcinoma, scatted mononuclear cells in interstitial tissues were
positive. In articular cartilage, chondrocytes were positive. Other
tissues tested including normal ovary and a colon adenocarcinoma
were negative.
[0468] In summary, the in situ data was consistent with expression
data described above for the Zcytor16. Zcytor16 expression was
observed predominately in mononuclear cells, and a subset of
epithelium was also positive. These results confirmed the presence
of Zcytor16 expression in immune cells and point toward a role in
inflammation, autoimmune disease, or other immune function, for
example, in binding pro-inflammatory cytokines, including but not
limited to IL-TIF. Moreover, detection of Zcytor16 expression can
be used for example as an marker for mononuclear cells in
histologic samples.
[0469] Zcytor16 is expressed in mononuclear cells, including normal
tissues (lymph nodes, spleen, thymus, pancreas and fetal liver,
lung), and abnormal tissues (inflamed appendix, lung carcinoma,
ovary carcinoma, pancreatitis, inflamed skin, and prostate
carcinoma). It is notable that plasma cells in the lymph node,
intestine, and lung carcinoma are positive for Zcytor16. Plasma
cells are immunologically activated lymphocytes responsible for
antibody synthesis. In addition, IL-TIF, is expressed in activated
T cells. In addition, the expression of Zcytor16 is detected only
in activated (but not in resting) CD4+ and CD19+ cells (Example
12). Thus, Zcytor16 can be used as a marker for or as a target in
isolating certain lymphocytes, such as mononuclear leucocytes and
limited type of activated leucocytes, such as activated CD4+ and
CD19+.
[0470] Furthermore, the presence of Zcytor16 expression in
activated immune cells such as activated CD4+ and CD19+ cells
showed that Zcytor16 may be involved in the body's immune defensive
reactions against foreign invaders: such as microorganisms and cell
debris, and could play a role in immune responses during
inflammation and cancer formation.
[0471] Moreover, as discussed herein, epithelium form several
tissues was positive for Zcytor16 expression, such as hepatocytes
(endoderm-derived epithelia), lung alveolar epithelium
(endoderm-derived epithelia), and ovary carcinoma epithelium
(mesoderm-derived epithelium). The epithelium expression of
Zcytor16 could be altered in inflammatory responses and/or
cancerous states in liver and lung. Thus, Zcyto16 could be used as
marker to monitor changes in these tissues as a result of
inflammation or cancer. Moreover, analysis of Zcytor16 in situ
expression showed that normal ovary epithelium is negative for
Zcytor16 expression, while it is strongly positive in ovary
carcinoma epithelium providing further evidence that Zcytor16
polynucleotides, polypeptides and antibodies can be used as a
diagnostic marker and/or therapeutic target for the diagnosis and
treatment of ovarian cancers, and ovary carcinoma, as described
herein.
[0472] Zcytor16 was also detected in other tissues, such as acinar
cells in pancreas (normal and pancreatitis tissues), trophoblasts
in placenta (ectoderm-derived), chondrocytes in cartilage
(mesoderm-derived), and ganglia cells in intestine
(ectoderm-derived). As such, Zcytor16 may be involved in
differentiation and/or normal functions of corresponding cells in
these organs. As such, potential utilities of Zcytor16 include
maintenance of normal metabolism and pregnancy, bone
formation/homeostasis, and physiological function of intestine, and
the like.
Example 22
In vivo affects of IL-TIF polypeptide
[0473] Mice (female, C57B1, 8 weeks old; Charles River Labs,
Kingston, N.Y.) were divided into three groups. An adenovirus
expressing an IL-TIF polypeptide (SEQ ID NO:15) was previously made
using standard methods. On day 0, parental or IL-TIF adenovirus was
administered to the first (n=8) and second (n=8) groups,
respectively, via the tail vein, with each mouse receiving a dose
of 1.times.10.sup.11 particles in 0.1 ml volume. The third group
(n=8) received no treatment. On days 12, mice were weighed and
blood was drawn from the mice. Samples were analyzed for complete
blood count (CBC) and serum chemistry. Statistically significant
elevations in neutrophil and platelet counts were detected in the
blood samples from the IL-TIF adenovirus administered group
relative to the parental adenovirus treated group. Also, lymphocyte
and red blood cell counts were significantly reduced from the
IL-TIF adenovirus administered group relative to the parental
adenovirus treated group. In addition, the IL-TIF adenovirus
treated mice decreased in body weight, while parental adenovirus
treated mice gained weight.
[0474] The results suggested that IL-TIF affects hematopoiesis,
i.e., blood cell formation in vivo. As such, IL-TIF could have
biological activities effecting different blood stem cells, thus
resulting increase or decrease of certain differentiated blood
cells in a specific lineage. For instance, IL-TIF appears to reduce
lymphocytes, which is likely due to inhibition of the committed
progenitor cells that give rise to lymphoid cells. IL-TIF also
decreases red blood cells. This finding agrees with the inhibitory
effects of IL-TIF on the proliferation and/or growth of myeloid
stem cells (Example 23), supporting the notion that IL-TIF could
play a role in anemia, infection, inflammation, and/or immune
diseases by influencing blood cells involved in these processes.
Antagnists against IL-TIF, such as antibodies or its soluble
receptor Zcytor16, could be used as therapeutic reagents in these
diseases.
[0475] Moreover, these experiments using IL-TIF adenovirus in mice
suggest that IL-TIF over-expression increases the level of
neutrophils and platelets in vivo. Although this may appear
contradictory to the finding seen in K562 cells (Example 23), it is
not uncommon to observe diverse activities of a particular protein
in vitro versus in vivo. It is conceivable that there are other
factors (such as cytokines and modifier genes) involved in the
responses to IL-TIF in the whole animal system. Nevertheless, these
data strongly support the involvement of IL-TIF in hematopoiesis.
Thus, IL-TIF and its receptors are suitable reagents/targets for
the diagnosis and treatment in variety of disorders, such as
inflammation, immune disorders, infection, anemia, hematopoietic
and other cancers, and the like.
Example 23
The IL-TIF Polypeptide Lyses K-562 Cells in Cytotoxicity Assay
[0476] The K-562 cell line (CRL-243, ATCC) has attained widespread
use as a highly sensitive in vitro target for cytotoxicity assays.
K-562 blasts are multipotential, hematopoietic malignant cells that
spontaneously differentiate into recognizable progenitors of the
erythrocytic, granulocytic and monocytic series (Lozzio, B B et
al., Proc. Soc. Exp. Biol. Med. 166: 546-550, 1981).
[0477] K562 cells were plated at 5,000 cells/well in 96-well tissue
culture clusters (Costar) in DMEM phenol-free growth medium (Life
Technologies) supplemented with pyruvate and 10% serum (HyClone).
Next day, human recombinant IL-TIF (Example 19), BSA control or
retinoic acid (known to be cytotoxic to K562 cells) were added.
Seventy-two hours later, the vital stain MTT (Sigma, St Louis,
Mo.), a widely used indicator of mitochondrial activity and cell
growth, was added to the cells at a final concentration of 0.5
mg/ml. MMP is converted to a purple formazan derivative by
mitochondrial dehydrogenases. Four hours later, converted MMP was
solubilized by adding an equal volume of acidic isopropanol
(0.04NHC1 in absolute isopropanol) to the wells. Absorbance was
measured at 570 nm, with background subtraction at 650 nm. In this
experimental setting, absorbance reflects cell viability. Results
shown in Table 12 are expressed as % cytotoxicity.
TABLE-US-00012 TABLE 12 Agent Concentration % Cytotoxicity BSA
Control 1 ug/ml 1.3 Retinoic acid 100 uM 62 IL-TIF 100 ng/ml 16.2
IL-TIF 300 ng/ml 32
[0478] The results indicate that IL-TIF may affect myeloid stem
cells. Myeloid stem cells are daughter cells of the universal blood
stem cells. They are progenitors of erythrocytes, platelets
megakaryocytes, monocytes (or migrated macrophages), neutrophil and
basophil, etc. Since K-562 blasts spontaneously differentiate into
progenitors of the erythrocytic, granulocytic and monocytic series,
it can be considered as myeloid stem cells. Thus, the results
demonstrate that IL-TIF has an inhibitory activity on the
proliferation and/or growth of myeloid stem cells. Thus IL-TIF
could play a role in anemia, infection, inflammation, and/or immune
diseases. In addition, an antaganist against IL-TIF, such as
antibodies or its soluble receptor Zcytor16, could be used to block
IL-TIF's activity on myeloid stem cells, or as therapeutic reagents
in diseases such as anemia, infection, inflammation, and/or immune
diseases.
Example 24
IL-TIF-Expressing Transgenic Mice
A. Generation of Transgenic Mice Expressing Mouse IL-TIF
[0479] DNA fragments from a transgenic vector containing 5' and 3'
flanking sequences of the lymphoid specific E.mu.LCK promoter,
mouse IL-TIF (SEQ ID NO:40), the rat insulin II intron, IL-TIF cDNA
and the human growth hormone poly A sequence were prepared using
standard methods, and used for microinjection into fertilized
B6C3f1 (Taconic, Germantown, N.Y.) murine oocytes, using a standard
microinjection protocol. See, Hogan, B. et al., Manipulating the
Mouse Embryo. A Laboratory Manual, Cold Spring Harbor Laboratory
Press, 1994.
[0480] Twenty-five mice transgenic for mouse IL-TIF with the
lymphoid-specific E.mu.LCK promoter were identified among 154 pups.
Eleven of the transgenic pups died within hours of birth, 9
transgenic pups with a shiny appearance were necropsied the day of
birth, and 2 grew to adulthood. Expression levels were low in one
adult animal. Tissues from the necropsied pups were prepared and
histologically examined as described below.
[0481] The shiny appearance of the neonate pups appeared to be
associated with a stiffening of the skin, as if they were drying
out, resulting in a reduction of proper nursing. Their movements
became stiffened in general.
B. Genotypic and Expression Analysis from Transgenic Mice
[0482] From the mouse IL-TIF transgenic line driven by the E.mu.Lck
promoter, described above, newborn pups were observed for
abnormalities on day one (day of birth) and sacrificed for tissue
collection. All pups were given a unique ear tag number, and those
designated as having a shiny skin phenotype at the time of
sacrifice were noted. Of the twelve pups, six were observed to have
the shiny skin phenotype, with two designated as "severe"
phenotypes. Severe phenotypes were defined as small pups with
little mobility whose skin was especially shiny and very dry. Skin
was collected from the left lateral side of each pup, and frozen in
Tissue-Tek embedding medium.
[0483] Genotyping confirmed that shiny skin was a good indicator of
transgenic status, although no expression data was collected.
Frozen skin blocks were sectioned to 7 microns on a cryostat and
stained to look for the presence of CD3, CD4, CD8, mouse
macrophages, B-cells, CD80, and MHC class II. The staining protocol
involved binding of commercially available antibodies to the
tissue, detection with a peroxidase labeled secondary antibody, and
DAB chromogen reaction to visualize staining.
[0484] Transgenic animals were found to be higher in MHC class II
and CD80, which stain for antigen-presenting cells and dendritic
cells respectively. The macrophage marker also detected more cells
in the severe and non-severe transgenics than in the wild type
animals, although the distribution of these cells was very
localized in the high dermis. Animals classified as severe
phenotypes had the most robust staining with all three of these
markers, showing a dramatic increase in cell intensity and number
when compared to the wild type. This variability may be due to a
difference in expression level of IL-TIF in these transgenic
founder pups. The MHC class II positive cells were located in the
lower dermis arranged in loose open clusters, while the CD80
positive cells were predominantly below the dermis either in or
just above the muscle/fat layer. These two cell populations do not
appear to overlap. All other markers were of equivalent staining in
all animals. Toluidine blue staining for mast cells revealed slight
to no difference between wild type and transgenic animals.
C. Microscopic Evaluation of Tissues from Transgenic Mice: IL-TIF
TG with EuLck Promoter has a Neonatal Lethal-Histology
[0485] On the day of birth, pups from litters containing IL-TIF
transgenics were humanely euthanized and the whole body immersion
fixed in 10% buffered formalin. Six transgenic and two
non-transgenic pups were submitted for further workup. Four of the
six transgenics were noted to have shiny skin at the time of
euthanasia. The fixed tissues were trimmed into 5 sections
(longitudinal section of the head and cross sections of the upper
and lower thorax and upper and lower abdomen). The tissues were
embedded in paraffin, routinely processed, sectioned at 5 um (Jung
2065 Supercut microtome, Leica Microsystems, Wetzlar, Germany) and
stained with H&E. The stained tissues were evaluated under a
light microscope (Nikon Eclipse E600, Nikon Inc., Melville, N.Y.)
by a board (ACVP) certified veterinary pathologist.
[0486] On microscopic examination, the epidermis of two of the
transgenic pups was observed to be thicker than the epidermis of
the other six mice including the controls. No other abnormalities
were noted in the skin and other tissues of any of the mice.
Representative areas of skin from corresponding regions of the
thorax and abdomen were imaged with the 40.times. objective lens
and with a CoolSnap digital camera (Roper Scientific, Inc., San
Diego, Calif.) that was attached to the microscope. The thickness
of the epidermis was then determined using histomorphometry
software (Scion Image for Windows (NIH Image), Scion Corp.,
Frederick, Md., v. B4.0.2). The results were as follows:
TABLE-US-00013 Average thoracic skin Average abdominal skin
Genotype/phenotype thickness (.mu.m) thickness (.mu.m)
Non-transgenic/normal 5.2 5.4 Transgenic/non-shiny 5.0 6.7
Transgenic/shiny 8.2 7.4 Transgenic/all 7.1 7.1
[0487] There were insufficient numbers of mice to determine
statistical significance; however, the transgenics, especially
those with shiny skin, tended to have a thicker epidermis than the
non-shiny transgenics and non-transgenic controls. The shiny
transgenics may have a higher expression level of IL-TIF than the
non-shiny transgenics; however, expression levels were not
determined for these mice.
[0488] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
4912149DNAHomo sapiensCDS(1)...(693) 1atg atg cct aaa cat tgc ttt
cta ggc ttc ctc atc agt ttc ttc ctt 48Met Met Pro Lys His Cys Phe
Leu Gly Phe Leu Ile Ser Phe Phe Leu 1 5 10 15act ggt gta gca gga
act cag tca acg cat gag tct ctg aag cct cag 96Thr Gly Val Ala Gly
Thr Gln Ser Thr His Glu Ser Leu Lys Pro Gln 20 25 30agg gta caa ttt
cag tcc cga aat ttt cac aac att ttg caa tgg cag 144Arg Val Gln Phe
Gln Ser Arg Asn Phe His Asn Ile Leu Gln Trp Gln 35 40 45cct ggg agg
gca ctt act ggc aac agc agt gtc tat ttt gtg cag tac 192Pro Gly Arg
Ala Leu Thr Gly Asn Ser Ser Val Tyr Phe Val Gln Tyr 50 55 60aaa ata
tat gga cag aga caa tgg aaa aat aaa gaa gac tgt tgg ggt 240Lys Ile
Tyr Gly Gln Arg Gln Trp Lys Asn Lys Glu Asp Cys Trp Gly 65 70 75
80act caa gaa ctc tct tgt gac ctt acc agt gaa acc tca gac ata cag
288Thr Gln Glu Leu Ser Cys Asp Leu Thr Ser Glu Thr Ser Asp Ile Gln
85 90 95gaa cct tat tac ggg agg gtg agg gcg gcc tcg gct ggg agc tac
tca 336Glu Pro Tyr Tyr Gly Arg Val Arg Ala Ala Ser Ala Gly Ser Tyr
Ser 100 105 110gaa tgg agc atg acg ccg cgg ttc act ccc tgg tgg gaa
aca aaa ata 384Glu Trp Ser Met Thr Pro Arg Phe Thr Pro Trp Trp Glu
Thr Lys Ile 115 120 125gat cct cca gtc atg aat ata acc caa gtc aat
ggc tct ttg ttg gta 432Asp Pro Pro Val Met Asn Ile Thr Gln Val Asn
Gly Ser Leu Leu Val 130 135 140att ctc cat gct cca aat tta cca tat
aga tac caa aag gaa aaa aat 480Ile Leu His Ala Pro Asn Leu Pro Tyr
Arg Tyr Gln Lys Glu Lys Asn145 150 155 160gta tct ata gaa gat tac
tat gaa cta cta tac cga gtt ttt ata att 528Val Ser Ile Glu Asp Tyr
Tyr Glu Leu Leu Tyr Arg Val Phe Ile Ile 165 170 175aac aat tca cta
gaa aag gag caa aag gtt tat gaa ggg gct cac aga 576Asn Asn Ser Leu
Glu Lys Glu Gln Lys Val Tyr Glu Gly Ala His Arg 180 185 190gcg gtt
gaa att gaa gct cta aca cca cac tcc agc tac tgt gta gtg 624Ala Val
Glu Ile Glu Ala Leu Thr Pro His Ser Ser Tyr Cys Val Val 195 200
205gct gaa ata tat cag ccc atg tta gac aga aga agt cag aga agt gaa
672Ala Glu Ile Tyr Gln Pro Met Leu Asp Arg Arg Ser Gln Arg Ser Glu
210 215 220gag aga tgt gtg gaa att cca tgacttgtgg aatttggcat
tcagcaatgt 723Glu Arg Cys Val Glu Ile Pro225 230ggaaattcta
aagctccctg agaacaggat gactcgtgtt tgaaggatct tatttaaaat
783tgtttttgta ttttcttaaa gcaatattca ctgttacacc ttggggactt
ctttgtttat 843ccattctttt atcctttata tttcatttta aactatattt
gaacgacatt ccccccgaaa 903aattgaaatg taaagatgag gcagagaata
aagtgttcta tgaaattcag aactttattt 963ctgaatgtaa catccctaat
aacaaccttc attcttctaa tacagcaaaa taaaaattta 1023acaaccaagg
aatagtattt aagaaaatgt tgaaataatt tttttaaaat agcattacag
1083actgaggcgg tcctgaagca atggtttttc actctcttat tgagccaatt
aaattgacat 1143tgctttgaca atttaaaact tctataaagg tgaatatttt
tcatacattt ctattttata 1203tgaatatact ttttatatat ttattattat
taaatatttc tacttaatga atcaaaattt 1263tgttttaaag tctactttat
gtaaataaga acaggttttg gggaaaaaaa tcttatgatt 1323tctggattga
tatctgaatt aaaactatca acaacaagga agtctactct gtacaattgt
1383ccctcattta aaagatatat taagcttttc ttttctgttt gtttttgttt
tgtttagttt 1443ttaatcctgt cttagaagaa cttatcttta ttctcaaaat
taaatgtaat ttttttagtg 1503acaaagaaga aaggaaacct cattactcaa
tccttctggc caagagtgtc ttgcttgtgg 1563cgccttcctc atctctatat
aggaggatcc catgaatgat ggtttattgg gaactgctgg 1623ggtcgacccc
atacagagaa ctcagcttga agctggaagc acacagtggg tagcaggaga
1683aggaccggtg ttggtaggtg cctacagaga ctatagagct agacaaagcc
ctccaaactg 1743gcccctcctg ctcactgcct ctcctgagta gaaatctggt
gacctaaggc tcagtgcggt 1803caacagaaag ctgccttctt cacttgaggc
taagtcttca tatatgttta aggttgtctt 1863tctagtgagg agatacatat
cagagaacat ttgtacaatt ccccatgaaa attgctccaa 1923agttgataac
aatatagtcg gtgcttctag ttatatgcaa gtactcagtg ataaatggat
1983taaaaaatat tcagaaatgt attggggggt ggaggagaat aagaggcaga
gcaagagcta 2043gagaattggt ttccttgctt ccctgtatgc tcagaaaaca
ttgatttgag catagacgca 2103gagactgaaa aaaaaaaaat gctcgagcgg
ccgccatatc cttggt 21492231PRTHomo sapiens 2Met Met Pro Lys His Cys
Phe Leu Gly Phe Leu Ile Ser Phe Phe Leu 1 5 10 15Thr Gly Val Ala
Gly Thr Gln Ser Thr His Glu Ser Leu Lys Pro Gln 20 25 30Arg Val Gln
Phe Gln Ser Arg Asn Phe His Asn Ile Leu Gln Trp Gln 35 40 45Pro Gly
Arg Ala Leu Thr Gly Asn Ser Ser Val Tyr Phe Val Gln Tyr 50 55 60Lys
Ile Tyr Gly Gln Arg Gln Trp Lys Asn Lys Glu Asp Cys Trp Gly65 70 75
80Thr Gln Glu Leu Ser Cys Asp Leu Thr Ser Glu Thr Ser Asp Ile Gln
85 90 95Glu Pro Tyr Tyr Gly Arg Val Arg Ala Ala Ser Ala Gly Ser Tyr
Ser 100 105 110Glu Trp Ser Met Thr Pro Arg Phe Thr Pro Trp Trp Glu
Thr Lys Ile 115 120 125Asp Pro Pro Val Met Asn Ile Thr Gln Val Asn
Gly Ser Leu Leu Val 130 135 140Ile Leu His Ala Pro Asn Leu Pro Tyr
Arg Tyr Gln Lys Glu Lys Asn145 150 155 160Val Ser Ile Glu Asp Tyr
Tyr Glu Leu Leu Tyr Arg Val Phe Ile Ile 165 170 175Asn Asn Ser Leu
Glu Lys Glu Gln Lys Val Tyr Glu Gly Ala His Arg 180 185 190Ala Val
Glu Ile Glu Ala Leu Thr Pro His Ser Ser Tyr Cys Val Val 195 200
205Ala Glu Ile Tyr Gln Pro Met Leu Asp Arg Arg Ser Gln Arg Ser Glu
210 215 220Glu Arg Cys Val Glu Ile Pro225 2303693DNAArtificial
SequenceThis degenerate nucleotide sequence encodes the amino acid
sequence of SEQ ID NO2 3atgatgccna arcaytgytt yytnggntty ytnathwsnt
tyttyytnac nggngtngcn 60ggnacncarw snacncayga rwsnytnaar ccncarmgng
tncarttyca rwsnmgnaay 120ttycayaaya thytncartg gcarccnggn
mgngcnytna cnggnaayws nwsngtntay 180ttygtncart ayaarathta
yggncarmgn cartggaara ayaargarga ytgytggggn 240acncargary
tnwsntgyga yytnacnwsn garacnwsng ayathcarga rccntaytay
300ggnmgngtnm gngcngcnws ngcnggnwsn taywsngart ggwsnatgac
nccnmgntty 360acnccntggt gggaracnaa rathgayccn ccngtnatga
ayathacnca rgtnaayggn 420wsnytnytng tnathytnca ygcnccnaay
ytnccntaym gntaycaraa rgaraaraay 480gtnwsnathg argaytayta
ygarytnytn taymgngtnt tyathathaa yaaywsnytn 540garaargarc
araargtnta ygarggngcn caymgngcng tngarathga rgcnytnacn
600ccncaywsnw sntaytgygt ngtngcngar athtaycarc cnatgytnga
ymgnmgnwsn 660carmgnwsng argarmgntg ygtngarath ccn
693416PRTArtificial SequencePeptide linker. 4Gly Gly Ser Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 155699DNAHomo
sapiens 5gagcccagat cttcagacaa aactcacaca tgcccaccgt gcccagcacc
tgaagccgag 60ggggcaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat
gatctcccgg 120acccctgagg tcacatgcgt ggtggtggac gtgagccacg
aagaccctga ggtcaagttc 180aactggtacg tggacggcgt ggaggtgcat
aatgccaaga caaagccgcg ggaggagcag 240tacaacagca cgtaccgtgt
ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 300ggcaaggagt
acaagtgcaa ggtctccaac aaagccctcc catcctccat cgagaaaacc
360atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc
cccatcccgg 420gatgagctga ccaagaacca ggtcagcctg acctgcctgg
tcaaaggctt ctatcccagc 480gacatcgccg tggagtggga gagcaatggg
cagccggaga acaactacaa gaccacgcct 540cccgtgctgg actccgacgg
ctccttcttc ctctacagca agctcaccgt ggacaagagc 600aggtggcagc
aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac
660tacacgcaga agagcctctc cctgtctccg ggtaaataa 699632DNAArtificial
SequenceOligonucleotide primer ZC29181 6gcggatccac tcagtcaacg
catgagtctc tg 32733DNAArtificial SequenceOligonucleotide primer
ZC29182 7gcagatcttg gaatttccac acatctctct tca 338108DNAHomo
sapiensCDS(1)...(108) 8atg gat gca atg aag aga ggg ctc tgc tgt gtg
ctg ctg ctg tgt ggc 48Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val
Leu Leu Leu Cys Gly 1 5 10 15gcc gtc ttc gtt tcg ctc agc cag gaa
atc cat gcc gag ttg aga cgc 96Ala Val Phe Val Ser Leu Ser Gln Glu
Ile His Ala Glu Leu Arg Arg 20 25 30ttc cgt aga tcc 108Phe Arg Arg
Ser 35936PRTHomo sapiens 9Met Asp Ala Met Lys Arg Gly Leu Cys Cys
Val Leu Leu Leu Cys Gly 1 5 10 15Ala Val Phe Val Ser Leu Ser Gln
Glu Ile His Ala Glu Leu Arg Arg 20 25 30Phe Arg Arg Ser
35106PRTArtificial SequenceGlu-Glu (CEE) Tag amino acid sequence
10Glu Tyr Met Pro Met Glu 1 5118PRTArtificial SequenceFLAG Tag
amino acid sequence 11Asp Tyr Lys Asp Asp Asp Asp Lys 1
5126PRTArtificial SequenceHis Tag amino acid sequence 12His His His
His His His 1 513210PRTHomo sapiens 13Thr Gln Ser Thr His Glu Ser
Leu Lys Pro Gln Arg Val Gln Phe Gln 1 5 10 15Ser Arg Asn Phe His
Asn Ile Leu Gln Trp Gln Pro Gly Arg Ala Leu 20 25 30Thr Gly Asn Ser
Ser Val Tyr Phe Val Gln Tyr Lys Ile Tyr Gly Gln 35 40 45Arg Gln Trp
Lys Asn Lys Glu Asp Cys Trp Gly Thr Gln Glu Leu Ser 50 55 60Cys Asp
Leu Thr Ser Glu Thr Ser Asp Ile Gln Glu Pro Tyr Tyr Gly65 70 75
80Arg Val Arg Ala Ala Ser Ala Gly Ser Tyr Ser Glu Trp Ser Met Thr
85 90 95Pro Arg Phe Thr Pro Trp Trp Glu Thr Lys Ile Asp Pro Pro Val
Met 100 105 110Asn Ile Thr Gln Val Asn Gly Ser Leu Leu Val Ile Leu
His Ala Pro 115 120 125Asn Leu Pro Tyr Arg Tyr Gln Lys Glu Lys Asn
Val Ser Ile Glu Asp 130 135 140Tyr Tyr Glu Leu Leu Tyr Arg Val Phe
Ile Ile Asn Asn Ser Leu Glu145 150 155 160Lys Glu Gln Lys Val Tyr
Glu Gly Ala His Arg Ala Val Glu Ile Glu 165 170 175Ala Leu Thr Pro
His Ser Ser Tyr Cys Val Val Ala Glu Ile Tyr Gln 180 185 190Pro Met
Leu Asp Arg Arg Ser Gln Arg Ser Glu Glu Arg Cys Val Glu 195 200
205Ile Pro 210141116DNAHomo sapiensCDS(21)...(557) 14tcgagttaga
attgtctgca atg gcc gcc ctg cag aaa tct gtg agc tct ttc 53 Met Ala
Ala Leu Gln Lys Ser Val Ser Ser Phe 1 5 10ctt atg ggg acc ctg gcc
acc agc tgc ctc ctt ctc ttg gcc ctc ttg 101Leu Met Gly Thr Leu Ala
Thr Ser Cys Leu Leu Leu Leu Ala Leu Leu 15 20 25gta cag gga gga gca
gct gcg ccc atc agc tcc cac tgc agg ctt gac 149Val Gln Gly Gly Ala
Ala Ala Pro Ile Ser Ser His Cys Arg Leu Asp 30 35 40aag tcc aac ttc
cag cag ccc tat atc acc aac cgc acc ttc atg ctg 197Lys Ser Asn Phe
Gln Gln Pro Tyr Ile Thr Asn Arg Thr Phe Met Leu 45 50 55gct aag gag
gct agc ttg gct gat aac aac aca gac gtt cgt ctc att 245Ala Lys Glu
Ala Ser Leu Ala Asp Asn Asn Thr Asp Val Arg Leu Ile 60 65 70 75ggg
gag aaa ctg ttc cac gga gtc agt atg agt gag cgc tgc tat ctg 293Gly
Glu Lys Leu Phe His Gly Val Ser Met Ser Glu Arg Cys Tyr Leu 80 85
90atg aag cag gtg ctg aac ttc acc ctt gaa gaa gtg ctg ttc cct caa
341Met Lys Gln Val Leu Asn Phe Thr Leu Glu Glu Val Leu Phe Pro Gln
95 100 105tct gat agg ttc cag cct tat atg cag gag gtg gtg ccc ttc
ctg gcc 389Ser Asp Arg Phe Gln Pro Tyr Met Gln Glu Val Val Pro Phe
Leu Ala 110 115 120agg ctc agc aac agg cta agc aca tgt cat att gaa
ggt gat gac ctg 437Arg Leu Ser Asn Arg Leu Ser Thr Cys His Ile Glu
Gly Asp Asp Leu 125 130 135cat atc cag agg aat gtg caa aag ctg aag
gac aca gtg aaa aag ctt 485His Ile Gln Arg Asn Val Gln Lys Leu Lys
Asp Thr Val Lys Lys Leu140 145 150 155gga gag agt gga gag atc aaa
gca att gga gaa ctg gat ttg ctg ttt 533Gly Glu Ser Gly Glu Ile Lys
Ala Ile Gly Glu Leu Asp Leu Leu Phe 160 165 170atg tct ctg aga aat
gcc tgc att tgaccagagc aaagctgaaa aatgaataac 587Met Ser Leu Arg Asn
Ala Cys Ile 175taaccccctt tccctgctag aaataacaat tagatgcccc
aaagcgattt tttttaacca 647aaaggaagat gggaagccaa actccatcat
gatgggtgga ttccaaatga acccctgcgt 707tagttacaaa ggaaaccaat
gccacttttg tttataagac cagaaggtag actttctaag 767catagatatt
tattgataac atttcattgt aactggtgtt ctatacacag aaaacaattt
827attttttaaa taattgtctt tttccataaa aaagattact ttccattcct
ttaggggaaa 887aaacccctaa atagcttcat gtttccataa tcagtacttt
atatttataa atgtatttat 947tattattata agactgcatt ttatttatat
cattttatta atatggattt atttatagaa 1007acatcattcg atattgctac
ttgagtgtaa ggctaatatt gatatttatg acaataatta 1067tagagctata
acatgtttat ttgacctcaa taaacacttg gatatccta 111615179PRTHomo sapiens
15Met Ala Ala Leu Gln Lys Ser Val Ser Ser Phe Leu Met Gly Thr Leu 1
5 10 15Ala Thr Ser Cys Leu Leu Leu Leu Ala Leu Leu Val Gln Gly Gly
Ala 20 25 30Ala Ala Pro Ile Ser Ser His Cys Arg Leu Asp Lys Ser Asn
Phe Gln 35 40 45Gln Pro Tyr Ile Thr Asn Arg Thr Phe Met Leu Ala Lys
Glu Ala Ser 50 55 60Leu Ala Asp Asn Asn Thr Asp Val Arg Leu Ile Gly
Glu Lys Leu Phe65 70 75 80His Gly Val Ser Met Ser Glu Arg Cys Tyr
Leu Met Lys Gln Val Leu 85 90 95Asn Phe Thr Leu Glu Glu Val Leu Phe
Pro Gln Ser Asp Arg Phe Gln 100 105 110Pro Tyr Met Gln Glu Val Val
Pro Phe Leu Ala Arg Leu Ser Asn Arg 115 120 125Leu Ser Thr Cys His
Ile Glu Gly Asp Asp Leu His Ile Gln Arg Asn 130 135 140Val Gln Lys
Leu Lys Asp Thr Val Lys Lys Leu Gly Glu Ser Gly Glu145 150 155
160Ile Lys Ala Ile Gly Glu Leu Asp Leu Leu Phe Met Ser Leu Arg Asn
165 170 175Ala Cys Ile1623DNAArtificial SequenceOligonucleotide
primer ZC25963 16agtcaacgca tgagtctctg aag 231723DNAArtificial
SequenceOligonucleotide primer ZC28354 17accaacaaag agccattgac ttg
231823DNAArtificial SequenceOligonucleotide primer ZC21195
18gaggagacca taacccccga cag 231923DNAArtificial
SequenceOligonucleotide primer ZC21196 19catagctccc accacacgat ttt
232025DNAArtificial SequenceOligonucleotide primer ZC14063
20caccagacat aatagctgac agact 252121DNAArtificial
SequenceOligonucleotide primer ZC17574 21ggtrttgctc agcatgcaca c
212224DNAArtificial SequenceOligonucleotide primer ZC17600
22catgtaggcc atgaggtcca ccac 242322DNAArtificial
SequenceOligonucleotide primer ZC27659 23tcaagctgag ttctctgtat gg
22242831DNAHomo sapiensCDS(34)...(1755) 24tagaggccaa gggagggctc
tgtgccagcc ccg atg agg acg ctg ctg acc atc 54 Met Arg Thr Leu Leu
Thr Ile 1 5ttg act gtg gga tcc ctg gct gct cac gcc cct gag gac ccc
tcg gat 102Leu Thr Val Gly Ser Leu Ala Ala His Ala Pro Glu Asp Pro
Ser Asp 10 15 20ctg ctc cag cac gtg aaa ttc cag tcc agc aac ttt gaa
aac atc ctg 150Leu Leu Gln His Val Lys Phe Gln Ser Ser Asn Phe Glu
Asn Ile Leu 25 30 35acg tgg gac agc ggg cca gag ggc acc cca gac acg
gtc tac agc atc 198Thr Trp Asp Ser Gly Pro Glu Gly Thr Pro Asp Thr
Val Tyr Ser Ile 40 45 50 55gag tat aag acg tac gga gag agg gac tgg
gtg gca aag aag ggc tgt 246Glu Tyr Lys Thr Tyr Gly Glu Arg Asp Trp
Val Ala Lys Lys Gly Cys 60 65 70cag cgg atc acc cgg aag tcc tgc aac
ctg acg gtg gag acg ggc aac 294Gln Arg Ile Thr Arg Lys Ser Cys Asn
Leu Thr Val Glu Thr Gly Asn 75 80 85ctc acg gag ctc tac tat gcc agg
gtc acc gct gtc agt gcg gga ggc 342Leu Thr Glu Leu Tyr Tyr Ala Arg
Val Thr Ala Val Ser Ala Gly Gly 90 95 100cgg tca gcc acc aag atg
act gac agg ttc agc tct ctg cag cac act 390Arg Ser Ala Thr Lys Met
Thr Asp Arg Phe Ser Ser Leu Gln His Thr 105 110
115acc ctc aag cca cct gat gtg acc tgt atc tcc aaa gtg aga tcg att
438Thr Leu Lys Pro Pro Asp Val Thr Cys Ile Ser Lys Val Arg Ser
Ile120 125 130 135cag atg att gtt cat cct acc ccc acg cca atc cgt
gca ggc gat ggc 486Gln Met Ile Val His Pro Thr Pro Thr Pro Ile Arg
Ala Gly Asp Gly 140 145 150cac cgg cta acc ctg gaa gac atc ttc cat
gac ctg ttc tac cac tta 534His Arg Leu Thr Leu Glu Asp Ile Phe His
Asp Leu Phe Tyr His Leu 155 160 165gag ctc cag gtc aac cgc acc tac
caa atg cac ctt gga ggg aag cag 582Glu Leu Gln Val Asn Arg Thr Tyr
Gln Met His Leu Gly Gly Lys Gln 170 175 180aga gaa tat gag ttc ttc
ggc ctg acc cct gac aca gag ttc ctt ggc 630Arg Glu Tyr Glu Phe Phe
Gly Leu Thr Pro Asp Thr Glu Phe Leu Gly 185 190 195acc atc atg att
tgc gtt ccc acc tgg gcc aag gag agt gcc ccc tac 678Thr Ile Met Ile
Cys Val Pro Thr Trp Ala Lys Glu Ser Ala Pro Tyr200 205 210 215atg
tgc cga gtg aag aca ctg cca gac cgg aca tgg acc tac tcc ttc 726Met
Cys Arg Val Lys Thr Leu Pro Asp Arg Thr Trp Thr Tyr Ser Phe 220 225
230tcc gga gcc ttc ctg ttc tcc atg ggc ttc ctc gtc gca gta ctc tgc
774Ser Gly Ala Phe Leu Phe Ser Met Gly Phe Leu Val Ala Val Leu Cys
235 240 245tac ctg agc tac aga tat gtc acc aag ccg cct gca cct ccc
aac tcc 822Tyr Leu Ser Tyr Arg Tyr Val Thr Lys Pro Pro Ala Pro Pro
Asn Ser 250 255 260ctg aac gtc cag cga gtc ctg act ttc cag ccg ctg
cgc ttc atc cag 870Leu Asn Val Gln Arg Val Leu Thr Phe Gln Pro Leu
Arg Phe Ile Gln 265 270 275gag cac gtc ctg atc cct gtc ttt gac ctc
agc ggc ccc agc agt ctg 918Glu His Val Leu Ile Pro Val Phe Asp Leu
Ser Gly Pro Ser Ser Leu280 285 290 295gcc cag cct gtc cag tac tcc
cag atc agg gtg tct gga ccc agg gag 966Ala Gln Pro Val Gln Tyr Ser
Gln Ile Arg Val Ser Gly Pro Arg Glu 300 305 310ccc gca gga gct cca
cag cgg cat agc ctg tcc gag atc acc tac tta 1014Pro Ala Gly Ala Pro
Gln Arg His Ser Leu Ser Glu Ile Thr Tyr Leu 315 320 325ggg cag cca
gac atc tcc atc ctc cag ccc tcc aac gtg cca cct ccc 1062Gly Gln Pro
Asp Ile Ser Ile Leu Gln Pro Ser Asn Val Pro Pro Pro 330 335 340cag
atc ctc tcc cca ctg tcc tat gcc cca aac gct gcc cct gag gtc 1110Gln
Ile Leu Ser Pro Leu Ser Tyr Ala Pro Asn Ala Ala Pro Glu Val 345 350
355ggg ccc cca tcc tat gca cct cag gtg acc ccc gaa gct caa ttc cca
1158Gly Pro Pro Ser Tyr Ala Pro Gln Val Thr Pro Glu Ala Gln Phe
Pro360 365 370 375ttc tac gcc cca cag gcc atc tct aag gtc cag cct
tcc tcc tat gcc 1206Phe Tyr Ala Pro Gln Ala Ile Ser Lys Val Gln Pro
Ser Ser Tyr Ala 380 385 390cct caa gcc act ccg gac agc tgg cct ccc
tcc tat ggg gta tgc atg 1254Pro Gln Ala Thr Pro Asp Ser Trp Pro Pro
Ser Tyr Gly Val Cys Met 395 400 405gaa ggt tct ggc aaa gac tcc ccc
act ggg aca ctt tct agt cct aaa 1302Glu Gly Ser Gly Lys Asp Ser Pro
Thr Gly Thr Leu Ser Ser Pro Lys 410 415 420cac ctt agg cct aaa ggt
cag ctt cag aaa gag cca cca gct gga agc 1350His Leu Arg Pro Lys Gly
Gln Leu Gln Lys Glu Pro Pro Ala Gly Ser 425 430 435tgc atg tta ggt
ggc ctt tct ctg cag gag gtg acc tcc ttg gct atg 1398Cys Met Leu Gly
Gly Leu Ser Leu Gln Glu Val Thr Ser Leu Ala Met440 445 450 455gag
gaa tcc caa gaa gca aaa tca ttg cac cag ccc ctg ggg att tgc 1446Glu
Glu Ser Gln Glu Ala Lys Ser Leu His Gln Pro Leu Gly Ile Cys 460 465
470aca gac aga aca tct gac cca aat gtg cta cac agt ggg gag gaa ggg
1494Thr Asp Arg Thr Ser Asp Pro Asn Val Leu His Ser Gly Glu Glu Gly
475 480 485aca cca cag tac cta aag ggc cag ctc ccc ctc ctc tcc tca
gtc cag 1542Thr Pro Gln Tyr Leu Lys Gly Gln Leu Pro Leu Leu Ser Ser
Val Gln 490 495 500atc gag ggc cac ccc atg tcc ctc cct ttg caa cct
cct tcc ggt cca 1590Ile Glu Gly His Pro Met Ser Leu Pro Leu Gln Pro
Pro Ser Gly Pro 505 510 515tgt tcc ccc tcg gac caa ggt cca agt ccc
tgg ggc ctg ctg gag tcc 1638Cys Ser Pro Ser Asp Gln Gly Pro Ser Pro
Trp Gly Leu Leu Glu Ser520 525 530 535ctt gtg tgt ccc aag gat gaa
gcc aag agc cca gcc cct gag acc tca 1686Leu Val Cys Pro Lys Asp Glu
Ala Lys Ser Pro Ala Pro Glu Thr Ser 540 545 550gac ctg gag cag ccc
aca gaa ctg gat tct ctt ttc aga ggc ctg gcc 1734Asp Leu Glu Gln Pro
Thr Glu Leu Asp Ser Leu Phe Arg Gly Leu Ala 555 560 565ctg act gtg
cag tgg gag tcc tgaggggaat gggaaaggct tggtgcttcc 1785Leu Thr Val
Gln Trp Glu Ser 570tccctgtccc tacccagtgt cacatccttg gctgtcaatc
ccatgcctgc ccatgccaca 1845cactctgcga tctggcctca gacgggtgcc
cttgagagaa gcagagggag tggcatgcag 1905ggcccctgcc atgggtgcgc
tcctcaccgg aacaaagcag catgataagg actgcagcgg 1965gggagctctg
gggagcagct tgtgtagaca agcgcgtgct cgctgagccc tgcaaggcag
2025aaatgacagt gcaaggagga aatgcaggga aactcccgag gtccagagcc
ccacctccta 2085acaccatgga ttcaaagtgc tcagggaatt tgcctctcct
tgccccattc ctggccagtt 2145tcacaatcta gctcgacaga gcatgaggcc
cctgcctctt ctgtcattgt tcaaaggtgg 2205gaagagagcc tggaaaagaa
ccaggcctgg aaaagaacca gaaggaggct gggcagaacc 2265agaacaacct
gcacttctgc caaggccagg gccagcagga cggcaggact ctagggaggg
2325gtgtggcctg cagctcattc ccagccaggg caactgcctg acgttgcacg
atttcagctt 2385cattcctctg atagaacaaa gcgaaatgca ggtccaccag
ggagggagac acacaagcct 2445tttctgcagg caggagtttc agaccctatc
ctgagaatgg ggtttgaaag gaaggtgagg 2505gctgtggccc ctggacgggt
acaataacac actgtactga tgtcacaact ttgcaagctc 2565tgccttgggt
tcagcccatc tgggctcaaa ttccagcctc accactcaca agctgtgtga
2625cttcaaacaa atgaaatcag tgcccagaac ctcggtttcc tcatctgtaa
tgtggggatc 2685ataacaccta cctcatggag ttgtggtgaa gatgaaatga
agtcatgtct ttaaagtgct 2745taatagtgcc tggtacatgg gcagtgccca
ataaacggta gctatttaaa aaaaaaaaaa 2805aaaaaaaaaa atagcggccg cctcga
283125574PRTHomo sapiens 25Met Arg Thr Leu Leu Thr Ile Leu Thr Val
Gly Ser Leu Ala Ala His 1 5 10 15Ala Pro Glu Asp Pro Ser Asp Leu
Leu Gln His Val Lys Phe Gln Ser 20 25 30Ser Asn Phe Glu Asn Ile Leu
Thr Trp Asp Ser Gly Pro Glu Gly Thr 35 40 45Pro Asp Thr Val Tyr Ser
Ile Glu Tyr Lys Thr Tyr Gly Glu Arg Asp 50 55 60Trp Val Ala Lys Lys
Gly Cys Gln Arg Ile Thr Arg Lys Ser Cys Asn65 70 75 80Leu Thr Val
Glu Thr Gly Asn Leu Thr Glu Leu Tyr Tyr Ala Arg Val 85 90 95Thr Ala
Val Ser Ala Gly Gly Arg Ser Ala Thr Lys Met Thr Asp Arg 100 105
110Phe Ser Ser Leu Gln His Thr Thr Leu Lys Pro Pro Asp Val Thr Cys
115 120 125Ile Ser Lys Val Arg Ser Ile Gln Met Ile Val His Pro Thr
Pro Thr 130 135 140Pro Ile Arg Ala Gly Asp Gly His Arg Leu Thr Leu
Glu Asp Ile Phe145 150 155 160His Asp Leu Phe Tyr His Leu Glu Leu
Gln Val Asn Arg Thr Tyr Gln 165 170 175Met His Leu Gly Gly Lys Gln
Arg Glu Tyr Glu Phe Phe Gly Leu Thr 180 185 190Pro Asp Thr Glu Phe
Leu Gly Thr Ile Met Ile Cys Val Pro Thr Trp 195 200 205Ala Lys Glu
Ser Ala Pro Tyr Met Cys Arg Val Lys Thr Leu Pro Asp 210 215 220Arg
Thr Trp Thr Tyr Ser Phe Ser Gly Ala Phe Leu Phe Ser Met Gly225 230
235 240Phe Leu Val Ala Val Leu Cys Tyr Leu Ser Tyr Arg Tyr Val Thr
Lys 245 250 255Pro Pro Ala Pro Pro Asn Ser Leu Asn Val Gln Arg Val
Leu Thr Phe 260 265 270Gln Pro Leu Arg Phe Ile Gln Glu His Val Leu
Ile Pro Val Phe Asp 275 280 285Leu Ser Gly Pro Ser Ser Leu Ala Gln
Pro Val Gln Tyr Ser Gln Ile 290 295 300Arg Val Ser Gly Pro Arg Glu
Pro Ala Gly Ala Pro Gln Arg His Ser305 310 315 320Leu Ser Glu Ile
Thr Tyr Leu Gly Gln Pro Asp Ile Ser Ile Leu Gln 325 330 335Pro Ser
Asn Val Pro Pro Pro Gln Ile Leu Ser Pro Leu Ser Tyr Ala 340 345
350Pro Asn Ala Ala Pro Glu Val Gly Pro Pro Ser Tyr Ala Pro Gln Val
355 360 365Thr Pro Glu Ala Gln Phe Pro Phe Tyr Ala Pro Gln Ala Ile
Ser Lys 370 375 380Val Gln Pro Ser Ser Tyr Ala Pro Gln Ala Thr Pro
Asp Ser Trp Pro385 390 395 400Pro Ser Tyr Gly Val Cys Met Glu Gly
Ser Gly Lys Asp Ser Pro Thr 405 410 415Gly Thr Leu Ser Ser Pro Lys
His Leu Arg Pro Lys Gly Gln Leu Gln 420 425 430Lys Glu Pro Pro Ala
Gly Ser Cys Met Leu Gly Gly Leu Ser Leu Gln 435 440 445Glu Val Thr
Ser Leu Ala Met Glu Glu Ser Gln Glu Ala Lys Ser Leu 450 455 460His
Gln Pro Leu Gly Ile Cys Thr Asp Arg Thr Ser Asp Pro Asn Val465 470
475 480Leu His Ser Gly Glu Glu Gly Thr Pro Gln Tyr Leu Lys Gly Gln
Leu 485 490 495Pro Leu Leu Ser Ser Val Gln Ile Glu Gly His Pro Met
Ser Leu Pro 500 505 510Leu Gln Pro Pro Ser Gly Pro Cys Ser Pro Ser
Asp Gln Gly Pro Ser 515 520 525Pro Trp Gly Leu Leu Glu Ser Leu Val
Cys Pro Lys Asp Glu Ala Lys 530 535 540Ser Pro Ala Pro Glu Thr Ser
Asp Leu Glu Gln Pro Thr Glu Leu Asp545 550 555 560Ser Leu Phe Arg
Gly Leu Ala Leu Thr Val Gln Trp Glu Ser 565 5702639DNAArtificial
SequenceOligonucleotide linker ZC13252 26ggcctgaaag cttcggataa
tgaaggtacc tgttagaaa 392727DNAArtificial SequenceOligonucleotide
linker ZC13453 27ttaggatccg gcccttcccc agatact 272836DNAArtificial
SequenceOligonucleotide primer ZC28590 28ttgggtacct ctgcaatggc
cgccctgcag aaatct 362933DNAArtificial SequenceOoligonucleotide
primer ZC28580 29ttgggatcca atgcaggcat ttctcagaga cat
333023DNAArtificial SequenceOligonucleotide primer ZC25963
30agtcaacgca tgagtctctg aag 233123DNAArtificial
SequenceOligonucleotide primer ZC25964 31gttcttgagt accccaacag tct
233218DNAArtificial SequenceOligonucleotide primer ZC14666
32agccaccaag atgactga 183322DNAArtificial SequenceOligonucleotide
primer ZC14742 33tgcatttggt aggtgcggtt ga 2234211PRTHomo sapiens
34Pro Glu Asp Pro Ser Asp Leu Leu Gln His Val Lys Phe Gln Ser Ser 1
5 10 15Asn Phe Glu Asn Ile Leu Thr Trp Asp Ser Gly Pro Glu Gly Thr
Pro 20 25 30Asp Thr Val Tyr Ser Ile Glu Tyr Lys Thr Tyr Gly Glu Arg
Asp Trp 35 40 45Val Ala Lys Lys Gly Cys Gln Arg Ile Thr Arg Lys Ser
Cys Asn Leu 50 55 60Thr Val Glu Thr Gly Asn Leu Thr Glu Leu Tyr Tyr
Ala Arg Val Thr65 70 75 80Ala Val Ser Ala Gly Gly Arg Ser Ala Thr
Lys Met Thr Asp Arg Phe 85 90 95Ser Ser Leu Gln His Thr Thr Leu Lys
Pro Pro Asp Val Thr Cys Ile 100 105 110Ser Lys Val Arg Ser Ile Gln
Met Ile Val His Pro Thr Pro Thr Pro 115 120 125Ile Arg Ala Gly Asp
Gly His Arg Leu Thr Leu Glu Asp Ile Phe His 130 135 140Asp Leu Phe
Tyr His Leu Glu Leu Gln Val Asn Arg Thr Tyr Gln Met145 150 155
160His Leu Gly Gly Lys Gln Arg Glu Tyr Glu Phe Phe Gly Leu Thr Pro
165 170 175Asp Thr Glu Phe Leu Gly Thr Ile Met Ile Cys Val Pro Thr
Trp Ala 180 185 190Lys Glu Ser Ala Pro Tyr Met Cys Arg Val Lys Thr
Leu Pro Asp Arg 195 200 205Thr Trp Thr 21035199PRTHomo sapiens
35Met Val Pro Pro Pro Glu Asn Val Arg Met Asn Ser Val Asn Phe Lys 1
5 10 15Asn Ile Leu Gln Trp Glu Ser Pro Ala Phe Ala Lys Gly Asn Leu
Thr 20 25 30Phe Thr Ala Gln Tyr Leu Ser Tyr Arg Ile Phe Gln Asp Lys
Cys Met 35 40 45Asn Thr Thr Leu Thr Glu Cys Asp Phe Ser Ser Leu Ser
Lys Tyr Gly 50 55 60Asp His Thr Leu Arg Val Arg Ala Glu Phe Ala Asp
Glu His Ser Asp65 70 75 80Trp Val Asn Ile Thr Phe Cys Pro Val Asp
Asp Thr Ile Ile Gly Pro 85 90 95Pro Gly Met Gln Val Glu Val Leu Ala
Asp Ser Leu His Met Arg Phe 100 105 110Leu Ala Pro Lys Ile Glu Asn
Glu Tyr Glu Thr Trp Thr Met Lys Asn 115 120 125Val Tyr Asn Ser Trp
Thr Tyr Asn Val Gln Tyr Trp Lys Asn Gly Thr 130 135 140Asp Glu Lys
Phe Gln Ile Thr Pro Gln Tyr Asp Phe Glu Val Leu Arg145 150 155
160Asn Leu Glu Pro Trp Thr Thr Tyr Cys Val Gln Val Arg Gly Phe Leu
165 170 175Pro Asp Arg Asn Lys Ala Gly Glu Trp Ser Glu Pro Val Cys
Glu Gln 180 185 190Thr Thr His Asp Glu Thr Val 19536211PRTHomo
sapiens 36Ser Asp Ala His Gly Thr Glu Leu Pro Ser Pro Pro Ser Val
Trp Phe 1 5 10 15Glu Ala Glu Phe Phe His His Ile Leu His Trp Thr
Pro Ile Pro Asn 20 25 30Gln Ser Glu Ser Thr Cys Tyr Glu Val Ala Leu
Leu Arg Tyr Gly Ile 35 40 45Glu Ser Trp Asn Ser Ile Ser Asn Cys Ser
Gln Thr Leu Ser Tyr Asp 50 55 60Leu Thr Ala Val Thr Leu Asp Leu Tyr
His Ser Asn Gly Tyr Arg Ala65 70 75 80Arg Val Arg Ala Val Asp Gly
Ser Arg His Ser Asn Trp Thr Val Thr 85 90 95Asn Thr Arg Phe Ser Val
Asp Glu Val Thr Leu Thr Val Gly Ser Val 100 105 110Asn Leu Glu Ile
His Asn Gly Phe Ile Leu Gly Lys Ile Gln Leu Pro 115 120 125Arg Pro
Lys Met Ala Pro Ala Asn Asp Thr Tyr Glu Ser Ile Phe Ser 130 135
140His Phe Arg Glu Tyr Glu Ile Ala Ile Arg Lys Val Pro Gly Asn
Phe145 150 155 160Thr Phe Thr His Lys Lys Val Lys His Glu Asn Phe
Ser Leu Leu Thr 165 170 175Ser Gly Glu Val Gly Glu Phe Cys Val Gln
Val Lys Pro Ser Val Ala 180 185 190Ser Arg Ser Asn Lys Gly Met Trp
Ser Lys Glu Glu Cys Ile Ser Leu 195 200 205Thr Arg Gln
210372464DNAMus musculusCDS(8)...(697) 37ggcaacc atg atg cct aag
cat tgc ctt cta ggt ctc ctc atc ata ctc 49 Met Met Pro Lys His Cys
Leu Leu Gly Leu Leu Ile Ile Leu 1 5 10ttg agc agt gca aca gaa ata
caa cca gct cgt gta tct ctg acg ctc 97Leu Ser Ser Ala Thr Glu Ile
Gln Pro Ala Arg Val Ser Leu Thr Leu 15 20 25 30cag aag gtc cga ttt
cag tcc aga aat ttc cac aat att ttg cac tgg 145Gln Lys Val Arg Phe
Gln Ser Arg Asn Phe His Asn Ile Leu His Trp 35 40 45caa gca ggg agc
tct ctc ccc agc aac aac agc atc tac ttt gtg cag 193Gln Ala Gly Ser
Ser Leu Pro Ser Asn Asn Ser Ile Tyr Phe Val Gln 50 55 60tac aag atg
tat gga cag agc caa tgg gaa gat aaa gtt gac tgc tgg 241Tyr Lys Met
Tyr Gly Gln Ser Gln Trp Glu Asp Lys Val Asp Cys Trp 65 70 75ggg acc
acg gcg ctc ttc tgt gac ctg acc aat gaa acc tta gac cca 289Gly Thr
Thr Ala Leu Phe Cys Asp Leu Thr Asn Glu Thr Leu Asp Pro 80 85 90tac
gag ctg tat tac ggg agg gtg atg acg gcc tgt gct gga cgc cac 337Tyr
Glu Leu Tyr Tyr Gly Arg Val Met Thr Ala Cys Ala Gly Arg His 95 100
105 110tct gcc tgg acc agg aca ccc cgc ttc act cca tgg tgg gaa aca
aaa 385Ser Ala Trp Thr Arg Thr Pro Arg Phe Thr Pro Trp
Trp Glu Thr Lys 115 120 125cta gat cct ccg gtc gtg act ata acc cga
gtt aac gca tct ttg cgg 433Leu Asp Pro Pro Val Val Thr Ile Thr Arg
Val Asn Ala Ser Leu Arg 130 135 140gtg ctt ctc cgt cct cca gag ttg
cca aat aga aac caa agt gga aaa 481Val Leu Leu Arg Pro Pro Glu Leu
Pro Asn Arg Asn Gln Ser Gly Lys 145 150 155aat gca tcc atg gaa act
tac tac ggc tta gta tac aga gtt ttc aca 529Asn Ala Ser Met Glu Thr
Tyr Tyr Gly Leu Val Tyr Arg Val Phe Thr 160 165 170atc aac aat tca
cta gag aag gag caa aaa gcc tat gaa gga act cag 577Ile Asn Asn Ser
Leu Glu Lys Glu Gln Lys Ala Tyr Glu Gly Thr Gln175 180 185 190aga
gct gtt gaa att gaa ggt ctg ata cct cat tcc agc tac tgc gta 625Arg
Ala Val Glu Ile Glu Gly Leu Ile Pro His Ser Ser Tyr Cys Val 195 200
205gtg gct gaa atg tac cag ccc atg ttt gac aga aga agc cca aga agc
673Val Ala Glu Met Tyr Gln Pro Met Phe Asp Arg Arg Ser Pro Arg Ser
210 215 220aag gag aga tgt gtg cag att cca tgaactggtc tgaggcgcta
aaaccggaag 727Lys Glu Arg Cys Val Gln Ile Pro 225 230catattgaga
acaggatgtc ttctgcctag aacagcttac taaacttctg ttttgatttt
787cttagagcaa tgtctaccca cacttttcaa ggatttcttt gtcagtactt
tcttttctgt 847ttatttcagt tataaagtaa atttgaacag attcaaaaat
agaatttaaa gagaaggtaa 907agaataaaga tcttgtggta aggcacatct
ttaatcccag cattgaaagg cagaatcagt 967cagatctctg acttttgggc
caacctggtc tgcatagaga gttccaggct agccagggcc 1027acacaatgag
accatgcatc aacatgataa gatgttaaga tagaaatgtt ctataaaaaa
1087tggaagctta tctgaatata tcaaccctaa caataatctt ttttctggtt
taaccctcaa 1147ataagaatca attaatagta ggcatttaat aaatgttgta
ataagtttta aggtattata 1207tatagaaaat cctgcaacaa taattttata
ttctattaag ccttttattt tgccagagat 1267atactgattt aatattttgt
gtgaatgagt atacttctgt gcatttttat tttatattaa 1327tgtctttttt
atttcttata aggtatttct acttagtgaa ctatctctct aaaaataaac
1387ttgtggctct gaattgatct ctagaataag attttcaata atagcatgta
aatatcgaag 1447aaaatcttca taatgcaatt tcccatcact taacacataa
tcttagtaag aatacccttt 1507ctggttttgc ttttaatagt gccttagaat
ttaacctatg ttgtctaaat gcagcatata 1567tttttcagca gcaaagacag
aagaaacata taagccacag tgataactca gccattcagg 1627caaagtggcc
tctctgtcca cagaatgaga gttgggctgg agacactgga gtccatacag
1687gaaccttgac ttgaagccaa aggcacacag tggatgctta aattatttct
gagattataa 1747ggaaattcac agccctgtgg actacactgc atctcctgag
caatgatcag gtggcctaag 1807gctcaaaaag tgaccagctt ttctaagcct
ttctctagct cttgagctta gactatcagc 1867tactgaaaat acatgtagga
gagaacattt gtgatcttta tgaaagatgc tccaaaatat 1927aactctgaaa
agtatagtcg gtacgtctgt cttcttacgt tttttaatgg taaatggtca
1987cagaccatta agaacataag agacatgtga aaagcaagag cattgtcctg
gctgtcctgt 2047gtgttcagaa aacactggcc ttcattctta tttttgaatt
attcaatttt tatttctttt 2107gtggcttagt acctgcttgg gaattctttg
ggttttttgt tcgttgtttg tttattttct 2167attttgtact ttgggaaggg
tgagatttga gaaggataac tcctctttgg gaatcaggac 2227catgtgtggt
cccagcattt aggttgtaag actcagtagt ctaattcctc ataatacaaa
2287aggcaattgt tggtttatga gtgttgtcag caatcagagt tttgctttga
ctttgtttgc 2347ttgggttttc cctcacacaa gtataagatg ctttgaaata
tattacgatt ttcacattct 2407ggatcatgtt aagaatatat tgtctaagta
cttcaataaa tcttactttg tacttta 246438230PRTMus musculus 38Met Met
Pro Lys His Cys Leu Leu Gly Leu Leu Ile Ile Leu Leu Ser 1 5 10
15Ser Ala Thr Glu Ile Gln Pro Ala Arg Val Ser Leu Thr Leu Gln Lys
20 25 30Val Arg Phe Gln Ser Arg Asn Phe His Asn Ile Leu His Trp Gln
Ala 35 40 45Gly Ser Ser Leu Pro Ser Asn Asn Ser Ile Tyr Phe Val Gln
Tyr Lys 50 55 60Met Tyr Gly Gln Ser Gln Trp Glu Asp Lys Val Asp Cys
Trp Gly Thr65 70 75 80Thr Ala Leu Phe Cys Asp Leu Thr Asn Glu Thr
Leu Asp Pro Tyr Glu 85 90 95Leu Tyr Tyr Gly Arg Val Met Thr Ala Cys
Ala Gly Arg His Ser Ala 100 105 110Trp Thr Arg Thr Pro Arg Phe Thr
Pro Trp Trp Glu Thr Lys Leu Asp 115 120 125Pro Pro Val Val Thr Ile
Thr Arg Val Asn Ala Ser Leu Arg Val Leu 130 135 140Leu Arg Pro Pro
Glu Leu Pro Asn Arg Asn Gln Ser Gly Lys Asn Ala145 150 155 160Ser
Met Glu Thr Tyr Tyr Gly Leu Val Tyr Arg Val Phe Thr Ile Asn 165 170
175Asn Ser Leu Glu Lys Glu Gln Lys Ala Tyr Glu Gly Thr Gln Arg Ala
180 185 190Val Glu Ile Glu Gly Leu Ile Pro His Ser Ser Tyr Cys Val
Val Ala 195 200 205Glu Met Tyr Gln Pro Met Phe Asp Arg Arg Ser Pro
Arg Ser Lys Glu 210 215 220Arg Cys Val Gln Ile Pro225
23039690DNAArtificial SequenceDegenerate polynucleotide sequence of
SEQ ID NO38 39atgatgccna arcaytgyyt nytnggnytn ytnathathy
tnytnwsnws ngcnacngar 60athcarccng cnmgngtnws nytnacnytn caraargtnm
gnttycarws nmgnaaytty 120cayaayathy tncaytggca rgcnggnwsn
wsnytnccnw snaayaayws nathtaytty 180gtncartaya aratgtaygg
ncarwsncar tgggargaya argtngaytg ytggggnacn 240acngcnytnt
tytgygayyt nacnaaygar acnytngayc cntaygaryt ntaytayggn
300mgngtnatga cngcntgygc nggnmgncay wsngcntgga cnmgnacncc
nmgnttyacn 360ccntggtggg aracnaaryt ngayccnccn gtngtnacna
thacnmgngt naaygcnwsn 420ytnmgngtny tnytnmgncc nccngarytn
ccnaaymgna aycarwsngg naaraaygcn 480wsnatggara cntaytaygg
nytngtntay mgngtnttya cnathaayaa ywsnytngar 540aargarcara
argcntayga rggnacncar mgngcngtng arathgargg nytnathccn
600caywsnwsnt aytgygtngt ngcngaratg taycarccna tgttygaymg
nmgnwsnccn 660mgnwsnaarg armgntgygt ncarathccn 690401050DNAMus
musculusCDS(50)...(589) 40aacaggctct cctctcactt atcaactttt
gacacttgtg cgatcggtg atg gct gtc 58 Met Ala Val 1ctg cag aaa tct
atg agt ttt tcc ctt atg ggg act ttg gcc gcc agc 106Leu Gln Lys Ser
Met Ser Phe Ser Leu Met Gly Thr Leu Ala Ala Ser 5 10 15tgc ctg ctt
ctc att gcc ctg tgg gcc cag gag gca aat gcg ctg ccc 154Cys Leu Leu
Leu Ile Ala Leu Trp Ala Gln Glu Ala Asn Ala Leu Pro 20 25 30 35atc
aac acc cgg tgc aag ctt gag gtg tcc aac ttc cag cag ccg tac 202Ile
Asn Thr Arg Cys Lys Leu Glu Val Ser Asn Phe Gln Gln Pro Tyr 40 45
50atc gtc aac cgc acc ttt atg ctg gcc aag gag gcc agc ctt gca gat
250Ile Val Asn Arg Thr Phe Met Leu Ala Lys Glu Ala Ser Leu Ala Asp
55 60 65aac aac aca gac gtc cgg ctc atc ggg gag aaa ctg ttc cga gga
gtc 298Asn Asn Thr Asp Val Arg Leu Ile Gly Glu Lys Leu Phe Arg Gly
Val 70 75 80agt gct aag gat cag tgc tac ctg atg aag cag gtg ctc aac
ttc acc 346Ser Ala Lys Asp Gln Cys Tyr Leu Met Lys Gln Val Leu Asn
Phe Thr 85 90 95ctg gaa gac att ctg ctc ccc cag tca gac agg ttc cgg
ccc tac atg 394Leu Glu Asp Ile Leu Leu Pro Gln Ser Asp Arg Phe Arg
Pro Tyr Met100 105 110 115cag gag gtg gtg cct ttc ctg acc aaa ctc
agc aat cag ctc agc tcc 442Gln Glu Val Val Pro Phe Leu Thr Lys Leu
Ser Asn Gln Leu Ser Ser 120 125 130tgt cac atc agt ggt gac gac cag
aac atc cag aag aat gtc aga agg 490Cys His Ile Ser Gly Asp Asp Gln
Asn Ile Gln Lys Asn Val Arg Arg 135 140 145ctg aag gag aca gtg aaa
aag ctt gga gag agc gga gag atc aaa gcg 538Leu Lys Glu Thr Val Lys
Lys Leu Gly Glu Ser Gly Glu Ile Lys Ala 150 155 160atc ggg gaa ctg
gac ctg ctg ttt atg tct ctg aga aat gct tgc gtc 586Ile Gly Glu Leu
Asp Leu Leu Phe Met Ser Leu Arg Asn Ala Cys Val 165 170 175tga
gcgagaagaa gctagaaaac gaagaactgc tccttcctgc cttctaaaaa
639gaacaataag atccctgaat ggactttttt actaaaggaa agtgagaagc
taacgtccac 699catcattaga agatttcaca tgaaacctgg ctcagttgaa
agagaaaata gtgtcaagtt 759gtccatgaga ccagaggtag acttgataac
cacaaagatt cattgacaat attttattgt 819cattgataat gcaacagaaa
aagtatgtac tttaaaaaat tgtttgaaag gaggttacct 879ctcattcctc
tagaagaaaa gcctatgtaa cttcatttcc ataaccaata ctttatatat
939gtaagtttat ttattataag tatacatttt atttatgtca gtttattaat
atggatttat 999ttatagaaaa attatctgat gttgatattt gagtataaag
caaataatat t 105041179PRTMus musculus 41Met Ala Val Leu Gln Lys Ser
Met Ser Phe Ser Leu Met Gly Thr Leu 1 5 10 15Ala Ala Ser Cys Leu
Leu Leu Ile Ala Leu Trp Ala Gln Glu Ala Asn 20 25 30Ala Leu Pro Ile
Asn Thr Arg Cys Lys Leu Glu Val Ser Asn Phe Gln 35 40 45Gln Pro Tyr
Ile Val Asn Arg Thr Phe Met Leu Ala Lys Glu Ala Ser 50 55 60Leu Ala
Asp Asn Asn Thr Asp Val Arg Leu Ile Gly Glu Lys Leu Phe65 70 75
80Arg Gly Val Ser Ala Lys Asp Gln Cys Tyr Leu Met Lys Gln Val Leu
85 90 95Asn Phe Thr Leu Glu Asp Ile Leu Leu Pro Gln Ser Asp Arg Phe
Arg 100 105 110Pro Tyr Met Gln Glu Val Val Pro Phe Leu Thr Lys Leu
Ser Asn Gln 115 120 125Leu Ser Ser Cys His Ile Ser Gly Asp Asp Gln
Asn Ile Gln Lys Asn 130 135 140Val Arg Arg Leu Lys Glu Thr Val Lys
Lys Leu Gly Glu Ser Gly Glu145 150 155 160Ile Lys Ala Ile Gly Glu
Leu Asp Leu Leu Phe Met Ser Leu Arg Asn 165 170 175Ala Cys
Val4220DNAArtificial SequenceOligonucleotide primer ZC10651
42agcttttctg cagcagctct 204323DNAArtificial SequenceOligonucleotide
primer ZC10565 43tttgcagaaa aggttgcaaa tgc 234420DNAArtificial
SequenceOligonucleotide primer ZC38001 44ccgttcgtga ctataacccg
204519DNAArtificial SequenceOligonucleotide primer ZC38022
45agccgtagta agtttccat 19464PRTArtificial SequenceSXWS polypeptide
motif 46Ser Xaa Trp Ser 147707DNAMus musculusCDS(2)...(691) 47c atg
atg cct aag cat tgc ctt cta ggt ctc ctc atc ata ctc ttg agc 49Met
Met Pro Lys His Cys Leu Leu Gly Leu Leu Ile Ile Leu Leu Ser 1 5 10
15agt gca aca gaa ata caa cca gct cgt gta tct ctg acg ccc cag aag
97Ser Ala Thr Glu Ile Gln Pro Ala Arg Val Ser Leu Thr Pro Gln Lys
20 25 30gtc cga ttt cag tcc aga aat ttc cac aat att ttg cac tgg caa
gca 145Val Arg Phe Gln Ser Arg Asn Phe His Asn Ile Leu His Trp Gln
Ala 35 40 45ggg agc tct ctc ccc agc aac aac agc atc tac ttt gtg cag
tac aag 193Gly Ser Ser Leu Pro Ser Asn Asn Ser Ile Tyr Phe Val Gln
Tyr Lys 50 55 60atg tat gga cag agc caa tgg gaa gat aaa gtt gac tgc
tgg ggg acc 241Met Tyr Gly Gln Ser Gln Trp Glu Asp Lys Val Asp Cys
Trp Gly Thr65 70 75 80acg gcg ctc ttc tgt gac ctg acc aat gaa acc
tta gac cca tac gag 289Thr Ala Leu Phe Cys Asp Leu Thr Asn Glu Thr
Leu Asp Pro Tyr Glu 85 90 95ctg tat tac ggg agg gtg atg acg gcc tgt
gct gga cgc cac tct gcc 337Leu Tyr Tyr Gly Arg Val Met Thr Ala Cys
Ala Gly Arg His Ser Ala 100 105 110tgg acc agg aca ccc cgc ttc act
cca tgg tgg gaa aca aaa cta gat 385Trp Thr Arg Thr Pro Arg Phe Thr
Pro Trp Trp Glu Thr Lys Leu Asp 115 120 125cct ccg gtc gtg act ata
acc cga gtt aac gca tct ttg cgg gtg ctt 433Pro Pro Val Val Thr Ile
Thr Arg Val Asn Ala Ser Leu Arg Val Leu 130 135 140ctc cgt cct cca
gag ttg cca aat aga aac caa agt gga aaa aat gca 481Leu Arg Pro Pro
Glu Leu Pro Asn Arg Asn Gln Ser Gly Lys Asn Ala145 150 155 160tcc
atg gaa act tac tac ggc tta gta tac aga gtt ttc aca atc aac 529Ser
Met Glu Thr Tyr Tyr Gly Leu Val Tyr Arg Val Phe Thr Ile Asn 165 170
175aat tca cta gag aag gag caa aaa gcc tat gaa gga act cag aga gct
577Asn Ser Leu Glu Lys Glu Gln Lys Ala Tyr Glu Gly Thr Gln Arg Ala
180 185 190gtt gaa att gaa ggt ctg ata cct cat tcc agc tac tgc gta
gtg gct 625Val Glu Ile Glu Gly Leu Ile Pro His Ser Ser Tyr Cys Val
Val Ala 195 200 205gaa atg tac cag ccc atg ttt gac aga aga agc cca
aga agc aag gag 673Glu Met Tyr Gln Pro Met Phe Asp Arg Arg Ser Pro
Arg Ser Lys Glu 210 215 220aga tgt gtg cac att cca tgaactggtc
tgaggc 707Arg Cys Val His Ile Pro225 23048230PRTMus musculus 48Met
Met Pro Lys His Cys Leu Leu Gly Leu Leu Ile Ile Leu Leu Ser 1 5 10
15Ser Ala Thr Glu Ile Gln Pro Ala Arg Val Ser Leu Thr Pro Gln Lys
20 25 30Val Arg Phe Gln Ser Arg Asn Phe His Asn Ile Leu His Trp Gln
Ala 35 40 45Gly Ser Ser Leu Pro Ser Asn Asn Ser Ile Tyr Phe Val Gln
Tyr Lys 50 55 60Met Tyr Gly Gln Ser Gln Trp Glu Asp Lys Val Asp Cys
Trp Gly Thr65 70 75 80Thr Ala Leu Phe Cys Asp Leu Thr Asn Glu Thr
Leu Asp Pro Tyr Glu 85 90 95Leu Tyr Tyr Gly Arg Val Met Thr Ala Cys
Ala Gly Arg His Ser Ala 100 105 110Trp Thr Arg Thr Pro Arg Phe Thr
Pro Trp Trp Glu Thr Lys Leu Asp 115 120 125Pro Pro Val Val Thr Ile
Thr Arg Val Asn Ala Ser Leu Arg Val Leu 130 135 140Leu Arg Pro Pro
Glu Leu Pro Asn Arg Asn Gln Ser Gly Lys Asn Ala145 150 155 160Ser
Met Glu Thr Tyr Tyr Gly Leu Val Tyr Arg Val Phe Thr Ile Asn 165 170
175Asn Ser Leu Glu Lys Glu Gln Lys Ala Tyr Glu Gly Thr Gln Arg Ala
180 185 190Val Glu Ile Glu Gly Leu Ile Pro His Ser Ser Tyr Cys Val
Val Ala 195 200 205Glu Met Tyr Gln Pro Met Phe Asp Arg Arg Ser Pro
Arg Ser Lys Glu 210 215 220Arg Cys Val His Ile Pro225
23049690DNAArtificial SequenceDegenerate polynucleotide sequence of
SEQ ID NO48 49atgatgccna arcaytgyyt nytnggnytn ytnathathy
tnytnwsnws ngcnacngar 60athcarccng cnmgngtnws nytnacnccn caraargtnm
gnttycarws nmgnaaytty 120cayaayathy tncaytggca rgcnggnwsn
wsnytnccnw snaayaayws nathtaytty 180gtncartaya aratgtaygg
ncarwsncar tgggargaya argtngaytg ytggggnacn 240acngcnytnt
tytgygayyt nacnaaygar acnytngayc cntaygaryt ntaytayggn
300mgngtnatga cngcntgygc nggnmgncay wsngcntgga cnmgnacncc
nmgnttyacn 360ccntggtggg aracnaaryt ngayccnccn gtngtnacna
thacnmgngt naaygcnwsn 420ytnmgngtny tnytnmgncc nccngarytn
ccnaaymgna aycarwsngg naaraaygcn 480wsnatggara cntaytaygg
nytngtntay mgngtnttya cnathaayaa ywsnytngar 540aargarcara
argcntayga rggnacncar mgngcngtng arathgargg nytnathccn
600caywsnwsnt aytgygtngt ngcngaratg taycarccna tgttygaymg
nmgnwsnccn 660mgnwsnaarg armgntgygt ncayathccn 690
* * * * *
References